Retinyl Palmitate Hydrolase Activity Research Articles

Identification of microsomal rat liver carboxylesterases and their activity with retinyl palmitate.

Retinyl esters are a major endogenous storage source of vitamin A in vertebrates and their hydrolysis to retinol is a key step in the regulation of the supply of retinoids to all tissues. Some members of nonspecific carboxylesterase family (EC 3.1.1.1) have been shown to hydrolyze retinyl esters. However, the number of different isoenzymes that are expressed in the liver and their retinyl palmitate hydrolase activity is not known. Six different carboxylesterases were identified and purified from rat liver microsomal extracts. Each isoenzyme was identified by mass spectrometry of its tryptic peptides. In addition to previously characterized rat liver carboxylesterases ES10, ES4, ES3, the protein products for two cloned genes, AB010635 and D50580 (GenBank accession numbers), were also identified. The sixth isoenzyme was a novel carboxylesterase and its complete cDNA was cloned and sequenced (AY034877). Three isoenzymes, ES10, ES4 and ES3, account for more than 95% of rat liver microsomal carboxylesterase activity. They obey Michaelis-Menten kinetics for hydrolysis of retinyl palmitate with Km values of about 1 micro m and specific activities between 3 and 8 nmol.min-1.mg-1 protein. D50580 and AY034877 also hydrolyzed retinyl palmitate. Gene-specific oligonucleotide probing of multiple-tissue Northern blot indicates differential expression in various tissues. Multiple genes are highly expressed in liver and small intestine, important tissues for retinoid metabolism. The level of expression of any one of the six different carboxylesterase isoenzymes will regulate the metabolism of retinyl palmitate in specific rat cells and tissues.

Retinyl palmitate hydrolase activity in human liver

Retinyl palmitate hydrolase (RPH) activity was studied in human liver after the optimal conditions of the assay in normal human liver homogenates were determined. The mean activity was 118 +/- 66 nmol.min-1.g-1 (mean +/- SD) protein in liver homogenates from six children and seven adults; no correlation was found between liver hydrolase activity and the enzyme endogenous substrate, ie, liver vitamin A concentrations. RPH activity was also studied in a human model of vitamin A deficiency represented by 22 children with biliary atresia: 11 patients with vitamin A deficiency (liver vitamin A concentration less than 70 nmol/g wet wt of liver) and 11 patients with normal vitamin A status after vitamin A treatment. The enzymatic assay had to be conducted after the removal of endogenous bile salts by cholestyramine because bile salts are accumulated in the liver of children with biliary atresia. No correlation was found between RPH activity and vitamin A status.

Distributions of retinoids, retinoid-binding proteins and related parameters in different types of liver cells isolated from young and old rats.

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.

Hydrolysis of retinyl esters by non-specific carboxylesterases from rat liver endoplasmic reticulum.

The four most important non-specific carboxylesterases from rat liver were assayed for their ability to hydrolyse retinyl esters. Only the esterases with pI 6.2 and 6.4 (= esterase ES-4) are able to hydrolyse retinyl palmitate. Their specific activities strongly depend on the emulsifier used (maximum rate: 440 nmol of retinol liberated/h per mg of esterase). Beside retinyl palmitate, these esterases cleave palmitoyl-CoA and monoacylglycerols with much higher rates, as well as certain drugs (e.g. aspirin and propanidid). However, no transacylation between palmitoyl-CoA and retinol occurs. Retinyl acetate also is a substrate for the above esterases and for another one with pI 5.6 (= esterase ES-3). Again the emulsifier influences the hydrolysis by these esterases (maximum rates: 475 nmol/h per mg for ES-4 and 200 nmol/h per mg for ES-3). Differential centrifugation of rat liver homogenate reveals that retinyl palmitate hydrolase activity is highly enriched in the plasma membranes, but only moderately so in the endoplasmic reticulum, where the investigated esterases are located. Since the latter activity can be largely inhibited with the selective esterase inhibitor bis-(4-nitrophenyl) phosphate, it is concluded that the esterases with pI 6.2 and 6.4 (ES-4) represent the main retinyl palmitate hydrolase of rat liver endoplasmic reticulum. In view of this cellular localization, the enzyme could possibly be involved in the mobilization of retinol from the vitamin A esters stored in the liver. However, preliminary experiments in vivo have failed to demonstrate such a biological function.

The effect of 3,4,3′,4′-tetrachlorobiphenyl on plasma retinol and hepatic retinyl palmitate hydrolase activity in female Sprague-Dawley rats

A single ip dose of 1, 5, or 15 mg/kg 3,4,3′,4′-tetrachlorobiphenyl (TCB) caused a dose-dependent depression of plasma retinol levels 24 hr after treatment of female Sprague-Dawley rats. The loss of plasma retinol appeared to be a function of depressed levels of the retinol-retinol-binding protein (RBP)-transthyretin ternary complex. No free retinol-RBP was observed in plasma from treated animals. Hepatic retinyl palmitate hydrolase (RPH) activity was also depressed and highly and positively correlated to the plasma retinol levels. TCB was determined to be a noncompetitive inhibitor of partially purified RPH with a K I of 91 μ m. Incubation of TCB with liver microsomes and NADPH decreased the inhibition of RPH. Doses of either 2,4,5,2′,4′,5′-hexachlorobiphenyl (HCB) or 3,4,5,3′,4′,5′-HCB equimolar to the 15 mg/kg TCB dose failed to cause a similar depression of plasma retinol in treated female rats. We conclude that, unlike other polychlorinated biphenyl congeners, TCB causes a depression of plasma retinol by inhibition of hepatic RPH.

Decreased hepatic retinyl palmitate hydrolase activity in protein-deficient rats

28-day-old weanling rats were fed a diet containing 3% casein as the only source of protein for eight weeks to induce protein deficiency. When compared to control animals (fed a diet containing 25% casein), these rats had significantly lowered body (5.2-fold reduction) and liver (2.5-fold reduction) weights. The circulatory level of retinol (nmol per ml plasma) as well as retinol (nmol per g tissue) in the liver of these protein-deficient animals were also reduced significantly, although their liver concentration of retinyl palmitate (nmol per g tissue) was comparable to that of the control group. Assay of liver tissue for retinyl palmitate hydrolase activity revealed a 4-fold reduction (compared to that of control animals) of specific enzyme activity (nmol retinol formed per g protein per h). These findings suggest that severe protein deficiency results in a decreased hydrolysis of retinyl esters in the liver, which may be in part responsible for the reduced level of metabolically ‘active’ retinoids available for normal physiological functions.

The effects of nonadecafluoro‐n‐decanoic acid on serum retinol and hepatic retinyl palmitate hydrolase activity in male sprague‐dawley rats

The effects of nonadecafluoro-n-decanoic acid (NDFDA) on serum retinol levels and hepatic retinyl palmitate hydrolase (RPH) activity were investigated in male Sprague-Dawley rats given a single intraperitoneal (IP) dose of 0, 50, or 100 mg/kg NDFDA and sacrificed at two, eight, or 11 days. Treated animals exhibited depressed serum retinol levels, lymphoid involution, and failure to gain weight in proportion to the dose. Hepatic RPH activities were depressed in both treatment groups at all time points and correlated with serum retinol levels. Hepatic retinol levels were also depressed by Day 11. Extraction of hepatic homogenates with acetone removed NDFDA and increased RPH activities twofold and threefold for the low- and high-dose groups, respectively. Analysis of partially purified RPH showed both NDFDA and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to be noncompetitive inhibitors: KI = 450 and 750 microM, respectively. We conclude that NDFDA causes a decrease in the mobilization of vitamin A from the liver by noncompetitive inhibition of RPH.

Retinyl palmitate hydrolase activity in the bovine retina

Retinyl palmitate hydrolase (RPH) activity of bovine tissues was estimated from retinol formation following incubation of tissue homogenates with all- trans retinyl palmitate. The quantity of retinol produced in the incubation mixture was analyzed by high-performance liquid chromatography. RPH activities of retinal pigment epithelium (RPE), liver, retina, muscle and brain were 194.2, 138.0, 72.5, 25.0 and 5.1 units/gm protein respectively. The RPH activity in the retina was far above that attributable solely to RPE contaminations. The presence of RPH in the retina suggests that retina can utilize retinyl esters for the formation of visual pigments and/or cellular metabolism.

Immunologic and enzymatic comparisons between human and bovine lipoprotein lipase

Studies were conducted to compare human and bovine lipoprotein lipase (LPL) preparations with regard to immunological cross-reactivity and substrate specificity. LPL was partially purified from human milk. An antiserum against the human LPL preparation was produced in a goat. This antiserum inhibited LPL enzymatic activity in human milk and in human post-heparin plasma. Neither bovine milk nor bovine post-heparin plasma LPL enzymatic activity was inhibited by this antiserum. These findings suggest that there are significant structural differences between the human and bovine enzymes in domains that are involved in enzymatic activity. Human and bovine post-heparin plasma and partially purified preparations of LPL from human and bovine milk were compared with regard to substrate specificity, by comparing their lipolytic activities against triglyceride, cholesteryl esters, and retinyl esters. Only the partially purified bovine milk LPL preparation possessed retinyl palmitate hydrolase activity. The results suggest that this latter activity may be the result of a previously unrecognized contaminant in the commonly used LPL preparations from bovine milk.

Retinoids, retinoid-binding proteins, and retinyl palmitate hydrolase distributions in different types of rat liver cells.

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.

Spatial Distribution of Retinol-Binding Protein and Retinyl Palmitate Hydrolase Activity in Normal and Vitamin A-Deficient Rat Liver

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 µg/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 µg/g wet weight. For RPH, the mean level was 136 pmol free fatty acids (FFA) formed/(min · 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.

Rat liver retinyl palmitate hydrolase activity. Relationship to cholesteryl oleate and triolein hydrolase activities

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.

Inhibition of rat liver retinyl palmitate hydrolase activity by ether analogs of cholesteryl esters and acylglycerides

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 > oleyl = palmitoyl > n-butyl = n-propyl > 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 μM. The acylglyceride ether analogs were much weaker inhibitors of the three hydrolase activites, 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.

Association of acylglyceride and retinyl palmitate hydrolase activities with zinc and copper metalloproteins in a high molecular weight lipid-protein aggregate fraction from chick liver cytosol

A lipid-protein aggregate fraction of molecular weight approx. 1.8·10 6 was isolated by gel filtration from chick liver cytosol. This aggregate fraction had a hydrated density range of 1.06–1.13, was 45% lipid, contained zinc and copper and was associated with triolein, phosphatidylcholine and retinyl palmitate hydrolase activity. Hydrolytic activities were stimulated by albumin and cholate, but not by dihydroxy bile acids, and inhibited by serine esterase and sulphydryl inhibitors. Incubation of the aggregate with fatty acid-labelled acylglycerides resulted in protein-binding fatty acid fractions with molecular weights of 150000, 60000, 13000 and approx. 2000. Incubation of the aggregate with [ 3H]retinyl palmitate yielded retinol-containing fractions with molecular weights of 150 000, 60 000 and 15 000. The latter peak appears, on the basis of amino acid composition, to be similar to the cellular retinol-binding protein. In addition, on incubation of the aggregate fraction, zinc and copper peaks are found with molecular weights of 150000, 60000 and 12000–8000. The latter were further purified to yield a copper-rich metalloprotein similar to ‘copper-chelatin’ and a zinc-rich metalloprotein, possibly zinc-metallothionein. Both these metalloprotein fractions had acyl hydrolase activity which was depressed in zinc-depleted animals. This may provide a possible explanation for the documented nutritional interactions between zinc and retinol.

Serum and intracellular retinol in the equine.

1. Serum and intracellular distribution of retinol was determined in equines maintained on four levels of vitamin A intake. 2. The form of retinol transported in serum was determined by gel filtration and chromatography to be a complex of retinol bound to a protein of molecular weight (MW) of approximately 20000, which was in turn complexed probably with prealbumin to yield a complex with a MW of 75000 to 80000. 3. Increasing dietary vitamin A levels enhanced the concentration of lipoprotein-bound retinyl esters in the plasma. 4. Vitamin A in the liver cytosol was found predominantly as retinyl esters in a lipid-protein aggregate of MW approximately 2 X 10(6) and hydrated density of 1.063-1.111. In the kidney and adrenal gland, two lipid-protein entities were found with MW of approximately 1.8 X 10(6) and 1.7 X 10(6) respectively. These fractions contained approximately 40 and 20% lipid respectively and had densities of 1.063-1.111 and approximately 1.21. 5. All lipid-protein aggregates were associated with retinyl palmitate hydrolase activity and guanidine treatment released a 15000 MW material, presumably intracellular retinol-binding protein. 6. Increasing dietary vitamin A enhanced the proportion of retinol in the 1.7 X 10(6) fraction. 7. Findings in equine plasma and liver resemble previous observations in other species. The characterization of two new lipid-protein aggregates in equine kidney and adrenal glands, which have hydrolase activity, may be important in intracellular retinal transport and metabolism, especially in animals subjected to high intakes of vitamin A.

Association of cellular retinol-binding protein and several lipid hydrolase activities with a vitamin A-containing high-molecular-weight lipid-protein aggregate from rat liver cytosol

A fluorescent, high-molecular-weight, lipid-protein aggregate was partially isolated from the cytosol fraction of rat liver by gel filtration on columns of Sepharose 4B or 6B. This aggregate was composed of approximately equal parts of protein and of lipid (mainly triglycerides), and was found to contain approximately 19% of the total liver vitamin A (predominantly as retinyl esters). Most of the liver cellular retinol-binding protein (CRBP) was found associated with the fluorescent, lipid-protein aggregate, along with much of the retinyl palmitate hydrolase activity present in the liver cytosol. The lipid-protein aggregate, and its several vitamin A-related components, all displayed an apparent hydrated density between 1.052 and 1.090 in the ultracentrifuge. CRBP in association with the lipid-protein aggregate was not immunoreactive in the CRBP radioimmunoassay. CRBP was, however, released from this aggregate and rendered immunoreactive by addition of detergents (e.g., Triton X-100). Three other lipid hydrolytic activities were also found in association with the lipid-protein aggregate, namely, triolein, cholesteryl oleate, and dipalmitoyl phosphatidylcholine hydrolase activities. These several hydrolytic activities were all found to be stimulated optimally by the addition of either sodium cholate or bovine serum albumin. With the information available, it is not clear whether this lipid-protein aggregate is formed in vitro, during liver homogenization, or whether it represents a specific lipoprotein with a significant functional role that exists in vivo in the liver cell.

Retinyl palmitate hydrolase activity in normal rat liver.

Retinyl palmitate hydrolase activity in normal rat liver.

Unusual properties of retinyl palmitate hydrolase activity in rat liver.

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.