Factors influencing the cholesterol esterification rate in lecithin cholesterol acyltransferase radioassay.
Factors affecting the esterification rate of cholesterol by lecithin cholesterol acyltransferase (LCAT E.C. 2.3.1.43) in native cold labelled substrates (human, rabbit, rat serum, plasma, VLDL, LDL depleted serum, rabbit intraocular fluids) repaired by use of ready-made 14C-cholesterol discs (Cholesterol kinetics LCAT-test, UVVVR, Czechoslovakia) were investigated. EDTA added to the serum during the cold incubation (18 h, 0 degrees C-4 degrees C) increased the rate of esterification due to elimination of Ca2+ ions. The similar stimulating effect was found in the presence of mercaptoethanol (ME) in the serum, while in the plasma already stimulated by EDTA no additional effect by ME could be noticed. Freezing and thawing did not affect the fractional esterification rate (FER-per cent of total serum unesterified cholesterol esterified per hour) in normolipidaemic sera, whereas in hyperlipidaemic sera, particularly those with high levels of VLDL, FER was stimulated. Esterification partially proceeded during the cold incubation of serum or plasma with 14C-cholesterol ready-to-use discs, attaining the values of about 0.3%/h and 2-6%/h, respectively, in human sera and in rabbit and rat sera. The starting level of esterification did not affect the linearity of LCAT reaction during warm incubation (30 min at 37 degrees C), neither was the absolute value of FER changed as compared with cold labelled sera with those inhibited by DTNB and reactivated by ME. Substantial LCAT activity was also detected in extremely diluted substrates--such as intraocular fluid collected from rabbits with induced uveitis or after preceding paracentesis.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D001826:Body Fluids / Q000201:enzymology; D002788:Cholesterol Esters / Q000097:blood*; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D004951:Esterification; D006801:Humans; D006949:Hyperlipidemias / Q000097:blood; D007700:Kinetics; D008074:Lipoproteins / Q000097:blood; D008623:Mercaptoethanol / Q000494:pharmacology; D009584:Nitrogen / Q000494:pharmacology; D009799:Ocular Physiological Phenomena; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000097:blood* / Q000378:metabolism; D010949:Plasma / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D011919:Rats, Inbred Strains
D002788:Cholesterol Esters; D008074:Lipoproteins; D008623:Mercaptoethanol; D004228:Dithionitrobenzoic Acid; D004492:Edetic Acid; D007862:Phosphatidylcholine-Sterol O-Acyltransferase; D009584:Nitrogen