Age and gender dependent alterations in the activities of glutathione related enzymes in healthy subjects.

OBJECTIVES: Oxidative stress as a result of increased free radical production is implicated in the pathogenesis of several diseases. Specific antioxidant enzymes have a crucial role in the prevention of these deleterious effects. Since the activities of these enzymes differ significantly in different populations and seem to be affected by various environmental factors, in this study we aimed to determine the reference values of glutathione related antioxidant enzyme activities in the erythrocytes of healthy subjects and to investigate the possible variations as a function of age and gender in a healthy Turkish Mediterranean population. DESIGN AND METHODS: 130 healthy subjects (12-90 yr, 82 females, 48 males) were divided into six different age groups. Erythrocyte glutathione peroxidase (GSH-PX), glutathione reductase (GR) and glutathione-s-transferase (GST) activities were measured on a Hitachi 704 autoanalyser by the modification of previously described manual UV spectrophotometric methods. RESULTS: No significant differences were observed in erythrocyte GSH-PX, GR and GST activities between different age groups. Overall, GST activities were significantly higher in females compared with males (8.08 +/- 1.39, 6.88 +/- 1.51 U/g Hb respectively, mean +/- SD, p < 0.001). A significant positive correlation between GSH-PX and GR activities was observed (r = 0.49, p < 0.001). CONCLUSION: The results of this study suggested that the activities of GSH-PX, GR and GST did not depend. GST activities overall were higher in females. The reference values that we obtained were different than the previous reports. This situation implies that each population should determine its own reference values and should investigate the influence of environmental factors and life style habits on the activities of these enzymes that constitute a major part of the antioxidant defense system in the human organism.

Association between homocysteine and neopterin in healthy subjects measured by a simple HPLC-fluorometric method.

OBJECTIVES: Neopterin and homocysteine promote vascular smooth muscle cell proliferation through the activation of nuclear factor(kappa) B. The aim of this study was to investigate the relation between these two compounds in healthy subjects by a rapid HPLC-fluorometric method which simplifies sample pretreatment for the measurement of neopterin in serum. DESIGN AND METHODS: In 40 healthy subjects (45.9 +/- 2.1 yr, mean +/- SEM, 10 males, 30 females) serum neopterin concentrations were measured by HPLC-fluorometry and enzyme-linked immunusorbant assay-ELISA and the results were compared. Urinary neopterin and plasma total homocysteine concentrations were assayed by HPLC-fluorometry. RESULTS: Serum neopterin concentrations measured by HPLC and ELISA were 7.5 +/- 0.4 and 7.4 +/- 0.3 nmol/L, respectively, r = 0.92, p < 0.01. Urinary neopterin level was 163.9 +/- 11.0 nmol/mmol creatinine and plasma total homocysteine 7.6 +/- 0.4 micromol/L. A significant positive correlation was observed between serum neopterin and plasma total homocysteine (r = 0.59, p < 0.01). CONCLUSIONS: A simple and rapid sample pretreatment for the measurement of neopterin in serum has been introduced. The significant positive correlation between neopterin and homocysteine implies that, interference with leukocyte function might be a new possible mechanism for the deleterious effects of homocysteine on vascular function.

Beta2-microglobulin and cystatin C in type 2 diabetes: assessment of diabetic nephropathy.

BACKGROUND: Changes in glomerular filtration rate (GFR) provide a valuable indicator of the progression of diabetic nephropathy (DN). This study was designed to demonstrate the clinical values of serum cystatin C (Cys C) and beta2-microglobulin in the assessment of renal function in type 2 diabetics by comparing them with the GFR, estimated from the uptake phase of 99 m technetium dimetiltriamino pentaacetic acid renogram (GFR-DTPA) and creatinine clearances. MATERIALS AND METHODS: 68 type 2 diabetic patients with (urinary albumin excretions (UAE) 30 - 300 mg/24 h) (n = 39) and without (UAE < 30 mg/24 h) (n = 29) microalbuminuria and 32 controls were enrolled in the study. Serum Cys C, beta2-microglobulin, creatinine, urinary microalbumin levels, creatinine clearances and GFR-DTPA values were determined in all groups. Non-parametric ROC curves, using a cut-off GFR-DTPA of 60 mL/min/1.73 m (2), were obtained for these markers. RESULTS: Serum Cys C, beta2-microglobulin, glucose and HbA1c concentrations were significantly higher in the group with diabetes compared to controls. In the patients with microalbuminuria, serum Cys C and glucose concentrations increased significantly in comparison to patients with normoalbuminuria, while no differences were observed for beta2-microglobulin levels. Serum creatinine concentrations, GFR-DTPA values and creatinine clearances were not different between both diabetic groups and controls. Cys C was positively correlated with beta2-microglobulin and creatinine and negatively with GFR values; beta2-microglobulin was also positively correlated with serum creatinine in microalbuminurics. A significant inverse correlation was found between beta2-microglobulin and GFR values in both microalbuminurics and normoalbuminurics. CONCLUSIONS: Increased Cys C and beta2-microglobulin in diabetics may be early indicators of incipient DN. The diagnostic accuracies of Cys C and beta2-microglobulin are superior to that of serum creatinine in distinguishing between mild and moderately reduced GFR.

Perfluorooctyl bromide dispersions in aqueous media for biomedical applications.

In studying perfluorooctyl bromide (PFOB) dispersions in aqueous media, we have used two types of surfactant: egg yolk phospholipids (EYP) and polyglycerol esters (PGE). Our interest in these dispersions arises from their potential biomedical applications as imaging solutions and oxygen-carrying solutions (i.e., blood substitutes). For EYP systems, we have identified the dispersion structure as consisting of (a) PFOB droplets (250-nm diameter) stabilized by a phospholipid monolayer adsorbed irreversibly at the o/w interface and (b) small empty phospholipid vesicles. With both surfactants commercial preparations yielded stable systems, while purified samples, being non-dispersible, could not be made to act as emulsifiers. In both cases, minor components in the commercial surfactant were found to be necessary for the formation of a stable dispersion, enabling the transport of the pure surfactant to the PFOB/water interface.

Concentrated emulsions of perflubron in aqueous media.

Flocculation of o/w emulsions consisting of a perfluorochemical (PFC) emulsified by either phospholipids or decaglyceroldioleate (10-2-O) was assessed both by direct observation and through photon correlation spectroscopy (PCS) and viscoelasticity measurements in unsteady oscillatory flow. Flocculation gives rise to emulsion instability but can be prevented (a) by the addition of a negatively charged surfactant to either phosphatidylcholine (PC) or 10-2-O, respectively the zwitterionic phospholipid and the nonionic surfactant used as primary emulsifiers, or (b) by using a saccharide solution as the continuous phase. The study indicates that both electrostatic (Coulombic) repulsive forces and hydration (steric) forces play a role in preventing flocculation. Various minor components of the egg yolk phospholipids (EYP) used in commercial emulsion preparation probably stabilize the emulsion by increasing both electrostatic and hydration repulsion.

Spuriously elevated inorganic phosphate level in a multiple myeloma patient.

We report the case of a patient with IgG multiple myeloma and pseudohyperphosphatemia. The patient had no clinical features of hyperphosphatemia. Subsequent investigations demonstrated that this hyperphosphatemia was spurious and was caused by a high concentration of the paraprotein. Deproteinization of the serum samples by sulfosalicylic acid resulted in normalization of the elevated phosphate values. This pseudohyperphosphatemia resulted from an increase in optic density because of interference between monoclonal immunoglobulin and the molybdic reagent used to determine phosphate in serum. These data indicate that the finding of marked hyperphosphatemia in multiple myeloma patients should always prompt an assay carried out on a deproteinized sample. In addition, knowledge of this phenomenon may avoid confusion, unnecessary testing and obviate confusion in the clinical evaluation of patients with multiple myeloma.

Plasma homocysteine levels in acute coronary syndromes.

Hyperhomocysteinemia is currently regarded as an independent and modifiable risk factor for ischemic vascular diseases and thrombosis. We measured fasting plasma total homocysteine levels by HPLC with fluorescence detection in 30 patients presenting with acute coronary syndromes and 30 age and sex-matched control subjects. Demographic data, classical risk factors (systolic blood pressure, diabetes mellitus, smoking, ethanol intake, family history of ischaemic heart disease) and life-style habits were recorded. Lipid fractions including total cholesterol, triglycerides, HDL-cholesterol, total cholesterol/HDL-cholesterol ratio, serum creatinine, LDL-cholesterol and vitamins involved in the metabolism of homocysteine, folic acid and vitamin B12 were also assessed. Total fasting homocysteine concentrations were significantly higher in the patient group (12.2 +/- 1.01 micromol/l) than in the control subjects (7.05 +/- 0.36 micromol/l; p < 0.0001). Homocysteine correlated positively with age (r = 0.617; p < 0.01) and serum creatinine (r = 0.457; p < 0.01) in the patient group. Hyperhomocysteinemia was not associated with vitamin B12 or folate deficiency states. Vitamin B12 concentration was 273 +/- 16.4 ng/l in the control group and 284.3 +/- 32.2 ng/l in the patient group (p = NS). Serum folate concentration also was not significantly different between controls and patients; 7.57 +/- 0.58 microg/l and 8.05 +/- 0.72 microg/l, respectively. Since no significant difference was observed in the lipid parameters between patients and controls, the hyperhomocysteinemia in the patient group supports the view that homocysteine is an independent risk factor for cardiovascular diseases. Our results strongly suggest that elevated homocysteine levels are among the interacting factors in the complex, multifactorial pathophysiology of ischemic heart disease.

Age-associated changes in nitric oxide metabolites nitrite and nitrate.

Aging is an important determinant of vascular disease. Endothelial dysfunction accompanying vascular disease may be related to cardiovascular risk factors such as aging, hypertension, and atherosclerosis. Experimental models suggest that endothelium-derived nitric oxide is reduced with aging, and this reduction is implicated in atherogenesis. The aim of this study was to determine whether increased age resulted in altered serum nitrite and nitrate levels, end-products of nitric oxide, in healthy subjects. Sixty-nine healthy individuals were divided into five different age groups: group I (6-15 years), group II (16-30 years), group III (31-45 years), group IV (46-60 years), and group V (>61 years). In these subjects, serum nitrite was measured by the Griess reaction and nitrate by the nitrate reductase method. Statistical analysis showed that serum nitrite levels were not significantly different in any of the groups, while serum nitrate concentrations exhibited significant differences (P<0.001). These findings suggest that nitric oxide synthesis and/or secretion is reduced with age and consequently endothelium-dependent vasodilation is impaired.

Urinary hypoxanthine and xanthine levels in acute coronary syndromes.

Ischemia leads to impaired ATP metabolism, with increased production of purine degradation products, such as hypoxanthine and xanthine, which are useful markers of tissue hypoxia. These extracellular markers of ischemia have been studied extensively in many clinical conditions of oxidative stress, including perinatal asphyxia, acute respiratory distress syndrome, cerebral ischemia, and preeclampsia. The aim of this study was to explore the usefulness of urinary hypoxanthine and xanthine as ischemia markers in acute coronary syndromes. Urinary excretion of hypoxanthine and xanthine was assessed by high-performance liquid chromatography in 30 patients with acute coronary syndromes and in 30 age- and sex-matched controls. Serum and urine uric acid, creatinine, and urea concentrations were also determined. Hypoxanthine excretion was significantly elevated in patients compared with healthy controls (84.37+/-8.63 and 42.70+/-3.97 nmol/mg creatinine, mean+/-SEM, P<0.0001). Urinary xanthine levels were also increased in patients with acute coronary syndromes (100.13+/-12.14 and 34.74+/-4.07 nmol/mg creatinine patients and controls, respectively; P<0.0001). Hypoxanthine and xanthine excretion showed a strong positive correlation in both groups. Significant negative correlations between urinary hypoxanthine and uric acid and xanthine and uric acid were observed in the patients, but not in controls. In conclusion, increased levels of ATP degradation products hypoxanthine and xanthine are observed in various hypoxic clinical conditions. This study suggests that these parameters may be useful markers of ischemia in patients with acute coronary syndromes.

Effects of defibrotide on aorta and brain malondialdehyde and antioxidants in cholesterol-induced atherosclerotic rabbits.

The effects of a high-cholesterol diet in the presence and absence of defibrotide, a single-stranded polydeoxyribonucleotide compound, on the lipid peroxidation product malondialdehyde, endogenous antioxidant enzymes catalase, glutathione peroxidase, and the antioxidant thiol compound GSH were investigated. Forty male New Zeland white rabbits were divided into four groups each consisting of 10 rabbits. Group I received a regular rabbit chow diet and group II 1% cholesterol plus regular chow, group III was given defibrotide (60 mg/kg per day p.o. in water) and was fed with regular chow, and group IV received defibrotide plus 1% cholesterol for 9 weeks. Blood cholesterol and malondialdehyde, catalase, glutathione peroxidase, and GSH were determined before starting the experimental diet regimen (basal). After 9 weeks, the same parameters were determined in blood, aorta, and brain tissues (end -experiment). Aortic tissue was examined under a light microscope for morphological alterations indicative of atherosclerosis. The increase in serum total cholesterol was greater in group II than group IV. Plasma malondialdehyde in group II was higher than in group III. Brain malondialdehyde in group II was higher than all other groups, and aortic malondialdehyde in this group was higher than group I and III. Serum catalase activity decreased in group II and increased in group III, compared with basal values. Brain catalase activity in group I was higher than group II, and aorta catalase in group IV was higher than in group I and III. Blood glutathione peroxidase activity in group III and IV was higher than basal. GSH concentrations decreased significantly in the cholesterol-fed groups (group II and IV). Histological alterations in the cholesterol-fed groups were more pronounced in group II. The increased levels of malondialdehyde in plasma, aorta, and brain tissue of group II suggest a role of oxygen free radicals in the pathogenesis of cholesterol-induced atherosclerosis. The higher malondialdehyde values in the brain tissues of animals in group II compared with group IV suggest a protective role of defibrotide in the brain against lipid peroxidation in the oxidant stress of cholesterol-induced atherosclerosis. Increased catalase activities in the blood and aortic tissues and increased glutathione peroxidase activities in the blood of rabbits receiving defibrotide suggest an induction of these antioxidant enzyme activities by defibrotide. These results imply that anti-atherosclerotic, anti-ischemic effects of this drug may be due to the beneficial effects on the oxidant-antioxidant balance of various tissues.