The fluorescence reaction described by Brown (1952) was reinvestigated for use with 0.05 - 0.1 jig. pure oestrogen and with blood extracts prepared in the same manner as urine extracts as described by Brown (1955). The fluorescence reaction proved
satisfactory for the measurement of these small amounts of oestriol, oestrone and oestradiol in pure solution, but it was not specific enough for use with blood extracts prepared by Brown's method.
Extracts which should have contained no oestrogens gave as much fluorescent activity as extracts from blood which contained large amounts of oestrogens.The Kober reaction is a highly specific reaction of the natural oestrogens, but at the time of commencement of this work was normally carried out with several microgrammes of oestrogens
in a final volume of reagent of 3-4ml. The 'micro'-Kober reaction was therefore developed so that 0.05 - 1 jig. oestrogen could be measured in a final volume of 0.4 ml., optical density measurements being made in a cuvette with a light -path of 10 mm. The 'micro' - Kober reaction proved to be sufficiently sensitive and specific for
use with extracts of blood obtained from the umbilical cord and from the pregnant woman. It was not sensitive enough to measure
with any degree of accuracy the amount of oestrogens present in blood from non-pregnant subjects, although on a few occasions small amounts of oestrone were detected in blood from this source. Contamination with dust particles, etc. had to be rigorously excluded from the tubes during the 'micro'-Kober reaction, since
this gave yellow colours which interfered in the measurement of the optical densities. The wavelength absorption curves of the colours
developed in the Kober reaction indicated the presence of impurities which were derived from the benzene and benzene -ethanol
used for chromatography. The impurities came in part from the quinol and in part from the benzene and the ethanol. Several attempts to purify the benzene were unsuccessful and the smallest
amount of quinol necessary to give consistent readings was used. The absorption spectra of extracts of blood from pregnant women
were almost identical to those of the corresponding pure oestrogen methyl ethers dissolved in the same volumes of solvents as those used for eluting them from the alumina columns, indicating that the blood extracts contained few impurities other than those derived
from the solvents themselves. The absorption spectrum of 'pure' benzene and extracts from non -pregnant subjects were practically linear between 480 and 560 mμ. The use of the Allen correction formula to correct for the optical densities given by the impurities was therefore justifiable. This conclusion was further supported
by the following findings. By the use of the Allen correction formula it was possible to calculate the optical densities given by the impurities. This was done for a series of standards (with and without added solvent) and for a series of blood extracts. The amounts of impurities present in the blood extracts were the same as those present in the series of pure oestrogens and solvents.The method finally adopted was almost identical to that described by Brown (1955) for the measurement of urinary oestrogens.
Acid hydrolysis of whole blood proved the most satisfactory means of getting the maximum yields of oestrogens. Diluted, whole blood was hydrolysed by boiling with 15 vol. % concentrated HCl, the hydrolysed blood was extracted with ether, and the ether extracts
were processed exactly as described by Brown, except that chromatography was performed with 0.7 g. alumina and the final measurement was made by the 'micro'-Kober reaction. Troublesome emulsions formed during the initial ether extractions and at partition between benzene /light petroleum and 1.6% NaOH, but
these were handled by careful manipulation and centrifugation.The accuracy of the method was less than that of the urinary method. The recovery of oestrogens added after hydrolysis was 60 -7%, and the recovery of oestrogens added before hydrolysis
was some 5 -20% lower. The sensitivity of the method was such that 0.18 μg. oestriol, 0.11 μg. oestrone and 0.08 μg. oestradiol could
be detected in 100 ml. blood. This proved satisfactory for the measurement of oestrogens in blood from women from the 12th week of pregnancy to term.The problem of extracting oestrogens from blood was investigated. Blood was separated into plasma and red cell fractions, and in some instances the cells were washed with 0.9¡o
saline. The plasma was found to contain practically all the oestrogens present in whole blood although traces of oestriol were
sometimes found in the red cell fractions, particularly if the cells were not washed with saline. This is in agreement with the work of Wall and Migeon (1959) who showed that successive saline or plasma washes removed all the oestrogen from the red cell fraction, which they found to contain up to 20% of the total oestrogen content.Direct ether extraction of whole blood, or extraction with ethanol -ether or ethanol removed 10 -20% of the total oestriol and 80 -90% of the oestrone and oestradiol (yield given by boiling with 15 vol. % HCl being taken as 100%). The ethanol or ethanol -ether fraction yielded a further 30-40% of the oestriol on hydrolysis by boiling with HC1 or by incubating with ß- glucuronidase. The remainder of the oestriol was recovered from the protein precipitate by acid hydrolysis followed by ether extraction. Hydrolysis of the whole blood by boiling with HCl in concentrations varying from 2.5 to 20 vol.% gave the highest yields of 'oestrogens, the acid
concentration not being critical. There was no increase in the amount of oestriol which was extractable with ether after incubation of the blood with ß-glucuronidase. These experiments
indicate that almost all of the oestrone and oestradiol are in the free form in blood, 10-20% of the oestriol is in the free form, 30-40% is either protein-bound or conjugated or possibly both
protein-bound and conjugated, and the remainder is more firmly protein- bound.Blood oestrogen levels were measured in normal, healthy, pregnant women, patients who were delivered by Caesarean section and patients suffering from pre-eclampsia. There were no differences in the levels in primigravid and parous subjects. There was a very wide scatter of results in the normal patients studied. Since there were no significant diurnal or daily variations in the concentration of blood oestrogens, as judged by the results obtained at various times of the day, and on consecutive days in four patients studied in the last trimester of pregnancy, this scatter was due to real differences in the individual levels. The concentrations of oestrogens rose from the 12th week of pregnancy to term. This rise was usually continuous, but in some patients the levels sometimes fell temporarily, usually to rise again. The three oestrogens rose at about the same rate until the 32nd week of pregnancy when the oestriol began to increase more rapidly than the oestrone and oestradiol. These rises in blood oestrogen levels closely
paralleled the corresponding rises in urinary oestrogen output. Using the mean values in Series A and the figures for urinary output (Brown, 1956) of 5,100; 510 and 140 μg. oestriol, oestrone and oestradiol at 20 weeks, and 33,500; 1,550 and 570 μg. at 40
weeks, the factors for the increases in oestrogen concentrations between the 20th and 40th week were (urine /blood) 6.5/6.0, 3.0/3.3
and 4.1/4.4 for oestriol, oestrone and oestradiol respectively. These calculations showed that the increases in the concentrations of all three oestrogens in urine closely paralleled the increases in blood, even to the extent of the more rapid increase in oestriol concentration after the 32nd week. Although the increases in blood and urine followed one another closely, the ratio of the relative concentrations of oestriol, oestrone and oestradiol were markedly
different for blood and urine. Using the same values as in the above calculations, the ratio of oestriol: oestrone: oestradiol were 22:1:0.37 and 1.8 :1:0.22 for blood and urine respectively.
The difference in ratio of oestriol to oestrone suggested that oestriol is cleared very much more rapidly than oestrone, but, on the other hand it might be the results of the difference in the relative proportions of free, conjugated and "protein- bound" oestriol and oestrone in blood.Since the blood levels showed no significant diurnal variation, and the ratio of blood concentration to urinary oestrogen output per hour remained more or less constant, it appears that the
rate of excretion of oestrogens must also remain constant. These ratios were used as a basis for speculation on the levels one might expect to find in blood from non -pregnant subjects, and on
possible modes of excretion of the oestrogens.The concentration of oestrogens in amniotic fluid,
umbilical venous and arterial blood and uterine venous and arm venous blood of the mother at Caesarean section were measured. Liquor contained very large amounts of conjugated oestriol and only 4% of free oestriol; in other words its oestrogen content resembled that of urine rather than that of blood. There was no significant difference in the concentration of the three oestrogens in umbilical venous and arterial blood. Small differences might have been expected if the foetus were an important site for the metabolism of oestrone and oestradiol (Diczfalusy and Magnusson, 1958). The concentrations in maternal uterine venous blood were significantly higher than those in the maternal peripheral blood, sometimes by as much as 25%. This difference probably represents
the gradient caused by entry of oestrogens into the maternal circulation from the placenta and loss through maternal metabolism and renal and faecal excretion of the oestrogens. The fact that a difference is detectable with the high uterine blood flow of approximately 600 ml/minute indicates a considerable production of oestrogens by the placenta. The peripheral oestrone levels were significantly lower in the women undergoing Caesarean section than in the normal series. This difference was shown not to be due to any premedication administered to the patients, and the possibility that it is due to rest in bed is under investigation. In support of this possibility it is interesting to note that the blood oestrone levels in a series of patients admitted to hospital with a variety of minor ailments were also significantly lower than those of the normal out-patients and that the urinary output of oestrogens falls overnight in pregnant and non-pregnant subjects (Brown, unpublished observations); similarly Fotherby and Strong (1960) showed that the urinary output of the metabolites of a number of adrenal steroids fell between 9 p.m. and 6.30 a.m.In the majority of cases of pre-eclampsia, there was a decrease in the concentration of oestriol and oestrone in blood, but little or no change in the oestradiol concentration, except in some cases of intra-uterine death. In all cases of still-birth
the oestrone concentration was below the normal minimum and in almost all these cases the oestriol concentration was also below the normal minimum. These low concentrations of blood oestrogens
were presumably due to placental dysfunction. In a few cases of severe pre -eclampsia where the symptoms were of short duration and had appeared after the 34th week of pregnancy, the oestriol
concentration was above the normal mean value and in one case was higher than the maximum value observed in the normal series, although the oestrone values were low. It was suggested that these higher levels might be the result of the renal damage which is known to occur in pre-eclampsia.The results obtained by using the present method agreed well with those obtained by Preedy and co- workers and Oertel et al, while the oestrone values of Svendsen were lower, but again there
was good agreement in the oestradiol values. There was very close agreement between the results for cord blood quoted here and those
of Diczfalusy and Magnusson. The specificity of these methods and the fluorimetric methods of Veldhuis and Varangot et al were discussed.Very small amounts of oestriol and oestrone were found in samples of blood taken from non-pregnant subjects, and the mean values were below the limits of sensitivity of the method.
Preliminary observations using the more sensitive fluorimetric method of Ittrich (1958) suggest that the levels are even lower than those indicated by the results of the 'micro'-Kober reaction and are near the limits of sensitivity of the Ittrich reaction
