Immunoreactive /8-Endorphin and Adrenocorticotropinin Human Cerebrospinal Fluid

KAZUWANAKAO, SHOGOOKI, ISSEY TANAKA, KAZUKOHoRII, YOSHIKATSu NAKAI,TomIoo FuRuI, MASANORIFUKUSHIMA, AKIo KUWAYAMA,NAOKI KAGEYAMA,and HIROOIMURA, Secotnd Division, Department of Medicine, Kr oto Uniiversitrj,Schlool of lMedicitne, Krjoto 606; Department ofNeurosurgery, Nagotya Uniiversity ,

Schlool of ledicine, Nagoya 466; Department of Itnternial Medicine,Aiclii Catncer Cetnter, Nagoya 464, Japanmm

A B S T RA C T To eltucidate the significance of 8-endorphin in htumilan cerebrospinial fluid (CSF), CSFlevels of ,8-endorphini-like immunoreactivity (,3-EP-LI)in various diseases were determined by a specificradioimmunoassay and compared with simultaneouslydetermined ACTH-like immunoreactivity (ACTH-LI)levels in CSF. CSF p-EP-LI and ACTH-LI in thecontrol group, consisting of 5 normal subjects and 19patients with nonendocrine diseases, were 22.2±1.3and 14.6±0.4 fmol/mIl, respectively. CSFlevels of thesepeptides in patients with schizophrenia (n = 19) andacromegaly (tl = 10) were not significantly differentfrom those in the control group. Patients with Cushing'sdisease (it = 7) had significantly lower CSF 8-EP-LIand ACTH-LI levels than those in the control group.Four of them showed a parallel increase in CSF 8-EP-LI and CSF ACTH-LI levels after the completeremoval of pituitary microadenomas (P < 0.05).

Gel chromatography of CSF p-EP-LI from a normalvolunteer, a control patient, and one patient each withcatatonia, Nelson's syndrome, Cushing's syndrome(adrenal adenoimia), and acromegaly gave similarpatterns consisting of three peaks with the elutionpositions comparable to those of authentic a-en-dorphin, 8-lipotropin, and possibly their precursormolecule.

Gel chromatographic patterns of CSF ,3-EP-LI andACTH-LI were compared in a normal volunteer. Thefirst peaks of 8-EP-LI and ACTH-LI eluted at thesame position and the second peak of ACTH-LIcoincided with the elution position of authentic ACTH.

CSF,-EP-LI and ACTH-LI levels determined every5 min over a period of 80 min in three normal volunteersdid not show moment-to-moment variability.

A significant correlation (r = 0.75, P < 0.001) was

Receivedfor publication 21 April 1980 anid itn revised form4 Autgu.st 1980.

seen betweeni CSF ,-EP-LI and ACTH-LI levels innormal sulbjects and patients studied (n = 73). This.suggests that f-endorphin and ACTH in human CSFshare the common regulatory mechanism in normaland pathologic conditions.

INTRODUCTION

,B-Endorphin, which corresponds to 61-91 amino acidsof ,-lipotropin (,8-LPH),' is a potent opioid peptideand has profound effects on mood and behavior (1-3).Since 8-endorphin exists not only in the pituitary butalso in the brain (4-9), it has been proposed thatschizophreniia is related to dysfunctions in the en-dorphin system (10). However, the origin of brain8-endorphin is controversial, and both the pituitaryand brain have been postulated as possible sites (4, 9, 11).

Previous studies have demonstrated that ,-en-dorphin, distinct from B-LPH, exists in normal humanplasma (12-14) and that it increases in parallel withACTH after metyrapone administration (12, 13, 15)or insulin-induced hypoglycemia (15, 16). High plasma,B-endorphin levels were observed in patients withNelson's syndrome, Addison's disease, and Cushing'sdisease in whom plasma ACTH levels were alsoelevated (14, 17-19). These observations suggestthe possible pituitary origin of /3-endorphin in periph-eral blood and are consistent with the discovery thatACTH, 3-LPH, and /3-endorphin are derived from acommon precursor molecule in a mouse pituitary cellline AT-20/D-16V (20) and in the bovine pituitary (21).

On the other hand, little is known about hormonesin the cerebrospinal fluid (CSF). ACTH-like immuno-

I Abbreviatiotns uised in this paper: ACTH-LI, ACTH-likeimmunoreactivity; CSF, cerebrospinal fluid; 8-EP-LI, 8-enidorphin-like immunoreactivity; B-LPH, 3-lipotropin; RIA,radioi mnininoas saxv.

J. Clin. Inlvest. (© The American Society for Clinical Inwvestigation, Inc. (0021-9738/80112/1383/08 $1.00Volume 66 DecemSber 1980 1383-1390

1383

reactivity (ACTH-LI) has been demonstrated in humanCSF (22, 23) in concentrations which did not correlatewith plasma ACTH-LI levels (23). The presence of,8-endorphin-like immunoreactivity (p8-EP-LI) in humanCSF was suggested by the difference between f8-EP-LI measured by an antiserum directed towards theCOOH-terminal portion of ,3-LPH and f8-LPH-likeimmunoreactivity measured by a NH2-terminal anti-serum (24). However, the nature of, -EP-LI in CSFwas niot clear because of the cross-reactivity of -LPHand /3-endorphin with the COOH-terminal antiserum.

Utilizing a sensitive radioimmunoassay (RIA) for,8-endorphin, combined with gel exclusion chroma-tography, we have demonstrated that ,B-EP-LI inhumnan CSF consists of 8-endorphin, ,-LPH, andpossibly the precursor molecule and that there is amarked dissociation between plasma and CSF ,8-EP-LIlevels (25). However, the relationship between 8-endorphin and ACTH in the CSF is not clear. Thepresent study was designed to clarify the relationshipof ,3-endorphin and ACTH in human CSF.

METHODS

Subjects'Five normal volunteers and 19 patients without endocrine

abniornmalities (intervertebral disk hernia and cerebrovascularaccident), 19 patients with schizophrenia (11 with hebe-phrenia, 6 with catatonia, 2 with paranoia), 1 with depression,1 with atypical psychosis, 1 with undifferentiated psychosis,7 with Cushing's disease, 10 with active acromegaly, 2 withNelson's syndrome, 1 with Addison's disease, 1 with Cush-ing's syndrome due to adrenal adenoma, 2 with isolatedACTH deficiency, and 4 under glucocorticoid treatment(prednisolone 15-20 mg/d) were studied. The patientswith intervertebral disk hernia anid those with cerebrovascularaccident were studied before medication. The patientswith schizophrenia and those with other psychoses hadbeen miaintained on the routine dosage of neuroleptic drugs.Five of seven patients with Cushing's disease and one oftwo patients with Nelson's syndrome were studied bothbefore and after transsphenoidal microsurgery. Four of thefive patients with Cushing's disease showed normal urinaryexcretion of 17-hydroxysteroids and 17-ketosteroids andnormiial plasma cortisol levels after the adenomectomy.Their clinical manifestations due to Cushing's diseasedisappeared conmpletely. One patient with Cushing's diseaseshowed n1o improvement of laboratory findings and clinicalmanifestations after the operation. One patient with Nelson'ssyndromiie mainitainied on 30 mg/d of cortisol showed aremnarkable improvement of pignmentation after the operationwith a miarked diminutioni of plasmiia ACTH levels.

Lumiiibar puncture was performied under local anesthesiaat 9:00 a.m. after an overnight fast. None of the subjectscomplained of pain. CSFwas collected into chilled siliconizeddisposable glass tubes, stored at -20°C and thawed onlyonce at the time of hormone assay.

Blood samples were withdrawn into chilled plasticsyringes immediately before lumbar puncture and trans-ferred to chilled siliconized disposable glass tubes con-taining Trasylol (500 kallikrein inactivator U/ml DelbayPharmaceuiticals, Div. of Schering Corp., Bloomfield, N. J.)an(l EDTA (1 mg/ml). Plasma was immediately separated by

centrifugation in a refrigerated centrifuge, and stored at-20°C until assayed.

For gel exclusion chromatographic studies of CSF ,pEP-LI,5 ml of CSF from a normal volunteer, and one patient eachwith cerebrovascular accident, catatonia, Nelson's syndrome,Cushing's syndrome, and acromegaly were immediatelydivided into 1-ml aliquots and lyophilized. The recoveryof human 13-LPH (5-20 fmol/ml) and human 8-endorphin(3-12 fmol/ml) added to CSF was 80 and 65%, respectively,during the procedure of lyophilization and reconstitution.

To compare chromatographic patterns of both CSF f3-EP-LIand ACTH-LI, 8 ml of CSF from a normal volunteer wasdivided into 1-ml aliquots and lyophilized.

To clarify the moment-to-moment variability in ,3EP-LIor ACTH-LI levels, lumbar CSF was collected every 5 minfrom three normal volunteers over a period of 80 min.

RIA

RIAfor ,l3endorphin. RIA for ,-endorphin was performedusing the talc adsorption method as described (26, 27). TheRIA standard diluent consisted of 0.05 M phosphate buffercontaining 0.5% human serum albumin (fraction V, ICNNutritional Pharmaceuticals, Cleveland, Ohio), 500 kallikreininactivator U/ml of Trasylol (Delbay Pharmaceuticals) and0.4% 2-mercaptoethanol (Nakarai Chemicals, Ltd., Kyoto,Japan). A two-stage assay procedure was employed, incubat-ing 0.1 ml of unextracted CSF sample or standard humanp-endorphin, and antiserum (final dilution 1:500,000) in 0.3ml of the standard diluent at 40C for a day and then adding'25I-,8-endorphin in 0.1 ml of the standard diluent and incubat-ing for 3 d at 40C. The standard human ,3endorphin andhuman ,3-LPH were donated by Dr. C. H. Li. The minimaldetectable (quantity of 8-endorphin was 0.3 fmol. Human,-endorphin and p-LPH equally displaced 125I-,8-endorphinfrom the antiserum when compared on a molar basis.

RIA for ACTH. RIA for ACTHwas performed using thetalc adsorption method (28). The RIA procedure was identicalin all respects to that described in RIA for ,-endorphin,except that ACTH and the antiserumii of CEA-IRE-SORINassociation ACTHRIA kit (Atomic Energy Laboratory of Bio-medical Products, Gif-sur-Yvette, France) (29) were used in-stead of ,-endorphin and the 0-endorphin antiserumii, respec-tively. The standard ACTH was donated by the NationalPituitary Agenicy. The minimal detectable (juantity of ACTHwas 0.2 fmol. This antiserum directs toward the midportion ofACTH, sinice it reacted similarly with (1-18) and (1-24)ACTH,weakly with (18-39)ACTH (<l% on a weight basis) and notwith a-melanotropini.

Gel exclusiotn chromatographyLyophilized CSFwas reconstituted in 0.3 ml of the standard

diluent, applied on a 0.7 x 47-49 cm columnl of Bio-Gel P-60(Bio-Rad Laboratories, Richmond, Calif.), and eluted with thesame diluent at 40C. The flow rate was 2 ml/h and the fractionvol was 0.72 ml. The column was calibrated with bluedextran as a void volume, unlabeled human ,-LPH, human,-endorphin, and human ACTH, and 125I as salt peak. Re-coveries of human 8-LPH, human 8-endorphin, and humanACTHapplied on the column were 90-100% in both cases.

Statistical analysisThe statistical analysis was performed by Student's t test

for paired or unipaired data as appropriate.

1384 Nakao et al.

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FIGURE 1 p-EP-LI concentrations in CSF from all subjects studied. AD, Addison's disease;NS, Nelson's syndrome; CS, Cushing's syndrome (adrenal adenoma); D, ACTHdeficiency.

RESULTS

Figs. 1 and 2 illustrate CSF ,B-EP-LI and ACTH-LIlevels in all subjects studied. The mean (+SE) hormonelevels in each group are shown in Table I. CSF,-EP-LIand ACTH-LI levels in 5 normal subjects and 19patients without endocrine abnormalities were notsignificantly different (Table I). Therefore, all 24subjects together were included in the control group.

As shown in Table I and Figs. 1 and 2, CSF ,-EP-LIand ACTH-LI levels in patients with schizophreniawere not significantly different from those in controlgroup. Among the types of schizophrenia, no sig-nificant difference was observed in CSF hormonelevels, although the levels tended to be higher incatatonia. CSF ,3-EP-LI and ACTH-LI levels inpatients with active acromegaly were not significantlydifferent from those in the control group. In patientswith pituitary-adrenal diseases such as Cushing'sdisease, Nelson's syndrome, Addison's disease, iso-

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FIGURE 2 ACTH-LI concentrations in CSFfrom all subjects studied. AD, Addison's disease; NS,Nelson's syndrome; CS, Cushing's syndrome (adrenal adenoma); D, ACTHdeficiency.

/3-Endorphin and ACTH in Human Cerebrospinal Fluid

lated ACTH deficiency, and Cushing's syndrome(adrenal adenoma), those with Cushing's diseaseshowed significantly low CSF f3-EP-LI and ACTH-LIlevels, and one with Cushing's syndrome and one withAddison's disease showed low and high /8-EP-LI andACTH-LI levels, respectively. Two patients withNelson's syndrome and two patients with isolatedACTHdeficiency showed similar values to those in thecontrol group. CSF levels of these hormones in fourpatients under glucocorticoid treatment were notsignificantly different from those in the control group,

but one of them showed distinctly low CSF hormonelevels.

In four of the patients with Cushing's disease,plasma hormone levels were substantially decreasedafter the complete removal of pituitary microadenoma,but CSF hormone levels were significantly increased(P < 0.05, Table II). In one patient with Nelson'ssyndrome the complete removal of the pituitaryadenoma had no effect on the CSF hormone levels.

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1385

TABLE I,fEP-LI and ACTH-LI Levels in HumanCSF

n P-EP-LI ACTH-LI

fmollml fmollml

Control 24 22.2±1.3 14.6±0.4Normal 5 22.3±1.1 14.4±0.3Nonendocrine patients 19 22.1±1.6 14.6±0.5

Schizophrenia 19 22.2±1.6 15.7±0.7Catatonia 6 26.3±3.0 17.4±1.4Hebephrenia 11 21.5±1.7 14.7±0.8Paranoia 2 14.3±1.8 13.0±0.1

Acromegaly 10 21.1±2.0 15.9±1.4Cushing's disease 7 11.0±1.5* 10.9±1.1*Patients under glucocorticoid

treatment 4 16.4±4.3 14.2±3.8

* < 0.01.

one additional component with a larger molecularweight. Similar gel-filtration pattems were observedin six CSF samples from a normal volunteer, one pa-tient each with cerebrovascular accident, catatonia,Nelson's syndrome, Cushing's syndrome (adrenaladenoma), and acromegaly.

The elution profiles obtained by gel filtration ofCSF f3EP-LI and ACTH-LI from a normal volunteerare shown in Fig. 5. ACTH-LI in CSF consisted ofthree major components, and the second peak elutedin the position compatible for authentic human ACTH.The first peak of CSFACTH-LI near the void volumeco-eluted with that of CSF 13EP-LI. One additionalpeak of CSF ACTH-LI was observed in the elutionposition compatible for the molecule of the sizebetween authentic ACTHand 125I.

DISCUSSION

In the 73 subjects studied, there was a positivecorrelation between CSF ,3EP-LI and ACTH-LIlevels (r = 0.75, P < 0.001, Fig. 3). A significantcorrelation between CSF (-EP-LI and ACTH-LIlevels was also observed in each group such as control,schizophrenia, acromegaly, Cushing's disease, andother pituitary-adrenal diseases.

There were no significant fluctuations of CSF,&EP-LI and ACTH-LI levels studied by samplingevery 5 min over a period of 80 min in three normalvolunteers.

The elution profiles obtained by gel filtration ofCSF 13EP-LI from patients with various diseases areshown in Fig. 4. /3EP-LI in CSF consisted of twocomponents with elution positions compatible withthose of f-LPH and f3-endorphin, respectively, and

This study demonstrated that CSF ,&EP-LI andACTH-LI levels in control subjects were 22.2+1.3and 14.6+0.4 fmol/ml, respectively. These results arecomparable to previously reported values of CSF,l3EP-LI (24, 25) and ACTH-LI (23). In addition, asignificant positive correlation was observed between,3EP-LI and ACTH-LI in CSF in normal subjects andpatients with various diseases. These results suggestthat ,3EP-LI and ACTH-LI in human CSF are regu-lated by common mechanisms.

The origin of ,EP-LI and ACTH-LI in CSF is notclear at present. It has been demonstrated that f-endorphin and ACTH in plasma do not penetrate theblood-CSF barrier (23, 25). The analgesic electricalstimulation of periventricular gray matter is known toincrease CSF,fEP-LI levels (30,31). It is highly likely,

TABLE II,fEP-LI and ACTH-LI Levels of CSF and Plasma in Cushing's Disease and Nelson's Syndrome

before and after the Pituitary Adenomectomy

,-EP-LI ACTH-LI

Before After Before AfterAdeno-

No. Case Age Sex mectomy CSF Plasma CSF Plasma CSF Plasma CSF Plasma

fmollml fmol/mlCushing's

disease1 H.K. 22 M C 10.2 33.3 19.7 7.7 10.8 24.4 17.2 6.92 K.T. 25 F C 11.4 32.9 19.2 <6.0 10.0 34.8 16.4 <5.63 E.F. 49 F C 4.9 24.6 8.1 <6.0 6.0 24.4 7.4 6.84 Y.Y. 22 M C 10.9 74.5 25.2 21.8 12.8 38.4 22.1 <5.65 H. K. 24 M I 14.5 27.0 10.3 29.8 13.7 17.8 7.3 18.7

Mean+SE 9.4±1.5 41.3±11.2 18.0+3.6* 9.9+1.4 30.5±3.6 15.8t3.1*Nelson's

syndrome6 Y.A. 43 F C 23.1 1370 20.6 52.3 16.0 1100 16.6 43.3

Abbreviations used in this table: M, male; F, female; C, complete; I, incomplete.* P < 0.05.

1386 Nakao et al.

* S

0 M

.00* 0

B-Ernkxphn-like Imununoreactivity pg/ml

FIGuRE 3 Correlation between /8-EP-LI and ACT]concentrations in 73 CSF samples. A significant poscorrelation was observed between them (r = 0.75, P < 0.1

therefore, that ,3-EP-LI and ACTH-LI in CSForigifrom the brain and not from the pituitary or bliOur previous observation that plasma and CSJendorphin levels can be dissociated under pathologconditions (25) also supports the central nern

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system origin of CSF ,-endorphin and ACTH. Recentstudies by Liotta et al. (11) have shown that a commonmolecule of f3endorphin and ACTH with a highmolecular weight is synthesized by cultured bovinehypothalamic cells. Our present study also demon-strated the presence of f3EP-LI and ACTH-LI of ahigh molecular weight form, which was eluted on gelchromatography at the elution position of the commonprecursor molecule.

Another interesting finding in this study is thenoticeable changes of ,¢EP-LI and ACTH-LI levelsin plasma and CSF in Cushing's disease before andafter the operation. Both CSF 3-EP-LI and ACTH-LIlevels in Cushing's disease before the surgery weresignificantly lower than those in the control group,although plasma levels were significantly higher inCushing's disease than those in the control group.Plasma f-EP-LI and ACTH-LI levels were sub-

' stantially decreased after the complete removal of themicroadenomas, but their CSF levels were increasedconversely after the operation. These findings clearly

H-LI demonstrate a concomitant change of 3EP-LI and,itive ACTH-LI levels in human CSF and strongly suggestDOl). the presence of common regulatory mechanisms.

However, the cause of low CSF levels of these pep-nate tides in cases of Cushing's disease is not clear atood. present. This abnormality might be caused by over-F & produced glucocorticoid, and not by ACTH or /&;ical endorphin, because CSF hormone levels were lowvous in a patient with Cushing's syndrome due to adrenal

3 VoWPHI3EP I

I 4- 4

I,, 4

V. B-LPH n-EP I

4,

10 20 30 10 20 30 10 20 30Fraction Number

FIGURE 4 Elution profiles obtained by gel-filtration (Bio-Gel P-60, 0.7 x 49 cm) of six CSFsamples. The lyophilized CSF was reconstituted in 0.3 ml standard diluent and 0.25 ml of re-constituted solution was applied to the column. The fractions were assayed by RIA for 8-en-dorphin. A, normal volunteer; B, cerebrovascular accident; C, schizophrenia (catatonia); D, Nel-son's syndrome; E, Cushing's syndrome (adrenal adenoma); F, acromegaly. Vo, void volume;I, iodine.

,3-Endorphin and ACTHin Human Cerebrospinal Fluid

r,

1387

Fraction Number

FIGURE 5 Elution profiles obtained by gel-filtration (Bio-Gel P-60, 0.7 x 47 cm) of CSF from a normal volunteer. Thelyophilized CSF was reconstituted in a 0.3 ml of standarddiluent and 0.25 ml of the reconstituted solution was appliedto the column. The fractions were analyzed in two systemsof RIA: /3-endorphin and ACTH. Vo, void volume; I, iodine.

adenoma, high in a patient with Addison's disease,and within normal limits in two patients with Nelson'ssyndrome who were maintained on steroids. Inaddition, CSF ,&EP-LI and ACTH-LI levels wereunchanged in a patient with Nelson's syndrome evenafter the complete removal of the pituitary adenoma,although plasma levels of these hormones wereremarkably decreased (T.A. in Table II). However,CSF levels of these hormones in four patients underglucocorticoid treatment were not significantly dif-ferent from those in the control group, and only oneof them showed distinctly low CSF hormone levels.The discrepancy between CSF levels in patients withCushing's disease and those under glucocorticoidtreatment may be explained by the dose of gluco-corticoid, because the remaining three patients hadreceived only a single dose of prednisolone (15 mg)in the morning. It is also possible that other stillunknown factors may cause the low CSF ,8-EP-LI andACTH-LI levels in patients with Cushing's disease.

The present study also demonstrates that there is noepisodic change in /3EP-LI and ACTH-LI levels inhuman CSF because 5-min sampling over a period of80 min disclosed little moment-to-moment variability.The results also suggest that the effect of lumbarpuncture on /3EP-LI and ACTH-LI levels in CSF,if any, is minimal.

A hypothesis that schizophrenia is caused by dys-function of the endorphin system has been proposed

(10) because 8-endorphin injected intraventricularlyinduced catatonia in rats (32) and because naloxone,an opiate receptor antagonist, is reported to reduceauditory hallucination in schizophrenia (33, 34). Thisstudy shows that CSF ,(EP-LI levels in schizophreniado not differ from those in the controls and that a gelchromatographic pattern of CSF f8-EP-LI from acatatoniac is essentially similar to that of a normalvolunteer and to those of other patients. These resultsare in disagreement with preliminary findings ofDomschke et al. (35), who reported levels of CSF3-EP-LI about 10 times higher in acute paranoid

patients and two times lower in chronic schizophreniathan of the control group. Reason for the discrepancyof the results may be explained in part by the differencein phase and medication of schizophrenia. However,our study shows a correlation between CSF ,3-EP-LIand ACTH-LI levels in schizophrenia, and thereforeexcludes technical errors, although Domschke et al. didnot report CSFACTH-LI levels. Recently it has beenreported that plasma ,B-EP-LI levels in schizophreniado not differ from those in controls (36) and that intra-ventricular administration of f8-endorphin producedno psychiatric symptoms in patients (37). Moreover,contradictory results have been reported regardingthe effect of naloxone on symptoms in schizophrenia(38-41). It seems necessary, therefore, to reevaluateinvolvement of ,&endorphin in schizophrenia.

CSF /3-EP-LI and ACTH-LI levels in acromegalyare of interest, because intraventricular administrationof ,endorphin stimulates growth hormone secretionin animals (42). In this study, however, CSF ,3-EP-LIand ACTH-LI levels in acromegaly did not differ fromthose in the control group and the gel chromatographicpattern of CSF,3-EP-LI from a patient with acromegalywas similar to those of other subjects. Thus, our resultsare in disagreement with those of Wiedemann et al.(43, 44), who reported elevated CSF concentrationof f8-EP-LI in acromegaly. The reason for this dis-crepancy is not clear. It should be noted, however,that a significant positive correlation was demon-strated between CSF ,8-EP-LI and ACTH-LI levels inacromegaly and that CSFACTH-LI levels in this studyare in agreement with those of Allen et al. (23).

Recently, Zakarian and Smyth (45) reported thatimmunoreactive ,3-endorphin demonstrated on gelexclusion chromatography consists of C fragment(61-91 of ,¢LPH), C' fragment (61-87) and theirN-acetylated forms and that the processing of lipotropinis under different control in anatomically distinctregions in rat pituitary and brain. Their group alsoreported that C fragment, C' fragment and their N-acetylated forms were isolated from porcine pituitary(46). However, active and inactive forms of endorphinsin human pituitary and brain are not clear at present.The physiological significance and the nature of

1388 Nakao et al.

,8-EP-LI in humain cerebrospinal fluid must awaitfurther investigation.

ACKNOWLEDGMENTSWeare indebted to Professor Choh Hao Li, Hormone ResearchLaboratory, University of California, San Francisco, for thegenerous supply of human 8-endorphin and human ,-lipotropin; to the hormone distribution program, NationalInistitute of Arthritis, Metabolism, and Digestive Diseases,National Pituitary Agency for the generous supply of standardACTH; to Doctors T. Kitade and M. Hyodo, Department ofAnesthesiology, Osaka Medical College, Ibaragi, Osaka; toDoctors M. Nakura, 0. Hayashi, A. Higashimura, and S.Miyoshi, Department of Psychiatry, Aino Hospital, Ibaragi,Osaka; and to Doctors T. Konishi, H. Nishitani, Departmentof Neurology, National Utano Hospital, Kyoto, Japan whoprovided samples for our study. We also thank Mr. H. Araifor technical assistance, Miss Y. Mitsuda and Miss C. Tamurafor secretarial assistance, and Miss M. Ohara for commentson the manuscript.

This work was supported in part by research grants for"Specific Diseases" from the Japanese Ministry of Healthand Welfare and from the Ministry of Education, Scieniceand Culture.

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