Asbjørn Svardal

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.

Redox Status and Protein Binding of Plasma Homocysteine and Other Aminothiols in Patients With Early-Onset Peripheral Vascular Disease Homocysteine and Peripheral Vascular Disease

Elevated total homocysteine (Hcy) in plasma is an independent risk factor for early-onset vascular disease in the coronary, cerebral, and peripheral arteries. Different forms of Hcy, and their relation to other aminothiols in plasma, have not been investigated in patients with vascular disease. We therefore investigated 65 patients (35 men and 30 women) operated on for peripheral arterial disease at < 50 years of age and 65 age- and sex-matched control subjects. Total, reduced, oxidized, and protein-bound Hcy, cysteine (Cys), and cysteinylglycine (CysGly) were measured 0 to 11 years (mean, 6 years) postoperatively, in the fasting state, and after a standard methionine loading dose that caused a transient increase in reduced, oxidized, and protein-bound Hcy. All forms of Hcy and Cys, except reduced Cys, were higher in fasting patients than fasting control subjects. A similar difference between the groups was observed after methionine loading. The levels of most Hcy forms both during fasting and after methionine loading were related to smoking, but multivariate analysis showed that the difference between patients and control subjects could not be explained by smoking alone. Notably, reduced Cys and the reduced/total ratio for Cys were significantly higher in control subjects than in patients, both during fasting and after methionine loading. In both groups, the redox status and protein binding of the various aminothiols in plasma were interactive, as demonstrated by positive correlations between their reduced/total ratios and by a decrease in protein-bound Cys when protein-bound Hcy was elevated during methionine loading.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Microbiota‐dependent metabolite trimethylamine‐N‐oxide is associated with disease severity and survival of patients with chronic heart failure

Recent metabolomic, experimental and clinical studies have demonstrated that trimethylamine‐N‐oxide (TMAO), a microbiota‐dependent metabolite from dietary phosphatidylcholine and carnitine, is a strong predictor of coronary artery disease (CAD). This finding suggests a link between the gut microbiota and atherosclerosis. The potential impact of TMAO in chronic heart failure (HF) is unknown. We hypothesized that TMAO levels would provide prognostic information about adverse outcomes in chronic HF.

Decreased vitamin A levels in common variable immunodeficiency: vitamin A supplementation in vivo enhances immunoglobulin production and downregulates inflammatory responses

Vitamin A has a broad range of immunological effects, and vitamin A deficiency is associated with recurrent infections. Common variable immunodeficiency (CVI) is a group of B‐cell deficiency syndromes with impaired antibody production and recurrent bacterial infections as the major manifestations, but the immunological dysfunctions may also include T cells and macrophages. In the present study we examined the possible role of vitamin A deficiency in CVI.

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.

Disturbed Glutathione Metabolism and Decreased Antioxidant Levels in Human Immunodeficiency Virus-Infected Patients during Highly Active Antiretroviral Therapy—Potential Immunomodulatory Effects of Antioxidants

Oxidative stress has been implicated in the pathogenesis of human immunodeficiency virus (HIV) infection. We examined the effect of highly active antiretroviral therapy (HAART) on plasma levels of several antioxidants and intracellular glutathione-redox status in CD4+ T cells, in 20 HIV-infected patients. HAART was accompanied by both an improvement of glutathione-redox status and an increase in levels of antioxidant vitamins, without full normalization. Glutathione supplementation in vitro increases T cell proliferation and suppresses the spontaneous release of tumor necrosis factor-alpha from peripheral blood mononuclear cells, in HIV-infected patients receiving HAART. Our findings suggest that therapeutic intervention aimed at normalization of oxidative disturbances in HIV infection could be of interest, in addition to HAART.

Virological and immunological effects of antioxidant treatment in patients with HIV infection

Intracellular oxidative stress in CD4+ lymphocytes due to disturbed glutathione homeostasis may lead to impaired lymphocyte functions and enhanced HIV replication in patients with HIV infection, especially in those with advanced immunodeficiency. The aim of the present study was to assess whether short‐term, high‐dose antioxidant treatment might have effects on immunological and virological parameters in patients with HIV infection.

Ferrous fumarate deteriorated plasma antioxidant status in patients with Crohn disease.

Background: Iron deficiency anaemia is a frequent complication of Crohn disease. Treatment with ferrous iron (Fe[Formula: See Text]) compounds is often unsatisfactory and is associated with gastrointestinal side effects. Theoretically, oral iron supplementation may even be harmful, because iron may reinforce intestinal inflammation by catalysing production of reactive oxygen species. We investigated the effect of ferrous iron on disease activity and plasma antioxidant status in patients with active Crohn disease. Methods: Ten patients with Crohn disease and iron deficiency and 10 healthy controls were given ferrous fumarate 120 mg for 7 days. The Crohn Disease Activity Index, gastrointestinal complaints and blood samples for antioxidant status, anaemia, inflammation and iron absorption were investigated on day 1 and day 8. Results: During 1 week of ferrous fumarate supplementation, the Crohn Disease Activity Index tended to increase (P = 0.071). Patients experienced aggravation of diarrhoea, abdominal pain and nausea. Plasma-reduced cysteine was lower (P = 0.038) in patients than it was in controls. One week of ferrous iron supplementation further decreased reduced cysteine (P < 0.001) and significantly decreased plasma-reduced glutathione (P = 0.004) in the patients. Serum iron increased significantly in patients after an oral iron load test (from 5.8 ± 3.2 μmol/L to 30.9 ± 13.1 μmol/L). Conclusions: Treatment of iron deficiency with ferrous fumarate deteriorated plasma antioxidant status and increased specific clinical symptoms in patients with active Crohn disease. Plasma reduced cysteine may be a sensitive indicator for oxidative stress in the intestine.

Hyperhomocysteinemia in patients operated for lower extremity ischaemia below the age of 50—effect of smoking and extent of disease

Moderate hyperhomocysteinemia may be a risk factor for atherosclerotic peripheral vascular disease (PVD). In order to develop PVD at an early age risk factors are more strongly expressed and hyperhomocysteinemia may be one such factor. Homocysteine is derived from methionine and is metabolised by cystathionine-synthase to cystathionine or remethylated to methionine. Cystathionine-synthase activity is dependent on vitamin B6 while the remethylation of homocysteine is dependent on vitamin B12 and folate. The present study analyses homocysteine in patients operated on for lower extremity ischaemia before the age of 50. Homocysteine before and after loading with methionine, vitamin B6, B12 and folate were measured at follow-up. The patients were compared to age- and sex-matched controls. Significantly more patients than controls had hyperhomocysteinemia, 16/58 vs. 4/65, defined as fasting total homocysteine above 18.6 mumol/l. Loading with methionine did not further discriminate between patients and controls. Smoking patients had higher levels of homocysteine than non-smoking patients or smoking and non-smoking controls. Smoking patients also had lower levels of vitamin B6. When comparing patients with suprainguinal, infrainguinal and multilevel disease the highest homocysteine levels were seen in the latter group. Also, in this group smoking patients had higher homocysteine levels. Multivariate analysis revealed that homocysteine was associated with low levels of vitamin B12, folate and smoking. Smoking therefore seems to be connected to increased homocysteine levels in patients with early development of atherosclerosis, partly explained by decreased levels of B6, B12 and folate.