Ultracentrifugation Of Plasma Research Articles

Plasma-derived extracellular vesicles improve mice embryo development.

To investigate the effect of plasma-derived extracellular vesicles (EVs) or conventional medium in fertilization and early embryo development rate in mice. MII oocytes (matured in vivo or in vitro conditions) were obtained from female mice. The extracellular vesicles were isolated by ultracentrifugation of plasma and were analyzed and measured for size and morphology by dynamic light scattering (DLS) and transmission electron microscopy (TEM). By western blotting analysis, the EVs proteins markers such as CD82 protein and heat shock protein 90 (HSP90) were investigated. Incorporating DiI-labeled EVs within the oocyte cytoplasm was visible at 23h in oocyte cytoplasm. Also, the effective proteins in the early reproductive process were determined in isolated EVs by western blotting. These EVs had a positive effect on the fertilization rate (P < 0.05). The early embryo development (8 cell, morula and blastocyst stages) was higher in groups supplemented with EVs (P < 0.01). Our findings showed that supplementing in vitro maturation media with EVs derived- plasma was beneficial for mice's embryo development.

Separation of postprandial lipoproteins: improved purification of chylomicrons using an ApoB100 immunoaffinity method

Elevated levels of triglyceride-rich lipoproteins (TRLs), both fasting and postprandial, are associated with increased risk for atherosclerosis. However, guidelines for treatment are defined solely by fasting lipid levels, even though postprandial lipids may be more informative. In the postprandial state, circulating lipids consist of dietary fat transported from the intestine in chylomicrons (CMs; containing ApoB48) and fat transported from the liver in VLDL (containing ApoB100). Research into the roles of endogenous versus dietary fat has been hindered because of the difficulty in separating these particles by ultracentrifugation. CM fractions have considerable contamination from VLDL (purity, 10%). To separate CMs from VLDL, we produced polyclonal antibodies against ApoB100 and generated immunoaffinity columns. TRLs isolated by ultracentrifugation of plasma were applied to these columns, and highly purified CMs were collected (purity, 90-94%). Overall eight healthy unmedicated adult volunteers (BMI, 27.2 ± 1.4 kg/m2; fasting triacylglycerol, 102.6 ± 19.5 mg/dl) participated in a feeding study, which contained an oral stable-isotope tracer (1-13C acetate). We then used this technique on plasma samples freshly collected during an 8 h human feeding study from a subset of four subjects. We analyzed fractionated lipoproteins by Western blot, isolated and derivatized triacylglycerols, and calculated fractional de novo lipogenesis. The results demonstrated effective separation of postprandial lipoproteins and substantially improved purity compared with ultracentrifugation protocols, using the immunoaffinity method. This method can be used to better delineate the role of dietary sugar and fat on postprandial lipids in cardiovascular risk and explore the potential role of CM remnants in atherosclerosis.

High-density lipoprotein therapy inhibits Porphyromonas gingivalis-induced abdominal aortic aneurysm progression.

Clinical and experimental studies have highlighted the potential implication of periondontal bacteria contamination in the pathogenesis of abdominal aortic aneurysms (AAA). In addition to their role in reverse cholesterol transport, high-density lipoproteins (HDLs) display multiple functions, including anti-inflammatory and lipopolysaccharide scavenging properties. Low plasma levels of HDL-cholesterol have been reported in AAA patients. We tested the effect of a HDL therapy in Sprague-Dawley rat model of AAA, obtained by intraluminal elastase infusion followed by repeated injections of Porphyromonas gingivalis (Pg). HDLs, isolated by ultracentrifugation of plasma from healthy human volunteers, were co-injected intravenously (10 mg/kg) with Pg (1.107 Colony Forming Unit) one, eight and 15 days after elastase perfusion. Rats were sacrificed one week after the last injection. Our results show that Pg injections promote the formation of a persistent neutrophil-rich thrombus associated with increased aortic diameter in this AAA model. HDLs significantly reduced the increased AAA diameter induced by Pg. Histology showed the onset of a healing process in the Pg/HDL group. HDL injections also reduced neutrophil activation in Pg-injected rats associated with decreased cytokine levels in conditioned media and plasma. Scintigraphic analysis showed an intense uptake of 99mTc-HDL by the AAA suggesting that HDLs could exert their beneficial effect by acting directly on the thrombus components. HDL supplementation may therefore constitute a new therapeutic tool for AAA treatment.

Plasma anti-α-galactoside antibody binds to serine- and threonine-rich peptide sequence of apo(a) subunit in Lp(a).

Lipoprotein(a) immune complexes [Lp(a) IC] of varying particle density obtained by ultracentrifugation of plasma from normal healthy donors were markedly dominated by IgG. Lp(a) and immunoglobulins were liberated from plasma Lp(a) IC by treatment with melibiose, a sugar specific for circulating anti-α-galactoside antibody (anti-Gal). Upon incubation with plasma lipoprotein fraction anti-Gal but not the α-glucoside-specific antibody from human plasma formed de novo IC with Lp(a). Binding of Lp(a) sugar-reversibly enhanced the fluorescence of FITC-labeled anti-Gal as did binding of α-galactoside-containing glycoproteins. This effect apparently due to conformational shift in the Fc region of the antibody was also produced by apo(a) subunit separated from Lp(a) and de-O-glycosylated apo(a) but not by any other plasma lipoproteins or by Lp(a) pre-incubated with the O-glycan-specific lectin jacalin. O-Glycans and their terminal sialic acid moieties in apo(a) of circulating Lp(a)-anti-Gal IC, in contrast to those in pure Lp(a), were inaccessible to jacalin and anion exchange resin, respectively. Unlike other plasma lipoproteins, Lp(a) inhibited Griffonia simplicifolia isolectin B4 which also accommodates serine- and threonine-rich peptide sequence (STPS) as surrogate ligand to α-galactosides at its binding site. Results suggest that anti-Gal recognizes STPS in the O-glycan-rich regions of apo(a) subunit in Lp(a) which contains no α-linked galactose.

Tamsulosin shows a higher unbound drug fraction in human prostate than in plasma: a basis for uroselectivity?

The efficacy-tolerability profile of tamsulosin in patients with benign prostatic hyperplasia (BPH) is assumed to be associated both with the α1-adrenoceptor selectivity profile of the drug and a small peak : trough ratio in the plasma pharmacokinetic (PK) profile. Tamsulosin is highly bound to plasma proteins, notably α1-acid glycoprotein (AGP). This protein is a high-affinity binding protein and AGP plasma concentration was found to influence the therapeutic (unbound) plasma concentrations for high-AGP-binding drugs. The study actually assessed unbound tamsulosin concentrations in both blood plasma and prostate tissue and reported that the unbound tamsulosin concentrations after multiple dosing in men with BPH, were much higher in prostate than in blood plasma. The assumption is put forward that differential free drug concentrations in prostate and blood plasma may contribute to the relative ‘uroselectivity’ of tamsulosin. The aim of this small patient study was to investigate tamsulosin concentrations in prostate and plasma samples in order to identify potential differences in the pharmacokinetics (PK) in plasma and prostate contributing to its pharmacodynamic activity profile in patients. Forty-one patients with benign prostatic hyperplasia (BPH) scheduled for open prostatectomy were given tamsulosin 0.4 mg for 6-21 days in order to reach steady-state PK. Patients were randomized over four groups to allow collection of plasma and tissue samples at different time points after last dose administration. Samples were collected during surgery and assayed for tamsulosin HCl. The free fraction (f(u)) of tamsulosin was determined by ultracentrifugation of plasma and prostate tissue spiked with (14)C-tamsulosin. C(max) in plasma at 4.4 h for total tamsulosin was 15.2 ng ml(-1) and AUC(0,24 h) was 282 ng ml(-1) h, while for prostate C(max) at 11.4 h post-dose was 5.4 ng ml(-1) and AUC(0,24 h) was 120 ng ml(-1) h. AUC(0,24 h) for total tamsulosin in prostate was 43% of the plasma AUC(0,24 h). f(u) was 0.4 % for plasma and 59.1% for prostate. Therefore calculated on unbound tamsulosin, a ratio of 63 resulted for prostate vs. plasma C(max) concentrations. These data indicate that in patients with confirmed BPH the amount of tamsulosin freely available in the target tissue (prostate) is much higher than in plasma.

Possible widespread low-level occurrence of murine leukemia virus-related gene sequences in humans

The article by Lo and colleagues (1) is widely reported as a confirmation of the earlier report of Lombardi et al. (2). In fact, there are major differences, including the inability to detect xenotropic murine leukemia virus-related virus in their patient cohort, but rather finding evidence for polytropic murine endogenous γ-retroviruses. More significant is the clear difference between the absence, except in one patient with chronic fatigue syndrome (CFS), of virus envelope sequences in the publication of Lo et al. (1), versus a finding of envelope sequences in 7 of 11 tested patients with CFS in the study of Lombardi et al. (2). The results are also presented as a true qualitative distinction between CFS patients and healthy controls. The use of the nested PCR at near the limits of its sensitivity, however, does not exclude a lower than detectable level of mouse endogenous retroviruses in many healthy individuals. The numerous “nonspecific” bands generated in the nested PCR assays on control and patient samples, as reported by Lo et al. (1), mean that any retroviral sequences would need to compete for primers with the nonspecific reactions occurring with normal DNA. The primer cross-reactivity with DNA sequences other than those of murine retroviruses limits the possible detection of authentic murine retroviral sequences potentially present in DNA of healthy controls. Quantitative increases in the levels of various microbes are characteristic of many CFS cases, and this may also apply to murine retroviruses. It is noteworthy that the electrophoresis data illustrated on the control and patient samples (figures 1B and 2B in ref. 1) were obtained using different nested primers. The authors may wish to comment on whether the patterns of nonspecific bandings were essentially identical in the patients versus the control samples using the same secondary sets of nested primers. Filtering of plasma before ultracentrifugation of plasma would help eliminate the possibility of viral RNA being present in cellular debris rather than as cell-free virus particles. Finally, in concluding an association of murine retroviruses and CFS, it would have been preferable to test matched controls from the same geographic area as the CFS patients and to include patients with non-CFS-related chronic illnesses.

Distribution of PCB congeners, DDE, hexachlorobenzene, and methylsulfonyl metabolites of PCB and DDE among various fractions of human blood plasma.

The concentrations of chlorinated biphenyls (CBs), 1, 1-bis(4-chlorophenyl)-2,2-dichloroethene (DDE), hexachlorobenzene (HCB), and the methylsulfonyl metabolites of CBs (MeSO(2)-CBs) and DDE (MeSO(2)-DDE) were determined in human plasma samples and in the fractions obtained by ultracentrifugation of plasma into very-low-density (VLDL), low-density (LDL), high-density (HDL) lipoprotein and lipoprotein depleted (LPDP) fractions (containing primarily albumin). The concentrations of triacylglycerols, cholesterol, phospholipids, and apolipoprotein B (apoB) were determined. The organochlorine compounds were associated with all fractions, but predominantly with the LPDP fraction. On an average 44% of CBs, 61% of MeSO(2)-CBs, 73% of DDE, 77% of MeSO(2)-DDE, and 45% of HCB were distributed in the LPDP fraction. A tendency to greater association of 3-methylsulfonyl substituted than of corresponding 4-methylsulfonyl substituted chlorobiphenyls to the LPDP fraction was noticed. Among the lipoprotein fractions, LDL was the main carrier of HCB, DDE and CBs. MeSO(2)-DDE was predominantly found in HDL and MeSO(2)-CBs were distributed equally among the LDL and HDL fractions. Calculating the concentrations of organochlorine compounds in relation to the content of apoB, the levels were about 10 times higher in VLDL than in LDL.

Effects of vitamin C and of a cell permeable superoxide dismutase mimetic on acute lipoprotein induced endothelial dysfunction in rabbit aortic rings.

Low density lipoprotein (LDL) inhibits endothelium-dependent relaxation. The mechanism is uncertain, but increased production of superoxide anion O2- with inactivation of endothelium-derived NO and formation of toxic free radical species have been implicated. We investigated effects of the cell permeable superoxide dismutase mimetic manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin (MnTMPyP), the free radical scavenger vitamin C and arginine (which may reduce O2- formation) on acute LDL-induced endothelial dysfunction in rabbit aortic rings, using LDL prepared by ultracentrifugation of plasma from healthy men and aortic rings from New Zealand white rabbits. LDL (150 microg protein ml(-1) for 20 min) markedly inhibited relaxation of aortic rings (in Krebs' solution at 37 degrees C and pre-constricted to 80% maximum tension with noradrenaline) to acetylcholine 82+/-10% (mean percentage difference between sum of relaxations after each concentration of acetylcholine in the presence and absence of LDL, +/-s.e.mean, n=26, P<0.001) but not to the endothelium-independent agonist nitroprusside. MnTMPyP (10 microM) reduced inhibitory effects of LDL from 124+/-27 to 56+/-17% (n=6, P<0.05). Vitamin C (1 mM) reduced inhibitory effects of LDL from 59+/-8 to 22+/-5% (n=6, P<0.05). Inhibitory effects of LDL were similar in the absence or presence of arginine (84+/-12 vs 79+/-16%, n=14, P=0.55). Effects of L-arginine (10 mM) did not differ significantly from those of D-arginine (10 mM). Acute (20 min) exposure of aortic rings to LDL impairs endothelium-dependent relaxation which can be partially restored by MnTMPyP and vitamin C. This is consistent with LDL causing increased O2- generation.

Development of a method to separate lipoprotein-bound and lipoprotein-depleted tissue factor pathway inhibitor. Measurement of free tissue factor pathway inhibitor activity.

The aim of this study was to develop a method to separate lipoprotein-bound from lipoprotein-free tissue factor pathway inhibitor (TFPI) and to measure the TFPI chromogenic activity and antigen in both fractions. This was performed by ultracentrifugation of plasma, after increasing its density to 1.21 g/ml with potassium bromide. Blood was taken from nine volunteers before and after an injection of low-molecular-weight heparin. The ultracentrifugation procedure was adequate, since the mean cholesterol recovery was 91% and only 2% of the cholesterol remained in the lipoprotein-depleted fraction. No free TFPI protein was found in the lipoprotein-rich fraction. Moreover, the amount of free TFPI in the lipoprotein-depleted fraction was close to that found in plasma. Using this method, we confirmed that heparin does not induce an increase in bound TFPI and that the moderate increase in total TFPI antigen in plasma is entirely caused by the enhancement of free TFPI. We then applied the TFPI chromogenic assay to the lipoprotein-depleted fraction to assess the activity of free TFPI. The activity was 0.11+/-0.03 and 0.36+/-0.08 U/ml before and after heparin, respectively (a 3.6-fold increase) while the activity of bound TFPI did not increase at all. We suggest that this method may be an alternative to gel filtration for measuring free TFPI activity, and might be of value in the search for TFPI abnormalities in patients with thrombosis.

High correlation but lack of agreement between direct high-performance gel chromatography analysis and conventional indirect methods for determining lipoprotein cholesterol

Low-density lipoprotein-cholesterol (LDL-C) is currently estimated clinically by using the Friedewald formula, when plasma triglycerides are < 4000 mg/L, or as the difference between infranatant and high-density lipoprotein-cholesterol (HDL-C) values after ultracentrifugation of plasma at native density, when plasma triglycerides are > or = 4000 mg/L (beta quantification). HDL-C is measured by selective precipitation of apolipoprotein B-containing lipoproteins from whole plasma or from the density > 1.006 kg/L infranatant. We compared these conventional methods for LDL-C and HDL-C with "high-performance" gel chromatography (HPGC), a method that directly and simultaneously measures both LDL-C and HDL-C in a single, microliter volume of plasma. Not surprisingly, we found that the results by all these methods were highly correlated. However, LDL-C values were significantly higher and HDL-C values significantly lower by the direct HPGC method than by the conventional methods (paired t-test). In addition, both Bland-Altman plots and concordance correlation analyses indicated lack of agreement between the methods' results in the majority of patients' subgroups.

Human intestinal alkaline phosphatase—release to the blood is linked to lipid absorption, but removal from the blood is not linked to lipoprotein clearance

To evaluate a possible quantitative relationship between the rise in intestinal alkaline phosphatase (IAP) activity and triglyceride-rich lipoproteins in the blood after an oral fat intake, a specific and sensitive immunocatalytic assay was used. First, day to day variation of the basal IAP activity in the blood of eight volunteers was evaluated. One group of subjects with high basal IAP activity and great variations from one day to the other was distinguished from a group with low basal IAP activity and small day to day variations. The subjects with high basal IAP activities were all secretors of red blood cell antigens, while those with low basal IAP activities were non-secretors. The IAP activity in the blood rose after fat rich meals but not after fat free meals. To further investigate this, IAP activities were measured in the blood of 28 men before 3, 6, 9 and 12 h after a standardized oral fat load of a mixed meal type. The activity rose in all individuals and showed a similar time course as the rise in plasma TG concentration. The elevations of the IAP activity were, however, not quantitatively correlated with the elevations in TG concentration, nor were there any relations in time to peak levels. Subjects with high basal IAP increased their IAP activity more than those with low basal IAP, but within neither group there was any correlation between the basal IAP activity and the rise in IAP activity. IAP did not float with lipoproteins on ultracentrifugation of plasma, nor did IAP bind to lipid droplets from a fat emulsion added to plasma.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasma HIV-1 viremia in HIV-1 infected individuals assessed by polymerase chain reaction.

We established a method to estimate the amounts of HIV-1 particles in plasma from patients with HIV-1 infection by using polymerase chain reaction (PCR) following reverse transcription (RT) of viral RNA (RNA-PCR) and assessed the potential usefulness of this approach to monitor the changes of viral load in patients with AIDS or AIDS-related complex (ARC) receiving 2',3'-dideoxyinosine (ddI). Plasma samples were obtained from 77 patients with HIV-1 infection (49 AIDS/ARC and 28 asymptomatic seropositives). Following ultracentrifugation of plasma, RNA was extracted from the pelleted virus and subjected to RT and PCR. The number of HIV-1 virus particles in each sample was determined using known amounts of HIV-1 DNA as reference control for PCR. The current plasma RNA-PCR technique quantitatively detected HIV-1 particles in plasma from 76 of 77 (98.7%) HIV-1-infected individuals examined. The numbers of HIV-1 particles in plasma from patients with AIDS or ARC were markedly higher than those in plasma from asymptomatic seropositive individuals (p less than 0.0001). Higher levels of plasma HIV-1 particle numbers were detected in individuals with lower CD4+ T cell counts. Patients (n = 10) who received oral ddI at doses greater than or equal to 6.4 mg/kg/day for 8 to 14 weeks had a profound decrease in plasma HIV-1 particle numbers (p = 0.0051). Patients (n = 7) receiving ddI for 45 to 71 weeks also had a decrease (p = 0.018). It should be noted, however, that more research is required to evaluate the usefulness of this technique in assessing the disease status and monitoring the activity of antiretroviral therapy.

Modulation of TXA 2 generation of platelets by human lipoproteins

The lipoprotein (LP) fractions VLDL, LDL, HDL 2 and HDL 3 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 A 2 (TXA 2) during spontaneous clotting of whole blood under the influence of the lipoprotein fractions. In our experiments the serum concentration of TXB 2, reflecting the capacity of platelets to generate TXA 2 during clotting, depends on several factors: the type of LP fraction used, the blood used for generation of TXA 2, 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 TXA 2 formation of platelets from healthy volunteers (p < 0.05, resp.). LDL from CHD patients inhibited the TXA 2 formation of platelets from atherosclerotic patients (p < 0.01). The HDL subfractions HDL 2 and HDL 3 from healthy volunteers inhibated TXA 2 formation by platelets from healthy volunteers as well as those from atherosclerotic patients (p < 0.05; p < 0.01, respectively). HDL 2 from patients with CHD inhibited only the TXA 2 formation of platelets from healthy volunteers (p < 0.01), whereas HDL 3 from CHD patients inhibited only the TXA 2 formation of platelets from atherosclerotic patients (p < 0.01).

Responses of Plasma Apolipoproteins to Gonadectomy and Androgen Substitution in Male Rats

To obtain information on testosterone effects on plasma apolipoproteins, the amount and composition of apo-proteins in lipoproteins of 5 density classes (VLDL, LDL, HDL2b, HDL2a, HDL3) was estimated in 3 groups of adult male rats: normal control rats, castrated rats, and rats injected daily with testosterone propionate (200 micrograms/day) for one week after castration. Apoproteins were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis after ultracentrifugation of plasma, and determined colorimetrically. Total amount of apoprotein carried in LDL (d = 1.006-1.063 g/ml) and HDL2b (d = 1.063-1.100 g/ml) was higher in castrated than in control rats, but was not significantly different from controls in testosterone substituted rats. LDL apo B and HDL2b apo E were higher in castrated than in normal rats; control levels were observed in androgen substituted rats. Except for a greatly increased relative amount of HDL2b apo E, and a decreased percentage of HDL2b apo A-I in castrated rats, there were no significant alterations by castration of apoprotein composition of the lipoproteins. The results raise the question whether the androgenic state might affect processes related to the effects of plasma LDL apo B and HDL apo E.

Failure of ultracentrifugation as a means of separating plasma free insulin from immunoglobulin fraction prior to radioimmunoassay

It has been reported that ultracentrifugation of plasma will allow direct measurement of free insulin in the serum of diabetic subjects with insulin antibodies. To validate this method, we determined recovery of immunoreactive insulin and immunoglobulin G from the plasma of normal individuals after ultracentrifugation. The upper and middle fractions of plasma after ultracentrifugation were evaluated at several combinations of time and temperature (4°C, 25°C, or 37°C for 3, 4 or 5 hours). None of these conditions effectively removed all immunoglobulin G without causing concomitant loss of insulin. We conclude that ultracentrifugation of plasma prior to radioimmunoassay cannot be used to reliably determine free insulin concentration in the plasma from subjects with circulating insulin antibodies.

Separation of the high-molecular-mass "immunoreactive ACTH" measured with the CIS-Sorin Kit by ultracentrifugation of plasma.

Separation of the high-molecular-mass "immunoreactive ACTH" measured with the CIS-Sorin Kit by ultracentrifugation of plasma.

Side-chain oxidation of lipoprotein-bound [24,25-3H]cholesterol in the rat: comparison of HDL and LDL and implications for bile acid synthesis.

The purpose of the study was to test the hypothesis that high density lipoprotein (HDL) cholesterol would be more easily oxidized in vivo than low density lipoprotein (LDL) cholesterol. Homologous plasma was incubated with [24,25-3H]cholesterol and fractionated by ultracentrifugation to obtain HDL and LDL each labeled with [3H] free sterol. HDL and LDL labeled with [24,25-3H]cholesteryl esters were prepared by ultracentrifugation of plasma from donor rats injected 24 hr previously with [24,25-3H]cholesterol in propylene glycol. These four labeled lipoproteins were administered to recipient rats. It was found that more tritium oxide (3H2O) was produced after the HDL doses than after the corresponding LDL doses, from 2--3-fold more when lipoprotein free cholesterol was labeled and from 2--6-fold more when lipoprotein cholesteryl esters were labeled. More 3H2O was produced from free cholesterol-labeled lipoproteins than from cholesteryl ester-labeled lipoproteins. Since oxidation of cholesterol is a measure of bile acid formation, it is concluded that under the conditions of the study HDL-cholesterol is a better precursor of bile acids than LDL-cholesterol.

Plasma retinol and retinol binding protein concentrations in patients on maintenance haemodialysis with and without vitamin A supplements.

Plasma retinol and retinol-binding protein (RBP) concentrations have been estimated in patients on maintenance haemodialysis over a 4-year period. For the first 2 years multivitamin supplements containing vitamin A were taken, and for the second 2 years no vitamin A supplements were given. Mean plasma retinol concentrations decreased significantly but only from 3.8 times normal to 3.1 times normal after vitamin A supplements stopped. There was no significant change in th high plasma RBP levels. Ultracentrifugation of plasma at a salt density of 1.21 showed that nearly all the retinol was associated with RBP in the high density protein fraction, as it is with normal subjects. Column chromatography confirmed that there was no increase in plasma retinyl esters in the renal failure patients, as is found in hypervitaminosis A due to drug overdosage. The high plasma retinol and RBP levels remained remarkably stable in individual patients throughout the 4-year study. The increase in plasma RBP was possibly related to residual urine output. The results are compatible with a feedback mechanism whereby the extent of the increase in plasma RBP as renal failure develops controls the consequent high plasma level of retinol.

Radioimmunoassay studies of human apolipoprotein E.

This report describes the development and first applications of a sensitive and specific double antibody radioimmunoassay for human apoplipoprotein E (apoE). ApoE was purified from the very low density lipoproteins of hypertriglyceridemic patients by heparin-agarose affinity chromatography, DEAE-cellulose chromatography, and preparative polyacrylamide gel electrophoresis. The purified apoprotein had an amino acid composition characteristic of apoE and resulted in the production of monospecific antisera when injected into rabbits. The radioimmunoassay, which was carried out in the presence of 5 mM sodium decyl sulfate, had a working range of 0.8-12 ng. The withinassay coefficient of variation was 9% and the coefficient of variation for systematic between-assay variability was 3%. Prior delipidation of samples with organic solvents did not alter their immunoreactivity. In 26 normal volunteers, the mean plasma apoE concentration was 36 +/- 13 microgram/ml. Hyperlipidemic patients (n = 68) had higher mean apoE levels. A single patient with type III hyperlipoproteinemia had a plasma apoE level of 664 microgram/ml. The plasma apoE level was independently related to plasma cholesterol and triglyceride levels in a population of 108 normal and nonchylomicronemic hyperlipidemic patients. The multiple correlation coefficient for this relationship was 0.73. Thus, variation in plasma cholesterol and triglyceride concentrations described 53% of the variation in apoE concentrations in this population. The lipoprotein distribution of apoE was investigated by agarose column chromatography and ultracentrifugation of plasma. Agarose column chromatography demonstrated that all or nearly all plasma apoE is associated with lipoproteins. In plasma from normal volunteers and hypercholesterolemic patients, apoE was found in two discrete lipoprotein classes: very low density lipoproteins and a set of lipoprotein particles with size and density characteristics similar to HDL2. In hypertriglyceridemic patients, nearly all apoE was associated with the triglyceride-rich lipoproteins.

SINKING PRE-BETA LIPOPROTEIN AND CORONARY HEART DISEASE IN JAPANESE-AMERICAN MEN IN HAWAII

A "sinking" pre-beta lipoprotein was sought in a probability sample of 1854, 50--72-year-old men of Japanese ancestry in Honolulu by ultracentrifugation of plasma and electrophoresis of the bottom fraction (density greater than 1.006) in agarose. A definite electrophoretic band was found in 5.6% of the men and a trace band was found in 4.6% of them. The frequency of such a band increased with age and decreased with adiposity. The relative risk for coronary heart disease, based on prevalence cases was found to be 1.7 in men with a definite band, and 1.4 in those with a trace band, when compared to men without. This association could not be explained by the higher low density lipoprotein levels in men with trace or definite bands. These data are consistent with previous reports suggesting that the Lp antigen (for which a sinking pre-beta lipoprotein is probably an insensitive marker) is associated with coronary disease.