671 A RAPID METHOD FOR THE ESTIMATION OF ESTRIOL IN PLASMA DURING PREGNANCY
S. Mfklosf, J. S. G. Bfggs, N. Selvage, J. Canning and G. Lythal University of Queensland, Department of Obstetrics and Gipxaecology, Clinical Sciences Building, Royal Brisbane Hospital, Brfsbane, Queensland, 4029, Australia Received:
8/27!75
ABSTRACT
A rapid, reliable fluorimatric nasthodfor the determination of estriol (Eg in plasma is described. The method is eminently suitable for use in hospital laboratories and provides results within 5 hours of the reception of samples. It has several features not found in other methods. As little as 0.2 ml of plasma is required, all additions and aliquotfng procedures are semi-automated and no special technical skill is necessary. A technician can carry out 20 detezminations per day. Average recoveries of 70% are routinely achieved, and the accuracy (2.4%) and the precision (4.0%) of the method are remarkably good for an assay of this type. A spectrofluoromater of high sensitivity, counting equipment and a high temperature oven are the essential nnjor pieces of equipment.
INTROWCTION There are many advantages in measuring E
in plasma rather 3 than iu urine in late pregnancy, and a number of methods has been
published durfng recent years (1 - 5).
Hethods rmst allow results
to be obtained within a working day and still be accurate, precise and sensftive enough to be acceptable In clinical practice. The mathods of Nachtigall et al. (1) and Mathur et al. (5) seemed to fulfill these requirements. even
However, too frequent quenching -
in the standards - yielded poor precision in our hands and the
introduction of further purification steps made these methods too lengthy for clinical use.
volume 26, Number 5
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Estrogen in the urine of non-pregnant women is measured in this Department by the method of Brown et al. (6), using a spectrofluoronmter of high sensitivity and resolution. Efforts were directed to developing a method for Ej in other biological fluids, using similar principles. mentioned above.
The method devised satisfies the criteria
It involves acid hydrolysis in a steam sterilizer,
ether extraction, benzene-hexane partition and quantitation by fluorimetry.
Results are corrected for losses by the use of tritiated B5
as an internal standard.
The method is described in more detail
than usual, since it has been found that even slight changes have affected the precision. MATERIALS AND ME!WODS Blood samples were collected into lithium-heparin tubes and centrifuged inmediately. The separated plasma was processed at once or stored at -15O. Reagents
Rigorous purification of almost all of the reagents was necessary in order to obtain reproducible results. Sulfuric acid, purchased from Merck (GR), proved to be satisfactory for the preparation of ths Kober reagent. Diethyl ether (May d;Baker) was taken from freshlyAll brands of ether except opened 500~ml bottles just before use. M & B quenched the fluorescence of pure standards. Hydrochloric acid and ethanol were purchased from Merck. Ethanol was xefluxed over sodium hydroxide pellets and twice re-distilled. sym-Tetxachloroethane (AR) was used as received. To1 uene was scintillation grade, benzene and n-hexane were fluorimetry grade Bydroquinone was re-crystallized from water. obtained from Merck. p-Nitrophenol (Merck, BDH) was re-crystallized from benzene until near colorless. Sodium hydroxide and sodium hydrogen carbonate were of AR grade (BDB). Carbonate buffer, pi?10.5, was prepared by dissolving 21 g of NaOH and 70 g of sodium hydrogen carbonate in water and making up to 1 liter (10). Water was distilled in an all-glass apparatus and then passed through a deionizer. Kober reagent The Kober reagent contained 66% (v/v) sulfuric acid and 2% quinol and was prepared by dissolving finely powdered quinol in the
673
diluted acid on a magnetic stirrer plate at room temperature in the The reagent was kept in an amber bottle dark, without any heating. at room temperature and placed in a dark cupboard when not in use. It was discarded as soon as a slight brownish color developed. Ittrich reagent This reagent was prepared from 2% finely divided p-nitro-phenol in sym-tetrachloroethane (w/v) and was pale yellow in color. Solution was effected in about 5 min. without the addition of ethanol by vigorous The reagent was discarded when deepening of its yellow color shaking. was observed. Stand,ards -Estriol was purchased from Ikapharm, Israel, and purified by TLC on glass fiber plates impregnated with silica gel (ITLC-SA), (Gelman A stock Instr. Co.) using a system of chloroform : methanol (97 : 3). stangard solution containing 100 m/ml in ethanol was prepared and kept Estriol-16d-D(+)-glucosiduronate(E3-Gp) was obtained from at 5 . Estriol (6,7- H (N) ), 53.1 Ci/xnnol, Sigma Chem. Co., St. Louis, MO. was purchased from New England Nuclear, Boston, Mass. and from the Radiochemical Centre, Amersham, V .K. The radioactive compounds were checked for purity by TLC in the same manner as the non-radioactive estriol. An aliquot was diluted with ethanol to give approx. 20,000 cpm per 10 pl and used as an internal standard during the later phase of this work. Estriol-(6,7-3B)-16c-glucosiduronate, prepared according to Slauawhite et al. (7) with nwdifications as suggested by Goebelsmann (personal comm.). was used as an internal standard during the early phase of this work. Counting
Radioactivity was measured in a Nuclear Chicago liquid scintillation counter, Model 4649, Mark II, using the channels ratio method. Tritiated E3 was counted All samples were counted for 10,000 counts. in 1.5ml of toluene containing "Gmnifluor" (4 g/l, NEN, Boston, Mass.) and the radioactive glucosiduronate in the system of Herberg (8). Cleaning of glassware Glassware used for fluorimetry must be meticulously cleaned. The tubes used for hydrolysis and extraction and those used for the Kober reaction were washed with ethanol, 5 times with hot tap water and then 5 times with distilled water. All tubes which contained plasma were soaked in acetone at fortnightly intervals. The glassware was then placed in a self-cleaning oven (Westinghouse,Model PAW 306, Email. Ltd., Aust.) which has a I-hr. cycle of 540°. Sometimes, dark brown stains appeared on the plasma tubes due to undissolved lipid material. These were removed by soaking the tubes overnight in Chromate traces were destroyed by washing the tubes chromic acid.
with a 0.2% acidified solution of sodium sulfite. Measurement of fluorescence Fluorescence was measured in the round cuvettes described by Brown et al. (9). By this means the extraction of fluorescence, centrifuqation and fluorimatry can be done in one tube. The risk of contaminating the atmosphere of the laboratory with tetrachloroethane is thus avoided. The tube projects through the cover of the Brown cell adapter (Aminco-BowmanCat. No. 4-8244) and is protected from liqht by a cylindrical cover. Fluorescence was measured in an AmincoBowman 4-8203 D spectrofluorometer with a solid state, blank-substract microphotometer and an off-axis ellipsoidal mirror condensing system using a xenon lamp, a 1 P 21 potted photomultiplier tube and 2 mn slits throughout. Sensitivity was better than one part per trillion of quinine sulfate in 0.1 N H2S04 and resolution was 0.8 nm with the 600 line/mm grating. The spectrofluorometer was standardized with quinine sulfate before use. Even with 2 mm slits, some liqht from the source passed to the photomultiplier. This was overcome by inserting a Corning 3-67 sharp cut-off filter which in turn shifted the maximum of excitation from 532 to 537 nm and that of the emission from 552 to 563 nm. Some 30% of the sensitivity was lost with the use of this filter. Extractions and partitioning Ether extraction, following hydrolysis of plasma, was performed in an alkaline medium (pH 9 - 9.5). Lower pH often produced emulsions. After extraction (by.holdinq all the tubes down in a rack with a board and shaking) and centrifuqation the aqueous layer was quick-frozen in dry ice/ethanol and the ether decanted. Dry ice was obtained by the use of a mechanical attachment designed to fit any standard Co2 cylinThis simple device ("Hedi-ice" from der equipped with a syphon tube. Metallurgical Supply Co., Houston, Tex.) consists of a nozzle, cone and collection bag and is attached to the cylinder; the valve is opened and the collection bag is immediately filled with dry ice of flaked consistency, ready for use. Methods The assay was first carried out with 0.5 ml of plasma (Ml) and later with 0.2 ml (M2). In order to speed up operations, all reagents were added from automatic dispensers. Plasma in appropriate amount is measured into centrifuge tubes (Quickfit B 24, 115 x 29 mm, or B 14, 150 x 18 mm for M2). Water is added (2 ml and 0.8 ml, respectively, followed by 10 pl of 3H-B3 and 0.4 ml cont. IiCl (0.16 ml for M2), and some antibumping granules. The contents are well mixed and the tubes are covered $th aluminium foil and placed in a steam sterilizer for 15 min. at 120 . After hydrolysis, the tubes are cooled under tap water and 5 N NaOH added (0.7 ml for Ml; 0.28 ml for M2) followed by carbonate buffer,
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pfi 10.5 (2.0 m.2 for Ml; 0.8 ml in M2) and the contents mixed (14). The mixture is then extracted with diethyl ether (10 ml for Ml; 6 ml for M2) by shaking the tubes together 100 times. The tubes are centrifuged at 2000 rpm for 5 min. in a refrigerated centrifuge. The tubes are placed in a dry ice/ethanol mixture and after quickfreezing the diethyl ether layer is decanted into a set of B 14 test tubes. A piece of antibumping granule is placed in each and the ether is evaporated in a water bath at 40°. Distilled water (2 ml) is added and the tubes are rotated to dissolve the dry residue. Two ml of a benzene/hexane (1:l) mixture, freshly prepared, is then added. The tubes are shaken well to extract E , E and fatty substances (11). The W&es are centrifuged at 2000 rpm $5 or min. and placed in a deep freeze for 5 min. to freeze the aqueous layer; the upper organic phase is then sucked off with a Pasteur pipette attached to a vacuum line. After quick thawing, 6 ml of diethyl ether is added to the aqueous phase, which is extracted by shaking vigorously as before. After centrifuging for a few minutes, the tubes are placed in dry ice/ethanol, the water layer quick-frozen and the ether decanted into another set of tubes (B 14, 125 x 18 zmn). One antibumping granule is added, and the ether is evaporated in a hot water bath. The tubes are dried by suction and the residue dissolved in 0.5 ml of ethanol using a Vortex mixer and 0.05 ml is taken for counting of radioactivity. Ethanol solution (0.2 ml) containing 5 g quinol/lOO ml (2 g quinol/lOO ml for M2) is now added, mixed well and evaporated to dryness at 60° on a rotary evaporator. Complete dryness must be achieved by using a strong vacuum.
One ml of 66% Kober reagent is added to the tubes and solution is effected by warming. The tubes are then rotated so as to cover theoinside area by the Kober reagent, tightly stoppered and heated.at 120 for 10 min. then cooled in ice water. When completely cool, 1.5 ml ice water is added to each tube and the contents well mixed. The tubes are immersed again in ice water and the contents poured into the corresponding fluorimetry tubes containing 0.75 ml of Ittrich reagrent. This had been measured out from a dispenser in a fume cupboard. Several sets of these tubes are prepared in advance and kept at -5" until required. The fluorimetry tubes are then stoppered and shaken vigorously all at the game time about 100 times, centrifuged at 2000 rpm for 3 min. at 0 , allowed to demist for a few minutes, then wiped carefully. Fluorimetry at 537/563 nm must be performed immediately. From the addition of ice water onwards, the steps should be completed in less than 20 minutes to avoid fading of fluorescence. The E3 content is calculated by direct comparison with a standard of 5Opg (or 20 ,uy for M2) processed at the same time. The instrument is set to read the same value of the standard in day-to-day operations so that the formula used to calculate the results is greatly simplified. The results are corrected for losses from the radioactivity recovered.
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676
TZDEOTDm RESULTS
Recovery
The losses incurred in the method are corrected by the use of radioactive
internal standards.
The recovery of free tritiated E3
+ when used as the internal standard was 70 - 3.8% (S.D.) (200 tests). Accuracy and precision The results of duplicate experiments in which known amounts of estriol glucosiduronate
were added to male plasma are given in Table 1.
The results are corrected for blank male plasma values. Table 1.
Amount of E -Glu a dded (ng)
Recovery of Estriol Glucosiduronate Added to Male Plasma
Equivalent of free E3 (ng)
Average amount recovered as free E3 (ng)
Average S.D.
% recovery (accuracy)
Number of assays (n)
10
6.2
6.0
2 1.12
96.8
6
20
12.4
12.2
f 1.26
98.7
19
40
24.8
24.4
+, 1.72
96.0
19
60
37.2
37.1
f 5.57
99.5
15
80
49.6
50.0
+, 2.2
100.7
14
The corrected recoveries ranged from 96 to 100.7%, with a mean of 98 + 2.4 (S.D.) %.
The coefficient of variation was thus
2.4 x 100/98 = 2.4%. The precision of the assay was examined by measuring the E3 concentration
of pooled late pregnancy plasma or of male plasma
to which a known amount of E3 was added. For the method using 0.5 ml of plasma
(Ml) the coefficient
of variation was 8.8% (n = 20), while for method M2 using 0.2 ml of
S plasma, the Mlue
677
T=EOXDCI
was 4% (n = 25).
The between assay precision, measured as the C.V. of a control plasma in duplicates in each assay, was 11.6% (n = 88) for Ml and 7.4% for M2 (n = 84). Sensitivity The smallest amount of E3 which could be measured with pure standards was around 300 pg. When 0.2 ml of water was assayed in place of plasma, 10 ml was measured by the instrument.
w/
This solvent blank, however, can
be substracted by making use of the blank-substract device of the microphotometer. SpeciThe specificity of the method depends largely on the effectiveness of the water/benzene-hexanepartition step in removing other estrogens (1). Tritiated E 3, together with 20 ng of carrier E3, was added to water and partitioned between equal volumes of benzene-hexane. The water layer was found to contain 98% of the radioactivity. Wilkinson et al. (12) found that 87 - 91% of El and E2 were extracted into the organic layer in similar experiments.
In our
laboratory, estrone (El) and estradiol (E2) in concentrations ranging from 10 - 60 ng were partitioned in the above system and it was found that there was practically no detectable amount of either compound in the water layer. Comparison of Methods Ml and M2
Duplicate aliquots of 33 control plasmas were analysed by
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678
WIIEOXDm
the twr, method?? Ml and M2, carried out by two different workers. The results are plotted in Fig. 1 and show a good correlation between the two sets of data.
Normal values Fig. 2 shows the means and percentiles of estriol values obtained in 105 healthy subjects studied prospectively pregnancy.
throughout
It depicts clearly the general rising trend of E3
concentration as pregnancy progresses The results are given in nml/l
(17). by multiplying pg/lOO ml by a
factor of 34.7, in compliance with the recent adoption in Australia and the U.K. of SI Units (De Syst&ns International
d'Vnit&s).
DISCUSSION Plasma estriol determinations have several advantages over those in maternal urine.
The problems of lengthy and uncertain collections
are overcome, as well as the delay in obtaining a result.
Estriol
is measured as a circulating hormone rather than as an excretory product. The method described satisfies the requirements of reliability and has proved robust in daily use.
The accuracy and precision of
the method compare well with those of other published fluorimatric methods for the estimation of E
3'
More importantly, results are able to be obtained within 5 hours of the specimens being taken.
This was made possible - (a) by
the use of automated devices for the addition of all reagents, using quick-freezing
techniques in all extractions:
ib) by
this also resulted
679
Figure 1 - Correlation of results obtained by two different workers using 0.5 ml (&fl)and 0.2 ml (M2) plasma volumes, respectively.
1200
1000
800 g t-J 8
600
s In 4 a 400
24
26
32
34
36
36
40
WEEKS
Figure 2-
Norm31 values of estriol in m3ternal plasma.
in an optimal and constant 70% recovery, (c) by the use of a steam sterilizer for hydrolysis and the development of fluorescence, thus reducing the time intervals required to 15 and 10 mins, respectively. Two trained technicians are currently performing 150 determinations per week. The method has been in use for about two years in this Department for research purposes and has recently been adopted as a routine procedure in the Royal Women's Hospital. Using the above method, mean E3 levels of 270 nmol/l (7.8 pg/ 100 ml) at 24 weeks to 811 nmol/l (23.4 ug/lOO ml) at 40 weeks have been obtained.
These results are in close agreement with those
obtained by other fluorimetric, competitive protein binding and radioimmunoassay methods (13 - 15).
The full details of these studies
were published elsewhere (17). No significant differences in results were observed when the glucosiduronate was used rather than the free compound as the internal standard.
This observation agrees with those of other workers (5, 13)
who found little difference between the two labelled standards.
In
view of its simplicity, therefore, tritiated free estriol was used as the internal standard in the later phase of this work. Quenching is an important problem in fluorescence procedures. All glassware used in the present method was heated to 540° which is high enough to destroy organic impurities (16). ACKNOWLBDGEMENTS We wish to thank the patients at the Mater Misexicordiae Mothers' Hospital, South Brisbane, and the Royal Women's Hospital, Brisbane, from whom plasma samples were obtained, and the staff of the Obstetrics and Gynaecology Department for their assistance.
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TDSOXDS
681
This work was supported by a grant to J.S.G.B. from the National Health and Medical Research Council of Australia. REFERENCES 2.
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
Nachtigall, L., Bassett, M., Hogsander, V., Slagle, S., and Levitz, M., J. CLIN. ENDOCRINOL. 26, 941 (1966). Gurpide, E., Glebenhain, M. E., Tseng, L., and Kelly, W. G., AM. J. OBSTET. GYNECOL. 109, $97 (1971). F.,J. OBST. GYNAECOL. BRIT. CWLTH. Corker, C. S., and Naftox, 78, 330 (1971). Tulchinsky, D., and Abraham, G. E., J. CLIN. ENDOCRINOL. 33, 775 (1971). Mathur, R. S., Learning,A. B., and Williamson, Ii.O., AM. J. OBSTET. GYNECOL. 113, 1120 (1972). Brown, J. B., McLeod, S. C., Macnaughtan, C., Smith, M. A., and Sniyth,B., J. ENDOCRINOL. 42, 5 (1968). Slaunwhite, W. R. Jr., Lic%man, H. A., and Sandberg, A. A., J. CLIN, ENDCCRINOL.24, 638 (1964). Herberg, R. J., ANAL. CHEM. 32, 42 (1960). Brown, J. B., Macnaughtan, C., Smith, M. A., and Smyth, B., J. ENDOCRINOL. 40, 175 (1968). Brown, J. B., BIGCHEM. J. 60, 185 (1955). Pinkelstein, M., Hestrin, S., and Koch, W., PROC. SOC. EXPTL. BIOL. MED. 69, 181 (1948). Wilkinson, M., Sffer, S. B., and Gupta, K., AM. J. OBSTET. GYNECOL. 114, 867 (1972). Nachtigall, L., Bassett, M., Hogsander, V., and Levitz, M., AM, J. OBSTET. GYNECOL. 101, 638 (1968). Macourt, D., Corker, C. S., and Naftolin, F., J. OBSTET. GYNAECOL. BRIT. CWLTH. 78, 335 (1971). Masson, G. M., J. OBSTET. GYNAECOL. BRIT. CWLTH. 80, 201 (1973). Kushinsky, S., and Paul, W., ANAL. BIOCHEM. 30, 465 (1969). Biggs, J. S. G., Miklosi, S., and Morrison, J., A.N.Z. J. OBSTET. GYNAECOL. (in press).
S. Mfklosf, J. S. G. Bfggs, N. Selvage, J. Canning and G. Lythal University of Queensland, Department of Obstetrics and Gipxaecology, Clinical Sciences Building, Royal Brisbane Hospital, Brfsbane, Queensland, 4029, Australia Received:
8/27!75
ABSTRACT
A rapid, reliable fluorimatric nasthodfor the determination of estriol (Eg in plasma is described. The method is eminently suitable for use in hospital laboratories and provides results within 5 hours of the reception of samples. It has several features not found in other methods. As little as 0.2 ml of plasma is required, all additions and aliquotfng procedures are semi-automated and no special technical skill is necessary. A technician can carry out 20 detezminations per day. Average recoveries of 70% are routinely achieved, and the accuracy (2.4%) and the precision (4.0%) of the method are remarkably good for an assay of this type. A spectrofluoromater of high sensitivity, counting equipment and a high temperature oven are the essential nnjor pieces of equipment.
INTROWCTION There are many advantages in measuring E
in plasma rather 3 than iu urine in late pregnancy, and a number of methods has been
published durfng recent years (1 - 5).
Hethods rmst allow results
to be obtained within a working day and still be accurate, precise and sensftive enough to be acceptable In clinical practice. The mathods of Nachtigall et al. (1) and Mathur et al. (5) seemed to fulfill these requirements. even
However, too frequent quenching -
in the standards - yielded poor precision in our hands and the
introduction of further purification steps made these methods too lengthy for clinical use.
volume 26, Number 5
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2975
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672
‘!B?FsEOXDCI
Estrogen in the urine of non-pregnant women is measured in this Department by the method of Brown et al. (6), using a spectrofluoronmter of high sensitivity and resolution. Efforts were directed to developing a method for Ej in other biological fluids, using similar principles. mentioned above.
The method devised satisfies the criteria
It involves acid hydrolysis in a steam sterilizer,
ether extraction, benzene-hexane partition and quantitation by fluorimetry.
Results are corrected for losses by the use of tritiated B5
as an internal standard.
The method is described in more detail
than usual, since it has been found that even slight changes have affected the precision. MATERIALS AND ME!WODS Blood samples were collected into lithium-heparin tubes and centrifuged inmediately. The separated plasma was processed at once or stored at -15O. Reagents
Rigorous purification of almost all of the reagents was necessary in order to obtain reproducible results. Sulfuric acid, purchased from Merck (GR), proved to be satisfactory for the preparation of ths Kober reagent. Diethyl ether (May d;Baker) was taken from freshlyAll brands of ether except opened 500~ml bottles just before use. M & B quenched the fluorescence of pure standards. Hydrochloric acid and ethanol were purchased from Merck. Ethanol was xefluxed over sodium hydroxide pellets and twice re-distilled. sym-Tetxachloroethane (AR) was used as received. To1 uene was scintillation grade, benzene and n-hexane were fluorimetry grade Bydroquinone was re-crystallized from water. obtained from Merck. p-Nitrophenol (Merck, BDH) was re-crystallized from benzene until near colorless. Sodium hydroxide and sodium hydrogen carbonate were of AR grade (BDB). Carbonate buffer, pi?10.5, was prepared by dissolving 21 g of NaOH and 70 g of sodium hydrogen carbonate in water and making up to 1 liter (10). Water was distilled in an all-glass apparatus and then passed through a deionizer. Kober reagent The Kober reagent contained 66% (v/v) sulfuric acid and 2% quinol and was prepared by dissolving finely powdered quinol in the
673
diluted acid on a magnetic stirrer plate at room temperature in the The reagent was kept in an amber bottle dark, without any heating. at room temperature and placed in a dark cupboard when not in use. It was discarded as soon as a slight brownish color developed. Ittrich reagent This reagent was prepared from 2% finely divided p-nitro-phenol in sym-tetrachloroethane (w/v) and was pale yellow in color. Solution was effected in about 5 min. without the addition of ethanol by vigorous The reagent was discarded when deepening of its yellow color shaking. was observed. Stand,ards -Estriol was purchased from Ikapharm, Israel, and purified by TLC on glass fiber plates impregnated with silica gel (ITLC-SA), (Gelman A stock Instr. Co.) using a system of chloroform : methanol (97 : 3). stangard solution containing 100 m/ml in ethanol was prepared and kept Estriol-16d-D(+)-glucosiduronate(E3-Gp) was obtained from at 5 . Estriol (6,7- H (N) ), 53.1 Ci/xnnol, Sigma Chem. Co., St. Louis, MO. was purchased from New England Nuclear, Boston, Mass. and from the Radiochemical Centre, Amersham, V .K. The radioactive compounds were checked for purity by TLC in the same manner as the non-radioactive estriol. An aliquot was diluted with ethanol to give approx. 20,000 cpm per 10 pl and used as an internal standard during the later phase of this work. Estriol-(6,7-3B)-16c-glucosiduronate, prepared according to Slauawhite et al. (7) with nwdifications as suggested by Goebelsmann (personal comm.). was used as an internal standard during the early phase of this work. Counting
Radioactivity was measured in a Nuclear Chicago liquid scintillation counter, Model 4649, Mark II, using the channels ratio method. Tritiated E3 was counted All samples were counted for 10,000 counts. in 1.5ml of toluene containing "Gmnifluor" (4 g/l, NEN, Boston, Mass.) and the radioactive glucosiduronate in the system of Herberg (8). Cleaning of glassware Glassware used for fluorimetry must be meticulously cleaned. The tubes used for hydrolysis and extraction and those used for the Kober reaction were washed with ethanol, 5 times with hot tap water and then 5 times with distilled water. All tubes which contained plasma were soaked in acetone at fortnightly intervals. The glassware was then placed in a self-cleaning oven (Westinghouse,Model PAW 306, Email. Ltd., Aust.) which has a I-hr. cycle of 540°. Sometimes, dark brown stains appeared on the plasma tubes due to undissolved lipid material. These were removed by soaking the tubes overnight in Chromate traces were destroyed by washing the tubes chromic acid.
with a 0.2% acidified solution of sodium sulfite. Measurement of fluorescence Fluorescence was measured in the round cuvettes described by Brown et al. (9). By this means the extraction of fluorescence, centrifuqation and fluorimatry can be done in one tube. The risk of contaminating the atmosphere of the laboratory with tetrachloroethane is thus avoided. The tube projects through the cover of the Brown cell adapter (Aminco-BowmanCat. No. 4-8244) and is protected from liqht by a cylindrical cover. Fluorescence was measured in an AmincoBowman 4-8203 D spectrofluorometer with a solid state, blank-substract microphotometer and an off-axis ellipsoidal mirror condensing system using a xenon lamp, a 1 P 21 potted photomultiplier tube and 2 mn slits throughout. Sensitivity was better than one part per trillion of quinine sulfate in 0.1 N H2S04 and resolution was 0.8 nm with the 600 line/mm grating. The spectrofluorometer was standardized with quinine sulfate before use. Even with 2 mm slits, some liqht from the source passed to the photomultiplier. This was overcome by inserting a Corning 3-67 sharp cut-off filter which in turn shifted the maximum of excitation from 532 to 537 nm and that of the emission from 552 to 563 nm. Some 30% of the sensitivity was lost with the use of this filter. Extractions and partitioning Ether extraction, following hydrolysis of plasma, was performed in an alkaline medium (pH 9 - 9.5). Lower pH often produced emulsions. After extraction (by.holdinq all the tubes down in a rack with a board and shaking) and centrifuqation the aqueous layer was quick-frozen in dry ice/ethanol and the ether decanted. Dry ice was obtained by the use of a mechanical attachment designed to fit any standard Co2 cylinThis simple device ("Hedi-ice" from der equipped with a syphon tube. Metallurgical Supply Co., Houston, Tex.) consists of a nozzle, cone and collection bag and is attached to the cylinder; the valve is opened and the collection bag is immediately filled with dry ice of flaked consistency, ready for use. Methods The assay was first carried out with 0.5 ml of plasma (Ml) and later with 0.2 ml (M2). In order to speed up operations, all reagents were added from automatic dispensers. Plasma in appropriate amount is measured into centrifuge tubes (Quickfit B 24, 115 x 29 mm, or B 14, 150 x 18 mm for M2). Water is added (2 ml and 0.8 ml, respectively, followed by 10 pl of 3H-B3 and 0.4 ml cont. IiCl (0.16 ml for M2), and some antibumping granules. The contents are well mixed and the tubes are covered $th aluminium foil and placed in a steam sterilizer for 15 min. at 120 . After hydrolysis, the tubes are cooled under tap water and 5 N NaOH added (0.7 ml for Ml; 0.28 ml for M2) followed by carbonate buffer,
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TBEOXD=
675
pfi 10.5 (2.0 m.2 for Ml; 0.8 ml in M2) and the contents mixed (14). The mixture is then extracted with diethyl ether (10 ml for Ml; 6 ml for M2) by shaking the tubes together 100 times. The tubes are centrifuged at 2000 rpm for 5 min. in a refrigerated centrifuge. The tubes are placed in a dry ice/ethanol mixture and after quickfreezing the diethyl ether layer is decanted into a set of B 14 test tubes. A piece of antibumping granule is placed in each and the ether is evaporated in a water bath at 40°. Distilled water (2 ml) is added and the tubes are rotated to dissolve the dry residue. Two ml of a benzene/hexane (1:l) mixture, freshly prepared, is then added. The tubes are shaken well to extract E , E and fatty substances (11). The W&es are centrifuged at 2000 rpm $5 or min. and placed in a deep freeze for 5 min. to freeze the aqueous layer; the upper organic phase is then sucked off with a Pasteur pipette attached to a vacuum line. After quick thawing, 6 ml of diethyl ether is added to the aqueous phase, which is extracted by shaking vigorously as before. After centrifuging for a few minutes, the tubes are placed in dry ice/ethanol, the water layer quick-frozen and the ether decanted into another set of tubes (B 14, 125 x 18 zmn). One antibumping granule is added, and the ether is evaporated in a hot water bath. The tubes are dried by suction and the residue dissolved in 0.5 ml of ethanol using a Vortex mixer and 0.05 ml is taken for counting of radioactivity. Ethanol solution (0.2 ml) containing 5 g quinol/lOO ml (2 g quinol/lOO ml for M2) is now added, mixed well and evaporated to dryness at 60° on a rotary evaporator. Complete dryness must be achieved by using a strong vacuum.
One ml of 66% Kober reagent is added to the tubes and solution is effected by warming. The tubes are then rotated so as to cover theoinside area by the Kober reagent, tightly stoppered and heated.at 120 for 10 min. then cooled in ice water. When completely cool, 1.5 ml ice water is added to each tube and the contents well mixed. The tubes are immersed again in ice water and the contents poured into the corresponding fluorimetry tubes containing 0.75 ml of Ittrich reagrent. This had been measured out from a dispenser in a fume cupboard. Several sets of these tubes are prepared in advance and kept at -5" until required. The fluorimetry tubes are then stoppered and shaken vigorously all at the game time about 100 times, centrifuged at 2000 rpm for 3 min. at 0 , allowed to demist for a few minutes, then wiped carefully. Fluorimetry at 537/563 nm must be performed immediately. From the addition of ice water onwards, the steps should be completed in less than 20 minutes to avoid fading of fluorescence. The E3 content is calculated by direct comparison with a standard of 5Opg (or 20 ,uy for M2) processed at the same time. The instrument is set to read the same value of the standard in day-to-day operations so that the formula used to calculate the results is greatly simplified. The results are corrected for losses from the radioactivity recovered.
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676
TZDEOTDm RESULTS
Recovery
The losses incurred in the method are corrected by the use of radioactive
internal standards.
The recovery of free tritiated E3
+ when used as the internal standard was 70 - 3.8% (S.D.) (200 tests). Accuracy and precision The results of duplicate experiments in which known amounts of estriol glucosiduronate
were added to male plasma are given in Table 1.
The results are corrected for blank male plasma values. Table 1.
Amount of E -Glu a dded (ng)
Recovery of Estriol Glucosiduronate Added to Male Plasma
Equivalent of free E3 (ng)
Average amount recovered as free E3 (ng)
Average S.D.
% recovery (accuracy)
Number of assays (n)
10
6.2
6.0
2 1.12
96.8
6
20
12.4
12.2
f 1.26
98.7
19
40
24.8
24.4
+, 1.72
96.0
19
60
37.2
37.1
f 5.57
99.5
15
80
49.6
50.0
+, 2.2
100.7
14
The corrected recoveries ranged from 96 to 100.7%, with a mean of 98 + 2.4 (S.D.) %.
The coefficient of variation was thus
2.4 x 100/98 = 2.4%. The precision of the assay was examined by measuring the E3 concentration
of pooled late pregnancy plasma or of male plasma
to which a known amount of E3 was added. For the method using 0.5 ml of plasma
(Ml) the coefficient
of variation was 8.8% (n = 20), while for method M2 using 0.2 ml of
S plasma, the Mlue
677
T=EOXDCI
was 4% (n = 25).
The between assay precision, measured as the C.V. of a control plasma in duplicates in each assay, was 11.6% (n = 88) for Ml and 7.4% for M2 (n = 84). Sensitivity The smallest amount of E3 which could be measured with pure standards was around 300 pg. When 0.2 ml of water was assayed in place of plasma, 10 ml was measured by the instrument.
w/
This solvent blank, however, can
be substracted by making use of the blank-substract device of the microphotometer. SpeciThe specificity of the method depends largely on the effectiveness of the water/benzene-hexanepartition step in removing other estrogens (1). Tritiated E 3, together with 20 ng of carrier E3, was added to water and partitioned between equal volumes of benzene-hexane. The water layer was found to contain 98% of the radioactivity. Wilkinson et al. (12) found that 87 - 91% of El and E2 were extracted into the organic layer in similar experiments.
In our
laboratory, estrone (El) and estradiol (E2) in concentrations ranging from 10 - 60 ng were partitioned in the above system and it was found that there was practically no detectable amount of either compound in the water layer. Comparison of Methods Ml and M2
Duplicate aliquots of 33 control plasmas were analysed by
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678
WIIEOXDm
the twr, method?? Ml and M2, carried out by two different workers. The results are plotted in Fig. 1 and show a good correlation between the two sets of data.
Normal values Fig. 2 shows the means and percentiles of estriol values obtained in 105 healthy subjects studied prospectively pregnancy.
throughout
It depicts clearly the general rising trend of E3
concentration as pregnancy progresses The results are given in nml/l
(17). by multiplying pg/lOO ml by a
factor of 34.7, in compliance with the recent adoption in Australia and the U.K. of SI Units (De Syst&ns International
d'Vnit&s).
DISCUSSION Plasma estriol determinations have several advantages over those in maternal urine.
The problems of lengthy and uncertain collections
are overcome, as well as the delay in obtaining a result.
Estriol
is measured as a circulating hormone rather than as an excretory product. The method described satisfies the requirements of reliability and has proved robust in daily use.
The accuracy and precision of
the method compare well with those of other published fluorimatric methods for the estimation of E
3'
More importantly, results are able to be obtained within 5 hours of the specimens being taken.
This was made possible - (a) by
the use of automated devices for the addition of all reagents, using quick-freezing
techniques in all extractions:
ib) by
this also resulted
679
Figure 1 - Correlation of results obtained by two different workers using 0.5 ml (&fl)and 0.2 ml (M2) plasma volumes, respectively.
1200
1000
800 g t-J 8
600
s In 4 a 400
24
26
32
34
36
36
40
WEEKS
Figure 2-
Norm31 values of estriol in m3ternal plasma.
in an optimal and constant 70% recovery, (c) by the use of a steam sterilizer for hydrolysis and the development of fluorescence, thus reducing the time intervals required to 15 and 10 mins, respectively. Two trained technicians are currently performing 150 determinations per week. The method has been in use for about two years in this Department for research purposes and has recently been adopted as a routine procedure in the Royal Women's Hospital. Using the above method, mean E3 levels of 270 nmol/l (7.8 pg/ 100 ml) at 24 weeks to 811 nmol/l (23.4 ug/lOO ml) at 40 weeks have been obtained.
These results are in close agreement with those
obtained by other fluorimetric, competitive protein binding and radioimmunoassay methods (13 - 15).
The full details of these studies
were published elsewhere (17). No significant differences in results were observed when the glucosiduronate was used rather than the free compound as the internal standard.
This observation agrees with those of other workers (5, 13)
who found little difference between the two labelled standards.
In
view of its simplicity, therefore, tritiated free estriol was used as the internal standard in the later phase of this work. Quenching is an important problem in fluorescence procedures. All glassware used in the present method was heated to 540° which is high enough to destroy organic impurities (16). ACKNOWLBDGEMENTS We wish to thank the patients at the Mater Misexicordiae Mothers' Hospital, South Brisbane, and the Royal Women's Hospital, Brisbane, from whom plasma samples were obtained, and the staff of the Obstetrics and Gynaecology Department for their assistance.
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TDSOXDS
681
This work was supported by a grant to J.S.G.B. from the National Health and Medical Research Council of Australia. REFERENCES 2.
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
Nachtigall, L., Bassett, M., Hogsander, V., Slagle, S., and Levitz, M., J. CLIN. ENDOCRINOL. 26, 941 (1966). Gurpide, E., Glebenhain, M. E., Tseng, L., and Kelly, W. G., AM. J. OBSTET. GYNECOL. 109, $97 (1971). F.,J. OBST. GYNAECOL. BRIT. CWLTH. Corker, C. S., and Naftox, 78, 330 (1971). Tulchinsky, D., and Abraham, G. E., J. CLIN. ENDOCRINOL. 33, 775 (1971). Mathur, R. S., Learning,A. B., and Williamson, Ii.O., AM. J. OBSTET. GYNECOL. 113, 1120 (1972). Brown, J. B., McLeod, S. C., Macnaughtan, C., Smith, M. A., and Sniyth,B., J. ENDOCRINOL. 42, 5 (1968). Slaunwhite, W. R. Jr., Lic%man, H. A., and Sandberg, A. A., J. CLIN, ENDCCRINOL.24, 638 (1964). Herberg, R. J., ANAL. CHEM. 32, 42 (1960). Brown, J. B., Macnaughtan, C., Smith, M. A., and Smyth, B., J. ENDOCRINOL. 40, 175 (1968). Brown, J. B., BIGCHEM. J. 60, 185 (1955). Pinkelstein, M., Hestrin, S., and Koch, W., PROC. SOC. EXPTL. BIOL. MED. 69, 181 (1948). Wilkinson, M., Sffer, S. B., and Gupta, K., AM. J. OBSTET. GYNECOL. 114, 867 (1972). Nachtigall, L., Bassett, M., Hogsander, V., and Levitz, M., AM, J. OBSTET. GYNECOL. 101, 638 (1968). Macourt, D., Corker, C. S., and Naftolin, F., J. OBSTET. GYNAECOL. BRIT. CWLTH. 78, 335 (1971). Masson, G. M., J. OBSTET. GYNAECOL. BRIT. CWLTH. 80, 201 (1973). Kushinsky, S., and Paul, W., ANAL. BIOCHEM. 30, 465 (1969). Biggs, J. S. G., Miklosi, S., and Morrison, J., A.N.Z. J. OBSTET. GYNAECOL. (in press).
