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Determination of urinary estriol by radioimmunoassay. Modification of a commercial kit for serum analyses
325
Clinica Chimica Acta, 79 (1977) 325-330 0 Elsevier/North-Holland Biomedical Press
CCA 8619
DETERMINATION OF URINARY ESTRIOL BY RADIOIMMUNOASSAY. MODIFICATION OF A COMMERCIAL KIT FOR SERUM ANALYSES
ANDERS KALLNER
* and OLLE LANTTO
Department of Clinical Chemistry, Huddinge University Hospital, Karolinska Znstitutet, Stockholm (Sweden) (Received January 2&h, 1977)
Summary A simple and rapid method for urinary estriol determination is described. Modification of a commercially available kit for serum estriol gives a method for urinary estriol determination with a capacity of approximately 75 determinations a day. A high degree of accurary was revealed by indirect comparison with a mass fragmentographic method. Introduction Determination of urinary estriol has become a common tool in monitoring complicated pregnancies. Spectrophotometric methods have successively been exchanged for more specific methods e.g. GLC and RIA. The latter method is sensitive enough to determine estriol in plasma from pregnant women, whereas GLC has been named by many the method of choice for urinary analyses. In order to have value in the clinic it is essential that analysis of urinary estriol is made promptly. When the number of analyses increases in the laboratory, the RIA method therefore becomes more attractive than GLC in spite of the higher cost of reagents. In the present report a commercially available RIA kit, designed for determination of estriol in serum, was modified for use in urinary analyses. The modified method was compared with a GLC method, the accuracy of which has been checked against a mass fragmentographic method [l].
* To whom correspondence should be addressed. Postal address: Huddinge University Hospital. S-141 66 Huddinge. Sweden.
326
Materials and methods Materials
RIA-kits for S-estriol determination were obtained from Radiochemical Centre, Amersham, England. The critical components of this kit are the hydrolyzing enzymes, the antiserum and the labeled estriol. According to the specifications, glucuronidase and sulfatase from Helix pomatia are used. The minimum activity of these enzymes at 37°C are specified to at least 3.3 pkal/l determined with phenolphtalein-mono-fl-glucuronide [2] and 4-nitrocatechol sulfate [3] as substrates in a pH 5 acetate buffer. The antiserum is raised in rabbits using estriol-6_carboxymethyloxime-BSA as antigen. It is buffered to pH 6.9 with a phosphate saline buffer and 10% serum to a dilution of 1 : 25 000. Cross-reactivity towards estetrol (13%), 16-epi-estriol (5.6%), estradiol (1.6%), estrone (0.4%) and 17-epi-estriol (0.4%) is reported. Eighteen other tested steroids, among them progesterone, have been tested, none of which gave but negligible cross-reaction. The labelled compound is a high purity ‘*‘Icontaining derivative of estriol. About 60 to about 100 nCi of this compound was added to each tube as labelled competitor to the endogenous estriol to be determined. Estriol-3fl-D-glucuronide, estriol 3-sulfate and estriol-16cY$-D-glucuronide, all sodium salts, were obtained from Sigma, St. Louis, MO., U.S.A. All other chemicals were from Merck, G.F.R. Urine and serum samples 24-h urine samples and serum samples were collected from pregnant women (30-40 weeks of gestation). Methods RIA procedure
Standards were prepared by dissolving estriol-3fl-D-glucuronide in water to concentrations of about 20, 40, 100 and 180 E.tmol/l. Water was used as zero standard. The original enzyme solution was diluted 1 : 50 with 0.15 M acetate buffer, pH 4.6, corresponding to a minimum of 66 nkat/l. The standards or centrifuged urine samples, 6 ~1, were diluted with 5 ml enzyme solution using a semi-automatic diluter (Auto Chem Instrument AB, Lidingo, Sweden) and incubated 3 h at 37°C. The standards were run in duplicate and the samples in singlets. After mixing on a Vortex mixer, 50 /..dof the hydrolysed solution was mixed with 200 ,ul of the ‘251-labelled estriol solution and 200 ~1 of the anti-estriol serum. The tubes were left at room temperature for 45 min. Ammonium sulphate solution, 500 ~1, was added to all tubes, and the tubes were mixed thoroughly. After centrifugation, the supernatant was poured off and the radioactivity of the precipitate determined by measuring for 1 min in a gamma counter (‘251, gross count). GLC procedure
U-estriol was determined as described by van de Calseyde et al. [4] and Rosenthal and Yaseen [5] with some modifications comprising carrying the
327
standard activity.
through
the whole
procedure
and doubling
the amount
of enzyme
Statistics Means, standard deviations, Student’s t-test and correlation coefficients were calculated according to conventional methods [8]. Regression lines were calculated by “the line of best fit” [9]. Results and discussion Estriol in human urine is mainly excreted as glucuronic and sulfuric acid esters and hydrolysis is necessary to determine the total estrogens. Also, in serum estriol occurs in conjugated form together with free estriol. It was found practical to dilute the urine to a convenient concentration of estriol with prediluted enzyme solution. By this procedure complete hydrolyses was usually achieved and a constant level of determined estriol obtained (Fig. 1). At very high levels (above 130 pmol/l), longer incubation time became necessary. However, in practical work a further dilution of the sample is necessary at this level because of the unfavorable course of the standard curve at such concentrations (Fig. 2). Incubation at higher temperatures, which has been recommended, [6], gave incomplete hydrolyses. Possibly this is due to inactivation of the enzymes at these temperatures. The original freezedried standard solutions of the kit were substituted with solutions of eStI”iOl-3/%Dglucuronide in water in order to obtain standard solutions of concentrations comparable to pregnancy urine. Different conjugates of estriol are hydrolyzed at different rates by the enzymes included in the kit. 240
1 x 37Y
200-
o 63%
? 160‘, f. ‘z .g 120Zi
W iI
80-
40-
Incubation time (hours) Fig. 1. Rate of enzymatic hydrolysis of natural conjugates in urine. Circles refer to urines of final concentration of about 50 and >200 pmol/l. respectively.
328
40
80
120
U-Estrlol Fig. 2. Standard curve represent S.D. (n = 8).
160
200
(pmol/O
of U-estriol
with different
kits within
the same production
batch.
Vertical
bars
Thus the 3/3-D-glucuronide and 3-sulfate were almost completely hydrolyzed within 30 min whereas about 120 E.tmol/l 16a-fl-D-glucuronide required about 3 h with a time course similar to that of native urine shown in Fig. 1. This is in accordance with the reported composition of pregnancy urine [lo]. To be able to use the 3@D-glucuronide as the primary standard it is thus necessary to achieve a complete hydrolysis of the estriol derivatives in urine. Recovery of added test substances was similar on addition to both urine and water. This implies that the effect of possible inhibitor8 in urine is eliminated by the extreme dilution (l/834) used. The protein content of urine is very low and the precipitation of antigenantibody complex might be enhanced by addition of a protein solution when diluting the sample. No advantage of such an addition could be shown in the present modification. One kit allows the determination of estriol in 60 tubes. In order to increase the yield of the kit and economize, standard curves were compared from several kits within the same batch. It was found that with the present system the same standard curve could be used from one series of analyses to the other, provided corrections for decay of radioactivity were made. The coefficient of variation of the individual points on the standard curve was about 4% between runs (Fig. 2). In order to check the level of the standard curve, one standard solution was routinely included in each series. The standard curve was shown to coincide with that obtained for serum determinations according to the instructions in the kit and accordingly estriol in serum could be assayed by either standard curve (Fig. 3). Further, serum samples and urinary samples could be assayed according to the same standard curve. The standard curve was plotted as percent bound radioactivity versus the logarithm of the amount of EUbEtaIKe. This curve showed the well known “S”shape but the inflection point appeared to be at 60% (Fig. 4). A logit transformation doe8 not yield a straight line unless the percent bound in the logit expression is decreased by 10%. This can be justified by assuming a nonspecific binding of the antibody amounting to about 10%. The straight logit/log standard curve was used for computerized estimation of estriol. A ma8s fragmentographic method (MID) for determination of U-estriol has recently been described from this laboratory [ 11. The present modification of the
329 12001
a
z L
iz
W
23
400
Y=0.94X-10.2 r = 0.997 n=23
I
50
10
S-Estriot (nmol II) B
U-Estriol
100
200
(pmol/I)
Fig. 3. S-Es&lo1 evaluated by standard curve according to kit (B) and standard curve obtained with estrlol3@-D-glueuronide as described in the present method (A). Fig. 4. Standard curve of e~ol-3~-D~luc~onide.
Additional points to secure position of inflexion point.
RIA method was compared to the GLC method in routine tory which agreed with this MID method (y = 0.93x + 6.9; good agreement was also found between the GLC and RIA The paired t-test showed no statistically significant different
Y=l.OOX
+ 0.90
r=096
1
40 U-Estriol
80
120 fpmol/ 24h)
160 RIA
Fig. 5. Regression line for U-estriol GLC = f (U-estriol RIA).
200
use in the laborar = 0.98). A very methods (Fig. 5). between samples
330 1601
I2 ;:
120-
x RIA
-36-t-37
-38-39
----c40--+Week
of
gestation
Fig. 6. U-Estriol course during pregnancy determined by GLC (0) and present method (X). In the lower pattern, the baby was still-born.
analyzed by the two methods (t = 1.5). The day-to-day precision was found to be 8.5% over a period of 30 days. To compare the methods further, several consecutive pregnancy urines were determined by both methods. As shown in Fig. 6 a very good agreement between methods was obtained at different levels of urinary estriol concentrations during pregnancy. The cross reaction of the antibody and various steroids is specified in the kit and significant for estetrol and 16-epi-estriol. These compounds do not occur to any large extent in pregnancy urine and any contribution of them or other substances to the antigen-antibody reaction does not seem to have any practical influence in determining estriol. In spite of the higher costs of reagents, the RIA dete~inations were found preferable because of the high speed of analyses. Thus, up to 75 determinations are easily handled in one working day and the normal batches of 20 to 30 samples can be ready in about 5 h. The main portion of this time is used for incubations and counting in the gamma counter. Additional samples add but little to this analysis time. Although it has been questioned lately [ 7 J, there may be value in determining serum estriol instead of or together with urinary estriol. It was considered advantageous to have similar methods for both types of analyses. References 1 Bjiirkhem, I.. Blomstrand. R.. Svensson, L., Tietz. F. and Carlstrom. K. (1975) Clin. Chim. Acta 62, 365 2 Fishman. W.H. (1970) in Metoden der Enzyrnatischen AnaIyse (Bergmayer. H.V., ed.), PP. 964-972, Verlag Chemie, Weinheim 3 Milazzo, F.H., Fitzgerald. J.W. (1966) Can. J. Microbial. 12, 735 4 van de Calseyde, J.F.. Scholties. R.J.H., Schmidt. N.A. and Kuypers. A.M.J. (1966) CIin. Chim. Acta 25,345 5 Rosenthal, A.F. and Yaseen, A. (1971) Clin. Chim. Acta 31, 171 6 SehoIIer, R., MCtay, S.. Herbon, S. and Jayle, M.F. (1966) Eur. J. Steroids 1. 373 7 Lindberg. B.S. and CarIstrBm, K. (1976) Acta Obstet. Gynaecol. Stand. 55. 163 8 Snedecor, G.W. and Cochran. W.G. (1972) Statistical Methods, Iowa State University Press, Iowa 9 Strewhart, W.A. (1931) Economic Control of Manufactured Products. D. van Nostrand, New York 10 Levitz, M., Jirku, H.. Kadner, S. and Young, B.K. (1975) J. Steroid. Biochem. 6,663
Clinica Chimica Acta, 79 (1977) 325-330 0 Elsevier/North-Holland Biomedical Press
CCA 8619
DETERMINATION OF URINARY ESTRIOL BY RADIOIMMUNOASSAY. MODIFICATION OF A COMMERCIAL KIT FOR SERUM ANALYSES
ANDERS KALLNER
* and OLLE LANTTO
Department of Clinical Chemistry, Huddinge University Hospital, Karolinska Znstitutet, Stockholm (Sweden) (Received January 2&h, 1977)
Summary A simple and rapid method for urinary estriol determination is described. Modification of a commercially available kit for serum estriol gives a method for urinary estriol determination with a capacity of approximately 75 determinations a day. A high degree of accurary was revealed by indirect comparison with a mass fragmentographic method. Introduction Determination of urinary estriol has become a common tool in monitoring complicated pregnancies. Spectrophotometric methods have successively been exchanged for more specific methods e.g. GLC and RIA. The latter method is sensitive enough to determine estriol in plasma from pregnant women, whereas GLC has been named by many the method of choice for urinary analyses. In order to have value in the clinic it is essential that analysis of urinary estriol is made promptly. When the number of analyses increases in the laboratory, the RIA method therefore becomes more attractive than GLC in spite of the higher cost of reagents. In the present report a commercially available RIA kit, designed for determination of estriol in serum, was modified for use in urinary analyses. The modified method was compared with a GLC method, the accuracy of which has been checked against a mass fragmentographic method [l].
* To whom correspondence should be addressed. Postal address: Huddinge University Hospital. S-141 66 Huddinge. Sweden.
326
Materials and methods Materials
RIA-kits for S-estriol determination were obtained from Radiochemical Centre, Amersham, England. The critical components of this kit are the hydrolyzing enzymes, the antiserum and the labeled estriol. According to the specifications, glucuronidase and sulfatase from Helix pomatia are used. The minimum activity of these enzymes at 37°C are specified to at least 3.3 pkal/l determined with phenolphtalein-mono-fl-glucuronide [2] and 4-nitrocatechol sulfate [3] as substrates in a pH 5 acetate buffer. The antiserum is raised in rabbits using estriol-6_carboxymethyloxime-BSA as antigen. It is buffered to pH 6.9 with a phosphate saline buffer and 10% serum to a dilution of 1 : 25 000. Cross-reactivity towards estetrol (13%), 16-epi-estriol (5.6%), estradiol (1.6%), estrone (0.4%) and 17-epi-estriol (0.4%) is reported. Eighteen other tested steroids, among them progesterone, have been tested, none of which gave but negligible cross-reaction. The labelled compound is a high purity ‘*‘Icontaining derivative of estriol. About 60 to about 100 nCi of this compound was added to each tube as labelled competitor to the endogenous estriol to be determined. Estriol-3fl-D-glucuronide, estriol 3-sulfate and estriol-16cY$-D-glucuronide, all sodium salts, were obtained from Sigma, St. Louis, MO., U.S.A. All other chemicals were from Merck, G.F.R. Urine and serum samples 24-h urine samples and serum samples were collected from pregnant women (30-40 weeks of gestation). Methods RIA procedure
Standards were prepared by dissolving estriol-3fl-D-glucuronide in water to concentrations of about 20, 40, 100 and 180 E.tmol/l. Water was used as zero standard. The original enzyme solution was diluted 1 : 50 with 0.15 M acetate buffer, pH 4.6, corresponding to a minimum of 66 nkat/l. The standards or centrifuged urine samples, 6 ~1, were diluted with 5 ml enzyme solution using a semi-automatic diluter (Auto Chem Instrument AB, Lidingo, Sweden) and incubated 3 h at 37°C. The standards were run in duplicate and the samples in singlets. After mixing on a Vortex mixer, 50 /..dof the hydrolysed solution was mixed with 200 ,ul of the ‘251-labelled estriol solution and 200 ~1 of the anti-estriol serum. The tubes were left at room temperature for 45 min. Ammonium sulphate solution, 500 ~1, was added to all tubes, and the tubes were mixed thoroughly. After centrifugation, the supernatant was poured off and the radioactivity of the precipitate determined by measuring for 1 min in a gamma counter (‘251, gross count). GLC procedure
U-estriol was determined as described by van de Calseyde et al. [4] and Rosenthal and Yaseen [5] with some modifications comprising carrying the
327
standard activity.
through
the whole
procedure
and doubling
the amount
of enzyme
Statistics Means, standard deviations, Student’s t-test and correlation coefficients were calculated according to conventional methods [8]. Regression lines were calculated by “the line of best fit” [9]. Results and discussion Estriol in human urine is mainly excreted as glucuronic and sulfuric acid esters and hydrolysis is necessary to determine the total estrogens. Also, in serum estriol occurs in conjugated form together with free estriol. It was found practical to dilute the urine to a convenient concentration of estriol with prediluted enzyme solution. By this procedure complete hydrolyses was usually achieved and a constant level of determined estriol obtained (Fig. 1). At very high levels (above 130 pmol/l), longer incubation time became necessary. However, in practical work a further dilution of the sample is necessary at this level because of the unfavorable course of the standard curve at such concentrations (Fig. 2). Incubation at higher temperatures, which has been recommended, [6], gave incomplete hydrolyses. Possibly this is due to inactivation of the enzymes at these temperatures. The original freezedried standard solutions of the kit were substituted with solutions of eStI”iOl-3/%Dglucuronide in water in order to obtain standard solutions of concentrations comparable to pregnancy urine. Different conjugates of estriol are hydrolyzed at different rates by the enzymes included in the kit. 240
1 x 37Y
200-
o 63%
? 160‘, f. ‘z .g 120Zi
W iI
80-
40-
Incubation time (hours) Fig. 1. Rate of enzymatic hydrolysis of natural conjugates in urine. Circles refer to urines of final concentration of about 50 and >200 pmol/l. respectively.
328
40
80
120
U-Estrlol Fig. 2. Standard curve represent S.D. (n = 8).
160
200
(pmol/O
of U-estriol
with different
kits within
the same production
batch.
Vertical
bars
Thus the 3/3-D-glucuronide and 3-sulfate were almost completely hydrolyzed within 30 min whereas about 120 E.tmol/l 16a-fl-D-glucuronide required about 3 h with a time course similar to that of native urine shown in Fig. 1. This is in accordance with the reported composition of pregnancy urine [lo]. To be able to use the 3@D-glucuronide as the primary standard it is thus necessary to achieve a complete hydrolysis of the estriol derivatives in urine. Recovery of added test substances was similar on addition to both urine and water. This implies that the effect of possible inhibitor8 in urine is eliminated by the extreme dilution (l/834) used. The protein content of urine is very low and the precipitation of antigenantibody complex might be enhanced by addition of a protein solution when diluting the sample. No advantage of such an addition could be shown in the present modification. One kit allows the determination of estriol in 60 tubes. In order to increase the yield of the kit and economize, standard curves were compared from several kits within the same batch. It was found that with the present system the same standard curve could be used from one series of analyses to the other, provided corrections for decay of radioactivity were made. The coefficient of variation of the individual points on the standard curve was about 4% between runs (Fig. 2). In order to check the level of the standard curve, one standard solution was routinely included in each series. The standard curve was shown to coincide with that obtained for serum determinations according to the instructions in the kit and accordingly estriol in serum could be assayed by either standard curve (Fig. 3). Further, serum samples and urinary samples could be assayed according to the same standard curve. The standard curve was plotted as percent bound radioactivity versus the logarithm of the amount of EUbEtaIKe. This curve showed the well known “S”shape but the inflection point appeared to be at 60% (Fig. 4). A logit transformation doe8 not yield a straight line unless the percent bound in the logit expression is decreased by 10%. This can be justified by assuming a nonspecific binding of the antibody amounting to about 10%. The straight logit/log standard curve was used for computerized estimation of estriol. A ma8s fragmentographic method (MID) for determination of U-estriol has recently been described from this laboratory [ 11. The present modification of the
329 12001
a
z L
iz
W
23
400
Y=0.94X-10.2 r = 0.997 n=23
I
50
10
S-Estriot (nmol II) B
U-Estriol
100
200
(pmol/I)
Fig. 3. S-Es&lo1 evaluated by standard curve according to kit (B) and standard curve obtained with estrlol3@-D-glueuronide as described in the present method (A). Fig. 4. Standard curve of e~ol-3~-D~luc~onide.
Additional points to secure position of inflexion point.
RIA method was compared to the GLC method in routine tory which agreed with this MID method (y = 0.93x + 6.9; good agreement was also found between the GLC and RIA The paired t-test showed no statistically significant different
Y=l.OOX
+ 0.90
r=096
1
40 U-Estriol
80
120 fpmol/ 24h)
160 RIA
Fig. 5. Regression line for U-estriol GLC = f (U-estriol RIA).
200
use in the laborar = 0.98). A very methods (Fig. 5). between samples
330 1601
I2 ;:
120-
x RIA
-36-t-37
-38-39
----c40--+Week
of
gestation
Fig. 6. U-Estriol course during pregnancy determined by GLC (0) and present method (X). In the lower pattern, the baby was still-born.
analyzed by the two methods (t = 1.5). The day-to-day precision was found to be 8.5% over a period of 30 days. To compare the methods further, several consecutive pregnancy urines were determined by both methods. As shown in Fig. 6 a very good agreement between methods was obtained at different levels of urinary estriol concentrations during pregnancy. The cross reaction of the antibody and various steroids is specified in the kit and significant for estetrol and 16-epi-estriol. These compounds do not occur to any large extent in pregnancy urine and any contribution of them or other substances to the antigen-antibody reaction does not seem to have any practical influence in determining estriol. In spite of the higher costs of reagents, the RIA dete~inations were found preferable because of the high speed of analyses. Thus, up to 75 determinations are easily handled in one working day and the normal batches of 20 to 30 samples can be ready in about 5 h. The main portion of this time is used for incubations and counting in the gamma counter. Additional samples add but little to this analysis time. Although it has been questioned lately [ 7 J, there may be value in determining serum estriol instead of or together with urinary estriol. It was considered advantageous to have similar methods for both types of analyses. References 1 Bjiirkhem, I.. Blomstrand. R.. Svensson, L., Tietz. F. and Carlstrom. K. (1975) Clin. Chim. Acta 62, 365 2 Fishman. W.H. (1970) in Metoden der Enzyrnatischen AnaIyse (Bergmayer. H.V., ed.), PP. 964-972, Verlag Chemie, Weinheim 3 Milazzo, F.H., Fitzgerald. J.W. (1966) Can. J. Microbial. 12, 735 4 van de Calseyde, J.F.. Scholties. R.J.H., Schmidt. N.A. and Kuypers. A.M.J. (1966) CIin. Chim. Acta 25,345 5 Rosenthal, A.F. and Yaseen, A. (1971) Clin. Chim. Acta 31, 171 6 SehoIIer, R., MCtay, S.. Herbon, S. and Jayle, M.F. (1966) Eur. J. Steroids 1. 373 7 Lindberg. B.S. and CarIstrBm, K. (1976) Acta Obstet. Gynaecol. Stand. 55. 163 8 Snedecor, G.W. and Cochran. W.G. (1972) Statistical Methods, Iowa State University Press, Iowa 9 Strewhart, W.A. (1931) Economic Control of Manufactured Products. D. van Nostrand, New York 10 Levitz, M., Jirku, H.. Kadner, S. and Young, B.K. (1975) J. Steroid. Biochem. 6,663