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We employed 3D reconstruction of CT images for evaluation of hepatic injuries in a series of eight trauma patients. One had additional reconstruction of a renal injury. 3D imaging provided precise anatomical delineation of damaged areas, particularly in relation to major vessels. Moreover, the imaging agreed with operative findings in every case. The 3D reconstruction facilitated decisions regarding intraoperative, reoperative and non-operative management. The improved imaging provided by 3D reconstruction may allow hepatic CT scans to be interpreted with greater ease and accuracy than conventional CT. We believe this is the first report of its use in liver trauma. Current indications may include postoperative confirmation of the extent of hepatic injuries, assessment before reoperation, and contribution to decisions regarding non-operative management. A limitation at present is the time taken for image production, but we suggest that in the future 3D imaging might contribute to evaluation before emergency surgery in patients with abdominal injuries.

The enzymatic activities unique to the glyoxylate cycle of higher plants and certain lower invertebrates, isocitrate lyase and malate synthase, have been demonstrated in homogenates prepared from human liver. Human liver can also carry out cyanide-insensitive fatty acid oxidation from palmitate. Utilizing light microscopic immunocytochemistry with an antibody produced against Euglena malate synthase, this enzyme localizes in numerous ovoid granules in human hepatocytes. Also, immunocytochemistry using antibodies produced against rat fatty acyl-CoA oxidase showed that this enzyme was localized in similar structures. With routine cytochemistry, catalase was seen in identical granular bodies. Both catalase and fatty acyl-CoA oxidase are peroxisomal enzymes. The presence of malate synthase in liver homogenates was further confirmed by Western blot analysis. These data suggest that the human liver may be capable of utilizing the carbon backbone of fatty acids for carbohydrate synthesis since the glyoxylate cycle in lower organisms subserves this anabolic function.

Information regarding the presence of the glyoxylate cycle in chick liver was sought. This metabolic pathway has long been thought to be absent from vertebrate tissues. Previous studies in other tissues have shown that, when present, this pathway is sensitive to vitamin-D. Thus, the effect of long-term vitamin-D deficiency and subsequent vitamin-D replacement on liver structure was studied by light microscopy. In addition, specific biochemical assays for the presence of glyoxylate cycle enzymes were performed. Light microscopy of lipid extracted tissues, light microscopic histochemistry, and quantitative histochemistry showed that the hepatocytes from vitamin-D-deficient animals contained primarily lipid. Hepatocytes from normal and vitamin-D-replete livers contained primarily carbohydrate as judged by their staining with periodic acid-Schiff (PAS). Also, malate synthase positive peroxisomes were seen in hepatocytes from normal and vitamin-D-treated chicks. Structures positive for this glyoxylate cycle enzyme were rarely seen in the hepatocytes from vitamin-D-deficient animals. Biochemical analyses showed the presence of the two unique glyoxylate cycle enzymes, isocitrate lyase and malate synthase, in chick hepatocytes. The activity of these enzymes was markedly increased in the vitamin-D-replete livers. In addition, chick hepatocytes demonstrated the capacity to oxidize fatty acid in the presence of cyanide. This activity, which is characteristic of peroxisomal B-oxidation rather than mitochondrial, was stimulated by vitamin-D treatment. Lastly, incubation of chick liver in the presence of a fatty acid substrate (palmitate) led to higher tissue glycogen content. The latter was further increased in liver from vitamin-D-replete animals. These data show the presence of glyoxylate cycle enzymes in a higher vertebrate and indicate that this tissue is endowed with the capacity to convert lipid to carbohydrate.

The immunocytochemical localization of cellular retinol-binding protein (CRBP), of plasma retinol-binding protein (RBP), and of plasma transthyretin (TTR) was studied in rat liver and kidney. The studies employed normal rats, retinol-deficient rats, and rats fed excess retinol. Antisera were prepared in rabbits against purified rat CRBP, RBP, and TTR. The primary antibodies and goat anti-rabbit IgG were purified by immunosorbent affinity chromatography, using the respective pure antigen coupled to Sepharose as the immunosorbent. This procedure effectively removed cross-reactive and heterophile antibodies, which permitted the specific staining and localization of each antigen by the unlabeled peroxidase-antiperoxidase method. CRBP was found to be localized in two cell types in the liver, the parenchymal cells and the fat-storing cells. Diffuse cytoplasmic staining for CRBP was seen in all the parenchymal cells. Much more intense staining for CRBP was seen in the fat-storing cells. The prominence of the CRBP-positive fat-storing cells changed markedly with vitamin A status. Thus, these cells were most prominent, and appeared most numerous, in liver from rats fed excess retinol. Both RBP and TTR were localized within liver parenchymal cells. The intensity of RBP staining increased markedly in retinol-deficient rat liver, consistent with previous biochemical observations. With the methods employed, specific staining for RBP or TTR was not seen in cells other than the parenchymal cells. In the kidney, all three proteins (CRBP, RBP, and TTR) were localized in the proximal convoluted tubules of the renal cortex. Staining for RBP was much more intense in normal kidney than in kidney from retinol-deficient rats. These findings reflect the fact that RBP in the tubules represents filtered and reabsorbed RBP. The pattern of specific staining for CRBP among the various tubules was very similar to that seen for RBP on adjacent, serial sections of kidney. The function of CRBP in the kidney is not known.

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)

The levels of retinol-binding protein, prealbumin, and several 'marker' enzymes were determined in homogenates, crude subcellular fractions and isolated Golgi apparatus prepared from the livers of vitamin A-deficient and control rats. Vitamin A deficiency led to a marked increase (3.5-fold) in hepatic retinol-binding protein concentration and to slight increases in hepatic pre-albumin levels, without affecting the levels of a number of marker enzymes localized in various subcellular compartments. The distributions of total protein and marker enzymes among various subcellular fractions were nearly identical in control and vitamin A-deficient preparations. In particular, vitamin A deficiency had no effect on the yield or enzymatic composition of isolated Golgi-rich fractions. In vitamin A-deficient rats, where the normal secretion of retinol-binding protein was blocked, a maximum of less than 10% of the total liver retinol-binding protein was accounted for in the Golgi. In contrast, in control rats, where the secretion of retinol-binding protein was proceeding at the normal rate, the relative amount of retinol-binding protein in Golgi increased to about 23% of the total liver pool. The data suggest that the Golgi apparatus is involved in the pathway of retinol-binding protein secretion from the liver, but demonstrate that the Golgi is not the major subcellular locus for retinol-binding protein in either normal or vitamin A-deficient rats.

Tetrachlorvinphos was fed at 8,000 or 16,000 ppm in diets to male and female (C57BL/6N X C3H/HeN)F1 mice for 80 weeks. Surviving mice were killed at 92 weeks, and all mice were completely necropsied. A high incidence of unusual nonneoplastic hepatic lesions in treated mice was present and characterized by pericellular fibrosis, hepatocyte nuclear pleomorphism, and intrasinusoidal foci of macrophages with intracytoplasmic crystalline structures. From 84 to 94% of the treated male mice and from 21 to 23% of the treated females had hepatocellular neoplasms. Only 17% of the control males and 7% of the control females had liver tumors. The induced tumors were frequently multiple in the liver, whereas the tumors in the controls were usually singular. The morphology of 241 liver tumors in 110 treated mice was different from that of tumors in controls. Liver tumors in control mice were generally composed of small basophillic hepatocytes. In treated mice, tumors were hepatocellular carcinomas composed of solid sheets of large basophilic or eosinophilic hepatocytes. Foci of prominent trabecular formation were seen in 51 tumors. Fifteen tumors were composed of small basophilic hepatocytes with oval cells interposed among them. Foci of capillary formation were noted in 3 of these tumors. In addition, 7 more typical hemangiosarcomas forming sinusoids and with thrombosis were observed.

Studies were conducted to characterize the localization and the concentration of cellular retinol-binding protein (CRBP) in rat liver during perinatal development. The studies employed both a specific immunohistochemical staining procedure and a sensitive and specific radioimmunoassay for CRBP. Dramatic changes were seen in both the levels and localization of CRBP. Marked increases in hepatic CRBP levels were seen during the last week of gestation and the first postnatal week, resulting in a quadrupling of CRBP levels in this 2-week interval. During the second postnatal week CRBP levels remained very high (approximately 110 to 120 micrograms/gm of wet weight, with peak values at about day 11 of age). During the third postnatal week, CRBP levels declined, followed by a further decline to adult levels (40 to 50 micrograms/ml) postweaning. A changing pattern of immunohistochemical localization of CRBP was seen that correlated with the changes in CRBP levels. In fetal livers at days 11 to 13 of gestation, CRBP was selectively localized in perisinusoidal cells that resembled stellate (fat-storing) cells in their location and shape but that lacked the lipid droplets usually seen in these cells in adult liver. During the final week of fetal development and the first postnatal week, a progressive increase in CRBP in parenchymal cells was seen. By the second postnatal week, very strong staining for CRBP was seen in all parenchymal cells. The staining of parenchymal cells for CRBP then declined, and by weaning (day 21) the pattern of localization of CRBP in liver resembled that seen in the adult, with low to medium staining seen in parenchymal cells and strongly intense staining in perisinusoidal stellate cells. Thus, the rise and then decline in intensity of CRBP staining in parenchymal cells paralleled the pattern of changes seen in CRBP levels during the perinatal period. In the placenta, CRBP was selectively localized in the trophoblast layer of the chorioallantoic placenta and in the entodermal layer of the yolk sac placenta. These findings suggest that either retinol is metabolized or plays a functional role at these placental sites.

Evidence for the glyoxylate cycle in the mammalian rat liver was sought. Activity of two unique glyoxylate cycle enzymes, isocitrate lyase and malate synthase, was found in rat liver homogenates. Vitamin D3 treatment of rachitic animals produced a five- and fourfold increase, respectively, in the activity of these enzymes. Vitamin D3 also increased the peroxisomal fatty acid oxidation and the accumulation of glycogen in liver slices in the presence of palmitate. These data suggest that the mammalian rat liver can convert fatty acid carbon to carbohydrate carbon directly.

Hepatic stellate cells play a quantitatively important role in hepatic retinoid metabolism and storage in rats maintained under normal nutritional conditions. Studies were conducted to further explore the biochemical characteristics of hepatic stellate cells. Stellate cells were isolated in high purity and yield from the livers of normal rats. The isolated cells had the morphology expected (on electron micrographs) for stellate cells, and were enriched in retinoids and in the intracellular retinoid-binding proteins. The composition of the stellate cell lipid droplets was examined. These lipid droplets were isolated in high purity and integrity from frozen and thawed stellate cell preparations by differential centrifugation. We estimate that the lipid composition of stellate cell lipid droplets consisted of approximately 42% retinyl ester, 28% triglyceride, 13% cholesterol (total) and 4% phospholipid. Thus, stellate cell lipid droplets contain substantial levels of both cholesterol and triglyceride, in addition to retinyl esters. Stellate cell homogenates were assayed for both retinol-binding protein and transthyretin by specific radioimmunoassays. Within the detection limits of these radioimmunoassays, we were unable to detect the presence of either retinol-binding protein (less than 9 ng per 10(6) cells) or transthyretin (less than 11 ng per 10(6) cells) in the stellate cell preparations. Total RNA, prepared from the isolated stellate cells, was examined by Northern blot analysis for retinol-binding protein mRNA and transthyretin mRNA, using cDNA probes for retinol-binding protein and transthyretin. Within the sensitivity of these assays, retinol-binding protein mRNA and transthyretin mRNA were not detected in stellate cells. These findings suggest that stellate cells do not synthesize or accumulate retinol-binding protein.

Hepatic stellate cells store the majority of the liver's retinoid (vitamin A) reserves as retinyl esters in stellate cell lipid droplets. A study was conducted to explore the effects of differences in dietary retinoid and triglyceride intake on the composition of the stellate cell lipid droplets. Weanling rats were placed on one of five diets that differed in retinoid or triglyceride contents. The dietary groups were: 1) control (2.4 mg retinol (as retinyl acetate)/kg diet and 20.5% of the calories supplied by triglyceride (as peanut oil]; 2) low retinol (0.6 mg retinol/kg diet and control triglyceride levels); 3) high retinol (24 mg retinol/kg diet and control triglyceride levels); 4) low triglyceride (2.4 mg retinol/kg diet and 5% of the calories supplied by triglyceride); and 5) high triglyceride (2.4 mg retinol/kg diet and 45% of the calories supplied by triglyceride). Stellate cells were isolated using the pronase-collagenase method and stellate cell lipid droplets were isolated by differential centrifugation. The levels of retinoids and other lipids were measured by high performance liquid chromatography. The stellate cells from control rats contained 113 micrograms total lipid/10(6) cells. Control stellate cell lipid droplets had the following mean percent lipid composition: 39.5% retinyl ester; 31.7% triglyceride; 15.4% cholesteryl ester; 4.7% cholesterol; 6.3% phospholipids; and 2.4% free fatty acids. Both the concentration of stellate cell lipids and the composition of stellate cell lipid droplets were markedly altered by changes in dietary retinoid. The low and high retinol groups contained, respectively, 82 and 566 micrograms total lipid/10(6) cells, with retinyl ester representing, respectively, 13.6% and 65.4% of the lipid present in the stellate cell lipid droplets. Low and high triglyceride groups were similar to controls in both stellate cell lipid content and the composition of the stellate cell lipid droplets. These findings indicate that the composition of stellate cell lipid droplets is strongly regulated by dietary retinoid status but not by dietary triglyceride intake.

A study was conducted to determine the levels and distributions of retinoids, retinol-binding protein (RBP), retinyl palmitate hydrolase (RPH), cellular retinol-binding protein (CRBP), and cellular retinoic acid-binding protein (CRABP) in different types of isolated liver cells. Highly purified fractions of parenchymal, fat-storing (stellate), endothelial, and Kupffer cells were isolated in high yield from rat livers. The retinoid content of each fraction was measured by HPLC analysis. RBP, CRBP, and CRABP were measured by sensitive and specific radioimmunoassays, and RPH activity was measured by a sensitive microassay. The concentrations of each parameter expressed per 10(6) parenchymal or fat-storing cells were, respectively: retinoids, 1.5 and 83.9 micrograms of retinol equivalents; RBP, 138 and 7.4 ng; RPH, 826 and 1152 pmol FFA formed hr-1; CRBP, 470 and 236 ng; and CRABP, 5.6 and 8.7 ng. When these data were expressed on the basis of per unit mass of cellular protein, the concentrations of RPH, CRBP, and CRABP in the fat-storing cells, which contain 10-fold less protein than the large parenchymal cells, were seen to be greatly enriched over parenchymal cells. The parenchymal cells contained approximately 9% of the total retinoids, 98% of the total RBP, 90% of the total RPH activity, 91% of the total CRBP, and 71% of the total CRABP found in the liver. The fat-storing cells accounted for approximately 88% of the total retinoids, 0.7% of the total RBP, 10% of the RPH activity, 8% of the total CRBP, and 21% of the CRABP in the liver. The endothelial and Kupffer cell fractions contained very low levels of all of these parameters. Thus, the large and abundant parenchymal cells account for greater than 70% of the liver's RBP, RPH, CRBP, and CRABP; but the much smaller and less abundant fat-storing cells contain the majority of hepatic retinoids and greatly enriched concentrations of RPH, CRBP, and CRABP.

Studies were conducted to explore the role of retinol in the control of the rate of synthesis of plasma retinol-binding protein (RBP) in the liver of the rat. Previous studies have shown that nutritional retinol status strongly influences RBP secretion from the liver cell. Both the in vivo relative rate of RBP synthesis and the in vitro translatable level of RBP-specific mRNA were examined in normal vitamin A, retinol-depleted, and retinol-repleted rats. The relative rate of RBP synthesis was estimated by measuring the extent of incorporation of [3H]leucine, [3H]lysine, and [3H]phenylalanine after a 12-min pulse label into immunoprecipitable RBP, relative to the incorporation of these amino acids into total liver trichloroacetic acid-precipitable protein. The level of translatable RBP-specific mRNA was quantitated in vitro by translation of rat liver poly(A+)RNA in the rabbit reticulocyte lysate protein-synthesizing system. The amount of newly synthesized RBP was determined relative to the amount of newly synthesized total protein. Both the relative rate of RBP synthesis (approximately 0.26%) and the translatable level of RBP-specific mRNA (approximately 0.14%) were found to be constant regardless of the retinol status of the rats. These results indicate that retinol, the molecule that RBP specifically binds and transports, does not appear to control the rate of synthesis of RBP or the translatable level of RBP-specific mRNA in the liver of the rat. Regulation of plasma RBP levels by retinol must be exercised at a locus beyond that of RBP synthesis.

A study was performed to identify the first translated product of messenger RNA for retinol-binding protein (RBP), the specific plasma transport protein for vitamin A. Poly(A)+RNA was isolated from rat liver and translated in the rabbit reticulocyte in vitro protein-synthesizing system. RBP was identified and separated from other translated products by immunoprecipitation with specific rabbit anti-rat RBP antiserum. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography of the immunoprecipitate consistently revealed one major product which migrated more slowly than purified rat serum RBP. The protein (preRBP) had an approximate molecular weight of 24,000. When dog pancreas microsomal membranes were cotranslationally present, the newly synthesized preRBP was processed to a protein which migrated coincidentally with purified rat serum RBP, approximately 20,500 daltons. These results indicate that RBP is initially synthesized as a larger molecular weight precursor (preRBP) which is rapidly processed by the removal of a peptide of approximately 3,500 daltons to the size of the final RBP molecule that circulates in the plasma.

The localization of immunoreactive retinol-binding protein (RBP) in rat liver was studied by immunofluorescence microscopy. The study employed specific antisera to rat RBP prepared in a rabbit and in a sheep. The indirect, two-stage method of localizing tissue antigens was employed, and livers of both normal and vitamin A-deficient rats were examined. Fab' fragments of immunoglobulins were used, to minimize non-specific labelling of the frozen sections of liver. With these techniques, the specific immune staining of RBP was observed within liver parenchymal cells. This staining appeared as both particulate and diffuse within the cytoplasm of the parenchymal cells, and was not concentrated within one region of the liver cell or lobule. Staining for RBP was not observed in nuclei or in cells other than parenchymal cells. Similar particulate and diffuse immune staining for RBP was observed in liver sections from both vitamin A-deficient and normal rats. More intense immune staining appeared to be present in the sections of vitamin A-deficient animals, in good correlation with the expected higher levels of RBP in deficient as compared to normal liver. When liver sections were exposed to an antiserum to rat albumin, instead of one to rat RBP, immune cytoplasmic staining was observed which was entirely of a diffuse nature, and did not appear particulate or granular. The findings suggest that RBP, unlike albumin, is localized in part within cytoplasmic vesicles or granules which are large enough to be detected with immunofluorescence, and which are present in livers of both normal and vitamin A-deficient animals. The nature of these putative RBP-containing particles remains to be explored.

Studies were conducted to explore the effects of colchicine on the secretion and metabolism of retinol-binding protein (RBP) by the liver. In the vitamin A-deficient rat, the rate of secretion of RBP from the liver into the serum is greatly reduced, and RBP accumulates in the liver. Injection of retinol (dispersed in a 20% Tween 40 solution) into deficient rats stimulated a rapid secretion of RBP from the liver into the serum. Colchicine treatment markedly inhibited the retinol-stimulated secretion of RBP from the liver into the serum. The effect of colchicine was most pronounced during the early period after retinol injection, particularly during the first 30 to 60 minutes. Ninety minutes after retinol injection, the serum RBP level of colchicine treated rats was only 36% as great as that of the control rats. In parallel experiments, a quantitatively similar inhibition of very low density lipoprotein (VLDL) secretion by colchicine was observed in the retinol-deficient rats. In contrast, colchicine did not affect the overall rate of hepatic protein synthesis, as estimated from the incorporation of [(3)H]leucine into total liver and serum protein; the secretion of newly synthesized protein was, however, inhibited. When rats were treated with colchicine and then injected with retinol, the level of RBP in a Golgi-rich fraction obtained from the liver homogenate increased markedly as compared to the level of prealbumin. The inhibition of RBP secretion by colchicine suggests that the microtubules play a role in RBP secretion. By analogy to studies on VLDL and albumin, these data provide presumptive evidence that the Golgi apparatus and secretory vesicles are involved in RBP secretion.-Smith, J. E., D. D. Deen, Jr., D. Sklan, and D. S. Goodman. Colchicine inhibition of retinol-binding protein secretion by rat liver.

Studies were conducted to explore the regulation of retinol-binding protein (RBP) metabolism in cultured primary hepatocytes from retinol-deficient rats. Newly isolated hepatocytes from retinol-deficient rats contained elevated levels (3.4-fold) of RBP, compared to hepatocytes from normal (retinol-adequate) rats. Addition of retinol to retinol-depleted hepatocytes stimulated RBP secretion by the cells in a concentration-dependent manner. Maximal stimulation of RBP secretion was seen with a retinol level of 0.3 micrograms/ml. The effect of retinol was quite rapid, and was evident by 20 minutes after addition of retinol to the medium. Stimulation of RBP secretion was only seen during the first few hours after retinol addition. The effect of retinol was specific for RBP; thus, retinol had no effect on the secretion rates of transthyretin or albumin. Addition of retinoic acid also stimulated RBP secretion by retinol-deficient hepatocytes. Addition of dexamethasone to retinol-deficient cells did not maintain the initial rate of RBP secretion. Dexamethasone also had no effect on the secretion of transthyretin or albumin by these cells. The effects of retinol and of dexamethasone seen here with retinol-depleted cells differed dramatically from effects seen in other studies with normal (retinol-adequate) hepatocytes. Thus, with normal cells, dexamethasone maintains RBP, TTR, and albumin production and secretion rates close to initial rates. Also in normal hepatocytes, with ample retinol available within the cell, addition of exogenous retinol does not appear to influence RBP secretion. In contrast, and as shown previously in intact rats, in retinol deficiency the availability of retinol specifically regulates the secretion of RBP by hepatocytes.

In previous studies, retinyl palmitate hydrolase activity in rat liver was partly characterized and was found to correlate and to partially copurify with hydrolytic activities against cholesteryl oleate and triolein. The present studies were designed to further explore relationships between these three lipid ester hydrolase activities, by use of non-hydrolyzable ether analogs of cholesteryl esters and acylglycerides. Cholesteryl ether analogs were potent inhibitors of all three hydrolase activities with relative potencies for the series of ethers of: linoleyl greater than oleyl = palmitoyl greater than n-butyl = n-propyl greater than ethyl = methyl. Retinyl palmitate hydrolase activity was most strongly inactivated by this series of analogs, with 48-86% of the activity inhibited at cholesteryl ether levels of 1 microM. The acylglyceride ether analogs were much weaker inhibitors of the three hydrolase activities, with the triolein, diolein and dipalmitin analogs showing similar inhibitory potencies, greater than that of the monolein and monopalmitin analogs. The data demonstrate the potential usefulness of ether analogs of cholesteryl esters and acylglycerides for exploring some of the characteristics of lipid ester hydrolase activities.

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.

A retinol-binding protein (RBP) cDNA clone was used to examine the effect of retinol status on the level of RBP mRNA in the liver, and to explore whether extrahepatic tissues contain RBP mRNA. In the first series of experiments, poly(A+) RNA was isolated from the livers of normal, retinol-depleted, and retinol-repleted rats and the levels of RBP mRNA in these samples were determined by both Northern blot and RNA Dot blot analyses. The levels of RBP mRNA in liver were similar in all three groups of rats. These findings confirm and extend previous studies which showed that retinol did not alter the in vivo rate of RBP synthesis or the translatable levels of RBP mRNA. In a second series of experiments, the RBP cDNA clone was used to survey poly (A+) RNA isolated from 12 different rat tissues for RBP mRNA by Northern blot analysis. We found that, along with the liver, many extrahepatic tissues contained RBP mRNA. Kidney contained RBP mRNA at a level of 5-10% of that of the liver, and the lungs, spleen, brain, stomach, heart, and skeletal muscle contained 1-3% of that of the liver. Translation of kidney poly (A+) RNA in rabbit reticulocyte lysates and immunoprecipitation of the translation products with anti-RBP antiserum resulted in a protein band of the same size as liver preRBP. These data suggest that RBP is synthesized in many extrahepatic tissues. It is possible that this extra-hepatically synthesized RBP may function in the recycling of retinol from these tissues back to the liver or to other target organs.

Studies were conducted to explore the synthesis of retinol-binding protein and transthyretin by embryonic and extraembryonic tissues during fetal development in the rat. The levels of retinol-binding protein mRNA and transthyretin mRNA were measured in fetal liver and in extraembryonic tissues by RNA gel blot analysis and hybridization with specific cDNA probes. Retinol-binding protein mRNA and transthyretin mRNA were both detected in the liver of fetuses at 14 days of gestation. The relative levels of these two transcripts increased during later fetal development; by the 20th day of gestation retinol-binding protein mRNA levels were comparable to those of the adult liver, while the levels of transthyretin mRNA were only 46% of those of the adult liver. Examination of the extraembryonic membranes for retinol-binding protein mRNA and transthyretin mRNA showed that these two transcripts were present specifically and only in the visceral yolk sac. The relative levels of retinol-binding protein mRNA and transthyretin mRNA in visceral yolk sac were constant from 14 to 20 days of gestation, averaging 58% and 51%, respectively, of the adult liver levels of these two transcripts. Both retinol-binding protein mRNA and transthyretin mRNA in the visceral yolk sac were found to be specifically localized in the endodermal layer. Finally, both immunoprecipitable retinol-binding protein and transthyretin were found to be synthesized and secreted by explant cultures of visceral yolk sac tissue. These data show that the visceral yolk sac is a major source of both retinol-binding protein and transthyretin in the developing fetus. Since the visceral yolk sac is a site of true placentation and nutrient transfer in the rodent, this raises the possibility that visceral yolk sac-derived retinol-binding protein and transthyretin may play important roles in the transport and delivery of retinol from the maternal blood to the developing fetus.

Studies were conducted to explore relationships in rat liver between retinyl palmitate hydrolase activity and the hydrolytic activities against cholesteryl oleate and triolein. Previous studies have shown positive correlations between these three lipid ester hydrolase activities. In order to extend this work, the hydrolase activities were further purified and characterized. The activities against cholesteryl oleate and triolein resembled retinyl palmitate hydrolase activity in showing great variability from rat to rat as assayed in vitro. The relative levels of the three activities were highly correlated with each other over a 50-fold range of activity in a series of 66 liver homogenates. Partial purification (approx. 200-fold) in the absence of detergents was achieved by sequential chromatography of an acetone powder extract of liver on columns of phenyl-Sepharose, DEAE-Sepharose and heparin-Sepharose. The three hydrolase activities copurified during each of these chromatographic steps. The properties of the three copurifying activities were similar with regard to stimulation of activity by trihydroxy bile salts, pH optimum (near 8.0), and observance of Michaelis-Menten-type saturation kinetics. The three activities were different in their sensitivity towards the serine esterase inhibitors diisopropylfluorophosphate and phenylmethanesulfonyl fluoride, and in their solubility properties in 10 mM sodium acetate, pH 5.0. Thus, triolein hydrolase activity was much less sensitive than the other two activities to the two inhibitors. In addition, the activity against cholesteryl oleate could be separated from the other two activities by extraction of an acetone powder with acetate buffer, pH 5.0. These results indicate that the three lipid hydrolase activities are due to at least three different catalytically active centers, and at least two distinct and separable enzymes. It is likely that three separate but similar enzymes, that appear to be coordinately regulated, are involved.

Studies were conducted to explore the structural features of retinoids that may be required to stimulate the secretion or production of retinol-binding protein (RBP) by H4II EC3 rat hepatoma cells in culture. Sixteen retinoids, that differed from all-trans-retinol in the cyclohexene ring, the polyene side chain, and/or the functional end group, were each incubated with H4II EC3 cells, and RBP secretion and accumulation were determined by radioimmunoassay. A number of retinoids, in addition to retinol, effectively stimulated RBP secretion. The results suggest that an intact cyclohexene ring may be necessary for the stimulation of RBP secretion. In contrast the system did not exhibit much specificity with regard to either the structure of the side chain or the nature of the end group. No relationship was found between the ability of a retinoid to stimulate RBP secretion and production and its biological activity. The biologically active retinoid, 13-cis-retinoic acid, was inactive in the present system, whereas the biologically inactive perhydromonoeneretinol was moderately effective in stimulating both RBP secretion and accumulation. In contrast, there appeared to be some relationship between the ability of different retinoids to stimulate RBP secretion and their ability to bind to RBP. In general, retinoids that had previously been shown to bind to RBP produced a greater stimulation of RBP secretion than those that did not bind to RBP. The secretion of RBP obtained with a given retinoid was not well correlated with the net accumulation of RBP. For example, retinoyl amide did not stimulate RBP secretion but was moderately effective in stimulating RBP accumulation. Thus, the secretion of RBP does not appear to be necessary for the stimulation of the net accumulation of RBP.

Propoxyphene and morphine lowered hepatic glutathione and increased serum glutamic-pyruvic transaminase (SGPT) activity when administered to male mice. Maximal changes were seen at 3 to 6 hr after administration, but the effects lasted for as long as 18 hr. Morphine-induced hepatic changes potentiated both acetaminophen and cocaine-induced hepatotoxicity. Naltrexone, a narcotic antagonist, abolished the glutathione depletion produced by both propoxyphene and morphine, but did not alter the propoxyphene-induced elevations of SGPT. Naltrexone also was tested against other narcotic agonists we have previously demonstrated to be hepatotoxic. Naltrexone pretreatment of antagonized L-alpha-acetylmethadol (LAAM)-induced depletion of glutathione and elevations of SGPT. Similarly, naltrexone antagonized norLAAM-induced depletion of glutathione and elevations of SGPT, but only lessened the magnitude of the changes induced by SKF525-A. The narcotic agonists morphine, LAAM, norLAAM and propoxyphene lower hepatic glutathione and induce hepatocellular damage, but these two effects appear to be unrelated.

A study was conducted to explore the spatial distribution within rat liver of two proteins importantly involved in retinoid metabolism in liver, namely, retinol-binding protein (RBP) and the enzyme retinyl palmitate hydrolase (RPH). The study was conducted with both vitamin A-sufficient (control) and vitamin A-deficient rats. Livers were carefully and reproducibly dissected into 11 sections each, and RBP levels and RPH activities were measured for each section homogenate. Both RBP and RPH activity displayed highly significant spatial heterogeneity in their distributions in liver. For control rats, the mean level of RBP was 39.0 micrograms/g wet weight, with a section-to-section variation of 14.5. For deficient rats, the corresponding RBP mean and variation values were 283 and 56 micrograms/g wet weight. For RPH, the mean level was 136 pmol free fatty acids (FFA) formed/(min X mg) with a section-to-section variation of 178. Both inspection of the data and analysis of variance indicated that this significant section-to-section variation (spatial heterogeneity) did not follow a consistent anatomic pattern from rat to rat. Thus, no one specific anatomic location in the liver was consistently high or low with regard to either RBP or RPH. Since the spatial distributions of both RBP and RPH activity did not follow a consistent anatomic pattern, it is not possible to obtain an accurate measure of the total liver levels for either parameter in a homogenate made from a small section. Finally, the patterns of distribution of RBP and RPH activity observed in the liver sections from both vitamin A-sufficient and deficient rats were not significantly correlated, either directly or inversely, as determined by chi-square analysis. Thus, RBP and RPH activity levels vary independently of each other in their heterogeneous anatomic distributions in rat liver.

The levels of retinoids, retinol-binding protein, cellular retinol-binding protein, cellular retinoic-acid-binding protein, transthyretin and the activities of retinyl palmitate hydrolase and cholesteryl oleate hydrolase were determined in purified parenchymal, fat-storing, endothelial and Kupffer cell preparations, and in liver homogenates from young adult (6-month-old) and old (36-month-old) rats. Retinoid levels were also determined in the plasma from young and old rats. Retinoid contents were determined by HPLC. The binding proteins and transthyretin were measured by specific radioimmunoassays; retinyl palmitate and cholesterol oleate hydrolases were measured by sensitive microassays. The retinoid content of both the liver homogenates and of the fat-storing, and parenchymal cell preparations increased between 6 months and 36 months of age. The cellular distribution of retinoids was similar for the two age groups analyzed with the fat-storing cells being the main retinoid storage sites in the rat liver. Concentrations of retinol-binding protein and transthyretin were high in parenchymal cell preparations. Cellular retinol-binding protein was enriched both in parenchymal and in fat-storing cell preparations; the highest concentrations of cellular retinoic-acid-binding protein were present in fat-storing cell preparations. No major differences were observed between the two age groups in the cellular concentrations and distributions of any of these binding proteins. High activity of cholesterol oleate hydrolase was measured in parenchymal and in Kupffer cell preparations; endothelial cell preparations also contained considerable activities. The distribution of this activity over the various cell types reflects their role in lipoprotein metabolism. Retinyl palmitate hydrolase activity was specifically enriched in parenchymal and in fat-storing cell preparations, consistent with the roles of these cells in retinoid metabolism. No major differences were observed between the two age groups in the cellular distributions of the two hydrolase activities. This study indicates that no major changes occur in the retinoid-related parameters analyzed with age, suggesting that rat liver retinoid metabolism does not change dramatically with age and that retinoid homeostasis is maintained.

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 explore hormonal and nutritional factors that might be involved in the regulation of retinol-binding protein (RBP) synthesis and secretion by the liver. The studies employed primary cultures of hepatocytes from normal rats. When cells were cultured in Dulbecco's modified Eagle's medium alone, a high rate of RBP secretion was observed initially, which declined and became quite low by 24 hr. Supplementing the medium with amino acids maintained RBP and albumin secretion at moderate (but less than initial) rates for at least 3 days. Further addition of dexamethasone maintained the production and secretion rates of RBP, transthyretin, and albumin close to the initial rates for up to 3-5 days in culture. The effects of dexamethasone were not rapid and were not specific for RBP; half-maximal effects were seen at 10(-9) to 10(-8) M levels. Hormonally treated hepatocytes produced and secreted RBP, transthyretin, and albumin at both absolute and relative rates similar to physiological values, as estimated from rates reported by others from studies in vivo (with both rats and humans) and with perfused livers. Glucagon addition partially maintained the secretion rates of these 3 proteins, but less effectively than did dexamethasone. A number of other hormones, added singly or in combination, did not affect RBP production or secretion. Addition of retinol to the cultured normal hepatocytes was without effect upon RBP secretion. These studies show that supplementing the culture medium of hepatocytes with amino acids and dexamethasone maintains RBP production and secretion for several days. In normal hepatocytes, with ample supply of retinol available within the cell, addition of exogenous retinol does not appear to influence RBP metabolism or secretion by the cells.

These studies report the hydrolysis of retinyl palmitate with liver homogenates and homogenate fractions from retinol-depleted rats. The studies utilized an effective in vitro assay for retinyl palmitate hydrolase (RPH) activity, in which microgram amounts of retinyl palmitate were employed as substrate, followed by the chromatographic separation and fluorescence assay of free and esterified retinol. RPH activity was maximal near pH 8 in Tris-maleate buffers, and required a bile salt for stimulation. Both cholate and taurocholate stimulated the reaction, whereas a number of other detergents tested were ineffective. The enzymatic activity showed an unusual subcellular distribution, with about 40% of total RPH activity recovered in the washed "nuclear" fraction (1,500 g pellet) and about 30--35% in the 105,000 g supernatant. This unusual distribution was not observed for marker constituents for plasma membranes, nuclei, mitochondria, lysosomes, Golgi apparatus, or endoplasmic reticulum. Despite its enrichment in the "nuclear" fraction, RPH activity was not enriched in purified preparations of nuclei or plasma membranes. Thus, RPH activity was not localized in any single, characterized subcellular structure. Another striking feature of the hepatic RPH activity was its extreme variability from rat to rat as assayed in vitro. Both the unusual subcellular distribution and the marked variability in activity were not observed for a variety of other hepatic ester hydrolase activities examined. Of ten lipid and nonlipid esters tested as substrates, only the hydrolytic activities against cholesteryl oleate and phytyl oleate correlated with, and partly resembled, RPH activity in these respects. The results suggest that the observed RPH activity is relatively specific for the hydrolysis of retinyl palmitate, and may therefore be significantly involved in hepatic retinyl ester metabolism.

Studies were conducted to examine the in vivo transfer of chylomicron (dietary) retinoid from rat liver parenchymal to stellate cells. We specifically addressed the question of whether chylomicron retinyl ester is transferred directly from hepatic parenchymal to stellate cells without first undergoing hydrolysis. [14C]Retinyl palmitate and its non-hydrolyzable ether analog, retinyl [3H]hexadecyl ether, were utilized to answer this question. Chylomicrons labeled with these retinoids were injected intravenously into rats. Liver cell fractions, highly enriched in parenchymal or in stellate cells, were isolated 0.5 h, 4.5 h and 24 h after chylomicron injection. The ratio of 3H: 14C found in parenchymal cell preparations 4.5 h after injection was 1.8 times the ratio for the injected chylomicrons, and 24 h postinjection the ratio had increased to 2.5 times that of the chylomicrons. In the stellate-cell-enriched preparations the 3H: 14C ratio was found to be 0.39, 0.29, and 0.23 times the ratio found in the injected labeled chylomicrons at 0.5 h, 4.5 h and 24 h after injection respectively. From the levels of 14C observed in the isolated stellate cells, it is estimated that 0.5 h postinjection the stellate cells contained approximately 34% of the 14C (i.e. the retinol injected as chylomicron retinyl ester) present in the liver. By 4.5 h the 14C present in isolated stellate cells had risen to approximately 41% of that present in the total liver, and 24 h after injection approximately 55% of hepatic total 14C was found in the stellate cells. These findings suggest that chylomicron retinyl ester is not transferred directly from the parenchymal to stellate cells without first undergoing hydrolysis to retinol.

Chylomicrons containing labeled cholesterol, mainly (70%) present as cholesteryl ester, were injected intravenously into intact rats, and samples of liver were obtained 27-210 min later. Most (58-75%) of the injected label was recovered in the liver after 27-75 min. Hepatic uptake occurred without hydrolysis of the labeled cholesteryl ester. In separate experiments, in vitro perfusion of livers of similarly treated rats for 30-35 min washed out only 3-9% of the labeled sterol. Samples of liver and small intestine were prepared for electron microscopy with Aquon as the dehydrating agent. Good retention (70% or more) of labeled cholesterol and satisfactory preservation of ultrastructure were obtained. After 30 min, the radioautographic reaction was localized mainly over the region of the cell boundary of the parenchymal liver cells, with fewer grains being present over intracellular organelles. At later time intervals, when considerable hydrolysis of the labeled cholesteryl ester had occurred, the radioautographic reaction was more evenly distributed. Phagocytosed labeled lipid was seen in Kupffer cells after the larger lipid load; phagocytosis by parenchymal cells was not seen. In other experiments, cholesteryl ester hydrolase activity was found in all subcellular fractions, the microsome and plasma membrane fractions showing the highest activity per mg protein. The mechanism of cholesteryl ester transport into the liver cell may involve: (1) hydrolysis at the cell surface; or (2) slow entry of intact molecules followed by intracellular hydrolysis of the ester bond.

The chemical and anatomical pathways of absorption of dietary retinal, retinoic acid, and retinol were examined in rats containing lymph, bile, and duodenal cannulae. The experiments were designed to maintain physiological conditions to the greatest possible extent. In each rat an uninterrupted flow of bile into the duodenum was maintained by connecting the duodenal cannula to the bile duct of a second rat. Labeled vitamin A compounds were introduced into the duodenum in very small amounts (7-14 micrograms) in the form of a bile-lipid mixture resembling normal intestinal contents. Under these conditions, most (70-80%) of the radioactivity recovered after the feeding of labeled retinol or retinal was found in the lymph, predominantly in saturated retinyl esters. In contrast, 92-95% of the radioactivity recovered after the feeding of labeled retinoic acid was found in the bile, and was contained in a mixture of polar metabolites, most of them more polar than free retinoic acid. Two-thirds of the small amount of radioactivity found in lymph after retinoic acid-(14)C feeding was in the form of free retinoic acid. The results indicate that under normal conditions the major pathway of retinal absorption involves its reduction to retinol, which is then esterified and transported via the lymphatics in a manner similar to that of dietary retinol. A small proportion of retinal is apparently normally oxidized, and is then transported via the portal vein and excreted in the bile in a manner similar to that of dietary retinoic acid. The relative importance, in quantitative terms, of these two pathways of retinal metabolism can vary, depending on the status of the animal.