Patent GB-1438862-A (GB)
Title: Quantitative determination fo cholesterol in body fluids
IPC: G01N33/92
Filed: 1973-05-17
Abstract: 1438862 Quantitative determination of cholesterol EASTMAN KODAK CO 17 May 1973 [22 May 1972] 23556/73 Heading G1B Total cholesterol, i.e. "free" cholesterol plus cholesterol esters, present in a body fluid, for example serum, is determined quantitatively by a method which comprises contacting a sample of the fluid with bromine in an amount in excess of that amount which reads therewith thereby to convert cholesterol and cholesterol esters in the sample to the 5, 6-dibromo derivatives thereof, removing excess bromine from the sample, contacting the sample with an iodide thereby to convert the 5, 6-dibromo derivatives to the corresponding 5, 6-di-iodo derivatives, allowing the 5, 6-di-iodo derivatives to decompose (optionally at elevated temperature for example of 50-60C) to regenerate the cholesterol and cholesterol esters with liberation of iodine and thereafter determining the amount of iodine liberated in any convenient known manner. The iodide may be an alkali metal iodide, for example sodium iodide or potassium iodide, and may be used when in the form of a solution in acetone. According to one alternative procedure, bromine water is added to the sample of body fluid and unreacted (excess) bromine is removed by use of a phenolic material, for example sulphosalicylic acid. According to further alternative procedures, the sample of body fluid is applied either to a coated test medium, the medium thereafter being contacted with bromine vapour, or to a coated test medium which comprises a bromine compound, for example silver bromide, decomposable upon irradiation to release bromine, the medium thereafter being subjected to radiation to effect release of bromine. The coated test medium, which may be in the form of a strip or tape, either may consist of an inert support coated with a layer of polyvinyl alcohol or starch, each of which materials reacts with iodine to give a colour, or may be a thin layer chromatographic element in the form of pre-coated sheet material consisting of a flexible film support, for example made of polyester, carrying a coating which comprises a sorbent, for example silica gel or cellulose, optionally dispersed in a polymeric binder, for example polyvinyl alcohol, reactive with iodine to give a colour; when the coated test medium does not comprise such a binder, a solution of an iodide to be applied to the medium may additionally comprise a reagent such as 4, 4'-bis (dimethylamino) thiobenzophenone (DMEATB) which reacts with iodine to give a colour. According to another alternative procedure, the sample of body fluid is applied to material such as filter paper which then is fumed with bromine vapour, contacted in sandwich manner with a test medium comprising an iodide and such a reagent which reacts with iodine to give a colour and thereafter treated with a solvent for the iodide. Detailed procedures involve (i) chloroform: solutions of cholesterol, of cholesteryl myristate and (comparison) of oleic acid, thin layer chromatographic pre-coated sheet material comprising silica gel sorbent and polyvinyl alcohol binder, bromine vapour, acetone solution of sodium iodide and reflection density measurement (optionally after heating to 60C) of the brown colour formed; (ii) chloroform solutions as per (i) supra, thin layer chromatographic precoated sheet material comprising cellulose sorbent and no binder, bromine vapour, acetone solution of sodium iodide and DMEATB and reflection density measurement (optionally after heating to 60C) of the blue colour formed; (iii) chromatographic separation of a lipid extract of human serum using n-hexane/ethyl ether/acetic acid developing solution, pre-coated sheet material as per (i) supra, bromine vapour and acetone solution of sodium iodide; (iv) chloroform solution of cholesterol, thin layer chromatographic pre-coated sheet material comprising cellulose sorbent, no binder and silver bromide, ultraviolet radiation and acetone solution of sodium iodide and DMEATB; and (v) human serum and (as standard) aqueous suspension of cholesterol, acetic acid anti-precipitant, bromine water, sulphosalicylic acid in crystal form, aqueous acetone solution of potassium iodide and visual comparison of the colour formed. Mention also is made of a continuous procedure involving a test tape of thin layer chromatographic sheet material comprising polyvinyl alcohol binder, bromine vapour, removal of excess bromine by an air jet and measurement of the intensity of the colour formed by means of a densitometer; in respect of this procedure, reference is made to Specification 1049364.