W. Carl Breckenridge

Hypertriglyceridemia Associated with Deficiency of Apolipoprotein C-II

A 59-year-old man with severe hypertriglyceridemia and no post-heparin lipolytic activity was studied because of a marked fall in plasma triglyceride concentrations after a blood transfusion. An apolipoprotein activator (apolipoprotein C-II) for lipoprotein lipase could not be detected by polyacrylamide-gel electrophoresis of apoproteins, immunodiffusion of the plasma against anti-apolipoprotein CII or activation assays for lipoprotein lipase. Furthermore, the patients triglyceride-rich lipoproteins would not serve as substrate for lipoprotein lipase. The patient had latent post-heparin lipolytic activity, which appeared after the addition of apolipoprotein CII to the post-heparin plasma. After a transfusion of 1 unit of plasma from a normal subject the patients plasma triglycerides fell, within one day, from 1000 to 250 mg per deciliter and remained below preinfusion concentrations for six days. We conclude that this patients hyperlipoproteinemia resulted from a deficiency of apolipoprotein C-II.

Inheritance of apolipoprotein C-II deficiency with hypertriglyceridemia and pancreatitis.

A study of the relatives of a patient with apolipoprotein C-II deficiency showed that the defect is inherited as an autosomal recessive trait. The kindred studied originated from an isolated population in which considerable inbreeding has occurred for 140 years. Seven homozygotes had marked fasting chylomicronemia and triglyceridemia, and lacked detectable apolipoprotein C-II by several assay methods. Five homozygotes had experienced one to many attacks of pancreatitis from as early as six years of age. Obligate heterozygotes had apolipoprotein C-II concentrations about 30 to 50 per cent of normal values and had normal plasma triglyceride concentrations. This metabolic defect should be considered in patients with markedly elevated plasma triglycerides who have apparent lipoprotein lipase deficiency, and usually also have pancreatitis.

Lipolysis of ApoC-II deficient very low density lipoproteins: Enhancement of lipoprotein lipase action by synthetic fragments of ApoC-II

Abstract Enzymic hydrolysis of triacylglycerol has been studied with very low density lipoproteins from an individual with a genetically determined absence of apoC-II, the activator apoprotein for lipoprotein lipase. Normal rates of ester cleavage by purified bovine milk lipoprotein lipase can be achieved in vitro with native apoC-II and by three shorter synthetic peptides, apoC-II(55–78), apoC-II(50–78) and apoC-II(43–78), which contain part of the carboxyl terminal third of the native apoprotein. At 0.5 μM concentration, all peptides produced a 7-fold activation. ApoC-II(43–78), but not apoC-II(50–78) or apoC-II(55–78), could bind VLDL as shown by separation of unbound 125I peptides and the lipoproteins. Thus, residues 43–50 of apoC-II are part of a lipid binding region. High affinity binding of apoC-II peptides to the lipoprotein substrate is not obligatory for activation of lipoprotein lipase.

Apolipoprotein and lipoprotein concentrations in familial apolipoprotein C-11 deficiency☆

Lipoprotein and apolipoprotein concentrations were determined in 11 homozygous and 9 heterozygous subjects for familial apolipoprotein C-II (Apo C-II) deficiency. Apo C-II was not detectable in the homozygotes, with the exception of 1 subject who possessed immunochemically detectable quantities in one of two samples. Apolipoproteins C-III (Apo C-III) and E (Apo E) were elevated 2-3-fold in 9 of 11 homozygotes. Apo C-III, but not Apo E, correlated with triglyceride levels (1500-4100 mg/dl). However, both Apo C-III and Apo E correlated with the cholesterol levels and one another. Apolipoproteins A-I (Apo A-I), A-II (Apo A-II) and B (Apo B) were reduced to approximately 50-60% of normal values in association with very low levels of cholesterol in high density (HDL; 11 +/- 2 mg/dl) and low density (LDL; 19 +/- 6 mg/dl) lipoproteins in the homozygous subjects. These alterations were associated with a marked decrease in the proportion of plasma Apo C-III associated with HDL. The levels of apolipoprotein D (Apo D) were within the normal range. Nine obligate heterozygotes had Apo C-II concentrations (mean 1.8 +/- 0.5 mg/dl; range 1.2-2.7 mg/dl) which were approximately 40-50% of normal values (mean 2.9 +/- 0.9 mg/dl; range 1.7-5.6 mg/dl). The reduction in absolute amounts of Apo C-II was also reflected in a reduction of the ratio Apo C-II/Apo C-III in very low density lipoproteins (VLDL) and in a reduction in the ability of the sera to activate skim milk lipoprotein lipase. The concentrations of Apo A-II, Apo B, Apo C-III and Apo E were normal. Apo A-I concentrations were normal or slightly low in association with slightly reduced concentrations of HDL cholesterol and a low proportion of plasma Apo C-III in HDL in relation to LDL and VLDL in some heterozygotes. It is concluded that the marked alterations in the apolipoprotein levels in homozygous subjects are primarily a reflection of the deficiency of Apo C-II which results in severe hypertriglyceridemia. In heterozygotes, the partial deficiency of Apo C-II appears to result in a minor disturbance of the clearance of the triglycerides and Apo C-III rich particles but no marked changes in the concentrations of total lipids, lipoproteins and apolipoproteins in fasting plasma.

Stereospecific Analysis of Triacylglycerols

Natural triacylglycerols consist of complex mixtures of fatty acids esterified to specific hydroxyl groups of the glycerol molecule. Although glycerol has a plane of symmetry, asymmetrical triacylglycerols are formed when the primary hydroxyl groups are esterified with different fatty acids. Furthermore, the primary hydroxyl groups can be distinguished by stereospecific enzymes.

Relation of genetic polymorphisms of apolipoprotein E, angiotensin converting enzyme, apolipoprotein B-100, and glycoprotein IIIa and early-onset coronary heart disease.

OBJECTIVE Apolipoprotein E (APOE) E4, apolipoprotein B-100 (APOB) Q3611 allele, the angiotensin converting enzyme (ACE) deletion (D) allele and glycoprotein IIIa (GP3A) P33 mutant allele are reported to predispose to early-onset coronary heart disease (CHD). These associations were not all confirmed in more recent studies. To determine the impact of these alleles on CHD, we examined the prevalence of these mutations in patients presenting with early-onset CHD and compared them to those manifesting CHD later in life. The delayed-onset was considered a sign of longevity and would serve as a comparative group to assess prevalence of the biochemical and genetic risk factors. METHODS 300 patients with a history of myocardial infarction or angina pectoris and angiographically documented CHD were studied. Patients were divided into two groups: group 1 (G1 = 150 patients) presenting with these findings under the age of 50 years; while group 2 (G2 = 150 patients) were patients presenting for the first time over the age of 65 years. Prevalence of the alleles of APOE, APOB, ACE and GP3A was assessed by molecular analysis. An association of any of these genotypes with early onset CHD could lead to a higher prevalence in the younger age group. RESULTS AND CONCLUSIONS None of the suspected alleles namely APOB Q3611 [G1: 10.7% vs. G2: 9.0%, p = 0.57], ACE D (G1: 52.0% vs. G2: 49.7%, p = 0.57), or the GP3A P33 (G1: 17.3% vs. G2: 15.7%; p = 0.58) showed any significant difference between the two groups. Subjects with APOE E4 were more frequent in the younger age group (G1: 18.3% vs. G2: 13.7%; p = 0.047), while APOE E2 was more frequent in G2 (G2: 10.0% vs. G1: 2.7%; p = 0.0002). Multivariate analysis showed an odds ratio of APOE E2 allele in G1 of 0.27 with a confidence interval of 0.10-0.73.

Quantification of sinking pre beta lipoprotein in human plasma

Two low density protein carriers of plasma cholesterol (beta-lipoprotein and sinking pre-beta-lipoprotein -- a genetic variant of beta-lipoprotein with pre-beta electrophoretic mobility) were isolated from human plasma by ultracentrifugation and gel filtration. They were subjected to agarose gel electrophoresis, followed by staining with Sudan Black B, and their concentration was determined by optical densitometry. The staining of the low lipoproteins was proportional to their total cholesterol the low lipoproteins was proportional to their total cholesterol content over a range of 2-50 mg/dl for sinking pre-beta-lipoprotein (SPB) and 50-250 mg/dl for beta-lipoprotein. Mixing experiments of the two purified lipoprotein preparations indicated that the cholesterol in the SPB could be estimated by the formula: SPB cholesterol = TBC x OD of SPB/(OD of SPB + OD of beta cholesterol), where OD is optical density and the TBC was either the total cholesterol content of mixtures of purified beta- and SPB-lipoproteins, or was determined as the total plasma low density lipoprotein cholesterol content measured by ultracentrifugation and polyanionic precipitation of beta- and SPB-lipoproteins from plasma. The SPB cholesterol values calculated by the above formula deviated from the true value (amount added) by a mean of 6% when beta-lipoprotein cholesterol was present at concentrations of 100, 165, and 216 mg/dl. The deviation did not correlate with the concentration of beta-lipoprotein cholesterol. The coefficient of variation for the determination of SPB lipoprotein by the scanning procedure wa 5-10% for SPB cholesterol concentrations of 6-30 mg/dl. SPB concentrations of 1-2 mg/dl cholesterol could be detected, but the method was unsatisfactory for quantification below 4 mg/dl cholesterol. By providing an estimate of the distribution of cholesterol between the subfractions of low density lipoprotein, the method should prove helpful in assessing risk of atherosclerosis.

Comparison of the effects of tamoxifen and of a tamoxifen analogue that does not bind the estrogen receptor on serum lipid profiles in the cockerel

Administration of the nonsteroidal antiestrogen tamoxifen to cockerels results in dose- and time-dependent decreases in the levels of free and esterified cholesterol, phospholipids, and triglycerides in serum and in very low density and low density lipoprotein fractions. Similar changes can be elicited using a tamoxifen analogue, N,N-diethyl-2-[(4-phenylmethyl)phenoxy]ethanamine.HCl (DPPE). Like tamoxifen, this compound is capable of binding antiestrogen binding sites and exhibits a relative binding affinity of 90% compared with tamoxifen (Ki approximately 4-5 nM). Unlike tamoxifen, DPPE shows no measureable affinity for the cockerel liver nuclear estrogen receptor. Further, DPPE exhibits no estrogen agonist or antagonist activity as measured at the level of synthesis of apolipoprotein II of very low density lipoprotein by liver, synthesis of ovalbumin by oviduct, or growth of the oviduct. Although it is possible that the lipid-lowering effects of tamoxifen result from the opposition of endogenous estrogen action in the cockerel, the similarity of the effects of tamoxifen and DPPE on the lipid profiles suggests common mechanisms that do not involve the estrogen receptor.

Structural Characterization of Gangliosides from Normal and Atherosclerotic Human Thoracic Aortas

A proliferation of cells, with characteristics of medial smooth muscle cells, occurs in the intimal region of the artery wall and has been considered important in the early phases of atherosclerosis (1,2). In addition, it has been claimed that cells on the surface of advanced lesions appear to be of monoclonal origin and different from the same cells in the media of adjacent normal tissue (3). These observations suggest that some form of alteration or transformation of the medial smooth muscle cell may occur in the atherosclerotic lesion.

The biochemistry of ϵ-amino groups of lysine residues from apolipoprotein b of human low density lipoprotein☆

Abstract The availability of E-lysine residues of apolipoprotein B in LDL for chemical or enzymic modification was investigated. Amino acid analyses of detergent-solubilized apolipoprotein B, following cyanoethylation with acrylonitrile, revealed that 10% of the lysine in apolipoprotein B were unreactive. The unreactive residues were associated with the most hydrophobic subfraction of apolipoprotein B. Since apolipoprotein B has a high molecular weight a study was undertaken to determine whether lysine residues were crosslinked to glutamic acid via ϵ-(γ-glutamyl)lysine as demonstrated for fibrin. Apolipoprotein B was digested exhaustively with proteases. The content of ϵ-(γ-glutamyl) lysine was determined by chromatography and isotope dilution. In contrast to earlier reports for serum LDL the data showed that less than 0.01 moles of lysine/mole of LDL apolipoprotein B were present as ϵ-(γ-glutamyl)lysine in plasma LDL. It was determined also that the crosslinks were not found in apolipoprotein B during clotting since LDL was not a substrate for clotting factor XIII which forms the bond in fibrin. Furthermore, the lipoprotein contained no inherent transglutaminase activity. It is concluded that the lysine residues in LDL, which are unreactive to cyanoethylation, can not be detected in the digests as ϵ-(γ-glutamyl)lysine.