A New Method Simplifying Collection of Serial Specimens for Gonadotropin Determinations JAMES W. HANSEN AND GRIFF T. ROSS National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland ABSTRACT. Patterns of gonadotropin levels have been compared in specimens of serum, 24-h urine collections, and first morning urine voidings collected throughout a menstrual cycle from 8 sexually mature, young women. Quantities of gonadotropins in first morning voidings were found to provide a reliable estimate of 24-h excretion of these hor-
mones and demonstrate the same patterns of change as do serum and 24-h urine specimens. Thus first morning voidings satisfactorily overcome logistical problems inherent in serial serum and 24-h urine sampling for studying gonadotropin excretion patterns. (J Clin Endocrinol Metab 41: 241, 1975)
F
IRST morning voiding urine specimens (FMV) are easily collected without discomfort by any toilet-trained patient and can be stored with little inconvenience until delivered periodically to the physician or laboratory. This study was undertaken to determine whether or not gonadotropin levels in such specimens estimate 24-h urine levels reliably and thus provide information similar to that derived from studies of serum of 24-h urine collections which are intrinsically more difficult to obtain, especially serially over several weeks. Materials and Methods Eight normal young women 18 to 22 yr of age with regular menstrual cycles, no evidence of endocrine disease, and who had never taken birth control pills volunteered for study. Specimens of blood and urine were obtained daily between two consecutive episodes of genital bleeding in each subject. The blood was collected each morning and the first morning voiding collected and kept separate from urine voided throughout the remainder of the day. Using a 50 ml aliquot of each urine specimen, the pH was adjusted to 4.5 with acetic acid and the gonadotropins precipitated by adding 100 ml of acetone. After discarding the supernatant, the precipitate was resuspended in 10 ml radioimmunoassay buffer which dissolved the gonadotropins (1). An aliquot of the resultant Received December 2, 1974.
solution was diluted and assayed for LH and FSH by radioimmunoassays using antibodies generated to the intact pituitary hormones. Tests of these antisera with isolated a and /3 subunits of hCG (human chorionic gonadotropin) revealed no significant cross reaction. Since purified subunits of FSH were not available to us, the possibility of antigenic recognition of human FSH /3 subunits cannot be excluded. All specimens from a single subject (serum, first morning voiding, and urine from the remainder of the day for each day) were assayed in the same radioimmunoassay using IRP-2-HMG as the reference preparation for dose interpolation performed by computer analysis (2). Urine creatinine was measured (3) and the value divided into urine hormone estimates to reduce collection errors resulting from variable intake and output and from variable time periods of collection. First morning voiding hormone levels (Ham) are obtained by dividing the gonadotropin concentration by the creatinine concentration. Daily 24-h urine levels (H24) are calculated from the volumes (V), hormone concentrations (H), and creatinine concentrations (Cr) in FMV and urine from the remainder of the day (RD): (H F M V ) (V FMV ) + ( H R P ) (V RD ) H04 =
(Cr F M V ) (V F MV) + (Cr R D ) (V HD )
Serum levels are measured directly and expressed as mlU/ml. Patterns of change in serum, first morning voidings, and 24-h urine gonadotropin concentration were evaluated by visual inspection. The reliability of measurements in FMV as an estimate of levels in 24-h urines was evaluated by
241
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JCE & M • 1975 Vol 41 • No 2
HANSEN AND ROSS
242 3000 LUTEINIZING HORMONE I.U PER GRAM CREATININE 1000
FIG. 1. Correlation between luteinizing hormone values from first morning voiding (FMV) and 24-h urine specimens collected throughout a menstrual cycle in each of 8 young women. The large tolerance ellipse encompasses 95% of the data and the small ellipse is the 95% confidence profile of the mean. The broken line is the line of identity between FMV and 24-h values.
300
|
100
30
10
10
correlation and linear regression analysis (4). To achieve more homogeneity of variance, the data was transformed logarithmically. Results The correlations between FMV and 24-h urine gonadotropin/creatinine values are 000 -
3000
1000
100 300 24-HOUR URINE
30
shown in Fig. 1 (LH) and 2 (FSH). The respective correlation coefficients are 0.84 (n = 199) and 0.87 (n = 199) both with P < 0.001. The slopes of the regression lines are 0.91 for LH and 0.99 for FSH. The lines of identity shown in Figs. 1 and 2 pass through the 95% confidence ellipse for the
FOLLICLE STIMULATING HORMOM" I.U. PER GRAM CREATININE -
^
300
?' FIG. 2. Correlation between follicle-stimulating hormone values from first morning voiding (FMV) and 24-h urine specimens collected throughout a menstrual cycle in each of 8 young women. The large tolerance ellipse encompasses 95% of the data and the small ellipse is the 95% confidence profile of the mean. The broken line is the line of identity between FMV and 24-h values.
100 /
30
10
/
sJ»
- .•
/
3
1 a
i
i
30
i
100
i
1
300
1000
24-HOUR URINE
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GONADOTROPINS IN FIRST MORNING URINES FOLLICLE 20.0 ' STIMULATING HORMONE
LUTE1NIZING HORMONE
20.0
243
• • •
•
• • • •
•
*
FMV
FlG. 3. Correlations of first morning voiding (FMV) and serum hormone values for luteinizing hormone and follicle-stimulating hormone throughout a menstrual cycle in each of 8 young women.
•
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2.0
. i f ! SOLS i «.~/ .
•
•
.2
••
•
• •
• •
#
I
3
10
30
100
•
10
20
40
SERUM
mean of FMV and 24-h urine hormone data indicating that the means of measurements of a gonadotropin using either FMV or 24-h urine specimens are indistinguishable at the P = 0.05 level. The 95% tolerance ellipses shown in the figures encompass 95% of the population of data points. The FMV hormone/creatinine ratio is compared to serum gonadotropin levels in Fig. 3. The correlation coefficient for LH is 0.54 (P < 0.001) and for FSH is 0.71 (P < 0.001). The patterns of gonadotropin levels in FMV, 24-h urine, and serum specimens throughout a typical menstrual cycle are compared in Fig. 4; similar patterns were seen in 6 of our 8 subjects. One of our subjects had a short (8 day) luteal phase; her patterns are compared in Fig. 5. Our other subject had an atypical cycle with no clear midcycle hormone surge, no welldefined luteal phase, and a 45-day interval between consecutive episodes of genital bleeding despite a history of regular 30-day cycles previously.
200 SERUM
100
0L 12
JSJL
FMV
8
g
4 24-HOUR
JU. 9
10
Discussion
20 30 DAYS
40
The patterns of gonadotropin levels FlG. 4. Comparison of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) patterns in serum, throughout a typical menstrual cycle in first morning voidings (FMV), and 24-h urine specisexually mature young women do not ap- mens throughout a menstrual cycle in a normal pear to differ significantly whether meas- woman.
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JCE & M • 1975 Vol 41 • No 2
HANSEN AND ROSS
244
20 DAYS FIG. 5. Comparison of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) patterns in serum, first morning voidings (FMV), and 24-h urine specimens throughout a menstrual cycle in a woman with a short luteal phase.
urements are made in serum, 24-h urine, or first morning voiding specimens. The midcycle surges are within one day of each other indicating that first morning voidings are as reliable as either serum or 24-h urine specimens in estimating the time of the midcycle LH surge. It is of interest that the LH surges appear to persist slightly longer in urine than in serum, perhaps reflecting the integrative function inherent in urine collections. In 4 of our subjects a small LH peak was noted about 5 days prior to the midcycle LH surge; the possible physiologic significance of this remains to be elucidated. The fortuitous occurrence of a short luteal phase in 1 subject afforded an opportunity to compare gonadotropin patterns in the various specimens in this patho-
physiologic state. Again, the patterns were similar regardless of the type of specimen used in this study. Thus in at least this one atypical state, the first morning voidings appeared to provide a reliable indicator of gonadotropin secretion pattern. The validity of first morning voidings for evaluating gonadotropin patterns is supported by the high correlation between levels in the various types of specimens. Furthermore, it also appears that levels in first morning voidings give a reliable estimate of levels in 24-h urine specimens. This is shown by the nearness to unity of the slopes in first morning voiding vs 24-h urine data, 0.90 and 0.99 for LH and FSH respectively, and by the fact that the line of identity passes through the 95% confidence ellipse encompassing the mean of the data. Thus, measurements of gonadotropin and creatinine in a single first morning voiding provide a reliable estimate of total 24-h urine hormone excretion. From these studies we conclude that in normal adult menstrual cycles, patterns of gonadotropin excretion in first morning voidings correspond to patterns in serum and 24-h urine specimens. The ratio of gonadotropin to creatinine in the first morning voiding also satisfactorily estimates 24-h urinary gonadotropin excretion. Thus for studies requiring serial gonadotropin measurements,firstmorning voidings provide a satisfactory alternative to either serial serum or 24-h urine specimens in adult women. References 1. Reiter, E. O., H. E. Kulin, and S. M. HamwoodJ Clin Endocrinol Metab 36: 661, 1973. 2. Rodbard, D. A., Clin Chem 20: 1255, 1974. 3. Chasson, A. L., H. J. Grady, and M. A. Stanley, AmJ Clin Pathol 35: 86, 1961. 4. Diem, K., Documenta Geigy, Scientific Tables, ed. 6, 1962, Geigy Pharmaceuticals, Ardsley, New York, 1962, pp. 145-198.
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mones and demonstrate the same patterns of change as do serum and 24-h urine specimens. Thus first morning voidings satisfactorily overcome logistical problems inherent in serial serum and 24-h urine sampling for studying gonadotropin excretion patterns. (J Clin Endocrinol Metab 41: 241, 1975)
F
IRST morning voiding urine specimens (FMV) are easily collected without discomfort by any toilet-trained patient and can be stored with little inconvenience until delivered periodically to the physician or laboratory. This study was undertaken to determine whether or not gonadotropin levels in such specimens estimate 24-h urine levels reliably and thus provide information similar to that derived from studies of serum of 24-h urine collections which are intrinsically more difficult to obtain, especially serially over several weeks. Materials and Methods Eight normal young women 18 to 22 yr of age with regular menstrual cycles, no evidence of endocrine disease, and who had never taken birth control pills volunteered for study. Specimens of blood and urine were obtained daily between two consecutive episodes of genital bleeding in each subject. The blood was collected each morning and the first morning voiding collected and kept separate from urine voided throughout the remainder of the day. Using a 50 ml aliquot of each urine specimen, the pH was adjusted to 4.5 with acetic acid and the gonadotropins precipitated by adding 100 ml of acetone. After discarding the supernatant, the precipitate was resuspended in 10 ml radioimmunoassay buffer which dissolved the gonadotropins (1). An aliquot of the resultant Received December 2, 1974.
solution was diluted and assayed for LH and FSH by radioimmunoassays using antibodies generated to the intact pituitary hormones. Tests of these antisera with isolated a and /3 subunits of hCG (human chorionic gonadotropin) revealed no significant cross reaction. Since purified subunits of FSH were not available to us, the possibility of antigenic recognition of human FSH /3 subunits cannot be excluded. All specimens from a single subject (serum, first morning voiding, and urine from the remainder of the day for each day) were assayed in the same radioimmunoassay using IRP-2-HMG as the reference preparation for dose interpolation performed by computer analysis (2). Urine creatinine was measured (3) and the value divided into urine hormone estimates to reduce collection errors resulting from variable intake and output and from variable time periods of collection. First morning voiding hormone levels (Ham) are obtained by dividing the gonadotropin concentration by the creatinine concentration. Daily 24-h urine levels (H24) are calculated from the volumes (V), hormone concentrations (H), and creatinine concentrations (Cr) in FMV and urine from the remainder of the day (RD): (H F M V ) (V FMV ) + ( H R P ) (V RD ) H04 =
(Cr F M V ) (V F MV) + (Cr R D ) (V HD )
Serum levels are measured directly and expressed as mlU/ml. Patterns of change in serum, first morning voidings, and 24-h urine gonadotropin concentration were evaluated by visual inspection. The reliability of measurements in FMV as an estimate of levels in 24-h urines was evaluated by
241
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JCE & M • 1975 Vol 41 • No 2
HANSEN AND ROSS
242 3000 LUTEINIZING HORMONE I.U PER GRAM CREATININE 1000
FIG. 1. Correlation between luteinizing hormone values from first morning voiding (FMV) and 24-h urine specimens collected throughout a menstrual cycle in each of 8 young women. The large tolerance ellipse encompasses 95% of the data and the small ellipse is the 95% confidence profile of the mean. The broken line is the line of identity between FMV and 24-h values.
300
|
100
30
10
10
correlation and linear regression analysis (4). To achieve more homogeneity of variance, the data was transformed logarithmically. Results The correlations between FMV and 24-h urine gonadotropin/creatinine values are 000 -
3000
1000
100 300 24-HOUR URINE
30
shown in Fig. 1 (LH) and 2 (FSH). The respective correlation coefficients are 0.84 (n = 199) and 0.87 (n = 199) both with P < 0.001. The slopes of the regression lines are 0.91 for LH and 0.99 for FSH. The lines of identity shown in Figs. 1 and 2 pass through the 95% confidence ellipse for the
FOLLICLE STIMULATING HORMOM" I.U. PER GRAM CREATININE -
^
300
?' FIG. 2. Correlation between follicle-stimulating hormone values from first morning voiding (FMV) and 24-h urine specimens collected throughout a menstrual cycle in each of 8 young women. The large tolerance ellipse encompasses 95% of the data and the small ellipse is the 95% confidence profile of the mean. The broken line is the line of identity between FMV and 24-h values.
100 /
30
10
/
sJ»
- .•
/
3
1 a
i
i
30
i
100
i
1
300
1000
24-HOUR URINE
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GONADOTROPINS IN FIRST MORNING URINES FOLLICLE 20.0 ' STIMULATING HORMONE
LUTE1NIZING HORMONE
20.0
243
• • •
•
• • • •
•
*
FMV
FlG. 3. Correlations of first morning voiding (FMV) and serum hormone values for luteinizing hormone and follicle-stimulating hormone throughout a menstrual cycle in each of 8 young women.
•
.?#-.•
Hi 2.0 9
2.0
. i f ! SOLS i «.~/ .
•
•
.2
••
•
• •
• •
#
I
3
10
30
100
•
10
20
40
SERUM
mean of FMV and 24-h urine hormone data indicating that the means of measurements of a gonadotropin using either FMV or 24-h urine specimens are indistinguishable at the P = 0.05 level. The 95% tolerance ellipses shown in the figures encompass 95% of the population of data points. The FMV hormone/creatinine ratio is compared to serum gonadotropin levels in Fig. 3. The correlation coefficient for LH is 0.54 (P < 0.001) and for FSH is 0.71 (P < 0.001). The patterns of gonadotropin levels in FMV, 24-h urine, and serum specimens throughout a typical menstrual cycle are compared in Fig. 4; similar patterns were seen in 6 of our 8 subjects. One of our subjects had a short (8 day) luteal phase; her patterns are compared in Fig. 5. Our other subject had an atypical cycle with no clear midcycle hormone surge, no welldefined luteal phase, and a 45-day interval between consecutive episodes of genital bleeding despite a history of regular 30-day cycles previously.
200 SERUM
100
0L 12
JSJL
FMV
8
g
4 24-HOUR
JU. 9
10
Discussion
20 30 DAYS
40
The patterns of gonadotropin levels FlG. 4. Comparison of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) patterns in serum, throughout a typical menstrual cycle in first morning voidings (FMV), and 24-h urine specisexually mature young women do not ap- mens throughout a menstrual cycle in a normal pear to differ significantly whether meas- woman.
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JCE & M • 1975 Vol 41 • No 2
HANSEN AND ROSS
244
20 DAYS FIG. 5. Comparison of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) patterns in serum, first morning voidings (FMV), and 24-h urine specimens throughout a menstrual cycle in a woman with a short luteal phase.
urements are made in serum, 24-h urine, or first morning voiding specimens. The midcycle surges are within one day of each other indicating that first morning voidings are as reliable as either serum or 24-h urine specimens in estimating the time of the midcycle LH surge. It is of interest that the LH surges appear to persist slightly longer in urine than in serum, perhaps reflecting the integrative function inherent in urine collections. In 4 of our subjects a small LH peak was noted about 5 days prior to the midcycle LH surge; the possible physiologic significance of this remains to be elucidated. The fortuitous occurrence of a short luteal phase in 1 subject afforded an opportunity to compare gonadotropin patterns in the various specimens in this patho-
physiologic state. Again, the patterns were similar regardless of the type of specimen used in this study. Thus in at least this one atypical state, the first morning voidings appeared to provide a reliable indicator of gonadotropin secretion pattern. The validity of first morning voidings for evaluating gonadotropin patterns is supported by the high correlation between levels in the various types of specimens. Furthermore, it also appears that levels in first morning voidings give a reliable estimate of levels in 24-h urine specimens. This is shown by the nearness to unity of the slopes in first morning voiding vs 24-h urine data, 0.90 and 0.99 for LH and FSH respectively, and by the fact that the line of identity passes through the 95% confidence ellipse encompassing the mean of the data. Thus, measurements of gonadotropin and creatinine in a single first morning voiding provide a reliable estimate of total 24-h urine hormone excretion. From these studies we conclude that in normal adult menstrual cycles, patterns of gonadotropin excretion in first morning voidings correspond to patterns in serum and 24-h urine specimens. The ratio of gonadotropin to creatinine in the first morning voiding also satisfactorily estimates 24-h urinary gonadotropin excretion. Thus for studies requiring serial gonadotropin measurements,firstmorning voidings provide a satisfactory alternative to either serial serum or 24-h urine specimens in adult women. References 1. Reiter, E. O., H. E. Kulin, and S. M. HamwoodJ Clin Endocrinol Metab 36: 661, 1973. 2. Rodbard, D. A., Clin Chem 20: 1255, 1974. 3. Chasson, A. L., H. J. Grady, and M. A. Stanley, AmJ Clin Pathol 35: 86, 1961. 4. Diem, K., Documenta Geigy, Scientific Tables, ed. 6, 1962, Geigy Pharmaceuticals, Ardsley, New York, 1962, pp. 145-198.
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