0021-972X/79/4801-0176$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1979 by The Endocrine Society

Vol. 48, No. 1 Printed in U.S.A.

MEASUREMENT OF 3-ENDORPHIN IN HUMAN PLASMA Sharon L. Wardlaw and Andrew G. Frantz Departments of Medicine, Columbia University College of Physicians and Surgeons, and The Presbyterian Hospital, New York, N.Y. 10032. ABSTRACT. 3-endorphin has been identified in human plasma by means of gel filtration and a sensitive radioimmunoassay for human 3-endorphin (3u-endorphin). Mean baseline plasma 3h~endorphin concentration in 5 individuals was 2 1 + 7 . 3 (SD) pg/ml (6.2 + 2.2 (SD) fmole/ml). Following metyrapone stimulation mean plasma concentration increased to 55.4 + 10.1 (SD) pg/ml (16.3 + 3.1 (SD) fmole/ml). The molar ratio of human 3-lipotropin (3h~L:pH) to 3h~ endorphin was 2.2 in baseline plasma and 2.4 after metyrapone stimulation. fr-endorphin is the 31 amino acid Cterminal end of 3-lipotropin (3-LPH) with potent opiate-like activity (1). Both 3h~LPH and ACTH are contained in pituitary corticotrophic cells and are secreted in parallel in response to metyrapone, insulin-induced hypoglycemia and vasopressin stimulation (2,3). Several sensitive and specific assays have been developed for measuring plasma 3ft-LPH levels (3-5). However, the measurement of plasma 3^-endorphin has been impeded by the fact that antisera to 3h~endorphin crossreact with 3n-LPH. We have approached the measurement of 3n-endorphin in human plasma by using gel filtration of plasma extracts followed by a radioimmunoassay employing an antibody with relatively high sensitivity for 3h~endorphin. MATERIALS AND METHODS Peptides: Peptides were obtained from the following sources: synthetic 3h~endorphin, 3c~endorphin (camel), a-endorphin, y-endorphin, and methionine-enkephalin — Peninsula Labs; 3n-LPH and 30-LPH (ovine) — Dr. C.H. Li; ACTH^SS — Third International Standard. Submitted September 7, 1978 Supported by USPHS grants CA-11704 and T32 AM-07271

Antiserum: 0.5 mg 3^-endorphin was conjugated to 2 mg of thyroglobulin with 50 mg of carbodiimide (6). Rabbits were immunized monthly with 0.8 mg of the conjugate in complete Freund's adjuvant. Iodination: 3-endorphin was labeled with 1 3 1 I by the lactoperoxidase method (7). The tracer was purified by Sephadex G-50 chromatography (1.7x30 cm) in .04M pH 7.5 phosphate buffer containing .05M NaCl and 0.5% bovine serum albumin (BSA). Plasma Extraction: 5 to 200 ml of plasma was extracted with one 50 mg talc tablet (Goldleaf Pharmacal Co.) per 5 ml of plasma. The adsorbed 3h~endorphin and 3h~LPH were eluted from the talc with 1 ml of 1:1 acetone-O.lN HC1 per 50 mg talc followed by a second wash with 0.5 ml acetoneHC1 per 50 mg talc. The acetone-HCl extracts were evaporated under a stream of air at 20C. The residue was either dissolved in 0.5-1 ml of the assay buffer for direct assay or in 0.1N acetic acid containing 0.1% BSA for chromatography. Chromatography: 20 ml plasma samples were extracted as described above and chromatographed on 0.9x45 cm Sephadex G-50 (fine) columns in 0.1N acetic acid with 0.1% BSA. Wider columns (1.7x40 cm) were used for chromatographing extracts of 100-200 ml

176

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RAPID COMMUNICATIONS 50

I 40Q.

CC O

\

Q

ui 3 0 •

io

-

20

Q

10-

25

250

500

1000

/3h-END0RPHIN-pg/tube

Fig.

1. Standard Curve

of plasma, rractions were lyophilized and dissolved in assay buffer. Assay: 8^-endorphin standards (25-1000 pg) or sample extracts (50-200 yl) were pre-incubated with antibody (final concentration 1:5000) and carrier rabbit y-globulin (50 yg) in a total of 0.45 ml assay buffer (.04M pH 7.5 phosphate with .05M NaCl and 0.5% BSA) for 24 hr at 4C. 50 yl 131 I-8 n -endorphin was then added and incubation continued for 24 hr at 4C. Bound and free peptides were separated by precipitation with sheep anti-rabbit Y~gl°bulin. Subjects: 34 normal volunteers (17 men, 17 women, age 23-51) had blood samples drawn between 8-10 A.M. 6 normal volunteers were given metyrapone, 2.25 g orally at 9 A.M.,with blood samples drawn beforehand and after 4 hr and 8 hr. The 4 and 8 hr samples were pooled and assayed together. In all cases blood was collected in heparinized tubes, promptly centrifuged at 4C, and stored frozen at -20C if not assayed immediately. Short periods of frozen storage (up to 1 week) did not alter 3n-endorphin or 3^-LPH concentrations. RESULTS Assay Characteristics: Assay sensitivity was 25 pg/tube with half

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maximal displacement of tracer at 150-200 pg (Fig. 1 ) . The inter-assay coefficient of variation was 12.7%; the intra-assay coefficient of variation was 8.3%. There was no crossreactivity withmethionine-enkephalin or ACTH at 1000 ng/tube. Displacement of 131I-3h-endorphin by 3 h ~ L P H w a s parallel to that with 3h~ en dorphin, the potency of 3n-LPH by weight being 10.8% that of 3h~endorphin (29% on a molar basis) (Fig. 2 ) . The mean recovery for unlabeled 3h~endorphin added to plasma and extracted was 74% + 10.6 (SD) for 23 determinations in 9 different assays. The recovery of 3h"LPH was identical to that of 3h" endorphin. Chromatography: As shown in Fig. 3, elution from a 0.9x45 cm Sephadex G-50 column gives a sharp separation of 3n-endorphin and 8n-LPH standards. In plasma chromatography, the pooled fractions 15-20 and 21-26, representing the 8h~LPH and 3h~endorphin peaks, respectively, were used for assay. Mean overall recovery for extraction and chromatography of unlabeled material was 44% for 3n-endorphin and 43% for 3h~LPH. 3h~endorphin was not generated from 3h~LPH as an artifact of the extraction and chromatographic procedures. When large amounts (250300 ng) of 3 h ~ L P H were added to plasma and extracted immediately or incu-

? 100-

o i 80 oa. u. o o

? 40

Q

m < 20

.025

.05

I

.25 .5

1.0 2.0 ng /tube

5

10

Fig. 2. Displacement of 1 3 1 I-3h~ e n dorphin tracer from anti-3h~ en d° r phi n antiserum by various peptides.

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178 9.0 H

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Table 1. Baseline and metyrapone stimulated plasma levels of 3n~endorphin (3-EP) and 3-LPH in 5 subjects. Subj. Baseline (pg/ml) Post-Metyrapone 3-LPH

3-EP

54

46

86

19

136

43

334

3. 14

67

59

409

25

161

67

459

5. 15

151

62

517

3-EP

1. 32 2. 4. 18

20

22

24

28

3-LPH

30

FRACTION NUMBER

3h~endorphin in plasma was further examined by the extraction and chromatography of 200 ml of pooled plasma from 3 subjects. Each fraction bated at 37C for h, 3 and 24 hr, foleluted was assayed separately. Siglowed by extraction and chromatogranificant immunoreactivity was seen phy, no significant increase in immuonly in two peaks, corresponding to noreactivity was noted under the 3^those of 3j1-endorphin and 3^-LPH endorphin peak. standards, as shown in Fig. 4. AcBaseline Plasma Levels of 3^-entivity under the two peaks, corrected dorphin: 5 ml of plasma from each of for recovery and crossreactivity of 34 normal volunteers was extracted 3h-LPH, corresponded to plasma conand assayed without chromatography. centrations of 26 pg/ml (7.6 fmoleMl) The mean "8-endorphin-like" immuno172 pg/ml (18.7 fmole/ml) for 3^and reactivity was 33 pg/ml + 15 (SD) < endorphin and 3n-LPH, respectively. with a range of 17 to 73 pg/ml. Responses to Metyrapone: 3h~endorFurther characterization of this "3~ phin levels in extracts of 20 ml of endorphin-like" immunoreactivity was undertaken to determine the actual amounts of 3n-endorphin and 3h~LPH in baseline plasma. Table 1 gives the results of chromatography on a 0.9x45 cm Sephadex G-50 column of extracts of 20 ml of baseline plasma from 5 subjects. The measured 3h~ endorphin concentration is the total immunoreactivity contained in fractions 21-26, corresponding to the elution of 3n-endorphin standard. The 3h~LPH concentration is the immu70 80 90 noreactivity contained in fractions ELUTION VOLUME 15-20, multiplied by 9.3 to correct for the 10.8% crossreactivity of 3nFig. 4. Elution pattern of 3-endorphinLPH compared to 3h~endorphin standard. like immunoreactivity in 200ml baseMean baseline 3h-endorphin was 21 + line and 100ml post-metyrapone plasma. 7.3 (SD) pg/ml (6.2 + 2.2 fmole/ml). All fractions measured against 3-EP Mean baseline 3n-LPH was 114 + 50 standard. No correction factor applied (SD) pg/ml (12.4+5.4 fmole/ml). to fractions in LPH peak. Fig. 3. Elution pattern of $^-endorphin and 3h~LPH standards.

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1979 Nol

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RAPID COMMUNICATIONS Table 2. Assay of pooled fractions representing 3-LPH and &-EP peaks, respectively. 3 -LPH peak was measured against both 3-LPH and 3-EP standards. 3-EP peak was measured against 3-EP standards only. Concentrations remain constant when samples are assayed at different dilutions, indicating parallelism between curves obtained with extracts and standards. Vol. of Extr . 50 yl 100 yl 150 yl 200 yl

3-LPH peak LPH ng/ml EP 10.0 0.90 10.7 1.02 9.9 0.99 9.8 1.10

3-EP peak EP ng/ml 1.00 1.00 0.87 0.90

plasma from 5 volunteers before and after metyrapone rose from 21 + 3.3 (SEM) pg/ml (6.2 + 0.99 fmole/ml) to 55.4 + 4.5 (SEM) pg/ml (16.3 + 1.4 fmole/ml) (Table 1 ) . The mean 3h~LPH/3^-endorphin molar ratio was 2,2 before and 2.4 after metyrapone. 100 ml of serum from one subject after metyrapone was extracted, chromatographed and each fraction assayed separately (Fig. 4 ) . Again two distinct peaks of immunoreactivity corresponding to 3h~LPH and 3h~endorphin standards were obtained. Total activity under the 3h~endorphin and 3h~ LPH peaks corrected for recovery, was 57 pg/ml (16.8 fmole/ml) and 564 pg/ml (61.3 fmole/ml, respectively, after metyrapone. In a separate experiment extracts of 75 ml of post-metyrapone plasma were chromatographed. Fractions representing 3h~endorphin and 3^-LPH peaks were pooled and assayed against their respective standards at multiple dilutions. The 3h-LPH peak was also assayed against a 3h""endorphin standard. Results (Table 2) showed parallelism between curves obtained for plasma extracts and standards. DISCUSSION In the rat 3-endorphin has been reported to be secreted concomitantly with ACTH and 3-LPH in pituitary

monolayer culture and to rise in serum in response to stress (8). Other assays have been used to examine 3-endorphin in human plasma and CSF, utilizing an antiserum directed against the N-terminal end of 3^-LPH which does not crossreact with 3h~endorphin and one directed against the C-terminal end crossreacting with 3h~LPH and 3^-endorphin (4,9). By the use of such antisera no significant amounts of 3h~ en dorphin were reported in basal plasma or following vasopressin and insulininduced hypoglycemia (4,9). The basal levels of 3h~endorphin we have measured are low, comprising only 15% by weight of the total 3-LPH-3-EP immunoreactivity, and might not have been detected with the above methods using smaller plasma samples. It has very recently been reported that 3h~ endorphin was undetectable in chromatographed extracts of baseline plasma and following vasopressin administration but was detectable in plasma of patients with Cushing's and Addison's disease(10). The 3-LPH/3-EP molar ratio reported in Cushing's disease was similar to the ratio we have reported in baseline and metyrapone stimulated plasma. Our finding of measurable quantities of 3-endorphin in normal plasma and the demonstration of a rise in plasma following an ACTH-releasing stimulus, raises further questions regarding the physiology of this peptide, whose ultimate role remains to be determined. ACKNOWLEDGMENT: We thank Dr.C.H.Li for his kind gift of 3-LPH. REFERENCES 1. Goldstein, A. ,Opioid peptides in pituitary and brain. Science 193; 1081, 1976. 2. Pelletier, G., R. LeClerc, F. Labrie, J. Cote, M. Chretien, and M. Lis, Immunohistochemical localization of 3-LPH in the pituitary gland. Endocrinology 100: 770, 1977. 3. Krieger, D.T., A. Liotta, and C.H. Li, Human plasma immunoreactive 3-LPH; Correlation with basal and stimulated

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RAPID COMMUNICATIONS plasma ACTH concentration. Life Sciences 21: 1771, 1977. 4. Jeffcoate, W.J., P. J.Lowry,L.H.Iees, J.Hope, and G.M.Besser, 3-LPH and 3-ffidorphin in human plasma and CSF. Program of 60th meeting of The Endocrine Society, Miami, Abst. No.142, 1978. 5. Wiedemann,E., T.Saito, J.A.Linfoot, and C.H.Li, Radioimmunoassay of human 3-LPH in unextracted plasma. J Clin Endocrinol Metab 45.: 1108, 1977. 6. Goodfriend, T.L.,L.Levine,and G.D. Fasman, A use of carbodiimides in immunology. Science 144: 1344, 1964. 7. Thorell,J.I., and B.G.Johansson, Enzymatic iodination of polypeptides.

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Biochim Biophys Acta: 251:363, 1971. 8. Guillemin,R., T.Vargo, J.Rossier, S.Minick, N.Ling,C.Rivier, W.Vale,and F.Bloom, 3-endorphin and ACTH are secreted concomitantly by the pituitary gland. Science JL9J7.: 1367, 1977. 9. Liotta, A.S., S. Toshihiro,and D.T. Krieger, 3-lipotropin is the major opioid-like peptide of human pituitary and rat pars distalis. Proc Natl Acad Sci USA T5\ 2950, 1978. 10. Suda,T., A.S.Liotta, and D.T.Krieger, 3-endorphin is not detectable in plasma from normal human subjects. Science 202: 221, 1978.

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1979 Nol