Application of the Ittrich Extraction Procedure to the Fluorimetric Measurement of Oestrogens in Blood

Abstract

The fluorescence reaction described by Ittrich (1958) for the quantitative estimation of oestrogens has been investigated, and applied to the measurement of oestriol, oestrone and oestradiol in fractions from blood prepared by the procedure of Roy & Brown (1960). The results obtained by this method from blood taken during pregnancy agreed closely with those given by the less sensitive Kober colour reaction. Fluorescing impurities derived from solvents and reagents gave 'base-line' values equivalent to approximately 0\m=.\02 \g=m\g. of each oestrogen per 100 ml. blood. These background values masked the oestriol and the majority of the oestradiol levels when the procedure was applied to peripheral venous blood obtained during the menstrual cycle. However, positive values for oestrone were encountered reaching 0\m=.\14 \ g=m\ g. / 100 ml. at the peak; these values correlated closely with the urinary oestrone output. The ratio of blood: urinary concentration of oestrone was the same during the menstrual cycle as during pregnancy.

Fluctuations in Human Serum Lipoproteins During the Normal Menstrual Cycle

“…Average values were then determined for each subject at the following periods during the cycle: days 1-6, menstruation; days 7-12, follicular or preovulatory phase; days 13-18, ovula-1965 tion; days 19-24, progestational phase; days 25-30, beginning of menstruation in the next cycle. The observations of Brown (1959) and Roy (1962) show that blood oestrogen concentrations are highest at day 14 and lowest during menses. The intra-subject variability and the lipoprotein values during the phases of the cycles were analysed statistically.…”

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

“…were obtained, (a) reagent blanks: k = 2-24 + 0-27 (n = 100); (b) Kober samples containing large amounts of oxidized quinol: k = 1-94 + 0-12 (n = 8); (c) reaction performed directly on 0-1 ml. urine from individuals excreting small amounts of oestrogens: k = 1-96 + 0-26 (n = 58); (d) crude phenolic extracts of urine from individuals excreting negligible amounts of oestrogens: k = 2-19 + 0-24 (n = 38, 15 patients); (e) sheep plasma (wethers and anoestrous ewes) extracted by the method of Roy (1962): k = 2-04 + 0-26 (n = 43). A value of approximately 2 was obtained in each case.…”

“…The Department of Obstetrics and Gynaecology, University of Melbourne, Australia (Received 13 June 1967) sensitivity (approx. 10~9 g.) is limited by fluorescence of residual impurities present in the extracts (Roy, 1962). Lunaas (1964) introduced a correction for these impurities based on the Allen (1950) correction applied to the emitted light.…”

Fluorometric Analysis

“…For estrogens in urine or blood, greater specificity is achieved with the Kober-Ittrich procedure, which involves extraction after sulfuric acid treatment into a 2% solution of p-nitrophenol in methylene chloride or tetrachloroethane (275,295,410,473). Total bile acids in serum can be determined, if cholesterol is removed, by fluorometry after reaction with sulfuric acid (279).…”