Disappearance of human chorionic gonadotropin ... - Clinical Chemistry

Clinical Chemistry 43:11 2155–2163 (1997)

Endocrinology and Metabolism

Disappearance of human chorionic gonadotropin and its a- and b-subunits after term pregnancy Juha Korhonen,* Henrik Alfthan, Pekka Ylo¨stalo, Johannes Veldhuis,1 and Ulf-Håkan Stenman We have used high-specificity and precision immunofluorometric assays to measure the elimination half-times of human chorionic gonadotropin (hCG), hCGa, and hCGb in serum over 21 days after delivery in six women with term pregnancies. Baseline concentrations and half-times were calculated with the use of a curve-fitting algorithm for multiexponential decay. In contrast to the two-component model, a three-component exponential function with baseline provided a fit for which predicted values could not be distinguished from the observed values by analysis of variance. Median half-times were 3.6, 18.0, and 53.0 h for hCG; 1.0, 23.4, and 194 h for hCGb; and 0.6, 6.2, and 21.9 h for hCGa. The mean ratio of hCGa to hCG decreased rapidly from 36.9% to 3.3% on day 3; thereafter it increased to 64.3% 21 days after delivery because of a higher baseline concentration of hCGa. hCGb had the slowest total elimination rate, and the ratio of hCGb to hCG in serum increased from 0.8% before delivery to 26.7% after 21 days. If the metabolism of hCG and hCGb is similar in patients with trophoblastic disease, the ratio of hCGb to hCG must be evaluated with caution in samples taken several days after initiating therapy. We conclude that the disappearance of hCGb from plasma is slower than previously recognized and that the ratios of hCGb or hCGa to intact hCG vary as a function of postpartum time. Such information may be important in clinical studies of pregnancy disorders.

Human chorionic gonadotropin (hCG)2 is a heterodimer composed of two highly glycosylated subunits, called a

Department of Obstetrics and Gynecology and Department of Clinical Chemistry, Helsinki University Central Hospital, Haartmaninkatu 2, FIN00290 Helsinki, Finland. 1 Department of Internal Medicine, University of Virginia Health Sciences Center, and National Science Foundation Center for Biological Timing, Charlottesville, VA 22908. * Author for correspondence. Fax 358-9-4714801; e-mail ulf-hakan, [email protected]. 2 Nonstandard abbreviations: hCG, human chorionic gonadotropin; hCGa and hCGb, highly glycosylated subunits of hCG; AUC, area under the curve. Received January 22, 1997; revision accepted July 3, 1997.

and b, that are noncovalently joined. The a-subunits of all glycoprotein hormones of pituitary origin, including follicle-stimulating hormone, luteinizing hormone, and thyroid-stimulating hormone, are virtually identical, but the b-subunits are different and thus confer biological specificity of the hormones. In early pregnancy the concentrations of hCG in serum start to increase 7–11 days after ovulation, corresponding to 21–25 days after the last menstrual period [1, 2]. After in vitro fertilization and embryo transfer, an increase of serum hCG can be observed 9 days after ovum retrieval, corresponding to 7 days after embryo transfer [3]. The increase is exponential, with a doubling time of 1.5 days during the first 6 weeks [4]. Serum hCG reaches peak concentrations of ;100 000 IU/L (in relation to the First International Reference Preparation) at 8 –10 weeks after the last menstrual period. The concentrations start to decrease after week 12 and stay fairly constant at about ;30 000 IU/L from the 20th week until term [5]. In addition to hCG, serum and urine from pregnant women and patients with trophoblastic disease contain free a-subunits (hCGa) and b-subunits (hCGb). The profile of the serum concentrations of hCGb during pregnancy resembles that of hCG, but the concentrations are lower. During gestation, the molar ratio of hCGb to hCG is 1.5– 4% in early pregnancy and decreases to 0.2–1% after the 10th week [3, 6]. In patients with benign trophoblastic disease, the ratio is similar to that in pregnancy, whereas higher ratios are observed in trophoblastic cancer. Thus the ratio may aid in differentiating between malignant and benign trophoblastic tumors [7–10]. The concentrations of hCGa increase throughout pregnancy from ,1 mg/L (69 pmol/L) [11] to 100 –300 mg/L (6900 –20 700 pmol/L) in the third trimester [12, 13]. The hCGa to hCG ratio is ,10% during the first trimester and increases to 30 – 60% at term [6]. When hCG is injected into humans, it reportedly has a biphasic disappearance curve with an initial fast half-time of ;5 h and a slow one of 24 –32 h [14 –16]. Injected hCGa and hCGb are cleared from circulation much more rapidly than hCG [16, 17]. hCGa has a rapid half-time of 13

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Korhonen et al.: Disappearance of hCG after term pregnancy

min and a slow one of 76 min. The corresponding times for hCGb are 41 and 236 min, respectively [18, 19]. Serial hCG estimations are used for detecting pregnancy-related disorders such as spontaneous abortion and ectopic pregnancy as well as following-up patients with ectopic pregnancy. In ectopic pregnancy patients selected for expectant management, decreasing concentrations usually indicate spontaneous resolution, but one-third of these still require surgery within 1–24 days [20]. Thus a decreasing hCG concentration alone is not a reliable indicator for spontaneous resolution of an ectopic pregnancy. Because hCGa and hCGb have been reported to have much shorter half-times than hCG, they might more rapidly reflect changes in placental function such as an impending abortion. Changes in the ratio of the subunits to intact hCG potentially could be used to evaluate trophoblastic activity in pregnancy-related disorders, e.g., a low subunit to hCG ratio might indicate the cessation of hCG production because the ratio may be expected to decrease rapidly when the production ceases such as during an abortion or after delivery.

Materials and Methods patients Six women with term pregnancies were included in the study, which was approved by the local institutional review committee. One of the patients had a twin pregnancy, one had gestational diabetes (non-insulin-dependent glucose intolerance of pregnancy), and one had diabetes mellitus (insulin-dependent diabetes with nephropathy). The mean age was 37 years (range 32– 48 years) and mean gestational age 38 6 4 weeks (range 37 1 3 to 39 1 5 weeks). In singleton pregnancies the mean birth weight was 3862 g (range 3270 – 4360 g) and the mean placental weight 678 g (range 600 –770 g). In the twin pregnancy the birth weights were 2970 g and 3325 g and placental weights 560 g and 685 g, respectively. The first blood sample was drawn from a cannula inserted in the right cubital vein 15 min before an elective caesarean section, performed under spinal anesthesia. Infusion of saline solution was started in the opposite arm to stabilize the hemodynamics during anesthesia before the beginning of spinal anesthesia. The infusion was continued in some individuals, depending on the blood pressure. At the time of the first sample, ;500 mL of saline solution had been infused and at the time of delivery ;1000 mL. The effect of saline infusion on the serum concentration of hCG and its subunits and consequently on the half-times was analyzed in one case (patient 5 in Table 3), in which blood samples were drawn before fluid infusion and after infusion of 1 L of saline. After delivery, blood samples were drawn at 5, 10, 15, 20, 30, 40, 60, 120, 180, 360, and 720 min during the first day and every 24 h thereafter for 6 days. Additional blood samples were obtained from all patients 14 to 16 days after delivery and from 4 of those patients 21 days after delivery. Serum samples were stored at 220 °C before assay.

immunoassays

Serum hCG, hCGb, and hCGa were determined by timeresolved immunofluorometric assays (DELFIA®, Wallac). The detection limit for hCG was 0.3 IU/L. The assays for hCGb and hCGa are newly developed time-resolved immunofluorometric assays and are described in detail elsewhere. The detection limit for hCGb was 0.3 pmol/L (0.01 mg/L) (conversion factor: 1 mg/L 5 45.5 pmol/L) and that for hCGa 2.8 pmol/L (0.04 mg/L) (conversion factor: 1 mg/L 5 69 pmol/L). Cross-reaction of hCG in the subunit assays was studied after separation of hCG and the subunits in pregnancy serum [3]. On a molar basis, the cross-reaction of hCG in the hCGb assay was 0.05% and that in the hCGa assay ,0.1%. The upper reference limits of hCG, hCGb, and hCGa in nonpregnant premenopausal women were 2.9 IU/L, 1.6 pmol/L [21], and 31 pmol/L, respectively [H. Alfthan, unpublished finding]. The assays measure equally intact and nicked forms of hCG and hCGb [H. Alfthan, unpublished finding].

calibrator preparations and conversion factors The hCG assay was calibrated against the WHO 3rd International Standard (75/569), 1 mg corresponding to 9286 IU, and hCGb and hCGa against the 1st International Standard, 75/551 and 75/569, respectively. For conversion of IU to molar units, molecular masses of 36 700, 22 000, and 14 500 Da were used for hCG, hCGb, and hCGa, respectively [22]. The conversion factor of hCG to SI unit was 1 IU/L 5 2.93 pmol/L.

calculations and statistical analysis The ratio of the subunits (hCGa or hCGb) to total hCG was calculated on the basis of the molar concentration of each subunit relative to the concentration of subunit 1 hCG. Percentiles, means, SDs, and 95% confidence limits for the concentrations of hCG, hCGb, and hCGa and the ratios of the concentrations to the initial values were calculated for each time point. The individual subject’s concentration values were used for calculations of baseline concentrations and half-times for hCG, hCGb, and hCGa with the use of a curve-fitting algorithm for multiexponential decay (Microsoft Excel®). An iterative approach was used to calculate the combination of basal concentration and two or three exponential functions providing the best fit of the observed concentration values. The algorithm minimizes the fitted variance, which is the sum of the squares of the differences between the calculated curve and the observed values. The mathematical model for the decay curve is defined as

OC z e n

f~t! 5

i

2R i z t

1A

(1)

i51

where Ci is a coefficient (decay amplitude) and Ri is a rate constant (min21). t is the time after delivery, and A is the baseline concentration. By choosing n between 1 and 3,

Clinical Chemistry 43, No. 11, 1997

sums of up to 3 exponential terms can be used [23]. Untransformed concentration data over time are fit, such that a triphasic decay curve is defined as f~t! 5 C 1 z e ~2R 1t! 1 C 2 z e ~2R 2t! 1 C 3 z e ~2R 3t! 1 A

(2)

and a biphasic decay curve is defined as f~t! 5 C 1 z e ~2R 1t! 1 C 2 z e ~2R 2t! 1 A

(3)

The half-times are calculated as t1/2 5 (ln2/Ri). The integrated contribution of each exponential component to the overall disappearance curves of hCG, hCGb, and hCGa was calculated on the basis of the area under the curve (AUC) by AUC 5 2

Ci 1 A z @~e 2R i z t ! 2 1# Ri

(4)

Significant differences between triphasic and biphasic half-time fits were evaluated by comparing the fitted variances (above) via F ratio testing with Bonferroni/ Dunn correction. We estimated whether the time course over which the measurements were made affected the half-times by analyzing time intervals of 0 – 6 days, 0 –1 day, 0 –12 h, and 0 – 6 h. The longest half-times were not calculated when the time interval was shorter than the longest half-time component.

Results The mean serum concentration of hCG before delivery was 32 275 IU/L (94 566 pmol/L) (range 1631– 84 995 IU/L). The corresponding value for hCGb was 729 pmol/L (range 48 –2198 pmol/L) and for hCGa 35 862 pmol/L (range 17 432– 68 145 pmol/L). The mean concentrations of hCG, hCGb, and hCGa are shown in Figs. 1–3, and the ratios of hCGb and hCGa to total hCG and the ratios of hCGa to hCGb are shown in Table 1. A three-component exponential function gave the best

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fit to the observed hCG decay curves (Fig. 1). In contrast to a biphasic model, this resulted in very small deviations from the observed values, and the differences between triphasic and biphasic models and between the biphasic model and the observed values were significant (Table 2). The curve for hCGb was also optimally fit to a threecomponent model (Fig. 2), with significant differences from the biphasic model (Table 2). The calculated biphasic curve of hCGb fell below the observed values during the first hour after delivery, rose above the observed values during the next 12 h, and also deviated thereafter. The curve for hCGa was also best fit to a three-component model (Fig. 3). It fit to a two-component model only when values from days 0 –2 were included in the calculations. When days 3– 6, 14, and 21 values were also included, the biphasic model gave a poor fit. There were no significant differences between calculated values of the triphasic half-time model and the observed values of hCGa, whereas calculated values of the biphasic model differed significantly from the observed ones and those of the triphasic model by analysis of variance (Table 2). The half-times were virtually identical when calculated for shorter time intervals. The median half-time of the most rapid component of hCG was ;6 times longer than that of hCGa and 4 times longer than that of hCGb (Table 3). The median half-time of the second component of hCG was 30% shorter than that for hCGb, but for hCGa the second component was 3 times shorter. The median half-time of the third component of hCGb was almost fourfold that of hCG, and for hCGa it was half of that for hCG (Table 3). The algorithm used was set to calculate the baseline concentrations of hCG and its subunits limiting the highest possible value to the upper reference limit of nonpregnant premenopausal women. Without this limitation two patients would have had baseline concentrations for hCG of 3.5

Fig. 1. Observed disappearance of hCG (mean 1 SD) and estimated triphasic exponential half-times of hCG and their combined curve.

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Table 1. Ratios (percentage) of hCG subunit to total hCG and ratio of hCGa to hCGb after delivery. Mean (SD) ratio Time after delivery

n

hCGb/(hCGb 1 hCG), %

hCGa/(hCGa 1 hCG), %

hCGa/hCGb

0 min 5 min 10 min 15 min 20 min 30 min 40 min 60 min 120 min 180 min 360 min 720 min 1 day 2 days 3 days 4 days 5 days 6 days 14 days 21 days

6 5 6 5 6 6 6 6 6 6 6 6 6 6 6 6 5 5 4 4

0.8 (0.2) 0.8 (0.2) 0.8 (0.2) 0.8 (0.2) 0.7 (0.2) 0.7 (0.2) 0.7 (0.1) 0.7 (0.1) 0.6 (0.1) 0.6 (0.1) 0.6 (0.1) 0.7 (0.1) 0.9 (0.1) 1.2 (0.2) 1.5 (0.3) 1.7 (0.3) 2.1 (0.3) 2.8 (0.5) 15.8 (7.4) 26.7 (7.6)

36.9 (21.7) 38.8 (23.2) 34.2 (22.2) 35.6 (23.5) 31.7 (21.7) 29.6 (21.3) 27.8 (21.4) 23.5 (18.8) 17.2 (15.6) 14.7 (13.1) 11.9 (11.4) 9.2 (9.5) 5.5 (5.3) 3.4 (3.1) 3.3 (2.3) 4.1 (2.7) 5.2 (3.2) 7.0 (4.5) 45.1 (19.7) 64.3 (20.6)

110.9 (116.2) 103.5 (102.6) 97.4 (102.2) 95.0 (102.3) 90.2 (96.1) 82.2 (86.6) 75.3 (80.9) 59.5 (60.8) 43.8 (44.7) 34.1 (30.9) 25.0 (23.6) 15.6 (15.1) 6.9 (6.4) 3.0 (2.5) 2.4 (1.6) 2.5 (1.6) 2.8 (1.7) 2.8 (1.2) 5.0 (1.9) 6.7 (4.7)

and 5.2 IU/L, two patients baseline concentrations for hCGb of 2.8 and 2.5 pmol/L, and one patient a baseline concentration for hCGa of 34.2 pmol/L. In one patient (number 5 in Table 3) the serum concentrations of hCG, hCGb, and hCGa were measured before and after induction of spinal anesthesia and infusion of 1 L of saline. The infusion lowered the serum concentrations by ;22%. When the postinfusion concentrations were used as initial values, the calculated half-times increased, but the median effect was small (2.2%, range 0 –24%). The mean concentrations of hCG, hCGb, and hCGa on day 21 after delivery were 2.6 IU/L (7.6 pmol/L) (range 0.7– 4.3 IU/L), 3.2 pmol/L (range 1.2–5.1 pmol/L), and 16 pmol/L (range 9.3–25.0 pmol/L), respectively. hCGb had the slowest total elimination rate, and decreased to a mean 6 SD concentration of 1.3% 6 1.0% of the initial value after 14 days and 0.9% 6 0.8% after 21 days. The hCG concentration decreased to 0.05% 6 0.02% of the initial value at 14 days and 0.02% 6 0.01% within 21 days. hCGa reached a concentration of 0.07% 6 0.03% within 14 days, decreasing only slightly thereafter to 0.05% 6 0.03% of the initial value on day 21 (Fig. 4).

The contributions of the various components to the disappearance curves were calculated on the basis of AUCs. For hCG, the fastest component represented 13.8% of all hCG and two slower ones were 68.8% and 17.4%, respectively. For hCGb, the corresponding numbers were 3.1%, 78.1%, and 18.9%, and for hCGa they were 23.9%, 59.7%, and 16.4%, respectively (Table 4). hCG contained much more of the most rapid component than hCGb. Therefore, the total disappearance of hCGb was clearly slower than that of hCG (Fig. 4), and after a transient decrease during the first hour after delivery, the ratio of hCGb to hCG increased gradually from 0.8% (range 0.6 –1.0%) before delivery to 15.8% (range 7.7–28.3%) after 14 days and 26.7% (range 20.6 –36.9%) after 21 days (Fig. 5). hCGa had the highest proportion of the rapid component and the lowest proportion of the slow component, explaining its most rapid total disappearance (Table 4 and Fig. 4). The mean ratio of hCGa to hCG before delivery was 36.9% (range 15.3–78.5%). Initially the ratio decreased rapidly with a nadir of 3.3% (range 1.4 –7.9%) on day 3, but thereafter it increased to 64.3% (range 42.5– 83.5%) 21 days after delivery (Fig. 6). This resulted from a constant

Table 2. Significance of the fitted variances of triphasic vs biphasic half-time curves of hCG, hCGb, and hCGa. P-valuea Form of hCG

hCG hCGb hCGa a

Triphasic vs observed

Biphasic vs observed

Triphasic vs biphasic

0.9138 0.8531 0.7063

,0.0001 ,0.0001 ,0.0001

,0.0001 ,0.0001 ,0.0001

Comparisons by analysis of variance are significant when the corresponding P is ,0.0167.


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Fig. 2. Observed disappearance of hCGb (mean 1 SD) and estimated triphasic exponential half-times of hCGb and their predicted overall decay curve.

background concentration of hCGa with decreasing hCG concentrations.

Discussion With the use of highly sensitive and specific immunofluorometric assays, we found in this study that the disappearance of endogenous hCGb from plasma after delivery is much slower than that observed in studies with the use of RIA and purified hCGb infused intravenously [18, 19]. Various possible explanations exist for this. Chemical differences, mainly in the carbohydrate side chains [24 – 26], have been shown to exist between circulating hCGb and that purified from pregnancy urine, which has been used for injection [18, 19]. The experimental setting in the present study is not directly comparable with that after infusion of purified hCGb; i.e., the half-time was mea-

sured for hCGb over a course of 3 weeks, whereas in earlier reports the time span has been only hours or a few days. However, for comparison, we also calculated halftimes for shorter time frames that were comparable with those in earlier studies. This did not affect the results substantially. After pregnancy, some release of small amounts of hCGb (and hCG) sequestered in tissues could cause an apparent increase in half-time. Another possibility is that trophoblastic cells remaining in the body continue to produce hCGb and very little or no hCG. Trophoblasts persist in the lungs for extended periods of time after pregnancy [27], but the mass of these cells is very small in comparison with that of the placenta. Therefore, any production of hCG and hCGb by persisting trophoblastic cells would not contribute significantly to the serum concentrations observed the first week after

Fig. 3. Observed disappearance of hCGa (mean 1 SD) and estimated triphasic exponential half-times of hCGa and their algebraically summed curve.

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Table 3. Individual baselines and triphasic half-times (h) of hCG, hCGb, and hCGa after delivery. Half-times, h Base

Rapid (I)

Medium (II)

2.90 2.90 2.35 0.21 0.66 1.22 1.71 1.17 1.79

2.08 4.88 3.71 3.46 2.04 6.17 3.73 1.61 3.58

17.26 18.64 19.16 16.66 12.82 25.32 18.31 4.10 17.95

50.88 48.72 64.10 55.02 38.11 60.86 52.95 9.31 52.95

hCGb, pmol/L Patient 1 Patient 2a Patient 3 Patient 4 Patient 5 Patient 6 Mean SD Median

1.60 1.59 1.60 0.65 0.00 0.93 1.06 0.66 1.26

1.12 0.79 1.08 0.93 0.80 1.25 0.99 0.19 1.01

23.50 26.83 23.26 21.81 22.26 24.33 23.67 1.79 23.38

191.56 196.60 134.78 214.86 462.12 102.64 217.09 127.27 194.08

hCGa, pmol/L Patient 1 Patient 2a Patient 3 Patient 4 Patient 5 Patient 6 Mean SD Median

5.01 25.61 9.35 31.00 10.88 22.96 17.47 10.43 16.92

0.70 0.54 0.85 0.35 0.63 0.62 0.62 0.17 0.63

8.79 4.74 8.49 3.28 6.64 5.67 6.27 2.15 6.16

126.26 21.38 100.12 15.25 17.69 22.45 50.53 49.31 21.92

hCG, IU Patient 1 Patient 2a Patient 3 Patient 4 Patient 5 Patient 6 Mean SD Median

a

Slow (III)

Patient with diabetes mellitus (white F).

delivery, when the difference in half-times was already apparent in an increasing ratio of hCGb to hCG (Fig. 5). Dissociation into subunits of the hCG remaining in circulation could also affect the estimated half-time of hCGb. This mechanism could be important if the disappearance of hCGb were more rapid than that of hCG, but the opposite was actually true. Intact hCG is quite stable, whereas nicked hCG dissociates more rapidly. Especially in trophoblastic disease [10, 28], nicking may increase the dissociation of hCG into subunits, which may contribute to a high ratio of hCGb to hCG. Long incubation times during the assay of hCGb can cause dissociation of hCG, thus increasing the apparent concentration of hCGb in the sample. However, with the incubation times used in the present assay for hCGb, this effect is negligible [3]. Although all these mechanisms could increase the halftime of hCGb, they probably do not explain why hCGb in all subjects studied disappeared substantially more slowly than hCG. Therefore, other explanations need to be considered. The most likely explanation for the longer half-time of hCGb (vs intact hCG) is that hCGb circulating in plasma differs from that isolated by dissociation of urinary hCG into subunits. hCG in urine is known to be less glycosylated than that in serum [24]. The carbohydrate composition, and especially the presence of terminal sialic acid, is known to affect the in vivo half-time of hCG [26]. Furthermore, the b-chain of both hCG and hCGb in crude urinary hCG preparations has been found to be partially cleaved or nicked between residues 47 and 48 [25, 29]. In addition, under the potentially harsh chemical conditions required to dissociate the subunits, denaturation and appearance of components with shortened half-times could occur. The hCG heterodimer is unusual in that it is held together by a loop of the b-chain embracing the

Fig. 4. Disappearance of hCG, hCGb, and hCGa after delivery. The values are given as the proportion (mean 1 SD) of the concentration before delivery. The SD bars not visible are too small to be seen at this scale.

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Table 4. Proportions of the various components of hCG, hCGa, and hCGb estimated on the basis of the AUC of each component. AUCa Form of hCG

hCG hCGb hCGa a

Rapid (I)

Medium (II)

Slow (III)

Total

0.48 (13.8) 0.14 (3.1) 0.13 (23.9)

2.41 (68.8) 3.45 (78.3) 0.32 (59.7)

0.61 (17.4) 0.82 (18.6) 0.09 (16.4)

3.50 (100) 4.41 (100) 0.54 (100)

Given in parentheses is the percent of the total.

a-chain [30]. Disrupting the dimer in vitro might therefore change the structure of hCGb in comparison with the circulating form, which probably never has been involved in heterodimer formation. Free hCGa in serum does not reassociate with hCGb [31, 32]. An increased ratio of hCGb to hCG, in most studies

.10%, has been observed in trophoblastic cancer, and this has been used to differentiate between malignant and benign trophoblastic disease [7–9, 33]. In the present study, ratios .10% were observed in 5 of the 6 patients studied 14 days after delivery, and in all 4 patients studied 21 days after delivery. If the metabolism of hCG

Fig. 5. Changes in the ratio of hCGb to total hCG (mean 1 SD) after delivery. The values are given as percentages.

Fig. 6. The ratio of hCGa to total hCG (mean 1 SD) after delivery. The values are given as percentages.

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and hCGb is similar in patients with trophoblastic disease, our findings suggest that the ratio of hCGb to hCG must be evaluated with caution in samples taken several days after initiation of therapy. An increased ratio of hCGb to hCG has actually been observed several weeks after treatment of trophoblastic disease [34, 35]. The two most rapid components of hCG had half-times similar to those observed for hCG injected into humans, i.e., 3.6 and 18 h as compared with 5 and 24 –36 h, respectively [14 –16]. In an earlier study three components with half-times of 15, 27, and 168 h were estimated for hCG after abortion [36]. The two latter half-times support our calculations about the third component with the half-time of several days (median 53 h for hCG). This component represented only 17% of total hCG. Therefore, it may not be detectable after injection unless a large amount of hCG is injected, or an ultrasensitive assay method is used, or very prolonged observations are carried out. However, it is possible that this component represents hCG produced by residual, gradually dying trophoblasts or that is less abundant in urinary hCG than in plasma. The half-times of the two most rapid components of disappearance of hCG, hCGb, and hCGa were similar in all the patients studied, but there was more individual variation in the half-times of the longest component, i.e., from 38 to 64 h for hCG, 103 to 462 h for hCGb, and 15 to 126 h for hCGa. Renal clearance accounts for 20% of the total disposal of hCG after injection of purified hCG preparations [19]. The slightly impaired kidney function in our patient with diabetes mellitus (patient 2 in Table 3) seemed to have no effect on the half-times of hCG and its subunits. The algorithm used for calculation of half-times in the present study was based on the principles described in the EXPFIT program [23]. A two-component model has been used in most earlier studies to calculate disappearance half-times of hCG and its subunits. However, the fit of a two-component model was unsatisfactory for hCG, hCGb, and hCGa, whereas a three-component model with baseline yielded a statistically preferred fit. In contrast to the two-component model, three exponentials with baseline provided a fit for which predicted values could not be distinguished from the observed values by analysis of variance. The baseline concentrations obtained with the algorithm were in most cases well within the range of the reference values for nonpregnant premenopausal women (Table 3) [21]. However, when the follow-up time is insufficient for analysis of the baseline, it can be restricted in the algorithm. In conclusion, we have developed a three-component exponential model with a baseline for calculation of half-times of hCG and its subunits. Disappearance of endogenous hCGb from plasma after delivery is slower than previously observed, and the ratios of hCGb or hCGa to intact hCG vary as a function of postpartum time. If the metabolism of hCG and hCGb is similar in

patients with trophoblastic disease, the ratio of hCGb to hCG must be evaluated with caution in samples taken several days after initiation of therapy. Ratios of hCGb to hCG .10%, which are indicative for chorionic cancer, were observed in all patients 21 days after delivery. However, this needs to be evaluated in patients with trophoblastic disease. Additional studies will also reveal whether the half-times are similar in early pregnancy and whether this can be used to diagnose pregnancy-related disorders.

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