Vitamin C and E supplementation blunts increases in total lean body mass in elderly men after strength training.

The aim of this study was to investigate the effects of vitamin C and E supplementation on changes in muscle mass (lean mass and muscle thickness) and strength during 12 weeks of strength training in elderly men. Thirty-four elderly males (60-81 years) were randomized to either an antioxidant group (500 mg of vitamin C and 117.5 mg vitamin E before and after training) or a placebo group following the same strength training program (three sessions per week). Body composition was assessed with dual-energy X-ray absorptiometry and muscle thickness by ultrasound imaging. Muscle strength was measured as one-repetition maximum (1RM). Total lean mass increased by 3.9% (95% confidence intervals: 3.0, 5.2) and 1.4% (0, 5.4) in the placebo and antioxidant groups, respectively, revealing larger gains in the placebo group (P = 0.04). Similarly, the thickness of m. rectus femoris increased more in the placebo group [16.2% (12.8, 24.1)] than in the antioxidant group [10.9% (9.8, 13.5); P = 0.01]. Increases of lean mass in trunk and arms, and muscle thickness of elbow flexors, did not differ significantly between groups. With no group differences, 1RM improved in the range of 15-21% (P < 0.001). In conclusion, high-dosage vitamin C and E supplementation blunted certain muscular adaptations to strength training in elderly men.

The aminothiol redox status in haemodialysis patients does not improve with folate therapy.

BACKGROUND: Patients on haemodialysis suffer from high cardiovascular morbidity and mortality, and oxidative stress may play a role in the pathophysiology of cardiovascular disease in these patients. Hyperhomocysteinemia is common in dialysis patients and may have pro-oxidant effects. Moreover, the redox status of the major plasma aminothiols (homocysteine [Hcy], cysteine and cysteinylglycine) may be regarded as a biomarker of oxidative stress. In the present study, we investigated the aminothiol redox status during a period of homocysteine-lowering therapy with folinic acid. MATERIAL AND METHODS: In the first part of the study, 32 stable patients receiving maintenance haemodialysis were compared with 32 reference subjects. In the second part, the patients were given folinic acid intravenously for 3 months. RESULTS: Before intervention with folinic acid, the patients had elevated concentrations of all redox species of Hcy. The aminothiol redox ratios were low. Folinic acid therapy lowered the concentrations of all Hcy redox species; however, the redox ratios did not improve. CONCLUSIONS: The low aminothiol redox ratios indicate the presence of oxidative stress in haemodialysis patients. Therapy with folinic acid lowered total Hcy concentrations, but did not improve the redox status. Thus, hyperhomocysteinemia appears to be of little importance in regard to the total level of oxidative stress in uraemia.

Holotranscobalamin and methylmalonic acid as prognostic markers following an acute myocardial infarction.

OBJECTIVE: To evaluate whether low levels of holotranscobalamin (holoTC) or elevated levels of methylmalonic acid (MMA), both indicators of vitamin B(12) deficiency, might predispose to new cardiovascular events following an acute myocardial infarction (MI). DESIGN: A prospective prognostic study. SETTING: One hospital center in Stavanger, Norway. SUBJECTS: A total of 300 patients admitted with an acute MI. METHODS: Registration of new TnT positive coronary events (defined as TnT>0.05 microg/l and a typical MI pattern) and/or cardiac death during a median follow-up time of 45 months. RESULTS: We compared the recurrence of events in the lowest quartile of holoTC (Q1<73.9 pmol/l) to the event rate above the 25% percentile (Q2-4). For methylmalonic acid (MMA) the same comparison was carried out for the upper quartile (Q4 > or =0.24 micromol/l) as compared with the event rate below the 75% percentile (Q1-3). After 18 and 45 months of follow-up, the odds ratio (OR) for Q1 vs Q2-4 for holoTC was 1.15 (95% confidence interval (CI) 0.91-1.46, P=0.25) and 1.05 (95% CI 0.86-1.29, P=0.64), respectively. For MMA the OR for Q4 vs Q1-3 was 0.95 (95% CI 0.76-1.19, P=0.67) after 18 months and 1.01 (95% CI 0.83-1.23, P=0.90) after 45 months. CONCLUSION: This study showed no increased risk of future cardiovascular events associated with low levels of holoTC or high levels of MMA following an acute MI.

Redox status and protein binding of plasma homocysteine and other aminothiols in patients with hyperhomocysteinemia due to cobalamin deficiency.

We determined reduced, oxidized, and protein-bound homocysteine, cysteine, and cysteinylglycine in plasma from 13 patients with hyperhomocysteinemia (total homocysteine in the range 30.6-159.8 mumol/L) due to cobalamin deficiency. Reduced homocysteine (means +/- SD: 1.87 +/- 2.06 mumol/L) was markedly above normal (0.24 +/- 0.12 mumol/L) in most patients, and the reduced fraction increased as an exponential function of the total homocysteine concentration. The ratio of reduced homocysteine to total homocysteine was positively correlated with the reduced-total ratio for cysteine and cysteinylglycine, suggesting redox equilibrium between different aminothiol species. The free oxidized and the protein-bound forms of homocysteine account for most of the homocysteine in plasma of these patients. The amount of protein-bound homocysteine was negatively correlated with the concentrations of both protein-bound cysteine and cysteinylglycine, indicating displacement of these aminothiols by homocysteine.

Atherothrombogenic risk modulation by n-3 fatty acids was not associated with changes in homocysteine in subjects with combined hyperlipidaemia.

Favourable effects of n-3 fatty acids on the atherogenic risk profile were recently demonstrated in subjects with combined (type IIb) hyperlipidaemia, not responding to a therapeutic diet. Re-examination of a previous patient material was performed to assess the influence of n-3 fatty acids on homocysteine and several coagulation factors. Subjects were randomly allocated to receive either a concentrated compound of 85% eicosapentaenoic acid (EPA)/docosahexaenoic acid (DHA) (n = 28), or corn oil (n = 29), in a daily dose of 4g for 12 weeks. The intervention was double-blind. Homocysteine remained unchanged in both groups after 12-week treatment. N-3 fatty acids supplementation did not affect the levels of fibrinogen, coagulation factor VII or tissue factor pathway inhibitor (TFPI), while plasminogen activator inhibitor (PAI) increased significantly (Student's t-test; p <0.05). Total blood platelets were significantly reduced in subjects receiving n-3 fatty acids (Student's t-test; p <0.05), whereas bleeding times increased non-significantly.

Redox status and protein binding of plasma homocysteine and other aminothiols in patients with homocystinuria.

Elevations of homocyst(e)ine levels in the blood of patients with homocystinuria may explain the high cardiovascular morbidity. We determined levels of reduced, oxidized, and protein-bound homocyst(e)ine, cyst(e)ine, and cyst(e)inylglycine in plasma from eight patients with homocystinuria. The technique used involved trapping of reduced thiols by collecting blood directly into tubes containing sulfhydryl-reactive reagents. All patients had high levels of homocysteine (range, 1.9 to 91.2 mumol/L), and among the aminothiols investigated, this species showed the most drastic elevation compared with trace levels (< 0.4 mumol/L) found in healthy subjects. The ratio between free homocysteine and total homocyst(e)ine (reduced to total ratio) was above normal and positively correlated to the reduced to total ratio for cyst(e)ine, suggesting that an equilibrium exists between these species through sulfhydryl disulfide exchange. The other homocyst(e)ine species (oxidized and protein-bound) were also markedly increased in patients with homocystinuria. Plasma cysteine and cysteinylglycine levels were moderately increased, whereas plasma concentrations of protein-bound cyst(e)ine, protein-bound cyst(e)inylglycine, and free cystine were below normal. Homocysteine in particular and other homocyst(e)ine species are markedly increased in plasma of homocystinurics, and these changes are associated with pronounced alterations in the level and the redox status of other aminothiols. This should be taken into account when considering homocyst(e)ine as an atherogenic agent, and the role of various homocyst(e)ine species in the pathogenesis of homocystinuria.

Endothelial cell adhesion molecules in healthy adults during acute hyperhomocysteinemia and mild hypertriglyceridemia.

OBJECTIVE: Hyperhomocysteinemia and hypertriglyceridemia are independently associated with atherosclerotic disease. The process of atherogenesis involves inflammation and endothelial dysfunction. We tested whether concurrent acute hyperhomocysteinemia and mild hypertriglyceridemia increase the concentrations of circulatory cellular adhesion molecules in healthy subjects. STUDY DESIGN AND METHODS: Twelve healthy volunteers aged 37.5 years (range, 25-51) participated in the present study. The concentrations of plasma total homocysteine (p-tHcy), serum triglycerides, circulatory cellular adhesion molecules (CAMs), and concentrations of nitrate were measured at 0 (fasting), 2, 4, and 6 h after loading with (1) methionine, (2) fat, (3) methionine + fat, and (4) water (control). Wash out period between each loading was >or=1 week. RESULTS: Percent relative changes from baseline in the concentrations of p-tHcy, 2, 4, and 6 h after methionine and methionine + fat were significantly different from after water and fat loading. Changes in the concentrations of serum triglycerides 2 h after fat loading were significantly different from water loading, whereas methionine + fat loading caused a significant difference after 2, 4, and 6 h. We detected a synergistic increase in the triglyceride area response to methionine + fat loading. We detected also a significant difference in percent relative changes in the concentrations of P-selectin (PSEL) (P = 0.02), E-selectin (ESEL) (P = 0.003), and vascular cell adhesion molecule-1 (VCAM-1) (P = 0.005) 6 h after methionine + fat loading as compared to water loading. There was an additive increase in the PSEL area response to methionine + fat loading. Furthermore, area response to VCAM was greater to methionine loading than water loading (P = 0.01). A decrease in the concentration of NO(3) was more pronounced after methionine + fat loading and a significantly decreased area response of nitrate to methionine + fat loading was detected than to area response to water loading (P = 0.002). CONCLUSION: Inflammatory activation of the endothelium takes place during concurrent transient hyperhomocysteinemia and mild hypertriglyceridemia.

Antiepileptic drugs as independent predictors of plasma total homocysteine levels.

Low folate levels have consistently been reported in patients with epilepsy on phenytoin (PHT), phenobarbital (PB) and primidone (PRD), while data on valproate (VPA) are conflicting. Furthermore, antiepileptic drugs (AEDs) may be associated with high levels of plasma total homocysteine (p-tHcy). Therefore, we have investigated the levels of p-tHcy, serum folate (S-FA) and erythrocyte folate (E-FA) in patients on PHT, PB and PRD (Group 1, n=21) and VPA (Group 2, n=24). Both groups had their own matched controls. Blood samples were drawn fasting and 6 h post methionine loading (6 h-PML). The Group 1 patients had fasting and 6 h-PML p-tHcy levels significantly higher than their controls (P=0.05 and <0.0001, respectively), and patients without dietary multivitamin supplementation (n=14), had lower fasting S-FA and E-FA levels than their controls (P=0.02 and 0.0003, respectively). The Group 2 patients had fasting and 6 h-PML levels of p-tHcy, S-FA and E-FA not different from their controls. In a multiple stepwise regression model comprising all subjects (n=90), the AEDs of Group 1 and the S-FA levels were independent predictors of p-tHcy levels. Thus, PHT, PB and PRD are associated with high p-tHcy and low folate levels, whereas VPA does not influence S-FA, E-FA and p-tHcy levels in adult patients.

Increased lipid peroxidation during long-term intervention with high doses of n-3 fatty acids (PUFAs) following an acute myocardial infarction.

OBJECTIVE: To assess the oxidative burden of a highly concentrated compound of n-3 PUFAs as compared to corn oil by measuring thiobarbituric acid-malondialdehyde complex (TBA-MDA) by HPLC. We also studied the influence on TBA-MDA of statins combined with n-3 PUFAs or corn oil. DESIGN: A prospective, randomised, double-blind, controlled study. SETTING: One hospital centre in Stavanger, Norway. SUBJECTS: A total of 300 subjects with an acute myocardial infarction (MI). INTERVENTIONS: Gelatine capsules, containing 850-882 mg EPA and DHA as concentrated ethylesters, or 1 g of corn oil, were ingested in a dose of two capsules twice a day for at least 1 y. Alpha-tocopherol (4 mg) was added to all capsules to protect the PUFAs against oxidation. RESULTS: After 1 y TBA-MDA increased modestly in the n-3 PUFA group (n=125), as compared to the corn oil group (n=130), P=0.027. Multiple linear regression analyses of fatty acids in serum total phospholipids (n=56) on TBA-MDA measured after 12 months intervention, showed no dependency. Performing best subsets regression, serum phospholipid concentration of arachidonic acid (20:4 n-6 PUFA) was identified as a predictor of TBA-MDA at 12 months follow-up, P=0.004. We found no impact of statins on TBA-MDA. CONCLUSION: TBA-MDA increased modestly after long-term intervention with n-3 PUFAs compared to corn oil post-MI, suggesting biological changes induced by n-3 PUFAs, rather than simply reflecting their concentration differences. The peroxidative potential of n-3 PUFAs was not modified by statin treatment. SPONSORSHIP: : Pharmacia A/S and Pronova A/S, Norway.

Relationship between methylmalonic acid, homocysteine, vitamin B12 intake and status and socio-economic indices, in a subset of participants in the British National Diet and Nutrition Survey of people aged 65 y and over.

OBJECTIVE: Assessment of functional vitamin B(12) status in a subset of the respondents in the British National Diet and Nutrition Survey of people aged 65 y and over. SETTING: National Diet and Nutrition Survey: a British nationwide cross-sectional sample of people aged 65 y and over, living either in the community or in institutions such as nursing homes, during one calendar year spanning 1994-1995. METHODS: Methylmalonic acid (MMA) concentrations were measured in plasma samples from 313 subjects (ca 14% of those originally enrolled in the survey). The results were compared with those for serum vitamin B(12), vitamin B(12) intakes and other status and intake estimates and with socio-demographic indices. RESULTS: Of the NDNS participants overall, 20% had serum vitamin B(12) concentrations<150 pmol/l. In the subset studied here, 24% of free-living and 46% of institution-living participants had MMA>0.5 micromol/l. Geometric mean MMA increased with age, from 0.25 micro mol/l in people aged 65-74 y to 0.38 micro mol/l in people aged 85+y. There was little evidence for any gender difference in MMA. It was inversely correlated with serum vitamin B(12) and with red blood cell folate; it was positively correlated directly with total homocysteine, but not significantly with serum folate or with vitamin B(12) intake. Among respondents with high MMA, a subgroup had normal serum vitamin B(12) but higher-than-average plasma urea and creatinine. Socio-demographic co-variates of MMA included receipt of State income benefits, social class of head of household, and educational attainment. These indices were not correlated with serum vitamin B(12). CONCLUSIONS: The progressive increase in MMA with age is metabolic evidence for increasing risk of functional vitamin B(12) deficiency with increasing age in older people. There is evidence that renal function is linked to high MMA in some older people. Age and renal function are thus both important when establishing upper reference limits for MMA. The socio-demographic observations suggest a link between poverty and poor functional vitamin B(12) status in older British people.

Plasma total homocysteine in a representative sample of 972 British men and women aged 65 and over.

OBJECTIVES: To provide a reference range for plasma total homocysteine (tHcy), an independent risk factor for vascular disease, and to explore relationships with nutritional indices for people aged 65 y and over, in the UK National Diet and Nutrition Survey (NDNS). DESIGN: The survey procedures described in the National Diet and Nutrition Survey Report (1997) included a health-and-lifestyle interview, a four-day weighed diet record, anthropometric and blood pressure measurements and a fasting blood sample for biochemical indices, including tHcy. SETTING: Eighty randomly selected postcode sectors from mainland Britain during 1995-1996. SUBJECTS: Of 2060 people interviewed, 1527 were visited by the nurse, 1276 gave a blood sample and 972 had tHcy measured. About 80% were in their own homes and the remainder were in nursing homes or similar institutions. RESULTS: Significant cross-sectional relationships, both univariate and multivariate were found between tHcy and index concentrations of folate and vitamin B12 (P < 0.0001), and between tHcy and plasma creatinine, urea, calcium, zinc, alpha 1-antichymotrypsin, lutein and cysteine (P = 0.013 to < 0.0001). Dietary nutrient analyses showed an association with folate intake. tHcy was also correlated with age and with domicile (free-living or institution), with history of vascular disease and with use of four classes of drugs, two of which are prescribed for vascular diseases. There was a north-south gradient in tHcy (P = 0.005), and also in food choices, blood micronutrient indices and vascular disease prevalence. CONCLUSIONS: The concentrations of tHcy found in this study provide a reference range for people aged 65 y and over, in mainland Britain. tHcy is a valuable functional index of micronutrient status and intakes for British people aged 65 y and over, which can assist the development of health-promotion strategies.

Haemostasis in ischaemic stroke and vascular dementia.

Abnormalities of coagulation and fibrinolysis may play an important role in the pathogenesis of ischaemic stroke and vascular dementia. We aimed to determine whether haemostatic function is altered in acute recent-onset or chronic ischaemic cerebrovascular disease. We studied consecutive patients with ischaemic stroke (n = 74) and vascular dementia (n = 42) compared with healthy controls (n = 40) in a case-control study. The ischaemic stroke group was assessed twice, 3-10 days after the acute stroke and at 1-3 months. Fibrinogen, fibrin D-dimer (marker of fibrin turnover) and von Willebrand factor (vWF) (marker of endothelial disturbance) were elevated acutely (P < 0.0001) and in the convalescent phase after ischaemic stroke (P < 0.0001, P < 0.0001, and P < 0.01 respectively, compared with controls). Similar results were seen in the vascular dementia group. Stepwise multivariate regression analyses showed that cerebrovascular disease correlated independently with fibrinogen (P < 0.001) and fibrin D-dimer levels (P < 0.001), while vWF correlated independently with electrocardiograph evidence of ischaemic heart disease (P = 0.004). Changes between acute and convalescent phases in ischaemic stroke were slightly inconsistent. However, in the acute stage there were tendencies for fibrinogen, D-dimer and vWF to be increased, and factor VIII was significantly higher. Abnormalities of haemostasis, including increased fibrin turnover and endothelial disturbance, are found in both acute and chronic cerebral ischaemia. Many of these patients have co-existent ischaemic heart disease and this may contribute to some of these changes. Acute ischaemic stroke is associated with transient changes in haemostatic factors; however, most abnormalities persist into the convalescent phase, and are also demonstrable in subjects with vascular dementia.

Modulation of phosphatidylcholine biosynthesis by peroxisome proliferating fatty acid analogues.

The modulation of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) biosynthesis by sulfur-substituted fatty acid analogues has been investigated in rats. We have compared the effects of two non-beta-oxidizable fatty acid analogues, 3-thiadicarboxylic acid and tetradecylthioacetic acid, which induce proliferation of peroxisomes, with those of the analogue tetradecylthiopropionic acid, which is a weak peroxisome proliferator. Repeated administration of 3-thiadicarboxylic acid for seven days resulted in increased hepatic concentrations of both PC and PE, but the PC/PE ratio was decreased. PC synthesis was increased, as evidenced by increased incorporation of [3H]choline into PC and an increased activity of cytidinetriphosphate (CTP): phosphocholine cytidylyltransferase. This was accompanied by a reduction in the pool sizes of choline and phosphocholine. The S-adenosylmethione/S-adenosylhomocysteine ratio (AdoMet/AdoHcy) was marginally affected, indicating no increase in the rate of methylation of PE to PC. Administration of tetradecylthioacetic acid also resulted in increased hepatic phospholipid levels, increased AdoMet/AdoHcy ratios and in slightly elevated activity of CTP:phosphocholine cytidylyltransferase. The most striking effect observed after tetradecylthiopropionic acid treatment was the development of fatty liver. The activity of CTP:phosphocholine cytidylyltransferase and the incorporation of [3H]choline into PC was reduced compared to 3-thiadicarboxylic acid treatment. Although the rate of methylation of PE seemed to be increased at an elevated AdoMet/AdoHcy ratio, this resulted in only minor changes in the hepatic PC and PE levels, and the PC/PE ratio remained unchanged. Furthermore, the hepatic levels of choline and phosphocholine were reduced in these rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Geographical variations in nutrition-related vascular risk factors in the UK: National Diet and Nutrition Survey of People Aged 65 Years and Over.

BACKGROUND: There is a strong north-south gradient of vascular disease in Britain, whose aetiology is not fully understood. OBJECTIVE: To test the hypothesis, in a cross-sectional survey of older people, that intakes and status indices for protective micronutrients, particularly those for which fruit and vegetables are rich sources, also vary on a north-south axis. DESIGN: The 1994-5 National Diet and Nutrition Survey of People Aged 65 Years and Over has provided a uniquely appropriate data-set for this purpose. The analysis, confined to free-living participants, compared nutrient intakes and status between people living in the north of Britain, from Scotland to Humberside, with those living south of the Wash, excluding the Midlands and Wales. RESULTS: Highly significant north-south differences, especially for vitamin C, but also to a significant extent for B-vitamins and carotenoids, indicated a more vitamin-rich diet, with more frequent use of vitamin supplements, in the south. Vitamin D status and fibre intakes were also higher in the south; sodium intake was greater in the north. Blood lipid indices did not, however, differ between north and south. North-south differences in the likelihood of receiving income support, of having manual socio-economic status and of smoking habit, appeared to be significant underlying socio-demographic factors. CONCLUSION: These findings are consistent with the hypothesis that for older British people, differences in nutrient intake and status indices between the north and south of Britain run parallel with, and may contribute to, the north-south axis of vascular disease risk.

Comparison of Abbott IMx total homocysteine assay with a high pressure liquid chromatography method for the measurement of total homocysteine in plasma and serum from a Norwegian population.

In this study 189 samples were analysed for the measurement of homocysteine by the Abbott IMx homocysteine assay and an HPLC method. A strong correlation was obtained between the homocysteine measurements performed by the Abbott IMx homocysteine assay and the HPLC method (coefficient of correlation, r2 = 0.947, p < 0.0001). The plot of the difference for the homocysteine measurements between the two methods against the average of the two measurements resulted a mean difference of 0.80 +/- 6.66 (mean +/- 2SD), and 0.008 +/- 0.126 (mean +/- 2SD) for log converted values of homocysteine. The concentrations of homocysteine measured in all the samples by the two methods were not significantly different. However, the Abbott IMx homocysteine assay measured the concentrations of total homocysteine in hyperhomocysteinemia as significantly higher than the HPLC method (25.00 micromol/l vs. 23.12 micromol/l, p < 0.0001). More studies may be required to explore factors that may influence measurements of homocysteine by the Abbott IMx homocysteine assay and the HPLC method.

Endogenous glutathione and platelet function in platelet concentrates stored in plasma or platelet additive solution.

Platelet function was studied in platelet concentrates by assay of the thrombin-induced release of endogenous serotonin and presence of the swirling phenomenon in relation to endogenous glutathione (GSH) and cysteine. In platelets stored in plasma, addition of cysteamine resulted in only a moderate fall in GSH after 5 days of storage, from an average of 14.91 to 11.46 nmol per 109 platelets. Exogenously added GSH had no effect, and addition of buthionine sulfoximine (BSO) resulted in almost complete depletion of GSH, to an average of 0.65 nmol per 109 platelets. Addition of cysteamine or GSH resulted in increased endogenous cysteine whereas BSO had no effect. In platelets stored in a platelet additive solution (T-sol), complete depletion of GSH was found in the presence of cysteamine, GSH and BSO. Endogenous serotonin was unchanged during storage both in plasma and in additive solution (2.8 nmol per 109 platelets). Despite almost total depletion of endogenous GSH, the thrombin-induced release of serotonin after 5 days' storage was significantly affected only in the presence of BSO in platelets stored in additive solution (mean values 72.3% vs. 63.3% of endogeneous serotonin, P < 0.05). Similarly, addition of cysteamine or GSH had no significant effect on swirling but BSO reduced the swirling score after 5 days' storage in platelet additive solution compared with plasma. After 10 days' storage, there was a significant reduction in swirling in the concentrates where BSO was added (P < 0.05).

Determination of reduced, oxidized, and protein-bound glutathione in human plasma with precolumn derivatization with monobromobimane and liquid chromatography.

This assay measures reduced (GSH), oxidized (GSSG, GSSR), and protein-bound (glutathione-protein mixed disulfides, ProSSG) glutathione in human plasma. Oxidized glutathione and ProSSG are converted to GSH in the presence of NaBH4, and, after precolumn derivatization with monobromobimane, GSH is quantitated by reversed-phase liquid chromatography and fluorescence detection. The NaBH4 concentration is optimized so that total recovery of oxidized glutathione is obtained and no interference with the formation/stability of the GSH-bimane adduct occurs. The presence of 50 microM dithioerythritol prevents reduced recovery at low concentrations of GSH, and the standard curve for GSH is linear over a wide concentration range and is super-imposed upon that obtained with GSSG. Selective determination of oxidized glutathione exploits the fact that N-ethylmaleimide (NEM) blocks free sulfhydryl groups and excess NEM is inactivated by the subsequent addition of NaBH4. To measure total glutathione including the protein-bound forms, the protein is solubilized with dimethyl sulfoxide, which is compatible with the other reagents and slightly increases the yield of the fluorescent GSH derivative. The assay is characterized by a sensitivity (less than 2 pmol) sufficiently high to detect the various forms of glutathione in plasma, by an analytical recovery of GSH and GSSG close to 100%, and by a within-day precision corresponding to a coefficient of variation of 7%. The assay was used to determine the dynamic relationships among various glutathione species in human plasma.

Determination of the in vivo redox status of cysteine, cysteinylglycine, homocysteine, and glutathione in human plasma.

An assay that measures the reduced, oxidized, and protein-bound forms of cysteine, cysteinylglycine, homocysteine, and glutathione in human plasma is described. Oxidized and protein-bound thiols are converted to their reduced counterparts by the use of NaBH4, and, following derivatization with monobromobimane (mBrB), the thiol-bimane adducts are quantified by reversed-phase ion-pair liquid chromatography and fluorescence detection. The presence of 50 microM dithioerythritol provides linearity of the standard curves at very low thiol concentrations. Selective determination of the oxidized forms was accomplished by blocking free sulfhydryl groups with N-ethylmaleimide (NEM) and excess NEM is inactivated by the subsequent addition of NaBH4. The reduced forms of the thiols in plasma were trapped with minimal oxidation by derivatizing blood samples at the time of collection. This was attained by drawing blood directly into tubes containing isotonic solutions of mBrB or NEM. The assay is sufficiently sensitive (less than 2 pmol) to detect the various forms of the four thiol compounds in human plasma. The analytical recovery of cysteine, cysteinylglycine, homocysteine, and glutathione was close to 100%, and the within-day precision corresponded to a coefficient of variation of 7, 8, 6, and 7%, respectively. The assay has been used to determine the various forms of the four thiol compounds in human plasma.

Modulation of glutathione content and the effect on methionine auxotrophy and cellular distribution of homocysteine and cysteine in mouse cell lines.

The inability of cells in culture to grow in medium where methionine is replaced by its metabolic precursor, homocysteine, has been linked to neoplastic transformation and termed 'methionine dependence' or 'methionine auxotrophy'. The present investigation was undertaken to establish the influence of intracellular glutathione level on methionine auxotrophy in different mouse cell lines. A non-transformed, methionine-independent fibroblast cell line with essential normal growth rate in methionine-deficient, homocysteine-supplemented medium (Met-Hcy+), showed only a slight initial lag and then the same growth as control when glutathione was reduced to less than 5% by the glutathione synthesis inhibitor buthionine sulfoximine (BSO). Increasing cellular glutathione by cystamine in a completely methionine-dependent leukemia cell line did not stimulate the cells to proliferate in Met-Hcy+ medium. A partly methionine-dependent transformed fibroblast cell line with reduced capacity to proliferate in Met-Hcy+ medium showed increased growth potential when the cells were depleted of glutathione by a non-toxic concentration of BSO. An even higher growth potential of these cells in Met-Hcy+ medium was obtained by addition of a non-toxic concentration of cystamine, while only a transient increase of glutathione content was observed under these conditions. Both BSO and cystamine increased the fraction of protein-bound cysteine and homocysteine in the partly methionine-dependent cells. These metabolic alterations correlated with the increased ability of these cells to utilize homocysteine for growth. Our results suggest that methionine auxotrophy is a metabolic defect that is not related to the cellular glutathione status, but may be related to the intracellular distribution between free and protein-bound forms of other thiols as cysteine and homocysteine.