On the isolation and identification of homocitrulline from urine

On the isolation and identification of homocitrulline from urine

BIOCHEMICAL Vol.4,No.5,1961 AND BIOPHYSICAL RESEARCH COMMUNICATIONS ONTHE ISOLATION ANDIDENTIFICPTION OF HOMOCITRULLINE FROMURINE* Theo Gerritsen, ...

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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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