Similarities in serum acylcarnitine patterns in type 1 and type 2 diabetes mellitus and in metabolic syndrome.

BACKGROUND/AIMS: In type 1 diabetes (T1D), type 2 diabetes (T2D) and metabolic syndrome (MetS), the associated complex metabolomic changes in the involvement of carnitine metabolism in total carnitine ester level has already been documented; here we extended the investigations to the individual acylcarnitines. METHODS: The fasting serum acylcarnitine concentrations were determined in 49 T1D, 38 T2D and 38 MetS patients and 40 controls by isotope dilution electrospray ionization tandem mass spectrometry. RESULTS: The acylcarnitine profiles of the 3patient groups shared elements with the controls. Considerably higher levels of almost all short-chain acylcarnitines (p < 0.05) and lower levels of some long-chain acylcarnitines were detected in T2D and MetS patients. The amounts of C3 and C4 carnitine were higher and most of the medium-chain and long-chain acylcarnitine levels were lower (p < 0.05) in T1D and MetS patients than in the controls. In T1D and T2D, the levels of C3 and C4 acylcarnitines were markedly elevated and some long-chain acylcarnitines were lower than the controls (p < 0.05). Moreover, significantly lower concentrations of free- and total carnitine were observed in T1D patients (p < 0.05). CONCLUSIONS: Profound alterations were detected in acylcarnitine profiles in the 3 patient groups. Similarities in the patterns suggest different degrees of involvement of the same metabolic systems in a systems biology approach.

Common functional variants of APOA5 and GCKR accumulate gradually in association with triglyceride increase in metabolic syndrome patients.

The common functional variants of the apolipoprotein A5 (APOA5) and the glucokinase regulatory protein genes (GCKR) have been shown to associate with increased fasting triglyceride (TG) levels. Albeit the basic association has been extensively investigated in several populations of different origin, less is known about quantitative traits of them. In our study accumulation rates of four APOA5 (T-1131, IVS3 + G476A, T1259C and C56G) and two GCKR (C1337T and rs780094) functional SNPs were analyzed in patients stratified into four TG quartile groups. Randomly selected 325 metabolic syndrome patients were separated into four quartile (q) groups based on the TG levels as follows q1: TG <1.38 mmol/l; q2: 1.38-1.93 mmol/l; q3: 1.94-2.83 mmol/l; and q4: TG >2.83 mmol/l. We observed significant stepwise increase of prevalence rates of minor allele frequencies in the four plasma TG quartiles for three APOA5 SNPs: -1131C (q1: 4.94%; q2: 8.64%; q3: 11.6%; q4: 12.3%), IVS3 + 476A (q1: 4.32%; q2: 7.4%; q3: 10.36%; q4: 11.1%), and 1259C (q1: 4.94%; q2: 7.41%; q3: 10.4%; q4: 11.7%). The haplotype analysis revealed, that the frequency of APOA5*2 haplotype gradually increased in q2, q3 and q4 (q1: 9.87%; q2: 14.8%; q3: 18.3%; q4: 21%). The distribution of the homozygotes of the two analyzed GCKR variants resembled to the APOA5 pattern. Contrary to the hypothetically predictable linear association coming from the current knowledge about the APOA5 and GCKR functions, the findings presented here revealed a unique, TG raise dependent gradual accumulation of the functional variants of in MS patients. Thus, the findings of the current study serve indirect evidence for the existence of rare APOA5 and GCKR haplotypes in metabolic syndrome patients with higher TG levels, which contribute to the complex lipid metabolism alteration in this disease.

Resveratrol improves insulin sensitivity, reduces oxidative stress and activates the Akt pathway in type 2 diabetic patients.

Although resveratrol has widely been studied for its potential health benefits, little is known about its metabolic effects in humans. Our aims were to determine whether the polyphenol resveratrol improves insulin sensitivity in type 2 diabetic patients and to gain some insight into the mechanism of its action. After an initial general examination (including blood chemistry), nineteen patients enrolled in the 4-week-long double-blind study were randomly assigned into two groups: a resveratrol group receiving oral 2 × 5 mg resveratrol and a control group receiving placebo. Before and after the second and fourth weeks of the trial, insulin resistance/sensitivity, creatinine-normalised ortho-tyrosine level in urine samples (as a measure of oxidative stress), incretin levels and phosphorylated protein kinase B (pAkt):protein kinase B (Akt) ratio in platelets were assessed and statistically analysed. After the fourth week, resveratrol significantly decreased insulin resistance (homeostasis model of assessment for insulin resistance) and urinary ortho-tyrosine excretion, while it increased the pAkt:Akt ratio in platelets. On the other hand, it had no effect on parameters that relate to ß-cell function (i.e. homeostasis model of assessment of ß-cell function). The present study shows for the first time that resveratrol improves insulin sensitivity in humans, which might be due to a resveratrol-induced decrease in oxidative stress that leads to a more efficient insulin signalling via the Akt pathway.

GCKR gene functional variants in type 2 diabetes and metabolic syndrome: do the rare variants associate with increased carotid intima-media thickness?

BACKGROUND: Recent studies revealed that glucokinase regulatory protein (GCKR) variants (rs780094 and rs1260326) are associated with serum triglycerides and plasma glucose levels. Here we analyzed primarily the association of these two variants with the lipid profile and plasma glucose levels in Hungarian subjects with type 2 diabetes mellitus and metabolic syndrome; and also correlated the genotypes with the carotid intima-media thickness records. METHODS: A total of 321 type 2 diabetic patients, 455 metabolic syndrome patients, and 172 healthy controls were genotyped by PCR-RFLP. RESULTS: Both GCKR variants were found to associate with serum triglycerides and with fasting plasma glucose. However, significant association with the development of type 2 diabetes mellitus and metabolic syndrome could not be observed. Analyzing the records of the patients, a positive association of prevalence the GCKR homozygous functional variants and carotid intima-media thickness was found in the metabolic syndrome patients. CONCLUSIONS: Our results support that rs780094 and rs1260326 functional variants of the GCKR gene are inversely associated with serum triglycerides and fasting plasma glucose levels, as it was already reported for diabetic and metabolic syndrome patients in some other populations. Besides this positive replication, as a novel feature, our preliminary findings also suggest a cardiovascular risk role of the GCKR minor allele carriage based on the carotid intima-media thickness association.

Storage at -80 degrees C decreases the concentration of HPLC-detected urinary albumin: possible mechanisms and implications.

BACKGROUND: Urinary albumin is now measured by high-performance liquid chromatography (HPLC) which also detects albumin missed by traditional immunochemical methods. A predictive effect of HPLC-detected albuminuria on mortality has just been reported in the AusDiab study, measuring albuminuria with HPLC after 7 years of -80 degrees C storage. However, there are already some data suggesting that HPLC-detected albuminuria is affected by -80 degrees C storage. We aimed to measure changes in HPLC-detected albuminuria after 2.5 years and find the factors which may be responsible for this alteration. METHODS: Urinary albumin was measured by the US Food and Drug Administration approved HPLC Accumin kit. Total free sulfhydryl groups (TFSG) of urine samples were measured by Ellman's reagent. RESULTS: We found a significant 24% average decrease in HPLC-detected albuminuria and a correlation between the magnitude of decrease and urinary pH. We found a correlation between changes of urinary albumin dimeric to monomeric ratio of stored urine and pH; however, only changes of monomeric form were found to be significant. A correlation was also found between the TFSG of fresh urine samples and pH. Less TFSG could be detected, and a correlation between TFSG and pH was absent in stored urine. CONCLUSIONS: We conclude that measurement of albuminuria by HPLC in long-term -80 degrees stored urine gives unreliable results. Decrease of HPLC-detected albuminuria is pH-dependent and may be due to the reducing capacity of urine. Prospective studies need to decide whether the predictive properties of HPLC-detected albuminuria decrease during longterm storage.

Serum total LDH activity and LDH-2 isozyme in nephrotic syndrome.

BACKGROUND/AIMS: Proteinuria, hypoproteinaemia, hypoalbuminaemia and oedema are major characteristics of nephrotic syndrome. Aims of this study were to detect serum total LDH activity and its isozymes in nephrotic syndrome. METHODS: In a cross-sectional study, clinical parameters were compared in three cohorts, namely kidney patients with or without nephrotic syndrome and hypoalbuminaemic controls (NEPHR, NON-NEPHR, CONTR, respectively). RESULTS: Serum total LDH activity in the NEPHR group was increased compared with the NON-NEPHR and CONTR groups (p < 0.001) and correlated with serum total protein (r = -0.549, p < 0.001), serum albumin (r = -0.596, p < 0.001), proteinuria (r = 0.456, p < 0.001) and serum total cholesterol (r = 0.523, p < 0.001). LDH isozyme pattern was analysed in three subgroups of the patients. Serum LDH-2 activity was higher in the NEPHR subgroup compared with the NON-NEPHR and CONTR subgroups (p < 0.001). Serum LDH-2 activity correlated with serum total protein (r = -0.665, p < 0.001), serum albumin (r = -0.615, p < 0.001), proteinuria (r = 0.694, p < 0.001), and serum total cholesterol (r = 0.723, p < 0.001). Linear regression analysis revealed that serum total protein proved to be an independent predictor of serum total LDH activity, while serum total protein and proteinuria were predictors of LDH-2. CONCLUSIONS: These findings suggest that serum total LDH activity might be a marker of the activity of the nephrotic syndrome.

[Analysis of microalbuminuria with immunonephelometry and high performance liquid chromatography. Evaluation of new criteria]. / Immunnefelometria és nagy teljesítményu folyadékkromatográfia a microalbuminuria vizsgálatában. Ujonnan javasolt határértékek vizsgálata.

INTRODUCTION: Hypertension as well as type 2 diabetes mellitus is a major factor in population mortality. Both diseases damage the endothelium, the early sign of which is microalbuminuria, which can be screened by dipstick and can be diagnosed by using immuno-based and high performance liquid chromatography methods. Using high performance liquid chromatography, the non-immunoreactive albumin can be detected as well. AIMS: The authors aimed at the examination of albuminuria in the case of immunonephelometrically negative patients with high performance liquid chromatography, in diabetic and hypertensive and non-diabetic hypertensive populations. The authors also wanted to compare the present (albumin-creatinine ratio: male: > or =2.5 mg/mmol, female: > or =3.5 mg/mmol) and a new criteria of the Heart Outcomes Prevention Evaluation study (patients without diabetes: immunological method, > or =0.7 mg/mmol; high performance liquid chromatography, > or =3.1 mg/mmol; individuals with diabetes: immunological method, > or =1.4 mg/mmol; high performance liquid chromatography, > or =5.2 mg/mmol) of microalbuminuria. METHODS: Examination of fresh urines of 469 microalbuminuria negative patients by dipstick were performed by immunonephelometry. Patients, who were microalbuminuria negative by immunonephelometry as well, were further analyzed by high performance liquid chromatography using the Accumintrade mark Kit, based on size-exclusion chromatography. RESULTS: Three times higher albuminuria were found with high performance liquid chromatography than with immunonephelometry. The intraindividual coefficient of variation did not differ in the two methods (37 +/- 31% vs. 40 +/- 31%, p = 0.869; immunonephelometry vs. high performance liquid chromatography; mean +/- standard deviation). Using the present criteria for microalbuminuria, 43% of immunonephelometrically negative patients proved to be microalbuminuric by high performance liquid chromatography. Using the new criteria of the Heart Outcomes Prevention Evaluation study, the rate of microalbuminuria positivity among the immunonephelometrically negative patients decreased to 14.5% by high performance liquid chromatography and the decrease in the number of microalbuminuria positive cases by high performance liquid chromatography could be observed mainly in the diabetic and hypertensive group (49% vs. 7.5%), while slighter decrease could be observed in the non-diabetic hypertensive group (37% vs. 26.5%). Applying the traditional criteria, the strongest predictor was the male gender by the logistic regression analysis. In 28% of microalbuminuria negative patients by immunonephelometry the diagnosis of microalbuminuria can be established using high performance liquid chromatography. CONCLUSIONS: Almost in one-third of microalbuminuria negative patients by immunonephelometry the diagnosis of microalbuminuria can be established by high performance liquid chromatography for which diagnosis three constitutive urine examinations are still needed. New criteria determined by the Heart Outcomes Prevention Evaluation study can be used neither in case of diabetic and hypertensive patients, nor in the case of non-diabetic hypertensive patients. The gender as the most important predictor of microalbuminuria cannot be ignored.

[The pleiotropic effects of losartan--the importance of decreasing uric acid level]. / A losartan pleiotrop hatásai-- a húgysavszintcsökkentés jelentosége.

Nowdays the goal of the antihypertensive treatment is to decrease the cardiovascular risk of the patients by treating the associated diseases and possible hypertensive end-organ damages. Losartan is an antihypertensive drug with all these capabilities and placebo-like side-effect profile. Besides the most important effects of losartan, which are mediated by blocking the angiotensin II. type 1. receptors, it also has other, molecule specific beneficial effects. The most important, well documented pleiotropic effect of losartan is its uricosuric effect. The pleiotropic effects together with the blockade of the angiotensin II. receptors are considered more and more important in the hypertensive end-organ protection and in the treatment of associated diseases in hypertensive patients.

Apolipoprotein A5 T-1131C variant confers risk for metabolic syndrome.

The -1131C is a naturally occurring variant of the apolipoprotein A5 (ApoA5) gene, which has been shown to associate with increased triglyceride levels. This variant has also been shown to confer risk for development of ischemic heart disease and stroke. The gene is in linkage disequilibrium with factors known to correlate with impaired glucose homeostasis. These observations prompted us to study the prevalence of the ApoA5 -1131C allele in patients with metabolic syndrome. A total of 201 metabolic syndrome patients and 210 controls were studied. In both groups the triglyceride levels of patients with -1131C allele were significantly increased compared to the subjects with -1131T allele (3.22+/-0.43 mmol/l vs. 2.24+/-0.12 mmol/l, p<0.01 in the metabolic syndrome patients; 2.10+/-0.19 mmol/l vs. 1.22+/-0.05 mmol/l, p<0.01 in the controls). In metabolic syndrome patients the prevalence of the ApoA5 -1131C variant was increased compared to the healthy controls (11% vs. 6.20%). Multiplex regression analysis model adjusted for age, gender, serum total cholesterol levels, acute myocardial infarction and stroke events revealed that the examined ApoA5 variant confers risk for the development of metabolic syndrome: the odds ratio at 95% confidence interval was 3.622 (1.200-10.936), p=0.02. Our findings strongly suggest that this variant is a risk factor for the development of hypertriglyceridemia and metabolic syndrome.

Stepwise positive association between APOA5 minor allele frequencies and increasing plasma triglyceride quartiles in random patients with hypertriglyceridemia of unclarified origin.

Apolipoprotein A5 (ApoA5) gene and its protein product play a central role in the complex regulation of circulating triglyceride levels in humans. Naturally occurring variants of the apolipoprotein A5 gene have been associated with increased triglyceride levels and have been found to confer risk for cardiovascular diseases. In our study, four polymorphisms, the T-1131C, IVS3+G476A, T1259C, and C56G alleles of APOA5 were analyzed in a total of 436 patients by polymerase chain reaction-restriction fragment length polymorphism methods. The randomly selected patients were classified into four quartile (q) groups based on triglyceride levels (q1: TG<1.31 mmol/l; q2: 1.31-2.90 mmol/l; q3: 2.91-4.85 mmol/l; q4: TG>4.85 mmol/l). We observed significant stepwise increasing association between the four APOA5 minor allele carrier frequencies and plasma triglyceride quartiles: -1131C (q1: 4.44%; q2: 8.95%; q3: 12.9%; q4: 20.6%), IVS3 + 476A (q1: 4.44%; q2: 5.79%; q3: 11.1%; q4: 19.7%), 1259C (q1: 4.44%; q2: 6.84%; q3: 11.1%; q4: 20.6%) and 56G (q1: 5.64%; q2: 6.31%; q3: 11.16%; q4: 11.9%). The serum total cholesterol and high density lipoprotein-cholesterol levels also showed allele-dependent differences in the quartiles. The findings presented here revealed a special arrangement of APOA5 minor alleles in patients with different serum triglyceride ranges in Hungarians.

Functional variants of glucokinase regulatory protein and apolipoprotein A5 genes in ischemic stroke.

Both the natural variants of the apolipoprotein A5 (APOA5) and the glucokinase regulatory protein gene (GCKR) have been shown to associate with increased fasting triglyceride levels. Here, we investigated the possible association of the functional variants of these two genes with non-fasting triglyceride levels and their susceptibility nature in ischemic stroke. A total of 513 stroke patients and 172 healthy controls were genotyped. All the APOA5 variants (T-1131C, IVS3 + G476A, C56G, and T1259C) were associated with increased triglyceride levels in all stroke patients and controls; except for T1259C, they all conferred risk for the disease. No such association was found for the examined GCKR rs1260326 (C1337T) variant. Furthermore, we examined the effects of specific combinations of the GCKR rs1260326 and APOA5 polymorphisms. Our findings confirmed the previous results regarding the association of APOA5 variants with triglyceride-level increase and stroke susceptibility of these alleles. By contrast, we could not detect any association of the studied GCKR allele with triglyceride levels or with the susceptibility of stroke in the same cohort of patients. In addition, the effect of APOA5 did not change significantly when specific combinations of the two genes were present.

Evidence of O-linked N-acetylglucosamine in diabetic nephropathy.

AIMS: There is increasing evidence that O-linked N-acetylglucosamine (O-GlcNAc) plays an important role in cell signaling pathways. It has also been reported that increases in O-GlcNAc contribute to the development of diabetes and diabetic complications; however, little is known about O-GlcNAc levels in diabetic nephropathy (DNP). Therefore the goal of this study was to determine whether O-GlcNAc could be detected in human kidney biopsy specimens, and if so to examine whether O-GlcNAc levels were increased in the kidneys of patients with DNP compared to the non-diabetic individuals. MAIN METHODS: Kidney biopsy specimens were obtained from type-2 diabetic patients (n=6) and patients diagnosed with thin basement membrane nephropathy (n=7) were used as non-diabetic controls. O-GlcNAc levels were assessed by immunohistochemistry using the anti-O-GlcNAc antibody CTD110.6. KEY FINDINGS: We show that O-GlcNAc modification of proteins can be detected in the human kidney biopsy specimens. Furthermore, in diabetic patients, we found significantly increased numbers of O-GlcNAc positive cells in the glomeruli and significantly elevated staining in the tubuli (both in the nucleus and in the cytosol). In addition we also observed an intense, granular O-GlcNAc staining specifically in diabetic tubuli. SIGNIFICANCE: In light of the increase in O-GlcNAc staining in the diabetic patients, we propose that increased O-GlcNAc levels might contribute to the development of diabetic nephropathy.

Effects of pentoxifylline and pentosan polysulphate combination therapy on diabetic neuropathy in type 2 diabetes mellitus.

Vascular dysfunction, including impaired perfusion has a pivotal role in the pathogenesis of microvascular complications in diabetes mellitus. Both pentoxifylline (PF) and pentosan polysulphate (PPS) are known to improve microcirculation. Antioxidant and antiproteinuric effects of PF are also known. In a placebo-controlled study, we determined the possible efficacy of PF-PPS combination therapy on diabetic neuropathy and nephropathy in type 2 diabetic patients. Patients in Verum group (n = 77) received PF-PPS infusions (100-100 mg/day) for 5 days. Control diabetics (Placebo group; n = 12) were given only saline infusions. Specialized cardiovascular autonomic reflex tests, vibration threshold values and urinary albumin excretion were assessed before and after therapy. In Verum group, autonomic score, indicating the severity of cardiac autonomic dysfunction, decreased after therapy (p < or = 0.001). Of the reflexes, deep breath and handgrip tests also improved after therapy (p < or = 0.001). Vibration threshold values, an indicator of the loss of sensory nerve function, were increased after therapy (p < or = 0.001). Results of cardiac autonomic tests and vibration threshold values remained unaltered in Placebo group. Majority of patients had normalbuminuria, which was not affected by PF-PPS. In conclusion, short-term PF-PPS therapy was effective on cardiovascular autonomic function and vibration perception, whereas it failed to reduce albuminuria within normal range in type 2 diabetic patients.

Apolipoprotein A5 IVS3+476A allelic variant associates with increased trigliceride levels and confers risk for development of metabolic syndrome in Hungarians.

BACKGROUND: Metabolic syndrome consists of multiple risk factors that are increasing the cardiovascular mortality. The T-1131C variant of the apolipoprotein A5 gene, associated with increased triglycerides, has been found to confer risk for cardiovascular diseases and metabolic syndrome. Because other naturally occurring variants of the gene also correlate with elevated triglycerides, the possible role of 2 common variants, the IVS3+G476A and T1259C, with metabolic syndrome was investigated. METHODS AND RESULTS: A total of 213 metabolic syndrome patients and 142 healthy controls were genotyped by polymerase chain reaction-restriction fragment length polymorphism. Serum triglycerides were increased in carriers compared with non-carriers in both groups (p<0.001); serum cholesterol levels were similar in all genotypes. The IVS3+476A allele frequency was increased in metabolic syndrome patients compared with controls (8.05 vs 2.47%; p<0.05), whereas the 1259C allele frequency did not differ between the groups. Multiple logistic regression analyses adjusted for age, gender, serum total cholesterol, acute myocardial infarction and stroke revealed that the IVS3+476A variant confers risk for development of metabolic syndrome (odds ratio =3.529, 95% confidence interval 1.308-9.029, p=0.009), but the 1259C allele had no such an effect. CONCLUSIONS: Carrying the IVS3+473A allele is associated with elevated triglycerides and confers risk for development of metabolic syndrome, a combination that represents increased risk for development of atherogenic vascular diseases.

Urinary ortho-tyrosine excretion in diabetes mellitus and renal failure: evidence for hydroxyl radical production.

BACKGROUND: Phenylalanine is converted to para- and ortho-tyrosine by hydroxyl free radical, or to para-tyrosine by the phenylalanine hydroxylase enzyme. The aim of this study was to measure para- and ortho-tyrosine in the urine and plasma of patients with chronic renal disease and/or diabetes, to obtain information on the renal handling of the different tyrosine isomers and, furthermore, to measure urinary levels of 8-epi-prostaglandin-F(2alpha), a marker of lipid peroxidation. METHODS: In our cross-sectional study we measured para-, ortho-tyrosine, and phenylalanine levels, using high performance liquid chromatography and 8-epi-prostaglandin-F(2alpha) with enzyme-linked immunosorbent assay (ELISA). We compared 4 groups: (1) controls (CONTR, N = 14), (2) patients with chronic kidney disease (CKD, N = 12), (3) patients with type 2 diabetes mellitus (DIAB, N = 17), (4) patients with chronic kidney disease and type 2 diabetes (DIAB-CKD, N = 19). RESULTS: We found a decreased plasma para-tyrosine level and decreased urinary para-tyrosine excretion in CKD patients, while the fractional excretion of para-tyrosine was similar in all 4 groups, approximately 1%. There was no difference in the plasma ortho-tyrosine levels between the groups. However, urinary ortho-tyrosine excretion was higher in all 3 groups of patients than in the CONTR group, and higher in DIAB and in DIAB-CKD patients than in CKD patients. The fractional excretion of ortho-tyrosine was significantly higher in DIAB and in DIAB-CKD patients than in the CONTR group. The fractional excretion of ortho-tyrosine exceeded 100% in the 2 diabetic groups. Urinary 8-epi-prostaglandin-F(2alpha)/creatinine ratio did not correlate with urinary ortho-tyrosine excretion. CONCLUSION: The difference between para-tyrosine levels of the groups is probably due to renal impairment, while there is indirect evidence for an increased tubular secretion or production of ortho-tyrosine in the kidney in diabetic patients with or without CKD.