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Inadequate laboratory technique for amino acid analysis resulting in missed diagnoses of homocystinuria.

To the Editor:

The diagnosis of cystathionine [beta]-synthase (CBS) deficiency and disorders of methionine remethylation depend on the demonstration of homocyst(e)inemia and homocystinuria. To be detectable in the usual analysis of amino acids, homocystine must remain as a free disulfide in the plasma or serum. However, homocystine undergoes an exchange reaction with free sulfhydryls, including cysteine, readily forming covalent disulfide bonds with these thiols, and gradually disappears as a free amino acid (1). Because preparation for quantitative amino acid analysis usually includes deproteinization, protein-bound homocysteine is not detected by standard methods (2-4). Homocysteine-cysteine mixed disulfide, also increased in the homocystinurias, undergoes a similar exchange.

This phenomenon is still not appreciated by many laboratories that perform amino acid analyses, and lack of awareness of the phenomenon continues to result in missing the diagnosis of homocystinuria. This was brought to our attention by the case of an infant with CBS-deficient homocystinuria. Routine newborn screening had identified an increased concentration of blood methionine (5). Quantitative plasma amino acid analysis 1 week later disclosed isolated hypermethioninemia. Subsequent plasma and urine amino acid analysis in specimens promptly delivered to our laboratory, however, revealed the presence of homocystine and the mixed disulfide in addition to the increase in methionine. We have had similar experiences with two other patients (Levy HL, Korson MS, Smith KL, unpublished data). In each instance, misdiagnoses caused a delay in initiating treatment.

During a period of 11 months, 14 specimens were obtained from five patients with CBS-deficient homocystinuria as part of routine follow-up. Each specimen consisted of 5 ml of venous blood collected in sodium heparin anticoagulant. The specimens were kept on ice and, within 2-3 h of collection, the plasma was separated by centrifugation at 3000 rpm for 10 min. The plasma was then removed and divided into three aliquots (A, B, and C) representing three different methods for processing the specimen.

In aliquot A, proteins were precipitated from 0.5 ml of the aliquot by adding 0.5 ml of 40 g/L sulfosalicylic acid containing S-2-aminoethyl-Lcysteine as internal standard. The sample was centrifuged at 10,000 rpm at room temperature for 1 min. The clear supernatant was filtered through a 0.2 [micro]m Millipore filter and divided into two parts. One part, A-1, was immediately analyzed for amino acids on a Beckman 7300 amino acid analyzer (Beckman Instruments). The second part, A-2, was stored at -20[degrees]C for 7 days and then thawed and analyzed for amino acids. Aliquots B and C were stored at -20[degrees]C without prior deproteinization. After 24 h of storage for aliquot B and 7 days for aliquot C, 0.5 ml was deproteinized and analyzed for amino acids as described above. Plasma from aliquots B and C was also prepared as described by Andersson et al. (6) and analyzed for total homocysteine (tHcy) on the amino acid analyzer; tHcy was also measured in five A-1 samples.

The processing protocol of A-1 (i.e., immediate deproteinization and analysis) was considered optimal for a clinical laboratory. Consequently, recovery of the sulfur amino acids in each of the other preparations was compared with those of the A-1 aliquot. The percentage of recovery of the measured sulfur amino acids (Table 1) was calculated using this formula:

% recovery = (X/Y)(100)

X = amino acid concentration of aliquot A-2, B, or C

Y = amino acid concentration of aliquot A-1

Methionine was stable regardless of storage or deproteinization. However, homocystine and the mixed disulfide and, to a lesser extent, cystine, were unstable when stored without prior deproteinization. This was especially striking after storage for 7 days. Immediate deproteinization allowed almost full recovery of these three compounds. Total homocysteine was essentially unaffected by storage without prior deproteinization.

It is clear from our results and those of others (1, 2, 7, 8) that homocystine is unstable in plasma or serum, even when frozen at -20[degrees]C. Homocystine and the homocysteine-cysteine mixed disulfide may become undetectable after only 1 day of sample storage without deproteinization, which is likely the shortest duration of time required for samples to be transported to a laboratory for amino acid analysis. Thus, the diagnosis of a disorder in which the accumulation of homocyst(e)ine is the defining feature may be missed without special precautions to compensate for the instability of thiol compounds in blood.

One method for avoiding this mistake is to deproteinize the plasma or serum specimen before transport. In this study, rapid deproteinization preserved the disulfides as free analytes for at least 7 days in storage at -20[degrees]C. However, plasma tHcy measurement is an even more effective method for assuring accurate diagnosis of the homocystinurias. After a week of storage without deproteinization, we found that virtually all tHcy could be recovered by a method of preparation that includes a reducing agent such as dithiothreitol (9). This procedure accommodates specimens transported by mail or courier at room temperature, as well as those in which the homocystine disulfide might not be detectable for biologic reasons, without the danger of a missed diagnosis.

We gratefully acknowledge the assistance we received from the Department of Laboratory Medicine at Children's Hospital, Boston, and Dr. David E. Fahrney of the Department of Biochemistry and Molecular Biology at Colorado State University.


(1.) Sturman JA, Applegarth DA. Automated amino acid analysis. In: Boulton AA, Baker GB, Wood JD, eds. Neuromethods, Vol. 3, Amino acids. Clifton, NJ: Humana Press, 1985;1-27.

(2.) Perry TL, Hansen S. Technical pitfalls leading to errors in the quantitation of plasma amino acids. Clin Chim Acta 1969;25:53-8.

(3.) Schaefer A, Piquard F, Haberey P. Plasma amino-acids analysis: effects of delayed sample preparation and of storage. Clin Chim Acta 1987;164:163-9.

(4.) Fiskerstrand T, Refsum H, Kvalheim G, Ueland PM. Homocysteine and other thiols in plasma and urine: automated determination and sample stability. Clin Chem 1993;39:263-71.

(5.) Levy HL. Screening of the newborn. In: Taeusch HW, Ballard RA, Avery ME, eds. Diseases of the newborn, 6th ed. Philadelphia: Saunders, 1991;111-9.

(6.) Andersson A, Brattstrom L, Isaksson A, Israelsson B, Hultberg B. Determination of homocysteine in plasma by ion exchange chromatography. Scand J Clin Lab Invest 1989;49:445-9.

(7.) Carson NAJ, Cusworth DC, Dent CE, Field CMB, Neill DW, Westall RG. Homocystinuria: a new inborn error of metabolism associated with mental deficiency. Arch Dis Child 1963;38: 425-36.

(8.) Kang S-S, Wong PWK, Becken N. Protein-bound homocyst(e)ine in normal subjects and in patients with homocystinuria. Pediatr Res 1979; 13:1141-3.

(9.) Brattstrom LE, Israelsson B, Jeppsson JO, Hultberg BL. Folic acid-an innocuous means to reduce plasma homocysteine. Scand J Clin Lab Invest 1988;48:215-21.

Kameo L. Smith [1]

Loetta Bradley [1]

Harvey L. Levy [2, 3]

Mark S. Korson [2, 3] *

[1] Dept. of Lab. Med.

[2] Div. of Genet.

Children's Hosp.

300 Longwood Ave.

Boston, MA 02115

[3] Dept. of Pediatr.

Harvard Med. School

Boston, MA 02115

* Author for correspondence. Fax 617-277-5933; e-mail
Table 1. Average percentage of recovery of sulfur amino acids in
aliquots relative to recovery in aliquot A-1 (optimal).

Aliquot Methionine Free Homoc (a) Free Cys

A-1 100 100 100
A-2 99 [+ or -] 2 99 [+ or -] 2 99 [+ or -] 8
B 99 [+ or -] 2 55 [+ or -] 24 85 [+ or -] 14
C 98 [+ or -] 2 8 [+ or -] 10 33 [+ or -] 17

Aliquot mixed disulfide tHcy

A-1 100 100
A-2 99 [+ or -] 1
B 60 [+ or -] 20 98 [+ or -] 3
C 7 [+ or -] 9 98 [+ or -] 3

Values are means [+ or -] 1 SD.

(a) Free Homoc, free homocystine disulfide; Free Cys, free cystine
disulfide; Homoc-Cys, homocysteine-cysteine.
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Title Annotation:Letters
Author:Smith, Kameo L.; Bradley, Loetta; Levy, Harvey L.; Korson, Mark S.
Publication:Clinical Chemistry
Article Type:Letter to the editor
Date:Apr 1, 1998
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