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.

Effect of ethynodiol diacetate and mestranol on serum folic acid and vitamin B12 levels and on tryptophan metabolism in baboons.

The effect of an oral contraceptive agent on serum folate and vitamin B12 levels and on some aspects of tryptophan metabolism in the baboon has been investigated. Folate levels were not affected by this treatment. Vitamin B12 levels were lowered during oral contraceptive treatment (P smaller than 0.1) and increased during a 30-day period of pyridoxine supplementation (P smaller than 0.01). Trends indicating deterioration of glucose tolerance and abnormal tryptophan metabolism were also observed. Definite conclusions concerning the effect of oral contraceptive agents on nutritive parameters could not be made. This is in keeping with the vast volume of literature published on the effects of oral contraceptive agents in humans and other species. It is concluded that perhaps, due to the biologically variable responses to oral contraceptive agents by individuals of any species, studies of serum levels of vitamins and other superficial biochemical parameters. Further, we conclude that the baboon could be a useful model for the study of endocrine influences on nutritive parameters, first because endocrine parameters resemble those of humans and second, because experimental protocols can be rigidly adhered to.

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.

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.

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.

Estrogen stimulation of conjugase activity in the uterus of ovariectomized rats.

Recent observations have indicated that uterine folic acid conjugase (pterolypolyglutamyl hydrolase) undergoes rhythmic variation during the reproductive cycle of the rat. In the present study conjugase activity and ratios of "total" folate to "free" folate were determined in uterine homogenates of ovariectomized rats, with ane without estrogen stimulation. An increase in conjugase levels and a relative increase in the "free" folate component occurred in castrated animals 12 to 18 hours following injection of 10 mug of estradiol-17beta.

PIP: Estrogen stimulation of conjugase activity in the uterus was studied in ovariectomized rats. The rats were sacrificed either without estrogen replacement or at varying time intervals after the sc administration of 10 mcg of estradiol 17-beta. The uterine horns were processed for folate determinations or for conjugase assay. A significant increase (p less than .001) of conjugase activity was seen in the animals sacrifieced 20 hours after administration of the hormone. The ratio of total-to-free folate was significantly lower (p less than .001) than the control 20 hours after injection of the hormone. The drop in both free and total folates could not be explained.

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.

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.

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.

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.

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.

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.

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.

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.

Diurnal and menstrual cycle variations in serum vitamin B-12 and folacin activity in baboons.

Serum levels of vitamin B-12 and folacin were determined every 6 hours over a 24-hour period in five baboons and every other day throughout complete menstrual cycles in seven baboons. All baboons were normally cycling females exhibiting serum estrogen, progestin, and luteinizing hormone levels during the follicular, ovulatory and, luteal phases, respectively, of their menstrual cycles of 217.9 plus or minus 115.6, 420.3 plus or minus 113.9, 207.3 plus or minus 88.6 pg/ml; 1.71 plus or minus 0.84, 2.72 plus or minus 1.50, 5.92 plus or minus 4.38 ng/ml; 2.85 plus or minus 2.3, 38.4 plus or minus 20.5, 6.0 plus or minus 6.6 mg/ml. Serum vitamin B-12 levels did not vary significantly over a 24-hour period although higher mean levels were observed at 1200 and 1800 hours, whereas the lowest mean levels were observed at 2400 and 0600 hours. Folacin activity varied significantly (P smaller than 0.01) over a 24-hour period with the mean level at 1800 hours significantly higher (P smaller than 0.01) than that at all other times. The overall means and standard deviations for vitamin B-12 and folacin, respectively, during this study were 742.2 plus or minus 785.6 pg/ml and 14.48 plus or minus 6.74 ng/ml. Serum levels of vitamin B-12 and folacin did not vary significantly between the follicular or luteal phases or show any significant correlation with the day of the menstrual cycle when analyzed by linear regression procedures. The overall means and standard deviations for vitamin B-12 and folacin, respectively, during the menstrual cycle study were 438.5 plus or minus 137.9 pg/ml and 14.8 plus or minus 4.27 ng/ml.

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.

A study of the effect of oral contraceptive agents on the absorption, metabolic conversion and urinary excretion of a naturally-occurring folate (citrovorum factor).

PIP: 31 individuals, 18-38 years of age (10 females taking oral contraceptives - (OC's) - 3 months or longer, 11 females without a history of OCs, and 10 males) were observed regarding a fasting serum folate, peak serum folate after ingestion of 6 mg of N 5 formyl-tetrahydrofolate and 5 hour urinary excretion of folate, in order to determine whether the system involved in converting a formyl derivative of folate to the methyl form during intestinal absorption was affected by OC. Assays were carried out using either S. fecalis or L. casei as the test organism. The conversion of formyl to methyl tetrahydrofolate in the intestines was unaffected by OC use. 2 theories explaining the reported lowered serum and red cell folate among OC users were presented. There may be an individual variation in metabolic handling of folate.^ieng