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.

Association of paraoxonase 55 and 192 gene polymorphisms on serum homocysteine concentrations in preeclampsia.

Paraoxonase 1 (PON1) is thought to influence serum homocysteine concentrations, at least in part, due to its homocysteine thiolactonase activity and to play a role in preeclampsia and atherosclerosis. We investigated the effects of PON 55 and PON 192 polymorphisms on plasma total homocysteine (tHcy) concentrations in preeclamptic and healthy pregnants among Turkish population (N = 106). PON 55 and 192 genotypes were determined by PCR RFLP techniques. Plasma tHcy concentrations were measured by high-performance liquid chromatography. No differences were observed in the distribution of PON 1 55/192 genotypes and allele frequencies between the preeclamptic and healthy pregnants. tHcy level in the plasma of preeclamptic women was found to be increased in comparison with healthy pregnants (P < 0.01). Preeclamptic women bearing the mutated PON 192 RR and wild-type PON1 55 LL genotypes had higher tHcy levels than those of the healthy pregnants with the corresponding genotypes, supporting the possibility that the hyperhomocysteinaemia seen in preeclamptic women is associated with the PON genotypes. However, no influence of the allelic distribution on plasma tHcy concentrations was detected in either group. Our results suggest that PON1 55 and 192 genotypes might have an important role in developing hyperhomocysteinaemia and may also have a role in the pathogenesis of preeclampsia in a Turkish population.

Unchanged asymmetric dimethylarginine levels in non-diabetic, premenopausal obese women who have common risk factors for cardiovascular disease.

This study was performed to test whether plasma asymmetric dimethylarginine (ADMA) concentrations are related to obesity and obesity complications including decrement in insulin sensitivity and adiponectin levels, dyslipidemia and low-grade inflammation. Asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) concentrations were analyzed by HPLC in 17 overweight (BMI > or = 25 kg/m2) and 40 obese (BMI > or = 30 kg/m2) premenopausal women. Age-matched healthy women were studied as controls. Obesity did not give rise to a significant change in circulating ADMA levels but reduced in SDMA levels. As compared with control subjects (0.441 +/- 0.102 microM), ADMA values in overweight and obese subjects were found to be as 0.412 +/- 0.102 and 0.436 +/- 0.093, respectively. No Pearson's association of ADMA with relevant risk variables for cardiovascular disease, including blood pressure, insulin sensitivity, inflammatory markers, lipid and adiponectin levels. However, in linear regression analysis, BMI, diastolic blood pressure, glucose, insulin, and IL-8 emerged as significant predictors of ADMA. In spite of obese women have elevated hs-CRP, triglyceride levels and decreased insulin sensitivity, adiponectin and HDL-cholesterol levels, all of which is closely linked risk factors for cardiovascular disease, circulating ADMA levels remained unchanged in obese individuals as compared with controls.

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.

Bone marker levels in gingival crevicular fluid during orthodontic intrusive tooth movement: a preliminary study.

The aim of the present study is to observe the changes in bone turnover markers, deoxypyridinoline (Dpd), osteocalcin, n-telopeptide (NTx), and bone alkaline phosphatase (balp) during the experimental orthodontic intrusion of maxillary premolar teeth. The study population required fixed appliance therapy involving the extraction of the maxillary first premolar teeth. Gingival crevicular fluid (GCF) samples were collected from each patient by using paper strips before the appliances were fitted and 1, 24, and 168 hours after the activation of appliances. After the second activation on the 21st, 22nd, and 28th days of the study, samples were collected. Enzyme-Linked Immunosorbent Assay (ELISA) tests were performed following manufacturer's recommendations. The results of the study indicate Dpd, osteocalcin, and balp values decrease with force application. Among the tested parameters only Dpd values showed statistically significant changes through time. One, 7, 22, and 28 day results show a significant amount of decrease when compared to 0 days. The extra decrease on the 22nd day (the day after the second activation) is also significantly lower. NTx crosslink values could not be detected in the experimental samples.

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.