Amount Of HDL Research Articles

A-209 Application of Bilirubin Oxidase for Improvement of False Positive Error Caused by Bilirubin on Cholesterol Efflux Capacity Assay by Immobilize Liposome-Bound Gel Beads method

Abstract Background High density lipoprotein (HDL) exerts various atheroprotective functions including cholesterol efflux capacity (CEC). HDL can attenuate lipid burden at atherosclerotic lesion by CEC. Based on the HDL`s role for moderate atherosclerosis, HDL-cholesterol level which indicates the amount of HDL has been evaluated to assess the risk of cardiovascular disease (CVD). Nevertheless, many clinical studies demonstrated that HDL-cholesterol level does not always reflect the risk of CVD, and CEC which represents the quality of HDL is considered as surrogate biomarker for CVD. CEC is prospective biomarker for CVD risk assessment, however, CEC measurement encounters barriers for clinical application because of its requirements of cultured cell and radioactive substance for conventional CEC assay. To overcome these problems, we invented a novel CEC assay called immobilized liposome-bound gel beads (ILG) method. This method is cell-free assay and utilizes a fluorescence substance as cholesterol tracer. Although the ILG method is useful assay for clinical application, previous study revealed that bilirubin caused false positive error on CEC value. Hence, current ILG method might produce incorrect CEC evaluation for patients with hyperbilirubinemia and even healthy subjects within the range of biological variation of bilirubin. In this study, we improved the influence of bilirubin for ILG method by using bilirubin oxidase (BOD). Methods BOD (Asahi Kasei Corp.) was added to 10 mM Tris-HCl (pH 7.4) containing 150 mM of NaCl and 1 mM of Na2EDTA (Buffer A). Bilirubin from ‘Interference check A Plus’ (Sysmex Inc.) was added to serum (free-: 10 mg/dL, conjugated-:9.7 mg/dL), and apoB-depleted serum (BDS) was derived from the bilirubin additive serum using polyethylene glycol. After the prepared bilirubin additive BDS was incubated with BOD-containing Buffer A for 16 h, the absorbance spectrum was analyzed. In addition, the time course of bilirubin decrement was monitored during 16 h-incubation. We further measured the fluorescence intensity of bilirubin additive BDS incubated with BOD-containing Buffer A. Using the newly developed BOD-containing Buffer A for ILG method, CEC of bilirubin additive serum was evaluated. Finally, we measured CEC of 4 healthy individuals by conventional and the improved ILG method. The CEC value was standardized by fluorescence intensity of reference sample which was measured in every assay. Results BOD contained in Buffer A could convert bilirubin to biliverdin from results of absorbance spectrum. According to the time course of bilirubin decrement, both free- and conjugated-bilirubin were oxidized to biliverdin in approximately 6 h. By converting bilirubin to biliverdin, fluorescence light was weakened greatly. We confirmed that there was no significant difference of CEC values between non bilirubin-additive serum and bilirubin additive serum. Compared healthy individual CEC values measured by conventional and improved ILG method, all subjects showed significant decrease of CEC values from the improved method, and false positive error caused by bilirubin was ameliorated. Conclusion In this study, we achieved to improve the positive error caused by bilirubin on CEC assay using ILG method. This improvement would contribute to more accurate CEC assessment by ILG method and advance the clinical application of this method.

Abstract 396: A Unique Case of Artefactual and Real HDL Disappearance Due to Isolevuglandins Adducts

We report a case of acquired, progressive, and severe HDL cholesterol (HDL-C) deficiency that emerged in an individual with multiple myeloma. An 81 year-old white male presented with extremely low HDL-c (≤5 mg/dL). Between the ages of 69 - 73 years old, his annual lipid panels demonstrated HDL-c >70 mg/dL. At that point, his HDL-C levels progressively decreased until their virtual disappearance over a period of 12 years. During this time, he developed anemia, an IgG-kappa M-spike, and increased plasma cells on bone marrow biopsy, compatible with a diagnosis of multiple myeloma. Review of his medications, medical history, and metabolic status failed to identify a cause for his progressive decine of HDL-C. Parallel analyses of plasma samples using detergent to solubilize and release HDL-associated cholesterol (SYNCHRO LX, Beckman) and separation of non-HDL precipitated by dextran sulfate yielded HDL-C values of 5 mg/dL and 47 mg/dL, respectively. Furthermore, HDL-C levels of 52 mg/dL and 54 mg/dL were confirmed using ultracentrifugation and NMR-based methods, respectively. Serum apoAI concentration was 153 mg/dL, suggesting normal levels of HDL, in contrast with the ultra-low HDL-C reported in one method. The patient’s alpha migrating band on lipoprotein gel electrophoresis also appeared normal, suggesting the presence of roughly normal amounts of HDL. Methodological artifact alone, however, did not explain the significant “real” decrease in HDL-C (from the upper 70’s mg/dL to the 40’s mg/dL) recorded during 12 years of monitoring. Isolevuglandins (IsoLG) are highly reactive ketoaldehydes that interact with lysine residues of proteins and head groups of phosphatidylethanolamine. It was recently proposed that IsoLG adducts on HDL can result in deleterious structural and functional changes in the particle. Interestingly, the patient’s serum showed high titers of autoantibodies to IsoLG-apoAI, but low titers against native apoAI. We further studied the turnover of IsoLG-apoAI and native apoAI in mice. This case of artefactual and real HDL-C disappearance in a patient with multiple myeloma is likely due to paraprotein-related interference of HDL-C assays and increased plasma clearance of modified apoA1.

Does Serum lipid profile differ in anemia and non-anemic older subjects?

Background:There is some evidence that shows the symptoms of anemia are fewere in overweight and obese people, so, the purpose of this research was to study the relationship between anemia and iron deficiency anemia (IDA) with lipid profile status of the elderly.Methods:This cross-sectional study came from the Amirkola Health and Ageing Project (AHAP). A demographic questionnaire was given to the older people and a blood sample was obtained to assay their lipid indexes (triglyceride, cholesterol, HDL and LDL) and the parameters related to anemia after 12 hours fasting. The data were analyzed by chi-square test, t-test and Pearson correlation using SPSS. A p<0.05 was considered as the significance level of the tests.Results: The average age of the people was 68.95±7.43 years old. In this study, the prevalence of anemia and IDA was 31% and 9%, respectively. The mean concentration of serum triglyceride, cholesterol and LDL in the anemia group and the IDA group was less than the control groups. The amount of HDL in different groups was almost the same, although the difference was statistically significant with respect to variables like age and obesity (P=0.001).Conclusion: The study showed that the amount of lipid profile (triglyceride and cholesterol) in the elderly with anemia and IDA was less compared to other people. This result was achieved in some other research studies too, but further research is suggested to find possible mechanisms.

Immune and metabolic responses of Chilo suppressalis Walker (Lepidoptera: Crambidae) larvae to an insect growth regulator, hexaflumuron

Efficient control of Chilo suppressalis Walker is always controversial due to highly economic damage, resistance to insecticides and environmental pollutions. So, combination of safe pest controls e.g. biocontrol agents and insect growth regulators seems to be promising via integrated pest management program. Bioassay of hexaflumuron on 4th larval instars revealed concentrations of 44.34, 179.74 and 474.94µg/ml as LC10-50 values. Numbers of total hemocytes, plasmatocytes and granulocytes as well as phenoloxidase activity increased in the different time intervals following treatment by hexaflumuron. Combined effects of hexaflumuron and Beauveria bassiana Vuillemin also increased hemocyte numbers and phenoloxidase activity at different time intervals using all concentrations. Activities of general esterases assayed by α- and β-naphtyl acetate and glutathione S-transferase using CDNB and DCNB increased 1-12h post-treatment. Activities of aspartate aminotransferase, alanine aminotransferase, γ-glutamyl transferase and aldolase increased in the larvae treated by hexaflumuron. However enhanced activity of lactate dehydrogenase was only obtained by treating 180 and 470µg/ml concentrations of hexaflumuron. Activities of ACP and ALP were found to be higher than control for all time intervals even 1-12h post-treatment. The amounts of HDL and LDL increased in the highest concentrations of hexaflumuron after 12-24h of post-treatment. Amount of triglyceride was higher than that of control after 1 and 3h but it was lower in other time intervals. Amounts of glycogen and protein were lower than those of control for all time intervals except for 6 and 12h of post-treatment in case of protein. Results of the current study revealed negative effects of hexaflumuron on intermediary metabolism of Chilo suppressalis but it increased the number of hemocytes and activity of phenoloxidase which are responsible for spore removal from hemolymph. It can be concluded that hexaflumuron is able to decrease survival and biological performance of C. suppressalis via intervening in intermediary metabolism but the given results showed incompatibility of the IGR with possible microbial control.

Study on the Effect of Paliurus spina-christi on Cholesterol, Triglyceride and HDL Levels in Diabetic Male Rats Fed a High Cholesterol Diet

Paliurus spina-christi Mill (Rhamnaceae) has been used in folk medicine as a hypocholesterolemic agent. In this study, the effect of total fruit extract on lowering lipid content of serum was investigated. Male rats were introduced streptozocin, subsequently fed with a high cholesterol diet and concomitantly treated with various doses of the extract. After 8 days, serum levels of cholesterol, triglyceride and HDL were measured. It was found that the administration of 100 mg/kg/day of the extract had reduced the amount of total cholesterol by 45.5% and the administration of 50 mg/kg/day reduced the amount of triglyceride by 48.73%. No significant enhancement was found in the amount of HDL. It was concluded that of Paliurus spina-christi extract can inhibit the enhancement of cholesterol and triglyceride.

Incubation of porcine high-density lipoprotein with the apical surface of LLC-PK1 renal tubular cells sustains the properties of orientated monolayers

HDL is present in the urine of patients with the nephrotic syndrome. The amount of HDL is directly related to the protein selectivity and therefore to the renal prognosis. Urinary HDL may therefore be involved in the pathogenesis of progressive renal impairment in proteinuric renal disease. LLC-PK1 cells were grown as orientated monolayers on filters. Uptake of HDL and permeability to inulin were measured. The influence of HDL in the growth medium on monolayer resistance, protein content, and sodium dependent glucose transport was studied. The effects of tetranitromethane (TNM) nitrosylation of HDL and of albumin, mevalonate, or simvastatin were investigated. Confluent LLC-PK1 monolayers took up fluorescently labelled HDL from either epithelial surface and formed a significant diffusion barrier to inulin. Monolayers incubated in 300 micrograms/ml HDL achieved a protein content and plateau of resistance equal to those in 10% fetal calf serum (FCS); 30-1000 micrograms/ml HDL applied to the apical surface of confluent monolayers maintained a plateau of resistance as well as 10% FCS and significantly better than serum-free medium. Sodium-dependent glucose transport was preserved in monolayers exposed to HDL. Simvastatin completely, and nitrosylation partially, removed the stimulatory properties of HDL. These were partly reproduced by albumin or mevalonate. HDL can enter renal epithelial cells from the apical surface. HDL added to this surface at confluence, reproducing the conditions found in the nephrotic syndrome, had a measurable positive effect on monolayer resistance. Results with nitrosylated HDL and HMG-CoA blockade suggest that these effects may be mediated via receptors and this enzyme system.

ポリアクリルアミドゲル電気泳動法によるHDL亜分画法についての2, 3の検討

An improved method was devised to estimate separately HDL2 and HDL3 by emproying a polyacrylamide-gel electrophoresis (PAGE).To serum or plasma was added a equal volume of the goat antiserum against human β-lipoprotein followed by centrifugation, and the supernatant which contains α-lipoprotein was prestained with Sudan-Black B. The stained material was subjected to PAGE with 7.75% gel for about 60 minutes at 4mA/one column. Our modification has two significant advantages; the method enables better separation of the two subfractions and it prevents the loss of the Sudan-Black B staining. It was noticed that a considerable amount of HDL was lost when Sudan-Black was added directly to serum (as in the original method by Miyahara) for some unknown reason, but when the staining was performed with the pre-separated α-fraction, the loss was much smaller. Also the use of 7.75% gel gave rise to a more distinct separation of subfractions as compared with those obtained with 6.75% gel. The application of the newly devised method provided the following findings. 1) The ratio of HDL2/HDL3 in women was twice as much that in men. Difference in ratio between young and old, however, was not significantly. 2) Cases showing a high HDL2/HDL3 ratio were found only in women. Higher HDL-C and lower TG were demonstrated in the cases with high HDL2/HDL3 ratio as compared with those in the low ratio group. 3) Convention of HDL3 to HDL2 was attempted by incubating the serum for 24hrs. at 37°C and it was found that the mobilities of HDL3 and HDL2 were decreased to 80 and 85%, respectively, as compared to their mobilities in the untreated serum. When the serum was added with PCMPS, a inhibitor for LCAT, before the incubation, the decrease of the mobilities was smaller; the extent of decrease was less than 10%.

Plasma Alpha-Lipoprotein Pattern and LCAT Activity in Acute Viral Hepatitis in Children

The alpha-lipoprotein (a-Lp) electrophoretic pattern on agarose gel was studied serially in 65 children with acute viral hepatitis. In seven cases the high-density lipoprotein fractions (HDL) were also separated by ultracentrifugation and studied in relation to lecithin: cholesterol acyltransferase activity (LCAT) during the acute phase and recovery. The a-Lp band was initially absent on electrophoresis in 36 cases and never appeared in 3 of the children who died. In those who survived a-Lp reappeared with clinical improvement even before a definite increase in S-ASAT and bilirubin was evident. In the seven children in whom it was measured LCAT activity ranged during the acute phase from 17.4 to 57 nmol h-1 ml-1. In patients with initially 'normal' LCAT activity (group I, no. = 3) HDL was normal both in amount and composition. In patients with 'low' LCAT (group II, no. = 4) HDL was reduced and of abnormal composition. In both groups the a-Lp band was absent or considerably reduced on electrophoresis. In the latter group LCAT activity increased significantly on recovery, as did the amount of HDL which became normal in composition. These results suggest that in acute viral hepatitis in children changes in composition and concentration of HDL depend on the degree of hepatic functional disturbance, as judged by plasma LCAT activity. Nevertheless, it appears that HDL electrophoretic behaviour may be abnormal during the early stage of the disease while plasma LCAT activity is not significantly affected.

Serum lipoproteins increase testosterone production in hCG-desensitized Leydig cells.

The effects of high density and low density lipoproteins (HDL and LDL) on testosterone synthesis by Leydig cells from rats treated three days earlier with 50 IU human chorionic gonadotropin (hCG) or from saline-injected control rats were investigated. HDL caused a significant, dose-dependent increase in both basal and hCG-stimulated testosterone production by Leydig cells from hCG-treated rats, but had no marked effect on Leydig cells from control rats. The serum testosterone concentration of hCG-treated rats was three times higher than in controls. Basal testosterone production by Leydig cells from hCG-treated rats was elevated two-fold in comparison to controls, while hCG-stimulated testosterone production was significantly lower than in controls. Both basal and hCG-stimulated testosterone production by Leydig cells from hCG-treated rats were significantly increased by the addition of HDL or LDL. Furthermore, HDL restored the hCG-stimulated testosterone production to the maximal amount produced by hCG-stimulated Leydig cells from control animals. When equivalent amounts of HDL or LDL cholesterol (140 microgram/ml) were added to the incubation, HDL caused a greater increase in testosterone production than did LDL. These results suggest that hCG-treatment, in vivo, caused a prolonged stimulation of the Leydig cells which resulted in depletion of the cholesterol available for steroidogenesis, since the addition of cholesterol in the form of HDL or LDL caused an increase in the testosterone production of these Leydig cells.

Plasma lipoprotein changes in experimental cholestasis in the dog

Plasma lipoproteins were compared before and after surgical induction of extrahepatic biliary obstruction in dogs by immunochemical techniques, zonal ultracentrifugation and gel filtration. A considerable increase in LDL was found one week after operation, with a zonal ultracentrifugal pattern which was more complex than that found before operation. The lipid composition was characterized by an increase of phospholipids and a decrease of triglycerides in cholestatic LDL fractions. None of the fractions displayed the inverse cholesterol-cholesterol ester ratio, characteristic for human LP-X. The apoprotein composition of cholestatic LDL was also markedly changed. Apoprotein A I, already present in the normal LDL 2 subclass, increased and two apoproteins with apparent molecular weights of 35 000–40 000 appeared in considerable amounts. The amount of HDL was decreased during cholestasis without any appreciable changes in composition.

HIGH DENSITY LIPOPROTEIN (HDL) POLYMORPHISMS IN RABBIT III. QUANTITATIVE DETERMINATION OF HDL AND THE INHERITED H1 1 AND R 67 ANTIGENS

Immunological quantification of HDL and the inherited antigens Hl 1 and R 67 was performed on serum samples from two rabbit populations (Dutch and Albino) comprising a total of 106 animals. The female animals were found to have a higher concentration of both antigens and of HDL than the male animals. The amounts of antigens and of HDL were not obviously normally distributed, but appeared to be more bimodally distributed. The relationship between the amount of antigens and amount of HDL were tested for each animal. A significant positive correlation was found between the amount of R 67 antigen and HDL both for male and female animals in the Albino rabbit population, and for male animals in the Dutch rabbit population. A significant correlation was also found between amounts of Hl 1 and R 67 antigens in the Dutch rabbit population.