Localization of carotenoids in plasma low-density lipoproteins studied by surface-enhanced resonance Raman spectroscopy.

Surface-enhanced resonance Raman scattering (SERRS) spectra were measured for the beta-carotene and lycopene carotenoids present in low-density lipoproteins (LDLs), which were isolated from human plasma and adsorbed on roughened silver surfaces. The silver surface was modified by formation of a self-assembled monolayer (SAM) of carboxylate-terminated linear alkanethiols in order to simulate the LDL binding region of the cellular LDL receptor. Thiols of different chain length were used to produce SAMs of varying thicknesses. It was shown that carotenoids are not released from the LDL particle upon adsorption onto the bare and thiol modified silver surfaces. The SERRS studies indicated that beta-carotene and lycopene were present in the shell of the LDL particle. The dependence of SERRS on the distance from the silver surface was different for beta-carotene and lycopene in LDL. This observation suggests that the two carotenoids are located in different places of the LDL particle.

Intestinal absorption of beta-carotene ingested with a meal rich in sunflower oil or beef tallow: postprandial appearance in triacylglycerol-rich lipoproteins in women.

BACKGROUND: Evidence indicates that different types of fat have different effects on the postprandial plasma triacylglycerol response. Therefore, the type of fat may influence the appearance of beta-carotene in postprandial triacylglycerol-rich lipoproteins, which is used as an indicator of intestinal beta-carotene absorption. OBJECTIVE: We compared in female subjects the appearance of beta-carotene in plasma triacylglycerol-rich lipoproteins after beta-carotene was ingested with a meal containing sunflower oil or beef tallow. DESIGN: Women (n = 11) each ingested 2 different vitamin A-free, fat-rich meals that were supplemented with beta-carotene (47 micromol) and contained equivalent amounts (60 g) of sunflower oil or beef tallow. Blood samples were collected hourly from 0 to 10 h; additional samples were collected at selected intervals until 528 h. In a subgroup of the women (n = 7), plasma chylomicrons and 3 subfractions of VLDLs were separated by cumulative rate ultracentrifugation. RESULTS: The appearance of beta-carotene in chylomicrons and in each VLDL subfraction was lower after ingestion with the meal containing sunflower oil than after ingestion with the meal containing beef tallow (P < 0.03). In chylomicrons, the area under the concentration-versus-time curve (AUC) for beta-carotene was 38.1 +/- 13.6% lower (P < 0.03); in contrast, the AUC for triacylglycerol was higher (P < 0.05) after the sunflower-oil-rich meal than after the beef-tallow-rich meal. CONCLUSIONS: Ingestion of beta-carotene with a meal rich in sunflower oil as compared with a meal rich in beef tallow results in lower appearance of beta-carotene and greater appearance of triacylglycerol in triacylglycerol-rich lipoproteins.

Use of a 13C tracer to quantify the plasma appearance of a physiological dose of lutein in humans.

Increased intake of lutein from vegetables promotes increased density of the macular pigment and therefore may protect against age-related macular degeneration. Our objective was to use a 13C tracer and high-precision gas chromatography-combustion interfaced-isotope ratio mass spectrometry (GC-C-IRMS) to investigate metabolism of a lutein dose equivalent to that absorbed from vegetables. Biosynthetic per-labeled (>99% 13C) lutein was purified from a commercially available extract of algal biomass. Subjects (n = 4) ingested 3 mg of [13C]lutein with a standardized low-carotenoid breakfast. Blood samples were collected at baseline and then hourly for 12 h; additional blood samples were drawn at 16, 24, 48, 72, 96, 192, 360, and 528 h. To produce perhydro-beta-carotene suitable for analysis by GC-C-IRMS, the plasma lutein fraction was hydrogenated on palladium-on-carbon catalyst with acid-catalyzed hydrogenolysis. The stable carbon isotope (13C/12C) ratio measured by GC-C-IRMS was used to calculate the plasma concentration of [13C]lutein. There was a rapid increase in [13C]lutein in plasma until peak enrichment at 16 h followed by a decline to the next measurement at 24 h. At 528 h, small changes in 13C enrichment from baseline could still be measured in plasma lutein. High-precision GC-C-IRMS enables complete definition of the appearance and disappearance of [13C]lutein in plasma after ingestion of a dose similar to that absorbed from foods.

Use of high-precision gas isotope ratio mass spectrometry to determine natural abundance 13C in lutein isolated from C3 and C4 plant sources.

A method was developed for high-precision stable carbon isotope ratio analysis of lutein isolated from a C3 (marigold flower) and a C4 (corn gluten meal) plant source using gas chromatography-combustion interfaced isotope ratio mass spectrometry. The natural abundance of 13C (expressed as delta 13C versus the international standard, Pee Dee Belemnite, in per mil units, denoted /1000) in lutein isolated from marigold flower and corn gluten meal was determined to be -29.90 +/- 0.20/1000 and -19.77 +/- 0.27/1000 (mean +/- S.D.), respectively. The high precision of gas isotope ratio mass spectrometry is potentially applicable to detect differences of isotopic composition of lutein in the blood, tissues, or excreta of animal models or humans that result from differences in the natural abundance of 13C in C3 and C4 plant foods.

Interactions in the postprandial appearance of beta-carotene and canthaxanthin in plasma triacylglycerol-rich lipoproteins in humans.

We investigated the plasma appearance of beta-carotene and canthaxanthin, an oxycarotenoid, in normolipidemic premenopausal women (n = 9) who ingested beta-carotene alone, canthaxanthin alone, and a combined dose. Blood samples were taken hourly for 12 h; additional blood samples were collected over 528 h. In a subset of the women (n = 5), plasma lipoproteins were separated into chylomicrons, very-low-density-lipoproteins (VLDL) subfractions, intermediate-density lipoproteins (IDLs), and low-density lipoproteins (LDLs). The appearance of beta-carotene in plasma was biphasic, with a minor peak at 5 h followed by a sustained peak at 24-48 h. The plasma appearance of canthaxanthin was monophasic, with a rapid increase to the final hourly measurement at 12 h and a steady decrease from the next measurement at 24 h. At 6 h, 23.4 +/- 2.9% of the increase in plasma canthaxanthin was associated with LDL, in contrast with 2.4 +/- 1.4% of the increase in plasma beta-carotene (P < 0.005). Ingestion of a combined dose of beta-carotene and canthaxanthin inhibited the appearance of canthaxanthin in plasma, chylomicrons, and each VLDL subfraction (P < 0.05), but did not significantly affect the rapid accumulation of canthaxanthin in LDL within 10 h. In contrast, ingestion of the combined dose did not significantly affect the appearance of beta-carotene in plasma or plasma lipoproteins. These findings suggest distinct mechanisms of incorporation into lipoproteins and specific interactions of beta-carotene and canthaxanthin during intestinal absorption in humans.

Intestinal absorption, serum clearance, and interactions between lutein and beta-carotene when administered to human adults in separate or combined oral doses.

Single equimolar doses (0.5 mumol/kg body wt) of lutein and/or beta-carotene in true solution in oil were given to eight adult subjects and 13 blood samples were taken during the subsequent 840 h. Whereas the mean serum concentration of lutein showed a single maximum at 16 h, that of beta-carotene peaked at 6 h and then again at 32 h. Subsequently, lutein and beta-carotene were cleared at approximately the same rate from the serum. The mean (+/- SEM) areas under the curve (AUCs) for lutein and beta-carotene during the first 440 h differed significantly: 59.6 +/- 9.0 and 26.3 +/- 6.4 mumol.h/L, respectively (P < 0.005). AUC values did not correlate with initial serum concentrations of the given carotenoid or with the order of dosing. When combined in the same dose, beta-carotene significantly reduced the serum AUC values for lutein to 54-61% of control values (P < 0.025), whereas lutein reduced the AUC value for beta-carotene in five subjects but enhanced it in three subjects. Effects of lutein on the AUC for beta-carotene were inversely related to the AUC for beta-carotene alone. Thus, carotenoids clearly interact with each other during intestinal absorption, metabolism, and serum clearance, although individual responses can differ markedly.

Pharmacokinetics of beta-carotene and canthaxanthin after ingestion of individual and combined doses by human subjects.

OBJECTIVE: This study investigated effects of ingestion of a combined dose of beta-carotene and canthaxanthin on their individual pharmacokinetics in serum. METHODS: During three 5-day study periods, two subjects ingested either a 25 mg dose of beta-carotene, a 25 mg dose of canthaxanthin, or a combined dose of 25 mg each of beta-carotene and canthaxanthin. Pharmacokinetics of the individual and combined doses were compared within subjects. RESULTS: Ingestion of a concurrent beta-carotene dose reduced the peak serum canthaxanthin concentration by 38.8 +/- 6.5%, and the 24- and 72-hour areas under the serum canthaxanthin concentration-time curves by 38.1 +/- 6.4 and 34.4 +/- 7.4%, respectively. The suggested antagonism between beta-carotene and canthaxanthin was not reciprocal; beta-carotene inhibited the appearance of canthaxanthin in serum but canthaxanthin did not inhibit the appearance of beta-carotene. CONCLUSION: Our data suggest that ingestion of a combined pharmacologic dose of beta-carotene and canthaxanthin reduces the bioavailability of the canthaxanthin dose.

Interactions of oral beta-carotene and canthaxanthin in ferrets.

Interactive effects of an oral dose of equal quantities of beta-carotene and either canthaxanthin or lycopene on serum and tissue beta-carotene accumulations were investigated in domestic ferrets. Like humans, ferrets absorb a substantial portion of ingested beta-carotene intact and accumulate it in tissues. After the ferrets ingested a low carotenoid purified diet for 13 d, they were randomly assigned to one of two groups of six animals. One group was dosed with beta-carotene (10 mg/kg body weight) and the other with beta-carotene and either canthaxanthin (Experiment 1) or lycopene (Experiment 2) (10 mg/kg body weight for each). In Experiment 1, ferrets that received a combined dose of beta-carotene and canthaxanthin had serum beta-carotene concentrations that were significantly lower at 8, 12 and 24 h post-dosing (P < 0.05), compared with those that received an individual dose of beta-carotene; liver, adrenal and kidney beta-carotene concentrations were also significantly reduced. In Experiment 2, ferrets that received a combined dose of lycopene and beta-carotene had lower serum and tissue beta-carotene concentrations than in those that received beta-carotene alone; the differences were not statistically significant with the exception of serum beta-carotene concentrations at 24 h post-dosing. The results suggest that, at the doses given, a concurrent oral canthaxanthin dose has a specific antagonistic effect on the bioavailability of a beta-carotene dose in ferrets.

The ferret as a model for evaluation of the bioavailabilities of all-trans-beta-carotene and its isomers.

The objective was to develop the ferret as a model for evaluation of the bioavailabilities of natural and synthetic beta-carotenes in foods. For these studies, a low carotenoid purified diet was formulated that produced excellent food intake and adequate growth. After consuming the diet for 16 d, ferrets were randomly assigned to one of three groups. For a 10-d period, they ingested a standardized amount of all-trans-beta-carotene (18 mumol/L) from either carrot juice, a test beverage of beta-carotene beadlets dispersed in fruit juices, or a control beverage of beta-carotene beadlets dispersed in water. Accumulations of all-trans-beta-carotene in the sera, livers and adrenals of ferrets that consumed the carrot juice were significantly lower (P < 0.02) compared with those of ferrets that consumed the test or control beverages. The content of a cis-isomer component relative to that of all-trans-beta-carotene was higher in each beta-carotene beadlet-fortified beverage than in the liver and adrenal tissues of ferrets that ingested the beverage; the cis-isomer was not measurable in sera. The content of all-trans-beta-carotene relative to that of all-trans-alpha-carotene, a structural isomer, was higher in carrot juice than in sera of ferrets that ingested the juice. We conclude that: 1) all-trans-beta-carotene is less bioavailable from carrot juice than from beta-carotene beadlet-fortified beverages, and 2) there are apparent bioavailability differences between isomers of beta-carotene in ferrets.

Beta-carotene uptake and tissue distribution in ferrets (Mustela putorius furo).

Ferrets accumulate beta-carotene in liver and adipose tissue after chronic feeding. This study was designed to further evaluate the time course of uptake and depletion of beta-carotene in ferret serum and tissues. Male ferrets (n = 15; 1000-1200 g) were given a single dose of beta-carotene (10 mg/kg body wt) with a meal. Animals were killed at various time points over an 11-d period. Blood and tissue samples were extracted and analyzed for beta-carotene by HPLC. Peak serum beta-carotene levels (0.68 +/- 0.18 mumol/L) were observed 8 h after the test meal. beta-Carotene was essentially cleared from the blood by 76 h. Peak beta-carotene concentrations (nmol/g) were observed between 8 and 16 h after ingestion for liver (1.20 +/- 0.04), lung (0.042 +/- 0.012), kidney (0.090 +/- 0.015) and spleen (0.076 +/- 0.012). Ferret liver also seemed to contain a variety of other polar and nonpolar carotenoids. Ferrets were shown to absorb beta-carotene from a meal and have a consistent serum response pattern. Absorbed beta-carotene is differentially distributed in a variety of tissues. The ferret seems to be a useful model for the study of beta-carotene absorption and metabolism.

Ultraviolet light-induced reductions in plasma carotenoid levels.

The effects of ultraviolet (UV) irradiation on plasma levels of carotenoids and vitamin A in human subjects were investigated in two crossover trials. UV exposures were given on 11 r 12 days of a 2-wk period. The 12 female and 12 male subjects received mean cumulative UV-A (320-400 nm) doses of 17.8 +/- 1.9 J/cm2 and 21.0 +/- 3.3 J/cm2 to the anterior and posterior sides of the body, respectively. UV-B (280-320 nm) doses were equivalent to 10% of UV-A doses given. Significant reductions in plasma total carotenoid levels were observed in both female (p less than 0.004) and male (p less than 0.05) subjects after repeated irradiation. There was no significant effect on plasma vitamin A levels. It was concluded that UV treatment can reduce plasma carotenoid levels in vivo.

Folic acid metabolism in vitamin B12-deficient sheep. Effects of injected methionine on liver constituents associated with folate metabolism.

1. The effects of injected l-methionine (2g every second day for 28 days) on liver folates and other constituents of liver associated with folate metabolism were studied in vitamin B(12)-deficient ewes and their pair-fed controls receiving vitamin B(12). The dose rate of methionine used was sufficient to restore almost to normal the elevated excretion in the urine of formiminoglutamate in the deficient animals. 2. Liver folates active for Lactobacillus casei, Streptococcus faecalis R and Pediococcus cerevisiae were severely depressed in deficient livers and were partly restored by methionine. Analysis of the folates after ion-exchange chromatography showed that the major effect of methionine was to increase the concentrations of tetrahydrofolates and formyltetrahydrofolates. Methyltetrahydrofolates were also increased, but there was no effect of methionine on the small amounts of incompletely reduced folates present in deficient livers. The folates present were predominantly penta-, hexa- and hepta-glutamates whether or not animals received vitamin B(12) or methionine. 3. Concentrations of ATP, NAD(+), NADH and NADPH were lower in freeze-clamped liver from vitamin B(12)-deficient sheep than in liver from pair-fed, vitamin B(12)-treated sheep. These changes were not affected by methionine which was also without effect on the elevated K(+)/Na(+) ratios found in deficient livers. 4. The livers of vitamin B(12)-deficient animals contained lower concentrations of choline and higher concentrations of lipid than their pair-fed controls. These effects were reversed by methionine.

Identification and measurement of the folates in sheep liver.

1. Methods are described for the extraction, separation by ion-exchange chromatography and estimation by microbiological assay of the folates in sheep liver. 2. Injection of [2-(14)C]-pteroylglutamate into a sheep fed on a stock diet led to extensive labelling of chromatographically separable liver folates. About 12% of the label in the liver could not be extracted by the method used. 3. Liver folates were examined in five ewes fed on restricted amounts of a diet of wheaten hay-chaff and gluten and injected weekly with vitamin B(12). Chromatographic separation was followed by microbiological assay with Lactobacillus casei, Streptococcus faecalis R. and Pediococcus cerevisiae both before and after treatment of fractions with conjugase (gamma-glutamylcarboxypeptidase). Evidence was obtained that the folates present were predominantly polyglutamate forms of tetrahydropteroylglutamate, 5-methyltetrahydropteroylglutamate and 5- (and 10-) formyltetrahydropteroylglutamates. Differences in the responses of the assay organisms permitted quantitative distinction between these three main classes of folates. 4. Methyltetrahydrofolates were eluted in seven successive peaks that were separated by constant increments in the logarithm of eluant [P(i)]. A similar relationship existed for seven successive peaks of tetrahydrofolate and may also have existed for each of the two series of formyltetrahydrofolates. 5. Based on these and other observations it is proposed that sheep liver folates consist predominantly of the mono- to hepta-glutamates of each of the reduced pteroates identified. The methods employed allowed quantitative determinations to be made of most of the folates present. The predominant forms were hexaglutamates. 6. Four components active for L. casei were detected that could not be identified. Three of them were polyglutamates.