High density lipoprotein subfractions, apolipoprotein A-I containing lipoproteins, lipoprotein (a), and cholesteryl ester transfer protein activity in alcoholic women before and after ethanol withdrawal

Abstract. We studied 11 female alcoholics before and after ethanol withdrawal of 2 weeks and 10 healthy normolipidaemic, nonalcoholic women of similar age. In alcoholic women the HDL₂ mass was increased by 63% ( P<0.01 ) on admission and normalized ( P <0.01) during abstention. The concentrations of HDL₃ cholesterol and its mass remained unchanged throughout the study. Consistently with the fall of HDL₂ gradient gel electrophoresis analyses also demonstrated decrease of the cholesterol concentration of HDL_2b and HDL_2a (P<0.05) during alcohol withdrawal. On admission the apo A-II concentration was increased by 48% ( P < 0.01) and it was normalized (P< 0.001) during abstention. Among apo A-I containing lipoproteins the most prominent change occurred in Lp A-1: A-11, which fell by 32% ( P<0.01 ) during 1 week's alcohol withdrawal. During abstention the lipoprotein (a) concentration increased in 10 out of 11 women. In patients cholesteryl ester transfer (CETP) activity increased by 35% (P<0.01) during 1 week of ethanol withdrawal. On admission post-heparin plasma lipoprotein (LPL) and hepatic lipase activities were increased by 25% (P = NS); during 1 week's abstention they both returned to the control level ( P < 0.05–< 0.01). In conclusion, chronic alcoholic women display multiple changes of lipoprotein metabolism which are rapidly reversed during abstinence. In contrast to alcoholic men, studied previously by us using the same study design and methods, there was no significant elevation of HDL₃ cholesterol and apo A-I. The data suggest that alcohol interferes with several regulatory steps of HDL metabolism which are partly gender dependent.     

Sleep apnoea in alcoholic patients after withdrawal

Respiration during sleep was studied in 16 withdrawn alcoholic patients and in 12 control subjects. The alcoholic patients had increased numbers of central (P less than 0.01) and of obstructive (P less than 0.05) apnoea and of hypopnoea episodes (P less than 0.01) as compared with controls. Significant positive associations were found between the frequencies of central apnoea (P less than 0.05) or hypopnoea (P less than 0.01) and clinical evidence of central nervous system damage in the alcoholic patients. Hypopnoea also showed a significant association with vagal neuropathy (P less than 0.05), assessed by tests of cardioreflexes. We conclude that abnormal respiratory events are common in abstinent alcoholic patients and that they are likely to be at least partly related to nervous damage.     

Effect of simvastatin on apoprotein B—containing lipoproteins in patients with diabetic nephropathy

Background: Diabetic patients with nephropathy usually have a more atherogenic lipoprotein profile than those without nephropathy, which may be associated with the substantially higher incidence of coronary heart disease (CHD) in this population. Simvastatin has been shown to significantly reduce the incidence of CHD events in diabetic patients.Objective: The purpose of this study was to evaluate the effect of simvastatin (10 mg/d) on atherogenic apoprotein (apo) B—containing lipoproteins in type 2 diabetic patients with nephropathy.Methods: Diabetic patients with nephropathy and a group of healthy control subjects matched for age, sex, and body weight were enrolled. Diabetic patients were administered simvastatin 10 mg/d for 6 months. Apo B—containing lipoproteins were sequentially separated by ultracentrifugation to yield very low-density lipoprotein (VLDL) (density <1.006 g/mL), intermediate-density lipoprotein (IDL) (1.006-1.019 g/mL), light low-density lipoprotein (LDL) (1.019-1.044 g/mL), and dense LDL (1.044-1.063 g/mL) fractions. Apo B in lipoproteins was measured by a sensitive enzyme-linked immunosorbent assay at baseline and after 6 months of simvastatin treatment.Results: A total of 18 patients with diabetic nephropathy and 36 matched controls were enrolled. The diabetic patients had significantly higher levels (P < 0.01) of total cholesterol, LDL cholesterol, triglycerides, and apo B compared with age- and weight-matched control subjects at baseline. The diabetic patients also had significantly higher levels (P < 0.05) of cholesterol and apo B in the VLDL, light LDL, and dense LDL fractions. Treatment with simvastatin for 6 months significantly reduced plasma total cholesterol by 21%, LDL cholesterol by 30%, and apo B by 25% (P < 0.001), but did not affect urinary albumin excretion. Simvastatin significantly decreased both triglyceride and cholesterol levels in VLDL by 18% (P < 0.05), and cholesterol and apo B in IDL by 22% (P < 0.05) and 26% (P < 0.01). Simvastatin decreased both the light and dense LDL subfractions to a similar extent, reducing cholesterol and apo B in light LDL by 27% (P < 0.001) and in dense LDL by 28% (P < 0.01) and 18% (P < 0.05), respectively. The light LDL/dense LDL ratio for apo B and for cholesterol were not altered by simvastatin therapy.Conclusions: The results of this study suggest that simvastatin may reduce levels of atherogenic apo B—containing lipoproteins and small dense LDL in diabetic patients with nephropathy.     

Metabolism of apoprotein B of plasma very low density lipoproteins in the rat.

As an extension of metabolic studies of the cholesteryl ester component of rat very low density lipoproteins, we have studied the metabolism of the B apoprotein component labeled by intravenous injection of [3H]lysine. The B apoprotein separated from other apoproteins by delipidation and selective precipitation with tetramethylurea could not be distinguished from B apoprotein prepared by the conventional gel filtration technique. After injection of [3H]lysine, specific activity of B apoprotein was maximal in very low density and low density lipoproteins 1 and 11/2-h later, respectively, in a manner consistent with a precursor-product relationship. When protein-labeled very low density lipoproteins were injected into rats, the relationships of specific activity again indicated that B apoprotein of very low density lipoproteins may be the sole precursor of that of low density lipoproteins. However, less than 10% of the B apoprotein that disappeared from very low density lipoproteins appeared in density lipoproteins. To evaluate the sites of removal of B aproprotein of very low density lipoproteins from plasma, protein-labeled very low density lipoproteins were incubated with unlabeled high density lipoproteins to reduce radioactivity in non-B apoproteins selectively by molecular exchange. Most of the B apoprotein was rapidly removed by the liver. The extensive hepatic uptake of both the cholesteryl ester and B apoprotein components of rat very low density lipoproteins may explain the characteristically low concentrations of plasma low density lipoproteins in the rat.     

Effects of ethanol on platelet thromboxane formation after ethanol withdrawal in chronic alcoholics: An in vitro study

The effect of ethanol on the formation of platelet thromboxane B2 (TXB2), a stable metabolite of thromboxane A2 (TXA2) was studied in vitro in six chronic alcoholics, admitted for detoxification, and in six healthy volunteers. Immediately after cessation of heavy drinking platelet count and ADP-induced TXB2 formation were lower in alcoholics than in nonalcoholic volunteers (P less than 0.05). Ten days after withdrawal of ethanol platelet count increased, and skin bleeding time shortened (P less than 0.05). Ethanol had no effect on arachidonate-induced platelet TXB2 formation, whereas ethanol added to platelet suspension prior to stimulation by ADP resulted in a concentration-related inhibitory tendency in both alcoholics and nonalcoholic control subjects. This effect of ethanol may be of significance for primary hemostasis in alcoholics.     

Defining the atherogenicity of large and small lipoproteins containing apolipoprotein B100

Apo-E-deficient apo-B100-only mice (APOE:(-/-)APOB:(100/100)) and LDL receptor-deficient apo-B100-only mice (LDLR:(-/-)APOB:(100/100)) have similar total plasma cholesterol levels, but nearly all of the plasma cholesterol in the former animals is packaged in VLDL particles, whereas, in the latter, plasma cholesterol is found in smaller LDL particles. We compared the apo-B100-containing lipoprotein populations in these mice to determine their relation to susceptibility to atherosclerosis. The median size of the apo-B100-containing lipoprotein particles in APOE:(-/-)APOB:(100/100) plasma was 53.4 nm versus only 22.1 nm in LDLR:(-/-)APOB:(100/100) plasma. The plasma levels of apo-B100 were three- to fourfold higher in LDLR:(-/-)APOB:(100/100) mice than in APOE:(-/-)APOB:(100/100) mice. After 40 weeks on a chow diet, the LDLR:(-/-)APOB:(100/100) mice had more extensive atherosclerotic lesions than APOE:(-/-)APOB:(100/100) mice. The aortic DNA synthesis rate and the aortic free and esterified cholesterol contents were also higher in the LDLR:(-/-)APOB:(100/100) mice. These findings challenge the notion that all non-HDL lipoproteins are equally atherogenic and suggest that at a given cholesterol level, large numbers of small apo-B100-containing lipoproteins are more atherogenic than lower numbers of large apo-B100-containing lipoproteins.     

Lipoproteins containing apoprotein B are a major regulator of neutrophil responses to monosodium urate crystals.

The inflammatory response to intraarticular urate crystals is known to be variable in gouty arthritis. One source of variability may be the modulation of cellular responses by crystal-bound proteins. We have identified three apolipoproteins among the polypeptides bound to urate crystals exposed to plasma. Identification was first based on their coelectrophoresis with polypeptides from isolated lipoproteins and diminution in the protein coat of crystals exposed to lipoprotein-depleted plasma. The apoproteins were immunochemically identified by the Western blotting technique as apoprotein A-I, apoprotein B (apo B), and apoprotein E. Because neutrophils play a central role in acute gout, we investigated the potential effects of lipoproteins on neutrophil-urate crystal interactions. Plasma profoundly inhibited urate crystal-induced neutrophil luminol-dependent chemiluminescence (CL). Lipoprotein depletion by KBr density gradient centrifugation completely abrogated the inhibitory effect of plasma on urate-induced CL. The inhibitory activity of lipoprotein-depleted plasma was restored by adding back the d less than or equal to 1.25 g/cm3 lipoprotein fraction. Plasma also inhibited urate crystal-induced neutrophil superoxide generation and cytolysis (lactic dehydrogenase loss). This inhibition was significantly diminished by lipoprotein depletion, indicating that the lipoprotein effect was not limited to CL. Lipoprotein-depleted plasma reconstituted with very low, intermediate, and low density lipoproteins (LDL) inhibited crystal-induced CL. High density lipoprotein reconstitution was without effect. Immunodepletion from plasma of all apo B lipoproteins by agarose-bound apo B-specific antibody also removed all inhibitory activity for urate-induced CL. Thus, apo B lipoproteins were shown to be the inhibitory species in plasma. Binding of apo B lipoproteins to urate crystals and inhibition of CL was also seen in the absence of other plasma proteins. In addition, the binding of whole lipoprotein particles to the crystals was verified by detection of crystal-associated cholesterol in addition to the apoprotein. The effects of LDL on urate crystal-induced CL were stimulus specific. Coincubation of urate crystals and neutrophils in the presence of 10 micrograms/ml LDL resulted in 83% inhibition. In contrast, CL responses to a chemotactic hexapeptide, opsonized zymosan, and Staphylococcus aureus were not inhibited by LDL. The effects of depletion of apo B lipoproteins on plasma suppression of urate crystal-induced CL appeared to be unique. Plasma or sera depleted of other urate crystal-binding proteins including fibrinogen, fibronectin, C1q, and IgG retained virtually all their CL inhibitory activity. Lipoproteins containing apo B are thus a major regulator of neutrophil responses to urate crystals. These lipoproteins are present in variable concentration in synovial fluid and may exert an important influence on the course of gout.     

Multiple myeloma in alcoholic liver cirrhosis

A case of multiple myeloma (Bence Jones, lambda) associated with alcoholic liver cirrhosis is reported. A 56-year-old Japanese male died of hepatic failure and hypercalcemia. Autopsy revealed alcoholic liver cirrhosis and plasma cell myeloma. Immunoelectrophoretic analysis of his reserved serum disclosed the presence of M component of lambda Bence Jones protein. IgA and lambda light chain were demonstrated in the cytoplasm of the myeloma cells. Complications such as generalized amyloidosis, metastatic calcification, myeloma kidney and hemorrhagic pancreatitis were noted. The coexistence of multiple myeloma and liver cirrhosis has rarely been reported. On the basis of a review of the reported cases, a possible association between both diseases was discussed.     

Noradrenergic sensitivity of the cerebral cortex after chronic ethanol ingestion and withdrawal.

The effects of both chronic ethanol treatment and 3 days of ethanol withdrawal on the cyclic adenosine 3':5'-monophosphate (cAMP) response of cerebral cortical slices to norepinephrine (NE) were studied. ED50 cAMP responses for each group were determined using graded doses of NE. Chronic ethanol fed rats displayed subsensitivity to NE. There was a 4.3-fold shift of the dose-response curve to the right. Rats chronically fed ethanol and then put on 3 days of ethanol withdrawal developed supersensitivity. There was a 2.4-fold shift of the dose-response curve to the left. There was no difference in the maximal cAMP response observed in either the chronic alcohol or the chronic alcohol three day withdrawal experiments. It is possible that the biphasic modifications of effectors by ethanol ingestion and withdrawal from a subsensitive to a supersenitive is the result of one basic phenomenon.     

Radioimmunoassay of human arginine-rich apolipoprotein, apoprotein E. Concentration in blood plasma and lipoproteins as affected by apoprotein E-3 deficiency.

A radioimmunoassay for apolipoprotein E in human blood serum has been developed that measures equally the major polymorphic species of the protein (apolipoproteins E-1, E-2, E-3, and E-4) and the apo E in the dimer of apolipoproteins E and A-II. The assay is specific and yields values for apolipoprotein E in very low density lipoproteins that agree closely with those obtained by a quantitative electrophoretic method. Apolipoprotein E is also present in at least one species of high density lipoprotein, but the content of apolipoprotein E in the lipoprotein fractions of plasma is uncertain owing to dissociation during ultracentrifugation. The concentration of apolipoprotein E is higher in serum of normolipidemic, premenopausal women than in men of comparable age and is a direct function of the serum triglyceride level. Apolipoprotein E levels are increased out of proportion to triglyceride levels in hyperlipidemic patients with familial dysbetalipoproteinemia (homozygotes for lack of apolipoprotein E-3). Heterozygous relatives of homozygotes have significantly higher apolipoprotein E levels in serum than unaffected relatives. The concentration of partially degraded (remnant) triglyceride-rich lipoproteins also appears to be increased in heterozygotes, who comprise about 15% of the population.     

High ethanol intake and malnutrition in alcoholic cerebellar shrinkage

To determine the influence of chronic ethanol intake and nutritional status on cerebellar shrinkage in alcoholism, we studied 12 undernourished patients with acute Wernicke's encephalopathy (WE), 12 undernourished and 24 well-nourished asymptomatic chronic alcoholics, and 24 age-matched well-nourished controls, using morphometric analysis of MRI scans with volumetry of the cerebellum. Alcoholics reported a mean daily intake of ethanol of 177+/-8 g over a period of 27+/-1 years. Most undernourished alcoholics and half of the well-nourished alcoholics, compared to one-tenth of the controls, showed a significant reduction in cerebellar volume (p< or =0.01, both). Alcoholics with cerebellar shrinkage (n=33) were older (p=0.05) and tended to report greater daily ethanol intake than alcoholics without cerebellar shrinkage (n=15), although not significantly so (p=0.09). Cerebellar volume correlated negatively with age in controls and asymptomatic alcoholics (r> or =0.52, p< or =0.01, both), with a significantly greater shrinkage for age in the latter (p=0.003). Logistic regression analysis showed that malnutrition (OR 6.6 [95%CI 1.7-25.6], p=0.005) and a daily ethanol intake of more than 140 g over ten years (OR 6.1 [95%CI 1.8-20.5], p=0.003) were independently associated with the development of cerebellar shrinkage.     

Effects of venlafaxine on ethanol withdrawal syndrome in rats

The present study was designed to investigate the effects of venlafaxine, a serotonin and noradrenaline reuptake inhibitor (SNRI), on ethanol withdrawal syndrome in rats. Adult male Wistar rats (187-319 g) were used for the study. Ethanol (7.2%, v/v) was given to rats by a liquid diet for 21 days. Control rats were pair-fed an isocaloric liquid diet containing sucrose as a caloric substitute to ethanol. Venlafaxine (5, 10, 20 and 40 mg/kg) and saline were injected to rats intraperitoneally just before ethanol withdrawal. After the 2nd, 4th and 6th hour of ethanol withdrawal, rats were observed for 5 min, and withdrawal signs that included locomotor hyperactivity, agitation, stereotyped behaviour and wet dog shakes were recorded or rated. A second series of injections was given at the 6th hour after the first one, and rats were then tested for audiogenic seizures. Venlafaxine produced some inhibitory effects on locomotor hyperactivity, stereotypic behaviours and wet dog shakes. However, a two-way anova of the data did not indicate any significant effect. It reduced the incidence of the audiogenic seizures at the 6th hour of ethanol withdrawal. Venlafaxine (20 mg/kg) also prolonged the latency of the seizures significantly. Our results suggest that acute venlafaxine treatment has limited beneficial effects on ethanol withdrawal syndrome in rats.     

Differential change in neuroactive steroid sensitivity during ethanol withdrawal

The progesterone metabolite 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-P or allopregnanolone) is a potent positive modulator of gamma-aminobutyric acid(A) (GABA(A)) receptors. Although it is well documented that chronic ethanol (EtOH) administration produces cross-tolerance to the positive modulatory effect of benzodiazepines and GABA at GABA(A) receptors, recent findings suggest that sensitivity to 3alpha,5alpha-P is enhanced during EtOH withdrawal. In addition, EtOH-naive inbred strains of mice, which differ in EtOH withdrawal severity (DBA/2 > C57BL/6), had marked differences in behavioral sensitivity to 3alpha,5alpha-P. Therefore, the present study was conducted to determine whether C57BL/6 (B6) and DBA/2 (D2) mice would be differentially sensitive to several of the pharmacological effects of 3alpha,5alpha-P during EtOH withdrawal. Male mice were exposed to EtOH vapor or air for 72 h. During withdrawal from EtOH, animals were injected with 3alpha,5alpha-P (0, 3.2, 10, or 17 mg/kg i.p.) and tested for activity and anxiolysis on the elevated plus maze, muscle relaxation, ataxia, and seizure protection following pentylenetetrazol. Sensitivity to the anticonvulsant effect of 3alpha,5alpha-P was enhanced during EtOH withdrawal in B6, but not D2 mice. In contrast, sensitivity to the muscle relaxant effects of 3alpha,5alpha-P was reduced in EtOH-withdrawing B6 and D2 mice, with a suggestion of decreased sensitivity to the anxiolytic effect of 3alpha,5alpha-P during EtOH withdrawal in B6. These results suggest that sensitization to the anticonvulsant effect of 3alpha,5alpha-P during EtOH withdrawal does not generalize across all genotypes nor does it generalize to all of the pharmacological effects of 3alpha,5alpha-P.     

Quantitative characterization of the main lipid and apoprotein components of plasma lipoproteins in men with types I and II diabetes mellitus with and without coronary heart disease

The paper is devoted to the characterization of the spectrum of lipoproteins (LP) including the determination of the content of apoproteins AI and B by quantitative "rocket" immunoelectrophoresis in 75 male patients aged 40 to 59 with normal body mass, with types I and II diabetes mellitus, in the state of compensation of carbohydrate metabolism, with and without signs of coronary heart disease. Sixty healthy men were controls. It was shown that in diabetic (types I and II) patients with CHD changes in LP spectrum values were similar to those in CHD patients without diabetes. In patients with type I diabetes without clinical signs of CHD, LP spectrum values including the content of apo-AI, apo-B and the ratio of apoB/apo-AI did not differ from those of the controls. Changes in LP-containing apo-AI expressed not only in the reduction of the total content of CH-HDIP but also in change of the composition of HDLP particles, were found in diabetic (type II) patients without clinical signs of CHD.     

A monoclonal-antibody-based immunoradiometric assay for apoprotein B in low-density lipoprotein

Five different mouse monoclonal antibodies were raised against apoprotein B, the major protein in human low-density lipoproteins (LDL). Competitive binding studies suggested that these antibodies recognized four different epitopes on the lipoprotein particle. All antibodies bound to very-low-density and intermediate-density lipoproteins, which are the metabolic precursors of LDL and also contain apoprotein B. One antibody (P1C1) displayed a relative specificity for apoprotein B in LDL as compared with that in very-low-density lipoprotein and was used as a radiolabeled first antibody in an immunoradiometric assay for this lipoprotein. Measurements, either on isolated LDL or directly on plasma samples, demonstrated that this assay provides a rapid and precise method of quantification.     

Ethanol withdrawal in mice bred to be genetically prone or resistant to ethanol withdrawal seizures

We are engaged in a selective breeding program developing lines of mice which differ in severity of withdrawal convulsions after ethanol treatment. Withdrawal seizure prone (WSP) mice show greater handling-induced convulsion scores than withdrawal seizure resistant (WSR) mice after 3 days of ethanol intoxication. In the present experiments, we sought to characterize these mice further as a model of genetic susceptibility to ethanol dependence and withdrawal. During withdrawal after chronic treatment with ethanol, WSP mice displayed more severe handling-induced convulsions and tremor than WSR mice, and tended to show greater reduction of exploratory activity. WSP and WSR mice did not differ in ethanol metabolism after acute treatment with ethanol alone or after chronic treatment with ethanol and pyrazole, an alcohol dehydrogenase inhibitor. Six to 10 hr after an acute injection of ethanol, WSP and WSR mice showed elevated handling-induced convulsions. This elevation was more pronounced in WSP mice than in WSR mice. WSP mice also showed slightly more severe convulsions than WSR mice when treated with saline or pyrazole alone. In summary, WSP and WSR mice treated with identical doses of ethanol differ in several symptoms of withdrawal, whereas not differing in ethanol metabolism. These mice constitute a useful population in which to study the molecular mechanisms of ethanol dependence and withdrawal.     

Concentrations of apoprotein CII, CIII, and E in total serum and in the apoprotein B-containing lipoproteins, determined by a new enzyme-linked immunosorbent assay

We describe a new enzyme-linked immunosorbent assay (ELISA) that permits direct determination of apoprotein (apo) CII, CIII, and E in total serum as well as in apo B-containing lipoprotein particles. To validate this ELISA technique, we studied several aspects of the assay: its specificity, the influence of the conditions of conservation of plasma and of lipoprotein fractions, the effect of delipidation, and its reproducibility. We measured the concentrations of apo CII, CIII, and E in total serum and in apo B-containing lipoproteins from a pool of normal sera and in sera from 75 healthy subjects. After sequential ultracentrifugation, the content of apo CII, CIII, and E in the major lipoprotein fractions was also determined. Total serum or plasma could be stored at -20 or -50 degrees C for at least six weeks and the isolated lipoprotein fractions for as long as four weeks, which suggests a protective effect of total serum on lipoprotein particle structure. Advantages of this ELISA include (a) its specificity, sensitivity, and reliability; (b) better discrimination than determination of total serum apoprotein; (c) easier application and greater rapidity; and (d) the possibility of application to population screening.