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A method for analytical isoelectric focusing (IEF) of apolipoprotein E (apoE) in immobilized pH gradients (IPG) and immunodetection of the separated isoforms has been developed for use with either very low density lipoproteins (VLDL) or whole plasma. Both VLDL and plasma were sequentially delipidated with 1,4-dioxane, acetone-ethanol, and ether. Neuraminidase treatment preceded the delipidation when required. Using preformed plates, pH 5.0-6.0 (LKB, Bromma) after rehydration with 6 M urea and dextran T-10, the IPG focusing pattern of the common isoforms (E2, E3, E4) was found to be equivalent to conventional IEF with the added resolution of the E4 disialo form. The use of self-poured narrower gradients permitted the further resolution of the E4 monosialo form, a previously unrecognized heterogeneity of the E2, E3, and E4 monosialo isoforms and differentiation of the apoE2** mutant; all of these forms comigrate with the common isoproteins in conventional IEF. Finally, the conditions for IPG of whole plasma using apoE monoclonal antibodies and enzyme-conjugated anti-mouse IgG for detection were established. Thus, IPG focusing is shown to be a powerful method for resolution of the apoE sialoforms and apoE mutant forms. The method has important implications in accurate and diagnostic phenotyping. Moreover, it is a convenient method for phenotyping which requires only very small volumes of plasma.

In a phase I safety and immunogenicity study, 112 healthy adult volunteers were randomly allocated to receive a new bivalent (A/Texas/36/91[H1N1-like], B/Harbin/7/94) split virion influenza vaccine propagated in Madin-Darby Canine Kidney cell culture or an identical vaccine manufactured using currently licensed egg propagated virus technology. Soreness at the injection site was common but generally mild (75% of the cell culture-derived vaccine group and 62.5% of the egg-derived vaccine group; p = not significant). General reactions were less common; headache was the most frequently reported adverse effect (26.8 and 30.4%, respectively; p = not significant). Geometric mean haemagglutination inhibition titres post-immunization against the A/Texas strain were 1012 reciprocal dilution in the cell culture-derived vaccine group and 790 in the egg-derived vaccine group; against the B/Harbin strain titres were 420 and 447, respectively (all comparisons, p = not significant). It is concluded that the cell culture-derived split virion influenza vaccine is safe and immunogenic in healthy adult volunteers.

A method for analytical isoelectric focusing (IEF) of apolipoprotein E (apoE) in immobilized pH gradients (IPG) and immunodetection of the separated isoforms has been developed for use with either very low density lipoproteins (VLDL) or whole plasma. Both VLDL and plasma were sequentially delipidated with 1,4-dioxane, acetone-ethanol, and ether. Neuraminidase treatment preceded the delipidation when required. Using preformed plates, pH 5.0-6.0 (LKB, Bromma) after rehydration with 6 M urea and dextran T-10, the IPG focusing pattern of the common isoforms (E2, E3, E4) was found to be equivalent to conventional IEF with the added resolution of the E4 disialo form. The use of self-poured narrower gradients permitted the further resolution of the E4 monosialo form, a previously unrecognized heterogeneity of the E2, E3, and E4 monosialo isoforms and differentiation of the apoE2** mutant; all of these forms comigrate with the common isoproteins in conventional IEF. Finally, the conditions for IPG of whole plasma using apoE monoclonal antibodies and enzyme-conjugated anti-mouse IgG for detection were established. Thus, IPG focusing is shown to be a powerful method for resolution of the apoE sialoforms and apoE mutant forms. The method has important implications in accurate and diagnostic phenotyping. Moreover, it is a convenient method for phenotyping which requires only very small volumes of plasma.

An underlying cause of type III hyperlipoproteinemia is the presence of variant forms of apolipoprotein (apo) E that are defective in binding to apo B,E low density lipoprotein receptors. This disorder is associated almost exclusively with the apo E2/2 phenotype. However, structural and functional heterogeneity have been demonstrated within this phenotype. The apo E2(Arg158----Cys) variant, displaying 1% of normal apo E3 binding activity, is the most defective known form. In this study, we describe a method in which a pair of 19-mer synthetic oligonucleotide probes were used to distinguish between DNA coding for arginine or cysteine at position 158 in apo E. The specificity of the probes was demonstrated by using DNA from subjects whose apo E protein sequence or phenotype was known. The probes were used to screen a French-Canadian population of 34 apo E2/2 subjects to determine the frequency of the apo E2(Arg158----Cys) variant. All 34 subjects, most of whom displayed clinical or biochemical features of type III hyperlipoproteinemia, were found to be homozygous for apo E2(Arg158----Cys), strongly suggesting that this variant is the most common form of apo E2 within this ethnic and clinical population. In addition, the utility of this approach in detecting new apo E mutants was demonstrated when DNA from one of the apo E3/3 control subjects, whose family has a history of hyperlipidemia and coronary artery disease, reacted with both probes. This result suggests that this subject is heterozygous for normal apo E3 and a new apo E3 variant that is likely to be functionally equivalent to apo E2(Arg158----Cys).

Observation of a markedly depressed HDL-cholesterol (5 mg/dL) in a patient with familial hypercholesterolemia (FH) receiving probucol (1 g/day) and clofibrate (2 g/day) prompted a review of all cases treated by this combination at our lipid clinic. Hypoalphalipoproteinemia (HDL-C less than 15 mg/dL) developed in 19 of 28 (70%) hyperlipidemic subjects who received this combination for an average of 1.5 years. This effect was sustained and reversible; it did not occur on either drug alone and was manifested on average 17 weeks after the combination was started. Plasma triglycerides increased significantly in most of those patients susceptible to this reduction in HDL-C. Plasma apolipoprotein A-I was decreased 82%, in proportion to the HDL-C fall, whereas apo A-II was lowered 65%. Since apo C-III concentrations tended to be high, the apo A-I/C-III ratio was markedly depressed. Apo E levels were unchanged and apo B levels reflected the high LDL concentrations of the underlying disease. An intermediate response was observed in subjects whose HDL-C remained well above 15 mg/dL on the combination. No deleterious side-effects could be attributed directly to the administration of the combined drugs in this high-risk group. One patient actually showed complete regression of xanthelasma and extensor tendon xanthomas of the finger on the combination. A parallel is drawn with Fish-Eye disease and the presence of the apolipoprotein A-I Milano variant, where similar HDL-C levels are observed in the absence of an increased atherogenic risk. It is mandatory to monitor plasma HDL-C in hypercholesterolemic patients treated with this combination, otherwise the pronounced HDL-deficiency could go unnoticed.

Implantation of MtT-F4 tumor, a mammotropic tumor that secretes large quantities of ACTH, GH and prolactin, into male Fisher rats induced the development of hyperlipidemia. Free fatty acid, triglyceride and cholesterol levels in the plasma were significantly increased at 31 days after tumor implantation. Blood glucose and glycerol levels remained normal, while uric acid concentration in the blood was significantly decreased. The concentrations of the serum lipoproteins were significantly increased, while, only small changes in the distribution of the serum lipids and the composition of the lipoproteins were observed. Following stimulation of isolated adipose tissue cells with ACTH, the lipolytic response and the accumulation of cyclic AMP was higher in cells derived from the rats with the tumor, although the accumulation of cyclic GMP was not different from control adipocytes. Further, when the isolated adipose tissue cells were stimulated with dibutyryl cyclic AMP no difference was observed between the control and tumor bearing groups. Clofibrate administered in the diet resulted in a complete elimination of the tumor effect on serum triglycerides and to a great extent prevented the rise in serum cholesterol. The tumor-induced increase in the concentration of the high density lipoproteins was not affected, but the elevation of the d less than 1.063 lipoproteins was not affected, but the elevation of the d less than 1.063 lipoproteins was partially reversed. The increased lipolytic response and accumulation of cyclic AMP following stimulation by ACTH was not altered in adipocytes derived from tumor bearing rats. However, clofibrate treatment resulted in a significantly greater accumulation of cyclic GMP in fat cells stimulated with ACTH from both control and tumor bearing rats. Clofibrate in the diet did not alter the levels of GH or prolactin or serum lipids in the control rats nor were the elevated hormone levels of the tumor bearing rats changed.

A systematic study of plasma lipids and lipoproteins was carried out in 11 cases of Friedreich's ataxia and 6 cases of familial spastic ataxia (Charlevoix-Saguenay disease) using 11 healthy normolipidemic volunteers of comparable age and sex as controls. No differences were noted in the fatty acid profile of the total lipid fraction, in the total cholesterol and phospholipids or in the percentage distribution of the individual phospholipid classes. The triglycerides were significantly higher in Friedreich's ataxia, but remained within the normal range. Although no systematic abnormalities could be detected in the electrophoretic pattern of plasma lipoproteins or in the apolipoprotein profile on polyacrylamide gel electrophoresis, major differences were found in the high density lipoprotein (HDL) fraction. Their total amount was reduced and their composition was abnormal in both neurological diseases. In Friedreich patients, the relative proportion of cholesterol and triglycerides was increased while the relative protein content was greatly reduced. In Charlevoix disease, a similar abnormality was seen except for the excess of triglycerides. The proportion of phospholipids in HDL was the same in the three groups of patients. In addition, the low density lipoprotein (LDL) fraction was slightly reduced in both diseases. This anomaly of the HDL fraction could indicate that the HDL apolipoprotein moiety has a greater affinity for cholesterol and triglycerides in Friedreich's ataxia than its normal counterpart.

Observation of a markedly depressed HDL-cholesterol (5 mg/dL) in a patient with familial hypercholesterolemia (FH) receiving probucol (1 g/day) and clofibrate (2 g/day) prompted a review of all cases treated by this combination at our lipid clinic. Hypoalphalipoproteinemia (HDL-C >15 mg/dL) developed in 19 of 28 (70%) hyperlipidemic subjects who received this combination for an average of 1.5 years. This effect was sustained and reversible; it did not occur on either drug alone and was manifested on average 17 weeks after the combination was started. Plasma triglycerides increased significantly in most of those patients susceptible to this reduction in HDL-C. Plasma apolipoprotein A-I was decreased 82%, in proportion to the HDL-C fall, whereas apo A-II was lowered 65%. Since apo C-III concentrations tended to be high, the apo A-I/C-III ratio was markedly depressed. Apo E levels were unchanged and apo B levels reflected the high LDL concentrations of the underlying disease. An intermediate response was observed in subjects whose HDL-C remained well above 15 mg/dL on the combination. No deleterious side-effects could be attributed directly to the administration of the combined drugs in this high-risk group. One patient actually showed complete regression of xanthelasma and extensor tendon xanthomas of the finger on the combination. A parallel is drawn with Fish-Eye disease and the presence of the apolipoprotein A-I Milano variant, where similar HDL-C levels are observed in the absence of an increased atherogenic risk. It is mandatory to monitor plasma HDL-C in hypercholesterolemic patients treated with this combination, otherwise the pronounced HDL-deficiency could go unnoticed.

The incorporation of [3H]leucine in vivo into very low density lipoproteins (VLDL) from the rat hepatic Golgi apparatus and serum was studied. A Golgi-rich fraction isolated on a discontinuous sucrose gradient between 0.5 and 1.1 M was found to contain VLDL having common antigenic determinants with serum VLDL. The incorporation of the [3H]leucine into the Golgi VLDL and serum VLDL suggested a precursor–product relationship. Analysis of the apoproteins of the Golgi VLDL by polyacrylamide gel electrophoresis revealed protein bands with similar mobility to those of serum VLDL, except that the former contained virtually no rapidly migrating peptides with the mobility of serum apo-C-II and apo-C-III. The pattern of incorporation of the [3H]leucine into the apoproteins was similar in VLDL from Golgi apparatus and serum, except for the absence of radioactivity in the area of the gel of Golgi apo-VLDL corresponding to apo-C-II and apo-C-III. The radioactive amino acid was incorporated predominantly into the Golgi apo-VLDL bands with similar mobility to apo-B and an apoprotein or group of apoproteins containing the arginine-rich peptide of serum VLDL. In vitro incubation of the Golgi VLDL with [3H]leucine-labeled HDL resulted in the acquisition of a number of proteins, including the rapidly migrating proteins. Administration of colchicine prior to the injection of [3H]leucine resulted in the appearance of gel bands and radioactivity in the apo-C-II and apo-C-III areas of Golgi apo-VLDL, suggesting that these can be acquired if secretion of VLDL is slowed or inhibited. The hepatic Golgi apparatus was then divided into fractions of predominantly forming face (GF3) or secretory granules (GF1). After polyacrylamide gel electrophoresis of the apo-VLDL from GF3, no visible bands or incorporation of [3H]leucine was found in the region of apo-C-II or apo-C-III. However VLDL from GF1 showed visible and radioactive bands in the apo-C-II and apo-C-III area although they represented a much smaller proportion of the total apoprotein than was found in the corresponding serum apo-VLDL. In the isolated perfused liver the percentage incorporation of [3H]leucine into the rapidly migrating apoproteins of Golgi VLDL was considerably less than that found in the corresponding apoproteins of perfusate VLDL, where circulating C lipoproteins are virtually absent.The data indicate that nascent VLDL begins to acquire the C-II and C-III apoproteins during its passage through the Golgi apparatus but that the main acquisition occurs during or after secretion into the space of Disse.

The predisposition for the development of hyperlipidemia rests almost equally on genetic and environmental factors and their interplay. Because one of the least understood factors is the duration of exposure to risk, the authors have chosen to review here some of the genetic factors and some of the relatively long-term environmental factors, such as diet and drug therapies, that are known to increase the risk of hyperlipidemia and likely the predisposition to cardiovascular disease.Some of the genetic factors and environmental factors such as diet and drug therapies that are known to increase the risk of hyperlipidemia and possibly the predisposition to cardiovascular disease are reviewed. The cholesterol associated with the low-density lipoproteins (LDL), accounting for 60-75% of the plasma levels, is responsible for the powerful and direct relationship which exists between plasma cholesterol and coronary heart disease. Also, the cholesterol that accumulates in atheromatous lesions is derived primarily from plasma LDL. Hyperlipidemia is defined by elevated levels of the plasma levels; the risk for atherosclerosis is associated with the classification types IIa, IIb, III, and possibly IV, a classification system based on phenotypic manifestations of increased lipoprotein fractions. The Lipid Research Clinics Program reports data on plasma lipid and lipoprotein cholesterol distributions of a large-scale screening of white men and women (both with and without sex hormone usage) aged 20-59 years in the US. They found age-related trends for rising triglycerides and cholesterol with differences between the sexes clearly demonstrated. It has been established that normolipemic individuals are not immune to the development of atherosclerosis. The recent focus on the apolipoprotein moieties has revealed a number of normolipemic dyslipoproteinemias associated with tissue lipid infiltration. Multifactorial population studies provide a strong case for the powerful role of the environment, i.e., predominantly dietary intake of total fat, saturated fat, saturated fats, and calories, in hyperlipidemia. According to the Seven Countries Study, populations with higher levels of cholesterol and LDL cholesterol (and increased atherosclerosis) have saturated fat intakes of 10% or more of calories. Migration studies of Japanese populations in Japan and in the US also show the influence of diet. As was shown early on, oral contraceptive (OC) use predisposes to the development of hyperlipidemia. OCs also predispose to other cardiovascular risk factors that, when combined with smoking, bring about a greatly magnified risk for myocardial infarction. Also reviewed in terms of their effect on the lipoprotein profile are antihypertensive therapies, retinoids, and hypolipidemic agents. Regarding genetic predisposition, single-gene mutations in apoproteins, lipoproteins, and some of the enzymes involved in lipoprotein may underlie disorders of hyperlipoproteinemia or hypolipoproteinemia.

Large Achilles tendon xanthomas of the type found in severe familial hypercholesterolemia were the first manifestation of cholestanolosis (cerebrotendinous xanthomatosis) in our patient, an otherwise asymptomatic normolipidemic 21-year-old woman. Extensive laboratory investigation disclosed the presence of hyperapobetalipoproteinemia which did not resolve with the administration of probucol. Immunofluorescent studies revealed marked accumulation of apolipoprotein B in a xanthoma excised from the tricipital area. This was in contrast to the spotty and weak fluorescence observed in a tuberous xanthoma, from the same anatomical area and histologically otherwise identical, obtained from a patient with heterozygous familial hypercholesterolemia. Hyperapobetalipoproteinemia has been reported before in association with sitosterolemia but not with cholestanolosis. We suggest that cholestanol, like sitosterol, may interfere with the normal uptake and degradation of low-density lipoproteins by peripheral cells.

Isolated livers from fed rats were perfused for 1 h with a medium consisting of Krebs–Ringer bicarbonate buffer with added albumin and glucose. Rates of perfusate and bile flow, differences in [Formula: see text], [Formula: see text], and pH, and glucose and potassium efflux were measured. Rewarmed slices of liver taken before the surgical preparation and before the perfusion were found to be able to reverse a cation shift imposed by cold incubation. Slices of liver taken after one perfusion were not able to effect this expected cation transport. It is proposed that the perfusate used was not ideal as evidenced by the altered membrane function of slices after perfusion.

The ability of liver slices prepared from normal rats and from isolated perfused livers following a 1 h control perfusion to reverse a cold-imposed shift in cations when rewarmed was confirmed as a control parameter of rat liver tissue. Three hours after normal rats had received a standard dose of CCl4 via a gastric tube, a decreased ability for the membrane transport of sodium was found. The infusion of CCl4 directly into the perfusate line was shown to cause acute toxic damage in isolated livers. Further, at the end of the 1 h perfusion, the infusion of CCl4 was shown to result in an increased tissue sodium level and a decreased capacity for the membrane transport of sodium and potassium by slices. It is suggested that a generalized change in plasmalemma function, as evidenced in alterations in the membrane transport of cations and tissue cation level, may be an early manifestation of CCl4 hepatotoxicity.

Isolated livers from fed rats were perfused for 1 h with a basic Krebs–Ringer bicarbonate buffer solution containing albumin and glucose and added (1) α-ketoglutarate, (2) pyruvate, fumarate, and glutamate, or (3) washed red blood cells. Perfusate flow rate, [Formula: see text], [Formula: see text], and pH changes across the liver, and glucose efflux in the perfusate, were measured during perfusion. Rewarmed slices of liver taken at the beginning of perfusion were found to be able to reverse a cation shift imposed by cold incubation. Slices of liver taken after 1 h of perfusion were not able to effect this cation shift unless red blood cells were included in the perfusate. It is proposed that noncellular perfusates containing metabolic substrates are not ideal for isolated rat liver perfusion studies as evidenced in the altered membrane transport capacity of slices after perfusion.

We have found a novel apoE5 mutation, using isoelectric focusing (IEF), in two apparently unrelated French-Canadian subjects. Co-dominant inheritance was demonstrated in the family of the first proband, a healthy male subject. The presence of the apoE5 form was not associated with lipid abnormalities or cardiovascular disease in this family. The second proband was a hyperlipidemic female patient suffering from angina, with no informative relatives available for study. In both individuals, monoclonal antibody studies demonstrated that the mutation was associated with the loss of two overlapping epitopes at the amino terminus of the protein. Cysteamine treatment of the very low density lipoproteins indicated that the mutant apoE contained only one cysteine residue, suggesting that apoE3 was the parental form. Two-dimensional electrophoresis suggested that the mutated protein had a slightly lower molecular weight (by 1-2 kDa). However, DNA sequencing of the third exon of the apoE gene in both probands revealed a single G to A substitution at the 48th nucleotide, changing the amino acid at position 13 from glutamic acid to lysine. These results were confirmed by oligo melting experiments with allele-specific probes in relatives of the probands. The study of this apoE variant should provide additional insight into the structure-function relationship of apoE.