Simultaneous radioimmunoassay of secretin and gastrin

ANALYTICAL

BIOCHEMISTRY

Simultaneous

74, 12- 24 ( 1976)

Radioimmunoassay

HSIN-HSIUNG TAI’ The Isaac Gordon Department

of Secretin and Gastrin

AND WILLIAM

Y. CHEY

Center of Gastroenterology, The Genesee Hospital, of Medicine, University of Rochester School of Medicine

and Dentistry, Rochester, New York 14607

Received August 6, 1975; accepted March 23, 1976 Radioimmunoassay techniques for the measurement of gastrointestinal hormones have been modified to allow simultaneous assay of both secretin and gastrin in the same plasma samples. The system employs antibodies with high specificity for these two hormones and both 1251-labeted secretin and 1311-labeled gastrin as markers. Separation of the free from the bound form of the hormones was achieved by plasma- and dextran-coated charcoal. The unique separation method has been found to be independent of the uniformity of plasma samples. The sensitivity and the reliability of the simultaneous assays were comparable to those of the single assays. The fasting levels of human plasma secretin and gastrin obtained by the simultaneous assay were 62.9 f 3.6 and 43.5 2 2.8 pg/ml respectively.

Both secretin and gastrin play important roles in a variety of gastrointestinal functions. Secretin stimulates exocrine pancreatic secretion rich in bicarbonate, inhibits gastric acid secretion, and relaxes smooth muscle of the gastrointestinal tract, while gastrin stimulates gastric acid secretion as well as enzyme secretion, inhibits small intestinal electrolyte and glucose absorption, and stimulates gastric motility. Radioimmunoassays of both hormones have been developed in various laboratories (l-7). Measurements of the plasma levels of both hormones are often needed for better assessment of gastrointestinal functions. We have recently developed a simultaneous radioimmunoassay system to determine the levels of both hormones concurrently in the same assay tube by using highly specific antisera and labeled hormones of different isotopes, lz51 and 1311. Such a modification can be performed not only without loss of the sensitivity, specificity, and precision of the individual assays, but it can more than double the efficiency with which both hormones may be measured. MATERIALS Anfibodies.

previously

Antibodies to secretin were produced and evaluated as described (4). Antibodies to gastrin were generated in a fashion

1 Correspondence to: Dr. Hsin-Hsiung Tai, Isaac Gordon Center of Gastroenterology, The Genesee Hospital, 224 Alexander Street, Rochester, N. Y., 14607. 12 Copyright 0 1976 by Academic Press. Inc. All rights of reproduction in any form reserved.

RADIOIMMUNOASSAY

OF

SECRETIN

AND

GASTRIN

13

similar to that for secretin (4) except that synthetic human gastrin I residues 2 through 17 (SHG: 2- 17) conjugated to bovine serum albumin was used as immunogen. The antibodies to gastrin did not cross react significantly with any other known gastrointestinal hormones including secretin, glucagon, motilin, CCK, VIP, and GIP. Labeled hormones. Synthetic porcine secretin was iodinated with Nalz51 by a modification of the chloramine T method and purified by SP-Sephadex C-25 column chromatography as described by Tai et al. (8). The specific activity of *251-labeled secretin was 500-550 ,zCi/~g. SHG: l- 17 was iodinated with Na1311 and purified by aminoethyl cellulose column chromatography as described by Stadil and Rehfeld (9). The specific activity of 1311-labeled gastrin was 600 &i/pg. Standards. Synthetic porcine secretin (obtained through the courtesy of Dr. Miguel Ondetti, Squibb Institute for Medical Research) and SHG: l-17 (Imperial Chemical Industries, England) were used as standard secretin and gastrin preparations, respectively. Other chemicals. Carrier-free Na1251 in NaOH was purchased from New England Nuclear Corporation. Trasylol (100,000 KIU/ampule) was purchased from FBA Pharmaceuticals. Norit A and dextran (MW 86,000) were products of Amend Drug and Chemical Company and K & K Laboratories, Inc. Bovine serum albumin (fraction V) and bovine y-globulin were from Sigma Chemical Company. Heparin was obtained from Fisher Scientific Company. METHODS Conditions of incubations. Both labeled hormones and unlabeled standards were respectively dissolved in 10% hormone-free human plasma (prepared by charcoal adsorption) in saline containing 500 KIU of Trasylol/ml of diluted plasma. The presence of a minute amount of plasma was found to be necessary to prevent secretin from glassware adsorption. Antisera of both hormones were diluted in the standard buffer, 50 mM sodium phosphate, pH 7.5, containing 0.1% bovine serum albumin (BSA). For routine measurements the procedure was the following: 0.2 ml of standard of varied concentrations of both secretin and gas&in or 0.2 ml of plasma sample was preincubated with 1.1 ml of diluted antisera of both secretin and gastrin (final dilutions were 300,000 and 100,000, respectively) for 24 hr before the addition of 0.2 ml of labeled hormones (about 5000 cpm each of 1251-secretin and 1311-gastrin). Postincubation was continued for another 48 hr. Both pre- and postincubation were performed at 4°C. All assays were set up in triplicate in 10 x 75-mm glass culture tubes. Separation procedure. Free and bound antigens were separated by dextran- and plasma-coated charcoal. Methods of preparing dextran- and plasma-coated charcoal were as follows. Dextran was first dissolved in

14

TAI AND CHEY

A o-o-oGASTRIN

d.l-

0.5

5

50

rig/TUBE

FIG. 1. Cross reactivity of secretin and gastrin antibodies with gastrin and secretin. Antibodies were respectively incubated with hormones at a series of concentrations under the conditions described in Methods. Ratio (B/T) of antibody-bound (B) to total (T) labeled hormone is plotted against the concentration of unlabeled hormone. (A) Secretin antibody; (B) gastrin antibody.

standard buffer (50 mM sodium phosphate, pH 7.5, with 0.1% BSA) at a concentration of 9 mg/ml. Not-it A was added to the dextran solution at a concentration of 90 mg/ml. The mixture was stirred overnight at 4°C. To the dextran-charcoal solution was then added an equal volume of 50% human plasma dialyzed against saline, and the mixture was further stirred overnight. When separation of the free from the bound antigens was required, 0.2 ml of human plasma dialyzed against water was added to the standard tubes for better uniformity of plasma protein concentration between standard and sample tubes. Immediately, 0.4 ml of the plasma-dextran-charcoal suspension was added to each tube and left for 20 min at 4°C. The tubes were then centrifuged at IOOOg for 10 min at 4°C. The supematant containing the antibody-bound hormones was decanted from the hard charcoal pellet containing the free hormones. When comparison of different separation methods was required, the dextran-

RADIOIMMUNOASSAY

OF SECRETIN

I

I

AND GASTRIN

,

15

#

o 6 B.GASTRIN 0.5 + 0.4

I \

T 0.3

¶

0.2 0.1 1

I

kT-T&x-

200

pg/TUBE FIG. 2. Superimposability of individual assays and simultaneous assay. Incubation and separation of bound from free labeled antigens were as described in Methods. (0) Represents values obtained by individual radioimmunoassay; (A) represents values obtained by the simultaneous assay method.

charcoal solution was either coated with an equal volume of 0.75% bovine y-globulin in saline (w/v) or mixed with an equal volume of saline under the same conditions used for coating with dialyzed plasma. Separation of the free from the bound form of labeled hormones was carried out as described above. Analysis of samples. Both the supernatant and the hard charcoal pellet were counted for 2 min in a Packard two-channel Auto Gamma spectrometer with window setting adjusted to 18 to 50 keV for monitoring lz51 radioactivity, and 275-375 keV for monitoring 1311radioactivity. At these settings the overlap from the 1311region into the region monitoring lz51 radioactivity remained relatively constant at 22.5%. Accordingly, counts in the lz51 channel were corrected by subtracting 22.5% of the counts recorded in the 1311 channel. The corrected bound to total ratio was calculated for each sample in both channels after subtracting the bound to total ratio of the control tube. A straight-line standard curve is fitted by the

16

TAI AND CHEY

A.SECRETlN

I

1

I

I

1

&LGASTRIN 0.5

kK: 0.2 0.1 I

I

I

,

50

100

150

200

pg/TUBE FIG. 3. Effect of preincubation of antibodies with unlabeled hormones of the sensitivity of radioimmunoassay. Antibody was either incubated directly with labeled and unlabeled hormone for 24 (- 0 - 0 -) and 48 hr (- A - A -) or preincubated with unlabeled hormones for 24 hr followed by postincubation with labeled hormones for 24 (- 0 - 0-) and 48 hr (- A - A -).

method of unweighted least squares to the logit transform function of BIB, plotted against the logarithm of the concentration of unlabeled hormones. The logit transform function is defined as follows: logit(B/&J

= log

BIB,

1 - BIB0

’

where B is the bound to total ratio of each sample and B,, is the same ratio obtained in the absence of unlabeled hormones. Computer programs for use on a Monroe Model 1860 computer were developed for the analysis of calculating the bound to total ratio and the logit transformation. Sample concentrations of each hormone were determined from the equations of the curves so derived and were printed out by the computer. Collection of human plasma samples. Samples of peripheral venous blood were drawn into heparinized tubes after an overnight fast. Plasma

RADIOIMMUNOASSAY

OF SECRETM

AND GASTRIN

17

A. SECRETIN 0.6 05

1

04-

VW==---8

4T 0.3-

./

/-

YA

0.20.1-

A/.

B.GASTRIN 0.6 0.5-

1

0,4$0.3-

7--. A'

.---

0.1

0.15

/ 0.20.1-

/ A I

3

I

0.05

0.20

0.25

VOLUMEOFPLASMA (ml) FIG. 4. Effect of plasma concentration on separation of bound from free labeled hormones by different methods. Antibody was incubated with iabeted hormone in a final volume of 1.5 ml of standard buffer containing 0.02 ml of plasma for 24 hr. Plasma was then added to the indicated concentration before carrying out separation by (- A - A -) dextran-charcoal, (- W - W -) dextran-charcoal coated with bovine y-globulin, and (- 0 - 0 -) dextran charcoal coated with dialyzed plasma. The coating procedure and conditions were as described in Methods.

was separated rapidly and treated with Trasylol at a concentration KIU/ml and kept frozen at -20°C until the time of the assay.

of 500

RESULTS

Antibody

Specificity

For the simultaneous assay of secretin and gastrin, it is most important that the antibody to one hormone exhibits minimal cross reactivity with the other hormone. Figure 1 shows that little cross reactivity (
18

TAI AND CHEY

a s k Q

150-

IOO-

50-

1 0.05

I 0.10

I

0.20 VOLUMEO’FPLASMA(ml) 0.15

I

0.25

FIG. 5. Linearity of the simultaneous radioimmunoassay. A plasma sample of high concentration of exogenously added secretin and gas&in was assayed at a series of dilutions of plasma under the conditions described in Methods. The measured concentrations are plotted against the volumes of plasma assayed in 1.5 ml of final incubation mixture. (-•l -) Represents secretin concentration; (- A - A -) represents gastrin concentration. Superimposability

of Individual Assays and Simultaneous Assay

Standard curves were determined from a set of samples in which the concentrations of secretin and gastrin were increased simultaneously over their respective concentration ranges. Hand-drawn curves derived from such an assay, together with those from a concurrent single assay for comparison, are illustrated in Fig. 2. The curves for individual and simultaneous radioimmunoassay are indistinguishable when antibody dilutions and marker concentrations are identical. Conditions of Incubations

Antibodies were either preincubated with unlabeled hormones for a period of time followed by the addition of the labeled hormones or directly incubated with both labeled and unlabeled hormones. The sensitivity of the assay was found to be greatly increased by preincubation, particularly for the assay of secretin. Figure 3 shows that preincubation of 24 hr followed by 48 hr of postincubation gave the best sensitivity of the simultaneous assay. Extension of either preincubation of postincubation, or both, for another 24 hr did not significantly increase the sensitivity of the assay. Therefore, for routine assays 24 hr of preincubation followed by 48 hr of postincubation were favorably used.

RADIOIMMUNOASSAY

OF SECRETIN TABLE

PRECISION

OF SIMULTANEOUS

19

AND GASTRIN

1

MEASUREMENTS

OF PLASMA

SECRETIN

AND GASTRIN~ Coefticient of variation (%)

Plasma

I

2

3

4

5

6

7

8

9

IO

Mean + SEM Wml)

Secretin Gastrill

58.2 83.3

63.0 87.2

69.9 79.7

60.7 89.9

56.0 80.8

60.9 84.6

64.8 81.9

69.5 85.6

64.0 87.2

68.1 84.9

63.5 k I.5 84.5 r 1.0

7.4 3.7

Secretin Gash

91.7 228.4

98.9 215.7

99.8 238.3

95.2 241.0

99.3 221.4

99.5 231.4

97.2 236.3

97. I 219.3

80.7 234.8

97.6 213.6

95.7 t 1.8 228 f 3.1

6.1 4.3

Sample

” Ten “standards”

made from two plasma samples

number

were assayed

simultaneously

in triplicate.

Separation The plasma- and dextran-coated charcoal effectively separated the free from the bound form of both hormones. The background radioactivity not bound by the charcoal in control tubes was, in most cases, between 4 and 7%. When the background radioactivity reached lo%, the labeled hormones were usually discarded. Figure 4 shows that separation of the free from the bound form of hormones by commonly used dextrancharcoal was affected by the plasma concentrations. If the dextrancharcoal was further coated with y-globulin, the dependency on the uniformity of plasma was reduced and more complete separation of the bound from the free form was achieved. However, a total independence was observed with dialyzed plasma- and dextran-coated charcoal as evidenced by the flat type of relationship. A variety of other factors including sodium chloride (150 mM), urea (100 mM), creatinine (10 mM), glucose (10 mM), and heparin (25 U/ml) also did not affect the separation procedure carried out by plasma- and dextran-coated charcoal. Although dextran did not appear to be necessary for total independence, inclusion of dextran always gave better precipitation of charcoal for clear decantation. Reliability

of the Assay

(i) Linearity of the assay. A plasma sample of high concentration of both secretin and gastrin produced by adding exogenous synthetic hormones was assayed at a series of plasma dilutions. The measured concentrations fell linearly with dilution of the plasma as shown in Fig. 5. (ii) Precision ofthe intraassay. Aliquots of two pooled human plasmas were tested 10 times in triplicate in the same assay. The coefficient of variation are shown in Table 1. (iii) Reproducibility ofth e interassay. Three human plasmas of different secretin and gastrin concentrations were kept frozen and assayed on four different occasions. The coefficients of variation are shown in Table 2.

20

TAI AND CHEY TABLE

REPRODUCIBILITY

OF SIMULTANEOUS

MEASUREMENTS

Plasma 1 (pg/ml) Assay number

Mean f SEM Coefficient of variation (%)

Secretin

2 OF PLASMA

Plasma 2 (pg/ml)

SECRETIN

AND GASTRIN~

Plasma 3 (pg/ml)

Gastrin

Secretin

Gastrin

Secretin

Gastrin

95.6 95.6 86.6 63.2

105.3 86.2 93.5 100.9

171.1 193.6 186.5 174.3

244.1 214.0 241.8 236.3

363.6 368.5 371.8 362.0

429.8 355.1 460.5 431.6

85.3 f 7.7

96.5 t 4.2

181.4 2 5.3

234.2 -t 7.0

366.5 + 2.3

419.3 + 22.5

17.9

8.7

5.8

5.9

1.2

10.7

o Three different plasma samples were assayed four times in triplicate during a period of 1 week. Plasmas 2 and 3 were produced to contain high concentrations of secretin and gastrin by exogenously adding these two hormones.

Determination of Plasma Secretin and Gastrin Normal Human Subjects

Concentration

in Fasting

The secretin and gas&in concentrations in plasma of 44 fasting normal human subjects were determined by simultaneous radioimmunoassay. Variations between individuals of plasma secretin and gastrin levels are shown in Fig. 6. The mean values f SEM were 62.9 ? 3.6 (ranging from 19.5 + 139.70) p&ml for secretin and 43.5 + 2.8 (ranging from 22.3 to 109.1) pg/ml for gastrin. DISCUSSION The results of these studies suggest that the simultaneous radioimmunoassay of secretin and gastrin can be performed without loss of the sensitivity, specificity, and precision of the individual assays. When used in conjunction with the computer program, the method more than doubles the efficiency with which both hormones may be measured. The sensitivity of the simultaneous assay apparently was greatly increased by preincubation of antibodies and unlabeled hormones, particularly secretin. Preincubation for 24 hr followed by 48 hr of postincubation gave the best sensitivity for secretin assay. The improvement is not due to the longer total incubation time of 72 hr since incubation directly for 72 hr without preincubation resulted in a displacement curve comparable to that of 48 hr of incubation shown in Fig. 3. Improvement of the assay sensitivity obtained by delayed addition of the labeled antigen has also been reported in the assay of a variety of hormones, such as luteinizing hormone (lo), oxytocin (1 l), and growth hormone (12). A theoretical analysis of the kinetics of radioimmunoassay system in these aspects has been dealt with by Robard et al. (13).

RADIOIMMUNOASSAY

OF SECRETIN

AND GASTRIN

21

. * .

100 i 80 * B

.A :;; -p!!i.

60-

40200'

.

SECRETIN

GASTRIN

FIG. 6. Plasma secretin and gastrin concentrations in fasting normal human subjects as assayed by the simultaneous method.

Current sensitivity of the radioimmunoassay for secretin and gastrin can be assessed from the sharp displacement curves for both hormones obtained under the optimal incubation conditions. The fall in bound/total ratio produced by 5 pg/tube of either hormone was much greater than the variation in replicates at 0 and 5 pg/tube. It is evident that concentrations of both hormones lower than 5 pg/tube are easily measurable. Since the fasting plasma levels of either hormone are of the order of 50 pg/ml, an aliquot of 0.2 ml of plasma routinely used for assay definitely contains a sufficient hormone concentration to falf into the sensitive range of the standard curves. Various methods have been employed to separate the free from the bound form of the labeled secretin or gastrin. These methods include ion-exchange resin absorption (1,6,7), dextran-charcoal adsorption (7) and second antibody precipitation (23). Since an acidic peptide, gas&in, and a basic peptide, secretin, represent two extremes of ionic properties, a single type of resin separation cannot be used for the simultaneous purposes. Methods which precipitate or exclude the antigen-antibody complex from binding to the adsorbents are naturally the methods of choice. Among these methods, second antibody precipitation, polyethylene glycol-KI precipitation (14), and dextran-charcoal have been tried. However, second antibody precipitation has been troubled by the inconsistent avidity of the second antibody supplied by commercial sources, dependence on uniform protein concentrations, and other factors. Similar difficulties have also been reported by several laboratories using second antibody precipitation (15- 17). Although second antibody derived from various sources may behave differently in the degree of dependence

22

TAI

AND

CHEY

on uniformity, it is advisable to check on this aspect before use. Polyethylene glycol-KI precipitation initially advanced by Desbuquois and Aurbach (14) has been used in the assay of various hormones. In our hands this type of separation still possessed a limited range of protein concentrations for consistency of precipitation. Dextran-charcoal adsorption was also found to be dependent on the uniformity of plasma samples as illustrated in this paper. Our studies indicated that the dextran coating was not effectively preventing the antigen-antibody complex from being bound to the charcoal. However, if the dextran charcoal was further coated with dialyzed plasma or y-globulin, the binding of this complex was minimized and little dependence on the uniformity of plasma samples could be demonstrated. The fact that the plasma-dextran-charcoal separation is independent of the presence of a wide range of urea, heparin, glucose, sodium chloride, and proteins allows this unique separation method to be used in various plasma samples, including those from patients with uremia and diabetes mellitus. The linearity of the assay performed at a series of plasma dilutions indicated that no other substances in the plasma significantly affect the antigen-antibody interactions. Although sodium chloride has been reported to affect the antigen-antibody interactions in the assay of gastrin (7) and ACTH (18), preparation of standard curves as well as dilution of plasma were always carried out in saline so that the concentrations of NaCl were approximately the same in both samples and standards. The level of plasma gastrin in fasting normal human subjects measured by the simultaneous assay agreed well with the values obtained by single assay reported by several laboratories (5-7). Most of these laboratories reported values ranging from 32 to 105 pg/ml. Slight variations could be due to the use of the control hospital patients as normal subjects, the inclusion of a varying number of persons with hypochlorhydria and gastritis in the normal group, and the use of very small groups. However, conflicting secretin levels have been presented by different investigators. Levels ranged from well below 100 pg/ml(3) to 642 ? 60 pg/ml(l9). The secretin level of 62.9 pg/ml in fasting normal subjects reported in this communication was much lower than our previously reported level of 467 pg/ml. The significant drop in the secretin level was in part attributed to the separation technique employed. Previously we used second antibody to precipitate the hormone-antibody complex. It was found that the bound/total ratios varied significantly in different hormone-free plasma when precipitated was carried out by second antibody. However, if separation was affected by the current technique, bound/total ratios showed little variation in different hormone-free plasma. These observations suggest that the primary antigen-antibody interactions are not significantly altered by the plasma factors, while the second antibody precipitation of the hormoneantibody complex is significantly affected. It is possible that the

RADIOIMMUNOASSAY

OF

SECRETIN

AND

GASTRIN

23

hormone-free plasma used then for preparing standard curves had an intrinsically higher bound/total ratio so that the plasma samples yielded falsely high concentrations of secretin. It is advisable to check the linearity of the plasma dilution curve as demonstrated in Fig. 5 to validate the applicability of the standard curve. Other explanations may be related to the improvement in the quality of the labeled secretin as described previously (8). The great discrepancies in secretin level presented by different investigators could also be related to different synthetic and natural standards, to the antisera, to the separation technique, and to the quality of labeled secretin. Since pure human secretin has not been available for cross-reactivity studies, the reported level of human plasma secretin should be regarded as porcine secretin equivalents. The wide range over which both assays may be conveniently used as demonstrated by this paper provides a great deal of latitude with respect to the ratios of two hormones which can be measured in a single aliquot of a particular sample. The simultaneous radioimmunoassay, therefore, allows the measurement of both hormones in certain cases of hormone stimulation where the concentration of one hormone increases substantially while the concentration of the other hormone changes slightly or remains constant. In fact, the working ratio of the two hormones could be further enlarged by employing a different antibody with intrinsically greater or less sensitivity. Such an alteration would be particularly helpful in studying certain aspects of the interaction of the two hormones by infusing one hormone and following the levels of both hormones kinetically. Recent application of these ideas by our laboratory has demonstrated that the plasma level of gastrin was elevated following ingestion of a meat meal while that of secretin was not significantly changed both in dogs (20) and in humans (21). On the contrary, the plasma level of secretin was elevated after infusion of acid into the duodenum while that of gastrin remained unchanged (20,21). Although meat meal studies and intraduodenal acid infusion studies indicated that secretin and gastrin appeared to be released by separate mechanisms, there is still a wide variety of test agents that are needed to confirm this observation and to elucidate further the mechanism of release. Application of the simultaneous assay will certainly facilitate the progress of these studies. Moreover, secretin and gastrin are widely distributed in the entire gastrointestinal tract. Plasma and tissue levels of these two hormones in relation to various states of gastrointestinal diseases have not been widely investigated. Whether there is any correlation between hormone levels and pathological states remains to be elucidated. The technique of the simultaneous assay will be a valuable tool to achieve these objectives. Although secretin was routinely labeled with lz51 and gastrin with 1311 because of the scarce supply of the synthetic secretin, either hormone

24

TAI AND CHEY

could be labeled with lz51 or 1311 and used for the simultaneous radioimmunoassay. Furthermore, it should be possible, in principle, to perform the simultaneous radioimmunoassay of any combination of two hormones provided that each antibody has a high degree of specificity and the hormones are labeled with different isotopes. Such a possibility, in fact, has been demonstrated by the simultaneous radioimmunoassay of insulin and growth hormone (22) and of cyclic AMP and cyclic GMP (23). REFERENCES 1. Young, J. D., Lazarus, L., Chisholm, D. J., and Atkinson, F. F. V. (1%8)3. Nucl. Med. 6, 641. 2. Boden, G., and Chey, W. Y. (1973) Endocrinology 92, 1617. 3. Bloom, S. R., and Ogawa, 0. (1973) J. Endocrinol. 58, 24. 4. Boehm, M., Lee, Y., and Chey, W. Y. (1974) in Endocrinology of the Gut (Chey, W. Y., and Brooks, F. P., eds.), p. 310, Charles B. Slack, Thorofare, N. J. 5. McGuigan, J. E., and Trudeau, W. L. (1970) Gastroenterology 58, 139. 6. Yalow, R. S., and Berson, S. A. (1970) Gastroenterology 58, 1. 7. Stadil, F., and Rehfeld, J. F. (1973) Sand. J. Gastroenterol. 8, 101. 8. Tai, H. H., Korsch, B., and Chey, W. Y. (1975)Anal. Biochem. 69, 34. 9. Stadil, F., and Rehfeld, J. F. (1972) Stand. J. Clin. Lab. Invest. 30, 361. 10. Midgley, A. R. (1966) Endocrinology 79, 10. 11. Chard, T., Forsling, M. L., James, A. R. Kitan, M. J., and Landon, J. (1970). J. Endocrinol.

46, 533.

12. Jacobs, H. S. (1%9)J. Clin. P&o/. 22, 710. 13. Rodbard, D., Ruder, H. J., Vaitukaitis, J., and Jacobs, H. S. (1971)3. C/in. Endocrinol. 33, 343.

14. Desbuquois, B., and Aurbach, G. D. (1971)5. Clin. Endocrinol. 33, 732. 15. Buckler, J. M. H. (1971) in Radioimmunoassay Methods (Kirkham, K. E., and Hunter, W. M., eds.), p. 273, Churchill & Livingston, London/Edinburgh. 16. dourt, G., and Hum, B. A. L. (1971) in Radioimmunoassay Methods (Kirkham, K. E., and Hunter, W. M., eds.), p. 283, Churchill & Livingstone, London/Edinburgh. 17. Burr, I. M., Grant, D. B., Sizonenko, P. C., Kaplan, S. L., and Grumbach, M. M. (1%9) J. Clin.

18. 19. 20. 21. 22.

Endocrinol.

29, 948.

Berson, S. A., and Yalow, R. S. (1968) J. C/in. Invest. 47, 2725. Kolts, B. E., and McGuigan, J. E. (1974) Gastroenterology 66, 849. Lee, K. Y., Tai, H. H., and Chey, W. Y. (1976) Amer. J. Physiol. 230, 784. Rhodes, R. A., Tai, H. H., and Chey, W. Y. (1976) Amer. J. Dig. Dis. In press. Morgan, C. R. (1966) Proc. Sot. Exp. Biol. Med. 123, 230. 23. Wehmann, R. E., Blonde, L., and Steiner, A. L. (1972) Endocrinology 90, 330.