Homocysteine elevation in sickle cell disease.

OBJECTIVE: Ischemic complications are common in patients with sickle cell disease. Hyperhomocysteinemia is a risk factor for arteriosclerosis and venous thrombosis, and given the propensity of patients with sickle cell disease to develop ischemic complications, we hypothesized that they might have elevated plasma homocysteine concentrations. METHODS: Plasma concentrations of homocysteine, vitamin B12 and folate were measured in 49 adults with sickle cell disease and 16 normotensive Black controls. All subjects with sickle cell disease had been prescribed folic acid 1 mg by mouth daily. RESULTS: The median plasma concentration of homocysteine of subjects with sickle cell disease was approximately 1.5-fold higher than that of controls (p=0.0008). This difference persisted, even when subjects with renal insufficiency were excluded. Plasma folate levels were 1.5-fold higher in subjects with sickle cell disease than in controls (p=0.0498). There was no significant difference in plasma vitamin B12 concentrations between the two groups. There was no difference in plasma homocysteine concentrations between transfused and non-transfused sickle cell subjects. CONCLUSIONS: Patients with sickle cell disease have elevated plasma concentrations of homocysteine in spite of elevated plasma folate levels and vitamin B12 concentrations similar to those observed in controls. Based on these data, we hypothesize that the concentration of folate required to normalize plasma homocysteine levels in patients with sickle cell disease may be higher than that of normal controls and that patients with sickle cell disease have a higher nutritional requirement for folic acid than the general population.

The accumulation of ascorbic acid by squamous cell carcinomas of the lung and larynx is associated with global methylation of DNA.

BACKGROUND: Cigarette smokers are known to have lower concentrations of circulating ascorbic acid than nonsmokers. In contrast, there is evidence that the extracellular fluid lining of the alveolus, which comes in close contact with cigarette smoke, and the alveolar macrophages of smokers are enriched with ascorbic acid. The clinical significance of these observations is unknown. METHODS: The authors measured the ascorbic acid concentrations and radiolabeled methyl incorporation (which is inversely related to the degree of DNA methylation in vivo) of paired samples of squamous cell carcinoma (SCC) and adjacent uninvolved mucosa of the lung and larynx (n = 22). RESULTS: Cancerous tissues had significantly higher ascorbic acid concentrations (mean +/- standard deviation [SD, 485 +/- 77; median, 483 ng/mg protein) compared with their matched uninvolved tissues (mean +/- SD, 151 +/- 52; median, 72 ng/mg protein; P < 0.0001). The radiolabeled methyl incorporation was significantly higher in cancerous tissues (mean +/- SD, 31,419 +/- 2629; median, 31,416 counts per minute [CPM]/microg DNA) compared with their matched uninvolved tissues (mean +/- SD, 11,883 +/- 1567; median, 11,444 CPM/microg DNA; P < 0.0001). The Spearman correlation between ascorbic acid concentrations and radiolabeled methyl incorporation by DNA in SCCs was inverse and statistically significant (r = -0.58, P = 0.008), indicating a beneficial effect of accumulated ascorbic acid in global methylation of DNA. In the uninvolved tissues, this correlation was inverse but statistically not significant (r = -0.20, P =0.35). CONCLUSIONS: Cancerous tissues of the lung and larynx demonstrated their ability to accumulate ascorbic acid. The accumulation of ascorbic acid by these tissues seemed to facilitate global methylation of DNA.

Correlates of total plasma homocysteine: folic acid, copper, and cervical dysplasia.

We examined correlates of total plasma homocysteine (tHcy) in 294 subjects with cervical intraepithelial neoplasia and 170 control subjects. Associations of tHcy with risk factors for cervical intraepithelial neoplasia and 24-h intakes and biochemical indices of nutrients were examined. Plasma and red blood cell folate and plasma B(12) were strong inverse correlates of tHcy (r = -0.35, -0. 31, and -0.27, respectively). Plasma copper and severity of dysplasia were positively correlated with tHcy (r = 0.14 and 0.21, respectively). A stepwise regression model that included red blood cell folate, plasma copper, grade of dysplasia, ethnicity, intake of polyunsaturated fatty acids, plasma vitamin B(12), intake of fat, and oral contraceptive use explained 29% of the variation in tHcy. Two hundred thirty-five subjects with cervical intraepithelial neoplasia were randomized to receive folic acid (10 mg/d) or placebo for 6 mo. After 2, 4, and 6 mo, mean tHcy in the folate-supplemented group (7.2 +/- 1.8, 7.0 +/- 1.9, and 7.0 +/- 2.3 micromol/L, respectively) was significantly lower than baseline and the placebo group at 2, 4, and 6 mo (8.9 +/- 3.1, 8.4 +/- 3.0, and 8.9 +/- 3.1 micromol/L, respectively). Supplementation lowered tHcy even in subjects in the highest quintile of baseline folate. Folate, vitamin B(12), copper, and severity of dysplasia are associated with tHcy. Folate supplementation significantly lowers tHcy even in folate-replete subjects.

Effect of total plasma homocysteine on cervical dysplasia risk.

We investigated whether total plasma homocysteine (tHcy) is associated with risk for cervical intraepithelial neoplasia (CIN). tHcy was evaluated, along with numerous risk factors for CIN and biochemical indexes of nutrients, in a previously reported study population of 294 subjects with CIN and 170 female controls without CIN. tHcy was significantly higher in cases than in controls (9.1 vs. 8.3 mumol/l, p = 0.002). Human papillomavirus type 16 infection [odds ratio (OR) = 6.7], oral contraceptive use (OR = 6.0), parity (OR = 2.2), and cigarette smoking (OR = 1.9) were significantly associated with CIN after adjustment for each other and for age, number of sexual partners, and plasma tHcy, folate, iron, and zinc. Human papillomavirus type 16 positivity increased risk for CIN more when tHcy was > 9.12 mumol/l (OR = 4.7) than when it was < or = 9.12 mumol/l (OR = 3.0). Cigarette use increased risk for CIN when tHcy was > 9.12 mumol/l (OR = 3.9), but not when tHcy was < or = 9.12 mumol/l (OR = 1.5). Parity increased risk for CIN more when tHcy was > 9.12 mumol/l (OR = 4.0) than when tHcy was < or = 9.12 mumol/l (OR = 2.0). These results suggest that elevated plasma tHcy is a risk factor for cervical dysplasia and that it enhances the effects of other risk factors. It is unknown whether tHcy is serving as a marker of folate deficiency or is acting through other mechanisms.

Iron compounds catalyze the oxidation of 10-formyl-5,6,7,8 tetrahydrofolic acid to 10-formyl-7,8 dihydrofolic acid.

We have previously demonstrated that 10-formyl-7,8-dihydrofolic acid (10-HCO-H2folate) is a better substrate for mammalian aminoimidazolecarboxamide ribotide transformylase (EC 2.1.2.3) than is 10-formyl-5,6,7,8-tetrahydrofolic acid (10-HCO-H4folate) (J.E. Baggott, G.L. Johanning, K.E. Branham, C.W. Prince, S.L. Morgan, I. Eto, W.H. Vaughn, Biochem. J. 308, 1995, 1031-1036). Therefore, the possible metabolism of 10-HCO-H4folate to 10-HCO-H2folate was investigated. A spectrophotometric assay for the oxidation of 10-HCO-H4folate to 10-HCO-H2folate which measures the disappearance of reactant (decrease in absorbance at 356 nm after acidification of aliquots of the reaction solution), is used to demonstrate that iron compounds catalyze the oxidation of 10-HCO-H4folate to 10-HCO-H2folate in the presence and absence of ascorbate. Chromatographic separation of the 10-HCO-H2folate product from the reaction mixture, its UV spectra, a microbiological assay and an enzymatic assay established that the iron-catalyzed oxidation product of 10-HCO-H4folate was 10-HCO-H2folate; without substantial side reactions. The inhibition of this iron-catalyzed oxidation by deferoxamine, apotransferrin and mannitol and the stimulation by citrate and EDTA indicated of a mechanism involving a reaction of 10-HCO-H4folate with hydroxyl radicals (*OH) generated by Fenton chemistry. The presence of "free iron" (e.g., Fe3+ citrate) in bile, cerebrospinal fluid and intracellularly suggest that this oxidation could occur in vivo and that 10-HCO-H4folate may be a *OH scavenger.

Decreased fat and nitrogen losses in patients with AIDS receiving medium-chain-triglyceride-enriched formula vs those receiving long-chain-triglyceride-containing formula.

OBJECTIVE: The purpose of this study was to compare two enteral formulas, differing only in fat source, for product acceptance, tolerance, and effect on fat malabsorption and nutritional status in subjects with acquired immune deficiency syndrome (AIDS). DESIGN: The double-blind, randomized 15-day trial was divided into a 3-day period in which solid food was consumed followed by a 12-day experimental period in which liquid formulas were consumed. SETTING/SUBJECTS: Twenty-three men and one woman with AIDS and fat malabsorption completed the study. The study was conducted in the General Clinical Research Center, University of Alabama Hospital, University of Alabama at Birmingham. Laboratory assays were performed in the Department of Nutrition Sciences. INTERVENTIONS: After 3 days of consuming a controlled, solid food diet containing 100 g fat per day from mixed sources to document fat malabsorption, subjects were randomly assigned to one of two groups. Each group received a liquid formula containing 35% of energy as fat for 12 days. One group received a formula containing 85% medium-chain triglycerides (MCTs) and the control group received a formula containing 100% long-chain triglycerides. MAIN OUTCOME MEASURES: Determinations included stool number, consistency, weight, and fat and nitrogen content; urine nitrogen and creatinine levels; and body weight. STATISTICAL ANALYSIS PERFORMED: Subject demographic and other baseline characteristics were compared using two-sample t tests; stool and urine assessments were compared between groups at the initial experimental period using two-sample t tests; changes from initial to final experimental periods were assessed by means of analysis of covariance; changes in pooled intake, body weight, and the number and consistency of bowel movements were also assessed using analysis of covariance. All statistical tests were two-tailed and considered significant at P < .05. RESULTS: Within-group comparisons indicated that subjects fed the MCT formula showed significantly decreased stool fat and stool nitrogen content (P = .01 and P = .03, respectively) and increased fat absorption (P = .03), whereas those fed the control formula did not. Differences in stool fat between the groups were not statistically significant. However, the difference in fat absorption from the initial to final formula period was significant (P = .04). Subjects consuming the MCT formula also tended to have a decreased number of bowel movements and abdominal symptoms, whereas subjects fed the control formula showed no improvement. All subjects maintained their body weights. APPLICATIONS: There may be advantages to using an MCT-based formula in the treatment of AIDS-associated malabsorption.

Modification of a high-performance liquid chromatographic method for assay of homocysteine in human plasma.

A modification of a previously published method for analysis of total homocysteine in human serum is presented. The modification was implemented to allow use of a different derivatizing agent (i.e., 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonamide) which reacts much faster than the original derivatizing reagent and at a lower temperature. Shorter reaction time and lower temperature lead to less destruction of some biological thiols. In order to retain an isocratic mobile phase with the new derivatizing agent, a different concentration of acetonitrile was found that affords a 7-8 min retention time.

Specific nutrient abnormalities in asymptomatic HIV-1 infection.

OBJECTIVE: To determine whether specific nutrient abnormalities occur in earlier stages of HIV-1 infection, thereby preceding the marked wasting and malnutrition that accompany later stages of the infection. DESIGN: A longitudinal investigation to determine biological, psychological and social factors thought to influence the progression and outcome of HIV-1 infection. Nutritional status was assessed using biochemical measurement of nutrient levels, dietary history, anthropometry and clinical examination for the signs and symptoms of nutritional deficiency or excess. SETTING: The study was performed on an outpatient basis at the University of Miami School of Medicine. PARTICIPANTS: One hundred homosexual men, aged between 20 and 55 years, who were asymptomatic other than persistent generalized lymphadenopathy (Centers for Disease Control stage III) and 42 age-matched homosexual men demonstrated to be free of HIV-1 infection at two 6-month intervals. MAIN OUTCOME MEASURES: Biochemical measurement of nutrient status, dietary history, anthropometry, clinical signs or symptoms of nutritional excess or deficiency were obtained for all participants. RESULTS: Despite few differences in mean blood levels of specific nutrients, prevalence of specific nutrient abnormalities was widespread among HIV-1-infected subjects, compared with non-infected male homosexual controls. Overtly and marginally low blood levels of vitamins A (18%), E (27%), riboflavin (26%), B6 (53%), and B12 (23%), together with copper (74%) and zinc (50%) were documented in HIV-1-seropositive subjects. With the exception of riboflavin, zinc, and copper, a similar prevalence of abnormalities among HIV-1-seronegative controls was not observed. CONCLUSION: Specific nutrient abnormalities occur with relative frequency in asymptomatic HIV-1 infection and may contribute to the rate and form of HIV-1 disease progression.

Cobalamin deficiency associated with methylmalonic acidemia in a cat.

A 9-month-old sexually intact male longhair cat was examined because of lethargy, anorexia, cold intolerance, and failure to thrive since acquisition at an early age. Clinical signs of disease were less pronounced when the cat was fed a low-protein diet. Anemia, hypoglycemia, low total CO2 content, and hyperammonemia were detected. The cat was euthanatized. Urine obtained immediately before euthanasia contained a large amount of methylmalonic acid. Total serum cobalamin concentration was low. Hepatic methylmalonic-CoA mutase activity, with and without the addition of coenzyme adenosylcobalamin, was consistent with a cobalamin deficiency. Methylmalonic acidemia secondary to a putative defect in cobalamin absorption was diagnosed.

Association of vitamin B6 status with parameters of immune function in early HIV-1 infection.

Nutritional deficiencies have been documented to affect immune function. The present study indicates that vitamin B6 deficiency is prevalent in CDC stage III HIV-1-infected subjects, despite adequate dietary vitamin B6 intake. As vitamin B6 deficiency has been previously shown to affect immune function, these relatively asymptomatic HIV-1-infected patients were examined for evidence of a relationship between vitamin B6 deficiency and immune dysregulation. Vitamin B6 status in HIV-1-infected subjects was significantly associated with functional parameters of immunity [multivariate F(3,36) = 3.70, p less than or equal to 0.02]. Additional analyses indicated that overtly deficient participants exhibited significantly decreased lymphocyte responsiveness to the mitogens phytohemagglutinin and pokeweed, and reduced natural killer cell cytotoxicity, compared to subjects with clearly adequate vitamin B6 status (chi 2 = 8.78, df = 3, p less than 0.04). Vitamin B6 status was not related to immune cell subpopulations, e.g., CD4, CD8 cell number, or level of serum immunoglobulins. The results of this study indicate that while vitamin B6 status is not a primary etiological factor in HIV-1-related immunological dysregulation, it appears to be an important cofactor of immune function.

Chronic toxicity of dichloroacetate: possible relation to thiamine deficiency in rats.

The chronic use of dichloroacetate (DCA) for diabetes mellitus or hyperlipoproteinemias has been compromised by neurologic and other forms of toxicity. DCA is metabolized to glyoxylate, which is converted to oxalate and, in the presence of adequate thiamine levels, to other metabolites. DCA stimulates the thiamine-dependent enzymes pyruvate dehydrogenase and alpha-ketoacid dehydrogenase. We postulated that the neurotoxicity from chronic DCA administration could result from depletion of body thiamine stores and abnormal metabolism of oxalate, a known neurotoxin. For 7 weeks, rats were fed ad lib. Purina chow and water or chow plus sodium DCA (50 mg/kg or 1.1 g/kg) in water. A portion of the DCA-treated animals also received intraperitoneal injections of 600 micrograms thiamine three times weekly or 600 micrograms thiamine daily by mouth. Thiamine status was assessed by determining red cell transketolase activity and, in a blinded manner, by recording the development of clinical signs known to be associated with thiamine deficiency. At the 50 mg/kg dose, chronic administration of DCA showed no clinical toxicity or effect on transketolase activity. At the 1.1 g/kg dose, however, DCA markedly increased the frequency and severity of toxicity and decreased transketolase activity 25%, compared to controls. Coadministration of thiamine substantially reduced evidence of thiamine deficiency and normalized transketolase activity. Inhibition of transketolase by DCA in vivo was not due to a direct action on the enzyme, however, since DCA, glyoxylate, or oxalate had no appreciable effects on transketolase activity in vitro. After 7 weeks, plasma DCA concentrations were similar in rats receiving DCA alone or DCA plus thiamine, while urinary oxalate was 86% above control in DCA-treated rats but only 28% above control in DCA plus thiamine-treated animals. No light microscopic changes were seen in peripheral nerve, lens, testis, or kidney morphology in either DCA-treated group, nor was there disruption of normal sperm production in the DCA-treated group. We conclude that stimulation by DCA of thiamine-requiring enzymes may lead to depletion of total body thiamine stores and to both a fall in transketolase activity and an increase in oxalate accumulation in vivo. DCA neurotoxicity may thus be due, at least in part, to thiamine deficiency and may be preventable with thiamine treatment.

Zinc concentration in plasma and erythrocytes of subjects receiving folic acid supplementation.

It has been suggested that oral supplements of folic acid interfere with the intestinal absorption of zinc and may have toxic side effects. The concentrations of Zn and folate in blood were monitored in a group of women with cervical dysplasia randomly assigned to receive 10 mg/d of either folic acid (pteroylglutamic acid) or ascorbate. Fifty subjects were evaluated after 2 mo; 21 of the same subjects were evaluated again after 4 mo. No untoward clinical effects were observed. Significant elevation of erythrocyte folate above the baseline value was observed in the supplemented group but not in the placebo group (p less than 0.001). The concentration of Zn in plasma and erythrocytes did not change significantly in either the folate-treated or placebo groups after 2 and 4 mo. It is concluded that carefully controlled clinical intervention trials of this type do not impose a risk of depleting the concentration of Zn in erythrocytes and plasma.

Quantitation of xanthurenic acid in rabbit serum using high performance liquid chromatography.

Xanthurenic acid (XA) has been quantified in the serum of normal and vitamin B6-deficient rabbits using high performance liquid chromatography. The concentration of XA in the serum of normal and B6-deficient rabbits was 141 and 2275 ng/ml, respectively. The coefficient of variation for a series of dilutions of standard XA (3.9 to 1000 ng) ranged from 45.5% at the lower limit of the curve to 10.9% at the higher range of the curve. The minimum detectable level was 3.9 ng/ml. Serum samples spiked with reference XA exhibited a parallel dose response. The percentage recovery of XA from serum samples was 80.8%. The procedure, which requires 1 to 2 ml of serum, is sensitive and may be a useful tool for assessing B6 nutritional parameters as well as the physiological role of XA. It offers advantages over urinary procedures because it is more sensitive, more specific, and allows the study of blood levels of XA.

Vitamin fluctuations in the blood of female baboons in relation to normal menstrual cycles, treatments with Lo-Ovral or Depo-Provera and a selected vitamin supplement.

Both endogenous and exogenous sex steriods may induce changes in plasma vitamin levels by altering availability, transport, binding, or use of vitamins. This study investigated some of those mechanisms by observing in a primate model (baboon), the blood levels of carotene, folate, vitamins A, B12, and C, and the status of vitamin B6, riboflavin, and thiamin. The latter three vitamins were studied by determining their relationship to asparate aminotransferase, glutathione reductase, and thiamin transketolase, respectively. The vitamin screen was obtained throughout normal menstrual cycles in 10 baboons and weekly for 55 wk in five baboons receiving Lo-Ovral and in four baboons receiving Provera. During the last 16 wk of hormonal treatment, all baboons received a vitamin supplement containing pyridoxine, riboflavin, and thiamin. Only carotene (p less than 0.0001), vitamin A (p less than 0.05), glutathione reductase (p less than 0.05), and thiamin transketolase (p less than 0.05) levels fluctuated significantly during normal menstrual cycles. Long-term treatment with Lo-Ovral and Provera resulted in numerous changes but there were very few differences between the two hormone treatments. Compared to control levels, vitamin C was elevated during treatment while all three enzyme activities were lowered. Vitamin supplementation raised asparate aminotransferase and glutathione reductase activity and the levels of folic acid, vitamin A, and carotene. This study demonstrates that interactions between hormones and vitamins and among vitamins themselves, are complex but it is likely that the treatments used here caused no physiologically significant vitamin alterations.

Selected coenzymes and vitamin values in the female baboon.

Laboratory assessment of folic acid, vitamin B12, ascorbic acid, carotene, vitamin A, thiamin, riboflavin, and vitamin B6 levels in the plasma or erythrocytes of normally cycling female baboons is reported. The laboratory methods are discussed and comparative data from humans are presented.

Transcervical endometrial biopsy in the baboon: Effects on the menstrual cycle and relations to protein and dry matter content and histology.

The baboon has been used increasingly for reproductive studies. While hormonal regulation of the menstrual cycle and ovulation as well as the endocrinology of gestation have been reported, little information is available describing endometrial parameters. It is the purpose of this paper to describe the ease with which repeated transcervical biopsies can be performed, to describe baseline endometrial protein and dry weight data and to demonstrate that the biopsy procedure itself does not significantly affect the baboons' ability to continue normal menstrual cycle function. Endometrial biopsy samples were taken throughout the menstrual cycle under light ketamine anesthesia. Protein and dry weight contents were determined. Endometrial biopsies Protein and dry weight contents were determined. Endometrial biopsies averaged 25 mg (wet weight) and contained 7.54% protein and 16.3% dry matter. The formulas (Y = a + bx) which expressed the linear relationships between wet weight (mg), protein (µg) and dry matter (µg) content and the correlation coefficients (r) were as follows: between wet weight and protein content - wet weight = 5.58 + 10.0 (protein), Sxy = 4.83, r = 0.883; between wet weight and dry weight - wet weight = 1.99 + 7.94 (dry weight), Sxy = 4.52, r = 0.904; between protein and dry weight - protein = 0.446 + 0.446 (dry weight), Sxy = 4.82, r = 0.870. All three linear regression coefficients were statistically significant (P < 0.001). No significant cyclical patterns in either protein or dry matter content were demonstrable throughout the menstrual cycles. The average length of all nonbiopsy cycles was 32.4 ± 2.7 days and 32.8 ± 3.6 days for those in which biopsies were taken. Similarly, follicular and luteal phase lengths for nonbiopsy and biopsy cycles were 15.4 ± 2.3 and 15.5 ± 2.8 days and 16.9 ± 2.2 and 17.2 ± 3.2 days, respectively. The time required for sex-skin swelling to decrease from maximum to minimum during the luteal phase was shorter, but the quiescent stage was equally lengthened. It was concluded that the endometrium of the baboon was easily accessible for study without causing serious alterations in menstrual cycle function. These studies further demonstrate the potential of the baboon as a model o reproductive studies. In fact, the baboon may well the only practical primate model available for endometrial studies.

Cyclic variations in folate composition and pteroylpolyglutamyl hydrolase (conjugase) activity of the rat uterus.

The "free" and "total" folate content and the activity of conjugase (pteroylpolyglutamyl hydrolase) were determined in homogenates of rat uteruses from animals sacrificed at specific stages of the reproductive cycle. Among 47 animals, conjugase activity was approximately twice as great during proestrus as in any other stage (P less than 0.001). A significant increase in total folate content (P less than 0.01) was observed in these animals, associated with a relatively greater increase in the free component than in the polyglutamyl component during proestrus. A similar decline in the ratio of total to free folate was observed (P less than 0.02) in a second group of 43 animals in which conjugase was inactivated even more rapidly than in the first group. Vascular engorgement was excluded as an explanation for the changes observed in proestrus. Since certain polyglutamyl derivatives of folate are potent inhibitors of thymidylate synthetase, the observed shift in ratio between total and free folates could be conducive to enhanced activity of this rate-limiting reaction of cellular proliferation. The data suggest that cycles of uterine cell growth and involution may be mediated through hormonally induced changes in enzymes governing the length of gamma-glutamyl folate chains. It is postulated that the mechanism involves the conversion of metabolic inhibitors into active coenzymes for one-carbon transfer reactions, and vice versa.