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UBC Theses and Dissertations

Immunochemical detection of human plasma enzyme lecithin:cholesterol acyltransferase Hon, Kenneth K.


A simple and specific electroimmunoassay has been developed for the human plasma enzyme lecithin:cholesterol acyltransferase (LCAT; E.C. This enzyme catalyzes the transfer of an acyl group from the C:2 position of lecithin to the 3-hydroxyl group of free cholesterol to form cholesterol ester and lysolecithin. The initial rate of this reaction is commonly measured by various radiochemical techniques and serves to reflect the enzyme level in plasma. However, such measurement of the enzymatic activity fails to separate the influences of enzyme, substrate and cofactor on the esterification rate. A direct measurement of the protein mass through an immunoassay coupled with a determination of enzymatic activity may help to provide information that would allow differentiation of enzyme abnormalities from that of other factors. The electroimmunoassay described in this thesis involved the production of a monospecific antibody against the enzyme and the subsequent use of an immuno-electrophoretic procedure. Electrophoresis was performed in an agarose gel containing anti-LCAT antibody. Quantitation of LCAT protein level was based on measurement of the area of the precipitate (rocket) in the samples and the pure LCAT standards. Specificity of the antibody produced was assessed as follows: (i) the antibody formed a single precipitin arc with both purified LCAT enzyme and normal human plasma, (ii) the antibody showed no reaction with most of the major lipoproteins very low density lipoproteins, low density lipoproteins and high density lipoproteins. (iii) complete inhibition of LCAT activity was achieved when the antibody was incubated with either human plasma or purified enzyme, (iv) the antibody did not react with plasma of patients with LCAT deficiency. (v) pre-incubation of antibody with normal human plasma led to a significant decrease in the antibody's anti-LCAT activity in a subsequent assay. No such decrease, however, was observed after similar immunoabsorption of the antibody with LCAT deficient plasma. The concentration (mean ± S.D.) of LCAT protein in normal subjects was 5.40 ± 0.54 mg/1 (n = 12). Two patients with inherited LCAT deficiency also showed no detectable level of the enzyme in their plasma. An investigation of the family members revealed that the patients' parents and two of their siblings had sub-normal amount of LCAT enzyme (3.46 ± 0.48 mg/1, n = 4). Their enzymatic activities, as measured by an artificial substrate assay (phosphotidylcholine:free cholesterol liposomes), were also lower than normal (26-37 versus 45-85 nmole free cholesterol esterified/ml plasma/hr.). This immunoassay appears to be a suitable procedure for the quantitation of plasma LCAT. Moreover, this quantitation correlated well with the enzyme activity in a study of the LCAT deficient family (r = 0.93, n = 6).

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