Brown, James B.

2018-05-22T12:43:25Z

2018-05-22T12:43:25Z

1952

When studying the behaviour of a substance in the body, once the qualitative responses have been investigated, a quantitative method for estimating the substance in the presence of body fluids is necessary before further information can be obtained. In the oestrogen field, a considerable amount is known about the qualitative response of the body to the oestrogens, but little is known concerning the actual mechanism by which they are metabolized. In clinical medicine, gross disorders of oestrogen production are easily recognized and if they are accompanied by a greatly increased production of oestrogens, they can be investigated by the crude quantitative methods at present available. However, little is known about the less obvious disorders. It is hoped that a more sensitive and precise method than is now available for estimating "natural" oestrogens in body fluids, will facilitate the investigation of oestrogen metabolism and of disorders which now are only suspected to be due to abnormalities of oestrogen metabolism. áuch a method is probably the most important single requirement in the oestrogen field today. That this is universally appreciated is shown by the number of laboratories actively engaged in research on oestrogen methods, and it was this need which has also prompted the investigation described in this thesis. A method for estimating natural oestrogens in urine was obviously the first to be developed, because urine is easily obtained in large amounts. However, methods are required for estimating oestrogens in blood and tissues and it is hoped that these will be developed in the near future. methods for estimating oestrogens can be divided into two classes, one biological, the other'chemical. Biological methods, 2 though specific, are tedious and lack the accuracy and convenience which are possible with chemical methods. iethods are required to isolate, identify and measure the small amounts of oestrogens which occur in urine. Even in pregnancy the urinary excretion seldom exceeds 20 mg. per day, and in non pregnancy the normal levels seem to be between 5 and 100 pg. per day. To measure the latter concentration, a method must be able to estimate one part of oestrogen in 500 million parts of urine, which appears at first to be an impossible task for a chemical method. Fortunately, however, when the natural oestrogens are heated with sulphuric acid under certain conditions, an intense red ( "Kober ") colour is produced which is almost specific for the natural oestrogens; under other conditions an even more intense greenish fluorescence is produced which, though not as specific as the red colour, is sensitive enough to estimate even the small amounts of oestrogens present in normal urine. A chemical method for measuring these amounts is therefore theoretically possible. Unfortunately, the methods which produce this colour and fluorescence also produce brown colours and bluish-white fluorescences with other urinary materials and these interfere in the measurement of the oestrogens. The limits of any method based on these colour or fluorescence reactions therefore depend upon the extent to which the interfering urine component can be removed. The investigation to be described deals with the development of a chemical method, and is divided into two sections. The first section describes the search for a stable colour method which is minimally affected by interfering urinary material; the other deals with the purification of urine extracts. In outline, the method finally adopted consists of the following steps: acid hydrolysis of the urine, extraction of this with ether, separation of the phenolic fraction containing the oestrogens from the acid, polyhydroxy phenol and neutral fractions, methylation of the phenolic fraction, and chromatography of the phenol methyl ethers on alumina columns. Oestrogens in the purified urine extract were estimated colorimetrically. A spectrophotometric procedure was used to correct for residual interfering urinary material. Experiments to determine the recovery of oestrogens added to hydrolysed male urine indicated that this method is satisfactory for estimating oestriol, oestrone and oestradiol -117(3 at concentrations greater than 100 pg. of each in a 24 hour specimen of urine.

http://hdl.handle.net/1842/30349

The University of Edinburgh

Annexe Thesis Digitisation Project 2018 Block 19

Chemical estimation of oestrogens in urine

The chemical estimation of oestrogens in urine

Thesis or Dissertation

Doctoral

PhD Doctor of Philosophy