Ivano Testa

High-performance liquid chromatographic assay of asymmetric dimethylarginine, symmetric dimethylarginine, and arginine in human plasma by derivatization with naphthalene-2,3-dicarboxaldehyde

Asymmetric dimethylarginine (ADMA) is an emerging cardiovascular risk factor. Its increased levels have been hypothesized to be a cause of endothelial dysfunction in pathological conditions such as hypertension, dyslipidemia, renal failure, hyperglycemia, and hyperhomocysteinemia. It acts as a potent competitive inhibitor of nitric oxide synthase. Methods using ortho-phthaldialdehyde (OPA) as derivatization reagent are widely performed in HPLC determination of ADMA, but they produce derivatives whose fluorescence rapidly decreases during time. Moreover, these methods do not allow a clear separation of ADMA from its stereoisomer symmetric dimethylarginine (SDMA). Our work describes a new method to determine ADMA, SDMA, and arginine that uses, as derivatizing reagent, naphthalene-2,3-dicarboxaldehyde (NDA). Chromatograms with low background, showing a complete separation of ADMA and SDMA, are obtained. NDA derivatives are considerably more stable than the OPA derivatives. The calibration curves of ADMA and SDMA are linear within the range of 0.01-16.0 microM. Coefficients of variation are less than 1.7% for within day and less then 2.3% for day to day. Absolute mean recoveries from supplemented samples are between 100 and 104%. These characteristics make this method reliable and easily manageable for large routine analyses.

Mitochondrial DNA backgrounds might modulate diabetes complications rather than T2DM as a whole.

Mitochondrial dysfunction has been implicated in rare and common forms of type 2 diabetes (T2DM). Additionally, rare mitochondrial DNA (mtDNA) mutations have been shown to be causal for T2DM pathogenesis. So far, many studies have investigated the possibility that mtDNA variation might affect the risk of T2DM, however, when found, haplogroup association has been rarely replicated, even in related populations, possibly due to an inadequate level of haplogroup resolution. Effects of mtDNA variation on diabetes complications have also been proposed. However, additional studies evaluating the mitochondrial role on both T2DM and related complications are badly needed. To test the hypothesis of a mitochondrial genome effect on diabetes and its complications, we genotyped the mtDNAs of 466 T2DM patients and 438 controls from a regional population of central Italy (Marche). Based on the most updated mtDNA phylogeny, all 904 samples were classified into 57 different mitochondrial sub-haplogroups, thus reaching an unprecedented level of resolution. We then evaluated whether the susceptibility of developing T2DM or its complications differed among the identified haplogroups, considering also the potential effects of phenotypical and clinical variables. MtDNA backgrounds, even when based on a refined haplogroup classification, do not appear to play a role in developing T2DM despite a possible protective effect for the common European haplogroup H1, which harbors the G3010A transition in the MTRNR2 gene. In contrast, our data indicate that different mitochondrial haplogroups are significantly associated with an increased risk of specific diabetes complications: H (the most frequent European haplogroup) with retinopathy, H3 with neuropathy, U3 with nephropathy, and V with renal failure.

Leukocyte telomere length is associated with complications of type 2 diabetes mellitus.

Diabet. Med. 28, 1388–1394 (2011)

Modifications induced by diabetes on the physicochemical and functional properties of erythrocyte plasma membrane

Abstract. Increasing evidence suggests that in experimental diabetes an impairment in Na+, K+–ATPase activity plays a central role in the pathophysiology of diabetic complications, while only a few data are available with regard to human subjects. We studied the erythrocyte membrane Na+, K+‐ATPase activity and membrane fluidity in insulin‐dependent and non‐insulin‐dependent diabetic subjects. A significant decrease in the enzyme activity and in fluorescence polarization values was found in both groups compared with normal subjects. Neither Na+, K+‐ATPase activity nor membrane fluidity was found to be related to metabolic control, assessed by means of fasting blood glucose levels and HbA1c. On the contrary, a significant correlation was observed between Na+, K+‐ATPase activity and membrane fluidity in both insulin‐dependent and non‐insulin‐dependent diabetic subjects. The present work provides evidence that a reduction in the Na+, K+‐ATPase activity is present in the plasma membranes of insulin‐dependent and non‐insulin‐dependent diabetics. Furthermore, it suggests that the change in enzyme activity might be related to modifications in membrane fluidity.

Determination of plasma metformin by a new cation-exchange HPLC technique.

Metformin is an oral antihyperglycemic agent used in the therapy of noninsulin-dependent diabetic patients. This biguanide can induce dangerous complications such as lactic acidosis when its plasma concentration is too high. For this reason, the determination of plasma metformin should always be done during treatment. We developed a new HPLC method, for the routine determination of plasma metformin, with good reliability, rapid execution, and low costs. Sample preparation involved precipitation of the plasma proteins containing the internal standard buformin with a mixture of methanol, zinc sulfate, and ethylene glycol; the diluted supernatant was injected into a cation-exchange column. The mobile phase was potassium dihydrogenphosphate buffer-containing acetonitrile. The eluent was monitored at 236 nm. The calibration curve is linear within the range of 20-4000 ng/mL; the within-day coefficients of variation were less than 2.2% for metformin and 1.5% for buformin; the day-to-day coefficients of variation were less than 2.5% for metformin and 1.9% for buformin. The mean recoveries obtained from supplemented samples were included between 99.4 and 104.2% for metformin. Many characteristics make this method useful and easily accessible to all clinical laboratories equipped with HPLC instrumentation.

Relationship between plasminogen activator inhibitor type-1 plasma levels and the lipoprotein(a) concentrations in non-insulin-dependent diabetes mellitus.

The first part of the paper deals with the relationship between two inhibiting factors of the complex enzyme cascade regulating fibrinolysis, namely plasminogen activator inhibitor type-1 (PAI-1) and lipoprotein(a) (Lp(a)). Blood concentrations of Lp(a), PAI-1 antigen (PAI-1 AG) and activity (PAI-1 AT), and the main parameters of lipo- and glyco-metabolic balance were studied in 80 type II diabetic patients. Roughly hyperbolic patterns have been found between PAI-1 and Lp(a). Negative statistically significant linear correlation can be elicited when Log PAI-1 AG and Log PAI-1 AT values are plotted versus Lp(a) values, the first one being particularly tight. These findings suggest a nearly on/off control of the two parameters, limiting the risk of hypofibrinolysis. The second part of the paper was aimed at verifying this hypothesis. A group of 30 diabetic patients were treated for 3 months with metformin, an antidiabetic biguanide compound which has been reported to reduce PAI-1 levels both in diabetic and in non-diabetic patients. Metformin significantly reduced PAI-1 AG and PAI-1 AT but did not influence plasma Lp(a) levels. A clear linear correlation between the basal Lp(a) values and the changes in PAI-1 AG levels was found. An even tighter correlation was elicited between the decrease in PAI-1, and PAI-1 pretreatment values.

Age-Dependent Changes of Serum Oxygen Radical Scavenger Capacity and Haemoglobin Glycosylation in Non-Insulin-Dependent Diabetic Patients

Background: Contradictory results have been reported in the literature concerning the correlation between glycosylated haemoglobin (HbA1c) and peroxidation level in serum of diabetic patients. Objective: To evaluate this correlation in type 2 diabetic patients by comparing the level of HbA1c with the oxygen radical absorbance capacity (ORACOH) of serum. Methods: One hundred and five type 2 diabetic patients were enroled for the study. After having obtained informed consent, venous blood samples were drawn after overnight fast at the time of routine diabetic check-ups. The blood was collected in plain and EDTA (1 mg/ml) tubes. Glycosylated haemoglobin (HbA1c) was determined by cation-exchange chromatography (HPLC), and spectrophotometric detection (Diamat Analyzer, BioRad). Serum was used for biochemical determinations performed by standard laboratory procedures and for ORACOH analysis. This last parameter was determined measuring the loss of β-phycoerytrin fluorescence due to oxidation by hydroxyl radicals generated by Cu2+ and H2O2, in the presence and absence of serum. Seventy-eight control age-matched subjects were obtained from the personnel staff of our Research Department and old healthy subjects, selected on the basis of Senieur Protocol, were relatives of the above mentioned personnel. Results: When the population of diabetic patients was taken as a whole, a decrease of ORACOH has been observed compared to the controls. Moreover, negative correlations were found comparing ORACOH either with HbA1c (r = –0.213; p = 0.029) and with the age of patients (r = –0.27; p = 0.005). To better understand the effect of age, the data were re-examined dividing the diabetics into two populations, i.e. under and over 65 years of age. An age-dependent decrease of ORACOH and an increase in HbA1c levels has been observed comparing these two populations; however, the correlation between the two parameters remained statistically significant only in the oldest group (r = –0.31; p = 0.026). Conclusions: Present data point to an involvement of oxidative stress in the glycation of haemoglobin especially in old diabetic patients, and provide support for the potential use of an antioxidant therapy in these patients, irrespective of their glycaemic control.

Asymptomatic Helicobacter pylori Infection Increases Asymmetric Dimethylarginine Levels in Healthy Subjects

Background.  Chronic infections have been demonstrated to be early factors of atherosclerosis and cardiovascular diseases, and their relevance increases when they are caused by agents with extremely broad spectrum of disease outcome such as Helicobacter pylori. The consequent endothelial impairment leads to a reduced bioavailability of nitric oxide. Increasing evidences have pointed out that the endogenous inhibitor of nitric oxide synthase, asymmetric dimethylarginine, defined as a risk factor for cardiovascular disease, may increase in infections and plays an important role impairing the vascular functions of the endothelium. Starting from these findings, we aim to investigate whether H. pylori may affect asymmetric dimethylarginine levels.

Helicobacter pylori masks differences in homocysteine plasma levels between controls and type 2 diabetic patients

Background Data in the literature have not clarified whether type 2 diabetes mellitus affects homocysteine plasma levels. Different variables able to influence homocysteine could be the cause of these controversial findings. An important but neglected confounding factor is Helicobacter pylori, which has been demonstrated to be a cause of elevated levels of homocysteine and which is prevalent in the Caucasian population, ranging from 30 to 40% incidence. Starting from these findings we wanted to verify whether differences in homocysteine levels exist between a type 2 diabetic population and a control group, taking into account the presence/absence of Helicobacter pylori.

GLYCOSYLATED HEMOGLOBIN AND FRUCTOSAMINES : DOES THEIR DETERMINATION REALLY REFLECT THE GLYCEMIC CONTROL IN DIABETIC PATIENTS ?

The present experiment was designed to determine whether scavenging capacity of serum, in addition to glucose level, influences hemoglobin and serum protein glycosylation in non-insulin dependent diabetic patients. For this purpose forty-seven patients homogeneous for age, disease duration, therapy and glyco-metabolic control were selected. Fasting and post-prandial glycemia and insulinemia as well as glycosuria were weekly analysed during the sixty days preceding glycosylated hemoglobin (HbA1c), fructosamines and serum scavenging capacity determination. This last parameter has been evaluated by a method based on the property of beta-phycoerythrin (beta-PE) to loss its fluorescence when damaged by oxygen radicals, that were produced by Cu++ and H2O2. The oxygen radical absorbance capacity (ORACOH) of serum was assayed as the ability of serum to delay the loss of beta-PE fluorescence. As expected, a statistically significant positive correlation was found comparing both fructosamines and HbA1c levels with mean fasting glycemia measured over twenty and sixty days, respectively. The key result of this study is represented by the finding that both HbAlc and fructosamines levels show a statistically significant negative correlation with ORACOH values. This correlation can explain a large percent of the data dispersion occurring when ORACOH is not taken into account. In order to better describe the role of ORACOH, patients were separated into two sub-groups with an ORACOH lower (L-ORACOH) and greater (H-ORACOH) than 100 U/ml. Examining the correlation between mean fasting glycemia and the two glycosylated proteins considered in these two sub-groups, curves with different slopes were obtained, supporting that the rate of glycosylation of both proteins was higher in L-ORACOH patients as compared to those with H-ORACOH. Present data suggest that for a proper interpretation of the HbA1c and fructosamines data in diabetic patients, the scavenging capacity level of serum should be taken into account.