Physico-Chemical Techniques in the Study of Organic Molecules of Biological Importance

Steel, G (1969) Physico-Chemical Techniques in the Study of Organic Molecules of Biological Importance. PhD thesis, University of Glasgow.

Abstract

The work described in this Thesis consists of a study of the lipids present in the fatty nodules (plaques) which occur during the fatty degeneration of the human aorta associated with the disease atherosclerosis. For many years studies of the chemistry of these lesions have concentrated on the major classes of lipid constituents, viz. cholesterol, cholesterol esters, phospholipids and triglycerides: the minor lipid groups havebeen largely neglected. We have attempted to remedy this omission, since quantity is not necessarily a measure of biological significance. Early studies on the subject were severely handicapped by the absence of suitable techniques for the separation and identification of microgram quantities of material and as a result had to use large quantities of tissue requiring, in some cases, several years for collection. The necessarily prolonged nature of these investigations introduced the possibility of gross artefact formation and cast doubt on the authenticity of certain compounds identified as arterial constituents. In the present wort, thin-layer and gas-liquid chromatography (TLC and GLC) and gas chromatography-mass spectrometry (GC-MS) were used to enable each investigation to be restricted to plaque tissue from one aorta. Lesions from severely diseased aortas were dissected and solvent extracted within 24 hours post mortem. Division of the extract into its major components was effected by column chromatography on silicic acid. Repeated crystallisation of the sterol fraction from methanol enabled the concentration of several sterols chromato-graphically more polar than cholesterol. The most prominent of these was tentatively identified on the basis of its chromatographic behaviour as 26-hydroxycholesterol (cholest-5-ene-3beta,26-diol), a previously unknown tissue constituent. Confirmation of this structure was obtained by GC-MS, making use of the ability of trimethylsilyl ether derivatives to promote characteristic mass spectral fragmentations. Using a similar approach,7-ketocholesterol (3beta-hydroxycholest-5-ene-7-one); 7beta-hydroxycholesterol (cholest-5-ene-3beta,7beta-diol) and 5alpha-cholestane-3beta,5,6beta-triol were confirmed as constituents of this fraction as had been suggested by earlier investigators. Examination of the hydrocarbons, which had been neglected by previous workers, revealed that the major component was the isoprenoid hydrocarbon squalene. The initial identification wasmade by TLC, GLC and GC-MS and was corroborated by infrared spectrometry. Preparative TLC on silica gel impregnated with silver nitrate enabled the separation of two unsaturated steroidal hydrocarbons, later shown by mass spectrometry to be a cholestene and a cholestadiene, from a series of n-alkanes. Hydroxylation of the cholestene and GC-MS of the resulting diol in the form of the trimethylsilyl ether derivative has established that the compound present is largely cholest-5-ene. Work is in progress to determine the double bond positions in the cholestadiene. TLC analysis of silicic acid column eluates of polarityintermediate between triglycerides and cholesterol revealed theexistence of several unidentified esters which were furtherpurified by extensive chromatography. Acetylation demonstratedthat these esters contained unesterified hydroxyl groups. Theprincipal neutral compounds generated on hydrolysis were shown tobe cholesterol, 26-hydroxycholesterol and 7beta-hydroxycholesterol. The presence of the latter two sterols was not unexpected:however, the identification of cholesterol as a hydrolysis productnecessitated the postulation of the existence of an ester ofcholesterol with a hydroxy fatty acid. TLC and GLC studies onthe fatty acid portion of the molecule have confirmed this andGC-MS examination has shown that the major compounds are isomeric C18-monohydroxy acids each with two double bonds. TLC of sterol fractions (as propionate esters) on silica gel impregnated with silver nitrate has permitted the separation of desmosterol from an overwhelming mass of cholesterol. The relatively unsatisfactory behaviour of sterol propionates on GLC and GC-MS has led to the preferred use of trifluoroacetates. Using this approach we have been able to identify cholestanol in plaque extracts. It is difficult to assess the significance of these findings in relation to the development of human atherosclerosis, as only one stage of the disease has been examined. However, the methods developed will form the basis of a further investigation which will involve the study of plaques at various stages of evolution in an attempt to relate chemical changes within the artery wall to the pathogenesis of atherosclerosis.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: Organic chemistry, Physical chemistry, Analytical chemistry
• Date of Award: 1969
• Depositing User: Enlighten Team
• Unique ID: glathesis:1969-78526
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 30 Jan 2020 15:12
• Last Modified: 30 Jan 2020 15:12
• URI: https://theses.gla.ac.uk/id/eprint/78526