Evaluating lyophilized human serum preparations for suitability as proficiency testing materials for high-density lipoprotein cholesterol measurement.

OBJECTIVE: To evaluate the suitability of various commercial preparations for use by the College of American Pathologists as survey materials in assessing high-density lipoprotein cholesterol measurement performance. DESIGN: Lyophilized human serum preparations from six vendors (vendors A through F) were evaluated to determine which material(s) best mimicked the commutability of fresh human serum. Two freshly collected unfrozen pools prepared from donor specimens were analyzed concurrently with the vendor materials to identify sources of variation and possible matrix bias. Each material was evaluated using 5 common precipitation reagents (phosphotungstate-magnesium, phosphotungstic acid, dextran sulfate [50K and 500K], and heparin-manganese). To evaluate how each reagent separates lipoproteins in each material, the lipoprotein separation patterns were profiled using high-pressure liquid chromatography and compared with separation patterns observed for the fresh human serum pools. MAIN OUTCOME MEASURES: Similarities in performance characteristics of vendor material(s) were compared with fresh human serum. RESULTS: Two of the six materials gave separation profiles for the lipoproteins similar to the typical patterns observed for human serum. Material from vendor B showed the best commutability across all of the precipitation reagents and had the best combination of low overall variability (10% for level 1 and 9.4% for level 2) and minimal concentration differences among reagents. CONCLUSIONS: Vendor B was selected by the College of American Pathologists to provide materials for use in assessing performance of lipid and lipoprotein testing in the 1994 Comprehensive Chemistry Surveys. This study demonstrates the great variability that different vendor preparations introduce into the measurement of high-density lipoprotein cholesterol. It also emphasizes the effort required to evaluate the suitability of processed materials for use in proficiency testing.

Proteolytic digestion in the elucidation of the structure of low density lipoprotein.

The apoprotein (apoB) of low density lipoprotein (LDL) is reported to be a large polypeptide, and it is proposed that there are two similar-sized subunit proteins in LDL (Smith, Dawson, and Tanford. 1972. J. Biol. Chem. 247: 3376-3381.). When apoB is isolated under conditions that minimize artifactual proteolysis, only a single, large molecular weight protein appears on polyacrylamide gel electrophoresis in SDS. To investigate the organization of apoB as it exists within native LDL, limited proteolysis with trypsin has been used as a structural probe. Tryptic digestion for 1 hr at pH 7.6 with enzyme-to-protein ratios of 1:100 and 1:5 results in the liberation of approximately 10% and 30% of apoB as smaller, water-soluble peptides. These peptides may be separated from the partially digested but still intact tryptic core (T-core) of the lipoprotein by chromatography on Sephadex G-75. Repeatedly, the 1:5 T-core of native LDL is found to contain a family of polypeptides of 14,000-100,000 molecular weight. Although they have lost significant quantities of apoprotein, these T-cores sustain an appearance of homogeneity, as studied by analytical ultracentrifugation. Their measured molecular weights do not differ appreciably from those of the native LDL, and the carbohydrate content of the 1:5 tryptic T-core of LDL is similar to that of the native LDL. In normolipemic individuals, LDL generally exists in a monodisperse state, but, in different individuals, monodisperse LDL may range in molecular weight from 2.4 to 3.9 x 10(6). Limited tryptic digestions were used to probe the organization of apoB in these different molecular weight LDL. As assayed by SDS-acrylamide gel electrophoresis of the larger polypeptides and fingerprinting of the smaller released peptides, those regions of LDL exposed to trypsin digestion are identical in monodisperse LDL of 2.5 and 3.4 x 10(6) molecular weight. Thus, the different quantities of lipid bound in these various LDL must interact with apoB so that the same regions of the apoprotein are exposed to the action of trypsin in these different molecular weight lipoproteins.

Species variability in the modification of erythrocyte surface proteins by enzymatic probes.

Bovine and equine erythrocytes have been studied by three different surface modification techniques to investigate the accessibility of the surface components to the external medium. Lactoperoxidase labeling of equine erythrocytes results in a significant labeling of only one membrane component, a 100 000-mol.wt polypeptide corresponding to the membrane-spanning Component III of human erythrocytes. The major sialoglycoprotein of the equine erythrocyte is not labeled. This is in contradistinction to the situation for human and bovine cells, where both components are labeled. The equine membrane sialoglycoprotein is also not markedly affected by pronase, chymotrypsin or trypsin treatment of whole cells under the treatment conditions used, although it can be cleaved by pronase in isolated membranes. Experiments with the isolated glycoprotein show that its cleavage by trypsin is quite selective, whereas cleavage by pronase and chymotrypsin is much more extensive. Labelling of bovine red cells by galactose oxidase treatment followed by reduction with 3H-labeled borohydride yields radioactivity in only one major peak, that corresponding increase in labeling. Equine erythrocytes don not show significant labeling by this technique unless a neuraminidase pretreatment has been performed. Then only the major glycoprotein is labeled. Thus the equine glycoprotein is apparently inaccessible to the cell surface by standard surface modification methods, although it is clearly a surface component. These experiments point out some of the limitations of surface labeling and proteolysis methods in probing the accessibility of membrane components. The results suggest that apparent inaccessibility of the equine glycoprotein is due partially to its structure and partially to its localization in the membrane.

Calcium-promoted aggregation of erythrocyte membrane proteins.

Introduction of Ca2+ (greater than 1 mM) into erythrocytes during hemolysis causes formation of an aggregate which is highly resistant to disruption by sodium dodecyl-sulfate and other denaturing agents. The process is temperature dependent, but it does not require incubation in isotonic medium. Aggregation can be prevented but not reversed with chelating agents such as ATP or EDTA. The aggregate can be isolated by chromatography in dodecylsulfate on Sepharose 4B. Its amino acid composition indicates that it contains spectrin as the primary, but not exclusive, polypeptide component. Aggregate formation does not require increased Ca2+ binding to the membranes, and no 45Ca2+ could be detected in the aggregate which had been separated by acrylamide electrophoresis on sodium dodecylsulfate. This indicates that the Ca2+ is important in the formation of the aggregate, but not in its stabilization or maintenance once it has been formed.