BIOCHEMICAL
Vol.4,No.5,1961
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ONTHE ISOLATION ANDIDENTIFICPTION OF HOMOCITRULLINE FROMURINE* Theo Gerritsen, Samuel H. Lipton, F. M. Strong and Harry A. Waisman From the Departments of Pediatrics and Biochemistry, University of Wisconsin, Madison, Wisconsin Received March 17, 1961 During column chromatographic analysis of free amino acids in urine by meansof the Moore and Stein procedure (1951), the appearance of an unidentified component in the form of a peak between valine and the buffer position
has been observed by several workers (Fowler
Jagenburg, 1959a; Spa&man, 1960).
color tests and Rf values as pipecolic
one on the basis of
acid.
In the present study on urines of infants following
and young children with the essentially
outlined by Spa&man -et al. (1958), an unidentified ohserved at, or just prior
to the buffer
change position
minutes (to 11 l/4 hrs), this peak clearly
after
It was still
acid hydrolysis
the procedure as
peak was frequently
on the 150 cm column chromatogram. By delaying the buffer
380 and LOOml.
1957;
Jagenburg obtained evidence that the
peak contained four components of which he identified
Beckman/Spinco Amino Acid Analyzer,
et al.,
change
(380 - 390 ml) change for 16
separated from cystine between
present and emerged at the sameposition
of the urine or after
performic acid oxidation.
Isolation Urine of a 3 month old normal infant,
found to excrete the unidenti-
fied compoundin unusually large amounts, was used as starting The urine was obtained under stertle
conditions,
under toluene during the 24 hr. collection
material.
and was kept in ice and
period.
Fifty
ml
WWJ
oxidized
*This work was supported in part by grants W-2730 and A-l.498 from the National Institutes of Health, U.S. Public Health Service. 379
BIOCHEMICAL
Vol. 4, No. 5,1961
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1.954) in order to convert cystine to
with Performic acid (Schram et&,
cysteic urine
acid which appears at a different was then hydrolyzed
remove interfering column together
place in the chromatogram.
in 20% HCl for 24 hours under refJ.ux in order to
polypeptides
which in unhydrolysed
Batches of hydrolysate
came off the developed little
reagent. equivalent
the 0.9 x 150 cm Amberlite
to 15 ml of urine were absorbed onto
IR I.20 (Na+) column of the Amino Acid Analyzer,
and the amino acids were eluted with 0.2 M citrate normal speed of 30 ml per hour.
The temperature
from 30' to 50' after
The first
11 hours.'
120 ml of eluate
predominantly
urine
with the unknown compound, but apparently
or no color with the ninhydrin
following
pH 3.25, at the
of the column was changed
330 ml were discarded,
were collected.
the unidentified
buffer,
The eluate,
containing
IR 120 (H+>.
column was washed with 200 ml water, the amino acids were eluted ml 4% ammonium hydroxide. equivalent
After concentration
to 150 ml of urine
column of the Amino Acid Analyzer, ninhydrin
acid,
alanine
A 100 ml fraction
positions. fraction
glycine,
was desalted
was obtained,
and eluted
as leucine. lyophilizing,
Crystals
with GO
in vacua, combined concen-
as before.
330 ml of eluate
and valine
were visible
coming off after valine
on an Amberlite
containing
After the
were again absorbed onto the d0 cm
reagent was added to the first
glutamic
and the
compound with traces of other amino acids,
was absorbed onto a 1.8 x 25 cm column of Amberlite
trates
This time and peaks of in the usual
was collected.
This
IR 120 column and a 10 ml concentrate
about 30 mg of the unknown compound, calculated were isolated
dissolving
of absolute
ethanol,
persisted.
Crystallization
the residue
from 2 ml of this
concentrate by
(9.6 mg) in 0.1 ml 1N HCl and 2 ml
and adding absolute
ether until
of the hydrochloride
a faint
yielded
turbidi*
needles,
m.p.
and Derformic
acid
177 - 178' dec. Identification The compound was stable oxidation
as stated
The
above.
during
acid hydrolysis
On a paper chromatogram in BuAc (n-butanol: 380
BIOCHEMICAL
Vol. 4, No. 5,196l
acetic acid:water
AND BIOPHYSICAL
= 120:30:50) the RP value was found to be 0.28 - 0.30;
in PhAm (phenol:water:anunonia 38% = l.50:50:1) The compoundreacted with ninhydrin at room temperature.
the Rf value was 0.68 - 0.71.
to give a purple-red color in 20 minutes
The color deepened considerably upon heating at 95'
A pink color resulted with isatin
for 5 minutes.
immediate yellow color with Ehrlich's crystalline
RESEARCH COMMUNICATIONS
reagent.
(0.3
in acetone),
In aqueous aohtinn
and an the
material gave a peach color reaction with diacetylmonoxime,
according to the method of Archibald
(l9&)
The absorption spectrum of this reaction
for the estimation of citr!Uine.
product had a maximumat 490 mu,
which can be considered as evidence for the presence of a ureido group. These color reactions were identical 3-ureidopropionic
with those of citrulline
and 2-amino-
acid, but both these compoundswere ruled out by the
Rf values and positions
on the column chromatogram. Pipecolic
acid was
likewise ruled out, as were's number of other rare amino acids. Alkaline
hydrolysis,
by refluxing
~JNNaOHfor 16 hours, or
in boiling
in saturated Ba (OH)2 for 3 hours, decomposedthe substance forming another nimrin
positive
compoundwhich showedan Rf value of 0.12 in BuAc and
about 0.74 in PhAmand did not give a yellow color with Ehrlich's This decomposition product was easily identified
reagent.
as lysine on the Amino Acid
Analyzer and by paper ChromatograpkgT. On the basis of lysine formation during alkaline hydrolysis, above mentioned color reactions, was 2-amino-&reidocaproic
and the
it was concluded that the unknown substance
acid (C-carbamyllysine)
or homocitrulli.ne.
'This amino acid was synthesized from lysine according to the method of Kurtz
(19119) as modified by Smith (l955), unidentified paper* additive
and was found to be identical
with the
compoundbv co-chromatography on the Amino Acid Anaxrzer and on
The unknown, and the synthetic
homocitrulline
gave a single and
symmetrical peak on the column chromatogram at two different
values (3.25 and 3.30).
Furthermore, the infrared
of the isolated material and the -thetic to be identical,
and different
381
spectra (KBr pellet)
homocitrulline-Wl
from the infrared
pR
were found
spectrum of citrulline-HCl.
BIOCHEMICAL
Vol. 4, No. 5,196l
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Discussion In this investigation, onlyinthe
urine
of infant8
excreted by the infant 50 mg per 24 hours. trations during
homocitrulline
an earlier
and young children.
whose urine
absent,
of 33 male adults
investigation
The approximate
was used in this
It was either
in the urine
was found in measurable amounts
--et al.
urine,
and its
investigation
was 30 -
or present in very low concen-
examined by one of the authors
(Gerritsen
and Horwitt).
in agreement with the presence of the unidentified in infant
amount
absence in adult urine,
This finding
peak eluted
after
as demonstrated
is valine
by Dustin
(lY55). In view of the report
hydrolysis
by Stark --et al. (1960) that homocitrulline of ribonuclease inactivated by 8M urea at b", it
product
conceivable
that the urinary
homocitrulline
in the sense that it could have been formed by
interaction
and cyanate in the bladder.
low urea concentration excretion
in urine
of homocitrulline
comparable to that of infants majoritg
of the urines
catheterization,
in this
However, the comparatively
(0.3M, both in infants
b adults
despite
(Jagenburg, investigation
do not support this
is
observed in the present work
might be an artifact, of lysine
is a
and adults),
a urinary
1959b.),
lysine
the low
output
and the fact that the
were collected
possibility.
by 2b hour
In addition,
Fowler
et al. (1957) observed that infants fed synthetic amino acid diets excreted -a urinary lysine, but failed to demonstrate the unidentified peak No. 9 detected
in the 100 cm column chromatogram of urine
containing
urines
in the urines
The fact that glutamine
that the precautions
In earlier
observed to result (Gerritsen,
that the hcmocitrullino
infection.
indicated
adequate.
on protein
diets.
It is unlikely to bacterial
from infants
was usually
unpublished
present in the
taken to prevent bacterial
work any growth of micro-organisms in the conversion
can be attributed
of glutamine
into
growth were
in urine was glutandc
acid
data). 382
,
Vol. 4, No. 5,1961
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
It has not yet been demonstrated that homocitrulline animal or humanmetabolism (Lowenstein and Cohen 1956;
plays a role in
Smith 1955) and we
are unaware of a previous report on its natural occurrence. on this subject, and on the origin of homocitrulline
Further work
in urine is in progress.
References: Archibald, R.M., J.%ol.Chem., 15;6. 121, 194&. 4, 75, 19%. Bustin, J.P., Moore, S. and Bigwood, E.J., Metabolism, :: Fowler, D.I., Norton, P.K., Cheung, M.??. and Pratt, E-L., Arch.Piochem., 68. U2, 1957. Gerritsen, T. and Horwitt, M.K., Unpublished. 11, sup& no. L3, 1959; 5": Jagenburg, O.R., Scand.J.Clin.Lab.Invest., a) pp. 93 - 96; b) PP. U-3, 128. 6. Kurtz, A.C., J.Biol.Chem., I& 1253, 1949. Lowenstein, Y.M. and Cohen, P.P., J.Biol.Chem., 220, 57, 1956. ;: Moore, S. and Stein, !J.H,, J.Biol.Chem., 192, 663,1951. 9. S&ram, E., Moore, S. and Bigwood, E.J., Bibchem.J., 2, 33, 19% 10. Smith, L.H., Jr., J.An.Chem.Soc., & 6691, 1955. D.H., Stein, Y.H. and Moore, S., Anal.Chem., 30,1190, 1958. 11. Spa&man, 12. Spackman, D.H., Perscnal communication, 1960. 13. Stark, G.R., Stein, N.E. and Moore, S., J.Biol.Chem., 235, 3177, 1960. 1.
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