Sensitivity, range, and reliability of a method for the bioassay of gastrin

Journal of Surgical Research

SENSITIVITY,

J.

Volume

9 N,umber 10, October 1969

WILXEN, c.

THE

and Laboratory

RANGE, FOR THE B.

AM.

Clinical

D.

METHOD

F.

TURNER,

FOH

AND RELIABILITY BIOASSAY OF M.D.,

LOWE,

F.R.C.S., M.D.,

PH.D.,

THE

Investigation

0.

IV. AND

BIOASSAY

J.

ot

gastrin-like activity in tissue extracts, reported by Lai [ 11, was based on a technique originally described by Ghosh and Schild [2]. Although purified gastrin was used for determining dose response relationships, the results obtained in Lai’s experiments suggested that the sensitivity of the method was low and that very small amomts of gastrin were unlikely to be detected. Barrett and his co-workers [3] investigated the effects of various anaesthetic agents on the secretory response of the perfused rat stonlach to gastrin, gastrin pentapeptide (ICI 50, 123) and histamine. They found that the smallest dose of pure gastrin required to produce a response was 0.1 rig/kg and that the activity of gastrin was approximately ten times that of the pentapeptide on a molar basis. They further reported a response to gastrin and gastrin pcntapeptide with intraperitoneal urethane, but no such effect was seen with intramuscular urethane or with any of the other agents used. The value of the rat bioassay method in the preoperative diagnosis of the Zollinger-Ellison svndrome was clearly demonstrated b!T Moore From the Department of Surgery and University Hospital, University of Mississippi hledical Center, Jackson, Mississippi 39216. Aided by U.S.P.H.S. Grant No. All-05122. Submitted for publication Feb. 10, 1969.

A. D.

BILLUPS, HARDY,

OF

A

METHOD

GASTRIN G.

HUNT,

M.D., M.D.,

M.D.,

F.A.C.S.

and his associates [4]. They reported ten patients giving a positive response for gastrinlike activity in the serum, urine, gastric juice or tumor extract, but gave no details of the method used for calculating the response. Using a similar method of assay, Ellison and Wilson [5] reported the occurrence of several false negative results in patients known to have the Zollinger-Ellison syndrome. In earlier studies, Bonfils and associates [6] noted an increased gastric secretion of hydrochloric acid in gastric fist& rats which were given intravenous doses of urine from two patients with the Z-E syndrome. Such an increase was not produced when urine from a healthv subject was administered. The demonstration by Tracy and Gregory [7] that the C-terminal tetrapeptide of gastrin possessesall the physiologic properties of the total molecule resulted in the synthesis of an active pentapeptide [ICI 50, 123; Ayerst Laboratories: AY 66083. The availability of this synthetic gastrin pentapeptide permits precise study of its action in laboratory animals and allows more direct comparison with the results obtained by other workers. In view of the conflicting reports concerning the value of the bioassay method, it was felt necessary, in establishing a technique for gastrin assay in our laboratory, to investigate its range, reliabilit!,, and sensitivity, using the synthetic pentapeptide AY:6608. This paper

JOURNAL

OF

SURGICAL

RESEARCH

VOL.

9,

h-0.

10,

Table 1. Dose Response to Gastrin Pentapeptide in Anesthetized Rat in Microequivalents of HCZ

reports the results of a series of experiments designed for this purpose. METHODS

AND

Dose of Pentapeptide (pg./kg.)

MATERIALS

The method used was based on that described by Lai and has been previously described in detail, together with an account of the factors which affect it. In brief, the stomach of an anesthetized total gastric-fistula rat was continuously perfused with warm saline, at a constant rate of 0.5 ml./min. The gastric effluent was collected in 20-minute samples of approximately 10 ml. and the acid content of each sample determined by titration to pH 7. A continuous intravenous infusion of saline at 1 ml./hr. was commenced and the preparation allowed to stabilize for a period of one hour before three 20-minute baseline collections were obtained. Thereafter, a test solution was substituted for the saline and a volume of 0.5 ml. infused over a period of 30 minutes. An interval of at least 90 minutes was allowed to elapse before a second test substance was given to the same animal. During the course of a single experiment it was possible to run three or four tests on the same preparation, Method Statistical

of Cakulution

of Response

(B)

(C)

15 3-42

25 22 7-50

lo-93

34 25-75

t 11

112

*17

i21

t

-t

(A)” 1.0 Mean

Range Standard deviation Standard error 04 Error

2.3

15.3

5.0 34

2.6

11.6

3.6 10.6

(D) 10.0

i

4.5 13.2

a Four doses of gastrin pentapeptide, A, B, C, and D were tested in all possible sequences in four groups of six rats each. Mean values given here are the average response for all rats ignoring the order in which the doses were given. A sienificant difference in response was achievzd only wi;h five- to ten-fold dose increments.

2.5, 5.0, and 10 pg./kg. were assayed in four groups of six rats each. Each animal received all four doses and all possible sequenceswere employed. The calculation of response to each dose was made difficult by overlap, and it was clearly affected by the order in which the doses were given; however, the mean results are reported in Table 1. A comparison of the

IO-

and

Analysis

Results were expressed in micro-equivalents of hydrochloric acid per 20 minutes and the response to a given stimulus calculated as total excess acid, by subtracting the mean baseline acid from that produced following the infusion of the test substance. In the great majority of cases the response was complete within 1 hour and 20 minutes, but occasionally it continued over a period of 1 hour and 40 minutes. The significance of this response was analyzed by comparing the mean baseline acid secretion with the mean test acid secretion, employing the t test for paired data. Dose-Response

1.0 GASTRIN PENTAPEPTIDE dw 0.1 -

O.O1 Ib pEq HU.

Relationships

Dose response relationships were investigated by two methods: (1) Four doses of gastrin pentapeptide 1.0, 552

1969

OCTOBER

Fig.

1. Dose response tide. Dose response curve of excess acid against log peptide.

for gastrin pentapepof acid output in pEq. dose of gastrin penta-

\VILKEK

Table 2.

Dose Response to Gastrin Pentapeptidc

No. of Rats

Standard Deviation

h4ean

0.01”

1.2 6 0.05 7 4.1 0.1 10 9.3 10 12.8 1.0 2.5 10 16.1 5.0 9 20.2 10.0 8 26.0 25.0 8 55.0 50.0 7 51.4 i* The response to this dose of gastrin pentapeptide

25-

w

w

5PJm t

w

t

OF

GASTRIN

MS

of Excess HCl Standard Error

Range -5to -1to -1 5 8 10 13 11 10

to to to to to to to

ll. 9 35 22 26 40 36 124 92

6 RATS)

PENTAPEPTIDE:

w9m

-

-

20PEq HCL

BIOASSAY

2Y 2.2 F 5.4 t 3.7 -c 1.4 F10.8 ir 3.2 i $5.3 -c 1.6 T!! 1.8 i 5.6 .” 3.1 ir 9.4 ir 6.8 I 2.4 k33.5 111.8 125.8 rt 9.8 was not statistically significant.

(MEANSOF GASTRIN IraN

AL.:

in Anesthetized

Micro-equivalents

Gastrin Pentapeptide (pg./kg. )

ET

t

*

-

*

-

t

r.~.

15-

:

SUCCESSIVE

TWENTY

MINUTE

COLLECTION

PERIODS

Fig. 2. Effect of repeating the same dose of gastrin pentapeptide. For method of calculating excess HCI, see the Methods section, page 552. Dose Gastrin Pentapeptide I%%~ 1.0 5.0 10.0

Mean Excess HCI -1. Standard Deviation (6 Rats) 11.9 1 4.6 30.2 t 25.1 24.6 + 8.3

16.9 2 7.9 35.2 +- 23.3 25.8 I 9.2

20.8 I 10.7 26.6 I 26.3 28.0 _+ 10.4

There was no significant difference in response to successive doses at any dose level of gastrin pentapeptide.

ranges and standard deviations with those given in Table 2 make the overlapping effects obvious. (2) In the second method, gastrin pentapeptide in doses ranging from 0.01 pg./kg. to 50 pg./kg. was assayed in separate groups of six to ten rats. The results obtained are detailed

in

Table

2. When

these

results

were

plotted on semilog paper, the relationship of response to incremental dose was approximately linear until doses greater than 10 pg./

kg. were given (Fig. 1) . A significant increase in response was obtained only with doses differing by a factor of 10. Although the 0.05pg./kg. dose of gastrin pentapeptide gave a significant response, this was invariably small and the lowest dose of gastrin pentapeptide which can be reliably detected in this preparation is 0.1 pg./kg. (total dose of 30 mugm. in a 300-gm. rat). No response to the 0.01~ug. dose could be detected, even in very sensitive animals. 553

JOURNAL

OF

SURGICAL

RESEARCH

VOL.

9,

NO.

10,

OCTOBER

1969

12 ‘kg G. II ICI 9 a pEq

7

MEAN

MEAN

RESPONSE OF 8 RATS

RESPONSE

HCL 6 5 4

TWENTY

MINUTE

COLLECTION

PERIODS

Fig. 3. Double responseto low dose of gastrin pentapeptide. Efect of Repeating the Same Dose of Gas&in Pentapeptide The effect of repeating the same dose of gastrin pentapeptide was investigated in three groups of six rats. Doses of 1, 5, and 10 pg./ kg. were each given on three occasions to the same preparation, with an interval of 1 hour 20 minutes between them. The results are depicted in Fig. 2. At the S- and lo-pg./kg.dose levels, there was no significant difference in the mean response to successive doses. At 1 pg./kg. the mean response increased with the second and third doses, but the increase was not statistically significant.

Response to Histamine No detectable response was obtained when a dose of 10 pg./kg. of histamine base was assayed in a group of eight rats. A dose of 50 ug./kg. of histamine, in the same number of animals, produced a small, but significant increase in acid output (Fig. 4). When the histamine was preceded by 0.5 ml. of serum from

MEANOF 6 RATS FOR EACH

Double Response to Gastrin Pentapeptide Gastrin pentapeptide, in dosesof 0.1, 1, 25, and 50 pg./kg., was infused in four groups of S-10 rats and collections continued for at least 3 hours after administration. With the lowest dose of gastrin pentapeptide, a clear, double response occurred, the second peak appearing 1 hour 40 minutes after onset of the infusion. This double response was seen only in small measure with the l-pg./kg. dose and not at all with the higher doses (Fig. 3). 554

SUCCESSIVE ~HISTAMINE +?I?PRETREATED

Fig. 4. the rat.

20 MINUTE

COLLECTIONS

ONLY WITH SERUM

OF Z.E. PATIENT

Responseto intravenous histamine in

II’ILEEK

Table 3. Response to Casttrin Pentapeptide in Serum: Effect of Prolonged Freezing

excess HCl;’ Standard deviation

IMean

Gastrin Pentapeptide in Serum: 1 pg./kg. Freshly Frozen Prepared 6 Months 9.7 14.2 +- 6.4 k6.9

a Mean of four determinations. ference between means P > 30.

No

significant

dif-

a patient with proven ulcerogenic tumor of the pancreas, the response to both doses was increased, but that to the lo-pg./kg. dose was still not statistically significant. An interval of at least 90 minutes was allowed between the administration of the serum and the administration of the histamine. Gustrin

Pentapeptide

in Serum

Gastrin pentapeptide was added to normal (fasting) human serum so that 0.5 ml. of the serum contained a dose of 1 pg./kg. when given to a 300-gm. rat. The response to gastrin pentapeptide in serum was similar to that produced by the pentapeptide alone, and the response was unchanged after four months of frozen storage (Table 3).

DISCUSSION The pattern of response to a known dose of gastrin pentapeptide was predictable. The response commenced within 10-15 minutes of starting the test infusion, but since the animal received little of the pentapeptide in the first IO minutes, the increase in acid content in the first 20-minute collection was usually small. The peak response occurred in the second or third 20-minute collection with almost equal frequency. Thereafter the response fell sharply and was complete within 1 hour and 20 minutes to 1 hour and 40 minutes. This pattern was so standard that we would expect any gastrin-containing material to produce a similar response, and in this preparation, such a response may be regarded as indirect evidence of gas&in-like activity. The smallest dose of gastrin pentapeptide

ET

AL.:

BIOASS.+Y

OF

GASTRIX

that could be reliably detected was 0.1 pg./ kg. but a dose as low as 0.05 pg./kg., (equivalent to 15 mpg. total dose in 3OO-gm. rat) significantly increased acid output above baseline levels. These results indicate a greater sensitivity of the rat bioassay method than suggested bv the small responses, to even large doses of purified gastrin, obtained by Lai [ 11. Grossman [8] estimated that human post-prandial serum contains approximately 13 mpg. of gastrin per milliliter. Since the activity of gastrin has been variously reported as 4 to 11 times greater than that of the pentapeptide, the estimated serum level of gastrin and the limits of sensitivity of the bioassay methods are about the same. It may bc anticipated, therefore, that small doses of circulating gastrin can be reliably detected by this method. The sensitivity of the bioassay method does not, however, approach that of the immunoassay technique, recently described by McGuigan [9]. Although immunoassay offers obvious advantages, it cannot so easily be set up. We have also found the bioassay method to be useful in determination of biologic activity of synthetic peptides prepared in our laboratory. On this basis, the continued use of bioassay would appear justified. Lai reported an increasing response to successive doses of gastrin extract, reaching a maximum with the fourth or fifth dose. He further found that the first response was irregular and disregarded it. With gastrin pentapeptide, the first response is invariably satisfactory and no significant increase or decrease was seen with repetition of the same dose. Any apparent increment with repeated administration of identical doses may be a consequence of the double response demonstrated with small doses of gastrin pentapeptide. Although this double response is clear cut, its significance is uncertain. Employing the bioassay method in the diagnosis of the Zollinger-Ellison syndrome, Moore and his co-workers found that approximately one in twelve rats failed to respond to gastrin. Of 200 rats, in the present series, in which the response to 1 pg./kg. of gastrin pentapeptide was assayed, only four failed to

JOURNAL

OF

SURGICAL

RESEARCH

VOL.

9

NO.

demonstrate a significant increase in acid output. When higher doses of gastrin pentapeptide were used, a significant response was obtained in almost every animal. In dose-response experiments, incremental responses were obtained to increasing doses of gastrin pentapeptide, but a mere doubling or trebling of the dose did not produce a significant change. Once a detectable level of gastrin pentapeptide was reached, it was necessary to increase this amount by a factor of 5, or even 10, before a significant increase in acid output was obtained. This point is further illustrated by the very small difference in mean response to doses of gastrin pentapeptide of 0.1 pg./kg. and 1 pg./kg. (Table 1). In considering the amount of circulating gastrin necessary for detection by the bioassay method, the large increase above threshold levels required to produce a significant increment in response is of importance. It follows that if 1 ml. of serum contains sufficient gastrin to be detected, then the amount contained in 0.5 ml. of the same serum will also be detectable. This permits the use of small volumes of serum and thus reduces the possible toxic and volume effects consequent on giving unaltered serum to a small animal. Barrett and his associates reported that intraperitoneal urethane was associated with failure of the anesthetized rat preparation to respond to dose levels of gastrin or gastrin pentapeptide above 8 pg./kg. In our experiments, in which 0.5 ml./100 gm. of intraperitoneal urethane (25% w/v) was used routinely, a dose of 50 ‘pg./kg. of gastrin pentapeptide gave a good response and inhibition was not evident. There was, however, no significant difference between the response to 25 pg./kg. and that to 50 pg./kg. We found low temperature levels (2733°C) to be associated with a decreased response to higher doses of gastrin pentapeptide. The preparation in Barrett’s experiments was maintained at 31°C and a combination of low temperature and larger dose of intraperitoneal urethane may have been responsible for their findings. Since a good response was obtained with high doses of gastrin pentapeptide an 556

10,

OCTOBER

1969

inhibitory effect is unlikely to be seen when assaying serum, unIess the serum contains very large amounts of gastrin. Inhibition is therefore unlikely to be the explanation for the negative bioassay results obtained in certain cases of the Zollinger-Ellison syndrome and with some tumor extracts obtained from such patients. The response to gastrin pentapeptide was not altered by adding it to serum and remained unchanged after the serum had been frozen for 4 months. This gives no indication of how gastrin itself behaves in the serum, but does demonstrate the stability of the pentapeptide under these conditions and the lack of obvious toxic effects with 0.5ml. volumes of serum. There was a four- to five-fold variation in the response to a given dose of gastrin pentapeptide from rat to rat. In view of this variation, it is necessary to carry out the assay in at least four and ideally eight animals so that reproducible and reliable results may be obtained. The response obtained in a single rat cannot be regarded as significant. The lack of response to histamine in the anesthetized rat preparation has been well documented by other workers. Barrett et al. found a dose of 80 pg./kg. necessary to produce a plateau of acid secretion in their preparation. This accords closely with the dose of 50 pg./kg. of histamine base required to produce a significant response in our experiments. If the histamine was preceded by a serum demonstrating gastrin-like activity, the response was more marked. This was probabl! due to a “priming” effect of the previous dose of gastrin-like material. The amount of histamine in the serum of Zollinger-Ellison patients is small [lo] and at least a IOOO-fold increase would be required to give a response equal to that produced by the smallest detectable amount of gastrin. Since gastrin and histamine act by different mechanisms, it is reasonable to assume that any response obtained to serum in this preparation is due to gastrin. In this context, the refractoriness of the rat’s gastric mucosa to histamine is of positive value.

WILKES

SUMMARY CONCLUSIONS

AND

The pattern of response to single and repeated doses of gastrin pentapeptide, doseresponse relationships, the effects of infusing gastrin pentapeptide in serum, and the response to histamine have been investigated in the anesthetized, total gastric-fist& rat. These experiments were designed to explore the range, sensitivity, and reliability of the gastrin bioassay method, and to define its potential usefulness in the diagnosis of the Zollinger-Ellison syndrome and in elucidating the role of gastrin in other states associated with gastric hypersecretion. 1. The lowest dose of gastrin pentapeptide reliably detected bv this method was 0.1 pg./kg., corresponding to a total body dose of 30 mug. in a 300-gm. rat. A dose of 0.05 pg./kg. (total d ose 15 mpg. in a 300-gm. rat) produced a significant increase in acid output over baseline levels, but a dose of 0.01 \kg./ kg. could not be detected. 2. Incremental responses were obtained with increasing doses of gastrin pentapeptide up to, and including, the highest dose used, i.e., 50 pg./kg., but significant differences in response were only obtained with 5- to lofold dose increments. 3. There was no significant difference in the response obtained with successive similar doses of gastrin pentapeptide in the same preparation. 4. A clear-cut double response was obtained with the lower doses of gastrin pentapeptidc, the second peak occurring approxinmtely 1 hour after the first. This double response was not seen with higher doses and its significance is uncertain. When testing substances for gastrin-like activity, at least 2 hours should be allowed to elapse between successive doses. ,5. A dose of 50 pg./kg. of histamine base was necessary to produce a significant response, confirming the refractoriness of this preparation to histamine. A lOOO-fold increase

ET

AL.:

HIOASSI~-

OF

G-\STRIS

in the serum content of histamine would be necessary before the response could be confused with that produced by even the smallest detectable dose of gastrin. 6. The bioassay method is considered of value as a means of detecting small amounts of gastrin pcntapeptide and of gastrin pentapeptide in serum. The sensitivity of the method is close to the estimated level of gastrin believed to be present in the serum of patients with the Zollinger-Ellison svndrome. In view of the variation in response. between animals, the mean results of four to tight experiments should be reported when a substance is assaved for gastrin-like activitl-.

REFERENCES 1.

Lai, K. S. of biological

Part Gut

1: A method 5:327, 1961.

2.

Ghosh, M. N., and Schild, H. 0. recording of acid gastric secretion Brit. J. Phrmacol. 13:54, 1958.

Continuous in the rat.

3.

Barrett, A. hl., Raventos, J., and Siddall, R. A. Influence of some anesthetics on pharmacologically stimulated gastric secretion in the rat. Brit. J. Pharmacd. 28:51, 1966.

4.

?\loore, F. T., hlurat, 1. E., Endahl, G. L., Baker, J. L., and Zollingkr, R. M. Diagnosis of ulcerogenic tumor of the pancreas bv bioassav-. Amer. J. Swrg., 113:735, 1967. ’

5.

Ellison, Ellison Amer.

6.

Bon&, S., Bader, J. I’., Dubrasquet, \I., and Lambling, A. Detection in human urine of a substance stimulating gastric secretion in the rat. C. 2% Sot. Bid. (Paris) 157:259, 1963.

7.

Tracy, H. J., and Gregory, R. A. Physiological properties of a series of synthetic peptides structurally related to Gastrin I. Natwe (London) 204:935, 1964. Grossman, M. I. Antibodies to gastrin. Gastroenterology 53:805, 1967. McGuigan, J. E. Antibodies to the carboxylterminal tetrapeptide of gastrin. Gastroenterolog~/ 53:697, 1967. Sircus, W. Evidence for a gastric secretagogue in the circulation and gastric juice of patients with the Z-E syndrome. Lancct 2:671, 1964.

8. 9.

10.

Studies assay

on Gastrin, of gastrin.

E. H., and Wilson, syndrome updated. 47:1115, 1967.

S. D. The ZollingerSwg. Ch. North