0021-972X/79/4905-0770$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1979 by The Endocrine Society
Vol. 49, No. 5 Printed in U.S.A.
Radioimmunoassay of a Basic Somatomedin: Comparison of Various Assay Techniques and Somatomedin Levels in Various Sera* R MARVIN BALA AND BANANI BHAUMICK Section of Endocrinology and Metabolism, Division of Medicine, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada T2N 1N4
ABSTRACT. Purification of a basic somatomedin (SM), with similarity to SM-C and insulin-like growth factor, from human plasma Conn fraction IV-1 enabled development of a RIA based on this SM. SM antiserum was produced by immunizing rabbits with partially purified SM. This antiserum (final dilution, 1:50,000) specifically bound approximately 40% of added [125I]SM in this RIA. The RIA sensitivity was 2 X 10~4 U immunoreactive SM (IRSM). Highly purified SM-C, insulin-like growth factor 1, and our SM revealed parallel and approximately equipotent dose-response curves in this RIA; rat SM and multiplication stimulation activity revealed less cross-reactivity. IRSM was detected in sera of all species tested except fish. Acidification of sera, without subsequent chromatography,
before assay permitted measurement of total IRSM with either an equilibrium or nonequilibrium RIA technique. Acidification of serum appears to increase SM-binding capacity while decreasing binding affinity of the 20,000-50,000 mol wt proteins in serum. The mean (±SEM) IRSM concentrations in sera from normals and patients with acromegaly, hypopituitarism, GH deficiency before/after treatment, and Laron dwarfism were 1.45 ± 0.17, 5.49 ± 0.48, 0.19 ± 0.07, 0.10 ± 0.02/0.64 ± 0.45, and 0.25 ±0.11 U/ml, respectively, compared to a pooled normal human serum reference standard which was designated to contain 1 IRSM U/ml. Measurements of total IRSM (bound and free) in serum may not accurately reflect SM bioactivity and will require interpretative caution. (J Clin Endocrinol Metab 49: 770,1979)
S
OMATOMEDIN (SM), a collective designation for GH-dependent biological growth factors (1), has been extensively investigated since the pioneer studies by Salmon and Daughaday (2). Utilizing different assay techniques to monitor SM activity during purification, a number of seemingly different SMs have been purified. SM-A (3), SM-C (4), insulin-like growth factor [IGF; previously designated nonsuppressible insulin-like activity-soluble (5)], and multiplication stimulation activity (MSA) (6) have somewhat similar SM activities, as measured by various bioassays and cell membrane receptorbinding assays (4, 7, 8), but differ on comparison by RIA (9, 10). The majority of SM in serum is bound to larger proteins (6, 7, 9-12); therefore, measurement of total SM in serum by RIA has required prior acidic chromatography of serum or nonequilibrium RIA techniques (9). We have purified a basic SM from human plasma Gohn fraction IV-1. This SM is similar to SM-C in terms of isolation procedures and SM activity (4), while the preliminary partial amino acid sequence studies suggest
similarity to IGF-1 (5). This SM was used to develop a highly sensitive RIA. Acidification of serum before equilibrium RIA permitted measurement of total immunoreactive SM (IRSM) in sera from normals and patients with relevant clinical conditions. Materials and Methods Purification ofSM1 Human plasma Cohn fraction IV-1 (Connaught Laboratories, Ontario, Canada) was extracted by the acid-ethanol-acetone procedure described by Hall (13). Subsequent purification consisted of sequential alternating protein separation techniques based on molecular size or charge analogous to those described by Van Wyk et al. (4). The purified SM revealed a single protein band on sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the preliminary amino acid sequence determination indicated that this purified SM was more than 75% pure. Isoelectric focusing indicated a pi near 8.6. The molecular weight was approximately 7500, as determined by gel filtration, sodium dodecyl sulfate polyacrylamide electrophoresis, and amino acid analysis. The preliminary determination of the amino acid sequence of this purified SM revealed that the first five N-terminal amino acids were identical to those reported for IGF-1 (5). Immunoreactive insulin was not detected in this SM preparation. The potency of this purified SM was approx-
Received November 3, 1978. Address requests for reprints to: Dr. R. M. Bala, Health Science Centre, Faculty of Medicine, University of Calgary, 2920 24th Avenue N. W., Calgary, Alberta, Canada T2N 1N4. * This work was supported by grants from the Medical Research Council of Canada.
Detailed report to be published. 770
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BASIC SM RIA imately 4000 U/mg, as measured by a hypophysectomized rat cartilage bioassay (14), where 1 U SM activity was equivalent to the SM activity present in 1 ml pooled serum obtained from normal adult male volunteers. SM antiserum Partially purified basic SM (590 bioactivity U/mg) was conjugated with ovalbumin (Sigma Chemical Co., St. Louis, Mo) utilizing glutaraldehyde (Electron Microscopy Sciences, Pittsburgh, PA) as the coupling agent (15). The conjugate contained 1 mg ovalbumin and 3 mg partially purified SM. Four male New Zealand White rabbits, weighing approximately 3 kg each, were immunized using the multiple site immunization technique described by Vaitukaitis et al. (16) except that Bordetella pertussis vaccine was omitted. Each rabbit received 1 mg ovalbumin-SM conjugate emulsified in complete Freund's adjuvant, followed by similar amounts of conjugate in incomplete Freund's adjuvant 10 days later. The two rabbits with detectable SM antibodies received additional booster injections of the ovalbumin-SM conjugate at 6-week intervals. The rabbits were bled from the central ear artery before immunization and at weekly intervals for measurement of serum SM antibody titers and collection of antiserum from the appropriately responding rabbits. The antiserum from one rabbit showing the greatest SM antibody titer was used for the studies reported here. Radioisotope- labeled SM The most highly purified basic SM (described above) was labeled with 125I according to the method of Hunter and Greenwood (17), followed by gel filtration on a column of Sephadex G-25 with Tris-HCl buffer (pH 7.4, 0.05 M). The eluted 125Ilabeled protein peak was collected in the Tris-HCl buffer containing 1% bovine serum albumin (fraction V, Sigma) and 0.02% sodium azide (referred to as Tris-BSA buffer), aliquoted, and stored at -40 C. Estimates of I25I specific activity were 50-200 /iCi//ig protein. Before use in the RIA, [125I]SM was chromatographed on columns of Sephadex G-50 equilibrated and eluted with the Tris-HCl buffer. The [125I]SM used for the RIA was eluted with a midpeak Kav (partition coefficient) corresponding to an indicated molecular weight of 7000-8000. SM reference standard Either the partially purified SM, which was also used for the primary immunization of rabbits (described above), or pooled serum were used as reference SM standards. The pooled serum was obtained from 10 healthy adult male volunteers who were taking no medication. This pooled serum, arbitrarily designated to contain 1 U IRSM activity/ml, was aliquoted and stored at -40 C until used. Reference or test serum samples The reference or test serum samples were acidified to pH 2.3 with a 1%finalformic acid concentration, left for approximately 4 h at 4 C, and then lyophilized. These serum samples were reconstituted to the original volume with Tris-HCl buffer just before use in the RIA. Tris-BSA buffer was used for dilution of reference or test samples.
771
RIA procedures First, 0.2 ml Tris-BSA buffer was added to all tubes. This was followed by the addition of 0.1 ml of the diluted reference or test samples. One hundred microliters of the Tris-BSA buffer containing approximately 10,000 cpm [125I]SM were added to each tube, and the reaction was initiated by the addition of 100 jtxl SM antiserum (final dilution, 1:50,000). After incubation for 48 h at 4 C, 100 pil 1:200 diluted normal rabbit serum (Grand Island Biological Co., Grand Island, NY), and 100 /ul 1:10 diluted goat antirabbit y-globulin (dilutions carried out with Tris-BSA buffer) were added to each tube. The tubes were further incubated for 24 h at 4 C before separation of bound and free hormones by centrifugation and supernatant aspiration, followed by counting of the radioactivity in the precipitate. Nonspecific binding (NSB) was determined by the percent of [125I]SM bound (%B) in the absence of SM antiserum or in the presence of an excess of partially purified SM. Since these were generally similar, the former was routinely used to calculate %NSB, which was then subtracted from total %B in all tubes to calculate the percentage of specific binding. Results were expressed as B/Bo, in which B represented specific binding at each dilution of added standard or test sample and Bo was the specific binding of [I25I]SM in the absence of reference standard or test sample. The reference standards and test samples were assayed in triplicate at all dilutions. Test samples were assayed in at least three dilutions. The above procedures were used in all assays (equilibrium technique) unless otherwise noted. For comparative studies, the nonequilibrium RIA technique was similar, except for incubation of reference standard or test samples and SM antiserum for 72 h at 4 C before the addition of the [I25I]SM, followed by another 24-h incubation before the addition of the second antibody (9). Gel filtration of serum at neutral pH [125I]SM (-10,000 cpm) was added to 1 ml nonacidified or acidified (final concentration, 1% formic acid) normal reference pooled serum, lyophilized, reconstituted to the original volume with 0.05 M sodium phosphate buffer (pH 7.5), and then incubated for 16 h at 4 C. The incubates were chromatographed on 1.6 X 90-cm columns of Sephadex G-200 with 0.05 M sodium phosphate buffer. The eluant was collected in consecutive small volume fractions over equal Kav intervals, and the radioactivity in the fractions was counted. In other experiments, 1-ml aliquots of the same nonacidified or acidified serum without added [l2r>I]SM were similarly lyophilized, reconstituted, and chromatographed, and the IRSM eluted in each consecutive 0.2-Kav interval was determined by RIA. Gel filtration of serum at acid pH One milliliter of normal pooled serum was acidified to a final formic acid concentration of 1% (pH 2.3) and was then chromatographed on a column of Sephadex G-75 with 1% formic acid. The eluant was pooled into consecutive equal volume fractions corresponding to 0.2-Kav intervals. The eluted fractions as well as an aliquot of the acidified starting serum were lyophilized and reconstituted with Tris-HCl or Tris-BSA buffer, respectively, before assay for IRSM.
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BALA AND BHAUMICK
772
Serum samples from patients with various clinical conditions Serum samples were obtained from 15 healthy adult volunteers (8 males and 7 females) who were not on any medication; paired samples at 0800 h (after 12 h of fasting) and 2000 h were obtained from 10 of these volunteers. After the blood had clotted, the serum was separated and stored at -40 C until assayed. Seventeen patients with acromegaly had elevated basal serum GH levels (range, 15-160 ng/ml) which remained greater than 10 ng/ml over 3 h after the ingestion of glucose (1 g/kg BW). Nine adult patients had panhypopituitarism before or after surgery for pituitary tumors and were receiving physiological replacement doses of L-T4 and glucocorticoids. Eight GH-deficient prepubertal children had normal TSH, PRL, and ACTH reserves on provocative testing. Five GH-deficient patients who were treated with GH were receiving 2 U human GH (hGH; Medical Research Council of Canada) im 3 times/ week. Serum samples were obtained approximately 16-24 h after the last GH injection. Four Laron dwarfism patients (18) had very low levels of SM bioactivity and high levels of GH in serum.
Results Evaluation of the SM RIA Only one of four rabbits showed a favorable response to immunization; binding of [125I]SM ( 0.05, by Student's twotailed t test)]. Even though the RIA dose-response curves of partially purified rat SM, IGF-2, and partially purified MSA did not appear parallel with our SM dose-response curve, the difference between slopes was not significant (limited number of doses tested and relatively large SEM).
0.6
0.4
0.2
0.0
0.05 0.1
0.5
1.0
5.0
10
ul SERUM ADDED
FIG. 5. Comparison of SM RIA dose-response curves of acidified sera obtained from different species.
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BALA AND BHAUMICK
774
tions are shown in Fig. 6. Acidification of serum before gel filtration resulted in a slight change in the protein elution profile, with a relative increase in the very large indicated molecular size (IMS) area proteins (Fig. 6A). Comparison of the elution profiles of radioactivity after gel filtration of [l25I]SM added to nonacidified or acidified serum (Fig. 6B) revealed that a greater proportion of recovered [125I]SM added to acidified serum was eluted in the 50,000-20,000 IMS area (peak III area) while very little was eluted in the peak IV area. Considering the minimal molecular sieving of the smaller (50,000, and free 125I, respectively). Total IRSM activities eluted in various 0.2-Kav intervals after similar gel filtration of 1 ml nonacidified or acidified serum are compared in Fig. 6C. After gel filtration of nonacidified serum, approximately 75%, 20%, and 5% of the total recovered IRSM were eluted in the >50,000 IMS area, peak III, and peak IV areas, respectively. After gel filtration of acidified serum, approximately 20%, 65%, and 15% of the total recovered IRSM were eluted in the >50,000 IMS area, peak III, and peak IV areas, respectively. The overall recoveries of IRSM after gel filtration of nonacidified and acidified serum were 42% and 102%, respectively. Measurement of IRSM was carried out without prior acidification of the eluted fractions. After gel filtration of acidified serum, the majorities of added [I25I]SM (Fig. 6B) and endogenous IRSM (Fig. 6C) were eluted in approximately similar (50,000-20,000 mol wt) IMS areas, whereas after gel filtration of nonacidified serum, the relative proportions of the total recovered [I25I]SM and endogenous IRSM which were eluted in the various IMS intervals were quite different. As shown in Fig. 7, after gel filtration of 1.0 ml acidified sera obtained from normals or acromegaly and hypopituitary patients on columns of Sephadex G-75 (equilibrated and eluted with 1% formic acid, pH 2.3), greater than 95% of the total recovered IRSM was eluted in the IMS area less than 20,000. The overall recoveries of IRSM after acidic gel filtration of sera from normal or acromegaly and hypopituitary patients were 106%, 96%, and 93%, respectively, when compared to the total IRSM measured in the applied acidified sera. Comparison of levels of IRSM in sera from normals and patients with various clinical entities
0.4
0.6
0.8
1.0
KAV
FIG. 6. Comparison of elution profiles of protein as optical density (O.D.) at 280 nM, cpm of [125I]SM added to serum, and IRSM after gel filtration of 1.0 ml acidified or nonacidified normal serum on Sephadex G-200 with 0.05 M, sodium phosphate buffer, pH 7.5. Protein and [125I]SM elution profiles in A and B, respectively, are shown ( ) for acidified serum ( ) and for nonacidified serum experiments ( )• C, The total IRSM eluted in each 0.2-Kav interval is shown for acidified serum (S3) and for nonacidified serum experiments (IsS).
The mean serum IRSM concentration in normal adult volunteers (eight males and seven females) was 1.45 ± 0.17 (SEM) U/ml (range, 0.80-2.5 U/ml) based on the pooled serum (designated to contain 1 U IRSM/ml) as reference standard. The IRSM levels in paired serum samples obtained at 0800 and 2000 h were not significantly different (P > 0.05, by paired t test). The IRSM levels in the sera of the males and females in the group were not significantly different. As shown in Fig. 8, the mean serum IRSM concentrations (units per ml ± SEM) were 5.49 ± 0.48, 0.19 ± 0.07, 0.10 ± 0.02, and 0.24 ±0.11 in patients with acromegaly, panhypopituitarism, isolated GH deficiency, and Laron dwarfism, respectively. The mean serum IRSM levels in acromegalic and panhypopituitary patients were significantly different from normals (P < 0.001) but were not different (P > 0.05) in
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BASIC SM RIA
775 (17)
2 5
I
4
13 2 cc
1 KAV INTERVAL FRACTIONS
FIG. 7. Comparison of the percent of the total recovered IRSM eluted in various Knv interval fractions after gel filtration of acidified hypopituitary, normal, or acromegalic human sera on Sephadex G-75 (1% formic acid, pH 2.3). Relatively negligible amounts of IRSM recovered in other Kov intervals ( 0.05), as assessed by one-way analysis of variance, followed by Duncan's multiple range test (20).
SM-C and IGF are measuring similar rather than separate IRSM proteins in serum which have similar SM bioactivities. This RIA measured relatively minimal amounts of IRSM activity (50,000 IMS) serum proteins, and some of this IRSM may then bind to smaller (
Vol. 49, No. 5 Printed in U.S.A.
Radioimmunoassay of a Basic Somatomedin: Comparison of Various Assay Techniques and Somatomedin Levels in Various Sera* R MARVIN BALA AND BANANI BHAUMICK Section of Endocrinology and Metabolism, Division of Medicine, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada T2N 1N4
ABSTRACT. Purification of a basic somatomedin (SM), with similarity to SM-C and insulin-like growth factor, from human plasma Conn fraction IV-1 enabled development of a RIA based on this SM. SM antiserum was produced by immunizing rabbits with partially purified SM. This antiserum (final dilution, 1:50,000) specifically bound approximately 40% of added [125I]SM in this RIA. The RIA sensitivity was 2 X 10~4 U immunoreactive SM (IRSM). Highly purified SM-C, insulin-like growth factor 1, and our SM revealed parallel and approximately equipotent dose-response curves in this RIA; rat SM and multiplication stimulation activity revealed less cross-reactivity. IRSM was detected in sera of all species tested except fish. Acidification of sera, without subsequent chromatography,
before assay permitted measurement of total IRSM with either an equilibrium or nonequilibrium RIA technique. Acidification of serum appears to increase SM-binding capacity while decreasing binding affinity of the 20,000-50,000 mol wt proteins in serum. The mean (±SEM) IRSM concentrations in sera from normals and patients with acromegaly, hypopituitarism, GH deficiency before/after treatment, and Laron dwarfism were 1.45 ± 0.17, 5.49 ± 0.48, 0.19 ± 0.07, 0.10 ± 0.02/0.64 ± 0.45, and 0.25 ±0.11 U/ml, respectively, compared to a pooled normal human serum reference standard which was designated to contain 1 IRSM U/ml. Measurements of total IRSM (bound and free) in serum may not accurately reflect SM bioactivity and will require interpretative caution. (J Clin Endocrinol Metab 49: 770,1979)
S
OMATOMEDIN (SM), a collective designation for GH-dependent biological growth factors (1), has been extensively investigated since the pioneer studies by Salmon and Daughaday (2). Utilizing different assay techniques to monitor SM activity during purification, a number of seemingly different SMs have been purified. SM-A (3), SM-C (4), insulin-like growth factor [IGF; previously designated nonsuppressible insulin-like activity-soluble (5)], and multiplication stimulation activity (MSA) (6) have somewhat similar SM activities, as measured by various bioassays and cell membrane receptorbinding assays (4, 7, 8), but differ on comparison by RIA (9, 10). The majority of SM in serum is bound to larger proteins (6, 7, 9-12); therefore, measurement of total SM in serum by RIA has required prior acidic chromatography of serum or nonequilibrium RIA techniques (9). We have purified a basic SM from human plasma Gohn fraction IV-1. This SM is similar to SM-C in terms of isolation procedures and SM activity (4), while the preliminary partial amino acid sequence studies suggest
similarity to IGF-1 (5). This SM was used to develop a highly sensitive RIA. Acidification of serum before equilibrium RIA permitted measurement of total immunoreactive SM (IRSM) in sera from normals and patients with relevant clinical conditions. Materials and Methods Purification ofSM1 Human plasma Cohn fraction IV-1 (Connaught Laboratories, Ontario, Canada) was extracted by the acid-ethanol-acetone procedure described by Hall (13). Subsequent purification consisted of sequential alternating protein separation techniques based on molecular size or charge analogous to those described by Van Wyk et al. (4). The purified SM revealed a single protein band on sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the preliminary amino acid sequence determination indicated that this purified SM was more than 75% pure. Isoelectric focusing indicated a pi near 8.6. The molecular weight was approximately 7500, as determined by gel filtration, sodium dodecyl sulfate polyacrylamide electrophoresis, and amino acid analysis. The preliminary determination of the amino acid sequence of this purified SM revealed that the first five N-terminal amino acids were identical to those reported for IGF-1 (5). Immunoreactive insulin was not detected in this SM preparation. The potency of this purified SM was approx-
Received November 3, 1978. Address requests for reprints to: Dr. R. M. Bala, Health Science Centre, Faculty of Medicine, University of Calgary, 2920 24th Avenue N. W., Calgary, Alberta, Canada T2N 1N4. * This work was supported by grants from the Medical Research Council of Canada.
Detailed report to be published. 770
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BASIC SM RIA imately 4000 U/mg, as measured by a hypophysectomized rat cartilage bioassay (14), where 1 U SM activity was equivalent to the SM activity present in 1 ml pooled serum obtained from normal adult male volunteers. SM antiserum Partially purified basic SM (590 bioactivity U/mg) was conjugated with ovalbumin (Sigma Chemical Co., St. Louis, Mo) utilizing glutaraldehyde (Electron Microscopy Sciences, Pittsburgh, PA) as the coupling agent (15). The conjugate contained 1 mg ovalbumin and 3 mg partially purified SM. Four male New Zealand White rabbits, weighing approximately 3 kg each, were immunized using the multiple site immunization technique described by Vaitukaitis et al. (16) except that Bordetella pertussis vaccine was omitted. Each rabbit received 1 mg ovalbumin-SM conjugate emulsified in complete Freund's adjuvant, followed by similar amounts of conjugate in incomplete Freund's adjuvant 10 days later. The two rabbits with detectable SM antibodies received additional booster injections of the ovalbumin-SM conjugate at 6-week intervals. The rabbits were bled from the central ear artery before immunization and at weekly intervals for measurement of serum SM antibody titers and collection of antiserum from the appropriately responding rabbits. The antiserum from one rabbit showing the greatest SM antibody titer was used for the studies reported here. Radioisotope- labeled SM The most highly purified basic SM (described above) was labeled with 125I according to the method of Hunter and Greenwood (17), followed by gel filtration on a column of Sephadex G-25 with Tris-HCl buffer (pH 7.4, 0.05 M). The eluted 125Ilabeled protein peak was collected in the Tris-HCl buffer containing 1% bovine serum albumin (fraction V, Sigma) and 0.02% sodium azide (referred to as Tris-BSA buffer), aliquoted, and stored at -40 C. Estimates of I25I specific activity were 50-200 /iCi//ig protein. Before use in the RIA, [125I]SM was chromatographed on columns of Sephadex G-50 equilibrated and eluted with the Tris-HCl buffer. The [125I]SM used for the RIA was eluted with a midpeak Kav (partition coefficient) corresponding to an indicated molecular weight of 7000-8000. SM reference standard Either the partially purified SM, which was also used for the primary immunization of rabbits (described above), or pooled serum were used as reference SM standards. The pooled serum was obtained from 10 healthy adult male volunteers who were taking no medication. This pooled serum, arbitrarily designated to contain 1 U IRSM activity/ml, was aliquoted and stored at -40 C until used. Reference or test serum samples The reference or test serum samples were acidified to pH 2.3 with a 1%finalformic acid concentration, left for approximately 4 h at 4 C, and then lyophilized. These serum samples were reconstituted to the original volume with Tris-HCl buffer just before use in the RIA. Tris-BSA buffer was used for dilution of reference or test samples.
771
RIA procedures First, 0.2 ml Tris-BSA buffer was added to all tubes. This was followed by the addition of 0.1 ml of the diluted reference or test samples. One hundred microliters of the Tris-BSA buffer containing approximately 10,000 cpm [125I]SM were added to each tube, and the reaction was initiated by the addition of 100 jtxl SM antiserum (final dilution, 1:50,000). After incubation for 48 h at 4 C, 100 pil 1:200 diluted normal rabbit serum (Grand Island Biological Co., Grand Island, NY), and 100 /ul 1:10 diluted goat antirabbit y-globulin (dilutions carried out with Tris-BSA buffer) were added to each tube. The tubes were further incubated for 24 h at 4 C before separation of bound and free hormones by centrifugation and supernatant aspiration, followed by counting of the radioactivity in the precipitate. Nonspecific binding (NSB) was determined by the percent of [125I]SM bound (%B) in the absence of SM antiserum or in the presence of an excess of partially purified SM. Since these were generally similar, the former was routinely used to calculate %NSB, which was then subtracted from total %B in all tubes to calculate the percentage of specific binding. Results were expressed as B/Bo, in which B represented specific binding at each dilution of added standard or test sample and Bo was the specific binding of [I25I]SM in the absence of reference standard or test sample. The reference standards and test samples were assayed in triplicate at all dilutions. Test samples were assayed in at least three dilutions. The above procedures were used in all assays (equilibrium technique) unless otherwise noted. For comparative studies, the nonequilibrium RIA technique was similar, except for incubation of reference standard or test samples and SM antiserum for 72 h at 4 C before the addition of the [I25I]SM, followed by another 24-h incubation before the addition of the second antibody (9). Gel filtration of serum at neutral pH [125I]SM (-10,000 cpm) was added to 1 ml nonacidified or acidified (final concentration, 1% formic acid) normal reference pooled serum, lyophilized, reconstituted to the original volume with 0.05 M sodium phosphate buffer (pH 7.5), and then incubated for 16 h at 4 C. The incubates were chromatographed on 1.6 X 90-cm columns of Sephadex G-200 with 0.05 M sodium phosphate buffer. The eluant was collected in consecutive small volume fractions over equal Kav intervals, and the radioactivity in the fractions was counted. In other experiments, 1-ml aliquots of the same nonacidified or acidified serum without added [l2r>I]SM were similarly lyophilized, reconstituted, and chromatographed, and the IRSM eluted in each consecutive 0.2-Kav interval was determined by RIA. Gel filtration of serum at acid pH One milliliter of normal pooled serum was acidified to a final formic acid concentration of 1% (pH 2.3) and was then chromatographed on a column of Sephadex G-75 with 1% formic acid. The eluant was pooled into consecutive equal volume fractions corresponding to 0.2-Kav intervals. The eluted fractions as well as an aliquot of the acidified starting serum were lyophilized and reconstituted with Tris-HCl or Tris-BSA buffer, respectively, before assay for IRSM.
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BALA AND BHAUMICK
772
Serum samples from patients with various clinical conditions Serum samples were obtained from 15 healthy adult volunteers (8 males and 7 females) who were not on any medication; paired samples at 0800 h (after 12 h of fasting) and 2000 h were obtained from 10 of these volunteers. After the blood had clotted, the serum was separated and stored at -40 C until assayed. Seventeen patients with acromegaly had elevated basal serum GH levels (range, 15-160 ng/ml) which remained greater than 10 ng/ml over 3 h after the ingestion of glucose (1 g/kg BW). Nine adult patients had panhypopituitarism before or after surgery for pituitary tumors and were receiving physiological replacement doses of L-T4 and glucocorticoids. Eight GH-deficient prepubertal children had normal TSH, PRL, and ACTH reserves on provocative testing. Five GH-deficient patients who were treated with GH were receiving 2 U human GH (hGH; Medical Research Council of Canada) im 3 times/ week. Serum samples were obtained approximately 16-24 h after the last GH injection. Four Laron dwarfism patients (18) had very low levels of SM bioactivity and high levels of GH in serum.
Results Evaluation of the SM RIA Only one of four rabbits showed a favorable response to immunization; binding of [125I]SM ( 0.05, by Student's twotailed t test)]. Even though the RIA dose-response curves of partially purified rat SM, IGF-2, and partially purified MSA did not appear parallel with our SM dose-response curve, the difference between slopes was not significant (limited number of doses tested and relatively large SEM).
0.6
0.4
0.2
0.0
0.05 0.1
0.5
1.0
5.0
10
ul SERUM ADDED
FIG. 5. Comparison of SM RIA dose-response curves of acidified sera obtained from different species.
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BALA AND BHAUMICK
774
tions are shown in Fig. 6. Acidification of serum before gel filtration resulted in a slight change in the protein elution profile, with a relative increase in the very large indicated molecular size (IMS) area proteins (Fig. 6A). Comparison of the elution profiles of radioactivity after gel filtration of [l25I]SM added to nonacidified or acidified serum (Fig. 6B) revealed that a greater proportion of recovered [125I]SM added to acidified serum was eluted in the 50,000-20,000 IMS area (peak III area) while very little was eluted in the peak IV area. Considering the minimal molecular sieving of the smaller (50,000, and free 125I, respectively). Total IRSM activities eluted in various 0.2-Kav intervals after similar gel filtration of 1 ml nonacidified or acidified serum are compared in Fig. 6C. After gel filtration of nonacidified serum, approximately 75%, 20%, and 5% of the total recovered IRSM were eluted in the >50,000 IMS area, peak III, and peak IV areas, respectively. After gel filtration of acidified serum, approximately 20%, 65%, and 15% of the total recovered IRSM were eluted in the >50,000 IMS area, peak III, and peak IV areas, respectively. The overall recoveries of IRSM after gel filtration of nonacidified and acidified serum were 42% and 102%, respectively. Measurement of IRSM was carried out without prior acidification of the eluted fractions. After gel filtration of acidified serum, the majorities of added [I25I]SM (Fig. 6B) and endogenous IRSM (Fig. 6C) were eluted in approximately similar (50,000-20,000 mol wt) IMS areas, whereas after gel filtration of nonacidified serum, the relative proportions of the total recovered [I25I]SM and endogenous IRSM which were eluted in the various IMS intervals were quite different. As shown in Fig. 7, after gel filtration of 1.0 ml acidified sera obtained from normals or acromegaly and hypopituitary patients on columns of Sephadex G-75 (equilibrated and eluted with 1% formic acid, pH 2.3), greater than 95% of the total recovered IRSM was eluted in the IMS area less than 20,000. The overall recoveries of IRSM after acidic gel filtration of sera from normal or acromegaly and hypopituitary patients were 106%, 96%, and 93%, respectively, when compared to the total IRSM measured in the applied acidified sera. Comparison of levels of IRSM in sera from normals and patients with various clinical entities
0.4
0.6
0.8
1.0
KAV
FIG. 6. Comparison of elution profiles of protein as optical density (O.D.) at 280 nM, cpm of [125I]SM added to serum, and IRSM after gel filtration of 1.0 ml acidified or nonacidified normal serum on Sephadex G-200 with 0.05 M, sodium phosphate buffer, pH 7.5. Protein and [125I]SM elution profiles in A and B, respectively, are shown ( ) for acidified serum ( ) and for nonacidified serum experiments ( )• C, The total IRSM eluted in each 0.2-Kav interval is shown for acidified serum (S3) and for nonacidified serum experiments (IsS).
The mean serum IRSM concentration in normal adult volunteers (eight males and seven females) was 1.45 ± 0.17 (SEM) U/ml (range, 0.80-2.5 U/ml) based on the pooled serum (designated to contain 1 U IRSM/ml) as reference standard. The IRSM levels in paired serum samples obtained at 0800 and 2000 h were not significantly different (P > 0.05, by paired t test). The IRSM levels in the sera of the males and females in the group were not significantly different. As shown in Fig. 8, the mean serum IRSM concentrations (units per ml ± SEM) were 5.49 ± 0.48, 0.19 ± 0.07, 0.10 ± 0.02, and 0.24 ±0.11 in patients with acromegaly, panhypopituitarism, isolated GH deficiency, and Laron dwarfism, respectively. The mean serum IRSM levels in acromegalic and panhypopituitary patients were significantly different from normals (P < 0.001) but were not different (P > 0.05) in
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BASIC SM RIA
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2 5
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13 2 cc
1 KAV INTERVAL FRACTIONS
FIG. 7. Comparison of the percent of the total recovered IRSM eluted in various Knv interval fractions after gel filtration of acidified hypopituitary, normal, or acromegalic human sera on Sephadex G-75 (1% formic acid, pH 2.3). Relatively negligible amounts of IRSM recovered in other Kov intervals ( 0.05), as assessed by one-way analysis of variance, followed by Duncan's multiple range test (20).
SM-C and IGF are measuring similar rather than separate IRSM proteins in serum which have similar SM bioactivities. This RIA measured relatively minimal amounts of IRSM activity (50,000 IMS) serum proteins, and some of this IRSM may then bind to smaller (
