Clinical Endocrinology (1977) 7,343-347 SHORT COMMUNICATION
T H E CHARACTERIZATION OF GROWTH HORMONE RELEASE INHIBITING HORMONE-LIKE IMMUNOREACTIVITY IN NORMAL URINE S . KRONHEIM, M. BERELOWITZ A N D B. L. PIMSTONE
Isotope and Immunoassay Laboratory, Department of Medicine, University of Cape Town Medical School, South Africa (Received I4 February 1977; revised 19 April 1977; accepted 20 April 1977)
SUMMARY. Using a previously described radioimmunoassay for growth hormone release inhibiting hormone (GH-RIH), the presence of GH-RIH-like immunoreactivity in urine has been characterized by demonstrating mobility identical to synthetic GH-RIH standard on two sephadex gel chromatographic systems, and parallelism of dilutions of the sephadex fractions with synthetic GH-RIH. Furthermore, 74% of the sephadex fraction cross-reacting in the immunoassay bound to antibody conjugated to sepharose and could be eluted by 1 M acetic acid. This immunospecific eluate showed identity with synthetic GH-RIH on both ion exchange and thin layer chromatography. Thus GH-RIH-like immunoreactivity is present in normal urine; this may have potential relevance in the search for a physiological role for this peptide. Apart from suppressing growth hormone release, the pharmacological administration of synthetic growth hormone release inhibiting hormone (GH-RIH) inhibits a wide range of endocrine and exocrine secretions from pancreas, gut and elsewhere (Plmstone et al., 1976). In addition, GH-RIH, or a peptide showing similar immunological identity, is widely distributed in CNS, gut, pancreas and thyroid (Hokfelt et al., 1975; Arimura el al., 1975; Kronheim et al., 1976) as well as being present in CSF (Pate1 et al., 1975; Kronheim et al., 1977). In this paper we characterize GH-RIH-like immunoreactivity found in the urine of normal subjects using our previously reported radioimmunoassay (Kronheim et al., 1976).
Methods and results Radioimmunoassay. The radioimmunoassay (RIA) of GH-RIHwas performed as previously described (Kronheim et al., 1976) using rabbit anti GH-RIH serum (final concentration 1 : 125,000), Tyr' GH-RIH radioiodinatedwith "'I by the Chloramine T technique, synthetic GH-RIH (Ayerst 249 10) as reference standard, and dextran-coated charcoal to separate antibody-bound 'I Tyr' GH-RIH from the free fraction. Studies were performed on early morning urine samples from twelve normal volunteers. The pH of all specimens was initially adjusted to 7.0. Correspondence: Professor B. L. Pimstone, Department of Medicine, Medical School, Observatory 1925, Cape Province, South Africa.
343
344
S. Kronheim, M. Berelowitz and B. L. Pimstone
In preliminary experiments the chemical integrity of '"I Tyr' GH-RIH after incubation with urine for 20 h at 4°C was established by paper chromatoelectrophoresis on three occasions. 87 f 3% (mean f SEM) of the label remained intact at the site of origin, similar to values found afterincubationin assay buffer (90 f 2% mean k SEM). Immunological integrity of the tracer after incubation with urine was tested on three occasions by immunoprecipitation using 100 pl of rabbit anti GH-RIH (final concentration 1 : 500) and dextran-coated charcoal to separate antibody bound from free tracer. 81.5 2.1% (mean SEM) of tracer was precipitable after incubation with urine, compared to 80.7 k 1.7% (mean f SEM) in assay buffer. The level of GH-RIH found by RIA of urine was 66 f 7 pg/mg creatinine (mean f SEM). Recovery experiments: 51 -100% (mean, 81%) recovery of GH-RIH was obtained from urine samples assayed before and after the addition of 0.5 ng synthetic GH-RIH on four occasions. Gel chromatography: 20 ml samples of urine were lyophilised, made up to 1 ml in 1 M acetic acid, centrifuged to remove any precipitate, and eluted off two sephadex (Pharmacia) systems with 1M acetic acid: (a) Sephadex GI0 in a column 1.6 X 65 cm at a flow rate of 30 ml/h and (b) Sephadex G25 in a column 2.5 X 90 cm at a flow rate of 37 ml/h. Columns were calibrated with synthetic GH-RIH (measured by RIA), dextran blue (void volume) and urea. A single peak of immunoreactivity showing identity with synthetic GH-RIH was eluted from both systems (Fig. 1). Serial dilutions of the immunoreactive peaks showed linearity and were parallel to the standard curve on logit-log plot (Fig. 2). Affinity chromatography: The specific antibody binding of GH-RIH eluted from the Sephadex G10 column was further studied on ten occasions using cyanogen bromide activated sepharose 4B (Pharmacia) linked to rabbit anti-GH-RIH or control columns as described elsewhere (Kronheim et ul., 1977). Unbound material was eluted by 10 mM phosphosaline buffer pH 7.6 (PBS) and antibody bound GH-RIH by 1 M acetic acid. 88.7 k 4.9%(mean +_
+_
+_
-z
-1 150
100 Elution volume (mll
LT
1 Oe3r
t
v0
OG2
I
200
250
I
I
300 350 400 Elution volume (rnl)
450
500
Fig. 1. Gel chromatography of concentrated urine on Sephadex G10 (upper) and G25f (lower) showing identity between the elution volume of synthetic GH-RIH (horizontal hatching) and urine immunoreactivity (vertical-hatching).Overlap of the two peaks indicated by cross-hatching in this Fig and in Figs 3 and 5. V, = void volume.
GH-RIH-likeimmunoreactivity in normal urine
-2
t
Synthetic GH-RIH(ng/ml) 0.25 06
0.125 I
I
I
1:8
1:4
l:z
1.0
I
1:1
Urine dilution
Fig. 2. Logit-log plot of serial dilutions of urine immunoreactivity eluted from Sephadex G I 0 ( 0 ) (rn = -2.71, n = 3) showing parallelism with synthetic GH-RIH standard ( 0 ) (rn = -2.59, n = 13). Means * SD are shown.
IPBS
1
Acetic acid
4.0
2.0
.
c E
0
3 4.0 2.0
r1
BH 10
20
30
40
Elution volume (ml)
Fig. 3. Elution profile of synthetic GH-RIH (horizontal hatching) and urine immunoreactivity eluted from Sephadex G-10 (vertical-hatching) when passed (a) through sepharose conjugated to non-immune rabbit serum control (upper) showing 89% of urine extract unbound and eluted by PBS,and (b) through sepharose-GH-RIH antibody conjugate (lower) on which 74% of urine fraction was bound and eluted off with acetic acid (see text).
345
S. Kronheim, M . Berelowitz and B. L. Pimstone
346
O*i"
O-i"
n
I
I (3
Elution volume (mll Fig. 4. Ion exchange chromatography (see text) of specific immunoreactive GH-RIH eluted from sepharose-antibody column. 90% of this immunoreactivity elutes with 0.2M ammonium acetate, coinciding with the pattern of synthetic GH-RIH standard. Bold line, GH-RIH; dotted line, urine. Arrows indicate ammonium acetate concentrations.
R, value Fig. 5. Thin layer chromatography (see text) of specific immunoreactive GH-RIH eluted from sepharose-antibody column. Note similar Rf values of synthetic GH-RIH (horizontal hatching, Rf, 0.6 1) and immunoreactive urinary GH-RIH (vertical-hatchingRf, 0.59).
SEM) of the immunoreactive GH-RIH was eluted from the control sepharose column with PBS, whilst 25.9 k 5.4% (mean k SEM) was similarly eluted from the antibody-sepharose column (Fig. 3). The residual GH-RIH (74.1%) remained bound to this column and was only eluted with 1~ acetic acid. This eluate was divided into two, freeze dried, and further characterized on ion exchange and thin layer chromatography. Ion exchange chromatography: 200 pl of eluted GH-RIH in 2 mM ammonium acetate buffer pH 4.65 was applied to a carboxymethyl cellulose (CM52 Whatman) column 5 X 1 cm prepared in the same buffer and pre-coated with 2% bovine serum albumin. Stepwise buffer gradients of 2 mM, 0.1M and 0.2M ammonium acetate pH 4.65 were used to elute, 10 X 5 ml samples being collected with each gradient. The samples were freeze dried and later reconstituted in assay buffer for assay of GH-RIH. In this system 100% of synthetic GH-RJH applied as a marker elutes with the 0.2M ammonium acetate as did 90% of the urinary CH-RIH separated by affinity chromatography (Fig. 4). Thin layer chromutogruphy: 25 pl of CH-RIH separated by affinity chromatography in 0.1M acetic acid was applied to 0.25 mm cellulose precoated plates (Eastman Kodak) and run in butanol :acetic acid :water (4 : 1 :5 ) for 4 h (Kronheim et al., 1977). Similar Rf values were obtained for synthetic GH-RIH (Rf, 0.61) and urinary GH-RIH immunoreactivity (Rf, 0.59) (Fig. 5 ) .
Discussion Using a sensitive and specific RIA we have demonstrated the presence of GH-RIH-like immunoreactivity in normal urine. That this does not merely represent damage to '1 Tyr' GH-
GH-RIH-like immunoreactivity in normal urine
347
RIH with consequent interference with binding in the assay system has been excluded by demonstrating chromatographic integrity of the label following incubation with urine. Furthermore, the degree of immunoprecipitation with excess antibody was identical after incubation with urine or assay buffer, although the high concentration of antibody used in this system may not necessarily be directly applicable to the assay situation in which antibody is used at a much greater dilution. Identical mobility of the urine immunoreactivity and synthetic GH-RIH on sephadex suggests that the two have similar molecular weights, whde parallelism of serial dilutions with synthetic standard also suggests a similar identity. However the material in urine causing displacement of tracer from antibody in our assay may not all be truly immunoreactive, as 26% failed to bind to antibody in our affinity system. Ion exchange and thin layer chromatography of the immunoreactive material eluted off tht antibody -sepharose column support its identity with synthetic GH-RIH. The characterization of GH-RIH-like immunoreactivity in urine may allow the study of physiological changes in GH-RIH secretion in man, although the possible presence of nonimmunoreactive material interfering with the assay could cause difficulty with interpretation.
Acknowledgments Synthetic somatostatin was a kind gift of Dr E. Polakow, Ayerst Laboratories (Pty) Limited, Johannesburg, South Africa. Financial support for this study was obtained from the South African Medical Research Council and Atomic Energy Board, International Atomic Energy Agency Grant No. 1806/RB, University of Cape Town Staff Research Fund, Harry Crossley Foundation and Nellie Atkinson Bequest. References ARIMURA, A., SATO, H., DUPONT, A., NISHI, N. & SCHALLY, A.V. (1975) Somatostatin: abundance of immunoreactive hormone in rat stomach and pancreas. Science, 189, 1007-1009. HOKFELT, T., EFENDIC, s., HELLERSTROM, c., JOHANSSON, o., LUFT, R. ARIMURA, A. (1975) Cellular localisation of somatostatin in endocrine-like cells and neurons of the rat with special references to the A,-cells of the pancreatic islets to the hypothalamus. Acta Endocrinologica (Kbh.), 80, Suppl. 200, 5-41. KRONHEIM, S., BERELOWITZ, M. & PIMSTONE, B.L. (1976) A radioimmunoassayfor growth hormone release inhibiting hormone: method and quantitative tissue distribution. Clinical Endocrinology, 5, 619-630.
KRONHEIM, S., BERELOWITZ, M. & PIMSTONE, B.L. (1977) The presence of immunoreactive g o w t h hormone release inhibiting hormone in normal cerebrospinal fluid. Clinical Endocrinology 6, 4 11 416.
PATEL, Y.C., RAO, K. & REICHLIN, S. (1975) Immunoreactive somatostatin (SRIF) in human cerebrospinal fluid (CSF). Clinical Research, 23, 389A. PIMSTONE, B.L., BERELOWITZ, M. & KRONHEIM, S. (1976) Somatostatin-1976. South African Medical Journal, 50, 1471-1473.
T H E CHARACTERIZATION OF GROWTH HORMONE RELEASE INHIBITING HORMONE-LIKE IMMUNOREACTIVITY IN NORMAL URINE S . KRONHEIM, M. BERELOWITZ A N D B. L. PIMSTONE
Isotope and Immunoassay Laboratory, Department of Medicine, University of Cape Town Medical School, South Africa (Received I4 February 1977; revised 19 April 1977; accepted 20 April 1977)
SUMMARY. Using a previously described radioimmunoassay for growth hormone release inhibiting hormone (GH-RIH), the presence of GH-RIH-like immunoreactivity in urine has been characterized by demonstrating mobility identical to synthetic GH-RIH standard on two sephadex gel chromatographic systems, and parallelism of dilutions of the sephadex fractions with synthetic GH-RIH. Furthermore, 74% of the sephadex fraction cross-reacting in the immunoassay bound to antibody conjugated to sepharose and could be eluted by 1 M acetic acid. This immunospecific eluate showed identity with synthetic GH-RIH on both ion exchange and thin layer chromatography. Thus GH-RIH-like immunoreactivity is present in normal urine; this may have potential relevance in the search for a physiological role for this peptide. Apart from suppressing growth hormone release, the pharmacological administration of synthetic growth hormone release inhibiting hormone (GH-RIH) inhibits a wide range of endocrine and exocrine secretions from pancreas, gut and elsewhere (Plmstone et al., 1976). In addition, GH-RIH, or a peptide showing similar immunological identity, is widely distributed in CNS, gut, pancreas and thyroid (Hokfelt et al., 1975; Arimura el al., 1975; Kronheim et al., 1976) as well as being present in CSF (Pate1 et al., 1975; Kronheim et al., 1977). In this paper we characterize GH-RIH-like immunoreactivity found in the urine of normal subjects using our previously reported radioimmunoassay (Kronheim et al., 1976).
Methods and results Radioimmunoassay. The radioimmunoassay (RIA) of GH-RIHwas performed as previously described (Kronheim et al., 1976) using rabbit anti GH-RIH serum (final concentration 1 : 125,000), Tyr' GH-RIH radioiodinatedwith "'I by the Chloramine T technique, synthetic GH-RIH (Ayerst 249 10) as reference standard, and dextran-coated charcoal to separate antibody-bound 'I Tyr' GH-RIH from the free fraction. Studies were performed on early morning urine samples from twelve normal volunteers. The pH of all specimens was initially adjusted to 7.0. Correspondence: Professor B. L. Pimstone, Department of Medicine, Medical School, Observatory 1925, Cape Province, South Africa.
343
344
S. Kronheim, M. Berelowitz and B. L. Pimstone
In preliminary experiments the chemical integrity of '"I Tyr' GH-RIH after incubation with urine for 20 h at 4°C was established by paper chromatoelectrophoresis on three occasions. 87 f 3% (mean f SEM) of the label remained intact at the site of origin, similar to values found afterincubationin assay buffer (90 f 2% mean k SEM). Immunological integrity of the tracer after incubation with urine was tested on three occasions by immunoprecipitation using 100 pl of rabbit anti GH-RIH (final concentration 1 : 500) and dextran-coated charcoal to separate antibody bound from free tracer. 81.5 2.1% (mean SEM) of tracer was precipitable after incubation with urine, compared to 80.7 k 1.7% (mean f SEM) in assay buffer. The level of GH-RIH found by RIA of urine was 66 f 7 pg/mg creatinine (mean f SEM). Recovery experiments: 51 -100% (mean, 81%) recovery of GH-RIH was obtained from urine samples assayed before and after the addition of 0.5 ng synthetic GH-RIH on four occasions. Gel chromatography: 20 ml samples of urine were lyophilised, made up to 1 ml in 1 M acetic acid, centrifuged to remove any precipitate, and eluted off two sephadex (Pharmacia) systems with 1M acetic acid: (a) Sephadex GI0 in a column 1.6 X 65 cm at a flow rate of 30 ml/h and (b) Sephadex G25 in a column 2.5 X 90 cm at a flow rate of 37 ml/h. Columns were calibrated with synthetic GH-RIH (measured by RIA), dextran blue (void volume) and urea. A single peak of immunoreactivity showing identity with synthetic GH-RIH was eluted from both systems (Fig. 1). Serial dilutions of the immunoreactive peaks showed linearity and were parallel to the standard curve on logit-log plot (Fig. 2). Affinity chromatography: The specific antibody binding of GH-RIH eluted from the Sephadex G10 column was further studied on ten occasions using cyanogen bromide activated sepharose 4B (Pharmacia) linked to rabbit anti-GH-RIH or control columns as described elsewhere (Kronheim et ul., 1977). Unbound material was eluted by 10 mM phosphosaline buffer pH 7.6 (PBS) and antibody bound GH-RIH by 1 M acetic acid. 88.7 k 4.9%(mean +_
+_
+_
-z
-1 150
100 Elution volume (mll
LT
1 Oe3r
t
v0
OG2
I
200
250
I
I
300 350 400 Elution volume (rnl)
450
500
Fig. 1. Gel chromatography of concentrated urine on Sephadex G10 (upper) and G25f (lower) showing identity between the elution volume of synthetic GH-RIH (horizontal hatching) and urine immunoreactivity (vertical-hatching).Overlap of the two peaks indicated by cross-hatching in this Fig and in Figs 3 and 5. V, = void volume.
GH-RIH-likeimmunoreactivity in normal urine
-2
t
Synthetic GH-RIH(ng/ml) 0.25 06
0.125 I
I
I
1:8
1:4
l:z
1.0
I
1:1
Urine dilution
Fig. 2. Logit-log plot of serial dilutions of urine immunoreactivity eluted from Sephadex G I 0 ( 0 ) (rn = -2.71, n = 3) showing parallelism with synthetic GH-RIH standard ( 0 ) (rn = -2.59, n = 13). Means * SD are shown.
IPBS
1
Acetic acid
4.0
2.0
.
c E
0
3 4.0 2.0
r1
BH 10
20
30
40
Elution volume (ml)
Fig. 3. Elution profile of synthetic GH-RIH (horizontal hatching) and urine immunoreactivity eluted from Sephadex G-10 (vertical-hatching) when passed (a) through sepharose conjugated to non-immune rabbit serum control (upper) showing 89% of urine extract unbound and eluted by PBS,and (b) through sepharose-GH-RIH antibody conjugate (lower) on which 74% of urine fraction was bound and eluted off with acetic acid (see text).
345
S. Kronheim, M . Berelowitz and B. L. Pimstone
346
O*i"
O-i"
n
I
I (3
Elution volume (mll Fig. 4. Ion exchange chromatography (see text) of specific immunoreactive GH-RIH eluted from sepharose-antibody column. 90% of this immunoreactivity elutes with 0.2M ammonium acetate, coinciding with the pattern of synthetic GH-RIH standard. Bold line, GH-RIH; dotted line, urine. Arrows indicate ammonium acetate concentrations.
R, value Fig. 5. Thin layer chromatography (see text) of specific immunoreactive GH-RIH eluted from sepharose-antibody column. Note similar Rf values of synthetic GH-RIH (horizontal hatching, Rf, 0.6 1) and immunoreactive urinary GH-RIH (vertical-hatchingRf, 0.59).
SEM) of the immunoreactive GH-RIH was eluted from the control sepharose column with PBS, whilst 25.9 k 5.4% (mean k SEM) was similarly eluted from the antibody-sepharose column (Fig. 3). The residual GH-RIH (74.1%) remained bound to this column and was only eluted with 1~ acetic acid. This eluate was divided into two, freeze dried, and further characterized on ion exchange and thin layer chromatography. Ion exchange chromatography: 200 pl of eluted GH-RIH in 2 mM ammonium acetate buffer pH 4.65 was applied to a carboxymethyl cellulose (CM52 Whatman) column 5 X 1 cm prepared in the same buffer and pre-coated with 2% bovine serum albumin. Stepwise buffer gradients of 2 mM, 0.1M and 0.2M ammonium acetate pH 4.65 were used to elute, 10 X 5 ml samples being collected with each gradient. The samples were freeze dried and later reconstituted in assay buffer for assay of GH-RIH. In this system 100% of synthetic GH-RJH applied as a marker elutes with the 0.2M ammonium acetate as did 90% of the urinary CH-RIH separated by affinity chromatography (Fig. 4). Thin layer chromutogruphy: 25 pl of CH-RIH separated by affinity chromatography in 0.1M acetic acid was applied to 0.25 mm cellulose precoated plates (Eastman Kodak) and run in butanol :acetic acid :water (4 : 1 :5 ) for 4 h (Kronheim et al., 1977). Similar Rf values were obtained for synthetic GH-RIH (Rf, 0.61) and urinary GH-RIH immunoreactivity (Rf, 0.59) (Fig. 5 ) .
Discussion Using a sensitive and specific RIA we have demonstrated the presence of GH-RIH-like immunoreactivity in normal urine. That this does not merely represent damage to '1 Tyr' GH-
GH-RIH-like immunoreactivity in normal urine
347
RIH with consequent interference with binding in the assay system has been excluded by demonstrating chromatographic integrity of the label following incubation with urine. Furthermore, the degree of immunoprecipitation with excess antibody was identical after incubation with urine or assay buffer, although the high concentration of antibody used in this system may not necessarily be directly applicable to the assay situation in which antibody is used at a much greater dilution. Identical mobility of the urine immunoreactivity and synthetic GH-RIH on sephadex suggests that the two have similar molecular weights, whde parallelism of serial dilutions with synthetic standard also suggests a similar identity. However the material in urine causing displacement of tracer from antibody in our assay may not all be truly immunoreactive, as 26% failed to bind to antibody in our affinity system. Ion exchange and thin layer chromatography of the immunoreactive material eluted off tht antibody -sepharose column support its identity with synthetic GH-RIH. The characterization of GH-RIH-like immunoreactivity in urine may allow the study of physiological changes in GH-RIH secretion in man, although the possible presence of nonimmunoreactive material interfering with the assay could cause difficulty with interpretation.
Acknowledgments Synthetic somatostatin was a kind gift of Dr E. Polakow, Ayerst Laboratories (Pty) Limited, Johannesburg, South Africa. Financial support for this study was obtained from the South African Medical Research Council and Atomic Energy Board, International Atomic Energy Agency Grant No. 1806/RB, University of Cape Town Staff Research Fund, Harry Crossley Foundation and Nellie Atkinson Bequest. References ARIMURA, A., SATO, H., DUPONT, A., NISHI, N. & SCHALLY, A.V. (1975) Somatostatin: abundance of immunoreactive hormone in rat stomach and pancreas. Science, 189, 1007-1009. HOKFELT, T., EFENDIC, s., HELLERSTROM, c., JOHANSSON, o., LUFT, R. ARIMURA, A. (1975) Cellular localisation of somatostatin in endocrine-like cells and neurons of the rat with special references to the A,-cells of the pancreatic islets to the hypothalamus. Acta Endocrinologica (Kbh.), 80, Suppl. 200, 5-41. KRONHEIM, S., BERELOWITZ, M. & PIMSTONE, B.L. (1976) A radioimmunoassayfor growth hormone release inhibiting hormone: method and quantitative tissue distribution. Clinical Endocrinology, 5, 619-630.
KRONHEIM, S., BERELOWITZ, M. & PIMSTONE, B.L. (1977) The presence of immunoreactive g o w t h hormone release inhibiting hormone in normal cerebrospinal fluid. Clinical Endocrinology 6, 4 11 416.
PATEL, Y.C., RAO, K. & REICHLIN, S. (1975) Immunoreactive somatostatin (SRIF) in human cerebrospinal fluid (CSF). Clinical Research, 23, 389A. PIMSTONE, B.L., BERELOWITZ, M. & KRONHEIM, S. (1976) Somatostatin-1976. South African Medical Journal, 50, 1471-1473.
