The work of Ackroyd and Hopkins (1)

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SYNTHESIS OF AMINO ACIDS IN THE ANIMAL BODY.
IV. SYNTHESIS OF HISTIDINE.*
BY BENJAMIN HARROW AND CARL P. SHERWIN.
(From the Chemical Research Laboratory of Fordham University, New York,
and the Laboratory of Biological Chemistry of Columbia University
at the College 0,~ Physicians and Surgeons, New York.)
(Received for publication, August 27, 1926.)
The work of Ackroyd and Hopkins (1) leads to the conclusion
that a diet must contain either arginine or histidine; they are,
as the authors term them, “interchangeable” amino acids. This
view receives support from the work of Ceiling (2). The com-
plete removal of histidine from a diet can be accomplished with-
out much difficulty, but, with the present methods at our dis-
posal, there is no equal assurance that arginine can be completely
removed. This makes it a diflicult, if not impossible matter, at
present, to study the arginine problem with any definite assurance
of success. The recent work of Vickery and Leavenworth (3)
holds out hope that this shortcoming may soon be overcome.
However, the question, Is histidine an essential amino acid? can
be answered with definiteness, as Rose and Cox (4) have shown,
and, as we are able to confirm. The absence of histidine from a
diet produces marked loss of weight and general decline; and this
irrespective of the amount of arginine which may be present.
Stewart (5) has pointed out that on a diet deficient in histidine
and arginine, young rats lose weight and their allantoin excretion
is markedly decreased. The addition of histidine to the defi-
cient diet causes a resumption of growth and an increased allan-
toin excretion, suggesting, as Ackroyd and Hopkins had pointed
out, that histidine functions as a precursor of purines in the
* This communication was presented in abstract form before the Federa-
tion of American Societies for Experimental Biology at Cleveland, Ohio,
December, 1925. See Novello, N. J., Harrow, B., and Sherwin, C. P., J.
Biol. Chem., 1926, lxvii, p. liv.
683
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Amino Acid Synthesis. IV
animal body. The addition of arginine to the deficient diet did
net give rise to resumption of growth nor to any increased allan-
toin excretion.
Obviously, histidine is an “essential” amino acid; and further-
more, histidine and arginine are not interchangeable. The latter
statement receives added support from previous work by one of us
(6). The statement was then made that “the ingestion of amino
acids other than arginine [by a fowl along with benzoic acid] does
not seem to increase greatly the output of ornithuric acid, ap-
parently contradictory to the theory that histidine is convertible
quite easily into arginine, for ingested histidine does not augment
ornithuric acid while arginine does.” The synthesis of ornithine
by the hen would indicate the possible synthesis of arginine in
the mammalian body and might, indeed, lead to the conclusion
that arginine is not to be classed as an “essential” amino acid
at all.
If we must regard histidine as an essential amino acid, we are
still interested in the question, To what extent may some of the
probable metabolic products of histidine replace this amino acid?
Is it, indeed, possible to replace what has hitherto been regarded
as an essential amino acid by some other compound? With the
view that light may also be thrown on the possible catabolism of
histidine in the animal body-whether the changes take place by
way of the a-hydroxy acid, the c+keto acid, or the a,~ unsaturated
compound (acrylic acid)-we selected d,l-imidazol lactic acid,
imidazol pyruvic acid, and imidazol acrylic acid. We also tried
imidazol itself.1
1 The imidazol used in these experiments was purchased from Kahl-
baum, and the amino acids were obtained from American firms, but they
were thoroughly tested before use. The imidazol derivatives were prepared
by us (results unpublished).
In the June number of the Journal (1926, Ixviii, 781) Cox and Rose re-
port experiments of much the same character as ours and come to much
the same conclusion. Their statement (p. 796) that, “The above experi-
ments constitute the first successful attempt by means of growth studies to
replace an ‘indispensable’ amino acid by any other compound whatso-
ever” is oontrary to the facts, since a summary of our work had already
been published. We reported the results of our experiments simultaneously
with Cox and Rose (Cox, G. J., and Rose W. C., J. Biol. Chem., 1926, lxvii,
p. iii).
B. Harrow and C. P. Sherwin 685
EXPERIMENTAL.
The experiments were performed on young rats. The methods
followed were essentially those described by Rose and Cox (4).
The source of nitrogen for the preliminary tests was unhydrolyzed
TABLE I.
Diets of Unhydrolyzed Casein and Completely Hydrolyzed Casein.
Diet No.
- -
Composition.
100 101 102 103
- .-
per cent per cent per cent per cent
Unhydrolysed casein .............. 14.7 14.7
Completely hydrolyzed casein ..... 14.2 14.05
Cystine ........................... 0.3 0.3 0.3 0.3
Tyrosine .......................... 0.3 0.45
Tryptophane ..................... 0.2 0.2
Dextrin .......................... 34.0 40.0 40.0 40.0
Sucrose ........................... 15.0 15.0 15.0 15.0
Lard ............................. 25.0 19.0 19.0 19.0
Salt mixture ...................... 4.0 4.0 4.0 4.0
Agar ............................. 2.0 2.0 2.0 2.0
Cod liver oil ..................... 5.0 5.0 5.0 5.0
Yeast ............................
- -
TABLE II.
Diet of Completely Hydrolyzed Casein from Which the Histidine and Arginine
Had Been Removed.
-
Composition. Diet 201.
.
Completely hydrolyzed casein minus histidine and per cent
arginine ................................................ 14.05
Cystine .................................................. 0.3
Tyrosine ................................................. 0.45
Tryptophane ............................................. 0.20
Dextrin .................................................. 400
Sucrose .................................................. 15.0
Lard .................................................... 19.0
Salt mixture ............................................. 4.0
Agar ..................................................... 2.0
Cod liver oil ............................................ 5.0
Yeast ....................................................
686 Amino Acid Synthesx IV
casein and completely hydrolyzed casein. Subsequently, in the
test experiments, a hydrolyzed casein from which the histidme
and arginine had been removed, was used. To this mixture of
unsatisfactory amino acids, definite quantities of the imidazol
compounds, including histidine, were added. The diets are given
in Tables I and II.
TABLE III.
Food Consumption and Body Weight Changes on Unhydroluzed Casein.
-
Weight at
Rat No. ecxopmmemreimneent ncet. -o f Days. Diet No. Abvocdehyr aanggeew eigdihanit ly. Aooved ragtoieoo nn.a umdapil-y
9m. 9m.
A 83 l-30 100 +:I2 3.6
31-60 101 $1.42 4.8
61-90 101 +0.96 5.0
91-120 101 +0.79 5.9
121-150 101 $0.55 6.6
_ .
B ( 79 l-18 100 $1.44 4.6
19-36 100 +1.09 3.3
37-60 101 +1.50 4.7
61-90 101 +o. 92 5.9
91-120 101 +0.90 7.0
121-150 101 +o.so 7.0
C 68 l-28 100 +1.0 3.6
2S60 101 f1.39 4.7
61-90 101 +0.96 5.2
91-120 101 +o.so 6.0
121-150 101 +0.70 8.0
1
D 73 1-28 100 $1.13 3.3
29-61 101 +1.52 5.0
62-90 101 +1.01 5.5
91-120 101 $0.80 6.5
121-150 101 +0.70 7.75
-
Preliminary experiments showed that 30 mg. of commercial
yeast-as a source of vitamin B-per rat per day were sufficient
for normal growth. The fat-soluble vitamin-the vitamin A
and the antirachitic substance-was supplied by the addition of
cod liver oil (5 per cent) to the diet. The nitrogen components
of the diet made up about 15 per cent.
B. Harrow and C. P. Sherwin 687
In Table III are summarized the results with unhyrolyzed
casein. These results indicate quite clearly normal growth devel-
opment in R.ats A, B, C, and D. Rats X, Y, and Z were placed
on completely hydrolyzed casein and the results are recorded in
Table IV. While the rate of growth in the earlier stages is not
so marked as when unhydrolyzed casein is used, nevertheless the
TABLE IV.
Food Consumption and Body Weight Changes on Diet of Completely
Hydrolyzed Casein.
- - -
Weight at
oommence- Average daily Average daily
Rat No. axpmereimrite nt.o f Days. Diet No. bocdhy angew eigihnt . ood ctioonna. ump-
-- _ -
gm.
X 81 l-30 100 +:lS 541
31-50 102 +0.51 4.2
51-65 103 +0.60 5.0
66-90 103 $0.80 5.7
91-120 103 +0.60 6.2
121-150 103 $0.91 8.1
.- . _ -_
Y 77 1-28 100 +0.98 4.6
2945 102 +0.33 5.2
46-66 103 f0.61 5.6
67-93 103 +0.69 6.0
94120 103 $0.77 6.6
121-150 103 $0.79 8.0
-- -- - -
Z 73 l-30 100 +1.02 4.4
31-50 102 $0.47 4.5
51-70 103 +o. 39 4.8
71-90 103 +0.80 5.9
91-120 103 $0.86 6.6
121-150 103 $0.81 8.3
- - -
results are sufhciently conclusive to substantiate Abderhalden’s
contention (7) that life can be supported where the sole source of
nitrogen is the amino acids obtained by the hydrolysis of a pro-
tein. In this respect, our results are in entire agreement with
those of Rose and Cox (4).
In the attempt to substitute for an essential amino acid such as
histidine a compound closely related to it, we first selected imid-
688 Amino Acid Synthesis. IV
TABLE V.
Food Consumption and Body Weight Changes when Imidazol Lactic Acid (A)
Is Used in Place of Histidine.
Rat No. emWxmmpeemeirginmehttn e cneto - fa t Diet No. Days. d aAiilnwyv eeimgbchgohtdea. y n ge !n&pde igrioohdft . eacal t ; ’
--
gm. gm. gm.
21 76 101 l-10 $2.8 104
201 1 l-33 -0.73 88
(201 + 34-50 +1.25 109
0.2 per
cent
A.)
201 51-66 -0.60 100 2.7
(201 -F 67-90 +1.26 129 6.3
0.4per
cent
A.)
201 91-120 -0.77 106 2.4
(201 + 121-150 $1.45 135 7.8
0.2 per
cent
histi-
dine.)
68 101 l-10 +3.33 101.3 7.0
201 i l-30 -1.15 78.3 2.5
(201 + 31-50 $0.88 96.0 4.8
0.1 per
cent
A.)
201 51-70 -0.75 81 2.3
(201 + 71-85 +2.07 112 6.7
a.3per
cent
A.)
201 86100 -0.77 97 2.2
(201 + 101-130 $0.70 119 7.4
0.1 per
cent
histi-
dine.)
-
B. Harrow and C. P. Sherwin 689
101 = Diet 101. A = imidaaol lactic acid.
201 = Diet 201. H = histidine.
CHART II.
101 = Diet 101. B = imidazol pyruvic acid.
201 = Diet 201. H = histidine.
690 Amino Acid Synthesis. IV
TABLE VI.
Food Consumption and Body Weight Changes when Imidazol Pyruvic Acid (B)
Is Used in Place if Histidine.
-
Rat No. ec :e oWxmmpeeemigrriheimtt n ecncto-. af t Diet No. Days. !aAiinwlyv ee irgabohgohted.a yn g mWdep iegrhioot df . ema1t dco$n?tsgioudnm . p-
--
vm. vm. vm. vm.
26 70 101 l-12 +1.7 90 4.4
201 13-30 -0.8 76 2.0
(201 -t 31-52 +0.3 83 3.3
0.3 per
cent
B.)
201 53-71 -0.5 74 2.1
(201 + 72-96 $0.3 79 3.4
0.5 per
cent
B.)
(201 + 97-126 +0.75 101 5.0
0.2 per
cent
histi-
dine.)
. ..-
40 82 101 l-10 +3.8 120 7.7
201 1 l-26 -2.45 81 2.2
(201 + 2740 -to.7 91 4.1
0.2 per
cent
B.)
201 41-60 -0.7 78 2.1
:201 + 61-77 +0.5 86 4.4
0.4 per
cent
B.)
201 78-92 -0.45 80 2.0
‘201 f 93-120 $1.6 125 6.6
0.2per
cent
histi-
dine.)
- -
B. Harrow and C. P. Sherwin 691
as01 lactic acid. The rat was first placed upon a diet the nitro-
gen components of which consisted of unhydrolyzed casein
(No. lOl), Diet 101 being next replaced by completely hydro-
TABLE VII.
Food Consumption and Body Weight Changes when Imidazol Acrylic Acid (C)
Is Used in Place of Histidine.
- -
Rat No. a0 7’rWo pmmeemriegimrheitten ncet.-o af t Diet No. Days. d MAyiwnv eeirgabhcgohted.a yn ge Wndpe eigroihoftd .e acaht l:AaOivllylet* ioUran~g.p efo- od
_-
gm. gm. om. 0m.
31 82 101 1-12 +2.6 113 7.1
201 X3-30 -0.7 101 2.2
(201 + 31-45 -0.07 100 3.5
0.3per
cent
c.1
201 46-56 -1.0 90 2.0
(201 + 57-70 -0.1 88 3.3
0.5 per
cent
c.1
--
96 77 101 1-15 $3.1 123.5 7.1
201 16-28 -0.9 112 2.4
(201 + 29-50 -0.1 110 3.3
0.5 per
cent
c.1
(201 + 51-62 f3.5 152 8.5
0.2 pe
cent
histi-
dine.)
(201 + 63-80 -0.15 149 2.9
1 per
cent
C.)
- -
lyzed casein from which the histidine and arginine had been
removed (Diet 201), and the imidazol lactic acid added to Diet
201 after the latter diet had shown unmistakable evidence of
insufficiency. The results are given in Table V (see also Chart I).
692 Ammo Acid Synthesis. IV
CHART III.
101 = Diet 101. C = imidanol acrylic acid.
201 = Diet 201. H = histidine.
CHART IV.
101 = Diet 101. D = imidasol.
201 = Diet 201. H = histidine.