Investigation of lipoprotein metabolism in persons with abstinence

Lipoprotein (LP) metabolism was investigated in the blood serum of persons with abstinence after ethanol intake in a single dose of (0.7-1.0) g/kg. Content of LP was determined with the method of gradient gel-electrophoresis and use of software for quantitative analysis. A LP-lipase and cholesteroletherificazing activity of blood serum were also examined. It was found out that the patients with abstinence were characterized by the slowing down of LP metabolism as compared with donors, who represented general population. A single action of ethanol causes a short-time inhibition of LP metabolism. After 3 days processes of apoA-containing LP transformation were renewed. In contrast to this, one could observe an increase of the level of apoB-containing fractions accompanied by incompatibility between quantitative LP changes and activities of enzymes, which catalyzed their transformation. This evidences for the impairment of coordination of processes of cholesterol and triacylglyceroles transport by ethanol.     

Lipoprotein metabolism in patients with diabetes of type II

Changes in contents of blood serum lipoproteins (LP) and activity of enzymes of their transformation were investigated in patients with diabetes mellitus of type II. It was found out that mechanisms of increasing of apoB-containing LP in these patients were on the one hand in enhancing of the processes of their new-formation in the liver. On the other hand, retardation of their arrival to peripheral tissues might be observed, that as a whole resulted in continuous LPLD circulation in blood. Above-mentioned processes were associated with a reciprocal decreasing of the amount of all the apoA-containing LP fractions examined. To a certain extent mechanisms of disturbance of LP metabolism in the circulation bed were stipulated by changes of function of the enzyme catalyzing their transformation.     

Modulation of TXA2 generation of platelets by human lipoproteins

The lipoprotein (LP) fractions VLDL, LDL, HDL2 and HDL3 were prepared by ultracentrifugation of plasma from healthy volunteers and from patients with coronary heart disease (CHD). We investigated the capacity of platelets from healthy volunteers and patients with atherosclerosis to generate thromboxane A2 (TXA2) during spontaneous clotting of whole blood under the influence of the lipoprotein fractions. In our experiments the serum concentration of TXB2, reflecting the capacity of platelets to generate TXA2 during clotting, depends on several factors: the type of LP fraction used, the blood used for generation of TXA2, and for the same LP fraction whether it was taken from plasma of healthy volunteers or patients with CHD. VLDL prepared from plasma of healthy volunteers inhibited but VLDL prepared from plasma of patients with CHD enhanced the TXA2 formation of platelets from healthy volunteers (p less than 0.05, resp.). LDL from CHD patients inhibited the TXA2 formation of platelets from atherosclerotic patients (p less than 0.01). The HDL subfractions HDL2 and HDL3 from healthy volunteers inhibited TXA2 formation by platelets from healthy volunteers as well as those from atherosclerotic patients (p less than 0.05; p less than 0.01, respectively). HDL2 from patients with CHD inhibited only the TXA2 formation of platelets from healthy volunteers (p less than 0.01), whereas HDL3 from CHD patients inhibited only the TXA2 formation of platelets from atherosclerotic patients (p less than 0.01).     

Lipid peroxidation--the factor promoting cholesterol accumulation in cells in atherogenesis

The cholesterol transfer between human erythrocytes and main classes of serum lipoproteins (LP) from healthy donors and artery-coronary disease patients was studied (artery-coronary disease is the main manifestation of atherosclerosis). It is shown that low-density lipoproteins (LDL) are capable of transporting cholesterol to erythrocytes, which lack the specific receptors for LDL. The cell cholesterol content in comparison with erythrocytes incubated without LDL was increased by 11.4%. The effect was even higher in case of LDL, isolated from serum of artery-coronary subjects (the cell cholesterol content was increased by 33.8%). High-density lipoproteins (HDL) accept cholesterol from cell membranes. However, cholesterol-accepting properties of HDL from artery-coronary disease patients were suppressed as compared with normal HDL. Both discovered events must promote the cholesterol accumulation in cell membranes in atherosclerosis. As it is shown by the spin probe method, lipid peroxidation (LPO) causes the disturbance of the structural organization of LP and as the consequence of that--the increase of LDL cholesterol-donating ability and the decrease of HDL cholesterol-accepting ability. The greater LDL are oxidized, the more cholesterol they transport to erythrocytes during incubation. The greater is the level of HDL peroxidation, the stronger their cholesterol-accepting function is suppressed. These results suggest that LPO can play an important role in LP modification, the disturbance of their interaction with cell surface and the cholesterol accumulation in cells in atherosclerosis.     

The effect of a 3-hydroxypyridine-class antioxidant on disorders of the microcirculatory system in experimental dyslipoproteinemia and its alimentary correction

The effect of antioxidant (AO) of the 3-hydroxypyridin class on microcirculatory system (MC) disorders of rabbits with dyslipoproteinemia (DLP) and its alimentary correction (the standard ration--9 months) was studied. Aorta, the intestinal mesentery microcirculatory bed, the microvessels adrenergic innervation, erythrocyte morphology were examined. The various lipoprotein fractions and lipid peroxidation (LP) products were studied in venous blood. After AO including into atherogenic diet (ATD) the lipid homeostasis disorders, the LP activation, MC disturbances, anomalous erythrocyte form appearance and atherosclerosis changes in aorta were less pronounced. Under the AO influence when DLP was corrected the initial level of lipid metabolism and LP restored, the MC disorders and the atherosclerosis changes in aorta regressed in a shorter period of time. The interrelation was observed between the DLP level, the structural and functional MC disorders and the extent of aorta atherosclerosis. Thus, DLP and LP correction at the early stages of pathological process can lead to the disappearance of MC disorders, what seems important for the prevention of atherosclerosis. The role of the LP activation in MC disorders in DLP and mexidol effect on them, especially in complex with DLP correction were evaluated.     

Endotoxin-lipoprotein complex formation as a factor in atherogenesis: associations with hyperlipidemia and with lecithin:cholesterol acyltransferase activity

A potential role of endotoxin–lipoprotein (bacterial lipopolysaccharide–lipoprotein, LPS–LP) complex formation as a pathogenic factor for atherosclerosis has not been studied yet. The aim of this study was to test the hypothesis that in endotoxinemia in humans hyperlipidemia associated with atherosclerosis development can favor an excessive LPS–LP complex formation, and endotoxin presented in blood can inhibit lecithin:cholesterol acyltransferase (LCAT), one of the key enzymes of reverse cholesterol transport. Endotoxin-binding capacity of lipoproteins (LP) in patients with normolipidemia and hyperlipidemia types IIa and IV was estimated from label incorporation into different LP fractions isolated by means of sequential ultracentrifugation following serum preincubation with Salmonella minnesota R595 ¹²⁵I-labeled LPS. The effect of varied concentrations of S. minnesota R595 LPS on LCAT activity was evaluated from the overall esterifying activity of serum using [1,2-³H₂]cholesterol-labeled substrate. The elevation of low density LP (LDL) and very low density LP (VLDL) contents in blood serum in hyperlipidemia types IIa and IV, respectively, resulted in significant elevation of LPS binding to these fractions. LPS added to the blood serum leads to the dose-dependent decrease in LCAT activity. The revealed phenomena of elevated LPS binding to atherogenic LP fractions in hypercholesterolemia and endotoxin-induced LCAT inhibition suggest the pathogenic role of LPS–LP complexes in atherogenesis.     

Lipoproteins from normolipidemic and dyslipidemic subjects modify the thromboxane A2 generation by platelets in clotting human blood

The study was performed to investigate the influence of lipoproteins (LP) on the thromboxane (TX) A₂ formation capacity of platelets in clotting whole blood in vitro. The different lipoprotein fractions VLDL, LDL, HDL₂ and HDL₃ were isolated from blood of normo- or dyslipidemic volunteers by ultracentrifugation. These lipoproteins were incubated in blood with different levels of serum total cholesterol (TC) taken from normolipidemics (TC < 200 mg/dl), moderate hypercholesterolemics (TC: 200–250 mg/dl) or subjects with high cholesterol level (TC > 250 mg/dl), respectively. The amount of serum TXA₂ formed within 60 min at 37°C was measured by enzyme immunoassay. The results obtained show that the efficacy of separate LP fractions to influence the TXA₂ production depends not only on the type of LP fraction but also on the source of plasma used for isolation of LP and on the cholesterol level in the blood for incubation: LDL taken from normolipidemics or moderate hyperlipidemics inhibited the TXA₂ formation in blood from normolipidemics (P < 0.02, respectively), but enhanced it in blood from persons with moderate hypercholesterolemia (P < 0.05). LDL from hyperlipidemics enhanced TXA₂ production in blood from hyperlipidemics (P < 0.05). The HDL₂ fractions inhibited the TXA₂ formation in blood from normo- and hypercholesterolemics (P < 0.02, resp.), but there was no effect of HDL₂ in clotting blood from persons with moderate hypercholesterolemia. All HDL₃ fractions tested inhibited the TXA₂ formation in all types of blood used for clotting (P < 0.02, resp.), probably due to their great cholesterol accepting capacity.     

Modifications to the surface of T-lymphocytes in chronic candidiasis of the skin and mucous membranes

27 patients with chronic candidiasis of the skin and mucous membranes have been examined with a view to study the expression of E-receptors and its changes after the incubation of T-lymphocytes with theophylline, levamisole, thymaline and Candida albicans antigen. This study has revealed that, in comparison with healthy persons, most patients show decreased expression of E-receptors, and the character of their changes after incubation with the above-mentioned preparations is essentially different. The deviations revealed in this study have been regarded as the modification of T-cell membranes, which is seemingly one of the reasons of the weak competence of T-cells. In its turn, the expression of E-receptors may be considered as an indicator of the influence of different factors on T-lymphocytes. Substances altering the level of cyclic nucleotides in the cell, the antigenic fractions of the fungal causative agent, the suppression or blocking the action of thymic hormone-like substances may serve as such factors.     

Serum zinc and copper in hyperlipoproteinemia

Starting from experimental observations demonstrating that a high ratio of zinc to copper led to hypercholesterolemia in rats, serum Zn and Cu levels were measured by atomic absorption spectrophotometry in 65 normolipemic controls and in 100 subjects with various types of hyperlipoproteinemia (HLP). Serum Zn levels did not significantly differ from control values in any type of HLP. However, hyperlipoproteinemic patients with obvious clinical atherosclerosis displayed significantly lower serum-Zn concentration than hyperlipo-proteinemic subjects without clinical symptoms. On the other hand, when compared to control subjects, serum Cu levels were not found to be decreased, but rather increased, in hyperlipoproteinemic patients with or without atherosclerosis. As a result, the Zn∶Cu ratio appeared to be lower than normal in hyperlipoproteinemic patients with cardiovascular disease. It is conceivable that changes of these trace elements should be rather connected to vessel injury and associated disease than to HLP which, at least in humans, is not accompanied by a high Zn∶Cu ratio.     

Increased Level of Lipid Peroxidation Products as a Risk Factor of Death in a Prospective Study

The peroxidation-based theory concerning the pathogenesis of atherosclerosis suggests that this disease is a result of the activation of free-radical oxidation and the accumulation of lipid peroxidation (LP) products in the body. This theory needs to be verified by prospective observation data to correlate the latter phenomenon with the increased prevalence of morbidity and mortality among patients with this and related diseases. The authors analyzed the mortality of 35- to 60-year-old males (n = 339) in a ten-year period depending on the initial level of LP fluorescent products (LPFP). Cardiovascular diseases were the main fatal factor in the examined group (47.4%). Mean LPFP levels were initially higher in the subjects who died before the end of the study than in those who survived. Using Coxe's proportional risk model, it was demonstrated that an increased LPFP level is an independent mortality risk factor. An increase in the LPFP level above 15 mmol of fluorene equivalents per mol cholesterol of apo-B-lipoproteins was associated with a nearly 2.4-fold increase in the mortality risk, and its significance was comparable with that of arterial hypertension. The prospective study showed that an increased level of LP products in blood plasma is a major mortality risk factor, being no less strong than the first order (etiological) factors.     

用转染EB病毒基因片段的真核细胞检测鼻咽癌病人的抗EB病毒核抗原-1(EBNA-1)抗体

用转染EBV DNA Bam HI K片段后稳定表达EBNA-1的K_4细胞作为靶细胞,检测50份鼻咽癌病人和38份健康对照者血清中的IgG/EBNA-1抗体;阳性率分别为100％和92％。前者的平均几何滴度为89.4,后者为18.3,前者约为后者的5倍。同一批被检血清经SPA吸收去除IgG竞争性抑制后,鼻咽癌病人的IgA/EBNA-1抗体阳性率达78％(GMT 20.9),健康人的IgA/EBNA-1抗体阳性率仅5.3％,效价亦很低(GMT 5.2)。表明IgA/EBNA-1抗体对鼻咽癌是比较特异的,可考虑作为鼻咽癌血清学诊断的指标之一。     

细胞因子TNF-a和黏附分子siCAM-1在嗜肺军团菌感染患者血清中的表达

目的探讨细胞因子TNF-a和黏附分子si CAM-1在嗜肺军团菌（LP）感染患者血清中的表达情况。方法选取2012年1月到2013年1月于沈阳军区总医院就诊的LP感染患者共45例作为观察组,同期选取45例健康者作为对照组,采用ELISA检测法,对血清中的细胞因子TNF-a和黏附分子si CAM-1的表达进行对比。结果观察组血清中的TNF-a和黏附分子si CAM-1表达在急性期浓度明显高于对照组,组间治疗效果比较差异有统计学意义（P〈0.05）。结论 LP感染患者血清中的TNF-a和黏附分子si CAM-1表达的检测,能为临床了解病情发展,制定科学预后方案提供准确的信息,对预后判断都有着重要的意义。     

老年急性脑梗死患者血清CRP, FG, LP(a)变化研究

目的:探讨老年急性脑梗死患者检测血清C反应蛋白(CRP)、纤维蛋白原(FG)及脂蛋白(a)(LP(a))的变化及意义。方法:选择2013年12月-2014年12月在我院就诊的年龄≥60岁的急性脑梗死患者90例作为实验组,根据中国脑卒中患者临床神经功能缺损程度评分标准(NDS),分为轻型、中型和重型;根据病灶直径及Adama分型法分为腔隙性脑梗死、小梗死和大梗死,另外选择90例健康老人为对照组。测定所有受试者血清中的CRP、FG及LP(a)的水平,并比较它们在实验组与对照组、实验组不同严重程度和不同病变类型的患者中的差异。结果:实验组血清CRP、FG和LP(a)的水平高于对照组,且差异具有统计学意义(P0.05);重型患者血清CRP、FG和LP(a)水平明显高于中型患者,中型患者显著高于轻型患者,且差异均有统计学意义(P0.05);大梗死患者血清CRP、FG和LP(a)水平明显高于小梗死患者,小梗死患者显著高于腔隙性梗死患者,且差异均有统计意义(P0.05)。结论:血清CRP、FG和LP(a)水平异常升高与老年急性脑梗死的发生发展密切相关。     

The lymphocytic basophilia after incubation of blood smears in distilled water

The incubation of peripheral blood smears in distilled water frequently used for the control of the digestion with ribonuclease produced distinct changes of basophilic ribonucleic acid containing structures in peripheral lymphocytes. The alteration of these structures was apparently produced by the activity of the endogenous ribonuclease since it was reduced or prevented by the lowering of the temperature or addition of the ribonuclease inhibitor. In addition, marked differences were found between peripheral blood lymphocytes of healthy persons and patients suffering from chronic lymphocytic leukaemia with respect to the sensitivity of lymphocytic basophilia to the incubation in distilled water.     

Age-related changes of serum lipoprotein oxidation in rats

Oxidation of low-density lipoprotein (LDL) may be a prelude to atherogenesis and directly age related. To assess whether there may be relationship between age and plasma lipoprotein (LP) oxidation, we studied copper-mediated LP oxidation isolated from the blood of 2 months, 7 months, and 15 months old rats. We determined whether the susceptibility of LP to oxidation might be related to vitamin C levels in serum, vitamin E levels in LP, or the total antioxidant capacity (TAC) of serum or LP. Serum vitamin C content was inversely related to age, malondialdehyde (MDA) propagation rate, and maximum change of MDA concentrations. However, there were no significant relationships between age and serum TAC, LP TAC, serum vitamin E, or the ratio of LP vitamin E to serum vitamin C content. The lag phase of MDA formation was significantly decreased with age and the ratio of LP vitamin E content to serum vitamin C content, increased with age. Maximum change of MDA concentration was positively correlated with the ratio of LP vitamin E contents to serum vitamin C concentration. Thus, as the rat ages, vitamin C status decreases with an increased LP susceptibility to oxidation. It is tempting to speculate that enhanced LP oxidation in older rats may reflect a reduced amount of recycling of LDL vitamin E by serum vitamin C.     

Serum and Antibodies of Glaucoma Patients Lead to Changes in the Proteome,Especially Cell Regulatory Proteins,in Retinal Cells

Purpose

Previous studies show significantly specifically changed autoantibody reactions against retinal antigens in the serum of glaucoma and ocular hypertension (OHT) patients in comparison to healthy people. As pathogenesis of glaucoma still is unknown the aim of this study was to analyze if the serum and antibodies of glaucoma patients interact with neuroretinal cells.

Methods

R28 cells were incubated with serum of patients suffering from primary open angle glaucoma (POAG), normal tension glaucoma (NTG) or OHT, POAG serum after antibody removal and serum from healthy people for 48 h under a normal or an elevated pressure of 15000 Pa (112 mmHg). RGC5 cells were additionally incubated with POAG antibodies under a normal pressure. Protein profiles of the R28 cells were measured with Seldi-Tof-MS, protein identification was performed with Maldi-TofTof-MS. Protein analysis of the RGC5 cells was performed with ESI-Orbitrap MS. Statistical analysis including multivariate statistics, variance component analysis as well as calculating Mahalanobis distances was performed.

Results

Highly significant changes of the complex protein profiles after incubation with glaucoma and OHT serum in comparison to healthy serum were detected, showing specific changes in the cells (e.g. Protein at 9192 Da (p<0.001)). The variance component analysis showed an effect of the serum of 59% on the cells. The pressure had an effect of 11% on the cells. Antibody removal led to significantly changed cell reactions (p<0.03). Furthermore, the incubation with POAG serum and its antibodies led to pro-apoptotic changes of proteins in the cells.

Conclusions

These studies show that the serum and the antibodies of glaucoma patients significantly change protein expressions involved in cell regulatory processes in neuroretinal cells. These could lead to a higher vulnerability of retinal cells towards stress factors such as an elevated IOP and eventually could lead to an increased apoptosis of the cells as in glaucoma.     

Free radical lipid oxidation affects cholesterol transfer between lipoproteins and erythrocytes

Human erythrocytes were incubated for 5 h at 37 degrees C with lipoproteins (LP), preliminary oxidized to different extent, as assessed by thiobarbituric acid (TBA) test. Cholesterol content in the cells was increased by 12-14% after incubation with low-density lipoproteins (LDL) along with augmentation of order parameter and rotational correlation time of spin-labeled stearic acids incorporated into membranes. If erythrocytes were incubated with oxidized LDL, containing 2.5-4 times more TBA-reactive material than native ones, cellular content of cholesterol was increased by 24-28%. In contrast, high-density lipoproteins (HDL2 and HDL3) removed cholesterol from cell membranes, when incubated with erythrocytes. This was followed by increased fluidity of membrane lipid phase as detected by the spin probe method. Oxidation of HDL2 and HDL3 decreased their ability to accept cholesterol from cell membranes. No detectable accumulation of TBA-reactive material was observed in the samples during the incubation. The antioxidant, butylated hydroxytoluene (BHT), in the concentration of 10(-5) M did not influence the cholesterol transfer between LP and erythrocytes. Hence, the effects of lipid peroxidation (LPO) on the cholesterol transfer seem to result from LP alterations by oxidation rather than from free radical reactions occurring during the incubation. By increasing cholesterol-donating ability of LDL and inhibition of cholesterol-accepting capacity of HDL lipid peroxidation in LP may activate cholesterol accumulation in blood vessel cells and thus contribute to atherosclerosis.     

Multiple forms of human gamma-glutamyltransferase: preparation and characterization of different molecular weight fractions

Eight fractions of human gamma-glutamyltransferase were prepared from liver tissue, serum and bile by gel filtration. Bile, pooled serum from patients with high gamma-glutamyltransferase activities and serum in which liver tissue had been incubated, each contained an enzyme fraction with molecular weight greater than 10(6). A fraction of about 80,000 molecular weight was obtained from bile, and by incubation of liver tissue in serum or sodium chloride solution, but not from the serum pool. The main enzyme fraction in native serum had a molecular weight of about 300,000, and the molecular weight of gamma-glutamyltransferase partially purified from liver was initially 160,000. The fractions had similar Km and Ki values, and differences in heat stability and binding to concanavalin A were not marked.     

Isoelectric spectra of human serum albumin in healthy subjects and in ischemic heart disease

Isoelectric spectra of serum albumins isolated from blood of patients with heart ischemia were studied using isoelectric focusing in borate-polyolic systems in a polyacrylamide gel. In patients with heart ischemia the amount of fractions with pI 4.3-4.9 and 5.2-7.4 is found to increase with a simultaneous decrease in the fraction with pI 4.9-5.2 as compared with these indices in healthy people from the control group. Especially pronounced changes in isoelectrophoregrams were observed for blood albumins of patients with transmural myocardium infraction.