The amount of tryptophane required for maintenance and growth is small (2). When an excess is supplied to some animals, kynurenic acid is excreted (3) and, under certain conditions, kynurenine also (4). Although these are quantitatively the most important of the known metabolic products of tryptophane, the amounts excreted in extensive tests in the dog and rabbit have usually accounted for less than half, more often for less than a third, of the tryptophane administered (4-6)

Berg, Clarence P.

Borchers, Raymond

Whitman, Newton E.

English

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Biochemistry -- Faculty Publications Biochemistry, Department of
1942
Tryptophane Metabolism X. The Effect Of Feeding
1(-)-, Dl-, And D(+)-Tryptophane, D(-)And Dl-
(β-3-Indolelactic Acid,(β-3-Indolepyruvic Acid,
And L(-)Kynurenine Upon The Storage Of Liver
Glycogen And The Urinary Output Of Kynurenic
Acid, Kynurenine, And Total Acetone Bodies
Raymond Borchers
State University of Iowa
Clarence P. Berg
State University of Iowa
Newton E. Whitman
State University of Iowa
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Borchers, Raymond; Berg, Clarence P.; and Whitman, Newton E., "Tryptophane Metabolism X. The Effect Of Feeding 1(-)-, Dl-, And
D(+)-Tryptophane, D(-)And Dl-(β-3-Indolelactic Acid,(β-3-Indolepyruvic Acid, And L(-)Kynurenine Upon The Storage Of Liver
Glycogen And The Urinary Output Of Kynurenic Acid, Kynurenine, And Total Acetone Bodies" (1942). Biochemistry -- Faculty
Publications. 266.
http://digitalcommons.unl.edu/biochemfacpub/266
J. BioI. Chem. 1942 145: 657-666. 
TRYPTOPHANE METABOLISM 
X. THE EFFECT OF FEEDING 1(-)-, dl-, AND d(+)-TRYPTOPHANE, d(-)-
AND dl-(j-3-INDOLELACTIC ACID,(j-3-INDOLEPYRUVIC ACID, AND l( -)-
KYNURENINE UPON THE STORAGE OF LIVER GLYCOGEN AND THE 
URINARY OUTPUT OF KYNURENIC ACID, KYNURENINE, AND TOTAL 
ACETONE BODIES· 
By RAYMOND BORCHERS, CLARENCE P. BERG, AND NEWTON E. 
WHITMAN 
(From the Biochemical Laboratory, State University of Iowa, Iowa City) 
(Received for pubJication, August 6, 1942) 
The amount of tryptophane required for maintenance and growth is 
small (2). When an excess is supplied to some animals, kynurenic acid is 
excreted (3) and, under certain conditions, kynurenine also (4). Although 
these are quantitatively the most important of the known metabolic prod-
ucts of tryptophane, the amounts excreted in extensive tests in the dog and 
rabbit have usually accounted for less than half, more often for less than a 
third, of the tryptophane administered (4-6).1 
Whether tryptophane is glycogenic or ketogenic is not clear. A number 
of years ago Dakin (7) observed that the injection of 14.5 gm. of l( - )-
tryptophane into the phlorhizinized dog yielded 2.7 gm. of extra glucose, 
3 gm. of kynurenic acid, and a precipitate with mercuric sulfate equivalent 
to about 3 gm. of tryptophane. He considered the yield of glucose too 
small to warrant concluding that it was formed from tryptophane. Per-
fusion of a surviving liver with 1.5 gm. of l( - )-tryptophane yielded no 
acetoacetic acid or acetone. 
The studies recorded in this communication were directed toward deter-
mining whether a change could be shown in acetone body output upon 
feeding tryptophane to the fasted, but otherwise normal, rat, or to the rat 
fed sodium butyrate, and whether such feeding would affect the storage of 
liver glycogen. We were also interested in determining how the dl and 
* The experimental data in this paper are taken from a dissertation submitted by 
Raymond Borchers in May, 1942, in partial fulfilment of the requirements for the 
degree of Doctor of Philosophy in Biochemistry in the Graduate College of the State 
University of Iowa. A preliminary report has been published (1). 
Some of the procedures employed are based on data from a thesis submitted by 
Newton E. Whitman for the degree of Master of Science in August, 1939. 
The assistance afforded by Grant 311 of the Committee on Therapeutic Research, 
Council on Pharmacy and Chemistry, American Medical Association, is gratefully 
acknowledged. 
1 Berg, C. P., and Rowe, V. K., unpublished data. 
657 
658 TRYPTOPHANE METABOLISM. X 
d(+) forms of tryptophane, indolepyruvic, dl- and d( -)-indolelactic acids, 
and I( - )-kynurenine would compare with Z( - )-tryptophane in these 
respects and in their effect upon the excretion of kynurenine and kynurenic 
acid in this species. 
EXPERIMENTAL 
The compounds tested were prepared by methods previously employed 
in this laboratory. Total nitrogen was determined by a semimicro- 
TABLE I 
Physical Properties of Compounds Studied 
I Melting point 
Compound Found 
(UllCW 
rected) 
; “C. 
I(-)-Trypt,ophane 1274-281 
(8) * 
dl-Tryptophane (11) 280 
d (+)-Tryptophanc 280 
(9) 
dl-Indolelactic 143 
acid (12) 
d(-)-Indolelactic 99 
acid (12) 
p-3.Indolepyruvic 205 
acid (13, 14) 
I(+)-Kynurenine 175 
sulfate? (4)j 185 
Recorded Found 
“C. 
277 (8) 
281-282 (9) 
282-283 (12) 
281-282 (9) 
145 (12) 
100-101 (12) 
211 (12) 
180 (4)$ 
Total nitrogen 
per cent 
3.4-13.’ 
3.6 
3.6 
6.7 
6.7 
6.9 
7.8 -8. 
( 
71 
II 8.38!+10.1$ 
MCU- 
[a]; Iound 
lated 
per cent degrees 
3.72 -32.0 to 
-33.5 
3.72 0.0 
3.72+32.1 
6.83 0.0 
6.83 -5.3 
6.89 
degrees 
-33.0 to 
-33.6 (10) 
+32.45 (9) 
-5.36 (12) 
+10.7 
+10.03 (4)$ 
* The references which follow the names of compounds are to methods of prepa- 
ration. 
t In the free form, kynureninc is lcvorotatory ((4) and foot-note 1). 
$ See foot-note 1. 
$ The concentration was 1.0 gm. per 100 cc. of solution in water; in all other in- 
stances the concentrations were 0.5 gm. per 100 cc. of aqueous solution. 
Kjeldahl procedure. Optical rotations were read in a Schmidt-Haensch 
polariscope with an electric sodium lamp. Pertinent analytical data and 
comparisons with the literature are summarized in Table I. 
Kynurenic acid, which is produced from several of these compounds, 
separates from the urine upon strong acidification, Kynurenine, trypto- 
phane, and indole derivatives form sparingly soluble complexes with the 
mercuric sulfate reagent, used in the Va,n Slyke procedure for determining 
total acetone bodies (16). Hence the following routines were devised to 
BORCHERS, BERG, AND WHITMAN 659 
determine kynurenic acid, “kynurenine,” and total acetone bodies in 
each 24 hour sample of urine. 
Kynurenic acid was precipitated by acidifying the urine to Congo red 
with sulfuric acid. After 24 hours in the refrigerator the precipitate was 
separated by centrifugation and washed by suspending it in 10 cc. of 5 per 
cent (by volume) sulfuric acid and recentrifuging. The supernatant fluid 
and washings were decanted into a 100 cc.. volumetric flask. The 
kynurenic acid was redissolved, reprecipitated, and washed with water- 
saturated butyl alcohol to remove extraneous indole derivatives, essentially 
as directed elsewhere (6). The supernatant urine and washings were 
diluted to volume with water and reserved for estimating “kynurenine” 
and total acetone bodies. 
“Kynwenine” refers collectively to all substances precipitable as the mer- 
curic sulfate complex from urines previously freed of kynurenic acid, ace- 
tone, and acctoacetic acid; for convenience in making comparisons, the 
precipitates were calculated as kynurenine. For the determination, 20 
cc. of the reserved supernatant urine and washings were mixed with 1 cc. 
of 50 per cent (by volume) sulfuric acid and boiled to expel the preformed 
acetone and the acetone produced by the decomposition of the acetoacetic 
acid. The solution was cooled, adjusted to 10 cc., and mixed with 20 cc. 
of 10 per cent mercuric sulfate in 5 per cent (by volume) sulfuric acid solu- 
tion. After 48 hours, the precipitate which formed was filtered off on 
tared, sintered glass crucibles, washed with 10 cc. of 5 per cent sulfuric 
acid, then with 10 cc. of water, dried at llO”, and weighed. Similar pre- 
cipitation of known weights of kynurenine with mercuric sulfate indicated 
that 1 mg. of the complex was equivalent to approximately 0.23 mg. of 
kynurenine. This factor was used in calculating ‘<kynurenine.” 
Total acetone bodies were determined on 50 cc. of the reserved supernatant 
urine and washings, clarified as directed by Van Slyke (16) and made up to 
a volume of 250 cc. for filtration. 25 cc. of the filtrate were mixed, in a 500 
cc. distilling flask, with 5 cc. of 17 N sulfuric acid and 100 cc. of water. 
The distilling flask was connected, through ground glass joints, to a con- 
denser and a delivery tube extending nearly to the bottom of a 500 cc. 
Erlenmeyer flask which contained a mixture of 5 cc. of 17 N sulfuric acid 
and 17.5 cc. of 10 per cent mercuric sulfate in 4 N sulfuric acid solution. 
Preformed acetone and acetone produced by the decomposition of aceto- 
acetic acid were distilled over until the volume of the solution in the dis- 
tilling flask had been reduced by half. The residue was then cooled, 17.5 
cc. of the 10 per cent mercuric sulfate reagent were added, and the mixture 
was set aside for 48 hours to allow the “kynurenine” to precipitate. The 
precipitate was filtered off and washed with 5 to 10 cc. of water. The 
filtrate and washings, which still cont’ained /%hydroxybutyric acid, were 

BORCHERS, BERG, SND WHITMAN 661 
yieldc I less, dl-tryptophane only a small amount, and the indolelactic acids 
and d(f)-tryptophane little or none. 
TABLE 11 
Average Total Urinary Output of Kynurenic Acid, “Kynurenine,” and Acetone Bodies 
in Female Rats Fed Tryptophane and Related Compounds, with or without 
S 
8 
8 
-f 
L 
2 
24 
24 
8 
8 
6 
6 
10 
10 
3 
3 
4 
4 
4 
12 
D;y 
feedingi 
3 
3 
3 
3 
3 
3 
3 
3 
3 
3 
3 
3 
3 
3 
3 
3 
2 
2 
2 
2 
2 
2 
Sodium Butyrate, a 
<YNl- 
renic 
acid 
?w. w. 
106 
14 
49 
35 
49 
76 
111 
27 
473 
43 
660 
87 
166 
37 
39 
35 
29 
31 
175 
34 
2973 
41 
589s 
87 
22 7245 
24 81 
26 7973 
88 262 
104 113 
19 49 
Kynu- 
mine’ 
b 
’ c 
L 
Acetone 
odies cal- 
ulated as 
acetone 
gm. *er 
spm. 
0.58 
0.35 
0.45 
0.34 
0.62 
0.18 
0.10 
0.13 
1.30 
4.35 
0.92 
4.96 
1.36 
4.71 
2.81 
4.80 
2.29 
4.01 
2.26 
0.91 
0.68 
3.98 
?r Preliminary ~24 Hour Fast 
Substances fed in gum tragacanth suspension% 
I(-)-Tryptophane + NaCl 
N&l 
dl-Tryptophane + NaCl 
NaCl 
d(+)-Tryptophane + NaCl 
NaCl 
Ammonium chloride 
None 
l(-)-Tryptophane + Na butyrate 
Na butyratc 
dl-Tryptophane + Na butyrate 
Na butyrate 
d(f)-Tryptophane + Na butyrate 
Na butyrate 
NH&l + Na butyrate 
Na butyrate 
dl-Indolelactic acid + Na butyrate 
Na butyrate 
d(-)-Indolelactic acid + Na butyrate 
Indolepyruvic acid + Na butyrate 
I(-)-Kynurenine + Na butyrate 
Na butyrate 
* The rats weighed 160 to 190 gm. each. Meeh’s formula was used in calculating 
surface area. 
7 An extra day of fasting was allowed to insure collection of all of the urine voided 
during the metabolism period. 
$ Per day each rat received 0.053 gm. of gum tragacanth and 0.6 gm. of sodium 
butyrate or its equivalent of sodium chloride. In addition, each experimental ani- 
mal was fed 0.6 gm. of tryptophane or its equivalent of other test material. 
$ These precipitates responded to the glyoxylic acid test. See the text. 
Since the composition of t,he “kynurenine” precipitates was obscure, these 
were analyzed for total nitrogen. Estimation of amino nitrogen failed to 
give reproducible results and was abandoned, as a differential test, in favor 
of the Shaw and McFarlane quantitative adaptation of the glyoxylic acid 
662 TRYPTOPHANE METABOLISM. X 
color reaction (17) for tryptophane. Mercuric sulfate precipitates ob- 
tained from pure solutions of tryptophane, kynurenine, and indolelactic 
acid showed nitrogen contents of 2.79,2.70, and 1.23 per cent, respectively. 
Similar precipitates isolated after I(-)-, dl-, and d(+)-tryptophane and 
I(-)-kynurenine were fed contained 2.4 to 2.9 per cent of nitrogen; those 
obtained after indolepyruvic acid and d( - )-indolelactic acid were fed con- 
tained 1.9 and 1.4 per cent, respectively. Previous tests of the Shaw and 
McFarlane procedure had shown that kynurenine, indolepyruvic acid, 
and indole do not respond typically, but t.hat skatole and indolepropionic 
acid produce colors qualitatively similar to the color formed with trypto- 
phane.2 The latter was found to be true also of indolelactic acid. Ap- 
plication of the method to pooled mercuric sulfate precipitates formed in 
the urines voided after I( -)-tryptophane, I( - )-kynurenine, or indolepy- 
ruvic acid was fed yielded no color; the “kynurenine” excreted after dl- 
tryptophane feeding produced a color equivalent to a content of 10 per 
cent of tryptophane; after d(+)-tryptophane feeding, to 33 per cent. 
The color produced by similar precipitates after d( -)-indolelactic acid feed- 
ing was compared wit.h that developed in an indolelactic acid standard; ap- 
parently 88 per cent of the “kynurenine” was in this form. Unfortunately 
similar quantitative tests were not made after dl-indolclactic acid was fed. 
The calorimetric assays and the nitrogen data suggest that the substance 
precipitated from the urines of rats fed I(-)-tryptophane was chiefly ky- 
nurenine, that some kynurenine was probably produced from indolepyruvic 
acid, but that none was formed from d(-)-indolelactic acid. Subtraction 
of kynureninc equivalent to the estimated t,ryptophane content from the 
Ma1 “kynureninc” precipitated after dl- and d+)-tryptophanc were fed 
leaves a larger balance of “kynurcnine” in each instance than was obtained 
after I( -)-tryptophane was fed. The precipitates probably contained 
d(+)-kynurenine which is formed from d(+)-tryptophane (15),’ but can- 
not be converted into kynurenic acid (18), and hence must either be ex- 
creted as such or undergo further metabolism by some other route. 
Products resulting from intestinal putrefaction and subsequent detoxica- 
tion may have been present, but probably in no greater amount than after 
Z( - )-tryptophane was fed. 
The average total acetone body excretion was slightly greater in the 
fasted rats fed tryptophane and sodium chloride than in their controls, but 
in rats fed sodium butyrate to augment the ketonuria, supplementation 
with tryptophane decreased the output markedly. Analysis showed that 
the acetone body precipitates from the urines of rats fed tryptophane con- 
tained an appreciable amount of nitrogen (an average of 0.084 per cent, as 
* Buck. D. 31. and Berg, C. I’., unpublished data. 


BORCHERS, BERG, AND WHITMAN 665 
suggest that production of ammonia and formation of urea from the other 
compounds tested may have been partly responsible for their effect in 
decreasing the acetone body output. However, since the decrease pro- 
duced by indolepyruvic acid, which contains only indole nitrogen, was ap- 
proximately equivalent to that induced by tryptophane, which contains 
both indole and amino nitrogen, and to the decrease induced by kynurenine, 
which contains two amino groups, other factors must also have been 
involved. 
In rats fasted for 48 hours, then fed 0.3 gm. of I(-)-, dl-, or d(+)-trypto- 
phane (or its equivalent of dl-indolelactic acid, indolepyruvic acid, or 
I( - )-kynurenine) at 4 hour intervals for periods of 4,8, 12, or 20 hours, no 
evidence for storage of liver glycogen was obtained. This would seem to 
indicate that the effect of these substances upon the acetone body output 
in the rats fed sodium butyrate cannot be ascribed to their conversion 
into glucose or a glycogenic intermediate, unless acetone body formation 
or excretion can be affected by amounts too small to induce glycogen 
storage. 
The yields of kynurenic acid and kynurenine from I(-)-tryptophane 
were proportionately the sameas from I( - )-kynurenine, but did not account 
for more than a fourth of the total ingested. Proportionate depressions 
in acetone body output indicate that the metabolism of I( -)-kynurenine is 
not limited to its conversion to kynurenic acid; quite likely I( -)-kynurenine 
may be an intermediate also in the metabolism of the large portion of ex- 
ogenous tryptophane which is not eliminated as kynurenic acid. Little 
kynurenic acid was produced from dl-tryptophane and apparently none 
from d(+)-tryptophane; the mercuric sulfate precipitates obtained after 
these were fed contained appreciable amounts of tryptophane, but prob- 
ably consisted chiefly of d(f)-kynurenine. A little over half of the d(-)- 
indolelactic acid fed was excreted; the accompanying decrease in acetone 
body output suggests that the rest was metabolized, but the data do not 
warrant concluding that either kynurenic acid or appreciable kynurenine 
was produced. Indolepyruvic acid was converted to kynurenic acid, ap- 
parently more readily than previously noted in the rabbit, though less 
readily than I(-)-tryptophane; the high nitrogen content of the mercuric 
sulfate precipit’ate and its negative response to the test for tryptophane 
suggest that it was composed in part of indolepyruvic acid and in part of 
kynurenine. 
BIBLIOGRAPHY 
1. Borchers, R., and Berg, C. P., Federation Proc., 1, pt. 2, 102 (1942). 
2. Rose, W. C., Science, 86, 298 (1937). 
3. Gordon, W. G., Kaufman, R. E., and Jackson, R. W., J. Biol. Chem., 113, 125 
(1936). 
666 TRYPTOPHANE METABOLISM. X 
4. Kotake, Y., and Iwao, J., Z. physiol. Chem., 196, 139 (1931). 
5. Correll, J. T., Berg, C. P., and Cowan, D. W., J. Biol. Chem., 123, 151 (1938). 
6. Berg, C. P., 1. Biol. Chem., 91, 513 (1931). 
7. Dakin, H. D., J. Biol. Chem., 14, 321 (1913). 
8. Cox, G. J., and King, H., in Clarke, H. T., et al., Organic synthcscs, New York, 
10, 100 (1930). 
9. Berg, C. P., J. BioZ. Chem., 199, 79 (1933). 
10. Berg, C. P., and Potgieter, M., J. BioZ. Chem., 94,661 (1931-32). 
11. du Vigneaud, V., and Sealock, R. R., J. BioZ. Chem., 96, 511 (1932). 
12. Bauguess, L. C., and Berg, C. P., J. BioZ. Chem., 104, 675 (1934). 
13. Boyd, W. J., and Robson, W., Biochem. J., 29, 555 (1935). 
14. Berg, C. P., Rose, W. C., and Marvel, C. S., J. BioZ. Chem., 86, 219 (1929-30). 
15. Kotake, Y., and Ito, N., J. Biochem., Japan, 26, 71 (1937). 
16. Van Slyke, D. D., J. BioZ. Chem., 32, 455 (1917). 
17. Shaw, J. L. D., and McFarlane, W. D., J. BioZ. Chem., 132, 387 (1940). 
18. Kotake, Y., and Ito, N., J. Biochem., Japan, 26, 161 (1937). 
19. Edson, N. L., Biochem. J., 29, 2082,2498 (1935). 
20. Good, C. A., Kramer, H., and Somogyi, M., J. BioZ. Chem., 100, 485 (1933). 
21. Somogyi, M., J. BioZ. Chem., 70, 599 (1926). 


Tryptophane Metabolism
X. The Effect Of Feeding 1(-)-, Dl-, And D(+)-Tryptophane, D(-)And Dl-(β-3-Indolelactic Acid,(β-3-Indolepyruvic Acid, And L(-)Kynurenine Upon The Storage Of Liver Glycogen And The Urinary Output Of Kynurenic Acid, Kynurenine, And Total Acetone Bodies

https://digitalcommons.unl.edu/cgi/viewcontent.cgi?article=1266&context=biochemfacpub

Tryptophane Metabolism X. The Effect Of Feeding 1(-)-, Dl-, And D(+)-Tryptophane, D(-)And Dl-(β-3-Indolelactic Acid,(β-3-Indolepyruvic Acid, And L(-)Kynurenine Upon The Storage Of Liver Glycogen And The Urinary Output Of Kynurenic Acid, Kynurenine, And Total Acetone Bodies

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