Stability of high-density-lipoprotein cholesterol in frozen sera.

Studies of the stability of cholesterol content of high-density lipoprotein (HDL-chol) in stored sera have had mixed findings: increases, decreases, and no change in values have been reported (1-7). Bachorik et al. (4) demonstrated that values for samples with low HDL-chol tend to increase during storage and those for samples with above-normal HDL-chol tend to decrease. Lipoproteins containing apolipoprotein B (apo-B)-i.e., lowand very-low-density lipoproteins (LDL and VLDL)-reportedly became progressively difficult to precipitate and HDL became progressively easier to precipitate after storage, at least with the heparinfMn2 precipitation method(4). Earlier, we reported (8) that values for two serum pools with below-normal and above-normal HDL-chol concentrations were stable when the pools were stored for more than 18 months at -20 #{176}C, when determined by heparin! Mn2 precipitation. Because these findings apparently did not support the results of Bachorik et al., we studied this problem further. We prepared two new serum pools with below-normal and above-normal concentrations of IIDL-chol (as in ref. 8), stored them at -20 #{176}C, and determined the concentrations almost each week, by precipitation with polyethylene glycol (PEG) (9), during a year (n = 49). Again, we found no significant correlation between HDL-chol concentration and duration of storage (r = -0.22, p> 0.10 for the low-value pool, and r = -0.26, 0.05 < p < 0.10 for the high-value pool). The CVs were 4.4% and 3.0%, respectively. We therefore wondered whether there was any difference between the stability of the serum pools and that of clinical serum samples. To test this, we determined HDL-chol by the PEG precipitation method in serum from apparently healthy persons and from patients with different types of hyperlipoproteinemia. We assayed 10 sera for serum lipids and HDL-chol within four days of blood sampling, with storage meanwhile at 4 #{176}C. After these determinations we then stored the sera at -20 #{176}C for a week and repeated the analyses after thawing. This procedure was repeated the second and third week with two other series of 10 sera. Thus, the values before and after storage were the mean of three different runs (to minimize the influence of the imprecision). Similarly, we studied the stability at -20 #{176}C during storage for three, six, eight, and 10 months for three series of sera. Total serum cholesterol remained constant throughout the study period (Table 1). HDL-chol tended to decrease (p = 0.07) after storage for one week and was significantly lower (p < 0.00 1) after storage for three to 10 months. The decrease of HDL-chol was greatest in the first three months and was independent of the concentrations of triglycerides and HDL-chol. Thus, at least as determined with the PEG method, apparent HDL-chol in sera decreases after storage at -20 #{176}C. These findings support the data obmined with the heparin/Mn2 procedure: when apo-B-containing lipoproteins, especially VLDL and intermediate-density lipoproteins (IDL), are completely removed, then decreased HDL-chol values can be expected, even when serum is stored at -20 #{176}C. The complete removal of apo-B-containing lipoproteins is most likely in sera with above-normal values of HDL-chol, which usually also have the smallest amounts of VLDL and IDL. Using the PEG method for HDL, we did not notice that after storage the removal of apo-B-containing lipoproteins from lipemic sera was any more difficult or incomplete than was the case for unfrozen sera. Evidently, the results for stability of serum pools at -20 #{176}C do not necessarily represent the stability of patients’ sera, because the serum pools had already been stored and thawed and they had also undergone other manipulations (ultracentrifugation, dilution) before the first HDL-chol determination was done.