This thesis is based on a worldwide overview (meta-analysis) of prospective observational studies of blood pressure and cholesterol, involving a centralised collection of data on over one million individuals from 59 studies, which I have co- ordinated since its inception. Analytically, the aim has been to develop and to use appropriate statistical techniques to assess the age- and sex-specific associations of usual blood pressure and of usual cholesterol with cause-specific mortality. Since the data set is uniquely large, and because appropriate methods of analysis (with full account taken of the time-dependent nature of the regression dilution bias) have been developed and used, these associations have been established more reliably. An integral part of the methodological element of the thesis has been to investigate the systematic underestimation of associations between risk factor and disease that are obtained when only a single baseline measurement is used to assess levels of such risk factors (the regression dilution bias). The extent of this bias has been investigated in each study that had repeat measurements of risk factors during follow-up. One particularly novel aspect has been the emphasis on, and methods developed to account for, the regression dilution bias in several studies simultaneously and in an appropriately time-dependent way. This thesis illustrates the extent to which random error and inappropriate statistical analysis lead to misleading conclusions concerning the importance of blood pressure and blood cholesterol, particularly in premature death. Only by studying adequate numbers of deaths (136,000 deaths among 1 million adults during 13 million person- years of follow-up) and by using appropriate statistical techniques - taking proper account of (a) the regression dilution bias; (b) the full range of blood pressure and cholesterol; (c) the opposing effects of HDL.and the remaining non-HDL cholesterol; and (d) age at death - did it become possible to provide reliable results on the true relationships between blood pressure, cholesterol fractions and vascular and other causes of death. These analyses have demonstrated reliably that, as causes of IHD death in early middle age, blood pressure and blood lipids are three to five times more important than suggested by inappropriate analyses, with no clinically relevant inverse associations with cancer or other non-vascular mortality (except, surprisingly, COPD).

The physico-chemical properties of lecithin:cholesterol acyltransferase were investigated. The amino acid composition analysis showed a relatively high content of glutamic acid, aspartic acid, glycine and leucine. The spectrophotometric titration of phenolic groups in the enzyme showed a large increase in absorbance at 295 nm with an apparent pK of about 12.0. The largest change in molar ellipticity at 222 nm was also observed above pH 11. Circular dichroism studies revealed that human lecithin:cholesterol acyltransferase has a relatively high content of β-pleated sheet structure (48%) with 20% α-helix, and 32% remaining structure. Human lecithin:cholesterol acyltransferase has a high extinction coefficient at neutral pH. Microsequencing of the amino terminal residues of the enzyme revealed a hydrophobic character. Inactivation of lecithin:cholesterol acyltransferase activity was observed using diisopropylfluorophosphate with a stoichiometry of 1 mole of diisopropylphosphate incorporated per mole of enzyme. This suggests the involvement of a serine residue in the active site of the enzyme, possibly for the formation of an acyl-intermediate. A new quicker assay method for lecithin:cholesterol acyltransferase was developed. This assay involved coupling reaction with acyl CoA synthetase, ΡΡᵢ-dependent phosphofructokinase, aldolase, triosephosphate isomerase and α-glycerol-3-phosphate dehydrogenase monitoring a change in the absorbance or fluorescence intensity due to the oxidation of NADH. The activity of each coupling enzyme was accurately determined to establish the optimum assay condition for lecithin:cholesterol acyltransferase. The coupled enzyme assay for lecithin:cholesterol acyltransferase by spectrofluorometry showed a significant change in relative fluorescence intensity whereas a UV absorption spectroscopy method showed no significant absorbance change for the initial rate of lecithin:cholesterol acyltransferase reaction.