SUPPRESSIONOF URIC ACID SYNTHESIS IN THE GOUTYHUMANBY THE USE OF 6-DIAZO-5-OXO-L-

NORLEUCINE*

By ARTHURI. GRAYZEL, J. E. SEEGMILLERAND ETHEL LOVE

(Fromt. the National Inistitute of Arthritis and Metabolic Diseases, National Institutes ofHealth, Bethesda, Md.)

(Submitted for publication September 23, 1959; accepted October 30, 1959)

A substantial number of patients with gout hasbeen shown to produce excessive amounts of uricacid (1-4). This overproduction has been dem-onstrated by the excretion of greater than normalamounts of uric acid in the urine, or an excessiveincorporation of isotopically labeled glycine intourinary uric acid. The present day treatment oftophaceous gout consists primarily in the ad-ministration of uricosuric agents to increase therenal excretion of the uric acid formed, with useof colchicine, ACTH gel, or phenylbutazone formanagement of the acute attacks. Control of theproduction of uric acid has been of necessity con-fined to limiting the purine and protein contentof the diet (5).

A logical approach to the treatment of thosepatients who overproduce uric acid would be toinhibit the excessive production by pharmacologicagents. In recent years several compounds havebeen reported to interfere with purine biosyn-thesis. Two such compounds, azaserine (o-diazo-acetyl-L-serine) and 6-diazo-5-oxo-L-norleucine(DON), have been shown to suppress the forma-tion of purines in bacteria (6), and Krakoff andKarnofsky have shown that DON inhibits uricacid production in the pigeon (7). Both com-pounds are structural analogs of glutamine (seeFigure 1) and both have been used in the humanas potential chemotherapeutic agents against can-cer (8. 9). Using partially purified enzyme sys-tems, Levenberg, Melnick and Buchanan (10)demonstrated that these agents do indeed preventthe synthesis of inosinic acid by blocking theutilization of glutamine in the enzymatic con-version of formylglycinamide ribotide to formyl-glycinamidine ribotide (Figure 2). DONwasfound to be the more potent inhibitor of the en-zyme system, requiring only 0.025 the concentra-

* A preliminary report of this work has been publishedin abstract form (J. clin. Invest. 1959, 38, 1008).

tion of azaserine to obtain the same degree of in-hibition. Although glutamine is also requiredin the first step in purine biosynthesis, theseagents are less effective inhibitors of this earlierreaction. Zuckerman, Drell and Levin (11) haveshown that azaserine reduced the incorporation ofglycine-1-C14 into urinary uric acid in gouty hu-mans, but did not alter either the serum uratelevels or the urinary excretion of uric acid. Thepurpose of our study was to observe the effectof the administration of DONon uric acid produc-tion in gouty patients, as measured by changes inthe serum urate levels and urinary excretion ofuric acid, and by the incorporation of glycine-l-C'4into urinary uric acid.

METHODS

Six male patients with gout and one with urate renallithiasis and essential hyperuricemia were studied duringhospitalization. They were all maintained on a standard2,600 calorie, 70 g protein diet, essentially free of purines.

H H H HN=N=C-C-C-C-C-COOH

1I H H /0 NH2 DON

(6-diazo-5-oxo-L-nor leucine)

_H H HN=N=C-C-0-C-C-COOH

11 H /0 NH2 AZASERINE(o-diazoacetyl- L-se rine)

H H HH2N-C-C-C-_C-COOH

11 H H /0 NH2 GLUTAMINE

FIG. 1. DON, AZASERINE AND GLUTAMINE, SHOWING

STRUCTURALRELATIONSHIPS.

447

ARTHURI. GRAYZEL, J. E. SEEGMILLERANDETHEL LOVE

0OHO-P-O-P-O JHO-I-OH

OH OH H | Glutomine

HNH21 2 Glycln

H + 'OHHN-RP ATP

PP-RP

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Glycinomide ribotide(GAR)

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Formylglycinomidine ribotide

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5 aminow4-imidozole-N-succino-corboxomide ribotide

OH

cRP

OtherPurines

5amino-4-imidozole-carboxomide ribotide(AIC-ribotide)

5-Formomide 4-imidazole-corboxamide-ribotide(formyl AIC-ribotide)

Inosinic Acid

(Hypoxonthine ribotide)

FIG. 2. PATHWAYFOR THE BIOSYNTHESIS OF URIC ACID. Asterisk indicates the enzyme that is inhibited by DON.

At least five days were allowed for equilibration beforestudies were begun. Daily 24 hour urine collections were

preserved with toluene at room temperature. The uricacid determinations of both urine and serum were madeby means of an enzymatic spectrophotometric assay (12).Urinary creatinine was determined by the method ofTaussky (13). DONprovided by the Cancer Chemo-

therapy National Service Center was freshly made up

in solution at the beginning of each study and stored inthe frozen state. It was given by the intramuscularroute, either 10 mg daily or in divided doses of 2.5 or 5mg every six hours. Isotopically labeled compounds were

administered on the third day of DONtherapy immedi-ately following DONinjection. Glycine-l-C' (obtained

TABLE I

Serum urate levels and 24 hour urinary uric acid excretion in patients receiving 6-diazo-5-oxo-L-norleucine (DON)

Serum urate

Average of 2 Excretion of uric aciddays prior to Lowestadministra- while Before DON Lowest while

Dose of DON tion of receiving (average of receivingPatient Age Diagnosis per day DON DON 3 days) DON

yrs mg/100 ml mg/100 ml mg/24 hr mg/24 hrT. B. 39 Essential 10 mg 9.5 6.5 1,280 730

hyperuricemiaand renal uratelithiasis

S. M. 53 Tophaceous gout 10 mg 9.4 9.9 393 313R. J. 41 Tophaceous gout 20 mg 10.2 9.0 1,140 716

in divideddoses

F. J. 34 Tophaceous gout 5 mg 10.9 11.4 1,250 1,110N. M. 49 Gout 5 mg 10.6 9.7 570 408J. S. 53 Gout 10 mg 7.9 7.5 598 467A. H. 48 Tophaceous gout 10 mg 7.5 6.9 494 417

NH

"Formyl" CH2 C-H

RP

* ~~NHCH2 C

Glutomine I 0c

ATP HeP\NH\RP

/C\ /ATP HP2N IR

448

DONSUPPRESSIONOF URATESYNTHESIS

IPatient RFJ. 41, Wd'

20 _ DON 2.5mg. m6g.20 IOmg./day 6 h.0 IIol;~ -J

15 Serum UricAcid

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Patient T.B., 39,Wd

Serum Uric Acid DmN/dIv

hI0 5 10 15 20 25 30

DAYS

FIG. 3. THE EFFECT OF DONON SERUMURATE LEVELSAND URINARY URIC ACID EXCRETION.

from Tracerlab Company of Boston), with a specificactivity of 1.33 mc per mmole, was given orally in a

dose of 5 /Ac with the breakfast milk. Simultaneously,36 mg of uric acid N1", containing 28.6 atom per centexcess of N', was administered intravenously as the

lithium salt to measure the urate pool and to allow cor-rections to be made for altered dynamics of this pool(14).1 The glycine pool, contributing to hippuric acid,was sampled by administering 450 mg of sodium benzoateintravenously, collecting urine for a two hour periodimmediately following the injection, and isolating hip-puric acid from the specimen. Such sampling was donesix hours after the initial administration of glycine-l-C1'and during the first two hours of the collection periodon the succeeding second, third, fifth and seventh days.A period of at least three months was allowed betweencontrol and DON studies to allow for elimination ofmost of the C1' and correction was made for residual C14.Hippuric acid was isolated by extraction with ether andrecrystallization from hot water (15). Urinary cre-atinine, as the double zinc salt (16), was isolated fromurine. Uric acid was isolated from aliquots of urineby adsorption on activated charcoal (17), or by pre-liminary precipitation as a copper salt when urine vol-umes exceeded 1,500 ml per day (18). Radioactivitywas determined using an end-window gas flow counter(Nuclear, Chicago) from planchets plated at infinitethickness with sufficient time for counting to producean error of not more than 3 per cent. The N15 contentof uric acid was determined in a Consolidated-Nier massspectrometer following preliminary digestion (19).

RESULTS

The effect of the administration of DON onthe serum urate levels, and on the 24-hour urinaryuric acid excretion values in the two patients whodemonstrated the greatest response to DON, areshown in Figure 3. The results obtained in allpatients are presented in Table I. Even thoughthe data in this table are presented in a mannerthat would emphasize small differences, i.e., bycomparing the average of several days during thepretreatment period with the lowest values ob-tained during treatment, the differences are notgreat. In four patients the difference in urinaryuric acid excretion was of the order of 20 per cent.Two patients showed a slight increase in serumurate levels during DON therapy. These twopatients both had tophaceous gout and the in-creased miscible urate pool may have preventedthe decline of their serum urate levels and urinaryuric acid excretion which might otherwise haveresulted from a decrease in urate production.

A more sensitive index of purine biosynthesisis provided by the incorporation of glycine-l-C14

1 Weare indebted to Miss Jean Benedict for providingthe uric acid-1,3-N"5.

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TIVITY, IN DISINTEGRATIONS PER MINUTE PER MILLIMOLE

OF URINARY URIC ACID, RESULTING FROM THE ORAL AD-

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FIG. 5. THE EFFECT OF DON ON THE CUMULATIVEINCORPORATION OF GLYCINE-1-C14 INTO URINARY URIC

ACID. All values are normalized to the same dose ofadministered glycine-1-C14.

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DONSUPPRESSIONOF URATESYNTHESIS

TABLE II

Suppression of uric acid synthesis from glycine-l-C'4 by DON

Per cent recovery of administered isotope in urinary uric acid in 7 days

C14 corrected for Effect ofGlycine-l-CI4 Uric acid N16 recovery of N1 DON

(a) (b) (a/b X 100) Per centof control

Patient Control DON Control DON Control DON incorporation

T. B. 1.11 0.26 77.2 75.2 1.44 0.35 24F. J. 0.99 0.61 80.6 72.8 1.23 0.84 68W. M. 0.30 0.05 * 35.7 17

* No N"6 urate administered.

into urinary uric acid. Figure 4 shows the effectof DONtherapy on the specific activity of C14in urinary uric acid; Figure 5 shows the cumula-tive incorporation of C14 into urinary uric acid inthree patients. From Table II it is apparent thatPatients W. M. and F. J., who failed to show adecline in serum urate during therapy, neverthe-less showed a substantially decreased incorpora-tion of glycine-1-C14 into uric acid indicative ofa depressed purine biosynthesis accompanyingDONtherapy.

In two of these patients corrections were madefor the dynamics of the urate pool, as evidenced

TABLE III

Toxic manifestations in patients receiving DON

Dose of Dura-Patient DON tion Toxic manifestations

mg/day daysT. B. 10 5 Erosions of oral mucosa on day 5

of therapyWV. M. 10 5 Erosions of oral mucosa on day 5

of therapyR. J. 10 5 Erosions of oral mucosa on day 3

of higher dose20 3 Asymptomatic duodenal ulcer dem-

onstrated by X-ray(Films taken at another hospital 2

months previously showed a de-formed duodenum)

F. J. 5 5 NoneN. M. 5 9 Erosions of oral mucosa on day 5

of higher dose10 6 Asymptomatic duodenal ulcer dem-

onstrated on X-rayJ. S. 10 6 Erosions of oral mucosa on day 6

Negative upper GI X-rayA. H. 10 6 Shallow erosion of tip of tongue on

day 6Negative upper GI X-ray

by the recovery of injected uric acid N15 in uri-nary uric acid (Table II). By seven days theenrichment of urinary uric acid with N15 in eachstudy had declined in a logarithmic manner tovalues approximating 1.0 per cent of those ob-served on the first day, except in Patient W. M.,in whom the value was 5 per cent. The fractionof N15 not recovered in urinary uric acid repre-sents urate removed from the body pool by meansother than renal excretion. These would includedeposition in tophi, losses via perspiration andgastrointestinal uricolysis. Thus, an estimationcan be made of the amount of C14 actually de-livered to the body's urate pool which did notappear as urinary uric acid. Such a corrected fig-ure represents the best approximation of the ex-tent of purine synthesis de novo. In PatientT. B., who received 10 mg of DONper day, thecorrected cumulative incorporation was reducedto 24 per cent of the control value. In PatientF. J., 5 mg of DONper day resulted in a reduc-tion to 68 per cent of the control value. Thespecific activity of the glycine pools sampled byhippuric acid and by creatinine was not signifi-cantly altered following DONadministration.

The toxic effects consisting of a sore mouth,occasionally with evidence of shallow erosions ofthe mucous membrane of the under lip, were ob-served at the end of five days in five patients(Table III). Two patients who received DONfor 14 and 7 days, respectively, developed asymp-tomatic duodenal ulcers which healed promptlyon medical therapy once the DONwas stopped.One patient, F. J., had no symptoms on a doseof 5 mg per day, although there was an effect onhis incorporation of glycine into urinary uric

451

ARTHURI. GRAYZEL, J. E. SEEGMILLERAND ETHEL LOVE

acid. No depression of the formed elements of theblood, changes in liver function, including theserum glutamic-oxalacetic transaminase level, ab-normalities of the urinary sediment, or occultblood in the stools were noted.

DISCUSSION

DONhad a marked effect on purine biosyn-thesis in the human, as shown by a substantialreduction in the incorporation of glycine-1-C14into urinary uric acid in all three patients studied.Such findings are consistent with the interpreta-tion that DONexerts an effect in vivo in thehuman similar to the effect observed in other bio-logical systems. Presumably, such effects resultfrom its action in blocking the enzymatic con-

version of formylglycinamide ribotide to formyl-glycinamidine ribotide. That DONhas an effecton other glutamine-requiring reactions (20)leaves open the possibility that other, more specificinhibitors of purine biosynthesis may be foundhaving fewer undesirable side effects.

DON administration failed to affect signifi-cantly the specific activity of the glycine poolsampled by hippuric acid and by creatinine. Thisis evidence that absorption of glycine-1-C14 fromthe intestine was unaffected by DON, and fur-ther supports the view that the action of DON,in the dosage used in this study, is primarily a

suppression of purine synthesis. The failure ofbalance studies to demonstrate a marked reduc-tion in serum urate levels or urinary uric acid ex-

cretion in five of the seven patients studied hasseveral possible explanations. With the short pe-

riods of drug administration and the presence of a

large miscible urate pool, a balance study may failto reflect brief changes in uric acid synthesis be-cause of the dampening effect of such a pool.The failure to obtain any decrease in serum uratelevels in Patients R. J. and W. N., despite a

substantial suppression of purine biosynthesis, as

shown by reduced glycine incorporation, might beexplained by the fact that these two patients hadthe largest tophaceous deposits of all the patientsstudied. Another possible explanation may berelated to the relatively short half-life of DONin the blood (approximately three to four hoursfollowing its intramuscular administration) (9).

Thus, maximal effects of DONwere obtained ata time when glycine-1-C14 in the body had itshighest specific activity. A compensatory over-production of uric acid during the remainder ofthe day may have masked such a suppression at agross level.

Upon discontinuing DON therapy, the serumurate showed a rebound, similar to that shown inFigure 3 (Patient R. J.), to values higher thanthose of the control period in five of the sevenpatients studied. Such transient elevations maywell be related to the operation of a homeostaticcontrol of purine synthesis (21, 22) similar tothat described for pyrimidine synthesis in bac-teria (23). A derangement of such homeostaticcontrol can be produced by the drug 2-ethylamino-1,3,4-thiadiazole (24, 25). A constitutional de-rangement in such a controlling mechanism hasbeen proposed to account for the increased uricacid synthesis seen in some cases of gout. A sup-pression of purine biosynthesis has also been pro-duced by administering 4-amino-5-imidazolecar-boxamide (21). This substance, however, is it-self converted to uric acid and so is of no valuein treatment of the excessive production of uricacid seen in gout.

A considerable variability in responsiveness ofdifferent patients to a given dose of DONmakesevaluation of its therapeutic index difficult. DONgiven for five days at a dose of 5 mg per day toPatient F. J. had no untoward side effects. Thesame dose continued for nine days in the secondpatient, N. M., again produced no untoward sideeffects, but when the dose was increased to 10 mgper day, an asymptomatic duodenal ulcer devel-oped after five days. Although DONadminis-tered in higher doses as chemotherapy for cancerresults in a significant incidence of leukopenia,thrombocytopenia, nausea and vomiting (9), nosuch toxic effects were observed during this study.The relatively high incidence of oral toxicity andpeptic ulceration, even at relatively low doses,makes the use of DONimprudent in* managingpatients who overproduce uric acid. This ap-proach, however, of reducing excessive uric acidproduction by means of inhibitors of purine bio-synthesis appears to be possible, and warrants afurther search for a more specific inhibitor witha more favorable therapeutic index.

452

DONSUPPRESSIONOF URATESYNTHESIS

SUMMARY

DON (6-diazo-5-oxo-L-norleucine) has beenadministered to six gouty patients and to one pa-

tion with renal urate lithiasis and essential hyper-uricemia. 1) Studies performed on three patientsdemonstrated that DON suppressed the incor-poration of glycine-1-C0 into urinary uric acid.2) Balance studies demonstrated that in two pa-

tients there was a significant decrease in serum

urate levels and 24-hour urinary uric acid excre-

tion following the administration of DON. 3)From the above studies, it is concluded that DONsuppresses uric acid biosynthresis, but such sup-

pression is not always reflected in a short-term bal-ance study, especially in the presence of an ex-

panded urate pool. 4) The use of pharmacologicagents to suppress synthesis in those gouty pa-

tients who overproduce uric acid represents a

new and rational approach to their therapy. Thehigh incidence of side effects following DONad-ministration prevents the practical use of this par-

ticular drug.ACKNOWLEDGMENT

The authors wish to express their appreciation to Mrs.Dolores Anderson and Miss Emma Lou Hendricksonfor technical assistance, and to Mr. William Comstockfor the N' determinations.

REFERENCES

1. Benedict, J. D., Roche, M., Yii, T. F., Bien, E. J.,Gutman, A. B., and Stetten, DeW., Jr. Incorpora-tion of glycine nitrogen into uric acid in normal andgouty man. Metabolism 1952, 1, 3.

2. Wyngaarden, J. B. Overproduction of uric acid as

the cause of hyperuricemia in primary gout. J.clin. Invest 1957, 36, 1508.

3. Seegmiller, J. E., Laster, L., and Liddle, L. V. Fail-ure to detect consistent overincorporation of gly-cine-1-C1' into uric acid in primary gout. Me-tabolism 1958, 7, 376.

4. Gutman, A. B., Yui, T. F., Black, H., Yalow, R. S.,and Berson, S. A. Incorporation of glycine-1-C14,glycine-2-C'4, and glycine-N' into uric acid innormal and gouty subjects. Amer. J. Med. 1958,25, 917.

5. Bien, E. J., Yu, T. F., Benedict, J. D., Gutman, A. B.,and Stetten, DeW., Jr. The relation of dietarynitrogen consumption to the rate of uric acid syn-

thesis in normal and gouty man. J. clin. Invest.1953, 32, 778.

6. Maxwell, R. E., and Nickel, V. S. 6-Diazo-5-oxo-L-norleucine, a new tumor-inhibitory substance. V.

Microbiologic studies of mode of action. Antibiot.and Chemother. 1957, 7, 81.

7. Krakoff, I. H., and Karnofsky, D. A. Inhibition ofuric acid biosynthesis in birds by o-diazoacetyl-L-serine (azaserine) and 6-diazo-5-oxo-L-norleucine(DON). Amer. J. Physiol. 1958, 195, 244.

8. Ellison, R. R., Karnofsky, D. A., Sternberg, S. S.,Murphy, M. L., and Burchenal, J. H. Clinicaltrials of o-diazoacetyl-L-serine (azaserine) in neo-plastic disease. Cancer 1954, 7, 801.

9. Magil, G. B., Myers, W. P. L., Reilly, H. C., Putnam,R. C., Magil, J. W., Sykes, M. P., Escher, G. C.,Karnofsky, D. A., and Burchenal, J. H. Pharma-cologic and initial therapeutic observations on6-diazo-5-oxo-L-norleucine (DON) in human neo-plastic disease. Cancer 1957, 10, 1138.

10. Levenberg, B., Melnick, I., and Buchanan, J. M.Biosynthesis of the purines. XV. The effect ofaza-L-serine and 6-diazo-5-oxO-L-norleucine on ino-sinic acid biosynthesis de novo. J. biol. Chem.1957, 225, 163.

11. Zuckerman, R., Drell, W., and Levin, M. H. The ef-fect of azaserine on the incorporation of glycine-C1'into uric acid in the gouty human (abstract).Ninth International Congress on Rheumatic Dis-eases, Toronto, 1957, vol. II, p. 204.

12. Liddle, L., Seegmiller, J. E., and Laster, L. Theenzymatic spectrophotometric method for deter-mination of uric acid. J. Lab. clin. Med. 1959, 54,903.

13. Taussky, H. H. A microcolorimetric determinationof creatine in urine by the Jaffe reaction. J. biol.Chem. 1954, 208, 853.

14. Benedict, J. D., Forsham, P. H., and Stetten, DeW.,Jr. The metabolism of uric acid in the normal andgouty human studied with the aid of isotopic uricacid. J. biol. Chem. 1949, 181, 183.

15. Soloway, S., and Stetten, DeW., Jr. The metabolismof choline and its conversion to glycine in therat. J. biol. Chem. 1953, 204, 207.

16. Benedict, S. R. Studies in creatine and creatininemetabolism. I. The preparation of creatine andcreatinine from urine. J. biol. Chem. 1914, 18,183.

17. Geren, W., Bendich, A., Bodansky, O., and Brown,G. B. The fate of uric acid in man. J. biol. Chem.1950, 1l33, 21.

18. Hawk, P. B., Oser, B. L., and Summerson, W. H.Practical Physiological Chemistry, 13th ed. NewYork, McGraw-Hill Book Company, 1954, p. 911.

19. Rittenberg, D. The preparation of gas samples formass spectrographic isotope analysis in Prepara-tion and Measurement of Isotopic Tracers, D. W.Wilson, A. 0. C. Nier and S. P. Reimann, Eds.Ann Arbor, Edward Brothers, 1946, p. 31.

20. Eidinoff, M. L., Knoll, J. E., Marano, B., and Cheong,L. Pyrimidine studies. I. Effect of DON (6-di-azo-5-oxo-L-norleucine) on incorporation of pre-

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ARTHURI. GRAYZEL, J. E. SEEGMILLERAND ETHEL LOVE

cursors into nucleic acid pyrimidines. Cancer Res.1958, 18, 105.

21. Seegmiller, J. E., Laster, L., and Stetten, DeW., Jr.Incorporation of 4-amino-5-imidazolecarboxamide-4-C' into uric acid in the normal human. J. biol.Chem. 1955, 216, 653.

22. Wyngaarden, J. B., and Ashton, D. M. The regula-tion of activity of phosphoribosylpyrophosphateamidotransferase by purine ribonucleotides: A po-tential feedback control of purine biosynthesis. J.biol. Chem. 1959, 234, 1492.

23. Yates, R. A., and Pardee, A. B. Control of pyrimi-dine biosynthesis in Escherichia coli by a feed-backmechanism. J. biol. Chem. 1956, 221, 757.

24. Seegmiller, J. E., Grayzel, A. I., and Liddle, L. Ex-cessive uric acid production in the human inducedby 2-ethylamino-1,3,4-thiadiazole. Nature (Lond.)1959, 183, 1463.

25. Krakoff, I. H., and Balis, M. E. Studies on the uri-cogenic effect of 2-substituted thiadiazoles in man.J. clin. Invest. 1959, 38, 907.

ANNOUNCEMENTOF MEETINGS

The Fifty-Second Annual Meeting of THEAMERICANSOCIETY FORCLINICAL INVESTIGATION will be held in Atlantic City, N. J., on Sundayafternoon and evening, May 1, 1960, in the Chalfonte-Haddon Hall (jointly withthe AFCR); and on Monday, May 2, at 9:00 A.M., at the Casino Theater onthe Steel Pier.

The Seventeenth Annual Meeting of THEAMERICANFEDERATIONFORCLINICAL RESEARCHwill be held in Atlantic City, N. J., on Sunday,May 1, 1960, at 9:00 A.M., at the Casino Theater on the Steel Pier. On Sun-day afternoon and evening, May 1, 1960, a joint sectional meeting with TheAmerican Society for Clinical Investigation will be held in rooms in theChalfonte-Haddon Hall.

THE ASSOCIATION OF AMERICANPHYSICIANS will hold itsSeventy-Third Annual Meeting in Atlantic City, N. J., at the Casino Theateron the Steel Pier on Tuesday, May 3, 1960, at 9:30 A.M., and in the VernonRoom, Chalfonte-Haddon Hall, on Wednesday, May 4, 1960, at 9:30 A.M.

454