Purification and characterization of human lipoprotein lipase and hepatic triglyceride lipase. Reactivity with monoclonal antibodies to hepatic triglyceride lipase

Human lipoprotein lipase and hepatic triglyceride lipase were purified to homogeneity from post-heparin plasma. These enzymes were purified 250,000- and 100,000-fold with yields of 27 +/- 15 and 19 +/- 6%, respectively. Molecular weight determination by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and reducing agents yielded Mr of 60,500 +/- 1,800 and 65,200 +/- 400, respectively, for lipoprotein lipase and hepatic triglyceride lipase. These lipase preparations were shown to be free of detectable antithrombin by measuring its activity and by probing of Western blots of lipases with a monospecific antibody against antithrombin. In additions, probing of Western blots with concanavalin A revealed no glycoproteins corresponding to the molecular weight of antithrombin. Four stable hybridoma-producing distinct monoclonal antibodies (mAb) to hepatic triglyceride lipase were isolated. The specificity of one mAb, HL3-5, was established by its ability to immunoprecipitate hepatic triglyceride lipase catalytic activity. Interaction of HL3-5 with this lipase did not inhibit catalytic activity. The three other mAb interacted with hepatic triglyceride lipase only after denaturation of the enzyme with detergents. The relatedness of these two enzymes was examined by comparing under the same conditions the thermal inactivation, the sensitivity to sulfhydryl and reducing agents, amino acid composition, and the mobility of peptide fragments generated by cyanogen bromide cleavage. The results of these studies strongly support the view that the two enzymes are different proteins. Immunological studies confirm this conclusion. Four mAb to hepatic triglyceride lipase did not interact with lipoprotein lipase in Western blots, enzyme-linked immunosorbent assay, and immunoprecipitation experiments. These immunological studies demonstrate that several epitopes of the hepatic triglyceride lipase protein moiety are not present in the lipoprotein lipase molecule.     

Cardiac-specific knock-out of lipoprotein lipase alters plasma lipoprotein triglyceride metabolism and cardiac gene expression

Fatty acids are the primary energy source for the heart. The heart acquires fatty acids associated with albumin or derived from lipoprotein lipase (LpL)-mediated hydrolysis of lipoprotein triglyceride (TG). We generated heart-specific LpL knock-out mice (hLpL0) to determine whether cardiac LpL modulates the actions of peroxisome proliferator-activated receptors and affects whole body lipid metabolism. Male hLpL0 mice had significantly elevated plasma TG levels and decreased clearance of postprandial lipids despite normal postheparin plasma LpL activity. Very large density lipoprotein-TG uptake was decreased by 72% in hLpL0 hearts. However, heart uptake of albumin-bound free fatty acids was not altered. Northern blot analysis revealed a decrease in the expression of peroxisome proliferator-activated receptor alpha-response genes involved in fatty acid beta-oxidation. Surprisingly, the expression of glucose transporters 1 and 4 and insulin receptor substrate 2 was increased and that of pyruvate dehydrogenase kinase 4 and insulin receptor substrate 1 was reduced. Basal glucose uptake was increased markedly in hLpL0 hearts. Thus, the loss of LpL in the heart leads to defective plasma metabolism of TG. Moreover, fatty acids derived from lipoprotein TG and not just albumin-associated fatty acids are important for cardiac lipid metabolism and gene regulation.     

Effects of diet and age on lipoprotein lipase and hepatic triglyceride lipase activities in the rat

Plasma clearance of triglyceride-rich lipoproteins appears decreased in aged humans and rats and may be due to lowered activities of the lipases responsible for lipid degradation. This study was designed to examine differential effects of age and diet on lipoprotein lipase (LPL) activity of adipose and heart tissue and hepatic triglyceride lipase (HTGL) activity. LPL and HTGL activities were examined in 3- and 13-month-old Sprague-Dawley rats after they had consumed either a high-carbohydrate or a high-fat diet for 14 days. The data were analyzed for age and diet differences by two-way analysis of variance. Although animals in the two age groups consumed diets of equal caloric content, the older rats gained less weight. Rats on the high-carbohydrate diet consumed less calories and gained less weight than the fat fed rats in both age groups. Neither heart nor adipose tissue LPL activity differed when examined for age or diet. HTGL activity levels, while not affected by age, were higher in the carbohydrate fed rats (P = 0.014). Regardless of age group, fasting plasma cholesterol levels were significantly higher in the carbohydrate-fed rats than fat-fed rats (P = 0.002). Thus, the diet effect was much stronger than the age effect for HTGL and plasma cholesterol levels.     

Differential characteristics of purified hepatic triglyceride lipase and lipoprotein lipase from human postheparin plasma.

Evidence is presented that hepatic triglyceride lipase (H-TGL) and lipoprotein lipase (LPL), purified from human postheparin plasma, can each hydrolyze both glyceryl trioleate and palmitoyl-CoA. The average ratio of glyceryl trioleate/palmitoyl-CoA hydrolase activities, obtained with enzyme preparations from 15 human postheparin plasma samples was 1.30 (1.18-1.52) for H-TGL and 8.75 (7.45-10.25) for LPL. Albumin was identified as the serum cofactor required for the hydrolysis of palmitoyl-CoA by H-TGL. It protected this enzyme from inactivation by this substrate. In contrast, palmitoyl-CoA activated and protected LPL from denaturation by dilution and incubation at 25 degrees C. The effects of other detergents were investigated on glyceryl trioleate hydrolase activities of both enzymes. Sodium dodecyl sulfate (0.4 mM) and Trisoleate (0.4 mM), which also effectively activated and protected LPL against inactivation, had only moderate protective effect on H-TGL. Sodium dodecyl sulfate at a higher concentration (1 mM) produced little or no inhibition of LPL, while completely inactivating H-TGL. Conversely, sodium taurodeoxycholate (0.4 mM) protected and activated H-TGL, but had only moderate protective effect on LPL. Triton X-100 (0.1-0.8 mM) and egg lysolecithin (0.05-2 mM) also protected H-TGL, but not LPL. The very dissimilar effects of detergents on preparations on H-TGL and LPL may form the basis for the direct assay of each enzyme in the presence of the other.     

Effects of dietary saturated and polyunsaturated fat on lipoprotein lipase and hepatic triglyceride lipase activity

The effects of saturated and polyunsaturated dietary fat on the lipolytic activity of post-heparin plasma, lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) were studied in the rat. The lipolytic activity was studied from 0 to 60 min using labelled chylomicrons as the substrate. Triacylglycerol hydrolysis rate was higher for the plasma of rats fed high fat diets (14% fat by weight). Chylomicrons of rats fed saturated or unsaturated fats were hydrolyzed at the same rate within the first 15 min but afterwards hydrolysis of chylomicrons of rats fed saturated fat was slower. The activities of LPL and HTGL were increased by high fat diets. Unsaturated fat increased more LPL activity than saturated fat conversely, HTGL activity was enhanced more by saturated fat than by unsaturated fat.     

A comparison of molecular properties of hepatic triglyceride lipase and lipoprotein lipase from human post-heparin plasma

Hepatic triglyceride lipase was isolated from human post-heparin plasma by the method of Ehnholm et al. using modifications which increased the specific activity 12-fold to approximately 3,000 mumol of free fatty acid/h/mg of protein. Lipoprotein lipase with similar specific activity was prepared from the same plasma samples using heparin and concanavalin A affinity chromatography. The molecular weight of hepatic triglyceride lipase (69,000) was slightly greater than that of lipoprotein lipase (67,000) as determined by polyacrylamide electrophoresis in sodium dodecyl sulfate-containing buffers. These proteins had identical amino acid compositions, terminal amino acid residues, and tryptic peptide maps. However, the differences previously described regarding optima of pH and ionic strength and the requirement for apolipoprotein CII (only for lipoprotein lipase) were maintained in the highly purified state. It was found that both proteins contain approximately 8% carbohydrate. Antisera prepared in goats selectively precipitated each activity. Other antisera prepared in chickens reacted with both enzymes, suggesting a common antigenic determinant.     

Lipid metabolism in endotoxic rats: decrease in hepatic triglyceride lipase activity

Studies were conducted to investigate the effect of E. coli endotoxin administration on hepatic triglyceride lipase (H-TGL) activity in rats, since H-TGL activity is known to behave differently from lipoprotein lipase (LPL) activity in various situations. Plasma triglyceride and free fatty acid concentrations were markedly elevated in animals after injection of endotoxin. Cholesterol and phospholipids were also increased significantly. Lipoprotein analysis by ultracentrifugation showed that the most pronounced increase of lipoproteins was in the VLDL and IDL fractions. Triglyceride lipase activities in post-heparin plasma were markedly decreased. A selective assay for H-TGL activity using a specific antibody revealed that this enzyme as well as LPL is significantly decreased (26% of control) in endotoxic animals. Thus, the increase of VLDL and IDL appears to result from the decrease of both of LPL and H-TGL.     

Role of lipoprotein lipase in plasma triglyceride removal.

Intravenous injections of anti-lipoprotein lipase serunis quantitatively block the catabolism of very low density lipoprotein (VLDL) and portomicron triglyceride and specifically inhibit triglyceride transport into ovarian follicles. The immunological studies presented provide information on the site of action of lipoprotein lipase (LPL). In the anti-LPL serum-treated animals initial plasma triglyceride accumulation occurs at the time of antiserum injection. This instantaneous inhibition of triglyceride removal provides direct evidence that the functional LPL responsible for VLDL and portomicron triglyceride hydrolysis is located in sites within the plasma compartment readily accessible to immunoglobulins. In vitro immunological studies show that the adipose, heart, ovarian, and liver LPL share common immunological determinants. Biochemical studies on highly purified heart and adipose LPL suggest that these enzymes have identical protein moieties.     

Appraisal of hepatic lipase and lipoprotein lipase activities in mice

A variety of methods are currently used to analyze HL and LPL activities in mice. In search of a simple methodology, we analyzed mouse preheparin and postheparin plasma LPL and HL activities using specific polyclonal antibodies raised in rabbit against rat HL (anti-HL) and in goat against rat LPL (anti-LPL). As an alternative, we analyzed HL activity in the presence of 1 M NaCl, a condition known to inhibit LPL activity in humans. The assays were validated using plasma samples from wild-type and HL-deficient C57BL/6 mice. We now show that the use of 1 M NaCl for the inhibition of plasma LPL activity in mice may generate incorrect measurements of both LPL and HL activities. Our data indicate that HL can be measured directly, without heparin injection, in preheparin plasma, because virtually all HL is present in an unbound form circulating in plasma. In contrast, measurable LPL activity is present only in postheparin plasma. Both HL and LPL can be measured using the same assay conditions (low salt and the presence of apolipoprotein C-II as an LPL activator). Total lipase activity in postheparin plasma minus preheparin HL activity reflects LPL activity. Specific antibodies are not required.     

Purification and characterization of lipoprotein lipase and hepatic triglyceride lipase from human postheparin plasma: production of monospecific antibody to the individual lipase

Lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) were purified to homogeneity from human postheparin plasma. Molecular, catalytic and immunological properties of the purified enzymes were investigated. The native molecular weights of LPL and HTGL were 67,200 and 65,500, respectively, by gel chromatography. The subunit molecular weights of LPL and HTGL were 60,600 and 64,600, respectively, suggesting that these enzymes are catalytically active in a monomeric form. In addition, the purified LPL and HTGL each gave a single protein band when they were detected as glycoproteins with a probe of concanavalin A. The purified enzyme preparations were free of detectable antithrombin III by Western blot analysis. Catalytic properties of the purified enzymes were examined using triolein-gum arabic emulsion and triolein particles stabilized with phospholipid monolayer as substrates. LPL catalyzed the complete hydrolysis of triolein to free oleate and monooleate in the presence of apolipoprotein C-II. Apparent Km values for triolein and apolipoprotein C-II were 1.0 mM and 0.6 microM, and Vmax was 40.7 mmol/h per mg. HTGL hydrolyzed triolein substrate at a rate much slower than LPL, and produced mainly free oleate with little monooleate. Apparent Km and Vmax values were 2.5 mM and 16.1 mmol/h per mg, respectively. Polyclonal antibodies were developed against the purified LPL and HTGL. The purity and specificity of these antisera were ascertained by immunotitration, Ouchterlony double diffusion and Western blot analyses. The anti-human LPL and anti-human HTGL antiserum specifically reacted with the corresponding either native or denaturated enzyme, indicating that two enzymes were immunologically distinct. We developed an assay system for LPL and HTGL in human PHP by selective immunoprecipitation of each enzyme with the corresponding antiserum.     

Structure of the human hepatic triglyceride lipase gene

The structure of the human hepatic triglyceride lipase gene was determined from multiple cosmid clones. All the exons, exon-intron junctions, and 845 bp of the 5' and 254 bp of the 3' flanking DNA were sequenced. Comparison of the exon sequences to three previously published cDNA sequences revealed differences in the sequence of the codons for residues 133, 193, 202, and 234 that may represent sequence polymorphisms. By primer extension, hepatic lipase mRNA initiates at an adenine 77 bases upstream of the translation initiation site. The hepatic lipase gene spans over 60 kb containing 9 exons and 8 introns, the latter being all located within the region encoding the mature protein. The exons are all of average size (118-234 bp). Exon 1 encodes the signal peptide, exon 4, a region that binds to the lipoprotein substrate, and exon 5, an evolutionarily highly conserved region of potential catalytic function, and exons 6 and 9 encode sequences rich in basic amino acids thought to be important in anchoring the enzyme to the endothelial surface by interacting with acidic domains of the surface glycosaminoglycans. The human lipoprotein lipase gene has been recently reported to have an identical exon-intron organization containing the analogous structural domains [Deeb & Peng (1989) Biochemistry 28, 4131-4135]. Our observations strongly support the common evolutionary origin of these two lipolytic enzymes.     

Enzyme-linked immunosorbent assay for rat hepatic triglyceride lipase

A noncompetitive enzyme-linked immunosorbent assay to measure rat hepatic triglyceride lipase (H-TGL) was developed. Antibodies to rat H-TGL were purified from goat antisera by immunoadsorption on an H-TGL-Sepharose 4B column. Routinely, Immulon 2 Removawell strips were coated with the purified antibody overnight at 4 degrees C. After blocking the wells with bovine serum albumin (BSA) for 2 hr at room temperature, standards (0.85 ng/ml-13.1 ng/ml) or samples were added to the wells and were incubated with the bound anti-rat H-TGL overnight at 4 degrees C. The standards and samples had been pretreated with 5-20 mM SDS for 30 min at room temperature and were then diluted so that the final SDS concentration in the assay was 1 mM or less. The pretreatment with SDS was necessary to achieve maximal immunoreactivity. The sample incubation was followed by an overnight incubation at 4 degrees C with an anti-rat H-TGL-horseradish peroxidase conjugate. Rat H-TGL was detected by the color development after the addition of 0.4 mg/ml of o-phenylenediamine in 0.01% H2O2, 0.1 M citrate phosphate, pH 5.0. A linear relationship was obtained between absorbance at 490 nm and the amount of highly purified rat H-TGL used as a standard. Inclusion of 1 M NaCl in the assay buffer (1% BSA, 0.05% Tween 20, 10 mM phosphate, pH 7.4) during the sample and conjugate incubations minimized non-specific interactions. Recoveries of purified rat H-TGL added to a rat liver perfusate sample ranged from 98.6% to 103%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Insulin-mediated modifications of myocardial lipoprotein lipase and lipoprotein metabolism

Recirculating organ perfusion in vitro was conducted with hearts from control rats, animals given a single dose of streptozotocin (65 mg/kg) 48 h earlier, and streptozotocin-treated rats administered insulin (5 units), 2 h prior to organ perfusion. During 45-min perfusions, the lipolysis of very low density lipoprotein (VLDL) triglyceride was significantly less in hearts from diabetics than in controls (41.9 +/- 7.3% of control). This was associated with significant reductions in heparin-releasable (functional) lipoprotein lipase and tissue lipoprotein lipase of perfused hearts. The decreases in VLDL triglyceride metabolism and the levels of myocardial lipoprotein lipase were completely reversed by treatment of diabetic rats with insulin 2 h prior to study. Similar improvement of VLDL triglyceride metabolism and increases in myocardial lipoprotein lipase activity were observed in hearts from diabetic rats by direct addition of 100 milliunits/ml of insulin to the recirculating perfusion media. Under these conditions, the increase in both fractions of lipoprotein lipase in response to insulin was completely inhibited, and utilization of VLDL triglyceride was partially inhibited by pre-perfusion with cycloheximide for 10 min. The data derived from either VLDL triglyceride lipolysis in organ perfusion or direct measurement of myocardial lipoprotein lipase demonstrate a direct effect of insulin on myocardial lipoprotein lipase activity, and suggest that the response to insulin may be due in part to effects on protein synthesis.     

Relationship of lipoprotein lipase activity to triglyceride uptake in adipose tissue

Fasted rats injected with actinomycin or fed glucose show increased lipoprotein lipase activity of epididymal adipose tissue. Data from the actinomycin-treated animals showed a direct correlation between the lipoprotein lipase activity and the uptake of lipoprotein triglyceride by the epididymal fat pad in vitro and in vivo. Data from the animals fed glucose confirmed these findings in vitro. These data strongly suggest that lipoprotein lipase plays a major role in triglyceride deposition in adipose tissue.     

ANGPTL3 decreases very low density lipoprotein triglyceride clearance by inhibition of lipoprotein lipase

KK/San is a mutant mouse strain established in our laboratory from KK obese mice. KK/San mice show low plasma lipid levels compared with wild-type KK mice despite showing signs of hyperglycemia and hyperinsulinemia. Recently, we identified a mutation in the gene encoding angiopoietin-like protein 3 (Angptl3) in KK/San mice, and injection of adenoviruses encoding Angptl3 or recombinant ANGPTL3 protein to mutant KK/San mice raised plasma lipid levels. To elucidate the regulatory mechanism of ANGPTL3 on lipid metabolism, we focused on the metabolic pathways of triglyceride in the present study. Overexpression of Angptl3 in KK/San mice resulted in a marked increase of triglyceride-enriched very low density lipoprotein (VLDL). In vivo studies using Triton WR1339 revealed that there is no significant difference between mutant and wild-type KK mice in the hepatic VLDL triglyceride secretion rate. However, turnover studies using radiolabeled VLDL revealed that the clearance of (3)H-triglyceride-labeled VLDL was significantly enhanced in KK/San mice, whereas the clearance of (125)I-labeled VLDL was only slightly enhanced. In vitro analysis of recombinant protein revealed that ANGPTL3 directly inhibits LPL activity. These data strongly support the hypothesis that ANGPTL3 is a new class of lipid metabolism modulator, which regulates VLDL triglyceride levels through the inhibition of LPL activity.     

A sandwich-enzyme immunoassay for the quantification of lipoprotein lipase and hepatic triglyceride lipase in human postheparin plasma using monoclonal antibodies to the corresponding enzymes

We have developed a sandwich-enzyme immunoassay (EIA) for the quantification of lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) in human postheparin plasma (PHP) using monoclonal antibodies (MAbs) directed against the corresponding enzymes purified from human PHP. The sandwich-EIA for LPL was performed by using the combination of two distinct types of anti-LPL MAbs that recognize different epitopes on the LPL molecule. The immunoreactive mass of LPL was specifically measured using a beta-galactosidase-labeled anti-LPL MAb as an enzyme-linked MAb, an anti-LPL MAb linked with the bacterial cell wall as an insolubilized MAb, and purified human PHP-LPL as a standard. The sandwich-EIA for HTGL was carried out by using two distinct anti-HTGL MAbs that recognize different epitopes on HTGL. The limit of detection was 20 ng/ml for LPL and 60 ng/ml for HTGL. Each method yielded a coefficient of variation of less than 6% in intra- and inter-assays, and a high concentration of triglyceride did not interfere with the assays. The average recovery of purified human PHP-LPL and -HTGL added to human PHP samples was 98.8% and 97.5%, respectively. The immunoreactive masses of LPL and HTGL in PHP samples, obtained at a heparin dose of 30 IU/kg, from 34 normolipidemic and 20 hypertriglyceridemic subjects were quantified by the sandwich-EIA. To assess the reliability of the measured mass values, they were compared with the corresponding enzyme activities measured by selective immunoinactivation assay using rabbit anti-human PHP-LPL and -HTGL polyclonal antisera. Both assay methods yielded a highly significant correlation in either normolipidemic (r = 0.945 for LPL; r = 0.932 for HTGL) or hypertriglyceridemic subjects (r = 0.989 for LPL; r = 0.954 for HTGL). The normal mean (+/- SD) level of lipoprotein lipase mass and activity in postheparin plasma was 223 +/- 66 ng/ml and 10.1 +/- 2.9 mumol/h per ml, and that of hepatic triglyceride lipase mass and activity was 1456 +/- 469 ng/ml and 26.4 +/- 8.7 mumol/h per ml, respectively. The present sandwich-enzyme immunoassay methods make it possible to study the molecular nature of LPL and HTGL in PHP from patients with either primary or secondary hyperlipoproteinemia.     

The fatty liver dystrophy (fld) mutation. A new mutant mouse with a developmental abnormality in triglyceride metabolism and associated tissue-specific defects in lipoprotein lipase and hepatic lipase activities

An autosomal recessive mutation, termed fatty liver dystrophy (fld), can be identified in neonatal mice by their enlarged and fatty liver (Sweet, H. O., Birkenmeier, E. H., and Davisson, M. T. (1988) Mouse News Letter 81, 69). We have examined the underlying metabolic abnormalities in fld/fld mice from postnatal days 3-40. Serum and hepatic triglyceride levels were elevated 5-fold in suckling fld/fld mice compared to their +/? littermates but abruptly resolved at the suckling/weaning transition. Blot hybridization analysis of liver and intestinal RNAs revealed a liver-specific increase in apolipoprotein (apo) A-IV and C-II mRNA concentrations (100- and 6-fold, respectively) that was limited to the suckling and early weaning stages in fld/fld mice. Resolution of these differences during the weaning period could not be delayed by prolonging suckling to the 20th postnatal day nor could the mutant phenotype be elicited in young adult animals with a high fat diet. Lipoprotein lipase (LPL) activity was reduced 16-fold in the white adipose tissue of fld/fld mice until the onset of weaning. Heart activity was decreased less than 2-fold, but there were no deficits in brown adipose tissue or liver. Hepatic lipase (HL) mRNA levels and activity were significantly reduced in fld/fld livers and sera, respectively, during the suckling period. Mapping studies show the fld locus to be distinct from loci encoding LPL, HL, and apoA-IV, and those responsible for the combined lipase deficiencies in cld/cld and W/Wv mice. These data suggest that the fld mutation is associated with developmentally programmed tissue-specific defects in the neonatal expression of LPL and HL activities and provide evidence for a new regulatory locus which affects these lipase activities. This mutation could serve as a useful model for (i) analyzing the homeostatic mechanisms controlling lipid metabolism in newborn mice and (ii) understanding and treating certain inborn errors in human triglyceride metabolism.     

A new method for the measurement of lipoprotein lipase in postheparin plasma using sodium dodecyl sulfate for the inactivation of hepatic triglyceride lipase.

Lipoprotein lipase (LPL) and hepatic triglyceride lipase (H-TGL) are lipolytic activities found in postheparin plasma. A simple and precise method for the direct determination of LPL in postheparin plasma is described. Pre-incubations of this plasma (45--60 min at 26 degrees C) with sodium dodecyl sulfate (35--50 mM) in 0.2 M Tris-HCl buffer, pH 8.2, results in the inactivation of H-TGL, while leaving LPL fully active. Direct determination of H-TGL is done in a separate aliquot of the same postheparin plasma sample using previously reported assay conditons that do not measure LPL. The sodium dodecyl sulfate-resistant lipolytic activity has the characteristics of LPL as judged by a) its activation by serum and by apolipoprotein C-II; b) its inactivation (over 90%) by 0.75 M NaCl; and c) its inactivation by a specific antiserum. No sodium dodecyl sulfate-resistant activity was found in postheparin plasma from a patient with LPL deficiency (primary type I hyperlipoproteinemia). An excellent correlation of values was obtained (r = 0.99) for 30 samples assayed after sodium dodecyl sulfate treatment and after immuno-inactivation of H-TGL. The intra-assay coefficient of variation was +/- 11% and 4% before and after normalization of values, respectively.     

Lipoprotein metabolism in hepatic lipase deficiency: studies on the turnover of apolipoprotein B and on the effect of hepatic lipase on high density lipoprotein

Hepatic lipase deficiency produces significant distortion in the plasma lipoprotein profile. Particles with reduced electrophoretic mobility appear in very low density lipoprotein (VLDL). Intermediate density lipoprotein (IDL) increases markedly in the circulation and plasma low density lipoprotein (LDL) levels fall. At the same time there is a mass redistribution within the high density lipoprotein (HDL) spectrum leading to dominance in the less dense HDL2 subfraction. The present study examines apolipoprotein B turnover in a patient with hepatic lipase deficiency. The metabolism of large and small very low density lipoproteins was determined in four control subjects and compared to the pattern seen in the patient. Absence of the enzyme did not affect the rate at which large very low density lipoproteins were converted to smaller particles within this density interval (i.e., of VLDL). However, subsequent transfer of small very low density lipoproteins to intermediate density particles was retarded by 50%, explaining the abnormal accumulation of VLDL in the patient's plasma. Despite this, intermediate density particles accumulated to a level 2.4-times normal because their subsequent conversion to low density lipoprotein has been almost totally inhibited. Consequently, the plasma concentration of low density lipoprotein was only 10% of normal. On the basis of these observations, hepatic lipase appears to be essential for the conversion of small very low density and intermediate density particles to low density lipoproteins. The pathways of direct plasma catabolism of these species were not affected by the enzyme defect. In vitro studies were performed by adding purified hepatic lipase to the patient's plasma.(ABSTRACT TRUNCATED AT 250 WORDS)