The effect of nephropathy on plasma sphingosine 1-phosphate concentrations in patients with type 2 diabetes.

OBJECTIVES: Sphingosine 1-phosphate (S1P) is carried in plasma by the HDL particles and albumin. It mediates several protective functions of HDL. Because of its barrier-enhancing effect, it has attracted attention in diseases associated with endothelial dysfunction. We examined the impact of circulating levels of S1P in diabetic nephropathy together with apoprotein M, a S1P-binding protein in HDL. Plasma levels of dimethylarginines were evaluated in this context. DESIGN AND METHODS: Patients with type 2 diabetes mellitus were divided into three groups according to daily albumin excretion: normoalbuminuria, microalbuminuria and macroalbuminuria (n=30 in each). In addition to routine analysis, S1P and apo M in plasma were measured using the enzyme-linked immunosorbent assays. Asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA) and l-arginine were determined by HPLC. Tukey's or Mann-Whitney U-test was used for the statistics. RESULTS: Plasma S1P levels showed a significant decline in parallel to kidney dysfunction. The highest significance was detected in the macroalbuminuric group. Although a significant increase in plasma SDMA in albuminuric groups was observed, apo M, l-arginine and ADMA levels were similar between the groups. CONCLUSION: Low plasma levels of S1P seemed to be associated with diabetic nephropathy. The main reason for the decreased S1P levels in our patients seems to be severe urinary albumin loss due to nephropathy. Low levels of S1P in patients with nephropathy may adversely affect the endothelial integrity and barrier function, thus causing a vicious circle.

Hyperhomocysteinemia in chronic renal failure patients: relation to tissue factor and platelet aggregation.

BACKGROUND: A moderate increase in plasma total homocysteine (t-hcy) is considered to be an independent risk factor for cardiovascular disease (CVD) in general population. One of the mechanisms by which hyperhomocysteinemia contributes to cardiovascular risk has been explained to be the increased thrombotic potential. Elevated t-hcy levels were also reported in chronic renal failure patients because the renal function is a major determinant of serum t-hcy levels. PATIENTS AND METHODS: We measured serum hcy and ADP-induced platelet aggregation and plasma tissue factor as a major activator of the coagulation cascade in hemodialysis (HD), peritoneal dialysis (PD) and early stage chronic renal failure (early stage CRF) patients who are not receiving dialysis and compared with those of control. In addition, we also determined serum vitamin B12 and folat levels which are the important factors regulating the metabolism of t-hcy. RESULTS: Hcy levels in all patient groups were significantly higher (HD: 20.42 +/- 1.91 micromol/l, PD: 35.47 +/- 6.30, early stage CRF: 24.39 +/- 3.06) than the normal levels (10.74 +/- 0.74) in spite of standard multivitamin supplementation. The highest t-hcy values were found in peritoneal dialysis patients. Vitamin B12 levels in hemodialysis/peritoneal dialysis patients and folat levels in hemodialysis/early stage CRF patients were also significantly above those of control. On the other hand, the significant elevations in plasma tissue factor concentration were found in all patient groups (HD: 331.4 +/- 31.3 pg/ml, PD: 306.0 +/- 30.0, early stage CRF: 277.2 +/- 25.5 and CONTROL: 69.5 +/- 13.5). t-hcy levels were positively correlated with creatinine (r: 0.791 p < 0.002) and tissue factor levels (r: 0.526 p < 0.05) in only early stage CRF group. The association between t-hcy and tissue factor persisted after these two parameters were adjusted for creatinine (r: 0.649 p < 0.05). On the other hand the same correlations were not observed in dialysis patient groups. In spite of the high tissue factor levels, ADP-induced platelet aggregations were found to be lower in all patient groups (HD: 102.6 +/- 6.7, PD: 98.6 +/- 7.6 and Early stage CRF: 84.9 +/- 7.6) than controls (154.9 +/- 13.7). CONCLUSION: These results suggest that hyperhomocysteinemia and increased tissue factor level are present in patients with renal failure, despite supplementation with vitamin B6 and B12 and folat. However, elevated levels of these thrombogenic factors are not linked with platelet aggregation.

Insulin resistance is not related to plasma homocysteine concentration in healthy premenapausal women.

This study was performed to test whether plasma homocysteine concentrations are related to insulin resistance in healthy premenopausal women. For this purpose, the relationship between insulin resistance (as assessed by HOMA index) and fasting plasma homocysteine level was determined in 83 healthy volunteers. The results indicated that homocysteine concentrations did not vary as a function of HOMA index (r = -0.147). Plasma homocysteine concentrations also did not vary as a function of other parameters of insulin resistance such as HDL-cholesterol and triglycerides, which they correlated inversely with body mass index (BMI). Furthermore, when individuals were classified according to quartiles of insulin resistance (HOMA index), plasma homocysteine concentrations from the lowest to the highest quartiles were not significantly different. On the other hand, the HOMA index correlated significantly with triglyceride concentrations (r = 0.377, p< 0.001), HDL-cholesterol (r = -0.310, p< 0.01) and BMI (r = 0.468, p< 0.001). These results suggest that plasma homocysteine concentrations are not related to insulin resistance and/or metabolic abnormalities associated with it in premenopausal women.

Evaluation of nitrosative and oxidative stress in Behçet disease.

Plasma nitrotyrosine and nitrite/nitrate levels as markers of nitrosative stress and plasma malondialdehyde (MDA) and protein carbonyl as markers of oxidative stress were determined in patients with Behcet disease (BD). To evaluate the balance between oxidant and antioxidant systems in these patients, we measured erythrocyte lysate CuZn superoxide dismutase (CuZn SOD) activity, plasma sulfhydryl (SH) values and total antioxidant activity. We also determined levels of plasma C-reactive protein (CRP), a key marker of inflammation, and compared them with those of healthy subjects. We found plasma nitrotyrosine levels of BD patients to be increased, indicating that nitrosative stress may occur in these patients. Plasma MDA and CRP levels in BD patients were found to be significantly higher than those in control group. However, plasma SH levels were decreased. No changes were observed in the other measured parameters of the patient group compared with the controls. These data suggest the possible involvement of nitric oxide (NO) together with reactive oxygen substances (ROS) in the pathogenesis of BD.

The effect of aminoguanidine on diabetes-induced inactivation of kidney Na(+),K(+)- ATPase in rats.

We studied the effect of aminoguanidine (AG), an inhibitor of advanced glycation product formation, on diabetes-induced oxidative damage. Renal cortex Na(+),K(+)- ATPase was chosen for study as a potential cellular target of oxygen radicals. In this study, the enzyme activity was reduced while malondialdehyde (MDA) and carbonyl levels were enhanced but sulphydryl (SH) level remained unchanged in the renal cortex in diabetic animals. Treatment of diabetic rats with AG had no significant effect on diabetes-induced impairments of enzyme activity and MDA but the carbonyl level readjusted to control level in the kidney. These results show that AG treatment at that dose did not exhibit profound antioxidant properties even if carbonyl stress was ameliorated by this treatment.

Testis glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase activities in aminoguanidine-treated diabetic rats.

Severe steroidogenic and spermatogenic alterations are reported in association with diabetic manifestations in humans and experimental animals. This study was planned to determine whether oxidative stress is involved in diabetes-induced alterations in the testes. Diabetes was induced in male rats by injection of 50 mg/kg of streptozotocin (STZ). Ten weeks after injection of STZ, levels of selenium and activities of selenium dependent-glutathione peroxidase (GPx) and phospholipid hydroperoxide glutathione peroxidase (PHGPx) were measured in rat testis. Lipid and protein oxidations were evaluated as measurements of testis malondialdehyde (MDA) and protein carbonyl levels, respectively. Testis sulfydryl (SH) levels were also determined. The control levels of GPx and PHGPx activities were found to be 46.5 +/- 6.2 and 108.8 +/- 19.8 nmol GSH/mg protein/min, respectively. Diabetes caused an increase in testis GPx (65.0 +/- 21.1) and PHGPx (155.9 +/- 43.1) activities but did not affect the levels of selenium or SH. However, the testis MDA and protein carbonyl levels as markers of lipid and protein oxidation, respectively, did not increase in the diabetic group. Aminoguanidine (AG) treatment of diabetic rats returned the testis PHGPx activity (136.5 +/- 24.9) to the control level but did not change the value of GPx activity (69.2 +/- 17.4) compared with diabetic group. MDA and protein carbonyl levels in testis were not affected by AG treatment of diabetic rats, but interestingly AG caused SH levels to increase. The results indicate that reactive oxygen radicals were not involved in possible testicular complications of diabetes because diabetes-induced activations of GPx and PHGPx provided protection against oxidative stress, which was reported to be related to some diabetic complications.

The effect of aminoguanidine on urinary heparan sulphate levels in experimental diabetes.

In the present study, we investigated whether the ameliorating effect of aminoguanidine on diabetes-related proteinuria and nephropathy is associated with glomerular basement membrane heparan sulphate contents. STZ-induced diabetic rats developed proteinuria (at the tenth week: diabetic rats, 713 +/- 418 mg protein per millimole creatinine; control rats, <30) and increased urinary heparan sulphate excretion (diabetic rats, 1,400 +/- 83 microg/mmol creatinine; control rats, 41 +/- 13; p < 0.001), suggesting loss of glomerular basement membrane charge. Aminoguanidine treatment of diabetic rats diminished urinary heparan sulphate levels (196 +/- 52), suggesting high incorporation of heparan sulphate-associated charge into glomerular basement membrane. Aminoguanidine administration to diabetic rats also relatively improved proteinuria (456 +/- 255). It is concluded that aminoguanidine treatment has a relative beneficial effect by restoring the diabetes-induced change in renal basement membrane heparan sulphate levels.

Impaired relaxation in aorta from streptozotocin-diabetic rats: effect of aminoguanidine (AMNG) treatment.

AIM: The effect of 8 weeks' streptozotocin (STZ)-induced diabetes and aminoguanidine (AMNG), the inhibitor of advanced glycosylation reaction, treatment on arteriolar reactivity to vasoactive substances was investigated in vitro. MATERIALS AND METHODS: Studies were performed in untreated control rats (n=10), STZ-induced (60 mg/kg i.v.) diabetic rats (n=10), AMNG-treated (600mg/l given in drinking water throughout 8 weeks) control rats (n=10) and AMNG-treated (600mg/l given in drinking water, beginning at 72h after STZ and throughout 8 weeks of diabetes) diabetic rats (n=10). Results are expressed as the mean +/-s.e. Relaxant responses are expressed as a percentage (%) relaxation of noradrenaline-induced tone. Statistical comparisons were made by one-way analysis of variance (ANOVA) followed by Tukey-Kramer multiple comparisons test. RESULTS: 1. The decreased body weights (205 +/- 6g) and increased blood glucose levels (583 +/- 8 mg/dl) of diabetic rats were partially restored by treatment of aminoguanidine (253 +/- 6 g, p < 0.05 and 480 +/- 14 mg/ dl, p < 0.001, respectively). 2. Diabetes caused a 71% deficit in maximal endothelium-dependent relaxation to acetylcholine for noradrenaline precontracted aortas (p<0.001). AMNG treatment prevented the diabetes-induced impairment in endothelium dependent relaxation (58 +/- 8%) to acetylcholine, maximum relaxation remaining in the non-diabetic range (78 +/- 4%). 3. Neither diabetes nor treatment affected endothelium-independent relaxation (pD2 and max. Relax.) to sodium nitroprusside. 4. Vasoconstrictor responses (pD2 and Max. Contraction) to noradrenaline and KCl were not influenced by the diabetic state and treatment. CONCLUSION: Our data suggest that 8 weeks of experimental diabetes is associated with a decreased endothelium-dependent vasodilatation. AMNG treatment may prevent diabetes-induced endothelial dysfunction. This may be mediated via the prevention of advanced glycosylation end product formation, the enhanced release of vasodilator substances such as prostacyclin, the increased elasticity of blood vessels, the antioxidant activity and inhibitor activity of enzyme aldose-reductase by AMNG.

Ebselen as protection against ethanol-induced toxicity in rat stomach.

The mucosal protective effect of ebselen was examined in an ethanol-induced rat gastric lesion model. Examination of gastric tissue samples by light microscopy showed that i.g. exposure to 50% ethanol induced gastric injury, which was more prominent in female rats. Ethanol did not effect the gastric acid secretion examined by means of H(+)-K+ATPase, the increment of which might be harmful in the stomach. But ebselen with or without ethanol kept H(+)-K+ATPase below control levels. Gastric alcohol dehydrogenase (ADH) was mainly responsible for oxidation of ethanol in the stomach before it enters the bloodstream. I.g. ethanol exposure inhibited the ADH activity but ebselen eliminated the ethanol-induced inhibition of this enzyme. Therefore, ebselen exhibited a beneficial effect by increasing the gastric ethanol metabolism and by ameliorating the possible tissue toxicity of ethanol. Consistently, we also found that ebselen diminished the blood ethanol level. A gender difference in the blood ethanol levels existed following the same dose of ethanol but there was no difference in ADH activity. Histologically, mucosal injury following ebselen exposure together with ethanol was less severe compared with ethanol treatment alone. We concluded that the decrease in ethanol-induced mucosal injury following ebselen may have contributed to the inhibition of H(+)-K+ATPase and the activation of ADH by ebselen.

Nitric oxide-mediated regulation of gastric H+, K+ -ATPase and alcohol dehydrogenase following ethanol-induced injury in rats.

The mucosal protective effect of nitric oxide (NO) was examined by using N(G)-nitro-L-arginine methyl ester (L-NAME) as nitric oxide synthase (NOS) inhibitor and nitroprusside (NP) as NO donating agent, in ethanol-induced rat gastric lesion model. The results are summarized as follows: (1) As gastric tissue samples were examined by light microscopy, intragastric exposure of ethanol was demonstrated to induce gastric injury, which was more prominent in female rats. The depletion of NO by L-NAME treatment exacerbated the ethanol-induced gastric lesion but NP together with ethanol promoted repair of the mucosal injury, especially in female rats. (2) Gastric H+, K+ -ATPase enzyme activity, which was responsible for acid secretion, seemed not to be effected by ethanol treatment. Together with ethanol, L-NAME treatment activated, whereas NP treatment inhibited, the enzyme activity in female rats. (3) Ethanol treatment inhibited gastric alcohol dehydrogenase (ADH) activity, which was responsible for the first-pass metabolism of ethanol. Together with ethanol, L-NAME did not effect the enzyme activity whereas NP treatment disappeared the inhibitory effect of ethanol in both gender. Hydroxyl radical (OH*) scavenger activity was found to increase in ethanol and ethanol + NP groups in both sexes, but superoxide radical (O2-*) scavenger activity did not change. The results indicate that NO may ameliorate the damaging effect of ethanol possibly by regulating acid secretion, ethanol metabolism, and antioxidant content in rat gastric mucosa.

Influence of selenium supplementation in non-toxic doses on testis lipid peroxide and antioxidant levels in chronic alcohol-fed rats.

Prolonged consumption of alcohol leads to peroxidative damage in testicular tissues and gonadal dysfunction. Selenium (Se) deficiency also gives rise to testicular structural and functional disturbances similar to those caused by alcohol. We examined the possibility that Se supplementation might, at least partially, prevent the testicular disorders induced by alcohol. Rats were fed alcohol and alcohol with 3 p.p.m. Se in drinking water for 5 months. Ethanol administration decreased vitamin C and glutathione levels in testicular tissue, but caused no alterations in vitamin E and polyunsaturated fatty acid levels. However, lipid peroxide levels were increased by alcohol. Selenium supplementation diminished both the depletion of vitamin C and the production of lipid peroxides, but did not affect the depletion of glutathione induced by alcohol in testicular tissue. In addition, Se supplementation ameliorated the decrement of serum testosterone levels induced by alcohol.

Selenium-induced activation of brain Na(+)-K(+)-ATPase in rats.

The influence of selenium supplementation on the activity of Na(+)-K(+)-ATPase and on the degree of free radical generation was studied in brain tissue of rats. Selenium was administered for 5 months in drinking water, and was measured in plasma by the fluorometric method at the end of the experimental period. Animals were sacrificed and brain tissue homogenates were used for enzyme assay and for assessment of lipid peroxide formation. Brain tissue from rats who received selenium showed significantly increased Na(+)-K(+)- ATPase activity but also significantly decreased lipid peroxide farmation. Since this enzyme is known to be inhibited by oxygen free radicals, selenium supplementation appears to exert a beneficial effect on the Na(+)-K(+)-ATPase activity by preventing free radical-induced damage.

Relationship of some endogenous sex steroid hormones to leukocyte arylsulphatase A activities in pre- and postmenopausal healthy women.

This study was planned to investigate whether the physiological variations in endogenous gonadal steroid hormones during fertile period to menopause might affect the Arylsulphatase A (ARS-A) activities in leukocytes in healthy women. In premenopausal women, means of the leukocyte ARS-A activity were significantly highest at ovulatory phase, and lowest at early follicular phase. In contrast, the mean leukocyte ARS-A activity in postmenopausal women was approximately equivalent to the mean leukocyte enzyme in cycling women at early follicular phase in whom the lowest enzymatic activity was observed. The results of our study, therefore, concluded that gonadal steroid released physiologically into the bloodstream might be expected to stimulate the lysosomal system thereby leading to an enhancement in leukocyte ARS-A activity.

Comparative effects of chronic administration of some psychotropic drugs on rat brain cortex acetylcholinesterase activity.

The response of central cholinergic neurotransmission to the chronic administration of some psychotropic drugs to rats was investigated using brain acetylcholinesterase activity as a neurochemical marker for cholinergic neurons. Rats were divided into four groups. Three experimental groups were given chlorpromazine, amitriptyline, or diazepam respectively, for a period of 30 days; and the control group received physiological saline only. Long-term treatment of chlorpromazine and amitriptyline resulted in significant increases in rat brain cortex enzyme activity, whereas only a slight increase was observed in the diazepam-treated group. These results indicate that the chronic treatment with some psychotropic drugs causes changes in central cholinergic transmission.

Alterations in some lipid components and Ca2+ ATPase activity in brain of rats fed an atherogenic diet.

Male Wistar rats were fed an atherogenic diet for four months to investigate possible diet-induced lipid alterations and brain Ca2+ ATPase activity. Total cholesterol and triglyceride levels were found to be increased significantly in both serum and brain while the phospholipid level was decreased in both. The distribution of serum cholesterol between high-density and low-density lipoproteins was altered when compared to control rats with a decrement in HDL-cholesterol and a pronounced increment in LDL-cholesterol. The atherogenic diet resulted in about 50% depression in brain Ca2+ ATPase activity. It is concluded that alterations in ion transport and neurotransmitter release may be expected due to pronounced inhibition of brain Ca2+ ATPase activity in rats fed an atherogenic diet.

Influence of eicosapentaenoic acid and vitamin E on brain cortex Ca2+ ATPase activity in cholesterol-fed rabbits.

The influence of eicosapentaenoic acid (EPA) and vitamin E on brain cortex Ca2+ ATPase activity was examined in rabbits receiving cholesterol-rich diets for a period of 45 days. Rabbits were divided as control (A) and cholesterol-fed groups (B, C, and D). Group C received 80 mg of EPA and group D received 100 IU of vitamin E every day in addition to the cholesterol-rich (2%, w/w) diet which was solely given to Group B. Rabbits receiving cholesterol alone had a significant reduction in brain microsomal phospholipid level. Microsomal free cholesterol and polyunsaturated fatty acids (PUFA) were significantly increased in all experimental groups. Cortex microsomal Ca2+ ATPase activity was found to be inhibited in all cholesterol-fed rabbits as compared to controls, but the highest inhibition was seen in rabbits fed cholesterol alone. Additions of EPA or Vitamin E to the cholesterol-rich diets resulted in a recovery of the enzymatic activity. It is concluded that cholesterol feeding without any addition of PUFA or antioxidant agent might cause an inhibition of brain Ca2+ ATPase activity in rabbits, thereby leading to the dysfunction in ion transport and neurotransmitter release.