Effect of C677T methylenetetrahydrofolate reductase gene polymorphism on plasma homocysteine levels in ethnic groups

The objective of this study was to examine plasma homocysteine levels and C677T methylenetetrahydrofolate reductase (MTHFR) gene polymorphism in two ethnic groups from Slovakia. The samples consisted of general Slovak-Romany population (68 men and 81 women) from Southwestern Slovakia and the Slovak-Caucasians (174 men and 177 women) who participated in the CINDI project. The homocysteine levels were examined by HPLC, the analysis of MTHFR genotypes was done by PCR. The Slovak-Romany men (12.0+/-5.6 (S.D.) micromol/l) and women (9.2+/-2.6 microol/l) have significantly lower plasma homocysteine levels (p<0.024 and p<0.00001) when compared to Caucasians (13.3+/-5.1 micromol/l in men and 11.3+/-4.3 micromol/l in women). The genetic equilibrium is assumed for the gene frequencies of the MTHFR polymorphism in both samples. The distribution of MTHFR genotypes did not differ between the two populations (TT 13 vs. 10.6 %; CT 46.6 vs. 41.7 %; CC 40.4 vs. 47.7 %, chí(2)2 = 2.315, df=2, ns). The effect of MTHFR genotypes on homocysteine levels was not confirmed in the Slovak-Romanies and TT homozygosity significantly increased plasma homocysteine levels only in Slovak-Caucasians (11.5+/-4.4 micromol/l, ns; vs. 14.8+/-4.8 micromol/l, p 0.002, respectively). To our knowledge, this is the first epidemiological study in the Romany population examining distribution of the MTHFR genotypes and their effect on homocysteine levels. Further studies are needed to establish the variety of cardiovascular risk factors among Romanies in order to evaluate the significance of particular factors.     

Central composite design as a powerful optimisation technique for enantioresolution of the rac-11-dihydrooracin--the principal metabolite of the potential cytostatic drug oracin

Three types of chiral stationary phase were used to achieve chromatographic resolution of enantiomers of rac-11-dihydrooracin (DHO), the principal metabolite of a potential cytostatic drug oracin. Chiralcel OD-R as a chiral stationary phase with mobile phase comprising acetonitrile (modifier) and sodium perchlorate (buffering component) proved to be the most suitable system. Chemometric optimisation based on the Box-Wilson central composite design was employed to find the optimum resolution. The optimum factor space was defined by three parameters: temperature, modifier concentration and buffer concentration. Newly designed chromatographic response functions based on a combination of resolution R(S) and retention time of the last component eluted t(RL) were employed to evaluate the resolution with regard to quality and quantity. Optimum values predicted from those models of response surfaces were in excellent agreement with the experimental results. The chromatographic resolution of DHO enantiomers is suitable for xenobiochemical studies on stereoselectivity and stereospecificity of biotransformation enzymes.     

Stereoselective pharmacokinetics and metabolism of flobufen in guinea pigs

Stereoselective aspects of pharmacokinetics and metabolism of a chiral nonsteroidal antiinflammatory drug, flobufen, 4-(2', 4'-difluorobiphenyl-4-yl)-2-methyl-4-oxobutanoic acid, were studied in male guinea pigs after p.o. administration of racemic flobufen (rac-flobufen) at a dose of 10 mg/kg. Blood samples were collected at intervals over 16 h after the administration of rac-flobufen for the quantification of flobufen enantiomers and their respective metabolites in plasma by chiral high-performance liquid chromatography (HPLC). Compartmental pharmacokinetic analysis was used to determine pharmacokinetic parameters of R- and S-flobufen. The plasma concentrations of the S- and R-enantiomers differed significantly during the experimental period. The S/R-enantiomeric ratio in 7plasma reached a maximum value of 10.1 at 240 min postdose. The oral clearance value of R-flobufen was five times higher than S-flobufen. The other pharmacokinetic parameters (K(e), T(1/2), V(SS)/F, MRT) of the enantiomers also differed substantially. All four stereoisomers of the dihydrometabolite of flobufen were detected in plasma with varying concentrations. Metabolite 17203 [4-(2,4-difluorophenyl)-phenylacetic acid] exhibited a relatively longer residence time compared to that noted for the enantiomers of the parent compound. Pharmacokinetics of the flobufen enantiomers were stereoselective in guinea pigs. The metabolism of flobufen was complex. However, metabolite 17203 seemed to be the main metabolite of flobufen that may be responsible for its relatively long-lasting antiphlogistic and immunomodulatory effects.     

Chiral aspects of metabolism of antiinflammatory drug flobufen in human hepatocytes

The metabolism of the nonsteroidal antiinflammatory drug flobufen, 4-(2',4'-difluorobiphenyl-4-yl)-2-methyl-4-oxobutanoic acid, was studied in primary cultures of human hepatocytes prepared by two-step collagenase perfusion of livers from four donors. Racemic flobufen or its individual enantiomers, R-(+)- and S-(-)-flobufen were used as substrates. Aliquots of culture medium were collected during 24-h incubation. The time-dependent disappearance of flobufen enantiomers and the formation of metabolites (stereoisomers of dihydroflobufen (DHF)) in hepatocytes were measured by chiral HPLC. The reduction of flobufen in human hepatocytes was stereoselective ((+)-R-flobufen was preferentially metabolized) and stereospecific ((2R;4S)-DHF and (2S;4S)-DHF stereoisomers were mostly formed). Although the structure of flobufen is different from the profens (2-arylpropionates), flobufen undergoes chiral inversion in human hepatocytes. The inversion of R-(+)-flobufen to S-(-)-flobufen predominates. The individual DHF stereoisomers were incubated in hepatocyte cultures and their biotransformation studied. The unidirectional chiral inversion of (2S;4S)-DHF to (2R;4S)-DHF and (2R;4R)-DHF to (2S;4R)-DHF was observed. Stereoselective oxidation of the DHFs to flobufen was also detected. Thus, flobufen metabolism in primary cultures of human hepatocytes is much more complicated (via chiral inversion and DHF re-oxidation) than was presumed from a preliminary achiral point of view.     

Relapsed follicular lymphoma sequentially treated with rituximab and 90Y-ibritumomab tiuxetan. Case report

BACKGROUND: Monoclonal antibodies have dramatically changed the treatment possibilities for follicular lymphoma. (90)Y-ibritumomab tiuxetan (Zevalin) is the first radioimmunotherapy agent approved for the treatment of relapsed and resistant follicular lymphoma patients. Long-term benefit was observed especially for patients achieving CR after radioimmunotherapy. METHODS AND RESULTS: A 65-year-old female patient with the second relapse of CD20 positive follicular lymphoma and multiple concomitant diseases was treated with four weekly doses of rituximab (375 mg/m(2)). (18)F-fluoro-deoxyglucose positron emission tomography combined with computed tomography (PET-CT) demonstrated only partial response to therapy with persistent PET scan positivity in enlarged abdominal lymph nodes. Therefore, it was decided to treat her with a 1200-MBq (32-mCi) dose of (90)Y-ibritumomab tiuxetan radioimmunotherapy. No acute complications were noted afterwards. Hematological nadirs were reached 4 weeks later, with a platelet count of 24 x 10(9)/l that normalized within the next 2 weeks. The patient had neither infection nor bleeding complications. Eight weeks after radioimmunotherapy, the PET-CT scans documented only 3 lymph nodes around the abdominal aorta, maximum size 2 x 1 cm. The PET scan analysis proved no accumulation of (18)F-fluoro-deoxy-glucose in any lymph nodes or other organs and tissues. CONCLUSIONS: Sequential treatment with rituximab and (90)Y-ibritumomab tiuxetan may be an interesting alternative in cases of relapsed follicular or other indolent lymphomas in pretreated or older patients with other concomitant diseases.     

A nonerythropoietic peptide that mimics the 3D structure of erythropoietin reduces organ injury/dysfunction and inflammation in experimental hemorrhagic shock

Recent studies have shown that erythropoietin, critical for the differentiation and survival of erythrocytes, has cytoprotective effects in a wide variety of tissues, including the kidney and lung. However, erythropoietin has been shown to have a serious side effect-an increase in thrombovascular effects. We investigated whether pyroglutamate helix B-surface peptide (pHBSP), a nonerythropoietic tissue-protective peptide mimicking the 3D structure of erythropoietin, protects against the organ injury/ dysfunction and inflammation in rats subjected to severe hemorrhagic shock (HS). Mean arterial blood pressure was reduced to 35 ± 5 mmHg for 90 min followed by resuscitation with 20 mL/kg Ringer Lactate for 10 min and 50% of the shed blood for 50 min. Rats were euthanized 4 h after the onset of resuscitation. pHBSP was administered 30 min or 60 min into resuscitation. HS resulted in significant organ injury/dysfunction (renal, hepatic, pancreas, neuromuscular, lung) and inflammation (lung). In rats subjected to HS, pHBSP significantly attenuated (i) organ injury/dysfunction (renal, hepatic, pancreas, neuromuscular, lung) and inflammation (lung), (ii) increased the phosphorylation of Akt, glycogen synthase kinase-3β and endothelial nitric oxide synthase, (iii) attenuated the activation of nuclear factor (NF)-κB and (iv) attenuated the increase in p38 and extracellular signal-regulated kinase (ERK)1/2 phosphorylation. pHBSP protects against multiple organ injury/dysfunction and inflammation caused by severe hemorrhagic shock by a mechanism that may involve activation of Akt and endothelial nitric oxide synthase, and inhibition of glycogen synthase kinase-3β and NF-κB.     

The relationship between micronuclei in human lymphocytes and selected micronutrients in vegetarians and non-vegetarians

A vegetarian diet results in higher intake of vitamins and micronutrients, which - although providing antioxidant defence - may lead to deficiency in other micronutrients involved in DNA metabolism and stability (such as vitamins belonging to the B group). The principal difference among various vegetarian diets is the extent to which animal products are avoided. We have performed a pilot study to determine the relationship between the micronucleus frequency in lymphocytes and diet, and we compared the levels of Vitamins C and E, beta-carotene, B(12), folic acid, homocysteine and total antioxidant capacity in healthy vegetarians and non-vegetarians. The vegetarian group, consisting of 24 volunteers (13 women and 11 men), were matched for age and sex with 24 volunteers (12 women and 12 men) with a traditional dietary habit. Among the vegetarians were 13 lacto-ovo-vegetarians with average duration of vegetarian diet 10.8 years (ranging from 5 to 26 years) and 11 lacto-vegetarians with average duration of vegetarian diet 8.2 years (ranging from 3 to 15 years). Homocysteine, Vitamins C and E and beta-carotene levels in plasma were assayed by HPLC, and serum folate and Vitamin B(12) were determined with Elecsys Immunoassay tests. The total antioxidant capacity of plasma was estimated by measuring the ferric-reducing activity in a spectrophotometric assay. Micronuclei were measured in cytokinesis-blocked lymphocytes. Vegetarians had significantly higher levels of Vitamin C and beta-carotene (but not Vitamin E) in plasma compared with non-vegetarians (P<0.001). There were no significant differences in serum levels of folic acid and Vitamin B(12) between the monitored groups. Levels of folic acid in vegetarians correlated with length of vegetarianism (r=0.62, P=0.001, N=24). Vegetarians had elevated levels of homocysteine compared with non-vegetarians (P=0.007), as did vegetarian women compared with non-vegetarian women (P=0.031). We did not find any differences in total antioxidant capacity or in micronucleus frequency between the groups. Micronuclei correlated with age (r=0.62, P<0.001, N=48), women having higher frequencies than men. Multifactorial regression analysis showed significant effects of age, sex and total antioxidant capacity on micronucleus frequency (N=48, P<0.001).     

The influence of polymorphism of -493G/T MTP gene promoter and metabolic syndrome on lipids, fatty acids and oxidative stress

The aim of this study was to investigate the effect of the microsomal triglyceride transfer protein (MTP) -493G/T polymorphism on clinical and biochemical parameters in relation to the presence of metabolic syndrome (MS). A group of 270 participants, 143 men and 127 women [50 men/36 women fulfilled the International Diabetes Federation (IDF) criteria of MS], was categorized on the basis of the MTP -493G/T polymorphism: GG homozygotes (Group GG) and carriers of the T allele (Group TT+TG). In men with MS, the presence of the T allele was associated with elevated concentrations of plasma insulin (by 48%, P<.01) and nonesterified fatty acids (by 49%, P<.05); homeostasis model assessment for insulin resistance index was higher by 64% (P<.05). Carriers of the T allele were further characterized by elevated plasma concentrations of total cholesterol (by 14%, P<.05) and by increased triglycerides in plasma (by 95%, P<.01) and in very low-density lipoprotein (by 106%, P<.01). They also had lower concentrations of n-6 polyunsaturated fatty acids in plasma phospholipids (by 3.5%, P<.05), lower Delta5-desaturase activities (by 18%, P<.05) and elevated concentrations of conjugated dienes in low-density lipoprotein (by 29%, P<.01). No significant differences between Groups GG and TT+TG were found in men without MS and in women with and without MS. Our results imply evidence for interactive effects of genetic, metabolic and gender-specific factors on several components of metabolic syndrome, which can increase the risk for cardiovascular disease.     

Fluid consumption, electrolyte excretion and heart remodeling in rats with myocardial infarct maintained on regular and high sodium intake.

The purpose of the study was to determine effect of high sodium intake on fluid and electrolyte turnover and heart remodeling in the cardiac failure elicited by myocardial infarction (MI). The experiments were performed on four groups of Sprague Dawley rats maintained on food containing 0.45% NaCl and drinking either water (groups 1, 2) or 1% NaCl (groups 3, 4). Groups 1 and 3 were sham-operated while in groups 2 and 4 MI was produced by the coronary artery ligation. In each group food and fluid as well as sodium intake, urine (Vu), sodium (UNaV), potassium (UKV) and solutes (UosmV) excretion were determined before and four weeks after the surgery. Size of the infarct, left ventricle (LV) weight and diameter of LV and right ventricle (RV) myocytes were determined during post-mortem examination. Before the surgery groups 3 and 4 ingested significantly more fluid and sodium, had higher Vu, UNaV, UKV and UosmV than the respective groups 1 and 2. In groups 2 and 4 MI resulted in significant decrease in Vu, UNaV and UosmV in comparison to the pre-surgical level. In Group 4 MI resulted also in a significant decrease of food and sodium intake. The MI size did not differ in groups 2 and 4 while diameter of LV myocytes was significantly greater in groups 2 and 4 than in groups 1 and 3, and in group 4 than in group 2. The study reveals that prolonged high sodium consumption increases fluid and electrolyte turnover both in the sham and in the MI rats and that the MI causes decrease in food and sodium intake in rats on high but not on regular sodium intake. In addition high sodium diet promotes development of greater post-MI hypertrophy of the LV myocytes.