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Objective: This study was designed to estimate the prevalence of and evaluate risk factors for subclinical vitamin A deficiency in Arab-Bedouin children at age 18 months, followed from birth. Design: Community-based, prospective, cohort study conducted in Rahat, a large Arab-Bedouin township, located near the city of Beer Sheva in the Negev region of southern Israel. Subjects: Healthy Bedouin infants (n=117) from the township, born at Soroka University Medical Center (SUMC) in Beer Sheva, were randomly recruited at birth. Enrollment was restricted to well infants born weighing >2500 g at birth. Results: More than 15% of the children had serum retinol concentrations below 0.7 μmol/l. Male sex (odds ratio (OR) 4.17 [1.14–15.32], P=0.031), stunting at age 12 months (OR 10.09 [2.00–50.97], P=0.05) and warm season at age 18 months (OR 6.20 [1.36–28.28], P=0.018) were associated with vitamin A deficiency. Maternal education decreased the risk of vitamin A deficiency (OR 0.81 [0.68–0.95], P=0.011). Conclusions: Study results indicate a significant vitamin A deficiency problem among Bedouin children. Deficiency may be prevented by increasing dietary intake of vitamin A, especially during the warm season. Other interventions include preventing and controlling diarrheal diseases in order to avert nutritional stunting, and providing nutritional education to women of childbearing age. Sponsorship: This study received financial support from the National Institute of Allergy and Infectious Diseases (AI-26497), the US–Israel Bi-national Science Foundation (BSF 90-00257), and the National Academy of Sciences/Institute of Medicine (AID/ANE 0158-G-SS-9035-00).

Lipocytes have been classified as vitamin A-storing, desmin-positive cells. In hepatic fibrogenesis, lipocytes transform into myofibroblasts, which express alpha-smooth muscle actin (alpha-SMA) and produce increased amounts of collagen. We isolated a population of vitamin A-poor lipocytes (VAPL) from normal rat liver and examined the morphological and biochemical differences between VAPL and vitamin A-replete lipocytes (VARL). Desmin and alpha-SMA expression were determined by Western blot in quiescent cells and in cells activated by culture on uncoated plastic. Both cell types were alpha-SMA-negative; however, in contrast to VARL, freshly isolated VAPL did not contain desmin. Desmin expression was induced in VAPL on activation. With time in culture, both VAPL and VARL expressed alpha-SMA and produced collagen, indicative of transformation to myofibroblasts. Ferritin receptor expression was demonstrated in cultured VARL after 1 day and in VAPL after 5 days, indicating that this is an early marker of lipocyte activation. After 7 days, VARL and VAPL were indistinguishable in terms of desmin, ferritin receptor expression, and collagen production. This study demonstrates the first isolation and characterization of two distinct quiescent subpopulations of lipocytes from normal rat liver: desmin-negative VAPL and desmin-positive VARL. Both populations of cells can be activated to myofibroblasts, the phenotype associated with hepatic fibrogenesis.

Oxidative DNA damage and antibodies to that damage have been implicated in lung, breast, and colorectal cancer. In this observational validation study, the relationship between anti-5-hydroxymethyl-2'-deoxyuridine (HMdU) autoantibody (aAb) and plasma micronutrients was assessed in 140 heavy smokers by ELISA. Anti-HMdU aAbs were 50% higher in women after adjustment for cigarettes/day (CPD; P = 0.002), although men smoked more and had higher plasma cotinine levels. The women reported taking more vitamin C (P0.005) and had higher plasma levels of alpha-carotene and beta-carotene (P0.001) and cryptoxanthin (P0.01) than men. Neither CPD nor cotinine was associated with aAb titers. Anti-HMdU aAbs were associated inversely with alpha-tocopherol (P = 0.10), retinol (P = 0.06), and age (P = 0.04) in women but not in men. In contrast to the men, womenor=50 years of age had significantly higher aAbs than those50 years of age (P = 0.05). Given the same duration of exposure, women had higher anti-HMdU aAbs and also reached peak levels at a lower cumulative smoking exposure (30 years) compared with male smokers (40 years). Subjects smoked an average of 28.9 +/- 0.81 CPD and initiated smoking at 17.2 +/- 0.33 (SE) years of age. Therefore, smokers who reported smoking for 30 years were typically50 years old. Womenor=50 years in the younger age group that smoked 21-30 years had significantly higher levels of aAbs than did men of the same age and smoking history (P = 0.012). Gender difference in aAbs was also evident in 29 persons who gave serial samples before and after quitting smoking (P0.028). In women, aAbs remained elevated for 14 months after smoking cessation but decreased significantly by 20.5 months (P0.032) by paired t tests. In men, aAbs increased with time since quitting smoking but not significantly. The finding of significantly elevated aAbs to oxidized DNA in femalesor=50 compared with male smokers of the same age and exposure suggests a possible interaction with hormones (e.g., estrogens) and may explain a heightened risk of smoking-induced lung cancer in women compared with men.

We have identified a retinol dehydrogenase (cRDH) that catalyzes the oxidation of 9-cis- but not all-trans-retinol and proposed that this enzyme plays an important role in synthesis of the transcriptionally active retinoid, 9-cis-retinoic acid. There is little information regarding either the biochemical properties of cRDH or how its 9-cis-retinol substrate is formed. We now report studies of the properties and expression of human and mouse cRDH and of the characteristics and location of the murine cRDH gene. Additionally, we report mouse hepatic 9-cis-retinol concentrations and demonstrate that 9-cis-retinol is formed in a time- and protein-dependent manner upon incubation of all-trans -retinol with cell homogenate. Human and mouse cRDH display similar substrate specificities for cis-isomers of retinol and retinaldehyde. Moreover, human and mouse cRDH show marked sensitivity to inhibition by 13-cis-retinoic acid, with both being inhibited by approximately 50% by 0.15 microm 13-cis-retinoic acid (for substrate concentrations of 10 microm). Lesser inhibition is seen for 9-cis- or all-trans-retinoic acids. Immunoblot analysis using antiserum directed against human cRDH demonstrates cRDH expression in several tissues from first trimester human fetuses, indicating that cRDH is expressed early in embryogenesis. Adult mouse brain, liver, kidney, and to a lesser extent small intestine and placenta express cRDH. The murine cRDH gene consists of at least 5 exons and spans approximately 6 kb of genomic DNA. Backcross analysis mapped the mouse cRDH gene to the most distal region of chromosome 10. Taken together, these data extend our understanding of the properties of cRDH and provide additional support for our hypothesis that cRDH may play an important role in 9-cis-retinoic acid formation.

To study the role of carboxyl ester lipase (CEL) in hepatic retinoid (vitamin A) metabolism, we investigated uptake and hydrolysis of chylomicron (CM)-retinyl esters (RE) by rat hepatoma (McArdle-RH7777) cells stably transfected with a rat CEL cDNA. We also studied tissue uptake of CM-RE in CEL-deficient mice generated by targeted disruption of the CEL gene. CEL-transfected cells secreted active enzyme into the medium. However, both control and CEL-transfected cells accumulated exogenously added CM-RE or CM remnant (CMR)-derived RE in equal amounts. Serum clearance of intravenously injected CM-RE and cholesteryl ester were not different between wild-type and CEL-deficient mice. Also, the uptake of the two compounds by the liver and other tissues did not differ. These data indicate that the lack of CEL expression does not affect the uptake of dietary CM-RE by the liver or other tissues. Moreover, the percentage of retinol formed in the liver after CM-RE uptake, the levels of retinol and retinol-binding protein in serum, and retinoid levels in various tissues did not differ, indicating that CEL deficiency does not affect hepatic retinoid metabolism and retinoid distribution throughout the body. Surprisingly, in both pancreas and liver of wild-type, heterozygous, and homozygous CEL-deficient mice, the levels of bile salt-dependent retinyl ester hydrolase (REH) activity were similar. This indicates that in the mouse pancreas and liver an REH enzyme activity, active in the presence of bile salt and distinct from CEL, is present, compatible with the results from our accompanying paper that the intestinal processing and absorption of RE were unimpaired in CEL-deficient mice.

Approximately 25% of postprandial retinoid is cleared from the circulation by extrahepatic tissues. Little is known about physiologic factors important to this uptake. We hypothesized that lipoprotein lipase (LpL) contributes to extrahepatic clearance of chylomicron vitamin A. To investigate this, [3H]retinyl ester-containing rat mesenteric chylomicrons were injected intravenously into induced mutant mice and nutritionally manipulated rats. The tissue sites of uptake of 3H label by wild type mice and LpL-null mice overexpressing human LpL in muscle indicate that LpL expression does influence accumulation of chylomicron retinoid. Skeletal muscle from mice overexpressing human LpL accumulated 1.7- to 2.4-fold more 3H label than wild type. Moreover, heart tissue from mice overexpresssing human LpL, but lacking mouse LpL, accumulated less than half of the 3H-label taken up by wild type heart. Fasting and heparin injection, two factors that increase LpL activity in skeletal muscle, increased uptake of chylomicron [3H] retinoid by rat skeletal muscle. Using [3H]retinyl palmitate and its non-hydrolyzable analog retinyl [14C]hexadecyl ether incorporated into Intralipid emulsions, the importance of retinyl ester hydrolysis in this process was assessed. We observed that 3H label was taken up to a greater extent than 14C label by rat skeletal muscle, suggesting that retinoid uptake requires hydrolysis. In summary, for each of our experiments, the level of lipoprotein lipase expression in skeletal muscle, heart, and/or adipose tissue influenced the amount of [3H]retinoid taken up from chylomicrons and/or their remnants.

The Carotene and Retinol Efficacy Trial (CARET), a randomized, placebo-controlled lung cancer chemoprevention trial of 30 mg of beta-carotene and 25,000 IU of retinyl palmitate, was prematurely terminated when a 46% excess lung cancer mortality was found in subjects on the active arm. Before the CARET intervention ended, 21 men were recruited to participate in a 6-month biomarker study using the same intervention as CARET that determined the effect of this supplementation on lung nutrient levels. Plasma and bronchoalveolar lavage (BAL) cell nutrient levels were measured before and after the intervention. The group in the active arm (n = 10) had plasma carotene level increases of over 10-fold, with a small increase in plasma retinol levels BAL cell levels of beta-carotene in the active group also increased 10-fold, from 4.5 to 46.3 pmol/10(6) cells (P = 0.0008), with no change in BAL cell retinol levels. Surgically obtained lung tissue from three CARET subjects in the active arm showed elevated carotene lung tissue levels but no increase in lung retinol levels compared to a group of surgical controls. Combined with our previous work showing a strong correlation between BAL and lung tissue nutrient levels, these findings suggest that supplementation with beta-carotene and vitamin A results in increased lung tissue as well as BAL cell levels of beta-carotene, with little change in lung retinol.

All-trans- and 9-cis-retinoic acid are active retinoids for regulating expression of retinoid responsive genes, serving as ligands for two classes of ligand-dependent transcription factors, the retinoic acid receptors and retinoid X receptors. Little is known, however, regarding 9-cis-retinoic acid formation. We have obtained a 1.4-kilobase cDNA clone from a normalized human breast tissue library, which when expressed in CHO cells encodes a protein that avidly catalyzes oxidation of 9-cis-retinol to 9-cis-retinaldehyde. This protein also catalyzes oxidation of 13-cis-retinol at a rate approximately 10% of that of the 9-cis isomer but does not catalyze all-trans-retinol oxidation. NAD+ was the preferred electron acceptor for oxidation of 9-cis-retinol, although NADP+ supported low rates of 9-cis-retinol oxidation. The rate of 9-cis-retinol oxidation was optimal at pHs between 7.5 and 8. Sequence analysis indicates that the cDNA encodes a protein of 319 amino acids that resembles members of the short chain alcohol dehydrogenase protein family. mRNA for the protein is most abundant in human mammary tissue followed by kidney and testis, with lower levels of expression in liver, adrenals, lung, pancreas, and skeletal muscle. We propose that this cDNA encodes a previously unknown stereospecific enzyme, 9-cis-retinol dehydrogenase, which probably plays a role in 9-cis-retinoic acid formation.

Amyloid fibrils derived from the Japanese, Portuguese, and Swedish types of familial amyloidotic polyneuropathy all consist of a variant transthyretin (TTR) with a substitution of methionine for valine at position 30 (TTR Met 30). In an attempt to establish an animal model of TTR Met-30-associated homozygous familial amyloidotic polyneuropathy and to study the structural and functional properties of human TTR Met 30, we generated a mouse line carrying a null mutation at the endogenous ttr locus (ttr-/-) and the human mutant ttr gene (6.0-hMet 30) as a transgene. In these mice, human TTR Met-30-derived amyloid deposits were first observed in the esophagus and stomach when the mice were 11 months of age. With advancing age, amyloid deposits extended to various other tissues. Because no significant difference was detected in the onset, progression, and tissue distribution of amyloid deposition between the ttr-/- and ttr+/+ transgenic mice expressing 6.0-hMet 30, endogenous normal mouse TTR probably does not affect the deposition of human TTR Met-30-derived amyloid in mice. TTR is a tetramer composed of four identical subunits that binds thyroxine (T4) and plasma retinol-binding protein. The introduction of 6.0-hMet 30 into the ttr-/- mice significantly increased their depressed serum levels of T4 and retinol-binding protein, suggesting that human TTR Met 30 binds T4 and retinol-binding protein in vivo. The T4-binding ability of human TTR Met 30 was confirmed by the analysis of T4-binding proteins in the sera of ttr-/- transgenic mice expressing 6.0-hMet 30. The T4-binding studies also demonstrated the presence of hybrid tetramers between mouse and human TTR subunits in the ttr+/+ transgenic mice expressing 6.0-hMet 30.

Ito cells, vitamin A-storing perisinusoidal cells, are believed to undergo myofibroblastic transformation in liver fibrogenesis. Our previous studies have shown that a diet high in polyunsaturated fat was key for induction of experimental alcoholic liver fibrosis. To investigate the cellular basis for this fibrogenic effect of a high-fat diet, we analyzed the content of vitamin A and cellular retinol binding protein (CRBP), the steady-state mRNA levels of procollagen-alpha1(I), transforming growth factor-beta1 (TGF-beta1), interleukin-6 (IL-6), and smooth muscle alpha-actin (alpha-SM) in freshly isolated Ito cells from rats given isocaloric amounts of ethanol and a low- or high-fat diet. After 10 wk, the Ito cell content of retinyl palmitate was severely reduced in both the high- and low-fat diet-ethanol-fed animals to 13-17% of those measured in respective pair-fed controls. On the other hand, the content of CRBP was reduced in the high-fat-ethanol rats but not in the low-fat-ethanol group. The cells from the high-fat-ethanol but not low-fat ethanol rats showed an 18-fold increase in procollagen-alpha1(I) mRNA at 17 wk, which was accompanied by 2.8- and 2.3-fold enhancement of TGF-beta1 and alpha-SM transcripts. IL-6 mRNA was not detected in the cells from any groups. These results demonstrate 1) myofibroblastic activation of Ito cells is evident in rats given a high-fat diet and ethanol but not in the low-fat-ethanol animals; 2) vitamin A depletion of Ito cells is the early and general effect of chronic ethanol intake but does not necessarily predict subsequent myofibroblastic activation; 3) reduced CRBP level is more closely associated with the subsequent cellular activation seen under the high-fat-ethanol regimen; and 4) IL-6 is not expressed in vivo by Ito cells from either normal livers or livers with alcoholic liver fibrosis.

Our laboratory has reported that adipose tissue and adipocytes are importantly involved in retinoid storage and metabolism. To gain further insight, we examined factors that may regulate CRBP mRNA levels in primary cultures of rat epididymal and murine BFC-1 beta adipocytes. Northern blot analysis revealed that retinoic acid is a potent inducer of CRBP mRNA, causing a 7.5-fold rise in mRNA levels in primary adipocytes and a 9.5-fold rise in BFC-1 beta adipocytes. This induction of CRBP mRNA was dose-dependent at retinoic acid concentrations ranging between 10(-8) and 10(-5) M. Retinoic acid induction of CRBP mRNA levels showed a short lag period (6 h) and reached a maximal level of induction by 12 h in BFC-1 beta adipocytes and by 48 h in primary epididymal adipocytes. Nuclear run-on transcription assays of retinoic acid-induced BFC-1 beta adipocytes indicated that the rate of CRBP gene transcription is enhanced 3.6- to 4.3-fold by retinoic acid. In contrast, dexamethasone markedly down-regulated CRBP expression in a dose-dependent manner at concentrations ranging between 10(-9) and 10(-6) M. CRBP mRNA levels in primary and BFC-1 beta adipocytes declined, respectively, by 90% and 80% when adipocytes were exposed to 10(-6) M dexamethasone for 24 h. Studies of mRNA half-life indicated that dexamethasone acts to lessen CRBP expression through the specific destabilization of CRBP mRNA. Treatment of both primary and BFC-1 beta adipocytes with triiodothyronine alone had no effect on CRBP mRNA levels; however, when adipocytes were treated with a mixture of triiodothyronine and retinoic acid, the induction of CRBP mRNA levels by retinoic acid was reduced. In summary, these studies indicate that CRBP gene expression is regulated by retinoic acid, dexamethasone, and triiodothyronine; thus suggesting that retinol uptake, intracellular transport, and metabolism are dynamically regulated in adipocytes.

Adipose tissue contains substantial stores of retinoid (retinol+retinyl ester) that, quantitatively, are second only to retinoid stores in the liver. Our studies show that retinoid levels in adipose tissue are markedly influenced by dietary retinoid intake. Because lipoprotein lipase (LPL) increases the uptake of lipoproteins and lipid emulsion particles by many cell types including adipocytes, we investigated whether LPL also increases retinoid uptake by adipocytes from lipid-containing particles. Addition of LPL (10 micrograms/ml) to BFC-1 beta adipocytes produced a 2-fold increase in cellular uptake of [3H]retinoid from a lipid emulsion containing [3H]retinyl ester. Heparin, which displaces LPL from binding sites on cell surface proteoglycans, increased [3H]retinoid uptake by an additional 2-fold. High performance liquid chromatography analyses showed that greater than 75% of the media and 85% of the cellular radioactivity was present as retinol. The conversion of retinyl ester to retinol by LPL was then assessed using model retinyl ester containing lipid emulsions. Although triglyceride appears to be the preferred substrate for LPL, after greater than 25% of the triglyceride was hydrolyzed, significant amounts of retinyl ester were hydrolyzed by LPL. Retinyl ester hydrolysis was increased approximately 20-fold in the presence of a source of apolipoprotein C-II. The physiologically significant palmitate, stearate, oleate, and linoleate esters of retinol were all hydrolyzed by LPL. When LPL was incubated with [3H]retinyl ester containing rabbit mesenteric chylomicrons and in the presence of heparin and apolipoprotein C-II, the LPL was able to completely hydrolyze the retinyl ester to retinol. Thus, LPL is able to catalyze the hydrolysis of retinyl esters and, through the process of hydrolysis, may facilitate uptake of retinoid by adipocytes.

The inability of the 'ethanol/high vitamin A Lieber-DeCarli diet' to induce liver fibrosis in two different rat strains was further evaluated by determining changes in parameters of liver cell damage and of retinoid and lipid metabolism. In the ethanol/vitamin A-treated group, slight but constant hepatic cell damage, as indicated by elevated alanine aminotransferase, aspartate aminotransferase and glutamate dehydrogenase activities in blood, was already observed at 6 months and maintained until the time of death at 16 months. Serum gamma-glutamyl transaminase activities were not raised. Moderate parenchymal liver cell damage was not accompanied by fibrosis. Hypertriglyceridemia or hypercholesterolemia were observed at 6-16 months of chronic alcohol administration. This response was strain dependent. In ethanol-treated rats of both strains, total liver retinoids and serum retinol concentrations were not altered. Therefore, the hypothesis that interaction between alcohol and retinoids is a major factor in the pathogenesis of alcoholic liver disease, needs to be reconsidered.

Studies were conducted to explore the effects of differences in retinoid nutritional status and of sex on the tissue distribution and levels of cellular retinol-binding protein (CRBP) and of cellular retinoic acid-binding protein (CRABP) in the rat. Sensitive and specific radioimmunoassays were developed and employed to measure the levels of both CRBP and CRABP. Four groups of six male rats each were fed experimental diets that differed greatly in the amount and kind of retinoids provided, but were otherwise identical. These groups were comprised of rats that were normal controls, retinoid-deficient, retinoic acid-fed, and excess retinol-fed. A fifth group of six female rats was fed the control diet. Immunogens identical with rat testis CRBP and CRABP, as assessed by radioimmunoassay displacement curves, were found in every rat tissue examined (21 tissues in males, 18 in females). The highest levels of CRBP were found in the proximal portion of the epididymis, the liver, and kidney. The highest levels of CRABP were found in the seminal vesicles, vas deferens, and skin. A significant (p less than 0.01) inverse relationship was found between CRBP and CRABP levels in the different tissues of the male reproductive tract. In both males and females, CRBP levels were highest in the gonads and proximal portion of the reproductive tract and decreased distally, whereas the opposite was true for CRABP. Retinoid-deficient rats showed reduced tissue levels of CRBP; thus, tissue CRBP levels are influenced by diet and retinoid availability. No differences in tissue CRBP levels were found in the rats fed the control, the retinoic acid, or the excess retinol diets. Female control rats had higher CRBP levels than male controls in 4 of 15 tissues compared (liver, lung, thymus, and fat). In contrast, tissue CRABP levels showed no diet- or sex-dependent differences. Only in one tissue, the skin, were differences observed (lower CRABP in retinoid-deficient and in female rats). Thus, CRABP metabolism and levels appear to be minimally influenced by the amount or kind of retinoid ligand available or by sex.

The distribution and levels of cellular retinol-binding protein (CRBP) and cellular retinoic acid-binding protein (CRABP) were measured in rat testicular peritubular and Sertoli cells and in isolated rat pachytene spermatocytes and spermatids. Two Sertoli cell preparations, one containing some germ cells and another that had been osmotically shocked to destroy germ cells, were examined. CRBP and CRABP levels were measured by specific and sensitive radioimmunoassays. Testicular peritubular cell cytosol preparations were found to contain high levels of CRBP (1.48 +/- 0.87 microgram CRBP/mg protein) but CRABP could not be detected. The mean CRBP level in Sertoli cell preparations that contained some germ cells was 0.93 +/- 0.24 microgram CRBP/mg protein; this value was similar to the level of 1.11 +/- 0.20 microgram CRBP/mg protein measured for Sertoli cells free of germ cells. The level of CRABP found in Sertoli cell preparations containing germ cells (0.81 +/- 0.32 microgram CRABP/mg protein) was approximately five times greater than was observed in Sertoli cells free of germ cells (0.16 +/- 0.03 microgram CRABP/mg protein). CRBP and CRABP levels in cultured Sertoli cells were not affected by time in culture for up to five days of culture. Pachytene spermatocytes and spermatids were very enriched in CRABP (0.72 +/- 0.26 microgram CRABP/mg protein for spermatocytes and 0.65 +/- 0.21 microgram CRABP/ml protein for spermatids). A search for a high molecular weight retinol-binding protein did not demonstrate the existence of such a protein in Sertoli cell-conditioned medium. In summary, these studies provide quantitative information about the distribution of the cellular retinoid-binding proteins in the cell types that compose the rat testis.(ABSTRACT TRUNCATED AT 250 WORDS)

A study was conducted to explore the effects of retinoic acid, fed to retinol-deficient rats, on the tissue distribution and levels of cellular retinol-binding protein (CRBP) and cellular retinoic acid-binding protein (CRABP). Sensitive and specific radioimmunoassays were employed to measure the levels of both CRBP and CRABP. Two groups of six male rats each were fed a purified retinoid-deficient diet supplemented with either: i) retinyl acetate (control group); or ii) retinoic acid (30 mg/kg diet) (retinol deficient-retinoic acid group). The retinoic acid supplementation was begun after 38 days on the retinoid-deficient diet alone, and was continued for 52-54 days. Analysis of the data indicated that only the CRBP level of the proximal epididymis in the retinol-deficient/retinoic acid group differed significantly from (was lower than) the corresponding control level, at the 1% confidence level. CRABP tissue levels did not differ significantly between the two groups. Thus, a moderately large intake of retinoic acid, as the only source of retinoids, had very little effect on the tissue distribution or levels of either its own cellular binding protein (CRABP) or of CRBP. This study provides further information showing that the tissue levels of the cellular retinoid-binding proteins are highly regulated and maintained in rats, even in the presence of marked changes in retinoid nutritional status.

An established cell line (TM-4) derived from murine Sertoli cells, the major supportive cell type of the testes, secretes a protein that binds retinol when grown in serum-free chemically defined medium. The protein that binds retinol is trypsin-sensitive and has an apparent Kd for retinol of 54 nM. Cholesterol, retinyl acetate, or UV-irradiated retinol at levels 100-fold in excess of retinol are poor competitors of [3H]retinol binding. Retinoic acid at a 100-fold molar excess inhibited [3H]retinol binding by 71%. In contrast, excess unlabeled retinol completely inhibits [3H]retinol binding. More than 80% of the total retinol-binding activity in confluent cultures is found in the culture medium. Prior to incubation with retinol, the protein that binds retinol has an apparent Mr of less than 150,000 by column chromatography; however, after incubation with retinol the protein that binds retinol exhibits an apparent Mr of 2 X 10(6) or greater and a sedimentation coefficient greater than 4 S. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals that the major iodinatable component of the aggregated protein that binds retinol has an apparent Mr of 70,000. The secreted protein that binds retinol is not immunologically cross-reactive with either serum or cellular retinol-binding protein or transferrin. These findings suggest that Sertoli cells may secrete a protein that binds retinol. Such a protein could be involved in the transport of retinol either to the lumen of the seminiferous tubules or to the developing germ cells themselves.

The main retinoids and some binding proteins and enzymes involved in retinol metabolism have been quantified in different types of rat liver cells. Hepatic perisinusoidal stellate cells contained 28-34 nmol of retinoids/10(6) cells, and parenchymal liver cells contained 0.5-0.8 nmol of retinoids/10(6) cells, suggesting that as much as 80% of more of total liver retinoids might be stored in stellate cells with the rest stored in parenchymal cells. Isolated endothelial cells and Kupffer cells contained very low levels of retinoids. More than 98% of the retinoids recovered in stellate cells were retinyl esters. Isolated parenchymal and stellate cell preparations both contained considerable retinyl palmitate hydrolase and acyl-CoA:retinol acyltransferase activities. Parenchymal cells accounted for about 75-80% of the total hepatic content of these two enzyme activities, with the rest located in stellate cells. On a cell protein basis, the concentrations of both of these activities were much greater in stellate cells than in parenchymal cells. In contrast, cholesteryl oleate and triolein hydrolase activities were fairly evenly distributed in all types of liver cells. Large amounts of cellular retinol binding proteins were also found in parenchymal and stellate cells. Although parenchymal cells accounted for more than 90% of hepatic cellular retinol binding protein, the concentration of the protein in stellate cells (per unit protein) was 22 X greater than that in parenchymal cells. Stellate cells were also enriched in cellular retinoic acid binding protein. Thus, both parenchymal and stellate cells contain substantial amounts of retinoids and of the enzymes and intracellular binding proteins involved in retinol metabolism. Stellate cells are particularly enriched in these several components.

Transthyretin (TTR) is involved in the plasma transport of both retinol and thyroid hormones. TTR is synthesized in the liver and choroid plexus, and in small amounts in several other tissues. A study was conducted to determine the tissue sites of degradation and turnover of TTR in the rat. The study employed TTR labeled with tyramine cellobiose (TC) and the trapped ligand method. Samples of purified rat TTR were labeled either with 125I-TC or directly with 131I. A mixture of the two labeled TTRs was injected intravenously into six rats. Blood samples were collected via a venous catheter for kinetic (turnover) analysis. After 24 or 48 h, the rats were killed, and 23 different tissues/organs were assayed as possible sites of TTR degradation. Derivatization of TTR with TC did not appreciably alter TTR plasma kinetics. Plasma turnover data were best fit by a three-pool model. The mean fractional turnover of plasma TTR was 0.15/h, and of total body TTR 0.04/h. The major sites of TTR degradation were the liver (36-38% of total body TTR degradation, almost all in hepatocytes), muscle (12-15%), and skin (8-10%). Tissues that were sites of 1-8% of body TTR degradation included kidneys, adipose tissue, testes, and the gastrointestinal tract. Less than 1% of total TTR degradation occurred in the other tissues examined. A second study was conducted in which labeled TTR was injected intraventricularly into the cerebrospinal fluid in order to explore the degradation of TTR of choroid plexus origin. The kinetics of the appearance and disappearance of such labeled TTR in plasma were physiologically reasonable, with an estimated turnover of cerebrospinal fluid TTR of the order of 0.33/h. The major tissue sites of degradation of labeled TTR injected into cerebrospinal fluid and into plasma were approximately the same. No specific degradation of TTR was found in the nervous system tissues. The most active organs of TTR catabolism, per gram wet weight, were liver and kidneys. These studies demonstrate that many tissues participate in TTR turnover and degradation; the studies provide quantitative information about the tissue sites of TTR catabolism.

Studies were conducted to define the subcellular localization of endogenous retinoids (vitamin A), retinoid-binding proteins, and acyl-CoA:retinol acyltransferase (ARAT) in liver and to determine whether their distributions were affected by hepatic vitamin A content. Quantitative subcellular fractionation techniques were used. Rats were fed purified diets either containing or lacking vitamin A to obtain animals with total retinoid stores ranging from 0.5 to 172 micrograms of retinol equivalent per gram of liver. Liver homogenates were fractionated by differential centrifugation to yield nuclear (N), mitochondrial-lysosomal (ML), microsomal (P), and high-speed supernatant (S) fractions. N, ML, and P were washed two more times by resuspension and centrifugation to remove constituents bound nonspecifically. S was further resolved into "floating lipid" and underlying "cytosol" by prolonged ultracentrifugation. The distributions of marker constituents were not affected by vitamin A status. Most of the retinyl ester in the liver was recovered in the S fraction where it was entirely (greater than 95%) associated with floating lipid. About half of the total free retinol was also recovered in the S fraction, but it was mostly (2/3) associated with cytosol per se. A substantial portion (30%) of the free retinol was recovered in the 3 X -washed microsomal (P) fraction. Sufficient binding capacity for retinol was present in both P (as retinol-binding protein) and S (as cellular retinol-binding protein) to quantitatively account for the amounts of free retinol present in the two fractions. ARAT activity in the liver was distributed among the subcellular fractions in a manner identical with an endoplasmic reticulum marker enzyme (NADPH-cytochrome C reductase).(ABSTRACT TRUNCATED AT 250 WORDS)