COMPARISON OF DIFFERENT METHODS OF ISOTOPICALLY LABELING THE ESTERIFIED CHOLESTEROL IN HUMAN HIGH-DENSITY LIPOPROTEINS

Different methods of incorporating esterified [3H]cholesterol into human high density lipoproteins (HDL) were assessed in terms of their influence on the subsequent rate at which esterified [3H]cholesterol was transferred from HDL to other plasma lipoproteins in 37.degree. C incubations containing tracer amounts of the labeled preparations added to aliquots of a common pool of unlabeled human plasma. Two basic methods were used to label the HDL esterified cholesterol: by the action of lecithin:cholesterol acyltransferase in incubations of plasma containing exogenous [3H]cholesterol and by exchange in incubations of plasma containing preparations of prelabeled LDL [low density lipoprotein]. The exchange labeling approach was also used to examine the effects of various HDL compositional changes on the subsequent behavior of its esterified [3H] cholesterol. The source of the label, per se, whether from the esterification of exogenous [3H]cholesterol or from exchange with labeled LDL, had no influence on the subsequent behavior of HDL esterified [3H]cholesterol. However, modification of the HDL composition during the labeling process had profound effects; e.g .; in preparations in which the esterified cholesterol content of the labeled HDL was increased by the action of lecithin:cholesterol acyltransferase, there was a reduced rate of transfer of esterified [3H]cholesterol out of the HDL fraction in subsequent incubations of the labeled HDL with fresh, unlabeled plasma. Conversely, when the esterified cholesterol content of the labeled HDL had been decreased by pre-incubation with Intralipid or very low density lipoproteins (VLDL), the subsequent rate of transfer of esterified [3H]cholesterol out of the HDL fraction was increased markedly. To obtain a biologically valid, labeled tracer of human HDL esterified cholesterol, the labeling should be achieved with minimal modification to the HDL composition. This means labeling by exchange in incubations in which lecitin:cholesterol acyltransferase is inhibited in plasma from which VLDL has been removed.