Clinica Chimica Acta, 211 (1992) 175-179

175

0 1992 Elsevier Science Publishers B.V. All rights reserved. 0009-8981/92/$05.00 CCA 05417

Short Communication

A screening method for cystine and homocystine in urine Le Phuc Thuy and W.L. Nyhan Department of Pediatrics and Institute for Molecular Genetics, University of California. San Diego, L.u Jolla, CA 92093 (USA)

(Received 6 June 1992; revision received 13 August 1992; accepted 17 August 1992)

Key words: Cystine; Cystinuria; Homocystine; Homocystinuria

Introduction Cystinuria is the most common of the specific aminoacidurias. It is a heritable disorder of amino acid transport in which large amounts of cystine, arginine, lysine and ornithine are excreted in urine. Homocystinuria is an inborn error of metabolism in which a defective activity of cystathionine synthetase causes an increase in urinary excretion of homocystine. The most common screening method for the detection of cystine and homocystine in urine was described by Brand et al. in 1930 [l]. Compounds containing disulfide bonds were first reduced by sodium cyanide to sulfhydryl compounds. Cysteine and homocysteine then reacted with sodium nitroprusside to develop a red color in the presence of an alkali [ 1,2]. Other methods using dithiothreitol [3], hydrogen [4], silver nitrate [5] and sodium borohydride [6] as reducing agents have been described. The extreme toxicity of sodium cyanide and the instability of the color developed in alkali prompted us to explore other methods to detect sulfhydryl compounds in urine. A simple, sensitive and safe method was based on the reduction of disulfide compounds by nascent hydrogen gas produced by the reaction of HCl with zinc. Cysteine and homocysteine react with nitroprusside and form a stable magenta ring in contact with an alkaline drop of ammonium hydroxide in sucrose.

Correspondence to: Le Phuc Thuy, Department of Pediatrics, University of California, San Diego, La Jolla, CA 92093, USA.

Materials and Methods Materials

Granular zinc was purchased from Aldrich Chemical Co. (Wisconsin). DLCystine, dihydrochloride (#C5632), DL-cysteine, hydrochloride (#C9768), DLhomocystine (#H0501) and DL-homocysteine thiolactone, hydrochloride (#H4628) and sodium nitroprusside (SOSOl) were obtained from Sigma Chemical Co. (Missouri). L-Cystine (#816853) was purchased from Calbio Pharmaceuticals, California; DL(+)-meso-homocystine (#3846) from Biochemical Research (California); D-Cysteine, hydrochloride (#2420) and L-homocysteine thiolactone hydrochloride (#3838) from Calbiochem (California). Thiola was from Mission Pharmacal Company (Texas) and penicillamine from Pierce Chemicals (Illinois). Homocysteine-cysteine mixed disultide and homocysteine-penicillamine mixed disulfide were gifts from Dr. Jerry Schneider (University of California, San Diego). Control urine was obtained from CIBA-Coming Co. (California). Other reagents of analytical grade were purchased from commercial sources. Preparation of reagents

Granular zinc was washed several times with 6 N HCl, rinsed four times with water then stored under water to protect the zinc from oxidation by air. Standard solutions of cystine A (1.5 mM) and B (0.3 mM) were prepared by dissolving 469.80 pg and 93.96 rg DL-cystine, dihydrochloride per ml of water. Sodium nitroprusside (5% w/v, 50 mg in 1 ml of water) was prepared daily. One gram of sucrose was dissolved in 1 ml of 30-38% ammonium hydroxide. This viscous solution of alkali was stored in a small flask with a ground-glass stopper. Assay

A few grains of zinc were mixed with 1 ml of urine or standard solution and one drop (50 ~1) of 6 N HCl in a test tube. Bubbles of nascent hydrogen were observed immediately. The mixture was let stand for 30-60 min at room temperature. Treated urine (100 ~1) was transferred to a spot plate and then mixed with 20 ~1 of 5% nitroprusside. Ten microliters of ammonium hydroxide in sucrose was delivered carefully into the bottom of the well without disturbing the reaction mixture. The color of the ring, if any, was compared with negative controls (water and control urine) and positive controls (standard solutions A and B of cystine). Results The development of a bright magenta ring indicated the presence of sulthydryl compounds in the urine. A pale brown circle was observed with normal control urine, while no color ring was formed with the water control. Control A (1.5 mM cystine) gave a deep red ring and control B (0.3 mM cystine) presented a magenta ring. The color ring of control B was used as reference to be compared with color rings developed in other tests.

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Interpretation

Spot tests giving no color ring were rated ‘negative’. Color rings lighter than standard B were rated ‘trace’. When color rings showed the same intensity as standard B, the rating, namely ‘normal’, ‘trace’ and ‘elevated’ were taken from Table I which took into account the creatinine concentration in urine. All urines which gave positive results were submitted to quantitative amino acid analysis. Sensitivity

The lower limit of the sensitivity of the assay was 0.3 mM for D-cysteine, Lhomocysteine and homocysteine-penicillamine mixed disultide. The limits were 0.2 mM for L-cystine, DL-CyStin& DL-meso-homocystine and homocysteine-cysteine mixed disuhide. Specificity

The assay was specific for sulfhydryl groups. Before reduction, the test was negative in the presence of disullide compounds (cystine, homocystine and mixed disulfides). Without reduction, the test was also positive for ketones, such as acetone (> 0.3 mM) and acetoacetate (> 3 mM). However, after the reduction step, the assay was negative for ketones which were reduced to alcohols. The test was negative for creatinine, penicillamine (< 1 mM), Thiola (< 10 mM), ampicillin and 3hydroxybutyrate (< 100 mM). There was no difference between cysteine and homocysteine or when compounds of D, L or DL configurations were used. The pH of acidified urine had no effect on the test. A survey of 250 screening tests for cystinuria and homocystinuria have been conducted using both the modified Brand test [2] and this method for each urine specimen. The amino acid content of all samples of positive results and most of the samples of negative results were determined on the amino acid analyzer. The results of this survey are shown in Table II. Nine out of 10 false positives by the modified Brand test were due to the presence of ketones in urine. Our assay showed only one false ‘trace’ result. The modified Brand test failed to identify 2 homocystinuric and

TABLE

I

Use of creatinine Creatinine

concentrations

in the interpretation

of results

Cystine equivalentsa (mmoI/mol creatinine)

Interpretation

(mM) 1-4 4-8 >8

75-300 38-75

A screening method for cystine and homocystine in urine.

Clinica Chimica Acta, 211 (1992) 175-179 175 0 1992 Elsevier Science Publishers B.V. All rights reserved. 0009-8981/92/$05.00 CCA 05417 Short Commu...
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