Interference by sucrose in the chemical determination of citrulline with diacetyl monoxime

Interference

by Sucrose in the Chemical Determination of Citrulline with Diacetyl Monoxime

The chemical determination of citrulline with diacrtyl mcmwime i4 subject to interference by sucrose and fr-uctose. Glucose. sorhitol. and mannitol do not interfere. Likewise. the presence of EDTA. 2.mercaptoethanol. and HEPES (N-2-hydroxyethylpipzr-azine-iv’-?-ethanc wlphonic :icid) at concentr;ltic)ns normally employed in mitochondrial isolation media does not affect the assay. The use of this method to estimate citrulline formation by mitochondria prepared in wcrcw containing medium mny thus be whject ta error unless the effect of sucrose is taken into account. For such btudies of mitachondrial citrulline of citrulline ;~ssay. formation employing diacetyl monoxime ~1s the method the u&e of a mannital-h;tsed isolation medium ib recommended.

The chemical determination of citrulline with diacetyl monoxime was developed by Archibald (1) from the work of Fearon (3) and Gornall and Hunter (3). This method has been widely used in studies of urea cycle activity and the measurement of urea cycle intermediates where the full amino acid spectrum afforded by the amino acid analyser has not been required (4-10). Although it has long been known that the method detects a wide range of substituted amides and that the color produced is light sensitive (3,3), there appears to be little information in the literature concerning the problem due to interference by carbohydrates, notably sucrose and fructose. Early recognition of the problem and the observation that mannitol does not interefere were made by Leuthardt and Miiller (9). More recently, Charles lot trl. (IO) also commented upon the interference caused by sucrose. As yet. however, there appear to be no data in the literature illustrating the nature or magnitude of the effect. During preliminary studies in our laboratory on the formation of citrulline by isolated mitochondria, we also noted that the sucrose isolation medium used in the preparation of the mitochondria interfered with the assay of the citrulline formed. The present paper reports briefly on the nature of this effect and illustrates the hazards of not allowing for this interference. MATERIALS Citrulline as possible.

AND METHODS

was determined by the method of Archibald (1). AS far light was excluded from the tubes during the incubations

552

PRESCOTT

AND

MANCNALL

FIG. 1. The effect of increasing concentrations of sucrose on the color development from citrulline. Standard curves were prepared from a stock citrulline solution. To a final sample volume of 4.0 ml were added 2.0 ml of concentrated H,SO,:H,,PO, (1:3 v/v). followed by 0.25 ml of 3”r (w/v) aqueous diacetyl monoxime. Tubes were incubated at 100°C for 15 min and, after cooling. the extinction concentration of sucrose in the total reaction mixture (A) 4.48 mM: (W) 8.96 mM: and (a) 13.44 mM.

at 490 nm was measured. was (0) no sucrose: (0)

The 2.24

final mtvr:

and subsequent cooling, and the tubes were brought into the light only just before reading the absorbance at 490 nm. The intensity of color developed may depend upon the presence of trace amounts of heavy metal ions, probably copper. and the use of technical-grade sulphuric acid has been advocated (7.11). In the present study, both the sulphuric and phosphoric acids were of technical grade. Although we have used Analar quality acids (Hopkins and Williams, England) without encountering any loss of sensitivity, this may not be true for all batches of Analar or higher quality reagents, and the addition of small quantities of copper sulfate may be necessary if extremely pure reagents are used. Rat liver mitochondria were prepared from male. fed rats weighing about 250 g. After stunning and cervical dislocation, the livers were flushed through with ice-cold isolation medium (280 mM sucrose containing 1.O mM HEPES (N-2-hydroxyethylpiperazine-N’-2-ethane sulphonic acid), 0.1 mM EDTA, and 0.2 mM mercaptoethanol, (pH 7.4). via a cannula introduced into the portal vein and were then homogenized in 10 vol of the same medium. The homogenate was centrifuged at 750,e (max) for 10 min in the 8 x .50-m] fixed-angle rotor of an M.S.E. highspeed 25 centrifuge, and the supernatant was respun at 19OOOg (max)

SUCROSE

FIG. zucrose.

2.

Absorption For each

scan.

EFFECTS

spectra the

of solid

ON

CITRULLINE

citulline standards line is the spectrum

in

the presence in the absence

the dotted line is the spectrum in the presence of sucrose (X.96 rnhl final the amounts of citrulline were (a) 0 pmol. (b) 0.3 Fmol. and (c) 0.X were performed as described in Materials and Methods and the legend spectra

were

recorded

using

a Pye

Unicam

SP

IX00

553

ASSAY

and of

absence s~croxe

of and

concentration). pmol. Assays to Fig. I. The

spectrophotometer.

for 10 min in the same rotor. After resuspending the pellet, these two spins were repeated and the final pellet was washed twice in isolation medium. RESULTS

AND DISCUSSION

The effect of increasing amounts of sucrose on the citrulline standard curve is shown in Fig. 1. Sucrose was found to decrease the sensitivity of the assay. not only by decreasing the amount of color produced at high citrulline concentrations but also by the production of a colored product, which was independent of the presence of citrulline but which also absorbs at 490 nm. In contrast to the pink coloration produced by citrulline. the product due to sucrose was yellowish-brown. Despite the precautions taken to exclude light. it is apparent from Fig. 1 that some fading of color may have occurred, since the curve obtained in the absence of sucrose was not a straight line. and Archibald t 1) reported that the color was lost most readily from the lower concentrations of citrulline. Figure ?, shows the absorption spectrum of the citrulline reaction product in the presence and absence of sucrose. This illustrates that the loss of sensitivity was associated with an absolute decrease of the peak height at the higher citrulline concentrations and did not result from a shift in the spectrum. so that 490 nm was no longer the maximum. Thus the sensitivity of the assay could not be restored

554

PRESCOTT

AND

MANGNALI

II

Orntthlne

FIG. citrulline

3. The upon

effect the

of not apparent

ornithine. Mitochondria and Methods and were

5 concentration

correcting dependence

for

sucrose interference of mitochondrial

were isolated suspended in

in the

sucrose medium same medium.

9.2 mg of mitochondrial protein) The final ornithine concentration the addition of carbamyl phosphate

10 (m mol/l

in the citrulline as described Mitochondria

were incubated in a total ranged from 0 to IO mM. (5 mM final concentration)

incubation at 37°C for 5 min by the addition of 300 ~1 of 0.33 tion. samples of the supernatant were taken for citrulline experiments showed the reaction with carbamyl phosphate for for

at least sucrose

5 min under these conditions. interference and open symbols

I

Solid symbols (0) (0) show uncorrected

reaction Reactions and

determination formation

of from

in Materials (approximately

volume of 300 ~1. were started by were stopped after

N HCIO,. After centrifugadetermination. Preliminary and ornithine to be linear indicate results.

results

corrected

by reading at a different wavelength. Studies with other sugars show that fructose produced a similar effect to sucrose and, on a molar basis, was as potent as sucrose in displacing the standard curve. Glucose. sorbitol, and mannitol on the other hand were without any effect. Likewise, EDTA, mercaptoethanol, and HEPES did not interfere. It seems likely, therefore, that the interference by sucrose was due to the fructose moiety rather than the glucose portion of the molecule. Since mannitol did not interfere with the assay, the use of a mannitolbased isolation medium appears preferable to a sucrose mixture when isolating mitochondria for subsequent studies of citrulline formation. If a sucrose isolation medium is to be used, it is important that the interference due to sucrose should be corrected for. This may be conveniently achieved by the inclusion of the appropriate amounts of sucrose in the standard curve. Figure 3 illustrates the potential hazard of not correcting for the sucrose interference in studies of citrulline formation by mitochondria isolated and incubated in sucrose-containing medium. Without correction for the effect of sucrose, the dependence

SUCROSE

EFFECTS

ON

CITRCILLINE

ASSAY

555

of citrulline formation upon the ornithine concentration appears to yield a nonlinear double reciprocal plot which might be taken as an indication that the system displays complex kinetics. If correction fog the effect of sucrose is made. the shape of the velocity-concentration curve is considerably altered and the corrected curve corresponds to a linear Lineweaver-Burk plot. Thus. in addition to previously reported problems of light sensitivity ( l-3) and interference by compounds such as glutathione (5), the chemical determination of citrulline with diacetyl monoxime is also sensitive to interference by sucrose and fructose. This may be of particular importance in studies of citrulline formation by cell fractions isolated in sucrose-containing medium and is of general relevance to the use of this method for the estimation of any other substituted amide under conditions where fructose or sucrose may also be present. ACKNOWLEDGMENTS of

The the

thanked

financial Department for

support of

pl-eparation

of The Medical of the

Wellcomc Illustration.

‘I‘ru~t i\ Northern

gratefully General

ackn~>wledged. Hospital.

The Sheffield

\tafl‘ are

diagl-am\.

REFERENCES I.

Archibald.

7. 3. 4. 5.

Fearon. Gornall. Brown. McLean.

6. McLean. 7. Rntner. eds.). X.

McCivan.

R. M.

(1944)

./. Bb~l.

C/~v,,r.

156.

I21 - 1-l’.

W. R. (1939) Bir~lre,,~. .I. 33, 90-907. A. G.. and Hunter. A. (1941) Bioc./lc,,,~. .I. 35, 6X-658. G. W.. and Cohen. P. P. (1959) .1. Birjl. Clrou. 234, l769P.. and Gurney. M. W. t 1963) Bio(,/~(,m. .I. 87, Y6- 104. P.. and Rossi. F. (I9641 Bio(./~(l~u. .I. 91, 261 -270. S. (1955) irr Methods in Enzymology tColowick. Vol. 2. pp. 359-367. Academic Press, London-New J. D..

Bradford,

N. M.,

Crompton,

M..

and

1774.

S. P., and York.

Chappell.

Kaplan.

J. B. t 1973)

N.

Bi~~/r(,rtl.

0.. J.

134.309%21.5, 9.

Leuthardt,

IO. Charles. I I. Grirolia. ed\.

F..

and

Miillcr.

4.

F. t 1948)

,5.q)rl-i~,~fitr

4, 17X-480.

R.. Tager. J. M.. and Slater, E. C. (1967) B;~K/I~,,I. S. (1955) i/r Method5 in Enzymology (Colowick. ). Vol. 2. pp. 350-355. Academic Press. London-New

Bio~~h~\. A(.zcr 131, S. P.. and Kaplan. York.

29-4 I. N. 0..