#Measuring cellular Glutathione concentration

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A Review: Oxidative Stress during Lactation in Dairy Cattle-Juniper Publishers

JUNIPER PUBLISHERS-OPEN ACCESS JOURNAL OF DAIRY & VETERINARY SCIENCES

Keywords

Keywords:  Oxidative stress; Lactation; Hydrogen peroxidase

Abbreviations :  SOD: Superoxide Dismutase; GPX: Glutathione Peroxidase; ROS: Reactive Oxygen Species; GPX: Glutathione Peroxidase

Introduction

Lactation is an important period in dairy cows from the point of view of physiological changes taking place, which in turn produce measurable changes in the diagnostic parameters of the blood. Oxidative stress plays a key role in the onset or progression of numerous human and animal diseases. High metabolic demands during lactation can impact the oxidative status of dairy cows. Researchers have reported higher oxidative stress in high producing dairy cows when compared to average producing dairy cows. Stage of lactation has also been found to affect the oxidative status of the animal. Dairy cows undergo oxidative stress mainly during the peripartal period. The period of transition between late pregnancy and early lactation is associated with lipid and protein metabolic changes [1]. Oxidative stress is also considered a physiological stress on the secretary activity of the parenchyma and the onset of milk secretion is accompanied by high-energy demand and increased oxygen demand.

Excessive production of free radicals together with damage at the cellular level is controlled by cellular antioxidant defense systems. Antioxidants can be defined as defense substances that delay, prevent or eliminate oxidative damage to a target molecule. Antioxidant enzyme systems can be includes (e.g., superoxide dismutase, glutathione peroxidase and catalase) and non-enzymatic systems (e.g. vitamin E and selenium). SOD and GPx shows significant variation between breed, seasonal effect and breeding seasons with or without supplement of antioxidant.

Superoxide Dismutase

Superoxide dismutase (SOD) accelerates the dismutation of the toxic superoxide radical to hydrogen peroxide and is considered the first intracellular defense against reactive oxygen species. The cytosol of all eukaryotic cells contains CuZn-SOD. Determination of SOD is important in the evaluation of antioxidant status, under physiological or pathological conditions [2]. SOD is one of the components of intrinsic antioxidant system. It is responsible for dissemination of superoxide radicals. SOD catalyses the conversion of superoxide radical to hydrogen peroxidase.

Significantly higher level of plasma concentration of SOD has been observed in mid lactating cows than early lactation [3]; and from 3 weeks before parturition to 9 weeks after parturition has also been reported [4]. Higher erythrocyte SOD activity during lactation than pregnancy has been also observed [5]. The increased SOD actually observed in the animals of mid-lactation showed high individual variation, possibly difference of age, parity, level of milk production and other inherent stress, might have caused such as high SOD level; further it is to be considered that SOD enzyme is transient in its action and actively shows a spurt on spontaneous induction of stress which may possible until other homeostasis enzyme have removed the association ROS and vice versa. The rise in SOD activity during early and mid lactation is a marker of oxidative stress. Higher serum SOD activity might be due to physiological upgrading of this enzyme in an attempt to neutralize/mitigation of superoxide radical challenges and adaption of animals to oxidative stress in an attempt to improve the antioxidant status.

Superoxide dismutase (SOD) accelerates the dismutation of the toxic superoxide radical to hydrogen peroxide and is considered the first intracellular defense against reactive oxygen species. The cytosol of all eukaryotic cells contains Cu- Zn-SOD. Determination of SOD is important in the evaluation of antioxidant status, under physiological or pathological conditions [2].

Glutathione Peroxidase

Glutathione peroxidase (GPX) plays an important role in cellular antioxidant defense. Determination of GPX activity might be beneficial in the evaluation of selenium status and in the evaluation of antioxidant status [2].

The enhanced lipid mobilization to satisfy the increased energy requirement for milk production disrupts several inflammatory and immune functions [6] and promotes free radicals production by leukocytes and endothelial cells [7]. In physiological conditions, the antioxidant defence system, provided by enzymes and antioxidants, scavenges reactive oxygen species (ROS), thus limiting or preventing oxidative damage. An increased production of free radicals or deficiencies of antioxidants may lead to oxidative stress, which impairs physiological functions, thus contributing to health disorders in lactating animals [8]. As a matter of the fact, oxidative stress can increase the susceptibility of dairy cattle to several diseases and metabolic disorders, particularly during the transition period.

Gpx catalyzed the conversion of H2O2 to H2O produced in the course of normal cellular events. It is also catalyses the reduction of fatty acid hydroperoxides and 1-monoacylglycerol hydroperoxides. Another GPx in RBCs termed phospholipid hydroperoxides glutathione peroxidase participate in reduction of more complex phospholipid hydroperoxides using GSH. Selenium deficiency can be diagnosed by measuring decresed RBC GPx activity in some species ,however caution indicated in using this activity as a direct indicator of selenium status because polymorphism in GPx activity may be present [9].

The maximum stress during early lactation is attributed due to peak lactation and more colostrum secretion by mammary gland. There may be further decrease in GPx level at mid lactation as compared to control values. Possible reason for this might be the physiological adjustment and adaption of animal body to lesser production during mid-lactation and supply of nutrients. A decrease of mean blood GSH-Px in dairy cows during mid-lactation might be due to as a loss of homeostatic control in the postpartal period [10].

In contrast, opposite patterns, by means of, higher GPx level during late gestation as compared to both stages of lactation [10], and, in terms of a significant higher glutathione peroxidase (GPx) level in mid lactating cows than early lactation, and a decrease in GPX level during early lactation than during advanced pregnancy but no significant difference have been observed [3,11]. So it can be concluded that the numbers of factors including various physiological and environmental factors may be responsible for the oxidative stress. Superoxide dismutase catalyses the dismutation of superoxide radical to hydrogen peroxide which is further metabolized to water by GSH-Px enzyme.

This review gives evidence that the lactating animals undergo substantial metabolic and physiology adaptation during transition from non-lactation and early lactation to mid lactation that contribute to dysfunctional host inflammatory responses [12]. The rise of oxidative stress markers could be due to pregnancy and early lactation which are considered as stressful stages accompanied by a high metabolic demand and elevates the requirements for tissue oxygen [13] and causes an increase of reactive oxygen species production. During lactation in order to sustain lactogenesis and fatty acid consumption from the mother's fat reserve and production of hydrogen peroxide that has been enhanced by intense lipolysis and mobilization of fatty acids from the body deposits [2].

The Se status is most frequently assessed either directly from Se concentrations, or indirectly from GSH-Px activity assessment [14]. Selenium is a very important essential trace element for proper intrauterine and postnatal development of calves. Although Se passes both placental and mammary barriers, placental transfer is more effective than the transport of Se into milk [15].

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Why Drink Hydrogenated Water?

"Don't forget to drink water and get sunshine. You are basically a houseplant with complicated emotions. "  -- Anonymous

A Tall Drink of Water

Last week we discussed what antioxidants are and why are they important for good health.

For nearly 15 years there has been a lot of focus on hydrogenated water to neutralize the effects of oxidative stress. I have been following this in the research for the last few years. To date, molecular hydrogen has been shown to have therapeutic potential in over 170 human and animal models and in essentially every organ of the body. A great resource for these studies and other information on molecular hydrogen can be found at the Molecular Hydrogen Institute website. Recent studies have shown that hydrogen water provides antioxidant properties, supports cellular energy production, and may help with inflammation. 

Hydrogen is the first and the lightest in the periodic table of chemical elements consisting of one proton and one electron. Hydrogen is the most abundant chemical substance in the universe, constituting roughly 75% of all normal matter. Hydrogen is a tasteless, colorless, and odorless gas. When added to water, it becomes hydrogen water or hydrogenated water. Regardless of which naming convention is used, studies have shown that hydrogen water supports cellular energy production and provides antioxidant and anti-inflammatory properties.

It is interesting to note that hydrogenated water has been associated with natural springs around the world that are famous for healing and longevity.

Oxidative stress occurs when free radicals and the body's antioxidants are out of balance. Factors such as poor diet, a sedentary lifestyle, air pollution, exposure to industrial chemicals, pesticides, and environmental toxins can increase free radicals within the body. Hydrogen water helps combat oxidative stress to neutralize the effects of free radicals.

It is interesting to note that hydrogenated water has been associated with natural springs around the world that are famous for healing and longevity. Waters with documented therapeutic benefits and contain hydrogen gas include Nordenau, Germany; Tiacote, MX;  Hita Tentyosui, Japan; and Nadone, India just to name a few.

Five simple reasons why hydrogenated water is effective.

1.     Hydrogen is the smallest molecule and easily enters the cell and cellular organelles like mitochondria easier than any other antioxidant.

2.     Molecular hydrogen can convert toxic oxygen radicals to water.

3.     Hydrogen triggers an increase in our body’s own antioxidant system like glutathione and other cytoprotective enzymes.

4.     Hydrogen exerts a beneficial effect on cell signaling, cell metabolism, and gene expression, resulting in anti-inflammatory, anti-allergy, and anti-obesity effects.

5.     Hydrogenated water has a high safety profile and doesn’t perturb homeostasis.

This excess of free radicals can be neutralized with antioxidants. Molecular hydrogen has been found to be effective against free radicals. Studies have shown that drinking hydrogen-enriched water can be a good way to help reduce oxidative stress and restore balance to the body.

Just recently Nikken introduced an exciting new product, the PiMag® PiDrogen Hydrogen Generator. It is a mini-generator accompanied by its own 16 fl oz / 475 ml glass bottle. The generator produces hydrogen-rich water that is rich in antioxidants and the glass bottle allows you to drink directly from it. The device is compact and lightweight and produces 800-1200 ppb (parts per billion), the measurement of molecular hydrogen dissolved in water.

PiDrogen system transforms PiMag water alkaline in a source of highly dissolved hydrogen gas concentrated. When consumed, the water in the device vo PiDrogen provides a large number of antioxidants to help relieve oxidative stress and restore balance to the body. Its exclusive state-of-the-art technology and compact design allow you to take it everywhere. The PiMag Hydrogen Generator ® PiDrogen by NIKKEN ® has been exclusively designed to provide all the benefits of hydrogen. Use the PiMag ® PiDrogen with  PiMag Waterfall® water and enjoy all its benefits.

Be Healthy by Choice

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Vitamin c and photo damage in skin..

Several reports have indicated that vitamin C levels are lower in aged or photodamaged skin [25,26,27]. Whether this association reflects cause or effect is unknown, but it has also been reported that excessive exposure to oxidant stress via pollutants or UV irradiation is associated with depleted vitamin C levels in the epidermal layer [33,34].

vitamin c serum for face

Indeed, more vitamin C is found in the epidermal layer than in the dermis, with differences of 2–5-fold between the two layers being consistently reported (Table 1 and [25,26]). Levels of vitamin C in skin are similar to the levels of other water soluble antioxidants such as glutathione [25,26,27,35]. There is a suggestion that vitamin C in the stratum corneum layer of the epidermis exists in a concentration gradient [36]. The lowest vitamin C concentration was present at the outer surface of the epidermis of the SKH-1 hairless mouse, a model of human skin, with a sharp increase in concentration in the deeper layers of the stratum corneum, possibly reflecting depletion in the outer cells due to chronic exposure to the environment [36]. 3.1. The Bioavailability and Uptake of Vitamin C into the Skin 3.1.1. The Sodium-Dependent Vitamin C Transporters

Vitamin C uptake from the plasma and transport across the skin layers is mediated by specific sodium-dependent vitamin C transporters (SVCTs) that are present throughout the body and are also responsible for transport into other tissues. Interestingly, cells in the epidermis express both types of vitamin C transporter, SVCT1 and SVCT2 (Figure 2) [37]. This contrasts with most other tissues, which express SVCT2 only [37,38,39]. SVCT1 expression in the body is largely confined to the epithelial cells in the small intestine and the kidney and is associated with active inter-cellular transport of the vitamin [40,41]. The specific localisation of SVCT1 in the epidermis is of interest due to the lack of vasculature in this tissue, and suggests that the combined expression of both transporters 1 and 2 ensures effective uptake and intracellular accumulation of the vitamin. Together with the high levels of vitamin C measured in the epidermal layer, the dual expression of the SVCTs suggests a high dependency on vitamin C in this tissue.

vit c serum

Both transporters are hydrophobic membrane proteins that co-transport sodium, driving the uptake of vitamin C into cells. Replacement of sodium with other positively charged ions completely abolishes transport [42]. SVCT1 and SVCT2 have quite different uptake kinetics reflecting their different physiological functions. SVCT1 transports vitamin C with a low affinity but with a high capacity (Km of 65–237 µmol/L) mediating uptake of vitamin C from the diet and re-uptake in the tubule cells in the kidney [41]. SVCT2, which is present in almost every cell in the body, is thought to be a high-affinity, low capacity transporter, with a Km of ~20 µM meaning it can function at low concentrations of vitamin C [41]. As well as transporter affinity, vitamin C transport is regulated by the availability of the SVCT proteins on the plasma membrane. 3.1.2. Bioavailability and Uptake

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Assessment of Growth, Lipid Peroxidation and Reactive Oxygen Species Scavenging Capacity of Ten Elite Cassava Cultivars Subjected to Heat Stress-Juniper Publishers

Cassava is an important source of energy-giving food in the developing countries [1]. Cassava productivity is stable and reliable, making the crop a candidate for reducing food insecurity, hunger and poverty in developing countries [2]. Under normal growth conditions, the crop gives high tuber yield and when the growth conditions are sub-optimal, cassava tuber yield is satisfactory [1]. For these reasons, cassava production in developing countries is expanding, a situation that makes the crop suitable for meeting Sustainable Development Goals (SDG) [2]. However, empirical evidence from climate change studies suggested that most cassava production areas would experience global warming and temperature extremes [3]. Indeed, heat wave (high temperatures) has been reported during growing period of cassava in Africa, Asia and Latin America [3]. As a warm temperate crop, cassava has best shoot and root growth and development at 25-32 ˚C [2]. Tempera tures above the normal optimum are sensed as heat stress. Heat stress upsets cellular equilibrium and lead to severe retardation of growth and development, and even result in plant death [4]. One of the physiological damages of oxidative stress caused by heat stress is lipid peroxidation. Peroxidation results in the breakdown of lipids and membrane functions by causing loss of fluidity, lipid cross-linking, and inactivation of membrane enzymes [5]. The extent of lipid peroxidation can be evaluated by measuring thiobarbituric acid reactive substances (TBARS) content, which is a secondary breakdown product of lipid peroxidation [6]. Hydrogen peroxide is the product of the first detoxification process of superoxide radical by SOD before scavenging by CAT and other peroxidases. Hydrogen peroxide production invariably measures ROS scavenging ability of plants under heat stress. Environmental stresses such as heat stress induce the accumulation of proline in many plant species [4]. Proline plays a role in cellular osmoregulation and also exhibits many protective effects; plants with elevated proline levels were reported to exhibit enhanced tolerance to abiotic stresses [7]. Levels of proline can be increased either by stimulation of its biosynthesis by 1-pyrroline-5-carboxylate synthetase(s) (P5CS) or by inhibition of its degradation by proline dehydrogenases [7].

Heat stress triggered an upsurge in production of reactive oxygen species (ROS) such as superoxide radical (O2 −), singlet oxygen (1O2), hydrogen peroxide (H2O2) and hydroxyl radicals (OH•) [8]. The ROS are produced from different sources in plants. Heat stress causes ROS production in chloroplasts and mitochondria by disturbing membrane stability and biochemical reactions such as the activity of ribulose-1,5-bisphosphate carboxylase/oxygenase [9]. In addition, ROS are produced in mitochondria from membrane instability, resulting in photorespiration and enzymes involved in cellular respiration such as complex I and III in the mitochondrial electron transport chain [10]. Furthermore, ROS are produced from NADPH oxidases (NOX) in the plasma membrane, amine oxidase in the apoplast and xanthine oxidase in peroxisomes, which are all induced by environmental stimuli including heat [11,12]. Excessive production of ROS under heat stress damages plant cells and tissues permanently by oxidation of cellular components such as lipids, proteins and DNA [13]. To remove excessive ROS, plants have developed detoxifying enzymes such as superoxide dismutase (SOD), catalase (CAT), peroxidases (POX), glutathione reductase (GR), ascorbate peroxidase (APX), and non-enzymatic antioxidants such as ascorbate, glutathione, carotene and tocopherols [13,14]. Apart from their destructive effects in cells, ROS can also act as signaling molecules in many biological processes such as stomatal closure, growth, development, and stress signaling [15]. Due to this dual role of ROS, plants are able to fine-tune their concentrations between certain thresholds by means of production and scavenging mechanisms. Since this ROS homeostasis is disrupted under stress in favour of production, constitutive and induced enzymatic antioxidant defenses are considered a crucial component of plant stress tolerance [8,14].

Physiological, antioxidant defence capacity and molecular responses of cassava to drought stress have been reported [16- 18]. In the same vein, the antioxidant defence capacities of wheat [19], rice [20], maize [20] have been investigated in response to heat stress. However, responses and antioxidant defence capacity of cassava to heat stress has not been reported. Equally, genetic improvement of cassava for heat tolerance has not been given adequate research attention. The objective of this study was to assess growth, lipid peroxidation and reactive oxygen species scavenging ability of ten commercial cultivars of cassava.

    Materials and Methods

Planting materials and growth conditions

Stem cuttings of cassava cultivars TMS 4 (2) 1425, TMS 97/3200, TMS 91/02324, TMS 98/0505, TMS 98/0510 TME 419, TME 12, UMUCASS 36, UMUCASS 37 and UMUCASS 38. were obtained from the International Institute of Tropical Agriculture (IITA), Ibadan. A stem cutting (10cm long), with more than two nodes, was planted per plastic pot containing 8 kg sterilized sandy loam soil with: рH оf 7.2 аnd саtiоn еxсhаngе сарасitу оf 15.3 сmоlkg-1. Daily, each plant was irrigated manually with 600mL to water holding capacity bу tар wаtеr, рH 6.8. Plants were grown at an average temperature of 26±2 ˚C under 65±5% relative humidity and 7-9 hours of daylight before and after heat treatment.

Heat treatment and experimental design

Four weeks after planting, temperature was raised from 26 ˚C and maintained at 40 ˚C for 30 minutes. The experimental design was randomized complete-block in three replications. Fifteen uniform plants were used per cultivar.

Measurement of growth parameters

At four weeks after planting and before heat treatment, number of leaf, ѕhооt hеight, leaf аrеа, number of root and drу wеight (biomass) of plants were dеtеrminеd. This was repeated four weeks after the heat treatment and the differences recorded as growth after exposure to heat stress. For drу wеight, plants were carefully removed to obtain intact roots. Adhering soil particles on roots were removed by dipping them in water before dried in аn оvеn at 80 ˚C to a constant weight. Lеаf area was mеаѕurеd by a leaf area meter.

Lеаf рrоlinе соntеnt

To examine the osmotic adjustment of plants, proline content of the third fully expanded leaf from the top was determined according to Bates et al. [21] 24 hours after heat treatment. Leaf tissues (3g) were extracted in 2ml of sulphosalicylic acid. The same volume of ninhydrin solution and glacial acetic acid was added. The samples were heated at 100 ˚C for 10 minutes, cooled in an ice bath and 5 ml of toluene was added. At 528 nm, absorbance by toluene was measured.

Phеnоliсѕ соntеnt

The method of Julkunen-Titto [22] was used to determine leaf total phenolics content 24 hours after heat treatment. Briefly, fresh tissues (0.5g) of third fully expanded and matured leaves from shoot tip were ground in 80% acetone and the homogenized mixture collected. Thereafter, a mix of Folin-Ciocalteu reagent (1ml), water (2ml) and the supernatant (0.1ml) were homogenized and vigorously shook for 10 minutes. To the mix was added 5ml of Na- 2CO3 and the volume was brought to 10ml using distilled water. Absorbance was read at 750nm wavelength.

Antioxidant enzyme assays

Enzyme activities were assayed from the fourth fully expanded leaves from the shoot tip 24 hours after heat treatment. After washing with distilled water, leaf sample (0.5g) was ground in cold 0.1mol/l phosphate buffer (pH 7.5) containing 0.5mmol/l EDTA. The homogenized mixture was centrifuged at 4 ˚C for 15 minutes at 15,000 x g. The supernatant served as enzyme assay in this study.

Ascorbate peroxidase

Determination of activity of ascorbate peroxidase (APX) as outlined by Nakano et al. [23] was followed. The 3ml-reaction mixture contained 50mmol/l potassium phosphate (pH 7.0), 0.2mmol/l EDTA, 0.5mmol/l ascorbic acid, 2% H2O2 and 0.1ml of enzyme extact. For one minute, a drop-in absorbance at 290 nm was noted. Oxidation of ascorbate was calculated using the extinction coefficient Ɛ =2.8/mmol/l/cm. One unit of APX activity was defined as one mmol ascorbate oxidized /ml /min at 25 ˚C.

Superoxide dismutase

The method of Dhindsa and Dhindsa [24] was followed for determination of activity of superoxide dismutase (SOD). In this study, a unit of SOD was the enzyme extract that caused photo-reduction of a half of inhibition of nitro-blue tetrazolium and SOD activity expressed as unit/mg protein.

Catalase

Activity of catalase (CAT) was measured as described by Aebi [25]. A 3ml-reaction mixture containing 0.1ml enzyme extract, 50mmol. /l phosphate buffer (pH 7.0 and 30mmol/l hydrogen peroxide was conducted. Activity of CAT was determined by recording absorbance of hydrogen peroxide at 240nm.

Peroxidase

The method of Hemeda and Klein [26] was used to determine activity of реrоxidase (POD) in a reaction mixture that contained enzyme extract, 0.05% guaiacol, 25mmol/l phosphate buffer (pH 7.0), 10mmol/l hydrogen proxide. The POD activity was determined by absorbance at 470nm [ε = 26.6/(mmol/l cm).

Statistical analysis

A one-way analysis of variance was performed on data to determine significance of the treatment effect using Statistical Analysis Systems 9.1.3. At 5% level of probability, treatment means were separated by Ducan’s Multiple Range Test.

    Results

Analysis of variance showed that cultivars differed for all growth traits at 1% level of probability (Table 1). The R2 ranged from 91.4 to 96.3%, while coefficient of variation ranged between 7.3 and 15.8%. All cultivars increased their shoot height, number of leaf, leaf area, root number and dry weight after exposure to heat stress (Table 2). Shoot height ranged from 17.6cm in TME 419 and 3.7cm in TMS 98/0510. The highest shoot (17.6cm) was more than triple the least shoot heights (10.1-11.9cm) observed in TMS 98/0510, TMS 98/0505, TMS 91/02324 and TMS 97/3200. Leave production was ranged from 5.7 per plant in TME 419 and TME 12 to 2.5 per plant in TMS 98/0510 (Table 2). Leaf area varied from 44.3 to 25.6cm2/plant the highest leaf area was observed in TME 419, followed by UMUCASS 37, UMUCASS 38 and TME 419. The number of roots ranged from 5.5 per plant in TME 419 to 2.6 per plant in TMS 4 (2) 1425, TMS 97/3200, TMS 91/02324, TMS 98/0505 and TMS 98/0510. Similarly, TME 419 recorded highest (12.6g per plant) dry weight but TMS 97/3200 had the lowest (Table 2).

Analysis of variance showed that cultivars differed for all physiological traits measured at 1% level of probability (Table 3). The R2 ranged from 85.4 to 95.1%, while coefficient of variation ranged between 12.3 and 23.1%. Lipid peroxidation ranged from 30.1 to 16.7mg/g of TBARS. The highest lipid peroxidation was observed in TMS 4 (2) 1425, TMS 97/3200, TMS 91/02324, TMS 98/0505 and TMS 98/0510 and least in TME 419 and TME 12 (Figure 1). Hydrogen peroxide production ranged from 24.6 to 38.5μg/g. Hydrogen peroxide production was highest in TMS 98/0505 and TMS 98/0510 and lowest in TME 12. Among the cultivars, proline content ranged 3.2 to 8.6mg/g while phenolic ranged from 14.0 to 24.0mg/g. While the highest proline and phenolic were produced by TME 419, the lowest proline was recorded in TMS 4 (2) 1425, TMS 97/3200, TMS 91/02324, TMS 98/0505 and TMS 98/0510 and lowest phenolic was found in TMS 97/3200, TMS 91/02324, TMS 98/0505, TMS 98/0510, UMUCASS 36, UMUCASS 37 and UMUCASS 38 (Figure 1).

Activity of ascorbate peroxidase ranged between 0.8 and 1.5mmol ascorbate/ ml/min while the activity of superoxide dismutase ranged between 4.0–6.7 unit/mg protein. TME 419, TME 12, UMUCASS 36, UMUCASS 37 and UMUCASS 38 had higher ascorbate peroxidase and superoxide dismutase activities than remaining cultivars (Table 4). In this study, catalase and peroxidase activities ranged from 25.4-34.3 unit/mg protein and 0.4-0.8 unit/mg protein, respectively. However, catalase activity of TMS 4 (2) 1425, TMS 97/3200 TMS 91/02324 TMS 98/0505, UMUCASS 36 and TMS 98/0510 was higher than that of TME 419, TME 12, UMUCASS 37 and UMUCASS 38. Cultivars were grouped into two by peroxidase (POD) activity: the POD activity of the first group (TMS 4 (2) 1425, TMS 97/3200, TMS 91/02324, TMS 98/0505, TMS 98/0510 and TME 419) doubled POD activity of the second group (TME 12, UMUCASS 36, UMUCASS 37 and UMUCASS 38, Table 4).

    Discussion

Like other crops, cassava plants experience heat stress on the field at all stages of its life cycle. Heat stress is exacerbated by climate change and long growth cycle of cassava [2,3]. Heat stress elicits molecular reactions in plants which triggers sequences of physiological responses that manifested in morphological alterations and adjustments [3]. In this study, it is noteworthy that exposure of cassava young plants to high temperature (heat) did not lead to total loss of growth in the ten cultivars investigated. Rather, cultivars displayed varying degree of adjustments of morphological traits as observed in shoot height, leaf area, root formation and dry matter accumulation as allowed by their genetic constituent. This implying that heat stress may not markedly reduce cassava productivity of these popular cassava cultivars in Africa as the plant may possess functioning heat tolerance mechanism. Variability in growth responses among cassava cultivars observed in this study agrees with previous report on morphological response to heat stress wheat, maize and rice [19,20]. For instance, after heat stress, cultivar 84-S had relative growth of 0.97g/g/day whereas M-503 had relative growth of 0.101 g/g/day in cotton [27].

Plants have developed several heat stress adaptive responses. In the present study, rapid increase in shoot height, to provide certain physiological and metabolic advantages, could be heat stress adaptive mechanism by TME 419 which is not present in other cultivars. Adjustment in leaf formation is a vital stress-adaptive mechanism in cassava to maintain metabolic processes. In the present study, all cultivars continued production of leaves at varying degree after exposure to heat stress indicating leaf formation cessation was not caused by heat stress in the cultivars. However, four cultivar displayed outstanding leaf production under heat stress suggesting their tolerance to heat stress. Furthermore, roots are essential organ of plants providing anchorage and extracting water and nutrients for plants. All cultivars retain their roots following exposure to heat stress indicating water and nutrient absorption might not be severely disrupted under heat stress in cassava. No plant was lost to heat stress. Dry matter accumulation of most cultivars was impressive, suggesting cassava has heat tolerance mechanism that allows dry matter production under heat stress.

Our data suggested that heat stress caused damage to lipids in cassava at varying magnitude across cultivars. Lipid oxidation by reactive oxygen species has been established to be produced by heat stress. For example, lipid peroxidation in cultivar 84-5 increased by 79.9% by heat stress [27]. Limited lipid peroxidation displayed in TME 419 and TME 12 could have resulted from low quantity of ROS generated by the cultivars or destruction of ROS by antioxidant enzymes. In addition, all cultivars produced hydrogen peroxide, an ROS generated by heat stress indicating negative metabolic machinery of the plants which must be removed to prevent damage of proteins, lipids and DNA. Limited amount of hydrogen peroxide observed in three cultivars (TMS 4 (2) 1425, TME 419, TME 12) indicated that the cultivars have capacity to remove hydrogen peroxide and thus tolerance of heat stress. While heat stress increased hydrogen peroxide production by 50.0% in drought-sensitive cultivar in cotton, heat stress has no effect on hydrogen peroxide release drount-tolerant cultivar [27].

Proline (av. 5mg/g) was detected in all cultivars after exposure to heat stress. Gathering of proline to high concentration is one of the early physiological reactions of plants experiencing abiotic stress to amelioriate its negative effects. After six hours of heat stress, high content (2.8-3.9pmol/g FW) of proline was observed in lower leaves of wild and transgenic tobacco [28]. Drought-sensitive cotton cultivar 84-S increased proline content by 5.9% after exposure to heat stress [27]. We suggest that TME 419 and TME 12 that recorded outstanding quantity (6-7mg/g) of proline to be exhibiting heat tolerance. Phenolics are produced by plants mainly for protection against biotic and abiotic stresses. All cultivars produced high amount of phenolic suggesting that they were capable of protecting themselves against adverse effects of heat stress.

Our results showed that APX, SOD, POD and CAT were active in all cultivars subjected to heat stress in this study. Heat stress has no effect on CAT activity in cotton [27]. Heat stress decreased POD activity in cotton.by 41.3%. APX activity increased by heat stress in some cultivar of cotton while heat stress had no effect on APX in other cultivars of cotton. This is important because toxicity of ROS to plants necessitated their immediate removal before destroying cellular components [29]. Thе ROS аrе rеmоvеd bу these аntiоxidаnt еnzуmеѕ which findings have suggested are involved in stress tolerance in plants.

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Formulation and Characterization of Glutathione-Loaded Bioadhesive Hydrogel for Ocular Delivery-Juniper Publishers

JUNIPER PUBLISHERS- ACADEMIC JOURNAL OF POLYMER SCIENCE

Abstract

Glutathione may prevent age-related, oxidative damage to ocular tissues but has poor corneal penetration. Hydrogel formulations were investigated to determine an optimized ocular delivery system. The rheology and texture profile of formulations were investigated at 25°C. The 1% w/v glutathione was incorporated into systems demonstrating desirable characteristics for ocular vehicles and physical characteristics re-determined. In vitro cumulative glutathione delivery across the bovine cornea was measured over 8 hours at 32ºC in Franz diffusion cells. Carbopol-containing Poloxamer systems exhibited shear-thinning behavior desirable for ocular formulations whilst Polyvinyl alcohol (PVA) and Polyacrylic acid (PAA) systems exhibited Newtonian behavior. Of the glutathione-containing systems, 0.2% w/v Carbopol 1342 was the most viscous with a viscosity of 960cP at a shear rate of 100sec-1. All formulations significantly increased the amount of glutathione delivered across the cornea (relative to an aqueous solution of glutathione) with the exception of 0.1% w/v Carbopol 940 (p=0.12). Formulations containing 0.1% w/v Carbopol 934, 0.1% w/v Carbopol 1342 and 0.2% w/v Carbopol 940 improved penetration dramatically (ca. 30%); but were not significantly different from each other. Therefore, Carbopol and Poloxamer formulations demonstrated enhanced penetration of glutathione across the cornea. The 0.2% w/v C940-containing-Poloxamer formulation was determined to be the most promising for ocular delivery of glutathione.

Keywords: Glutathione; Hydrogel; Ocular delivery; Rheology; Newtonian behavior; Sustained release; Permeation

Introduction

Glutathione (γ-glutamyl-cysteinyl-glycine; GSH) is a highly hydrophilic thiol tri-peptide with a low-molecular weight of 307.4Da [1,2] The reduced form of GSH serves as a strong intracellular antioxidant [1,3] and plays an important role in the protection of the eye from oxidative stress [4]. In addition to its antioxidant activity, glutathione serves as a cofactor of enzymes involved in the degradation of peroxides, such as glutathione peroxidises. Moreover, as a component of NADPH pathway, it prevents cell components from being oxidized thus promoting DNA regulation and protein synthesis [3]. Franco et al [5] also reported glutathione’s role in the regulation of cell-cycle, signal transduction and immune response [5]. Given that all ocular tissues contain GSH as a primary antioxidant, replenishing the amounts of GSH in the eye could reduce the incidence of ocular diseases related to oxidative stress and aging, such as cataracts. The cornea is particularly susceptible to chemical insults and damage because it is the outer-most layer of the eye and therefore exposed to the harsh environment. GSH maintains normal hydration, protects the cellular membrane and degrades xenobiotic agents [4] in the cornea. Therefore, GSH may play a role in the treatment of keratitis and other corneal diseases [4].

Due to anatomical and physiological characteristics, the eye is uniquely shielded and a highly protected organ that presents many barriers to effective ocular drug delivery [6]. The main physical barriers shielding the eye are tear film and cornea, which protect the anterior of the eye, Tear film is a protective layer that prevents the entry of foreign molecules into the eye. The blood-retina barrier, which protects the back of the eye [7]. The cornea is the main barrier against drug penetration to the inner tissues of the eye due to its small surface area and relative impermeability [8]. In addition, many ocular enzymes in the cornea could degrade glutathione and prevent its therapeutic effect. These enzymes include endopeptidases (plasmin, collagenase) and exopeptidases, such as the hydrolytic aminopeptidase, which degrade amino acids [9].

The main advantages of stopical administration are its ease and convenience of use and localized drug effects, thus, reducing systemic absorption and avoiding enzymatic degradation through hepatic first pass metabolism [10]. However, the natural ocular defense mechanisms lead to poor bioavailability. Blinking increases the production of the tear film’s aqueous layer which in turn increases tear turnover to approximately 1μL/min, which is equivalent to 16% of the tear film turnover [11]. Both the removal of excess fluid and tear turnover reduce the extent of drug absorption. Conventional eye drops require frequent administration, with each drop being more than 30μl, which is estimated to be the maximum volume the eye can accommodate without overflow. Drainage of instilled solution occurs within 15–30 seconds after administration [12], therefore the majority of the drugs administered are removed before absorption. Although ocular ointments have the viscosity to increase pre-ocular drug retention time, they often cause irritability to the eye, resulting in lower compliance. In addition, their oily medium is not compatible with water soluble drugs such as GSH. Consequently, an ocular formulation with desirable characteristics that shows promising potential would be required to effectively deliver glutathione. This delivery system should prevent enzymatic degradation of glutathione, enhance its pre-corneal retention and penetration.

Hydrogels can be formed by dispersing polymers in an aqueous medium where they undergo swelling to produce a viscous gel capable of increasing pre-corneal retention time, prolonging contact time by increasing mucoadhesion and controlling drug release [12]. These properties increase trans-corneal absorption and hence the amount of drug reaching the anterior chamber of the eye. Poloxamers are non-ionic triblock copolymers comprised of ethylene oxide and propylene oxide, which are non-irritating to the ocular surface. With favorable characteristics such as strong hydrogen-bonding, high MW and sufficient flexibility to interact with the mucus network contribute to excellent mucoadhesion. Another defining characteristic include their ability to increased stability of drugs, Carbopol (CP) is a synthetic high-MW polymer comprised of acrylic acid that are cross-linked with either allyl ether of pentaerythritol or allyl sucrose [3]. They are anionic and therefore offer increased mucoadhesion hence contact time [13]. In addition, CP is reported to open the cellular tight junctions and could promote trans-corneal penetration of glutathione [14]. PVA has been used extensively in ocular formulations due to its ability to maintain ocular osmotic pressure while PAA has been widely used as a viscosity enhancer [2]. In this project, we developed a novel hydrogel formulation which composed of CP, PAA and PVA, and used as a carrier system for ocular delivery of GSH. This delivery system is aimed to enhance the permeation of the GSH over the cornea, thus, to maximize the bioavailability of GSH via ocular administration.

Materials and Methods

L-glutathione reduced≥99% was purchased from Sigma-Aldrich (USA). P407 and P188 were purchased from BASF (Germany). C934NF were from Noveon (USA) while C940NF and C1342NF polymer were purchased from Lubrizol (USA). PVA came from Applichem (Germany) and PAA was from Polyscience Inc (USA). Sodium hydroxide (NaOH) was from Scharlau (Spain). All other chemicals and solvents were of analytical grade. Bovine eyes were obtained from Auckland Meat Processors (New Zealand) and stored at20°C until required.

Methods

Preparation of Formulations

The CP formulations were prepared by adding the required amount of CP to 35mL of milli-Q water and were continuously stirred until the CP was completely dissolved. The solution was cooled to 4°C and the required amount of Poloxamer 407 (P407) and Poloxamer 188 (P188) were added with gentle stirring. The formulation was stored in the refrigerator and stirred every 30 minutes until the Poloxamers were completely dissolved. Milli-Q water was added to make up to a final volume of 50mL. The pH of the formulations was then adjusted to 7.4 by the addition of a small amount of 1M of NaOH. (Table 1) lists the samples prepared using the method mentioned above.

PVA formulations (Table 2) were prepared by adding the required amount (w/v) to 35mL of milli-Q water, which was then heated to 50°C under constant stirring until all PVA was dissolved and made up to 50mL with milli-Q water. The pH was adjusted to 7.4 by adding a small amount of 5M NaOH. The corresponding concentration of PAA samples were added according to (Table 2) and dilutions were made with milli-Q water. The control used in the screening of the formulations was milli-Q water.

The polymer combinations were shown in (Table 3). The CP formulations containing glutathione were prepared by dissolving 0.5g of glutathione in milli-Q water. Once glutathione was dissolved, the required quantity of CP was added. The solutions were continuously stirred until CP was completely dispersed and the Poloxamers were prepared according to the method described above. The control for samples mentioned in (Table 3) was aqueous solution of glutathione. Simulated tear fluid (STF) was prepared based on the method used by Hagerstrom et al [15]. Based on the physical parameters obtained, several potential formulations with glutathione will be investigated in this study.

Characterization

Rheological Properties

The rheological properties were examined using the Brookfield DV-III+ rheometer (Brookfield Engineering Laboratories, Inc. USA) with spindle 40 for PVA and PAA samples and spindle 52 for the CP formulations. This was carried out in a temperature- controlled environment at 25±1°C to simulate average room conditions. Each sample went through 20–40 loops of shearing, in between which there was a 10 second delay. The speed of shearing increased by 5rpm at each loop and the torque was kept between 9 to 90% by manipulating the starting rpm to ensure accuracy of data. The range of rotational speed was between 1 and 394 rpm corresponding to a shear rate range between 2 and 788s-1.

Mechanical and Mucoadhesive Properties

TA-XT plus texture analyzer (Stable Micro Systems, UK) with a 5kg load cell at 25±1°C was used. Texture analyzer was calibrated. For the measurement of the mechanical properties, a 10mm diameter delrin cylinder probe was twice compressed into each formulation at the rate of 2 mms-1 to a depth of 5mm with a trigger force of 0.1g. A delay of 15 seconds was allowed between the compressions. Data collection and calculations were gathered from the Texture Exponent 3.0.5.0. The force-time plot measured mechanical properties such as hardness, compressibility, adhesiveness and cohesiveness. This experiment was conducted in triplicate.

Hardness is measured by the peak force of the first compression cycle. Compressibility is measured as the positive force area during the first compression of the probe [3,16]. Adhesiveness is the negative force area of the first compression [16]. Cohesive force of each sample is the ratio of the first and second positive force area of the two consecutive compressions.

Mucoadhesive force measurements were performed on freshly prepared bovine cornea using TA-XTplus texture analyzer (Stable Micro Systems, UK) with a 5kg load cell at 25±1°C. The cornea was stabilised on the mucoadhesion test rig. A small amount of sample was applied to the 10mm delrin cylinder probe surface, after which the probe was lowered at a constant speed of 2mms-1 and a trigger force of 5g. After 10 seconds of contact, the probe was moved away at a speed of 0.5mms-1, generating a curve. The area under the curve (AUC) was calculated by the Texture Exponent 3.0.5.0 program. The work of mucoadhesion was calculated through Equation 1[3]. Each measurement was conducted in triplicates.

where πr² is the surface area of the cornea.

Permeability Studies

Ex-vivo drug permeation studies were performed by Franz diffusion cell (VTC 200, Logan Instrument Corp). The thermostat of the Franz diffusion cells was set at 32±1°C to mimic the ocular surface temperature. Bovine cornea is placed between the donor and the receptor chamber. Twelve mL of STF served as the dissolution medium in the receptor chamber which functioned as the reservoir. One mL of formulation was placed in the donor chamber and the cap was covered with parafilm to prevent evaporation. Samples (0.4mL) were collected at predeterminate time intervals and replaced by 0.4mL of STF for the first four hours, after which a greater amount of sample was withdrawn and replaced in order to maintain sink condition. UV spectrometer was blanked with STF at the wavelength of 215 nm and the absorbance of each sample was obtained. The absorbance for samples presented in Table 3 was measured and concentrations were calculated. In order to evaluate the penetration rate, the apparent permeability coefficient (Papp) was calculated, using the following equations.

where ΔQ / Δt is the steady-state of the linear portion of the graph which presents the amount of drug in the receptor chamber versus time, A represents corneal area available for diffusion (1.766cm2), Co is the initial glutathione concentration in the donor chamber and 60 is the conversion factor from minute to second [2,17]. The linear branch of the permeation data was determined using correlation analysis. A minimum of six data points in the linear branch were taken to calculate the flux, J, (μg.cm-2s-1) by linear regression. The flux was then divided by the concentration in the donor (μg.cm-3) in order to calculate Papp (cm.s-1) [2,18].

Cryo-Scanning Electron Microscopy (Cryo-SEM)

Cryo-SEM was used to evaluate the hydrogel structure at swollen state. Images were obtained through Philips XL30S FEG (Field Emission Gun) SEM (Netherlands). Cryo unit, Gattan Alto 2500 was employed, including a fracture stage and a sputter coater, with a coating temperature of less than -120ºC. Samples were placed on a brass specimen holder and heated to allow thermogellation. The sample were frozen by using liquid nitrogen (-200ºC) and then cut and allowed to heat to -90ºC under vacuum. This allowed the frozen water in between gel pores to evaporate, generating the clear structure of polymer hydrogel. The surface of the sample was sputtered with gold for 4 minutes at -120ºC, to minimize any charge builds up, after which the samples were viewed under the cryo-SEM.

Statistical Analysis

Statistical data was analyzed using Microsoft Excel 2007, with two-way variance analysis (two-way ANOVA) and pair wise comparisons were performed using t-tests. p<0.05 indicates a significant difference.

Results

Characterization of Rheological Properties

Hydrogel ocular formulations should ideally have a viscosity of around 1000–5000 cP in order to maximize pre-ocular retention time and the delivery of glutathione. Formulations with similar viscosities were plotted together for clear and apparent analysis. The 0.3% C1342 formulation was not included as it was too viscous and the method that was used to evaluate rheological properties was not suitable [19]. All CP dispersions exhibited non-Newtonian shear-thinning (pseudoplastic) behavior (Figure 1) i.e. decreasing viscosity with increasing shear rate.

It was observed that the increase in viscosity with increasing CP concentration was proportionally similar at 0.1% and 0.2% for C934 and C940 at a shear rate of 40s-1. Conversely, C934 exhibited a much greater increase in viscosity at 0.3% compared to that of C940 (Figure 2). The viscosity difference appears to be proportional between 0.1% and 0.2% of C1342 and 0.2% and 0.3% C934 at the shear rate of 40s-1. Moreover, the 0.1% and 0.2% C940-containing formulations were the least viscous, while the respective C934 formulations appeared to be three-times more viscous than C940-containing formulations (Figure 2). The difference in viscosity between all formulations at different concentrations appears to be significant apart from that between 0.1% C934 and 0.2% C940 and between 0.1% C1342 and 0.3% C940.

The difference in viscosity at different concentrations for each CP was statistically significant (p<0.05) (Figure 3A, 3B & 3C). The C940 exhibited the most proportional increase in viscosity with increasing concentration (Figure 3B); while the viscosities of C934 and C1342 appeared to have been more dramatically affected by the change in concentration. The rheological characteristics of PVA and PAA formulations are shown in (Figure 4). PVA and PAA results were analyzed separately from that of the CP formulations because their viscosity range was vastly different. The PVA and PAA formulations demonstrated Newtonian flow, where a linear relationship between shear rate and shear stress was evident (Figure 4) [7]. Furthermore, the Newtonian flow properties showed that the viscosity of these formulations remained constant despite increasing shear rate. The 25% PAA is approximately eight-times more viscous than the diluted 12.5% PAA and has a much higher shear stress than all the other three formulations. Similar to the CP systems, the viscosity of PAA and PVA also increased with increasing concentration. Again, there was a significant difference between the viscosities of these simple chain polymers where PAA appears to be more viscous than PVA. The 25% PAA displays a viscosity which is comparable to 0.1% C940-Poloxamer formulation after shearing (Figure 2).

Based on the optimal ocular formulation characteristic described by existing literatures available, the formulation demonstrating the most desirable rheological properties contains the 0.2% C940, it exhibited the lowest viscosity at the lowest shear rate investigated.

Characterization of mechanical and mucoadhesive properties

The hardness and compressibility of the hydrogel system during formulation screening increased with an increase in CP concentration. This trend can be observed for all CP-containing formulations (Figure 5A & 5B). Significant increase in hardness for C940 and C1342-containing systems was observed at concentration of 0.2% compared to concentration of 0.1% and there was significant difference compared to control (p<0.05). However, no significant differences between the CP systems and control at 0.1% (p>0.05) were observed. At 0.3% and 0.5%, all CP systems were significantly different, compared to control (p < 0.05). Conversely, PVA showed no distinctive change compared to control and no significant differences between the different concentrations was observed (p>0.05). PAA exhibited a significantly lower hardness compared to control (p<0.05), however, there was no difference when concentration was doubled. The hardness of the hydrogel in the presence of PVA was no different when it was compared to all 0.1% CP formulations (p>0.05). The results obtained were comparable to the previous literature data [20]. The compressibility of the hydrogel in the presence of C1342 at 0.3% and 0.5%, as well as C940 0.5%, was significantly different when compared against control (p<0.05). Similar to hardness, PAA showed a significantly lower compressibility in comparison to control (p<0.05).

A relationship exists between the increase in adhesion and a decrease in cohesion of formulations (Figure 6A & 6B), with a concentration increase. At 0.1%, C1342-containing formulations were significantly higher compared to control (p<0.05). However, adhesion of all CP-containing formulations increased with an increase in concentration. Conversely, for PVA and PAA formulation, no such trend can be observed with increase in concentration, and statistical analysis showed no significant difference when compared to control (p > 0.05). At 0.1%, 0.2% and 0.3% of C934 and C94 containing systems, the cohesive forces were significantly higher when compared to control (p<0.05), but this was not demonstrated at a concentration of 0.5% (p>0.05). Cohesion of 0.1% and 0.2% of C1342-containing formulations were significantly higher to control (p < 0.05). The cohesion forces of 0.1%, 0.2% and 0.3% of C934-containing formulations were significantly different compared to 0.5% of C1342, C940 and 0.1% of C940 respectively (p<0.05). The PVA and PAA formulations demonstrated higher cohesive forces than control (p<0.05) and showed a concentration dependent relationship similar to the CP formulations. There was no significant difference in cohesion between PVA and control (p>0.05), but both PAA concentrations had significantly lower cohesion compared to control (p<0.05).

The measured apparent mucoadhesion showed a difference between the control and all formulations screened (Figure 7). It also showed a consistent increase in mucoadhesive force with increase in concentration, however, there was no significant difference between different CPs at concentrations 0.1% and 0.3%. At 0.2%, only apparent mucoadhesion of C940 was significantly different to that of other CPs. At 0.5% all CP formulations were significantly different from each other. PVA and PAA showed a similar trend, PAA at 25% obtained similar results to 0.5% C934 containing formulation.

Corneal permeability studies

The cumulative diffusion profiles of each various hydrogel formulations of glutathione over eight hours are shown in (Figure 8–10). All of the hydrogel systems showed a linear diffusion trend and showed a significantly higher diffusion when compared to the control (p<0.05). The permeation of 0.1% C940 formulation was significantly less than 0.1% C934, 0.1% and 0.2% C1342 (p<0.05). The remaining formulations did not show any differences when compared to each other. The ranking order of the different formulation groups in their ability to promote glutathione diffusion across bovine cornea is: 0.2% C940 and 0.1% C1342 > 0.1% C934, 0.2% C1342 and 0.2% C934 > 0.1% C940 > Control.

The apparent permeability coefficient (Papp) and Flux values were calculated and shown in (Figure 11). From the Papp of the different formulations measured, the permeability of glutathione was increased compared to water (p<0.01). The 0.2% C940 (p=0.03) and 0.1% C1342 (p=0.05) had an approximately 1.2-fold higher corneal permeability than 0.1% C940. All other hydrogel formulations did not show differences in their permeability (p > 0.05). The average Papp values of the hydrogels were in the range of 1.21–1.57 × 10–4 cm.s-1 compared to 0.64×10–4cm.s-1 produced by the control. This shows that the CP combinations enhance the permeability of drug by up to a 2.4-fold (p<0.05).

The Cryo-SEM images demonstrate the polymeric structure of the achieved CP hydrogel systems. Images of C934 and C1342 hydrogel systems with incorporated glutathione were obtained (Figure 12). Although, attempt was made to obtain images of C940 hydrogel structure, this could not be achieved due to experimental error. An air bubble was formed during preparation and, as it was cut, no formulation remained on the metallic rib for imaging; thus, the images of C940 hydrogel structure were not obtained and displayed.

Discussion

In order to increase the bioavailability of glutathione (and hence achieve post-corneal concentration), the pre-corneal residence time or penetration of the ocular formulation must be extended beyond that of the conventional formulations [21]. This may be achieved by increasing the viscosity, spreadability or mucoadhesion of the formulations; or by incorporating permeation enhancing materials into the formulation. The simplest approach is to enhance the viscosity because a two-fold increase in maximum bioavailability of a drug can be achieved. Therefore, viscosity is often considered as the major factor influencing ocular retention [12]. The increase in retention time of instilled formulations is apparent, once viscosity is greater than 10mPas but a very high viscosity results in ocular discomfort and epithelial damage can occur from blinking [22]. The difference in the effective number of polymers evaluated was likely the reason for the higher shear stress observed in this experiment. Upon the addition of NaOH solution to adjust pH to 7.4, carboxyl groups on the carbomers (pKa 3–5) are ionized, leading to electrostatic repulsion and dramatically increasing the rigidity of the formulation structure [23]. Thus, the ionized carboxylic groups could have also interacted with the small but very positive sodium ions, leading to a reduction in viscosity and shear stress. This theory could have been validated had the rheological characterization been carried out at both pH 5 and pH 7. Although Davies et al. [12] found the viscosity profile of PVA to be pseudoplastic in contrast to our findings; the conditions they used were extreme (85oC) and would not occur in the eye. Moreover, Davies et al. [12] prepared their PVA solution with PBS, introducing ions that possibly change the interactions between the molecules. Nevertheless, C934 was found to have greater viscosity-enhancing capability than PVA. Structurally, PAA are CPs without cross-link units and can be described as linear polymers (pKa~4.5) [24]. Although PAA has similar rigidity as the carbomers, it is less viscous because the strength provided by cross-links is much more robust.

The structures of hydrogels are highly dependent upon the interaction between the polymers and the swelling medium [25]. The mechanical properties of the hydrogel formulations were assessed through hardness, compressibility, adhesion and cohesion. The ability to prolong ocular contact time was measured through its strength of mucoadhesion [20,26,27]. The hardness is desired to be low in order for the formulation to be easily administered onto the ocular surface [27,28]. Therefore, lower concentrations of CP formulations (0.1% and 0.2%) are favoured. The “hardness” of anionic gels is dependent on the molecular network density and the degree of cross-linking, which is correlated to the polymer concentration, as well as viscosity [29–31]. C1342-containing hydrogels exerted the highest “hardness” out of all three CPs. This is likely to be due to increased cross-linking between its long alkyl methacrylate chain and allyl ethers of pentaerythritol and its high molecular weight compared to C934 and C940 [32]. Similarly, C934 with relatively lower molecular weight was the easiest formulation to deform due to its reduced rigidity. Additionally, surface molecules have a net inward force due to the cohesive forces being stronger than adhesive forces with the air, therefore a surface energy is created in parallel to the surface. This requires energy to break, which would have been measured as hardness [33–35]. The hardness of PAA was lower than the control. This may be explained through the accumulation of PAA molecules on the surface, which would push the liquid molecules into the bulk, leading to reduction in surface tension [33,34]. Compressibility claimed to measure the ease of spread of the formulations over the corneal surface; where a lower value was favorable. With increasing concentration, total resistance was greater hence more force was required as the parameter quantifies deformation under shear and compression [28]. The claimed “cohesion” is described as the spatial reformation of the hydrogel structure after successive compressions. It attempted to measure the relaxation of the polymers, which is dependent on time and the deformation [36]. The “cohesion” of the formulation hoped to examine the ability of the hydrogel to reform its structure after application to the ocular surface, where a high value was desired for maintenance of its structural integrity [27]. A general decline in cohesive force is shown with increase in concentration, which is due to the increase in mass of the polymers [37]. The adhesion is defined to be the interaction between different molecules within the hydrogel. This estimates the ability of the formulation to adhere to the target site [20]. At higher concentrations, there were more polymers present with greater capability to form chemical bonds [19]. The results showed a plateau at concentrations 0.3% and 0.5% for all CP-containing formulations. This may be explained by the high cohesive forces within the hydrogel structure to prevent adhesive forces forming with the probe [28]. The degree of mucoadhesion is dependent on the hydration, anionic charges, molecular weight and cross-linking of the polymer [32,38]. All CP-containing formulations were combinations of Poloxamers and CPs. This combination increased its mucoadhesive properties as the polyether components of Poloxamers were able to form tertiary carbon bonds with polyacrylic acid units of CPs, which may potentially expose different binding groups to increase mucoadhesive interactions [34,39]. Moreover, the CP-containing systems at 0.1% and 0.2% had a greater degree of swelling, therefore increased hydration. The polymers are flexible and free to diffuse to come in close contact with the corneal surface and interpenetrate with the mucus layer, to create a strong entangled network through non-covalent interactions [19,38].

The cumulative amount of glutathione that diffused across the cornea from the original administered dose (1mL), after eight hours ranged from 50.6% to 80.1% from the hydrogels compared to 45.0% from the control system. This is very high compared to the 5% or less, which is typically seen to reach the aqueous humour in living human eyes [40]. This is due to the absence of other ocular barriers such as tear turnover, tear dilution, lacrimation and nasal lacrimal drainage in the Franz diffusion cell apparatus [40]. The glutathione control group follows a linear trend across the corneal epithelial membrane and represents zero-order kinetics where diffusion was at a constant rate throughout the experiment. Conversely, the glutathione-loaded CP-containing hydrogels show a biphasic release profile. The initial phase was attributable to the release of glutathione from the outer pores of the gel surface and the second phase showed the sustained release of glutathione from the matrix of the polymer. This release profile shows benefits on application, rapid diffusion of glutathione through the corneal epithelial membrane will occur, followed by a sustained diffusion over time.

Conclusion

Glutathione is a potent anti-oxidant that is essential in the maintenance of tissue health. By replenishing the tissues with glutathione, it is hoped that the progression of ocular damages can be halted or even reversed. Hydrogels composed of CPPoloxamer, PAA and PVA were formulated and investigated as potential delivery systems for glutathione across the cornea. The CP-Poloxamer combinations exhibited favourable pseudoplastic behavior and cohesion/adhesion properties. These were further investigated for their ability to increase glutathione permeability across excised bovine corneas using a Franz diffusion cell. Penetration of glutathione across the cornea was significantly increased compared to the plain drug solution. The 0.2% C940- containing-Poloxamer formulation was determined to be the most promising for ocular delivery of glutathione. To study the efficacy of glutathione formulations on the reversibility of cataracts, in vivo tests can be performed in the future.

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[vitamindripstherapy]

The Nutrients You Required Right Now: Glutathione + B5 + High Dose B12

As devastating wildfires craze on in Northern as well as Southern California, the requirement to protect your system from the harmful impacts of smoke is more important than ever.

It goes without claiming that if exposure to smoke in the air is the closest you and I concern the fires that are swallowing up whole communities in both Northern and Southern California, then we should be greatly happy. That stated, the toxic results of smoke are actual ... as well as frequently dangerous. As smoke coverings the Bay Location, signals of "red" unhealthy air top quality have actually been issued for a week directly and also counting. Moreover, also if you're just going outside in short ruptureds, your body is still being revealed to significant quantities of poisonous chemicals. Consequently, you might see lack of breath, coughing, congestion, fatigue, itchy/dry eyes and also skin, migraine, nausea, mind fog, and sleeplessness, among various other signs.

But what about the impacts you don't observe? On a microscopic degree, the smoke in our air is triggering potentially long-lasting cellular damages. At best, it's maturing us ... at worst, it's establishing the stage for numerous chronic illness in the future (or aggravating existing ones).

During the California wildfires of 2017, B12 LOVE NDs, Elizabeth Korza and also Tara Levy, wrote about some easy steps you can take at home to shield yourself from smoke contaminants. However, if you wish to sustain your system past these day-to-day safety measures, there are 3 nutrients that ought to be top of mind when you visit your preferred regular B12 LOVE Delighted Hr (Pro Suggestion: You can request all three simultaneously as a "Glutathione Mixed drink" on our menu):.

Glutathione: Glutathione (GSH) is found normally in the body as a product of the liver. It's in fact a combination of three amino acids-- cysteine, glutamic acid, and glycine-- and also places as one of the most essential anti-oxidant produced by the body. That's right, the MOST essential! Especially, it prevents mobile damage brought on by cost-free radicals as well as peroxides, using security against oxidative stress and anxiety, mercury & toxic steels, alcohol, and natural contaminants.

" Every system in the body is influenced by the state of the glutathione system, especially the body immune system, the nerve system, the gastrointestinal system, as well as the lungs." Martin Gallagher, MD, DC, ABOIM.

Last month, Dr. Shannon as well as I attended a clinical conference which included a session titled "Glutathione: the most powerful antioxidant?" It ends up that's a rhetorical inquiry, as well as the answer is definitely of course, yes, as well as a lot more yes!

B5 (Dexpanthenol): Ideal understood for its energy-boosting results, B5 manufactures and metabolizes fats, healthy proteins, and also coenzyme-A (a vital component of cellular respiration). It's utilized as a treatment for exhaustion, adrenal stress, acne, as well as allergies.

High Dose B12 (5mg/mL Methylcobalamin or Hydroxocobalamin): As the base of every one of our nutrient injections, we can't claim enough good things concerning Vitamin B12 ... particularly in high dose concentrations (and btw, there's no such point as way too much B12!).

Moreover, the little extra dose of B12 makes a big difference in general well-being. It is an incredibly important vitamin for maintaining healthy nerve cells, as well as additionally helps in the manufacturing of DNA and RNA, the body's genetic product. In Addition, Vitamin B12 assists in red cell development and assists iron work better in the body. Vitamin B12 is not only increasing to the immune system, it helps battle fatigue, sleeping disorders, anxiety, persistent discomfort, anxiety, brain fog, and anemia, amongst various other things.

Got inquiries? Send us a message or come see us at one of our Happy Hrs. Remain healthy and safe out there!

~ Emily, R.N.

The post “ The Nutrients You Required Right Now: Glutathione + B5 + High Dose B12″ was appeared first on B12 Love by Emily Crichton

Know more about the benefits of iv vitamin therapy and how it can help you achieve optimum health. The IV Lounge is a highly recommended drip clinic in Toronto. Go check them out at www.theivlounge.ca

[paullassiterca]

Replace Dangerous Oils With Healthy Fats

30 Tips in 30 Days Designed to Help You Take Control of Your Health

This article is included in Dr. Mercola’s All-Time Top 30 Health Tips series. Every day during the month of January, a new tip will be added that will help you take control of your health.

Dietary fats are a crucial component of a healthy diet, but the devil’s in the details, and the type of fats you choose can make a world of difference. While the notion that saturated fats cause heart disease is a fallacy, some fats do cause cardiovascular problems and need to be avoided.

Replacing dangerous oils with healthy fats is one simple way to boost your health and reduce your risk of chronic disease. Here, I’ll review some of the key points to remember when adding more fat into your diet.

Trans Fat — A Hidden Culprit in Heart Disease for Decades

Before 1900, American housewives used lard and butter for cooking. It wasn’t until 1911 that our diets experienced a dramatic change with the introduction of trans fat in the form of Crisco, the first hydrogenated vegetable oil product to hit the market.

Hydrogenated vegetable oils and margarine quickly became the backbone of the food industry. In my new book “Superfuel: Ketogenic Keys to Unlock the Secrets of Good Fats, Bad Fats, and Great Health ,” cowritten with James DiNicolantonio, Pharm.D, we take a deep dive into this topic. In fact, our book reveals how the consumption of soybean oil has increased over a thousand percent from 1909 to 1999.

Trans fats became a staple dietary fat with the introduction of processed foods, and could be found in everything from cookies and crackers to french fries and frozen food. Unfortunately, it would take more than a century for the truth about trans fat to be fully recognized.

The U.S. Food and Drug Administration (FDA) didn’t remove partially hydrogenated oils from the generally recognized as safe (GRAS) list until 2015,1 based on evidence suggesting their removal could prevent thousands of heart attacks and deaths each year.

In reality, research by Fred Kummerow, dating back to 1957, showed that trans fat interferes with the basic functioning of cellular membranes. Even small amounts of manufactured trans fat have been shown to have adverse effects on your heart, insulin sensitivity and neurological system.

Processed Vegetable Oils Do More Harm Than Good

In response to research and public opinion, many restaurants have since turned from partially hydrogenated oils to 100 percent vegetable oil. However, while these oils do not have trans fats, they’re just as bad, if not worse. There are three significant reasons for this:

When heated, vegetable oils degrade to extremely toxic oxidation products, including cyclic aldehydes,2 which have been linked to neurodegenerative diseases and certain types of cancer. In her book, Teicholz cites research showing that aldehydes cause toxic shock in animals through gastric damage

Vegetable oils are a concentrated source of omega-6 linoleic acid, which has led to a severe imbalance between the omega-6 to omega-3 ratio in most people’s diets

Many of the vegetable oils produced today — especially corn and soy — are products of genetic engineering (GE) and a significant source of glyphosate exposure, and glyphosate has also been linked to gut damage and other health problems

In summary, processed vegetable oils (polyunsaturated fat) harm health by:

Creating high amounts of oxidation products when used in cooking (as they are very susceptible to heat), including aldehydes, which are what cause oxidized low-density lipoprotein (LDL) associated with heart disease. Aldehydes also crosslink tau protein and create neurofibrillary tangles, thereby contributing to the development of neurodegenerative diseases.

Damaging the endothelium (the cells lining your blood vessels) and causing an increase in penetration of LDL and very low-density lipoprotein (VLDL) particles into the subendothelium. In other words, these oils get integrated in your cell and mitochondrial membranes, and once these membranes are impaired, it sets the stage for all sorts of health problems.

Damaging your mitochondria and DNA by making your cell membranes more permeable, allowing things to enter that shouldn’t.

Making the cell membrane less fluid, which impacts hormone transporters in the cell membrane and slows your metabolic rate.

Inhibiting cardiolipin, an important component of the inner membrane of your mitochondria that needs to be saturated in DHA in order for it to function properly.

Cardiolipin can be likened to a cellular alarm system that triggers apoptosis (cell death) by signaling caspase-3 when something goes wrong with the cell. If the cardiolipin is not saturated with DHA, it cannot signal caspase-3, and hence apoptosis does not occur. As a result, dysfunctional cells are allowed to continue to grow, which can turn into a cancerous cell.

Inhibiting the removal of senescent cells, i.e., aged, damaged or crippled cells that have lost the ability to reproduce and produce inflammatory cytokines that rapidly accelerate disease and aging.

Stripping your liver of glutathione (which produces antioxidant enzymes), thereby lowering your antioxidant defenses.3

Inhibiting delta-6 desaturase (delta-6), an enzyme involved in the conversion of short-chained omega-3s to longer chained omega-3s in your liver.4

Exposing you to toxic 4-hydroxynonenal (4HNE), which forms during the processing of most vegetable oils, even if the oil is obtained from organic crops. 4HNE is highly toxic, especially to your gut bacteria, and consumption of 4HNE has been correlated with having an obesogenic balance of gut flora. It also causes DNA damage and instigates free radical cascades that damage your mitochondrial membranes.5

Exposing you to glyphosate residues, as most vegetable oils are made with genetically engineered crops. Glyphosate has been shown to disrupt the tight junctions in your gut and increase penetration of foreign invaders, especially heated proteins, which can cause allergies.

Address Your Omega-6 to Omega-3 Ratio to Protect Your Health

Marine-based omega-3 is one of the most important fats in the human diet, as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are actually key structural elements of cells, including your brain cells, and not just simple fuel. If you don’t have enough DHA and EPA, your body’s ability to repair and maintain healthy cell structures is seriously impaired.

Unfortunately, in the past 100 years, our omega-6 intake has nearly tripled while our intake of omega-3 has decreased 10fold, and this imbalance has also likely played a significant role in our skyrocketing disease rates. Eating too much damaged omega-6 fat — found in abundance in processed vegetable oils — and too little animal-based omega-3 sets the stage for diabetes, cardiovascular disease, rheumatoid arthritis, cancer, depression and Alzheimer’s, just to name a few.

Now, omega-6 fat in and of itself is not the problem. The problem is that most people get far too much of it, and insufficient amounts of omega-3, and that most of the omega-6 people eat has been damaged and oxidized through processing. Evidence implicating excessive consumption of omega-6-rich vegetable oils as a direct cause of heart disease include but is not limited to:6

The amount of linoleic acid in adipose tissue and platelets is positively associated with coronary artery disease, and studies7 measuring changes in linoleic acid concentrations in adipose tissue in Americans show concentrations increased from 9.1 percent in 1959 to 21.5 percent in 2008. This increase also paralleled increases in the prevalence of obesity, diabetes and asthma.

Conversely, the long-chained omega-3s docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) have been shown to protect against coronary artery disease, which is why maintaining a healthy balance between omega-3 and omega-6 is so important.

Patients with atherosclerosis have higher amounts of linoleic acid oxidation products in their plasma, low-density lipoprotein (LDL) and atherosclerotic plaques.

Oxidation of linoleic acid begins before any clinical signs of atherosclerosis become apparent.

When the endothelium (the interior lining of your blood vessels) is exposed to linoleic acid, LDL transfer across the endothelium is increased and this is an essential step in the atherosclerotic process.

Low linoleic acid diets reduce LDL oxidation.

A meta-analysis of randomized controlled trials in humans showed that when saturated fat and trans fat are replaced with omega-6 PUFAs, all-cause mortality, ischemic heart disease mortality and cardiovascular mortality increase.

Oxidation products of linoleic acid are found in infarcted tissue.

The linoleic acid metabolite 9-HODE is a strong promoter of inflammation, and may be both a marker for and inducer of atherosclerosis.

How to Balance Your Omega Fat Ratios

Ideally, you want to maintain a ratio somewhere in the range of (4-2)-to-1 of omega-6 and omega-3 fats. This, however, is nearly impossible if you’re regularly eating processed foods or restaurant fare, as these are loaded with omega-6 from industrial vegetable oils like corn oil and canola oil.

While you do need omega-6, it should be in its unprocessed form, not industrial vegetable oils. Good sources are whole, raw plant seeds and tree nuts. In summary, to correct your omega-6 to omega-3 ratio, you typically need to do two things:

1. Significantly decrease intake of damaged omega-6 by avoiding processed foods and foods cooked in vegetable oil at high temperatures. A number of studies8,9 have found that people who regularly eat deep-fried foods have a significantly increased risk of stroke and death.

Common sources of harmful omega-6 to avoid include corn oil, canola oil, soy oil, hydrogenated or partially hydrogenated fats, margarine and shortening.

2. Increase your intake of marine-based omega-3 fats. Ideal sources include small fatty fish such as sardines, anchovies and herring, along with wild-caught Alaskan salmon, or a supplement such as krill oil.

Healthiest Fats for Cooking

Getting back to cooking oils, if vegetable oils are “out,” what should you use to cook with? Healthy alternatives include:

• Coconut oil — This is what I believe is the best cooking oil. It has a number of valuable health benefits, including a positive effect on your heart and antimicrobial properties. It’s also a great source of energy, thanks to its medium-chain fatty acids (MCFAs). When consumed, the MCFAs are digested and converted by your liver into energy that you can immediately use. Coconut oil also helps stimulate your metabolism to encourage a healthy weight profile.

• Grass fed butter — Raw, organic butter made from healthy grass fed cows’ milk contains many valuable nutrients, including vitamins A, D, E and K2. Furthermore, it contains various minerals and antioxidants that support good health.

• Organic ghee, which has been used for cooking for thousands of years, is another good choice.

• Olive oil — This oil contains healthy fatty acids that can help lower your risk of heart disease. While the standard recommendation has been to avoid using olive oil for cooking and to only use it cold, recent research10 in which 10 popular cooking oils were compared, contradicts this advice, showing extra-virgin olive oil actually scored best for both oxidative stability and lack of harmful compounds produced when heated.

A word of caution is warranted, however. Fake olive oil abounds, so it’s important to take the time to investigate your sources. Tests reveal anywhere from 60 to 90 percent of the olive oils sold in American grocery stores and restaurants are adulterated with cheap vegetable oils or nonhuman-grade olive oils, which are harmful to health in a number of ways.11

For tips on how to assess the quality of your olive oil, see the short video below. For more information, see “Is Your Olive Oil Fake?” where I cover this topic in-depth.

Peanut oil and sesame oil are two other healthy options. While both are high in omega-6, peanut oil is high in antioxidants, and sesame oil has been shown to benefit diabetics. The caveat with these two oils is that you need to consume them unheated and in moderation, so as not to throw off your omega-6 to omega-3 ratio.

>>>>> Click Here <<<<<

Black Seed Oil — The Forgotten Gem

Black seed (Nigella Sativa) oil is another exceptional fat with a long history of use in traditional systems of medicine, including Ayurveda and Siddha. The most abundant active plant chemical in black seed is thymoquinone; other bioactive compounds include α-hederin, alkaloids, flavonoids, antioxidants and fatty acids.

As for its antioxidant activity, black cumin seed has been found to be far more potent than vitamin C.12 In modern times, researchers have confirmed Nigella Sativa may be helpful for:

Type 2 diabetes — In one study, Nigella sativa improved glucose tolerance as efficiently as metformin.13 It’s also been shown to improve the performance of antidiabetic medication14

Reducing asthma symptoms — In one study,15 thymoquinone was found to be instrumental, by reducing two inflammatory mediators of asthma and other inflammatory processes.

Another study16 found black cumin seed also acts as a relaxant, and displays both anticholinergic (reducing spasms in smooth muscle) and antihistaminic (blocking allergic reactions) effects. Here, thymoquinone was found to be superiorto the asthma drug fluticasone (a synthetic glucocorticoid)

Enhancing memory and reducing stress — The results showed black cumin seed inhibited stress-induced biochemical changes in a dose-dependent manner. Memory and cognition was also dose-dependent17

Reducing damage caused by cadmium poisoning18 — May also serve as prophylactic against chemical warfare agents

Protecting against and attenuating aflatoxicosis19

Alleviating symptoms of allergic rhinitis20

Candidiasis21

Rheumatoid arthritis22

Cancer23,24,25

Black seed oil has at least 20 different pharmacological actions, which helps explain how it can be useful for so many different and varying ailments, including:26

Antidiabetic

Anti-cancer

Immunomodulatory

Analgesic (pain relief)

Antimicrobial

Anti-inflammatory

Spasmolytic

Bronchodilator27

Hepatoprotective

Renal protective

Gastroprotective

Antioxidant

How to Use Black Seed Oil

Black seed oil is a highly undervalued and oft-forgotten kitchen staple. When used in cooking, it imparts a warm, slightly bitter flavor that tastes something like a blend of thyme, oregano and nutmeg.

A mixture of black seed oil, honey and garlic also makes for a powerful tonic that can help soothe coughs and boost immunity, especially during cold and flu season or if you feel like you’re coming down with an infection.28

Like all seeds, black seed oil is high in polyunsaturated fats. So, when taken in excess, it could make your mitochondrial membranes more susceptible to oxidation.

For this reason, I suggest limiting your daily intake to 1 to 2 tablespoons or less. A simple way to get a small amount of black seed oil into your diet on a regular basis is to use it in your homemade dressing. Here are a few suggestions:

Mix apple cider vinegar, black seed oil, fresh lemon juice, cilantro and tahini. Experiment with the ratios to enhance the flavor you enjoy the most

A simple and yummy dressing that goes particularly well with broccoli, asparagus or salad greens includes: 1 tablespoon apple cider vinegar, 1 tablespoon lemon juice, one-half teaspoon minced garlic, a dash of ground black pepper and a few fresh basil leaves, chopped

Alternatively, you can use apple cider vinegar and/or black seed oil as substitutes for other oils and vinegars in whatever dressing recipe you’re already using. Keep in mind that the black seed oil does have a spicy kick to it, so substituting the full amount may make it too spicy. Start by adding just a small amount, and experiment to find the ratio of vinegar, olive oil and black seed oil you enjoy

Optimize Your Health by Selecting the Right Fats

The list below, obtained from Dr. Cate Shanahan, author of “Deep Nutrition: Why Your Genes Need Traditional Food,” summarizes some of the best and worst fats found in our modern diet. Replacing the bad fats in your diet with ones from the “good” list is a simple way to safeguard your health without making any radical changes.

To learn more about the ins and outs of dietary fats, pick up a copy of my latest book, “Superfuel: Ketogenic Keys to Unlock the Secrets of Good Fats, Bad Fats, and Great Health,” cowritten with James DiNicolantonio, Pharm.D., which gives more in-depth specifics on how to discriminate between healthy and harmful dietary fats.

>>>>> Click Here <<<<<

Tip #6Avoid Becoming a Cancer Statistic

from Articles http://articles.mercola.com/sites/articles/archive/2019/01/07/replace-dangerous-oils-with-healthy-fats.aspx source https://niapurenaturecom.tumblr.com/post/181796702211

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Effect of Selenium and Vitamin E on Development and Viability of Preimplanted Mouse Embryo- Juniper Publishers

Abstract

In vitro culture results higher level of reactive oxygen species (ROS) oxygen than in vivo environments that cause lipid peroxidation of cellular membranes. Selenium (Se) and Vitamin E (Vit-E) are the important antioxidants that protect mammalian cells against lipid peroxidation. Therefore, the present study was conducted to investigate whether Se or Vit-E and Se+Vit-E overcome the the undesirable oxidative stress produced by hydrogen peroxide (H2O2) and enhance the development of pre implanted mouse embryo. Co-incubating the embryos with 60nM Se and/or 100nM Vit-E were increased (P<0.05) the blastocyst development rate.

The addition of H2O2 reduced the development of mouse embryo, but the addition of Vit-E, Se and Se+Vit-E reduced the detrimental effect of H2O2 and influenced the higher rate of development to blastocysts, compared to CZB alone (P<0.05). The incorporation and oxidation of 14C-glucose in the blastocysts developed by the medium supplemented with Se and/or Vit-E in the presence or absence of H2O2 were significantly higher (P<0.05) than that of the control. Moreover, Vit-E is more effective than Se and Se+Vit-E in reversing ROS-induced mouse embryo toxicity. Therefore, Vit-E may be supplemented in the CZB medium for better development and viability of pre implanted mouse embryo.

Keywords:  Mouse embryo; Selenium; Vitamin E

Abbrevations Ros: Reactive Oxygen Species; -Oh: Hydroxyl Radical; O2-: Superoxide Anion Radical; Se: Selenium; Gpx: Glutathione Peroxidase; Cpm :Counts Per Minutes

    Introduction

In vitro embryo culture suffers from excessive developmental failure. Its inefficiency is linked with the generation of reactive oxygen species (ROS), such as H2O2, hydroxyl radical (-OH) and superoxide anion radical (O2-), appears as the by-products of cell metabolism [1]. Superoxide may also spontaneously break down into oxygen and H2O2. As ROS are highly reactive molecules, their accumulation can lead to damage and breakage of DNA strands. There are many evidences have been found that ROS compromises embryo development in many species [2-6].

Selenium (Se), an essential trace element for mammals, is an integral part of anti- oxidant system [7]. Se dependent glutathione peroxidase (GPx) has an important role in free radical protective mechanisms. Vitamin E (Vit-E), the predominant lipid-soluble antioxidant in animal cells, protects cells from oxygen radical damage in vivo [8,9] and in vitro [10,11].Our previous study investigated that Se and Vit-E, as the integral parts of antioxidant systems which play important roles for the in vitro maturation, fertilization and culture of porcine oocyte [12]. However, there is a very limited studies were conducted with the effect of Se and Vit-E on the development of preimplanted mouse embryo. Therefore, the present study was conducted to investigate whether Se, Vit-E or Se+Vit-Eovercome the undesirable oxidative stress produced by hydrogen peroxide (H2O2) and enhance the development of preimplanted mouse embryo.

    Materials and Methods  

Chemicals and reagents

The basic embryo culture medium used in this study was CZB [13] , which contains 1mM glutamine, 0.1 mM EDTA and 5mg/ml BSA, and considered as control. A part of CZB was added with 30% H2O2 to get a final concentration of 0.0003% that considered as negative control. According to the experimental layout, CZB (with or without H2O2) was supplemented with30, 60, 90Nm Se (sodium selenite; Sigma-Aldrich, St. Louis, MO, USA), 50, 100 and150nM of Vit-E (α-tocopherolacetate) and their combination (Se+Vit-E). Selenium, Vit-E and H2O2 were equilibrated in culture medium under CO2 incubator for 8 hours before the start of culture. Vit-E was dissolved in ethanol, and an emulsion was formed by vortex mixing before adding it to the embryo. PMSG and HCG used in this study were obtained from Sankyo Chemical Industries Ltd., Tokyo, Japan. The copulation plug was checked 24 hours later. Radioactive 14C (U)-glucose was purchased from American Radio labeled Chemicals (St. Louis, Mo, USA). All other chemicals were of analytical grade and purchased from Nacalai Tesque (Kyoto, Japan) unless otherwise indicated.

Collection of embryos

Embryos were obtained from 7-8 weeks old female ICR mice. They were offered feed with a balanced standard diet and ad libitum clean drinking water. Animals were kept in polycarbonate cage with wood shavings under a 12h light: 12h dark regimen (light on at 6:00), at a temperature of 20±1 °C in accordance with the "Guideline for Regulation of Animal Experimentation, Faculty of Agriculture, Shinshu University.” Female mice were induced to superovulation with PMSG (5 1U, i.p.) followed 48h later by hCG (51U, i.p.) and met with male mice. Zygotes of 1-cell stages were collected at 2 5 hours HCG post injection by flushing out from the fallopian tubes. The embryos were subsequently incubated in CZB medium.

Experimental layout

In fact, there are four experiments were conducted under this study In experiment 1, effect of CZB supplanted with different levels of Se (0, 30, 60 and 90nM) and Vit-E (0, 50, 100 and 150nM) on the development of mouse embryo from 1-cell stage to the blastocyst were evaluated. The experiment 11 were conducted to investigate the effects of Se (60nM), Vit-E (100nM) and their combination (60nM Se+100nM Vit-E) on the development from 2-cell to blastocyst stages of embryo in the presence (1mM) or absence of H2O2 in CZB medium. Where, the experiment 111 was conducted to the effect evaluate Se (60nM), Vit-E (100nM) and their combination (60nM Se+100nM Vit-E) on the accumulation of ammonia (NH4) in presence or absence of H2O2 due to metabolism of embryo during the development up to blastocyst stages. 1ncorporation and oxidation of 14C-glucose at blastocysts developed by the supplementation of Se (60nM), Vit-E (100nM) and their combination (60nM Se+100nM Vit-E) in the presence or absence of H2O2 were evaluated by conducting the experiment IV. These experiments were conducted five times repeated.

Culture of embryos

The collected zygotes of 1-cell stages were transferred to the culture dishes for washing, and grown in-vitro to the developmental stages up to blastocyst. Embryos were cultured according to the standard techniques, in groups of 10 zygotes were placed into 35mm-diameter culture dishes (Nunc Co., Denmark) containing 30|il of each CZB medium under a layer of paraffin oil and equilibrated overnight in an atmosphere of 5% CO2 in air at 37 °C. The pH of all media was 7.4 after equilibration. The developing embryos seemed to be normal in their morphology, with almost no fragmentation.

Ammonia determination

During incubation period embryos which developed in the presence of Se and/or Vit-E with or without H2O2, the ammonia concentrations in the medium were assessed by using the Bertholot-indophenol method as described in our previous study [14] . To determine the ammonia concentration in the medium, 100μ1 of the culture medium was removed every 2-4h and frozen at -40 °C until measurement. The procedure was carried out five times for the analysis. A calibration curve in the range 0.0003� of ammonia was run with each experiment. The mean coefficient for determination of the calibration curve of five experiments was 0.994.

Incorporation and oxidation of 14C-glucose

The experiment was initiated with 14C-glucose 18.5kBq/0.1mol (specific activity 9.69MkBq/mol, Moravek Biochemicals, 1nc., USA). Each of the ten blastocysts which developed in the presence of Se and/ or Vit-E with or without H2O2 was transferred in a microtube of 50μ1 CZB medium drop containing 14C-glucose then overlaid with mineral oil. On the other hand, 1ml of 2.5Mm NaOH solution was transferred into a 1.5ml micro tube as a trap for the evolved 14 CO2. Both microtubes of NaOH and 14C-glucosewith embryos were confined into a scintillation vial using a rubber stopper. The scintillation vials were incubated for 5h in an incubator at 37 °C. After incubation period, the metabolic reactions of embryos were stopped with an injection of 100μ1 of 10% perchloric acid (PCA) kept at room temperature for 24h. The acid insoluble materials were carefully washed by millipore filtration (8.0|iM white SCWP, 47mm; Millipore Corporation, Bedford, MA, USA) with 5% PCA and the filter papers were kept overnight under a lamp. After drying, the filter papers were transferred into scintillation vials. The NaOH solution was transferred into a new scintillation vial by washing 3-4 times with cocktail (0.5% PPO+0.03% POPOP solution in toluene). All the scintillation vials with 5ml of cocktail were set in a Liquid Scintillation Counter (LS-6500, Beckman Instruments, Inc. USA) to determine the levels of radio activity [15]. This experiment was conducted ten times to improve its accuracy. The values of incorporation and oxidation were expressed directly as counts per minutes (CPM).

Statistical analysis

Data obtained from this stud were analyzed by one-way ANOVA using the GLM procedure of SAS (SAS Institute, Cary, NC). The data expressed as percentage were tested by Chi-square test. Data were presented as mean ±SEM of at least 5 replicates and differences were considered significant at the level of P<0.05.

    Results

The effects of supplementation of different levels of Se (0, 30, 60 and 90nM) in CZB medium on the development of mouse embryo are presented in (Table 1). The results revealed that 30 and 60nM of Se was more effective (P<0.05) for mouse embryo development than that of the control and 90nM of Se, where the level showed detrimental effect on the development of embryo as blastocyst. On the other hand, (Table 2) demonstrated that the supplementation of 100nM of Vit-E was the most effective (P<0.05) for embryonic development than that of the 0, (control), 50 and 150nM. The lowest (P<0.05) percentage of blastocyst was observed when zygotes were cultured in CZB supplemented with 150nM of Vit-E.

Detrimental effects of H2O2 and reducing or protecting ability of Se, Vit-E or their combination from the effects of H2O2 on the embryonic development were shown in (Table 3). The results demonstrated that the development of mouse embryos was reduced by the detrimental effect of H2O2 especially in CZB, the occurrence after 8 cell markedly decreased under H2O2 (P<0.05). However, the supplementation of Se, Vit-E and Se+Vit-E were able to reduce the detrimental effect of H2O2 and enhanced (P<0.05) the development of mouse embryo to be blastocyst. Highest percentage of blastocysts was obtained when the zygotes were cultured in the CZB medium supplemented with Vit-E in the presence or absence of H2O2.

During the development of mouse embryos up to the blastocyst stage, accumulation of metabolic NH4+ in the CZB medium supplemented with Se, Vit-E and Se+Vit-E in the presence or absence of H2O2 is showed in (Table 4). The results revealed that the lowest (P<0.05) accumulation of metabolic NH4+ was observed in the CZB medium supplemented with Vit-E in the presence or absence of H2O2. The effects of supplementation of Se, V1t-E and Se+Vit-E in the CZB medium with or without H2O2 on incorporation and oxidation of 14C-glucose at the blastocyst stage are shown in (Table 5). The incorporation of 14C-glucose at the blastocyst stage cultured with Se, Vit-E and Se+Vit-E in the CZB medium were not significantly differed (P>0.05), but higher rate of incorporation (P<0.05) occurred by the supplementation of Se or Vit-E in the presence of H2O2. On the other hand, in the presence of H2O2, the oxidation of 14C-glucose by the blastocysts were higher (P<0.05) when zygotes were cultured in the CZB medium supplanted with Vit-E or Se alone or Se+Vit-E than the basic CZB medium. There was a slightly higher rate of oxidation of 14C-glucoseoccured in the blastocyst stage cultured with Se, Vit-E and Se+Vit-E.

    Discussion

A number of intrinsic and extrinsic factors have been shown to influence in vitro survival of the embryos to the blastocyst stage in extended culture. Previous studies suggest that in vitro extrinsic factors such as prolonged culture conditions and the autocrine and paracrine activities of the embryos may also contribute to the failure of optimal embryo development. Among the factors that might affect in vitro development of embryos is the balance between oxidative stress, and the ability of the embryos to neutralize their effects [16] reported a sustained increase in oxygen, glucose and pyruvate uptake during in vitro embryo development. The embryos were dependent on oxidative phosphorylation for energy (ATP) production at all stages of pre-elongation development, with perhaps a shift in dependence towards glycolysis in conjunction with compaction. This enhancement in oxidative metabolism of the embryo could be linked to the detected increase in ROS, which are characterized by the presence of an unpaired electron [17] and free-radical intermediaries [18].

Free radicals are generated from leakage of high-energy electrons as they proceed down the electron transport chain. The free radicals have many harmful effects including DNA damage [19]. Many embryos under oxidative stress step into a transient cell cycle arrest which is activated by DNA damage response before apoptosis [20]. Legge & Sellens [21] were suggested that the 2-cell block in mouse embryo is at least in part, of free radical damage incurred by embryos during collection and culture, and that medium supplementation with the radical scavenger, reduced glutathione, can improve embryo development in vitro reported that enhanced oxidative metabolism of embryos may be associated with increased ROS levels detected. The gradual increase in ROS levels from the 2-cell embryo up to the late morula stage could depend on the metabolic change undergone by the embryo during its development. 1t is necessary to prevent ROS as much as possible during culture embryos. However, it is unclear as to which embryos may be adversely affected and to what extent.

The present study showed that Se, Vit-E and SE+Vit-E increased blastocyst formation compared to control. Especially, it suggested that the formation of mouse blastocysts cultured in the presence of 60nM/ml Se and 100nM/ml Vit-E were significantly higher than control. The trace element Se is a component of antioxidative seleno enzymes, Glutathione Peroxidase (GPx) and ThioredoxinReductase (ThxRed) that decrease oxidative stress. Se, as sodium selenite, has been reported as a co-factor for glutathione peroxidase and other proteins and used as an anti-oxidant in medium [22]. 1n cell culture system, sodium selenite protected cell from oxidative damage, free radicals and obstructed lipid peroxide products [23,24]. Se played a role in the antioxidant defense system in the formation of mouse blastocyst, which was essential for the catalytic activity of glutathione peroxidase. Glutathione, a thiol tripeptide component in all cell types has an important role in the transportation of amino acid, synthesis of the protein and DNA, and reduction of disulfide bonds [25].

In the present study, Se and Vit-E were used as combined supplements in the CZB medium for culture of mouse embryos, but this combination showed lower influence in the development of mouse blastocyst than Vit-E alone. Alpha-tocopherol (Vitamin E) is well known as an ROS scavenger in in vivo and in vitro conditions [26-28] and is the most important antioxidant present in ovarian tissue and follicular fluid. The antioxidant activity of a-tocopherol in preventing free-radical-induced tissue damage is accepted by most investigators and is believed to be the primary free radical scavenger and to inhibit lipid peroxidation in the mammalian cell membrane [26-29].The present study also demonstrated that the supplementation of Vit-E (specially, 100nM) played an important role in the development of mouse embryo. The results are in agreement with the results of our previous study and the study of [30] they also reported that the optimal concentration of Vit-E in embryo culture is 100nM.

There is a method to observe the effect of mild oxidative stress with retardation of embryo development in the medium supplemented with 1-5mM H2O2. The H2O2 used in this experiment was 1mM, and performed detrimental effects on the development embryo after 8 cells [31]. Reported that effects of H2O2 on blastocyst formation became more severe during the treatment of later stages of development. Embryos may also have different sensitivities to ROS at different developmental stages [32]. The exogenous oxidant H2O2 leads to over production of ROS, which may induce multiple cellular damages, including lipid peroxidation, nuclear DNA strand breaks, and mitochondrial alteration, consequently disturbing the development of pr implanted embryos in vitro [33-35]. Most of the embryos under oxidative stress step into a transient cell cycle arrest in vitro, which is activated by DNA damage response before apoptosis.

Vit-E(α-tocopherol)isapredominantlipid-solubleantioxidant that has been considered as a primary free radical scavenger in biological membranes [36-38]. a-tocopherolscavenges peroxyl radicals from polyunsaturated fatty acid in membrane phospholipids or lipoproteins that do not spread the radical chain, thereby protecting against lipid peroxidation [39]. Our previous study reported that supplementation of a-tocopherol maintains the development of mouse embryo and pig oocyte quality, fertilization rates and embryo development. Mouse preimplantation embryos can be cultured in a simple defined medium. Under such conditions energy substrates in the medium represent a major source of carbon for anabolism. Glucose is incorporated into macromolecules during in-vitro culture of cleaving mouse embryos and blastocysts [40]. 1n particular, both acid-soluble glycogen and desmoglycogen are rapidly synthesized from glucose presumably to act as a source of energy at implantation [41]. Overall metabolism, as assessed by oxygen consumption is low during the cleavage stages of development before rising sharply at the blastocyst stage. Moreover, metabolic activity has been shown to relate to developmental potential.

In this study, the accumulation of ammonia in the culture was measured regardless of the presence or absence of H2O2, the lower accumulation of ammonia in the blastocyst stage is better than the higher accumulation that occur incidence to the blastocyst [42] showed that high levels of ROS in culture media are associated with low rates of embryo development and blastocyst formation [43]. Suggested two possible mechanisms for the inhibitory effects of NH4+. Perturbation of intracellular pH requires the involvement of Na/K ATPase to transport NH4+ across membranes. Alternatively, may interact directly with enzymes, participating in a series of futile cycles which detoxify NH4+ and result in consumption of ATP. Thus, by whichever mechanism, inclusion of NH4+in culture media will divert ATP from growth to maintenance.

Azizimoghadam reported that pentose phosphate pathway activity of total glucose metabolism was increased at the compacted morula stage and was highest at the blastocyst stage [44]. 1n this study, incorporation and oxidation of 14C-glucose in the blastocyst was significantly influenced when cultured with Vit-E and Se in the presence of H2O2. The incorporation and oxidation of 14C-glucose at the blastocyst tended to resemble the development of embryo. The present study appeared that incorporation and oxidation of 14C- glucose was good for showing the activity of embryos.

Generation of ROS induced by glucose utilization was assumed to be caused by the activation of NADPH oxidase, an enzyme that catalyzes the oxidation of NADPH, generates NADP that serves as a coenzyme of the oxidative arm of the pentose phosphate pathway (PPP) [45]. The gradual increase in ROS levels from the 2-cell embryo up to the late morula stage could depend on the metabolic change undergone by the embryo during its development. 1t is necessary to prevent ROS as much as possible during culture embryos.

    Conclusion

In conclusion, our results showed that culture of mouse zygotes in the CZB medium supplemented with 60nM Se and/ or 100nM Vit-E improves the developmental rate of mouse blastocyst formation in the absence or presence of free radicals or their sources. Therefore, the present study reveals that selenium and vitamin E improves mice blastocyst viability by minimizing the level of free radicals that might occur during development of blastocyst in vitro and which may be useful for assisted reproductive techniques.

To know more about  journal of veterinary science impact factor: https://juniperpublishers.com/jdvs/index.php

To know more about Open Access Publishers: Juniper Publishers

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Age Reduction Breakthrough

If you eschew hyperbole and hang in for the long haul, maintaining a discipline of understatement in the midst of a flashy neon world, you may be offered a modicum of credence when you make an extraordinary announcement. No one is entitled to this courtesy twice. If the news that you trumpet to the moon does not pan out, your readers will be justified in discounting everything you say thereafter.  

Here goes.

I believe major rejuvenation has been achieved in a mammal, using a relatively benign intervention that shows promise of scaling up to humans. I’m going to stake my reputation on it.

Cartoon by Maddy Ballard

In the race to effect substantial, system-wide rejuvenation, Harold Katcher is a dark horse. He has the right academic credentials and a solid history of research. In fact, in earlier life he was part of a team that discovered the breast cancer gene, brca1. I asked Harold for a biographical sketch, and have printed it in a box at the end of this posting.

But Katcher has no research grants or university lab or venture capital funding, no team of grad students mining databases and screening chemicals in the back room.

One thing Katcher has going for him is the correct theory. Most of the explosion in aging research (and virtually all the venture capital startups) are looking to treat aging at the cellular level. Their paradigm is that aging is an accumulation of molecular damage, and they see their job as engineering of appropriate repair mechanisms.

The truth, as Katcher understands it, is that, to a large extent, aging is coordinated system-wide via signal molecules in the blood. It was our common realization of this vision that brought Katcher and me together more than a decade ago. Katcher briefly describes his 2009 epiphany below. It was the source of his 2013 essay (it took a few years to get it into print) on the significance of parabiosis experiments for the future of aging science.

Of course, Katcher was not the only one to get the message about the power of signal molecules in the blood to reprogram tissues to a younger state throughout the body. The problem is that there are thousands of constituents represented in tiny concentrations in blood plasma, but conveying messages that cells read. Which of these are responsible for aging? A small number of labs, including the Conboys at Berkeley, Amy Wager at Harvard, and Tony Wyss-Coray at Stanford have been searching for the answer over the last decade and more.

Katcher has been able to guess or intuit or experimentally determine the answer to this question. With seed funding from Akshay Sanghavi, he set up a lab in Bangalore two years ago, and tried to rejuvenate old lab rats, using a fraction extracted from the blood of younger rats. The first round of experiments were encouraging, published in this space a year ago. He obtained the next round of funding from a reader of this blog, and had enough rats to titrate dosages experimentally, and to see if treated rats who aged again over time could be re-treated successfully.

There is a hole in this story that awaits the resolution of intellectual property rights. Katcher and Sanghvi have not applied for patents and have not yet found a suitable partner to provide financing for human trials. They have not revealed any details of the treatment, besides the fact that it is in four intravenous doses, and that it is derived from a fraction of blood plasma. Katcher thinks that the molecules involved will not be difficult to manufacture, so that when a product is eventually commercialized, it will not require extraction from the blood of live subjects, rodent or human.

We’re still waiting for longevity curves of these treated rats. In the meantime, the best available surrogate measure of age comes from methylation clocks, as developed by Steve Horvath at UCLA, and other scientists as well. Crucially, Katcher found an ally in Horvath, who didn’t just test his rejuvenated rats, but did the needed statistical analysis to develop a set of six methylation clocks specialized to rats. FIve of the clocks are optimized for different tissues, and one is calibrated across species, so that it can measure age in humans as well as corresponding age in “rat years” (about 1/40 human year). The two-species clock was a significant innovation, a first bridge for translating results from an animal model into their probable equivalent in humans.

In a paper posted to BioRxiv on Friday, Katcher and Horvath report results of the methylation measurements in rejuvenated rats. “Crucially, plasma treatment of the old rats [109 weeks] reduced the epigenetic ages of blood, liver and heart by a very large and significant margin, to levels that are comparable with the young rats [30 weeks]….According to the final version of the epigenetic clocks, the average rejuvenation across four tissues was 54.2%. In other words, the treatment more than halved the epigenetic age.”

Human-rat clock measure of relative age defined as age/maximum species lifespan.

Besides the methylation clock, the paper presents evidence of rejuvenation by many other measures. For example:

IL-6, a marker of inflammation, was restored to low youthful levels

Glutathione (GSH), superoxide dismutase (SOD), and other anti-oxidants were restored to youthful levels

In tests of cognitive function (Barnes maze), treated rats scored better than old rats, but not as well as young rats.

Blood triglycerides were brought down to youthful levels

HDL cholesterol rose to youthful levels

Blood glucose fell toward youthful levels

A major question in blood plasma rejuvenation experiments has been how often the cure must be administered. Many of the components of blood plasma are short-lived, secreted into the blood and absorbed continuously throughout the day. The good news from Katcher’s results is that it seems only four injections are needed in order to achieve rejuvenation.

A second question which these experiments resolve is whether rejuvenation requires both adding and removing molecular species from the blood plasma. For example, pro-inflammatory cytokines are found in old blood at much higher levels. Irina and Mike Conboy, people who I regard as most credible in the field, have said that removing bad actors from the blood is probably more important than restoring youthful levels of beneficial signals. They were grad students at Stanford 15 years ago, when the modern wave of parabiosis science was initiated, and have pursued the subject continuously ever since. Katcher’s experiments have achieved their results only by adding blood components, not by removing or even neutralizing others.

In Katcher‘s experiments, molecular species were added, but nothing was removed. This suggests that he has found the necessary formula for re-programming epigenetics, so that lower levels of the bad actors occur as a result. But it remains to be seen whether even better results can be obtained if some plasma constituents are removed.

A question that remains unresolved concerns the location and mechanism of the aging clock. I have been undecided over the years between two models:

There is a central aging clock, perhaps in the hypothalamus, which keeps its own time and transmits signals throughout the body that coordinate methylation state of dispersed tissues

Information about epigenetic age is dispersed through the body, and the body’s clock is a feedback loop that is continually updating methylation age locally in response to signals received about the methylation age globally.

There is a suggestion in the data that the hypothalamus may be more difficult to rejuvenate than other tissues. Does it play a more important role than other tissues in coordinating the age of the entire body? Horvath (personal communication) counsels caution in drawing this inference until measurements are corroborated and more experiments are done.

The Bottom Line

These results bring together three threads that have been gaining credibility over the last decade. Mutually reinforcing, the three have a strength that none of them could offer separately.

The root cause of aging is epigenetic progression = changes in gene expression over a lifetime.

Methylation patterns in nuclear DNA are not merely a marker of aging, but its primary source. Thus aging can be reversed by reprogramming DNA methylation.

Information about the body’s age state is transmitted system-wide via signal molecules in the blood. Locally, tissues respond to these signals and adopt a young or an old cellular phenotype as they are directed.

Harold Katcher, Biographical Sketch

So, you might consider me a late bloomer.  While I have thousands of citations in the literature, with publications ranging from the discovery of the human ‘breast cancer gene’, to protein structure, bacteriology, biotechnology, bioinformatics, and biochemistry, there was no center or direction to my work as I had given up my personal goal of solving/curing aging when I learned that ‘wear and tear’ was the cause of it.  Yet something happened in year 1985 when I was in California working with Michael Waterman and Temple Smith (fathers of bioinformatics) that is inexplicable: I found myself in Intensive Care with a tube inserted into my trachea and the knowledge that I might not live.   And then I had a dream: I dreamed that somehow in the far future (and on another world), I was being feted for ‘bringing immortality to mankind’. Clearly, I survived that incident (started with an infected tooth).    I lived a wonderful life – becoming a computer programmer (which I loved), leaving that for the University of Maryland’s Asian division, becoming a full professor and then the Academic Director for the Sciences, in Tokyo, Japan.  By the time I left Japan in 2004, (my daughter Sasha was a fourth-grader, (yonensei), in the Japanese school system), I was teaching for U of M online – somewhat retired, and looking forwards to writing computer programs for fun and profit. Yet I never ever forgot that dream. It was clearly impossible; I had no lab – and really, there was no way to repair all damaged cells – it’d be like sweeping back the ocean. And then, in 2009, I read an old paper from 2005, a paper written by the Conboys, (Michael and Irina), Tom Rando and others, coming from Irv Weisman’s lab, that completely changed my life; that showed me that everything I believed about aging was wrong – that aging occurred at the organismic level, not at the cellular level and could be reversed. Well, the rest of the story is about persistence and the blessed intervention of Akshay Sanghvi who too saw there was another way and provided the structural, monetary, and emotional support (and some good ideas) that had me start a new career at age 72 in Mumbai, India.  I feel twenty years younger than I did three years ago, I guess that’s another hint about aging. Now the ‘mystical’ dream?  It wouldn’t be the first time in history that that happened – take that as a datum.

source https://joshmitteldorf.scienceblog.com/2020/05/11/age-reduction-breakthrough/

[healthtimetaylor]

Lung anti-oxidant depletion: a predictive indicator of cellular stress induced by ambient fine particles.

PMID:  Environ Sci Technol. 2020 Jan 21. Epub 2020 Jan 21. PMID: 31961142 Abstract Title:  Lung anti-oxidant depletion: a predictive indicator of cellular stress induced by ambient fine particles. Abstract:  Regulations on ambient particulate matter (PM) are becoming more stringent due to adverse health effects arising from PM exposure. PM-induced oxidant production is a key mechanism behind the observed health effects and is heavily dependent on PM composition. Measurement of the intrinsic oxidative potential (OP) of PM could provide an integrated indicator of PM bioreactivity, and could serve as a better metric of PM hazard exposure than PM mass concentration. The OP of two chemically-contrasted PM2.5 samples was compared through four acellular assays and OP predictive capability was evaluated in different cellular assays on two in vitro lung cell models. PM2.5 collected in Paris at site close to the traffic exhibited a systematically higher OP in all assays compared to PM2.5 enriched in particles from domestic wood burning. Similar results were obtained for oxidative stress, expression of anti-oxidant enzymes and pro-inflammatory chemokine in human bronchial epithelial and endothelial cells. The strongest correlations between OP assays and cellular responses were observed with the antioxidant (ascorbic acid and glutathione) depletion (OPAO) assay. Multivariate regression analysis from OP daily measurements suggested that OPAO was strongly correlated with PAH at traffic site while it was correlated with potassium for the domestic wood burning sample.

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[we-future-first]

[in-depth] How it's made: the science behind cultured/clean/cell-based meat, part 4a: the components of cell culture medium and fetal bovine serum

The Futurology subreddit frequently features highly upvoted posts on cell-based meat, reflecting the media attention and public interest that has followed the industry. There are many introductory resources to how cell-based meat is produced and what its benefits may be, however, there are no comprehensive resources that fully inform those interested in learning more. Below you’ll find the 5th post in a multi-part series that walks through the science driving the innovative technology of cell-based meat. These posts are intended to be educational but lengthy and best understood by those with science backgrounds.

Please check out the previous posts linked below. Each post is also formatted for easier reading here.

Series I: Cell Lines

Series II: Bioprocessing

Series III: Bioengineering 1 and 2

Series IV: Cell culture media 1, 2, and 3

Series V: Final products

Series VI: Impact (environment, human health, food security, animal welfare)

Introduction

Growing cells ex vivo requires the same fundamental inputs as required in vivo: a mixture of a carbon-based energy source, amino acids, salts, vitamins, water, and other components to support cell viability and vitality. This mixture, known as the cell culture medium, is the most important factor in cell culture technology. Although cell culture is routinely performed in academic labs and industrial bioprocesses, creating the biomass required for cell-based meat to achieve mass-market penetration at competitive prices will demand significant reductions in costs, innovations for serum removal, and optimization across a diverse set of species and cell types. An overview of cell culture medium composition and the factors at play to achieve price parity with conventional meat are discussed below.

Common Components of Cell Culture Medium

The first instance of culturing tissues outside of the body came from Sydney Ringer in 1882. By creating a balanced salt solution with similar pH, osmolarity, and salt concentration to that of an animal’s body, Ringer was able to keep various animal tissues alive outside of the body for several days. Subsequent work in the following decades first demonstrated that culturing cells in the presence of blood plasma (i.e. serum) or embryonic extracts assisted in cellular proliferation and viability, allowing tissues to survive for longer periods of time. Over time, researchers identified the importance of glucose, amino acids, glutathione, insulin, and vitamins in the sera being used.1 Once this was known, scientists aimed at uncovering the additional unknown essential components of serum and other extracts that permitted cell proliferation and viability.

In the 1940s and 50s, working with the first immortalized cell lines such as L cells2 and HeLa (discussed in Series I), scientists used iterative approaches to discover that low molecular weight dialyzed fractions of serum containing amino acids were necessary for cell survival. In 1955, Harry Eagle developed a Minimum Essential Medium by testing the amino acid requirements on several different cell lines, discovering that thirteen were indispensable. Eagle’s minimum essential medium additionally consists of glucose, six inorganic salts, eight water-soluble vitamins, and dialyzed serum. Variations on this medium were then derived using a variety of different cell lines as well as trial and error approaches that aimed at replacing serum with chemically defined components. These variations, including Dulbecco’s Minimum Essential Medium (DMEM), Iscove’s Modified DMEM, Ham’s F12, Medium 199, RPMI 1640, Leibovitz’s L-15, and others, still make up the majority of what are referred to as basal cell culture media in use for culturing the variety of cell types used today.3,4

What makes these formulations essential? Although formulations have been varied and optimized over time, the principal components of basal cell culture media have remained largely unchanged. Importantly, these variations may be cell-type specific, including for the cell types used in cell-based meat (described in Series I). Therefore, rather than discussing optimal conditions for a specific cell line or species, only the general roles of each component of common basal media including glucose, amino acids, inorganic salts, vitamins, and buffers are briefly discussed below.

Glucose

Glucose (specifically D-glucose) is the most common energy input used in cell culture, although some media formulations use galactose or a combination of glucose and its metabolite, pyruvate. Industrially, it is produced enzymatically using amylase enzymes to breakdown starches from maize, potato, wheat, and other crops into constituent sugars used in various downstream products such as industrialized food, fermentation processes, or in this case, culturing of cells. Glucose enters the cell via transporter proteins on the cell surface, using either passive transport down its concentration gradient (more common) or ATP-dependent active transport. Once inside the cell, it serves as a reducing agent against oxidative stress in the form of NADPH generation via the pentose phosphate pathway, as well as a primary source of energy in the form of ATP generation via glycolysis.

In cell culture, glucose is used at concentrations between 5.5 and 55 mM, where the lower end is more common and similar to fasting blood glucose levels in humans. Different cell types will require different amounts of glucose. During periods of rapid cell proliferation and growth, as typically maintained during bioprocessing, glucose metabolism is high and can yield lactic acid even in the presence of sufficient oxygen, leading to pH changes.5 Thus, glucose and lactic acid levels are commonly measured and tightly controlled throughout a bioprocess (discussed in Series II).

Amino Acids

Amino acids are necessary to create proteins and other low molecular weight compounds such as nucleotides and small peptides. Amino acids can be split into two groups: essential and non-essential. Non-essential amino acids (NEAAs) can be synthesized de novo by an animal, whereas essential amino acids (EAAs) must be obtained through the diet. Generally speaking, pathways for the de novo synthesis of NEAAs are conserved in vertebrate species.6 In humans and many other animals, the EAAs include histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine. NEAAs include alanine, arginine, asparagine, aspartate, cysteine, glutamate, glutamine, glycine, proline, serine, taurine, and tyrosine. However, EAA requirements can vary between species. For instance, dogs, cows, and pigs have the same EAA requirements as humans plus arginine, whereas cats and chickens require the same EAA as the former plus taurine and glycine, respectively.

Importantly, what is considered to be “essential” in cell culture is different than what is considered “essential” to a whole organism, as the diversity of cell types that may synthesize certain amino acids in vivo are not present in vitro. For instance, Eagle’s Minimum Essential Medium formulation lists 13 (L-enantiomer) amino acids as being essential across multiple cell lines in vitro: arginine, cysteine, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, tyrosine, and valine. As an example, arginine is essential in vitro as its biosynthesis in vivo primarily occurs between epithelial cells in the gut and proximal tubule cells of the kidney. Thus, arginine must be supplied in the absence of these cell types. Media that are particularly nutrient-rich (eg. DMEM/F12 or Medium 199) may contain all amino acids. Alternatively, NEAAs can be supplemented independently.

Industrialized production of amino acids can be obtained through bulk extraction from protein hydrolysates (discussed later), chemical synthesis, or microbial fermentation and purification, with the latter being the most common.7 Amino acids enter the cell through a variety of transporter proteins on the cell surface, at rates influenced by the cell’s state and consumption rates due to protein production levels, cell cycle state, and other parameters. Once inside the cell, amino acids serve as substrates for many biosynthetic pathways and optimal concentrations are important for maintaining metabolic equilibrium. The majority of carbon mass in proliferative cells is derived from bulk amino acids rather than glucose or L-glutamine, which are the most rapidly metabolized.8

Ultimately, the levels of amino acids required for cell culture are determined not only by their utilization by the growing cells, but also by individual amino acid solubility, stability, and interaction with other medium components such as metal cations, all of which can change once in a complex mixture.9 Consideration for all of these variables is highly complex and a full understanding of amino acid behavior, utilization, and optimization in a bioprocess has yet to be accomplished. Given the variety of biosynthetic pathways that involve amino acids, it is likely that amino acid content, concentration, and perfusion rate (when applicable) will need to be optimized for a particular bioprocess across species and cell types for parameters such as growth rates or protein content in the final product. Computational approaches to model specific utilization rates of amino acids and other basal media components are an active area of research10 (discussed later).

L-glutamine

L-glutamine deserves special consideration as one of the most important amino acids included in cell culture media, as it is readily transported into cells and becomes a major contributor to protein biomass. It is a notable precursor of carbon and nitrogen-containing biomolecules such as the intermediate molecules used in the synthesis of other amino acids and nucleotides11 and it can be added at concentrations 3-40x higher than other amino acids in the medium.12 During times of high cellular growth and proliferation, the demand for glutamine outpaces its supply, making it de facto an essential amino acid that can be readily metabolized as a replenishing alternative energy source (i.e. anaplerosis). At physiological pH in a cell culture medium solution, L-glutamine is unstable, resulting in its decomposition into pyroglutamate and ammonia, the latter of which is toxic to cells. Ammonia, therefore, is a tightly monitored and regulated metabolite in large scale bioprocesses that involve high densities of cells undergoing rapid growth (discussed in Series II).

In order to avoid some of these disadvantages of L-glutamine, glutamate — which is more stable in solution — can be substituted in when working with cells expressing high levels of glutamine synthetase, an enzyme which enables intracellular conversion of glutamate to glutamine while consuming ammonia in the process. A more common practice involves supplementation with L-glutamine as a stable dipeptide in the form of alanyl-glutamine (i.e. GlutaMAX) or glycyl-glutamine, which enable cells to endogenously cleave the dipeptide for more controlled usage of the amino acids in the dipeptide. There is still much to learn about amino acid metabolism in cell culture. For instance, recent discoveries suggest L-glutamine is entirely dispensable for the culture of pluripotent stem cells.13

Inorganic Salts

The inclusion of inorganic salts) is important in establishing and maintaining the osmolarity of the cell with its surrounding cell culture medium solution as well as serving as enzymatic cofactors and important components of receptor and extracellular matrix proteins. These inorganic salts are composed of cations and anions that fully dissociate in solution. The original minimal essential medium solution contained six inorganic salts (calcium chloride, potassium chloride, magnesium sulfate, sodium chloride, sodium phosphate, and sodium bicarbonate), which are based on Earle’s salt solution. Other formulations include additional inorganic salts containing zinc, copper, and iron, which have particular importance for a variety of cellular functions (discussed later).

Although all cells maintain a resting membrane potential, excitable cells such as neurons and skeletal muscle cells are particularly sensitive to changes in ionic concentrations that can readily affect their functionality and viability. Several basal medium formulations have thus been optimized for salt concentrations for neuronal14 and skeletal muscle cell culture that more accurately recapitulate the interstitial fluids surrounding these cell types. The osmolality or measurement of osmotic pressure within the medium is typically between 260 to 320 mOSM/kg (milliosmoles per kg of solute), although this can vary with cell lines that are particularly robust in varying solute concentrations such as insect cells.15 Changes in the salt concentration, either abruptly due to medium changing or slowly due to water evaporation, can lead to osmotic shock. Thus, maintenance of osmolarity is an important component of cell culture.

Vitamins

Vitamins are classes of organic compounds that serve as a critical component for the maintenance and growth of cells. Most vitamins are essential in that they need to be obtained directly from the diet or cell culture medium with few exceptions (e.g. vitamin D synthesized by fibroblasts and keratinocytes of the skin or some B vitamins produced in low levels by intestinal microbiota). Vitamins are classified as either fat-soluble or water-soluble and can serve broadly as enzymatic cofactors, antioxidants, and hormones. Vitamins are processed in a variety of ways in vivo following ingestion, often in a complex sequence that ends in absorption into intestinal cells via membrane surface transporters. This complex sequence involved in absorption can be largely avoided in vitro, as hostile environments (e.g. stomach acid) or barriers (e.g. the blood-brain-barrier) are absent.16 Thus, vitamins are typically included in a medium formulation as a single chemical compound that can be processed and absorbed directly by cells in vitro.

Vitamins can also effectively function as a group of compounds (i.e. vitamers) where each compound can serve the vitamin’s functional role, albeit with varying properties. The natural production of vitamins in microbes and plants has made industrial production of vitamins via microbial fermentation possible, however, improvements in metabolic engineering strategies are needed to increase yields and sustainability in the industry. For these reasons, some vitamins are produced more efficiently via chemical synthesis.17

Water-soluble vitamins including riboflavin (vitamin B2), nicotinamide (vitamin B3), pantothenic acid (vitamin B5), pyrodoxine and pyridoxal (vitamin B6), biotin (vitamin B7), i-inositol (vitamin B8), folic acid (vitamin B9), cyanocobalamin (vitamin B12), and choline are typically added to and “essential” in cell culture media, sometimes in various modified forms in order to provide stability. Fat-soluble vitamins A, D, E, and K are excluded in basal medium formulations but can be added if necessary when dissolved in an organic solvent. Similar to the different in vivo versus in vitro requirements of amino acids, fat-soluble vitamins play specific roles for certain cell types or bodily functions and are thus only “essential” when culturing a relevant cell type. For instance, a metabolite of vitamin A, retinoic acid, is an important developmental morphogen (discussed in detail later) and may be included as an additive in media to derive spinal motor neuron cells from pluripotent stem cells.18 Special consideration for stability must be taken when using serum-free medium formulations (discussed later) as the lack of stabilizing serum proteins can lead to rapid degradation via light, heat, oxidation, or pH fluctuations.19 These properties make it advisable to reconstitute powdered B vitamins immediately before use (discussed later).

Buffering Systems

Buffers are essential to cell culture systems as they serve to maintain pH at a constant level (for mammalian cells, generally 7.4 ± 0.4) despite changes in the composition of acids or bases that would otherwise alter the pH of the cell culture medium. Buffers are mixtures of a weak acid and its conjugate base or a weak base and its conjugate acid, where each mixture serves as a sponge to soak up free protons or hydroxide ions in solution, minimizing their effect on overall pH. Buffer systems in cell culture typically consist of either CO2-bicarbonate systems or buffering agents such as HEPES. As discussed in Series II, a CO2-bicarbonate system can be achieved by exogenous addition of 5-10% gaseous CO2 (often delivered in bioreactor systems via sparging)), which reaches equilibrium in solution with bicarbonate ions, forming a natural buffer system.

pH slowly changes over time due to the respiration of cells and the release of additional CO2, which forms carbonic acid in solution, in addition to the metabolism of glucose and the formation of lactic acid. The resultant decreasing pH changes are counteracted by the inclusion of sodium bicarbonate in the basal medium itself. Importantly, added sodium bicarbonate should be proportional to the atmospheric CO2 being used to maintain equilibrium. For instance, for media containing 1.5 to 2.2 g/L sodium bicarbonate, 5% CO2 is recommended, whereas 10% CO2 is recommended for media containing 3.7 g/L sodium bicarbonate.

HEPES is a zwitterionic buffer that can be used in cell culture systems as a supplemental buffer, especially in the absence of CO2 exposure. As one of Good’s buffers, its high solubility, low toxicity, and membrane impermeability have made it attractive for use in cell culture applications. In the scale-up of highly proliferative stem cell populations, dissolved CO2 due to high metabolism can reach levels that are deleterious for cell growth and nutrient utilization.21 Attempts have thus been made to limit dissolved CO2 by culturing cells in the presence of atmospheric CO2 levels with added buffering capacity from HEPES or other Good’s buffers.22 This strategy may be useful for future scale-up efforts in cell-based meat. Consideration for the cost of the buffer must also be weighed, as it may constitute the most expensive component of a basal media formulation at scale.

Preparation

Out of convenience, most academic and lab-scale cell culture is performed using commercially available premade liquid media. However, large volumes necessitate on-site preparation of liquid cell culture media from reconstituted powdered medium ingredients. Powdered medium is more efficiently transported and stored, resulting in cost savings and reduced degradation of fragile ingredients (e.g. B vitamins). Ideally, a powdered medium contains all of the components to be utilized and is created through a process known as micronization, where the average size of crystallized particles in the mix is reduced in order to increase solubility and homogeneity. When ready to use, the powder is typically reconstituted in a dedicated tank using high-quality water prepared by reverse osmosis, deionization, and filtration. The reconstituted medium is then itself sterilized by filtration (e.g. through a 0.22 µm filter), irradiation, or other methods discussed in Series II (e.g. pulsed electric fields). The use of sterilization involving high heat is precluded by some heat-labile ingredients that may be part of the formulation. Other preparation methods for additional ingredients are discussed throughout.

Serum

As previously mentioned, a basal medium formulation is often sufficient to keep cells alive for short periods of time, but in order for them to proliferate efficiently over extended periods of time, a variety of animal sera) (e.g. fetal bovine serum, horse serum, and others) and extracts (e.g. chick embryo extract) have historically been used (notably, on a volumetric basis, serum-free formulations are now more dominant in their usage although FBS is still often included in routine cell culture in academic settings). Serum is a high protein-containing mixture that contains growth and attachment factors, hormones, antioxidants, lipids, and other components (all described later) that mimic a proliferative, fetal-like state. Indeed, most sera used in cell culture are derived from fetal animals, which are rich in the necessary components and contain low immunoglobulin and complement content due to developmentally immature immune systems. As fetal bovine serum (FBS) is the most common sera used in cell culture, it will be used as a reference example throughout this section.

Originally employed in the late 1950s,24 FBS has become a mainstay in biomedical research because it can supplement the growth of virtually all common human, animal, and even insect cell lines. As an added supplement for many cell culture applications in amounts typically 5-20% of total medium volume, FBS — when used — is often the most expensive part of performing cell culture.

FBS is harvested from a fetal calf any time during the last two-thirds of gestation following the discovery of pregnant cows due for slaughter. It has been estimated that up to 8% of cows in the slaughter line may be pregnant, making FBS a byproduct of the meat processing industry.25 It is prepared by the sterile collection of fetal blood followed by coagulation at low temperatures and centrifugation to remove clotting factors and blood cells. The serum supernatant is then filtered and assessed for a variety of quality controls including residual microbial or viral contamination, endotoxin, immunoglobulin content, and total protein, before being bottled and sold commercially, at prices exceeding $1000 USD per liter (at time of writing, July 2019) depending on quality control parameters (some described later), which vary by industry and use-case.

Despite its long history of use, FBS has several well-described issues that have made its replacement a priority in recent years. First, FBS contains hundreds or even thousands of different components and the true composition and amounts of these components are unknown, making it a chemically undefined product. The composition also varies by geographic region where a cow’s diet can vary, by batch within the same geographic region, by seasonality of collection, by the quantity and identity of antibiotics or hormones received by the mother, and by the gestational age of the fetus. Variability can also stem from a single bottled product originating from fetuses of different sexes.26 This variability has led to a growing concern over serum’s contribution to irreproducibility of in vitro experiments within and between labs around the world.27 Rigorous quality control involving testing of serum batches across multiple cell lines or experiments prior to purchasing a specific, well-performing large batch is often performed in industry but can remain burdensome from a labor and economic perspective for smaller academic labs. Thus, the inherent variability and undefined nature of FBS use leads to compounding external costs in quality control testing, experimental irreproducibility or conflicting results, and follow-up research to dissect irreproducible signals.

Second, FBS is a potential source of contamination from multiple organisms, including Mycoplasma, viruses, and bovine spongiform encephalopathy. Mycoplasma are a class of parasitic bacteria that lead to metabolic and gene expression variations for infected cell lines. Mycoplasma are likely the most common cell line contaminant, with recent estimates showing 11% of cell lines being infected, and rates as high as 70% in geographical regions where testing is not routine.28 Although presently FBS is routinely filtered using 0.1 micron systems that should theoretically capture Mycoplasma, suppliers cannot make this guarantee. The common cell line contaminants M. arginini and A. laidlawii, in particular, have been linked in origin to FBS, and ongoing cross-contamination of cell lines has likely propagated this contamination in laboratories since the 1960s and 1970s when FBS batches were routinely positive for these bacteria.29 Additional methods to decontaminate serum from Mycoplasma include gamma irradiation, however, this can also damage growth factors and other proteins in the serum.30 Thus, the use of FBS is responsible for a non-trivial amount of bacterial contamination in cell lines today, leading to compounding problems concerning reproducibility and potential unknown variability stemming from some decontamination practices.

In addition to bacterial contamination, the threat of adventitious viral agents in FBS also persists. Regulations under USDA and the EU mandate the testing and/or treatment (via heat or irradiation) of eight viruses known to be present in FBS from all geographical regions of origin.31 Although modern production methods make the risk of contamination in a validated batch low, viral contamination is often still detectable in batches that manufacturer screens claim to be negative.32 Similarly, the threat of FBS containing the causative prion proteins involved in bovine spongiform encephalopathy (i.e. Mad Cow Disease, which manifests in humans as variant Creutzfeldt-Jakob Disease) is persistent and requires additional testing as well as documented traceability for the FBS origin. For instance, countries such as the USA, New Zealand, and Australia have no documented cases of bovine spongiform encephalopathy; thus FBS originating from these countries may be considered ‘safer,’ often commanding significantly higher prices and collectively comprises up to 90% of the serum supply for commercial therapeutics.33 This fact has also incentivized fraudulent activity in the field, where manufacturers may opt for fake labels from New Zealand in order to solicit higher prices.34 Industry associations have formed in an attempt to mitigate these concerns. Nevertheless, the inherent risk of contamination from FBS poses threats to experimental and bioprocess reproducibility, drives price fluctuations, and can even incentivize bad actors that value profit over safety. Contamination will be discussed further from a food safety perspective in Series V.

Third, there is a limited global supply of FBS and there exists competition for it from profitable, mature industries. For instance, while the vaccine and biologics industries have begun to move to serum-free formulations (discussed later), the rise of cell therapies and stem cell research more generally has ushered in an impending demand that exceeds current availability. Because FBS is a byproduct of a more lucrative product per animal (i.e. meat and dairy) and profits are retained by slaughterhouses rather than farmers, farmers have little incentive to increase cattle herds to meet a future FBS demand.35 It has thus been hypothesized that “peak serum” has been met, with serum availability relatively stagnant and serum demand increasing dramatically as cell therapies begin to be approved.36 The replacement of serum thus may be driven first by limited total availability followed by cost concerns that will spur replacement innovation in the field as non-pharmaceutical players are priced out. In the case of cell-based meat, this cost concern is already prohibitive, making FBS an economic nonstarter as meat products cannot be justified at prices that rival a cell-based therapeutic (currently at a cost of goods of approximately $50,000 and selling price of hundreds of thousands of dollars).

Lastly, the use of FBS carries ethical concerns, making its use inherently misaligned with one of the fundamental benefits of cell-based meat: animal welfare (discussed in Series VI). A single liter of serum requires 1-3 fetuses, with roughly 2 million fetal calves used in serum collection annually, totaling approximately 800,000 liters of FBS produced per year. The collection process involves removal of the fetus from the mother’s womb and aseptic collection of blood by a syringe placed directly into the beating heart as this contains unclotted blood, raising concerns that the fetus could consciously experience the event as painful.37 Thus, the search for serum-free formulations (discussed later) is in alignment with the cell-based meat industry and general animal welfare concerns, manifested by replacement, reduction, or refinement of animal experiments or animal-based products in science.

The next series on cell culture medium will explore the components of serum that have made it a near-universal cell culture supplement and approaches for replacing serum in a cost-effective manner.

About / Disclosure

Elliot Swartz, Ph.D. (/u/e_swartz) is the author and is employed by The Good Food Institute, a 501(c)3 nonprofit using markets and innovation to accelerate the plant-based and cell-based meat sectors.

Feel free to ask anything about the science discussed or how to get more involved in the future of food. Many questions will additionally be addressed in upcoming discussion topic series!

submitted by /u/goodfoodinstitute [link] [comments] source https://www.reddit.com/r/Futurology/comments/d6uup8/indepth_how_its_made_the_science_behind/

[juniperpublishers-agriculture]

Using Xyloglucan Oligosaccharides as Biostimulant to Enhance Tobacco Tolerance to Salt Stress- Juniper Publishers

Abstract

Xyloglucan oligosaccharides (XGOs) derived from the hydrolysis of plant cell wall xyloglucan, are a new class of naturally occurring biostimulants that exert a positive effect on plant growth and morphology and can enhance plant’s tolerance to stress. Here, we aimed to determine the influence of exogenous Tamarindus indica L. cell wall-derived XGOs on Nicotiana tabacum’s tolerance to salt stress by examining the plant’s morphology, physiological, and metabolic changes after XGO application. N. tabacum plants were grown in solid media for two months under salt stress with 100mM of sodium chloride (NaCl) ± 0.1μM XGO. Germination percentage (GP), number of leaves (NL), foliar area (FA), primary root length (PRL), and density of lateral roots (DLR) were measured. Also, 21-old-day N. tabacum plants were treated with a salt shock (100mM NaCl) ± 0.1μM XGOs. Proline, total chlorophyll, and total carbonyl contents in addition to lipid peroxidation degree and activities of four enzymes related to oxidative stress were quantified. Results showed that under saline conditions, XGOs caused a significant increase in NL and PRL, promoted lateral root formation, produced an increase in proline and total Chl contents, while reducing protein oxidation and lipid peroxidation. Although they modulated the activity of the enzymes analyzed, they were not statistically different from the salt control. XGOs may act as metabolic inducers that trigger the physiological responses for counteracting the negative effects of oxidative stress under saline conditions.

Keywords: Antioxidant system; Biostimulants; Nicotiana tabacum; Salt stress; Xyloglucan oligosaccharides

Abbreviations: CAT: Catalase; Chl: Chlorophyll; DLR: Density of Lateral Roots; FA: Foliar Area; GP: Germination Percentage; GPX: Peroxidase; GR: Glutathione Reductase; MS: Murashige and Skoog; NaCl: Sodium Chloride; NL: Number of Leaves; PCA: Principal Component Analysis; PRL: Primary Root Length; RL: Lateral Roots; ROS: Reactive Oxygen Species; SOD: Superoxide Dismutase; XGOs: Xyloglucan Oligosaccharides

    Introduction

Modern agriculture faces many challenges in order to meet the growing demand for worldwide food. The world’s population is growing at an accelerated rate. By the end of 2050 it is expected to reach 9.8 billion people and 11.2 billion in 2100 according to the “World Population Prospects: The 2017 Revision”, published by the United Nations Department of Economic and Social Affairs. However, food productivity and availability are decreasing as a result of the effects of several biotic and abiotic factors. Therefore, several actions are being taken to reduce these losses and to cope with the growing food need for the world’s population.

Soil salinity is a worldwide phenomenon that occurs under almost all climatic conditions and is a major impediment to achieving increased crop yields. Using the FAO/UNESCO soil map of the world (1970–1980), FAO estimated that 19.5% of irrigated land were salt-affected soils, and of the almost 1.5 billion ha of dryland agriculture, 32 million (2.1%) suffer from salinity problems [1]. Salt-affected soils are characterized by abundant quantities of neutral soluble salts that adversely affect plant uptake of nutrients in the soil and their growth [2]. Under salt stress, plants are also under other types of stresses, which have deleterious effects on them such as water stress, ionic toxicity, and nutritional deficiencies [2]. Altogether, these conditions confer oxidative stress and metabolic imbalance to plants [3]. Consequently, plants exposed to high saline conditions shown growth inhibition or retardation.

The morphology of plants exposed to salinity can be affected by soil salt concentrations, type of plant species, age, and plant stages (vegetative or flowering), and/or the type of salt present [4,5]. For example, there is a decrease in plant lengths, leaf (foliar) areas, leaf numbers and root systems under high concentrations of NaCl [4]. Also, many studies confirm the inhibitory effects of salinity on photosynthesis by changing chlorophyll content thus affecting Chl components and damaging the photosynthetic apparatus [5].

In addition, plants exposed to high NaCl concentrations (such as100-200mM) show rapid overproduction of reactive oxygen species, which have detrimental effects on the plants’ cells. ROS causes membrane lipid component peroxidation and oxidation of cellular components such as proteins and nucleic acids, which finally lead to programmed cell death [6,7]. ROS-initiated damage is reduced and repaired by a complex antioxidant system, which combines enzymatic and non-enzymatic components. It consists of low molecular weight antioxidant metabolites, including ascorbic acid, carotenoids, glutathione, α-tocopherol and enzymes such as catalase, peroxidase, superoxide dismutase, glutathione reductase and others. The degree of cellular damage will depend on the balance between ROS production and elimination by the antioxidant scavenging system [8].

Plants also accumulate compatible solutes in response to salt stress, which provides protection to them by participating in ROS detoxification and cellular osmotic regulation in addition to contributing to enzyme/protein stabilization and membrane integrity protection [6]. Among them, proline is one of the most important ones due to its multiple roles as part of the plant’s response to various types of stresses. It functions as an osmolyte for osmotic adjustment, buffering cellular redox potential under stress conditions, maintaining protein integrity, enhancing different enzymes activities, and free radical scavenging [9,10]. Its accumulation in leaves under salt stress has been correlated with stress tolerance in many plant species, allowing them to survive under this type of stress [6].

Many efforts have been done to overcome the problems associated with high soil salinity and salt stress in plants. However, the use of traditional physical and chemical methods for environmental restoration of salt contaminated soils demand significant investment of technological and economic resources [11]. In addition to these traditional approaches, different biostimulant classes have been used to increase crop performance under salt stress and to mitigate stress-induced limitations [12-14]. A plant biostimulant is any substance or microorganism that is applied to plants with the aim of enhancing nutrition efficiency, abiotic stress tolerance and/or crop quality traits regardless of its nutrients content. By extension, they also designate commercial products containing mixtures of such substances and/or microorganisms [15]. Plant biostimulants based on natural materials have received considerable attention by both the scientific community and commercial enterprises. According to Stratistics Market Research Consulting (MRC), the Global Biostimulants Market is accounted for $1.50 billion in 2016 and is expected to grow gradually to reach $3.79 billion by 2023 due to growing importance for organic products in agricultural industries [16]. However, understanding the mechanisms by which biostimulants act is critical to their widespread use for helping plants cope in saline-affected soils.

XGOs, derived from the breakdown of xyloglucans in plant cell walls, are emerging as a new class of naturally occurring biostimulants as a result of their positive effects on plant growth and morphology [17-19]. Plant-derived XGOs are also used as biotic pesticides and seed coating agents to maintain plant freshness in addition to capsule materials for synthetic seeds [20]. Xyloglucan is the quantitatively predominant hemicellulosic polysaccharide in the primary walls, which consists of ~20% (w/w) dicot and ~5% monocot primary cell walls [21]. Its backbone is composed of a β-(1,4)-D-glucan backbone that is quasi-regularly substituted with α-D-xylosyl residues linked to glucose through the O-6 position. In many species, the backbone has a regular pattern of three substituted glucose units followed by an unsubstituted glucose residue [22]. As a variety of complex structures can be formed, a code letter for each glucosyl residue has been defined to allow for the unambiguous naming of xyloglucan oligosaccharides. For example, XGOs can be classified as the XXXG-type of the XXGG-type, in which a capital G represents a unbranched Glcp residue and a capital F represents a Glcp residue that is substituted with a fucose- containing trisaccharide [23]. Soluble XGOs can be obtained from tamarind (Tamarindus indica L.) seeds after partial digestion with cellulase. A fraction of these XGOs have been shown to have physiologically active functions in plants and oligosaccharides, also known as oligosaccharins [19,24]. Their biological properties in plants depends on the fragmented structures and their concentrations, which need to be extremely low to get a variety of effects (10–9 - 10–8M) [17-19]. Few experimental data are available concerning the use of the XGOs as plant biostimulants for mitigating the damage imposed by salt stress conditions in plants [25]. Also, each new formulation requires a new biological evaluation to ensure that the effects are beneficial, consistent, and predictable.

For all of the above, the objective of this study was to determine the biostimulating effects of application of exogenous XGO derived from T. indica L. cell walls on N. tabacum seedlings grown under saline stress conditions with special attention to their influence on plant morphology, necessary physiological and metabolic changes to overcome stress, ROS detoxification, and antioxidant capacities.

    Materials and Methods

Oligosaccharin composition and concentration used

The XGO fraction used in this work was the same formulation previously reported and tested on plants [18,26]. Briefly, XGO was extracted and purified from tamarind (T. indica L.) seeds. The predominant composition of the XGO extracts consisted of XLLG and XXLG/XLXG with lower proportions of XXXG, XXGG, and XXG oligosaccharides as classified by Fry et al. [23]. Mass spectra obtained by matrix-assisted laser desorption ionisation-time of flight (MALDI-TOF) spectrometry [27] are shown in Supplementary Table 1&1A. Relative proportions of xyloglucan oligosaccharides obtained by MALDI and high-performance anion-exchange chromatography with pulsed amperometric detection analysis were similar (data not show). The isolated XGO fraction showed no cellulase activity, and protein could not be detected. The uniformity of the XGO mixture was confirmed by gel filtration analysis through a BioGel P2 (Bio-Rad Laboratories, Inc., Hercules, CA, USA). XGOs were used at a final concentration of 0.1μM which was selected based on previous experiments as an optimal concentration for stimulating root and leaf development in N. tabacum without causing changes in plants’ chromosome number [17,28].

Plant material and general growth conditions

Botanical seeds of N. tabacum Linn. were used as the plant model. Seeds were kindly provided by Dr. Alexis Acosta Maspons from the Institute of Biotechnology of National Autonomous University of Mexico (UNAM) (Cuernavaca, Morelos, Mexico). All seeds were harvested at the same time, kept at 4 °C in the dark, and grown under the same controlled conditions. Seeds were surface- sterilized and grown on MS [29] solid medium in a growth chamber (DAIHAN Scientific, model WISD, Korea) at 23 °C in longday conditions (16h light/8h dark) and 50% relative humidity. Sowing and the way in which each treatment was applied was according to the chosen evaluation (plant and root morphology measurements and biochemical analysis) and is explained in detail for each one in this section. Each experiment was repeated to generate three biological replicates.

Germination percentage and plant morphology measurements

Disinfected seed were sowed onto magenta boxes with MSagar- media, either alone as a negative control, supplemented with XGO at 0.1μM or 100mM NaCl to induce salt stress, or a combined (both NaCl+XGO). The concentration of the NaCl solution was determined based on experimental data (data not shown) in which it was considerable biomass decrease in the presence of 100mM NaCl was demonstrated. Each treatment consisted of nine seeds per magenta box and five boxes per each biological replicate. Germination percentage was calculated 10 days after sowing. Germination criteria were considered complete germination after the embryo emerged from the seed and a whole seedling was formed [30]. For plant morphology analysis, seedlings were grown for two months, and the number of leaves was then counted. Also, leaves were harvested by cutting three of the oldest ones to measure their foliar area, which were immediately photographed under a stereomicroscope (Olympus, Model CX31-RTSF) coupled to an Infinity Analyzer camera. FA was measured using the Infinity Analyze 3 software (Lumenera) according to the manufacturer instructions. For root length measurements and lateral root primordium frequency, sterilized seeds were plated on square Petri dishes in a vertical orientation containing MS-agar-media as control or supplemented with XGO at 0.1μM or 100mM NaCl to induce salt stress or a combination of XGO+NaCl. Each treatment consisted of six seeds per Petri square dishes and four dishes per each biological replicate. All measurements were performed on 3-week old plants that were fixed for 72h as previously described and had exhibited whole root systems [18]. The total number of lateral roots, which is the sum of the number of lateral root and number of primordia, were counted directly under a stereomicroscope (Olympus, Model CX31-RTSF). The fixed plants were then placed on a slide to allow the primary root extension and measurements by taking photographs and processing the images. A microscope (Olympus, Model SZ2-ILTS) coupled to an Infinity Analyzer camera was used, and primary roots lengths were measured using the Infinity Analyze 3 (Lumenera) software according to the manufacturer instructions. Lateral root density (DLR, represented as D in the formula) per mm of primary root length (PRL, represented as L in the formula) was calculated using the following equation: D = (RL+P)/L, where RL + P is the sum of the number of lateral root and number of primordia [31].

Induction conditions for biochemical analysis

For all of the biochemical analyses, a uniform induction experiment was designed in order to analyze XGO- (alone or in combination with salt shock) induced dynamic changes in N. tabacum seedlings. Specifically, we focused on the restitution phase (stage of resistance, continuing stress) of plants’ stress response phases [32]. Consequently, seeds were first placed in magenta boxes containing MS-agar-media to allow their homogeneous growth for 21 days. After that time, four treatments were administered:

a. One mL of sterile distilled and deionized water as a negative control.

b. A solution of 100 mM NaCl for salt shock.

c. 0.1μM XGO.

d. 0.1μM XGO+100mM NaCl were added to the top of the solid media.

Sampling leaves were taken at two and five days after induction, immediately frozen in liquid nitrogen, and stored at -80 °C until their use.

Quantification of proline and photosynthetic pigment content

After two and five days of XGO application with or without salt shock using NaCl 100mM, proline and total chlorophyll (Chl a+b) contents were measured. Proline extractions and quantifications were performed as previously described [33]. Briefly, the extract was prepared by mixing 20mg of ground leaves in 1mL of 80% ethanol, sonicated for 5min, and incubated for another 20min in the dark. The mixture was centrifuged at 20,000g for 5min, and 200μL of the supernatant were added to 400μL of reaction mix (ninhydrin 1% (w/v) in acetic acid 60 % (v/v), ethanol 20% [v/v]), and heated at 95 °C for 20min. Finally, absorbance was determined at 520nm using a FLU Ostar Omega Microplate Reader (BMG LABTECH GmbH, Germany). A calibration curve was used for proline concentration quantification and expressed as μg of proline per mg of plant fresh weight. For photosynthetic pigment content quantification, the Chl a and b and total chlorophyll (Chl a+b) contents were extracted with 80 % acetone and measured as described elsewhere [34].

Protein and lipid oxidative damage

Plant leaf tissue was ground to a fine powder with liquid nitrogen to ensure sample homogenization. Protein oxidation was measured using protein carbonyl content [35]. Briefly, 100μL of sodium phosphate buffer (PBS, pH 7.8) was added to 400mg of each sample, sonicated for 20min in the dark, centrifuged at 20,000g during 20min at 4 ºC, and the supernatant was used. Protein concentration in the supernatant was measured using a BCA Quantic Pro Sigma Kit to compare carbonyl content related to total protein content. Total carbonyl content was measured using dinitrophenylhydrazine (DNPH) reagent [35].

Lipid oxidation was analyzed with a thiobarbituric acid reactive substances (TBARS) method [36]. For the assay, 200mg of ground plant sample was submerged in 1000μL of acetone and sonicated for 5min. The mixture was then incubated for 10min in the dark and centrifuged at 4 °C and 20,000g for 5min. Then, 200μL of the supernatant was added to 300μL of a reaction mix containing 2:1 of 20 % (v/v) trichloroacetic acid (TCA) and 0.67% (w/v) of thiobarbituric acid (TBA). The reaction mix was heated at 95 °C for 15min, cooled at room temperature, and centrifuged at 20,000g at 4 °C for 20min. Lipid oxidation was measured by determining the absorbance at 532nm using a FLU Ostar Omega Microplate Reader (BMG LABTECH GmbH, Germany). The methylenedianiline (MDA) standard and standard curve for the estimation of total MDA were prepared as previously described [37]. Results were indicated as A532 per gram of plant sample.

Antioxidant enzyme activities

Plant leaves collected (0.4g) were homogenized in liquid nitrogen and 100μL of PBS (pH 7.8) containing protease inhibitor (Sigma Aldrich) concentration 5X. The mixture was then sonicated for 20 min in the dark and centrifuged at 20,000g at 4 ºC for 20min, after which time the protein content was measured using a bicinchoninic acid (BCA) Sigma® kit. Enzyme activities were determined immediately. Activities of the antioxidant enzymes, CAT (EC EC 1.11.1.6), GPX (EC 1.11.1.7), GR (EC 1.6.4.2, GR), and SOD (EC 1.15.1.1, SOD) were determined. The evaluation of enzymatic activities was performed by comparing equal amounts of total protein extracts from the samples collected.

CAT activity was measured by monitoring the enzyme-induced decomposition of an H2O2 solution at 240nm and calculated as H2O2 reduced per mg of protein per min [38]. GPX activity was assayed as previously described [39], in which the reaction mixture contained potassium phosphate buffer (100nM), guaiacol (15mM, pH 6.5), H2O2 0.05 % (v/v), and 60μL of protein extract. Guaiacol oxidation was monitored at 470nm and an enzyme unit was defined as the production of 1μm of oxidized guaiacol per mg of protein per min. SOD activity was measured by adapting the previously described chromogenic assay [40] for leaf tissue protein extract analysis. Briefly, for the reaction 225μL sodium pyrophosphate (pH 8.3, 0.025M), 18.8μL (186μM) phenazine methosulfate, 56.3μL (300μM) nitroblue tetrazolium, 93.7μL distilled water, and 5μL of protein extract were mixed. To initiate the reaction, 37.5μL (780μM) nicotinamide adenine dinucleotide was added, incubated for 1.5 minutes and 187.5μL glacial acetic acid were added to stop the reaction. The chromogen was extracted by addition of 700μL n-butanol followed by incubation for 10min, centrifugation at 20,000g for 5min, and absorbance measurement at 560nm. For GR activity measurement, the reaction was started by the addition of oxidized glutathione, and the decrease in absorbance at 340nm every min over a 3min period was read [41]. GR activity corresponded to the amount of enzyme required to oxidize 1μmol min-1 of nicotinamide adenine dinucleotide phosphate. For all enzyme activity analyses, results were expressed as U mg-1 protein.

Statistical analysis

For all variables analyzed, each experiment was performed in triplicate. The data were expressed as average ± standard deviation (SD) of the three independent replicates as a measure of dispersion. For the variable NL, FA, PRL, and DLR, the data were evaluated by an analysis of variance (ANOVA) by ranks (Kruskal Wallis test) and compared using a nonparametric multiple comparison test proposed by Conover [42] because the variables evaluated did not show a normal distribution and had heterogeneous variances. The adjustment to the premises was verified through the tests of Shapiro Wilk and Levene. PCA was performed with Pearson correlation matrices to represent a two-dimensional plane of treatment effects upon the five morphological traits [43]. The values of eigenvectors higher than the mean of the minor and the major values of the component were considered as significant.

Data obtained from biochemical analyzes were processed using a factorial ANOVA using a fixed effect model, in which the factors consisted of the treatments (XGO±NaCl) and the days after each treatment (two and five days). Previously, compliance with the normality and homogeneity premises were verified through the Shapiro-Wilk and Levene tests. All statistical analyses were performed with the InfoStat program [44].

Resultst

Effect of XGO on germination and growth of Nicotiana

The effects on GP and plant and root growth of N. tabacum seedlings, grown with MS ± 0.1μM XGO, salt stress with ± 100mM NaCl, or a combination of XGO and NaCl are shown in Table 1 and Figure 1. GP was statistically similar between the negative control and 0.1μM of XGO (close to 90%). However, GP was significantly reduced to 71% and 75% with 100 mM NaCl and 0.1μMXGO+100mM NaCl, respectively, compared to the MS negative control (P<0.05). Both salt stress control (MS+NaCl) and XGO+NaCl were statistically similar. On the other hand, 0.1μM XGO significantly caused a promotion in FA and PRL in two-month-old N. tabacum seedlings compared to negative control, but no statistical differences were observed in NL or in DLR. Moreover, when XGO was combined with 100 mM NaCl, there was a significant increase in NL, PRL, and DLR (P<0.05) related to salt control, but no statistical differences were observed in FA (Table 1 & Figure 1). Thus, addition of 100 mM NaCl caused an inhibition of NL and PRL by 36.3% and 43%, respectively, in N. tabacum seedlings compared to the MS negative control. Nevertheless, the inhibitory effects of salt stress on NL and PRL were reduced to 16.5% and 15.4%, respectively, compared to untreated control when XGO was incorporated in the media.

MS: Negative control; XGO: 0.1μM); MS+NaCl: Salt stress with 100mM NaCl; XGO+NaCl: 0.1μM XGO + 100mM NaCl; GP: Germination percentage; NL: Number of leaves; FA: Foliar area (mm2); PRL: Primary root length (mm); DLR: Density of lateral root.

The PCA explained the contribution percentage of each component to the total variation with the five quantitative characters evaluated (GP, NL, FA, PRL, and DLR) and each treatment (XGO and/or salt stress) (Table 2). The first two principal components (PC 1 and 2) justified 99.3 % of the total variation. The characters with the greatest contribution to variability consisted of NL, FA, and PRL in PC 1, and DLR and GP in the PC 2. The biplot chart of first and second component showed that PRL and DLR were significantly correlated (P<0.05) in addition to NL, FA, PG, and NL with PRL (Figure 2). There is a separation between treatments with NaCl either with or without XGO application in the first principal component, and in component 2 there was a clear separation of XGO treatments from those without XGO. The higher values in PRL, DLR, and NL correspond to treatments where XGO has applied alone, compared to those where it was combined with salt stress. In the second component, DLR and GP characters showed the highest positive and negative contribution, respectively. Also, there was a well-defined separation of XGO and XGO+NaCl from MS and MS+NaCl, with the higher average DRL seen with the XGO values.

Changes in proline and total chlorophyll content

Figure 3 shows the effect of XGO and salt stress on proline and chlorophyll contents of N. tabacum leaves measured after two and five days of induction. It can be noticed that treatments without salt (MS±XGO) exhibited no significant differences in proline content after two and five days of treatment (Figure 3a). Additionally, salinity stress in N. tabacum seedlings promoted significant proline accumulation compared to untreated control at both time points. However, the results obtained XGO+NaCl application reflects a gradual and significant increase in the proline content, which is 75.98% higher than salt stress control after five days of treatment (Figure 3a).

Total chlorophyll (Chl a+b) content was significantly higher after XGO application compared to the untreated control, which reached the highest levels among all treatments at both time points (Figure 3b). Also, as expected the Chl a+b content was significantly reduced by salinity stress in N. tabacum leaves compared to the untreated control and remained constant over time (P<0.05). On the other hand, XGO combined with NaCl produced a significantly higher Chl a+b content (29.58% upper) compared to salt stress control after five days of treatment, which reached levels similar to the negative control.

Changes in protein and lipid oxidative damage

The effects of XGO and salt stress on protein oxidation, measured as total carbonyl contents, in N. tabacum leaves at two and five days after induction is shown in Figure 4a. Plants treated with XGO exhibited no significant differences in total carbonyl content related to control plants after two and five days of treatment. On the other hand, NaCl application significantly increased its content by 67.10% compared to the untreated control after five days. In contrast, at the same time point, XGO application combined with salt stress significantly reduced protein oxidation in N. tabacum leaves by 98.45% compared to the salt stress control.

Lipid peroxidation was calculated in terms of MDA content as an indicator of lipid oxidative deterioration caused by severe oxidative stress in N. tabacum leaves at two and five days after induction (Figure 4b). XGO application caused the MDA amounts to remain constant over time. On the other hand, leaves from plants exposed to salinity stress demonstrated significantly higher MDA accumulation compared to untreated control at both time points. However, XGO application and salt stress also significantly caused a reduction in lipid peroxidation by 51.88% compared to salt stress control after five days of treatment.

Changes in activities of enzymes from the antioxidant system

We also examined four oxidative stress response enzyme markers in the context of their activities. Figure 5 shows the effect of XGO application and salt stress on the activities of the antioxidant enzymes, CAT, GPX, SOD, and GR in N. tabacum leaves after two and five days of induction. The results showed that five days after treatment with XGO alone resulted in a significantly higher CAT, GPX, and SOD activities over time compared to the negative control (P <0.05) (Figure 5a-c). GR activity was not significantly affected by the application of any treatment (Figure 5d). Salt shock with 100mM NaCl induced significantly higher CAT, GPX, and SOD activities two days after induction compared to the negative control. After five days in the presence of NaCl, GPX decreased, but CAT activity remained higher (P<0.05) than the untreated control. There was also an apparent increase in CAT, GPX and SOD activities after five days of treatment with XGO+NaCl, but their levels were statistically similar to those of salt control (Figure 5a–c).

Discussion

This study provides evidence concerning the effects of exogenous application of XGOs on enhancement of N. tabacum growth and development under saline conditions and salt shock in order to allow them to overcome the salt-stress limitations. Clearly, under the conditions used in the current work, XGO alone had a positive effect on NL, FA, and PRL although it was ineffective in promoting DRL. However, combined with continuous salt stress, this effect is rearranged, and XGO causes an increase in NL and PRL in addition to promoting lateral root formation although no changes in FA were observed compared to the salt control. Consequently, in the presence of XGO the plants managed to recover from limitations imposed by salt, so they display visible morphological characteristic improvement (Figure 1).

The analysis of eigenvalues corresponding to morphological changes is based on PCA analysis and explains >99% of the total experimental variability within the two first components. This is almost 100 % of the experimental variability that was achieved by reducing up to two principal components. The five evaluated traits showed a high contribution in one of the two first principal components; thus, each trait was very important to explain the variability observed in the experiment. PC 1, which explains the highest percent of variability (90.7%) allowed for separation of treatments under salt stress (100mM of NaCl) from those without NaCl. The highest values of NL, PRL, and FA were obtained in the medium with XGO and without salt stress, which were projected in the positive quadrant of the component. This result clearly indicates that XGO in the medium enhanced PLR and increased FA and NL in tobacco seedlings. PC 2, which explains the rest of the variability (8.6%), separates the treatments with XGO from the treatments without this biostimulant. In this component, the traits that showed the highest contribution were PG and DLR. According to these findings, the presence of XGO in the medium stimulated DLR, but the highest values of PG were obtained in the MS medium without NaCl.

These results confirmed that external application of 0.1μM XGO positively influenced plant growth and morphological features even under salt stress conditions and could be correlated with auxin-like activity. It is known that at approximately 1μM, at least four different cellotetraose-based XGOs (XXXG, XXLG, XXFG, and XLLG) mimic auxin by inducing growth [45]. In a previous study, we confirmed that auxin-like activity of the same XGO fraction mix and concentration used in this work on Arabidopsis thaliana seedlings [18]. These are important findings since the ability of a biostimulant to influence plant hormonal activity is one of their many important benefits because they can exert large influences that eventually will improve their health and growth. As plant growth regulators plays an important role as chemical messengers, they alert the plants when stressful environmental conditions exist so they can initiate or increase their stress response processes [46].

In this context, XGO may be acting as a “switches” that turn on the plants for stressful situations by altering hormonal balances. Zhang and Schmidt (1999) discuss the “switch” concept and give some examples of other types of biostimulants that reinforce our evidence. In this context, the results also suggested that exogenous XGO applications could be acting as “pre-stress conditioners” [46] and their effects are manifested by improving osmotic regulation, photosynthetic efficiency, or by causing an increase in antioxidant levels. This argument is based on the results in which it was shown that 21-day-old plants exposed to external application of 0.1μM XGO had higher proline accumulation and total Chl content in addition to higher CAT activity levels, GPX, and SOD compared to untreated plants. Of more interest was their effect when applied in combination with salt shock (XGO+NaCl) compared with those treated with NaCl alone (MS+NaCl). In this case, the highest recorded proline levels in addition to higher total Chl were observed after five days of treatments. Enhancement of this antioxidant machinery could be reflected in significant protein oxidation (total carbonyl content) and lipid peroxidation reduction. Therefore, it can be seen that the XGOs help the plant to cope with the effect of saline stress via proline accumulation.

Osmotic regulation is an important mechanism for plant cellular homeostasis under saline conditions in which proline is the most common osmolyte for osmoprotection [47]. The higher accumulations of proline recorded with XGO and NaCl at five days after treatment could be correlated with stress tolerance and may participate in the stress signal influence on adaptive responses (Figure 2) [6]. Proline also contributes to stabilization of sub-cellular structures (such as membranes and proteins), and its cytoplasmic accumulation could help reduce oxidative stress-generated plant protein and membrane damage after exposure to salinity [6]. This effect can be inferred because of lower total carbonyl content levels since protein-bound carbonyls represent a marker of global protein oxidation and lipid peroxidation products as biomarkers for oxidative stress that were observed in plants treated with XGO+NaCl (Figure 3). Therefore, XGO indirectly helps the cell cope with salt stress by maintaining cellular osmotic adjustment and protein and lipid integrity.

Another important result indicated that XGO seems to mitigate negative effects on photosynthesis in stressed plants by increasing Chl content. This parameter was used because of salinity- induced increase in chlorophyllase activity with the consequent degradation of chlorophyll (at least transiently). Consequently, we can deduce that the increase in exogenously XGO-induced total Chl content enabled tobacco plants to tolerate salt-stress in addition to promoting their development and growth. Similar results with photosynthetic pigments were obtained in our lab when the same XGO fraction mix was evaluated on A. thaliana seedling growth under saline stress [48].

Regarding the activity of antioxidative enzymes, exogenous application of 0.1μM XGO increased CAT, GPX and SOD enzyme activities compared to the negative control after five days of treatment. Due to the fact that no significant XGO+NaCl effects on enzymes’ activity that were analyzed in this work were observed, it would be advisable to analyze other antioxidant enzymes to expand the analysis in addition to determining its mode of action in the enzymatic antioxidant system.

Our study revealed that XGO seem to be working as metabolic inducers that trigger the physiological responses mentioned above. Some results support that xyloglucan fragments do not penetrate the cell, but instead, it has been suggested that the existence of specific receptors on the plasma membrane, which interact with the fragments, activate a signaling cascade inside the cell [49]. However, to date, no specific candidate has been identified as a possible receptor of these molecules [19]. Also, it has been suggested that they can promote modifications or integrate into the cell wall, which can affect not only the extracellular events in the wall but also intracellular events [50]. These authors demonstrated that incubation of pea stem segments partially bisected longitudinally with a xyloglucan oligosaccharide (9mM XXXG), accelerated the cell elongation by integration of xyloglucans as they were incorporated into the cell wall and became transglycosylated by xyloglucan endotransglycosylase (XET). According to this, XGO’s effects observed in our work may also result from a signal transduction XET-mediated or -induced cell wall modification cascade but not from the oligosaccarides’ direct actions.

Conclusion

Overall, we conclude that XGO can exert beneficial impacts on tobacco plants’ stress response either through hormone-like effects, osmotic regulation, photosynthetic efficiency improvement, and increase in antioxidant levels. They promote the proline accumulation as an organic osmolyte and increase total chlorophyll content and modify some antioxidative enzymes’ activities that eventually affect development of plant roots’ growth and development under salt stress. Further in vivo studies are needed to confirm the antioxidant effect of XGOs during salt stress in crop plants as well as to unravel their mechanism of action on oxidative responses. However, according to these results, the exogenous application of XGO as biostimulant at very low concentrations could be considered an alternative for improving the growth and productivity of crops of agronomic importance under salt stress.

To know more about Journal of Agriculture Research- https://juniperpublishers.com/artoaj/index.php

To know more about open access journal publishers click on Juniper publishers

[journalmaterialscience-blog]

Preliminary Studies on the Protective Effect of Rosmarinus Officinalis on Astrocytes Introduction Different pro-oxidant compounds in the form of reactive oxygen species (ROS) such as hydrogen peroxide, nitric oxide, superoxide and the highly reactive hydroxyl radicals and reactive nitrogen species (RNS) are naturally generated in biological systems. Its production is counteracted by the intrinsic antioxidant defense, both enzymatic and non-enzymatic, which protects against free radicals and the subsequent cell damage [1]. Oxidative damage occurs as an imbalance between the production of ROS and the ability of intrinsic antioxidant systems, to scavenge these radicals. Oxidation of macromolecules such as proteins, lipids and DNA may lead to cell degeneration and death due to an increase in the release of apoptotic inducing factors [2,3]. Brain is especially sensitive to oxidative stress because of the high proportion of unsaturated fatty acids, the high metabolic rate, the low antioxidants proportion and the slow cellular regeneration. Neurodegenerative diseases such as Alzheimer’s, Parkinson or amyotrophic lateral sclerosis have been found to be directly related to oxidative stress increase, elderly being the main risk factor for the development of these kind of diseases, together with toxic metabolic or infectious processes [4-7]. Rosmarinus officinalis L. (Lamiaceae) is an ever green plant spontaneously growing in the Mediterranean area. Aerial parts of rosemary are rich in polyphenolic compounds endorsed with antioxidant activity [8-12]. In continuation with our research line, R. officinalis methanolic extract was assayed on the human astrocyte glioblastoma, which is known as a useful model for the study of astrocyte functions under both physiological and pathological conditions, with the aim of assessing the mechanism of action of the antioxidant ability. In this study, the antioxidant capacity was first evaluated in the R. officinalis methanolic extracts by the oxygen radical absorbance capacity (ORAC) method [13]. Briefly, sample of Trolox was mixed with fluorescein in a 96-multiwell plate and the AAPH added. AAPH was used to generate peroxyl radicals that oxidize fluorescein, causing a decrease in fluorescence (excitation wavelength 485nm and emission wavelength 528nm) which is measured every 4 seconds for 90 minutes at 37 CºC. Then, the effect of Rosemary methanol extract on cell viability was tested in the MTT assay at different concentrations on the human astrocyte glioblastoma U373. Finally, GSH and GSSG/GSH ratio levels were tested to determine whether rosemary extract may influence on this antioxidant defence activity. Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), a water-soluble analogue of vitamin E, was chosen as a positive control in all the assays conducted in this work. Trolox is able to decrease ROS production, to prevent cytotoxicity in human cancer cell lines and to rescue cells from apoptotic death [14,15]. Go to Materials and Methods Plant material and extraction process Aerial parts of R. officinalis spontaneously growing in Spain were harvested during flowering in May, 2004. Samples were identified by the Department of Aromatic and Medicinal Plants Research, National Institute of Agricultural and Food Technology (INIA). A voucher specimen was deposited for internal control at the INIA (Madrid, Spain). Samples were dried in an oven at 35°C, grind down and sieved through a 2 mm mesh, and kept protected from light and moisture until use. 60 mg of each sample was extracted with 20 ml Methanol for one hour, under shaking. The suspension was then filtered through one filter paper; 10ml Methanol were added to the sample and filter again over the first methanolic extract. The extract was left for overnight to dry and stored at 5ºC and protected from light until use. Reagents Dulbecco´s modified Eagle´s medium (DMEM), RPMI1640 medium, Foetal bovine serum (FBS), PBS, Gentamicin were purchased from Gibco (Invitrogen, Paisley, UK). Dimethyl sulphoxide (DMSO), Hydrogen peroxide solution (30% w/w), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT), 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), 2,7-dichloro-dihydrofluorescein di acetate (DCFH-DA), AAPH were obtained from Sigma-Aldrich (St Louis, MO, USA). Cell culture Human astrocytoma U373 MG line was obtained from Cell Culture and Biological Resources Unit at Alcalade Henares University (Madrid, Spain). Cells were grown in a humidified incubator at 5% CO2 and 95% air at 37ºC in Dulbecco’s Modified Eagle’s medium (DMEM) piruvate free,from Invitrogen (Madrid, Spain),, supplemented with 10% fetal bovine serum (FBS) (Biowhitaker) and 50mg/l of each one of the following antibiotics: gentamicin, penicillin and streptomycin. ORAC assay Sample of Trolox was mixed with fluorescein in a 96-multiwell plate and the AAPH added. AAPH was used to generate peroxyl radicals that oxidize fluorescein causing a decrease in fluorescence (excitation wavelength 485nm and emission wavelength 528nm) which is measured every 4 seconds for 90 minutes at 37 °C in a multiwell plate reader (FLUO star OPTIMA fluorimeter, BMG LABTECH). Results calculate the relationship of the areas under the curve between blank and samples and are expressed as micromoles of Trolox equivalents per gram. MTT assay Cell viability (cell growth inhibition) was determined by MTT assay [16] with some modification. Cells were incubated in 96-well plates, at density of 5 x 10⁴ cells/well for 24h, then the cells were treated with different concentrations of the Romero extracts (range from 3.13 to 800 µg/ml) for another 24h. Triton X-100 5% was used as a negative control, finally 2mg/ml MTT was added and the plate were incubated for 1 h at 37 °C, then the form azan crystal formed were dissolved by adding DMSO and the absorbance was measured at 550 nm using Digiscan 340 microplate reader (ASYA Hitech GmbH, Eugendorf, Austria). For all the experiments, every sample was analyzed in triplicate, with four plates for each condition. Intracellular ROS production assay ROS production was evaluated by the DCFH-DA assay [17] with some modification, This assay is based on the oxidation of the non fluorescent compound 2′,7′-dichlorofluorescein (DCFH) into the fluorescent compound dichlorofluorescein (DCF) in presence of ROS. Cells were incubated in 96-well plate for 24h and 50µl of 2ʹʹ, 7ʹ-dichlorofluorescin diacetate (DCFH-DA) at a concentration of 10 µM were added for 30 min at darkness. Then, cells were treated with different concentrations of rosemary extract and the generation of ROS was measured for 2h in a microplate fluorescence reader (FLx800, Bio-Tek Instrumentation) with excitation at 480 nm and emission at 510 nm. Determination of the Glutathione levels The GSH and GSSG levels were determined according to the method of Hissin and Hilf [18]. Determination of GSH was performed by adding 50 μL of the sample to a mixture of150 μL of 0.1 M sodium phosphate buffer (pH 8.0) and 20 μL of o-phthaldehyde (1mg/mL methanol). The determination of GSSG was conducted by mixing 50 μL of the sample and 3 μL of N-ethylmaleimide for 30 min in darkness before adding 150 μL of 0.1 N NaOH (pH 12) and 20 μL of o-phthaldehyde (1mg/mL, methanol). Finally, both preparations were incubated for 15 min at room temperature in darkness, and fluorescence was measured at an emission wavelength of 485 nm and an excitation wavelength of 528 nm with a microplate fluorescence reader (FLx800, Bio-Tek Instrumentation). Statistical analysis Stat graphics Centurion 16.1.15 (XV) was used. One-way analysis of variance (ANOVA) followed to Fishers least significant difference (LSD) test was applied to obtain the differences between samples. p < 0.05 was considered as statistically significant. Go to Results and Discussion Results showed a showed a strong antioxidant activity by the ORAC method, with a value of 3.03±0.15 µmol TE/mg (value is mean ± SD, n=3). The direct effect of Rosemary extraction cell viability (MTT) showed no statistically significant differences on cell survival with respect to the control group (untreated cells) for concentrations between 12.5 and 200µg/mL; the lowest (3.13 and 6.25µg/mL) and the highest (400-800µg/ml) concentrations induced a decrease in cell survival although far away from the levels achieved with the toxic alone (Triton) (Figure 1A). Thus, concentrations ranging from 12.5 to 200µg/mL were chosen for the following assays. Pretreatment of cells with doses of 12.5, 25, 50 and 100 μg/mL of the extract for 24h before H2O2 exposure was able to significantly recover cell viability when compared to the negative control, Triton (Figure 1B). To test the effect of different concentrations on intracellular ROS levels, doses of 12.5, 25, 50 and 100 µg/mL of the extract were added and evaluated by the DCFH-DA assay (Figure 2A). H2O2 as the oxidant insult caused an increase in ROS levels by 117% when compared to control cells. Rosemary extract did not increase ROS concentration, this indicating no cellular stress or oxidative damage which could influence the functional conditions of cells. Pretreatment of the cells with the methanolic extract previous to oxidative insult, ROS levels were also inferior to those achieved by untreated cells, although no statistically significant differences were found (Figure 2B). Therefore, neuronal cells treated with the R. officinalis extract seem to be in a favourable condition to face an oxidative challenge. Then the protective effect of rosemary on GSH and GSSG concentration was determined in cells treated with 1mMH2O2 or 1 mM H2O2 plus noted concentrations of extract or Trolox as a positive control (Table 1). A slight depletion of intracellular GSH levels was observed when 1 mM H2O2 was added for 24 h to astrocytes; co-treatment with 0.5mM Trolox completely prevented the depletion of GSH. Co treatment with different rosemary concentrations partially recovered GSH levels, the strongest effect found with 50 µg/mL rosemary extract. Although the GSH recover was no statistically significant, the ratio GSSG/GSH was closer to untreated cells (0.46 vs 0.41, respectively). The role of reduced Glutathione (GSH) as the main non-enzymatic antioxidant defence is due to the reaction with free radicals and the repair of free radical induced damage through electron-transfer reactions. Moreover, the loss of cellular GSH seems to have an important role in apoptotic signalling [19-21]. Therefore, maintaining GSH concentration above a critical threshold while facing a stressful situation represents a crucial advantage for cell survival. In conclusion, the results obtained in this work support previous data on the antioxidant effect of R. officinalis [10,11]. Rosemary methanolic extract was not toxic on the assayed cell line and exerted moderate antiradical and antioxidant activities by partially recovering GSH levels. These results may contribute to the knowledge of the mechanism effect, althoughfurther experiments are needed to assess and define the molecular mechanism of action involved in such antioxidant effect in order to confirm R. officinalis as a potential therapeutics within those diseases in which oxidative stress plays a crucial role. For more Open access journals please visit our site: Juniper Publishers For more articles please click on: Journal Material Science juniper publishers material science composite materials

[conners-clinic]

Pro-Oxidants vs. Anti-Oxidants

A similar relationship exists in pro- vs. anti-oxidants. It seems we are overwhelmed with information about the importance of increasing anti-oxidants in our diet; the truth be told: we cancer care without pro-oxidation would prove fruitless. To understand this, let’s discuss the mechanisms.

A free radical is a reactive molecule that tends to damage cell parts so we tend to think that this is always ‘bad’… NOT true.  However, when DNA is damaged, this can mean damaged genes but this is precisely why our body replicates cells and the ‘old’ cell dies.  If the genes controlling cell multiplication are harmed, cell growth can get stuck in the “on” position and cause cancer – this is an example of imbalance.

Cancer cells are very active reproducing and growing causing an increased load of free radicals within the cancer cell. Cancer cells are not good at handling more free radicals, since they already have more in them than normal cells and they tend to spew them out of the cells forming a slime layer that makes it more difficult to penetrate. At first thought, maybe it would be wise to flood the cancer with anti-oxidants. Until we understand that, at the root, the cancer is growing without oxygen, fermenting its energy, we then arrive at a different solution.

Normally the body makes energy through oxidation and is able to quench free radicals produced with substances called antioxidants. Antioxidants lessen the amount of free radical-induced injury and bring balance. This ‘tug-o-war’ continues and may be illustrated in exercise. We go to the gym and work our muscles, breaking tissue down in the presence of a depleting supply of oxygen (almost a semi-cancerous state) for muscles to ‘recover’ stronger, bigger, more thrifty in their use of oxygen, healthier in what we call ‘better shape’.

Cancer is similar in that the cells have created a survival mechanism to grow and thrive in a hypoxic environment. Since pro-oxidant strategies increase free radicals, it may better seem that the way to kill cancer cells is by bombarding them with pro-oxidants. This would lead to more free radicals within cancer cells, and injury and eventual death to the cancer cells.

The mechanism of many chemotherapy drugs, as well as radiation, in destroying cancer cells is by causing free radical increase within cancer cells. This is the same mechanism in many of the apoptogens (cancer killers) in a nutritional approach.

Many nutraceuticals that are normally considered to be anti-oxidants actually have a pro-oxidant, cancer-killing effect on cancer cells.  Curcumin, for example is just one anti-oxidant that acts as a pro-oxidant to a cancer cell. It also elicits anti-inflammatory benefits that aide in breaking down the barrier for an immune assault on the growing cancer mass.  Selenium, EGCG (from Green Tea Extract), high dose Vitamin C and others also act like this. Some so-called antioxidants at high levels have other ways of killing cancer cells, like inhibiting certain enzymes (Curcumin inhibits topoisomerase II) that hinder normal apoptosis.

We have to be careful taking dogmatic stances in our nutritional approach.  The “anti-oxidants are good for you so excess amounts of anti-oxidants must be better” ignores the very principles of health that regulate homeostasis – balance.  Mixing different nutrition can be counterproductive as well; for instance, use of Curcumin along with Glutathione has shown to be a very wrong approach. Extensive research within the past half-century has indicated that Curcumin, the yellow pigment in curry powder, exhibits antioxidant, anti-inflammatory, and pro-apoptotic (aiding in normal cell death) activities.

Recent studies have investigated whether the anti-inflammatory and pro-apoptotic activities assigned to Curcumin are mediated through its pro-oxidant/anti-oxidant mechanism. Much data has revealed that TNF-mediated NF-κB (markers found to accelerate cancer) activation was inhibited by Curcumin – therefore Curcumin acts to slow cancer growth.  Glutathione, normally a great anti-oxidant (some would argue that it is the body’s greatest) reversed the inhibition – that means that Glutathione negated the cancer stopping benefits of Curcumin. This is just one example of what can go wrong with taking too many supplements!

Cellular pro-oxidants, called reactive oxygen species (ROS), are constantly produced in our body. As stated, excessive ROS can induce oxidative damage in the cell and promote a number of degenerative diseases including accelerated aging. Cellular antioxidants protect against the damaging effects of ROS and have long been the sales-pitch of health practitioners. However, we cannot ignore normal balance; in moderate concentrations, ROS are necessary for a number of protective reactions. ROS are essential mediators of antimicrobial phagocytosis (killing bio-toxins), detoxification reactions carried out by the cytochrome P-450 complex (the main liver detox pathway), and apoptosis which eliminates cancerous and other life-threatening cells. Can you say ‘balance’ again?

Excessive ingestion of antioxidants could dangerously interfere with these protective functions, while temporary depletion of antioxidants can enhance anti-cancer effects of apoptosis. This is just another lesson against ‘cookbook nutrition’. This is where practitioners educated in Kinesiology may have an advantage in determining the correct approach for the patient.  Functional medicine testing measuring ROS baselines may also prove effective on determining a nutraceutical attack, as each patient is different.

This was an excerpt from Dr Conners’ book, Stop Fighting Cancer and Start Treating the Cause.

Free Download Buy the Book

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[neckpaintherapy-blog1]

Core Support

New Post has been published on https://brownfamily-dc.com/product/core-support/

Core Support

The human body is exposed to a wide variety of toxins on a daily basis including chemicals found in foods, environmental toxins, and pharmaceuticals. The liver is the body’s main detoxification organ which provides enzyme systems that safely process and remove xenobiotics (foreign chemical substances) out of the body, as well as unhealthy hormone metabolites. These detoxification systems are very complex and require a variety of nutrients for optimal function. There are two main pathways of detoxification in the liver, known as phase I and phase II. In phase I, composed mainly of cytochrome P450 enzymes, non-reactive compounds undergo specific reactions which use oxygen to form a reactive site on the compound. Most pharmaceuticals are metabolized through Phase I biotransformation. This prepares the metabolite for the next step of detoxification. Phase II is a crucial step- if molecules from Phase I are not fully metabolized by Phase II conjugation they may cause free radical damage to proteins, RNA, and DNA within the cell. Phase II reactions result in the biotransformation of fat-soluble compounds into water-soluble compounds that can then be excreted in the urine, bile, or stool. The ingredients included in Core Support were chosen for their ability to support one of the six pathways of phase II detoxification. N-acetyl cysteine along with glycine and taurine are well known amino acids for their role in supporting the liver. Antioxidants such as lipoic acid, green tea, ellagic acid and the vegetable antioxidant blend provide a synergistic approach to liver support and promote enhanced detoxification.

Core Support was created as part of the Core Restore Kit. Core Restore supports Phase II liver detoxification by providing protein, fiber, and nutrients to help eliminate toxins from the body.

Suggested Use:

Mix 2 scoops (39.0 g) with 8 oz. of water or the beverage of your choice two times daily or as recommended by your health care professional

INGREDIENT BENEFITS

N-Acetyl Cysteine

N-acetyl cysteine (NAC) is a sulfhydryl-containing amino acid that is commonly used to support liver health. Though studies have shown the absorption of oral glutathione to be limited, supplementation with NAC has been shown to significantly increase circulating levels of glutathione, a primary antioxidant that plays a major role in protecting cellular health. Increasing glutathione levels in turn increases the production of specialized antioxidant enzymes such as glutathione peroxidase, glutathione reductase and detoxification enzymes such as glutathione S-transferase. Through the activity of these enzymes, NAC protects the body from oxidative damage, increases phase II detoxification, and enhances the normal breakdown of toxins and other metabolic by-products of the body.

Glycine

One of the six phase II detoxification pathways is amino acid conjugation (the attachment of amino acids to a toxin). Glycine is one of the amino acids used in this process. Glycine also aids in glutathione conjugation. Glycine preserves intracellular glutathione concentration and protects cells from oxidative damage. This process is mediated by a protein called glycine transporter 1 or GLYT1. Research has shown that glycine treatment of human intestinal cells against an oxidative agent, reduced the intracellular concentration of reactive oxygen species (ROS) when exposed to oxidative challenge.

Taurine

The sulfation pathway is another important phase II detoxification pathway. During the sulfation pathway, a sulfur-containing molecule is attached to the toxin in order to produce a compound that can be excreted out of the body. Studies show taurine effectively conjugates bile acids, and protects the liver against toxic heavy metals such as arsenic by supporting gluthathione levels in the liver.

Lipoic Acid

Lipoic acid is a potent antioxidant which has been shown to increase glutathione, vitamin E, and vitamin C levels in the body. Lipoic acid has been shown to support phase II detoxification by increasing the activity of enzymes including NAD(P)H, quinine oxidoreductase, and glutathione S-transferase. Lipoic acid has been used to detoxify mycotoxins (toxic by-products produced by fungi and molds). Some mycotoxins have been found to mimic xenobiotics (foreign chemical substances) in their effects on the body and in their routes of detoxification. Lipoic acid has also been shown to reverse age-related loss of glutathione synthesis.

Green Tea Extract

Green tea is one of the most widely consumed beverages throughout the world and is traditionally consumed in numerous cultures for its health- promoting benefits. One of the main polyphenols in green tea includes epigallocatechin- 3-gallate (EGCG). Green tea polyphenols have demonstrated significant antioxidant and inflammatory balancing effects. Green tea has also been shown to provide phase II stimulating properties. Studies have shown that green tea extract increases phase II enzymes such as glutathione transferase, NAD(P)H, quinine reductase, epoxide hydrolase, and UDP glucuronosyltransferase. EGCG potentiates cellular defense capacity against chemical toxins, ultraviolet radiation, and oxidative stress.

Rosemary

Rosemary contains polyphenols that are potent antioxidants, which provide a significant boost to immune response and up-regulate detoxification mechanisms of the liver. Carnosol, an antioxidant in Rosemary, induces glutathione-stransferase, as well as other important phase II enzymes. Rosemary essential oil and carnosol have also been shown to increase intracellular glutathione levels.

Vegetable Antioxidant Blend

Core Support includes VitaVeggie®, a blend of high concentration superfood vegetables with significant antioxidant potential. VitaVeggie® is high in ORAC value (oxygen radical absorbance capacity- a method of measuring antioxidant activity) and includes health promoting compounds including sulphoraphane and glucosinolates. Cruciferous vegetables including broccoli, kale, and Brussels sprouts increase the enzyme activity of both Phase I and Phase II detoxification pathways. Sulforaphane, a naturally occurring isothiocyanate derived from cruciferous vegetables, induces phase II detoxification enzymes and supports the body’s response to oxidative stress to promote balanced inflammation. Glucosinolates present in Brassica vegetables serve as precursors for biologically active metabolites, which induce Phase II enzymes via the activation of Nrf2, the master cellular switch responsible for antioxidant production.

Schizandra Berry Extract

Schizandra is an adaptogenic botanical used medicinally to help fight off the physical and mental effects of stress. Schizandra is also used to support liver health and neutralize the effects of toxin exposure. Schizandra enhances liver detoxification pathways by increasing the levels of reduced glutathione in the liver as well as glutathione reductase and glutathione S-transferase activity. In animal studies, schizandra has been shown to support phase I metabolism and protect the liver from free radical damage induced by toxic chemical exposure following ingestion of carbon tetrachloride.

Psyllium Husks

Psyllium husk is from the plant Plantago ovata and largely used for its fiber content. Psyllium husk contains a large amount of soluble fiber per volume. Psyllium is used to improve gastrointestinal transit time and for cardiovascular health by promoting normal cholesterol levels through the elimination of cholesterol-rich bile. Studies show psyllium husk powder up-regulates genes involved in bile acid synthesis and binds to bile acids in the intestines to gently remove them from the body.

#Detox, #Gastrointestinal, #Liver

[curegbm]

U.S. Sen. Richard Blumenthal (former CT Attorney General who was part of the 1998 litigation winning $246 Billion against tobacco companies for deceiving the public about the dangers of smoking) is leading a campaign to determine whether new “5G” wireless technology is safe and is asking the FCC for proof that 5G not pose health risks. “We need to know whether the radio frequencies can cause cancer,” Blumenthal said. Blumenthal and CA Rep. Anna Eshoo, both members of the Senate Commerce Committee that oversees the FCC, gave the FCC a Dec. 17 deadline to comply with their request for information.

Blumenthal's letter said “most of our current regulations regarding radiofrequency safety were adopted in 1996 and have not yet been updated for next generation equipment and devices.”

Blumenthal also cited a study released this month by the National Toxicology Program, an inter-agency program within the U.S. Department of Health and Human Service, that showed evidence of cancerous heart tumors, as well as some evidence of cancerous brain tumors, in male rats exposed to exposed to high levels of radiofrequency radiation like that used in 2G and 3G cell phones. https://ntp.niehs.nih.gov/results/areas/cellphones/ https://www.niehs.nih.gov/health/materials/cell_phone_radiofrequency_radiation_studies_508.pdf

“The stark, simple fact is that health hazards are unknown and unstudied,” Blumenthal said at his press conference. “That is a sign of neglect and disregard at the Federal Communications Commission that is unacceptable. We need to know whether the technology can cause cancer and other diseases.

Read more https://ctmirror.org/2018/12/03/blumenthal-wants-fcc-prove-5g-wireless-technology-safe/

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2 recent TV programs on wireless health effects:

1) Excellent Coverage of EHS on S African News Program Carte Blanche, originally aired in March 2018 - 8-minute video http://c4st.org/electromagnetic-hypersensitivity-disorder/

Includes interviews with people with EHS and a medical doctor knowledgable about EHS. EHS has been diagnosed with ICD-10 codes PDX, SDX, W99.9, T66, Z58.8

2) 5G Wireless Radiation Dangers - on Info Wars

https://www.dropbox.com/s/vbfmhjbfblubjwm/Info%20Wars%20-%20War%20Rooms.mp4?dl=0

Per Merriam Webster: https://www.merriam-webster.com/medical/microwave%20sickness

Microwave Sickness: a condition of impaired health reported especially in the Russian medical literature that is characterized by headaches, anxiety, sleep disturbances, fatigue, and difficulty in concentrating and by changes in the cardiovascular and central nervous systems and that is held to be caused by prolonged exposure to low-intensity microwave radiation.

To read more about electrohypersensitivity (EHS) aka microwave illness:

Physicans for Safe Technology: https://mdsafetech.org/science/es-science/

Environmental Health Trust: https://ehtrust.org/science/electromagnetic-sensitivity/

We Are The Evidence https://wearetheevidence.org/electromagnetic-sensitivity/

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3 very important scientific studies regarding EHS/microwave sickness:

1) 2014 DeLuca et al study which found statistically significant genetic and metabolic differences between EHS and non-EHS, is further evidence of EHS as a medical condition, and not a psychosomatic one https://www.hindawi.com/journals/mi/2014/924184/

2) 2016 Belyaev et al, EUROPAEM Guidelines for Prevention, Diagnosing and Treating EHS https://www.degruyter.com/downloadpdf/j/reveh.2016.31.issue-3/reveh-2016-0011/reveh-2016-0011.pdf

It covers all aspects of EHS, including skin effects (elevated mast cells in the upper dermis of the skin and histamine release from the mast cells), neurological symptoms, effects on endocrine system, liver and organ systems, and increased vulnerability of children.

Mechanisms also discussed: Increased peroxynitrite, malfunctioning cell membranes, mitochondrial dysfunction, decreased glutathione, cellular stress proteins.

It also gives treatment recommendations: These include a balanced homeostasis in order to increase the “resistance” to EMF. There is increasing evidence that a main effect of EMF on humans is the reduction of their oxidative and nitrosative regulation capacity.This hypothesis also explains observations of changing EMF sensitivity and the large number of symptoms reported in the context of EMF exposure. Based on currently available knowledge it appears useful to recommend a treatment approach, as those gaining ground for multisystem illnesses, that aims at minimizing adverse peroxynitrite effects. Measures that enhance the immune system and reduce stress in combination with detoxification will promote EHS recovery."

Supplements needed to combat the oxidative stress, like antioxidants, detoxifying agents, supportive nutrients, NAC, ALA, vitamin E, C, methylfolate, methylcobalamin, etc.

Also MTHFR and COM-T increases need for nutrients like methylated B vitamins to combat EHS.

3) 2015 Belpomme et al identified many biomarkers to objectively diagnose EHS and MCS. http://www.ehs-mcs.org/fichiers/1454070991_Reliable_biomarkers.pdf ,

Both disorders [EHS and MCS] were associated with hypoperfusion [low cerebral blood flow] in the capsulothalamic area, suggesting that the inflammatory process involve the limbic system and the thalamus. Our data strongly suggest that EHS and MCS can be objectively characterized and routinely diagnosed by commercially available simple tests. Both disorders appear to involve inflammation-related hyper-histaminemia, oxidative stress, autoimmune response, capsulothalamic hypoperfusion and BBB opening, and a deficit in melatonin metabolic availability; suggesting a risk of chronic neurodegenerative disease. Finally the common co-occurrence of EHS and MCS strongly suggests a common pathological mechanism