Normotriglyceridemic abetalipoproteinemia. absence of the B-100 apolipoprotein.

In the two genetic forms of abetalipoproteinemia described previously, recessive abetalipoproteinemia and homozygous hypobetalipoproteinemia, all lipoproteins that normally contain apolipoprotein B are absent from plasma. We describe here a new disorder in which normal low density and very low density lipoproteins are absent, but in which triglycerides are absorbed from the intestine and chylomicrons are present in plasma. The underlying molecular defect appears to be selective deletion of the hepatogenous B-100 apolipoprotein. The B-48 apolipoprotein found in chylomicrons is spared. These findings suggest that the two species of apolipoprotein B are under separate genetic control and that low density lipoproteins are not normally derived from chylomicrons.

Molecular and metabolic basis for the metabolic disorder normotriglyceridemic abetalipoproteinemia.

We have previously described a disorder, normotriglyceridemic abetalipoproteinemia, that is characterized by the virtual absence of plasma low density lipoproteins and complete absence of apoB-100, but with apparently normal secretion of triglyceride-rich lipoproteins containing apoB-48. The patient's plasma lipoproteins were shown on polyacrylamide gels and by antibody mapping to have a new truncated apoB variant, apoB-50, circulating along with her apoB-48. We have found this individual to be homozygous for a single C-to-T nucleotide substitution at apoB codon 2252, which produces a premature in-frame stop codon. Thus, this is a rare example of homozygous hypobetalipoproteinemia. Electron photomicrographs revealed that the diameters of particles in the d less than 1.006 g/ml lipoprotein fraction, in both the postprandial and postabsorptive state, are bimodally distributed. The molar ratio of apoE to apoB in these particles is 3.5:1, similar to normal VLDL. The plasma LDL interval contains both spherical and cuboidal particles. Autologous reinfusion of labeled d less than 1.006 g/ml lipoproteins showed exponential disappearance from plasma, with an apparent half-removal time of 50 min, somewhat slower than for normal chylomicrons but within the normal range for VLDL. The calculated production rate for apoB was within the normal range in this subject. A very small amount of label was found briefly in the IDL fraction, but none at any time in LDL or HDL. Therefore, because LDL particles that contain apoB-50 lack the putative ligand domain of the LDL receptor, we conclude that the very low level of LDL is due to the rapid removal of the abnormal VLDL particles before their conversion to LDL can take place.

Apolipoprotein and size heterogeneity in human umbilical cord blood low density lipoproteins.

Neonatal umbilical cord blood plasma low density lipoproteins (LDL, d = 1.019-1.063 g/ml) were subfractionated by density gradient ultracentrifugation into seven fractions (from 1.024 to 1.062 g/ml); the bulk of the LDL mass was in a density region of 1.034-1.042 g/ml. Apolipoprotein B by 10% SDS-polyacrylamide gel electrophoresis varied inversely with density, with only trace amounts present in the most dense fraction. The distribution of apolipoprotein B molecular weight forms was assessed by both 3% SDS-polyacrylamide gel electrophoresis and relative aminoacyl mass determination. Lower molecular weight forms of apolipoprotein B (B74 and B26) increased relative to apolipoprotein B100 with increasing density, ranging from undetectable in fraction 1 to apolipoproteins B26 and B74 comprising 30% of the total mass of apolipoprotein B in fraction 6. No apolipoprotein B48 was detectable in the LDL. Apolipoprotein E as determined by both SDS-polyacrylamide gel electrophoresis and radioimmunoassay increased with density with a maximum (14% of the protein) in the most dense fraction, fraction 7. Apolipoprotein A-I by SDS-polyacrylamide gel electrophoresis increased with increasing density and was the major apolipoprotein in fraction 7. Electron microscopic analysis revealed spherical particles whose diameters decreased with increasing density, ranging from 28.6 nm in the top fraction (fraction 1) to 15.6 nm in the bottom fraction (fraction 7). Gradient gel electrophoresis revealed that most of the fractions contained several different sized particles. The bottom fraction (fraction 7), enriched in apolipoproteins E and A-I, had a unique, poorly defined peak at 14.6 nm on gradient gel electrophoresis and showed a tendency to pack hexagonally upon electron microscopy. The unusual composition and apolipoprotein distribution in neonatal LDL fractions suggests that the LDL in the neonate are metabolically very diverse.

Effect of oxidized lipids in the diet on oxidized lipid levels in postprandial serum chylomicrons of diabetic patients.

OBJECTIVE: To determine whether humans with type 2 diabetes have increased levels of oxidized fatty acids in their serum chylomicron fraction after the ingestion of dietary oxidized fatty acids. RESEARCH DESIGN AND METHODS: The study was performed on 31 male type 2 diabetic patients and 24 age-matched control subjects. Among the diabetic patients, 22 had poor glycemic control, defined as HbA1 > 10% (normal value < 7.7%). Nine patients had good glycemic control (HbA1 < or = 10). Heated corn oil containing low or high levels of oxidized fatty acids was used as a test meal. At 2.5 h after the test meal, 50-ml blood samples were obtained from all subjects, and the chylomicron fraction (Sf > 1,000) was isolated. The degree of oxidation in chylomicrons was determined by measuring conjugated dienes. For determining the postprandial levels of triglycerides and of oxidized lipids in serum chylomicrons over an extended time period, blood samples were obtained at 0, 2.5, 5.0, and 7.5 h for isolation of chylomicrons and determination of fatty acid oxidation. RESULTS: We found that at 2.5 h after the consumption of the test meal containing either a low or high oxidized fatty acid content, conjugated dienes in serum chylomicrons in diabetic subjects in poor glycemic control were increased compared with those in control subjects. Diabetic patients in good glycemic control had similar levels of oxidized lipid in their chylomicrons when compared with control subjects. Additionally, in diabetic patients in poor glycemic control, the levels of oxidized lipids in chylomicrons remained elevated for an extended post-prandial period. CONCLUSIONS: In diabetic subjects with poor glycemic control, dietary oxidized lipids induce an exaggerated and sustained increase in the levels of oxidized lipids in chylomicrons when compared with either control subjects or diabetic patients with good glycemic control. These increased postprandial levels of potentially atherogenic oxidized lipids may contribute to the accelerated atherosclerosis associated with diabetes.

Heterogeneity of apolipoprotein B: isolation of a new species from human chylomicrons.

Low density lipoproteins and the triglyceride-rich lipoproteins of human serum each contain proteins of high molecular weight termed apolipoprotein B, which have previously been thought to be identical. We have isolated four species of apolipoprotein B with unique molecular weights and amino acid compositions. We have assigned numerical designations to these species in a centile system based upon their relative apparent Mr in NaDodSO4. One which we term B-100, with an apparent Mr of 549,000 +/- 7650 (SD) determined by NaDodSO4 gel electrophoresis, predominates in low density and very low density lipoproteins and is also present in chylomicrons from thoracic duct lymph or from plasma. Substantial amounts of two large proteins designated B-74 (apparent Mr 407,000 +/- 5790) and B-26 (apparent Mr 144,500 +/- 8970), which appear to be complementary fragments or constituents of the B-100 protein, are found in the low density lipoproteins of many individuals. A distinct protein, B-48, with an apparent Mr of 264,000 +/- 8150 is a major and constant constituent of chylomicrons from thoracic duct lymph or from plasma.

Carboxyl terminal analysis of human B-48 protein confirms the novel mechanism proposed for chain termination.

In this study we have confirmed the presence of a single base difference between intestinal mRNA coding for B-48 and hepatic mRNA coding for B-100, which results in the substitution of a stop codon (UAA) for a glutamine codon (CAA) at a point corresponding to amino acid residue 2153 in the B-100 sequence. Based on this finding, B-48 is predicted to terminate at residue 2152 with the sequence ... Met Ile. To confirm this finding at the protein level, B-48 and B-100 were each digested with cyanogen bromide and the digestion products were analysed for the presence of isoleucine. Isoleucine was found only in cyanogen bromide digests of B-48 confirming that only B-48 terminates with the predicted amino acid sequence ... Met Ile.

Exposure to cigarette smoke delays the plasma clearance of chylomicrons and chylomicron remnants in rats.

We investigated the effects of cigarette smoke exposure on the clearance of chylomicrons (CM) and CM remnants in rats after administration of a fat-containing meal. There was a decrease in clearance of both postprandial CM and exogenous radiolabeled CM in smoke-exposed animals. For exogenous CM, clearance (t1/2) increased significantly for both triglyceride and cholesterol labels and correlated with the delay in liver uptake. This decrease in lipid clearance could not be explained by decreased lipoprotein lipase (LPL) activity because smoke exposure resulted in a significant increase in LPL activity. When the hydrolysis of CM by endothelial LPL was tested in a heart perfusion system, there was no difference in CM hydrolysis between the two groups. Hepatic lipase activity was also unchanged in smoke-exposed animals. However, there was a significant delay in the CM remnant uptake into livers isolated from smoke-exposed rats. Thus the delay in CM clearance in smoke-exposed animals cannot be attributed to reduced lipase activities but results from impaired hepatic uptake of CM remnants.

Apolipoprotein is responsible for neutralization of xenotropic type C virus by mouse serum.

We have shown that the circulating lipoproteins of the mouse contain a potent inhibitor of infectivity of the xenotropic type C virus. This virus neutralization does not involve immunoglobulins or complement. After fractionation of the lipoproteins on the basis of particle size, flotation properties, and electrostatic charge, virus neutralizing activity is found primarily in the triglyceride-rich lipoproteins (predominantly the chylomicrons) and in the HDL(2) subfraction of the high density lipoproteins. In fasted animals, activity resides chiefly in the high density lipoproteins. Neutralization titers increase strikingly during alimentary lipemia in both the lipoproteins of the rho < 1.006 g/cm(3) fraction and the high density lipoproteins. Increased activity persists in the high density lipoproteins after the lipemia recedes. Virus neutralizing activity is completely eliminated in all fractions by antiserum against high density lipoproteins and, in the triglyceride-rich fractions, by antiserum to murine apolipoprotein B. Complete removal of lipids markedly reduces the neutralizing activity of both classes of lipoproteins. Apolipoproteins delipidated with tetramethylurea retain some activity, which is enhanced by binding to a phospholipid-stabilized triglyceride emulsion and which is abolished by proteolytic digestion. We have demonstrated in vitro transfer of activity between high density and very low density lipoproteins of the mouse. These data indicate that xenotropic virus neutralization by normal mouse serum depends upon a protein that transfers among lipoprotein particles in a fashion analogous to the C apolipoproteins of other mammalian species.

Congenital genitourinary anomalies. Is there a predilection for multiple primary malignant neoplasms?

A case of simultaneous uterine and renal cell carcinoma in an elderly woman who had a septate vagina, double cervix, uterus didelphys, and a single kidney secondary to contralateral renal agenesis is reported. She was treated for a period of 8 months, first with pelvic irradiation followed by total abdominal hysterectomy and bilateral salpingo-oophorectomy and subsequently with heminephrectomy. Her renal function was normal postoperatively. The patient died of congestive heart failure in June 1990 after being free of carcinoma for approximately 18 years. The authors believe that this is the only case of its kind currently reported in the literature. Four of her family members died of either gastric (n = 3) or lung (n = 1) cancer, and one sister is alive with colon cancer. Only 19 proven cases of this constellation of congenital anomalies have been reported in the literature, and none have been associated with genitourinary (GU) carcinomas. There is a 50% to 70% incidence rate of genital tract anomalies in female patients with unilateral renal agenesis, secondary to the intimate association of the mesonephric and müllerian ducts. It has been suggested that the GU tract is prone to multiple primary malignant neoplasms, and there are families genetically predisposed to the development of large bowel and GU carcinomas. No conclusions can be drawn concerning the development of carcinoma in patients with congenital GU anomalies because of the small number of patients and the lack of follow-up in the literature.

Scission of human apolipoprotein B-100 by kallikrein: characterization of the cleavage site.

Low density lipoprotein (LDL) from human plasma was digested with the specific endoprotease, kallikrein. Apolipoprotein B-100, the protein moiety of LDL, was cleaved by kallikrein into two fragments (K1 and K2) which we have compared to the naturally occurring fragments, B-74 and B-26. We have found that K1 and K2 precisely match B-74 and B-26 with respect to molecular weight, stoichiometry, and amino terminal amino acid sequence. These findings provide strong evidence that kallikrein is the agent responsible for the formation of B-74 and B-26 in human LDL.

Oxidized lipids in the diet accelerate the development of fatty streaks in cholesterol-fed rabbits.

Studies have indicated that oxidized lipoproteins may play a role in atherosclerosis. We have recently demonstrated that the levels of oxidized lipoproteins in the circulation can be directly correlated to the quantity of oxidized lipids in the diet. The present study tested the hypothesis that dietary oxidized lipids accelerate the development of atherosclerosis. For 12 to 14 weeks, 36 male New Zealand White rabbits were fed a low-cholesterol (0.25%) diet containing either 5% unoxidized corn oil (control diet) or 5% oxidized corn oil (oxidized-lipid diet). Serum cholesterol levels increased to a similar extent in both groups, with the majority of the cholesterol in the beta-migrating very low density lipoprotein (beta-VLDL) fraction. Beta-VLDL from control animals contained 3.86+/- 0.57 versus 9.07 +/- 2.14 nmol conjugated dienes per micromol cholesterol (P<.05) in rabbits fed the oxidized-lipid diet. No difference in oxidized lipid levels was detected in LDL. Most important, feeding a diet rich in oxidized-lipid resulted in a 100% increase in fatty streak lesions in the aorta. Additionally, rabbits that were fed the oxidized-lipid++ diet had a >100% increase in total cholesterol in the pulmonary artery that was primarily due to an increase in cholesteryl ester. Oxidized lipids are frequently present in the typical US diet, and our results suggest that consumption of these foods may be an important risk factor for atherosclerosis.

Apolipoprotein B (B-48) of rat chylomicrons is not a precursor of the apolipoprotein of low density lipoproteins.

We have found that, in chylomicrons from intestinal lymph fistulas in the rat, the sole molecular species of apolipoprotein B (apo B) is B-48. This protein is analogous to the B-48 apoprotein of human chylomicrons. In contrast, preparations of chylomicrons from blood serum are known to contain species of B apolipoproteins of higher molecular weight, presumably due to the presence of hepatogenous lipoproteins. We studied the removal of 125I-labeled apo B-48 of intestinal lymph chylomicrons from blood plasma of rats. The removal of [14C]cholesteryl esters of biologically labeled chylomicrons was unaffected by radioiodination. The labeled cholesteryl esters and apo B-48 disappeared rapidly from the density (P) less than 1.006 g/ml fraction of plasma. In contrast to the apo B of very low density lipoproteins (1.019 less then p less then 1.063 g/ml) after 1 hr. No 125I was found in the B-100 or B-95 apolipoproteins at any time. We conclude that, unlike the species of apo B found uniquely in hepatogenous very low density lipoproteins, the apo B-48 protein of chylomicrons is not a precursor of the B apoprotein of low density lipoproteins.

Remnants of lipoproteins of intestinal and hepatic origin in familial dysbetalipoproteinemia.

We used the low molecular weight form of apolipoprotein B (B-48) as a marker for the identification of remnant particles formed from chylomicrons in the plasma of patients with familial dysbetalipoproteinemia. In the serum of patients fasted 14 hours, the d less than 1.006 g/cm3 lipoproteins of prebeta mobility, separated by starch block electrophoresis, contained only the primary hepatogenous species of apolipoprotein B (B-100), and their lipid composition resembled that of normal prebeta very low density lipoproteins. In contrast, the fraction with beta mobility contained both the B-48 and B-100 proteins; the B-48 protein was found primarily among the largest particles. All fractions of beta mobility were greatly enriched with cholesteryl esters. The beta fraction thus contains remnant particles which appear to originate both from chylomicrons and hepatogenous very low density lipoproteins. It appears that these remnant particles share a common removal mechanism which is impaired in familial dysbetalipoproteinemia.

Pre-beta high density lipoprotein. Unique disposition of apolipoprotein A-I increases susceptibility to proteolysis.

Apolipoprotein A-I-containing lipoproteins (high density lipoproteins, HDL) can be separated into two subfractions, which have pre-beta and alpha electrophoretic mobilities, respectively. These fractions differ in both composition and structure. Some preparations of pre-beta-migrating HDL, but not alpha-migrating HDL, were found to contain two polypeptides with Mr of approximately 26 and 14 kDa, which are scission products of apolipoprotein (apo) A-I. They are recognized by monospecific antibodies to apo A-I and have N-terminal sequences identical to those of mature apo A-I. This proteolytic scission of apo A-I occurs primarily after venipuncture. Immediate addition of protease inhibitors minimized the appearance of the fragments in plasma. To study the relative susceptibilities of pre-beta and alpha HDL to proteolysis, the lipoproteins were incubated in vitro with plasmin. The apo A-I in pre-beta HDL was extensively degraded, but that in alpha-migrating HDL was degraded to a much lesser extent, indicating that the appearance of apo A-I fragments in pre-beta HDL was due to enhanced sensitivity to proteolysis. To varying degrees, thrombin, kallikrein, elastase, arginine C endoprotease, and chymotrypsin also appear to cleave pre-beta HDL faster than alpha HDL. Most of the proteases generated a 12 to 14 kDa peptide fragment under conditions of limited cleavage. These results suggest that the conformational state of apo A-I in pre-beta-migrating HDL or its spatial relationship to lipids is significantly different from that of apo A-I in alpha-migrating HDL.(ABSTRACT TRUNCATED AT 250 WORDS)

Properties of serum high-density lipoproteins in the crab, Cancer antennarius Stimpson.

Two classes of high-density lipoprotein (HDL) comprise virtually all the lipoprotein mass in female hemolymph. These lipoproteins have hydrated densities of 1.187 g/ml (HDL3) and 1.112 g/ml (HDL2). A third species (HDL1, density 1.080 g/ml) appeared in ovigerous crabs. The mean annual HDL protein concentration was 109 mg/dl of which 67% was HDL3. HDL proteins of both HDL2 and HDL3 were mostly insoluble in tetramethylurea. Three major components with apparent mol. wts of 185,000, 100,000 and 84,000 daltons were identified by gel electrophoresis in SDS. Amino acid compositions are reported. Electron microscopy indicated that the HDL are polymorphic and discoidal. Similarities in shape and differences in size of HDL3 and HDL2 particles were consistent with their lipid and protein composition. Phospholipids, mostly phosphatidylcholine, were the dominant lipid class (74%); no cholesteryl esters were detected. Palmitic and oleic acids were the major fatty acid components of esterified lipids.

Structural domains of human apolipoprotein B-100. Differential accessibility to limited proteolysis of B-100 in low density and very low density lipoproteins.

The structural domains of human apolipoprotein B-100 in low density lipoproteins (LDL) and the conformational changes of B-100 that accompany the conversion of very low density lipoproteins (VLDL) to LDL were investigated by limited proteolysis with 12 endoproteases of various specificities, and their cleavage sites were determined. In B-100 of LDL, we identified two peptide regions that are highly susceptible to proteolytic cleavage. One region encompassed about 40 amino acids (residues 1280-1320, designated as the NH2-terminal region) and the other about 100 amino acids (residues 3180-3280, designated as the COOH-terminal region). IN LDL, the cleavage sites in both susceptible regions of B-100 were readily accessible to limited proteolysis; but in VLDL, only sites in the COOH-terminal region were readily accessible. Moreover, B-100 in VLDL appeared less degraded than B-100 in LDL by all enzymes used. Reduction of disulfide bonds of B-100 in both LDL and VLDL before digestion by Staphylococcus aureus V8 protease and clostripain exposed additional cleavage sites and increased the rate of B-100 degradation, suggesting that disulfide bonds probably exert conformational constraints. These results indicate the presence of three principal structural domains in B-100 of LDL that are relatively resistant to limited proteolysis. These three domains are connected by the two susceptible peptide regions. Our results also demonstrate differential accessibility of cleavage sites in B-100 of LDL and VLDL to limited proteolysis. This differential accessibility suggests that substantial changes in the conformation or environment of B-100 accompany the conversion of VLDL to LDL.

Structural comparison of human apolipoproteins B-48 and B-100.

In this study we have investigated the structural relationship between human apolipoproteins B-48 and B-100 by comparing protein structure and by comparing nucleotide sequence from intestinal and hepatic cDNA clones. Sequences from intestinal and hepatic cDNA were identical over the entire distance analyzed (7194 bases), which is more than required to code for B-48. The amino-terminal amino acid sequences from intact B-48 and B-100 proteins were also identical over the entire distance analyzed (16 residues). Additional protein homology was evaluated by the combined techniques of peptide mapping and immunoblotting. Purified B-48 and B-100 were each digested with three different endoproteases, and the resulting peptides were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Peptide bands were then detected by silver stain and by Western blotting with antisera against specific regions of B-48 and B-100. The resulting patterns suggest that B-48 is extensively homologous with the amino-terminal portion of B-100. We have identified only four peptides from B-48 (at least one in each digest) that are absent from the parallel digests of B-100. These peptides appear to arise from the ultimate carboxyl terminus of B-48 and appear to be totally homologous with a region located near the center of B-100. Our observations suggest that mature, circulating B-48 is homologous over its entire length (estimated to be between 2130 and 2144 amino acid residues) with the amino-terminal portion of B-100 and contains no sequence from the carboxyl end of B-100.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation of a cDNA clone encoding the amino-terminal region of human apolipoprotein B.

A partial cDNA clone for the B-26 region of apolipoprotein B was isolated from an adult human liver DNA library by screening with an oligonucleotide probe derived from amino-terminal protein sequence obtained from purified B-26 peptide. Antisera against a synthetic 17-residue peptide whose amino acid sequence was encoded by the clone cross-reacts with apolipoproteins B-26, B-100, and B-48, but not with B-74. The nucleotide sequence immediately upstream from the amino terminus of B-26 codes for an apparent signal sequence, implying that the B-26 moiety is in an amino-terminal locus in the B-100 protein. That this sequence represents a 5' end region is further supported by primer extension analysis using a fragment of the cDNA clone and by S1 nuclease protection experiments using the corresponding region in a genomic clone.

Distribution of lipid-binding regions in human apolipoprotein B-100.

The distribution of lipid-binding regions of human apolipoprotein B-100 has been investigated by recombining proteolytic fragments of B-100 with lipids and characterizing the lipid-bound fragments by peptide mapping, amino acid sequencing, and immunoblotting. Fragments of B-100 were generated by digestion of low-density lipoproteins (LDL) in the presence of sodium decyl sulfate with either Staphylococcus aureus V8 protease, pancreatic elastase, or chymotrypsin. Particles with electron microscopic appearance of native lipoproteins formed spontaneously when detergent was removed by dialysis from enzyme digests containing fragments of B-100 and endogenous lipids, or from incubation mixtures of delipidated B-100 fragments mixed with microemulsions of exogenous lipids (cholesteryl oleate and egg phosphatidylcholine). Fractionation of the recombinant particles by isopycnic or density gradient ultracentrifugation yielded complexes similar to native LDL with respect to shape, diameter, electrophoretic mobility, and surface and core compositions. Circular dichroic spectra of these particles showed helicity similar to LDL but a somewhat decreased content of beta-structure. Most of the fragments of B-100 were capable of binding to lipids; 12 were identified by direct sequence analysis and 14 by reaction with antisera against specific sequences within B-100. Our results indicate that lipid-binding regions of B-100 are widely distributed within the protein molecule and that proteolytic fragments derived from B-100 can reassociate in vitro with lipids to form LDL-like particles.