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The comparative value of several sources of dietary folate in promoting growth of folate-depleted rats was determined in a folate depletion-repletion rat growth bioassay. Folate-depleted rats were fed an amino acid-based diet supplemented with 11 different concentrations of folate (227, 272, 317, 363, 408, 454, 499, 544, 590, 635 and 680 nmol/kg) from each of 12 different sources of folate (folic acid, fried beef liver, cooked pinto beans individually, or as 1/3, 1/1, or 3/1 combinations of folate from the folic acid/beans, folic acid/beef liver and beans/beef liver) for a total of 132 treatments. Growth response to folic acid and bean folate was linear, whereas that to beef liver folate was distinctly nonlinear, beef liver folate being more potent at lower dietary concentrations but less potent at higher concentrations compared with folic acid and bean folate. Folic acid and bean folate were equivalent to and exchangeable with one another in promoting growth. Beef liver folate and folic acid/bean folate had an interactive effect in promoting growth. The nature of the interaction was antagonistic in that the presence of folic acid and/or bean folate reduced the efficacy of beef liver folate and vice versa. Beef liver folate is not exchangeable with either folic acid or bean folate. We conclude that food folates generally are not exchangeable and do interact adversely. A statistical interaction model that predicted the growth-promoting effect of several sources of dietary folate was developed and validated.

The Tax protein from human T-cell leukemia virus type I (HTLV-I) is a 40-kDa phosphoprotein capable of activating transcription from its own long terminal repeat (LTR), as well as increasing the transcription of cellular genes. Transcriptional activation of the HTLV-I LTR has been demonstrated via a cyclic-AMP-responsive element within the 21-bp Tax-responsive elements of the LTR. Phorbol esters also upregulate expression via the LTR. Since phosphorylation of Tax may play a role in these processes, we investigated the relative effects of kinase-stimulating agents on 32P incorporation into Tax. Our studies demonstrated that the phorbol ester 4 beta-phorbol-12 beta-myristate-13 alpha-acetate greatly stimulated Tax phosphorylation in a time- and dose-dependent manner. In contrast, 8-bromoadenosine 3'-5'-cyclic monophosphate induced little stimulation of Tax phosphorylation. Tax phosphorylation occurred only on serine residues and was mapped to a single tryptic fragment in both Tax-producing human lymphocytes and mouse fibroblast cells.

The incidence of neural tube defects was studied in mouse embryos from dams fed an amino acid-based diet containing 45, 91, 136, 181, 227 or 453 nmol folic acid/kg diet (Experiment 1) or 227, 453, 566, 680, 906, 1132, 1698 or 2266 nmol folic acid/kg diet (Experiment 2). Reproductive tracts were examined 12 d postcoitum and gross and microscopic examination of all embryos was performed. A single implantation was found at levels less than or equal to 181 nmol folic acid/kg diet. With one exception, bred mice fed 227 or 453 nmol folic acid/kg diet in Experiment 1 had 100% resorptions. In Experiment 2, 100% of implantations in mice fed 227 nmol folic acid/kg diet and approximately 75% of implantations in mice fed 453 or 566 nmol folic acid/kg diet resorbed. The 906 nmol folic acid/kg diet was sufficient for successful pregnancy. Mice fed 227 nmol folic acid/kg diet in Experiment 2 weighed approximately 80% of mice fed higher levels of folic acid. Inadequate dietary folic acid resulted in fewer and smaller embryos (which developed normally). These results suggest that folate deficiency alone is insufficient to produce neural tube defects in Swiss-Webster mice. Because individual micronutrients (e.g., folate) can be omitted from the amino acid-based diet, the specific role of folic acid in neurulation can now be studied systematically.

Background Transgenic mice carrying the human T-lymphotropic virus type 1 tax1 (transactivator) gene develop peripheral nerve sheath tumors with well-characterized times of onset and tissue involvement. Purpose and methods To evaluate the effect of dietary folic acid on age at tumor onset and on the concentration of folate in tissues and tumors, we bred heterozygous transgenic mice and systematically assigned their offspring at weaning (within litters) to a 2 x 2 x 2 factorial arrangement. The three variables studied were 1) the tax1 gene (presence or absence), 2) gender (male or female), and 3) dietary level of folic acid (0.11 or 11.34 mumol folic acid per kilogram of controlled amino acid-based diet). Blood and tissues were collected from tumor-bearing transgenic mice (prior to cachexia) and from nontransgenic littermates, matched whenever possible for gender and diet. Results Transgenic mice fed a diet containing 0.11 mumol of folic acid per kilogram developed tumors significantly later (92.8 +/- 6.4 days) than did those fed a diet containing 11.34 mumol of folic acid per kilogram (71.9 +/- 3.9 days). Folate concentrations in tumors of mice fed the low-folate diet were approximately one third those in tumors of mice fed the higher folate diet. Brain folate concentrations in mice fed the low-folate diet were less than one half those in mice fed the higher folate diet. Conclusion Results show that the onset of spontaneous tumors can be delayed by feeding mice the lowest level of folate adequate to meet nutritional requirements for normal growth. Implication Transgenic animal models of human disease offer great potential for evaluating the role of micronutrients in human carcinogenesis.

Tissues, whole blood, serum, and tumor folate of a tumor-bearing transgenie (TBT) mouse model for human neurqfibromatosis were measured to characterize effects of neoplasia on concentration of folate among tissues and tumors. Tissues of 11 TBT mice (killed when tumor burden was estimated to be 1% of body weight to minimize possible t~ffects of eachexia) and eight nontransgenic controls (NTC) of similar age were analyzedJbr fi~late. TBT mice had 1.5 +- 0.2 tumors. Tumor mass was 0.35 +- O. I0 g/mouse, and overall tumor folate concentrations were 0.38 +_ O. I0 nmol/g. Overall total tumor Jolate was 0.13 +- 0.04 nmol/mouse. Body weights and erythrocyte and leukocyte counts of TBT and NTC mice were similar. Hematocrits and hemoglobin concentrations were lower in TBT than in NTC mice. Livers and kidneys from TBT mice weighed less than those of NTC mice while spleens weighed more. Both groups had similar folate concentrations in liver and spleen while concentrations in kidney, brain, and serum were lower in TBT than in NTC miee. Folate per liver, kidney, and brain was lower while that per spleen was higher in TBT than in NTC mice. Tumor folate concentrations were lower than those of most mouse nontumor tissues. Folate concentrations of tumors were variable and were ranked as follows: nose > tail >--.[bot >-- ear. The presettce of tumors was associated with reduced folate concentration of some nontumor tissues. Folate concentrations of kidney, brain, and serum decreased as the number and mass of tumors increased.

Two experiments were conducted to determine the feasibility of using a folate depletion/repletion protocol with rats fed an amino acid-based diet to measure the bioavailability of food folate. Growth, liver folate and serum folate of depleted rats that were fed test foods incorporated into a folate-free, amino acid-based diet were standardized against similar responses of rats fed known amounts of folic acid incorporated into the same diet. Bioavailability of folate of cooked broccoli, refried beans and orange juice concentrate in experiment 1 was 80-89, 113 and 62%, respectively, based on growth response; in experiment 2, values for cooked and raw broccoli, cooked cabbage and cantaloupe were 95, 103, 74 and 81%, respectively. The results demonstrate that in addition to serum and liver folate concentrations, growth may be a useful response criterion to evaluate the bioavailability of folates in foods. Further research is needed to determine the relevance of these bioavailability estimates to human nutrition.

Increased consumption of vegetable foods (cereals, legumes, fruits) and some beverages (tea, cider, wine) is associated with reduced risk of cancer. Polyphenols in these foods and beverages are thought to be responsible, based on data from in vitro assays and from in vivo studies that used animals pretreated with carcinogen and given tea or polyphenol-spiked water to drink. We tested the hypothesis that dehydrated-dealcoholized red wine (wine solids), when consumed as part of a precisely defined complete diet, would delay tumor onset in transgenic mice that spontaneously develop externally visible tumors without carcinogen pretreatment. Sibling transgenic mice were weaned onto an amino acid-based diet alone or supplemented with red wine solids. Mice were examined daily; the age at which a first tumor appeared was recorded as the age of tumor onset. The concentration of the major polyphenol of red wine (catechin) in blood serum was also measured at the end of the study. The supplemented diet was fed continuously for three generations to ensure that it supported normal growth and reproduction. We discovered that the wine solid supplement delayed tumor onset, that intact catechin was absorbed, and that the supplemented diet supported normal growth and reproduction for three generations. Also, our simple experimental protocol offers an alternate and/or complementary way to identify foods, beverages, and their constituents that delay tumor onset and to investigate possible mechanisms involved.

To improve standardization of a folate bioassay, folate-depleted rats were repleted with a folate-free amino acid-based diet supplemented with 29 levels of folic acid. Growth was the main response variable and body tissue folate concentrations were also assessed. Because a positive correlation was observed between low levels of dietary folic acid and growth and little or no correlation was observed between high levels and growth, six regression models with a steep slope for low levels and a shallow or zero slope for high levels of dietary folic acid were evaluated. The model referred to as the "two-phase regression" or "change-point" model best described the relationship. Depleted rats needed 674 +/- 71 nmol folic acid/kg diet to reach their full growth potential. This value is biologically sensible, and this regression model is well established in the statistical literature. The change-point model is highly recommended to characterize the growth response, because growth is a functional response and, in the range of 226 to 680 nmol folic acid/kg, this response is linear, which is an additional advantage. Linear responses are easier to interpret because of complicated issues of interpretation and confidence intervals with nonlinearities. Linear regressions described serum and liver folate responses, whereas exponentials described whole blood and carcass folate responses. Depleted rats needed 5920 and 5780 nmol folic acid/kg diet to maximize their whole blood and carcass folates, respectively.

Most commercially available tretrahydro and substituted folates are racemic mixtures of R and S isomers about pteridine-carbon 6. Some reports show the unnatural stereoisomers modify specific aspects of folate metabolism, whereas others show no significantly different biologic effects between isomers. The extent to which unnatural folate stereoisomers are inert in vivo is unclear. Possible interactions between isomers at normal dietary folate intakes were tested by comparing growth, liver and serum folate concentrations, and congener patterns of folate-depleted rats fed amino acid-based diets with 283, 566 or 1132 nmol folic acid (standards), or 566 nmol racemic 5-methyltetrahydrofolate, 5-formyltetrahydrofolate or 5,10-diformyltetrahydrofolate/kg diet for 3 wk. Growth and tissue folate levels increased with each increment in dietary folic acid. Growth and liver and serum folate concentrations of rats fed 566 nmol 5-formyltetrahydrofolate or 5-methyltetrahydrofolate/kg diet were similar to those of rats fed 283 nmol folic acid/kg diet. Rats fed 5,10-diformyltetrahydrofolate lost weight and had depressed serum folate levels, and most died. Results show that biologic activity of racemic 5-formyltetrahydrofolate and 5-methyltetrahydrofolate was half that of folic acid, and the R isomer did not affect growth at the levels fed. Addition of a second formyl group destroyed folate bioactivity. The folate-depleted rat model is useful for testing biologic effects of individual folate stereoisomers in vivo as they become commercially available.

Folate bioavailability of beef liver, lima beans, peas, spinach, mushrooms, collards, orange juice and wheat germ was estimated with a protocol of folate depletion-repletion using growth and liver, serum and erythrocyte folate of weanling male rats. Diets with 125, 250 and 375 micrograms folic acid/kg were standards. Individual foods were incorporated into a folate-free amino acid-based diet alone (250 micrograms folate/kg diet from food) or mixed with folic acid (125 micrograms folate from food + 125 micrograms folic acid) to evaluate folate bioavailability and effects of food matrix. Beef liver and orange juice folates were as available as folic acid, whereas those of wheat germ were less bioavailable. Folates of peas and spinach were also less available than folic acid using liver and serum folate concentrations and total liver folate as response criteria, but they were not lower when based on growth and erythrocyte folate concentrations. Lima bean, mushroom and collard folates were as available as folic acid using four of five response criteria. Folate bioavailability of all foods generally exceeded 70%. All response criteria gave approximately equivalent results, indicating that growth and tissue folate levels are appropriate criteria. No food matrix effects were observed for any food except lima beans. Foods rich in polyglutamyl folates were less bioavailable than those of foods rich in short-chain folates.

To clarify relationships between dietary folic acid intake, blood levels and body stores of folate, rats were fed an amino acid-based diet supplemented with 0, 0.125, 0.5, 1, 2 or 4 mg folic acid/kg diet for 25 d. Folate concentrations of carcass, liver, gastrointestinal (GI) tract, kidney, spleen, testes, heart and lung from rats fed the folate-free diet were 0.06 +/- 0.01, 0.73 +/- 0.08, 0.05 +/- 0.01, 0.39 +/- 0.01, 0.05 +/- 0.01, 0.17 +/- 0.01, 0.02 +/- 0.01 and 0.02 +/- 0.01 micrograms/g, respectively. Serum and erythrocyte concentrations and total body stores were 0.88 +/- 0.16 ng/mL, 0.30 +/- 0.01 micrograms/mL and 13.9 +/- 0.7 micrograms, respectively. Body folate distribution was carcass, 55.6 +/- 1.4%; liver, 26.0 +/- 1.9%; erythrocytes, 7.7 +/- 0.4%; kidney, 4.8 +/- 0.2%; GI tract, 3.0 +/- 0.2%; and testes, 2.5 +/- 0.2%. Carcass content dropped to 38% whereas liver content increased to 44% in rats fed the highest dietary level. Tissue concentrations were correlated with one another and with dietary folate levels. Under these experimental conditions total body folate could be predicted from serum folate, but the general applicability of this relationship requires further study.

Folate depletion and repletion protocols are not well standardized. Weanling rats were moderately depleted of folate in 28 d with a folate-free purified diet based on 17% amino acids as the nitrogen source. They were then folate repleted for 23 d with the amino acid diet supplemented with either 125, 250, 500, 1000 or 2000 micrograms folic acid/kg. Hematology, growth and tissue folate levels were measured in subsets of the rats when they were 24 (baseline), 52 (depleted) and 75 d old (repleted). The same measurements were made in control rats that had been fed 2 mg folic acid/kg of the amino acid diet for the same period of time. Our findings show that with repletion, growth of previously depleted rats is in direct proportion with the level of supplementation up to 1000 micrograms folic acid/kg diet. Serum folate levels of repleted rats also increased in proportion to supplementation between 500 to 2000 micrograms/kg diet, and liver folate levels increased proportionally with the level of supplement within the range of 125 to 2000 micrograms/kg diet. The 2000 micrograms/kg supplement was sufficient to restore liver folate levels equivalent to that of controls, but body weight and serum folate levels failed to catch up with that of controls in the 23-d repletion period. There was a nonlinear relationship between serum and liver folate levels: serum folate remained constant at about 6 micrograms/l as liver folate increased to about 7 micrograms/g, then serum folate diverged by increasing to 120 micrograms/l with only minor increases in liver folate.(ABSTRACT TRUNCATED AT 250 WORDS)

We describe a simple, inexpensive sample preparation method that involves the isolation of chloride as hydrogen chloride from serum and urine prior to chloride analysis with the chloride ion-selective electrode. Chloride analyses of clinical chemistry standards with the present method were found to be in good agreement with analyses reported by the manufacturer. Reliability of the method is also evident by complete recovery of chloride added to serum and urine, minimal day-to-day variation of analyses, and a coefficient of variation that generally is less than 2%. An evaluation of factors influencing the procedure is also reported. The usefulness of the chloride ion-selective electrode to determine chloride in serum or urine is greatly enhanced by the sample preparation method described since matrix interference by other sample components is removed prior to analysis.

A factorial experiment was conducted with weanling rats fed a purified diet to determine the influence of dietary chloride (0.02, 0.10 and 0.50%) as sodium chloride on fluoride bioavailability (2 or 10 ppm as sodium fluoride). After 6 wk, rats fed the lowest chloride-containing diets had significant reductions of plasma chloride, urinary chloride excretion and growth rate compared to other chloride groups. Depressed growth occurred in rats fed chloride-deficient diets despite the fact that food intake was similar for all treatments. Fluoride retention was greatest in chloride-deficient rats, which was reflected in enhanced skeletal uptake of fluoride. Fluoride absorption was not inhibited by high chloride intake. We therefore conclude that emphasis on the effect of chloride on fluoride bioavailability should be directed towards an enhancement of fluoride retention by low salt (sodium chloride) diets rather than in terms of a possible negative effect of a high salt diet on fluoride absorption.