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Department of Food Toxicology, School of Veterinary Medicine Hannover, D-30173 Hannover, Germany and * Center of Anatomy, Medical School Hannover, D-30623 Hannover, Germany
1To whom correspondence should be addressed. E-mail: thomas.arnhold{at}bc.boehringer-ingelheim.com.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • retinol • retinoic acid • vitamin A • liver • supplementation • intestine • metabolism
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) are essential for various biological processes, such as vision, reproduction, cell growth and differentiation, and embryonic development (1
). In dermatology and oncology, retinoids are effective therapeutics (2
). On the other hand, several case reports have indicated that vitamin A supplementation of 25,000 IU3
vitamin A or more by pregnant women could result in birth defects (3
). The epidemiologic study of Rothman et al. (4
) even suggests that intake of vitamin A at levels exceeding 10,000 IU/d may be associated with an increased teratogenic risk. A single meal of liver can contain a 10- to 30-fold higher amount of vitamin A. Our previous study (5
) as well as the study of Buss et al. (6
) showed that liver consumption led to a substantial increase of concentrations of active retinoid metabolites. However, in humans, systemic exposure to polar retinoids appeared remarkably different (6
) after excess vitamin A intake from high doses of supplements or consumption of fried liver. After the intake of 500,000 IU vitamin A as a supplement, the area under the concentration-time curve (AUC)4
values of all-trans-retinoic acid (RA) in plasma were significantly (P < 0.01, Student’s t test) higher than after consumption of a liver meal with equal amounts of vitamin A (Fig. 2C
). On the other hand, plasma AUC of 13-cis-RA were in the same range (Fig. 2
B). The observed differences could not be explained exclusively by an elevated bioavailability of vitamin A from supplements because plasma AUC values of retinyl palmitate were comparable (Fig. 2
A).
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). It is likely that vitamin A exerts most of the beneficial, but also the teratogenic actions via bioactivation to retinoic acid isomers, which may then bind to these receptors.
In addition to the liver, many tissues can convert retinol to RA including brain, kidney, spleen, pancreas, skin, testis and lung (8
–11
). Cytosolic alcohol dehydrogenases but also microsomal enzymes of the short-chain alcohol dehydrogenase family are involved in the first metabolic step, the oxidation of retinol to retinal (12
,13
). Subsequently, retinal is converted to RA either by cytosolic retinal dehydrogenases (14
,15
) or by cytochrome P450 isozymes (16
–18
). Whether small intestinal enzymes might be involved in the metabolism of excess vitamin A remains speculative. Ingested retinyl esters, the major form of orally administered vitamin A, are cleaved intraluminally to retinol, which is then absorbed into the intestinal cells (19
). There, retinol is immediately bound to a specific carrier protein, cellular retinol-binding protein type II (CRBP II), a protein that is expressed nearly exclusively in the intestine. Then, retinol is reesterified to retinyl esters by the specific enzymes lecithin-retinol acyl transferase and acyl CoA-retinol acyl transferase (20
,21
). Subsequently, retinyl esters are packed into chylomicrons and transferred into the lymphatic duct. Chylomicrons are reduced to chylomicron remnants in the systemic bloodstream (22
). These remnants are taken up by the liver and retinyl esters are subsequently stored in stellate cells of the liver (23
). The uptake and reprocessing steps in the intestine might be saturable when an excess amount of vitamin A is given as a bolus, thus leading to a faster absorption of vitamin A from a supplement compared with a liver meal. Therefore, other metabolic pathways such as oxidation or glucuronidation may contribute to vitamin A metabolism. It was demonstrated recently that cytosolic fractions of human enterocytes can convert retinol to RA in the presence of NADPH (24
).
The goal of the present study was to explore the differences in the metabolic pattern of vitamin A in humans after either supplementation or liver consumption by a participation of intestinal enzymes in the oxidative processing of excessive amounts of vitamin A. Therefore, we examined by an in vitro approach whether subcellular fractions of the intestine could oxidize retinol to all-trans-RA. Additionally, an in vivo experiment was performed to examine whether excess vitamin A is metabolized to polar retinoids in the intestine and whether the metabolites generated are released into the portal vein, thus possibly contributing to systemic retinoid levels. Vitamin A metabolites were determined in plasma of the portal vein and of a systemic vein (external jugular vein, hereafter called central vein). The pig was chosen because pigs are omnivorous and their intestinal tract resembles that of humans in many aspects (25
).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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HPLC solvents were of gradient grade quality from Merck (Darmstadt, Germany). Unless otherwise mentioned, all other chemicals and reagents were from Sigma (Deisenhofen, Germany). Dithiotreitol was supplied by Pharmacia (Freiburg, Germany). ß-Glucuronidase from Escherichia coli (EC 3.2.1.31; 2 x 105 IU/L) was purchased from Boehringer Mannheim (Mannheim, Germany). Retinol, all-trans-RA, 13-cis-RA and retinyl palmitate were from Sigma. Retinyl esters other than retinyl palmitate were synthesized in our laboratory. Glucuronides of retinol and all-trans-RA were kindly provided by Dr. J. A. Olson and Dr. A. B. Barua (Ames, IA). 14-Hydroxy-4,14-retro-retinol (14-HRR) and anhydroretinol were gifts from Dr. F. Derguini and Dr. J. Buck (New York, NY). All other reference retinoids were gifts from Hoffmann-La Roche (Basel, Switzerland and Nutley, NJ).
Isolation of porcine enterocytes and preparation of cytosolic and microsomal fractions.
Porcine small intestinal samples were obtained from a local slaughterhouse. Porcine enterocytes were isolated by the chelation-elution method according to the isolation of rat enterocytes described by Pinkus (26
). The binding of enterocytes to the basal membrane was loosened by complexation with EDTA; the cells obtained were then rinsed out with phosphate buffer (pH 7.4) by repeated extension and contraction of the intestine. The isolated enterocytes were resuspended in phosphate buffer (pH 7.4) containing potassium chloride (100 mmol/L), EDTA (1 mmol/L) and dithiotreitol (1 mmol/L). Cytosolic and microsomal fractions were isolated using a differential centrifugation procedure described by Guengerich (27
). The enterocytes were homogenized using an Elvehjem teflon potter and subsequent ultrasonication. The homogenate was centrifuged for 20 min at 10,000 x g, and the resultant supernatant was again centrifuged for 60 min at 100,000 x g. The supernatant of this centrifugation step (representing the cytosolic fraction of the enterocytes) was collected and frozen at -80°C until further usage. The sediment was resuspended in phosphate buffer and centrifuged for 70 min at 100,000 x g. The supernatant was discarded and the sediment (representing the microsomal fraction) was suspended in phosphate buffer (pH 7.4) containing glycerole (200 g/L) and EDTA (1 mmol/L). The protein concentration of cytosolic and microsomal fractions was determined by the bicinchoninic acid method according to Smith et al. (28
).
In vitro experiments.
The oxidative metabolism of all-trans-retinol to all-trans-RA was investigated by incubation of cytosolic or microsomal fractions of porcine enterocytes with various amounts of all-trans-retinol. In preliminary experiments, the incubation of all-trans-retinol with cytosolic fractions of porcine enterocytes was shown to be linear with respect to the duration of incubation (10–30 min) and the protein concentration (0.5–2 g/L). The incubations were performed in duplicate in a total volume of 500 µL containing NAD+ (2 mmol/L), potassium chloride (150 mmol/L), HEPES (20 mmol/L), dithiotreitol (2 mmol/L), cytosolic protein (2 g/L) or microsomal protein (1 g/L) and all-trans-retinol [0–750 µmol/L, predissolved in ethanol or dimethyl sulfoxide (DMSO)]. All incubations contained 1 vol % of solvent. The reactions were started with NAD+ and stopped after 30 min by addition of a threefold volume of isopropanol. Retinoid metabolites were determined by HPLC and UV detection as described below. The rate of all-trans-RA formation [V] was plotted vs. the substrate concentration [S] and data were fitted by nonlinear regression. Km and Vmax were determined from the Lineweaver-Burk plot ([1/V] vs. [1/S]).
In vivo experiments.
The trial protocol for the in vivo experiments complied with NIH guidelines (29
). A German Landrace pig (14 wk old; body weight, 23 kg) was supplied by the Center of Anatomy, Medical School Hannover (Hannover, Germany). After general anesthesia of the pig, the right external jugular vein was cannulated using a standard venous cannula (o.d., 2.1 mm; i.d., 1.4 mm) within a leading tube. After exteriorization, the cannula was fixed in the neck region. In a second step, a midline laparotomy was performed. In the upper jejunal mesentery a side branch of the upper mesenteric vein was exposed. Through this vein, a cannula leading to the portal vein was established. The portal vein cannula was exteriorized in the right flank of the abdomen and the laparotomy was closed by suturing the layers of the abdominal wall. The pig recovered from surgery within 3 d. Throughout the experiment, the pig was allowed to move in a wooden box (2.5 m2) without fixation during blood sampling. The pig received twice daily 
700 g of a pelleted pig maintenance diet (NAFAG No. 9103 minipig maintenance diet, NAFAG, Gossau, Switzerland, for detailed information visit http:\/\/www.nafag.ch). The pig was deprived of food for 12 h before the experiments. Food was offered again 5–6 h after dosing.
Two subsequent experiments with 4-d washout periods were performed. In the first, the pig was given a vitamin A supplement containing a retinyl palmitate oil in water emulsion (A-Mulsin, Mucos Pharma, Geretsried, Germany) mixed with a small amount of food. The total dose administered was 430,000 IU vitamin A. In the second, the pig was fed 600 g liver. The vitamin A content of the liver was determined by HPLC. The amount of liver consumed contained a total dose of 667,000 IU vitamin A. In both experiments, blood samples were taken from the portal and central veins at different time intervals up to 24 h. Plasma was prepared and stored at -80°C until analysis. Retinoid metabolites were determined in portal and central vein plasma by HPLC and UV detection as described below. Areas under the plasma-concentration time curve values (AUC) were determined by the trapezoidal method (30
) for 24 h after vitamin A intake. The maximum plasma concentrations (Cmax) and time to maximum plasma concentrations (Tmax) are the observed values.
HPLC analysis.
Plasma samples as well as in vitro incubation mixtures were extracted with a threefold volume of isopropanol and underwent solid-phase extraction according to a method previously described (31
). A modification of the HPLC method described by Eckhoff and Nau (32
) was used for determination of retinoids. This method uses a linear gradient formed from 57.5% methanol and 42.5% aqueous 60 mmol/L ammonium acetate to 95% methanol and 5% ammonium acetate within 11 min. To determine also retinol and retinyl esters in a single chromatographic run, the methanol percentage was increased to 100% at 11.2 min and further maintained at this level until 25 min (33
). The starting conditions of the gradient were reached again after 26 min. With this method, separation of retinyl palmitate and retinyl oleate was not possible. Detection was performed by monitoring the UV absorbance of the eluate at 340 and 356 nm simultaneously using a Shimadzu SPD 10 AV two wavelength detector (Kyoto, Japan). Peak eluates of putative retinoid glucuronides were collected, evaporated to dryness, redissolved in buffer, subjected to hydrolysis by ß-glucuronidase and the resulting retinoids were rechromatographed. The procedure was described in detail by Sass and Nau (34
).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The Michaelis-Menten kinetics of the all-trans-RA formation in cytosolic fractions of porcine enterocytes are presented in Figure 3A
. The formation of all-trans-RA (dissolved either in ethanol or in DMSO) was concentration dependent. As shown in the Lineweaver-Burk plot (Fig. 3
B), Km and Vmax of the reaction were determined to be 94–96 µmol/L and 7.9–8.6 pmol/(min · mg protein), respectively, depending on the solvent used. The incubation of microsomal fractions of porcine enterocytes with retinol did not form detectable levels of oxidative metabolites under the experimental conditions used. RA isomers other than the all-trans form were not identified. The intermediate product, retinal, was detectable in trace amounts only.
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Predose levels of retinoids in plasma from portal and central veins are shown in Table 1
. Only retinol and retinyl palmitate/oleate (16:0/18:1) were detected in all plasma samples. Additionally, all-trans-RA and 13-cis-RA were detected before supplementation, but only at low levels. Retinol concentrations were slightly elevated at the start of the second experiment (liver feeding). Retinyl palmitate/oleate levels were markedly lower in the portal vein than in the central vein.
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), 13-cis-4-oxo-RA, retinoyl ß-D-glucuronide (RAG) and retinyl ß-D-glucuronide (ROG). Table 2
summarizes the kinetic data. Considering the AUC and Cmax levels of retinol, retinyl palmitate/oleate and total retinyl esters in central vein plasma, it became apparent that the relative bioavailability of vitamin A from the liver feeding was 70% [relative bioavailability = AUCliver · dosesupplement/(AUCsupplement · doseliver)] of the supplement’s availability. After liver feeding, the AUC and Cmax of retinyl palmitate/oleate and total retinyl esters were lower in portal vein plasma than in central vein plasma, whereas AUC and Cmax were comparable in central and portal veins after supplementation. Major differences between supplementation and liver feeding occurred with respect to phase I and phase II metabolism of vitamin A. After vitamin A supplementation, all-trans-RA concentrations in the portal vein were much higher than in the central vein. The concentration-time curve peaked at 1.75 h, then declined rapidly; there was a second peak at 8 h, possibly reflecting enterohepatic circulation, whereas the all-trans-RA levels in central vein plasma were only marginally elevated at all time points (Fig. 4A
). Such a difference in all-trans-RA kinetics between central and portal veins did not occur after liver feeding (Fig. 4
B). On the other hand, the kinetics of 13-cis-RA did not exhibit major differences between central and portal veins or between supplementation and liver feeding (Fig. 4
C and D, respectively). Glucuronidation of retinoic acid moieties to RAG also occurred. AUC of RAG in portal vein plasma were higher than those in central vein plasma, although only marginally (Fig. 4
E and F, respectively). The major metabolic pathway of vitamin A, however, was the direct glucuronidation of retinol, yielding ROG. The AUC of ROG were approximately twofold higher after supplementation compared with after consumption of liver, whereas Cmax levels were five- to sevenfold higher (Fig. 4
G and H, respectively). Additionally, a marked difference in ROG kinetics was obvious between portal and central veins after supplementation. ROG levels were higher in portal vein than in central vein after supplementation, but not after liver feeding.
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and B, respectively. In Figure 5
A, HPLC chromatograms of a portal vein plasma sample (upper trace) and a central vein plasma sample (lower trace) both taken 1.75 h after supplementation (at Tmax for all-trans-RA) are overlaid. Portal vein levels of retinoid metabolites were greater, with the major differences apparent for all-trans-RA (peak 5) and ROG (peak 2). The peak eluting at 1 min after all-trans-RA in portal vein plasma could not be identified. In Figure 5
B, HPLC chromatograms of a portal vein plasma sample taken 1.75 h after supplementation (upper trace) and 2.5 h after liver feeding (lower trace), which are representative for the respective Tmax of all-trans-RA, are shown. Again, all-trans-RA and ROG levels were higher after supplementation compared with after liver consumption.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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,36
–38
). The formation of all-trans-RA from unbound retinol occurred in the presence of either cytosolic fractions or microsomal fractions, mainly from liver (10
,17
,36
,37
,39
,40
). Additionally, conversion of retinal to RA was described in presence of human cDNA-expressed P450 enzymes (18
). In most tissues, retinol does not occur in its unbound form, but bound to CRBP I. The oxidation of unbound as well as CRBP I bound retinol was examined with cytosolic fractions from calf liver (41
). The Km values for unbound and CRBP I bound retinol were 7.1 and 8.4 µmol/L, respectively. The Vmax value for the conversion of free retinol [15 pmol/(min · mg protein) was higher than that for CRBP I bound retinol [10 pmol/(min · mg protein)]. Because the dissociation constant for the retinol-CRBP I complex is 10-8 mol/L and Km values were in the same range, the dissociation of the retinol-CRBP I complex should not be a prerequisite for the oxidation of retinol. It was therefore concluded that CRBP I might serve as a channel protein for retinol metabolizing enzymes (42
). In the gastrointestinal (GI) tract, the functionally diverse CRBP type II exists almost exclusively (43
,44
). In vivo experiments in rats have shown that the molar ratio of unesterified retinol to CRBP II in the GI tract changed after oral administration of high vitamin A doses (given as retinyl acetate) from <1 in control groups to 3–19 in treated groups (45
). It can therefore be concluded that large amounts of unbound retinol are present in the GI tract after administration of a vitamin A bolus. This unbound fraction of retinol might be metabolized by phase I or phase II enzymes.
In the present work, the in vitro oxidation of retinol to all-trans-RA was demonstrated in cytosolic fractions of porcine enterocytes. A Km of 94–96 µmol/L and a Vmax of 7.9–8.6 pmol/(min · mg protein) was determined. The results of the in vitro experiments indicated that retinol is a substrate for cytosolic enzymes of the porcine intestinal mucosa, although with a weak affinity. Due to the high Km of the reaction, it can be concluded that nonspecific enzymes are involved, which metabolize retinol only at high local concentrations, but with a turnover comparable to those rates described in the literature (41
).
The differences in oxidative vitamin A metabolism in humans observed in the study of Buss et al. after intake of 500,000 IU vitamin A as a supplement or a liver meal raised the question whether intestinal metabolism of retinol to RA moieties and subsequent release of polar retinoids into the systemic circulation after a bolus of vitamin A as a supplement might be responsible for that phenomenon. In our in vivo experiment in a pig, amounts of vitamin A comparable to those of Buss et al. (6
) were administered either as a supplement (430,000 IU) or liver (667,000 IU). It was clearly demonstrated that retinol is metabolized to all-trans-RA already in the intestine. However, contrary to the human situation, other metabolic pathways dominated, yielding glucuronides of all-trans-RA and retinol. We assume that in humans, higher amounts of all-trans-RA would have been formed intestinally after vitamin A supplementation. The recent finding that human intestinal enterocytes can oxidize retinol to all-trans-RA with a Km of 15 µmol/L and a Vmax of 2 pmol/(min · mg protein) (24
) supports this hypothesis.
In contrast to retinyl esters, which are transported in chylomicrons via the lymphatic duct, polar metabolites of retinol are transported via the portal vein. As a consequence, portal vein plasma levels in the pig were higher than the corresponding levels in the central vein after vitamin A supplementation, at least for all-trans-RA and ROG.
To date, in vivo experiments describing the role of the GI tract in the metabolism of vitamin A have focused mainly on the esterification of absorbed retinol and the transport of resulting retinyl esters via the lymphatic duct (46
–48
). Only in some experiments was the role of the GI tract in the oxidative metabolism of vitamin A studied. In ferrets, all-trans-RA was detected in the intestinal mucosa after perfusion of small intestinal segments with either ß-carotene or retinal (49
). After daily doses of 20 mg retinyl acetate to rats over 14 d, all-trans-RA, 13-cis-RA, all-trans-RAG and more polar metabolites were detected in the intestinal mucosa (50
). Because the bile duct was cannulated shortly before termination of the experiment, the authors concluded that polar metabolites must have been generated in the intestine. However, it cannot be excluded that polar metabolites might have been generated in the liver and subsequently transported via the systemic circulation to intestinal tissues. Therefore, portal and central vein plasma levels of retinoids were compared in our in vivo study.
In summary, our study demonstrated that all-trans-RA is formed after incubation of cytosolic fractions of porcine enterocytes with retinol. The in vivo experiment in a pig showed that retinol is already metabolized in the intestine to polar phase I and phase II metabolites, which are subsequently released into the portal vein. The major metabolic pathways of vitamin A in porcine intestine are summarized in Figure 6
. These findings raise the question whether a threshold of vitamin A intake exists in humans at which the intestine contributes to systemic exposure of putative teratogenic vitamin A metabolites.
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FOOTNOTES |
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3 1 IU = 1 International Unit vitamin A = 1.05 nmol vitamin A alcohol (retinol) and/or esters of vitamin A.
4 Abbreviations used: AUC, area under the plasma concentration-time curve; CRBP, cellular retinol-binding protein; DMSO, dimethyl sulfoxide; GI, gastrointestinal; 14-HRR, 14-hydroxy-4,14-retro-retinol; RA, retinoic acid; RAG, retinoyl ß-D-glucuronide; RAR, retinoic acid receptor; ROG, retinyl ß-D-glucuronide; RXR, retinoid X receptor.
Manuscript received 2 July 2001. Initial review completed 27 July 2001. Revision accepted 22 October 2001.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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with cytosolic fractions from porcine intestine (duplicate incubations). The data were fit by nonlinear regression (Vt = Vmax · [S]/Km + [S] where Vmax is the maximum velocity, [S] is the substrate concentration and Km is the Michaelis-Menten constant).
of a German Landrace pig after either supplementation of 430,000 IU vitamin A or consumption of liver containing 667,000 IU vitamin A.
