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A Comparison of the Color Chemical Test with the Friedman Modification of the Aschheim-Zondek Test
A OOMPtiiiOlf OF .TJDl ·.t)QLOI. ·OBilliWAL
~
WPIB 'lD
Mcmlno&!faf 'op !a ............._. '.fDT
FtulmMAN
GRACE McCORMACK,
A.B., RocHESTER, N.Y.
(Fro-m the Depa.1·trnent of Obstetrics and Gynecology, o·mversity, of Rorhcster Medic{!.! 8oho()l and Stronp M em orictl Hospital)
rea~tion
pre~nan~iol
for test for pregnancy based on the color A CHEMICAL (Guterman may eventually replacE' that of the Friedman modification withintwo 2
)
of the Aschheim-Zondek test. The determination can be completed or three hours, and the results compm·e favorably with the Friedman modifi-. cation of the Aschheim-Zondek test. The chemical test has the distinct advantage over the Friedman test in not r·equiring rabbits, in heing quicke1· anfl less expensive. In our laboratory, we have been carrying out the chemical color reaetion test for pregnandiol simultaneously with the Friedman test for pregnanc.y. rhe color reaction test is based upon the method for pregnandiol extraction by Astwoorl and Jones 1 and the colm· reaction of Talbot and his associates,:1 which has been modified by Guterman 2 for the purpose of aseertaining- preg>llancy. The method consists essentially of the extraetion with toluene of tl1e :tcid-hydrolyzed pregnandiol complex of urine, followed by the purification [tnd precipitation of the pregnandiol. The characteristic color is developed hy mlfuric acid. The technique of Huterman 2 is as follows: A. Hydrolysis and extraction of pregnandiol: 1. One hundred cubic centimetet·s of urine, 50 c.c. toluene, chemienlly pure, 10 c.e. concentrate<'! hydrochloric acid, :mil 2 glass beads are ad <'led to a 1'100 e.e. fiat· bottomed ·Florence flask. ~- 'rhe flask is eonnected via a one-holed eork stopper to a vertical Liebig eondenser and the mixture is boiled vigorou~ly over nn electric hot-plate for fifte(m minutes. :). The flask an
water tap. 4. The mixture is transfent>d to a .iOII c.(~. 8eparator~· funnel, anrl the lower l!J,t>er is drawn off. 5. ·rhe toluene layer and th~t toluene-water emulsion are washed twice with 15 c.c. portions of 0.1 N sodium hydroxide ami then twice with lli 0.e. portions of distilled water. B. Precipitation of impurities: 1. The washed toluene and toluene-water emulsion ( A-5) are transJ.el'red to a 125 c.c. Erlenmeyer flask. ll. The mixture is boiled over an electric hot-plate (in tlre hood). 3. When the water has evaporat~ and the toluene mixture is bfriling smoothly, w,, c.c. of 2 per cent sodium hydroxide in absolute meth~mol are added.
10
MC CORMACK :
COLOR CHEMICAL TEST F'OR PRJ<;GNANCY
4. The mixture is evaporated until one-half of the original toluene volumt' i~ reached. n. The toluene mixture is then filtered, while hot, through a fritted glass filter (medium porosit~- Pyrex) with mild suction. (If the filtrate has ::tn orange, pink, or brown tinge, ~teps B-3, B-4, and B-5 mnst be repeated until thr• filtrato> is yellow or yellow green.) 6. The precipitate (B-5) is washed with 15 c.c. hot toluene. 7. 'rhe combined filtrates ( B-5 and B-6) are then evaporated to dryness over th.hot-plate (in the hood), a gentle air stream being used to drive off the last tmces of toluene. 'J'hi~ avoids charring of the residue. C. Precipitation of pregnaniliol: 1. Five cubic centimeters of acetone are added to the residue (B-7), and tht• mixture .is warmerl over a hot·Illate until the solution is complete. 2. Twenty cubic ~entimeters of 0.1 N sodium hyoroxirle are added slowly while the mixture is still on the hot-plate. :l. The flask i~ then placed in an ice-water bath for thirty minutes ot· in a rP· frigerator for one hour. D. lRolaLion of pregnandiol: 1. 'rhe mixture ( 1!-3) is filtered through a fritted glass filter. 2. The precipitate (D·l) is washed with 15 c.c. distilled water. 3. The receiving flask is rhanged, and 10 c.c. hot absolute alcohol is pasRerl through the fritted glass :filtel' to dissolve the precipitate. 4. The alcohol filtrate (D-3 1 is evaporated to drynes~ over an electric-hot plate. E. Color development: 1. Ten cubic eentimeters of concentrated sulfuric acid are added to the residut> (D 4), and the eo lor is observed in a test tube when solution is complete. Colorles!l to light yellow solution i~ read as negative. Orange to deep orange brown is read as positive.
We have made a few minor changes in the above technique so that it is never necessary to refilter the toluene mixture containing the impurities, as the filtrate is always yellow in color (B-5). We also find that in using an eggshaped porcelain crucible, Coors No. 2, that the mixture will evaporate to dryness without charring (B-7). The technical differences, as compared to the technique of Guterman/ are as :follows: A-4. Allow the toluene layer and toluene-water emulsion to stand until no won• urine can possibly be drawn off. A-5. If the mixture should turu blue litmus paper red after the second addition of alkali, repeat. Then, if the solution is not clear, centrifuge. The clear solution will appear at the top of the tube. After each addition of alkali and distilleil water, shake for two or three minutes, preferably in a shaker. B-1. Transfer to 125 c.c. Brlenrneyer flask the waslu:>d toluene and toluene-water emulsion; then pour the supernatant into a similar flask. B-4. ·The solution i~ evaporatect until approximately one-half o:f the original toluene volume is reached. After the addition of the sodium hydroxide in methanol, the solution becomes cloudy. When precipitation of the impurities is complete, tht> solution suddenly bubbles up; then bo·[Js smoothly. Allow the solution to continue boiling until approximately one-half of this vo:tume has evaporated, before removing from the hot-plate.
724
.\MERICA~
.JOURNAL OF OBSTETRIC:'\ A1\D \H"KECOLOGY
Results
The chemical color reaction test for pregllalldiol has been done simultallt'·· uusly with the Friedman test for preg·nancy on 304 patients. Of this numlwr, 262 tests were in perfect agreement: t:)O tests showPd positive resnlts for both pregnancy tests, and 132 tests showed negative results for both. Th0se findings were borne out Dy subsequent cHmcat nndings. T1H' results on the other patients did not coincide. Negative chemical tests and positive Friedman tests were obtained on 30 patients diagnosed aR threatt'IINl ahortion. From this group, 22 aborted; 10, however·, wen' given progestermw, and :wveral months. later, 8 of the patients were still pregnant. All persons wen~ hospitalized on account of mucn meeamg. Two patients gave posithe results for both tests; then suddenly aborte(l the following day. There hnrl been no lileeding prior to abortion. Two patients, we believe, had a persistent corpus luteum cyst that finally degenerated. We did a series of tests, each time obtaining positive chemical and negative rabbit results. After two months, amPnorr·hea ceased, and a few clays later the pregnandiol titer d1·opped, finally rt>aching the negative level. rrv/o cases of hydatid mole \Vere tested. In both instances the chemica! test was negative; the rabbit results, positive. In eight cases, pregnancy was indicated chemically two or three days before it showed biologically. The ehemiral rt>sults were read incorrectly in ten cases. The table below summarizes the results: 'PABLE I CHEMICAL TEST POSITIVE NEGATIVE
I
·FlllEDMAN TEST POSITIVE NEGATIVE
----~----------~~~---
----~~~~--------------------~--------
*Normal pregnancy Ectopic pregnancy Not pregnant Threatened abortion Corpus luteum cyst Hydatid mole
121
9 2
132
:w
121 9
132
~ 2 •Eight from this group gave positive chemical results two or three days before the remits were positive biologically.
----------
Discuasion and Oo:n.clution The results which were obtain:ed leave no doubt that there is an equivalent degree of accuracy between the chemical color reaction test and the Friedman test for pregnancy. The chemical test has the advantage over the Frie(}man test in that a result can be obtained-lvithin tv;o or three _hours, and -that _it is more economical. The chemical test plus the Friedman test are good indicators in cases of threatened abortion. The chemical test is negati've in threatened abortion due to the fact that there is little or no progesterone being r>roduced. This decrease or lack o:f progesterone, no doubt, is the cause of the threatening abortion. In contrast, .the Friednum test depends upon. the anterior-pituitary-like substance elaborated by chorionic villi. ·· A positive
MCCORMACK:
t'OI,OR
CH~:MICAL
'l'EST FOH PREGNANCY
725
chemical test will be obtained in cases of a corpus luteum cyst but, since such cases are not too numerous and can be differentiated from normal pregnancy by the Friedman test, it should not he a disturbing factor. I wish to expresR of this work.
m~· apprP~iation
to Dr. C. Arthur Eld<'n for hiR friendly help and
~riticism
References ,J. Biol. Chern. 137: 397, 1941. Guterman, H. S.: .T. Clin. Endocrinol. 4: 262, 1944 . .3. 'falbot, N. B., Berman, R. A., MacLachlan, B. A., and Wolfe, J. K.: 1: 668, 194]. l. Astwood, B. B., and Jones, G. E. S.: ~-
.r.
Clin. Endocrinol.
~
WPIB 'lD
Mcmlno&!faf 'op !a ............._. '.fDT
FtulmMAN
GRACE McCORMACK,
A.B., RocHESTER, N.Y.
(Fro-m the Depa.1·trnent of Obstetrics and Gynecology, o·mversity, of Rorhcster Medic{!.! 8oho()l and Stronp M em orictl Hospital)
rea~tion
pre~nan~iol
for test for pregnancy based on the color A CHEMICAL (Guterman may eventually replacE' that of the Friedman modification withintwo 2
)
of the Aschheim-Zondek test. The determination can be completed or three hours, and the results compm·e favorably with the Friedman modifi-. cation of the Aschheim-Zondek test. The chemical test has the distinct advantage over the Friedman test in not r·equiring rabbits, in heing quicke1· anfl less expensive. In our laboratory, we have been carrying out the chemical color reaetion test for pregnandiol simultaneously with the Friedman test for pregnanc.y. rhe color reaction test is based upon the method for pregnandiol extraction by Astwoorl and Jones 1 and the colm· reaction of Talbot and his associates,:1 which has been modified by Guterman 2 for the purpose of aseertaining- preg>llancy. The method consists essentially of the extraetion with toluene of tl1e :tcid-hydrolyzed pregnandiol complex of urine, followed by the purification [tnd precipitation of the pregnandiol. The characteristic color is developed hy mlfuric acid. The technique of Huterman 2 is as follows: A. Hydrolysis and extraction of pregnandiol: 1. One hundred cubic centimetet·s of urine, 50 c.c. toluene, chemienlly pure, 10 c.e. concentrate<'! hydrochloric acid, :mil 2 glass beads are ad <'led to a 1'100 e.e. fiat· bottomed ·Florence flask. ~- 'rhe flask is eonnected via a one-holed eork stopper to a vertical Liebig eondenser and the mixture is boiled vigorou~ly over nn electric hot-plate for fifte(m minutes. :). The flask an
water tap. 4. The mixture is transfent>d to a .iOII c.(~. 8eparator~· funnel, anrl the lower l!J,t>er is drawn off. 5. ·rhe toluene layer and th~t toluene-water emulsion are washed twice with 15 c.c. portions of 0.1 N sodium hydroxide ami then twice with lli 0.e. portions of distilled water. B. Precipitation of impurities: 1. The washed toluene and toluene-water emulsion ( A-5) are transJ.el'red to a 125 c.c. Erlenmeyer flask. ll. The mixture is boiled over an electric hot-plate (in tlre hood). 3. When the water has evaporat~ and the toluene mixture is bfriling smoothly, w,, c.c. of 2 per cent sodium hydroxide in absolute meth~mol are added.
10
MC CORMACK :
COLOR CHEMICAL TEST F'OR PRJ<;GNANCY
4. The mixture is evaporated until one-half of the original toluene volumt' i~ reached. n. The toluene mixture is then filtered, while hot, through a fritted glass filter (medium porosit~- Pyrex) with mild suction. (If the filtrate has ::tn orange, pink, or brown tinge, ~teps B-3, B-4, and B-5 mnst be repeated until thr• filtrato> is yellow or yellow green.) 6. The precipitate (B-5) is washed with 15 c.c. hot toluene. 7. 'rhe combined filtrates ( B-5 and B-6) are then evaporated to dryness over th.hot-plate (in the hood), a gentle air stream being used to drive off the last tmces of toluene. 'J'hi~ avoids charring of the residue. C. Precipitation of pregnaniliol: 1. Five cubic centimeters of acetone are added to the residue (B-7), and tht• mixture .is warmerl over a hot·Illate until the solution is complete. 2. Twenty cubic ~entimeters of 0.1 N sodium hyoroxirle are added slowly while the mixture is still on the hot-plate. :l. The flask i~ then placed in an ice-water bath for thirty minutes ot· in a rP· frigerator for one hour. D. lRolaLion of pregnandiol: 1. 'rhe mixture ( 1!-3) is filtered through a fritted glass filter. 2. The precipitate (D·l) is washed with 15 c.c. distilled water. 3. The receiving flask is rhanged, and 10 c.c. hot absolute alcohol is pasRerl through the fritted glass :filtel' to dissolve the precipitate. 4. The alcohol filtrate (D-3 1 is evaporated to drynes~ over an electric-hot plate. E. Color development: 1. Ten cubic eentimeters of concentrated sulfuric acid are added to the residut> (D 4), and the eo lor is observed in a test tube when solution is complete. Colorles!l to light yellow solution i~ read as negative. Orange to deep orange brown is read as positive.
We have made a few minor changes in the above technique so that it is never necessary to refilter the toluene mixture containing the impurities, as the filtrate is always yellow in color (B-5). We also find that in using an eggshaped porcelain crucible, Coors No. 2, that the mixture will evaporate to dryness without charring (B-7). The technical differences, as compared to the technique of Guterman/ are as :follows: A-4. Allow the toluene layer and toluene-water emulsion to stand until no won• urine can possibly be drawn off. A-5. If the mixture should turu blue litmus paper red after the second addition of alkali, repeat. Then, if the solution is not clear, centrifuge. The clear solution will appear at the top of the tube. After each addition of alkali and distilleil water, shake for two or three minutes, preferably in a shaker. B-1. Transfer to 125 c.c. Brlenrneyer flask the waslu:>d toluene and toluene-water emulsion; then pour the supernatant into a similar flask. B-4. ·The solution i~ evaporatect until approximately one-half o:f the original toluene volume is reached. After the addition of the sodium hydroxide in methanol, the solution becomes cloudy. When precipitation of the impurities is complete, tht> solution suddenly bubbles up; then bo·[Js smoothly. Allow the solution to continue boiling until approximately one-half of this vo:tume has evaporated, before removing from the hot-plate.
724
.\MERICA~
.JOURNAL OF OBSTETRIC:'\ A1\D \H"KECOLOGY
Results
The chemical color reaction test for pregllalldiol has been done simultallt'·· uusly with the Friedman test for preg·nancy on 304 patients. Of this numlwr, 262 tests were in perfect agreement: t:)O tests showPd positive resnlts for both pregnancy tests, and 132 tests showed negative results for both. Th0se findings were borne out Dy subsequent cHmcat nndings. T1H' results on the other patients did not coincide. Negative chemical tests and positive Friedman tests were obtained on 30 patients diagnosed aR threatt'IINl ahortion. From this group, 22 aborted; 10, however·, wen' given progestermw, and :wveral months. later, 8 of the patients were still pregnant. All persons wen~ hospitalized on account of mucn meeamg. Two patients gave posithe results for both tests; then suddenly aborte(l the following day. There hnrl been no lileeding prior to abortion. Two patients, we believe, had a persistent corpus luteum cyst that finally degenerated. We did a series of tests, each time obtaining positive chemical and negative rabbit results. After two months, amPnorr·hea ceased, and a few clays later the pregnandiol titer d1·opped, finally rt>aching the negative level. rrv/o cases of hydatid mole \Vere tested. In both instances the chemica! test was negative; the rabbit results, positive. In eight cases, pregnancy was indicated chemically two or three days before it showed biologically. The ehemiral rt>sults were read incorrectly in ten cases. The table below summarizes the results: 'PABLE I CHEMICAL TEST POSITIVE NEGATIVE
I
·FlllEDMAN TEST POSITIVE NEGATIVE
----~----------~~~---
----~~~~--------------------~--------
*Normal pregnancy Ectopic pregnancy Not pregnant Threatened abortion Corpus luteum cyst Hydatid mole
121
9 2
132
:w
121 9
132
~ 2 •Eight from this group gave positive chemical results two or three days before the remits were positive biologically.
----------
Discuasion and Oo:n.clution The results which were obtain:ed leave no doubt that there is an equivalent degree of accuracy between the chemical color reaction test and the Friedman test for pregnancy. The chemical test has the advantage over the Frie(}man test in that a result can be obtained-lvithin tv;o or three _hours, and -that _it is more economical. The chemical test plus the Friedman test are good indicators in cases of threatened abortion. The chemical test is negati've in threatened abortion due to the fact that there is little or no progesterone being r>roduced. This decrease or lack o:f progesterone, no doubt, is the cause of the threatening abortion. In contrast, .the Friednum test depends upon. the anterior-pituitary-like substance elaborated by chorionic villi. ·· A positive
MCCORMACK:
t'OI,OR
CH~:MICAL
'l'EST FOH PREGNANCY
725
chemical test will be obtained in cases of a corpus luteum cyst but, since such cases are not too numerous and can be differentiated from normal pregnancy by the Friedman test, it should not he a disturbing factor. I wish to expresR of this work.
m~· apprP~iation
to Dr. C. Arthur Eld<'n for hiR friendly help and
~riticism
References ,J. Biol. Chern. 137: 397, 1941. Guterman, H. S.: .T. Clin. Endocrinol. 4: 262, 1944 . .3. 'falbot, N. B., Berman, R. A., MacLachlan, B. A., and Wolfe, J. K.: 1: 668, 194]. l. Astwood, B. B., and Jones, G. E. S.: ~-
.r.
Clin. Endocrinol.