A case of hyperalphalipoproteinemia associated with albumin complexing.

We present a 3 year follow up of a new type of hyperalphalipoproteinemia with stable high density lipoprotein (HDL) concentrations around 6.0 mmol/l in a female with persistently elevated erythrocyte sedimentation rate (ESR). By density gradient ultracentrifugation, next to the intensively colored HDL2-fraction, an additional band between HDL3 and the serum proteins was seen consistently. The patient's plasma contained an unique complex of albumin with apoprotein A-I. Her IgM was 3- to 4-fold elevated was not complexed to HDL. Familial forms of hyperalphalipoproteinemia could be excluded. As a presumed reason for the existence of the HDL-albumin complex we found that the patient's post-heparin plasma lipoprotein lipase activity was over 2-fold increased, while that of hepatic lipase was over 2-fold decreased: no common cause for the existence of the albumin complex and the increased concentration of IgM was found.

Indications for the presence of circulating peroxidized low density lipoproteins in WHHL rabbits treated with antioxidants.

Low density apolipoprotein (apo) B-100 in homozygous WHHL rabbits appears heterogeneous on SDS-PAGE. At least four high molecular weight apolipoproteins were identified in contrast to the uniform apo B-100 band in the very low density lipoprotein (VLDL) fraction of WHHL-homozygotes and the apo B containing lipoproteins present in WHHL heterozygotes or New Zealand White rabbits. We studied whether these aberrations in homozygote WHHL-LDL were due to physical changes induced by lipid peroxidation. Therefore we treated WHHL rabbits for 28 days with a standard dose of 1% probucol or with a dose of 0.1% vitamin E and studied the parameters indicating lipid peroxidation in circulating LDL and in vitro in relation to its antioxidant content. The LDL of probucol-fed rabbits appeared completely resistant against oxidation in vitro with Cu2+ ions. LDL fluorescence and serum malondialdehyde concentrations, indicators of lipid peroxidized LDL, were lower than in the control WHHL rabbits. LDL of vitamin E-fed WHHL rabbits showed a twofold increased lag phase in comparison with LDL of control-fed animals; the maximal rate of oxidation was 2- to 3-fold lower while LDL fluorescence was between the values obtained in the two other groups. Malondialdehyde concentration in the vitamin E-treated group was also decreased when compared with controls. Despite these indications of increased lipid peroxidized circulating LDL in WHHL controls which could be reversed, at least partially, by the antioxidant treatments applied, these treatments were without effect on the physical structure of LDL as examined with agarose gel electrophoresis. Neither antioxidant treatment changed the typical apo B-100 pattern in WHHL-LDL.

Studies on the function of hepatic lipase in the cat after immunological blockade of the enzyme in vivo.

In order to investigate the in vivo function of hepatic lipase, cats were injected with anti-cat hepatic lipase antibodies which produced a complete and specific inhibition of heparin-releasable hepatic lipase. The cat was chosen as an animal model because it displays, like man, a relative deficiency of lipoprotein lipase compared to hepatic lipase and because the possession of two subfractions of high density lipoproteins, HDL2 and HDL3. In fasted cats no changes were observed in plasma triglycerides or phospholipids. In fed animals triglycerides increased considerably, indicating that hepatic lipase may have a function in the postprandial phase. In fat-loaded cats (6 g of fat/kg) triglycerides in the d less than 1.019 g/ml fraction increased from 4 h after the blockade due to accumulation of lipoproteins with pre-beta-mobility containing the apoproteins, apo B-100, apo E and apo A-I. Apo B-48 did not accumulate consistently. Phospholipids in the HDL2-fraction and those in the HDL3-fraction of the fat-loaded cats tended to increase and decrease from 6 and 9 h after the blockade, respectively. The absolute change in HDL2 phospholipids approximated that of HDL3-phospholipids. Overall, the density of HDL particles decreased, apparently secondary to the accumulation of apo A-I in the d less than 1.019 g/ml fraction. Our findings suggest that hepatic lipase is involved in the hydrolysis of a special class of apo A-I containing triglyceride-rich lipoproteins synthesised in the postprandial phase.

Evaluation of the dual-precipitation method for determination of cholesterol in high-density lipoprotein subfractions HDL2 and HDL3 in serum.

We compared the dual-precipitation method for measurement of cholesterol in high-density lipoprotein subfractions HDL2 and HDL3 (Gidez et al., J Lipid Res 1982;23:1206-33) with density-gradient ultracentrifugation in a swinging-bucket rotor (Demacker et al., Clin Chem 1983;29:656-63). The concentration of dextran sulfate 15,000 (DS) needed for optimal accuracy of the HDL2-chol and HDL3-chol values was established empirically. At a DS concentration of 0.87 g/L, the values for HDL2-chol as well as for HDL3-chol in 88 sera did not differ significantly from those obtained by ultracentrifugation. The precision of the method was satisfactory and was related to the concentration. Nevertheless, the dual-precipitation method lacks specificity inasmuch as it produces no fractions that contain only one HDL subfraction. HDL2 and HDL3 each contained an equivalent amount of cholesterol from the other. At increasing DS concentrations, some radiolabeled HDL3 appeared to have precipitated prior to complete precipitation of HDL2. This lack of specificity can be tolerated in large-scale epidemiological studies for screening, but not in small-scale intervention studies or in assay of clinical samples, where better accuracy is needed and ultracentrifugation is preferred.

Five methods for determining low-density lipoprotein cholesterol compared.

We evaluated three precipitation methods for determination of low-density lipoprotein cholesterol in serum and an indirect method involving the Friedewald formula (Clin Chem 18: 499-502, 1972) by comparison with results by ultracentrifugation. The results of all methods for 83 sera, including 59 hyperlipidemic type IIA, IIB, and IV sera agreed very well, at least for concentrations of serum triglycerides below 8 mmol/L. The accuracy of the Friedewald formula was confirmed in 285 other sera, including 66 sera with triglycerides content between 4.52 and 8.0 mmol/L. For type III sera, the precipitation methods produced similar values to those obtained with the Friedewald formula, all being much higher than the ultracentrifugation values. Density-gradient ultracentrifugation showed that the very-low-density lipoprotein remnants in type III sera almost completely coprecipitated with the low-density lipoproteins. The precipitation methods are not only accurate but also very precise (CV less than 5%); they can therefore be used in clinical laboratories to measure atherogenic low-density lipoproteins plus the remnants of very-low-density lipoproteins. However, when serum triglycerides and high-density lipoprotein cholesterol also are determined, the Friedewald formula is a reliable alternative.

Enzymic and chemical-extraction determinations of free fatty acids in serum compared.

We compared an enzymic test kit for determination of free fatty acids in serum (NEFA C-test, WAKO) with two modifications of a chemical extraction procedure, I (Clin. Chim. Acta 43: 317-320, 1973) and II (Clin. Chim. Acta 80: 327-332, 1977). All three procedures are specific for long- and medium-chain fatty acids. Short-chain fatty acids, some keto acids, and phospholipids did not interfere. Added fatty acid was quantitatively accounted for in all methods. Results obtained with the enzymic method and II did not differ significantly, whereas the results by I were about 10% lower. The concentration of NaCl in the copper reagent, but not the kind of solvent used to dissolve the standard, influenced the accuracy of the chemical methods. In the enzymic procedure, hydrolysis of triglycerides during incubation is unlikely to be the reason for too-high values. The precision of all three procedures is acceptable for use in clinical laboratories.

Stability of frozen liquid control sera for assay of cholesterol in high-density lipoprotein.

Liquid serum pools with low-, normal-, or above-normal concentration of high-density lipoprotein cholesterol were prepared by selection and dilution of sera having low lipoprotein content or by enrichment with concentrated high-density lipoprotein. Stability of the serum pools depended on storage conditions. On storage between -2 and -12 degrees C, the high-density lipoprotein cholesterol content decreased. At a constant temperature of -19 to -21 degrees C the concentrations remained stable for more than 18 months. The imprecision (CV) of the high-density lipoprotein cholesterol assay during this period as established with enzymic cholesterol analysis (Clin. Chem. 26: 1780-1786, 1980) of these serum pools was between 2.7 and 4.8% (n = 51). Serum pools prepared and stored as described are suitable for internal quality-control procedures. In external quality-control trials these sera may be superior to the commercially available lyophilized lipid control sera.

A study of the use of polyethylene glycol in estimating cholesterol in high-density lipoprotein.

We studied polyethylene glycol 6000 precipitation of lipoproteins other than high-density lipoproteins, before cholesterol is estimated in the supernate. Other lipoproteins in the supernatant fractions were detected by using rocket immunoelectrophoresis. A polyethylene glycol concentration of 75 g/L in the final mixture appeared to be optimal, and results agreed with those obtained by ultracentrifugation. Differences in serum pH, use of polyethylene glycol from different suppliers, or the presence of ethylenediaminetetraacetate resulted in values that differed significantly (by 40 to 60 mumol/L) from the reference values. Polyethylene glycol did not interfere in four different methods for determination of cholesterol. In combination with an enzymic cholesterol method, the polyethylene glycol method appeared to be very precise, even when lipemic sera (triglycerides up to 5.5 mmol/L) were analyzed that had diminished high-density lipoprotein cholesterol values. We consider this method a method of choice, especially when lipemic sera are tested and enzymic cholesterol analysis is used.