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Half-lives of α-tocopherol in plasma have been reported as 2-3 d, whereas the Elgin Study required >2 y to deplete α-tocopherol, so gaps exist in our quantitative understanding of human α-tocopherol metabolism. Therefore, 6 men and 6 women aged 27 ± 6 y (mean ± SD) ingested 1.81 nmol, 3.70 kBq of [5-(14)CH(3)]-(2R, 4'R, 8'R)-α-tocopherol. The levels of (14)C in blood plasma and washed RBC were monitored frequently from 0 to 460 d while the levels of (14)C in urine and feces were monitored from 0 to 21 d. Total fecal elimination (fecal + metabolic fecal) was 23.24 ± 5.81% of the (14)C dose, so feces over urine was the major route of elimination of the ingested [5-(14)CH(3)]-(2R, 4'R, 8'R)-α-tocopherol, consistent with prior estimates. The half-life of α-tocopherol varied in plasma and RBC according to the duration of study. The minute dose coupled with frequent monitoring over 460 d and 21 d for blood, urine, and feces ensured the [5-(14)CH(3)]-(2R, 4'R, 8'R)-α-tocopherol (the tracer) had the chance to fully mix with the endogenous [5-(14)CH(3)]-(2R, 4'R, 8'R)-α-tocopherol (the tracee). The (14)C levels in neither plasma nor RBC had returned to baseline by d 460, indicating that the t(1/2) of [5-CH(3)]-(2R, 4'R, 8'R)-α-tocopherol in human blood was longer than prior estimates. [ABSTRACT FROM AUTHOR]

Our objective was to quantify the absorption and conversion to retinoids of a 1.01-nmol, 3.7-kBq oral dose of (14)C-beta-carotene in 8 healthy adults. The approach was to quantify, using AMS, the elimination of (14)C in feces for up to 16 d after dosing and in urine for up to 30 d after dosing. The levels of total (14)C in undiluted serial plasma samples were measured for up to 166 d after dosing. Also, the levels of (14)C in the retinyl ester (RE), retinol (ROH), and beta-carotene fractions that were isolated from undiluted plasma using HPLC were measured. The apparent digestibility of the (14)C was 53 +/- 13% (mean +/- SD), based on the mass balance data, and was generally consistent with the area under the curve for zero to infinite period of (14)C that was eliminated in the feces collections made up to 7.5 d after dosing. Metabolic fecal elimination, calculated as the slope per day (% (14)C-dose/collection from d 7.5 to the final day), was only 0.05 +/- 0.02%. The portion of the (14)C dose eliminated via urine was variable (6.5 +/- 5.2%). Participants [except participant 6 (P6)] had a distinct plasma peak of (14)C at 0.25 d post-dose, preceded by a shoulder at approximately 0.1 d, and followed by a broad (14)C peak that became indistinguishable from baseline at approximately 40 d. Plasma (14)C-RE accounted for most of the absorbed (14)C early after dosing and P1 had the longest delay in the first appearance of (14)C-RE in plasma. The data suggest that plasma RE should be considered in estimating the ROH activity equivalent of ingested beta-carotene. [ABSTRACT FROM AUTHOR]

BACKGROUND: Excentral cleavage of beta-carotene to retinoids and apocarotenoids occurs in vitro and in animal models. Whether it occurs in humans is unclear. OBJECTIVE: We tested the hypothesis of whether humans can cleave beta-carotene excentrally. DESIGN: A healthy man was given an oral dose of all-trans [10,10',11,11'-(14)C]-beta-carotene (1.01 nmol; 100 nCi). Its fate and that of its metabolites were measured in serial plasma samples. Its fate in feces and urine was also measured over time. Selected plasma samples were spiked with reference standards of retinol, beta-apo-12'-carotenal, beta-apo-8'-carotenal, 13-cis-retinoic acid, all-trans-retinoic acid, beta-carotene-5,6-epoxide, all-trans-beta-carotene, and retinyl palmitate and subjected to reverse-phase HPLC fractionation. The plasma, plasma fractions, urine, and feces were measured for (14)C with the use of accelerator mass spectrometry. RESULTS: Sixty-five percent of administered (14)C was absorbed, and 15.7% was eliminated in urine during the first 21 d after dosing. (14)C-beta-carotene and (14)C-retinyl palmitate appeared in plasma 0.25 d after the dose. (14)C-beta-carotene and (14)C-retinol both appeared at 0.5 d only. On day 3 after the dose, 2 large (14)C peaks appeared in plasma: one matched the retention time of beta-apo-8'-carotenal, and the other did not match any of the reference standards used. The delayed appearance of (14)C-beta-apo-8'-carotenal in plasma suggests that the excentral cleavage occurred after the (14)C-beta-apo-8'-carotene was absorbed into the body. CONCLUSION: These data suggest that excentral cleavage of ingested beta-carotene occurs in vivo in humans. Confirmation of that possibility and further study to identify and characterize additional metabolites are needed. Copyright © 2007 American Society for Nutrition [ABSTRACT FROM AUTHOR]

BACKGROUND: Quantitation of human vitamin E metabolism is incomplete, so we quantified RRR- and all-rac-alpha-tocopherol metabolism in an adult. OBJECTIVE: The objective of the study was to quantify and interpret in vivo human vitamin E metabolism. DESIGN: A man was given an oral dose of 0.001821 micromol [5-14CH3]RRR-alpha-tocopheryl acetate (with 101.5 nCi 14C), and its fate in plasma, plasma lipoproteins, urine, and feces was measured over time. Data were analyzed and interpreted by using kinetic modeling. The protocol was repeated later with 0.001667 micromol [5-14CH3]all-rac-alpha-tocopheryl acetate (with 99.98 nCi 14C). RESULTS: RRR-alpha-tocopheryl acetate and all-rac-alpha-tocopheryl acetate were absorbed equally well (fractional absorption: approximately 0.775). The main route of elimination was urine, and approximately 90% of the absorbed dose was alpha-2(2'-carboxyethyl)-6-hydroxychroman. Whereas 93.8% of RRR-alpha-tocopherol flow to liver kinetic pool B from plasma was returned to plasma, only 80% of the flow of all-rac-alpha-tocopherol returned to plasma; the difference (14%) was degraded and eliminated. Thus, for newly digested alpha-tocopherol, the all-rac form is preferentially degraded and eliminated over the RRR form. Respective residence times in liver kinetic pool A and plasma for RRR-alpha-tocopherol were 1.16 and 2.19 times as long as those for all-rac-alpha-tocopherol. Model-estimated distributions of plasma alpha-tocopherol, extrahepatic tissue alpha-tocopherol, and liver kinetic pool B for RRR-alpha-tocopherol were, respectively, 6.77, 2.71, and 3.91 times as great as those for all-rac-alpha-tocopherol. Of the lipoproteins, HDL had the lowest 14C enrichment. Liver had 2 kinetically distinct alpha-tocopherol pools. CONCLUSIONS: Both isomers were well absorbed; all-rac-alpha-tocopherol was preferentially degraded and eliminated in urine, the major route. RRR-alpha-tocopherol had a longer residence time and larger distribution than did all-rac-alpha-tocopherol. Liver had 2 distinct alpha-tocopherol pools. The model is a hypothesis, its estimates are model-dependent, and it encourages further testing. Copyright © 2006 American Society for Nutrition [ABSTRACT FROM AUTHOR]

BACKGROUND: The vitamin A activity of beta-carotene is variable and surprisingly low in women. The reasons for this are not well understood. The vitamin A activity of beta-carotene in men is still uncertain. Contributions of dietary factors compared with individual traits are largely unknown. OBJECTIVE: Our objective was to measure the intrinsic variability in the vitamin A activity of beta-carotene among healthy, well-fed men living in a controlled environment. DESIGN: We used a double-tracer test-retest design. We dosed 11 healthy men orally with 30 micromol hexadeuterated (D6) retinyl acetate (all-trans-19,19,19,20,20,20-[2H6]retinyl acetate) and then with 37 micromol D6 beta-carotene (19,19,19,19',19',19'-[2H6]beta-carotene) 1 wk later. Doses were taken with breakfasts containing 16 g fat. We measured D6 retinol, D6 beta-carotene, and trideuterated (D3) retinol (derived from D6 beta-carotene) concentrations in plasma. Areas under the plasma concentration x time since dosing curves (AUCs) were determined for D6 retinol, D6 beta-carotene, and D3 retinol. RESULTS: All men had detectable D6 retinol concentrations in plasma. The mean (+/-SE) absorption of D6 beta-carotene in all subjects was 2.235 +/- 0.925%, and the mean conversion ratio was 0.0296 +/- 0.0108 mol retinol to 1 mol beta-carotene. Only 6 of 11 men had sufficient plasma concentrations of D6 beta-carotene and D3 retinol that we could measure. The mean absorption of D6 beta-carotene in these 6 subjects was 4.097 +/- 1.208%, and the mean conversion ratio was 0.0540 +/- 0.0128 mol retinol to 1 mol beta-carotene. CONCLUSION: The vitamin A activity of beta-carotene, even when measured under controlled conditions, can be surprisingly low and variable. [ABSTRACT FROM AUTHOR]

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

To date, 14 C tracer studies using accelerator mass spectrometry (AMS) have not yet resolved lipid-soluble analytes into individual lipoprotein density subclasses. The objective of this work was to develop a reliable method for lipoprotein separation and quantitative recovery for biokinetic modeling purposes. The novel method developed provides the means for use of small volumes (10-200 µL) of frozen plasma as a starting material for continuous isopycnic lipoprotein separation within a carbon- and pH-stable analyte matrix, which, following post-separation fraction clean up, created samples suitable for highly accurate 14 C/ 12 C isotope ratio determinations by AMS. Manual aspiration achieved 99.2 ± 0.41% recovery of [5- 14 CH 3 ]-(2R, 4'R, 8'R)-α-tocopherol contained within 25 µL plasma recovered in triacylglycerol rich lipoproteins (TRL = Chylomicrons + VLDL), LDL, HDL, and infranatant (INF) from each of 10 different sampling times for one male and one female subject, n = 20 total samples. Small sample volumes of previously frozen plasma and high analyte recoveries make this an attractive method for AMS studies using newer, smaller footprint AMS equipment to develop genuine tracer analyses of lipophilic nutrients or compounds in all human age ranges.

Background: In a marker-trait association study we estimated the statistical significance of 65 single nucleotide polymorphisms (SNP) in 23 candidate genes on HDL levels of two independent Caucasian populations. Each population consisted of men and women and their HDL levels were adjusted for gender and body weight. We used a linear regression model. Selected genes corresponded to folate metabolism, vitamins B-12, A, and E, and cholesterol pathways or lipid metabolism., Methods: Extracted DNA from both the Sacramento and Beltsville populations was analyzed using an allele discrimination assay with a MALDI-TOF mass spectrometry platform. The adjusted phenotype, y, was HDL levels adjusted for gender and body weight only statistical analyses were performed using the genotype association and regression modules from the SNP Variation Suite v7., Results: Statistically significant SNP (where P values were adjusted for false discovery rate) included: CETP (rs7499892 and rs5882); SLC46A1 (rs37514694; rs739439); SLC19A1 (rs3788199); CD36 (rs3211956); BCMO1 (rs6564851), APOA5 (rs662799), and ABCA1 (rs4149267). Many prior association trends of the SNP with HDL were replicated in our cross-validation study. Significantly, the association of SNP in folate transporters (SLC46A1 rs37514694 and rs739439; SLC19A1 rs3788199) with HDL was identified in our study., Conclusions: Given recent literature on the role of niacin in the biogenesis of HDL, focus on status and metabolism of B-vitamins and metabolites of eccentric cleavage of β-carotene with lipid metabolism is exciting for future study.

Kinetic models enable nutrient needs and kinetic behaviors to be quantified and provide mechanistic insights into metabolism. Therefore, we modeled and quantified the kinetics, bioavailability, and metabolism of RRR-α-tocopherol in 12 healthy adults. Six men and 6 women, aged 27 ± 6 y, each ingested 1.81 nmol of [5(-14)CH(3)]-(2R, 4'R, 8'R)-α-tocopherol; each dose had 3.70 kBq of (14)C. Complete collections of urine and feces were made over the first 21 d from dosing. Serial blood samples were drawn over the first 70 d from dosing. All specimens were analyzed for RRR-α-tocopherol. Specimens were also analyzed for (14)C using accelerator MS. From these data, we modeled and quantified the kinetics of RRR-α-tocopherol in vivo in humans. The model had 11 compartments, 3 delay compartments, and reservoirs for urine and feces. Bioavailability of RRR-α-tocopherol was 81 ± 1%. The model estimated residence time and half-life of the slowest turning-over compartment of α-tocopherol (adipose tissue) at 499 ± 702 d and 184 ± 48 d, respectively. The total body store of RRR-α-tocopherol was 25,900 ± 6=220 μmol (11 ± 3 g) and we calculated the adipose tissue level to be 1.53 μmol/g (657 μg/g). We found that a daily intake of 9.2 μmol (4 mg) of RRR-α-tocopherol maintained plasma RRR-α-tocopherol concentrations at 23 μmol/L. These findings suggest that the dietary requirement for vitamin E may be less than that currently recommended and these results will be important for future updates of intake recommendations.

Using linear regression models, we studied the main and 2-way interaction effects of the predictor variables gender, age, BMI, and 64 folate/vitamin B-12/homocysteine (Hcy)/lipid/cholesterol-related single nucleotide polymorphisms (SNP) on log-transformed plasma Hcy normalized by RBC folate measurements (nHcy) in 373 healthy Caucasian adults (50% women). Variable selection was conducted by stepwise Akaike information criterion or least angle regression and both methods led to the same final model. Significant predictors (where P values were adjusted for false discovery rate) included type of blood sample [whole blood (WB) vs. plasma-depleted WB; P < 0.001] used for folate analysis, gender (P < 0.001), and SNP in genes SPTLC1 (rs11790991; P = 0.040), CRBP2 (rs2118981; P < 0.001), BHMT (rs3733890; P = 0.019), and CETP (rs5882; P = 0.017). Significant 2-way interaction effects included gender × MTHFR (rs1801131; P = 0.012), gender × CRBP2 (rs2118981; P = 0.011), and gender × SCARB1 (rs83882; P = 0.003). The relation of nHcy concentrations with the significant SNP (SPTLC1, BHMT, CETP, CRBP2, MTHFR, and SCARB1) is of interest, especially because we surveyed the main and interaction effects in healthy adults, but it is an important area for future study. As discussed, understanding Hcy and genetic regulation is important, because Hcy may be related to inflammation, obesity, cardiovascular disease, and diabetes mellitus. We conclude that gender and SNP significantly affect nHcy.

Background: Excentral cleavage of beta-carotene to retinoids and apocarotenoids occurs in vitro and in animal models. Whether it occurs in humans is unclear., Objective: We tested the hypothesis of whether humans can cleave beta-carotene excentrally., Design: A healthy man was given an oral dose of all-trans [10,10',11,11'-(14)C]-beta-carotene (1.01 nmol; 100 nCi). Its fate and that of its metabolites were measured in serial plasma samples. Its fate in feces and urine was also measured over time. Selected plasma samples were spiked with reference standards of retinol, beta-apo-12'-carotenal, beta-apo-8'-carotenal, 13-cis-retinoic acid, all-trans-retinoic acid, beta-carotene-5,6-epoxide, all-trans-beta-carotene, and retinyl palmitate and subjected to reverse-phase HPLC fractionation. The plasma, plasma fractions, urine, and feces were measured for (14)C with the use of accelerator mass spectrometry., Results: Sixty-five percent of administered (14)C was absorbed, and 15.7% was eliminated in urine during the first 21 d after dosing. (14)C-beta-carotene and (14)C-retinyl palmitate appeared in plasma 0.25 d after the dose. (14)C-beta-carotene and (14)C-retinol both appeared at 0.5 d only. On day 3 after the dose, 2 large (14)C peaks appeared in plasma: one matched the retention time of beta-apo-8'-carotenal, and the other did not match any of the reference standards used. The delayed appearance of (14)C-beta-apo-8'-carotenal in plasma suggests that the excentral cleavage occurred after the (14)C-beta-apo-8'-carotene was absorbed into the body., Conclusion: These data suggest that excentral cleavage of ingested beta-carotene occurs in vivo in humans. Confirmation of that possibility and further study to identify and characterize additional metabolites are needed.

Optimizing folate status requires continued monitoring of erythrocyte (RBC) folate and folate intake. The accuracy of RBC folate assays remains a concern. Therefore, we measured RBC folate with 4 different assays, examined the interassay correlations, and compared RBC folate with folate intake as measured by an abbreviated folate-targeted food/supplement screener. The screener had 21 questions (19 diet, 2 supplement) and measured usual and customary intakes of dietary folate equivalents (DFEs). Our design was a 4 x 2 x 2 factorial, 4 assays in pregnant and nonpregnant women before and after each group received a folic acid supplement (1814 nmol/d) for 30-60 d. Folate assays included L. casei, chemiluminescence, GC-MS, and radioassay (RA). Baseline RBC folate levels ranked low to high by assay (mean +/- SE) were as follows: 1155 +/- 44 nmol/L (L. casei) < 1390 +/- 43 nmol/L (chemiluminescence) < 1531 +/- 39 nmol/L (GC-MS) < 1727 +/- 55 nmol/L (RA) (P < 0.0001). Supplementation raised RBC folate levels (mean +/- SE) as follows: 138 +/- 63 nmol/L (chemiluminescence) < 267 +/- 64 nmol/L (GC-MS) = 285 +/- 75 nmol/L (L. casei) < 351 +/- 87 nmol/L (RA). Pregnant women had higher RBC folate than nonpregnant women using chemiluminescence and RA. Interassay correlations (r) ranged from 0.4679 to 0.8261 (P < 0.001). Correlations of RBC folate with folate intake ranged from 0.2676 to 0.4622 (P < 0.0004). We conclude that RBC folate levels are assay dependent, as is the definition of optimized status; there continues to be a need for an accurate assay of RBC folate. RBC folate correlated with total folate intake using a folate-targeted food/supplement screener.

Background: A quantitative understanding of human folate metabolism is needed., Objective: The objective was to quantify and interpret human folate metabolism as it might occur in vivo., Design: Adults (n = 13) received 0.5 nmol [(14)C]pteroylmonoglutamate (100 nCi radioactivity) plus 79.5 nmol pteroylmonoglutamate in water orally. (14)C was measured in plasma, erythrocytes, urine, and feces for >/=40 d. Kinetic modeling was used to analyze and interpret the data., Results: According to the data, the population was healthy and had a mean dietary folate intake of 1046 nmol/d, and the apparent dose absorption of (14)C was 79%. The model predictions showed that only 0.25% of plasma folate was destined for marrow, mean bile folate flux was 5351 nmol/d, and the digestibility of the mix (1046 + 5351 nmol/d) was 92%. About 33% of visceral pteroylmonoglutamate was converted to the polyglutamate form, most of the body folate was visceral (>99%), most of the visceral folate was pteroylpolyglutamate (>98%), total body folate was 225 micromol, and pteroylpolyglutamate synthesis, recycling, and catabolism were 1985, 1429, and 556 nmol/d, respectively. Mean residence times were 0.525 d as visceral pteroylmonoglutamate, 119 d as visceral pteroylpolyglutamate, 0.0086 d as plasma folate, and 0.1 d as gastrointestinal folate., Conclusions: Across subjects, folate absorption, bile folate flux, and body folate stores were larger than prior estimates. Marrow folate uptake and pteroylpolyglutamate synthesis, recycling, and catabolism are saturable processes. Visceral pteroylpolyglutamate was an immediate precursor of plasma p-aminobenzoylglutamate. The model is a working hypothesis with derived features that are explicitly model-dependent. It successfully quantitated folate metabolism, encouraging further rigorous testing.

The Experimental Biology 2003 symposium entitled "New Technologies for Nutrition Research" was organized to highlight new and emerging technologies, including nanotechnology and proteomics, and to suggest ways for their integration into nutrition research. Speakers focused on topics that included accelerator mass spectrometry for ultra-low level radiolabel tracing, nanodevices for real-time optical intracellular sensing, mass spectrometric techniques for examining protein expression, as well as potential applications for nanotechnology in the food sciences. These technologies may be particularly useful in obtaining accurate spatial information and low-level detection of essential and nonessential bioactive food components (nutrients) and their metabolites, and in enhancing the understanding of the impact of nutrient/metabolite and biomolecular interactions. Highlights from this symposium are presented briefly herein.

We present the application of a nonparametric method to performing functional principal component analysis for functional curve data that consist of measurements of a random trajectory for a sample of subjects. This design typically consists of an irregular grid of time points on which repeated measurements are taken for a number of subjects. We introduce shrinkage estimates for the functional principal component scores that serve as the random effects in the model. Scatterplot smoothing methods are used to estimate the mean function and covariance surface of this model. We propose improved estimation in the neighborhood of and at the diagonal of the covariance surface, where the measurement errors are reflected. The presence of additive measurement errors motivates shrinkage estimates for the functional principal component scores. Shrinkage estimates are developed through best linear prediction and in a generalized version, aiming at minimizing one-curve-leave-out prediction error. The estimation of individual trajectories combines data obtained from that individual as well as all other individuals. We apply our methods to new data regarding the analysis of the level of 14C-folate in plasma as a function of time since dosing of healthy adults with a small tracer dose of 14C-folic acid. A time transformation was incorporated to handle design irregularity concerning the time points on which the measurements were taken. The proposed methodology, incorporating shrinkage and data-adaptive features, is seen to be well suited for describing population kinetics of 14C-folate-specific activity and random effects, and can also be applied to other functional data analysis problems.

The effect of vitamin A supplements on metabolic behavior of an oral tracer dose of [14C]beta-carotene was investigated in a longitudinal test-retest design in two adults. For the test, each subject ingested 1 nmol of [14C]beta-carotene (100 nCi) in an emulsified olive oil-banana drink. Total urine and stool were collected for up to 30 days; concentration-time patterns of [14C]beta-carotene, [14C]retinyl esters, and [14C]retinol were determined for 46 days. On Day 53, the subjects were placed on a daily vitamin A supplement (10000 IU/day), and a second dose of [14C]beta-carotene (retest) was given on Day 74. All 14C determinations were made using accelerator mass spectrometry. In both subjects, the vitamin A supplementation was associated with three main effects: 1). increased apparent absorption: test versus retest values rose from 57% to 74% (Subject 1) and from 52% to 75% (Subject 2); 2). an approximately 10-fold reduction in urinary excretion; and 3). a lower ratio of labeled retinyl ester/beta-carotene concentrations in the absorptive phase. The molar vitamin A value of the dose for the test was 0.62 mol (Subject 1) and 0.54 mol (Subject 2) vitamin A to 1 mol beta-carotene. Respective values for the retest were 0.85 and 0.74. These results show that while less cleavage of beta-carotene occurred due to vitamin A supplementation, higher absorption resulted in larger molar vitamin A values.

Carotenoids provide pigmentation to avian species, and also have immunomodulatory potential, although experimental results are often inconsistent. Therefore, dietary carotenoid deposition into immune tissue of growing chicks was examined in relation to their maternal carotenoid status (i.e., yolk carotenoid level). Single-comb white leghorn chicks were hatched from carotenoid-replete (C+) or carotenoid-deplete (C-) eggs. For 4 wk posthatch, chicks were fed diets whose carotenoid level ranged from 0 to 38 mg total carotenoid/kg. Carotenoid additions consisted of lutein + canthaxanthin at a ratio of 4:1. After 4 wk, the carotenoid concentration of thymus, bursa, liver, plasma and shank epithelium was measured by HPLC. Egg yolk-derived carotenoids were detectable in chicks fed 0 dietary carotenoids for 4 wk. Chicks hatched from C+ eggs had significantly greater tissue lutein, zeaxanthin and/or canthaxanthin for all tissues (P < 0.05), compared to chicks hatched from C- eggs. Only bursa carotenoids were not dependent on chick diet (P = 0.24); for all other tissues, C+ chicks incorporated dietary carotenoids in a dose-dependent manner (P < 0.01), whereas C- chicks never achieved the same level of carotenoid incorporation. This study demonstrated the importance of maternal carotenoid status on incorporation of yolk- and diet-derived tissue carotenoids in an avian model, and may explain some variability in carotenoid-based research, given that maternal carotenoid status is rarely controlled.

Background: Evidence exists that red wine, which contains a large array of polyphenols, is protective against cardiovascular disease and possibly cancer., Objective: We tested the hypothesis that catechin, the major monomeric polyphenol in red wine, can delay tumor onset in transgenic mice that spontaneously develop tumors., Design: Mice were fed a nutritionally complete amino acid-based diet supplemented with (+)-catechin (0-8 mmol/kg diet) or alcohol-free solids from red wine. Mice were examined daily; the age at which a first tumor appeared was recorded as the age at tumor onset. Plasma catechin and metabolite concentrations were quantified at the end of the study., Results: Dietary catechin significantly delayed tumor onset; a positive, linear relation was observed between the age at tumor onset and either the amount of dietary catechin (r(2) = 0.761, P < 0.001) or plasma catechin and metabolite concentrations (r(2) = 0.408, P = 0.003). No significant effects on tumor onset were observed when mice consumed a diet supplemented with wine solids containing <0.22 mmol catechin/kg diet, whereas a previous study showed that wine solids with a similar total polyphenol concentration but containing approximately 4 times more catechin significantly delayed tumor onset by approximately 30 d compared with a control diet. The catechin composition of the wines is directly related to processing conditions during vinification., Conclusions: Physiologic intakes of specific dietary polyphenols, such as catechin, may play an important role in cancer chemoprevention. Wines have different polyphenol concentrations and compositions; therefore, the overall health benefits of individual wines differ.

Background: The vitamin A activity of beta-carotene is variable and surprisingly low in women. The reasons for this are not well understood. The vitamin A activity of beta-carotene in men is still uncertain. Contributions of dietary factors compared with individual traits are largely unknown., Objective: Our objective was to measure the intrinsic variability in the vitamin A activity of beta-carotene among healthy, well-fed men living in a controlled environment., Design: We used a double-tracer test-retest design. We dosed 11 healthy men orally with 30 micromol hexadeuterated (D6) retinyl acetate (all-trans-19,19,19,20,20,20-[2H6]retinyl acetate) and then with 37 micromol D6 beta-carotene (19,19,19,19',19',19'-[2H6]beta-carotene) 1 wk later. Doses were taken with breakfasts containing 16 g fat. We measured D6 retinol, D6 beta-carotene, and trideuterated (D3) retinol (derived from D6 beta-carotene) concentrations in plasma. Areas under the plasma concentration x time since dosing curves (AUCs) were determined for D6 retinol, D6 beta-carotene, and D3 retinol., Results: All men had detectable D6 retinol concentrations in plasma. The mean (+/-SE) absorption of D6 beta-carotene in all subjects was 2.235 +/- 0.925%, and the mean conversion ratio was 0.0296 +/- 0.0108 mol retinol to 1 mol beta-carotene. Only 6 of 11 men had sufficient plasma concentrations of D6 beta-carotene and D3 retinol that we could measure. The mean absorption of D6 beta-carotene in these 6 subjects was 4.097 +/- 1.208%, and the mean conversion ratio was 0.0540 +/- 0.0128 mol retinol to 1 mol beta-carotene., Conclusion: The vitamin A activity of beta-carotene, even when measured under controlled conditions, can be surprisingly low and variable.

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.

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.

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

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.

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.

A kernel-smoothing method with locally varying bandwidths for the nonparametric estimation of derivatives of a function is proposed for highly nonequidistant data as they occur in pharmacokinetic response curves. We construct estimates having the particular property that the derivative estimates correspond exactly to the corresponding derivatives of the curve estimate even under locally varying bandwidth choice. The effects on the estimation of peak location (characteristic points) are investigated. In an example, characteristic points are estimated for a recently developed in vivo isotope dilution assay for vitamin A (retinol) nutritional status. The in vivo kinetics of appearance and disappearance of isotopically labeled retinol can be described with the proposed method.

Lipid composition of body fat can be a key indicator of nutritional status and a number of human disorders. In vivo 13C NMR provides for repeated, noninvasive analysis of fatty acyl chain composition on individuals, which circumvents classical problems of individual variation and repetitive invasive sampling. It also offers a unique opportunity to examine acyl chain organization in situ. This approach was used to examine the fatty acyl chain composition in the perirenal fat pads of rats fed olive, safflower, and menhaden oil-containing diets. These changes were then monitored during a diet switch between olive and menhaden oil-fed rats. The fatty acid composition of perirenal fat pads and livers was also analyzed using gas chromatography for comparison with the in vivo NMR analysis. Both tissues assumed the general characteristics of diet fatty acyl chain and fatty acid composition and the diet switch induced a switchover of the perirenal composition in 30-45 days. These results indicate that a large portion of the diet fatty acyl chains were incorporated directly into adipose and liver tissues although some were also metabolized, particularly in menhaden oil-fed rats. Furthermore, changes in the in vivo spin-lattice relaxation times (T1) of fatty acyl carbons in the perirenal fat pads and their lipid extracts were followed and effective correlation times (tau eff) were calculated from the T1 data. The result indicated that the in vivo segmental mobility of acyl carbons was sensitive to changes in diet-derived fatty acyl chain composition and that the central region of the acyl chain was more sensitive to these changes. There was a qualitative similarity but quantitative differences in the tau eff of acyl carbons acquired in vivo and from extracts. These results suggest that adipose triacylglycerides experience an overall liquid-like microenvironment in vivo but with more restriction in their mobility, and that different factors may exist in governing their organization in situ versus in extracts.

Three experiments were conducted with young chicks to determine whether dietary leucine affects the conversion of tryptophan to niacin. Either tryptophan or niacin improved growth and reduced perosis when chicks were fed a purified diet marginal in tryptophan and deficient in niacin. Addition of 4.8% L-leucine to the diet did not alter the growth and perosis prevention response obtained with tryptophan. Liver weight was slightly increased by the addition of 5.4% L-leucine to the diet. Plasma insulin was slightly reduced by leucine and by isoleucine and valine. Picolinic carboxylase in the kidney was reduced in chicks fed 0.2% tryptophan with no niacin and was also reduced when isoleucine and valine were added to the diets. Liver picolinic carboxylase activity was not influenced by diet. Plasma isoleucine and valine were reduced by the addition of leucine to the diet and were increased again when isoleucine and valine were added to the diet. Plasma leucine was increased by the addition of leucine but was not altered by valine and isoleucine. Plasma tryptophan was not influenced by dietary supplements of leucine or isoleucine and valine. The results show that in the chick there is no evidence for an effect of leucine on the tryptophan to niacin pathway.

The rate of synthesis and catabolism of sarcoplasmic- and myofibrillar-muscle protein was measured in operated, sham-operated and food-restricted rats by using Na2 14CO3. The food-restricted group underwent sham operations and were limited to the food intake of the operated animals. Protein synthesis and catabolism were increased in the sarcoplasmic-muscle fraction in operated rats compared with that in sham-operated or food-restricted rats. The rate of synthesis of the myofibrillar protein decreased in operated animals, but the rate of catabolism was not altered in the myofibrillar-muscle fraction of the operated animals compared with that in food-restricted and sham-operated animals. In the operated animals, there was a net loss of protein from the muscle. Thus the rats that underwent surgery lost muscle protein, primarily as a result of a decrease in synthesis of myofibrillar protein. The changes in protein turnover in operated animals were not due to decreases in food intake, since protein turnover in sham-operated animals that were restricted to the food intake of the operated rats was not different from that in sham-operated rats fed ad libitum.

Hamster liver post-nuclear membranes catalyze the transfer of mannose from GDP-mannose to endogenous dolichyl phosphate and to a second major endogenous acidic lipid. This mannolipid was believed to be synthesized from endogenous retinyl phosphate and was tentatively identified as retinyl phosphate mannose (Ret-P-Man) (De Luca, L. M., Brugh, M. R. Silverman-Jones, C. S. and Shidoji, Y. (1982) Biochem. J. 208, 159-170). To characterize this endogenous mannolipid in more detail, we isolated and purified the mannolipid from incubations containing hamster liver membranes and GDP-[14C]mannose and compared its properties to those of authentic Ret-P-Man. We found that the endogenous mannolipid was separable from authentic Ret-P-Man on a Mono Q anion exchange column, did not exhibit the absorbance spectrum characteristic of a retinol moiety, and was stable to mild acid under conditions which cleave authentic Ret-P-Man. The endogenous mannolipid was sensitive to mild base hydrolysis and mannose was released from the mannolipid by snake venom phosphodiesterase digestion. These properties were consistent with the endogenous acceptor being phosphatidic acid. Addition of exogenous phosphatidic acid, but not phospholipids with a head group blocking the phosphate moiety, to incubations containing hamster liver membranes and GDP-[14C]mannose resulted in the synthesis of a mannolipid with chromatographic and physical properties identical to the endogenous mannolipid. A double-labeled mannolipid was synthesized in incubations containing hamster liver membranes, GDP-[14C]mannose, and [3H]phosphatidic acid. Mannosyl transfer to exogenous phosphatidic acid was saturable with increasing concentrations of phosphatidic acid and GDP-mannose and specific for glycosyl transfer from GDP-mannose. Class E Thy-1-negative mutant mouse lymphoma cell membranes, which are defective in dolichyl phosphate mannose synthesis, also fail to transfer mannose from GDP-mannose to exogenous phosphatidic acid or retinyl phosphate. Amphomycin, an inhibitor of dolichyl phosphate mannose synthesis, blocked mannosyl transfer to the endogenous lipid, and to exogenous retinyl phosphate and phosphatidic acid. We conclude that the same mannosyltransferase responsible for dolichyl phosphate mannose synthesis can also utilize in vitro exogenous retinyl phosphate and phosphatidic acid as well as endogenous phosphatidic acid as mannosyl acceptors.

The degree of relatedness among mammalian xanthine oxidases (XO) was determined by microcomplement fixation. Rabbit anti bovine milk XO serum was tested against xanthine oxidase (homologous protein) and against the heterologous proteins of bovine liver, monkey liver, rat liver, lactating cow serum, nonlactating cow serum, and steer serum. The indices of dissimilarity for the heterologous proteins were expressed as units of immunological distance and the percent sequence differences among these proteins inferred from the y = 5x relationship where y is immunological distance and x is percent sequence difference. Rat liver XO differed by approximately 27% in amino acid sequence from bovine milk XO. In order of increasing immunological distance from bovine milk XO, the sources of XO ranked as follows: lactating cow serum less than nonlactating cow serum less than steer serum = beef liver less than monkey liver less rat liver. The monkey ranked much closer than the rat in order of phylogenetic kinship to the cow. Starch gel electrophoresis of liver, milk, and serum showed that the milk and the serum contained only cationic forms of xanthine oxidase while all the liver samples tested contained cationic as well as anionic forms of the enzyme. The electrophoretic mobility properties of xanthine oxidase confirmed the polymorphic nature of the enzyme as revealed by the immunological data.

The effects of several highly purified simple and mixed dietary triglycerides (TGs) on serum and liver cholesterol and on sterol excretion were studied in rats. The TGs contained 4- to 18-carbon fatty acids with melting points of -75 to 63.5 degrees C. Ratios of polyunsaturated to saturated fatty acids ranged from 0.1 to 105. Ratios of total unsaturated to saturated fatty acids ranged from 0.1 to 115. All diets contained 8% TG plus 0.82% safflower oil. Sterols were quantified directly by a new and improved high resolution gas chromatographic method and were identified by mass spectrometry. TG digestibilities correlated negatively with melting points above 30 degrees C (R = -0.9). Serum cholesterol was lower in rats fed tributyrin, tricaproin, tricaprylin, tricaprin, trielaidin, trilinolein or partially hydrogenated soybean oil (43-49 mg/dl) than in those fed trilaurin, trimyristin, tripalmitin, tristearin, triolein or corn oil (54-59 mg/dl). Liver lipid levels correlated (R = 0.65) with the degree of unsaturation of dietary TGs. Liver cholesterol levels correlated negatively with fecal excretion of coprostanol plus cholesterol (R = -0.4). Coprostanol plus cholesterol excreted in feces correlated weakly (R = 0.3) with intake of total sterol and of polyunsaturated TGs (R greater than or equal to 0.4 are at least 80% significant). The results demonstrate that consumption of polyunsaturated TGs was associated with higher hepatic lipid levels. Also, greater fecal excretion of coprostanol plus cholesterol was associated with lower hepatic cholesterol levels.