NITROPRUSSIDE TEST PROCEDURE

Intended As A Screening Method for Cystinuria and Homocystinuria

SUMMARY AND EXPLANATION

Cystinuria and homocystinuria represent inherited disorders characterized by the excretion in urine of large amounts of amino acids (1). Cystinuria results from an impaired renal tubular reabsorption of cystine and other amino acids which leads to urinary calculi and, possibly, mental retardation. At least three different types of this disorder hav ebeen described. Cystinuria appears to occur as frequently as phenylketonuria (2). Reportedly, 1 in 400 newborn may be classified as "cystinuria at risk" (3). Many of these cystinurias are transient, but subsequent studies show a significant number to be heterozygous for the disorder. Homocystinuria involves a deficiency of cystathoinine synthase which appears autosomal recessive in nature. Besides mental retardation, this condition results in osteoporosis, ectopia lentis and vascular disease. Metabolic anomalies involving vitamin B12 utilization also increase homocystine excretion (1).

Knox (4) has described a procedure for the non-quantitative determination of cystine and homocystine in urine. This method used cyanide to reduce the disulfide linkage in these amino acids and nitroprusside to form colored complexes with the resulting free sulfhydryl groups. The method is not completely specific for cystine and homocystine since other disulfides or sulfhydryl groups may react. Therefore, the procedure is recommended for screening purposes only. Any positive result would suggest further confirmatory testing for diagnosis.

The EAGLE NITROPRUSSIDE TEST PROCEDURE employs a modification of the Knox method. The cyanide and nitroprusside reagents are stabilized in liquid form. Interferences from creatinine, acetone and acetoacetate have been minimized.

PRINCIPLE

Cyanide reduces the disulfide linkage in cystine and homocystine to the free sulfhydryl groups. Nitroprusside forms a colored complex with the free sulfhydryl groups which appears pink to purple. The color intensity depends on urinary concentration.

REAGENTS: FOR IN-VITRO DIAGNOSTIC USE

Reagent Set Cat. No. N-11 includes:

CYANIDE REAGENT - (Cat. No. N-11)

REACTIVE INGREDIENTS:

1.7 M sodium cyanide. Stabilizer present.

PRECAUTIONS:

CAUTION - Do not mix with acid. If ingested, perform gastric lavage and call a physician. Discard by flushing with large amounts of water.

STORAGE AND STABILITY:

Store at 2 - 8º C. Stable until expiration date if sealed tightly.

DETERIORATION:

The reagent should be a clear, colorless solution. Turbidity would indicate deterioration, and the reagent should not be used.

NITROPRUSSIDE SOLUTION - (Cat. No. N-12)

REACTIVE INGREDIENTS:

36 mM sodium nitroprusside. Stabilizer added.

PRECAUTIONS:

Causes irritation. Avoid contact with skin, eyes and clothing. In case of contact, wash with large amounts of water.

STORAGE AND STABILITY:

Store at 2 - 8º C. Stable until expiration date if sealed tightly.

DETERIORATION:

The solution should be clear and reddish in color. Turbidity or a yellow coloration would indicate deterioration, and the solution should not be used.

CYSTINE SOLUTION - (Cat. No. N-13)

REACTIVE INGREDIENTS:

50 mg/dL cystine. Stabilizer added.

PRECAUTIONS:

For in-vitro diagnostic use.

STORAGE AND STABILITY:

Store at 2 - 8º C. Stable until expiration date if sealed tightly.

DETERIORATION:

The solution should be a clear, colorless solution. Turbidity would indicate deterioration, and the solution should not be used.

SPECIMEN COLLECTION

PRECAUTIONS:

1. Freshly collected urine is recommended.

2. The specimen should not be contaminated with feces.

3. For screening newborns, specimens should be collected at 10 - 14 days after birth, and at 3 - 4 weeks of age.

SAMPLE STORAGE:

Refrigerate the specimen as soon as possible after collection. If testing is delayed longer than 24 hours after collection, freeze the sample.

ADDITIVES:

No special additives or preservatives are needed.

INTERFERING SUBSTANCES:

Creatinine gradually developes an orange color in this procedure. Even at high creatinine levels, however, there should be no difficulty in distinguishing the pink - purple color of cystine. Acetone and acetacetate at 50 mM concentration do not interfere. Acetaminophen, salicylates, phenobarbital, ampicillin and tetracycline are unreactive. Patients receiving methionine of N-acetylcysteine may show a positive result. Urines from individuals receiving ascorbic acid may cause a reduced sensitivity in the procedure.

PROCEDURE

MATERIALS PROVIDED:

CYANIDE REAGENT (Cat. No. N-11), NITROPRUSSIDE SOLUTION (Cat. No. N-12), and CYSTINE SOLUTION (Cat. No. N-13).

MATERIALS REQUIRED BUT NOT PROVIDED:

1. Spot plate

2. Dropper pipets

3. Applicator sticks

PREPARATION OF CYSTINE REFERENCE SAMPLE:

A Cystine Reference Sample should be prepared by adding 1 drop of the CYSTINE SOLUTION to 10 drops of a normal urine and mixing well. The cystine concentration is approximately 5 mg/dL in the Regerence Sample. It is recommended that a normal urine and the Reference Sample be included in each test series.

MANUAL PROCEDURE:

1. Add 2 drops of urine specimen tot he spot plate cavity.

2. Add 1 drop of CYANIDE REAGENT . Use an applicator stick to mix well.

3. Wait 10 minutes. Add 1 drop of NITROPRUSSIDE REAGENT.

4. Observe for color change.

INTERPRETATION OF RESULTS

An immediate pink to purple color upon the addition of NITROPRUSSIDE REAGENT would indicate the presence of cystine or homocystine. Color intensity depends on urinarty concentration. Normal urine has a slight pink color due to the NITROPRUSSIDE REAGENT. The test should be considered positive if the color intensity equals or exceeds that of the 5 mg/dL Cystine Reference Sample.

LIMITATIONS

The method is not completely specific for cystine and homocystine since other disulfides and free sulfhydryl groups may react. The NITROPRUSSIDE TEST PROCEDURE is intended as a screening method only. A positive result would indicate further amino acid testing for diagnosis. Such services are available through many reference laboratories (5). With low protein intake and high urine volume, this procedure may fail to detect homocysitnuria (1).