Determining associations and assessing methodological issues in a case-control study of hazardous air pollutants and neural tube defects

Recent studies have reported positive associations between maternal exposures to air pollutants and several adverse birth outcomes. However, there have been no assessments of the association between environmental hazardous air pollutants (HAPs) such as benzene, toluene, ethylbenzene, and xylene (BTEX) and neural tube defects (NTDs) a common and serious group of congenital malformations. Before examining this association, two important methodological questions must be addressed: (1) is maternal residential movement likely to result in exposure misclassification and (2) is it appropriate to lump defects of the neural tube, such as anencephaly and spina bifida, into a composite disease endpoint (i.e., NTDs). ^ Data from the National Birth Defects Prevention Study and Texas Birth Defects Registry were used to: (1) assess the extent to which change of residence may result in exposure misclassification when exposure is based on the address at delivery; (2) formally assess heterogeneity of the associations between known risk factors for NTDs, using polytomous logistic regression; and (3) conduct a case-control study assessing the association between ambient air levels of BTEX and the risk of NTDs among offspring. ^ Regarding maternal residential mobility, this study suggests address at delivery was not significantly different from using address at conception when assigning quartile of benzene exposure (OR 1.0, 95% CI 0.9, 1.3). On the question of effect heterogeneity among NTDs, the effect estimates for infant sex P = 0.017), maternal body mass index P = 0.016), and folate supplementation P = 0.050) were significantly different for anencephaly and spina bifida, suggesting it is often more appropriate to assess potential risk factors among subgroups of NTDs. For the main study question on the association between environmental HAPs and NTDs, mothers who have offspring with isolated spina bifida are 2.4 times likely to live in areas with the highest benzene levels (95% CI 1.1, 5.0). However, no other significant associations were observed.^ This project is the first to include not only an assessment of the relationship between environmental levels of BTEX and NTDs, but also two separate studies addressing important methodological issues associated with this question. Our results contribute to the growing body of evidence regarding air pollutant exposure and adverse birth outcomes. ^

Colorectal cancer: An insight on dietary factors

Aim. To assess the relationships between dietary factors and colorectal cancer risk. ^ Methods. We looked at all the systematic reviews published in last ten years on the topic. ^ Results. For fruits-vegetables some studies1 were significant for heterogeneity and others2 were not. In study by Aune at al3 only fruits were significant, although all the studies had protective RR between 0.90 to 0.94. For folate only case-control group of studies, the study by Sanjoaquin et al4 was significant with p heterogeneity being 0.01 and all of them had protective effect with RR between 0.75 to 0.95, for dietary as well as total folate. For fiber study by Park et al5 p was insignificant at 0.14 an RR was 0.84. Vitamin B6 study by Larsson et al6 had significant p with RR 0.90. For dietary fat both Alexander7 and Liu8 concluded that there is insufficient evidence that dietary fat is an independent causative risk factor. Only one study by Norat et al9 out of three was able to achieve significant p heterogeneity for meat. All the studies reported RR between 1.14 to 1.35, clearly implicating meat as culprit for increasing the risk of colorectal cancer. ^ Conclusions. We would recommend the use of fruits and vegetables to be protective against colorectal cancer. Also meat consumption increases the risk of colorectal cancer.^ *Please refer to dissertation for references/footnotes.^

Evolution and treatment of vitamin B12 deficiency as a risk factor for (cognitive and functional) neurodegenerative diseases in institutionalized elderly = Evolución y tratamiento de la deficiencia de vitamina B12 como factor de riesgo de enfermedades neurodegenerativas (cognitivas y funcionales) en las personas mayores institucionalizadas.

Prevalence of vitamin B12 deficiency is very common in elderly people and can reach values as high as 40.5% of the population. It can be the result of the interaction among several factors. Vitamin B12 deficiencies have been associated with neurological, cognitive deterioration, haematological abnormalities and cardiovascular diseases that have an important influence on the health of the elderly and their quality of life. It is necessary to approach the problems arisen from the lack of data relative to them. The main objective of this thesis was to analyse the evolution of vitamin B12 status and related parameters, lipid and haematological profiles and their relationship to health risk factors, and to functional and cognitive status over one year and to determine the effect of an oral supplementation of 500 μg of cyanocobalamin for a short period of 28 days. An additional objective was to analyze the possible effects of medicine intakes on vitamin B status. Three studies were performed: a) a one year longitudinal follow-up with four measure points; b) an intervention study providing an oral liquid supplement of 500 μg of cyanocobalamin for a 28 days period; and c) analysis of the possible effect of medication intake on vitamin B status using the ATC classification of medicines. The participants for these studies were recruited from nursing homes for the elderly in the Region of Madrid. Sixty elders (mean age 84 _ 7y, 19 men and 41 women) were recruited for Study I and 64 elders (mean age 82 _ 7y, 24 men and 40 women) for Study II. For Study III, baseline data from the initially recruited participants of the first two studies were used. An informed consent was obtained from all participants or their mentors. The studies were approved by the Ethical Committee of the University of Granada. Blood samples were obtained at each examination date and were analyzed for serum cobalamin, holoTC, serum and RBC folate and total homocysteine according to laboratory standard procedures. The haematological parameters analyzed were haematocrit, haemoglobin and MCV. For the lipid profile TG, total cholesterol, LDL- and HDLcholesterol were analyzed. Anthropometric measures (BMI, skinfolds [triceps and subscapular], waist girth and waist to hip ratio), functional tests (hand grip, arm and leg strength tests, static balance) and MMSE were obtained or administered by trained personal. The vitamin B12 supplement of Study II was administered with breakfast and the medication intake was taken from the residents’ anamnesis. Data were analyzed by parametric and non-parametric statistics depending on the obtained data. Comparisons were done using the appropriate ANOVAs or non-parametric tests. Pearsons’ partial correlations with the variable “time” as control were used to define the association of the analyzed parameters. XIII The results showed that: A) Over one year, in relationship to vitamin B status, serum cobalamin decreased, serum folate and mean corpuscular volumen increased significantly and total homocysteine concentrations were stable. Regarding blood lipid profile, triglycerides increased and HDL-cholesterol decreased significantly. Regarding selected anthropometric measurements, waist circumference increased significantly. No significant changes were observed for the rest of parameters. B) Prevalence of hyperhomocysteinemia was high in the elderly studied, ranging from 60% to 90 % over the year depending on the cut-off used for the classification. LDL-cholesterol values were high, especially among women, and showed a tendency to increase over the year. Results of the balance test showed a deficiency and a tendency to decrease; this indicates that the population studied is at high risk for falls. Lower extremity muscular function was deficient and showed a tendency to decrease. A highly significant relationship was observed between the skinfold of the triceps and blood lipid profile. C) Low cobalamin concentrations correlated significantly with low MMSE scores in the elderly studied. No correlations were observed between vitamin B12 status and functional parameters. D) Regarding vitamin B12 status, holo-transcobalamin seems to be more sensitive for diagnosis; 5-10% of the elderly had a deficiency using serum cobalamin as a criterion, and 45-52% had a deficiency when using serum holotranscobalamin as a criterion. E) 500 μg of cyanocobalamin administered orally during 28 days significantly improved vitamin B12 status and significantly decreased total homocysteine concentrations in institutionalized elderly. No effect of the intervention was observed on functional and cognitive parameters. F) The relative change (%) of improvement of vitamin B12 status was higher when using serum holo-transcobalamin as a criterion than serum cobalamin. G) Antiaenemic drug intake normalized cobalamin, urologic drugs and corticosteroids serum folate, and psychoanaleptics holo-transcobalamin levels. Drugs treating pulmonary obstruction increased total homocysteine concentration significantly. H) The daily mean drug intake was 5.1. Fiftynine percent of the elderly took medication belonging to 5 or more different ATC groups. The most prevalent were psycholeptic (53%), antiacid (53%) and antithrombotic (47%) drugs.

Effects of physical exercise and hydration on homocysteine concentrations in physically active male adults = Efectos del ejercicio físico y la hidratación sobre las concentraciones de homocisteína en varones físicamente activos

La presente tesis analiza el efecto del ejercicio físico agudo y la hidratación sobre las concentraciones de homocisteína total (tHcy) y su relación con los parámetros implicados en el metabolismo de la homocisteína como el folato, la vitamina B12, y la creatina en una muestra de varones jóvenes físicamente activos. El trabajo se basa en los resultados del estudio realizado en la Facultad de Ciencias de la Actividad Física y del Deporte de la Universidad Politécnica de Madrid. Para el cual se contó con un total de 29 voluntarios sanos físicamente activos de la Comunidad de Madrid. Los principales resultados de esta tesis son: a) Las concentraciones de tHcy aumentaron después del ejercicio agudo tanto tras una prueba de intensidad máxima (VO2max) como una submáxima (65 % of VO2max) en varones físicamente activos independientemente de las sus concentraciones basales de tHcy. b) Las concentraciones de tHcy disminuyeron 2 h después del ejercicio físico aeróbico submáximo tras aplicar un protocolo de hidratación con una bebida para deportistas. c) Un adecuado protocolo de hidratación durante el ejercicio físico agudo previno el aumento de las concentraciones de tHcy hasta 2 h después del ejercicio. d) Las concentraciones de tHcy aumentaron a las 6 h tras la finalización del ejercicio únicamente en los test en los que no se siguió un protocolo de hidratación durante el ejercicio físico. e) A las 24 h tras el ejercicio, las concentraciones de tHcy volvieron a los niveles basales independientemente de si se aplicó un protocolo de hidratación durante el ejercicio o no. f) Es necesario aclarar si existen mecanismos subyacentes relacionados con el riesgo cardiovascular debido al aumento transitorio de las concentraciones de tHcy inducidas por el ejercicio agudo. Se necesitan más estudios que analicen la relación entre las concentraciones de tHcy después del ejercicio físico agudo y la implicación de la creatina, vitamina B12 y folato como parámetros relacionados en el metabolismo de la homocisteína. El efecto agudo del ejercicio físico aumenta las concentraciones de tHcy por encima de los valores recomendados; sin embargo, un adecuado protocolo de hidratación mantiene las concentraciones a niveles basales y previene el posterior aumento en una muestra de varones adultos físicamente activos. ABSTRACT The current thesis analyzes the effect of exercise and hydration on total homocysteine (tHcy) concentrations and the relationship with the implicated parameters, like folate, vitamin B12, and creatine in physically active male adults. The work is based on the results of the study conducted at the Faculty of Physical Activity and Sport Sciences of the Technical University of Madrid. A total of 29 physically active voluntary healthy males from the Region of Madrid were recruited. The main outcomes of this thesis are: a) tHcy concentrations increased after acute exercise with both, maximal (VO2max) and submaximal (65 % of VO2max) tests in physically active male subjects independently of their baseline tHcy status. b) After 2 h of rehydration with a sport drink, tHcy concentrations, which had previously increased during an acute exercise, decreased significantly, although they didn´t recover to baseline values. c) An adequate hydration protocol during acute aerobic submaximal exercise prevents the increase of tHcy concentrations and maintains these concentrations at baseline up to 2 h post-exercise. d) Serum tHcy concentrations increased after submaximal exercise when the hydration protocol during exercise was not applied. Furthermore, tHcy concentrations reached maximal values 6 h after the end of exercise. e) At 24 h, tHcy concentrations recovered baseline values independently whether or not there was a hydration protocol during exercise. f) There is a need to clarify the underlying mechanisms related to cardiovascular risk due to the transient increase of tHcy concentrations induced by acute exercise. Further research analayzing the relationship between tHcy concentrations after acute exercise and the implication of creatine, vitamin B12 and folate as related parameters in the homocysteine metabolism is needed. Finally, tHcy concentrations increased above the recommended values after an acute aerobic submaximal exercise; nevertheless, a good hydration protocol maintains tHcy concentrations at baseline and prevents the further increase in a sample of physically active male adults.

Polymorphisms in the methylenetetrahydrofolate reductase gene are associated with susceptibility to acute leukemia in adults

Reduction of 5,10-methylenetetrahydrofolate (methyleneTHF), a donor for methylating dUMP to dTMP in DNA synthesis, to 5-methyltetrahydrofolate (methylTHF), the primary methyl donor for methionine synthesis, is catalyzed by 5,10-methylenetetrahydrofolate reductase (MTHFR). A common 677 C → T polymorphism in the MTHFR gene results in thermolability and reduced MTHFR activity that decreases the pool of methylTHF and increases the pool of methyleneTHF. Recently, another polymorphism in MTHFR (1298 A → C) has been identified that also results in diminished enzyme activity. We tested whether carriers of these variant alleles are protected from adult acute leukemia. We analyzed DNA from a case–control study in the United Kingdom of 308 adult acute leukemia patients and 491 age- and sex-matched controls. MTHFR variant alleles were determined by a PCR-restriction fragment length polymorphism assay. The MTHFR 677TT genotype was lower among 71 acute lymphocytic leukemia (ALL) cases compared with 114 controls, conferring a 4.3-fold decrease in risk of ALL [odds ratio (OR = 0.23; 95% CI = 0.06–0.81]. We observed a 3-fold reduction in risk of ALL in individuals with the MTHFR 1298AC polymorphism (OR = 0.33; 95% CI = 0.15–0.73) and a 14-fold decreased risk of ALL in those with the MTHFR 1298CC variant allele (OR = 0.07; 95% CI = 0.00–1.77). In acute myeloid leukemia, no significant difference in MTHFR 677 and 1298 genotype frequencies was observed between 237 cases and 377 controls. Individuals with the MTHFR 677TT, 1298AC, and 1298CC genotypes have a decreased risk of adult ALL, but not acute myeloid leukemia, which suggests that folate inadequacy may play a key role in the development of ALL.

A common mutation in the methylenetetrahydrofolate reductase gene is associated with an accumulation of formylated tetrahydrofolates in red blood cells

A common mutation (C677T) in the gene encoding for methylenetetrahydrofolate reductase (MTHFR) (5-methyltetrahydrofolate:(acceptor) oxidoreductase, EC 1.7.99.5), a key regulatory enzyme in one-carbon metabolism, results in a thermolabile variant of the MTHFR enzyme with reduced activity in vitro. In the present study we used a chromatographic method for folate analysis to test the hypothesis that this mutation would be associated with altered distribution of red blood cell (RBC) folates. An alteration was found as manifested by the presence of formylated tetrahydrofolate polyglutamates in addition to methylated derivatives in the RBCs from homozygous mutant individuals. 5-Methyltetrahydrofolate polyglutamates were the only folate form found in RBCs from individuals with the wild-type genotype. Existence of formylated folates in RBCs only from individuals with the thermolabile MTHFR is consistent with the hypothesis that there is in vivo impairment in the activity of the thermolabile variant of MTHFR and that this impairment results in an altered distribution of RBC folates.

Homocysteine induces congenital defects of the heart and neural tube: Effect of folic acid

The biological basis or mechanism whereby folate supplementation protects against heart and neural tube defects is unknown. It has been hypothesized that the amino acid homocysteine may be the teratogenic agent, since serum homocysteine increases in folate depletion; however, this hypothesis has not been tested. In this study, avian embryos were treated directly with d,l-homocysteine or with l-homocysteine thiolactone, and a dose response was established. Of embryos treated with 50 μl of the teratogenic dose (200 mM d,l-homocysteine or 100 mM l-homocysteine thiolactone) on incubation days 0, 1, and 2 and harvested at 53 h (stage 14), 27% showed neural tube defects. To determine the effect of the teratogenic dose on the process of heart septation, embryos were treated during incubation days 2, 3, and 4; then they were harvested at day 9 following the completion of septation. Of surviving embryos, 23% showed ventricular septal defects, and 11% showed neural tube defects. A high percentage of the day 9 embryos also showed a ventral closure defect. The teratogenic dose was shown to raise serum homocysteine to over 150 nmol/ml, compared with a normal level of about 10 nmol/ml. Folate supplementation kept the rise in serum homocysteine to ≈45 nmol/ml, and prevented the teratogenic effect. These results support the hypothesis that homocysteine per se causes dysmorphogenesis of the heart and neural tube, as well as of the ventral wall.

Segregation of Heterotrimeric G Proteins in Cell Surface Microdomains: Gq Binds Caveolin to Concentrate in Caveolae, whereas Gi and Gs Target Lipid Rafts by Default

Select lipid-anchored proteins such as glycosylphosphatidylinositol (GPI)-anchored proteins and nonreceptor tyrosine kinases may preferentially partition into sphingomyelin-rich and cholesterol-rich plasmalemmal microdomains, thereby acquiring resistance to detergent extraction. Two such domains, caveolae and lipid rafts, are morphologically and biochemically distinct, contain many signaling molecules, and may function in compartmentalizing cell surface signaling. Subfractionation and confocal immunofluorescence microscopy reveal that, in lung tissue and in cultured endothelial and epithelial cells, heterotrimeric G proteins (Gi, Gq, Gs, and Gβγ) target discrete cell surface microdomains. Gq specifically concentrates in caveolae, whereas Gi and Gs concentrate much more in lipid rafts marked by GPI-anchored proteins (5′ nucleotidase and folate receptor). Gq, apparently without Gβγ subunits, stably associates with plasmalemmal and cytosolic caveolin. Gi and Gs interact with Gβγ subunits but not caveolin. Gi and Gs, unlike Gq, readily move out of caveolae. Thus, caveolin may function as a scaffold to trap, concentrate, and stabilize Gq preferentially within caveolae over lipid rafts. In N2a cells lacking caveolae and caveolin, Gq, Gi, and Gs all concentrate in lipid rafts as a complex with Gβγ. Without effective physiological interaction with caveolin, G proteins tend by default to segregate in lipid rafts. The ramifications of the segregated microdomain distribution and the Gq-caveolin complex without Gβγ for trafficking, signaling, and mechanotransduction are discussed.

Methylenetetrahydrofolate reductase (MTHFR) polymorphisms and risk of molecularly defined subtypes of childhood acute leukemia

Low folate intake as well as alterations in folate metabolism as a result of polymorphisms in the enzyme methylenetetrahydrofolate reductase (MTHFR) have been associated with an increased incidence of neural tube defects, vascular disease, and some cancers. Polymorphic variants of MTHFR lead to enhanced thymidine pools and better quality DNA synthesis that could afford some protection from the development of leukemias, particularly those with translocations. We now report associations of MTHFR polymorphisms in three subgroups of pediatric leukemias: infant lymphoblastic or myeloblastic leukemias with MLL rearrangements and childhood lymphoblastic leukemias with either TEL-AML1 fusions or hyperdiploid karyotypes. Pediatric leukemia patients (n = 253 total) and healthy newborn controls (n = 200) were genotyped for MTHFR polymorphisms at nucleotides 677 (C→T) and 1,298 (A→C). A significant association for carriers of C677T was demonstrated for leukemias with MLL translocations (MLL+, n = 37) when compared with controls [adjusted odd ratios (OR) = 0.36 with a 95% confidence interval (CI) of 0.15–0.85; P = 0.017]. This protective effect was not evident for A1298C alleles (OR = 1.14). In contrast, associations for A1298C homozygotes (CC; OR = 0.26 with a 95% CI of 0.07–0.81) and C677T homozygotes (TT; OR = 0.49 with a 95% CI of 0.20–1.17) were observed for hyperdiploid leukemias (n = 138). No significant associations were evident for either polymorphism with TEL-AML1+ leukemias (n = 78). These differences in allelic associations may point to discrete attributes of the two alleles in their ability to alter folate and one-carbon metabolite pools and impact after DNA synthesis and methylation pathways, but should be viewed cautiously pending larger follow-up studies. The data provide evidence that molecularly defined subgroups of pediatric leukemias have different etiologies and also suggest a role of folate in the development of childhood leukemia.

P-glycoprotein confers methotrexate resistance in 3T6 cells with deficient carrier-mediated methotrexate uptake.

P-glycoprotein (Pgp), a transmembrane efflux pump encoded by the MDR1 gene, transports various lipophilic drugs that enter the cell by passive diffusion through the lipid bilayer. Pgp-expressing multidrug-resistant cell lines are not usually cross-resistant to a hydrophilic antifolate methotrexate (MTX). MTX enters cells primarily through a folate carrier, but passive diffusion becomes the primary mode of MTX uptake in carrier-deficient cells. To test if a deficiency in MTX carrier would allow Pgp to confer resistance to MTX, a MTX carrier-deficient cell line (3T6-C26) was infected with a recombinant retrovirus expressing the human MDR1 gene. The infected 3T6-C26 cells showed increased survival in MTX relative to uninfected cells. Multistep selection of the infected cells with vinblastine led to increased Pgp expression and a concomitant increase in resistance to MTX. MTX resistance of Pgp-expressing 3T6-C26 cells was reduced by Pgp inhibitors, including a Pgp-specific monoclonal antibody UTC2. In contrast, the expression and the inhibition of Pgp had no effect on MTX resistance in 3T6 cells with normal carrier-mediated MTX uptake. Thus, a deficiency in the MTX carrier enables Pgp to confer resistance to MTX, suggesting that hydrophilic compounds may become Pgp substrates when such compounds enter cells by passive diffusion.

Simple tandem DNA repeats and human genetic disease.

The human genome contains many repeated DNA sequences that vary in complexity of repeating unit from a single nucleotide to a whole gene. The repeat sequences can be widely dispersed or in simple tandem arrays. Arrays of up to 5 or 6 nt are known as simple tandem repeats, and these are widely dispersed and highly polymorphic. Members of one group of the simple tandem repeats, the trinucleotide repeats, can undergo an increase in copy number by a process of dynamic mutation. Dynamic mutations of the CCG trinucleotide give rise to one group of fragile sites on human chromosomes, the rare folate-sensitive group. One member of this group, the fragile X (FRAXA) is responsible for the most common familial form of mental retardation. Another member of the group FRAXE is responsible for a rarer mild form of mental retardation. Similar mutations of AGC repeats give rise to a number of neurological disorders. The expanded repeats are unstable between generations and somatically. The intergenerational instability gives rise to unusual patterns of inheritance--particularly anticipation, the increasing severity and/or earlier age of onset of the disorder in successive generations. Dynamic mutations have been found only in the human species, and possible reasons for this are considered. The mechanism of dynamic mutation is discussed, and a number of observations of simple tandem repeat mutation that could assist in understanding this phenomenon are commented on.

Novel multiple sclerosis susceptibility loci implicated in epigenetic regulation.

We conducted a genome-wide association study (GWAS) on multiple sclerosis (MS) susceptibility in German cohorts with 4888 cases and 10,395 controls. In addition to associations within the major histocompatibility complex (MHC) region, 15 non-MHC loci reached genome-wide significance. Four of these loci are novel MS susceptibility loci. They map to the genes L3MBTL3, MAZ, ERG, and SHMT1. The lead variant at SHMT1 was replicated in an independent Sardinian cohort. Products of the genes L3MBTL3, MAZ, and ERG play important roles in immune cell regulation. SHMT1 encodes a serine hydroxymethyltransferase catalyzing the transfer of a carbon unit to the folate cycle. This reaction is required for regulation of methylation homeostasis, which is important for establishment and maintenance of epigenetic signatures. Our GWAS approach in a defined population with limited genetic substructure detected associations not found in larger, more heterogeneous cohorts, thus providing new clues regarding MS pathogenesis.

Efficacy of B vitamins in lowering homocysteine in older men - Maximal effects for those with B-12 deficiency and hyperhomocysteinemia

Background and Purpose - A higher plasma concentration of total homocysteine (tHcy) is associated with a greater risk of cardiovascular events. Previous studies, largely in younger individuals, have shown that B vitamins lowered tHcy by substantial amounts and that this effect is greater in people with higher tHcy and lower folate levels. Methods - We undertook a 2-year, double-blind, placebo-controlled, randomized trial in 299 men aged >= 75 years, comparing treatment with a daily tablet containing 2 mg of folate, 25 mg of B-6, and 400 mu g of B-12 or placebo. The study groups were balanced regarding age (mean +/- SD, 78.9 +/- 2.8 years), B vitamins, and tHcy at baseline. Results - Among the 13% with B12 deficiency, the difference in mean changes in treatment and control groups for tHcy was 6.74 mu mol/L (95% CI, 3.94 to 9.55 mu mol/L) compared with 2.88 mu mol/L (95% CI, 0.07 to 5.69 mu mol/L) for all others. Among the 20% with hyperhomocysteinaemia, the difference between mean changes in treatment and control groups for men with high plasma tHcy compared with the rest of the group was 2.8 mu mol/L (95% CI, 0.6 to 4.9 mu mol/L). Baseline vitamin B12, serum folate, and tHcy were significantly associated with changes in plasma tHcy at follow-up (r = 0.252, r = 0.522, and r = -0.903, respectively; P = 0.003, <0.001, and <0.001, respectively) in the vitamin group. Conclusions - The tHcy-lowering effect of B vitamins was maximal in those who had low B12 or high tHcy levels. Community-dwelling older men, who are likely to be deficient in B12 or have hyperhomocysteinemia, may be most likely to benefit from treatment with B vitamins.

Downstream effects on human low density lipoprotein of homocysteine exported from endothelial cells in an in vitro system

A model system is presented using human umbilical vein endothelial cells (HUVECs) to investigate the role of homocysteine (Hcy) in atherosclerosis. HUVECs are shown to export Hcy at a rate determined by the flux through the methionine/Hcy pathway. Additional methionine increases intracellular methionine, decreases intracellular folate, and increases Hcy export, whereas additional folate inhibits export. An inverse relationship exists between intracellular folate and Hcy export. Hcy export may be regulated by intracellular S-adenosyl methionine rather than by Hcy. Human LDLs exposed to HUVECs exporting Hcy undergo time-related lipid oxidation, a process inhibited by the thiol trap dithionitrobenzoate. This is likely to be related to the generation of hydroxyl radicals, which we show are associated with Hcy export. Although Hcy is the major oxidant, cysteine also contributes, as shown by the effect of glutamate. Finally, the LDL oxidized in this system showed a time-dependent increase in uptake by human macrophages, implying an upregulation of the scavenger receptor. These results suggest that continuous export of Hcy from endothelial cells contributes to the generation of extracellular hydroxyl radicals, with associated oxidative modification of LDL and incorporation into macrophages, a key step in atherosclerosis. Factors that regulate intracellular Hcy metabolism modulate these effects. Copyright © 2005 by the American Society for Biochemistry and Molecular Biology, Inc.