Clin. Biochem. 8, 293-297 (1975)

DIFFERENTIAL ASSAY FOR UREA AND CITRULLINE Automated and Manual Procedures

NELLY B L U M E N K R A N T Z and GUSTAV A S B O E - H A N S E N

University of Copenhagen Department of Dermatology (with Connective Tissue Research Laboratories), Rigshospital, Copenhagen, Denmark.

(Accepted April 1, 1975)

CLBIA, 8, (5) 293-297 (1975) Clin. Biochem.

Blumenkrantz, Nelly and Asboe, Hansen, Gustav

University of Copenhagen Department of Dermatology (with Connective Tissue Research Laboratories), Rigshospital, Copenhagen, Denmark.

D I F F E R E N T I A L ASSAY FOR U R E A AND C!TRULLINE. Automated and Manual Procedures.

1. A simple, quick and sensitive method, manual and automated, for the assay of urea and citrulline in urine or serum is presented. 2. I t may be used by clinical wards as a screening test for citrullinemia.

BOREL ET AL 1 demonstrated a substance in normal urine, which reacts directly with Ehrlich's reagent giving a chromogen with a peak of maximal absorption at 500 nm. This substance masked the reaction of the oxidation product of some hydroxyproline-containing peptides and free hydroxy- proline with Ehrlich's reagent 1. After treatment with urease the inferrer- ing substance disappeared. The idea that this qualitative f inding might be used for quantitative determination of urea led to this study. The fact that citrulline also reacts directly with Ehrlich's reagent, prompted us to study citrulline with the same procedure after digestion of urea with urease.

MATF_~RIAL AND METHODS

Ehrlich's reagent. Four grams of p-dimethylaminobenzaldehyde (pdmab) analytical reagent (E. Merck, A.G., Darmstadt) was dissolved in 10 ml perchloric acid, and 20 ml isopropanol was added. The reagent was prepared daily.

Urea (E. Merck A.B., Darmstadt) . An aqueous solution containing 20 mg /ml was prepared. Dilutions from 20 to 200 ~g urea /ml were used to assess the sensitivity of the reaction.

Citrulline (Sigma Chem. Co., St. Louis, Mo.). An aqueous solution of citrulline of the same concentration as mentioned above for urea was prepared.

Urease. (Worthington Biochem. Corp., N.Y.) Crude Enzyme URC 44 H092. A solution of urease containing 82 U (1 mg) in 2 ml citrate-phosphate buffer was used.

294 B L U M E N K R A N T Z and A S B O E - H A N S E N

Citrate-Phosphate buffer pH 7.0. 6.5 ml of 0.1 M citric acid was mixed with 43.6 ml of 0.2 M Na2HP04. The solution was di luted to 100 ml with disti l led water .

Urine. Twenty-four-hour urines were collected f rom individuals on a s tandard diet. The samples were kept frozen a t - 2 0 ° to the moment of analysis . Fo r the screening tes t a single sample of urine can be used.

Serum. Samples of blood were obtained by venous puncture, and the serum was separa ted f rom the clot by centr i fugat ion.

Perchloric acid solution. A 2 N aqueous solution of perchloric acid (E. Merck A.G., Darms tad t ) was used to deproteinize the serum.

Wetting agent solution. (Bri j 30-35% Technicon Chem. S.A., Belgium). A solution containing 1 ml Bri j per l i ter wa te r was prepared.

Preparation of samples

Serum. 0.5 ml serum was diluted with saline to 1 ml. 0.1 ml of the urease solution was added, and the samples were incubated a t 37 ° for 30 minutes. At the end of the incuba- tion period 0.2 ml 2N ttC104 was added. The prec ip i ta te obtained was separa ted from the superna tan t by centr i fugat ion a t 2000 rpm for 15 min. the superna tan t being used for the analysis for citrulline. A s imi lar al iquot of serum was submit ted to exact ly the same procedure wi th the only difference tha t 0.1 ml buffer was added instead of the urease solution. The supe rna t an t obtained a f t e r prec ip i ta t ion was used for the analys is for urea plus citrulline. The values of the urea content were determined by subtract ion of ci trul l ine f rom the to ta l (urea plus ci trul l ine) value.

Urine. 0.1 ml urine was diluted to 10 ml with disti l led water . 0.1 ml urease or buffer was added to two ml of this solution which was then incubated a t 37 ° for 30 minutes. The samples could now be analyzed.

Standards. Urea or ci truil ine in a concentrat ion of 200 ~g/ml was used as s tandard. When the determinat ions were performed on serum, the s tandards contained 0.2 ml 2N HCI04 adjus ted to the same acid conditions as the sample. When determined on urine the s tandards also contained 200 ~g/ml of the substances mentioned. When urease digest ion was prac t i sed on the samples, 0.1 ml buffer was added to the s tand- ards instead.

Manual assay. 0.8 ml serum, deproteinized superna tan t or diluted urine was diluted to 2 ml wi th dist i l led wa te r and added to 0.34 ml of Ehrl ich 's reagent . The samples were immedia te ly read a t 440 nm in a Beckman speetrophotometer .

Automated assay. Analyses were per formed in an auto-analyzer (Technicon Ins t ru- ments Division, Chauncey, N.Y.). The amounts of sample and reagen t delivered through the correspondent tubing are shown in the f low d iag ram (Fig . 1). The procedure can be run a t a speed of 60 determinat ions per hour. The react ion was carr ied out as fol- lows. The sample was mixed with Ehrl ich 's r eagen t throughout the s t ream. The com- bined solution was mixed during the passage through a double delay coil. As there is no avai lable 400 nm interference f i l te r for the Technicon Auto-Analyzer , a 450 nm f i l t e r was used. The color produced was measured in the colorimeter a t tached to the machine. The eolor imeter is f i t ted with a 15 mm tubular flow cell. All tubing was clear. Tygon except those used fo r the intake of wa te r and Ehrl ich 's reagent , which were made of Acidflex. The fo rmer was Acidflex due to the need of del ivery of 1.19 ml /min , only found in tha t tubing. Absorbaneies were reg is te red in the recorder a t tached to the machine. A t the end of the run, the tubing used for the in take of Ehrl ich 's r eagen t was washed with n-propanol, while all the res t of the sys tem was washed with 1 N HC1. Al te rna te cups of sample and wa te r were placed in the sampler . The react ion was performed a t room tempera ture .

ASSAY FOR U R E A AND C I T R U L L I N E 295

SINGLE MIXING

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Ac

DOUBLE MIXING

COIL

DELIVERY ml/min

0.6 AIR

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ONATING PUMP

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0.34 COLOR REAGENT WATER

BUBBLE-WASTE

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c ~ RECORDER

COLORIMETER 450 nm

Fi~. 1. Flow diagram for automated urea ci trul l inea~say.

,i Ac: ACIDFLEX

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RESULTS

The chromogen obtained had a peak of maximum absorpt ion at 440 nm. Assays performed with urea and citrulline s tandards showed that the absorbances were proport ional to concentrations between 25 and 1000 [ g (Figs. 2 and 3). Under the acid conditions wl-:'~h (o!2~-.,ed the deprote~n:=r.- tion with H C 1 Q a slightly lower sensiti~i::y wa; ob'miu2:l (Figs. 2 and 3). In consequence, HCI04 should be adde2 io the s tandard in case the assay for urea a n d / o r citrulline is per formed o_: :r'_'::m. The resu!ts we.:e highly reproducible. A comparison of the results obtained wi 'h the a~.to-~.aatcd assay presented herein with those obtained ~q~:l the auto:uated p!'o:~dure reported in the Technicon auto-analyzer met:~odolcgy sheet for the de- terminat ion of urea ni t rogen 2, appears f rom Ta.Lh 1. Amm~_nia, creatini_nc, creatine, ornithine, arginine or histidine did not inoerfere with the reaction.

TABLE 1

SERUM AND URINARY LEVELS OF UREA.

Comparison of results with two automated procedme.~.

Assay as indicated Sample No. Urea Assay proposed by Technicon Lab.

Serum 60 mM/1 5.47 ± 0.60 5.36 4- 0.61 Urine 60 mM/24 h

volume 216.00 4- 30.00 212.00 4- 33.00

296 B L U M E N K R A N T Z and A S B O E - H A N S E N

1.0 Q9

O.8

QT

Standard in buffer plus H C l O 4 0.6

. . . . . . . . . . . . . Slandard in HzO

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-

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OI

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t t

25 i

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I iO0 IO00 200 400

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Fig. 2. Recording of the chromogen obtained with different concentrations of urea in water or in HCI04 with Ehrlich's reagent by the automated procedure.

DISCUSSION

In contrast to Borel et al's findingZ, under our experimental conditions, the peak of maximum absorption Of the chromogen deve]oped at 440 nm. An automated assay for the urea nitrogen has been described by the Technicon Laboratory. However, this method is more complicated than that described in this paper. It re~luires dialysis of the sample and a heat- ing step at 95 ° after mixing with the reagent diacetyl monoxime in the presence of thiosemicarbazide under strong acid conditions. Several stock and working solutions are necessary, and the flow diagram is more com- plicated than that presented here. The sensitivity and the specificity of the reaction are not indicated~.

The advantages of quickness, simplicity arzd high reproducibility render the automated and manual procedures presented above useful for clinical chemistry laboratories. The use of urease digestion on an aliquot sample ensures specificity. The possibility to differentiate urea from citrulline by

ASSAY F~OR UREA AND CITRULLINE 297

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0.8

07

Q6 Slondord in buffer plus HCIO 4

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. i i " 0.4 .. ." ," :

t.

~" o.2

o., !i /[

0 25 50 I00 200 400 5C)0 F'O C I T R U L L I N E

=

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Fig. 3. Recording of the chromogen obtained with different concentrations of citrul- line in water or in HCIOt with Ehrlich's reagent by the automated procedure.

the step of urease digestion made the method useful as a screening test for neonatal citrullinemia. The values obtained without urease digestion ex- press the content of urea plus citrulline, while those obtained after urease digestion correspond to citrulline only. The need of a quick diagnosis of citrullinemia to establish an early supplement of arginine to the diet 3, makes the use of this assay important as a screening test for the condition.

REFERENCES

1. Borel, J. P., Caranjot, J. M. and Jayle, M. F. Clin. Chim. Acta 16, 500-416, 1967. 2. Technicon Laboratory: Method file. Urea nitrogen and micro-urea nitrogen. 3. Danks, D. M., Tippett, P. and Zentner, G. Arch. Dis. Child, 49, 579-581, 1974.