Delayed tumor onset in transgenic mice fed a low-folate diet.

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.

Adenine nucleotide metabolism in relation to purine enzymes in liver, erythrocytes and cultured fibroblasts.

To evaluate the regulation of adenine nucleotide metabolism in relation to purine enzyme activities in rat liver, human erythrocytes and cultured human skin fibroblasts, rapid and sensitive assays for the purine enzymes, adenosine deaminase (EC 2.5.4.4), adenosine kinase (EC 2.7.1.20), hyposanthine phosphoribosyltransferase (EC 2.4.28), adenine phosphoribosyltransferase (EC 2.4.2.7) and 5'-nucleotidase (EC 3.1.3.5) were standardized for these tissues. Adenosine deaminase was assayed by measuring the formation of product, inosine (plus traces of hypoxanthine), isolated chromatographically with 95% recovery of inosine. The other enzymes were assayed by isolating the labelled product or substrate nucleotides as lanthanum salts. Fibroblast enzymes were assayed using thin-layer chromatographic procedures because the high levels of 5'-nucleotidase present in this tissue interferred with the formation of LaCl3 salts. The lanthanum and the thin-layer chromatographic methods agreed within 10%. Liver cell sap had the highest activities of all purine enzymes except for 5'-nucleotidase and adenosine deaminase which were highest in fibroblasts. Erythrocytes had lowest activities of all except for hypoxanthine phosphoribosyltransferase which was intermediate between the liver and fibroblasts. Erhthrocytes were devoid of 5'-nucleotidase activity. Hepatic adenosine kinase activity was thought to control the rate of loss of adenine nucleotides in the tissue. Erythrocytes had excellent purine salvage capacity, but due to the relatively low activity of adenosine deaminase, deamination might be rate limiting in the formation of guanine nucleotides. Fibroblasts, with high levels of 5'-nucleotidase, have the potential to catabolize adenine nucleotides beyond the control od adenosine kinase. The purine salvage capacity in the three tissues was erythrocyte greater than liver greater than fibroblasts. Based on purine enzyme activities, erythrocytes offer a unique system to study adenine salvage; fibroblasts to study adenine degradation; and liver to study both salvage and degradation.

Animal models and analytical approaches for understanding the relationships between wine and cancer.

We used two approaches for studying the relationships between wine consumption, wine composition and cancer In the first approach, a transgenic mouse model of human neurofibromatosis, combined with the use of well-defined, chemically purified diets, showed that red wine contains nonalcoholic components that can delay tumor onset. In additional studies, catechin, the main monomeric polyphenol of red wine, delayed tumor onset in this mouse model in a positive, linear relationship when incorporated into the diet at levels of 0.5-4 mmol/kg diet. In the second approach, low doses of the chemical carcinogen 2-amino-1-methyl-6-phenylimidazo(4, 5-b)pyridine (PhlP) were administered to rats, and formation of DNA adducts was evaluated by accelerator mass spectrometry. Consumption of red wine solids (the residue from red wine remaining after removal of alcohol and water) and the wine polyphenol quercetin did not influence PhlP-DNA adduct levels or induce liver enzymes (glutathione-S-transferase and quinone reductase). However, quercetin did alter distribution of PhlP in the rat tissues compared to control animals and animals fed other potential dietary chemopreventive agents, including phenylethyl isothiocyanate and sulforaphane. These studies demonstrate the feasibility of these approaches for studying the chemopreventive potential of dietary components at physiologic levels in

Homogenized bovine milk xanthine oxidase: a critique of the hypothesis relating to plasmalogen depletion and cardiovascular disease.

A hypothesis has repeatedly been promoted that xanthine oxidase from homogenized bovine milk is absorbed intact, damaging cardiovascular tissue by depleting plasmalogens and initiating atherosclerotic changes that culminate in heart disease. In the light of recent experimental evidence, the present paper examines the validity of this hypothesis and associated claims. The evidence leads to the conclusion that 1) absorption of dietary xanthine oxidase has not been demonstrated; 2) a relationship between intakes of homogenized milk and levels of serum xanthine oxidase activity have not been established; 3) a direct role for xanthine oxidase in plasmalogen depletion has not been established; 4) neither liposome formation during homogenization of milk nor absorption of intact liposomes from the gastrointestinal tract has been demonstrated; and 5) data are lacking to support the claim that large doses of folic acid inhibit xanthine oxidase in vivo and/or are therapeutic in heart disease. Experimental evidence has failed to substantiate, and in many cases has refuted, the xanthine oxidase/plasmalogen depletion hypothesis.

Variability of the conversion of beta-carotene to vitamin A in women measured by using a double-tracer study design.

BACKGROUND: Blood beta-carotene and vitamin A responses to oral beta-carotene are variable in humans. Some individuals are characterized as responders and others as low- or nonresponders. A better understanding of the conditions that produce the variability is important to help design public health programs that ensure vitamin A sufficiency. OBJECTIVE: Our objective was to assess variability in absorption and conversion of beta-carotene to vitamin A in vivo in humans by using a novel double-tracer ¿hexadeuterated (D(6)) beta-carotene and D(6) retinyl acetate approach. DESIGN: Eleven healthy women were housed at the US Department of Agriculture Western Human Nutrition Research Center metabolic unit for 44 d, where they consumed diets adequate in vitamins and minerals except for carotenoids. After an adaptation period, the women were given 30 micromol D(6) retinyl acetate orally, followed 1 wk later with 37 micromol D(6) beta-carotene (approximately equimolar doses). Time-dependent plasma concentration curves were determined for D(6) retinol, D(6) beta-carotene, and trideuterated (D(3)) retinol (derived from D(6) beta-carotene). RESULTS: Mean (+/-SE) absorption of D(6) beta-carotene was 3.3 +/- 1.3% for all subjects. The mean conversion ratio was 0.81 +/- 0.34 mol D(3) retinol to 1 mol D(6) beta-carotene for all subjects. However, only 6 of the 11 subjects had plasma D(6) beta-carotene and D(3) retinol concentrations that we could measure. The mean absorption of D(6) beta-carotene in these 6 subjects was 6.1 +/- 0.02% and their conversion ratio was 1.47 +/- 0.49 mol D(3) retinol to 1 mol D(6) beta-carotene. The remaining 5 subjects were low responders with

Influence of folate status and polyphenol intake on thiamin status of Irish women.

The relationship of folate status and polyphenol intake to thiamin status was studied in 80 randomly selected elderly and young Irish women, with key variables affecting thiamin nutrition controlled for. Folate, thiamin, and polyphenol intakes were measured during a 4-wk baseline (elderly and young) and 6-wk double-blind (elderly) supplementation period. Only elderly subjects were randomly assigned to placebo, folic acid (400 micrograms), thiamin (10 mg), or folic-acid-plus-thiamin groups. Thiamin status (TPP effect) was not affected by folate status (plasma and erythrocyte folate) during the baseline period or with folic acid supplementation alone. Polyphenol intake was not correlated with thiamin status. Only thiamin intake and thiamin supplementation significantly affected thiamin status. Because the majority of subjects (102 of 160) were initially thiamin deficient, enrichment of Irish grain products with thiamin is recommended.

Delayed tumor onset in transgenic mice fed an amino acid-based diet supplemented with red wine solids.

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.

Estimating the concentration of beta-carotene required for maximal protection of low-density lipoproteins in women.

The reportedly inconsistent antioxidant protective effect of beta-carotene on plasma LDL may depend on LDL's beta-carotene concentration. We measured carbonyl production by CuSO4-challenged LDL from nine healthy women living at the US Department of Agriculture-Western Human Nutrition Research Center and consuming a natural food diet that provided only 0.14 micromol beta-carotene/d for 120 d. During the first 60 d, four women received a placebo and the remaining five women received too small a supplement (0.93 micromol beta-carotene/d) to increase plasma or LDL beta-carotene; therefore, the data for all nine women during this time were pooled. From days 61 to 120, all subjects received the small supplement. From days 101 to 120 they all received an additional, larger, mixed carotenoid supplement (6.16 micromol beta-carotene/d). Plasma beta-carotene dropped from 0.76 +/- 0.21 micromol/L (x +/- SEM) on day 2 to 0.33 +/- 0.08 on day 60 (P = 0.035) and rose to 1.73 +/- 0.18 (P = 0.001) on day 120. LDL beta-carotene dropped from 1.67 +/- 0.53 micromol/g LDL protein on day 2 to 1.27 +/- 0.28 micromol/g LDL protein on day 60 (P = 0.650) and rose to 10.04 +/- 1.07 micromol/g LDL protein (P = 0.001) on day 120. Plasma lycopene dropped from 0.20 micromol/L on day 2 to 0.02 micromol/L on day 60 and did not increase by day 120. Carbonyl production rose from 24 +/- 6 micromol/g LDL protein on day 2 to 42 +/- 4 micromol/g LDL protein (P = 0.001) on day 60 and dropped to 6 +/- 1 micromol/g LDL protein (P = 0.001) on day 120. LDL seemed fully protected with 9.7 +/- 2.5 micromol beta-carotene/g LDL protein, or 2.3 +/- 1.8 micromol beta-carotene/L plasma.

Vitamin A concentrations in liver determined by isotope dilution assay with tetradeuterated vitamin A and by biopsy in generally healthy adult humans.

The vitamin A status in 11 generally healthy surgical patients was estimated by measuring the dilution of a 45-mg oral dose of tetradeuterated retinyl acetate (99% pure). After purification of retinol by high-performance liquid chromatography, the ratio of 2H4-retinol:1H-retinol in plasma was measured by gas chromatography-mass spectrometry. On the basis of the observed ratios of [2H4]retinol:[1H]retinol over 19-47 d, the total body reserves and liver concentrations of vitamin A were calculated. Liver biopsy samples taken at surgery were directly analyzed for vitamin A. The correlation coefficient between calculated and measured liver vitamin A concentrations for 10 of the subjects was 0.88, and the Spearman's rank correlation coefficient was 0.95 (p less than 0.002). Thus, total body reserves of vitamin A in humans can be estimated validly in the marginal and satisfactory ranges by a benign, relatively noninvasive procedure.

Activities of purine pathway enzymes in gouty human fibroblasts aged in vitro.

The finite life-span of fibroblasts in culture may reflect aging at the cellular level and gout is clinical condition whose incidence also increases with age. In order to better understand the age-related changes in purine metabolism, activities of purine degrading (adenosine deaminase and 5'-nucleotidase) and reutilizing (adenine phosphoribosyltransferase, hypoxanthine phosphoribosyl-transferase and adenosine kinase) enzymes were measured in serially cultured skin fibroblasts from normal subjects and from gouty patients who overproduce uric acid. Serially cultured fibroblasts from gouty overproducers of uric acid displayed increased purine enzyme levels with increasing cell passage while fibroblasts from normal donors showed little change in activity. There was no alteration in relative degrading and reutilizing enzyme levels. The data suggest an increase in the rate of purine turnover in aging gouty fibroblasts compared with normal fibroblasts.

Behavioral and neurochemical changes in folate-deficient mice.

Weanling mice were fed an amino acid-based diet supplemented with 0 or 11.3 mumol folic acid/kg diet for approximately 38 days to study behavior and neurochemistry in folate deficiency. After approximately 5 wk, mice fed the unsupplemented diet weighted approximately 70% as much those fed the supplemented diet. After 2 wk, mice fed the unsupplemented diet consistently discarded (spilled) more food, and after approximately 5 wk, they had spilled 3 times more than mice fed the supplemented diet. Serum folate, brain folate and brain S-adenosylmethionine of mice fed the unsupplemented diet were 4, 53, and 60% as high, respectively, as those of mice fed the supplemented diet. Pathologic changes were not evident in brain, spinal cord, or skeletal muscle of folate-deficient mice. The hypothalamic 5-hydroxyindole acetic acid/serotonin ratio and caudate dopamine, homovanillic acid, and 3,4-dihydroxyphenylacetic acid concentrations were lower in deficient than control mice. Folate-deficient mice develop a behavioral activity, food spilling, which may have a neurochemical basis in the serotonin and dopamine systems.

Composition of and cholesterol in Araucana and commercial eggs.

Araucana eggs from six sources and commercial-type white eggs of two major supermarket brands and from the University of California flock were collected and analyzed for cholesterol content of the yolk. The yolks of Araucana eggs were significantly higher in cholesterol than those of commercial white eggs.

Comparative effects of thermal and surgical trauma on rat muscle protein metabolism.

A modified intraperitoneal pool flooding technique, employing L-3H-tyrosine, was developed for measuring muscle protein synthetic rates following traumatic injury. Sufficient radiolabeled tyrosine was injected intraperitoneally to effect a six-fold increase in plasma tyrosine concentration (124-800 microM) resulting in constant, sustained specific radioactivities in plasma- and intracellular-free tyrosine pools. Localized vs systemic effects of thermal and surgical trauma on gastrocnemius muscle protein turnover were assessed 2 and 4 days postinjury. Thermal trauma increased total, myofibrillar, and sarcoplasmic muscle protein synthesis (44%) and protein degradation (300%). Conversely, surgical trauma decreased synthesis of total (24%), myofibrillar (14%), and sarcoplasmic (43%) muscle proteins without altering protein degradation. Short-term restriction of pair-fed controls did not affect either aspect of protein turnover.

Muscle protein metabolism of rats in surgical trauma.

The effects of decreased food intake and degree of surgical trauma on total, myofibrillar and sarcoplasmic muscle protein synthesis and degradation were assessed in two experiments (A and B). Trauma consisted of an abdominal incision with or without hysterectomy. The degree of trauma in experiment B was increased relative to that in experiment A by extending the length of the incision, operative manipulation and time required to perform the surgery. To account for postoperative diminutions in food intake on protein turnover, a group of nonoperated rats were pair-fed to the level of food consumed by hysterectomized rats. Traumatized rats in experiment B lost more weight, ate less, and had a lower muscle total protein concentration than corresponding rats in experiment A, confirming a more severe trauma in experiment B. In both experiments, trauma depressed total protein content of muscle. Synthesis was measured by the incorporation of L-[U-14C] tyrosine from a single meal into total, sarcoplasmic and myofibrillar proteins of gastrocnemius muscle. Degradation was calculated as the difference between the growth rate and the synthetic rate. Synthetic rate (ks) of total protein was depressed by surgical trauma; the more severe the trauma, the greater the depression. In mild trauma, the depression in ks was due only to a decrease in sarcoplasmic protein synthesis (ke), whereas with more severe trauma, synthetic rates of both sarcoplasmic (kes) and myofibrillar (kem) proteins were decreased. Protein degradation (kd,) was increased on day 2 in experiments A and B, had returned to control values on day 4 in experiment A and had decreased below control values in experiment B.(ABSTRACT TRUNCATED AT 250 WORDS)

Postsurgical muscle protein turnover in perfused hindquarters of the rat.

Skeletal muscle protein synthesis and degradation were measured simultaneously in perfused hindquarters of adult female rats 0, 2, and 4 days after surgical trauma. In order to explore the role of decreased postoperative nutrient intake on muscle protein turnover, a group of rats were pair-fed to the level of food consumed by surgical traumatized animals. Protein synthesis was measured by the incorporation of 3H-L-phenylalanine into the myofibrillar (contractile) and sarcoplasmic (soluble) proteins of gastrocnemius muscle. Protein degradation rates were calculated from the release of myofibrillar 3-methyl histidine (3MH) during the perfusion. Surgical trauma significantly depressed myofibrillar and sarcoplasmic protein synthetic rates by 33 and 29%, respectively. Protein degradation, as assessed by 3MH release into perfusate, increased 25% on the second postoperative day but returned to control levels by the 4th day after surgery. Food restriction of the pair-fed control rats did not alter protein synthesis, however, protein degradation decreased significantly. In conclusion, the effect of trauma on protein turnover appears not to be due to decreased nutrient consumption.

Development and survival of Drosophila melanogaster fed a diet containing pyrimidine analogs.

A single generation of Drosophila melanogaster was raised on different media. One of the media was unsupplemented (the control) and the others were supplemented with pyrimidine analog at 10.3 mmol/kg culture medium. The relative numbers of larvae, pupae, and F1 adults reproduced from parent flies on each medium served as an indication of the relative toxicity of the supplements. The relative decreasing order of toxicity of the pyrimidines was as follows: 5-bromouracil < thymine < uracil = orotic acid = control = cytosine, control < UMP. The toxic effects of 5-bromouracil and thymine seem to be associated with the addition of a bromine or methyl group to carbon 5 of the pyrimidine ring. The UMP supplementation increased the number of adult F1 flies above the control group indicating that UMP was not only non toxic but also that it was beneficial.

Tissue folate binding protein levels in transgenic mice with tumors and in non-transgenic controls.

Localized folate deficiency may be a risk factor for cancer. Since, folate binding proteins (FBP) and reduced folate carrier proteins (RFC) mediate cellular transport of folate, we compared FBP concentrations in several organs from tumor-bearing transgenic (TBT) mice and tumor-free non-transgenic controls (NTC) of the same strain, age, and fed identical diets. Liver, spleen, brain, small intestine and kidney were individually homogenized in phosphate-buffered saline (PBS) and separated into membrane, cytoplasmic, mitochondrial/lysomal and nuclear fractions (confirmed with marker enzymes). Homogenates and fractions was analyzed for total protein, and FBP. We used rabbit anti-bovine milk antibody and ELISA to measure FBP. FBP concentrations in kidney, small intestine, and spleen of TBT mice were higher than those of NTC mice; the opposite was true in liver and lung. FBP seemed to be upregulated in kidneys (all fractions), small intestine (all fractions), and spleen (cytoplasmic and nuclear fractions only) of TBT mice compared to NTC mice; the opposite appeared true in liver (all fractions) and lung (all fractions). FBP concentrations in brain, heart, and muscle of TBT mice were not different from those in brain, heart and muscle of NTC mice. A longitudinal study will determine if these changes in FBP concentrations precede tumor onset.

Statistical models for quantitative bioassay.

We discuss various statistical approaches useful in the analysis of nutritional dose-response data with a continuous response. The emphasis is on the multivariate case with several predictors. The methods which will be discussed can be classified into parametric models, including change-point models, and nonparametric models, which rely on smoothing methods such as weighted local linear fitting. The methods will be illustrated with the analysis of data generated from a folate depletion-repletion bioassay experiment conducted on rats, where the measured growth rate of the rate is the response variable. We also discuss the biological conclusions that can be drawn from applying various statistical methods to this data set.

Protocol development for biological tracer studies.

Improved instrumentation and the increased availability of labeled compounds have democratized the application of isotope-dilution (tracer) methodology in nutrient metabolism. Still, the most challenging aspects of tracer experimentation reside in the steps that precede the measurement of an isotopically labeled tracer, i.e. the design of a suitably labeled tracer and its isolation and purification from complex biological matrices. Construction of useful mathematical models of nutrient dynamics require methodologies that guarantee that the integrity of the tracer is maintained across the entire sampling and analyte isolation protocol. The ability to provide accurate and reliable data highlights a need for analytical chemists to play a central role in these studies. In this regard, examples and discussion of issues relevant to stable-isotope experimentation are provided.

A method for improving the nutritional value of food proteins: covalent attachment of amino acids.

Casein was modified by use of a series of active N-hydroxy-succinimide esters of amino acids in order to study the effects of new covalently linked hydrophobic or hydrophilic groups on its physical and nutritional properties. Tryptophan was used to determine the best conditions for the chemical reaction and to study the stability of the newly formed amide linkage (isopeptide bond). Casein was also modified with glycine, alanine, methionine, N-acetyl-methionine and aspartic acid. In vitro hydrolysis studies using bovine chymotrypsin, pancreatine and rat bile-pancreatic juice indicated that digestibility of the modified casein derivatives was lower than that of the untreated protein. Since solubility was not significantly changed (except for tryptophyl-casein), the decreased in vitro digestibility is probably due to other factors such as steric hindrance as well as decrease in lysine residues available to trypsin in pancreatin and rat pancreatic juice. Plasma amino acid patterns for rats fed a 10% protein diet of highly modified glycyl-casein or methionyl-casein suggest that the epsilon-aminolysyl derivatives are readily hydrolyzed in vivo. This was confirmed by the growth response of rats fed the following isonitrogenous diets (protein source listed only): casein, casein + free methionine, methionyl-casein, casein + free N-acetyl-methionine, N-acety-methionyl-casein. Covalently attached methionine appeared to be as readily available as the free amino acid; bound N-acetyl-methionine was also available but to a slightly lower extent. Although this study is preliminary, the covalent attachment of amino acids to proteins appears to be a promising method for improving the biological value of food proteins.