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© 2004 The American Society for Nutritional Sciences J. Nutr. 134:276S-280S, January 2004

Supplement: Proceedings of Symposium to Honor the Memory of James Allen Olson

Vitamin A: Overlapping Delivery Pathways to Tissues from the Circulation1,2

Jisun Paik*, Silke Vogel*, Loredana Quadro{dagger}, Roseann Piantedosi*, Max Gottesman{dagger}, Katherine Lai*, Leora Hamberger{dagger}, Milena de Morais Vieira* and William S. Blaner*,**,3

* Department of Medicine, {dagger} Institute for Cancer Research and ** Institute of Human Nutrition, Columbia University, New York, NY 10032

3To whom correspondence should be addressed. E-mail: wsb2{at}columbia.edu.


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Although retinol bound to retinol-binding protein (RBP) is the most abundant retinoid form present in the circulations of humans and most mammals, other retinoid and proretinoid forms are also present in the blood. We are interested in understanding to what extent each of these circulating retinoid forms contributes towards retinoid actions within cells and tissues. Here we report two studies focused on this question. First, we examined retinoid transport and storage in RBP-deficient mice that lack circulating RBP. These mice under normal laboratory conditions are phenotypically normal except for a visual impairment early in life that is corrected if the mice are maintained on a vitamin A-sufficient diet throughout life. The RBP-deficient mice take up vitamin A from the diet into most tissues at least as well as wild type mice. Compared to wild type mice, mice lacking RBP accumulate excess vitamin A in the liver, since there is no RBP to facilitate mobilization of stored retinol from hepatic stores. In a second study, we explored in vitro the actions of carotene cleavage enzyme (CCE) in facilitating ß-carotene cleavage to retinoid in the testis. CCE is most highly expressed in the testis. Pull-down experiments coupled with MALDI-MS analysis showed that mouse testis CCE is able to interact with the testis-specific lactate dehydrogenase-C (LDH-C) isoform. This may suggest that CCE and LDH-C act in concert to catalyze ß-carotene cleavage.

Retinoids (vitamin A and its analogs) are needed to maintain normal growth and development, immunity, reproduction, vision and other essential physiologic processes (1–5). With the exception of vision, all-trans- and 9-cis-retinoic acid are the active retinoid forms needed to modulate retinoid-dependent functions (1–4). These isomers of retinoic acid act by binding members of the retinoic acid receptor (RAR) and retinoid X receptor (RXR) families of ligand-dependent transcription factors to influence the rates of transcription of retinoid-responsive genes (1–4). In vision, 11-cis-retinal acts as the chromophore for the visual pigment rhodopsin (5).

All retinoids must be acquired from the diet either as preformed vitamin A (primarily as retinol and retinyl ester) or as provitamin A carotenoid (usually as {alpha}-carotene, ß-carotene or ß-cryptoxanthin) (1,6–8). Within the small intestine a portion of the provitamin A carotenoid is cleaved by carotene cleavage enzyme (CCE) to retinal and, following its enzymatic conversion to retinyl ester, this carotene-derived retinoid is packaged along with dietary preformed retinoid in nascent chylomicrons (6–8). Some unconverted dietary provitamin A carotenoid is also incorporated into chylomicrons and absorbed intact (6–8). The majority of chylomicron retinoid and carotenoid is cleared from the circulation by the liver. However, a significant percentage, 25–30%, of chylomicron retinoid is cleared from the circulation by extrahepatic tissues (6–8).

To meet tissue needs for retinoid, the liver secretes retinol bound to retinol-binding protein (RBP) (9–11). In the blood, the retinol-RBP complex forms a protein-protein complex with transthyretin (TTR) (9–11). Blood levels of retinol-RBP-TTR are held relatively constant throughout life except during time of impaired dietary intake of retinoid or in certain disease states (9–11). In addition to retinol bound to RBP-TTR, other retinoid forms can be found in the fasting circulation, albeit at relatively low levels compared to retinol (9–11). The fasting circulation contains low levels of all-trans-retinoic acid bound to albumin (at 0.1 to 0.4% of the level of plasma retinol) and soluble retinoyl-ß-glucuronides and retinyl-ß-glucuronides (6–8). In addition, retinyl esters and provitamin A carotenoids are present in circulating lipoprotein fractions (10,11). These alternative circulating retinoids and their subsequent metabolism within cells are summarized diagramatically in Figure 1.



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  		FIGURE 1 Comprehensive view of how different forms of vitamin A may come to cells from the blood. Retinol bound to RBP and complexed to TTR is the predominant form of vitamin A present in the fasting circulation. The retinol taken up by a cell can either be oxidized to retinal by the actions of one of many retinol dehydrogenases (RolDH) or esterified to retinyl ester by the action of lecithin:retinol acyltransferase (LRAT). The retinyl esters present within cells can be hydrolyzed to retinol via the actions of a retinyl ester hydrolase (REH). In addition to retinol-RBP-TTR, following consumption of a vitamin A-rich meal, the circulation may also contain relatively large levels of retinyl esters in chylomicrons and their remnants. Although the liver takes up the majority of the dietary vitamin A, many tissues take up some dietary vitamin A from chylomicrons. Retinoic acid bound to albumin is present in the fasting and postprandial circulations, albeit at levels that are only 0.1 to 0.4% those of retinol-RBP. Similarly, relatively low levels of retinyl-ß-glucuronides, retinoyl-ß-glucuronides and retinyl esters in lipoproteins are also found in both the fasting and postprandial circulations. Provitamin A carotenoids can be absorbed intact from the diet and are found in the circulation both postprandially in chylomicrons and under fasting conditions in lipoproteins. Retinal formed either through the oxidation of retinol or through cleavage of provitamin A carotenoids by carotene cleavage enzyme (CCE) is subsequently oxidized by a retinal dehydrogenase (RALDH) to retinoic acid. Retinoic acid can act within the nucleus of a cell to regulate the transcription of vitamin A-responsive genes. Within cells, retinol can be bound to cellular retinol-binding protein, type I (CRBP-I) and retinoic acid may be found bound to cellular retinoic acid-binding proteins type I or type II (CRABP-I or CRABP-II).

 
We have asked how the different retinoid forms present in the circulation contribute to meeting the retinoid needs of different cell types and tissues. Earlier, we demonstrated that mice are phenotypically normal in the absence of either TTR or RBP (12–14). Thus, in the face of very low circulating retinol levels, these mutant mice maintained on a vitamin A-sufficient diet appear normal (12–14). We also demonstrated in the rat that circulating retinoic acid can contribute significantly to tissue retinoic acid pools (15). We report here studies of how the tissues of RBP-deficient mice acquire sufficient retinoid. In addition, we report biochemical studies of the enzymatic cleavage of ß-carotene to retinal and its subsequent conversion to retinol in the testis.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
RBP-deficient mice and mouse husbandry

The generation and physiology of RBP-deficient mice have been described in detail elsewhere (13). RBP-deficient and wild type mice from the same genetic background were maintained ad lib on a vitamin A-sufficient commercial chow diet (W.F. Fisher & Son, Piscataway, NJ). All mice were housed throughout life in a specific virus and pathogen free barrier environment in accordance with the NIH guidelines (16).

HPLC analysis of serum and liver vitamin A

Serum retinol and liver total retinol (retinol + retinyl ester) concentrations were determined by reverse-phase HPLC using a procedure we have previously described (17). Briefly, to an aliquot of serum (or liver homogenates) an equal volume of absolute ethanol containing a known amount of the internal standard retinyl acetate (Sigma Chemical Co., St. Louis, MO) was added. Endogenous retinol and retinyl esters and the internal standard were extracted into hexane. After one backwash with H2O, the hexane extract was evaporated to dryness under a gentle stream of N2. Immediately upon reaching dryness, the retinoid containing lipid film was redissolved in 40 µl benzene for injection onto the HPLC. Retinol and retinyl esters were analyzed on a 4.6 x 250 mm 5 µm Beckmann Ultrasphere C18 column (Beckmann Instruments, Fullerton, CA). The mobile phase consisted of acetonitrile/methanol/dichloromethane (70:15:15 v/v) delivered at a flow rate of 1.8 mL/min. Retinoids were detected by UV absorbance at 325 nm using a Waters 996 detector.

Expression and purification of CCE

An open reading frame for a CCE cDNA was amplified by PCR from the original clone (AW044715) and subcloned into the mammalian expression vector pcDNA3 (CCE/pcDNA3) (Invitrogen, San Diego, CA) and into the bacterial expression vector pGEX-3X (CCE/pGEX) (Amersham Pharmacia, Piscataway, NJ) (18). Both clones were sequenced to verify orientation and correct reading frame in the case of the pGEX-3X vector. This latter vector was used to express CCE as a fusion protein with bacterial glutathione-S-transferase (GST). The recombinant fusion protein was purified by affinity chromatography on glutathione-Sepharose (Amersham Pharmacia, Piscataway, NJ), according to the manufacturer’s instructions. Expression of a GST-containing fusion protein in E. coli was confirmed by Western blot analysis. CCE/pcDNA3 was transfected into CHO cells using calcium-phosphate transfection (19) and CCE expression was verified by Northern blot analysis and in vitro enzyme activity assay.

Assay of CCE activity

In vitro enzyme assays for CCE were carried out using a previously reported procedure (18,20). As an enzyme source, depending on the experiment, we employed either crude bacterial homogenate (CCE/pGEX), 12,000 x g supernatant obtained from the bacterial homogenate, purified GST-fusion protein of carotene cleavage enzyme or 10,000 x g supernatant obtained from CHO cells transfected with carotene cleavage enzyme. Protein concentrations were measured using Bio-Rad Bradford protein assay reagents according to the manufacturer’s instructions employing bovine serum albumin as standard. Specific activity was defined as pmol of retinal produced/mg protein/h. Enzyme kinetics data were analyzed using EnzFit 5.0 software (Perrella Scientific, Amherst, NH).

Retinal and retinol isomers and ß-carotene were analyzed by normal phase HPLC on a 4.6 x 150 mm Supelcosil LC-Si column (Supelco, St. Louis, MO) preceded by a silica guard column (Supelco) using hexane:ethyl acetate:butanol (96.9:3:0.1, v/v) as the mobile phase flowing at a rate of 0.8 mL/min. Isomers of retinol and retinal, and ß-carotene were detected by absorbance of 325, 365, and 450 nm, respectively, using a Waters 996 Photodiode array detector (Waters Associates, Milford, MA). Retinol and retinal peaks were identified by comparing retention times and spectral data for the experimental compounds with those of authentic standards. Each retinol and retinal isomer was quantitated by comparing its integrated area under the peak against those of known amounts of purified standards. The loss during extraction was accounted for by adjusting the recovery to that of the internal standards, either 13-cis-retinol or all-trans-9-(4-methoxy-2,3,6-trimethyl phenyl)-3,7-dimethyl-2,4,6,8-nonatetraen-1–01 (TMMP-ROH).

Pull-down assays

Mouse CCE fused to GST was purified using glutathione-Sepharose and incubated with a 10,000 x g supernatant from a mouse testis homogenate for 30 min at room temperature. Following incubation, the mixtures were centrifuged at 500 x g for 5 min at 4°C and unbound proteins were discarded. The testis protein(s) precipitating with the CCE-GST fusion protein bound to the glutathione-Sepharose was considered the pull-down product. The glutathione-Sepharose beads were then washed 3 times with PBS and proteins bound to the glutathione-Sepharose were eluted with 10 mmol/L glutathionine in 50 mmol/L Tris, pH 8.0. Proteins eluting from the affinity resin were separated by 15% SDS-PAGE to identify proteins that interact with the carotene cleavage enzyme fusion protein. Protein bands were subjected to matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) by the Protein Chemistry Core Facility of the Howard Hughes Medical Institute at Columbia University (18).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
Earlier we reported that mice totally lacking any circulating RBP (RBP-deficient mice) are phenotypically normal, displaying normal longevity and fertility if they are maintained throughout life on a vitamin A-sufficient diet (13,14). Clearly RBP is not essential for delivering vitamin A to tissues. However, these mutant mice do display impaired vision early in life. Electroretinagram analyses of visual function for weanling wild type, heterozygous and homozygous RBP-deficient mice indicate that the response of homozygous RBP-deficient mice to light is far less strong than either wild type or heterozygous RBP-deficient mice (14). Thus, the visual phenotype is a recessive trait.

Measures of serum retinol levels in the RBP-deficient mice indicate that small amounts of retinol (3–6 µg/dL) are present in the circulation of the mutant mice fed a control vitamin A-sufficient chow diet (13,14). However, when the RBP-deficient mice are placed on a totally vitamin A-deficient, but otherwise nutritionally complete diet, serum retinol levels quickly (within 6 d) drop to undetectable (13,14). Figure 2 shows the effect of age on hepatic total retinol (retinol + retinyl ester) concentrations for wild type and RBP-deficient mice maintained on a vitamin A-sufficient diet throughout life. As can be seen from Figure 2, at the time of weaning (postnatal days 19, 20 and 21) the wild type and RBP-deficient mice have nearly identical hepatic total retinol levels. However, by 5- and 8-mo of age, the levels of total hepatic retinol in the RBP-deficient mice are elevated compared to wild type controls. Thus, RBP-deficient mice take up dietary vitamin A efficiently. The accumulation of hepatic retinol in the mutants reflects their inability to mobilize hepatic retinol stores.



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  		FIGURE 2 The age-dependence of vitamin A stores in the livers of wild type (•) and RBP-deficient ({circ}) mice maintained on a vitamin A-sufficient diet throughout life. RBP-deficient mice progressively accumulate more vitamin A in their livers than do wild type mice that are able to synthesize RBP. This excess accumulation of vitamin A in the livers of RBP-deficient mice arises from the inability of RBP-deficient mice to mobilize vitamin A from liver stores. The error bars indicate standard deviations for mean values determined for 6 to 8 mice (livers) at each time.

 
Plasma concentrations of all-trans-retinoic acid in wild type and RBP-deficient mice are similar and very low (1.7–3.3 nmol/L). Fasting plasma levels of retinyl esters, present in lipoprotein particles, were low in both the RBP-deficient and wild type mice. Thus, neither retinoic acid nor retinyl ester transport in the circulations of RBP-deficient mice differs from wild type mice.

We are interested in understanding how tissues within the body acquire retinoid from the circulation and the recent identification of cDNA clones for CCE (18,21–26) has made it possible to explore molecular aspects of the delivery to and metabolism of provitamin A in tissues. Mouse testis expresses the highest levels of CCE mRNA of any tissue in the mouse, including the small intestine (Fig. 3). Since CCE is a soluble enzyme that utilizes highly insoluble hydrocarbons like ß-carotene as substrates, we hypothesized that CCE must act in concert with other cellular proteins if it is to catalyze efficiently ß-carotene cleavage. To this end, we carried out pull-down experiments using mouse testis 10,000 x g supernatants and recombinant mouse CCE-GST fusion protein. An SDS-PAGE gel for a representative pull-down experiment is shown in Figure 4. Of the several protein bands associated with the CCE-GST fusion protein, one with an apparent mass of ~32 kDa was a specific pull-down product (Fig. 4). The Coomassie stained band for this protein was removed from the SDS-PAGE gel (see Fig. 4), digested with trypsin and analyzed by MALDI-MS. The protein was identified as a testis specific isoform of lactate dehydrogenase, lactate dehydrogenase-C (LDH-C) (27,28). The MALDI-MS profile for the trypsin fragments and the sizes of predicted trypsin fragments of mouse LDH-C are provided in Figure 5. All 21 tryptic fragments labeled in Figure 5 agree within 0.5 mass-ion units with those predicted from database analysis for mouse LDH-C.



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  		FIGURE 3 Distribution of CCE mRNA in adult tissues. Expression of CCE mRNA was examined by RT-PCR. Primers spanning ~60% of the open reading frame of the mouse CCE cDNA sequence were used to amplify CCE mRNA in total RNA from mouse intestine, testes, kidney, and liver. This RT-PCR experiment was repeated 3 times, each giving similar results. One representative result is shown.

 


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  		FIGURE 4 Mouse testis contains a protein that interacts with CCE. Glutathione-Sepharose affinity purified CCE-GST fusion protein was incubated with 10,000 x g supernatant prepared from a mouse testis homogenate. Recombinant CCE and proteins interacting with CCE were eluted from the glutathione-Sepharose beads with 10 mmol/L glutathione, and were visualized by Coomassie staining following SDS-PAGE. The major protein band, indicated by the arrow, pulled-down upon incubation with the glutathione-Sepharose bound CCE-GST fusion protein with the testis supernatant, was analyzed by MALDI-MS. The * indicates the position of the CCE-GST fusion protein. Lanes: 1. Molecular weight markers labeled to the left in kDa; 2. Proteins pulled-down following incubation of the glutathione-Sepharose bound CCE-GST fusion protein with a 10,000 x g supernatant prepared from a mouse testis homogenate. The arrow points to the pulled-down testis protein band taken for study by MALDI-MS; 3. Proteins pulled-down following incubation of glutathione-Sepharose bound GST with the same 10,000 x g testis supernatant employed in lane 2; 4. Proteins pulled-down following incubation of glutathione-Sepharose bound CCE-GST fusion protein with a 10,000 x g supernatant prepared from a mouse testis homogenate different from the one employed in lanes 2 and 3. The gap in the SDS-PAGE gel represents the site where the testis protein was excised for MALDI-MS analysis (see Fig. 5 below); 5. Proteins pulled-down following incubation of glutathione-Sepharose bound GST with the 10,000 x g testis supernatant employed for the pull-down shown in lane 4; 6. Proteins pulled-down following incubation of the glutathione-Sepharose bound carotene-15,15'-dioxygenase-GST fusion protein with homogenate buffer alone; and 7. Proteins pulled-down following incubation of glutathione-Sepharose bound GST with homogenate buffer alone.

 


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  		FIGURE 5 MALDI-MS separation of tryptic fragments obtained for the mouse testis protein pulled-down with recombinant mouse CCE-GST fusion protein. (Panel A) Mass spectrometry profile for the protein band excised from the SDS-PAGE gel shown in Figure 4, lane 4, was submitted to tryptic digestion followed by MALDI-MS analysis. Based on the mass (m/z) values for the 21 numbered tryptic fragments, the excised protein was identified as the testis-specific isoform, lactate dehydrogenase C (LDH-C). The peaks labeled "S" represent internal standard and those labeled "T" reflect known peaks arising from trypsin fragments. (Panel B) The predicted mass (m/z) values for each of the numbered LDH-C fragments shown in Panel A. These predicted mass values for LDH-C fragments were obtained from protein sequence databases (ProFound at http://prowl.rockefeller.edu/cgi-bin/ProFound and MS-Fit at http://prospector.ucsf.edu).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
The delivery of retinoids to tissues through the circulation involves a variety of overlapping pathways (9–11). These pathways are outlined in Figure 1. Although it seems likely that most tissues rely on retinol-RBP as their major circulating source of vitamin A, the fasting circulation also contains low levels of retinoic acid, retinyl-ß-glucuronides, retinoyl-ß-glucuronides and retinyl esters and possibly provitamin A carotenoids in lipoprotein particles. In addition, after consumption of a vitamin A-containing meal, the circulation carries relatively high concentrations of retinyl esters in chylomicrons and their remnants. Our studies with RBP-deficient mice demonstrate that tissues can derive the vitamin A (as retinal or retinoic acid) they need to support vitamin A-dependent functions independent of RBP-retinol.

How are the RBP-deficient mice able to thrive? Neither retinoic acid transport nor retinyl ester transport in the circulations is upregulated. The chow and purified diets that we have employed in our studies of RBP-deficient mice contain no or only very little provitamin A carotenoid. We believe that the RBP-deficient mice are able to acquire sufficient vitamin A from its regular (daily) intake from the diet. Approximately one-quarter of dietary vitamin A is delivered to extrahepatic tissues (6–8). Since the mutant mice cannot mobilize hepatic retinol stores, it is this delivery of postprandial vitamin A to extrahepatic tissues that renders these mice phenotypically normal. We propose that RBP allows for the storage of dietary vitamin A in the liver and assures that tissues are able continuously to acquire vitamin A from liver stores even in times of insufficient dietary vitamin A intake. The primary function of RBP, therefore, is to relieve the organism of the obligate need for daily vitamin A intake from the diet.

Some provitamin A carotenoids escape cleavage to vitamin A in the intestinal mucosa and are absorbed intact into the body from the diet. It is possible that this provitamin A carotenoid can serve also as an important source of vitamin A in tissues. The recent cloning of CCE in Drosophila (21), chicken (22), mouse (18,24,25) and the human (26) has renewed interest in the biochemistry of tissues utilization of circulating provitamin A carotenoid. Since CCE is expressed in the mouse testis (Fig. 5), this allows the testis to cleave provitamin A carotenoids to vitamin A. Our in vitro studies indicate that mouse CCE can participate in protein-protein interactions with LDH-C. These data may suggest that CCE and LDH-C exist as a complex within the cytosol that acts physiologically to facilitate carotene cleavage. It is possible that other proteins participate in the complex since both CCE and LDH-C are soluble proteins. It remains to be established how insoluble substrates like ß-carotene associate with CCE and how retinal, which also is water insoluble, is removed from the CCE-LDH-C complex following its formation. The cloning of CCE makes it possible to approach these and other important questions regarding the conversion of provitamin A carotenoids.

Our data provide new insights into how the vitamin A needs of tissues throughout the body are satisfied. Although the experimental approaches that we have used for our studies are modern, many of the questions that we addressed are not new. Moreover, many of our findings can be explained and understood using data that have been available in the literature for decades. However, from the context of a memorial symposium honoring Professor James A. Olson, whose work on vitamin A and carotenoids has spanned five decades, this is probably not inappropriate. Ultimately, our investigations are but part of a larger picture that was first framed by Professor Olson and other earlier investigators. This work provides some new glimpses into vitamin A and carotenoid physiology but also reminds us of things these earlier investigators already knew.


   ACKNOWLEDGMENTS
 
We wish to acknowledge helpful conversations about this work with Professor James A. Olson (Iowa State University, Ames) prior to his death. We are thankful for having the opportunity to discuss our work with him. The late Professor Olson’s collegiality and wise advice will be missed by all of those who knew him.


   FOOTNOTES
 
1 Presented as part of the James Allen Olson Memorial Symposium, "Functions and Actions of Retinoids and Carotenoids" held at Iowa State University, June 21–24, 2001 to honor the memory of James Allen Olson. This conference was supported by the U.S. Department of Agriculture; National Institutes of Health; Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University (ISU); Department of Food Science and Human Nutrition, ISU; College of Liberal Arts and Sciences, ISU; F. Hoffmann-La Roche; Kemin Foods, L.C., Procter & Gamble Company; Lipton; Best Foods; BASF; SmithKline Beecham; Cognis Corporation; Allergen and INEXA. Guest editor for this symposium was Norman I. Krinsky, Department of Biochemistry, School of Medicine, and the Jean Mayer Human Nutrition Research Center on Aging, Tufts University, Boston, MA 02111-1837. Back

2 This work was supported by NIH grants R01 EY12858 and R01 DK52444 and a grant from the United States Department of Agriculture. Back

4 Abbreviations used: CCE, carotene cleavage enzyme; GST, glutathione-S-transferase; LDH-C, lactate dehydrogenase-C; retinoic acid receptor; MALDI-MS, matrix-assisted laser desorption ionization-mass spectrometry; RAR, retinoic acid receptor; RBP, retinol-binding protein; RXR, retinoid X receptor; TTR, transthyretin. Back


   LITERATURE CITED
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 

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