Evaluation of nutritional and genetic determinants of total homocysteine, methylmalonic acid and S-adenosylmethionine/S-adenosylhomocysteine values in Brazilian childbearing-age women

Background: Cobalamin (Cbl) and folate deficiencies and gene polymorphism of key enzymes or carriers can impair homocysteine metabolism and may change the serum values of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH). We investigated the nutritional and genetic determinants for total homocysteine (tHcy), methylmalonic acid (MMA) and SAM/SAH in healthy Brazilian childbearing-age women. Methods: Serum concentrations of Cbl, folate, red blood cell folate, ferritin, tHcy, MMA, SAM, SAH and other metabolites were measured in 102 healthy unrelated women. The genotypes for MTHFR C677T, MTHFR A1298C, MTR A2756G, MTRR A66G, TC2 C776G, TC2 A67G and RFCI A80G gene polymorphisms were identified by PCR-RFLP. Results: Serum folate and Cbl were inversely correlated with tHcy and serum MMA, respectively. Cbl deficiency was associated with increased MMA and reduced alpha-aminobutyrate, serine and N-methylglycine concentrations. No variable was associated with SAM/SAH ratio. In addition, gene polymorphisms were not selected as determinants for tHcy, MMA and SAM/SAH ratio. Iron, Cbl and folate deficiencies were found respectively in 30.4%, 22.5% and 2.0% of individuals studied. Conclusions: There was a high frequency of Cbl and iron deficiency in this group of childbearing-age women. Serum folate and Cbl were the determinants of serum tHcy and MMA concentration, respectively. (c) 2007 Elsevier B.V. All rights reserved.

Genetic polymorphisms involved in folate metabolism and concentrations of methylmalonic acid and folate on plasma homocysteine and risk of coronary artery disease

Objectives Alterations in the enzymes involved in homocysteine (Hcy) metabolism or vitamin deficiency could play a role in coronary artery disease (CAD) development. This study investigated the influence of MTHFR and MTR gene polymorphisms, plasma folate and MMA on Hcy concentrations and CAD development. MMA and folate concentrations were also investigated according to the polymorphisms. Methods Two hundred and eighty-three unrelated Caucasian individuals undergoing coronary angiography (175 with CAD and 108 non-CAD) were assessed in a case-control study. Plasma Hcy and MMA were measured by liquid chromatography/tandem mass spectrometry. Plasma folate was measured by competitive immunoassay. Dietary intake was evaluated using a nutritional questionnaire. Polymorphisms MTHFR and MTR were investigated by polymerase chain reaction (PCR) followed by enzyme digestion or allele-specific PCR. Results Hcy mean concentrations were higher in CAD patients compared to controls, but below statistical significance (P = 0.246). Increased MMA mean concentrations were frequently observed in the CAD group (P = 0.048). Individuals with MMA concentrations > 0.5 mu mol/l (vitamin B(12) deficiency) were found only in the CAD group (P = 0.004). A positive correlation between MMA and Hcy mean concentrations was observed in both groups, CAD (P = 0.001) and non-CAD (P = 0.020). MMA mean concentrations were significantly higher in patients with hyperhomocysteinemia in both groups, CAD and non-CAD (P = 0.0063 and P = 0.013, respectively). Folate mean concentration was significantly lower in carriers of the wild-type MTHFR 1298AA genotype (P = 0.010). Conclusion Our results suggest a correlation between the MTHFR A1298C polymorphism and plasma folate concentration. Vitamin B(12) deficiency, reflected by increased MMA concentration, is an important risk factor for the development both of hyperhomocysteinemia and CAD.

Mitochondrial energy metabolism in neurodegeneration associated with methylmalonic acidemia

Methylmalonic acidemia is one of the most prevalent inherited metabolic disorders involving neurological deficits. In vitro experiments, animal model studies and tissue analyses from human patients suggest extensive impairment of mitochondrial energy metabolism in this disease. This review summarizes changes in mitochondrial energy metabolism occurring in methylmalonic acidemia, focusing mainly on the effects of accumulated methylmalonic acid, and gives an overview of the results found in different experimental models. Overall, experiments to date suggest that mitochondrial impairment in this disease occurs through a combination of the inhibition of specific enzymes and transporters, limitation in the availability of substrates for mitochondrial metabolic pathways and oxidative damage.

Increased MMA concentration and body mass index are associated with spontaneous abortion in Brazilian women A pilot study

Background: The pathophysiology of spontaneous abortion is complex and may involve the interaction of genetic and environmental factors. We evaluated the predictors of spontaneous abortion in Brazilian pregnant women. The effects of age, gestational age. body mass index (BMI), cigarette smoking, alcohol ingestion, use of multivitamins and concentrations of vitamins (folate, cobalamin and vitamin 136) and vitamin-dependent metabolites were analyzed. Methods: Study population included 100 healthy women that attended pre-natal care in 2 health centers of Sao Paulo, Brazil, and in whom pregnancy outcome was known. Folate and cobalamin status was measured in blood specimens collected between 4 and 16 weeks. The genotypes for 8 gene polymorphisms were evaluated by PCR-RFLP. Results: Eighty-eight women had normal pregnancy outcome (Group 1), while 12 experienced a miscarriage after blood collection (Group 2). Increased methylmalonic acid (MMA) concentrations were found in Group 2 (median [25th-75th percentile]=274 [149-425] nmol/l) relative to Group 1 (138 [98-185]) (P<0.01). No differences between the groups were observed for serum cobalamin, serum or red cell folate, and serum total homocysteine or allele frequencies for 8 polymorphisms. In a conditional logistic regression analysis including age, gestational age, serum creatinine, MMA, cystathionine, body mass index (BMI), cigarette smoking, alcohol ingestion and use of multivitamins the risk of abortion was significantly associated with MMA (OR [95% CI] = 3.80 [1.36, 10.62] per quartile increase in MMA), BMI (OR [95% CI] = 5.49 [1.29,23.39] per quartile) and gestational age (OR [95% CI] = 0.10 [0.01, 0.77] per increase of interval in gestational age). Conclusions: Increased serum MMA and BMI concentrations are associated with spontaneous abortion in Brazilian women. (C) 2009 Elsevier B.V. All rights reserved.

Relationships between gene polymorphisms of folate-related proteins and vitamins and metabolites in pregnant women and neonates

Background: The methylenetetrahydrofolate reductase (MTHFR), glutamate carboxypeptidase II (GCPII) and reduced folate carrier (RFC1) gene polymorphisms were associated with folate status. We investigated the effects of these polymorphisms on serum folate (SF) and folate-related metabolites in mothers and their neonates. Methods: Cobalamin (Cbl), SF, total homocysteine (tHcy), methylmalonic acid (MMA), S-adenosylmethionine (SAM), and S-adenosylhomocysteine (SAH) were measured in 275 healthy women and their neonates. MTHFR C677T, GCPII C1561T and RFC1 A80G polymorphisms were determined by PCR-RFLP. Results: Maternal tHcy was affected individually by MTHFR C677T and GCPII C1561T polymorphisms and by combined genotypes MTHFR 677TT/GCPII 1561CC and MTHFR 677TT/RFC1 80AG. The MTHFR and RFC1 polymorphisms were not associated with variations in vitamins or SAM, SAH and MMA in neonates. Neonatal tHcy was predicted directly by maternal tHcy and inversely by maternal SF, neonatal Cbl and neonatal RFC1 80G allele (AG+GG genotypes). Maternal MMA and SAM/SAH were predicted by creatinine and Cbl, respectively. Neonatal MMA was predicted by maternal MMA and GCPII 1561T allele (CT+TT genotypes) and by neonatal Cbl. Conclusions: Maternal tHcy was affected by MTHFR C677T, RFC1 A80G and GCPII C1561T polymorphisms. Maternal GCPII C1561T variant was associated with neonatal MMA. Neonatal RFC1 A80G polymorphism influenced tHcy in neonates. (C) 2008 Elsevier B.V. All rights reserved.

Association between decreased vitamin levels and MTHFR, MTR and MTRR gene polymorphisms as determinants for elevated total homocysteine concentrations in pregnant women

Objectives: To examine the association between methylenetetrahydrofolate reductase (MTHFR) (C677T and A1298C), methionine synthase (MTR) A2756G and methionine synthase reductase (MTRR) A66G gene polymorphisms and total homocysteine (tHcy), methylmalonic acid (MMA) and S-adenosylmethionine/ S-adenosylhomocysteine (SAM/SAH) levels; and to evaluate the potential interactions with folate or cobalamin (Cbl) status. Subjects/ Methods: Two hundred seventy-five healthy women at labor who delivered full-term normal babies. Cbl, folate, tHcy, MMA, SAM and SAH were measured in serum specimens. The genotypes for polymorphisms were determined by PCR-restriction fragment length polymorphism ( RFLP). Results: Serum folate, MTHFR 677T allele and MTR 2756AA genotypes were the predictors of tHcy levels in pregnant women. Serum Cbl and creatinine were the predictors of SAM/SAH ratio and MMA levels, respectively. The gene polymorphisms were not determinants for MMA levels and SAM/SAH ratios. Low levels of serum folate were associated with elevated tHcy in pregnant women, independently of the gene polymorphisms. In pregnant women carrying MTHFR 677T allele, or MTHFR 1298AA or MTRR 66AA genotypes, lower Cbl levels were associated with higher levels of tHcy. Lower SAM/SAH ratio was found in MTHFR 677CC or MTRR A2756AA genotypes carriers when Cbl levels were lower than 142 pmol/l. Conclusions: Serum folate and MTHFR C677T and MTR A2576G gene polymorphisms were the determinants for tHcy levels. The interaction between low levels of serum Cbl and MTHFR (C677T or A1298C) or MTRR A66G gene polymorphisms was associated with increased tHcy.

DOUBLE ANEUPLOIDY (48,XXY,+21) OF MATERNAL ORIGIN IN A CHILD BORN TO A 13-YEAR-OLD MOTHER: EVALUATION OF THE MATERNAL FOLATE METABOLISM

Double aneuploidy, (48,XXY,+21) of maternal origin in a child born to a 13-year-old mother: evoluation of the maternal folate metabolism: The occurrence of non-mosaic double trisomy is exceptional in newborns. In this paper, a 48,XXY,+21 child, the parental origin of the extra chromosomes and the evaluation of the maternal folate metabolism are presented. The infant was born to a 13-year-old mother and presented with the typical clinical features of Down syndrome (DS). The origin of the additional chromosomes was maternal and most likely resulted from errors during the first meiotic division. Molecular analysis of 12 genetic polymorphisms involved in the folate metabolism revealed that the mother is heterozygous for the MTHFR C677T and TC2 A67G polymorphisms, and homozygous for the mutant MTRR A66G polymorphism. The maternal homocysteine concentration was 4.7 mu mol/L, a value close to the one considered as a risk factor for DS in our previous study. Plasma methylmalonic acid and serum folate concentrations were 0.17 mu mol/L and 18.4 ng/mL, respectively. It is possible that the presence of allelic variants for the folate metabolism and Hey concentration might have favored errors in chromosomal disjunction (hiring gametogenesis in this young mother. To our knowledge, this is the first patient with non-mosaic Down-Klinefelter born to a teenage mother, resulting from a rare fertilization event combining an abnormal 25,XX,+21 oocyte and a 23,Y spermatozoon.

Surface and subsurface erosion of primary enamel by acid beverages over time

This study evaluated the influence of a cola-type soft drink and a soy-based orange juice on the surface and subsurface erosion of primary enamel, as a function of the exposure time. Seventy-five primary incisors were divided for microhardness test (n=45) or scanning electron microscopy (SEM) analysis (n=30). The specimens were randomly assigned to 3 groups: 1 - artificial saliva (control); 2 - cola-type soft drink; and 3 - soy-based orange juice. Immersion cycles in the beverages were undertaken under agitation for 5 min, 3 times a day, during 60 days. Surface microhardness was measured at 7, 15, 30, 45 and 60 days. After 60 days, specimens were bisected and subsurface microhardness was measured at 30, 60, 90, 120, 150 and 200 µm from the surface exposed. Data were analyzed by ANOVA and Tukey’s test (a=0.05). Groups 2 and 3 presented similar decrease of surface microhardness. Regarding subsurface microhardness, group 2 presented the lowest values. SEM images revealed that after 60 days the surfaces clearly exhibited structural loss, unlike those immersed in artificial saliva. It may be concluded that erosion of the surfaces exposed to the cola-type soft drink was more accentuated and directly proportional to the exposure time.

Can previous acid etching increase the bond strength of a self-etching primer adhesive to enamel?

Because a greater research effort has been directed to analyzing the adhesive effectiveness of self etch primers to dentin, the aim of this study was to evaluate, by microtensile testing, the bond strength to enamel of a composite resin combined with a conventional adhesive system or with a self-etching primer adhesive, used according to its original prescription or used with previous acid etching. Thirty bovine teeth were divided into 3 groups with 10 teeth each (n= 10). In one of the groups, a self-etching primer (Clearfil SE Bond - Kuraray) was applied in accordance with the manufacturer's instructions and, in the other, it was applied after previous acid etching. In the third group, a conventional adhesive system (Scotchbond Multipurpose Plus - 3M-ESPE) was applied in accordance with the manufacturer's instructions. The results obtained by analysis of variance revealed significant differences between the adhesive systems (F = 22.31). The self-etching primer (Clearfil SE Bond) presented lower enamel bond strength values than the conventional adhesive system (Scotchbond Multipurpose Plus) (m = 39.70 ± 7.07 MPa) both when used according to the original prescription (m = 27.81 ± 2.64 MPa) and with previous acid etching (m = 25.08 ± 4.92 MPa).

The role of γ -aminobutyric acid (Gaba) in somatic embryogenesis of Acca sellowiana Berg. (Myrtaceae)

The γ-aminobutyric acid (Gaba) is a non-protein amino acid found in prokaryotes and eukaryotes. Its role in plant development has not been fully established. This study reports a quantification of the levels of endogenous Gaba, as well as investigation of its role in different stages of somatic embryogenesis in Acca sellowiana Berg. (Myrtaceae). Zygotic embryos were used as explants and they were inoculated into the culture medium contained different concentrations of Gaba (0,2, 4, 6, 8 and 10 µM). The highest concentrations of endogenous Gaba were detected between the third and nine days after inoculation, reaching the value of 12.77 µmol.g-1FW. High frequency of somatic embryogenesis was observed in response to 10 µM Gaba. This treatment also resulted in a large number of normal embryos, and the lowest percentage of formation of fused somatic embryos, phenotypic characteristic of most deformed embryos in all treatments. Also, all treatments promoted the formation of the somatic embryos with positive characteristics of development resumption, which however did not originate the seedlings.

Tensile bond strength and SEM analysis of enamel etched with Er:YAG laser and phosphoric acid: a comparative study In vitro

Er:YAG laser has been studied as a potential tool for restorative dentistry due to its ability to selectively remove oral hard tissue with minimal or no thermal damage to the surrounding tissues. The purpose of this study was to evaluate in vitro the tensile bond strength (TBS) of an adhesive/composite resin system to human enamel surfaces treated with 37% phosphoric acid, Er:YAG laser (lambda=2.94 mum) with a total energy of 16 J (80 mJ/pulse, 2Hz, 200 pulses, 250 ms pulse width), and Er:YAG laser followed by phosphoric acid etching. Analysis of the treated surfaces was performed by scanning electron microscopy (SEM) to assess morphological differences among the groups. TBS means (in MPa) were as follows: Er:YAG laser + acid (11.7 MPa) > acid (8.2 MPa) > Er:YAG laser (6.1 MPa), with the group treated with laser+acid being significantly from the other groups (p=0.0006 and p= 0.00019, respectively). The groups treated with acid alone and laser alone were significantly different from each other (p=0.0003). The SEM analysis revealed morphological changes that corroborate the TBS results, suggesting that the differences in TBS means among the groups are related to the different etching patterns produced by each type of surface treatment. The findings of this study indicate that the association between Er:YAG laser and phosphoric acid can be used as a valuable resource to increase bond strength to laser-prepared enamel.

Effects of prone and supine position on oxygenation and inflammatory mediator in a hydrochloric acid-induced lung dysfunction in rats

PURPOSE: To compare the effectiveness of mechanical ventilation of supine versus prone position in hydrochloric acid (HCl)-induced lung dysfunction. METHODS: Twenty, adult, male, Wistar-EPM-1 rats were anesthetized and randomly grouped (n=5 animals per group) as follows: CS-MV (mechanical ventilation in supine position); CP-MV (mechanical ventilation in prone position); bilateral instillation of HCl and mechanical ventilation in supine position (HCl+S); and bilateral instillation of HCl and mechanical ventilation in prone position (HCl+P). All groups were ventilated for 180 minutes. The blood partial pressures of oxygen and carbon dioxide were measured in the time points 0 (zero; 10 minutes before lung injury for stabilization), and at the end of times acid injury, 60, 120 and 180 minutes of mechanical ventilation. At the end of experiment the animals were euthanized, and bronchoalveolar lavages (BALs) were taken to determine the contents of total proteins, inflammatory mediators, and lungs wet-to-dry ratios. RESULTS: In the HCl+P group the partial pressure of oxygen increased when compared with HCl+S (128.0±2.9 mmHg and 111.0±6.7 mmHg, respectively) within 60 minutes. TNF-α levels in BAL do not differ significantly in the HCl+P group (516.0±5.9 pg/mL), and the HCl+S (513.0±10.6 pg/mL). CONCLUSION: The use of prone position improved oxygenation, but did not reduce TNF-α in BAL upon lung dysfunction induced by HCl.

Modulation of peritoneal macrophage activity by the saturation state of the fatty acid moiety of phosphatidylcholine

To determine the effects of saturated and unsaturated fatty acids in phosphatidylcholine (PC) on macrophage activity, peritoneal lavage cells were cultured in the presence of phosphatidylcholine rich in saturated or unsaturated fatty acids (sat PC and unsat PC, respectively), both used at concentrations of 32 and 64 µM. The treatment of peritoneal macrophages with 64 µM unsat PC increased the production of hydrogen peroxide by 48.3% compared to control (148.3 ± 16.3 vs 100.0 ± 1.8%, N = 15), and both doses of unsat PC increased adhesion capacity by nearly 50%. Moreover, 64 µM unsat PC decreased neutral red uptake by lysosomes by 32.5% compared to the untreated group (67.5 ± 6.8 vs 100.0 ± 5.5%, N = 15), while both 32 and 64 µM unsat PC decreased the production of lipopolysaccharide-elicited nitric oxide by 30.4% (13.5 ± 2.6 vs 19.4 ± 2.5 µM) and 46.4% (10.4 ± 3.1 vs 19.4 ± 2.5 µM), respectively. Unsat PC did not affect anion production in non-stimulated cells or phagocytosis of unopsonized zymosan particles. A different result pattern was obtained for macrophages treated with sat PC. Phorbol 12-miristate 13-acetate-elicited superoxide production and neutral red uptake were decreased by nearly 25% by 32 and 64 µM sat PC, respectively. Sat PC did not affect nitric oxide or hydrogen peroxide production, adhesion capacity or zymosan phagocytosis. Thus, PC modifies macrophage activity, but this effect depends on cell activation state, fatty acid saturation and esterification to PC molecule and PC concentration. Taken together, these results indicate that the fatty acid moiety of PC modulates macrophage activity and, consequently, is likely to affect immune system regulation in vivo.

Synthesis, screening for antiacetylcholinesterase activity and binding mode prediction of a new series of [3-(disubstituted-phosphate)-4,4,4-trifluoro-butyl]-carbamic acid ethyl esters

A series of nine new [3-(disubstituted-phosphate)-4,4,4-trifluoro-butyl]-carbamic acid ethyl esters (phosphate-carbamate compounds) was obtained through the reaction of (4,4,4-trifluoro-3-hydroxybut-1-yl)-carbamic acid ethyl esters with phosphorus oxychloride followed by the addition of alcohols. The products were characterized by ¹H, 13C, 31P, and 19F NMR spectroscopy, GC-MS, and elemental analysis. All the synthesized compounds were screened for acetylcholinesterase (AChE) inhibitory activity using the Ellman method. All compounds containing phosphate and carbamate pharmacophores in their structures showed enzyme inhibition, being the compound bearing the diethoxy phosphate group (2b) the most active compound. Molecular modeling studies were performed to investigate the detailed interactions between AChE active site and small-molecule inhibitor candidates, providing valuable structural insights into AChE inhibition.