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Renal clearances of estriol conjugates in normal human pregnancy at term
Renal clearances of estxiol conjugates in normal human pregnancy at term BRUCE
K. YOUNG,
HELMUT
JIRKU,
SUSAN
PH.D.
KADNER,
MORTlMER
M.D.*
B.S.
LEVITZ,
PH.D.
New York, New York
In late human pregnancy more than PO per cent of the total estriol (E3) in body,puids consists of four conjugates, estriol-3-sulfate (E3-3S), estriol-16glucosiduronate (E,-I 6G), estriol-3-glucuronide (E,-3G), and estriol-3-sulfate-16glucosiduronate (E,-3S-I 6G). Since the relative amounts of E3 in blued and urine would be determined by the kidney, the renal clearance of each conjugate was determined and compared with inulin and p-aminoh$$uric acid (PAH) clearance, as measures of glomerular.filtration rate (GFR) and the effective renal @.sma,pow. Five women were studied in the lateral decubitus position with in&in and PAH infusion. Samples of blood and urine were collected at 40 minute intervals and analyzed. The method for EB conjugates involved separation of the jour conjugates on Sephadex LH-20, enzyme hydrolysis, and radioimmunoassay. Rennl clearances for E,-3S and E,-3S-16G were below inulin. Es-3G approximated inulin; E,-16G exceeded inulin and approached PAH. In pLasma E,-3S-IGG repre.sented 48.4 ir 7.2 per cent; in urine E3-16G represented 69.5 +- 7.3 per cent of total ES. Thas, dfjferent conjugates predominate in blood and urine.
LATE HUMAN pregnancy estriol is synthesized mainly in the placenta from 16o-hydroxy-dehydroepiandrosterone sulfate, principally of fetal origin.‘* ’ An additional source of estriol is by conversion from estrone in the maternal and fetal livers? The estriol so produced is metabolized almost exclusively to conjugates which are excreted in the urine.” Since four principal conjugates are involved; estriol-3-sulfate (ES-3S), estriol- 16-glucosiduronate (ES-16G), estriol-3-glucuronide (E,-3G), and estriol3-sulfate- 16-glucosiduronate (&-3S- 16G), the relative amounts of estriol in the urine and blood would be
controlled to a large degree by the renal clearance of each of these conjugates. Since estriol levels in these fluids are accepted indicators of fetal-placental health,‘, 6 a study was undertaken to measure the renal clearances of each estriol conjugate in normal pregnancy at term. In four of the five studies comparisons were made with the clearances c)f inulin and p-aminohippuric acid (PAH) which reflect, respectively, the glomerular filtration rate (GFR). and the effective renal plasma flow.’
I N
From the Department of Obstetrics and Gynecolog, York University School of Medicine. Supported by a grant from the National Health (Grant CA 02071).
Institute
Part of this study was presented at the Fourth International Congress on Hormonal Steroids, City, Mexico, September, 1974. Received
for publication
Accepted October
October
Materials and methods Materials. The four radioactive estriol conjugates. used were synthesized in this laboratory from 2,4,6,‘i; 3H-estriol (New England Nuclear Co., Boston, Massachusetts) by published methods. Each conjugate was purified by gradient elution chromatography on Celite as described.’ Analytical methods. The procedure for the analysis of estriol conjugates in plasma and urine is similar to that described in detail for the analysis of amniatit. fluid.Y Briefly, about 2,000 c.p.m. of each tritiated estrio1 conjugate are added to 0.2 to 0.5 ml. of urine OI plasma. These serve both as markers for the endogenous conjugates and as tracers for the correction of methodological losses. After precipitation of the pro-
New of
Mexico
7, 1975.
30, 1975.
Reprint requests: Mortimer Levitt, Ph.D., Department of Obstetrics and Gynecology, New York University Medical Center, 550 First Ave., New York, New York, 10016. *Irma
T. Hirsch1
Career
Scientist.
38
Renal clearances of estriol conjugates
Table I. Concentration in Patient M
of estriol
conjugates
E,-3s*
in plasma
(P) and urine
E,-16G
(U) in three successive 40 minute
E,3G
E,-3.5-l
39
periods
IX T~ltU/
P-f) P-l P-2 P-3 U-l r-2 u-3
,ughl.
Rt
pglml.
0.017 0.016
14.5 13.6 13.9
0.018 0.017
0.017
na$ 0.44
15.4
14.4 19.7
0.024 0.023 3.05
7.0 8.8 7.7
0.33 0.84
c
65.7 61.2 65.1
5.79 3.72
j.ig/ml.
%
0.028 0.025
23.8 21.2
0.016
13.1
0.014 0.65 1.35 0.70
14.0 14.2 12.2
p&ml.
57c
0.054 0.060 0.065 0.074 0.62
46.2
1.49
15.8
0.86
15.0
il*glml.)
17 0.1 18 0. 122
0.1
50.8 .?I3 .:3 I:$.:<
4.64 9.46 5.70
*Abbreviations of estrogen conjugates indicated in text. +Indicates % contribution of the conjugate to the calculated total. $Not analvzed-sample lost Table II. Concentration periods in patient A*
of estriol
conjugates
E,-?S
P-O P-l P-2 P-3 IJ-1 U-2 u-3
E,-16G
pglrr1.1.
Q
iLgiml.
0.020 0.02 1
12.3 12.4 13.9
0.052 0.043 0.036 0.056
0.017
0.024 0.3” 0.45 0.4i
in plasma (P) and urine
12.5 2.4
2.8 3.0
9.85 11.18 11.85
(U) in three successive 40 minute
E,-3G
%
pglml.
31.9
0.028 0.032 0.016 0.033 1.83 2.88 2.18
25.3
29.5 29.2 76.2
71.0 75.6
Ey?S-26G
5% 17.2 18.8 13.1 17.2 14.1 18.3
13.9
7hlul f&+d.~
pglml.
R
0.063 0.074 0.053
38.6 43.5
0.163
433 41.1
0.122
0.079 0.94
1
0.170 0.192
1.24
7.3 7.9
12.94 15.75
1.18
7.5
13.68
*Foot.nores to Table I also apply here. reins with ethanol, the conjugates are converted to their triethylammonium salts and separated on a Sephadex LH-20 column. Each conjugate is hydrolyzed with Glusulase and the estriol is purified by solvent partitions. The estriol is submitted to radioimmunoassay using rabbit antisera (kindly supplied by Dr. Uwe Goebelsmann) highly specific toward estriol.” Subjects. Five women volunteered for the study. Each patient was about to undergo repeat cesarean section at 39 weeks’ gestation and there were no clinical or laboratory abnormalities. 4 Foley catheter was inserted for the collection of urine and an intravenous infusion was started. The patient received a priming close of 8 mg. per kilogram of PAH and 50 mg. per kilogram of inulin in normal saline. This was followed by a continuous drip of 500 ml. of normal saline containing 12.5 Gm. of mannitol. 8 Gm. of inulin, and 3 Gm. of PAH. at 2 ml. per minute. The patient remained on her left side as much as possible during the procedure. Blood samples were drawn at time zero and then at three 10 minute intervals, during which time three urine samples were collected. The bladder was emptied with suprapubic pressure. The plasma was separated promptly and stored together with the urine samples at -17” C. until processed. Aliquots were analyzed tar PAW and inulin.’
The renal clearances (RC) of PAH, inulin, and each estriol conjugate were calculated according to the formula: RC = UV/P, where U and P refer ro the concentrations in the urine and plasma, respectively. and V is the urinary output in milliliters per minute. The value for P is the average of the concentrations at the beginning and the end of the test period. All clearances were corrected for surface area using the standard nomogram based on height and weight. Rssults The concentrations of estriol conjugates in each plasma and urine sample collected from the five patients are shown in Tables I to V. The tables also include the per cent contribution of each conjugate to the calculated total. The following features are wort.hy of comment. (1) ES-16G and E,-3S-16G comprised about 80 per cent of the total conjugated estriol in both fluids, but Ea-3S-16G predominated iu the plasma whereas E,-16G was more concentrated in the urine. (2) The per cent contribution of I!.,-3G to plasma is similar to that in urine. (3) The per cent contribution of each conjugate to the total is far more stable in the urine than in the plasma. (4) In Patient-s M and E, the calculated total plasma estriol is remarkably stable but in the other three cases the differences between the
40
Young
September 1. 1976 Am. J. Ohster. Gvnecol.
et al.
Table III. Concentration in Patient E*
of estriol conjugates
E,-3S
P-O P-l P-3 U-l u-2 + 3t
in plasma (P) and urine
E,-16G
fqglml.
%
0.044 0.037 0.045 0.92 0.66
19.9 19.4 22.0 3.7 2.7
&ml.
E,-3G 5%
0.063 0.063 0.055 20.1 20.6
(U) in three successive 40 minute
pglml.
28.5 33.0 27.0 80.5 83.5
E,-3S-16G 9%
0.029 0.026 0.028 2.29 2.19
periods
13.1 13.6 13.7 9.2 8.9
pgiml.
0.085 0.065 0.076 1.64 1.20
7r
38.5 34.0 37.3 6.6 4.9
7’0td ( pglmi.)
0.22 I 0.191 0.204 24.9 24.6
*See Table I footnotes for pertinent explanations. iSample P-2 was lost, consequently urines 2 and 3 were combined. Table IV. Concentration in Patient C*
of estriol conjugates
E,-3.S
in plasma (P) and urine
Ea.1 6G
(U) in three successive 40 minute
periods
E,-3S-26C
E,-3G
T&d P-O P-l P-2 P-3 U-l
u-2 u-3
pglml.
5%
0.060 0.048 0.066 0.054 1.58 1.36 0.92
18.0 18.6 23.7 20.6 2.8 3.2 2.6
p&d.
0.052 0.065 0.049 0.061 45.1 33.7 27.9
%
15.7 25.2 17.6 23.3 79.5 79.9 79.3
&ml.
0.047 0.016 0.034 0.025 3.36 2.50 2.29
5%
14.2 6.2 12.2 9.5 5.9 5.9 6.5
&ml.
0.173 0.129 0.129 0.122 6.37 4.60 4.09
%
52.1 50.0 46.4 46.6 11.8 10.9 11.6
(wlmU
0.332 0.258 0.278 0.262 35.8 42.2 35.2
*Footnotes to Table I also apply here. extremes are from 18 per cent to 30 per cent. Table VI shows the calculated renal clearances of each conjugate. In four patients the renal clearances of PAH and inulin were also determined and the data are presented in the same table. In general, the renal clearance of E,-16G far exceeded that of Es-3G and was about 10 times that of E3-3S or E,-3%16G, which were similar. In Patients A and C the renal clearances of E,-16G were similar to that of PAH, whereas in the other subjects they were lower. Nevertheless, the values clearly exceeded those for inulin clearance. Generally, the RC of E,-3G was comparable to that of inulin. The RC of E,-3S or E,-3S-16G was significantly lower than the value for inulin.
Comment There are reports on the assay of estriol conjugates in plasma and urine in human pregnancy. Investigating plasma Goebelsmann and associates” separated the four conjugates by countercurrent distribution and quantitated the estriol fluorometrically. Tikkanen” combined separation on Sephadex with gas-liquid chromatography for the estimation of estriol conjugates in urine. Our method is the simplest reported, in that the conjugates are separated clearly on a single short Sephadex LH-20 column. Furthermore, only 0.5 ml. of fluid is required for analysis. A comparison between the average values obtained in our studies and
those reported by these investigators are presented in Table VII. This is the first study which focuses in detail on the concentrations of estriol conjugates in the urine and plasma in a way that permits the estimation of renal clearance rates of each conjugate. Studies on clearance are influenced by the fact that renal function of women in late pregnancy is altered by position. Measurements of GFR based on inulin clearance and renal blood flow. based on PAH clearance, show a 20 per cent decrease when the patient is supine, compared with measurements in the lateral decubitus position.‘3 Accordingly, the subjects were asked to maintain the lateral position throughout the experiment. There is another potential source of error in these determinations. The urine collections were virtually complete, so an aliquot represents the average excretion for that collection period. However, there were some rather wide changes in the plasma concentrations of estriol conjugates. Averaging values in the plasma at the start and end of the 40 minute period does not necessarily produce the mean for that period. In clearance studies multiple observations made on each subject usually compensates for this deficiency in such a difficult experimental protocol. In this connection it should be noted that undefined factors involving moment to moment biologic changes in mother and fetus may produce rapid alterations in the concentration of estriol conju-
Volume
126
Number
1
Renal
Table V. Concentration Patient G*
of estriol conjugates
in plasma (P) and urine
clearances
of estriol
41
conjugates
(U) in three successive 40 minute
periods
in
1 E,-3S
P-O P-l P-2 P-3 U-l u-2 u-3 *Footnotes
E,-16G
E,-3G
E,-3s16G
@ml.
%
pglml.
YO
pglml.
%
Ccglml.
%
Totd I w/ml.)
0.032 0.035 0.035 0.026 0.217 0.282 0.211
12.4 14.9 17.9 13.1 3.6 4.1 3.6
0.069 0.068 0.045 0.022 3.93 4.51 4.09
26.8 29.1 23.0 11.1 65.8 66.2 69.5
0.058 0.046 0.036 0.023 1.31 1.55 1.13
22.6 19.7 18.4 11.6 22.0 22.7 19.2
0.098 0.085 0.080 0.128 0.514 0.476 0.455
38.2 36.3 40.7 64.2 X.6 7.0 7.7
0.257 0.234 0.196 0.199 5.98 6.81 5.88
and inulin
in late human
to Table
I also apply
Table VI. Renal clearances* pregnancy
here.
of estriol conjugates,
p-aminohippuric
acid (PAH),
Renal clearance
Patient M
Time period$
Urine output (ml. lmin.)
1 2 3
1.2 1.6 2.8
1 2 3
E,-?G
23 76 50
201 409 405 338
274 96 119 163
12 35 31 26
483 874 736 69R
74 136 128 113
2.0 1.2 1.4
32 29 35 32
436 352 383 390
129 149 133 137
29 25 27 27
351 346 356 351
89 60 56 68
1 2+3
2.5 1.88
67 35 51
940 447 694
245 181 213
65 38 52
136Q 1440 1405
99 74 87
1 2 3
0.7 0.6 0.9
19 14 13 15
70 58 65 64
29 21 27 26
1.1 2.5 3.1
7 19 20 15
63’i 364 282 428 -. -_
107 80 71 86
1 2 3
508 343 428 426 61 194 361 205
Average E Average c
Average G
Average
E,-3.S
conjugate+
E,-16G
Average A
E&o1
27 91 113 77
Es-3S-16G
6 14 13 11
PAH
Aulin
-
*Values are in ml. Per minute. t Abbreviations of estriol conjugates in text. SSuccessive 40 minute collections except in Patient E, where 2 + 3 is for 80 minutes. gates in the blood. Similar changes may be masked by analyzing an aliquot from urine collected over a given time period. Consequently, we are presently unable to interpret the significance of the observation that the per cent contribution of each conjugate to the total varied in the plasma samples, but remained quite constant in the urines. Another variable meriting consideration is the possible change in renal function with gestation. Accordingly, it is emphasized that the data accumulated in this study pertain to subjects at term. Renal handling of the different estriol conjugates showed differences related to the presence of a sulfate moiety and to the sire of glucuronidation. E,-3S-16G
and E3-3S (except in Subject M) were far more concentrated in the plasma, whereas EB-16G predominated in the urine. &-3G contributed to both fluids about equally. Consistent with these data is the observation that the cakulated renal clearances of E3-16G and E,-3G clearly exceed those of E;-3S and &-3S-16G. These differences may be explained partly in terms of the relative binding to serum proteins. Goebelsmann and associates” studied the binding of each estriol conjugate to serum using the methods of equilibrium dialysis and gel filtration on Sephadex G-25 cotumns. By each method the binding of E&3!?, and E,-3S-16G was nearly 90 per cent, about twice that of Es-16G or E3-3G. The avidity of binding of the
42
Young et al.
September Am. J. Obstet.
Table VII. Estriol conjugates late human pregnancy
in plasma and urine
U&e Conjugates*
Reported”
E,-3S Es-16G Es-3G Es-3S- 16G
2.5 68.0 22.9 6.5
Plasma
This studyt 5.1 69.5 13.0 12.4
in
f f t ”
2.3 7.3 4.3 2.3
Reported” 30.1 17.7 11.8 40.8
This studyt 16.2 24.2 11.2 48.4
f t -c 1
5.2 2.8 2.3 7.2
Values are in per cent of total esuiol conjugates in the fluid indicated. They are calculated from the concentrations. *Abbreviations of estriol conjugates in text. tThe figures are the averages of the values from the five subjects studied. The average deviations are also shown.
sulfate esters of & to plasma proteins would reduce the GFR and probably tubular secretion, since it is postulated that the unbound conjugate is very likely the specie cleared. Based on these marked differences in binding to plasma proteins, the clearance rates of the simple glucosiduronates would be about 5 times greater than the sulfates. However, in the absence of information on the relative rates of release from binding such estimates are speculative. An interesting observation is that the calculated renal clearance of ES-16G approaches that of PAH and exceeds that of E,-3G. Apparently tubular secretion is a more important route for the disposition of ES-16G.
However, a complicating feature is that the kidney can convert E3 to E3-16G.14 Although the concentrations of unconjugated ES in the plasma are quite low, renal conversion to ES-16G introduces an error in the estimated renal clearance of ES-16G of unknown magnitude. There is no present evidence that the kidney is involved in the metabolism of the other 3 conjugates or that tubular reabsorption plays a role. These patterns indicate that measurements of blood estriol and urinary estriol are in effect, looking at different
species
of estriol.
Since
the
usual
assays
are
of‘
total Es, the fact that different conjugates predominate in various body fluids has not received adequate consideration. Complications of pregnancy such as preeclampsia and diabetes affect renal function. Furthermore, medical complications such as obstructive jaundice have been shown to alter the urinar) excretion of E,-3G, presumably the result of decreased production of this conjugate.‘” Further study of ED conjugates in abnormal pregnancies may reveal other specific differences in Es metabolism, as well as explain discrepancies between blood and urine estriol concentrations such as those observed in some cases of class D diabetes mellitus.16 The authors are Jerome Lowenstein and also to Dr. determinations of
REFERENCES
1. Ryan, K. J.: Metabolism of C-16-oxygenated steroids by human placenta: The formation of egtriol, J. Biol. Chem. 234: 2006, 1959. 2. Siiteri, P. K., and MacDonald, P. C.: Placental estrogen biosynthesis during human pregnancy, J. Clin. Endocrinol. 26: 751, 1966. 3. Schwers, J., Eriksson, G., and Diczfalusy, E.: Metabolism of oestrone and oestradiol in the human foeto-placental unit at mid-pregnancy; Acta Endocrinol. (Kbh.) 49: 65, 1965. 4. Levitz, M., and Katz, J.: The synthesis and some properties of estriol-1 5-3H, Steroids 5: 11, 1965. 5. Nachtigall, L. M., Bassett, M., Hogsander, U., and Levitz, M.: Plasma estriol levels in normal and abnormal pregnancies: An index of fetal welfare: AM. J. OBSTET. GYNECOL. 101: 638, 1968. 6. Greene, J. W., Jr., Smith, K., Kyle, G. C., Touchstone, J. C., and Duhring, J. L.: The use of urinary estriol excretion in the management of pregnancies complicated by diabetes mellitus, AM. J. OBSTET. GYNECOL. 91: 684, 1965. 7. Smith, H. W.: Principles of Renal Physiology, New York, 1956, Oxford University Press, Inc. 8. Young, B. K., Jirku, H., and Levitz, M.: Estriol conjugates in amniotic fluid at midpregnancy and term, J. Clin. Endocrinol. 85: 208, 1972. 9. Young, B. K., Jirku, H., Slyper, A. J., Levitz, M., Kelly, W.
1. IY’jrj Gynerol
10.
11.
2. 3.
1 4.
15.
16.
grateful to Drs. David Baldwin and for valuable discussions and advice, Marilyn Cozzens-Kessler for the p-aminohippuric acid and inulin.
G., and Yaverbaum, S.: Estriol conjugates in amniotic fluid of normal and Rh-isoimmunized patients, J. Clin. Endocrinol. 39: 842, 1974. Katagiri, H., Stanczyk, F. Z., and Goebelsmann, U.: Estriol in pregnancy. III. Development, comparison and use of specific antisera for rapid radioimmunoassay of unconjugated estriol in pregnancy plasma, Steroids 24: 225, 1974. Goebelsmann, U., Chen, L.-C., Saga, M., Nakamura, R. M., and Jaffe, R. B.: Plasma conc&tration and protein binding of oestriol and its conjugates in pregnancy, Acta Endocrinol. (Kbh.) 74: 592, 1973. Tikkanen, M. J.: Urinary excretion of estriol conjugates in normal pregnancy, J. Steroid B&hem. 4: 57, 1973. Chesley, L. C. and Sloan, D. M.: The effect of posture on renal function in late pregnancy, AM. J. OBSTET. GYNECOL. 89: 754, 1964. Kirdani, R. Y., Sampson, D., Murphy, G. P., and Sandberg, A. A.: Studies on phenolic steroids in human subjects. XVI. Role of the kidney in the disposition of estriol, J. Clin. Endocrinol. 34: 546, 1972. Adlercreutz, H., and Tikkanen, M. J.: Defects in hepatic uptake and transport and biliary excretion of estrogens: Some new concepts of liver metabolism, Med. Chir. Di. 2: 59, 1973. Levitz, M., and Selinger, M.: Plasma estriol levels in class D diabetes of pregnancy, AM. J. OBSTET. GYNECOL. 108: 82, 1970.
K. YOUNG,
HELMUT
JIRKU,
SUSAN
PH.D.
KADNER,
MORTlMER
M.D.*
B.S.
LEVITZ,
PH.D.
New York, New York
In late human pregnancy more than PO per cent of the total estriol (E3) in body,puids consists of four conjugates, estriol-3-sulfate (E3-3S), estriol-16glucosiduronate (E,-I 6G), estriol-3-glucuronide (E,-3G), and estriol-3-sulfate-16glucosiduronate (E,-3S-I 6G). Since the relative amounts of E3 in blued and urine would be determined by the kidney, the renal clearance of each conjugate was determined and compared with inulin and p-aminoh$$uric acid (PAH) clearance, as measures of glomerular.filtration rate (GFR) and the effective renal @.sma,pow. Five women were studied in the lateral decubitus position with in&in and PAH infusion. Samples of blood and urine were collected at 40 minute intervals and analyzed. The method for EB conjugates involved separation of the jour conjugates on Sephadex LH-20, enzyme hydrolysis, and radioimmunoassay. Rennl clearances for E,-3S and E,-3S-16G were below inulin. Es-3G approximated inulin; E,-16G exceeded inulin and approached PAH. In pLasma E,-3S-IGG repre.sented 48.4 ir 7.2 per cent; in urine E3-16G represented 69.5 +- 7.3 per cent of total ES. Thas, dfjferent conjugates predominate in blood and urine.
LATE HUMAN pregnancy estriol is synthesized mainly in the placenta from 16o-hydroxy-dehydroepiandrosterone sulfate, principally of fetal origin.‘* ’ An additional source of estriol is by conversion from estrone in the maternal and fetal livers? The estriol so produced is metabolized almost exclusively to conjugates which are excreted in the urine.” Since four principal conjugates are involved; estriol-3-sulfate (ES-3S), estriol- 16-glucosiduronate (ES-16G), estriol-3-glucuronide (E,-3G), and estriol3-sulfate- 16-glucosiduronate (&-3S- 16G), the relative amounts of estriol in the urine and blood would be
controlled to a large degree by the renal clearance of each of these conjugates. Since estriol levels in these fluids are accepted indicators of fetal-placental health,‘, 6 a study was undertaken to measure the renal clearances of each estriol conjugate in normal pregnancy at term. In four of the five studies comparisons were made with the clearances c)f inulin and p-aminohippuric acid (PAH) which reflect, respectively, the glomerular filtration rate (GFR). and the effective renal plasma flow.’
I N
From the Department of Obstetrics and Gynecolog, York University School of Medicine. Supported by a grant from the National Health (Grant CA 02071).
Institute
Part of this study was presented at the Fourth International Congress on Hormonal Steroids, City, Mexico, September, 1974. Received
for publication
Accepted October
October
Materials and methods Materials. The four radioactive estriol conjugates. used were synthesized in this laboratory from 2,4,6,‘i; 3H-estriol (New England Nuclear Co., Boston, Massachusetts) by published methods. Each conjugate was purified by gradient elution chromatography on Celite as described.’ Analytical methods. The procedure for the analysis of estriol conjugates in plasma and urine is similar to that described in detail for the analysis of amniatit. fluid.Y Briefly, about 2,000 c.p.m. of each tritiated estrio1 conjugate are added to 0.2 to 0.5 ml. of urine OI plasma. These serve both as markers for the endogenous conjugates and as tracers for the correction of methodological losses. After precipitation of the pro-
New of
Mexico
7, 1975.
30, 1975.
Reprint requests: Mortimer Levitt, Ph.D., Department of Obstetrics and Gynecology, New York University Medical Center, 550 First Ave., New York, New York, 10016. *Irma
T. Hirsch1
Career
Scientist.
38
Renal clearances of estriol conjugates
Table I. Concentration in Patient M
of estriol
conjugates
E,-3s*
in plasma
(P) and urine
E,-16G
(U) in three successive 40 minute
E,3G
E,-3.5-l
39
periods
IX T~ltU/
P-f) P-l P-2 P-3 U-l r-2 u-3
,ughl.
Rt
pglml.
0.017 0.016
14.5 13.6 13.9
0.018 0.017
0.017
na$ 0.44
15.4
14.4 19.7
0.024 0.023 3.05
7.0 8.8 7.7
0.33 0.84
c
65.7 61.2 65.1
5.79 3.72
j.ig/ml.
%
0.028 0.025
23.8 21.2
0.016
13.1
0.014 0.65 1.35 0.70
14.0 14.2 12.2
p&ml.
57c
0.054 0.060 0.065 0.074 0.62
46.2
1.49
15.8
0.86
15.0
il*glml.)
17 0.1 18 0. 122
0.1
50.8 .?I3 .:3 I:$.:<
4.64 9.46 5.70
*Abbreviations of estrogen conjugates indicated in text. +Indicates % contribution of the conjugate to the calculated total. $Not analvzed-sample lost Table II. Concentration periods in patient A*
of estriol
conjugates
E,-?S
P-O P-l P-2 P-3 IJ-1 U-2 u-3
E,-16G
pglrr1.1.
Q
iLgiml.
0.020 0.02 1
12.3 12.4 13.9
0.052 0.043 0.036 0.056
0.017
0.024 0.3” 0.45 0.4i
in plasma (P) and urine
12.5 2.4
2.8 3.0
9.85 11.18 11.85
(U) in three successive 40 minute
E,-3G
%
pglml.
31.9
0.028 0.032 0.016 0.033 1.83 2.88 2.18
25.3
29.5 29.2 76.2
71.0 75.6
Ey?S-26G
5% 17.2 18.8 13.1 17.2 14.1 18.3
13.9
7hlul f&+d.~
pglml.
R
0.063 0.074 0.053
38.6 43.5
0.163
433 41.1
0.122
0.079 0.94
1
0.170 0.192
1.24
7.3 7.9
12.94 15.75
1.18
7.5
13.68
*Foot.nores to Table I also apply here. reins with ethanol, the conjugates are converted to their triethylammonium salts and separated on a Sephadex LH-20 column. Each conjugate is hydrolyzed with Glusulase and the estriol is purified by solvent partitions. The estriol is submitted to radioimmunoassay using rabbit antisera (kindly supplied by Dr. Uwe Goebelsmann) highly specific toward estriol.” Subjects. Five women volunteered for the study. Each patient was about to undergo repeat cesarean section at 39 weeks’ gestation and there were no clinical or laboratory abnormalities. 4 Foley catheter was inserted for the collection of urine and an intravenous infusion was started. The patient received a priming close of 8 mg. per kilogram of PAH and 50 mg. per kilogram of inulin in normal saline. This was followed by a continuous drip of 500 ml. of normal saline containing 12.5 Gm. of mannitol. 8 Gm. of inulin, and 3 Gm. of PAH. at 2 ml. per minute. The patient remained on her left side as much as possible during the procedure. Blood samples were drawn at time zero and then at three 10 minute intervals, during which time three urine samples were collected. The bladder was emptied with suprapubic pressure. The plasma was separated promptly and stored together with the urine samples at -17” C. until processed. Aliquots were analyzed tar PAW and inulin.’
The renal clearances (RC) of PAH, inulin, and each estriol conjugate were calculated according to the formula: RC = UV/P, where U and P refer ro the concentrations in the urine and plasma, respectively. and V is the urinary output in milliliters per minute. The value for P is the average of the concentrations at the beginning and the end of the test period. All clearances were corrected for surface area using the standard nomogram based on height and weight. Rssults The concentrations of estriol conjugates in each plasma and urine sample collected from the five patients are shown in Tables I to V. The tables also include the per cent contribution of each conjugate to the calculated total. The following features are wort.hy of comment. (1) ES-16G and E,-3S-16G comprised about 80 per cent of the total conjugated estriol in both fluids, but Ea-3S-16G predominated iu the plasma whereas E,-16G was more concentrated in the urine. (2) The per cent contribution of I!.,-3G to plasma is similar to that in urine. (3) The per cent contribution of each conjugate to the total is far more stable in the urine than in the plasma. (4) In Patient-s M and E, the calculated total plasma estriol is remarkably stable but in the other three cases the differences between the
40
Young
September 1. 1976 Am. J. Ohster. Gvnecol.
et al.
Table III. Concentration in Patient E*
of estriol conjugates
E,-3S
P-O P-l P-3 U-l u-2 + 3t
in plasma (P) and urine
E,-16G
fqglml.
%
0.044 0.037 0.045 0.92 0.66
19.9 19.4 22.0 3.7 2.7
&ml.
E,-3G 5%
0.063 0.063 0.055 20.1 20.6
(U) in three successive 40 minute
pglml.
28.5 33.0 27.0 80.5 83.5
E,-3S-16G 9%
0.029 0.026 0.028 2.29 2.19
periods
13.1 13.6 13.7 9.2 8.9
pgiml.
0.085 0.065 0.076 1.64 1.20
7r
38.5 34.0 37.3 6.6 4.9
7’0td ( pglmi.)
0.22 I 0.191 0.204 24.9 24.6
*See Table I footnotes for pertinent explanations. iSample P-2 was lost, consequently urines 2 and 3 were combined. Table IV. Concentration in Patient C*
of estriol conjugates
E,-3.S
in plasma (P) and urine
Ea.1 6G
(U) in three successive 40 minute
periods
E,-3S-26C
E,-3G
T&d P-O P-l P-2 P-3 U-l
u-2 u-3
pglml.
5%
0.060 0.048 0.066 0.054 1.58 1.36 0.92
18.0 18.6 23.7 20.6 2.8 3.2 2.6
p&d.
0.052 0.065 0.049 0.061 45.1 33.7 27.9
%
15.7 25.2 17.6 23.3 79.5 79.9 79.3
&ml.
0.047 0.016 0.034 0.025 3.36 2.50 2.29
5%
14.2 6.2 12.2 9.5 5.9 5.9 6.5
&ml.
0.173 0.129 0.129 0.122 6.37 4.60 4.09
%
52.1 50.0 46.4 46.6 11.8 10.9 11.6
(wlmU
0.332 0.258 0.278 0.262 35.8 42.2 35.2
*Footnotes to Table I also apply here. extremes are from 18 per cent to 30 per cent. Table VI shows the calculated renal clearances of each conjugate. In four patients the renal clearances of PAH and inulin were also determined and the data are presented in the same table. In general, the renal clearance of E,-16G far exceeded that of Es-3G and was about 10 times that of E3-3S or E,-3%16G, which were similar. In Patients A and C the renal clearances of E,-16G were similar to that of PAH, whereas in the other subjects they were lower. Nevertheless, the values clearly exceeded those for inulin clearance. Generally, the RC of E,-3G was comparable to that of inulin. The RC of E,-3S or E,-3S-16G was significantly lower than the value for inulin.
Comment There are reports on the assay of estriol conjugates in plasma and urine in human pregnancy. Investigating plasma Goebelsmann and associates” separated the four conjugates by countercurrent distribution and quantitated the estriol fluorometrically. Tikkanen” combined separation on Sephadex with gas-liquid chromatography for the estimation of estriol conjugates in urine. Our method is the simplest reported, in that the conjugates are separated clearly on a single short Sephadex LH-20 column. Furthermore, only 0.5 ml. of fluid is required for analysis. A comparison between the average values obtained in our studies and
those reported by these investigators are presented in Table VII. This is the first study which focuses in detail on the concentrations of estriol conjugates in the urine and plasma in a way that permits the estimation of renal clearance rates of each conjugate. Studies on clearance are influenced by the fact that renal function of women in late pregnancy is altered by position. Measurements of GFR based on inulin clearance and renal blood flow. based on PAH clearance, show a 20 per cent decrease when the patient is supine, compared with measurements in the lateral decubitus position.‘3 Accordingly, the subjects were asked to maintain the lateral position throughout the experiment. There is another potential source of error in these determinations. The urine collections were virtually complete, so an aliquot represents the average excretion for that collection period. However, there were some rather wide changes in the plasma concentrations of estriol conjugates. Averaging values in the plasma at the start and end of the 40 minute period does not necessarily produce the mean for that period. In clearance studies multiple observations made on each subject usually compensates for this deficiency in such a difficult experimental protocol. In this connection it should be noted that undefined factors involving moment to moment biologic changes in mother and fetus may produce rapid alterations in the concentration of estriol conju-
Volume
126
Number
1
Renal
Table V. Concentration Patient G*
of estriol conjugates
in plasma (P) and urine
clearances
of estriol
41
conjugates
(U) in three successive 40 minute
periods
in
1 E,-3S
P-O P-l P-2 P-3 U-l u-2 u-3 *Footnotes
E,-16G
E,-3G
E,-3s16G
@ml.
%
pglml.
YO
pglml.
%
Ccglml.
%
Totd I w/ml.)
0.032 0.035 0.035 0.026 0.217 0.282 0.211
12.4 14.9 17.9 13.1 3.6 4.1 3.6
0.069 0.068 0.045 0.022 3.93 4.51 4.09
26.8 29.1 23.0 11.1 65.8 66.2 69.5
0.058 0.046 0.036 0.023 1.31 1.55 1.13
22.6 19.7 18.4 11.6 22.0 22.7 19.2
0.098 0.085 0.080 0.128 0.514 0.476 0.455
38.2 36.3 40.7 64.2 X.6 7.0 7.7
0.257 0.234 0.196 0.199 5.98 6.81 5.88
and inulin
in late human
to Table
I also apply
Table VI. Renal clearances* pregnancy
here.
of estriol conjugates,
p-aminohippuric
acid (PAH),
Renal clearance
Patient M
Time period$
Urine output (ml. lmin.)
1 2 3
1.2 1.6 2.8
1 2 3
E,-?G
23 76 50
201 409 405 338
274 96 119 163
12 35 31 26
483 874 736 69R
74 136 128 113
2.0 1.2 1.4
32 29 35 32
436 352 383 390
129 149 133 137
29 25 27 27
351 346 356 351
89 60 56 68
1 2+3
2.5 1.88
67 35 51
940 447 694
245 181 213
65 38 52
136Q 1440 1405
99 74 87
1 2 3
0.7 0.6 0.9
19 14 13 15
70 58 65 64
29 21 27 26
1.1 2.5 3.1
7 19 20 15
63’i 364 282 428 -. -_
107 80 71 86
1 2 3
508 343 428 426 61 194 361 205
Average E Average c
Average G
Average
E,-3.S
conjugate+
E,-16G
Average A
E&o1
27 91 113 77
Es-3S-16G
6 14 13 11
PAH
Aulin
-
*Values are in ml. Per minute. t Abbreviations of estriol conjugates in text. SSuccessive 40 minute collections except in Patient E, where 2 + 3 is for 80 minutes. gates in the blood. Similar changes may be masked by analyzing an aliquot from urine collected over a given time period. Consequently, we are presently unable to interpret the significance of the observation that the per cent contribution of each conjugate to the total varied in the plasma samples, but remained quite constant in the urines. Another variable meriting consideration is the possible change in renal function with gestation. Accordingly, it is emphasized that the data accumulated in this study pertain to subjects at term. Renal handling of the different estriol conjugates showed differences related to the presence of a sulfate moiety and to the sire of glucuronidation. E,-3S-16G
and E3-3S (except in Subject M) were far more concentrated in the plasma, whereas EB-16G predominated in the urine. &-3G contributed to both fluids about equally. Consistent with these data is the observation that the cakulated renal clearances of E3-16G and E,-3G clearly exceed those of E;-3S and &-3S-16G. These differences may be explained partly in terms of the relative binding to serum proteins. Goebelsmann and associates” studied the binding of each estriol conjugate to serum using the methods of equilibrium dialysis and gel filtration on Sephadex G-25 cotumns. By each method the binding of E&3!?, and E,-3S-16G was nearly 90 per cent, about twice that of Es-16G or E3-3G. The avidity of binding of the
42
Young et al.
September Am. J. Obstet.
Table VII. Estriol conjugates late human pregnancy
in plasma and urine
U&e Conjugates*
Reported”
E,-3S Es-16G Es-3G Es-3S- 16G
2.5 68.0 22.9 6.5
Plasma
This studyt 5.1 69.5 13.0 12.4
in
f f t ”
2.3 7.3 4.3 2.3
Reported” 30.1 17.7 11.8 40.8
This studyt 16.2 24.2 11.2 48.4
f t -c 1
5.2 2.8 2.3 7.2
Values are in per cent of total esuiol conjugates in the fluid indicated. They are calculated from the concentrations. *Abbreviations of estriol conjugates in text. tThe figures are the averages of the values from the five subjects studied. The average deviations are also shown.
sulfate esters of & to plasma proteins would reduce the GFR and probably tubular secretion, since it is postulated that the unbound conjugate is very likely the specie cleared. Based on these marked differences in binding to plasma proteins, the clearance rates of the simple glucosiduronates would be about 5 times greater than the sulfates. However, in the absence of information on the relative rates of release from binding such estimates are speculative. An interesting observation is that the calculated renal clearance of ES-16G approaches that of PAH and exceeds that of E,-3G. Apparently tubular secretion is a more important route for the disposition of ES-16G.
However, a complicating feature is that the kidney can convert E3 to E3-16G.14 Although the concentrations of unconjugated ES in the plasma are quite low, renal conversion to ES-16G introduces an error in the estimated renal clearance of ES-16G of unknown magnitude. There is no present evidence that the kidney is involved in the metabolism of the other 3 conjugates or that tubular reabsorption plays a role. These patterns indicate that measurements of blood estriol and urinary estriol are in effect, looking at different
species
of estriol.
Since
the
usual
assays
are
of‘
total Es, the fact that different conjugates predominate in various body fluids has not received adequate consideration. Complications of pregnancy such as preeclampsia and diabetes affect renal function. Furthermore, medical complications such as obstructive jaundice have been shown to alter the urinar) excretion of E,-3G, presumably the result of decreased production of this conjugate.‘” Further study of ED conjugates in abnormal pregnancies may reveal other specific differences in Es metabolism, as well as explain discrepancies between blood and urine estriol concentrations such as those observed in some cases of class D diabetes mellitus.16 The authors are Jerome Lowenstein and also to Dr. determinations of
REFERENCES
1. Ryan, K. J.: Metabolism of C-16-oxygenated steroids by human placenta: The formation of egtriol, J. Biol. Chem. 234: 2006, 1959. 2. Siiteri, P. K., and MacDonald, P. C.: Placental estrogen biosynthesis during human pregnancy, J. Clin. Endocrinol. 26: 751, 1966. 3. Schwers, J., Eriksson, G., and Diczfalusy, E.: Metabolism of oestrone and oestradiol in the human foeto-placental unit at mid-pregnancy; Acta Endocrinol. (Kbh.) 49: 65, 1965. 4. Levitz, M., and Katz, J.: The synthesis and some properties of estriol-1 5-3H, Steroids 5: 11, 1965. 5. Nachtigall, L. M., Bassett, M., Hogsander, U., and Levitz, M.: Plasma estriol levels in normal and abnormal pregnancies: An index of fetal welfare: AM. J. OBSTET. GYNECOL. 101: 638, 1968. 6. Greene, J. W., Jr., Smith, K., Kyle, G. C., Touchstone, J. C., and Duhring, J. L.: The use of urinary estriol excretion in the management of pregnancies complicated by diabetes mellitus, AM. J. OBSTET. GYNECOL. 91: 684, 1965. 7. Smith, H. W.: Principles of Renal Physiology, New York, 1956, Oxford University Press, Inc. 8. Young, B. K., Jirku, H., and Levitz, M.: Estriol conjugates in amniotic fluid at midpregnancy and term, J. Clin. Endocrinol. 85: 208, 1972. 9. Young, B. K., Jirku, H., Slyper, A. J., Levitz, M., Kelly, W.
1. IY’jrj Gynerol
10.
11.
2. 3.
1 4.
15.
16.
grateful to Drs. David Baldwin and for valuable discussions and advice, Marilyn Cozzens-Kessler for the p-aminohippuric acid and inulin.
G., and Yaverbaum, S.: Estriol conjugates in amniotic fluid of normal and Rh-isoimmunized patients, J. Clin. Endocrinol. 39: 842, 1974. Katagiri, H., Stanczyk, F. Z., and Goebelsmann, U.: Estriol in pregnancy. III. Development, comparison and use of specific antisera for rapid radioimmunoassay of unconjugated estriol in pregnancy plasma, Steroids 24: 225, 1974. Goebelsmann, U., Chen, L.-C., Saga, M., Nakamura, R. M., and Jaffe, R. B.: Plasma conc&tration and protein binding of oestriol and its conjugates in pregnancy, Acta Endocrinol. (Kbh.) 74: 592, 1973. Tikkanen, M. J.: Urinary excretion of estriol conjugates in normal pregnancy, J. Steroid B&hem. 4: 57, 1973. Chesley, L. C. and Sloan, D. M.: The effect of posture on renal function in late pregnancy, AM. J. OBSTET. GYNECOL. 89: 754, 1964. Kirdani, R. Y., Sampson, D., Murphy, G. P., and Sandberg, A. A.: Studies on phenolic steroids in human subjects. XVI. Role of the kidney in the disposition of estriol, J. Clin. Endocrinol. 34: 546, 1972. Adlercreutz, H., and Tikkanen, M. J.: Defects in hepatic uptake and transport and biliary excretion of estrogens: Some new concepts of liver metabolism, Med. Chir. Di. 2: 59, 1973. Levitz, M., and Selinger, M.: Plasma estriol levels in class D diabetes of pregnancy, AM. J. OBSTET. GYNECOL. 108: 82, 1970.