Further Observations on the Kober Colour and Ittrich Fluorescence Reactions in the Measurement of Oestriol, Oestrone and Oestradiol

Abstract

Higher temperatures allow lower sulphuric acid concentrations and shorter heating times to be used in the Kober colour reaction for oestrogens. A one-stage reaction which is completed in 5 min. at 120\s=deg\is described for oestrone and oestradiol, and a two-stage reaction which requires two periods of heating for 5 min. at 120\s=deg\is described for oestriol. The conditions were applied to the Ittrich fluorescence procedure. A spectrophotofluorimetric correction was developed in which fluorescence was measured at wavelengths for excitation and emitted light near the optima for the oestrogens and at another combination at which the oestrogens produced virtually no fluorescence whereas that of impurities was not diminished. Extraction, centrifugation and fluorimetry were performed in specially designed cells. The sensitivity is 0\ m=. \ 05\ p=n-\ 0\ m=. \ 1 ng./sample with a linear response up to 300 ng. and a precision better than 4% in the range 1\m=.\0\p=n-\100 ng.

Elevated levels of prolactin in nulliparous women

“…Aliquots of urine were coded and air-freighted frozen in dry ice to Melbourne, where urine levels of oestrone (uEl),oestradiol (uE2),and oestriol (uE3) were measured by J.B.B., using a method involving the use of spectrophotofluorimetry and internal radioactive standards (Brown et al, 1968). Urine hormone concentrations were converted into absolute amounts by multiplying the concentration by the total volume of urine collected.…”

Pituitary Function in the Empty Sella Syndrome

“…Testosterone in serum was determined by radioimmunoassay [8] and 17|)-estradiol by a fluorometric method [23]; total urinary es trogens by spectrofluorometry [6] and FSH, LH and prolactin by radioimmunoassay [23]. In 3 subjects total urinary gonadotropins were determined by the mouse uterine assay.…”

Oestrogenic Function in Postmenopausal Women

“…Thyssen & Veeman (1968), using gas liquid chromatography, examined thirty-five Dutch women and obtained a mean value for each oestrogen which was four times higher than that which we have demonstrated. In the present investigation, the technique of Brown et al (1968), which was used for oestrogen determination, had a sensitivity considerably higher than that of his original method (Brown, 1955). This might well be expected to yield estimates which approximate more closely to the true value.…”

“…In order to avoid inclusion of women Brown, MacNaughtan, Smith & Smyth (1968), including the saponification step described by Brown, Bulbrook & Greenwood (1957).…”

“…In one of the latter group, parallel assays using both the spectrophotometric and spectrophotofluorimetric end-points were carried out. It was, therefore, possible to compare the results obtained by the method of Brown et al (1957) Meisels (1966) and was used in a preliminary report by Dove (1968). As reported above, the MV has now been accepted as a standard by the second Tutorial on Hormonal Cytology (Weid, 1968) where it was shown that the MV, as computed from the MI, is constant for any one slide examined as measured by a number of different investigators.…”