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

Serum carotenoid depletion follows first-order kinetics in healthy adult women fed naturally low carotenoid diets.

Dietary intakes of carotenoids are highly variable in human populations as are serum carotenoid concentrations. However, there are few controlled data relating carotenoid intake to concentration. Most of the data that are available are from measurements of the absorption and decay of large pharmacologic doses of carotenoids, and are therefore of unknown physiologic relevance. Our objective was to determine the half-life (t(1/2)) of the most abundant carotenoids in blood serum from healthy adult women living under controlled conditions. As part of two carotenoid isotopic studies, we measured serum concentrations of beta-carotene, alpha-carotene, lutein, zeaxanthin, beta-cryptoxanthin and lycopene in 19 healthy young adult women that were fed controlled low carotenoid diets for approximately 10 wk. All other nutrients (vitamins A, E and C) were provided at 100-150% of the 1989 U.S. recommended dietary allowance levels. Exercise and activities were controlled throughout the studies to simulate usual activity patterns. Carotenoid concentrations were measured by reversed-phase HPLC. Serum carotenoid concentration decreases during depletion followed first-order kinetics. The half-lives determined in decreasing order were as follows: lutein (76 d) > alpha-carotene (45 d) = beta-cryptoxanthin (39 d) = zeaxanthin (38 d) = beta-carotene (37 d) > lycopene (26 d). Half-lives were unrelated to physical or demographic characteristics such as body mass, body fat, racial background or age in these relatively homogeneous groups. Carotenoids decreased by similar first-order mechanisms, although the rates differed for individual carotenoids.

Long-term kinetic study of beta-carotene, using accelerator mass spectrometry in an adult volunteer.

We present a sensitive tracer method, suitable for in vivo human research, that uses beta-[(14)C]carotene coupled with accelerator mass spectrometry (AMS) detection. Using this approach, the concentration-time course of a physiological (306 microgram 200 nCi) oral dose of beta-[(14)C]carotene was determined for 209 days in plasma. Analytes included beta-[(14)C]carotene, [(14)C]retinyl esters, [(14)C]retinol, and several [(14)C]retinoic acids. There was a 5.5-h lag between dosing and the appearance of (14)C in plasma. Labeled beta-carotene and [(14)C]retinyl esters rose and displayed several maxima with virtually identical kinetic profiles over the first 24-h period; elevated [(14)C]retinyl ester concentrations were sustained in the plasma compartment for >21 h postdosing. The appearance of [(14)C]retinol in plasma was also delayed 5.5 h postdosing and its concentration rose linearly for 28 h before declining. Cumulative urine and stool were collected for 17 and 10 days, respectively, and 57.4% of the dose was recovered in the stool within 48 h postdosing. The stool was the major excretion route for the absorbed dose. The turnover times (1/k(el)) for beta-carotene and retinol were 58 and 302 days, respectively. Area under the curve analysis of the plasma response curves suggested a molar vitamin A value of 0.53 for beta-carotene, with a minimum of 62% of the absorbed beta-carotene being cleaved to vitamin A.In summary, AMS is an excellent tool for defining the in vivo metabolic behavior of beta-carotene and related compounds at physiological concentrations. Further, our data suggest that retinyl esters derived from beta-carotene may undergo hepatic resecretion with VLDL in a process similar to that observed for beta-carotene.

Determination of blood folate using acid extraction and internally standardized gas chromatography-mass spectrometry detection.

Whole blood folate level is a superior indicator of folate nutritional status than serum/plasma level. Problems with and lack of confidence in results of current whole blood folate assays have limited its popularity for assessing folate nutritional status. Here, an acid extraction GCMS detection method that measures total folate whole blood is presented. Folates are released from the matrix of whole blood and cleaved to para-aminobenzoic acid (pABA) by acid hydrolysis in the presence of [(13)C(6)]pABA as internal standard (IS). The hydrolysate is passed over a C18 resin to remove heme. The pABA isotopomers are ethyl esterified, isolated on C18 resin, and trifluoroacetylated. Following normal-phase HPLC separation, the isotopomers are silylated to their tBDMS derivatives. The abundance of these derivatives are measured at m/z 324 for [(13)C(6)]pABA as IS and m/z 318 for pABA from whole blood folate. Our method uses readily available chemicals and our results agree well with those using Lactobacillus casei, the current gold standard reference assay. The presence of folate analogs (methotrexate) or antibacterials (sulfonamines) does not affect our method. This feature makes it useful in monitoring folate status of patients undergoing chemotherapy. Before using our method, pABA supplements must be discontinued for a few days.

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

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.

Intrinsic erythrocyte labeling and attomole pharmacokinetic tracing of 14C-labeled folic acid with accelerator mass spectrometry.

Long-term physiologic tracing of nutrients, toxins, and drugs in healthy subjects is not possible using traditional decay counting of radioisotopes or stable isotope mass spectrometry due to radiation exposure and limited sensitivity, respectively. A physiologic dose of 14C-labeled folic acid (35 microg, 100 nCi) was ingested by a healthy adult male and followed for 202 days in plasma, erythrocytes, urine, and feces using accelerator mass spectrometry. All samples and generated wastes were classified nonradioactive and the subject received a lifetime-integrated radiological effective dose of only 11 microSv. Radiolabeled folate appeared in plasma 10 min after ingestion but did not appear in erythrocytes until 5 days later. Approximately 0.4% of the erythrocytes were intrinsically labeled with an average of 130 (14)C atoms during erythropoiesis from the pulse of plasma [14C]folate. An appropriate radiocarbon-labeled precursor can intrinsically label DNA or a specific protein during synthesis and obtain limits of quantitation several orders of magnitude below that of stable isotope methods.

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.

The dynamics of folic acid metabolism in an adult given a small tracer dose of 14C-folic acid.

Folate is an essential nutrient that is involved in many metabolic pathways, including amino acid interconversions and nucleotide (DNA) synthesis. In genetically susceptible individuals and populations, dysfunction of folate metabolism is associated with severe illness. Despite the importance of folate, major gaps exist in our quantitative understanding of folate metabolism in humans. The gaps exist because folate metabolism is complex, a suitable animal model that mimics human folate metabolism has not been identified, and suitable experimental protocols for in vivo studies in humans are not developed. In general, previous studies of folate metabolism have used large doses of high specific activity tritium and 14C-labeled folates in clinical patients. While stable isotopes such as deuterium and 13C-labeled folate are viewed as ethical alternatives to radiolabeled folates for studying metabolism, the lack of sensitive mass spectrometry methods to quantify them has impeded advancement of the field using this approach. In this chapter, we describe a new approach that uses a major analytical breakthrough, Accelerator Mass Spectrometry (AMS). Because AMS can detect attomole concentrations of 14C, small radioactive dosages (nCi) can be safely administered to humans and traced over long periods of time. The needed dosages are sufficiently small that the total radiation exposure is only a fraction of the natural annual background radiation of Americans, and the generated laboratory waste may legally be classified non-radioactive in many cases. The availability of AMS has permitted the longest (202 d) and most detailed study to date of folate metabolism in a healthy adult human volunteer. Here we demonstrate the feasibility of our approach and illustrate its potential by determining empirical kinetic values of folate metabolism. Our data indicate that the mean sojourn time for folate is in the range of 93 to 120 d. It took > or = 350 d for the absorbed portion of small bolus dose of 14C-folic acid to be eliminated completely from the body.

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.

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.

Ion trap mass spectrometry for kinetic studies of stable isotope labeled vitamin A at low enrichments.

The role of beta-carotene in chemoprevention of cancers and other chronic diseases generated controversy when subpopulations taking beta-carotene supplements showed increased mortality in clinical trials. Determination of the dynamics of beta-carotene in individual human subjects has emerged as a high priority. Stable isotope labeled beta-carotene tracers can be employed to determine rates of conversion to retinol (vitamin A), but tracer doses must be small to minimize perturbation of endogenous retinoid and carotenoid pools. In such cases, ratios of labeled tracer/endogenous retinol are often low, and quantitative analysis at enrichments of < 1 mol% are unreliable owing to ion-molecule reactions that generate ions at the same mass as the labeled tracer even when no tracer is present. The current study demonstrates improved gas chromatography/mass spectrometry quantification of retinol-d4 and unlabeled retinol, as their tert-butyldimethylsilyl ethers, at low enrichments using an ion trap mass spectrometer operated in selected ion storage mode. Electron ionization of analyte takes place in the ion trap using conditions that eject ions outside the range m/z 390-420, and molecular ions at m/z 400 and 404 from retinol and retinol-d4 are quantified. Using this approach, unlabeled retinol yields a signal close to values calculated from natural isotopic abundances (approximately 0.13%), whereas several quadrupole instruments operated using selected ion monitoring yielded 2-5 times greater signal when no labeled retinol was present.

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.

Quantitative analysis by gas chromatography of volatile carbonyl compounds in expired air from mice and human.

Formaldehyde, acetaldehyde and acetone expired from tumor-bearing transgenic mice and formaldehyde exhaled from breast cancer patients were analyzed using gas chromatography. The tumor-bearing mice expired significantly more formaldehyde per unit metabolic size (1.43-2.98 micromol) than did control mice (0.77-1.01 micromol). There was no detectable difference in the levels of expired acetaldehyde and acetone between the two groups of mice. The exhaled formaldehyde levels from three women with breast cancer and from three healthy women were satisfactorily determined using the method developed in this study. The results suggest that these carbonyl compounds may be used as a biomarker.

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.

Statistical interaction model for exchangeability of food folates in rat growth bioassay.

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.

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.

Compartmental analysis of the dynamics of beta-carotene metabolism in an adult volunteer.

Metabolism of a 73 mumol oral dose of beta-carotene-d8 in olive oil was determined from plasma beta-carotene-d8 and retinol-d4 concentration-time curves in an adult male. beta-Carotene-d8 and retinol-d4 concentrations in serial plasma were measured using high performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS), respectively. Plasma beta-carotene-d8 and retinol-d4 concentration-time curves were described by a 5-term and a 3-term polyexponential equation, respectively, using an empirical description of beta-carotene metabolism. A physiologic compartmental model of beta-carotene metabolism was also constructed and tested. This model suggests that 22% of the beta-carotene dose is absorbed: 17.8% as intact beta-carotene and 4.2% as retinoid. Also, it suggests that both liver and enterocyte are important in converting beta-carotene to retinoid; 43% is converted in liver and 57% in enterocyte. Finally, it suggests that the mean residence time for beta-carotene is 51 days and that the 73 mumole dose does not alter the fractional transfer coefficients of the system after absorption takes place. The issue of central versus eccentric cleavage of beta-carotene in humans can be studied with further modeling combined with use of appropriately labeled beta-carotene.

Protocol for collection and HPLC analysis of volatile carbonyl compounds in breath.

This noninvasive method for collection and analysis of a wide range of aldehydes and ketones in human breath may enable assessment of lipid peroxidation and metabolic status in vivo. Breath samples are drawn through silica cartridges impregnated with 2,4-dinitrophenylhydrazine, which traps carbonyls as their hydrazone derivatives. The hydrazone derivatives are eluted from the cartridges with acetonitrile, separated by reversed-phase HPLC, and quantified spectrophotometrically. Using this method, we have measured formaldehyde, acetaldehyde, acetone, propanal, 2-butanone, butanal, pentanal, and hexanal. Recoveries of carbonyls added to Douglas bags were > 90%, except for 2-butanone, which was 86.2%. The overall CVs for sampling plus analyzing duplicate aliquots of breath were < 11%. The results indicate that this protocol can be used to monitor changes of carbonyl production by analyzing expired air, which may, with further study, indicate physiological and pathological status.