Hyperhomocystinemia in patients with coronary artery disease

Hyperhomocystinemia has been related to an increased risk of cardiovascular disease in several studies. The C677T polymorphism for the gene that encodes the methylenetetrahydrofolate reductase enzyme (MTHFR) and low plasma folate levels are common causes of hyperhomocystinemia. Due to differences in nutritional patterns and genetic background among different countries, we evaluated the role of hyperhomocystinemia as a coronary artery disease (CAD) risk factor in a Brazilian population. The relation between homocysteine (Hcy) and the extent of CAD, measured by an angiographic score, was determined. A total of 236 patients referred for coronary angiography for clinical reasons were included. CAD was found in 148 (62.7%) patients and 88 subjects had normal or near normal arteries. Patients with CAD had higher Hcy levels [mean (SD)] than those without disease (14 (6.8) vs 12.5 (4.0) µM; P = 0.04). Hyperhomocystinemia (Hcy >17.8 µM) prevalence was higher in the CAD group: 31.1 vs 12.2% (P = 0.01). After adjustment for major risk factors, we found an independent association between hyperhomocystinemia and CAD (OR = 2.48; 95% CI = 1.02-6.14). Patients with a more advanced coronary score had a higher frequency of hyperhomocystinemia and tended to have higher mean Hcy levels. An inverse relation between plasma folate and Hcy levels was found (r = -0.14; P = 0.04). Individuals with the MTHFR C677T polymorphism had a higher prevalence of hyperhomocystinemia than those without the mutated allele. We conclude that hyperhomocystinemia is independently associated with CAD, with a positive association between Hcy level and disease severity.

Effect of polymorphisms of the MTHFR and APOE genes on susceptibility to diabetes and severity of diabetic retinopathy in Brazilian patients

Diabetes mellitus (DM) is a highly prevalent complex genetic disorder. There has been a worldwide effort in the identification of susceptibility genes for DM and its complications, and the 5-10-methylenetetrahydrofolate reductase (MTHFR) and apolipoprotein-E (APOE) genes have been considered good candidate susceptibility genes to this condition. The objectives of the present study were to determine if the 677T MTHFR and epsilon2/epsilon3/epsilon4 APOE alleles are risk factors for DM and for severity of diabetic retinopathy (DR). A total of 248 individuals were studied: 107 healthy individuals and 141 diabetic patients (46 with type 1 diabetes and 95 with type 2 diabetes), who also had DR (81 with non-proliferative DR and 60 with proliferative DR). The polymorphisms were analyzed by PCR followed by digestion with restriction enzyme or the single-nucleotide primer extension method. No evidence of association between the 677TT genotype of MTHFR gene and DM [cases: TT = 10/95 (10.6%); controls: TT = 14/107 (13%)] or with severity of DR was observed [cases: TT = 5/60 (8.5%); controls: TT = 9/81 (11.1%); P > 0.05]. We also did not find evidence of an association between APOE alleles and proliferative DR (epsilon2, epsilon3 and epsilon4 in cases: 9, 76, and 15%, and in controls: 5, 88, and 12%, respectively) but the carriers of epsilon2 allele were more frequent among patients with type 2 DM and DR than in controls [cases: 15/95 (15.8%); controls: 7/107 (6.5%); P < 0.05]. Therefore, our results suggest that the epsilon2 allele/APOE might be a risk factor for diabetes in the Brazilian population.

The clinical impact of MTHFR polymorphism on the vascular complications of sickle cell disease

Sickle cell disease (SCD) is one of the most common inherited diseases in the world and the patients present notorious clinical heterogeneity. It is known that patients with SCD present activation of the blood coagulation and fibrinolytic systems, especially during vaso-occlusive crises, but also during the steady state of the disease. We determined if the presence of the factor V gene G1691A mutation (factor V Leiden), the prothrombin gene G20210A variant, and methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism may be risk factors for vascular complications in individuals with SCD. We studied 53 patients with SCD (60% being women), 29 with SS (sickle cell anemia; 28 years, range: 13-52 years) and 24 with SC (sickle-hemoglobin C disease; 38.5 years, range: 17-72 years) hemoglobinopathy. Factor V Leiden, MTHFR C677T polymorphism, and prothrombin G20210A variant were identified by PCR followed by further digestion of the PCR product with specific endonucleases. The following vascular complications were recorded: stroke, retinopathy, acute thoracic syndrome, and X-ray-documented avascular necrosis. Only one patient was heterozygous for factor V Leiden (1.8%) and there was no prothrombin G20210A variant. MTHFR 677TT polymorphism was detected in 1 patient (1.8%) and the heterozygous form 677TC was observed in 18 patients (34%, 9 with SS and 9 with SC disease), a prevalence similar to that reported by others. No association was detected between the presence of the MTHFR 677T allele and other genetic modulation factors, such as alpha-thalassemia, ß-globin gene haplotype and fetal hemoglobin. The presence of the MTHFR 677T allele was associated with the occurrence of vascular complications in SCD, although this association was not significant when each complication was considered separately. In conclusion, MTHFR C677T polymorphism might be a risk factor for vascular complications in SCD.

Polymorphisms in genes MTHFR, MTR and MTRR are not risk factors for cleft lip/palate in South Brazil

Non-syndromic cleft lip and palate (CL/P) occurs due to interaction between genetic and environmental factors. Abnormalities in homocysteine metabolism may play a role in its etiology due to polymorphisms in genes involved in this pathway. Because of the involvement of MTHFR, MTR and MTRR genes with folate metabolism and the evidence that maternal use of folic acid in early pregnancy reduces the risk for CL/P, we evaluated the influence of their polymorphisms on the etiology of CL/P through a case-control study. The analyses involved 114 non-syndromic phenotypically white children with clefts (case) and 110 mothers, and 100 non-affected (control) children and their mothers. The polymorphisms 677C>T of MTHFR, 2756A>G of MTR, and 66A>G of MTRR genes were analyzed by PCR-RFLP. Allelic frequencies did not differ from other studies conducted on white populations for MTHFR 677T allele (0.35) and for MTR 2756G allele (0.17), but MTRR 66G allele frequency (0.35) was lower than observed elsewhere. The genotypic distribution of the 677C>T polymorphisms under study did not show significant differences between CL/P patients, their mothers and controls. These results suggest that the alterations of folate metabolism related to these polymorphisms are not involved in clefting in the population under study.

The MTR A2756G polymorphism is associated with an increase of plasma homocysteine concentration in Brazilian individuals with Down syndrome

Individuals with Down syndrome (DS) present decreased homocysteine (Hcy) concentration, reflecting a functional folate deficiency secondary to overexpression of the cystathionine ß-synthase gene. Since plasma Hcy may be influenced by genetic polymorphisms, we evaluated the influence of C677T and A1298C polymorphisms in the methylenetetrahydrofolate reductase gene (MTHFR), of A2756G polymorphism in the methionine synthase gene (MTR), and of A80G polymorphism in the reduced folate carrier 1 gene on Hcy concentrations in Brazilian DS patients. Fifty-six individuals with free trisomy 21 were included in the study. Plasma Hcy concentrations were measured by liquid chromatography_tandem mass spectrometry with linear regression coefficient r² = 0.9996, average recovery between 92.3 to 108.3% and quantification limits of 1.0 µmol/L. Hcy concentrations >15 µmol/L were considered to characterize hyperhomocystinemia. Genotyping for the polymorphisms was carried out by polymerase chain reaction followed by enzyme digestion and allele-specific polymerase chain reaction. The mean Hcy concentration was 5.2 ± 3.3 µmol/L. There was no correlation between Hcy concentrations and age, gender or MTHFR C677T, A1298C and reduced folate carrier 1 A80G genotype. However, Hcy concentrations were significantly increased in the MTR 2756AG heterozygous genotype compared to the MTR 2756AA wild-type genotype. The present results suggest that the heterozygous genotype MTR 2756AG is associated with the increase in plasma Hcy concentrations in this group of Brazilian patients with DS.

Reduced folate carrier 1 (RFC1) is associated with cleft of the lip only

In this report, we have reanalyzed genotyping data in a collection of families from South America based on maternal origin. Genotyping analysis was performed at the Craniofacial Anomalies Research Center at the University of Iowa. These genotypes were derived from genomic DNA samples obtained from blood spots from children born with isolated orofacial clefts in 45 hospitals located in eight countries (Argentina, Bolivia, Brazil, Chile, Ecuador, Paraguay, Uruguay, and Venezuela) collaborating with ECLAMC (Latin American Collaborative Studies of Congenital Malformations) between January 1998 and December 1999. Dried blood samples were sent by regular mail to the Laboratory of Congenital Malformations, Federal University of Rio de Janeiro. Previous findings suggested that mitochondrial haplotype D is more commonly found among cleft cases born in South America. We hypothesized that association of certain genes may depend upon the ethnic origin, as defined by population-specific markers. Therefore, we tested if markers in MTHFR (5,10-methylenetetrahydrofolate reductase) and RFC1 (reduced folate carrier 1) were associated with oral clefts, depending on the maternal origin defined by the mitochondrial haplotype. Transmission distortion of alleles in MTHFR C677T and RFC1 G80A polymorphic variants was tested in 200 mother/affected child pairs taking into consideration maternal origin. RFC1 variation was over-transmitted to children born with cleft lip only (P = 0.017) carrying mitochondrial DNA haplotypes other than haplotype D. Our results provide a new indication that variation in RFC1 may contribute to cleft lip only. Future studies should investigate the association between oral clefts and RFC1 based on more discrete phenotypes.

The effect of 677C>T and 1298A>C MTHFR polymorphisms on sulfasalazine treatment outcome in rheumatoid arthritis

Despite the availability of several new agents for the treatment of rheumatoid arthritis (RA), sulfasalazine remains the mainstay because of both cost and experience with its use. Methylenetetrahydrofolate reductase (MTHFR) is involved in folate metabolism and several polymorphisms have been described in the MTHFR gene. Of these, the 677C>T and 1298A>C polymorphisms have been associated with altered enzyme activity. To examine the association between 677C>T and 1298A>C MTHFR polymorphisms and sulfasalazine efficacy for the treatment of RA, a total of 117 RA patients treated with sulfasalazine (1 g daily; duration of treatment 17 ± 5 months) were analyzed. The 677C>T and 1298 A>C polymorphisms were detected using a PCR-RFLP method. RA was diagnosed according to the criteria of the American College of Rheumatology (ACR). The remission of RA symptoms was evaluated according to the ACR 20% response criteria. Allele and genotype frequencies were compared by the two-sided Fisher exact test. The frequency of remission was 47.2% and 44.6% in carriers of 677T and 1298C alleles, compared to 40.7% and 42.0% in carriers of 677C and 1298A alleles, respectively. These differences were statistically non-significant. When the multivariate analysis was additionally adjusted for patients’ age, gender and RA duration, the association of the MTHFR 677T allele with increased frequency of remission was statistically significant. Although RA remission rate in carriers of the MTHFR 677T and 1298C alleles was more frequently observed, it does not seem that 677C>T and 1298A>C MTHFR polymorphisms have a major influence on treatment outcome in RA patients treated with sulfasalazine.

Glucose-6-phosphate dehydrogenase and glutathione reductase activity in methemoglobin reduction by methylene blue and cyst amine: study on glucose-6-phosphate dehydrogenase-deficient individuals, on normal subjects and on riboflavin-treated subjects

The authors have standardized methods for evaluation of the activity of the glucose-6-phosphate dehydrogenase and of glutathione reductase. The general principle of the first method was based on methemoglobin formation by sodium nitrite followed by stimulation of the glucose-6-phosphate dehydrogenase with methylene blue. Forty six adults (23 males and 23 females) were studied. Subjects were not G6PD deficient and were aged 20 to 30 years. The results showed that methemoglobin reduction by methylene blue was 154.40 and 139.90 mg/min (p<0.05) for males and females, respectively, in whole blood, and 221.10 and 207.85 mg/min (n.s.), respectively, in washed red cells. These data showed that using washed red cells and 0.7g% sodium nitrite concentration produced no differences between sexes and also shortened reading time for the residual amount of methemoglobin to 90 minutes. Glutathione reductase activity was evaluated on the basis of the fact that cystamine (a thiol agent) binds to the SH groups of hemoglobin, forming complexes. These complexes are reversed by the action of glutathione reductase, with methemoglobin reduction occurring simultaneously with this reaction. Thirty two adults (16 males and 16 females) were studied. Subjects were not G6PD deficient and were aged 20 to 30 years. Methemoglobin reduction by cystamine was 81.27 and 91.13 mg/min (p<0.01) for males and females, respectively. These data showed that using washed red cells and 0.1 M cystamine concentration permits a reading of the residual amount of methemoglobin at 180 minutes of incubation. Glutathione reductase activity was evaluated by methemoglobin reduction by cystamine in 14 females before and after treatment with 10 mg riboflavin per day for 8 days. The results were 73.69 and 94.26 jug/min (p<0.01) before and after treatment, showing that riboflavin treatment increase glutathione reductase activity even in normal individuals. Three Black G6PD-deficient individuals (2 males and 1 female) were also studied. The G6PD and glutathione reductase were partially activated, the change being more intense in males. On the basis of race and of the laboratory characteristics observed, it is possible to suggest that the G6PD deficiency of these individuals is of the African type and that the female is heterozygous for this deficiency. Analysis of the results as a whole permitted us to conclude that the methods proposed here were efficient for evaluating the activity of the glucose-6-phosphate dehydrogenase and of glutathione reductase. The latter is dependent on the pentose pathway, which generates NADPH, and on riboflavin, a FAD precursor vitamin.

Isoniazid acetylating phenotype in patients with paracoccidioidomycosis and its relationship with serum sulfadoxin levels, glucose-6-phosphate dehydrogenase and glutathione reductase activities

The authors evaluated the isoniazid acetylating phenotype and measured hematocrit, hemoglobin, glucose-6-phosphate dehydrogenase and glutathione reductase activities plus serum sulfadoxin levels in 39 patients with paracoccidioidomycosis (33 males and 6 females) aged 17 to 58 years. Twenty one (53.84%) of the patients presented a slow acetylatingphenotype and 18(46.16%) a fast acetylating phenotype. Glucose-6-phosphate- dehydrogenase (G6PD) acti vity was decreased in 5(23.80%) slow acetylators and in 4(22.22%) fast acetylators. Glutathione reductase activity was decreased in 14 (66.66%) slow acetylators and in 12 (66.66%) fast acetylators. Serum levels of free and total sulfadoxin Were higher in slow acetylator (p < 0.02). Analysis of the resultspermitted us to conclude that serum sulfadoxin levels are related to the acetylatorphenotype. Furthermore, sulfadoxin levels were always above 50 µg/ml, a value considered therapeutic. Glutathione reductase deficiency observed in 66% of patients may be related to the intestinal malabsorption of nutrients, among them riboflavin, a FAD precursor vitamin, inpatients with paracoceidioidomycosis.

Normal bone density in male pseudohermaphroditism due to 5alpha- reductase 2 deficiency

Bone is an androgen-dependent tissue, but it is not clear whether the androgen action in bone depends on testosterone or on dihydrotestosterone. Patients with 5alpha-reductase 2 deficiency present normal levels of testosterone and low levels of dihydrotestosterone, providing an in vivo human model for the analysis of the effect of testosterone on bone. OBJECTIVE: To analyze bone mineral density in 4 adult patients with male pseudohermaphroditism due to 5alpha-reductase 2 deficiency. RESULTS: Three patients presented normal bone mineral density of the lumbar column (L1-L4) and femur neck, and the other patient presented a slight osteopenia in the lumbar column. CONCLUSION: Patients with dihydrotestosterone deficiency present normal bone mineral density, suggesting that dihydrotestosterone is not the main androgen acting in bone.

Biomass accumulation, photochemical efficiency of photosystem II, nutrient contents and nitrate reductase activity in young rosewood plants (Aniba rosaeodora Ducke) submitted to different NO3-:NH4+ ratios

The rosewood (Aniba rosaeodora Ducke) is a native tree species of Amazon rainforest growing naturally in acidic forest soils with reduced redox potential. However, this species can also been found growing in forest gaps containing oxide soils. Variations in the forms of mineral nitrogen (NO3- or NH4+) may be predicted in these different edaphic conditions. Considering that possibility, an experiment was carried out to analyze the effects of different NO3-:NH4+ ratios on the growth performance, mineral composition, chloroplastid pigment contents, photochemical efficiency photosystem II (PSII), and nitrate redutase activity (RN, E.C.1.6.6.1) on A. rosaeodora seedlings. Nine-month-old seedlings were grown in pots with a washed sand capacity of 7.5 kg and submitted to different NO3-:NH4+ ratios (T1 = 0:100%, T2 = 25:75%, T3 = 50:50%, T4 = 75:25%, and T5 = 100:0%). The lowest relative growth rate was observed when the NO3-:NH4+ ratio was equal to 0:100%. In general, high concentrations of NO3- rather than NH4+ favored a greater nutrient accumulation in different parts of the plant. For the chloroplastid pigment, the highest Chl a, Chl b, Chl tot, Chl a/b and Chl tot/Cx+c contents were found in the treatment with 75:25% of NO3-:NH4+, and for Chl b and Cx+c it was observed no difference. In addition, there was a higher photochemical efficiency of PSII (Fv/Fm) when high NO3- concentrations were used. A linear and positive response for the nitrate reductase activity was recorded when the nitrate content increased on the culture substrate. Our results suggest that A. rosaeodora seedlings have a better growth performance when the NO3- concentrations in the culture substrate were higher than the NH4+ concentrations.

Comparison of the effect of two HMG CoA reductase inhibitors on LDL susceptibility to oxidation

OBJECTIVE: To study the differences between fluvastatin and pravastatin regarding LDL susceptibility to oxidation, plasma levels of total cholesterol (TC), HDL-C, LDL-C and triglycerides (TG) in hypercholesterolemic patients with established coronary heart disease (CHD). METHODS: A double-blind randomized parallel study was conducted that included 41 hypercholesterolemic outpatients with CHD treated at the Instituto de Cardiologia do Rio Grande do Sul. The inclusion criteria were LDL-C above 100 mg/dL and triglycerides below 400 mg/dL based on 2 measures. After 4 weeks on a low cholesterol diet, those patients that fullfilled the inclusion criteria were randomized into 2 groups: the fluvastatin group (fluvastatin 40 mg/day) and the pravastatin group (pravastatin 20 mg/day), for 24 weeks of treatment. LDL susceptibility to oxidation was analyzed with copper-induced production of conjugated dienes (Cu2+) and water-soluble free radical initiator azo-bis (2'-2'amidinopropanil) HCl (AAPH). Spectroscopy nuclear magnetic resonance was used for determination of lipids. RESULTS: After 24 weeks of drug therapy, fluvastatin and pravastatin significantly reduced LDL susceptibility to oxidation as demonstrated by the reduced rate of oxidation (azo and Cu) and by prolonged azo-induced lag time (azo lag). The TC, LDL-C, and TG reduced significantly and HDL-C increased significantly. No differences between the drugs were observed. CONCLUSION: In hypercholesterolemic patients with CHD, both fluvastatin and pravastatin reduced LDL susceptibility to oxidation.

Mutations in Plasmodium falciparum dihydrofolate reductase and dihydropteroate synthase of isolates from the Amazon Region of Brazil

Since the late 1970s pyrimethamine-sulfadoxine (PS; FansidarTM Hoffman-LaRoche, Basel) has been used as first line therapy for uncomplicated malaria in the Amazon basin. Unfortunately, resistance has developed over the last ten years in many regions of the Amazon and PS is no longer recommended for use in Brazil. In vitro resistance to pyrimethamine and cycloguanil (the active metabolite of proguanil) is caused by specific point mutations in Plasmodium falciparum dihydrofolate reductase (DHFR), and in vitro resistance to sulfadoxine has been associated with mutations in dihydropteroate synthase (DHPS). In association with a proguanil-sulfamethoxazole clinical trial in Brazil, we performed a nested mutation-specific polymerase chain reaction to measure the prevalence of DHFR mutations at codons 50, 51, 59, 108 and 164 and DHPS mutations at codons 436, 437, 540, 581 and 613 at three sites in the Brazilian Amazon. Samples from two isolated towns showed a high degree of homogeneity, with the DHFR Arg-50/Ile-51/Asn-108 and DHPS Gly-437/Glu-540/Gly-581 mutant genotype accounting for all infections in Peixoto de Azevedo (n = 15) and 60% of infections in Apiacás (n = 10), State of Mato Grosso. The remaining infections in Apiacás differed from this predominant genotype only by the addition of the Bolivia repeat at codon 30 and the Leu-164 mutation in DHFR. By contrast, 17 samples from Porto Velho, capital city of the State of Rondônia, with much in- and out-migration, showed a wide variety of DHFR and DHPS genotypes.

8-Methoxy-naphtho[2,3-b]thiophen-4,9-quinone, a non-competitive inhibitor of trypanothione reductase

The enzyme trypanothione reductase is a recognised drug target in trypanosomatids and has been used in the search of new compounds with potential activity against diseases such as leishmaniasis, Chagas disease and African trypanosomiasis. 8-Methoxy-naphtho [2,3-b] thiophen-4,9-quinone was selected in a screening of natural and synthetic compounds using an in vitro assay with the recombinant enzyme from Trypanosoma cruzi. Its mode of inhibition fits a non-competitive model with respect to the substrate (trypanothione) and to the co-factor (NADPH), with Ki-values of 5 and 3.6 µM, respectively. When tested against human glutathione reductase, this compound did not display any significant inhibition at 100 µM, indicating a good selectivity against the parasite enzyme.