J Clin Endocrinol Metab 41: 638, 1975
IMMUNOREACTIVE SOMATOMEDIN B IN URINE Rosalyn S. Yalow, Kerstin Hall, and Rolf Luft Solomon A. Berson Research Laboratory, Veterans Administration Hospital, Bronx, N.Y. 10468; Mt. Sinai School of Medicine, CUNY, 10029; and Department of Endocrinology and Metabolism, Karolinska Sjukhuset, Stockholm, Sweden. ABSTRACT. Urinary excretion by 8 normal adult subjects of immunoreactive somatomedin B was 27.6 + 4.4 yg between 1000 h and 1400 h compared to a mean plasma concentration at
1200 h of 5.9 + 0.9 yg/ml. Free somatomedin B in urine averaged 85.9$, although in the plasma of the same subjects all but less than 5% was bound to serum proteins.
Until now the presence of somatomedin in urine has not been reported. Immunoreactive somatomedin B in unextracted human plasma is associated with a serum protein at least as large as y-globulin (1) and it is unlikely that in subjects without proteinuria there is significant urinary excretion of this protein. It therefore was of interest to determine the rate of urinary excretion of immunoreactive somatomedin B and whether the excreted peptide was free or bound to a larger molecular weight protein.
was generally greater than
MATERIALS AND METHODS Somatomedin B was determined by radioimmunoassay (RIA) with only minor modifications of methods previously described (1). Plasma is assayed at a dilution of 1:20000 or greater and urine at a dilution of 1:250 or greater. For RIA the standard diluent is k% normal dog plasma in 0.02 M barbital buffer, guinea pig antiserum 1019-5 is used at a dilution of 1:7000, the incubation time is 6 days at 4°C and separation of bound and free 125lsomatomedin is effected by using 5 ing of uncoated charcoal. Fractionation of somatcmedin B in 5 to 50 yl plasma and urine was carried out at 4°C on Sephadex G-50 fine columns (1 x 50 cm). Marker molecules (131l-albumin and Na 131i) were added to all samples before application to the column. Recovery of immunoreactivity from the columns
Eight adult subjects, 5 male and 3 female, ranging in age from 27 to
54 years with no known abnormalities in GH secretion were studied. For each subject all urine was collected for 4 hours, generally from 1000 h to 1400 h, and heparinized blood was drawn about noon. Because the assay sensitivity permitted measurement of less than 25 pg somatomedin B/ml, plasma and urine samples were diluted 1:1000 or 1:10 respectively in standard diluent prior to addition to the incubation tubes. RESULTS The plasma and urinary levels of immunoreactive somatomedin B are given in Table 1. The mean plasma concentration at about noon In these 8 subjects was 5.9 + 0 . 9 (SEM) yg/ml. Mean urinary excretion was 27.6 + 4.4 (SEM) yg for the 4 hour period. In agreement with our earlier studies (1) somatomedin B in unextracted plasma eluted in the void volume on Sephadex G-50 filtration (Fig. 1 ) . If free somatomedin were 5$ or more of the total Immunoreactivity, it would have been detected in these studies. In contrast, in urine more than 85/5 of the Immunoreactive somatomedin B was not protein-bound (Fig. 1, Table 1).
Submitted June 12, 1975 638
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 12 November 2015. at 12:37 For personal use only. No other uses without permission. . All rights reserved.
RAPID COMMUNICATIONS Table 1: SUBJECT
Plasma and Urinary Somatomedin B i n Adult Subjects PLASMA CONC.
URINE
2.2
C.B. W.O. B.S. S.H. N.W. E.S. L.G. R.Y.
2.3 4.5 6.5 6.5 7.5 8.5 9-0
CONC. yg/ml
VOL. ml
0.230 0.213 0.158 0.110 0.105 0.080 0.080 0.158
200 200 80 150
185 280 320 225
5.9+0.9 30 PLASMA
CPM 100
88-
5,
URINE
0
20
639
40
60
80
100
PERCENT OF ELUTION VOLUME BETWEEN I3I I-ALBUMIN AND I 3 I I "
Pig. 1 - Sephadex G-50 gel filtration of Inmunoreactive somatomedin B in plasma and urine of subject R.Y.
TOTAL yg/TThrs
46.0 42.6 12.6 16.5 19.4 22.4
25.6 35.6 27T5+4.4
% FREE
84.8 85.7 84.5 84.9 85.3 89.5 89.7 82.5 B5T9+.9
The major fraction of urinary somatomedin B is in the free form, only about 1556 appearing to be protein-bound. Immunoreactive somatomedin in plasma is associated with a protein which is at least as large as Y-globulin and has an electrophoretic mobility on paper resembling the ctglobulins (1). Since it is unlikely that a serum protein of this molecular size is excreted in the urine of subjects without renal disease, further investigation is required to determine whether or not the urinary and plasma binding proteins are identical. There have been no reports evaluating whether the in vivo biologic activity of somatomedin parallels the total or the free somatomedin in plasma. Measurement of the daily urinary excretion of somatomedin, which may reflect the somatomedin not bound to plasma protein, may prove useful in such investigations. ACKNOWLEDGMENT
DISCUSSION The mean plasma concentration of somatomedin in this group of 8 adult subjects is somewhat lower than that earlier reported for 12 adult subjects in the basal state (9.8 +_ 1.5 yg/ml). Whether this represents diurnal variation or differences in the selection of the group was not resolved in this study.
The authors are greatly indebted to AB KABI, Stockholm for the somatomedin B used in these studies and to Mrs. Nancy Wu for technical assistance. REFERENCE: 1. Yalow, R. S., K. Hall, and R. Luft, J Clin Invest 55:127, 1975.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 12 November 2015. at 12:37 For personal use only. No other uses without permission. . All rights reserved.
IMMUNOREACTIVE SOMATOMEDIN B IN URINE Rosalyn S. Yalow, Kerstin Hall, and Rolf Luft Solomon A. Berson Research Laboratory, Veterans Administration Hospital, Bronx, N.Y. 10468; Mt. Sinai School of Medicine, CUNY, 10029; and Department of Endocrinology and Metabolism, Karolinska Sjukhuset, Stockholm, Sweden. ABSTRACT. Urinary excretion by 8 normal adult subjects of immunoreactive somatomedin B was 27.6 + 4.4 yg between 1000 h and 1400 h compared to a mean plasma concentration at
1200 h of 5.9 + 0.9 yg/ml. Free somatomedin B in urine averaged 85.9$, although in the plasma of the same subjects all but less than 5% was bound to serum proteins.
Until now the presence of somatomedin in urine has not been reported. Immunoreactive somatomedin B in unextracted human plasma is associated with a serum protein at least as large as y-globulin (1) and it is unlikely that in subjects without proteinuria there is significant urinary excretion of this protein. It therefore was of interest to determine the rate of urinary excretion of immunoreactive somatomedin B and whether the excreted peptide was free or bound to a larger molecular weight protein.
was generally greater than
MATERIALS AND METHODS Somatomedin B was determined by radioimmunoassay (RIA) with only minor modifications of methods previously described (1). Plasma is assayed at a dilution of 1:20000 or greater and urine at a dilution of 1:250 or greater. For RIA the standard diluent is k% normal dog plasma in 0.02 M barbital buffer, guinea pig antiserum 1019-5 is used at a dilution of 1:7000, the incubation time is 6 days at 4°C and separation of bound and free 125lsomatomedin is effected by using 5 ing of uncoated charcoal. Fractionation of somatcmedin B in 5 to 50 yl plasma and urine was carried out at 4°C on Sephadex G-50 fine columns (1 x 50 cm). Marker molecules (131l-albumin and Na 131i) were added to all samples before application to the column. Recovery of immunoreactivity from the columns
Eight adult subjects, 5 male and 3 female, ranging in age from 27 to
54 years with no known abnormalities in GH secretion were studied. For each subject all urine was collected for 4 hours, generally from 1000 h to 1400 h, and heparinized blood was drawn about noon. Because the assay sensitivity permitted measurement of less than 25 pg somatomedin B/ml, plasma and urine samples were diluted 1:1000 or 1:10 respectively in standard diluent prior to addition to the incubation tubes. RESULTS The plasma and urinary levels of immunoreactive somatomedin B are given in Table 1. The mean plasma concentration at about noon In these 8 subjects was 5.9 + 0 . 9 (SEM) yg/ml. Mean urinary excretion was 27.6 + 4.4 (SEM) yg for the 4 hour period. In agreement with our earlier studies (1) somatomedin B in unextracted plasma eluted in the void volume on Sephadex G-50 filtration (Fig. 1 ) . If free somatomedin were 5$ or more of the total Immunoreactivity, it would have been detected in these studies. In contrast, in urine more than 85/5 of the Immunoreactive somatomedin B was not protein-bound (Fig. 1, Table 1).
Submitted June 12, 1975 638
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 12 November 2015. at 12:37 For personal use only. No other uses without permission. . All rights reserved.
RAPID COMMUNICATIONS Table 1: SUBJECT
Plasma and Urinary Somatomedin B i n Adult Subjects PLASMA CONC.
URINE
2.2
C.B. W.O. B.S. S.H. N.W. E.S. L.G. R.Y.
2.3 4.5 6.5 6.5 7.5 8.5 9-0
CONC. yg/ml
VOL. ml
0.230 0.213 0.158 0.110 0.105 0.080 0.080 0.158
200 200 80 150
185 280 320 225
5.9+0.9 30 PLASMA
CPM 100
88-
5,
URINE
0
20
639
40
60
80
100
PERCENT OF ELUTION VOLUME BETWEEN I3I I-ALBUMIN AND I 3 I I "
Pig. 1 - Sephadex G-50 gel filtration of Inmunoreactive somatomedin B in plasma and urine of subject R.Y.
TOTAL yg/TThrs
46.0 42.6 12.6 16.5 19.4 22.4
25.6 35.6 27T5+4.4
% FREE
84.8 85.7 84.5 84.9 85.3 89.5 89.7 82.5 B5T9+.9
The major fraction of urinary somatomedin B is in the free form, only about 1556 appearing to be protein-bound. Immunoreactive somatomedin in plasma is associated with a protein which is at least as large as Y-globulin and has an electrophoretic mobility on paper resembling the ctglobulins (1). Since it is unlikely that a serum protein of this molecular size is excreted in the urine of subjects without renal disease, further investigation is required to determine whether or not the urinary and plasma binding proteins are identical. There have been no reports evaluating whether the in vivo biologic activity of somatomedin parallels the total or the free somatomedin in plasma. Measurement of the daily urinary excretion of somatomedin, which may reflect the somatomedin not bound to plasma protein, may prove useful in such investigations. ACKNOWLEDGMENT
DISCUSSION The mean plasma concentration of somatomedin in this group of 8 adult subjects is somewhat lower than that earlier reported for 12 adult subjects in the basal state (9.8 +_ 1.5 yg/ml). Whether this represents diurnal variation or differences in the selection of the group was not resolved in this study.
The authors are greatly indebted to AB KABI, Stockholm for the somatomedin B used in these studies and to Mrs. Nancy Wu for technical assistance. REFERENCE: 1. Yalow, R. S., K. Hall, and R. Luft, J Clin Invest 55:127, 1975.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 12 November 2015. at 12:37 For personal use only. No other uses without permission. . All rights reserved.
