Sulfur Amino Acid Deficiency Research Articles

Sulfur-containing amino acids and oxidative stress in chronic pancreatitis patients

Background: Chronic pancreatitis (CP) patients are at high risk of malnutrition due to malabsorption. Sulfur amino acids (SAAs) being important antioxidant could affect pancreatic function. In this study, we have analyzed blood SAAs and its relation to antioxidant levels. Methods: One hundred and seventy-five CP patients and 113 healthy normal controls were prospectively studied. Disease characteristics and imaging features were recorded. Plasma SAAs were estimated using high-performance liquid chromatography. Erythrocyte reduced glutathione (GSH), GSH peroxidase, superoxide dismutase, plasma Vitamin C, erythrocyte thiobarbituric acid reactive substance (TBARS), urinary inorganic sulfate, and creatinine were estimated by spectrophotometry. Results: Plasma SAAs, urinary inorganic sulfate/creatinine ratio, and blood antioxidant levels were lower, whereas TBARS was higher in CP patients as compared to controls. Plasma methionine and TBARS were inversely correlated, whereas plasma cysteine and GSH level were directly correlated. Plasma cysteine and Vitamin C levels were lower, whereas TBARS was higher in CP patients with atrophy as compared to patients without atrophy. No statistical difference in these parameters between diabetic and nondiabetic CP patients was observed. Conclusion: Deficiency of SAAs appears to be associated with oxidative stress in CP patients. Possible benefit of supplementation of SAAs needs to be elucidated.

Activating transcription factor 4‐dependent B cell translocation gene‐1 induction promotes ferroptosis

Ferroptosis is a special type of programmed cell death that is characterized by iron‐dependent lipid peroxidation. The present study investigated the role of B cell translocation gene‐1 (BTG‐1) on sulfur amino acid deficiency (SAAD)‐mediated cell death. SAAD in HepG2 cells depleted the reduced glutathione, accelerated lipid peroxidation, increased cyclooxygenase 2 expression, and promoted cell death. SAAD‐mediated cell death was specifically inhibited by pretreatment with ferrostain or defroxamine, chemical inhibitors of ferroptosis. In parallel with cell death, SAAD increased expression of microtubule‐associated protein light chain 3‐II and facilitated acridine orange positive autophagic flux. Inhibition of autophagy accelerated the SAAD‐mediated cell death. Moreover, SAAD induced BTG‐1 expression in HepG2 cells. By using BTG‐1 promoter haboring reporter gene and siRNA, activating transcription factor 4 (ATF4) was identified as an essential transcription factor for SAAD‐mediated BTG‐1 induction. Knock out of BTG‐1 gene by CRISPR/Cas9 system decreased the cytotoxicity by SAAD. Furthermore, BTG‐1 deficiency increased microtubule‐associated protein light chain 3‐II and autophagic flux. Thus, present study demonstrates that SAAD induces ferroptosis, and the ATF4‐dependent BTG‐1 induction contributes to accelerate ferroptosis by inhibiting autophagy.Support or Funding InformationThis study was supported by the National Research Foundation of Korea (NRF) funded by Korea government (MSIP)(Grant No. 2018R1A5A2025272 and 2018R1A2B6007514).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Upregulation of capacity for glutathione synthesis in response to amino acid deprivation: regulation of glutamate–cysteine ligase subunits

Using HepG2/C3A cells and MEFs, we investigated whether induction of GSH synthesis in response to sulfur amino acid deficiency is mediated by the decrease in cysteine levels or whether it requires a decrease in GSH levels per se. Both the glutamate-cysteine ligase catalytic (GCLC) and modifier (GCLM) subunit mRNA levels were upregulated in response to a lack of cysteine or other essential amino acids, independent of GSH levels. This upregulation did not occur in MEFs lacking GCN2 (general control non-derepressible 2, also known as eIF2α kinase 4) or in cells expressing mutant eIF2α lacking the eIF2α kinase Ser(51) phosphorylation site, indicating that expression of both GCLC and GCLM was mediated by the GCN2/ATF4 stress response pathway. Only the increase in GCLM mRNA level, however, was accompanied by a parallel increase in protein expression, suggesting that the enhanced capacity for GSH synthesis depended largely on increased association of GCLC with its regulatory subunit. Upregulation of both GCLC and GLCM mRNA levels in response to cysteine deprivation was dependent on new protein synthesis, which is consistent with expression of GCLC and GCLM being mediated by proteins whose synthesis depends on activation of the GCN2/ATF4 pathway. Our data suggest that the regulation of GCLC expression may be mediated by changes in the abundance of transcriptional regulators, whereas the regulation of GCLM expression may be mediated by changes in the abundance of mRNA stabilizing or destabilizing proteins. Upregulation of GCLM levels in response to low cysteine levels may serve to protect the cell in the face of a future stress requiring GSH as an antioxidant or conjugating/detoxifying agent.

Cross-species and tissue variations in cyanide detoxification rates in rodents and non-human primates on protein-restricted diet

We sought to elucidate the impact of diet, cyanide or cyanate exposure on mammalian cyanide detoxification capabilities (CDC). Male rats (∼8weeks old) (N=52) on 75% sulfur amino acid (SAA)-deficient diet were treated with NaCN (2.5mg/kg bw) or NaOCN (50mg/kg bw) for 6weeks. Macaca fascicularis monkeys (∼12years old) (N=12) were exclusively fed cassava for 5weeks. CDC was assessed in plasma, or spinal cord, or brain. In rats, NaCN induced seizures under SAA-restricted diet whereas NaOCN induced motor deficits. No deficits were observed in non-human primates. Under normal diet, the CDC were up to ∼80× faster in the nervous system (14ms to produce one μmol of thiocyanate from the detoxification of cyanide) relative to plasma. Spinal cord CDC was impaired by NaCN, NaOCN, or SAA deficiency. In M. fascicularis, plasma CDC changed proportionally to total proteins (r=0.43; p<0.001). The plasma CDC was ∼2× relative to that of rodents. The nervous system susceptibility to cyanide may result from a “multiple hit” by the toxicity of cyanide or its cyanate metabolite, the influences of dietary deficiencies, and the tissue variations in CDC. Chronic dietary reliance on cassava may cause metabolic derangement including poor CDC.

Changes in body composition in broilers by a sulfur amino acid deficiency during growth

In the factorial approach, amino acid (AA) requirements are determined using the AA composition of retained protein, which is assumed to be constant. However, this hypothesis may not be valid because the AA composition of body protein can be affected by the diet. The objective of this study was to quantify the changes in chemical body composition of broilers receiving diets either deficient (TSAA–) or sufficient (TSAA+) in TSAA. Diet TSAA+ was formulated according to the Ross recommendation. Diet TSAA– provided 36% true digestible Met:Lys and 64% true digestible TSAA:Lys, which were, respectively, 34 and 22% lower compared with diet TSAA+. Performance and tissue weight gain between 7 and 42 d of age were not affected by the TSAA supply. In TSAA– chickens, protein gain was lower in the carcass (P < 0.01) and tended to be lower in the empty body (P = 0.06) and pectoralis major muscle (P = 0.10). Compared with TSAA+ chickens, lipid gain in TSAA– chickens was 78% greater in the pectoralis muscle (P < 0.001), 28% greater in abdominal fat (P < 0.05), and 10% greater in the carcass (P = 0.10). In the pectoralis muscle, there was a tendency for an increase in the redness value (a*; P = 0.10). The TSAA supply affected the AA composition of tissues and tissue gain, but the Met and Cys concentrations were changed only in the offal (P = 0.08). The deficient TSAA supply resulted in an increase in the Ser concentration in the empty body, carcass, and pectoralis muscle (P < 0.05). In contrast, it resulted in a decrease in the concentrations of Lys and Glu in the empty body, of Phe, Tyr, Gly, and Glu in the pectoralis muscle, and of Ala in the offal (P < 0.05). This indicates that although chickens cope with a TSAA deficiency predominantly by changing the protein and lipid concentration in the body, the AA composition is also affected. This calls into question the use of a constant ideal AA profile in poultry nutrition.

A sulfur amino acid deficiency changes the amino acid composition of body protein in piglets

Experiments carried out to determine the amino acid requirement in growing animals are often based on the premise that the amino acid composition of body protein is constant. However, there are indications that this assumption may not be correct. The objective of this study was to test the effect of feeding piglets a diet deficient or not in total sulfur amino acids (TSAA; Met + Cys) on nitrogen retention and amino acid composition of proteins in different body compartments. Six blocks of three pigs each were used in a combined comparative slaughter and nitrogen balance study. One piglet in each block was slaughtered at 42 days of age, whereas the other piglets received a diet deficient or not in TSAA for 19 days and were slaughtered thereafter. Two diets were formulated to provide either 0.20% Met and 0.45% TSAA (on a standardized ileal digestible basis) or 0.46% Met and 0.70% TSAA. Diets were offered approximately 25% below ad libitum intake. At slaughter, the whole animal was divided into carcass, blood, intestines, liver, and the combined head, tail, feet and other organs (HFTO), which were analyzed for nitrogen and amino acid contents. Samples of the longissimus muscle (LM) were analyzed for myosin heavy chain (MyHC) and actin contents. Nitrogen retention was 20% lower in piglets receiving the TSAA-deficient diet (P < 0.01). In these piglets, the nitrogen content in tissue gain was lower in the empty body, carcass, LM and blood (P < 0.05) or tended to be lower in HFTO (P < 0.10), but was not different in the intestines and liver. The Met content in retained protein was lower in the empty body, LM and blood (P < 0.05), and tended to be lower in the carcass (P < 0.10). The Cys content was lower in LM, but higher in blood of piglets receiving the TSAA-deficient diet (P < 0.05). Skeletal muscle appeared to be affected most by the TSAA deficiency. In LM, the Met content in retained protein was reduced by 12% and total Met retention by more than 60%. The MyHC and actin contents in LM were not affected by the TSAA content of the diet. These results show that a deficient TSAA supply affects the amino acid composition of different body proteins. This questions the use of a constant ideal amino acid profile to express dietary amino acid requirements, but also illustrates the plasticity of the animal to cope with nutritional challenges.

Intestinal metabolism of sulfur amino acids

The gastrointestinal tract (GIT) is a metabolically significant site of sulfur amino acid (SAA) metabolism in the body and metabolises about 20 % of the dietary methionine intake which is mainly transmethylated to homocysteine and trans-sulfurated to cysteine. The GIT accounts for about 25 % of the whole-body transmethylation and trans-sulfuration. In addition, in vivo studies in young pigs indicate that the GIT is a site of net homocysteine release and thus may contribute to the homocysteinaemia. The gut also utilises 25 % of the dietary cysteine intake and the cysteine uptake by the gut represents about 65 % of the splanchnic first-pass uptake. Moreover, we recently showed that SAA deficiency significantly suppresses intestinal mucosal growth and reduces intestinal epithelial cell proliferation, and increases intestinal oxidant stress in piglets. These recent findings indicate that intestinal metabolism of dietary methionine and cysteine is nutritionally important for intestinal mucosal growth. Besides their role in protein synthesis, methionine and cysteine are precursors of important molecules. S-adenosylmethionine, a metabolite of methionine, is the principal biological methyl donor in mammalian cells and a precursor for polyamine synthesis. Cysteine is the rate-limiting amino acid for glutathione synthesis, the major cellular antioxidant in mammals. Further studies are warranted to establish how SAA metabolism regulates gut growth and intestinal function, and contributes to the development of gastrointestinal diseases. The present review discusses the evidence of SAA metabolism in the GIT and its functional and nutritional importance in gut function and diseases.

Metabolic profiling reveals diverse effects of sulfur amino acid deficiency

Previous data using bioinformatics show the ability to predict sulfur amino acid (SAA) intake without directly measuring any SAA metabolites. The purpose of this study is to use metabolic profiling to determine the metabolites that significantly change with dietary SAA. 10 healthy subjects were fed a semi‐synthetic, chemically defined diet containing no SAA for 5 days and then the same diet including SAA for 5 more days. Plasma was collected after the meal on the first and last day of each study period and analyzed using a mass spectrometry metabolomics profiling method. The results reveal that 1% of the detected metabolic profile (2,570 features) varies significantly with SAA intake. As expected, methionine and cysteine increase significantly when SAA is in the diet. No significant changes in any other known SAA metabolites were observed; however, 3 unknown sulfur containing metabolites differed significantly with dietary SAA. 2 of these unknowns were significantly decreased with adequate dietary SAA, suggesting an uncharacterized metabolic pathway that is upregulated in times of SAA deficiency. 16 non‐sulfur containing metabolites were altered significantly by dietary SAA, showing the wide ranging effects SAA have on metabolism. This study demonstrates a use for metabolic profiling techniques to better understand the effects of SAA deficiency on metabolism.Funding: NIH ES012870, DK066008, ES012929Grant Funding SourceT32‐ES012870

Sulfur amino acid deficiency upregulates intestinal methionine cycle activity and suppresses epithelial growth in neonatal pigs

We recently showed that the developing gut is a significant site of methionine transmethylation to homocysteine and transsulfuration to cysteine. We hypothesized that sulfur amino acid (SAA) deficiency would preferentially reduce mucosal growth and antioxidant function in neonatal pigs. Neonatal pigs were enterally fed a control or an SAA-free diet for 7 days, and then whole body methionine and cysteine kinetics were measured using an intravenous infusion of [1-(13)C;methyl-(2)H(3)]methionine and [(15)N]cysteine. Body weight gain and plasma methionine, cysteine, homocysteine, and taurine and total erythrocyte glutathione concentrations were markedly decreased (-46% to -85%) in SAA-free compared with control pigs. Whole body methionine and cysteine fluxes were reduced, yet methionine utilization for protein synthesis and methionine remethylation were relatively preserved at the expense of methionine transsulfuration, in response to SAA deficiency. Intestinal tissue concentrations of methionine and cysteine were markedly reduced and hepatic levels were maintained in SAA-free compared with control pigs. SAA deficiency increased the activity of methionine metabolic enzymes, i.e., methionine adenosyltransferase, methionine synthase, and cystathionine beta-synthase, and S-adenosylmethionine concentration in the jejunum, whereas methionine synthase activity increased and S-adenosylmethionine level decreased in the liver. Small intestine weight and protein and DNA mass were lower, whereas liver weight and DNA mass were unchanged, in SAA-free compared with control pigs. Dietary SAA deficiency induced small intestinal villus atrophy, lower goblet cell numbers, and Ki-67-positive proliferative crypt cells in association with lower tissue glutathione, especially in the jejunum. We conclude that SAA deficiency upregulates intestinal methionine cycle activity and suppresses epithelial growth in neonatal pigs.

Thiol/Disulfide Redox Status Is Oxidized in Plasma and Small Intestinal and Colonic Mucosa of Rats with Inadequate Sulfur Amino Acid Intake

Low molecular weight thiol/disulfide redox pools are dependent upon extracellular cysteine (Cys) availability. We determined whether dietary sulfur amino acid (SAA) deficiency induces oxidative stress in vivo, as determined by redox state of major thiol/disulfide couples in plasma [Cys/cystine (CySS)] and intestinal mucosa [glutathione (GSH)/glutathione disulfide (GSSG)]. Rats were fed isocaloric, isonitrogenous semipurified diets: either SAA-adequate (control), SAA-deficient, or SAA-supplemented, pair-fed to intake of the SAA-deficient group. Reference rats consumed standard rat food ad libitum. After 7 d, plasma and gut mucosal samples were analyzed for Cys, CySS, GSH and GSSG, and the redox potentials of Cys/CySS and GSH/GSSG were determined. Mean daily food intake in the pair-fed rats was similar (approximately one-half of reference-rat intake). Body weight decreased in all pair-fed groups, but rats fed the SAA-deficient diet lost significantly more body weight. Dietary SAA deficiency decreased GSH concentrations in both plasma and gut mucosa, increased plasma GSSG, and oxidized plasma and gut mucosal GSH/GSSG redox and plasma Cys/CySS redox. SAA supplementation resulted in a more reducing plasma Cys/CySS redox potential. Reference rats exhibited similar tissue and plasma GSH/GSSG redox as rats that ate semipurified SAA-adequate rat food, which provided similar net SAA intake. Our in vivo data show that inadequate dietary SAA intake oxidizes the thiol/disulfide redox status in rat-gut mucosa and plasma. Such oxidation of redox pools is associated with oxidative stress and the onset or progression of several pathological conditions. Thus, dietary SAA deficiency could contribute to the progression of disease by causing an oxidation of these components.

Repression by oxidative stress of iNOS and cytokine gene induction in macrophages results from AP-1 and NF-κB inhibition mediated by B cell translocation gene-1 activation

Previously, we reported that oxidative stress caused by sulfur amino acid deficiency (SD) induces B cell translocation gene-1 (Btg-1), which belongs to the Apro family, in hepatocytes. In view of the impairment of immune function by protein restriction that causes SD, this study investigated whether SD or other oxidative stress inhibits iNOS and cytokine expression and induces Btg-1 in macrophages and explored the causal relationship of Btg-1 induction and repression of the genes. When macrophages were incubated in sulfur amino acid-deprived medium, lipopolysaccharide induction of iNOS, TNFα, IL-1β, and IL-6 was significantly decreased compared to control. Because AP-1 and NF-κB are the common transcription factors that regulate the genes encoding iNOS and cytokines, we examined AP-1 and NF-κB DNA binding activities and transactivation of the iNOS gene containing the DNA binding elements. Induction of the reporter gene pGL-miNOS-1588 comprising the −1.6 kb iNOS promoter in lipopolysaccharide-activated macrophages was inhibited 30–70% by SD or treatment with pro-oxidants, including tert-butylhydroxyquinone, buthionine sulfoximine, and 3-morpholinosydnonimine. Oxidative stress increased Btg-1 mRNA. SD-induced oxidative stress activated Btg-1 in macrophages, as evidenced by nuclear translocation of endogenous or green fluorescent protein-tagged Btg-1, which localized in the cytoplasm in the resting state. Expression of Btg-1 inhibited lipopolysaccharide-inducible AP-1 and NF-κB activities, repressing transactivation of the target gene pGL-miNOS-1588. These results provide evidence that oxidative stress induced by SD or pro-oxidants inhibits the expression of iNOS and cytokines in macrophages with Btg-1 activation and that the gene repression by oxidative stress may result from Btg-1-mediated inhibition of AP-1 and NF-κB activities.

Cadmium-induced non-apoptotic cell death mediated by oxidative stress under the condition of sulfhydryl deficiency

Cadmium (Cd), which accumulates primarily in the liver and the kidney, induces apoptosis and also causes necrotic cell death in certain pathophysiologic situations. Previously, we have shown that Cd activated mitogen-activated protein kinases and that sulfur amino acid deficiency potentiated Cd-induced cytotoxicity via activation of mitogen-activated protein kinases. In the present study, we established the mechanistic basis of apoptotic and non-apoptotic cell death induced by Cd in H4IIE cells a rat-derived hepatocyte cell line. Cd at 0.3–10 μM decreased viability of cells in a concentration-dependent manner. Cd-induced cytotoxicity was enhanced by pretreatment with buthionine sulfoximine (BSO). Cd at 0.3 μM induced translocation of Bad to mitochondria, decreased the level of mitochondrial BcL XL with the release of cytochrome c, and induced procaspase-9 activation and poly(ADP-ribose) polymerase (PARP) cleavage. Sulfhydryl deficiency by BSO, however, blocked PARP cleavage in spite of the decrease in procaspase-9. Cytochrome c release, procaspase-9 activation and PARP cleavage were all increased by 1 μM Cd irrespective of BSO pretreatment. We also used H 2O 2 (10–100 μM) as a source of oxidative stress. Cd (0.3–1 μM) + H 2O 2 (70 μM) resulted in greater extents of cytochrome c release, procaspase-9 activation and PARP cleavage in H4IIE cells than Cd alone. Flow cytometric analysis confirmed apoptotic and non-apoptotic cell death by Cd depending on cellular glutathione (GSH) content. These results provide evidence that Cd at the physiologically obtainable concentration causes non-apoptotic cell death under the condition of sufhydryl deficiency, whereas Cd at the micromolar level induces apoptosis. The cell death mechanism involves cytochrome c release from mitochondria and decrease in the level of procaspase-9, but not PARP cleavage, implying that alterations in cellular sulfhydryls may be the major determining factor for the path of cell death in response to low level of Cd.

Chondroitin sulfate and other sulfate containing chondroprotective agents may exhibit their effects by overcoming a deficiency of sulfur amino acids

Dietary supplements derived from cartilage and medications containing sulfated amino sugars, are used with increasing frequency by individuals affected with osteoarthritis (OA). Our lack of knowledge regarding their mode of action is further complicated by the fact that many of these compounds have to be degraded by the intestinal flora for absorption. The metabolic studies performed in this work suggest that the sulfate moiety present in these molecules may be overcoming a dietary deficiency of sulfur amino acids (SAA; cysteine and methionine), our primary sources of sulfate. The compounds tested are rapidly converted into free sulfate before or after absorption, depending on their chemical nature, and excreted quantitatively when the intake of protein seems to exceed the dietary requirement for the SAA. At lower levels of protein intake, sulfur is retained and excretion considerably reduced. Cartilage is a unique tissue that after acquiring its full complement of cells during early development expands in size by deposition of collagen type II, and tissue specific glycosaminoglycans (GAG) that require a source of inorganic sulfate for their synthesis. After maturity, the turnover of these molecules is very limited. Classical studies by Mankin and Maroundas estimated a T1/2 of 3.5 years for some proteoglycan fractions and 25 years for most others 1–3 . Sub-optimal amounts of SAA, our major sources of inorganic sulfate used for the de novo synthesis of GAG, may not cause any significant problem to healthy cartilage, but when OA begins to progress could lead to serious consequences. During the onset of disease GAG turnover is greatly enhanced, as evidenced by the rapid loss of

Methionine as a methyl group donor in growing cattle.

Holstein steers were used in two 5 x 5 Latin square experiments to evaluate the sparing of methionine by alternative sources of methyl groups (betaine and choline). Steers were housed in metabolism crates and limit-fed a soybean hull-based diet high in rumen degradable protein. To increase energy supply, ruminal infusions of volatile fatty acids and abomasal infusions of glucose were provided. An amino acid mixture, limiting in methionine, was infused abomasally to ensure that nonsulfur amino acids did not limit protein synthesis. Treatments for Exp. 1 were abomasal infusion of 1) water, 2) 2 g/d L-methionine, 3) 1.7 g/d L-cysteine, 4) 1.6 g/d betaine, and 5) 1.7 g/d L-cysteine + 1.6 g/d betaine. Treatments for Exp. 2 were abomasal infusion of 1) water, 2) 2 g/d L-methionine, 3) 8 g/d betaine, 4) 16 g/d betaine, and 5) 8 g/d choline. In both experiments, nitrogen retention increased in response to methionine (P < 0.05), demonstrating a deficiency of sulfur amino acids. Responses to cysteine, betaine, and choline were all small and not significant. The lack of response to cysteine indicates that the response to methionine was not due to transsulfuration to cysteine or that cysteine supply did not alter the flux of methionine through transsulfuration. The lack of response to betaine suggests that the steers' needs for methyl groups were met by the dietary conditions or that betaine was relatively inefficient in increasing the remethylation of homocysteine to methionine and, thereby, reducing the synthesis of cysteine from homocysteine. Under our experimental conditions, responses to methionine were likely due to a correction of a deficiency of methionine per se rather than of methyl group donors.

The Effects of Dietary Sulfur Amino Acid Deficiency on Rat Brain Glutathione Concentration and Neural Damage in Global Hemispheric Hypoxia-Ischemia

Primary brain injury in stroke is followed by an excitotoxic cascade, oxidative stress and further neural damage. Glutathione is critical and depleted in oxidative stress. Since cysteine is limiting in glutathione synthesis, this study investigated the effect of dietary sulfur amino acid (SAA) deficiency on neural damage in a rat model of global hemispheric hypoxia-ischemia (GHHI). Animals were fed with SAA deficient ("deficient") or control diet for 3 days, subjected to right common carotid artery ligation and hypoxia, and diet continued for 3 more days. Histologically evaluated neural damage at 7 days post hypoxia-ischemia was greater in "deficient" rats, shown by mean (±SEM) global and hippocampal grid scores of 2.5±0.7 and 34.9±9.3%, respectively, vs. controls' scores of 0.1±0.1 and 0.1±0.1%, respectively. Mean brain (±SEM) reduced glutathione was not different between groups at 6 h post hypoxia-ischemia, but was decreased in "deficient" animals 3 days later in neocortex (1.46 μmoles/g wet weight±0.05 vs. 1.67±0.04 in controls) and thalamus (1.60 μmoles/g wet weight±0.05 vs. 1.78±0.03 in controls). Administration of a cysteine precursor to "deficient" animals did not ameliorate neural damage. These findings suggest that well-nourished but not "deficient" animals tolerate a mild brain insult. The decline in brain glutathione in the "deficient" animals may be one of several contributing mechanisms.

Alterations in cellular Ca 2+ and free iron pool by sulfur amino acid deprivation: the role of ferritin light chain down-regulation in prooxidant production

Deficiency of sulfur amino acids occurs in certain pathophysiological states such as protein-calorie malnutrition. Sulfur amino acid deprivation (SAAD) increases oxidative stress through a decrease in GSH. Ferritin expression is induced by oxidative stress, which confers resistance to oxidative insults. The effects of SAAD on the changes in cellular Ca 2+ and free iron pool, prooxidant production and the ferritin light chain (FLC) expression were comparatively evaluated in Hepa1c1c7 and Raw264.7 cells. [Ca 2+] i was rapidly increased by SAAD. Sulfhydryl-containing compounds prevented the increase in [Ca 2+] i in cells under SAAD, supporting the role of redox-state in the regulation of [Ca 2+] i. Thapsigargin or Ca 2+-free medium inhibited the increase in [Ca 2+] i, showing that Ca 2+ originated from endoplasmic reticulum as well as from extracellular source. Inhibition of Ca 2+ mobilization decreased the fluorescence of Phen Green SK inside cells, representing the inhibition of free iron release. Both inhibition of Ca 2+ mobilization and iron chelation decreased dichlorofluorescein oxidation, indicating the possibility that the increase in [Ca 2+] i affected that in cellular free iron and prooxidant production. FLC protein level was immunochemically detectable in Raw264.7 cells, but not in Hepa1c1c7 cells. SAAD alone (or in combination with FeSO 4) down-regulated FLC protein expression, while SAAD increased the FLC mRNA level in both Hepa1c1c7 and Raw264.7 cells. Calcium or iron chelators prevented increases in the FLC mRNA. These results provided evidence that changes in cellular Ca 2+ and iron pool by SAAD increased cellular oxidative stress and that the down-regulation of FLC protein by SAAD would further enhance prooxidant production in spite of the increase in FLC mRNA.

Potentiation of arsenic-induced cytotoxicity by sulfur amino acid deprivation (SAAD) through activation of ERK1/2, p38 kinase and JNK1: the distinct role of JNK1 in SAAD-potentiated mercury toxicity

Sulfur amino acid deficiency occurs in certain pathophysiological situations (e.g. protein-calorie malnutrition). Previous studies revealed that sulfur amino acid deprivation (SAAD) activated MAP kinases and potentiated cadmium-induced cytotoxicity by activation of ERK1/2 in conjunction with p38 kinase or JNK. The present study was designed to determine susceptibility of cells to a variety of heavy metals in combination with SAAD. Viability was assessed in H4IIE cells treated with sodium arsenite, mercuric chloride, sodium selenite, lead acetate, chromium trioxide or manganese chloride. SAAD potentiated the cytotoxicity of H4IIE cells by arsenic or mercury (i.e. EC50, 19 and 5 μM in SAAD vs. 401 and 42 μM in control medium, respectively). TUNEL assays revealed that the potentiated arsenic or mercury toxicity involved apoptotic cell death. Lead or selenite moderately elicited cell death, which was not enhanced by SAAD. Chromium or manganese caused no significant cytotoxicity. Treatment of cells with U0126 [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene] an ERK1/2 inhibitor or SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole] a p38 kinase inhibitor effectively prevented SAAD-potentiated arsenic toxicity. The potentiated arsenic toxicity was also inhibited in cells stably expressing a dominant negative mutant of c-Jun N-terminal kinase 1 [JNK1(−)]. The inhibitors of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 kinase failed to prevent mercury-induced toxicity enhanced by SAAD. JNK1(−) cells were minimally susceptible to mercury in SAAD medium. These results demonstrated that SAAD potentiated cytotoxicity induced by arsenic or mercury and that activation of ERK1/2, p38 kinase and JNK1 was responsible for the potentiated arsenic toxicity, whereas the mercury toxicity enhanced by SAAD was mediated with the activity of JNK1.

Bioactivation of cyanide to cyanate in sulfur amino acid deficiency: relevance to neurological disease in humans subsisting on cassava.

Neurological disorders have been reported from parts of Africa with protein-deficient populations and attributed to cyanide (CN-) exposure from prolonged dietary use of cassava, a cyanophoric plant. Cyanide is normally metabolized to thiocyanate (SCN-) by the sulfur-dependent enzyme rhodanese. However, in protein-deficient subjects where sulfur amino acids (SAA) are low, CN may conceivably be converted to cyanate (OCN-), which is known to cause neurodegenerative disease in humans and animals. This study investigates the fate of potassium cyanide administered orally to rats maintained for up to 4 weeks on either a balanced diet (BD) or a diet lacking the SAAs, L-cystine and L-methionine. In both groups, there was a time-dependent increase in plasma cyanate, with exponential OCN- increases in SAA-deficient rats. A strongly positive linear relationship between blood CN- and plasma OCN- concentrations was observed in these animals. These data are consistent with the hypothesis that cyanate is an important mediator of chronic cyanide neurotoxicity during protein-calorie deficiency. The potential role of thiocyanate in cassava-associated konzo is discussed in relationship to the etiology of the comparable pattern of motor-system disease (spastic paraparesis) seen in lathyrism.

Role of methionine as a methyl group donor in cattle (1999)

Holstein steers were used in two 5 × 5 Latin square experiments to evaluate the sparing of methionine by alternative sources of methyl groups (betaine or choline). Steers were housed in metabolism crates and limit fed a diet high in rumen degradable protein. To increase energy supply, volatile fatty acids were infused into the rumens, and glucose was infused into the abomasum. An amino acid mixture, limiting in methionine, was infused abomasally to ensure that non-sulfur amino acids did not limit protein synthesis. Treatments for Exp. 1 were abomasal infusion of 1) water (control), 2) 2 g/day additional L-methionine, 3) 1.7 g/day Lcysteine, 4) 1.6 g/day betaine, and 5) 1.7 g/day L-cysteine + 1.6 g/day betaine. Treatments for Exp. 2 were abomasal infusion of 1) water (control), 2) 2 g/day additional L-methionine, 3) 8 g/day betaine, 4) 16 g/day betaine, and 5) 8 g/day choline. In both experiments, nitrogen retention increased (P<.05) in response to methionine, demonstrating a deficiency of sulfur amino acids. Responses to cysteine, betaine and choline were small. The low response to cysteine indicates that either the response to methionine is not due to transsulfuration to cysteine, or that cysteine supply does not alter the flux of methionine through transsulfuration. The small responses to betaine and choline suggest that they do not substitute for methionine. Thus, under our experimental conditions, responses to methionine likely were due to a correction of a deficiency of methionine per se rather than of methyl group donors.

Neural damage in global hemispheric hypoxic ischemia is increased by sulfur amino acid (SAA) deficiency: [formula omitted]§, J.L. Franklin§, B.H.J. Juurlink‡, J.A. Thornhill†. §College of Pharmacy and Nutrition, ‡Dept. of Anatomy and Cell Biology, and †Dept. of Physiology, University of Saskatchewan, Saskatoon, SK, Canada S7N 5C9

Neural damage in global hemispheric hypoxic ischemia is increased by sulfur amino acid (SAA) deficiency: [formula omitted]§, J.L. Franklin§, B.H.J. Juurlink‡, J.A. Thornhill†. §College of Pharmacy and Nutrition, ‡Dept. of Anatomy and Cell Biology, and †Dept. of Physiology, University of Saskatchewan, Saskatoon, SK, Canada S7N 5C9