dilution · 26ring m. london the isotope dilution method affords a precise technique for...

THE USE OF STABLE ISOTOPES IN BIOLOGICAL AND MEDICALRESEARCH

By IRVING M. LONDON

(From the Department of Medicine, College of Physicians and Surgeons, Columbia University,and The Presbyterian Hospital in the City of New York)

During the past 15 years there has been a rapidand extensive development of the use of isotopesin biological and medical investigation. In 1923,Hevesy's pioneering studies, in which thorium Bwas used to investigate the absorption and locali-zation of lead by plants, revealed some of the pos-sibilities of the use of tracer technique for bio-chemical research. The exploitation of these pos-sibilities marked time, however, until isotopes ofelements more widely associated with biochemicalprocesses were made available. The discovery ofdeuterium by Urey in 1931 and the developmentof methods for production of heavy water werethe first steps toward providing stable isotopesfor tracer studies in biological systems.

There soon followed the preparation of theheavy isotopes of nitrogen, carbon and oxygen,which, with hydrogen, constitute a large portionof the elements of interest to the biologist. Thediscovery of artificial radioactivity by Joliot andCurie in 1934 led before long to the production ofa number of radioactive isotopes which are of valuein biological investigations. The past decade anda half have witnessed a swift and enthusiastic ap-plication of stable and radioactive isotopes to awide variety of biologic studies.

The reports of these studies form a voluminousliterature. Several reviews (1-7) which deal withvarious aspects of tracer research have appearedin recent years. The scope of the present discus-sion is limited to the use of stable isotopes and itis not the purpose of this paper to attempt an-other review, which would require far more space

.than is available. This paper is designed to indi-cate some of the types of investigative problemsfor which tracers are particularly suited and topresent illustrative examples of studies in whichstable isotopes have been used.

The discussion is presented in two parts. I.Types of problems for which isotope investiga-tion is suitable. A. Problems of dilution andtransport-the addition of isotopic substance A toa biologic system and the subsequent isolation ofsubstance A and the determination of its isotope

content. B. Problems of conversion-the additionof isotopic substance A to a biologic system andthe subsequent isolation of substance B and thedetermination of its isotope content. C. Kineticsof biochemical reactions. II. An illustration ofthe use of isotope techniques in the investigationof related problems in one field: the biosynthesisand metabolism of porphyrins.

Problems of Dilution and Transport

Shortly after Hevesy's first experiments withthorium B, one of his colleagues expressed thehope that a tracer would be found which couldpermit the determination of the fate of individualwater molecules such as those in the tea they wereabout to drink (8). Within a decade Urey's dis-covery of deuterium and preparation of heavy wa-ter had provided an indicator which could help inthe solution of such a problem. This type of prob-lem is characteristic of a number of questionswhich can be answered by a basically simple ap-plication of the isotope technique. This applica-tion consists in introducing a labeled substanceof known isotope concentration into a system andsubsequently measuring the isotope concentrationof the same substance in the system as a whole orin one or more of its constituent units. One cantrace the fate of the labeled substance, i.e., its dis-tribution and localization, and one can measurethe time relationships of its distribution. Interms of biological experimentation, this tech-nique is applicable to the study of mechanisms andrates of transfer of biological substances by absorp-tion, secretion or excretion and to the study ofdiffusion and membrane permeability. The sametechnique is applicable to the solution of quanti-tative analytic problems, whether primarily bio-logical or chemical. By determining the dilutionin isotope concentration of the labeled substanceafter its addition to the system one can determinethe amount of this substance which is present inthe system. In the animal organism precisemeasurements of a variety of body constituentsare made possible, and in a purely chemical system,

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26 RING M. LONDON

the isotope dilution method affords a precisetechnique for quantitative analysis.

A few examples which are representative ofthe various applications of this type of tracer workmay serve to illustrate the use of the technique.Hevesy and Hofer investigated the rate at whichwater is eliminated from the human body (8).They administered heavy water to a subject andsubsequently measured the deuterium concentra-tion in the urine. By determining the interval be-tween the time of maximal concentration of deu-terium in the urine and the time when the deu-terium concentration in the urine was one-halfthe maximal value, they) determined tl./2, the timerequired for one-half of the heavy water ingestedto be eliminated from the body. This value is ninedays. Inasmuch as the biologic behavior of heavywater of low isotope concentration is essentiallythe same as that of ordinary water, this value fort1/2 applies equally to ordinary light water. Theaverage time which a water molecule spends inthe body is t1/2 X In 2, or 13 days.

In the same study, these investigators appliedthe principle of isotope dilution to determine thewater content of the body. After the first daythe deuterium concentration in the water of urinemay be taken as the deuterium concentration intotal body water. This is based on the reasonableassumption that the heavy water becomes com-pletely mixed with the total body water and ishandled in the body in a manner indistinguishablefrom that of ordinary water. The total body wa-

ter volume is then equal to the product of the deu-terium concentration and the volume of the heavywater administered divided by the deuterium con-

centration in the urine. These calculations yieldeda value for total water volume of 43 + 3 liters or

63 + 4 per cent of the body weight. This methodwas modified by Moore (9) who determined thedeuterium concentration in plasma one hour af-ter the intravenous injection of heavy water. Com-plete equilibration of the injected D20 with thebody water was found to occur within one hour;consequently the deuterium concentration in theone-hour plasma sample could be consideredequivalent to the deuterium concentration in totalbody water. By this method, a value for totalbody water of 47.8 liters or 72 per cent of thebody weight was obtained in a normal adult male.

The ability of the tracer technique to follow thetransfer of a substance across a membrane has

permitted the study of a number of biological prob-lems which could not be satisfactorily. investigatedby other methods. Without the aid of isotopes,the measurement of such transfer would dependon the determination of the net change in thequantity of the substance on each side of the mem-

brane. Under physiological conditions, however,little or no net change in the quantity of a greatmany substances is found despite considerabletransfer, for the transfer takes place simultane-ously in both directions across the membrane.The isotope technique avoids this difficulty. Byintroducing a labeled substance on one side of themembrane one can follow the movement of thelabeled substance in a system at equilibrium or ina steady state. Inasmuch as the biochemical be-havior of labeled units is indistinguishable fromthat of unlabeled units of the same substance, thetransfer of the labeled substance is representativeof the transfer of the substance as a whole.

The studies of Gellhorn and Flexner (10) onthe transfer of water across the placenta illustratethis application of the isotope technique. DOwas injected into a pregnant guinea pig, and afterten minutes during which equilibration of D20between the various fluid compartments of themother was known to occur, the fetuses were de-livered by Caesarean section. By determining thetotal body water of the fetus and the deuteriumconcentrations in maternal and fetal plasma, it waspossible to calculate the quantity of water trans-ferred across the placenta per unit time. It wasfound that the rate of transfer of water across aunit weight of placenta from the 28th day of gesta-tion to term increased about nine times, and thatthe rate of tranfer of water to a unit weight offetus was parallel to the relative growth rate ofthe fetus.

The principle of isotope dilution has foundready application to quantitative analytic studies(11-13). Most commonly, a small knownamount of labeled substance is added to a systemcontaining an unknown amount of the same sub-stance unlabeled. By determining the dilution inthe isotope concentration of the substance isolatedfrom the system, the quantity of substance origi-nally present in the system can be calculated. Thistechnique permits accurate quantitative determina-tions of a large number of substances for whichother good quantitative methods may not be avail-able. In addition, the need for quantitative isola-

1256i

USE OF ISOTOPES IN BIOLOGICAL AND MEDICAL RESEARCH

tion of the substance is obviated. A pure sampleadequate in amount for isotope analysis is suffi-cient. For accurate results it is essential that thelabeled material which is added to the system andthe material isolated from the system be pure.

The isotope dilution method has been used todetermine quantitatively the optical isomers ofamino acids present in amino acid mixtures (14)and to determine the amino acid content of horsehemoglobin (15) and human and bovine albumin(16). Its use has demonstrated that the claim forthe presence of large quantities of D-glutamicacid in malignant tumor tissue is invalid (17).

A modification of this procedure has been de-scribed by Keston, Udenfriend, and Cannan (18)for amino acid analyses. A mixture of aminoacids is treated with a labeled substance, e.g., p-iodophenylsulfonyl chloride labeled with I131. Alarge amount of the non-isotopic p-iodophenyl-sulfonyl derivative of the amino acid to be meas-ured is added to the mixture and the compound issubsequently isolated and its specific activity (iso-tope concentration in the case of stable isotopes)determined. Inasmuchias stable derivatives maybe formed with other amino acids in the mixture,the isolated compound may require numerous re-crystallizations before purity is established. Itshould be noted that complete conversion of theamino acid to its derivative must occur if ac-curate results are to be obtained. This methodpermits the quantitative determination of micro-gram quantities of amino acids.

The principle of isotope dilution has been ap-plied in reverse by Bloch and Anker (19) to de-termine the isotope concentration of a metabo-lite present in too small an amount to permit directisolation. Since the amount of isotopic metaboliteis also unknown, the addition of a known amountof nonisotopic metabolite (carrier) merely facili-tates isolation, but calculation of the original iso-tope concentration of the metabolite is not possible.If two different quantities of normal carrierare added to separate aliquots of the metabolitesolution,' however, the two samples of metabo-lite subsequently isolated will have different iso-tope concentrations. Sufficient data are thenavailable for calculation of the original isotope con-centration. The error of this procedure, however,may be as high as 20 per cent.

Problems of ConversionIsotopes are particularly suited to the study of

conversion reactions. One of the principal typesof investigation in intermediary metabolism is con-cerned with determining the products of the bio-logical conversion of a substance or to determineits biologic precursors. Experiments performedwithout the aid of isotopes are for the most part oftwo types: (1) in vivo studies in which substanceA is administered and the quantity of substance Bwhich is formed in the tissues or is excreted isdetermined; and (2) in vitro studies in whichsubstance A is added to tissue slices or isolated or-gans and the quantity of substance B formed inthe system is measured. Although these types ofstudy have yielded much valuable information, theydo not yield conclusive evidence for the determina-tion of biologic precursors or conversion products.In vivo studies in which an increased excretion ofsubstance B following the administration of sub-stance A is interpreted as evidence that A is abiologic precursor of B, are open to the criticismthat increased excretion of B represents an in-creased loss of B from the organism rather thanincreased synthesis and that A is responsible forthis effect merely by virtue of its effect on the ex-cretory mechanism. An increased formation of Bin vivo or in vitro following the addition of A doesnot prove the conversion of A to B but may indi-cate rather that A is concerned in reactions yield-ing energy required for the synthesis of B. Fur-thermore, the absence of increased formation ofB following the administration of A does not pre-clude the role of A as a specific precursor for B.Most biologic syntheses involve a series of reac-tions. Only one of these reactions may be limitingand principally responsible for the rate of forma-tion of B. If the specific conversion of precursorA to substance B is not this limiting reaction, theadministration of A will probably not be reflectedby an increased formation of B. Clearly anothertechnique of investigation which offers unequivocalevidence for the specific utilization of structuralcomponents of one substance for the biologic syn-thesis of another is required. Such is the isotopetechnique.

A striking example of the use of isotopes in thestudy of conversion reactions is the investigationof the biological synthesis of creatine and creati-nine. Bloch and Schoenheimer (20) addressedtheir attention first to the relationship in vivo of

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IRVING M. LONDON

creatine to its anhydride, creatinine. Creatinelabeled with N15 was fed to rats and subsequentlycreatine was isolated from the muscles and creati-nine from the urine. The N15 concentrations inthe isolated creatine and creatinine were nearlyidentical and indicated that urinary creatinine isderived from body creatine. Similar studies werecarried out in which after the preliminary periodof feeding N15 labeled creatine, the rats were kepton. a creatine-free diet, during which time theisotope content of urinary creatinine was identicalwith that of body creatine. This finding indicatedthat body creatine is the sole precursor of urinarycreatinine in an animal on a creatine-free diet.Were any other nitrogenous source present, theisotope concentration in the creatinine would havebeen lower than that of the body creatine as aresult of dilution with non-isotopic nitrogen.

To determine whether the creatine ~:.creati-nine reaction is reversible in vizo as it is in vitro,isotopic creatinine was administered to rats andbody creatine was subsequently isolated. Therewas essentially no isotopic nitrogen in the creatine,a finding which indicates that in vivo creatinine isnot convertible to creatine.

The next step in these studies was the searchfor the biologic precursors of creatine. Clues tothe identity of these precursors were providedby Brand et al. (21, 22) who found that pa-tients with muscular dystrophy excrete morecreatine after administration of glycine, and byFisher and Wilhelmi (23) who reported that theaddition of arginine to the fluid perfusing rabbithearts resulted in a quantitative conversion tocreatine. In addition Borsook and Dubnoffshowed that guanidoacetic acid is methylated tocreatine in surviving liver slices and that thismethylation is markedly accelerated by the addi-tion of methionine; they concluded that the methylgroup of methionine is transferred to guanidoaceticacid to form creatine (24). Simultaneous and in-dependent investigations by Bloch and Schoen-heimer revealed that when glycine labeled with

NH2 NH2 NH2

CH2COOH + C==NH -- C=I

Glycine Amidine NH

From Arginine CH2

N15 was administered to normal rats, creatine witha high isotope content was formed (25). Simi-larlv sarcosine labeled with N" yielded creatinewith equally high isotope content. Inasmuch asthe ingestion of isotopic sarcosine was shown tolead to the deposition in proteins of isotopic gly-cine to the same extent as when glycine itself wasfed, it was concluded that sarcosine is very rapidlydemethylated after its ingestion and that it servesas a precursor of creatine only after demethylationto glycine (26). When guanidoacetic acid labeledwith N15 was fed, creatine was formed with anisotope content approximately the same as whenan equivalent amount of isotopic creatine was ad-ministered; clearly, methylation of guanidoaceticacid to form creatine must occur quickly (25).

With the role of glycine as a precursor estab-lished and with the demonstration of the rapidmethylation of guanidoacetic acid to form creatine,there y et remained the problem of determiningconclusively the biologic sources of the variousgroups in the creatine molecule. Working inde-pendently, Borsook and Dubnoff found that kid-ney slices could form guanidoacetic acid from argi-nine and glycine (27) And Bloch and Schoen-heimer showed that the administration of argininelabeled with N1 in the amidine group resulted inthe formation of creatine of high isotope concen-tration (28). By degradation studies the latterinvestigators were able to show that the amidinenitrogen of creatine is derived from the amidinenitrogen of arginine, and that the nitrogen of thesarcosine group originates from glycine (29).

The final link was provided when du Vigneaudet al. found that the administration to rats ofmethionine labeled with deuterium in the methylgroup results in the formation of creatine with ahigh deuterium concentration (30, 31).

These studies established the immediate bio-logical source of each part of the creatine mole-cule. The biosynthesis of creatine may be sum-marized as follows:

NH2

NH + CH3 L C=NH

Methyl N-CH3From Methionine

!COOH CH2COOH

Guanidoaceticacid

Creatine

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Recent studies on the biosynthesis of uric acidoffer another example of the use of the isotopetechnique in establishing the identity of the bio-logic precursors of an important chemical sub-stance. Sonne, Buchanan and Delluva (32-34)prepared a number of compounds labeled with C18,administered them to pigeons, isolated the uricacid from the excreta, and degraded the uric acidby procedures which permitted separate isotopeanalyses of each of the carbon atoms in the uricacid molecule. They found that the number 6carbon is derived from CO2, carbon atoms 2 and 8from the carboxyl carbon of acetate (CHC*OOH)or formate (HC*OOH), and carbon atom 4 fromthe carboxyl carbon of glycine (CH2NH2C*OOH).Shemin and Rittenberg (35) demonstrated thespecific utilization of the amino group of glycinefor nitrogen 7 in an experiment in which N15labeled glycine was fed to a human and the uricacid isolated from his urine was degraded. Ni-trogen atoms 1, 3 and 9 are apparently derivedfrom ammonia of the general metabolic pool.With glycine established in positions 4 and 7, itremained for Karlsson and Barker (36) usingglycine labeled with C14 in the a position to showthat the methylene carbon of glycine is incorpo-rated into position 5 (NH2C*H2COOH). Thesecomposite data derived from both avian and humanexperiments have established the immediate bio-logic precursors of each component of the uricacid molecule.

Progress in the delineation of the conversionreactions in intermediary metabolism has beenmarkedly facilitated by the use of isotopes. Limi-tations of space prevent discussion of the many

excellent studies which illustrate this applicationof tracer technique. The interested reader is re-

ferred to reviews or representative papers byWood (37), Evans (38), Gurin (39), Stetten(40), Weinhouse (41) and their colleagues for a

more thorough consideration of the use of tracersin the study of conversion reactions.

Kinetics of Biochemical Reactions

Within a few years after the start of their in-vestigations in intermediary metabolism with theaid of isotopes, Schoenheimer, Rittenberg andtheir colleagues concluded that the conventionalconcept of the division of metabolic processes intoexogenous and endogenous forms was untenable.

Their studies demonstrated that there is a con-

stant interchange of dietary constituents with bodyconstituents and that the body constituents are ina state of flux, of continuous synthesis, degrada-tion, and interchange. They termed this conceptof intermediary metabolism "the dynamic stateof body constituents." This concept not onlycomprises an appreciation of the interconversionsand synthetic and degradative reactions of bodyconstituents, but it emphasizes the importance ofthe rates at which these reactions are carried outin the living organism. The kinetics of processes

in intermediary metabolism has received relativelylittle attention, however, as compared with theattention focussed on delineating the pathways ofsynthesis and degradation. In the future, how-ever, the kinetic aspects of biologic processes may

be expected to receive considerably more empha-sis in isotope studies. An appreciation of the im-portance of a metabolic road requires a knowledgeof the extent of the traffic on that road. Whenmore than one pathway for the conversion of one

substance to another exists, the rates at whichthe reaction is carried out via the different routesindicate the relative significance of these routes.

Studies in reaction kinetics are pertinent to theinvestigation of chemical changes in biologicalfluids and tissues which occur in disease. By de-termining reaction rates, it is possible to ascertainwhether an abnormally high concentration of a

substance is the result of an increased rate of syn-thesis or a diminished rate of degradation, andwhether an abnormally low concentration results

from a diminished rate of synthesis or an in-

creased rate of degradation.

The considerations and calculations involved in the

measurement of reaction rates in isotope experimentshave been discussed by Zilversmit et al. (42), by Bran-

son (43), and by Radin (44). The principal considera-

tion is the basic assumption of biological isotope research,namely, that isotopic molecules are biologically indis-

tinguishable from non-isotopic molecules. In nearly all

cases the additional assumption may be made that the

appearance and disappearance of all molecules proceed at

random. The simplest type of problem of reactionkinetics is concerned with a biological system in thesteady state, i.e., the rate of appearance of the substanceunder study equals its rate of disappearance and, duringthe period of study, these rates remain constant. Underthese conditions, the total number of molecules, M, ofthe substance in the system is equal to m, the numberof molecules which enter (or leave) the system per unit

1259

IRVING M. LONDON

of time, multiplied by the turnover time, T, the time re-quired for the appearance in the system or disappearancefrom the system of M molecules of the substance.

One type of experiment in which the rate of synthesisand degradation of a substance may be calculated is thatin which a small amount of the labeled substance whichis normally synthesized in the system is added to thesystem and its rate of disappearance is measured. Inthe steady state the rate of disappearance is equal to therate of appearance and is consequently a measure of therate of turnover. The rate of creatine turnover wasstudied by this technique (45). Adult rats were givenisotopic creatine and were then placed on a creatine-freediet. Samples of creatinine were isolated at intervalsfrom the urine. The isotope content of the creatininewas shown in the studies described earlier to be the sameas that of the body creatine. The decrease in isotopeconcentration in the urinary creatinine indicates there-fore the disappearance of labeled creatine from the tis-sues and its replacement by newly synthesized non-isotopic creatine. The curve of isotope concentration inthe creatinine declines in exponential fashion, i.e., thedecline in isotope concentration at any moment is pro-portional to the isotope concentration at that moment.Since the slope, K, of the declining isotope concentrationcurve is equal to m . i.e., the fraction of the total sub-stance M which disappears from the system per unittime, and since i is the isotope concentration at anytime t, then d= ki.dt

On integration i = iLes t, in which i0 is the isotopeconcentration at the start of the experiment. On con-verting to logarithmic values, ln i = In i0 - k t. A plotof In i against t yields a straight line whose slope is k.Accordingly, by serial determinations of the isotope con-centration, k can be calculated, and t1/2, the half life-

time, can be determined from the equation t1/2 = ln 2

Since T = t1/2 X In 2, the turnover time is also readilycalculated. To express the rate of turnover in terms ofunits of weight per unit of time, M must be determinedquantitatively by an independent procedure.

In the case of creatine in rats, the turnover rate wasfound to be about 0.02 per day. By determining thetotal body creatine, and the daily urinary creatinine ex-cretion, it was found that the daily creatinine excretioncorresponds to about 2 per cent of the total bodycreatine. The close agreement of these values for dailysynthesis of creatine and excretion of creatinine precludesthe existence of any major catabolic pathway for creatineother than creatinine formation and excretion.

Hoberman et al. (46, 47) have utilized this technique tostudy the rate of turnover of body creatine in man. Intwo human male subjects on a creatine-free diet valuesof 42 and 48 days for the half lifetime of body creatine

obtained ~ In 2 0.69 0.69 48were obtained 1t2= l - 0.0164 = 42; 0.0143 -J

When methyl testosterone is administered to the normalsubject, the slope of the curve becomes steeper and re-flects a more rapid dilution of body creatine (47). By

appropriate calculations it can be shown that the morerapid dilution is the result of an increased rate of synthe-sis of creatine and an increase in the total body creatine.This study affords direct evidence that methyltestosteroneaccelerates creatine synthesis.

When it is very difficult to synthesize an iso-topic compound whose biologic turnover is to bestudied, it may be possible to prepare the com-pound by biosynthesis and to study its turnover inthe same or in another organism. An isotopic com-pound which is known to be a biologic precursorof the substance is administered to the subject andsubsequently the substance is isolated serially andits isotope concentration is determined. In thecase of substances in the dynamic state, the isotopeconcentration rises to a maximum shortly after theend of the administration of the isotopic precursorand then declines exponentially. The upwardslope of the curve reflects the appearance of newlysynthesized labeled molecules and their replace-ment of unlabeled molecules formed prior to theadministration of the isotopic precursor. Thedeclining portion of the curve results from thedisappearance of labeled molecules and their re-placement by newly synthesized molecules con-taining little or no isotope. Inasmuch as thedegradation of the labeled molecules may in somecases result in isotopic fragments which can bereutilized for the synthesis of new molecules ofthe same substance, the rate of decline in isotopeconcentration may be slower than the actual rateof degradation. This discrepancy is probably notmarked, however, for after the degradation of thelabeled molecule, the isotopic fragment commonlyenters a metabolic pool of similar fragments oflower isotope concentration. A representativesample of this pool is utilized for the synthesis ofnew molecules. These conditions approximateroughly the replacement of isotopic moleculesby non-isotopic molecules and provide a goodmeasure of the turnover rate of a substance inthe dynamic state. This technique has been usedto study the rates of turnover of serum andantibody proteins in the rabbit in whom it wasfound that the half lifetime of serum and antibodyprotein molecules is about two weeks (48). Sheminand Rittenberg have utilized this technique in anextensive study of the interrelationships involvedin nitrogen transfer and of the rates of turnoverof nitrogen in various tissues of the rat (49).

1260


0.400

U 0.300K

0.200

gi

£ 0.1000

2 4 6 S 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42TIME IN DAYS

FIG. 1. N15 CONCENTRATIONIN SERUMPROTEINS AFTERFEEDING N15 LABELED GLYCINE FOR Two DAYS

The kinetics of serum protein formation in thehuman have been investigated by this method(50). Glycine labeled with N15 was fed to a hu-man subject for two days and the N15 concentra-tions in the total protein were determined. Fig-ure 1 shows a curve representative of the data ob-tained in these studies. The maximal N15 con-centration is reached shortly after the cessation offeeding labeled glycine and thereafter the isotopeconcentration declines in a roughly exponentialmanner. The t1/2 value for total serum protein isabout ten days.

With a t,/2 of about ten days, the turnover time, T,for a serum protein molecule is t1/2 X In 2 or about 15days. The normal human adult male with a total plasmavolume of 45 ml. per kilogram of body weight has aserum protein content of about 3 Gms. per kilogram ofbody weight. Substitution in the equation M= mX Tyields a value for m of 02 Gm. of serum protein perkilogram of body weight per day. In a normal man of70 kilograms, approximately 14 Gms. of serum proteinare synthesized and degraded daily. It should be re-membered that t1/2 values are approximations and aresomewhat greater than the actual half lifetimes. Ac-cordingly, the shorter actual half lifetimes will be re-flected in proportionately greater rates of synthesis anddegradation.

A theory and a practical procedure for evalua-tion of the rate of protein synthesis have recentlybeen described by Sprinson and Rittenberg (51).A small amount of N15 labeled amino acid is ad-ministered to the subject and the N15 content inthe urine excreted after the administration of theamino acid is determined. The greater the utili-zation of the amino acid for protein synthesis, the

smaller the total excretion of N15 during the pe-riod of study. Mathematical treatment of the dataon the cumulative excretion of N15 during a pe-riod of 48 to 72 hours after ingestion of the iso-topic amino acid affords an estimation of the rateof protein synthesis and the size of the metabolicpool of nitrogen. The rate of protein synthesis perkilogram of body weight per day is approximately0.2 Gm. of nitrogen in adult man. The nitrogenpool, i.e., the nitrogenous compounds which arederived from the diet or from the degradation ofbody constituents and which are utilized for thesynthesis of tissue constituents, was found to beapproximately 0.4 - 0.6 Gm. per kilogram in nor-mal man. This procedure should prove to be val-uable in the study of a variety of metabolic dis-orders and in the investigation of endocrine ef-fects on nitrogen metabolism.

The rate of synthesis and degradation of a bio-logic substance can be determined by still anothertechnique which measures the rate of incorpora-tion of deuterium into the compound from thebody fluids. This technique is applicable only tometabolic reactions in which hydrogen, or deu-terium, is stably bound to carbon. By maintaininga steady deuterium concentration in the body fluids

0.5

I°t.4* 0.3

_ 0.2z0

0Iw 0.11F .0 9

.04X .03

.02hi

C

.01

NORMAL MANSERUM CHOLESTEROL

5 10 15 20 25 30 35 40 45 50 55 60 65TIME IN DAYS

FIG. 2. DEUTERIUM CONCENTRATIONIN SERUMCHO-LESTEROL PLOrTED SEMILOGARITHMICALLY

Ct =deuterium concentration at any time t. C.=maximum deuterium concentration which is attained atinfinite time,

1261

IRVING M. LONDON

and by serial determinations of the deuterium con-centration in the substance under study one canmeasure the rate at which the substance is syn-thesized. The maximum deuterium concentra-tion which is achieved in this substance is usu-ally lower than that of the body water since afraction of the hydrogen atoms of most compoundsis not derived from body water. The half life-time of a substance is the time required to reach50 per cent of this maximum concentration. Stud-ies on .the rate of formation of body cholesterolin the mouse have shown a half lifetime for thecholesterol molecule of 15 to 25 days (52). Therate of synthesis of serum cholesterol in a nor-mal man has recently been investigated by thistechnique. Figure 2 describes a curve obtainedby plotting the isotope concentration against time

lo aimmdeuterium concentration at time t vs. i[l.g (i maximumdeuterium concentrationa te tie]

The linear curve indicates that the change in iso-tope concentration is exponential in character andfrom the slope of the curve the half lifetime ofserum cholesterol can be determined. In this nor-mal male subject, the half lifetime is about ninedays, the turnover time about 13 days (53).

The Biosynthesis and Metabolism of Porphyrins

The preceding discussion has been concernedwith presenting examples of the main types of bio-logical problems which are readily investigatedwith the aid of isotopes. These studies are for themost part unrelated and are diffusely spread overa number of fields of research. It may be well,therefore, to present an account of studies whichillustrate the application of a variety of isotopemethods to the investigation of a group of relatedproblems in one field. The studies to be describedare concerned with the biosynthesis and metab-olism of porphyrins.

These studies were initiated with the finding byShemin and Rittenberg that glycine is specificallyutilized for the biologic formation of the proto-porphyrin of hemoglobin (54). When glycinelabeled with N15 is fed to a human, the hemin iso-lated from the red blood cells of the subject is foundto contain N15 (55). Serial determinations ofthe N15 concentration in the hemin describe acurve (Figure 3) (55, 56) which is strikinglydifferent from the type of curve of isotope con-

ez0.3 ig

,0.2

0

400 20 40 60 80 100 120 140 160 180 200 220

TIME IN DAYS

FIG. 3. N15 CONCENTRATIONIN HEMIN AFTER FEEDINGN15 LABELED GLYCINE FOR Two DAYS

centration observed in serum proteins after theadministration of labeled glycine (Figure 1). Thephenomenon observed in the case of serum pro-teins represents a dynamic process in which pro-tein molecules are continuously being degradedand resynthesized. If hemin were in the dynamicstate, even though the red cell itself were morpho-logically intact, the N15 concentration in the heminwould describe a similar curve. The curve of N15concentration in hemin is fundamentally different,however, and cannot be the result of a randomsynthesis and degradation of hemoglobin in theperipheral blood. The presence of hemoglobinlabeled with N15 in erythrocytes many weeks afterthe end of glycine administration must be due tosynthesis and incorporation of hemoglobin labeledwith N15 in the erythrocytes during formation ofthe cells in the bone marrow. The persistence oflabeled hemoglobin in the red blood cells formonths after its synthesis in the bone marrow indi-cates that once incorporated into the red cell thehemoglobin remains with the cell until the celldisintegrates. In brief, the persistence of thelabeled hemoglobin reflects the survival or life spanof the cells containing the hemoglobin.

The upward slope of the curve of isotope con-centration in hemin represents the release into thecirculation of erythrocytes containing isotopichemoglobin and their replacement of cells formedprior to the administration of glycine which con-tain no isotopic hemoglobin. The maximum iso-

1262


tope concentration is reached and maintained whencells with heme of insignificant N15 concentrationreplace cells formed before the glycine adminis-tration. The declining portion of the curve re-flects the destruction of cells containing labeledhemoglobin and the point of inflection representsthe period of .most marked destruction of thesecells. The moderately abrupt decline indicatesthat the heme is not significantly, if at all, re-utilized for new hemoglobin formation when thecell disintegrates. If the heme were reutilizedsignificantly, a slower decline in isotope concen-tration would occur.

It is clear that normal human red blood cells aredestroyed as a function of their age and not inindiscriminate fashion. The average life span ofthe human erythrocyte can be calculated from thecurve of isotope concentration in hemin. Thesecalculations are presented in detail elsewhere (55,56). Values of 127, 120 and 109 days have beenobtained in two normal men and one normalwoman. Since these subjects were in a steadystate, these values correspond to the production

POLYCYTHEMIA VERA

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and destruction of approximately 0.8 per cent ofthe red cells per day. Despite a rather wide rangein the ages at death of the red cell population, thetime span which encompasses the ages at death ofhalf (the second and third quarters) of the cellpopulation is relatively short. In the two normalmale subjects this time span was found to be 28and 35 days, in the normal female subject 32 days.

This isotope method possesses the unique ad-vantage of making possible the study of the lifespan and pattern of destruction of the red cells inthe same individual in whom the cells are madeand destroyed without altering the usual state ofthe individual. Accordingly, it is well suited tothe study of hemoglobin synthesis and red bloodcell dynamics in normal and pathologic states.

Polycythemia vera is one of the clinical disorderswhich has been investigated with the aid of thismethod (56). The study was performed in anattempt to determine the factors which might beoperative in the production of the characteristicabnormality of the disease, namely, the markedincrease in the total number of circulating erythro-cytes. Theoretically this increase might resultfrom one or both of these factors: (1) an in-creased rate of hemoglobin and erythrocyte syn-thesis and (2) prolonged life of the erythrocytes.A patient in a fully developed stage of the diseasewas given glycine labeled with N15 in a dosageequivalent to that used in the normal subjects.Figure -4 describes the curve of the isotope concen-tration in hemin. The pattern of red cell destruc-tion is normal and the average life span of the redcells is 131 days, a value close to the values ob-tained in the normal subjects. Inasmuch as theerythrocyte life span is essentially normal, themarked increase in the number of circulating redcells must be associated with an elevated rate ofred cell and hemoglobin synthesis. In this patientthe rate of synthesis was 2.5 times the normalvalue.

These findings demonstrate a functional hyper-activity of the blood-forming apparatus in polycy-themia vera in a fully developed stage of the dis-ease. It is likely that the development of thepolycythemia earlier in the disease is also char-acterized by increased hematopoietic activity withthe maintenance of a normal erythrocyte lifespan. Various theories have been proposed forthe etiology of polycythemia vera but conclusive

1263

IRVING M. LONDON

proof in support of any of them is lacking. Thefundamental cause of the functional hyperactivityof hematopoietic tissue remains unknown. Thetheory, first proposed by Minot and Buckman(57), that polycythemia vera is a neoplasm, ap-pears to be most compatible with available evi-dence. The persistent bone marrow hyperplasiainvolving all marrow elements, the developmentof leukemia in some cases of polycythemia vera andthe development of polycythemia in some casesof leukemia suggest that this is a neoplastic proc-ess. It appears to be a benign neoplasm whichcan develop malignant characteristics. If it is aneoplasm, it is an instance of neoplastic growthwhich is associated with an increase in syntheticactivity with no apparent diminution in degrada-tive activity. The fundamental cause, however,remains no less obscure than that of other formsof neoplasia.

Sickle cell anemia is another clinical disorderwhich has been investigated with the aid of theisotope method (56). A patient with the char-acteristic findings of this disease received labeledglycine in a dosage similar to that used in the nor-mal subjects. The curve of isotope concentra-tion in the hemin is shown in Figure 5. It ismarkedly different from the normal. Followinga rapid rise to a maximum on the seventh day,the curve declines exponentially. The exponentialdecline results from a random disappearance oflabeled heme from the circulating blood, i.e., theheme is removed from the circulation at a ratewhich is independent of the age of the heme atthe time of its degradation. A curve of this shapecould result from (1) a random destruction of the

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red blood cells and a consequent loss -of labeledheme from the circulating blood; or (2) a randomdegradation and synthesis of heme in circulatingred blood cells which are morphologically intact;or (3) random synthesis and degradation of hemein red blood cells which are themselves undergoingrandom destruction. The latter two possibilitiesseemed unlikely in the light of earlier studieswhich demonstrated that the hemoglobin of cir-culating erythrocytes is not in the dynamic state.However, when the whole blood of patients withsickle cell anemia is incubated with N'5 labeledglycine heme labeled with N15 is formed (58).This in vitro synthesis of heme indicates that theperipheral blood in patients with sickle cell ane-mia may synthesize heme. The synthesis in thein vitro experiments occurs at a rate of only 0.1- 0.2 per cent of the red cell heme in 24 hours. Ifall the hemoglobin in the circulating erythrocytesof sickle cell anemia were synthesized in the periph-eral blood at a rate of the same order of magnitudeas in the in vitro experiments, the hemoglobinturnover would be ten to 25 times slower thanthat which is actually observed in this case. Itwould seem that the random disappearance ofheme must be due for the most part to an indis-criminate destruction of erythrocytes. If somerandom synthesis of heme in the peripheral bloodof patients with sickle cell anemia does occur, itprobably plays a very minor role in the hemo-globin turnover in this disease.

Inasmuch as the erythrocytes of sickle cell ane-mia are destroyed indiscriminately, they do nothave a true life span and their survival is moreappropriately measured in terms of their halflifetime. The half lifetime (t1/2), i.e., the timerequired for the isotope concentration to declinefrom any given value on the declining portion ofthe curve to one-half that value, is 29 days inthis case. The mean survival time, or turnovertime (t,/2 X ln 2), is 42 days. With these dataand determinations of total circulating hemoglobinand red blood cell volumes, the rates of hemo-globin and red cell production may be calculated.The rates in this patient were found to be nearlythree times the normal.

The increased hematopoietic activity is mostlikely a compensatory response to the markedlyreduced number of erythrocytes. The diminishedsurvival time reflects a defect which is in all prob-

TIME IN DAYS

FIG. 5. N15 CONCENTRATIONIN HEMIN AFrER FEEDiNGN15 LABELED GLYcINE FOR Two DAYS

1264


ability intrinsic to the red cell. This defect, whichprobably involves the structure of the red cellmembrane, may be associated with the sicklingprocess but cannot be ascribed to this phenome-non alone, since the red cells of individuals withsickle cell trait but without anemia are not ab-normally susceptible to destruction (59, 60).

Pernicious anemia is another disease processwhich has been investigated (56). A patient withthe typical physical and laboratory findings of thedisease received N15 labeled glycine. No anti-anemia therapy had been given when the experi-ment was begun. The curve of isotope concen-tration in hemin, the red blood cell counts, hemo-globin values, and reticulocyte counts during thecourse of the study are shown in Figure 6. Theisotope concentration in hemin rose rapidly andwas approaching its maximum on the 16th day.It was considered inadvisable to withhold treat-ment longer and liver extract therapy in large

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dosage was begun. A satisfactory reticulocyte re-sponse and rise in hemoglobin and red blood cellcounts occurred.

The rise in hemoglobin and erythrocyte levelswas accompanied by a decline in the isotope con-centration in the hemin. This decline was antici-pated for a dilution of the isotope concentrationin the hemin of circulating erythrocytes shouldresult from the release into the circulation of largenumbers of new cells formed when the isotopeconcentration in the body glycine had fallen to alow value. The decline in isotope concentrationpersisted, however, after the erythrocyte andhemoglobin values approached normal levels anda mere dilution effect should have been minimal.To differentiate a dilution effect from actual de-struction of the cells containing labeled hemo-globin, the changes in the total amount of N15in the heme of circulating erythrocytes were de-termined. It has been shown (61) that the hemo-

a a . II1 I3

HEMIN

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FIG. 6. N15 CONCENTRATIONIN HEMIN AFTER FEEDING N'15 LABELED GLYCINE FORTWODAYS TO A SUBJECT WITH PERNICIOUs ANEMIA

1265

I a

IRVING M. LONDON

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globin concentration in the peripheral blood servesas a reliable index of the total hemoglobin in circu-lation before and after the start of liver treatmentin pernicious anemia. Accordingly, a curve repre-senting the changes in the total amount of hemeN15 in circulation could be obtained by multiply-ing the hemoglobin concentration in the peripheralblood by the isotope concentration in the hemin(Figure 7).

The initial rise in total heme N15 is similar tothe rise in isotope concentration noted in Figure6. After the start of liver therapy, an additionalrise occurs due to the influx of many new cells.Although these newly formed cells contain hemeof relatively low isotope concentration, their totalnumber represents a considerable increment inthe quantity of heme N15 in the circulating blood.The maximum value for total heme N15 is attainedabout two weeks after the start of liver therapy.If the cells of untreated pernicious anemia en-joyed a normal life span, the curve would havemaintained a plateau until the 40th to 60th day andthen would have begun to decline. The curve de-clines, however, in linear fashion almost immedi-ately after reaching its peak. If all the cells weredestroyed indiscriminately, the decline would havebeen exponential. The linear decline suggests thatthe cell population is mixed, many of the cells be-ing destroyed indiscriminately and others as a func-tion of their age. From the declining portion ofthe curve it is possible to calculate that the meansurvival time of the mixed cell population is 90days and that the mean survival time of the cellsformed prior to liver therapy is approximately 85days. These calculations are presented in detailelsewhere (56).

With these data and determinations of totalhemoglobin and red blood cell values, it can beshown that the rate of production of erythrocytescapable of reaching the peripheral blood is onlyabout 50 per cent of normal. This diminishedrate of production and the diminished survivaltime of the cells in circulation are consistent withthe view that in untreated pernicious anemia theerythrocytes are intrinsically defective. The ab-sence of an abnormal hemolytic factor in the plasmaof pernicious anemia patients is supported bystudies with the Ashby technique (62, 63) whichhave shown an essentially normal survival of nor-mal cells transfused to recipients with perniciousanemia. There is no marked deficiency in the rateof production of circulating hemoglobin which inthis case was 80 per cent of normal.

After treatment with liver extract for one year,the patient was again studied. The findings re-veal that the red blood cells are destroyed as afunction of age, not indiscriminately, and that theiraverage life span is 129 days, a normal value.Complete restoration to normal red cell dynamicshas occurred (Figure 8).

In the untreated state, this patient showed onlymild diminution in the rate of production of circu-*lating hemoglobin. But even a normal rate ofproduction and destruction of circulating red cellhemoglobin fails to provide an adequate explana-tion for the very large amounts of bile pigmentwhich are produced in pernicious anemia. To in-vestigate this and other problems of bile pigmentmetabolism, the biologic origin of bile pigment hasbeen studied with the isotope technique.

It has commonly been assumed that bile pigmentnormally is derived almost exclusively, if not com-

1266

tort of livertherapy

I I I I I I I I I--- I


pletely, from the degradation of hemoglobin ofmature circulating red blood cells. In accordancewith this assumption, the appearance of N15 in thebile pigment following the administration of N15labeled glycine should reflect the destruction ofred blood cells containing labeled hemoglobin.Since in the normal human no significant numberof red cells is destroyed for several weeks aftertheir release into the circulation, there should beno significant concentration of N15 in bile pigmentduring the early part of the experiments in thenormal subjects. Stercobilin isolated from thestools collected during the first eight days of theexperiment in one of the normal subjects wasfound, however, to have a high concentration ofN15 (64). This finding suggests that a portion ofbile pigment is derived from one or more of thefollowing sources: (1) hemoglobin of red bloodcells which are destroyed shortly after reachingthe peripheral blood or never reach it and aredestroyed in the bone marrow; (2) porphyrinswhich are not utilized for hemoglobin production;or (3) direct synthesis of bile pigment via a path-way which does not involve degradation of aporphyrin ring. From quantitative considerationsit appears unlikely that myoglobin or the respira-tory heme pigments serve as a significant source ofthis portion of bile pigment.

The finding of an additional source of bile pig-ment formation appeared to offer a reasonable ex-planation for the discrepancy in untreated perni-cious anemia between the very high levels of bile

PERNICIOUS ANEMIA, TREATED

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pigment production and the relatively moderatedestruction of circulating red cell hemoglobin.When a similar study was performed in the pa-tient with pernicious anemia, an extraordinarilyhigh concentration of N15 was found in thestercobilin isolated during the first several daysof the experiment (65). These findings indicatethat a very large portion of the bile pigment pro-duced in this disease is derived from one or moreof the additional sources which have been sug-gested.

The investigation of porphyrin metabolism wasfacilitated by the finding that heme synthesis oc-curs in vitro in the blood of patients with sicklecell anemia (58) and in avian nucleated erythro-cytes (66). Since significant heme synthesis isnot observed in normal human blood under similarexperimental conditions, the in vitro synthesis ob-served in the blood of sickle cell anemia was be-lieved to be due to the presence of numerous im-mature reticulated cells. On incubation of bloodsamples from other subjects with hematologic dis-orders characterized by elevated reticulocytecounts, however, significant heme synthesis wasnot observed (58). These findings indicated thatalthough the reticulocytes in the blood of sicklecell anemia might be responsible for the heme syn-thesis, the mere presence of numerous reticulocytesin the blood of subjects with other hematologicdisorders does not insure the ability of such bloodto synthesize heme in vitro. Further studies (67)have confirmed the finding that some blood sam-ples with elevated reticulocyte counts from patientswith other blood dyscrasias do not perform signifi-cant in vitro heme synthesis. These studies haveshown, however, that other blood samples withelevated reticulocyte counts from subjects withdisorders other than sickle cell anemia may syn-thesize heme in vitro. To investigate some of theproblems posed by these observations, the capacityfor heme synthesis of reticulocytes in the blood ofexperimental animals which had no disorder oferythrocyte or hemoglobin formation was studied.The blood of previously normal rabbits in wjomreticulocytosis was induced by bleeding or phenyl-hydrazine hemolysis was incubated aerobicallywith N15 labeled glycine for 24 hours. The N15concentrations in the hemin of these blood sam-ples revealed that approximately 0.3 - 0.9 percent of the total heme in the in vitro system was

0 20 40 60 80 100 120 140 160 o80 200TIME IN DAYS

1267

IRVING M. LONDON

newly synthesized during the 24 hour incubationperiod. These findings demonstrate that mam-malian reticulocytes possess a significant capacityfor heme synthesis and suggest that some hemesynthesis may occur in vivo in the reticulocyteafter its release into the peripheral blood.

The in vitro synthesis of heme by avian erythro-cytes has been utilized to determine the precur-sors which participate in the biosynthesis of pro-toporphyrin. It has been found, using C14 labeledcompounds, that both carbon atoms of acetic acid,the carbonyl carbon of pyruvic acid, and themethylene carbon of glycine are utilized in the syn-thesis of the carbon skeleton of protoporphyrin(68). The utilization of the amino group of gly-cine for the synthesis of both types of pyrrole ringsof protoporphyrin has been demonstrated by find-ing equal N15 concentrations in the two pyrroletypes which were obtained by appropriate degra-dation of hemin formed following N15 glycine feed-ing (69).

The biosynthesis of porphyrins and bile pigmenthas been investigated in a patient with congenitalporphyria who excretes large amounts of uro-porphyrin I and coproporphyrin I. Shortly afterthe feeding of N15 labeled glycine, very high con-centrations of N15 were found in the coproporphy-rin I, uroporphyrin I and stercobilin (65). Thesefindings indicate that glycine is specifically uti-lized in the biosynthesis of porphyrins of the I iso-mer configuration as well as of protoporphyrin IXwhich is of the etioporphyrin III isomer configura-tion. They furnish additional evidence in sup-port of earlier observations (64) that bile pigmentis derived in part from one or more sources otherthan the hemoglobin of mature circulating erythro-cytes.

CONCLUSION

Despite the relatively short period of time dur-ing which isotopes have been available, tracer tech-niques have exerted a pervasive influence on bio-cheqiical thought and practice. A definitive evalu-ation of this influence may have to await more ex-tensive development of isotope investigation. Itis possible, however, to indicate the two principaldevelopments in biochemical thought which havealready occurred and are due in part to the im-pact of tracer techniques.

Tracer studies have helped to develop a con-cept which provides insight into the mechanismsof biological synthesis. It has become evident thatcomplex organic substances to a large extent aresynthesized not from compounds of similar com-plex configuration but rather from small com-pounds of simple structure. The extensive par-ticipation of glycine and acetic acid in biosyntheticprocesses has. focussed attention on the biochemicalversatility and significance of small simple com-pounds.

The outstanding contribution of isotope method-ology to biochemical thinking has been the devel-opment of the concept of the dynamic state of thebody constituents. The experimental basis andtheoretical implications of this concept, which in-cludes an appreciation of the kinetics of biochem-ical reactions, represent a major advance towarda thorough understanding of the processes whichoccur in the living cell.

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21. Brand, E., Harris, M. M., Sandberg, M., and Ringer,A. L, Studies on the origin of creatine. Am. J.Physiol., 1929, 90, 296.

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from methionine to choline and creatine. J. Biol.Chem., 1940, 134, 787.

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43. Branson, H., The use of isotopes in an integralequation description of metabolizing systems, in:Cold Spring Harbor Symposium on QuantitativeBiology. Long Island Biological Laboratory, ColdSpring Harbor, Long Island, N. Y., 1948, 13, 35.

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46. Hoberman, H. D., Sims, E. A. H., and Peters, J. H.,Creatine and creatinine metabolism in the normalmale adult studied with the aid of isotopic nitro-gen. J. Biol. Chem., 1948, 172, 45.

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50. Unpublished data from this laboratory.51. Sprinson, D., and Rittenberg, D., The rate of inter-

action of the amino acids of the diet with thetissue proteins. J. Biol. Chem., in press.

52. Rittenberg, D., and Schoenheimer, R., Deuterium asan indicator in the study of intermediary metabo-lism. XI. Further studies on the biological up-take of deuterium into organic substances, withspecial reference to fat and cholesterol formation.J. Biol. Chem., 1937, 121, 235.

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utilization of glycine for the synthesis of theprotoporphyrin of hemoglobin. J. Biol. Chem.,1946, 166, 621.

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56. London, I. M., Shemin, D., West, R., and Ritten-berg, D., Heme synthesis and red blood cell dy-namics in normal humans and in subjects withpolycythemia vera, sickle-cell anemia, and per-nicious anemia. J. Biol. Chem., 1949, 179, 463.

57. Minot, G. R., and Buckman, T. E., Erythremia(polycythemia rubra vera). The development ofanemia; the relation to leukemia; considerationof the basal metabolism, blood formation anddestruction and fragility of the red cells. Am. J.M. Sc., 1923, 166, 469.

58. London, I. M., Shemin, D., and Rittenberg, D., ThemVitro synthesis of heme in the human red bloodcell of sickle cell anemia. J. Biol. Chem., 1948,173, 797.

59. Singer, K., Robin, S., King, J. C., and Jefferson,R. N., The life span of the sickle cell and thepathogenesis of sickle cell anemia. J. Lab. & Clin.Med., 1948, 33, 975.

60. Callender, S. T. E., Nickel, J. F., and Moore,C. V., Sickle cell disease: studied by measuringthe survival of transfused red blood cells. J. Lab.& Clin. Med., 1949, 34, 90.

61. Gibson, J. G., 2nd, Clinical studies of the blood vol-ume. VI. Changes in blood volume in perniciousanemia in relation to the hematopoietic responseto intramuscular liver extract therapy. J. Clin.Invest., 1939, 18, 401.

62. Loutit, J. F., The life and death of the red corpuscle.Proc. Roy. Soc. Med., 1946, 39, 757.

63. Mollison, P. L., Survival of transfused erythrocytes,with special reference to cases of acquired hmmo-lytic anemia. Clin Sc., 1947, 6, 137.

64. London, I. M., West, R., Shemin, D., and Ritten-berg D., On the origin of stercobilin in humans.Federation Proc., 1948, 7, 169.

65. London, I. M., Shemin, D., West, R., and Ritten-berg, D., Unpublished data.

66. Shemin, D., London, I. M., and Rittenberg, D., Thein vitro synthesis of heme from glycine by thenucleated red blood cell. J. Biol. Chem., 1948, 173,799.

67. London, I. M., Shemin, D., and Rittenberg, D.,Studies in hemoglobin formation with the aid ofthe isotope technique. J. Clin. Invest., 1949, 28,796.

68. Radin, N. S., Rittenberg, D., and Shemin, D., Bio-synthesis of heme. Federation Proc., 1949, 8,240.

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dilution · 26ring m. london the isotope dilution method affords a precise technique for...

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