Kelley E. Johnston

Low seminal plasma folate concentrations are associated with low sperm density and count in male smokers and nonsmokers

OBJECTIVE To measure folate levels in seminal plasma from smokers and nonsmokers and to evaluate relationships between seminal plasma folate levels and both folate nutriture and semen quality measures. DESIGN Observational study. SETTING United States Department of Agriculture, Western Human Nutrition Research Center, Presidio of San Francisco, San Francisco, California. PATIENT(S) Healthy male smokers (n=24) and nonsmokers (n=24). MAIN OUTCOME MEASURE(S) Blood levels of plasma folate and homocysteine, seminal plasma total, non-methyl- and 5-methyltetrahydrofolate concentrations, and total sperm count and density. RESULTS Total seminal plasma folate concentrations were on average 1.5 times higher than blood plasma folate concentrations in all men. Seminal plasma folates contained 5-methyltetrahyrdofolate (74% of total) and non-methyltetrahydrofolates (26% of total); all samples had less than four glutamyl residues. Total and 5-methyltetrahydrofolate concentrations correlated significantly with blood plasma folate and homocysteine concentrations. Seminal plasma non-methyltetrahydrofolate levels correlated significantly with sperm density and total sperm count. Seminal plasma of smokers contained a proportionally lower concentration of non-methyltetrahydrofolates compared with nonsmokers. CONCLUSION(S) Seminal plasma total folate and 5-methyltetrahydrofolate concentrations reflect folate nutriture. The non-methyltetrahydrofolate fraction of seminal plasma may be important for male reproductive function.

Elevated amniotic fluid interleukin-6 levels at genetic amniocentesis predict subsequent pregnancy loss

OBJECTIVE Our purpose was to determine the proportion of pregnancy loss after genetic amniocentesis that is related to preexisting subclinical intrauterine inflammation. STUDY DESIGN We accessed our bank of stored second-trimester amniotic fluid and maternal serum samples obtained from women undergoing genetic amniocentesis at our institution from 1988 to 1995 (N = 11,971). Interleukin-6 levels were measured by enzyme-linked immunosorbent assay in samples from every case resulting in spontaneous postprocedure loss (excluding fetal aneuploidy and anomalies) within 30 days after the procedure (n = 66) and from 66 normal control women delivered at term and matched for year of test, gestational age, maternal age, and indication for amniocentesis. RESULTS Mean maternal serum interleukin-6 levels were the same in each group (0.02 +/- 0.07 ng/ml for cases and 0.06 +/- 0.25 ng/ml for controls, p = 0.45). Mean amniotic fluid interleukin-6 levels were higher in cases (4.0 +/- 13.1 ng/ml) than in controls (0.5 +/- 0.7 ng/ml, p = 0.04). The higher mean amniotic fluid interleukin-6 levels in the cases resulted from the inclusion of eight very high values (> or = 3 SD or > or = 2.5 ng/ml). When these samples were excluded, the means and range of values were the same in each group (0.4 +/- 0.4 ng/ml for cases and 0.5 +/- 0.7 ng/ml for controls, p = 0.58). Twelve percent (8/66) of the cases and 3% (2/66) of the controls had amniotic fluid interleukin-6 levels > or = 2.5 ng/ml (p = 0.048, odds ratio 4.1, 95% confidence interval 1.0 to 31.2). Although the overall correlation between maternal serum and amniotic fluid interleukin-6 levels was good (r = 0.50, p < 0.002), only one of the eight cases would have been identified by a maternal serum interleukin-6 level > or = 3 SD above the mean (> or = 0.8 ng/ml). CONCLUSION Analysis of our complete unselected group of postamniocentesis pregnancy losses indicates that up to 12% may result from preexisting subclinical intrauterine inflammation. This inflammation is most likely localized and may not be identified by a maternal serum interleukin-6 level before the procedure.

Folate Status of Mothers During Pregnancy and Mental and Psychomotor Development of Their Children at Five Years of Age

Objective. There are limited data relating folate nutritional status of mothers during pregnancy to mental and psychomotor development of their offspring. Using an existing data set from a study on the effect of prenatal zinc supplementation on child neurodevelopment, we evaluated the association between folate nutritional status of mothers during pregnancy and neurodevelopment of their children. Methods. Maternal blood folate and total homocysteine (tHcy) concentrations were measured at 19, 26, and 37 weeks of gestation. At a mean of 5.3 years of age, 355 black children with low-socioeconomic background were given 6 tests: Differential Ability Scales, Visual and Auditory Sequential Memory, Knox Cube Test, Gross Motor Scale, and Grooved Pegboard. The scores of the tests between the 2 groups of mothers with poor versus adequate folate nutritional status classified by blood folate or tHcy concentrations were compared. Results. There were no differences in the test scores of neurodevelopment between the 2 groups. Conclusion. Folate nutritional status of mothers in the later half of pregnancy assessed by plasma and erythrocyte folate and plasma tHcy concentrations had no impact on neurodevelopment of their children at age 5. It is unknown whether our findings in a low-socioeconomic population can be readily extrapolated to other populations.

Role of amniotic fluid homocysteine level and of fetal 5,10-methylenetetrahydrafolate reductase genotype in the etiology of neural tube defects

A mutation in the gene 5,10-methylenetetrahydrofolate reductase (MTHFR), leading to altered homocysteine metabolism, has been identified in parents and fetuses with fetal neural tube defects. We sought to determine which is of greater importance in fetal neural tube defect formation: the fetal MTHFR mutation or elevated amniotic fluid homocysteine level. We retrieved stored amniotic fluid from cases of isolated fetal neural tube defect diagnosed from 1988 to 1998 (n = 80), and from normal controls matched for race, month and year of amniocentesis, and maternal age. The presence or absence of the 677C-->T mutation of MTHFR was determined and homocysteine levels were measured; cases and controls were compared. Significantly more cases than controls were heterozygous or homozygous for the 677C-->T MTHFR mutation (44% vs 17%, P < or = 0.001). Cases were also significantly more likely than controls to have an amniotic fluid homocysteine level above the 90th centile (>1.85 micromol per liter); 27% vs 10%, P = 0.02. Thirty one cases and 12 controls had an abnormal genotype; however, amniotic fluid homocysteine levels were not significantly different between these two groups (6/31, or 19% of cases had an elevated homocysteine compared to 1/12, or 8% of controls; P = 0.65). In contrast, 40 cases and 60 controls had a normal genotype; the neural tube defect cases had significantly higher homocysteine levels (13/40, or 32% of cases had an elevated homocysteine level compared to only 6/60, or 10% of controls; P = 0.008). Although both abnormal fetal MTHFR genotype and abnormal amniotic fluid homocysteine concentration are significantly associated with neural tube defects, the association with amniotic fluid homocysteine concentration is significant regardless of the fetal MTHFR genotype. The relationship between maternal and fetal homocysteine metabolism is complex.

Refrigeration of blood samples prior to separation is essential for the accurate determination of plasma or serum zinc concentrations.

An evaluation of refrigeration (7°C) to prevent falsely high plasma or serum zinc concentrations owing to elapsed time between blood collection and centrifugation was performed. At room temperature (23°C), both plasma and serum zinc concentrations increased significantly, if blood samples were stored uncentrifuged. Plasma zinc concentrations increased 6.3% at 1 h and 40.7% at 24 h, whereas serum zinc concentrations increased only 0.9% at 1 h and 12.5% at 24 h at room temperature. When blood samples were stored uncentrifuged in the refrigerator for up to 24 h, there were no significant increases in zinc concentrations in either plasma or serum. These findings suggest that plasma or serum separation should be performed immediately after blood drawing to obtain accurate zinc concentrations, and if this is not feasible, the samples should be immediately refrigerated and separation performed within eight hours.

Amniotic fluid homocysteine levels, 5,10-methylenetetrahydrafolate reductase genotypes, and neural tube closure sites.

A specific gene mutation leading to altered homocysteine metabolism has been identified in parents and fetuses with neural tube defects (NTDs). In addition, current animal and human data indicate that spine closure occurs simultaneously in five separate sites that then fuse. We sought to determine whether either this mutation or abnormal amniotic fluid homocysteine levels are associated with all five neural tube closure sites. We retrieved stored amniotic fluid from cases of isolated fetal neural tube defect diagnosed from 1988 to 1998 (n = 80) and from normal controls matched for race, month and year of amniocentesis, and maternal age. Cases were categorized according to defect site by using all available medical records. The presence or absence of the 677C-->T mutation of 5, 10-methylenetetrahydrafolate reductase (MTHFR) gene was determined, and homocysteine levels were measured; case and controls were compared. Significantly more cases than controls were heterozygous or homozygous for the 677C-->T MTHFR mutation (44% vs. 17%, P < or = 0. 001). Likewise, cases were significantly more likely than controls to have amniotic fluid homocysteine levels >90th centile (>1.85 micromol/L), 27% vs. 10%, P = 0.02. Most (83%) of control cases had both normal MTHFR alleles and normal amniotic fluid homocysteine levels (normal/normal), whereas only 56% of NTD case were normal/normal (P = 0.001). When evaluated by defect site, only defects involving the cervical-lumbar spine, lumbosacral spine, and occipital encephalocele were significantly less likely to be normal/normal than controls (P = 0.007, 0.0003, and 0.007, respectively), suggesting a strong association with the 677C-->T allele. In contrast, anencephaly, exencephaly, and defects confined to the sacrum included many cases that had both normal MTHFR alleles and normal homocysteine and were not significantly different from controls. The 677C-->T MTHFR mutation and elevated homocysteine levels appear to be disproportionately associated with defects spanning the cervical-lumbar spine, lumbosacral spine, and occipital encephalocele. In contrast, anencephaly, exencephaly, and defects confined to the sacrum may not be related to altered homocysteine metabolism.

Effect of angiotensin-converting enzyme gene polymorphism on pregnancy outcome, enzyme activity, and zinc concentration*

Objective To evaluate the effect of angiotensin-converting enzyme (ACE) genotypes on pregnancy outcome, the incidence of pregnancy-induced hypertension, and changes in blood pressure (BP) during pregnancy; and the relationship between plasma ACE activities and plasma and erythrocyte zinc concentrations in each genotype. Methods The subjects (n = 191) were selected from 580 indigent African-American pregnant women who enrolled toward the end of a trial to evaluate the effect of zinc supplementation on pregnancy outcome. This selection resulted in 93 subjects who received zinc and 98 who received placebo. Sample size was calculated with a 0.50 correlation coefficient between plasma ACE activities and zinc levels and a power of 80%. This calculation indicated that the sample size in each ACE genotype should be more than 28. Angiotensin-converting enzyme genotypes were identified using polymerase chain reaction. Blood pressure, plasma ACE activities, and plasma and erythrocyte zinc concentrations were measured at each prenatal clinical visit. Results Pregnancy outcome, the incidence of pregnancy-induced hypertension, and BP were not different among the three ACE genotypes. There was no significant correlation between plasma ACE activities and zinc concentrations. Zinc supplementation did not have a significant effect on either plasma ACE activities or zinc concentrations, probably because of the small sample size in our study. Conclusion There was no effect of ACE gene polymorphism on pregnancy outcome, the incidence of pregnancy-induced hypertension, or changes in BP during pregnancy. Among each ACE genotype, plasma ACE activities did not correlate significantly with plasma zinc concentrations.

Plasma folate conjugase activities and folate concentrations in patients receiving hemodialysis

Abstract Folate conjugase activities and folate concentrations in blood obtained from patients with chronic renal failure and healthy controls were measured by radioassay using pteroyldiglutamyl-[ 14 C]-glutamic acid as substrate and microbiologic assay, respectively. A total of 32 patients receiving hemodialysis for an average of 5.4 years participated in the study. Folate conjugase activities in posthemodialysis plasma were significantly higher than those in prehemodialysis samples and were similar to those of controls. Plasma folate conjugase was inhibited by the in vitro addition of the heated extract of prehemodialysis plasma. The heated extract of urine from a healthy control also inhibited the enzyme. Furthermore, plasma folate conjugase activities were reduced by the in vitro addition of sulfate, one of the major constituents in urine, although the addition of urea or uric acid had no such effect. Data indicate the presence of an unidentified heat-stable inhibitor(s) that is excreted by the normal kidney, and sulfate may be one of them. During hemodialysis, plasma folate concentrations decreased, while erythrocyte folate concentrations remained unchanged. Five of 32 patients demonstrated plasma folate concentrations lower than normal. These findings suggest that a careful evaluation of folate requirements is necessary in patients maintained with hemodialysis.

Plasma extracellular superoxide dismutase activity in healthy pregnant women is not influenced by zinc supplementation

We hypothesized that plasma extracellular superoxide dismutase (EC-SOD) activity reflects the zinc nutriture of healthy pregnant women. Sixty-three women were selected from 580 African-American women who participated in a clinical trial to evaluate the effect of prenatal zinc supplementation on pregnancy outcome. Half of the women received zinc (25 mg/d) and the other half was given a placebo from about 19 wk gestation to delivery. In the trial, a positive effect of zinc supplementation on birthweight was observed, indicating that the population as a whole had suboptimal zinc nutriture. Using plasma samples obtained during the trial, EC-SOD activities were measured and the values were compared with plasma zinc concentrations and plasma alkaline phosphatase activities. Plasma EC-SOD activities in our subjects were lower than previously published values for healthy adults in Korea. Although plasma EC-SOD activity may reflect severe zinc deficiency, it is not a sensitive marker for marginal deficiency status. Plasma EC-SOD activities did not prove to be a better indicator of zinc nutriture of pregnant women than either plasma zinc or plasma alkaline phosphatase activities.

Folate concentrations of fast foods measured by trienzyme extraction method

Abstract Folate concentrations in common fast foods containing beef were measured using a new trienzyme folate extraction method and compared to the values using traditional folate conjugase. A total of 56 fast foods were purchased from local restaurants after the 1998 mandate of folic acid fortification in enriched cereal grains. One serving of hamburger, sandwich, pizza (one eighth of a 30-cm pizza) and Mexican foods contained a mean of 314 (±98, S.D.), 401 (±115), 221 (±45) and 282 (±126) μg of folate, respectively. Breakfast items provided the lowest folate amount among the foods (165±89 μg per serving). These values are markedly higher than those in the literature most likely due to the mandate of folic acid fortification in cereal-grain products and the use of the new folate extraction technique. We hope that the information presented here is a useful means to accurately calculate dietary folate intake.