0021-972X/78/4706-1287$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1978 by The Endocrine Society

Vol. 47, No. 6 Printed in U.S.A.

Comparison of [125I]Somatomedin A and [125I]Somatomedin C Radioreceptor Assays for Somatomedin Peptide Content in Whole and Acid-Chromatographed Plasma* JAMES M. HORNER, FRANCES LIU, AND RAYMOND L. HINTZf Department of Pediatrics, Stanford University School of Medicine, Stanford, California 94305 ABSTRACT. The placental membrane radioreceptor assay was used to measure the levels of somatomedin (SM) peptides in plasma. Displacement of both [I25I]somatomedin A ([125I]SM-A) and [125I]somatomedin C ([125I]SM-C) by normal whole plasma, the peptide fraction of acid-chromatographed plasma, and a partially purified, insulin-free SM preparation were compared. The peptide fraction of plasma was isolated by acid chromatography over Sephadex G-50 in 0.25 M formic acid with a yield of >90%, as determined by bioassay and [125I]SM. In the case of [125I]SM-A, the dose-response curves for whole plasma, acid-chromatographed plasma, and the standard SM preparation were parallel (P > 0.2). In contrast, for [125I]SM-C, the dose-response curves for acid-chromatographed plasma and the puri-

fied SM preparation were parallel (P > 0.2), but both differed significantly from that of whole plasma (P < 0.001). In addition, there was less variability in the assay of acid-chromatographed plasma compared to whole plasma. The results indicate that radioreceptor assay of unextracted normal plasma using [125I]SM-A is a valid measure of SM peptide concentration, while radioreceptor assay of unextracted normal plasma using [125I]SM-C, in our hands, is not. Acid chomatography of plasma before its assay is an uncomplicated procedure which allows valid and precise measurement of SM peptide content using either [125I]SM-A or [125I]SM-C. (J Clin Endocrinol Metab 47: 1287, 1978)

T

HE SOMATOMEDINS are a group of peptide hormones under GH control. These peptides have both growth-promoting and insulin-like activities in target tissues (1), and include somatomedin A (SM-A) (2), somatomedin C (SM-C) (3), nonsuppressible insulin-like activity-soluble (NSILA-S) (4), and multiplication-stimulating activity (MSA) (5). Although the purified peptides all have molecular weights in the range of 5000-8000 daltons, the majority of bioassayable somatomedin activity in plasma appears at a molecular weight >60,000 daltons. This apparent discrepancy is explained by recent studies which show that each of the somatomedins circulates in plasma bound to a specific binding protein (6-11). For years, measurements of these hormones in plasma depended on cumbersome and imprecise bioassays of somatomedin activity

(12-18). Recently, radioreceptor assays (RRAs) (19-23) have been developed for their quantitation. In addition, RIAs have been reported for NSILA-S (24) and SM-C (25). The RRA for SM-A or SM-C (20) employs a particulate human placental membrane preparation which has been shown to have specific receptors for the somatomedins. The changes in SM-A and SM-C levels expected in hypopituitarism, acromegaly, and with GH treatment of hypopituitary dwarfism have been observed when unextracted serum was used in this placental membrane RRA (19, 20). NSILA-S, which is measured in a rat liver plasma membrane RRA (21) and a chick embryo fibroblast RRA (27), cannot be accurately determined in unextracted serum (9, 26), but its measurement after gel filtration of serum on Sephadex G-75 correlates directly with the GH status of the serum donor (28, Received December 13, 1977. 29). Separation of the NSILA-S peptide from * This work was supported in part by NIH Grant AMits carrier protein is the suggested explanation 19168 and Grant AM-07217 from the NIH and The Naof this phenomenon (28), but the removal of tional Foundation/March of Dimes. It was presented in part at the International Symposium on Somatomedin specific or nonspecific inhibitors or proteolytic and Related Peptides, Santa Margherita Ligure, Italy, enzymes which interfere with the assay has March 1-3, 1978. f To whom requests for reprints should be addressed. not been ruled out. 1287

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HORNER, LIU, AND HINTZ

Because of the discrepancy between the ability to validly assay levels of SM-A and SM-C us. NSILA-S in whole serum or plasma as well as numerous factors which might influence the placental membrane RRA when whole serum or plasma is assayed, we have compared the specific SM-A and SM-C activities in whole plasma and the peptide fraction of acid-chromatographed plasma from normal adult males using the placental membrane RRA and standard bioassays for somatomedin. Recovery of somatomedin peptide after chromatography was carefully assessed. In addition, displacement of radioactive SM-A and SM-C by whole plasma and separated plasma were compared to their displacement by a standard purified somatomedin preparation.

JCE & M • 1978 Vol47 • No 6

somatomedin activity/mg was used for determination of nonspecific binding and for standard curves in the RRA for SM-A and SM-C. This preparation contained equal amounts of neutral and basic somatomedin when assayed by either [125I]SM-A or [l25I]SM-C RRA after separation by TLIEF. The maximal specific binding (Bo; mean ± SD) for [125I]SM-A was 10.95 ± 0.55% of total counts added per tube; that for [125I]SM-C was 8.40 ± 0.42%. The nonspecific binding for each trace was comparable and less than 4% of the total counts added. There was no correlation of the Bo with time over the period of experimentation. Test plasma Heparinized plasma from 20 healthy young adult males was used for all experiments. It was collected, pooled, assigned 1 U somatomedin activity/ml, and frozen at — 70 C until used.

Materials and Methods Purification and iodination of somatomedin Highly purified neutral and basic preparations of somatomedin were isolated by a modification of the methods of Van Wyk et al. (3). The acid-ethanol extract of Cohn IV-I paste was dissolved in 0.25 M formic acid and chromatographed on Sephadex G50 with 0.25 M formic acid as the solvent. The active fractions were pooled, lyophilized, dissolved in 0.05 M NH4HCO3 (pH 8.1), and chromatographed on Sephadex G-50 in 0.05 M NH4HCO3. The active fractions of eluate were again pooled and lyophilized, and then further purified by thin layer isoelectric focusing (TLIEF). After IEF, the peptides were separated from the ampholytes by G-50 chromatography. A neutral peptide with a pi of 6.9 was isolated which was identical to SM-A by chemical characteristics and bioactivity. A basic peptide with a pi of 8.2, apparently identical to SM-C, was also isolated. These preparations were apparently homogeneous by the criteria of gel electrophoresis and TLIEF. Absolute identity of these peptides with the previously described SM-A of Hall (2) and SM-C of Van Wyk (3) could only be proven by amino acid-sequencing data. Both SM-A (pi 6.9) and SM-C (pi 8.2) had potencies of 600 U/mg dry weight by hypophysectomized rat bioassay. These peptides were iodinated with 125I by a modification of the chloramineT method (30, 31) to specific activities of 40-50 mCi/mg and stored at -20 C until used. Because of the relative scarcity of purified somatomedin, a partially purified, immunoreactive insulin-free somatomedin preparation with approximately 50 U

Chromatography Twenty 1.0-ml aliquots of the pooled plasma were placed over Sephadex G-50 columns (0.9 x 100 cm) in 0.25 M formic acid at room temperature. Fifteen milliliters of the effluent (Kd, 0.25-0.50), containing over 95% of the somatomedin activity and no detectable somatomedin-binding protein activity (7), were collected from each column in siliconized Pyrex tubes containing 1.0 ml bovine serum albumin (BSA), lyophilized, and stored at room temperature until assayed. Plasma treated in this way will be referred to as acid-chromatographed or separated plasma. Determination of recovery of somatomedin peptide after acid chromatography The percentage of somatomedin recovered in the 15-ml fractions of effluent was determined using radioactive somatomedin. Highly purified [125I]SMA and [125I]SM-C free of aggregate were incubated with plasma overnight at 4 C. Each was then placed on a Sephadex G-50 column (0.9 X 100 cm) in 0.05 M NH4HCO3, pH 8.1. The void volume, which has been shown to contain somatomedin bound to its binding protein under like conditions (7), was collected. Four 1.0-ml aliquots of this void volume were counted and chromatographed in a manner identical to the normal plasma samples. The fractions with Kds of 0.25-0.50 were collected in siliconized Pyrex tubes containing 1% BSA, lyophilized, redissolved in 1.0 ml 0.05 M Tris-HCl without BSA, pH = 7.4, and counted. The percentage yield was calculated by dividing the counts added to each

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J

COMPARISON OF [125I]SM-A AND [125I]SM-C RRAs FOR SM column into the counts recovered and multiplying by 100.

1289

and x = the corresponding dose of plasma or purified somatomedin. A two-tailed Student's t test was applied when significances were determined.

Placental membrane RRA The placental membrane used in the RRA was prepared from term placentae by the method of Cuatrecasas (32) and stored at —20 C until needed. Incubations were performed in 11 x 75-mm polypropylene tubes. Placental membrane suspension [200-250 jug protein/tube, as determined by the method of Lowry et al. (33)] was added to assay tubes containing graded doses of whole or separated plasma or unlabeled somatomedin, approximately 10,000 cpm radioactive somatomedin, and 0.05 M Tris-HCl with 1% BSA, pH = 7.4, at 4 °C. The final incubation volume was 0.5 ml. Plasma dosages ranged from 0.8 - 16 /il undiluted plasma. After incubation of the samples at 4 C for 16-20 h, 1.0 ml cold 0.05 M Tris-HCl with 1% BSA, pH = 7.4, was added to each tube. The tubes were centrifuged at 10,000 X g for 10 min, and the supernatants were removed by suction. The pellets were then counted in a y-counter. Specific binding was determined by subtracting from each tube the average of the counts bound in duplicate tubes having a large excess of unlabeled somatomedin (0.5 U somatomedin/ml. Assays using both whole and separated plasma and both [125I]SM-A and [I25I]SM-C were run in paralled. Standard displacement curves for both [125I]SM-A and [125I]SM-C were made using a partially purified, insulin-free somatomedin preparation with a potency of ~50 U/mg. Intraassay and interassay variability were determined for whole plasma and separated plasma with both SM-A and SM-C traces. The interassay variability was assessed from 8 independent assays. The intraassay variability was determined using 10 replicates in each system. Bioassay Bioassays of whole and separated plasma samples were performed by the hypophysectomized rat costal cartilage bioassay of Daughaday (16) and the porcine bioassay of Van den Brande (15). Statistical analysis Common statistical methods were employed in evaluation of the data. The slopes and intercepts of the dose-response curves were determined by the method of least squares regression, according to the equation y = a + b In x, where y = the percentage of maximum specific binding of radioactive ligand

Results Chromatographed separation of somatomedin from its binding protein using Sephadex G-50 gel and 0.25 M formic acid at room temperature results in high and reproducible yield of the somatomedin peptide. Figure 1 shows the chromatographic pattern of plasma which is equilibrated overnight at 4 C with [125I]SM-C peptide and placed on a Sephadex G-50 column at a neutral pH. A large portion of the radioactive somatomedin (53.7%) appears in the void volume, consistent with its binding to a large carrier protein. When this fraction containing the labeled somatomedin peptide-somatomedin-binding protein complex is rechromatographed over Sephadex G50 in 0.25 M formic acid at room temperature, virtually all of the radioactivity elutes off the column just before the salt peak in the small molecular weight peptide range, corresponding to the region of somatomedin activity according to Hintz and Liu (7) (Fig. 2). Table 1 shows the recovery of [12oI]SM-C from this area of somatomedin peptide activity after acid-chromatography. The mean (±SD) recovery of [125I]SM-C from the Sephadex G-50 column is 96.9 ± 1.3%. After lyophilization, the yield of [125I]SM-C averages 91.9 ± 2.1% of the total amount originally placed on the gel (Table 1). Chromatography of [125I]SM-A gave comparable results. This mean yield of 91.9% was used as the average recovery of native somatomedin peptide from whole plasma chromatographed in an identical fashion. Figure 3 shows the displacement curves of [125I]SM-A by whole plasma and acid-chromatographed plasma. The curves are clearly parallel and almost superimposable. Fitting the data to a natural logarithm-linear function, y = a + b In x, by least squares regression analysis, the slope of the whole plasma doseresponse curve is -19.08 ± 0.87; that of the separated plasma dose-response curve is -20.79 ± 1.13. The difference in slopes is 1.71,

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JCE&M • 1978 VoU7 • No 6

HORNER, LIU, AND HINTZ

1290

1

1

1

i

i



i

i

i

o—o

125

I-SM ALONE

•—•

125

I-SM PREINCUBATED WITH PLASMA

< • -

-

/

• • •

\

1 •

40 50 60 70 FRACTION NUMBER (1 ml)

110

FIG. 1. Sephadex G-50 chromatography of [125I]SM-C radioligand at 4 C in 0.05 M NH4HCO3, pH 8.1, before and after incubation with whole plasma. Sample volumes of 1.0 ml were placed on a 0.9 x 100-cm column in 0.05 M NH4 HCO3, pH 8.1, and 1.0-ml fractions (represented along the abscissa) were collected and counted in a y-detector (total counts per min/ml fraction are depicted on the ordinate). O—O, Pattern of [I25I]SM radioligand alone; • — # , pattern of the [125I]Somatomedin radioligand after its incubation with 1.0 cc plasma overnight at 4 C. There is a shift of over half of the radioactivity (53.7%) from the area of the small molecular weight peptide to the void volume after incubation with plasma, consistent with its binding to a large molecular weight binding protein. Almost identical results were obtained when the same experiments were performed with [125I]SM-A.

30

40

50

60

70

80

90

100

FRACTION NUMBER (1 ml)

FIG. 2. Sephadex G-50 chromatography of [125I]SM-C-somatomedin-binding protein complex at room temperature in 0.25 M formic acid, pH = 2.3. One milliliter of the void volume of the plasma + [I25I]SM-C sample chromatographed on Sephadex G-50 in 0.05 M NH4HCO3, pH = 8.1 (see Fig. 1), was placed on a 0.9 X 100-cm column in 0.25 M formic acid, pH = 2.3, and 1.0-ml fractions (represented along the abscissa) were collected and counted in a y-detector (the total counts per min/ml fraction are shown on the ordinate). Virtually all of the radioactive SM-C ligand elutes off the column in the area coincident with the small molecular weight peptides, demonstrating complete dissociation from the large molecular weight binding protein under acid conditions. Acid chromatography of [125I]SM-A-somatomedinbinding protein complex under the same conditions resulted in an identical elution pattern.

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COMPARISON OF [125I]SM-A AND [125I]SM-C RRAs FOR SM

1291

TABLE 1. Recovery of [125I]somatomedin (SM) peptide after acid chromatography of [I25I]SM peptide free of aggregate and bound to the large molecular weight protein fraction of plasma and lyophilization of the small molecular weight peptide fraction Total [125I]SM in fractions 51-65 after acid chromotography (cpm)

Column no.

I II III IV Mean ± SD

19,380 18,745 18,615 18,448

18,554 18,496 17,920 17,856

CHROMATOGRAPHED PLASMA

% Yield

95.7 98.7 96.3 96.8 96.9 ± 1.3

Total [125I]SM after lyophilization (cpm) 17,993 17,485 16.635 16,451

% Yield overall

92.8 93.0 89.4 89.2 91.1 ±2.1

WHOLE PLASMA

PURIFIED SOMATOMEDIN

20

1.0

5.0 10.0 20.0 20

UNDILUTED PLASMA EQUIVALENTS ul/ml

1.0

100 ISM] ng/ml

5.0 10.0 20.0

1000 2000 UNDILUTED PLASMA EQUIVALENTS ul/ml

FIG. 3. Comparison of the competitive binding curves for [I25I]-SM-A and whole and acid-chromatographed plasma with that for [125I]SM-A and partially purified somatomedin peptide in the placental membrane RRA. The doses of partially purified somatomedin peptide are expressed on the abscissa as nanograms per ml; doses of whole and acidchromatographed plasma are expressed as microliter equivalents of undiluted plasma per ml. The ordinate depicts the percentage of maximal binding at each dose. For the partially purified somatomedin peptide, each data point represents the mean (±SEM) of 2 experiments; for whole plasma, each data point represents the mean (±SEM) of 7 experiments; for acid-chromatographed plasma, each data point represents the mean (±SEM) of 21 experiments. Two-tailed Student's t tests of the slopes of all three of these dose-response curves obtained by least squares regression analysis reveal that they are not statistically different (P » 0.2), i.e. all three lines are parallel.

with a SE of the slopes of 1.43. Hence, these slopes are not statistically different (P > 0.2). Furthermore, displacement of [125I]SM-A trace by whole and separated plasma parallels its displacement by purified somatomedin (Fig. 3). The slope of the purified somatomedin dose-response curve is -18.91 ± 0.80. The difference between the slopes of purified SM and whole plasma is 0.17, with a SE of the slopes of 1.18. The difference between the slopes of purified SM and separated plasma is 1.88, with a SE of the slopes of 1.39. Thus, the slope of the dose-response curve for purified

SM is not statistically different from the slopes of the dose-response curves of whole or separated plasma for displacement of [125I]SM-A (P » 0.2). Furthermore, the relative potencies of whole and separated plasmas with regard to their capacity to displace [125I]SMA from placental membrane are statistically identical. Whole and extracted plasmas cause almost identical displacements of [125I]SM-A trace in the placental membrane RRA and their displacement curves are parallel not only to each other, but also to that of purified somatomedin. This suggests that the [125I]SM-

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HORNER, LIU, AND HINTZ

1292

A RRA may be useful as a valid measure of total somatomedin peptide concentration in whole plasma. The results of similar experiments using 125 [ I]SM-C do not lead to the same conclusion. Figure 4 illustrates the displacement of [125I]SM-C from placental membrane by graded doses of whole plasma and acid-chromatographed plasma. It is quite apparent that the displacement curves are not parallel. Increasing doses of the separated plasma have a greater ability to displace the SM-C trace than do equivalent doses of whole plasma. The slope of the separated plasma dose-response curve is -22.61 ± 1.21, while that of the whole plasma curve is -14.12 ± 1.19. The difference between the slopes is 8.49, with the SE of the slopes being 1.70. This difference in the slopes is statistically significant at P < 0.001. Displacement of [125I]SM-C by the purified somatomedin standard resulted in a dose-response curve with a slope of —24.11 ± 1.05. Statistical comparison of this dose-response curve to the separated plasma curve shows that they are parallel. The difference between the slopes is 1.50, with a SE of 1.60, so the

100

CHROMATOGRAPHED \ PLASMA

80 -

JCE&M • 1978 Vol47 • No6

difference is not significantly different from zero (P > 0.2). On the other hand, the whole plasma curve is not parallel to the purified SM standard. The difference between slopes is 9.99, with a SE of 1.59, so the difference is statistically significant at P < 0.001. Further separation of the peptide fraction of acid-chromatographed plasma by IEF reveals that there is peptide capable of displacing both [125I]SM-A and [125I]SM-C from the placental membrane receptor throughout the pH range from 5.6-8.4. As shown in Table 2, both radioligands are displaced by acid, neutral, and basic fractions from the TLIEF plate. Overall, the [125I]SM-A RRA detected 97.1% of the somatomedin peptide applied to the plate; the [125I]SM-C RRA detected 87.1%. No somatomedin peptide was detectable by either assay at a pH less than 5.6 or greater than 8.4. Furthermore, both assays detected comparable amounts of somatomedin peptide in each fraction. These results provide further evidence that the placental membrane RRA, using either [125I]SM-A or [125I]SM-C, is measuring total somatomedin content in plasma, not in just one particular species.

PURIFIED \^j SOMATOMEDIN

X,

-

WHOLE PLASMA

\

-

X

\ 60 -

\

\

I*

40 -

\

\

20 -

0 _

l l 1 1.0 5.0 10.0 20.0 1 1 1 30 50 100 [SM] ng/ml UNDILUTED PLASMA EQUIVALENTS ul/ml

\ \

-

_ 1 1 ) 5.0 10.0 20.0

1.0 1 1000 2000 UNDILUTED PLASMA EQUIVALENTS ul/ml

FIG. 4. Comparison of the competitive binding curves for [125I]-SM-C and whole and acid-chromatographed plasma with that for [I25I]SM-C and partially purified somatomedin peptide in the placental membrane RRA. The doses of partially purified somatomedin peptide are expressed on the abscissa as nanograms per ml; doses of whole and acidchromatographed plasma are expressed as microliter equivalents of undiluted plasma per ml. The ordinate depicts the percentage of maximal binding at each dose. For the partially purified somatomedin peptide, each data point represents the mean (±SEM) of 6 experiments; for whole plasma, each data point represents the mean (±SEM) of 7 experiments; for acid-chromatographed plasma, each data point represents the mean (±SEM) of 21 experiments.

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COMPARISON OF [125I]SM-A AND [125I]SM-C RRAs FOR SM TABLE 2. [125I]SM-A and [125I]SM-C RRA of fractions of acid-chromatographed plasma after TLIEF

pH Interval

4.5-5.6 5.6-6.5 6.5-7.3 7.3-7.9 7.9-8.4 8.4-9.0

SM peptide (mU) determined by [125I]SM-A

RRA

SM peptide (mU) determined by [125I]SM-C RRA

0 76 122 146 102 0

0 60 90 134 116 0

Five hundred microliters of the peptide fraction of acid-chromatographed plasma, equivalent to 460 mU somatomedin (SM) peptide were separated on a TLIEF plate. The fractions corresponding to the pH intervals listed in the first column were isolated, separated from ampholytes by column chromatography, lyophilized, redissolved, and assayed by both the [125I]SM-A and the [125I]SM-C RRAs. It is apparent that both assays detect SM peptides in the acid, neutral, and basic pH range and that they detect comparable amounts in each interval. Overall, the [125I]SM-A RRA detected 97.1% of the SM peptide applied to the plate; the [125I]SM-C RRA detected 87.1%.

Placental radioreceptor interassay variability for both SM-A and SM-C in both whole and separated plasma ranged from 8-10%. Intraassay variability, on the other hand, was more than twice as great for whole plasma (8-14%) as for separated plasma (4-6%) for both SM-A and SM-C. Both whole and separated plasmas were also assayed in the pig and rat costal cartilage bioassays. The mean potency of the separated plasma compared to that of whole plasma was 111% by procine bioassay and 137% by rat bioassay. Although the separated plasma seems to be slightly more potent than' the whole plasma in both bioassays, the variability of these assays does not allow the inference that separated plasma has more somatomedin activity per ml without more detailed studies. This is especially true in light of the evidence presented above that whole and separated plasmas are virtually equipotent in the placental membrane RRA using [125I]SM-A. Discussion The data presented here demonstrate a simple and valid means of more directly measuring the somatomedin peptide content in plasma using [125I]SM-C or [125I]SM-A. Acid

1293

chromatography of whole plasma before measurement of its somatomedin peptide content by the placental membrane RRA is practical and offers several advantages. The recovery data (Table 1) show that over 90% of somatomedin peptide in plasma is recovered after acid chromatography of whole plasma and lyophilization of the small molecular weight fraction. In addition, RRA of this lyophilized peptide fraction measures only displacement of radioactive trace from the membrane receptor by peptides whose molecular weights approximate that of the somatomedin peptide; larger and smaller molecular weight species are eliminated. The displacement curves of [125I]SM-A and [125I]SM-C by this peptide fraction are parallel to those of purified somatomedin peptide standard. Hence, one obtains a more precise measure of true plasma somatomedin peptide content without interference from specific or nonspecific inhibitors, proteolytic enzymes which might destroy the trace and/or membrane, or somatomedinbinding protein. This advantage might be especially important when plasmas from people with various diseases and GH states are assayed. The concentrations of such interfering substances may well vary from one person to another or from one disease to another. Studies are now in progress to substantiate this hypothesis. In the case of SM-A, our results indicate that, at least with normal plasmas, measurement of somatomedin peptide content in the placental membrane RRA using acid-chromatographed or separated plasma offers no advantage over its measurement using whole plasma, except for a slight decrease in variability. Both the whole plasma and the separated plasma dose-response curves are parallel to the dose-response curve obtained using the partially purified somatomedin standard (Fig. 3); and whole and separated plasma are almost equipotent in displacing the SM-A trace from the placental membrane. Takano (19, 34) has measured somatomedin in whole plasma in both normal and disease states using radioactive SM-A and has concluded that displacement of radiolabeled SM-A by whole plasma and purified nonradioactive SM-A are paral-

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HORNER, LIU, AND HINTZ

lei. Our results using the [125I]SM-A RRA with normal plasma concur with those of Takano. Whether our RRA for somatomedin using whole plasma and [125I]SM-A is a valid tool for the measurement of somatomedin peptide content in abnormal states is yet to be determined. In this study, measurement of somatomedin peptide by the placental membrane RRA and [125I]SM-C is valid only if the plasma is first separated by acid chromatography. As seen in Fig. 4, the separated plasma dose-response curve is parallel to the partially purified somatomedin dose-response curve. The relative potencies of whole and separated plasmas cannot be determined using [125I]SM-C because of the lack of parallelism of their dose-response curves. Furthermore, the variability in the measurement of somatomedin peptide is less when separated plasma is assayed. In the initial characterization of the [125I]SM-C placental membrane RRA (20), it was concluded that whole plasma may be accurately measured using this assay. The discrepancy between these earlier data and the present results may be due to a subtle difference in the [125I]SM-C preparations. Another possible explanation is that our data have been derived from dose-response curves for standard somatomedin and whole plasma for SM-C using a much larger number of samples, such that the difference in slopes becomes significant (P < 0.001). Although measurement of NSILA-S is invalid if unextracted serum is assayed (27), its measurement is accurate and correlates directly with the GH status of the donor if the serum if first extracted by acid gel filtration before its assay in the rat liver membrane RRA (29, 35), the fibroblast RRA, or the bioassay (28). These findings are very similar to our findings with the placental RRA for somatomedin using [125I]SM-C. Our measurements of somatomedin peptide content in acid-chromatographed plasma using [125I]SM-A and [125I]SM-C result in doseresponse curves that are not only parallel (P > 0.2) but virtually superimposable, consistent with both assays measuring the same substances (Fig. 3 and 4). Furthermore, it has

JCE&M • 1978 VoU7 • No 6

been shown that purified SM-A and SM-C cross-react comparably in the placental membrane RRA when either [125I]SM-A or [125I]-

Comparison of [125I]somatomedin A and [125I]somatomedin C radioreceptor assays for somatomedin peptide content in whole and acid-chromatographed plasma.

0021-972X/78/4706-1287$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1978 by The Endocrine Society Vol. 47, No. 6 Printed in U...
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