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Biomedical subjects

K R Norum

Publications and source records attributed to K R Norum.

At least 109 records · Page 6Linked to original sources
PubMed1983

Retinol esterification by microsomes from the mucosa of human small intestine. Evidence for acyl-Coenzyme A retinol acyltransferase activity.

The mechanism of the intestinal esterification of retinol has been obscure. Recently, an acyl-Coenzyme A (CoA):retinol acyltransferase (ARAT) was found in rat intestinal microsomes, and experiments were therefore conducted to determine whether a corresponding enzyme exists in human small intestine. When microsomes were incubated with [3H]retinol and palmitoyl-CoA, or retinol and [1-14C]palmitoyl-CoA, radioactive retinyl palmitate was formed as identified by alumina column chromatography and reverse-phase high-pressure liquid chromatography. Heating the microsomes for 30 min at 60 degrees C resulted in loss of activity. The esterification was negligible without exogenous acyl-CoA and markedly stimulated by palmitoyl-, oleoyl-, and stearoyl-CoA in concentrations up to 20 microM. The acyl-CoA was successfully replaced by an acyl-CoA generating system, but not by unactivated palmitate (2.5-200 microM). The assay was dependent on the presence of albumin with optimum activity at 2-10 mg/ml. The optimal retinol concentration was 20-30 microM and pH approximately 7.4. The esterifying activity was completely inhibited by 8 mM of taurocholate and to 90% by 1 mM of 5,5'-dithiobis(2-nitrobenzoic acid). Activity was found throughout the small intestine. In jejunum the rate of retinol esterification was: 3.44 +/- 2.24 nmol [3H]retinyl ester formed . mg microsomal protein-1 . min-1 (mean +/- SD, n = 12). The corresponding activity in whole homogenates of biopsies were 1.17 +/- 0.28 (n = 8). It is concluded that human small intestine contains a microsomal acyl-CoA:retinol acyltransferase. Due to its high activity in vitro this enzyme is likely to be responsible for the intestinal esterification of retinol.

Acyltransferases↗
PubMed1982

Intracellular transport and degradation of 125I-Labelled denatured serum albumin in isolated nonparenchymal rat liver cells.

The intracellular movement, following uptake of 125I-labelled denatured serum albumin into nonparenchymal liver cells, was followed by means of subcellular fractionation. Isolated nonparenchymal rat liver cells were prepared by means of differential centrifugation. The cells were homogenized in a sonifier and the cytoplasmic extract subjected to isopycnic centrifugation in a sucrose gradient. The intracellular movement of the labelled albumin was followed by comparing the distribution profile of radioactivity in the sucrose gradient with those of marker enzymes for plasma membrane and lysosomes. The distribution profiles for radioactivity after the cells had been exposed to the labelled denatured albumin for different time periods indicated that the radioactivity was first associated with subcellular fractions of lower modal densities than the lysosomes. With time of incubation the radioactivity moved towards higher densities. After prolonged incubations in the absence of extracellular labelled denatured albumin the radioactivity peak coincided with that of the lysosomal marker beta-acetylglucosaminidase. When the cells were treated with the lysosomal inhibitor leupeptin, degradation of the labelled albumin was decreased, resulting in a massive intracellular accumulation of radioactivity. The radioactivity peak coincided with the peak of activity for the lysosomal marker beta-acetylglucosaminidase, suggesting lysosomal degradation.

Animals↗
PubMed1982

Characterization of the stimulatory effect of high-fat diets on peroxisomal beta-oxidation in rat liver.

1. The effect on rat liver peroxisomal beta-oxidation of feeding diets containing various amounts of dietary oils was investigated. With increasing amounts (5-25%, w/w) of soya-bean oil an apparent, but not statistically significant, increase of 1.5-fold was found both in specific activity, and in total liver activity. Increasing amounts of partially hydrogenated marine oil revealed a sigmoidal dose-response-curve, giving a 4-6-fold increase in the peroxisomal beta-oxidation activity at 20% or more of this oil in the diet. 2. Addition of small amounts of soya-bean oil to the marine-oil diet had no effect on the peroxisomal beta-oxidation activity, but decreased the C20:3(5,8,11) fatty acid/C20:4(5,8,11,14) fatty acid ratio in liver phospholipids from 0.74 to 0.01. 3. Starvation for 2 days led to a 1.5-1.8-fold increase in the peroxisomal beta-oxidation activity in rats previously fed on a standard pelleted diet, but had no effect in rats given high-fat diets. 4. Feeding partially hydrogenated marine oil or partially hydrogenated rape-seed oil resulted in higher activities than the corresponding unhydrogenated oils. 5. No significant differences in the effect on peroxisomal beta-oxidation could be detected between diets containing rape-seed oils with 15 or 45% erucic acid respectively. 6. These findings are discussed in relation to the possible effects of C22:1 and trans fatty acids in the process leading to increased peroxisomal beta-oxidation activity in the liver.

Animals↗
PubMed1982

Cholesterol esterification and lipids in blood plasma of the char (Salmo alpinus L.) during sexual maturation.

1. The initial rate of cholesterol esterification and the concentration of triacylglycerols in plasma were generally lower in prespawning than in immature chars. No typical pattern of variation of plasma cholesterol could be observed. 2. The observed variations in plasma cholesterol esterification and lipids may be caused by reduced feeding in the prespawning period. 3. The initial rate of cholesterol esterification in plasma was positively correlated with the concentrations of triacylglycerols and unesterified cholesterol in plasma, respectively.

Animals↗
PubMed1982

In vivo uptake of chylomicron [3H]retinyl ester by rat liver: evidence for retinol transfer from parenchymal to nonparenchymal cells.

We have studied hepatic uptake of chylomicron retinyl ester. Chylomicrons were obtained from intestinal lymph of rats that were given retinol in groundnut oil by intraduodenal injection. When lymph was injected intravenously into normal rats, the radioactivity was cleared from blood with t1/2 approximately equal to 10 min. Retinyl ester was taken up initially by the liver, which, after 30 min, contained 80-90% of the radioactivity injected. Initially, most of the radioactivity was in hepatocytes, but after 30 min it disappeared from these cells and reappeared in nonparenchymal liver cells. After 2 hr these cells contained more radioactivity than the hepatocytes. When lymph was injected into vitamin A-deficient rats or rats given vitamin A in the form of retinoic acid, the plasma clearance and initial hepatic uptake of radioactivity were similar to that found in control animals. However, the nonparenchymal cells in these animals did not accumulate radioactivity. The current data suggest that vitamin A (in chylomicron remnants) is taken up initially by hepatocytes and then is released from these cells and delivered mainly to nonparenchymal liver cells in normal animals. In vitamin A-deficient rats, the vitamin is transferred from the hepatocytes to extrahepatic tissues.

Animals↗
PubMed1982

The effect of feeding and fasting on the activity of acyl-CoA: cholesterol acyltransferase in rat small intestine.

The purpose of the present study was to test if the microsomal acyl-CoA:cholesterol acyltransferase (ACAT) in rat small intestine is regulated under physiological conditions. Two previously described in vitro assays were used, both based on the esterification of endogenous cholesterol with exogenous acyl-CoA, performed or generated during the incubation. The important and consistent finding with rats on normal diet was an increase in ACAT activity with fasting and a decrease with feeding. Independent of the assay used, the ratio between ACAT activity in night-fasted and night-fed animals was about 2 (P less than 0.005). When the fasting period was extended to 36 h a corresponding difference was found whether the ACAT assay was based on preformed [1-14C]oleoyl- or [1-14C]palmitoyl-CoA as acyl-donor (P less than 0.05). The microsomal content of unesterified cholesterol was higher in fasted than fed animals, suggesting that availability of this substrate might be a factor in the regulation of rat intestinal ACAT activity.

Acyl Coenzyme A↗
PubMed1982

Metabolism of very long-chain monounsaturated fatty acids (22:1) and the adaptation to their presence in the diet.

Unadapted rats and other animal species have a limited capacity to metabolize monounsaturated fatty acids with 22 carbons (22:1). Excess amounts in the diet of fats containing these fatty acids cause a transient accumulation (lipidosis) of triacylglycerol in the heart and other tissues but not in the liver, which seems to export the 22:1 fatty acids as very low density lipoproteins to the blood plasma. The acute lipidosis most probably is explained by a slow oxidation of 22:1 acyl-CoA by the mitochondrial acyl-CoA dehydrogenase combined with an inhibitory effect of this CoA ester on the oxidation of acyl-CoA esters of a more "normal" chain length. Other fatty acid metabolizing enzymes also show slow reaction rates with the 22:1 fatty acids. Upon continued feeding of diets with 22:1 fatty acids, an adaptation takes place and the lipidosis disappears. This adaptation coincides with the development of an increased capacity to chain-shorten the 22:1 fatty acids, especially in the liver, but also in the heart. The chain-shortening seems to be due to a partial beta-oxidation of the 22:1 fatty acids by the peroxisomal beta-oxidation enzyme system which shows an increased activity in adapted rats. In such rats, less 22:1 fatty acids circulate in the plasma very low density lipoproteins than in unadapted rats. The drug clofibrate (ethyl-p-chlorophenoxyisobutyrate) which induces increased activity of the peroxisomal beta-oxidation enzymes, provides partial protection against the lipidosis in unadapted animals. Hydrogenated fish oil (containing different 22:1 isomers and many fatty acids with trans double bonds) is more efficient as an inducer of the chain-shortening of erucic acid in the liver than is rapeseed oil, which contains only one 22:1 fatty acid isomer and no fatty acids with trans double bonds. The hydrogenated fish oil causes less lipidosis than does rapeseed oil when diets containing the same amount of 22:1 fatty acids are fed. It is suggested that CoA esters that are poorly oxidized by the mitochondria (e.g., esters of erucic acid, of some fatty acids with trans double bonds, and of clofibric acid) may trigger the adaptation process.-Bremer, J., and K. R. Norum. Metabolism of very long-chain monounsaturated fatty acids (22:1) and the adaptation to their presence in the diet.

Acyl Coenzyme A↗
PubMed1982

Acyl CoA:retinol acyltransferase in rat small intestine: its activity and some properties of the enzymic reaction.

The present study was conducted to examine whether the intestinal esterification of retinol could be due to a microsomal acyl-CoA transferase. When the 'microsomal fraction' of rat mucosa was incubated with [3H]retinol and palmitoyl-CoA or oleoyl-CoA, [3H]retinyl esters were formed as identified by alumina column chromatography and reverse phase high-pressure liquid chromatography (HPLC). Unlabeled retinol and [1-14C]palmitoyl-CoA yielded retinyl[1-14C]palmitate. The esterifying activity was lost when microsomes were heated at 60 degrees C for 30 min. Only negligible activity was observed without exogenous acyl-CoA while 10-20 muM gave optimum activity provided that 2-5 mg/ml of albumin was present. Replacement of acyl-CoA by palmitate gave no esterification, indicating that the activity was not a reversed hydrolase reaction. Optimum pH was 7.1-7.6 and optimal concentration of retinol was 15 muM. With palmitoyl-CoA, the formation of retinyl ester was 1.00 +/- 0.26 nmol . mg protein-1 . min-1 (x +/- SD, n = 4) in rats killed postprandially versus 2.06 +/- 0.66 (n = 5) after 36 hr of fasting. Oleoyl-CoA gave lower activity: 0.52 +/- 0.14 and 1.41 +/- 0.36, respectively. The variation with feeding and fasting was significant (P less than 0.05) and corresponded to that of the intestinal acyl-CoA:cholesterol acryltransferase (ACAT). Inhibition of retinol esterification was observed with taurocholate and the thiol-blocking agent 5,5'-dithiobis (2-nitrobenzoic acid). The data show that rat intestinal microsomes catalyze the formation of retinyl esters by an acyl-CoA:retinol acyltransferase with several properties in common with ACAT located in the same subcellular fraction.

Acyltransferases↗
PubMed1981

The binding of high density lipoproteins to isolated rat hepatocytes.

The association of 125I-labelled rat high density lipoproteins (125-I-HDL) to suspended rat hepatocytes was studied at 4 degrees C. 125 I-HDL associated to isolated hepatocytes by two processes-one of high and one of low affinity. The cell-association of 125I-HDL exhibited saturation kinetics and was inhibited to varying degrees with both rat and human lipoproteins such as VLDL, LDL and HDL but not by lipoprotein deficient serum or asialo-fetuin. The cell-association of 125I-HDL did not require divalent cations and could be reduced by pronase treatment of the cells. The binding site was clearly different from the receptor for LDL in extrahepatic cells since heparin and apolipoprotein E did not compete and cholesterol ester labelled HDL. The number of binding sites for HDL at 4 degrees C was 2.2 X 10(6) per cell and the association constant (Ka) 8.2 X 10(6) (mol/l)-1. Experiments with HDL labelled with [3H] cholesterol by means of lecithin: cholesterol acyltransferase (LCAT, EC 2.3.1.43) in the cholesterol ester moiety suggested that the same mechanism was responsible for the cell-association of HDL prepared this way.

Animals↗
PubMed1981

Enzymic esterification of cholesterol in rat intestinal mucosa catalyzed by acyl-CoA: cholesterol acyltransferase.

Previous work has shown CoA-dependent esterification of cholesterol in rat intestinal mucosa. Using (1-(14)C)oleoyl-CoA as the labeled substrate, we have proved that the esterification is catalyzed by acyl-CoA: cholesterol acyltransferase (ACAT) existing in the 'microsomal fraction' of the mucosal cell. The apparent K(m) for oleoyl-CoA is 25 microM, the optimal pH 7.4-7.9, and the optimal concentration of albumin 10-20 mg/ml. The reaction is rectilinear for only 2 min. Increasing the microsomal cholesterol concentration by incubation with plasma from patients with familial lecithin: cholesterol acyltransferase deficiency leads to increasing ACAT activity. The ACAT was inhibited by taurocholate and by the thiol-blocking agent 5,5'-dithiobis(2-nitrobenzoic acid). The specific activity of the enzyme is high-that is, approximately 1 nmol cholesteryl oleate formed x mg microsomal protein(-1) x min(-1).

Animals↗
PubMed1981

Acyl-CoA:cholesterol acyltransferase in human small intestine: its activity and some properties of the enzymic reaction.

Esterification of endogenous cholesterol in human small intestinal mucosa by acyl-CoA:cholesterol acyltransferase (ACAT, EC 2.3.1.26) was studied using [1-14C]oleoyl-CoA as substrate. The reaction was linear for 2 min only. The esterification of cholesterol was stimulated by albumin, but this effect was dependent on the oleoyl-CoA concentration. When the albumin concentration was 5 g/liter, maximal esterification was obtained with 35 microM oleoyl-CoA. The pH optimum was 7.2-7.8. The ACAT specific activity was highest in microsomal preparations from jejunum (0.21 +/- 0.19 (n = 18) nmol cholesteryl oleate . mg microsomal protein-1 . min-1), and lower in proximal duodenum and distal ileum. Whole homogenates of biopsies had about 1/4 of the activity of the corresponding microsomal preparation. Microsomal preparations from jejunum contained acyl-CoA hydrolase (EC 3.1.2.2) which under the prevailing conditions had a maximal activity of 4.4 nmol oleate formed . microsomal protein-1 . min-1. The high activity of intestinal ACAT in man renders it possible that this enzyme plays a role in cholesterol absorption.

Acyl Coenzyme A↗
PubMed1981

Binding, uptake, and degradation of 125I-labelled high-density lipoproteins in isolated non-parenchymal rat liver cells.

Binding, uptake, and degradation of 125I-labelled HDL were measured in isolated rat non-parenchymal cells in vitro. The binding exhibited saturation kinetics and was inhibited to various degrees with other unlabelled lipoproteins such as VLDL, LDL, and HDL, but the uptake was not reduced by heat and formaldehyde-denatured albumin. The binding could be reduced by pronase treatment of the cells. Acetylated 125I-labelled HDL were more effectively taken up and degraded by non-parenchymal cells than unmodified 125I-labelled HDL. The Kupffer cells were able to take up two to three times as much HDL per cell as the endothelial cells. 125I-labelled HDL bind to the plasma cell membrane and can be dissociated by addition of unlabelled HDL. Relatively more labelled HDL dissociate at 4 degrees C than at 37 degrees C. The non-parenchymal cells also internalize 125I-labelled HDL at 37 degrees C, and the lysosomotropic drug chloroquine inhibits partly the degradation of accumulated 125I-labelled HDL.

Animals↗
PubMed1980

The intracellular distribution of high density lipoproteins taken up by isolated rat hepatocytes.

The subcellular distribution of 125I-labelled HDL taken up by rat hepatocytes in vivo and in vitro has been studied with subcellular fractionation techniques: differential centrifugation and isopycnic centrifugation in sucrose gradients. 125I-labelled HDL bind to plasma membranes both in vivo and in vitro and part of the membrane-bound 125I-labelled HDL can be dissociated by the addition of unlabelled HDL. The hepatocytes also internalize 125I-labelled HDL. The 125I-labelled HDL accumulate, however, at different intracellular sites in the in vivo and in vitro situation. The subcellular distribution pattern of 125I-labelled HDL taken up by the cells in vivo is similar to that of the lysosomal marker enzyme acid phosphatase. Peak activity was found at a density of 1.20 g/ml. In vitro 125I-labelled HDL accumulate in an organelle with a medium density of about 1.13 g/ml. This distribution was similar to that of the plasma membrane marker 5'-nucleotidase. The subcellular distribution of radioactivity taken up in vivo was changed to lower density by incubating the cells with chloroquine, a drug known to render the lysosomes more boyant. Chloroquine had no effect on the distribution of 125I-labelled HDL taken up by hepatocytes in vitro.

5'-Nucleotidase↗
PubMed1980

In vitro effects of lecithin:cholesterol acyltransferase on apolipoprotein distribution in familial lecithin:cholesterol acyltransferase deficiency.

Action of LCAT on the plasma of patients afflicted with familial LCAT deficiency shifts the distribution of C apolipoproteins from lipoproteins of d less than 1.019 g/ml to lipoproteins of d greater than 1.109 g/ml, and causes an opposite shift in the distribution of apolipoprotein E. The altered distribution of apolipoprotein E appears to depend primarily on enzyme-related effects on HDL. Loss of apolipoprotein E from HDL occurs as cholesteryl esters are formed and transfer to other lipoproteins; disc-shaped HDL, rich in apolipoprotein E, are converted into spherical particles; and the population of HDL as a whole is converted first into particles the size of HDL2 and HDL3 and then into intermediate-sized particles. Transfer of apolipoprotein E to artificially prepared triglyceride-rich particles occurs at a nearly linear rate that is slow than the rates of formation and transfer of cholesteryl esters or the rate of formation of "HDL2" and "HDL3." Transfer of apolipoprotein E is faster, however, when the patients' disc-shaped HDL are incubated with triglyceride-rich particles in the presence of normal plasma lipoproteins of d greater than 1.063 g/ml. Since the disc-shaped HDL, rich in apolipoprotein E, resemble particles reported to be released from perfused rat livers, they may be nascent lipoproteins of hepatic origin. If so, it appears that action of LCAT on these lipoproteins may be one of the factors that regulates the content of apolipoprotein E in VLDL.

Apolipoproteins↗
PubMed1980

Characterization of apolipoprotein E-rich high density lipoproteins in familial lecithin:cholesterol acyltransferase deficiency.

We have isolated and charachterized a subfraction of high density lipoproteins, rich in apolipoprotein E, from the plasma of patients afflicted with familial lecithin:cholesterol acyltransferase deficiency. Prepared by successive ultracentrifugal flotation, affinity chromatography on heparin-agarose, and affinity chromatography on conconavalin A-agarose, the subfraction contained disc-shaped lipoproteins that measured 14--40 nm in diameter and 4.4--4.5 nm in thickness. The major components were apolipoprotein E, phosphatidylcholine, and unesterified cholesterol, though other apolipoproteins and lipids were present in small amounts. A second subfraction of high density lipoproteins, isolated during the chromatography, contained apolipoproteins A-I and A-II, but no apolipoprotein E. This subfraction included disc-shaped lipoproteins, 13--24 nm in diameter, as well as small round particles, 5.7 nm in diameter. Both subfractions contained similar proportions of total protein relative to lipid, similar amounts of unesterified cholesterol relative to phosphatidylcholine, and a similar distribution of phosphatidylcholine fatty acid.

Adult↗
PubMed1980

Abnormalities in lipoproteins of d < 1.006 g/ml in familial lecithin:cholesterol acyltransferase deficiency.

Studies of different sized lipoproteins of d < 1.006 g/ml from patients with familial lecithin:cholesterol acyltransferase deficiency have yielded new evidence of abnormalities in this lipoprotein class. Lipoproteins of all sizes contain high amounts of unesterified cholesterol, low amounts of total protein, and particularly low amounts of apolipoproteins C-II and C-III. Lipoproteins 60 nm in diameter or larger include particles that show a notched appearance upon electron microscopy, and contain a) a high combined volume of phospholipid, unesterified cholesterol, and protein; b) high amounts of cholesteryl ester and apolipoproteins C-I and E, and c) two major tetramethylurea-insoluble proteins that can be separated by electrophoresis in the presence of sodium dodecylsulfate. In contrast, lipoproteins that are 40 nm in diameter or less appear to contain low amounts of cholesteryl ester, normal amounts of apolipoproteins C-I and E, and a single tetramethylurea-insoluble protein the size of that in control lipoproteins. Since these abnormalities occur in the lipoproteins of four different patients from four different families, they are probably effects of the enzyme deficiency. Most, however, appear to arise indirectly because in vitro experiments published earlier indicate that few are reversed by incubation in the presence of the enzyme and patient high density lipoproteins.

Adult↗