Polymorphisms in genes involved in folate metabolism as maternal risk factors for Down syndrome.

Down syndrome is a complex genetic and metabolic disorder attributed to the presence of three copies of chromosome 21. The extra chromosome derives from the mother in 93% of cases and is due to abnormal chromosome segregation during meiosis (nondisjunction). Except for advanced age at conception, maternal risk factors for meiotic nondisjunction are not well established. A recent preliminary study suggested that abnormal folate metabolism and the 677C-->T polymorphism in the methylenetetrahydrofolate reductase (MTHFR) gene may be maternal risk factors for Down syndrome. The present study was undertaken with a larger sample size to determine whether the MTHFR 677C-->T polymorphism was associated with increased risk of having a child with Down syndrome. Methionine synthase reductase (MTRR) is another enzyme essential for normal folate metabolism. A common polymorphism in this gene was recently associated with increased risk of neural tube defects and might also contribute to increased risk for Down syndrome. The frequencies of the MTHFR 677C-->T and MTRR 66A-->G mutations were evaluated in DNA samples from 157 mothers of children with Down syndrome and 144 control mothers. Odds ratios were calculated for each genotype separately and for potential gene-gene interactions. The results are consistent with the preliminary observation that the MTHFR 677C-->T polymorphism is more prevalent among mothers of children with Down syndrome than among control mothers, with an odds ratio of 1.91 (95% confidence interval [CI] 1.19-3.05). In addition, the homozygous MTRR 66A-->G polymorphism was independently associated with a 2. 57-fold increase in estimated risk (95% CI 1.33-4.99). The combined presence of both polymorphisms was associated with a greater risk of Down syndrome than was the presence of either alone, with an odds ratio of 4.08 (95% CI 1.94-8.56). The two polymorphisms appear to act without a multiplicative interaction.

Increase in plasma homocysteine associated with parallel increases in plasma S-adenosylhomocysteine and lymphocyte DNA hypomethylation.

S-Adenosylmethionine and S-adenosylhomocysteine (SAH), as the substrate and product of essential cellular methyltransferase reactions, are important metabolic indicators of cellular methylation status. Chronic elevation of SAH, secondary to the homocysteine-mediated reversal of the SAH hydrolase reaction, reduces methylation of DNA, RNA, proteins, and phospholipids. High affinity binding of SAH to the active site of cellular methyltransferases results in product inhibition of the enzyme. Using a sensitive new high pressure liquid chromatography method with coulometric electrochemical detection, plasma SAH levels in healthy young women were found to increase linearly with mild elevation in homocysteine levels (r = 0.73; p < 0.001); however, S-adenosylmethionine levels were not affected. Plasma SAH levels were positively correlated with intracellular lymphocyte SAH levels (r = 0.81; p < 0.001) and also with lymphocyte DNA hypomethylation (r = 0.74, p < 0.001). These results suggest that chronic elevation in plasma homocysteine levels, such as those associated with nutritional deficiencies or genetic polymorphisms in the folate pathway, may have an indirect and negative effect on cellular methylation reactions through a concomitant increase in intracellular SAH levels.

Abnormal folate metabolism and mutation in the methylenetetrahydrofolate reductase gene may be maternal risk factors for Down syndrome.

BACKGROUND: Down syndrome, or trisomy 21, is a complex genetic disease resulting from the presence of 3 copies of chromosome 21. The origin of the extra chromosome is maternal in 95% of cases and is due to the failure of normal chromosomal segregation during meiosis. Although advanced maternal age is a major risk factor for trisomy 21, most children with Down syndrome are born to mothers <30 y of age. OBJECTIVE: On the basis of evidence that abnormal folate and methyl metabolism can lead to DNA hypomethylation and abnormal chromosomal segregation, we hypothesized that the C-to-T substitution at nucleotide 677 (677C-->T) mutation of the methylenetetrahydrofolate reductase (MTHFR) gene may be a risk factor for maternal meiotic nondisjunction and Down syndrome in young mothers. DESIGN: The frequency of the MTHFR 677C-->T mutation was evaluated in 57 mothers of children with Down syndrome and in 50 age-matched control mothers. Ratios of plasma homocysteine to methionine and lymphocyte methotrexate cytotoxicity were measured as indicators of functional folate status. RESULTS: A significant increase in plasma homocysteine concentrations and lymphocyte methotrexate cytotoxicity was observed in the mothers of children with Down syndrome, consistent with abnormal folate and methyl metabolism. Mothers with the 677C-->T mutation had a 2.6-fold higher risk of having a child with Down syndrome than did mothers without the T substitution (odds ratio: 2.6; 95% CI: 1.2, 5.8; P < 0.03). CONCLUSION: The results of this initial study indicate that folate metabolism is abnormal in mothers of children with Down syndrome and that this may be explained, in part, by a mutation in the MTHFR gene.

A new HPLC method for the simultaneous determination of oxidized and reduced plasma aminothiols using coulometric electrochemical detection.

A new method has been developed that is capable of providing a complete profile of the most common monothiols and disulfides present in plasma or tissue extracts. The method utilizes reversed phase ion-pairing high performance liquid chromatography coupled with coulometric electrochemical detection to simultaneously quantify free oxidized and reduced aminothiols or total aminothiols after chemical reduction. The method is extremely sensitive, with limits of detection in the 5 fmol/mL range for monothiols and 50 fmol/mL for dithiols. The interassay and intraassay coefficients of variation for total and free aminothiols ranged between 1.2 and 5.8%. The mean recoveries for total and plasma aminothiols ranged between 97.1 and 102.8%. The aminothiols are quantified directly, without derivatization, and include methionine, homocysteine, homocystine, cystathionine, cysteine, cystine, cysteinylglycine, and oxidized and reduced glutathione. Because a complete aminothiol profile of metabolites in both the remethylation (anabolic) and transulfuration (catabolic) pathways of homocysteine metabolism can be determined simultaneously, this new method should be useful in determining the metabolic etiology of homocysteinemia and in designing appropriate nutritional intervention strategies. Basic research applications of this method should lead to an increased understanding of the metabolic pathology of aminothiol imbalance.

Anthropometric measures and metabolic rate in association with risk of breast cancer (United States).

To investigate whether cancer risk-reduction seen in calorie-restricted animals also applies to breast cancer in women, we have analyzed data from the first National Health and Nutrition Examination Survey in the United States and subsequent follow-up surveys. During the follow-up of one to 155 months, 182 out of 7,622 women developed breast cancer. Due to biased under-reporting of dietary intake, the analysis did not examine calorie intake as an exposure variable, but rather focused on anthropometric measures and metabolic rate as biomarkers of nutritional balance. Multiple Cox regression analysis showed elevated odds ratios (OR) for height, elbow width, and skinfolds among postmenopausal women. ORs for the fifth quintile were 2.0 (95 percent confidence interval [CI] = 1.0-3.8), 2.3 (CI = 1.2-4.7), and 2.0 (CI = 1.0-4.0), respectively. Weight (OR = 2.5, CI = 1.2-5.1) and resting metabolic rate (OR = 2.0, CI = 1.0-4.0) were significant relative to the second quintile. Bitrochanteric breadth, sitting height, body fat, body mass index, or combination variables were not associated with cancer risk. It was concluded that in the analysis of breast cancer data, skeletal measures ought to be considered as routine potential confounders, and that using measured rather than estimated metabolic rates may improve risk prediction.

Cigarette smoking, intracellular vitamin deficiency, and occurrence of micronuclei in epithelial cells of the buccal mucosa.

The study focuses on the assessment of chromosomal damage associated with folate and vitamin B12 deficiency, and with cigarette smoking in a tissue directly exposed to cigarette smoke (buccal mucosa) while controlling for potential confounding factors. A cross-sectional study was carried out among 39 current smokers (CSs) and 60 noncurrent smokers (NCSs). Buccal mucosal cells, saliva, and blood samples were collected from each subject. The Health Habits and History Questionnaire (Block et al., 1986) was modified to obtain dietary and other relevant information. Methods used to measure folate, vitamin B12 levels, and the frequency of micronucleated cells in buccal mucosal cells gave reproducible results. The study results suggest that CSs have buccal mucosal folate and vitamin B12 levels that are lower than those among NCSs. CSs were three times more likely to have micronucleated buccal mucosal cells compared to NCSs. There appeared to be no association between low buccal folate and vitamin B12 levels chromosomal damage. The salivary vitamin B12 concentrations and plasma vitamin C and E concentrations, however, seem to be marginally protective against the occurrence of buccal mucosal micronuclei, whereas plasma beta-carotene seems to increase the occurrence of micronuclei. Overall, the results do not support the concept that localized folate and vitamin B12 deficiencies in the buccal mucosal cells of smokers are associated with chromosomal damage in those cells. The presence of vitamin B12 deficiencies in the buccal mucosal cells of smokers are associated with chromosomal damage in those cells. The presence of vitamin B12 in the immediate environment (saliva) and vitamin C and E in the plasma, however, appear to be marginally protective against chromosomal damage in buccal mucosal cells.

Local and systemic effects of cigarette smoking on folate and vitamin B-12.

A cross-sectional study was carried out among 39 current smokers (CS) and 60 noncurrent smokers (NCS) to evaluate the effects of cigarette smoking on folate and vitamin B-12 concentrations in the circulation and in tissues directly exposed to cigarette smoke. Univariate analysis showed significantly lower plasma, red blood cell (RBC), and buccal mucosa (BM) folate and BM vitamin B-12 concentrations in CS compared with NCS. The association between smoking and folate and vitamin B-12 concentrations in plasma, RBCs, and BM cells was reduced after other variables were controlled for. Total folate intake and plasma vitamin C concentrations were significant predictors of plasma and RBC folate concentrations. The plasma and RBC concentrations of folate were significantly lower in subjects who had last smoked < 1 h before the blood sample was drawn than in subjects who had smoked earlier. At the current recommended dietary allowance (RDA) for folate, CS had 42% lower plasma folate concentrations than NCS, whereas at an intake three times the RDA, the plasma folate concentration was the same for CS and NCS. The results also suggested that CS have BM folate and vitamin B-12 concentrations that are lower than those of NCS.

Dietary intake and circulating vitamin levels of rheumatoid arthritis patients treated with methotrexate.

The nutrient intakes and circulating vitamin levels of 32 patients with rheumatoid arthritis who were treated with methotrexate were evaluated over a 6-month period. Dietary data were obtained and blood was drawn prior to the initiation of and following 12 and 24 weeks of methotrexate therapy. More than 50% of the patients had food intakes providing less than 67% of the recommended dietary allowance for zinc, vitamin E, folic acid, pyridoxine, and magnesium. Patients 51 years or older had better nutrient intakes than patients less than 51 years. Of the patients, 22% consumed vitamin supplements at the time they were recruited for the study. Mean circulating vitamin levels measured over the 6-month period were within normal limits. Our findings agree with previously published reports that patients with rheumatoid arthritis, particularly the subpopulation taking methotrexate, consume diets that are marginal in some nutrients. Additional research needs to be done to identify more sensitive nutrient assays and to establish more definitively the nutrient needs of patients with rheumatoid arthritis taking several therapeutic agents.

Effect of smoking on folate levels in buccal mucosal cells.

The objective of the study was to document the existence of localized deficiency of folate in a tissue exposed to cigarette smoke, by analysis of oral and circulatory levels of this vitamin in smokers and non-smokers. Buccal mucosal cells and blood samples were collected from 25 smokers and 34 non-smokers. The Health Habits and History Questionnaire was completed by each subject. A 96-well plate L. casei assay, along with preincubation with a folate-free chick pancreas pteroyl-gamma-glutamyl hydrolase, was used to quantitate total buccal mucosal cell folates. The reproducibility (CV 5 to 7%) and recovery (95 to 106%) of the folate assay were satisfactory. Smokers had significantly lower buccal mucosal cell folate levels than did non-smokers. The mean plasma folate level of smokers although within normal limits, was also significantly lower than that of non-smokers. There were no significant differences in mean dietary folate intake or in alcohol consumption between the 2 groups. The strength of the positive association between smoking and plasma and buccal mucosal cell folate deficiency (by any definition) was moderate to strong and statistically significant. Our results indicate that cigarette smoking may result in a localized folate deficiency in buccal mucosal cells, independent of the plasma folate levels.

Inhibition of folate-dependent enzymes by non-steroidal anti-inflammatory drugs.

Many non-steroidal anti-inflammatory drugs (NSAIDs) (including sulphasalazine, sulindac, indomethacin, naproxen, salicylic acid, ibuprofen, piroxicam and mefenamic acid) were found to be competitive inhibitors (with respect to folate) of avian liver phosphoribosylaminoimidazolecarboxamide formyltransferase (AICAR transformylase, EC 2.1.2.3) and bovine liver dihydrofolate reductase (EC 1.5.1.3). In contrast, aspirin and the antipyretic-analgesic drugs acetaminophen and antipyrine were weak inhibitors of these enzymes. Structure-activity correlation suggests that an aromatic ring with a side chain containing a carboxylic acid is a requirement for competitive inhibition of the transformylase. The above-listed NSAIDs also inhibited the folate-coenzyme-mediated biosynthesis of serine from glycine and formate (i.e., the C1 index) by human blood mononuclear cells (BMCs) in experiments where the drug was added to a culture of BMCs. Acetaminophen had a weak inhibitory effect on the C1 index. Consistent with the results obtained in vitro is the observation that the C1 index of BMCs from rheumatoid-arthritis patients treated with drugs which possess little antifolate activity (e.g. acetaminophen) is higher than the C1 index of BMCs from rheumatoid-arthritis patients treated with NSAIDs possessing more potent antifolate activity (e.g. sulindac, sulphasalazine, naproxen and ibuprofen). The mean activity of the transformylase in BMCs taken from healthy humans was 1.98 nmol of product/h per 10(6) cells and the activity was positively correlated with BMC folate levels. These results are consistent with the hypothesis that (1) the antifolate activity of NSAIDs, and hence cytostatic consequences, are important factors in producing anti-inflammatory activity and (2) aspirin exerts its anti-inflammatory effects after its conversion into salicylic acid, which possesses greater antifolate activity than its parent compound.

Effect of total parenteral nutrition on histamine in the rat brain and other tissues.

The effects of a constant infusion of total parenteral nutrition on the histamine content of the hypothalamus was studied in adult male rats. Histamine content of the stomach, kidney, liver, and heart was also determined. The relationship between tissue histamine levels and duration of the infusion was examined. Rats receiving intravenous infusions of total parenteral nutrition had significantly higher (p less than 0.001) histamine levels in the hypothalamus than did orally-fed control rats. No difference was observed in the histamine level in the remainder of the brain. Animals receiving parenterally infused nutrient solutions had a higher histamine content in the kidneys but lower histamine levels in the stomach than did the control rats. No changes in the histamine content of liver or heart were observed. It appears that increases in histamine levels in the kidney and hypothalamus of rats given total parenteral nutrition persist up to 5 days and 9 days, respectively. The mechanisms responsible for and the consequences of the alterations of histamine content of these tissues remain to be established.

Methotrexate therapy in rheumatoid arthritis patients diminishes lectin-induced mononuclear cell proliferation.

Methotrexate (MTX) is an anti-folate drug used in cancer chemotherapy because of its anti-proliferative effects. However, it is unclear whether the anti-proliferative effects of MTX contribute to the efficacy of low-dose MTX in the treatment of rheumatoid arthritis (RA). To date, either no change or a paradoxical increase in lectin-induced proliferation has been observed in cultures of peripheral blood mononuclear cells (PBMC) from MTX-treated RA patients (RA + MTX). In these earlier studies, high folate-containing media and tritiated thymidine (3H-TdR) were used. Our studies were designed to test the hypothesis that the use of a culture medium with a low folate content along with tritiated deoxyuridine (3H-UdR) permits detection of diminished phytohemagglutinin (PHA)-induced proliferative responses of PBMC from RA + MTX. The data demonstrate decreased PHA-induced cellular proliferation of cultured PBMC from RA + MTX compared with controls. When comparing the PBMC proliferative responses in high vs low folate medium, a significantly greater increase (P less than 0.05) in proliferation occurs in the cells from RA + MTX cultured in the high folate medium. This suggests that an in vivo folate-deficient state of the cells from RA + MTX may be corrected in vitro when a high folate medium is used in culture. We conclude that the use of 3H-UdR and a medium containing folate within the normal range of plasma folate levels eliminates artifacts associated with the use of high folate medium and 3H-TdR, which obscures the anti-proliferative effect of MTX in RA patients.

Early nutrition intervention services for children with special health care needs.

Dietitians must be responsive to the changing needs of their clients and employers, to societal concerns, and to legal mandates. A recently passed amendment (PL 99-457) to the Federal Education for the Handicapped Act gives nutrition professionals the opportunity to have a voice in establishing nutrition policy and standards of care for young handicapped and high-risk children. The new law extends preventive services to children as young as 3 years of age, and Part H of the law provides financial incentives for states to provide services to children with special health care needs from birth to 2 years of age. This article reviews relevant provisions of the new law and describes two projects undertaken by nutritionists from Alabama, Mississippi, and Texas. It also summarizes challenges to nutritionists that will result from the law's implementation.

Variations of brain histamine levels in germ-free and nephrectomized rats.

The influence of nephrectomy on brain and peripheral tissue histamine and on brain norepinephrine, dopamine, serotonin, and 5-hydroxyindoleacetic acid was studied in germ-free and conventionally housed rats. The conventional controls had higher levels of histamine in the hypothalamus than the germ-free control animals, but no differences existed for histamine in whole brain minus the hypothalamus or in peripheral tissues. Nephrectomy increased brain histamine and 5-hydroxyindoleacetic acid levels in both germ-free and conventional rats, but had no effect on norepinephrine, dopamine or serotonin. In contrast, the histamine level in the heart of the nephrectomized germ-free animals was lower than that for germ-free controls. There were no changes in the heart or liver histamine levels of the conventional nephrectomized rats.

Brain histamine levels in the hamster during the estrous cycle and on selected days of pregnancy.

Brain histamine levels were determined in golden hamster hypothalamus and 'brain minus hypothalamus' on each of the 4 days of the estrous cycle and on selected days of pregnancy. The highest histamine content of the hypothalamus was observed on day 3 of the estrous cycle. The highest histamine content of the hypothalamus was observed on day 3 of the estrous cycle, which is the day prior to recurrence of heat on day 4. Day 4 terminates in ovulation. The histamine level in the remainder of the brain peaked on day 2. During gestation the histamine content of the 'brain minus hypothalamus' was greatest on day 5, while the maximum content of histamine in the hypothalamus was not reached until day 8. After the 8th day of pregnancy, there was an overall decline in brain histamine that continued until parturition. The hypothalamic histamine level in nonpregnant females was not different from that of males. However, in the remainder of the brain, histamine levels in females on days 1 and 2 of the estrous cycle were higher than in males.

Changes in tissue histamine during the estrous cycle, pregnancy and pseudopregnancy in the golden hamster.

The histamine content of reproductive tissues and skeletal muscle was determined in the golden hamster during the estrous cycle, pregnancy, and pseudopregnancy. Histidine decarboxylase activity was measured in uterine implantation sites and intersites from Day 4 to Day 10 of pregnancy. Histidine decarboxylase was also measured in mesometria and placentas on selected days of gestation. During the estrous cycle, uterine and skeletal muscle histamine levels were highest on Day 2 and lowest on Day 4 of the cycle. The ovarian histamine content did not change significantly among the different stages of the cycle. While the histamine content of uterine implantation sites of attachment was high on Days 4 and measurable on Days 5 and 6 of pregnancy, the levels were below the limits of detection by Day 7. On the other hand, the highest levels of histamine were in the uterine interimplantation sites on Days 8 and 9. The ovarian levels of histamine were highest on Day 13 of pregnancy. Histamine in skeletal muscle did not change significantly during pregnancy. The histidine decarboxylase activity in the implantation sites began rising on Day 9 and increased dramatically on Day 10. Placental histidine decarboxylase activity was very high on Days 13 and 15. Overall, we observed changes in uterine and skeletal muscle histamine during the estrous cycle that may be explainable in light of previously reported changes in mast cell numbers and circulating estrogens. During pregnancy, histamine levels of implantation sites and implantation intersites varied, as did the histamine content of ovarian tissue. Histidine decarboxylase activity rises in the uterus and placental tissue after the formation of the placenta.