Metabolism of carotenoid analogs in humans.

Single oral doses (100 mumol) in peanut oil of 4,4'-dimethoxy-beta-carotene, ethyl beta-apo-8'-carotenoate, and beta-apo-8'-carotenal were administered to healthy adult male subjects (n = 4-6). Blood samples were taken frequently thereafter, and serum carotenoids and retinoids were analyzed by HPLC. The metabolism of the three analogs was very different; 4,4'-dimethoxy-beta-carotene was oxidized at the 4 and 4' positions but apparently not cleaved, whereas ethyl beta-apo-8'-carotenoate was not detectably metabolized, and beta-apo-8'-carotenal was extensively converted to its corresponding acid, alcohol, and fatty acyl ester and detectably converted to retinyl ester and possibly to two shorter beta-apocarotenals. Serum concentrations of endogenous retinoids and carotenoids, except as noted above, were not affected in any case. Kinetically, the maximum serum concentrations, areas under the curve, and mean sojourn times for the three analogs differed by 50-, 270-, and 5-fold, respectively. For any given analog, however, the fractional standard deviations for these parameters were only 0.2-0.5.

The correlation between liver vitamin A concentrations in micro- (needle biopsy) and macrosamples of human liver specimens obtained at autopsy.

A technique is described for the analysis by gradient-elution reverse-phase high-pressure liquid chromatography of vitamin A at 325 nm in small liver samples taken by needle biopsy. This assay, in contrast to previous procedures, allows rapid separation and quantitation of approximately 0.1 microgram retinol and retinyl esters, requires only 7 mg liver sample, and separates groups of retinyl esters. Vitamin A values determined in needle biopsy samples (7 to 70 mg) and in gross samples (1 to 6.3 g) of the same 24 autopsy specimens correlated well (R = 0.96) over the range 1 to 400 micrograms vitamin A per g liver. The ratio of total vitamin A values in micro- and macrosamples was 1.09 +/- 0.46 (SD). Total vitamin A values in macrosamples of liver determined by high-pressure liquid chromatography and by a differential spectrophotometric assay also correlated well (R = 0.91). Under suitable clinical conditions, the procedure should be useful in validating indirect techniques for assessing vitamin A status.

Iron and zinc supplementation improves indicators of vitamin A status of Mexican preschoolers.

BACKGROUND: The coexistence of multiple micronutrient deficiencies is a widespread public health problem in many regions of the world. Interactions between zinc deficiency and vitamin A metabolism have been reported but no longitudinal studies have evaluated the effect of iron deficiency on vitamin A. OBJECTIVE: The objective of this study was to investigate the effect of supplementation with iron, zinc, or both on vitamin A and its metabolically related proteins retinol binding protein (RBP) and transthyretin. DESIGN: The study was a longitudinal, double-blind, placebo-controlled trial in which 219 rural Mexican children aged 18-36 mo were randomly assigned to receive 20 mg Zn/d, 20 mg Fe/d, 20 mg Zn/d plus 20 mg Fe/d, or placebo. RESULTS: Six months after supplementation, plasma retinol increased in all supplemented groups. Compared with placebo, zinc supplementation was associated with significantly higher plasma retinol and transthyretin but the increase in RBP was not significant. Iron supplementation significantly increased plasma retinol, RBP, and transthyretin. Supplementation with zinc plus iron significantly increased plasma retinol but not RBP or transthyretin. Children deficient in zinc, iron, or vitamin A (as indicated by nutrient plasma concentration) at the beginning of the study had a significantly greater increase in retinol than did children with adequate nutrient status. CONCLUSIONS: Supplementation with zinc, iron, or both improved indicators of vitamin A status. The results of this study agree with previous observations of a metabolic interaction between zinc and vitamin A and suggest an interaction between iron and vitamin A metabolism.

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.

Properties of retinoids. Structure, handling, and preparation.

Retinoids are unstable compounds being readily oxidized and/or isomerized to altered compounds, especially in the presence of oxidants including air, light, and excessive heat. They are labile toward strong acids and solvents that have dissolved oxygen or peroxides. In this review, procedures for handling and storage of retinoids and biological samples containing them have been described. The physical and chemical properties of retinoids have been reported. Simplified procedures for derivatizations and purification, and methods for quantitation of retinoids have been presented.

Retinoids and carotenoids in bovine pineal gland.

The retinoids and carotenoids in bovine pineal gland were analyzed by high-performance liquid chromatographic methods. The major retinoid observed was all-trans retinol, with an average level of 579 pmol/g wet tissue. In agreement with a previous report, retinal was not detected; however, we could not detect any retinyl ester in bovine pineal gland. Methods similar to those used in studying metabolism of vitamin A in the bovine eye were used to study the esterification and isomerization reactions of retinoids in the pineal gland. Neither esterification nor isomerization reactions occurred. These results suggest that the key retinoids and enzymes involved in visual function are absent in the bovine pineal gland. The major carotenoid, which has not previously been reported in pineal gland, was beta-carotene at an average level of 1830 pmol/g wet tissue.

Use of the modified relative dose response (MRDR) assay in rats and its application to humans for the measurement of vitamin A status.

After an appropriate dose of 3,4-didehydroretinol (vitamin A2, DR) is given orally in corn oil to retinol (R)-depleted rats, the ratio of plasma DR/R values at 3.5 h is inversely related to the liver concentration of vitamin A (Tanumihardjo & Olson, 1988). In the present study, a similar procedure, termed the modified relative dose response (MRDR) assay, was employed with rats containing a much broader range of liver reserves; ie, less than 2 to 107 nmol of vitamin A per g wet weight of liver (less than 1 to 30 micrograms/g). The DR/R ratio for 15 of 16 rats with liver reserves less than 17 nmol/g (less than 5 micrograms/g) was greater than 0.15, whereas the ratio was less than 0.15 for 7 of 8 rats with liver reserves greater than 17 nmol/g. This distribution provides sensitivity, specificity and positive predictive values of 94, 88 and 94 per cent, respectively. The DR/R ratios reached a maximal plateau in two other groups of rats between 3.5 and 8 h. At all times up to 24 h, mean DR/R ratios for vitamin A-depleted rats (45 +/- 6 nmoles of vitamin A/g liver) were approximately twice those for vitamin A-sufficient rats (230 +/- 40 nmoles of vitamin A/g liver). In three well-nourished adults, presumably with liver reserves of greater than 300 nmol/g wet weight liver (greater than 80 micrograms retinol equivalents/g), and in two young (1- and 3-years old) well-nourished children, maximal DR/R ratios were less than 0.023. In these cases, peak DR/R ratios were observed, with one possible exception, between 8 and 12 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Absorption of canthaxanthin by the rat is influenced by total lipid in the intestinal lumen.

In this study the effect of luminal lipid on the absorption of canthaxanthin (CTX) was investigated using the lymph duct cannulated rat. Treatments were emulsions designed to deliver increasing amounts of olive oil (10, 30, 50, 70, or 90 mg/h) and CTX (12.5 nmol/h). Emulsions were continuously infused into the duodenum for 12 h, and lymph was collected during the final 6 h of infusion for analysis. As the amount of lipid in the emulsion increased, a linear increase in the absorption of CTX was observed. The recovery of CTX in the lymph when infused with 10 mg/h olive oil was 14.2 +/- 1.2% and with 90 mg/h was 26.9 +/- 5.7%. The efficiency of CTX absorption nearly doubled by increasing the amount of lipid infused with CTX. The correlation between lipid load and CTX absorbed was r= 0.85. We conclude that luminal lipid load affects CTX absorption.

A comparison of lycopene and canthaxanthin absorption: using the rat to study the absorption of non-provitamin A carotenoids.

The purpose of this study was to validate the use of the mesenteric lymph duct cannulated rat to study the absorption of carotenoids which do not have provitamin A activity. The absorption of two carotenoids, a hydrocarbon carotenoid (lycopene) and a xanthophyll carotenoid (canthaxanthin), were investigated. In the first experiment, lipid emulsions containing lycopene (LYC) or canthaxanthin (CTX) were continuously infused into the duodenum, and lymph was collected for analysis at 2-h intervals. The time course for absorption of carotenoids and triacylglycerol (TAG) was similar. Carotenoids and TAG reached steady-state concentrations in the lymph by 6 h. There was no evidence for a delayed release of either carotenoid from the intestine relative to TAG. During a second experiment, emulsions containing increasing concentrations of LYC or CTX (5, 10, 15, 20 mumol/L) were infused. The LYC and CTX in the lymph increased in a dose-dependent manner. The average efficiency of CTX absorption was 16% while the efficiency of LYC absorption averaged only 6%. Efficiency of carotenoid absorption was not related to concentration infused. Finally, to test whether LYC and CTX interact during absorption both were added to a lipid emulsion at equal concentrations (20 mumol/L) and infused. The carotenoids did not significantly affect each other's absorption. These results demonstrate the usefulness of the rat as an animal model to study the absorption of non-provitamin A carotenoids.

Excess vitamin E decreases canthaxanthin absorption in the rat.

The recent attention given to the possible role of alpha-tocopherol (alpha-Toc) and carotenoids in the prevention and treatment of a variety of illnesses resulted in segments of the population increasing their consumption of these nutrient/antioxidants. Once consumed, alpha-Toc and carotenoids are thought to follow the same absorptive pathway and may influence each other's absorption, particularly when taken in large doses. The purpose of this study was to determine if alpha-Toc and the carotenoid, canthaxanthin (CTX), interact during absorption. Rats were intraduodenally infused with corn oil emulsions containing combinations of alpha-Toc (0 or 300 mumol/L) and CTX (5, 10, 15, 20 mumol/L) in a 2 x 4 factorial arrangement. Absorption was determined by measuring recovery of CTX and alpha-Toc in the mesenteric lymph. The amount of CTX in the lymph increased significantly with the amount infused into the duodenum. The overall efficiency of CTX absorption from emulsions without alpha-Toc averaged 12% with individual animals having a range of 8 to 18%. Efficiency of absorption was not related to concentration of CTX infused. When alpha-Toc (300 mumol/L) was added to the oil emulsion, the absorption of CTX was decreased by at least 50%. Recovery of alpha-Toc in the lymph averaged ca. 10% and was not affected by CTX. These results suggest that concurrent consumption of a large dose of alpha-Toc may influence carotenoid bioavailability.

A comparison of lycopene and astaxanthin absorption from corn oil and olive oil emulsions.

The effect of different oils on the absorption of carotenoids was investigated in mesenteric lymph duct cannulated rats. Sixteen treatment emulsions containing increasing concentrations of either lycopene (LYC) or astaxanthin (AST) (5, 10, 15, 20 micromol/L) were prepared with olive oil or corn oil and continuously infused into the duodenum of the rat. Absorption of carotenoids into the mesenteric lymph duct was determined. Absorption of LYC and AST from both oils increased with the amount infused into the duodenum. The average recovery of AST in the lymph from the olive oil emulsion was 20% but was decreased to 13% from emulsions containing corn oil. Lycopene was not as well absorbed as AST. The average recovery of LYC was 6% from olive oil emulsions but only 2.5% when infused with corn oil. The LYC used in this study was isolated from tomato paste and was primarily in the all-trans form. We did not observe any significant isomerization of all-trans LYC to 9-cis LYC during absorption. We conclude that the type of oil with which a carotenoid is consumed can influence its absorption.

Bacterial catabolism of lipoic acid. Isolation and identification of a methyl ketone.

A soil bacterium, Pseudomonas putida LP, can be grown on lipoate as sole source of carbon, sulfur, and energy. In addition to the previously identified catabolites (bisnorlipoate, tetranorlipoate, beta-hydroxybisnorlipoate, lipoate thiolsulfinate, and two bisnorlipoate thiolsulfinates) isolated from cultures of the organism grown on [1,6-14C[lipoate, a methyl ketone (1,2-dithiolane-3-butyl-3'-one) has now been isolated and identified. This catabolite was isolated by solvent extraction and hydrophobic gel filtration and characterized by chromatographic mobilities and spray reactions and by UV, IR, PMR, and mass spectrometries. The methyl ketone presumably arises by decarboxylation of the beta-keto acid formed during the beta-oxidation of lipoate to bisnorlipoate by the microorganism.

Retinyl ester (vitamin A ester) and carotenoid composition in human liver.

Surgical liver biopsy samples from seven diseased and five healthy human subjects, 3-33 years of age, were analyzed by reversed-phase high-pressure liquid chromatography for retinol (vitamin A alcohol), retinyl esters (vitamin A esters), and carotenoids. Total liver vitamin A values ranged from 7.8 to 2860 nmol/g liver (2.2 to 817 micrograms/g). As a percentage, liver retinol decreased with increasing liver reserves of vitamin A. Retinyl palmitate was the predominant vitamin A ester (57 to 83 mole%) in all samples, with retinyl stearate (5.5 to 11.4%), oleate (4.9 to 17.2%), and myristate plus palmitoleate (pair not resolved; 3.3 to 11.9%) next most common. Lesser amounts of retinyl linoleate, linolenate, and arachidonate were found. Normal livers had significant amounts of several carotenoids: lutein (0.2 to 16.2 nmol/g), lycopene (10.2 to 55.1 nmol/g), alpha-carotene (3.0 to 7.3 nmol/g), and beta carotene (5.8 to 25 nmol/g). Total carotenoid values ranged from 26.5 to 67 nmol/g in normal liver samples. There was no correlation between liver vitamin A and individual or total carotenoids in normal livers.

Calculation of chromatographic dead times and retention indices with the aid of a computer program in BASIC.

A computer program is presented for calculating chromatographic dead time from the observed retention times of an homologous series of standards using the iterative approximation procedure of Guardino et al. The program is written in BASIC and is readily adaptable to most microcomputers capable of using BASIC. Corrected retention times and capacity factors of standards and of samples are also calculated by the program. Examples of applications in gas chromatography and reversed-phase high-pressure liquid chromatography are presented. A listing of the BASIC program is given.

Adsorption of Pu, Pb and Cd to mouth surfaces during oral administration to mice.

Substantial adsorption of Pu, Cd and Pb onto mouth surfaces of mice occurs during consumption of drinking water that contains these elements. If this adsorption is not taken into account, the amounts deposited in the eviscerated carcass (muscle plus skeleton) after gastrointestinal absorption are overestimated by 2-9 times for the experiments described. An evaluation of data indicate that significant adsorption can also occur during gavage administration. Methods are described for estimation of amounts deposited in the carcass after exposure of mice to these metals via drinking water or gavage.