THE METABOLISM OF DL-GLUTAMIC ACID-l-C4 IN MAN*

BY FRANK W. PUTNAM, AIKO MIYAKE, AND FRANZ MEYER

(From the Department of Biochemistry, College of Medicine, University of Florida, Gainesville, Florida, and the Argonne Cancer Research Hospital,

United States Atomic Energy Commission, and the Departmen,t of Riochemistry, University of Chicago, Chicago, Illinois)

(Received for publication, November 18, 1957)

Despite its alleged occurrence in human and animal tumors (l-3), D- glutamic acid is not utilized to any appreciable extent by experimental animals (4,5). The fate of n-glutamic acid in man, however, has not been fully investigated. In the course of study of the biosynthesis of abnormal proteins in multiple myeloma (6-9), nn-glutamic acid-l-C4 was injected in- travenously into a patient. Frequent blood and urine samples were taken, and the expiratory CO* was collected at intervals. Time curves were determined for the radioactivity of (1) expiratory CO*, (2) urinary urea, (3) urinary amino acids, (4) serum glutamic acid, (5) urinary Bence- Jones protein, and (6) serum y-globulin (the myeloma globulin). A comparison was also made of the radioactivity of the CO2 released by the reaction of urinary amino acids and of the hydrolyzed Bence-Jones pro- tein with ninhydrin versus glutamic acid decarboxylase. As might be ex- pected, the L-glutamic acid was rapidly metabolized. However, the D- glutamic acid was excreted apparently unchanged in the urine and was not significantly incorporated into the Bence-Jones protein, which is believed to be made in the tumor cells.

EXPERIMENTAL

Methods and Procedures

Radioactivity MeUSUTementsDL-GlUtamic acid-l-U4 with an activity of 10 PC. per mg. was obtained from the Isotopes Specialties Company, Inc., Burbank, California. This compound was chosen because the radio- activity is completely liberated from the L form on incubation with glu- tamic acid decarboxylase. This procedure not only permits a differentia- tion of the L and D isomers but also affords a direct method for measuring the specific activity of glutamic acid in protein hydrolysates without isola- tion of the amino acid. Prior to administration, the compound was dis- solved in a 0.9 per cent NaCl-0.5 per cent glucose solution, filtered through ultrafine sintered glass, and tested for toxicity and sterility. No carrier glutamic acid was administered.

* Aided by research grants from the National Cancer Institute, National Insti- tutes of Health (No. C-1331-(24), and from the American Cancer Society.

657

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658 METABOLISM OF DL-GLUTAMIC ACID

The precautions, techniques, and apparatus used in radioactivity meas- urements were similar to those described in a related experiment (8). The purified, lyophilized, salt-free protein samples were counted on planchet,s; the specific activity corrected for self-absorption is expressed as counts per minute (c.p.m.) per mg. of protein. Protein samples were hydrolyzed by autoclaving for 12 hours with 6 N HCl. The hydrolysntes were con- centrated in a flash evaporator with repeated washings to remove the acid. The amino acids in the hyclrolysates were then decarboxylated by reaction with ninhydrin at 100” or by incubation at 37” with successive aliquots of glutamic acid clecarboxylase? The amino acids in urine samples of 0.3 ml. or 3.0 ml. were likewise decarboxylated with ninhydrin but in the presence of 15 mg. of L-glutamic acid as carrier. Enzymatic decarboxylation of 1 .O ml. serum samples was also carried out in the presence of 15 mg. of carrier L-glutamic acid. In all the clecarboxylation reactions the system was first flushed with nitrogen to remove preexisting COZ; the COZ released by the reaction was trapped with Ba(OH) 2 and counted as BaCOs in aluminum planchets with an area of 3.47 cm?. The activities are given as counts per minute for idinitely thick samples. The specific activity of expiratory COS (expressed as millimicrocuries per millimole of COZ) was kindly measured by Dr. George Okita by means of a vibrating reed electrometer. Dr. Okita also determined the specific activity of urinary urea COZ (expressed as millimicrocuries per millimole of COZ); for this, the electrometer was used to measure the activity of the COZ released by the action of urease on urine samples. Because of the variety of methods employed, it was not feasible to reduce all radioactivity measurements to common units.

Serum and Urinary Proteins-Serum protein fractions were prepared either by starch zone electrophoresis or by salt precipitation (9). The Bence-Jones protein WM isolated by ammonium sulfate precipitation and prolonged dialysis against distilled water (8). These protein preparations were characterized by electrophoresis in the Tiselius apparatus and by sed- imentation in the analytical ultracentrifuge (6-8).

Subject and Protocol

Subject-The patient (H. H.), a 67 year-old male, was admitted to the Argonne Cancer Research Hospital for treatment and terminal care. For therapy, urethane was given throughout the experiment and radiation just prior to it. Laboratory study revealed Bence-Jones proteinuria, hyper- globulinemia, moderate anemia (12 gm. per cent of hemoglobin), and severely depressed renal function. Blood urea nitrogen rose progressively

1 An acetone-dried preparation of L-glutamic acid decarboxylase was prepared by t.he method of S. Mandeles (private communication) from a strain of Escherichia coli originally received from V. Najjar.

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F. W. PUTNAM, A. MIYAKE, AND F. MEYER 659

from 22 mg. per cent to 176 mg. per cent, and the urea cIearance fell ter- minally to a value of 2.0 as calculated by the square root method. The di- agnosis of multiple myeloma was confirmed upon postmortem examination. Kidney damage was extensive and typical of the disease.

Protocol-For the experiment approximately 450 PC. of nn-glutamic acid- l-Cl4 were given intravenously over a 15 minute interval to the fasting pa- tient? He had some discomfort because of nausea and vomiting, and there was a diuresis for the 1st hour.3 Fluids were given parenterally on sev- eral occasions during the first few days but no whole blood until the 4th day. In the first 24 hours, nineteen blood samples were obtained by in- travenous catheter, the first at 15 minutes after the start of the injection. A complete urine collection was maintained with twenty-four samples be- ing taken by urethral catheter on the 1st day. The catheters were then removed, and blood samples were discontinued; but complete urine collec- tion was kept up for a week with a total of forty-nine specimens. Blood was allowed to clot, and the serum specimens were frozen for later study. Aliquots of the urine were taken for immediate isoIation of the Bence- Jones protein, and the balance was stored in a deep freeze unit. The ex- piratory air was sampled at intervals for the first 8 hours. Except for the initial discomfort, there was no adverse result of the experiment. Ni- trogen intake studies were carried on for the following 2 months.

RESULTS AND DISCUSSION

Metabolism and Excretion of Glutamic Acid

Expiratory COz-n-Glutamic acid is readily deaminated to yield cr-ke- toglutarate by L-glutamic acid dehydrogenase or via the transaminase re- action, both of these enzymes being optically specific (12, 13). Thus, L-

glutamic acid-l-C4 would form a-ketoglutarate which on decarboxylation would yield CY402 and unlabeled succinic acid. Contrary to common as- sumption (14), no deaminating mechanism for n-glutamic acid is known in mammalian organisms (4,15). However, Ratner (5) has reported that pyrrolidonecarboxylic acid is excreted in urine as the quantitative end product of n-glutamic acid metabolism in the rat.

2 Dr. Milton Weiner aided in devising the protocol and in medical management of the patient under the direction of Dr. Robert Hasterlik and Dr. George V. LeRoy. Dr. Robert Wissler and Dr. Benjamin Spargo prepared the autopsy report.

8 Of twenty-two amino acids injected intravenously in different combinations into dogs, toxic reactions such as vomiting occurred only when glutamic acid was in the mixture (10). Glutamine is well tolerated under the same conditions, and it seems to be the physiological form of the amino acid in the circulation (11). On the other hand, glutamate is now being administered experimentally in the therapy of hepatic coma.

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660 METABOLISM OF DL-GLUTAMIC ACID

In view of the above known metabolic pathways a sharp early maximum in the specific activity of expiratory Cl402 was to be expected. Indeed, the maximum occurred at about 25 minutes and was followed by an exponential decline in radioactivity with a half time of 85 minutes (Fig. 1). On bal- ance, 3.1 per cent of the injected activity or 6.2 per cent of the activity in t,he L form was exhaled in the first 8 hours. In the 5 minute period at the time of the maximal specific activity of expiratory Cot, the subject ex-

40

30

20

0” v IO

%

-5

: E 5

3

,2

I 200 400

Minutes After Administration

FIG. 1. Specific activity and cumulative activity of expiratory CO2 after injec- tion of 450 pc. of DL-glutamic acid-l-C” into a fasting man.

haled 0.72 PC. of C402, but only 0.02 PC. in the 5 minute period at 8 hours after injection. At 24 hours the activity of expiratory COZ would have been negligible.

Urinary Amino Acids-From the primary data obtained after reaction of the urine samples with ninhydrin, curves have been plotted in Fig. 2 for the rate of urinary excretion of W-amino acids, both for the cumulative activity and the activity excreted per minute. The cumulative activity for the first 20 hour period was calculated from the thick sample count of the aliquot of urine decarboxylated, the volume of urine excreted, and the time

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F. W. PUTNAM, A. MIYAKE, AND F. MEYER 661

interval (see representative data in Table I). In the 1st hour there was a diuresis accounting for more than one-third of the total counts excreted on the 1st day, yet in this period the Bence-Jones protein had negligible ac- tivity. The total activity excreted in any single collection period was greatest for Sample 3 taken shortly after the injection.4+ 6 As illustrated in Fig. 2, the excretion of Cl4 via amino acids was slight after 5 hours. One would expect an exponential decline in the amount of Cl4 voided via uri- nary amino acids if the latter were freely excreted unchanged by metab- olism and if the output of urine per minute were constant. To correct for the variable output the rate of urinary excretion of Cl4 released by ninhy- drin is expressed in Fig. 2 as the total counts per minute voided per min-

ACTIVITY EXCRETED PER MINUTE

0 5 IO 15 HOURS AFTER ADMINISTRATION

FIG. 2. Urinary amino acid activity excreted per minute and cumulative activity excreted in the first 20 hours after injection of nL-glutamic acid-1-C14.

ute (e.g. data of Column 6 of Table I divided by the time interval of Col- umn 3). When plotted on this basis, which allows for the initial diuresis, the maximum in excretion occurs within the 1st half-hour. The rate of decline was exponential up to 8 hours and had a half time of about 80 minutes.

Comparison of the activity of the COz obtained by reaction of the urine with n-glutamic acid decarboxylase with that evolved by ninhydrin gave evidence that the U402 produced in the latter case was almost wholly ascribable to the n-glutamic acid injected. Thus, in urine Sample 4 the

4 Sample 2 is the exception, representing the injection period for which no urine remained for this test. Sample 3 is omitted from the curve for activity excreted per minute (Fig. 2) because the collection bottle overflowed owing to the diuresis.

6 Total activity equals the thick sample count per volume of sample times the total urine volume collected in the designated interval.

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662 METABOLISM OF DL-GLUTAMIC ACID

thick sample count was 6520 c.p.m. for the B&O3 obtained from 0.3 ml. of urine after reaction with ninhydrin in the presence of 15 mg. of carrier L-glutamic acid. Under the same conditions the thick sample count for the BaCO$ produced by enzymatic decarboxylation was only 5 c.p.m. Quantitatively, of course, the excretion of n-glutamic acid-l-Cl* is greater than is indicated by the above specific activity, for the natural amino acid is diluted by the body pool, whereas n-glutamic acid is not. Because there was only a limited uptake of Cl* into other n-amino acids (see later), their contribution can be neglected. Glutamine is decarboxylated by nin-

TABLE I Representative Data on Urinary Excretion of Cl4 Reactive and Unreactive

with Ninhydrin - - - ___- Sample No. Time* Time interval I I. Jrine volume Aliquott rota1 activity Urine residue

(1) (2) (3) (4) (9 (6) (7) --

hrs. nzin. W&l. c.p.m. 10s C.P.rn. c.p.m.

3 0.42 20 2501 43,500 36201 108 7 2.0 30 60 116,000 2320 719 9 3.5 60 50 110,000 1840 2950

11 5.75 60 60 30,400 610 2015 14 8.75 60 65 7,860 170 1319 16 11.25 120 135 4,000 180 1385 19 17.25 120 150 1,480 74 712

- * From the mid-point of the injection to the mid-point of the interval. i The aliquot activity is given as the thick sample count for the BaC03 obtained

from the ninhydrin reaction with 3 mI. of urine in the presence of 15 mg. of carrier nn-glutamic acid. Total activity = (aliquot activity X urine volume)/3.0 ml. Column 7 gives the thick sample count of the residue remaining after the ninhydrin reaction on the 3.0 ml. urine aliquot. The more extensive data plotted in Fig. 2 were obtained with 0.3 ml. urine samples.

# Volume incomplete because of diuresis.

hydrin (16), but not by the enzyme, and is probably the form in which L-glutamic acid is normally excreted in the urine (16, 17). However, since glutamic acid rises in standing urine, even in the frozen state (17), the specific activity of n-glutamine should have been reflected by the enzymatic treatment of the above sample which had been in the deep freeze for 8 months.

Unlike the rat (5) man does not appear to excrete pyrrolidonecarboxylic acid as the metabolic end product of n-glutamic acid. Since ninhydrin does not evolve CO2 from pyrrolidonecarboxylic acid (16), none of the urinary amino acid C’* is attributable to this compound. Moreover, an ethyl ace- tate extract of acidified urine (Sample 4), which should have contained the cyclic compound but not glutamic acid, had only 6 c.p.m., whereas the

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F. W. PUTNAM, A. MIYAKE, AND F. MEYER 663

treated urine sample had 2200 c.p.m. To be sure, because of the lability of glutamine and the reversibility of the cyclization of glutamic acid, it was not possible to establish unambiguously the nature of the initially excreted form of the injected n-glutamic acid from studies of the frozen urine.

No residual radioactivity after ninhydrin decarboxylation of the urine was observed that could not in major part be attributed to urea or Bence- Jones protein. As a crude measure, the residue from the 3.0 ml. urine samples subjected to the ninhydrin reaction was evaporated to dryness on planchets and counted directly. From the data of Table I it is clear that the radioactivity of the urine residue (Column 7) followed a different time- course and was of a lower order of magnitude than that released by ninhy- drin (Column 5). Although the thick sample count of the urine residue and of the BaC03 derived from the ninhydrin reaction cannot be equated in the same activity units, both are obtained from the same sample of urine. Yet, the BaC03 count is 400 times that of the residue in Sample 3 but only twice as great in Sample 19. The fact that the specific activity- time curve for the urine residue (not plotted) closely parallels the corre- sponding activity curves for urinary urea and Bence-Jones protein also suggests that the radioactivity of the residue is due to the latter substances.

An attempt was made to estimate the total amount of Cl4 excreted by way of n-glutamic acid by summation of the total activity (thick sample counts) released by ninhydrin decarboxylation of each specimen (see rep- resentative data of Column 6). Only 90 PC. of urinary amino acid could be accounted for when a factor was applied to convert thick sample counts to microcuries. Although this is equivalent to less than one-half of the D-

glutamic acid injected, the deficit is probably due to the assumptions in- volved in making the estimate.’ The contribution of Cl4 from urinary L-

amino acids can be disregarded in this calculation (see later). From the above data we conclude that the bulk of the radioactivity ex-

creted via urinary amino acids was eliminated as n-glutamic acid, as a nin- hydrin-reactive metabolite thereof, or was reconverted to n-glutamic acid during the course of storage and handling of the urine samples.

6 When 0.06 PC. of nn-glutamic acid was decarboxylated by ninhydrin in 0.3 ml. of the patient’s urine in the presence of 15 mg. of n-glutamic acid as carrier, the thick sample count was 15,250. Thus, 1 PC. was equivalent to approximately 250,000 counts under these conditions. (The amount of carrier added is sufficient to over- come any significant effect of urinary n-amino acids.)

7 A less likely possibility is that n-pyrrolidonecarboxylic acid is formed in uivo and can be metabolized, whereas the remaining uncyclised n-glutamic acid is ex- creted. In what appears to be the only recorded report on the administration of n-pyrrolidonecarboxylic acid to man, Abderhalden and Hanslian (18) assumed the compound was metabolized because of their inability to demonstrate it in the urine. Rabbits, however, excreted some of the D compound after administration of the racemic mixture.

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6G4 METABOLISM OF DL-GLUTAMIC ACID

Urinary Urea-According to the established mechanism of urea forma- tion a CY4-labeled precursor of carbon dioxide should contribute radio- activity both to expiratory CO2 and to urea. Hence, the specific activity of urinary urea was measured with the aid of urease (Fig. 3). The maximum in the specific activity of urinary urea occurred at about 2.5 hours, signi- ficantly later than for expiratory COZ (Fig. 1). The decline in radioactiv- ity of the urea was slower than that for either expiratory CO2 or urinary amino acids, the half time being about 280 minutes for urea and 80 to 96 minutes for expiratory CO2 and urinary amino acids. Taken together, these data confirm the rapidity of the metabolism of the ar-carboxyl car- bon of glutamic acid. It is to be noted that, although the urea carbon is

Fra. 3. Specific activity of urinary urea and of a soluble Bence-Jones (Fraction S) excreted after injection of DL-glutamic acid-l-W.

125

-G-

100: E 4

75 ; BENCE-JONES PROTEIN

FRACTION S P

503

2 25 6

7

3

t&RS IO

AFTER ADMINISTRATlb% 20

O@

(FROM MIDTIME OF INJECTION )

protein

in equilibrium with respiratory CO*, the activity of the urinary urea is considerably below that of the expiratory CO2 (compare the ordinate scales of Figs. 1 and 3). Furthermore, there is a delay in the maximal activity of the urinary urea, which is probably ascribable to the time requirement for metabolic incorporation of CO2 in the ornithine cycle and the subsequent excretory process.

Serum L-Glutamic Acid-The sharp maxima in the specific activity curves for expiratory CO2 and urinary urea suggest a rapid disappearance of labeled amino acid from the circulation. An attempt to measure the rate of decline in serum n-glutamic acid-l-Cl4 was made through use of the de- carboxylase. Although much of the glutamic acid in blood is present in the form of glutamine, considerable decomposition of the amide occurs on standing in the frozen state for a period of 8 months, as was the case in this experiment (11). An abrupt decline in serum n-glutamic acid activity

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F. W. PUTNAM, A. MIYAKE, AND F. MEYER 665

was noted during the first 2 hours (see Fig. 4 of Putnam and Miyake (9)). This accords with the evidence deduced above for the rapid loss of the cY-carboxyl carbon and indicates that the supply of L-glutamic acid- l-C4 available for protein synthesis diminished rapidly.

Incorporation of Glutumic Acid into Bence-Jones Protein

Although the rate of incorporation of radioactivity into Bence-Jones pro- tein will be discussed separately in connection with the biosynthesis of the myeloma globulin (9), it is pertinent to inquire, first, whether the Bence- Jones protein was labeled spuriously by adsorption of C”-amino acids from the highly active urine and, secondly, whether n-glutamic acid was in- corporated into the urinary protein. Four types of observations indicated that adsorbed amino acid made only a negligible contribution to the radio- activity of the BenceJones protein. These were (a) comparison of the rate activity curves of Figs. 2 and 3, (b) dialysis of labeled protein against non- isotopic glutamic acid and dialysis of a mixture of unlabeled protein and C”- glutamic acid against a solution of inactive amino acid, (c) reaction of nin- hydrin on intact labeled protein, and (d) action of glutamic acid decar- boxylase on hydrolyzed and unhydrolyzed labeled protein.

Tests for Adsorption-(a) Three successive samples of urine were obtained representing the period from zero time to 35 minutes after the injection ceased. As indicated in Fig. 3, there was only negligible radioactivity in the Bence-Jones protein prepared from these urine samples. In view of the high radioactivity of the urine in the 1st hour (Table I), these results attest the efficacy of prolonged dialysis in preventing spurious data owing to adsorption. (b) As illustrated in Table II, there was no change in the activity of the Benee-Jones protein after dialysis against a solution con- taining inactive glutamic acid. Moreover, all added glutamic acid-l-c” could be removed from a solution of weakly active Bence-Jones protein by a similar sequence of dialysis. (c) Ninhydrin released no detectable CrO, from the most active sample of intact Bence-Jones protein. (d) Glutamic acid decarboxylase releases CO2 equivalent to 99 per cent of the L anti- pode present in a mixture of nn-glutamic acid-l-C4 added to a solution of inactive Bence-Jones protein, provided several incubations are made. Yet, there is no significant change in the specific activity of labeled Bence-Jones protein incubated with the enzyme under similar conditions. By all these criteria, then, it appeared that the activity of the BenceJones protein was not attributable to adsorption of urinary amino acids.

Reaction with Glutamic Acid Decarboxylase-This experiment also pre- sented an opportunity to test the hypothesis of K6gl et al. that n-glutamic acid occurs characteristically in tumor proteins (l-3). Several investi- gators, using isotopic techniques and other methods, have failed to re-

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666 METABOLISM OF DL-GLUTAMIC ACID

produce the results of Kijgl et CL!. and attribute them to partial racemiza- tion during acid hydrolysis (19). However, the hypothesis has been re- vived recently on the basis that the feeding of rat or human tumor pro- teins to animals (which avoids racemization) results in the excretion of n-glutamic acid by the dog (2, 3) and of n-pyrrolidonecarboxylic acid by the rat (20, 21). Autopsy samples of tumor tissue were obtained 23 months after the administration of the labeled amino acids, but these proved to be devoid of detectable radioactivity. However, since the Bencedones protein is found only in association with a plasma cell tumor, the possibility was investigated that this abnormal protein might contain the unnatural amino acid. For this purpose, we compared the activity of

TABLE II Tests fog Adsorption of Ddi’lulamic Acid-IO4 by Bence-Jones Protein -

Experiment No. Dialysate !lo. of hanger

-

1 Cl

.-

-

C.p.m. Per q. pmte1n

-

I

--

-

I. Labeled protein (No. 16s)

Control II. WeakIy labeled pro-

tein (No. 61s) + glu- taniic acid-l-C”

Control -

10 mg. yc glutamic acid in 9% NaCl

10 “ $qc ‘I Distilled water

“ in 0.9% NaCl

No dialysis

10 mg. y0 glutamic acid in 0.9% NaCl 10 “ yc (‘ “ “ 0.9% “ Distilled water No dialysis

1 5

10

45 47

3700

120

1 2

the CO2 released by ninhydrin from an acid hydrolysate of the protein with that obtained by prior treatment with glutamic acid decarboxylase.

Preliminary experiments established that some of the activity of the Bence-Jones protein was present in a form other than L-glutamic acid; that is, successive incubation of the hydrolyzed protein with the enzyme failed to release all the 04 that was susceptible to ninhydrin. This re- sult could have occurred through the direct incorporation of n-glutamic acid into the protein, through the uptake of Cl4 into other L-amino acids later incorporated into the protein, or because of partial racemization dur- ing hydrolysis. In further investigation a protein hydrolysate was sepa- rated into three fractions by use of ion exchange columns. The acidic amino acids were adsorbed on the Amberlite resin IR-4B in the carbonate form at pH 6; the filtrate contained the neutral and basic amino acids.

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F. W. PUTNAM, A. MIYAKE, AND F. MEYER 667

The dicarboxylic amino acids were eluted at pH 2 and separated by use of a column of Dowex 50 in the sodium form at pH 3.4. The eluted fractions corresponding to glutamic and aspartic acids were verified by paper chroma- tography, The glutamie acid fraction was subjected to successive incu- bations with enzyme prior to reaction with ninhydrin as detailed in Table III. The other fractions were allowed to react only with ninhydrin.

By the above procedure n-glutamic acid could not be detected in phys- iologically significant amounts in the Bence-Jones protein. Only 3 per cent of the Cl4 of the glutamic acid fraction was unreactive with the enzyme. Since heating L-glutamic acid with boiling HCl for 24 hours causes race- mization of 3 to 6 per cent (22), no significance can be attached to the en-

TABLE III Radioactivity of CO2 Released from Amino Acid Fractions of Bence-Jones Protein by

Glutamic Acid Decurboxylase and Ninhydrid

Fraction No. Aliquot ElUylIW Ninhydrin

Thick sample counts BS BaCOt

1. Glutamic acid (with carrier)

2. Aspartic acid (no carrier) 3. Neutral and basic (no carrier)

C.).rn. c.p.m.

A 980 27 B 950 22 c 980 34

33 97

* Aliquot A was incubated with one batch of enzyme for 2 hours at room tempera- ture, and the residue allowed to react with ninhydrin. Aliquot B, two successive incubations with fresh enzyme (total of 3.5 hours) followed by ninhydrin. Aliquot C, three successive incubations totaling 3.5 hours, followed by ninhydrin. In all cases 15 mg. of carrier nn-glutamic acid were present initially.

zymatically unreactive glutamic acid. Moreover, the enzyme is some- what inhibited by the presence of the carrier D antipode.8 The occur- rence of significant radioactivity in the neutral and basic amino acids is attributable to the conversion of glutamic acid to other 5-carbon amino acids and to some fixation of respiratory Cl402 into arginine and other amino acids. It is apparent from the much greater activity of the glutamic acid in the presence of carrier, compared to the carrier-free amino acids, that the spread of Cl4 into other L-amino acids occurred to only a limited extent. As noted previously, this could not account for much of the uri- nary C402 released by ninhydrin but not by the enzyme.

Activity of Bence-Jones Protein-The time-course for the radioactivity of the Bence-Jones protein (Fig. 3) resembles that already published in

8 S. Mandeles (private communication).

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668 METABOLISM OF DL-GLUTAMIC ACID

other cases (6-8, 23). Moreover, the curves for urinary urea and protein also exhibit the striking conformity previously observed (7, 23). The latter indicates that Bence-Jones protein, despite its size, is eliminated like a low molecular weight, nitrogenous excretory product. Fig. 3 gives data only for the first 20 hours for a protein fraction soluble in distilled water. More extensive data for this material as well as for an insoluble fraction will be presented in the accompanying paper (9). For the present pur- pose it is sufficient to note the initial lag or “transit period” of about 40 minutes, the sharp maximum at 4.5 hours, and the rapid rate of decline in activity. The data seem to fit best a semilogarithmic plot with two ex- ponential functions. The half time of decline is 7.5 hours for the 1st day and about 21 hours for the next 3 days. An almost identical relationship has been found in another case (23) and somewhat lower slopes in other in- stances (6-8,24).

Serum Proteins-Specific activity curves as a function of time for the myeloma serum globulin of this patient, together with data on other se- rum proteins (9), reveal that the glutamic acid was incorporated after the same lag period as for the Bence-Jones protein. However, the activity of the myeloma globulin rose and fell more slowly. The maximum occurred at 10 to 12 hours and, in accord with previous studies (6, 7), was only about one-seventh that of the urinary protein. Although no quantitative measurement of the incorporation of the glutamic acid-C* into the total body protein was obtained, it is evident from the foregoing that this process was substantial. However, comparative studies with lysine uniformly la- beled with Cl* have shown that the essential amino acid is more slowly metabolized and is thus a more efficient precursor of the Bence-Jones pro- tein when administered under similar conditions (8).

SUMMARY

nn-Glutamic acid-l-Cl* was injected intravenously into a patient with multiple myeloma, frequent blood and urine samples were taken by cathe- ter, and the expiratory CO2 was collected. Radioactivity measurements were made on the following substances which are listed in the order of de- creasing rate of decline of Cl* activity, each with its time of maximal ac- tivity: (1) serum n-glutamic acid (mid-time of injection, 7.5 minutes), (2) expiratory CO2 (25 minutes), (3) urinary amino acids (40 minutes), (4) urinary urea (2.5 hours), (5) Bence-Jones protein (soluble fraction) (4.5 hours), (6) serum r-globulin (the myeloma globulin) (10 to 12 hours). The data reflect the ease of metabolism of the e-carboxyl group of glutamic acid and the rapidity of synthesis and excretion of Bence-Jones protein. Al- though an isotope balance was not established, the release of C’*OZ from urinary amino acids by ninhydrin, but not by glutamic acid decarboxylase,

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F. W. PUTNAM, A. MIYAKE, AND F. MEYER 669

indicated that much of the n-glutamic acid was excreted unchanged. The use of similar techniques failed to demonstrate any ‘significant incorpor- ation of n-glutamic acid into Bence-Jones protein.

BIBLIOGBAPHY

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MeyerFrank W. Putnam, Aiko Miyake and Franz

IN MAN14ACID-1-CTHE METABOLISM OF dl-GLUTAMIC

1958, 231:657-670.J. Biol. Chem. 

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