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A simple automated method for the measurement of oestrogens in the urine of pregnant women
201
CLINICA CHIMICA ACTA
A SIMPLE AUTOMATED OESTROGENS
METHOD FOR THE MEASUREMENT
IN THE URINE OF PREGNANT
I. R. HAINSWORTH
AND
University
of Steroid Biochemistry,
(Received
Department
OF
WOMEN
P. E. HALL
Royal Infirmary,
Glasgow (U.K.)
May 21, 1971)
SUMMARY
An automated method, using the ‘AutoAnalyzer” has been developed for the measurement of oestrogens in the urine of pregnant women. No pre-treatment of the urine is required. The Kober reaction is performed directly on a small sample of urine and the Kober chromogens are extracted into chloroform and measured fluorimetrically. Twenty samples are assayed per hour. The precision, accuracy, specificity and sensitivity of the method are satisfactory.
Since the introduction of relatively simple methods for the measurement of oestriol in late pregnancy urine (e.g.l?,, obstetricians have recognised the value of oestrogen measurements in certain disorders of pregnancy as an indication of foetal well-being3T4.This recognition has resulted in a great increase in the number of analyses required and, together with the requirement for a speedy return of information5, has necessitated the establishment of even quicker, simpler methodsa. A simple, automated procedure is now required. With regard to continuous flow methods, a rather slow and complicated, semiautomatic procedure has been described7. A quicker method, also based on Technicon “AutoAnalyzer” modules, has been demonstrated by Ua Conaill and Muira. This method has recently been elaborated upon by Barnard and Logang, who have attempted to measure the more polar oestrogens in pregnancy urine. These methods are still, however, complex and costly. Extraction of the Kober oestrogen chromogen into an organic solvent and its fluorimetric measurementlO was combined with the direct reaction of a small volume of urine with Kober reagentlr. This very simple method was automated by Van Kessel and ~011.~~. Consideration of these methods has enabled us to develop a rapid automated method based on the “AutoAnalyzer”.
Clin. Chim. Acta,
35
(1971)
ZOI
-208
202
HAINSWORTH.
HA4I.i
.MATERIALS
1. Reagents Concentrated Trichloracetic
sulphuric acid.
acid.
Chloroform. Quinol. all AnalaR grade (British Drug Houses, Ltd.). Rober reagent: Sulphuric acid (666 ml) ; Distilled Ittrich reagent: A 5% (w/v) solution Oestriol standard: A stock solution From this a working standard
water (333 ml) ; Quinol (20 g).
of trichloracetic
acid in chloroform.
of oestriol in ethanol
(I mg/ml) is made up.
(IO pg/ml) is made up in water.
2. Instrumedation
(a) Adoanalyzer (Technicon, Ltd.). Sampler II, cam-20 samples/h, sample to wash ratio, I : I. Proportioning pump. Oil bath, 120~ -coil, 40 ft. 1.6 mm I.D. Single pen recorder. (b) Fluorimeter. A MK.V
fluorimeter
(Locarte
Co., Ltd.,
24 Emperors
Gate,
London, S.W. 7) is used. The cheaper MK.IV is equally suitable. The source of exciting light is a Wotan thallium lamp (A.E.G. Ltd., 27 Chancery Lane, London, W.C.2.). This gives a very intense isolated spectral line at 535 nm. No primary filter is required. On the secondary side, a Balzers 562 nm interference filter (Optical & Electrical Coatings, Ltd., Howard Road, Reigate, Surrey) is fitted. The flow-cell, cell holder, and the apparatus for coupling the fluorimeter Technicon Recorder were supplied by Locarte Co., Ltd.
to the
(c) Miscellaneous. All transmission tubing carrying undiluted Kober reagent is glass, sleeved with acidflex. The Ittrich reagent is carried in P.T.F.E. tubing (1.5 mm I.D.). Both the Kober and Ittrich reagents are pumped by displacement with water. The Kober reagent and the displacing water are separated by a layer of dichloromethane. METHOD
The manifold is shown in Fig. I. The 24-h collections of urine are made up to 2 1, or if the initial volume is greater than this, to 3 1. Every sixth position on the Sampler is occupied by a standard. The sampling rate is 20/h and the sample to wash ratio is I :I. The urine is diluted with water, re-sampled and pumped into an air-segmented stream of Kober reagent. After passing through a single mixing coil, the mixture takes about 8 min to traverse the heating bath, which is at a temperature of 120'. Water is added to the Kober reagent, and the two are mixed and cooled in a single mixing coil immersed in a bath of crushed ice. After the stream is debubbled, it is joined by the Ittrich reagent. Extraction of the fluorogen takes place in the two double mixing coils, also immersed in crushed ice. Clin. Chim.
Acta,
35 (1971)
ZOI-zo8
OESTROGENS
IN PREGNANT
203
URINE PUMP -
SAMPLER
Fig. I. Manifold.
About 0.55 of the Ittrich reagent which enters the phase separator is passed to the fluorimeter flow-cell. The rest of the Ittrich reagent and the diluted Kober reagent go to waste. The sensitivity controls on the fluorimeter are so adjusted that a IO ,ug/ml oestriol standard gives a recorder-pen deflection of 0.7 of full scale. RESULTS
Precision Precision was determined by running replicate analyses of standard solutions of oestriol and by analysing a series of urine samples twice, in duplicate, on each occasion. For II analyses of a ro-,ug/ml oestriol standard solution, the coefficient of variation was 1.45% and for II analyses of a 20 ,ug/ml oestriol standard, the coefficient of variation was 3.22%. From 89 duplicate analyses of urine samples, the within-batch coefficient of variation was estimated at 1.50°$ and from 28 analyses in separate batches the between-batch coefficient of variation was estimated at 2.48%. Clin. Chim.
Acta,
35 (1971) 201-208
HAINSWOKTH.
HALL
Oestriol was added to 12 urine samples from non-pregnant women collections diluted to 2 1) to give a concentration of IO mg/l. The mean recovery was 91.5% with a standard deviation of 3.5%.
(z-11
204 Recovery of added oestviol
Speci;ficity Twelve urine samples from male and non-pregnant ysed. No sample caused any deflection of the recorder-pen Sensitivity Low oestrogen in the preceding
titre samples (less than 5 mg/q
position
way, the sensitivity
on the sampler.
was better
than
I
When
mg/q
female subjects were analfrom its base-line.
h) were run with a cup of water
carry-over
was eliminated,
in this
11.
Linearity of detector response This was investigated by running a series of oestriol solutions in ascending and then in descending order of concentration. Results are shown in Fig. 2. A series of
150
7
-
i i -
loo
/
-
z .o, .z Y & a
i
50-
/
/
/
oestm
concentration
Fig. 2. Detector response. Oestriol solutions:
(pg
/ ml
)
x , ascending order of concentration;
+, descending
order of concentration.
urine samples of known relative oestrogen concentration were prepared by mixing various proportions of pregnancy and non-pregnancy urine. This series was also run in the same manner. Results are shown in Fig. 3. czin. Chim. A&, 35 (1971) 201-208
OESTROGENS IN PREGNANT URINE 250
200
150
-
100
E
0
20
0
Parts
40
60
per $30 of pregnancy
80
100
urine
Fig. 3. Detector response. Mixed pregnancy and non-pregnancy
Approach
urines. Symbols as in Fig. 2.
to steady state conditions during analysis
The mean peak height at a sampling rate of 20 per hour was expressed as a percentage of the height of the plateau obtained during continuous sampling. This was 7q.2°/o when a ro-,ug/ml oestriol standard was run, and was 7q.5o/o when a zz-pg/ml oestriol standard was run. Carry-over
Carry-over was determined by running standard solutions of oestriol, IO ,ug/ml (H) and 2 ,ug/ml (L), in the following order: HHLLHHLLHHLL. If the peak heights are h,h,l,l,, h,h,l,l,, h,h,l,l,, the carry-over is calculated by the formula 1,-l, (ref. 13). The mean carry-over was 0.040. h,-1, Interfering
substances
Glucose was added, at various concentrations, to aliquots of a pool of urine from pregnant women. Each aliquot was then assayed for oestrogens. The results are presented in Table I. TABLE EFFECT
I OF GLUCOSE
Glucose content
Nil
2 gll
5 &Tll
10 g/l
20 gll
Oestrogen (mg/z 1) o/0 of initial value
18.4 -
16.7 90.8
16.2 88.0
15.5 84.2
14.1 76.7 C&z. Chim. Acta, 35 (1971) 201-208
A similar experiment are presented in Table II. Thymol interferes urine preservative. TABLE
was performed
with bovine
in the method to a marked extent
serum albumin.
The results
and must not be used as a
11
EFFECT OF
ALBUMIPi ~~___
Albumin
content
h’il
Oestrogen (mg/z 1) “/: of initial value
3 gll
18.4
19.0 103.3
5 sll
rogll
15.6 101.1
1X.j 1OO.i
Relatizje contribution of di$erent oestrogens The analytical system is not equally sensitive
.~
?O,g/l 18.5 100.5
to all oestrogens.
When solutions
of oestriol, oestradiol and oestrone (IO pg/ml) were run, the heights of the peaks obtained with the oestradiol and oestrone were about 0.3 of the height of the oestriol peaks. Com;harison with separate acid hydrolysis of oestrogen conjugates Seven urine samples (24-h collections diluted to 2 1) from pregnant subjected centrated
women were
to an acid hydrolysis procedure. To 2 ml of urine was added 0.3 ml of conhydrochloric acid and the mixture was heated to 134~ for 3.5 min in a (Surgical Equipment Supplies, Ltd., Westfields Road, Sister” autoclave
“Little London, W.3.). Total oestrogens were then measured by the present method. Aliquots (2 ml) of urine plus 0.3 ml water were also assayed. The experiment was repeated with three of the samples to which glucose (20 g/l) had been added. Results are presented in Table III. TABLE
III
EFFECT OF Oestrogen Without
A SEPARATE
ACID
HYDROLYSIS
STEP
(nag/l) hydrolysis
Without hydrolysis glucose 20 g/l _____~.
After
hydrolysis
After hydrolysis glucose 20 g/l
6.5
8.2
9.’
7.’
5.0
5.5 5.0 6.1 8.5
5.7
8.9
___~
8.0 7.8 __~
4.6 .j ‘7
7.9 ‘s.0
34 4.6 44
Acid hydrolysis depressed the measured oestrogen concentration to a mean of 86.8% of the control level. The addition of glucose reduced the mean measured oestrogen concentration to 91.5%, without acid hydrolysis, and to 52.7% after acid hydrolysis. Estimated running costs For a weekly workload of 300 samples, the average weekly expenditure be of the order of E 55.00. This is made up of: Clin. Chim.
Acta,
35 (1971)
201-208
would
OESTROGENS
IN PREGNANT
207
URINE
Chemicals Replaceable items Depreciation of equipment Technician’s salary, plus overheads The approximate cost of each assay is rg p.
L 7.00 : 10.00 8.00 L 30.00
DISCUSSION
The absence of a separate acid hydrolysis step results in higher measured oestrogen levels in comparison with methods in which this procedure is used. Oestrogens are destroyed, to some extent, by acid hydrolysis14, although this is more severe with some oestrogens than with others. Thus the “direct” Kober reaction minimises this loss. It is known that a high level of glucose in the urine increases this destruction of oestrogens during acid hydrolysis lb. The present method tolerates urinary glucose concentrations of up to 20 g/l with only a 23% loss of oestrogen. Thus the method should give a truer indication of the oestrogen production in the pregnant diabetic woman. In designing the system, it was found that the recovery of added oestriol was poorer when the size of the sample was increased. It is therefore necessary to dilute the urine, before it joins the Kober reagent, to obtain a sufficiently small sample. To achieve a satisfactory system, an efficient phase separation is essential. The use of a BO connector proved adequate but its function was dictated both by the size of the solvent segments reaching it and by the amount of Ittrich reagent directed to the flow-cell. Although a vigorous extraction of the fluorogen into the Ittrich reagent is necessary, the use of air segmentation and of beaded mixing coils was found to disrupt phase separation, and two double mixing coils were found to give an adequate extraction. The pumping of the flow-cell return was found to be optimal using blue pump-tubing (1.60 ml/min); this allowed 0.55 of the organic phase to pass through the flow-cell. In normal practice, the system is allowed to run for 20 min before any samples are analysed. In a typical run of 3 to 4 h duration, no change in the baseline is seen and little change in sensitivity occurs. If the amount of Ittrich reagent in the displacement bottle is allowed to run very low, there is some loss of sensitivity, presumably due to the trichloracetic acid being extracted from the chloroform into the displacing water. This problem can be minimised by displacing the Ittrich reagent with 2% sulphuric acid. The displacement bottle which contains the Kober reagent requires to be handled with care since heat will be generated if the contents are mixed and this will vaporize the dichloromethane, causing the bottle contents to be expelled explosively. Two precautions are observed in this laboratory; the bottle is not moved after being filled and it is emptied by suction, not by pouring out the contents. This method is considered to be a viable procedure for the measurement of urinary oestrogens in pregnancy, with the advantages of simplicity, speed and cheapness. NOTE ADDED
IN PROOF
The Kober and Ittrich
reagents may be pumped directly through acidflex Clin. Chim.
Acta,
35 (1971)
201-208
20s
HAISSWOIITH.
tubing (Kober--green bottle is then required used in the authors’
Ittricll-~purpleiblack acidflex). acidflex, only for the flowcell return. This modification
:I
HALL
displacement
is now routinel!.
laboratory.
ACKNOWLEDGEMEST
This (London) urine.
work was initiated
Working
by the Department
Party on the automation
of Health
and Social
of the analysis of oestrogens
Securitv
in pregnanci-
REFERIXCES I A. KLOPPER 2 J, B. BROWN
AND G. Ii.WILSOS, J. Obstet. Gynaecol. AND 31.G. COYLE, 1. Obsfct. Gynaecol.
Brit. Commonwralth, Brit. Commonwraith,
69 (1962) 533. 70 (1963) 219.
3 A. KLOPPER, Obstet. Gynecol. Surv., 23 (1968) 819. 4 A. C. TURNBULL, Brit. J. Hosp. Med., 3 (1970) 539. 5 R. T. BOOTH, >I.I. STERN, C. WOOD, &I.J. H. SHARPLES AXUU J. H. M. PISI(ERTOX, ,J. Obstef. Gynaecol. Brit. Commonwealth, 72 (1965) ~29. 6 R. E. OAKEY, L. R. A. BRADSHAW, S. S. ECCLES, S. R. STITCH AND R. I’. HEYS, Clir~. Chim. A&z, 15 (1967) 3j. 7 H. S. STRICKLER, S. S. HOLT, I-I. F. A~EVEUO,
E. SAIER ASU R. C. GRAUER,
Steroids,9
(1967)
‘93. 8 D. UA CONAILL APED G. G. MuIR,C!ZTZ.Chrnz., 14 (1968) 1010. 9 W’. P. BARSARD AND Ii.W. LOGAN, Clin. Chinz. Acta, 29 (1970) .+or. TO G. TTTRICH, Acta Endocriml., 35 (1960) 31. II I’. J, BROMRACHER, A. H. J. GIJZEX ASD P. E. \'ERHEESEN, Clin.C/cinz. Ada, 20 (1968)360. IL H. VAN I~ESSEL, R. SEITZIXGMR, J. SCHREURS AP\'D;I[. \:ERSTEEG, 1Yrd. Tijds~hv. Verlosk., 69
(1969) 81. 13 LEMAG WORKIP~G I'ARTY, J. Clin.Pathol., 22 (1969) 278. I.+ J. B. BROWN AND H. A. F. BLAIR, J. Endocrinol., 17 (1958)411. 1.5H. .\DLERCREUTZ ASD T. LUUKKAISES, in Ii. 1%.EIK-XES AND I<.C. I~ORNIX.(;(Etls.), Gas Phnsc Chrowzatogvafihy ofStwoids, Springer-Verlag. Berlin, 1968, p. 72. CZi*z. Chin?.Acfa.
3j
(1971) 201-20s
CLINICA CHIMICA ACTA
A SIMPLE AUTOMATED OESTROGENS
METHOD FOR THE MEASUREMENT
IN THE URINE OF PREGNANT
I. R. HAINSWORTH
AND
University
of Steroid Biochemistry,
(Received
Department
OF
WOMEN
P. E. HALL
Royal Infirmary,
Glasgow (U.K.)
May 21, 1971)
SUMMARY
An automated method, using the ‘AutoAnalyzer” has been developed for the measurement of oestrogens in the urine of pregnant women. No pre-treatment of the urine is required. The Kober reaction is performed directly on a small sample of urine and the Kober chromogens are extracted into chloroform and measured fluorimetrically. Twenty samples are assayed per hour. The precision, accuracy, specificity and sensitivity of the method are satisfactory.
Since the introduction of relatively simple methods for the measurement of oestriol in late pregnancy urine (e.g.l?,, obstetricians have recognised the value of oestrogen measurements in certain disorders of pregnancy as an indication of foetal well-being3T4.This recognition has resulted in a great increase in the number of analyses required and, together with the requirement for a speedy return of information5, has necessitated the establishment of even quicker, simpler methodsa. A simple, automated procedure is now required. With regard to continuous flow methods, a rather slow and complicated, semiautomatic procedure has been described7. A quicker method, also based on Technicon “AutoAnalyzer” modules, has been demonstrated by Ua Conaill and Muira. This method has recently been elaborated upon by Barnard and Logang, who have attempted to measure the more polar oestrogens in pregnancy urine. These methods are still, however, complex and costly. Extraction of the Kober oestrogen chromogen into an organic solvent and its fluorimetric measurementlO was combined with the direct reaction of a small volume of urine with Kober reagentlr. This very simple method was automated by Van Kessel and ~011.~~. Consideration of these methods has enabled us to develop a rapid automated method based on the “AutoAnalyzer”.
Clin. Chim. Acta,
35
(1971)
ZOI
-208
202
HAINSWORTH.
HA4I.i
.MATERIALS
1. Reagents Concentrated Trichloracetic
sulphuric acid.
acid.
Chloroform. Quinol. all AnalaR grade (British Drug Houses, Ltd.). Rober reagent: Sulphuric acid (666 ml) ; Distilled Ittrich reagent: A 5% (w/v) solution Oestriol standard: A stock solution From this a working standard
water (333 ml) ; Quinol (20 g).
of trichloracetic
acid in chloroform.
of oestriol in ethanol
(I mg/ml) is made up.
(IO pg/ml) is made up in water.
2. Instrumedation
(a) Adoanalyzer (Technicon, Ltd.). Sampler II, cam-20 samples/h, sample to wash ratio, I : I. Proportioning pump. Oil bath, 120~ -coil, 40 ft. 1.6 mm I.D. Single pen recorder. (b) Fluorimeter. A MK.V
fluorimeter
(Locarte
Co., Ltd.,
24 Emperors
Gate,
London, S.W. 7) is used. The cheaper MK.IV is equally suitable. The source of exciting light is a Wotan thallium lamp (A.E.G. Ltd., 27 Chancery Lane, London, W.C.2.). This gives a very intense isolated spectral line at 535 nm. No primary filter is required. On the secondary side, a Balzers 562 nm interference filter (Optical & Electrical Coatings, Ltd., Howard Road, Reigate, Surrey) is fitted. The flow-cell, cell holder, and the apparatus for coupling the fluorimeter Technicon Recorder were supplied by Locarte Co., Ltd.
to the
(c) Miscellaneous. All transmission tubing carrying undiluted Kober reagent is glass, sleeved with acidflex. The Ittrich reagent is carried in P.T.F.E. tubing (1.5 mm I.D.). Both the Kober and Ittrich reagents are pumped by displacement with water. The Kober reagent and the displacing water are separated by a layer of dichloromethane. METHOD
The manifold is shown in Fig. I. The 24-h collections of urine are made up to 2 1, or if the initial volume is greater than this, to 3 1. Every sixth position on the Sampler is occupied by a standard. The sampling rate is 20/h and the sample to wash ratio is I :I. The urine is diluted with water, re-sampled and pumped into an air-segmented stream of Kober reagent. After passing through a single mixing coil, the mixture takes about 8 min to traverse the heating bath, which is at a temperature of 120'. Water is added to the Kober reagent, and the two are mixed and cooled in a single mixing coil immersed in a bath of crushed ice. After the stream is debubbled, it is joined by the Ittrich reagent. Extraction of the fluorogen takes place in the two double mixing coils, also immersed in crushed ice. Clin. Chim.
Acta,
35 (1971)
ZOI-zo8
OESTROGENS
IN PREGNANT
203
URINE PUMP -
SAMPLER
Fig. I. Manifold.
About 0.55 of the Ittrich reagent which enters the phase separator is passed to the fluorimeter flow-cell. The rest of the Ittrich reagent and the diluted Kober reagent go to waste. The sensitivity controls on the fluorimeter are so adjusted that a IO ,ug/ml oestriol standard gives a recorder-pen deflection of 0.7 of full scale. RESULTS
Precision Precision was determined by running replicate analyses of standard solutions of oestriol and by analysing a series of urine samples twice, in duplicate, on each occasion. For II analyses of a ro-,ug/ml oestriol standard solution, the coefficient of variation was 1.45% and for II analyses of a 20 ,ug/ml oestriol standard, the coefficient of variation was 3.22%. From 89 duplicate analyses of urine samples, the within-batch coefficient of variation was estimated at 1.50°$ and from 28 analyses in separate batches the between-batch coefficient of variation was estimated at 2.48%. Clin. Chim.
Acta,
35 (1971) 201-208
HAINSWOKTH.
HALL
Oestriol was added to 12 urine samples from non-pregnant women collections diluted to 2 1) to give a concentration of IO mg/l. The mean recovery was 91.5% with a standard deviation of 3.5%.
(z-11
204 Recovery of added oestviol
Speci;ficity Twelve urine samples from male and non-pregnant ysed. No sample caused any deflection of the recorder-pen Sensitivity Low oestrogen in the preceding
titre samples (less than 5 mg/q
position
way, the sensitivity
on the sampler.
was better
than
I
When
mg/q
female subjects were analfrom its base-line.
h) were run with a cup of water
carry-over
was eliminated,
in this
11.
Linearity of detector response This was investigated by running a series of oestriol solutions in ascending and then in descending order of concentration. Results are shown in Fig. 2. A series of
150
7
-
i i -
loo
/
-
z .o, .z Y & a
i
50-
/
/
/
oestm
concentration
Fig. 2. Detector response. Oestriol solutions:
(pg
/ ml
)
x , ascending order of concentration;
+, descending
order of concentration.
urine samples of known relative oestrogen concentration were prepared by mixing various proportions of pregnancy and non-pregnancy urine. This series was also run in the same manner. Results are shown in Fig. 3. czin. Chim. A&, 35 (1971) 201-208
OESTROGENS IN PREGNANT URINE 250
200
150
-
100
E
0
20
0
Parts
40
60
per $30 of pregnancy
80
100
urine
Fig. 3. Detector response. Mixed pregnancy and non-pregnancy
Approach
urines. Symbols as in Fig. 2.
to steady state conditions during analysis
The mean peak height at a sampling rate of 20 per hour was expressed as a percentage of the height of the plateau obtained during continuous sampling. This was 7q.2°/o when a ro-,ug/ml oestriol standard was run, and was 7q.5o/o when a zz-pg/ml oestriol standard was run. Carry-over
Carry-over was determined by running standard solutions of oestriol, IO ,ug/ml (H) and 2 ,ug/ml (L), in the following order: HHLLHHLLHHLL. If the peak heights are h,h,l,l,, h,h,l,l,, h,h,l,l,, the carry-over is calculated by the formula 1,-l, (ref. 13). The mean carry-over was 0.040. h,-1, Interfering
substances
Glucose was added, at various concentrations, to aliquots of a pool of urine from pregnant women. Each aliquot was then assayed for oestrogens. The results are presented in Table I. TABLE EFFECT
I OF GLUCOSE
Glucose content
Nil
2 gll
5 &Tll
10 g/l
20 gll
Oestrogen (mg/z 1) o/0 of initial value
18.4 -
16.7 90.8
16.2 88.0
15.5 84.2
14.1 76.7 C&z. Chim. Acta, 35 (1971) 201-208
A similar experiment are presented in Table II. Thymol interferes urine preservative. TABLE
was performed
with bovine
in the method to a marked extent
serum albumin.
The results
and must not be used as a
11
EFFECT OF
ALBUMIPi ~~___
Albumin
content
h’il
Oestrogen (mg/z 1) “/: of initial value
3 gll
18.4
19.0 103.3
5 sll
rogll
15.6 101.1
1X.j 1OO.i
Relatizje contribution of di$erent oestrogens The analytical system is not equally sensitive
.~
?O,g/l 18.5 100.5
to all oestrogens.
When solutions
of oestriol, oestradiol and oestrone (IO pg/ml) were run, the heights of the peaks obtained with the oestradiol and oestrone were about 0.3 of the height of the oestriol peaks. Com;harison with separate acid hydrolysis of oestrogen conjugates Seven urine samples (24-h collections diluted to 2 1) from pregnant subjected centrated
women were
to an acid hydrolysis procedure. To 2 ml of urine was added 0.3 ml of conhydrochloric acid and the mixture was heated to 134~ for 3.5 min in a (Surgical Equipment Supplies, Ltd., Westfields Road, Sister” autoclave
“Little London, W.3.). Total oestrogens were then measured by the present method. Aliquots (2 ml) of urine plus 0.3 ml water were also assayed. The experiment was repeated with three of the samples to which glucose (20 g/l) had been added. Results are presented in Table III. TABLE
III
EFFECT OF Oestrogen Without
A SEPARATE
ACID
HYDROLYSIS
STEP
(nag/l) hydrolysis
Without hydrolysis glucose 20 g/l _____~.
After
hydrolysis
After hydrolysis glucose 20 g/l
6.5
8.2
9.’
7.’
5.0
5.5 5.0 6.1 8.5
5.7
8.9
___~
8.0 7.8 __~
4.6 .j ‘7
7.9 ‘s.0
34 4.6 44
Acid hydrolysis depressed the measured oestrogen concentration to a mean of 86.8% of the control level. The addition of glucose reduced the mean measured oestrogen concentration to 91.5%, without acid hydrolysis, and to 52.7% after acid hydrolysis. Estimated running costs For a weekly workload of 300 samples, the average weekly expenditure be of the order of E 55.00. This is made up of: Clin. Chim.
Acta,
35 (1971)
201-208
would
OESTROGENS
IN PREGNANT
207
URINE
Chemicals Replaceable items Depreciation of equipment Technician’s salary, plus overheads The approximate cost of each assay is rg p.
L 7.00 : 10.00 8.00 L 30.00
DISCUSSION
The absence of a separate acid hydrolysis step results in higher measured oestrogen levels in comparison with methods in which this procedure is used. Oestrogens are destroyed, to some extent, by acid hydrolysis14, although this is more severe with some oestrogens than with others. Thus the “direct” Kober reaction minimises this loss. It is known that a high level of glucose in the urine increases this destruction of oestrogens during acid hydrolysis lb. The present method tolerates urinary glucose concentrations of up to 20 g/l with only a 23% loss of oestrogen. Thus the method should give a truer indication of the oestrogen production in the pregnant diabetic woman. In designing the system, it was found that the recovery of added oestriol was poorer when the size of the sample was increased. It is therefore necessary to dilute the urine, before it joins the Kober reagent, to obtain a sufficiently small sample. To achieve a satisfactory system, an efficient phase separation is essential. The use of a BO connector proved adequate but its function was dictated both by the size of the solvent segments reaching it and by the amount of Ittrich reagent directed to the flow-cell. Although a vigorous extraction of the fluorogen into the Ittrich reagent is necessary, the use of air segmentation and of beaded mixing coils was found to disrupt phase separation, and two double mixing coils were found to give an adequate extraction. The pumping of the flow-cell return was found to be optimal using blue pump-tubing (1.60 ml/min); this allowed 0.55 of the organic phase to pass through the flow-cell. In normal practice, the system is allowed to run for 20 min before any samples are analysed. In a typical run of 3 to 4 h duration, no change in the baseline is seen and little change in sensitivity occurs. If the amount of Ittrich reagent in the displacement bottle is allowed to run very low, there is some loss of sensitivity, presumably due to the trichloracetic acid being extracted from the chloroform into the displacing water. This problem can be minimised by displacing the Ittrich reagent with 2% sulphuric acid. The displacement bottle which contains the Kober reagent requires to be handled with care since heat will be generated if the contents are mixed and this will vaporize the dichloromethane, causing the bottle contents to be expelled explosively. Two precautions are observed in this laboratory; the bottle is not moved after being filled and it is emptied by suction, not by pouring out the contents. This method is considered to be a viable procedure for the measurement of urinary oestrogens in pregnancy, with the advantages of simplicity, speed and cheapness. NOTE ADDED
IN PROOF
The Kober and Ittrich
reagents may be pumped directly through acidflex Clin. Chim.
Acta,
35 (1971)
201-208
20s
HAISSWOIITH.
tubing (Kober--green bottle is then required used in the authors’
Ittricll-~purpleiblack acidflex). acidflex, only for the flowcell return. This modification
:I
HALL
displacement
is now routinel!.
laboratory.
ACKNOWLEDGEMEST
This (London) urine.
work was initiated
Working
by the Department
Party on the automation
of Health
and Social
of the analysis of oestrogens
Securitv
in pregnanci-
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E. SAIER ASU R. C. GRAUER,
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‘93. 8 D. UA CONAILL APED G. G. MuIR,C!ZTZ.Chrnz., 14 (1968) 1010. 9 W’. P. BARSARD AND Ii.W. LOGAN, Clin. Chinz. Acta, 29 (1970) .+or. TO G. TTTRICH, Acta Endocriml., 35 (1960) 31. II I’. J, BROMRACHER, A. H. J. GIJZEX ASD P. E. \'ERHEESEN, Clin.C/cinz. Ada, 20 (1968)360. IL H. VAN I~ESSEL, R. SEITZIXGMR, J. SCHREURS AP\'D;I[. \:ERSTEEG, 1Yrd. Tijds~hv. Verlosk., 69
(1969) 81. 13 LEMAG WORKIP~G I'ARTY, J. Clin.Pathol., 22 (1969) 278. I.+ J. B. BROWN AND H. A. F. BLAIR, J. Endocrinol., 17 (1958)411. 1.5H. .\DLERCREUTZ ASD T. LUUKKAISES, in Ii. 1%.EIK-XES AND I<.C. I~ORNIX.(;(Etls.), Gas Phnsc Chrowzatogvafihy ofStwoids, Springer-Verlag. Berlin, 1968, p. 72. CZi*z. Chin?.Acfa.
3j
(1971) 201-20s