THE METABOLISM OF L-HISTIDINE

BY CHARLES TESAR* AND D. RITTENBERG

(From the Department of Biochemistry, College of Physicians and Surgeons, Columbia University, New York)

(Received for publication, May 5, 1947)

Shortly after the discovery of histidine, Abderhalden et al. (1) attempted to ascertain the fate of this amino acid when administered to dogs but could demonstrate only an increased excretion of urea and ammonia. How- ever, since these initial experiments were carried out, many investigators have presented evidence in support or refutation of the idea that histidine may be converted into various other metabolites. An extensive review of the subject is not attempted here, but some of the evidence for the possible conversion products will be mentioned briefly. The possibility that histi- dine and arginine were metabolically interconvertible and played a special role in purine synthesis was suggested by Ackroyd and Hopkins (2), based on the observation that when either of these amino acids was restored to a deficient diet of young rats growth was resumed and allantoin excretion returned to normal levels. The indispensability of histidine as a dietary constituent was confirmed by Rose and Cox (3), but arginine, however, was found to be ineffective for growth when substituted for histidine in a deficient diet. The conversion of histidine into creatine was suggested by Abderhalden and Buadze (4). The degradation of histidine to glutamic acid was proposed by Edlbachcr and Kraus (5), who isolated glutamic acid after the action of liver histidase upon histidine. The biological decar- boxylation of histidine to form histamine was indicated by the increase in the histamine content of guinea pig lungs following the injection of histidine (6). Though ergothioneine, carnosine, and anserine are structurally related to histidine, their metabolic derivation from histidine has not yet been demonstrated.

With the exception of a single isotope experiment which indicated that the imidazole ring of histidine is not synthesized in rats (7), our present knowl- edge of the metabolism of histidine is based largely on the results of balance experiments on animals or isolated tissues. To obtain further evidence as to the metabolic fate of this amino acid, synthetic L-histidine, containing a

* Submitted by Charles Tesar in partial fulfilment of the requirements for the degree of Doctor of Philosophy in the Faculty of Pure Science, Columbia University.

Present address, Brady Urological Institute, Johns Hopkins Hospital, Baltimore. 35

by guest on March 29, 2020

http://ww

w.jbc.org/

Dow

nloaded from

36 METABOLISM OF I.-HISTIDINE

high concentration of N15 in the ring nitrogen attached to the y-carbon atom, was fed to rats. A number of amino acids and other nitrogenous substances were isolated and the distribution of the isotope in the organs and some of their components determined.

EXPERIMENTAL

Isotopic histidine was synthesized by the procedure of Ashley and Harington (8, 9). N15 was introduced into the imidazole ring by the con- densation of isotopic thiocyanate with y-ketoornithine to form 2-thiol- histidine, which was subsequently oxidized to L-histidine. Since the y- ketoornithine was prepared from naturally occurring histidine, without racemization, the resynthesized histidine had the same steric configuration.

Isotopic Sodium Thiocyanate-The procedure of Schulze (10) for the synthesis of thiocyanate was modified to conserve the isotopic ammonia. The modified reaction is 3N”HdNOs + 3CSz + 2Fe(OH)s + GNaOH= 3NaCN”S + 2FeS + S + 3NaNOs + 12Hz0, where the symbol N” repre- sents nitrogen with a high concentration of N15. To a mixture of 25 cc. of carbon disulfide and 17 gm. of ferric hydroxide in 40 cc. of absolute methanol, 10.8 gm. of ammonium nitrate, containing 68.4 atom per cent excess N15 in the ammonium ion, were added and the resultant mixture shaken mechanically in a tightly closed bottle. Sodium hydroxide pellets, divided roughly in nine portions totaling 10.8 gm., were added at 2 hour intervals. After being shaken for an additiona 24 hours, the mixture was diluted with water and centrifuged. The deposit was washed several times by centrif- ugation; the combined supernatants were saturated with hydrogen sulfide, filtered, acidified to litmus (not to Congo red), and heated to boiling. On cooling, the solution was neutralized by the addition of dilute sodium hydroxide in an amount equivalent to the hydrochloric acid added, and evaporated to dryness in vucuo. The salts were extracted with several portions of absolute ethanol and the extract, containing the sodium thio- cyanate, evaporated on a steam bath. The residue was extracted with 100 cc. of absolute ethanol and the extract evaporated to dryness. 9.80 gm. (90 per cent of theory) of isotopic sodium thiocyanate were obtained. The product was 99 per cent pure as assayed calorimetrically against standard- ized normal thiocyanate.

y-Ketoornijhin.e-With minor modifications of the procedures of gshley and Harington (9), 200 gm. of L-hi&dine monohydrochloride monohydrate were degraded, in several runs, to 22 gm. of r-ketoornithine dihydrochloride. The best over-all yield was about 25 per cent. In this procedure the imi- dazole ring of the methyl ester of histidine was cleaved by exhaustive benzoylation to form methyl-or, y , 6-tribenzamido-AY-pentenoate. Renzoy- lation by the procedure recommended by Ashley and Harington gave sticky

by guest on March 29, 2020

http://ww

w.jbc.org/

Dow

nloaded from

C. TESAR AND D. RI!Fl’ENBERG 37

products with variable yields of 12 to 25 per cent. Better results were obtained by a two-phase benzoylation with yields of over 60 per cent. To an aqueous solution of the methyl ester hydrochloride of histidine and excess sodium carbonate, a S-fold excess of benaoyl chloride dissolved in benzene was added and the mixture stirred vigorously for 9 hours at 0”. After filtration, the benzene phase was worked up for the desired product. Hydrolysis of the methyl-a, y , 6-tribenzamido-AY-pentenoate in two steps gave y-ketoornithine dihydrochloride. This was obtained largely as a resin which was not purified further, for owing to its unstable nature such procedures invariably were accompanied by considerable decomposition. Approximately 3 gm. of crystalline product were obtained.

C&I~~N~OS.~HCI. Calculated, N 12.5, Cl 32.4; found, N 13.2, Cl 32.7

Isotopic Histidine-By the condensation (9) of 21.0 gm. of y-keto- ornithine with 8.0 gm. of isotopic sodium thiocyanate, 5.94 gm. (32 per cent of theory) of 2-thiolhistidine were obtained. This product was oxidized with 80 gm. of ferric sulfate to yield 2.64 gm. (52 per cent of theory) of histidine, which was purified as the monohydrochloride.

CeHpNaOy HCl.HaO Calculated, N (corrected for isotope content) 20.3; found, N 20.2

“ N16 22.8 atom %; found, N15 22.9 atom y0 [cxlt = f9.69” (2.2% in normal HCl)

The a-amino nitrogen, liberated as ammonia by ninhydrin (ll), contained no significant amount of excess N15.

Feeding Experiment-Three adult male rats, having a total weight of 890 gm., were fed an individual daily ration of 15 gm. of a stock diet consisting of 68 per cent corn-starch, 15 per cent casein, 5 per cent yeast, 4 per cent salt mixture (12), 6 per cent Wesson oil, and 2 per cent cod liver oil. After a preliminary feeding period of 2 weeks, a supplement of 1 m&I of the isotopic histidine was added to the daily ration of each rat for the experimental period of 3 days. During this period the rats consumed all of the diet and gained less than 2 per cent in weight. The urine and feces were collected daily. At the end of the 3rd day, the rats were killed by exsanguination and the blood collected in oxalate. The bodies were segregated in four groups, namely skin, liver, other internal organs, and carcass. These groups were processed separat,ely and the isotope concentrations of the various components determined (13).

Excreta-The daily collection of urine and feces was analyzed for total nitrogen and P. Tlrinary ammonia was adsorbed on permutit, recovered by addition of alkali, and distilled into acid. The urea of an ammonia-free

by guest on March 29, 2020

http://ww

w.jbc.org/

Dow

nloaded from

38 METABOLISM OF L-HISTIDINE

sample was decomposed by incubation with urease and the ammonia liberated analyzed for N15. The results appear in Table I.

The presence of a urinary component having a relatively high N15 con- centration was indicated by the observation that the total nitrogen had a

TABLE I

Distribution of Isotope in Excreta

Day of experiment

1st 2nd 3rd

Total

Source of nitrogen

Feces ‘I “

Nl6 concentration Total N Total N’s

atom fit7 cent emTeSS meq. m.eq.

0.298 6.31 0.0188

0.971 8.78 0.0852 1.095 8.35 0.0915

(0.834)* 23.44 0.1955

1st Whole urine 2nd “ “

3rd “ “

Total

1.23 59.6 0.734 1.44 62.2 0.895 1.62 59.6 0.965

(1.43)*

1st Urea 2nd “

3rd “

Total

1.236 46.0 0.569 1.39 46.4 0.645 1.578 46.4 0.73i

(1.40)*

1st Ammonia 2nd “

3rd ‘I

Total

0.466 3.90 0.0182 0.663 5.02 0.0333 0.752 5.01 0.0376

(0.640)*

Combined urine Allantoin 0.130 “ “ cz-Amino N 1.91-3.43

181.4

138.8

13.93

2.594

1.945

0.0891

* The figures in parentheses represent the average N’s concentration of the total excretory partitions, computed as follows:

Average N15 atom per cent = milliequivalents of total Nr5

milliequivalents total N x 100.

slightly higher isotope concentration than that of the urea. To obtain some information as to the nature of the high isotopic component, the imidazole content of the urine and the N15 concentration of the a-amino nitrogen of the urinary amino acids were determined. Assayed colori- metrically, the imidazole content of the combined 3 day urines, expressed

by guest on March 29, 2020

http://ww

w.jbc.org/

Dow

nloaded from

C. TESAR AND D. RITTENBERG 39

as histidine, was 22 mg.. and was not greater than that observed with rats on a normal stock diet. After removal of urea by incubation with urease, the a-amino nitrogen was liberated as ammonia by the action of ninhydrin by an adaptation of the procedures of Van Slyke et al. (14) and MacFadyen (11). The analyses of several determinations varied from 1.91 to 3.43 atom per cent excess N15, the variations being apparently due to differences in the alkali concentration employed and to prolongation of the aspiration period for the removal of ammonia. This effect was not in- vestigated further because of the desire to isolate the purine end-product, allantoin, from the limited volume of urine remaining. Allantoin was isolated by the procedure of Wiechowski (15), m.p. 235”.

CHNO 4 6 4 3. Calculated, N 35.4; found, N 35.0

Blood-The oxalated blood was centrifuged and the proteins of the plasma were precipitated with 6 per cent tricholoroacetic acid and hydro- lyzed in hydrochloric acid. Samples of the plasma non-protein fraction and protein hydrolysate were digested in concentrated sulfuric acid for N15 analysis. The cells were washed with isotonic saline, hemolyzed by the addition of water, and the cell residues deposited by centrifugation. Hemin was obtained as an amorphous precipitate by the addition of the hemolysate to a solution of acetic acid and sodium chloride (16). After two reprecipita- tions, the material was analyzed for Nls. The residual cell hemolysate was hydrolyzed in hydrochloric acid, an aliquot removed for total nitrogen analysis, and the balance processed for the isolation of histidine as the hydrochloride (17).

C~HON~O~*HCI.H~O. Calculated, N 20.1; found, N 19.6, N’s 0.504 atom y0

The a-amino nitrogen, liberated by ninhydrin (11) had an N15 concentration of 0.031 atom per cent excess. Since the a-nitrogen of the imidazole ring should contain no excess isotope, the concentration of the y-nitrogen was computed as (3 X 0.504) - 0.031 = 1.481 atom per cent excess N15. The results are given in Table II.

Creatinine--Muscle creatine was isolated as creatinine picrate from an alcoholic extract of half of the minced carcass (18) and purified as creatinine zinc chloride.

(C~HTN~O)~-ZIICI~. Calculated, N 23.2; found, N 22.6

A sample of the creatinine zinc chloride was refluxed in a 10 per cent solu- tion of barium hydroxide for 24 hours and the amidine nitrogen, liberated as ammonia, swept over into dilute acid by a stream of nitrogen gas. It amounted to 96 per cent of the calcuiated quantity. The isotope content of the sarcosine moiety was determined by removal of the barium with

by guest on March 29, 2020

http://ww

w.jbc.org/

Dow

nloaded from

40 METABOLISM OF L-HISTIDINE

TABLE II Distribulion of Isotopic Nitrogen in Blood

Source of nitrogen

Plasma Non-protein N Protein N

Erythrocytes “ “ Cell residues Hemin Histidine

“ Cd-N I‘ Y-N

Ne concentration Total N

atom $%r cent czccss meq.

0.780 0.49 0.612 10.8 0.096 19.6 0.051 21.3 0.042 0.504 0.031 1.4s*

Total Nn

m.eq.

0.0039 0.0662 0.0183 0.0109

* Calculated as atom per cent Nl6 of 7-N = (atom per cent N’s of histidine X 3) - atom per cent N’s of or-N.

TABLE III Distribution of Isotopic Nitrogen in Organ Components

Source of nitrogen

Histidine ......................... “ a-N ..................... ‘I 7-N ......................

Glutamic acid .................... Aspartic “ ..................... Arginine ..........................

“ amidine N ............... ‘I ornithine N .............

Tyrosine ......................... Proline ........................... Creatinine ........................

‘I amidine N ............... ‘I sarcosine N. ............

Adenine .......................... Guanine .......................... Amide N ......................... Total protein N .................. Non-protein N ...................

Trichloroacetic acid extract. .. Alcohol extract .................

* See foot-note to Table II.

--

(i

.

-

CWZiSS

rtom gcr cent erce.ss

1.264 0.033 3.759* 0.078 0.060 0.023 0.043 0.012 0.021 0.017 0.027 0.033 0.017

0.060 0.090

0.220

Liver

otonr jer cent e.m%?*~

4.50 0.058

13.44* 0.697 0.574 0.334 0.651 0.064 0.103

0.217 0.194 0.316 0.479

0.668 0.296

_ -

i I

-

Internal organs

rtom per cent czctx*

0.109t 0.096

0.110

0.269 0.243

t Calculated from the atom per cent N’s of adenine picrate (see the text).

excess sulfuric acid and digesting the concentrated filtrate. The results appear in Table III.

by guest on March 29, 2020

http://ww

w.jbc.org/

Dow

nloaded from

C. TESAR AND D. RIWNBERG 41

Carcass Non-Protein and Total Protein Nitrogen-The untreated half of the minced carcass was extracted with 6 per cent trichloroacetic acid to remove the non-protein nitrogen fraction and the residual tissue hydrolyzed in hydrochloric acid. Aliquots of the trichloroacetic acid extract and the hydrolysate were analyzed for nitrogen content and Nr5. Tyrosine, histi- dine, arginine, proline, glutamic acid, aspartic acid, and amide nitrogen were isolated by the usual methods. The analytical values appear in Table IV and their N15 concentrations in Table III.

Purines of Liver and Internal Organs’-The liver and internal organ fractions were processed separately for the isolation of adenine and guanine. The minced tissue was extracted with 6 per cent trichloroacetic acid, washed with ethanol, and dried. Aliquots of the trichloroacetic acid and ethanol extracts were analyzed for N15. The dried tissue was refluxed for 1 hour in normal hydrochloric acid in 50 per cent formic acid, liberating the readily hydrolyzed purines. After removal of the bulk of the acids by

TABLE IV Nitrogen Analyses of Isolated Carcass Components

Components analyzed

Tyrosine .............................. IIistidine 3,4-dichlorobenzenesulfonate Proline ................................ Glutamic acid hydrochloride. .......... Aspartic “ .......................... Arginine monohydrochloride. ..........

-

--

-

N calculated

9.9 cent

7.74 6.91

12.2 7.63

10.5 26.5

-

-

N found

ZQr cm1

7.71 6.94

12.0 7.41

10.5 26.4

vacuum distillation, the purines were precipitated from a hot solution of the syrup by the addition of a suspension of cuprous oxide. The precipitate was dissolved in hot 40 per cent trichloroacetic acid, boiled for 45 minutes, the pH adjusted to 5.0 with normal sodium citrate, and the purines re- precipitated by adding cuprous oxide. The precipitate wassuspended in normal hydrochloric acid, copper was removed with hydrogen sulfide, and the filtrate concentrated by vacuum distillation. Guanine was precipitated from the concentrated aqueous solution by adjusting the pH to 5.0 and was recrystallized as the sulfate.

CsHsNaO.HzS04.2HzO Calculated. N 32.1, Found. Liver guanine N 32.2, internal organ guanine N 31.0

1 The procedure for the isolation of purines was kindly suggested by Dr. Samuel Graff, College of Physicians and Surgeons, Columbia University, New York.

by guest on March 29, 2020

http://ww

w.jbc.org/

Dow

nloaded from

42 METABOLISM OF L-HISTIDINE

Adenine picrate was precipitated from the mother liquor by the addition of saturated picric acid. Adenine picrate from the liver fraction was dis- solved in normal hydrochloric acid, picric acid extracted with ether, and the adenine hydrochloride obtained from the concentrated aqueous solution recrystallized.

C~H~N~*HCl*+HzO. Calculated, N 38.8; found, N 36.8

Because very little adenine picrate was obtained from the internal organ fraction, it was analyzed directly after reduction with phosphorus and hydriodic acid.

C~H~N~*C~H~N~OT+H~O. Calculated, N 30.8; found, N 31.2, N16 0.068 atom To

The N16 concentration of the adenine was computed as 0.068 X S/5 = 0.109 atom per cent excess N15.

Soluble copper was precipitated from the hydrolysates of the liver and internal organ fractions with hydrogen sulfide, and the filtrates hydrolyzed in 20 per cent hydrochloric acid. Aliquots were taken for the determina- tion of total nitrogen and N15. The results appear in Table III.

Liver Amino Acids-From the liver hydrolysate, amide nitrogen and several amino acids were isolated by similar procedures used for the carcass constituents. The analytical values appear in Table V and the isotope concentrations are given in Table III. The low value of the nitrogen analysis of glutamic acid was attributed to the presence of sodium chloride introduced during a preliminary isoelectric precipitation from hydrochloric acid solution by the addition of sodium hydroxide. The amount of glutamic acid remaining was too small for further purification. If, as we believe, the low nitrogen value results from contamination by sodium chloride, the N15 values will not be in error, for no nitrogen-containing impurity is involved.

Skin-The rat skins were hydrolyzed in hydrochloric acid and a sample taken for total nitrogen and N15 analysis (Table VII).

Histidine Content of Liver and Carcass-In a separate experiment, the histidine content of liver and carcass of rats on a stock diet was determined by the isotope dilution method (19). The combined livers of three adult rats and a single carcass were minced separately, extracted with 6 per cent trichloroacetic acid, and hydrolyzed for 24 hours in 20 per cent hydro- chloric acid. Isotopic histidine, containing 22.9 atom per cent excess N15 (for all 3 N atoms), was added to each solution before hydrolysis, 0.0374 m&f to the liver fraction and 0.163 m&l to the carcass fraction. Aliquots of each hydrolysate were taken for total protein nitrogen, yielding 60.7 milliequivalents for liver and 284 milliequivalents for carcass hydrolysate. Excess hydrochloric acid was removed by vacuum distillation, and a, solution of the syrup made alkaline with barium hydroxide. Insoluble

by guest on March 29, 2020

http://ww

w.jbc.org/

Dow

nloaded from

C. TESAR AND D. RI’ITENBERG 43

humin was filtered off and aliquots analyzed for nitrogen content and N15. Amide nitrogen of the hydrolysate, evolved as ammonia, was aspirated jnto dilute acid by a stream of nitrogen gas for 12 hours and was analyzed for nitrogen content and Nlj. Barium was removed from the hydrolysate with sulfuric acid, and histidine was precipitated with alcoholic mercuric chloride (17) and purified as the dichlorobenzenesulfonate.

(C,H,O,SCl,),.C,H,N,Oz Calculated. N 6.91 Found. Liver N 6.79, Nr6 0.783 atom %; carcass N 6.82, N16 0.940 atom 70

The amount of histidine in each hydrolysate was computed from the N15 concentration and amount of the isotopic histidine added, and the N15 con- centration of the isolated histjdine: in liver ((22.9/0.788) - 1) X 0.0374 = 1.05 mM; and in carcass ((22.9/0.940) - 1) X 0.163 = 3.80 mM. The percentage of histidine nitrogen in the total protein nitrogen was (1.05 X

TABLE V

Nitrogen Analyses of Isolated Liver Components

Components analyzed

Tyrosine ................................... Histidine 3,4-dichlorobenzenesulfonate .... Glutamic acid hydrochloride. .............. Aspartic “ .............................. Arginine monohydrochloride ................

N calculated N found

per cent per cent

7.74 7.71 6.91 6.97 7.63 6.49

10.5 10.3 26.5 26.0

3 X 100)/60.7 = 5.2 per cent for liver and (3.80 X 3 X 100)/284 = 4.0 per cent for carcass.

Appreciable amounts of Nx5 were found in the humin and amide nitrogen fractions of each hydrolysate.

Liver humin 3.67 m.eq. N X 0.020 atom y. Nl6 =7.34 X 1Oe4 m.eq. Nls “ amide N 2.87 ” “ x 0.014 “ % “ =4.02 X lo-” “ ”

Carcass humin 5.07 “ “ x 0.011 “ % “ =5.5s x 10-n “ “ <‘

amide N 6.11 “ “ X 0.606 “ % “ =3.67 X lo+ “ “

The N15 in these fractions was derived from the added isotopic histidine and indicates a degradation or adsorption of histidine. The quantity and percentage of the isotopic histidine supplement, corresponding to the N15 found in each fraction, was computed from the data above.

Liver humin (7.34 X 10wa)/(0.229 X 3) = 10.7 X 10-4 m&r histidine (2.9%) “ amideN (4.02 X 1O-4)/(O.229X 3) = 5.85 X lo- “ “ (1.6%)

Carcass humin (5.58 x lo-4)/(0.229 x 3) = 8.12 x 10-4 “ (‘ (0.5%) “ amide N (3.67 X X0-+)/(0.229 X 3) = 5.34 x lo-” ‘( ” (0.4%)

by guest on March 29, 2020

http://ww

w.jbc.org/

Dow

nloaded from

44 METABOLISM OF L-HISTIDINE

The total quantity of isotopic histidine lost in these fractions was 16.6 X 10e4 mM (4.4 per cent) in liver hydrolysate and 13.5 X lo4 mM (0.8 per cent) in carcass hydrolysate.

DISCUSSION

Recovery of Isotope-During the feeding period the rats consumed 9 mM of isotopic histidine containing 68.4 atom per cent excess N15 in the y- nitrogen, or a total of 6.15 milliequivalents of N15. Only 82 per cent of the isotope was accounted for in the fractions analyzed (Table VI). In the preparation of the tissue fractions, the gastrointestinal contents were washed out and discarded without determining the N15 content. Since the diet of the preceding day contained 33 per cent of the administered isotope, this fraction may account for part or all of the missing 18 per cent. The feces gave a negative diazo test for histidine and contained only 3 per cent

TABLE VI

Distribution of Isotopic Nitrogen in Excreta and Animal Body

The diet contained a total of 6.15 milliequivalents of N15.

Source of nitrogen N1’ in fraction Amount recovered

Feces ....................................... Urine ....................................... Non-proteinN .............................. Total protein N ........... _. ................

?fZ.F.*. #er cent

0.20 3 2.59 42 0.32 5 1.98 32

Total N’s recovered., ..................... 5.09 82 ~-

of the total N15, indicating that the administered histidine was well ab- sorbed.

Urinary Ammonia and Urea-In all cases in which amino acids containing excess N15 in the a-amino group have been fed to animals in this laboratory (20-23), the isotope concentration of urinary ammonia was found to be consistently higher than that of urea. On the other hand, in this experi- ment the N15 concentration of the urinary ammonia was less than half of that of the urea (Table I). This difference in the relative isotope concen- trations of urinary components may be attributed to either the formation of urinary ammonia from cy-amino acids or a specific utilization of degraded imidazole nitrogen for the synthesis of urea. The formation of urinary ammonia from the deamination of a-amino acids by kidney deaminases offers a ready explanation for the higher isotope concentration found in ammonia in those experiments in which the excess N15 was located in the a-amino group of the amino acid fed. Although the nitrogen from moder-

by guest on March 29, 2020

http://ww

w.jbc.org/

Dow

nloaded from

C. TESAR AND D. BFITENBERG 45

ately large amounts of histidine administered in balance experiments is excreted largely as urea (l), the existing theories for the synthesis of urea do not lend support to a hypothesis for a specific conversion of imidazole nitrogen to urea. Urea might conceivably be produced by a direct cleavage of the imidazole ring or through the intermediate formation of citrulline, by a splitting of the ring between the y-carbon and the adjacent nitrogen. Citrulline may form urea either by way of the arginine cycle of Krebs and Henseleit (24) or by degradation to glutamic acid as proposed by Bach (25). If the conversion of isotopic imidazole nitrogen to urea proceeded directly through citrulline and arginine, a relatively high concentration of Nl5 would be expected in liver arginine. However, this was not observed. Glutamine formed from the degradation of histidine by the action of liver histidase (26) may play a special rBle in the production of urea (27) and urinary ammonia (28). However, glutamine formed by this mechanism from isotopic histidine would contain no excess NX5, for only the normal (Y- and b-nitrogens of histidine are retained in the conversion, and consequently the urea or ammonia subsequently produced would also lack excess N15.

Urinary Degradation Product of Hi&dine-The presence of a component in the urine of relatively high isotope concentration was indicated by the observation that the N15 concentration of the average total nitrogen (1.43 atom per cent) was significantly higher than that of urea (1.40) and am- monia (0.64) (Table I), which together made up 84 per cent of the urinary nitrogen. This component was not due to contamination of the urine with the dietary isotopic histidine supplement, for the urine contained only traces of spilled food. The imidazole content, assayed as 22 mg. of histi- dine, even if it contained the high N15 concentration of administered histidine, would influence only slightly the N15 concentration of the total nitrogen.

The N15 concentration of the a-amino nitrogen liberated from the urine by the action of ninhydrin ranged from 1.91 to 3.43 atom per cent excess N15. The most probable source of the urinary amino acids is the plasma, and the N15 concentration of the amino acids by the plasma may be expected to be higher than that found in the tissue proteins. However, it would be striking indeed if the average N15 value, as represented by the results ob- tained for the urinary amino acids, were almost 5 times that determined for the most metabolically active amino acid of the liver, namely glutamic acid with an N15 concentration of 0.697 atom per cent excess. It is not improbable that a degradation product of histidine, in which the imidazole ring had been ruptured and the y-nitrogen subject to cleavage by ninhydrin or alkali, was excreted in the urine and thereby contributed its high N15 content to Dhe a-amino nitrogen determination. A urinary degradation product of histidine is suggested by the observation of Eaton and Doty (29),

by guest on March 29, 2020

http://ww

w.jbc.org/

Dow

nloaded from

46 METABOLISM OF L-HISTIDINE

that following the administration of moderately large amounts of histidine to a dog a considerable amount of an unidentified non-imidazole substance was found in the urine. In rats after the injection of histidine there was an increase in the amino nitrogen of the urine which exceeded the increase of imidazole nitrogen (30). In the action of liver histidase on histidine, an intermediate product was obtained which was unstable to alkali, the amount of ammonia liberated being dependent upon the alkali concentration (5). Such degradation products in the urine may possibly account for the high N15 values obtained.

Isotope Distribution of Organ Proteins-The concentration of N15 in the various organ proteins (Table VII) was not uniform and may depend on (a) the concentration of N15 in the reactive substances in the environmental

TABLE VII

Distribution of Isotope in Tissue Proteins

Tissue proteins N’S concentration

Erythrocytes Cell residues. Hemoglobin.

Plasma............. Liver............... Internal organs. Carcass............. Skin................

0.051 21.3 0.0109 1 0.096 19.6 0.0188 1 0.612 10.8 0.0662 3 0.479 50.9 0.244 12 0.110 72.3 0.0800 4 0.090 1057 0.95 48 0.089 689 0.61 31

Total N

m.cq.

Total Nl’

m.q.

Total N’6 in tissue proteins. / 1.979

- Fraction of total N15 in

proteins

fier cent

100

plasma, (b) the concentration of exchangeable components in the organ proteins and environmental plasma, and (c) the relative chemical activity or rate of protein turnover. In contrast with the plasma proteins, which had the highest concentration of N15 of all the tissues investigated, the proteins of the erythrocytes displayed a relatively low value, which reflects the slow rate of synthesis of the red blood cell. The N15 concentration of the liver proteins was over 5 times that found in carcass and skin and may be attributed both to its great metabolic activity and to its favorable location for reaction with dietary histidine absorbed from the intestine by way of the portal vein. Although the proteins of the carcass and skin had the lowest Nls concentrations, these organs, because they contain the bulk of the body proteins, contained 80 per cent of the total N1j in the animal. The pooled internal organs were a mixture of several kinds of tissues, which

by guest on March 29, 2020

http://ww

w.jbc.org/

Dow

nloaded from

C. TESAR AND D. RITTENBERG 47

differ in function and composition, and the Nib concentration obtained was an average figure and does not indicate the relative activity of the separate organs. When other isotopic amino acids, as L-leucine (20) or L-proline (23), were fed to rats, the N15 concentration of several of the internal organs, notably the intestinal wall and kidney, differed little from that obtained for liver. The low value obtained here for the mixture of internal organs may indicate a very slow rate of exchange in some of the other internal organs.

Replacement of Organ Histidine-The high N15 concentrations of isolated histidine (Table III) are referable to the imidazole nitrogen, for the N15 concentration of the a-amino nitrogen was very low. Since the imidazole group is not synthesized in the animal (7), the isotope of t,he imidazole group of tissue histidine was derived directly from the dietary supplement and the N15 must be, therefore, solely in the y-nitrogen. In addition to the 1 mM of isotopic histidine supplement, the daily ration of each rat con- tained 2.250 gm. of casein with a histidine content of 3.0 per cent (31), or 0.435 mM of histidine, and 760 mg. of yeast with a histidine content of 1.03 per cent (32), or 0.050 mM of histidine, making a total of 1.48 mM. Since the y-nitrogen of the histidine supplement had a N15 concentration of 68.4 atom per cent excess, the N15 concentration of the y-nitrogen in the total dietary histidine was 68.4 X 1.00/1.48 = 46.2 atom per cent excess Nl5. The percentage of histidine replaced in the tissues can be computed from this figure and the N15 concentration of the y-nitrogen of the isolated histidine. In liver, 13.4/46.2 X 100 = 29 per cent of the total histidine was replaced by dietary histidine in 3 days. In a similar manner, it was computed that at least 8 per cent of carcass and 3 per cent of erythrocyte histidine had been replaced. When dietary L-leucine was fed, 24 per cent of liver leucine and 7 per cent of carcass leucine were replaced within the same experimental period (20). The close agreement of the turnover rate of histidine and leucine in the proteins of the liver and carcass may indicate that the regeneration of these proteins occurs by a process in which the entire molecule is synthesized from amino acids rather than by replacement of amino acid residues one at a time. The replacement rate discussed above is in accord with the finding that half of the total nitrogen of liver proteins is replaced by the nitrogen of the diet and other proteins in 7 days (22). Shemin and Rittenberg have recently measured the life span of the red blood cell in the human and found it to be about 127 days (33). The replacement rate found here for hemoglobin histidine indicates that about 1 per cent of the hemoglobin is synthesized per day in the red blood cell of the rat and is in agreement with their value.

The absolute amount of histidine replaced in the liver can be computed from the results of the determination for the percentage of histidine nitrogen in total liver protein nitrogen obtained by the isotope dilution method (see

by guest on March 29, 2020

http://ww

w.jbc.org/

Dow

nloaded from

48 METABOLISM OF L-HISTIDINE

“Experimental”). It is the product of one-third of the concentration of histidine nitrogen, the total liver protein nitrogen (Table VII), and the concentration of replaced histidine : $- X 0.052 X 50.9 X 0.29 = 0.25G m&r. The amount of N15 in the replaced histidine is computed as 0.256 X 0.464 = 0.119 milliequivalent, and its percentage of the total N15 of the liver (Table VII) is 0.119 X loo/O.244 = 49 per cent. By a similar com- putation, it can be shown that 1.17 milliequivalents of histidine had been replaced in the carcass tissue and contained 57 per cent of total Xl5 (Table VII). The balance of the isotope was distributed among all the other constituents of the respective organs. The replaced histidine of the liver and carcass was equivalent to 2 and 9 per cent, respectively, of the total histidine of the diet.

A Criterion for Possible Conversion Products-Measurable amounts of N15 were found in every component isolated from the tissues (Tables II and III), indicating a non-specific utilization of the y-nitrogen of histidine fol- lowing its metabolic degradation. The relative N’j concentrations of the amino acids (Table III) other than histidine are not unlike those observed after feeding isotopic ammonium citrate (16, 18, 34) or other amino acids (20-23). Since the imidazole nitrogen of histidine is not subject to nitrogen exchanges, the occurrence of N15 in all the other components must be due either to the direct conversion of histidine or to synthetic procedures and nitrogen exchanges subsequent to the liberation of the N15 from the imida- zole ring. Such nitrogen exchanges are supported by the mechanisms of transamination of Braunstein and Kritzmann (35) and the deamination and reamination systems of Knoop and Oesterlin (36) and von Euler et al. (37). Metabolites of which histidine is an immediate precursor may be expected to contain a high N15 concentration provided that (a) the N15 remains attached to the carbon chain in the conversion, (b) the metabolite is not subject to rapid nitrogen exchanges, producing a marked dilution of the isotope, and (c) the conversion is sufficiently rapid for the N15 concen- tration to reach levels higher than that observed following the administra- tion of isotopic substances which are not specific precursors.

In the utilization of N15 in tracer experiments there is no basic nitrogen fraction representative of a common “nitrogen pool,” having a uniform isotope concentration similar to that of “body water” in experiments in which deuterium is employed as a marker. In each organ, however, certain fractions, such as total protein nitrogen and non-protein nitrogen, whose N15 concentrations are an average of all the components they contain, may be selected as a basis for comparision of the relative N15 concentrations of possible conversion products. Thus, it may be possible to indicate the direct conversion of histidine into other compounds, if it can be demon- strated that the relative N15 concentration of the component under con-

by guest on March 29, 2020

http://ww

w.jbc.org/

Dow

nloaded from

C. TESAR AND D. RI’ITENBERG 49

sideration, when compared to such average nitrogen pools, is consistently greater than that obtained by feeding isotopic substances which are not immediate precursors. In Table VIII, in addition to the N15 concentrations of the total protein nitrogen and non-protein nit,rogen, the values for amide nitrogen and urea are also employed as comparative bases. Amide nitrogen is an ammonia type of moiety with an active turnover rate, and urea, since it is produced in relatively large amounts in the liver from ammonia, can be taken as a favorable basis for liver components with the exception of arginine. In Table VIII a series of ratios of the N15 concentrations of glutamic acid of the liver is compared with similar ratios obtained from other experiments in which the isotopic dietary supplement was not con- sidered an immediate precursor of the glutamic acid. In each column, the ratios obtained in the comparable experiments are similar to that in the histidine experiment. From this evidence histidine cannot be considered a specific precursor for gIutamic acid, for the N15 contributed to this com-

TABLE VIII

Ratio of Isotope Concentration in Glutamic Acid to Selected Nitrogen Fractions

Labeled compound fed Glutamic acid Glutamic acid Glutamic acid Glutamic acid T-protein Non-protein N Urea Amide N

L-Histidine 1.45 1.04 L-Lcucine (20) 1.99 1.49 Ammonium citrate (16) 2.09 1.39

0.50 0.76

2.20 2.38 2.09

pound by histidine is certainly not greater than that observed when other isotopic amino acids are administered.

The degradation of histidine to glutamic acid by the action of liver enzymes, according to Edlbacher and Neber (26), may occur by two hypothetical mechanisms. In the main route of the hypothesis, the imidazole ring is ruptured by liver histidase, followed by the loss of the y-nitrogen. In such a mechanism the glutamic acid formed would contain no N15. In the alternative route, a smaller proportion of the histidine is first deaminated to urocanic acid in which the imidazolc ring is subsequently cleaved by urocanase to yield glutamic acid, the y-nitrogen of histidine being retained as the a-amino nitrogen of glutamic acid. However, owing to the rapid turnover rate of the a-amino nitrogen of glutamic acid, it is doubtful whether the glutamic acid formed by such a conversion would ret,ain a sufficiently high N15 concentration to establish its derivation from histidine. This view is confirmed by the biological conversion of proline, synthesized with both deuterium and N15, into glutamic acid (23). This conversion was demonstrated by the high deuterium content of the isolated

by guest on March 29, 2020

http://ww

w.jbc.org/

Dow

nloaded from

50 METABOLISM OF L-HISTIDINE

glutamic acid, but the N15 concentration of the glutamic acid with respect to that of aspartic acid was not greater than the ratio obtained after feeding isotopic leucine (20). Apparently, if the N15 were not lost in the initial conversion of proline into glutamic acid, then the concentrat,ion was decidedly reduced by the rapid nitrogen exchanges of the a-amino group of the glutamic acid formed. Glut’amic acid, synthesized by transamination mechanisms from a-ketoglutaric acid available as an intermediate in carbohydrate metabolism, would also contribute to the dilution of N15 of the conversion product. From these considerations, the findings of this experiment do not exclude the mechanism proposed by Edlbacher. To test such a mechanism, histidine synthesized with the carbon isotope would be required. The metabolic conversion of ornithine into glutamic acid has been demonstrated with deuterium as a label (38).

Although histidine might conceivably be degraded to aspartic acid, the same comparative ratios of the N15 concentrations as calculated for glutamic acid do not lend support to such a hypothesis. The same holds for arginine; the relative isotope concentrations are not sufficiently high to indicate any specific contribution of isotope from histidine. The relatively higher N15 value of the amidine nitrogen is attributed t,o the participation of arginine in the synthesis of urea. In experiments of longer duration, the amidine nitrogen of arginine and urea had approximately equal N15 concentrations (22). The utilization of ornit,hine (39) and proline (23) for the synthesis of arginine in the rat has been demonstrated by means of deuterium as a tracer.

Both tyrosine and proline undergo comparatively slow nitrogen exchanges or syntheses as indicated by their low isotope concentrations. Histidine does not contribute N15 directly to the formation of these components, for the ratios of their Ni5 concentrations with respect to basic nitrogen fractions are not greater than that observed when ot,her isot.opic amino acids or ammonia was administered.

The low N15 concentration of creatinine, and its ratio with respect to the N15 concentrations of total protein nitrogen and non-protein nitrogen of carcass, compared to similar ratios obtained after feeding isotopic am- monium citrate do not indicate any direct conversion of histidine nitrogen into creatine.

Allantoin (Table I), the metabolic end-product of purines in the rat, has an nil5 concentration intermediate between those of the purines of the liver and of the other internal organs respec ive:y (Table III) and may be taken as representat’ve of the isotope values of total body purines. Barnes and Schoenheimer (34) found that following the feeding of isotopic ammonium &rate to rats the N15 concentration of allantoin was approximately equal to that of the tissue purines. The ratio of the N15 concentration of allan-

by guest on March 29, 2020

http://ww

w.jbc.org/

Dow

nloaded from

C. TESAR AND D. RIlYTENBERG 51

toin, with respect to that of urea, is not greater than that obtained with isotopic ammonium citrate. It can be concluded that the imidazole ring of histidine is not uMized for the direct synthesis of purines to any extent.

Hemin-The low value of Nl5 obtained for hemin is in agreement with the low values of isotopic nitrogen found in hemin after feeding isotopic ammonium citrate (16), proline (23), or leucine (20) and gives no indication that histidine plays a special role in its synthesis.

Humin and Amide Formation--In the determination of the histidine con- tent of rat liver and carcass by the isotope dilution method, humin and amide nitrogen, obtained from the hydrolysates to which isotopic hist.idine had been added before hydrolysis, were found to contain N15 in appreciable amounts. On refluxing histidine in concentrated hydrochloric acid in the presence of carbohydrate about 2 per cent of the histidine nitrogen was found in humin (40) and was attributed to adsorption rather than to modification of the histidine molecule (41). On the other hand, the presence of N15 in the amide nitrogen fraction must be due to decomposition. Under the vigorous conditions of acid hydrolysis employed here, it is evident that the imidazole group of histidine was slowly disrupted.

SUMMARY

1. L-Histidine was synthesized with a high concentration of N15 in the y-nitrogen of the imidazole ring.

2. The isotopic histidine was added to the stock diet of three adult rats for 3 days and was well absorbed. Only 3 per cent of the N15 was excreted in the feces, 42 per cent in the urine, and the major part of the balance incorporated in the tissue proteins. The highest concentrations of N15 were found in the blood plasma and liver proteins, while that in the muscle, skin, internal organ, and erythrocyte proteins was relatively low.

3. The isotope concentration of urinary ammonia, with respect to that of urea, was lower than that observed in experiments in which the N15 was situated in the a-amino nitrogen of the dietary amino acid. This dis- similarity in the distribution of the N15 is attributed to the role of a-amino nitrogen in the formation of urinary ammonia.

4. A relatively high Nl5 concentration was obtained in the determination of the a-amino nitrogen of urinary amino acids and may possibly indicate the presence of a degradation product of histidine.

5. During the 3 day experimental period, the minimal amount of histi- dine replaced in liver was 29 per cent, in carcass 8 per cent, and in erythro- cytes 3 per cent. Of the total N15 present in the respective organs, 49 per cent of the liver N15 and 57 per cent of the carcass N15 were present in the replaced histidine. The replaced histidine of the liver and carcass was 2 and 9 per cent, respectively, of the total histidine consumed in the diet.

by guest on March 29, 2020

http://ww

w.jbc.org/

Dow

nloaded from

52 METABOLISM OF L-HISTIDINE

6. From the relative concentrations of the isotope in the components isolated, it was apparent that the imidasole nitrogen was not selectively utilized upon rupture of the ring, but was redistributed in a manner similar to that of the nitrogen of ammonium salts or amino acids, which are not specific precursors. There is no evidence that histidine is a precursor of glutamic acid, arginine, creatine, or purines.

7. The concentration of histidine nitrogen in the total protein nitrogen of the whole organ of the rat, as determined by the isotope dilution method, is 5.2 per cent for liver proteins and 4.0 per cent for carcass proteins.

8. Evidence for the partial decomposition of histidine, during acid hydrolysis of normal proteins to which isotopic histidine has been added, is presented by the finding of iV5 in the amide nitrogen fraction.

BIBLIOGRAPHY

1. hbderhalden, E., Einbeck, H., and Schmid, S., Z. physiol. Chem., 68, 395 (1910). 2. Ackroyd, H., and Hopkins, F. G., Biochem. J., 10, 551 (1916). 3. Rose, W. C., and Cox, G. J., J. Biol. Chem., 61, 747 (1924). 4. Abderhalden, E., and Buadze, S., dfed. Klin., 25, I1 (1929). 5. Edlbacher, S., and Kraus, J., 2. physiol. Chem., 195, 267 (1931). 6. Bloch, W., and Pinosch, I-I., 2. physiol. Chem., 239, 236 (1936). 7. Schoenheimer, R., Rittenberg, D., and Keston, A. S., J. Biol. Chem., 127, 385

(1939). 8. Kossel, A., and Edlbacher, S., Z. physiol. Chem., 93, 396 (1914-15). 9. Ashley, J. N., and Harington, C. R., J. Chem. Sot., 2586 (1930).

10. Schulze, J., J. prakt. Chem., 27, 518 (1883). 11. MacFadyen, D. A., J. Biol. Chem., 153, 507 (1944). 12. Osborne, T. B., and Mendel, I,. B., J. Biol. Chem., 16,311 (1913). 13. Rittenberg, D., Keston, A. S., Rosebury, F., and Schoenheimer, R., J. BioZ.

Chem., 127, 291 (1939). 14. Van Slyke, D. D., MacFadyen, 1). A., and Hamilton, I’. B., J. Biol. Chem., 150,

251 (1943). 15. Wiechowski, W., Beitr. them. Physiol. u. Path., 11, 109 (1908). 16. Rittenberg, D., Schoenheimer, R., and Keston, A. S., J. BioZ. Chem., 128, 603

(1939). 17. Foster, G. L., and Shemin, D., in Organic syntheses, New York, 18, 43 (1938). 18. Foster, G. L., Schoenheimer, R., and Rittenberg, D., J. BioZ. Chem., 127, 319

(1939). 19. Rittenberg, D., and Foster, G. L., J. BioZ. Chem., 133, 737 (1940). 20. Schoenheimer, R., Ratner, S., and Rittenberg, D., J. Biol. Chem., 130,703 (1939). 21. Schoenheimer, R., Ratner, S., and Rittenberg, D., J. BioZ. Chem., 127, 333 (1939).

Ratner, S., Schoenheimer, R., and Rittenberg, D., J. BioZ. Chem., 134, 653 (1940). Ratner, S., Rittenberg, D., Keston, A. S., and Schoenheimer, R, J. BioZ. Chem., 134, 665 (1940). Bloch, K., and Schoenheimer, R., J. BioZ. Chem., 136, 99 (1940). Weissman, N., and Schoenheimer, R., J. BioZ. Chem., 140, 779 (1941). Stetten, D., Jr., J. BioZ. Chem., 144, 501 (1942). Ratner, S., Weissman, N., and Schoenheimer, R., J. BioZ. Chem., 147, 549 (1943).

22. Shemin, I)., and Rittenberg, D., J. BioZ. Chem., 163, 401 (1944).

by guest on March 29, 2020

http://ww

w.jbc.org/

Dow

nloaded from

C. TESAR AND D. RI!l.“J!ENBERG 53

23. Stetten, M. R., and Schoenheimer, R., J. Biol. Chem., 163, 113 (1944). 24. Krebs, H. A., and Henseleit, H., 2. physiol. Chem., 210, 33 (1932). 25. Bach, S. J., Biochem. J., 33, 1833 (1939). 26. Edlbacher, S., and Neber, M., 2. physiol. Chem., 224, 261 (1934). 27. Leuthardt, F., 2. physiol. Chem., 262, 238 (1938). 28. Van Slyke, D. D., Phillips, R. A., Hamilton, P. B., Archibald, R. M., Futcher,

P. H., and Hiller, A., J. BioZ. Chem., 160, 481 (1943). 29. Eaton, A. G., and Doty, J. R., J. Nutr., 21, 25 (1941). 30. Lelu, P., Bull. Sot. chim. biol., 19, 292 (1937). 31. Dunn, M. S., Shankman, S., and Camien, M. N., J. BioZ. Chem., 161, 669 (1945). 32. Woolley, D. W., and Peterson, W. H., J. BioZ. Chem., 122, 207 (1937-38). 33. Shemin, D., and Rittenberg, D., J. BioZ. Chem., 166, 627 (1946). 34. Barnes, F. W., *Jr., and Schoenheimer, R., J. BioZ. Chem., 161, 123 (1943). 35. Braunstein, A. E., and Kritzmann, M. G., Biokhimiya, 2, 260, 874 (1937). 36. Knoop, F., and Oesterlin, H., 2. physiol. Chem., 148, 294 (1925). 37. von Euler, H., Adler, E., Gunther, G., and Das, N. B., 2. physiol. Chem., 264,

61 (1935). 38. Roloff, M., Ratner, S., and Schoenheimer, R., J. BioZ. Chem., 136, 561 (1940). 39. Clutton, R. F., Schoenheimer, R., and Rittenberg, D., J. BioZ. Chem., 132, 227

(1940). 40. Roxas, M. L., J. BioZ. Chem., 27, 71 (1916). 41. Gortner, R. A., and Helm, G. E., J. Am. Chem. Xoc., 39, 2477 (1917).

by guest on March 29, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Charles Tesar and D. RittenbergTHE METABOLISM OF l-HISTIDINE

1947, 170:35-53.J. Biol. Chem. 

  http://www.jbc.org/content/170/1/35.citation

Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

alerts to choose from all of JBC's e-mailClick here

  ml#ref-list-1

http://www.jbc.org/content/170/1/35.citation.full.htaccessed free atThis article cites 0 references, 0 of which can be

by guest on March 29, 2020

http://ww

w.jbc.org/

Dow

nloaded from