STUDIES IN METHIONINE METABOLISM. II....

STUDIES IN METHIONINE METABOLISM. II. FASTING PLASMAMETHIONINE LEVELS IN NORMALANDHEPATOPATHIC

INDIVIDUALS IN RESPONSETO DAILY METHIONINEINGESTION 1

By LAURANCEW. KINSELL, HAROLDA. HARPER, GRACEK GIESE, SHELDONMARGEN,2DAVID P. McCALLIE,3 AND JEAN R. HESS

(From the Division of Medicine, University of California Medical School; Department ofBiology, University of San Francisco; Metabolic Research Unit, University of

California-U. S. Naval Hospital; and Department of Medicine, U. S. NavalHospital; San Francisco and Oakland, Calif.)

(Received for publication March 12, 1949)

In earlier methionine studies (1), it was notedthat the fasting plasma methionine 4 levels in somepatients with acute and chronic liver disease, re-ceiving DL-methionine, tended to be markedlyincreased. It was not too clear in all instanceswhether this elevation related to the methionineadministration, to the disease process, or to a com-bination of the two. It was definite, however, thatsevere liver disease in itself could account forsome elevation of the fasting plasma methioninelevel. Over an 11-month period we have at-tempted further to clarify this picture.

PROCEDURES

The actual quantitative assay procedures have beenpreviously described (2, 3). It might be emphasizedhere that one concept held earlier has been modified inthat the normal fasting plasma methionine level varieswithin a very narrow range, from 0.25 mgm. per 100 cc.to 0.48 mgm. per 100 cc., with a mean of 0.33 mgm. per100 cc. The previous concept of a wider normal rangerelated to a longer period intervening between the ob-taining of the blood sample and the preparation of theprotein-free filtrate. Considerable proteolysis apparentlyoccurs in a matter of a few hours in some plasma speci-mens. All determinations here reported have been per-formed on protein-free filtrates prepared within threehours from the time of withdrawal of blood.

The first patients and controls in this series were given3 gms. of DL-methionine three times daily by mouth ascompressed 0.5 gm. tablets, over varying periods of time.Prior to, during, and following such periods of adminis-

1 This work is supported by grants from the ResearchDivision of the Bureau of Medicine and Surgery, U. S.Navy (BuMed No. 007046), and from the Office ofNaval Research, under a contract between the latterand the University of California.

2Senior Research Fellow, U. S. Public Health Serv-ice, 1947-48; and Schering Research Fellow in Endocrin-ology, 194849.

8 Lieutenant (j.g.) MC., USNR.4Unless otherwise qualified, the word "methionine"

will refer to L-methionine, i.e., the natural isomer.

tration, fasting blood specimens were obtained for D- andL-methionine quantitation. In some of these individuals,urines collected over 24-hour and/or 72-hour periodswere also assayed for D- and L-methionine.

When it became apparent that a striking differenceexisted in the methionine retention pattern between nor-mal individuals and many patients with chronic liverdamage, a standard program was set up consisting ofthe following procedures:A. Studies prior to methionine administration:

1. Fasting blood specimens for plasma D- and L-methionine assay, Monday through Friday, during WeekNo. 1.

2. One or more three-day quantitative urine specimensduring Week No. 1 for D- and L-methionine.

3. No medication of any sort during this period, ex-cept for routine "multi-vitamin" dietary supplementationin all patients,5 and parenteral vitamin K in those indi-viduals with significantly low prothrombin levels. Suchmedication was constant throughout the entire periodof study.

4. A diet high in protein, moderate in fat, and ade-quate in calories, was administered throughout the study.The most severe cirrhotics received a semi-liquid, high-protein, low-salt intake.

B. Studies during methionine administration:1. On Sunday of Week No. 2, 3 gms. of DL-methionine

were administered at 1:00 p.m. and 8:00 p.m.2. Thereafter, 3 gms. of methionine were administered

at 9:00 a.m., 1:00 p.m. and 8:00 p.m., daily during WeeksNo. 2 and No. 3, up to and including 9:00 a.m. on Mon-day of Week No. 4.

3. Daily fasting bloods were obtained Monday throughFriday for D- and L-methionine during Weeks No. 2 andNo. 3.

4. Three-day quantitative urine specimens were ob-tained for methionine assay throughout this period.

C. Studies following the cessation of methionineadministration:

1. After the 9:00 a.m. dose on Monday of Week No. 4,as previously noted, methionine was discontinued.

5 "Hexavitamin Tablets" (Strong, Cobb & Co., Inc.),six tablets daily.

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L. KINSELL, H. HARPER, G. GIESE, S. MARGEN, D. MCCALLIE, AND J. HESS

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STUDIES IN METHIONINE METABOLISM. II

2. Daily fasting blood specimens, Monday throughFriday of Week No. 4, were continued.

3. Twenty-four hour quantitative urine specimens wereobtained and assayed for methionine during Week No. 4.(The change from a three-day to a one-day collectionwas made in order to determine more exactly the periodof time necessary to eliminate all excess methionine fromthe tissues and blood.)

During this entire period of study, the patients were

subjected to careful daily clinical evaluation by dif-ferent members of the staff in order to detect any observa-ble changes which might relate to the duration ofmethionine administration and the plasma level ofmethionine.

This procedure was found to be unsatisfactory forserial studies in patients with acute viral hepatitis. Suchindividuals have therefore received 9 gms. of methioninedaily for two-day periods, at intervals during the course

of their disease. The evaluation of the periods precedingand following methionine administration has been cor-

respondingly shortened.For purposes of description, comparison, and evalua-

tion, the patients have been divided into arbitrary diag-nostic categories (see below).

CHEMICAL EVALUATION

Normal Controls (Figure 1)

Six control studies have been obtained in fiveyoung adult males. In control subject MAR, twostudies were conducted-one on an average diet(essentially the same type of diet as supplied toall other controls) and the second on an 800-calorie, relatively high-protein diet. This latterstudy was carried out because of a question as towhether the abnormal values noted in patients withliver damage might refer to the relatively poor

dietary intake of one or two of the patients studiedin the early phase of this investigation. As willbe seen from the data which follow, there are no

significant differences in methionine retention inthis normal man while on the 800-calorie diet as

compared to an average isocaloric diet.

Fasting Plasma Methionine LevelsL-methionine

1. Prior to methionine administration: All val-ues were in the normal range previously noted.

2. During methionine administration: Elevationof the fasting level was noted in the first 24 hoursin all subjects. In no normal man was a valuenoted in excess of 2 mgm. per 100 cc. at any timeduring the period of methionine administration.

3. Following methionine administration: A fallto normal levels was noted within 24-48 hoursafter the cessation of methionine administrationin all normal control subjects.

D-methionine1. Prior to methionine ingestion: Within the

limits of the method, no D-methionine was foundin any instance in the pre-treatment state.

2. During methionine ingestion: An elevationof the fasting plasma D-methionine was noted inall control subjects within the first 24 hours. Max-imal values were 1.92, 2.54, 0.62, 2.39, 2.35 and2.11 mgm. per 100 cc., respectively.

3. Following methionine ingestion: Except forthe control subject MIC, all values returned toessentially zero within 24 hours after the cessationof the methionine.

Urinary D- and L-Methionine Excretion

Urinary excretion values were obtained in onlytwo of the normal controls. Maximal excretionrates of 1.52 and 1.25 mgm. per hour for L-methionine and rates of 102.5 and 55.8 mgm. perhour for D-methionine were observed in thesemen. The behavior of the two isomers as "highand low (renal) threshold substances," respec-tively, has been emphasized in previous reports.

Patients with Acute Hepatitis (Figure 2)Serial studies on two representative patients

are shown using a short (two-day methionine ad-ministration) program for the reasons previouslynoted. The relationship between the status of thedisease, as shown by the presence of icterus, andthe degree of methionine retention is obvious.In these and other similar patients, the cephalincholesterol flocculation and the methionine reten-tion procedures were usually the last of a panel ofliver function tests (which included the determina-tion of bromsulfalein retention, free and combinedbilirubin, and prothrombin time) to become nor-mal.

Patients with Chronic Viral Hepatitis (Figure 3)Studies in three patients are presented. All had

histories compatible with an attack of acute viralhepatitis one and one-half or more years beforethis study was undertaken. All have been exten-sively studied from the standpoint of liver func-

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tion (all of the usual tests were abnormal); pro-tein metabolism (balance studies); and liver his-tology (biopsy). The histological findings arethose of localized areas of round cell infiltration,evidence of hepatocellular abnormality and moder-ate amounts of periportal fibrosis. The actualproof of viral infection is unfortunately not pos-sible.

Patient CUM, aged 25-At the time of thisstudy his disease process appeared to be progres-sively less active, as evidenced by diminution inhepatomegaly (to approximately 3 cm. below therib margin, on deep inspiration), gradual disap-pearance of spider angiomata, and diminishingabnormality of liver function tests.

Patient RIC, aged 21, had had involvement ofthe liver for a period of one and one-half or moreyears, dating from an attack of acute hepatitisprior to his original admission to this Hospital.At the time this test was performed, he had majorabnormality of all liver function tests including aretention of 14 per cent bromsulfalein (5 mgm.per kgm. x 45 min.). He also had evidence ofportal hypertension including some splenomegaly,and had had bleeding from an esophageal varixabout two months previously.

Patient CUN, aged 50, in the spring of 1946noted gradual onset of painless jaundice. Sixweeks later he was admitted to the Veterans Hos-pital in San Francisco at which time he had amajor degree of ascites which required eight para-centeses. He was told at that time that he hadviral hepatitis.

On March 1, 1948 he had a severe hemorrhagefrom esophageal varices. On admission to thisHospital ten days later, he was found to have con-siderable ascites, an enlarged, tender liver, moder-ate jaundice, and abnormality of all tests of liverfunction above noted. He was also found to havecardiac valvular disease probably referable to anold attack of rheumatic fever.

On a high-protein, adequate-calorie diet pluscomplete bed rest, he rapidly mobilized his asciticfluid and showed considerable clinical improve-ment and some degree of improvement in liverfunction, although at no time did he have completenormality of any of the liver function tests used.

From March 18 to May 13, 1948, five fastingplasma methionine values were all significantly

above the normal, the highest being 1.51 mgm. per100 cc.

On July 9, 1948 he was placed on 9 gms. ofDL-methionine daily. By July 27 he had becomeobviously toxic, the toxicity including some de-gree of disorientation and a penetrating odor whichwas not entirely that of methionine and which sug-gested the so-called "hepatic fetor." The samedose of methionine, that is, 9 gms. daily, was con-tinued until the 2nd of August, 1948. From July21 until August 2, he also received 9 gms. of cho-line chloride daily. The first methionine valueshown in Study No. 1 in this patient was obtainedon July 21, that is, 12 days after the institutionof methionine in a dose of 9 gms. daily, and on thefirst day of choline chloride administration.

The second methionine study was carried on aspart of a protein balance study and was institutedon October 11, 1948. His liver function tests atthis time were still significantly abnormal althoughhe no longer had any detectable ascites. The ini-tial portion of the second study was interruptedbecause of the appearance of symptoms which wereattributed to salt deprivation referable to a pe-riod of hot weather and low-sodium intake. Whenthis condition was corrected the study was re-sumed with the results shown in Figure 3. Dur-ing this second period of methionine administra-tion, he again manifested the symptoms of majorand progressive mental clouding coupled with astrong fetor which was similar to, but probablynot identical with that which is commonly calledfetor hepaticus.

Following the completion of this balance study,the patient was permitted to return home and wasinstructed to come in for a further evaluation afterthe Christmas holidays. Unhappily, he had a fatalhemorrhage from esophageal varices about onemonth after leaving this hospital and his localphysician obtained no postmortem examination.

The deviations from normal of the fastingplasma L-methionine values in all three patientsare quite definite, and in the last two are moststriking. The degree of abnormality would appearto parallel the severity of the disease process. Itis also apparent that increased urinary L-methio-nine appears in those individuals with very highplasma levels.

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Patients with Chronic, Non-Viral Liver Damage(Cirrhosis) (Figure 4)

Two successive studies on two patients areshown inasmuch as these men showed markedclinical and chemical improvement in the intervalbetween these evaluations. Both men (DOHand MEY) had the common denominator of ex-cessive and prolonged use and abuse of alcoholplus inadequate dietary intake during some ofthese periods of alcoholic indulgence. PatientDOHwas 33 years of age and by virtue of hisrelative youth presumably had a more reversibledegree of liver damage. Patient MEYwas 54years of age and when first admitted showed allthe usual findings of the disease, including anenlarged, hard, irregular liver, multiple spiderangiomata, clinical icterus, and general evidenceof impaired nutrition. He had had several pre-vious episodes of jaundice, as well as one episodeof hematemesis, presumably from esophagealvarices.

These patients received the usual therapy for*this disease, that is to say, a high-protein, high-vitamin, adequate-calorie diet with supplementalcholine chloride and with complete bed rest dur-ing the first several weeks of treatment. Routineliver function tests, which initially were abnormalin both men, became progressively more normal;and in Patient DOH, at the time of the secondstudy shown in Figure 4, had all become normalexcept for slight elevation of the serum globulinand 3 + cephalin flocculation.

As in the patients with chronic viral liver dam-age, it appears that the degree of elevation of thefasting plasma L-methionine, under these condi-tions, bears some relation to the degree of hepaticdamage.

Patients with Unusual Forms of Liver Pathology(Figure 5)

In Figure 5 are shown the findings on three pa-tients. Patient DEP is a cirrhotic, 58 years ofage, who has a long history of alcoholism, hadantiluetic therapy with arsenicals in 1908-1910,and malaria in 1926. He entered this Hospitaltwo months before the first methionine studywith a red blood cell count of one million as thedirect result of hemorrhage from esophagealvarices. With adequate therapy he had rapid dis-

appearance of ascites and hepatomegaly, andstriking general clinical improvement. He hashad at all times a moderate degree of proteinuria.At the time of his initial study, he was far alongon the road to convalescence, having a barely pal-pable liver, no residual ascites, and clinically nocomplaints whatever. His other liver functiontests at this time were normal. The methionineretention study showed a plasma L-methioninelevel which was within normal limits throughout.There was, however, a very marked elevation ofthe plasma D-methionine, a maximal level of 16.16mgm. per 100 cc. being attained on the 11th dayof methionine administration, together with mostimpressive elevation throughout. On the fourthday post-methionine, the plasma D-methionine wasstill elevated, the level being 0.95 mgm. per 100cc. Evaluation of the urinary methionine levelsshowed a single peak excretion on the secondthree-day period during methionine administra-tion, the level reaching 115.5 mgm. per hour, butat all other times the D-methionine excretion wasso low as to approach the limits of accuracy of theprocedure. From this observatio* we postulatedthat the mechanism of the entire picture relatedto some intrinsic renal defect which caused thisman to reabsorb D-methionine at the same rateat which he normally would reabsorb L-methio-nine.

One month later the procedure was repeated inan identical fashion. The plasma D-methionineand L-methionine levels, as will be seen fromFigure 5, were not significantly different fromthat noted in the first studies, but the urinary D-methionine values were of a high rather than lowmagnitude on this occasion. Urinary L-methio-nine was minimal throughout both studies. Dur-ing these two periods of methionine administrationhe manifested symptoms and findings which maybe referable to methionine toxicity; namely, aprogressive anorexia coupled with malaise andwith a strong fetor approximating that noted inthe patients previously described. There was nomajor degree of disorientation, however, as wasobserved in Patient CUN (Figure 3).

Patient BIE, a 52-year-old man with a hugeliver as part of his hemachromatosis and withmarked abnormality of many liver function tests,proved to have a perfectly normal pattern of

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methionine retention. He represents the onlyman so far with unquestionably active liver diseasewho has shown such a picture. It may be thathemachromatosis represents an extremely specificform of liver disease in which the utilization ofmethionine is not impaired. It should be noted,

however, that he was undergoing rapid clinicalimprovement referable to a high-protein, low-so-dium intake, and hence that widespread proteintissue formation might account for such a finding.

Patient FER, aged 50, has chronic liver damage,presumably resulting from prolonged advanced nu-

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Halving the dosage still resulted in marked elevation of the fasting plasma L-methionine. "MTT" refers to intravenous methionine studies (1).

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tritional deficiency (he was a prisoner of war ofthe Japanese for four years). In addition to clini-cal and chemical evidence of moderate hepatic in-sufficiency, he has evidence of portal hypertensionin terms of recurrent hemorrhages from esophagealvarices and a considerable degree of splenomegaly.His plasma L-methionine pattern is similar to thatseen in patients with moderate chronic liver dam-age.

One additional study (Figure 6) is includedon a man with very severe cirrhosis, chiefly be-cause of the length of the study and the fact thatthe dosage of methionine was diminished by one-

half at one stage. It will be noted in this patient,TUC, that with reduction in dosage a temporaryfall in the plasma methionine level occurred, butthat with the continuance of the smaller dose a

rise approaching the previous levels was obtained.The one high value on the dosage of 9 gms. ofmethionine was not entirely comparable with thatobserved in other individuals on the same dosageinasmuch as this fasting level was obtained as partof an intravenous methionine study and relatedto a discontinuance of orally administered methio-nine for a 48-hour period prior to the test. It isprobable that had the fasting level been obtainedwithout any such two-day interval, the valuewould have been higher than that noted in any

other individual included in this report. Thefigure is presented chiefly to demonstrate that even

moderate dosage of methionine in individuals withextreme degrees of hepatic insufficiency resultsin fasting blood levels which are quite unphysio-logic.

DISCUSSION

From the preceding observations, it is ap-

parent that many patients with liver damage are

unable properly to utilize methionine, and thatsuch impairment of methionine metabolism is no

longer present when the liver damage disappears(in patients with acute hepatitis) or has becomequiescent (in individuals with cirrhosis).

If one compares the abnormalities in plasmamethionine in patients with liver disease withthe blood sugar findings in diabetics, there ap-

pears to be some analogy. The individual with

severe liver disease has a high fasting plasmamethionine level without previous methionine ad-ministration. The abnormality in methioninemetabolism in the patient with mild or moderateliver damage is only apparent when stress is ap-plied. In our experience thus far, a spontaneouslyhigh fasting level is always correlated with a pro-longed, difficult convalescence, whether the dis-ease be viral hepatitis or "cirrhosis."

The possible mechanisms of impairment inmethionine utilization provide material for specu-lation. Three metabolic pathways are normallyopen: (1) Incorporation of the methionine intoprotein tissue; (2) demethylation with resultanthomocysteine formation; and (3) oxidation of themethionine molecule with resultant excretion ofthe oxidized sulfur as urinary inorganic sulfate.It appears probable that the block occurs in (1)or (2), inasmuch as the excretion of urinary sul-Eate is considerably increased in 'some of the pa-tients with high plasma methionine levels. Simul-taneous quantitation of plasma cysteine will helpto rule in or out the presence of block in themethionine -- homocysteine -> cysteine sequence.Such studies are under way at the present time.

Also in the process of evaluation is the determi-nation of methionine utilization in patients withnon-hepatic disease. Should a high degree ofspecificity for liver disease be found, it is conceiv-able that the procedure may have some clinicalapplicability.

The appearance of a reproducible picture oftoxicity in patients whose ability to utilize methio-nine is impaired, suggests the need for caution inthe indiscriminate use of this material as a thera-peutic agent in individuals with liver disease. Itis probably best to regard dietary methionine asthe logical source of the amino acid in such pa-tients.

SUMMARY

Daily administration of 9 gms. of DL-methionineto patients with liver damage results in a signifi-cant elevation of the fasting plasma L-methionine,as compared to normal controls; such abnormalretention disappears when the hepatic status re-verts to normal.

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In some patients with severe liver damage,continued administration of methionine results intoxic manifestations.

ACKNOWLEDGMENTS

Acknowledgment is made to Wyeth, Inc., for suppliesof DL-methionine, "Meonine," used in this study; to theMead-Johnson Company, for supplies of "Amigen" and"Lonalac" (sodium-free, reconstituted milk); and toMerck & Co., Inc., for supplies of pure amino acids andvitamins.

Acknowledgment is made to Florence E. Olson, re-

search dietician, for her assistance; and to M. F. Jackfor editing and typing this paper.

BIBLIOGRAPHY

1. Kinsell, L. W., Harper, H. A., Barton, H. C., Hutchin,M. E., and Hess, J. R., Studies in methionine andsulfur metabolism. I. The fate of intravenouslyadministered methionine, in normal individuals andin patients with liver damage. J. Clin. Invest.,1948, 27, 677.

2. Harper, H. A., Kinsell, L. W., and Barton, H. C.,Plasma L-methionine levels following intravenousadministration in humans. Science, 1947, 106, 319.

3. Kinsell, L. W., Harper, H. A., Barton, H. C.,Michaels, G. D., and Weiss, H. A., Rate of disap-pearance from plasma of intravenously adminis-tered methionine in patients with liver damage.Science, 1947, 106, 589.

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STUDIES IN METHIONINE METABOLISM. II....

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