Comparison of zinc reduction with platinum reduction for analysis of deuterium-enriched water samples for the doubly labeled water technique.

OBJECTIVE: Isotope ratio mass spectrometry of hydrogen and oxygen is frequently used to determine total energy expenditure (TEE) using doubly labeled water. Conventionally, hydrogen isotope ratio is determined in hydrogen gas generated from water samples using zinc reduction. We compare this with a new automated platinum method to determine the ratios of hydrogen isotopes in deuterium-enriched water samples. RESEARCH METHODS AND PROCEDURES: The platinum method of sample preparation was compared with the zinc method in three ways: analytical variation in deuterium enrichment (within sample; n = 51), analytical variation in TEE estimates (within sample set; n = 10), and level of agreement of TEE estimates between both methods (n = 14). RESULTS: For the zinc method, the standard deviation for multiple sets of triplicate 2H2O sample analysis was +/-4.36 per thousand and +/-2.07 per thousand for platinum. The correlation between TEE estimates when sample sets were analyzed in duplicate was r = 0.89 for zinc and r = 0.83 for platinum. The intercept and slope of the regression line were significantly different from the line of identity for duplicate TEE estimates by zinc but were not different from the line of identity for platinum. After correction for the intra-assay variation of each method, the correlation between zinc and platinum for TEE was 0.77, and the intercept, but not the slope, of the regression was significantly different from the line of identity. The mean difference between the zinc method and the platinum method was 56 kcal/day, and the 95% confidence interval was -438 to 550 kcal/day. DISCUSSION: These data suggest that the platinum method is at least as reliable as the zinc method as a sample preparation technique for isotope ratio mass spectrometry of deuterium-enriched water samples. The platinum method is also less costly and less labor-intensive than the zinc method.

The use of an enteral expert system in the prescription of enteral formulas in a university hospital.

A personal computer-based expert system has been developed for the prescription of enteral formulas based on patient-need characteristics. Two hundred twelve inpatients in a university hospital setting were prospectively evaluated to compare the identity and cost of the enteral formula prescribed by the expert system with the identity and cost of the enteral formula prescribed by the ward team. Two hundred seven patients had complete data to allow analysis. There was a mean cost savings (+/-SD) of $1.18 +/- 7.69/d for each patient using the expert system compared with the MD-prescribed formula (P = 0.023). We project that the use of this program would save $27,564/y in our hospital (an average of 23,360 patient/days of enteral feeding per year). We conclude that the use of an expert system can be cost-effective in the prescription of enteral formulas for hospitalized patients.

Cofactor role for 10-formyldihydrofolic acid.

10-Formyl-7,8-dihydrofolic acid (10-HCO-H2folate) was prepared by controlled air oxidation of 10-formyl-5,6,7,8-tetrahydrofolic acid (10-HCO-H4folate). The UV spectra of the 10-HCO-H2folate preparation has lambda max. 234, 333 nm and lambda min. 301 nm at pH 7.4, and lambda max. 257, 328 nm and lambda min. 229, 307 nm at pH 1. 1H-NMR spectroscopy of 10-HCO-H2folate (in 2H2O; 300 MHz) suggested a pure compound and gave resonances for one formyl group proton, two protons on C-7 and C-9, and no evidence for a C-6 proton, which is consistent with the structure proposed. The spectral properties indicated that the 10-HCO-H2folate preparation is not appreciably contaminated with 10-HCO-H4folate, 5,10-methenyltetrahydrofolic acid (5,10-CH = H4folate) or 10-formylfolic acid (10-HCO-folate). The above data establish that the 10-HCO-H2folate prepared here is authentic. In contrast, a folate with a UV spectrum having lambda max. 272 nm and lambda min. 256 nm at pH 7, which was prepared by 2,6-dichloro-indophenol oxidation of 10-HCO-H4folate and reported to be 97% pure [Baram, Chabner, Drake, Fitzhugh, Sholar and Allegra (1988) J. Biol. Chem. 263, 7105-7111], is apparently not 10-HCO-H2folate. 10-HCO-H2folate is utilized by Jurkat-cell (human T-cell leukaemia) and chicken liver aminoimidazolecarboxamide ribonucleotide transformylase (AICAR T'ase; EC 2.1.2.3) in the presence of excess 5-amino-imidazole-4-carboxamide ribotide (AICAR) resulting in the appearance of approximately 1 mol of H2folate product for each mol of AICAR formylated. The present 10-HCO-H2folate preparation had a kinetic advantage over 10-HCO-H4folate resulting from a difference of approx. 5-fold in K(m) values when both folates were used as cofactors for Jurkat-cell and rat bone marrow AICAR T'ase. No substantial kinetic advantage was observed using chicken liver AICAR T'ase. 10-HCO-H2folate had little or no activity with Jurkat-cell or chicken liver glycinamide ribonucleotide transformylase (GAR T'ase, EC 2.1.2.2). The existence in vivo of 10-HCO-H2folate is suggested in mammals by several reports of detectable amounts of radiolabelled 10-HCO-folate in bile and urine after administration of radiolabelled folic acid.

Supplementation with folic acid during methotrexate therapy for rheumatoid arthritis. A double-blind, placebo-controlled trial.

OBJECTIVE: To determine the effect of two different weekly doses of folic acid on the toxicity and efficacy of low-dose methotrexate therapy for rheumatoid arthritis. DESIGN: Randomized, double-blind, placebo-controlled study. PATIENTS: 79 persons between 19 and 78 years of age who fulfilled the American Rheumatism Association's criteria for rheumatoid arthritis. INTERVENTION: Participants were randomly assigned to visually identical placebo or to 5 mg or 27.5 mg of folic acid each week. MEASUREMENTS: Duration, intensity, and clinical severity of toxic events; efficacy (indices of joint tenderness and swelling and grip strength); plasma and erythrocyte folate levels; and other laboratory variables. RESULTS: Folic acid supplementation at either dose did not affect the efficacy of methotrexate therapy as judged by joint indices and patient and physician assessments of disease. Patients given folic acid supplements had lower toxicity scores than did participants given placebo (P < or = 0.001). Low blood folate levels and increased mean corpuscular volumes were associated with substantial methotrexate toxicity, whereas daily dietary intakes of more than 900 nmol (400 micrograms) of folic acid were associated with little methotrexate toxicity. CONCLUSIONS: Folic acid, an inexpensive vitamin, is safe in a broad range of doses and protects patients with rheumatoid arthritis who are taking methotrexate from toxicity while preserving the efficacy of methotrexate.

Differences in methotrexate and 7-hydroxymethotrexate inhibition of folate-dependent enzymes of purine nucleotide biosynthesis.

7-Hydroxymethotrexate (7-OH-MTX) is the major and, frequently the only, pteridine metabolite found in bone-marrow aspirates of patients chronically treated with low-dose oral methotrexate (MTX) [Sonneveld, Schultz, Nooter and Hahlen (1986) Cancer Chemother. Pharmacol. 18, 111-116]. The Ki values for MTX and 7-OH-MTX for avian liver 5-amino-imidazole-4-carboxamide ribonucleotide transformylase differ by 4.5-fold in favour of 7-OH-MTX as the better inhibitor, while Ki values for avian liver glycinamide ribonucleotide transformylase differ by 1.9-fold favouring MTX as the better inhibitor. Thus 7-OH-MTX possesses a different enzyme-inhibiting repertoire from its parent drug and this information may be useful in explaining the mechanism of action of low-dose MTX therapies used to treat autoimmune disease.

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.

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.

Effects of folate deficiency and supplementation on methylnitrosourea-induced rat mammary tumors.

BACKGROUND: There are metabolic and epidemiologic data consistent with the hypothesis that folate deficiency increases the likelihood of cancer. Conversely, it is also known that folate is necessary for cancer growth, but few experiments in laboratory animals have evaluated the effects of folate deficiency on the development of chemically induced cancers. PURPOSE: Our purpose was to determine the effects of nutritional folate deficiency in female Fischer 344 rats on initiation and early promotion of methylnitrosourea (MNU)-induced mammary cancer. METHODS: Rats (age, 27 days) were fed a folic acid-deficient diet (AIN-76A) supplemented with glycine and succinylsulfathiazole [FA(0)]; the FA(0) diet supplemented with 2 or 40 mg of folic acid per kilogram [FA(2) or FA(40), respectively]; or the FA(0) diet supplemented with 20 mg of folinic acid per kilogram [FL(20)]. At 57 days of age, each diet-treated group (30 rats in each group) received MNU (50 mg/kg) by intravenous injection. Immediately after MNU treatment, all animals were fed the AIN-76A complete diet containing 2 mg of folic acid per kilogram. Control groups were fed the AIN-76A complete diet throughout the entire experiment. RESULTS: After 4 weeks, folate deficiency, but not anemia or growth suppression, was documented by lower folate levels in plasma and red blood cells in the group receiving the FA(0) diet. Cancer multiplicity (i.e., number of mammary cancers per number of tumor-bearing animals) at 180 days after MNU injection was 1.32, 1.90, 2.14, and 2.73 mammary cancers per tumor-bearing animal in the FA(0), FA(2), FA(40), and FL(20) groups, respectively; the value in the FA(0) group was statistically significant compared with the values in the other groups. The time required for 50% of the rats to develop palpable mammary cancer was 170, 142, 100, and 85 days, respectively. The value of 170 days for the FA(0) group was statistically significant compared with the values of 100 and 85 days. Mammary cancer incidence was 63%, 70%, 72%, and 73%, respectively; these percentages were not significantly different. CONCLUSIONS: Folate deficiency suppresses and folate supplementation enhances initiation or early promotion of MNU-induced mammary cancer in rats, even when the folate-deficient rats do not have anemia or growth suppression. IMPLICATION: Since the rat is relatively resistant to folate deficiency anemia, other animal models should be used to test the effect of folate nutriture on carcinogenesis.

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.

Long-term treatment of the MRL/lpr mouse with methotrexate and 10-deazaaminopterin.

Female MRL/lpr mice were treated with I.P. doses of methotrexate (MTX) and 10-deazaaminopterin (DAAM) in the range of 1 to 100 mg/kg body weight/week, in two equally divided doses. Treatment began at 7 weeks of age and continued to 30 weeks of age. Joint histopathology scores were tightly correlated with skin lesion-proteinuria scores at 30 weeks of age. MTX at levels of 5, 25, and 100 mg/kg body weight/week and DAAM at a level of 25 mg/kg body weight/week significantly reduced skin lesion-proteinuria scores below controls in a dose dependent manner. Animals receiving MTX at 25 mg/kg body weight/week had a significantly longer median life span and animals receiving MTX at 100 mg/kg body weight/week had a greater than 15% suppression of growth when compared with controls. Longevity and skin lesion-proteinuria scores appeared to be good indicators of drug efficacy while growth suppression appeared to be a good indicator of drug toxicity.

Nutrient levels in amniotic fluid from women with normal and neural tube defect pregnancies.

We analyzed nutrient levels in amniotic fluid obtained during the second trimester of normal, uncomplicated pregnancies from 221 women who delivered apparently healthy infants and from 8 with neural tube defect (NTD) pregnancies. Folate was measured by microbiological assay, vitamin B12 by a radiobinding method, and zinc, copper and iron by atomic absorption spectrophotometry. We found that the mean amniotic fluid nutrient levels of normal pregnancies were 24.7 nmol/l for folate, 600 pmol/l for vitamin B12, and 1.7, 1.9, and 9.0 mumol/l for zinc, copper and iron, respectively. Amniotic fluid folate, zinc, copper and iron levels of NTD pregnancies were similar to those found during normal pregnancy, however, vitamin B12 levels were markedly lower than those of normal pregnancies.

Detection of inhibition of 5-aminoimidazole-4-carboxamide ribotide transformylase by thioinosinic acid and azathioprine by a new colorimetric assay.

The colorimetric assay for 5-aminoimidazole-4-carboxamide ribotide (AICAR) transformylase (phosphoribosylamino-imidazolecarboxamide formyltransferase; EC 2.1.2.3) has been extensively modified. The modified assay is based upon the short-term permanganate oxidation of the folate product, tetrahydrofolate (H4folate) to p-aminobenzoyl glutamate (pABG). The modified assay was used to detect the transformylase activity in crude extracts of peripheral-blood mononuclear cells (PBMCs). Azathioprine and its metabolite, thioinosinic acid (tIMP), are competitive inhibitors (with respect to AICAR) of the chicken liver transformylase and the transformylase from PBMCs of the MRL/lpr mouse, an animal model of systemic autoimmune disease. The Ki values of tIMP and azathioprine for the chicken liver enzyme are 39 +/- 4 microM and 120 +/- 10 microM, whereas the Ki values for the enzyme from PBMCs of the MRL/lpr mouse are 110 +/- 20 microM and 90 +/- 14 microM respectively. The anti-inflammatory drugs ibuprofen and naproxen are also inhibitors of the transformylase.

Effect of miso (Japanese soybean paste) and NaCl on DMBA-induced rat mammary tumors.

An AIN-76A diet supplemented with miso (Japanese soybean paste) reduced the incidence (p = 0.05, Fishers exact test) and delayed the appearance (p = 0.04, log rank test) of dimethylbenz[a]anthracene (DMBA)-induced mammary adenocarcinomas in female Sprague-Dawley rats. A NaCl-supplemented AIN-76A diet (containing the same amount of NaCl as in the miso-supplemented diet) also delayed the appearance of tumors (p = 0.02, log rank test) compared with the AIN-76A control diet. The miso- and NaCl-supplemented diet treatment groups showed a trend toward a lower number of cancers per animal, a trend toward a higher number of benign tumors per animal, and a trend toward a lower growth rate of cancers compared with controls. However, no statistical differences in the total number of tumors per animal or growth rate of the cancers were observed in the miso, NaCl, and control groups. Both miso and NaCl supplementation resulted in increased water intake and urine output but no change in the growth of the animals. These data suggest that miso consumption may be a factor producing a lower breast cancer incidence in Japanese women.

The effect of folic acid supplementation on the toxicity of low-dose methotrexate in patients with rheumatoid arthritis.

Thirty-two patients with rheumatoid arthritis completed a 24-week, placebo-controlled, double-blind trial of folic acid (FA) supplementation during low-dose methotrexate (MTX) therapy. Administration of the daily FA supplement significantly lowered toxicity scores without affecting efficacy, as measured by joint counts, joint indices, and patient and physician evaluation of disease activity. Fifteen patients experienced some sort of toxicity; 67% were in the placebo group, and 33% were in the FA supplement group. Four patients in the placebo group had toxicity levels serious enough to require discontinuation of the MTX, while no patients in the FA supplement group discontinued MTX because of toxicity. Low-normal initial plasma and red blood cell folate levels were predictive of future toxicity with MTX therapy. We conclude that a daily supplement of 1 mg of FA during low-dose MTX therapy (median dose 7.5 mg/week [16.4 mumoles]) is usefull in lessening toxicity without altering efficacy during the first 6 months of treatment.

Zinc and copper balance in children on continuous ambulatory peritoneal dialysis.

We monitored serum zinc and copper levels for 4 months in six patients treated with continuous ambulatory peritoneal dialysis (CAPD). Zinc and copper fluxes were studied during a single dialysis exchange and over a 3-day period. Routine oral trace element supplements were then discontinued for 2 months. Serum zinc levels declined but serum copper levels remained unchanged. One month after oral supplements had been restarted, serum zinc levels returned to normal and serum copper levels rose above initial values. Zinc and copper concentrations in dialysis exchange indicated that the patients absorbed zinc and lost copper in significant amounts. The patients had poor dietary intakes of both minerals. These data suggest that patients treated with CAPD benefit from oral zinc supplementation.

Inhibition of 5-aminoimidazole-4-carboxamide ribotide transformylase, adenosine deaminase and 5'-adenylate deaminase by polyglutamates of methotrexate and oxidized folates and by 5-aminoimidazole-4-carboxamide riboside and ribotide.

With the use of a continuous spectrophotometric assay and initial rates determined by the method of Waley [Biochem. J. (1981) 193, 1009-1012] methotrexate was found to be a non-competitive inhibitor, with Ki(intercept) = 72 microM and Ki(slope) = 41 microM, of 5-aminoimidazole-4-carboxamide ribotide transformylase, whereas a polyglutamate of methotrexate containing three gamma-linked glutamate residues was a competitive inhibitor, with Ki = 3.15 microM. Pentaglutamates of folic acid and 10-formylfolic acid were also competitive inhibitors of the transformylase, with Ki values of 0.088 and 1.37 microM respectively. Unexpectedly, the pentaglutamate of 10-formyldihydrofolic acid was a good substrate for the transformylase, with a Km of 0.51 microM and a relative Vmax. of 0.72, which compared favourably with a Km of 0.23 microM and relative Vmax. of 1.0 for the tetrahydro analogue. An analysis of the progress curve of the transformylase-catalysed reaction with the above dihydro coenzyme revealed that the pentaglutamate of dihydrofolic acid was a competitive product inhibitor, with Ki = 0.14 microM. The continuous spectrophotometric assay for adenosine deaminase based on change in the absorbance at 265 nm was shown to be valid with adenosine concentrations above 100 microM, which contradicts a previous report [Murphy, Baker, Behling & Turner (1982) Anal. Biochem. 122, 328-337] that this assay was invalid above this concentration. With the spectrophotometric assay, 5-aminoimidazole-4-carboxamide riboside was found to be a competitive inhibitor of adenosine deaminase, with (Ki = 362 microM), whereas the ribotide was a competitive inhibitor of 5'-adenylate deaminase, with Ki = 1.01 mM. Methotrexate treatment of susceptible cells results in (1) its conversion into polyglutamates, (2) the accumulation of oxidized folate polyglutamates, and (3) the accumulation of 5-aminoimidazole-4-carboxamide riboside and ribotide. The above metabolic events may be integral elements producing the cytotoxic effect of this drug by (1) producing tighter binding of methotrexate to folate-dependent enzymes, (2) producing inhibitors of folate-dependent enzymes from their tetrahydrofolate coenzymes, and (3) trapping toxic amounts of adenine nucleosides and nucleotides as a result of inhibition of adenosine deaminase and 5'-adenylate deaminase respectively.