Vitamin A contained in the lipid droplets of rat liver stellate cells is substrate for acid retinyl ester hydrolase.

Vitamin A is stored in the lipid droplets of liver stellate cells (LSCs), as retinyl esters whose hydrolysis is necessary for the secretion of retinol into the blood. Here, we isolated these retinyl esters under their physiological form, i.e., in LSC lipid droplets, which had retained their morphological and biochemical characteristics. These retinyl esters are substrate for an hydrolytic enzyme, whose optimum pH is 4.1, and which is kinetically similar to the acidic retinyl ester hydrolase (aREH) we had previously described (Mercier et al., Biochim. Biophys. Acta (1994) 1212, 176-182). The cellular and subcellular localizations of aREH activity in rat liver suggest that this enzyme could be involved in the hydrolysis of the esterified vitamin A stores.

Retinol mobilization from cultured rat hepatic stellate cells does not require retinol binding protein synthesis and secretion.

Retinol mobilization from retinyl esters stores of hepatic stellate cells (HSCs) is a key step in the regulation of mammalian retinol homeostasis, but the precise mechanisms of such a mobilization are still poorly understood. Using primary cultures of HSCs, we first demonstrated that HSCs expressed immunoreactivity against retinol-binding-protein (RBP) when cultured in a medium containing RBP but were unable to synthesize RBP transcripts and proteins. Using pulse and chase-type experiments, we demonstrated that radioactive retinol was released in culture medium without binding proteins. Inhibition of protein secretion by brefeldin A did not modify quantitatively retinol release. This data ruled out, for the first time, the direct involvement of RBP in retinol mobilization from HSCs. Moreover, HSCs co-cultured with primary isolated hepatocytes displayed an increase of retinol transfer from HSCs to hepatocytes when they established direct physical contacts, as compared with co-cultures without contact. Based on this latter data, a mechanism of retinol mobilization from HSCs via the hepatocytes using retinol transfer through cellular membranes is proposed.

Plasma levels of 8-epiPGF2alpha, an in vivo marker of oxidative stress, are not affected by aging or Alzheimer's disease.

Free radicals are likely involved in the aging process and there is a growing body of evidence that free radical damage to cellular function is associated with a number of age-related diseases such as atherosclerosis, cancer, and neurologic disorders. The present study was designed to evaluate in a healthy population the evolution with age of 8-epiPGF2alpha plasma levels, a recently proposed marker of in vivo lipid peroxidation. Moreover we investigated this marker of oxidative stress in patients with Alzheimer's disease (AD), an age-related neurodegenerative disorder in the development of which free radicals have been involved. Our results show that in the healthy population studied, despite decreased antioxidant defenses with increasing age as monitored by antioxidant capacity measurement, plasma 8-epiPGF2alpha levels were not correlated with age. Moreover, we have demonstrated that AD patients presented no modification of plasma 8-epiPGF2alpha level and no major alteration of the antioxidant status. In conclusion, the measurement of plasma 8-epiPGF2alpha did not allow us to detect alterations in oxidative stress with aging or in AD.

Comparison of the postprandial plasma vitamin A response in young and older adults.

To assess the influence of age on vitamin A intestinal and liver metabolism in humans, the postprandial plasma concentrations of intestinal-originated vitamin A, i.e., retinyl esters, and liver-originated vitamin A, i.e., retinol, were compared in eight young (20-30 years old) and eight elderly (64-72 years old) healthy men. Plasma and chylomicron retinyl esters and retinol concentrations were measured for up to 24 h following the intake of a test meal that contained 23,300 RE retinyl palmitate. The chylomicron retinyl palmitate response (area under the curve) was not significantly different between the two groups, but its peak was slightly delayed (1 h) in the elderly men. The proportion of the different retinyl esters secreted in the chylomicrons was not significantly different between the two groups. The postprandial plasma retinol concentration did not change in the young participants, whereas it significantly increased in the elderly. These results suggest that vitamin A intestinal absorption and retinol intestinal esterification processes are not markedly modified in the elderly, whereas the chylomicron clearance and the regulation of postprandial plasma retinol concentration are apparently altered in these subjects.

Dietary triglycerides, up to 40 g/meal, do not affect preformed vitamin A bioavailability in humans.

OBJECTIVE: Assessing the effect of the dose of dietary triglycerides on preformed vitamin A (retinyl esters) bioavailability in humans. DESIGN: Four test meals containing 15,000 RE retinyl-palmitate and either 0, 15, 30 or 40 g added triglycerides were ingested by eight healthy volunteers, at different days and in a randomized order. SETTING: The study was done in the Hospital Sainte Marguerite, Marseille, France. SUBJECTS: Eight healthy male volunteers were recruited by advertisement. INTERVENTION: Blood samples were collected every hour for seven hours after the test meals intake. Serum and chylomicron (Svedberg flotation unit > 1000) were prepared by centrifugation and retinyl esters were measured by HPLC. RESULTS: The serum retinyl ester response was not significantly lower after the intake of the meal without added triglycerides (7944 +/- 3262 nmol/L h) than after the intake of the fat meals (10012 +/- 2182, 7869 +/- 3157 and 10777 +/- 2067 nmol/L h for the 15, 30 and 40 g-fat meal, respectively), indicating that the serum retinyl ester response was not related to the amount of meal triglycerides. Chylomicron retinyl linoleate response stepwise increased when the amount of meal triglycerides increased while retinyl palmitate and retinyl stearate responses reached a maximum since 15 g triglycerides. Postprandial serum retinol concentration did not change whatever the meal ingested. CONCLUSIONS: (i) a significant amount of preformed vitamin A is apparently absorbed when ingested with trace amount of meal triglycerides only; (ii) meal triglycerides, up to 40 g/meal, do not increase preformed vitamin A bioavailability; (iii) the retinyl ester pattern recovered in the chylomicrons, and probably the esterification process of retinol, is affected by the amount of meal triglycerides; (iv) postprandial retinol homeostasis is not affected by dietary triglycerides.

Relative contribution of foetal and post-natal nutritional periods on feeding regulation in adult rats.

AIM: The aim of this study was to assess the contribution of both foetal and/or post-natal nutritional periods on feeding regulation in adult rats. METHODS: Body weight gain, adipose tissue development, food preferences and feeding pattern under regular chow or Western diets were characterized on four experimental groups of rats: pups born from protein-restricted dams (R) and weaned by control (RC) or R dams (RR) and pups born from control dams weaned by C (CC) or R dams (CR). RESULTS: Rats born with intrauterine growth restriction (IUGR) and fed a Western diet at adulthood appeared predisposed to body weight gain and more fat accretion, whereas CR rats, despite their preference for high-fat diet and their hyperphagia for Western diet, did not show significant increase in fat tissue. Daytime food intakes, as well as their speed of ingestion, were found modified in RC and RR. Alterations in the hypothalamic appetite regulatory mechanisms were investigated through neuropeptide expression analysis. IUGR rats showed altered expression of key elements of leptin and NPY signalling, while CR rats exhibited lesser expression of enterostatin, MC4r and HT-1Br mRNA. CONCLUSION: Altogether, these results indicate that peri-natal nutrition has different lasting effects on feeding pattern and hypothalamic appetite regulation, depending on the time window insult.

In vitro and in vivo inhibition of beta-carotene dioxygenase activity by canthaxanthin in rat intestine.

beta-Carotene dioxygenase catalyzes the conversion of provitamin A carotenoids to vitamin A in mammalian tissues. Whether the enzyme can also cleave non-provitamin A carotenoids to retinoid analogs with biological activities is still unclear. We investigated (i) substrate specificities of beta-carotene dioxygenase toward provitamin A and non-provitamin A carotenoids and (ii) potential antagonistic effects of non-provitamin A carotenoids on beta-carotene conversion to vitamin A. Provitamin A substrates were 8 to 23% as active as beta-carotene. No polar metabolites were detected with canthaxanthin or zeaxanthin as substrates; these compounds efficiently inhibited the beta-carotene cleavage reaction by 71 and 40%, respectively. Kinetic studies indicated mixed inhibition for canthaxanthin (Ki = 1.6 microM) and non-competitive for zeaxanthin (Ki = 7.8 microM), suggesting that both compounds do not interact significantly with the active site of the enzyme. In vivo, dietary combinations of canthaxanthin and beta-carotene resulted in lower liver levels of both carotenoids and vitamin A and in a higher beta-carotene/vitamin A ratio as compared to groups supplemented with the compounds separately. This supports the view that canthaxanthin at high doses competes with beta-carotene for intestinal absorption and inhibits the conversion of beta-carotene to vitamin A. Thus, we suggest that although canthaxanthin is not a substrate for beta-carotene dioxygenase, it is likely to affect the activity of provitamin A carotenoids by direct interaction with the enzyme.

Rat intestinal beta-carotene dioxygenase activity is located primarily in the cytosol of mature jejunal enterocytes.

The purposes of this study were to determine the location of beta-carotene dioxygenase (EC 1.13.11.21) activity within the rat gastrointestinal tract, within the villus and within enterocytes, and to identify the metabolites produced in each intestinal fraction. In Wistar female rats, maximal activity was detected in the cytosol (74-93% of the total cellular activity) of mature functional enterocytes harvested from the jejunum (67% of the intestinal activity). The specific activity, expressed in pmol of retinoids/(h x mg protein) rose from 49 +/- 3 in the stem cells to 199 +/- 12 in the mature functional cells (P < 0.05). Thus the intestinal beta-carotene cleavage activity might be regulated during the enterocyte maturation process. By using HPLC with diode array and radioactive detectors, retinal, and in the presence of NAD+, retinoic acid, were identified as the only metabolites produced. No beta-12'-, 10'-, and 8'-apo-carotenals were detected, even when various enzyme sources were tested. These results suggest that the major, if not the sole, pathway for the formation of vitamin A from beta-carotene in the rat intestine is central cleavage.

The bioavailability of alpha- and beta-carotene is affected by gut microflora in the rat.

The present study examined whether the intestinal microflora could affect the bioavailability and vitamin A activity of dietary alpha- and beta-carotene in the rat. In the first set of experiments, we used conventional, germ-free (axenic), and human-flora-associated (heteroxenic) rats. In a second series, conventional rats were treated with either an antibiotic mixture or a potent inhibitor of gastric secretion (Omeprazole). All animals were first depleted of vitamin A over 4 weeks and then were fed on a sterilized diet supplemented with 14 mg beta-carotene and 3 mg alpha-carotene/kg for 2 weeks. In both experiments, a reduction in the intestinal microflora resulted in an increased storage of beta-carotene, alpha-carotene and vitamin A in the liver. Neither the nature of the metabolism of the intestinal microflora (aerobic or anaerobic) nor treatment with omeprazole, to modify intestinal pH, induced a significant effect on the measured variables. When incubated with 15 mumol beta-carotene/l for 72 h, neither the anaerobic nor the aerobic sub-fractions obtained from rat or human faeces contributed to beta-carotene degradation or to vitamin A synthesis. These findings suggest that reduction in gut microflora results in a better utilization of alpha- and beta-carotene by rats, although bacteria do not have a direct effect on the bioavailability of these pigments.

Simple method for clinical determination of 13 carotenoids in human plasma using an isocratic high-performance liquid chromatographic method.

We report a reversed-phase high-performance liquid chromatography method which resolves 13 identified carotenoids and nine unknown carotenoids from human plasma. A Nucleosil C18 column and a Vydac C18 column in series are used with an isocratic solvent system of acetonitrile-methanol containing 50 mM acetate ammonium-dichloromethane-water (70:15:10:5, v/v/v/v) as mobile phase at a flow-rate of 2 ml/min. The intra-day (4.5-8.3%) and inter-day (1.3-12.7%) coefficients of variation are suitable for routine clinical determinations.

Esterification of vitamin A by the human placenta involves villous mesenchymal fibroblasts.

Vitamin A (retinol) and its active derivatives (retinoic acids) are essential for growth and development of the mammalian fetus. Maternally derived retinol must pass the placenta to reach the developing fetus. Despite its apparent importance, little is known concerning placental transfer and metabolism of retinol, and particularly of placental production and storage of retinyl esters. To elucidate this metabolic pathway, we incubated, in the presence of retinol, 1) human full-term placental explants and 2) primary cultures of major cells types contributing to placental function: trophoblasts and villous mesenchymal fibroblasts. We used HPLC to determine the types and concentrations of retinyl esters produced by these explants and cells. About 14% of total cellular retinol in placental explants was esterified. The most abundant esters were myristate and palmitate. Primary cell cultures showed that fibroblasts efficiently produced retinyl esters, but trophoblasts did not. In both types of experiments, no retinyl esters were detected in the culture medium, suggesting that retinyl esters were produced for storage purpose. These results suggest that villous mesenchymal fibroblasts are primary sites of retinol esterification and storage in the placenta.

Postprandial chylomicron and plasma vitamin E responses in healthy older subjects compared with younger ones.

The effect of ageing on vitamin E bioavailability in humans was assessed by comparing chylomicron and plasma alpha-tocopherol postprandial concentrations after a dose of vitamin E (432 or 937 IU as d1-alpha-tocopherol acetate), in eight young (20-30 years old) and eight healthy elderly men (64-72 years old). The fasting plasma alpha-tocopherol concentration was significantly higher in the elderly (33 +/- 2 mumol L-1) than in the young (22 +/- 2 mumol L-1). In both groups, the plasma and chylomicron alpha-tocopherol postprandial concentrations were significantly, approximately twofold, higher after the 937-IU meal than after the 432-IU meal. For both test meals, the chylomicron alpha-tocopherol areas under the curve were significantly lower in the elderly than in the young subjects: 98.9 +/- 16.5 (young group) vs. 55.3 +/- 7.8 (elderly group) mumol L-1 h for the 937-IU test meal and 60.4 +/- 14.1 (young group) vs. 26.0 +/- 7.6 (elderly group) mumol L-1 h for the 432-IU test meal, whereas the plasma alpha-tocopherol area under the curve was significantly higher in elderly than in young subjects: 337.56 +/- 16.11 (937-IU test meal) vs. 159.81 +/- 35.55 (432-IU test meal) mumol L-1 h in the young group and 709.55 +/- 69.33 (937-IU test meal) vs. 436.39 +/- 41.08 (432-IU test meal) mumol L-1 h in the elderly group. We concluded that (a) the amount of vitamin E appearing in plasma is proportional to the dose ingested (up to 937 IU); (b) the intestinal absorption of vitamin E is not increased, even possibly decreased, in the elderly; and (c) the amount of vitamin E transported by non-chylomicron lipoproteins is apparently higher in the elderly. This suggests that vitamin E postprandial transport is affected by ageing, mainly as the consequence of age-related modifications of lipoprotein metabolism.

Low and high responders to pharmacological doses of beta-carotene: proportion in the population, mechanisms involved and consequences on beta-carotene metabolism.

The aim of this study was to assess the interindividual variability of chylomicron beta-carotene response to a pharmacological load of beta-carotene in the population, to identify the mechanisms responsible for this variability, and to evaluate its consequences on beta-carotene status and metabolism. The variability, as estimated by the 3-h chylomicron beta-carotene response to 120 mg beta-carotene in 79 healthy male volunteers, was high (CV = 61%), but it was unimodal and all the subjects had detectable chylomicron beta-carotene. In 16 subjects randomly selected among the 79, the interindividual variability of the triglyceride-adjusted chylomicron (beta-carotene + retinyl palmitate) response (0-12.5 h area under the curve) was high (CV = 54%), suggesting that there is a high interindividual variability in the efficiency of intestinal absorption of beta-carotene. The chylomicron beta-carotene response was correlated (r = 0.50, P < 0.05) with the chylomicron triglyceride response. The beta-carotene status, as assessed by beta-carotene concentration in buccal mucosal cells, was correlated (r = 0.73, P < 0.05) with the triglyceride-adjusted chylomicron beta-carotene response, i.e., with the ability to respond to beta-carotene. The triglyceride-adjusted chylomicron retinyl-palmitate response was correlated (r = 0.55, P < 0.05) with the triglyceride-adjusted chylomicron beta-carotene response. Plasma all-trans retinoic acid slightly, but significantly, increased (+40%) 3 h after the beta-carotene load, but this increase was not related to the triglyceride-adjusted beta-carotene response. In conclusion, the ability to respond to beta-carotene is highly variable, but there is probably a very small proportion of true non-responders to pharmacological doses of beta-carotene in the healthy population. This variability is apparently mainly due to interindividual differences in the efficiency of intestinal absorption of beta-carotene and in chylomicron metabolism. The ability to respond to beta-carotene can affect the beta-carotene status and the provitamin A activity of beta-carotene, but it has apparently no effect on the amount of retinoic acid appearing in the plasma after the ingestion of a pharmacological dose of beta-carotene.

Oxidative stress status and antioxidant status are apparently not related to carotenoid status in healthy subjects.

Several lines of evidence suggest that carotenoids may have a beneficial effect on health as a result of their antioxidant properties. In addition to beta-carotene, five other carotenoids are recovered in noticeable amounts from human plasma and tissues. Although the effect of beta-carotene on in vivo lipid peroxidation has been documented, few data are available on the effects of the other carotenoids. We evaluated the ability of the main human carotenoids to reduce lipid peroxidation by determining the correlations between plasma carotenoid concentration and plasma antioxidant capacity (in 79 healthy volunteers) and between carotenoid status and breath pentane excretion (in a subgroup of 24 subjects). Carotenoid intake was assessed by means of a 3-day food recall. Carotenoid status was evaluated by measurement of beta-carotene, lycopene, lutein/zeaxanthin, and alpha-carotene in plasma and buccal mucosal cells. Antioxidant status was evaluated by measurement of the total antioxidant capacity of the plasma. Oxidative stress status was evaluated by breath pentane measurements. Food recall data and the carotenoid concentrations in plasma and buccal mucosal cells showed that the subjects had normal carotenoid intake and normal carotenoid status. The total antioxidant capacity of the plasma was not related to the concentration of any specific carotenoid. The level of expired air pentane was not related to the carotenoid status of the subjects. These results show that normal concentrations of carotenoids in plasma and tissues are not correlated with these clinical markers of antioxidant and oxidative stress status.

Chylomicron beta-carotene and retinyl palmitate responses are dramatically diminished when men ingest beta-carotene with medium-chain rather than long-chain triglycerides.

The effect of the ingestion of beta-carotene with medium-chain triglycerides (MCT) or long-chain triglycerides (LCT) on the bioavailability and the provitamin A activity of beta-carotene was investigated in humans. Sixteen healthy young men ingested, on two different days, a test meal containing 120 mg beta-carotene incorporated into 40 g LCT (LCT meal) or 40 g MCT (MCT meal). This meal was followed 6 h later by a beta-carotene-free meal containing 40 g LCT. Chylomicron beta-carotene, retinyl palmitate and triglycerides were measured every hour for 12.5 h after the first meal. No significant increase in chylomicron triglycerides was detected for the 6 h after the MCT meal intake, whereas a significant increase in chylomicron triglycerides was observed after the LCT meal intake. The chylomicron beta-carotene and retinyl palmitate responses to the MCT meal (0-6 h area under the curves, AUC) were significantly (P < 0.05) lower [AUC = 68.1 +/- 26.8 and 43. 4 +/- 10.4 nmol/(L.h), for beta-carotene and retinyl palmitate, respectively] than those obtained after the LCT meal [301.4 +/- 64.0 and 166.0 +/- 29.0 nmol/(L.h), respectively]. The chylomicron beta-carotene and retinyl palmitate responses obtained after the beta-carotene-free meal (6-12.5 h AUC) were also significantly lower when the first meal provided MCT rather than LCT. The chylomicron (retinyl palmitate/beta-carotene) ratios were constant during the postprandial periods, whatever the meal ingested. We conclude that the chylomicron beta-carotene response is markedly diminished when beta-carotene is absorbed with MCT instead of LCT. This phenomenon is apparently due to the lack of secretion of chylomicrons in response to MCT; however, a lower intestinal absorption of beta-carotene or a higher transport of beta-carotene via the portal way in the presence of MCT cannot be ruled out. Finally, the data obtained show that MCT do not affect the rate of intestinal conversion of beta-carotene into vitamin A.

Processing of vitamin A and E in the human gastrointestinal tract.

We aimed to provide basic data on the processing of vitamin A and E in the human gastrointestinal tract and to assess whether the size of emulsion fat globules affects the bioavailability of these vitamins. Eight healthy men received intragastrically two lipid formulas differing in their fat-globule median diameter (0.7 vs. 10. 1 microm. Formulas provided 28 mg vitamin A as retinyl palmitate and 440 mg vitamin E as all-rac alpha-tocopherol. Vitamins were measured in gastric and duodenal aspirates, as well as in chylomicrons, during the postprandial period. The gastric emptying rate of lipids and vitamin A and E was similar. The free retinol/total vitamin A ratio was not significantly modified in the stomach, whereas it was dramatically increased in the duodenum. The proportion of ingested lipid and vitamins was very similar in the duodenal content. The chylomicron response of lipids and vitamins was not significantly different between the two emulsions. Our main conclusions are as follows: 1) there is no significant metabolism of vitamin A and E in the human stomach, 2) the enzyme(s) present in the duodenal lumen is significantly involved in the hydrolysis of retinyl esters, and 3) the size of emulsion fat globules has no major effect on the overall absorption of vitamin A and E.