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Technical Briefs |
Serv. de Bioquím., Hosp. Univ. Sant Joan de Déu, Passeig de Sant Joan de Dé 2, 08950-Esplugues (Barcelona), Spain
a author for correspondence: fax 34-3-2803626
Determination of plasma total homocysteine (tHcy; the sum of all forms of thiol derivatives that form homocysteine by reduction) (1) is essential for diagnosis and follow-up of homocystinuric patients (2) and for detection of moderate hyperhomocysteinemia, a risk factor for coronary, cerebral, and peripheral vascular disease (3). In childhood, moderate hyperhomocysteinemia may result from several genetic, nutritional (4), and iatrogenic factors (1), renal failure (5), HIV (6), and cancer (1). Plasma tHcy in children may be a useful biochemical marker for a genetic risk of premature atherosclerosis (1).
Various methods to measure tHcy have been described during the last decade, but HPLC with fluorescence detection is the most common (4)(7). Age- and sex-specific reference intervals for adults have been reported (4), considering even vitamin status (8), but data for children are sparse and inconsistent [9, 10], and no evidence is available in Mediterranean countries, where dietary vitamin content differs from that in Northern Europe or North America.
Our goal was to establish reference values for a pediatric population in our geographical area with our working conditions so that we could compare the values in further studies with those of children at risk for moderate hyperhomocysteinemia.
Specimens were collected from apparently healthy children (by history and analytical data) sent to our laboratory for analytical control before minor surgical interventions (n = 195; 112 males and 83 females, ages 2 months–18 years), in accordance with the Helsinki Declaration of 1975, as revised in 1983. We collected 1 mL of blood in Venoject (Terumo Corp., Leuven, Belgium) tubes containing EDTA, immediately cooled them on ice, and centrifuged them within 15 min (2000g for 10 min at 4 °C). The plasma was rapidly separated to limit release of homocysteine from blood cells, frozen at -40 °C, and analyzed within 14 days. Samples and calibrators were processed according to the method of Vester and Rasmussen (11), as modified by Spaapen et al. (12) and adapted to our working conditions. In short, we added 25 µL of 0.1 mmol/L ß-mercaptopropionylglycine (internal calibrator) to 100 µL of plasma or calibrator, and reduced the mixture with 25 µL of 150 mL/L tri-n-butylphosphine for 30 min at 4 °C. Deproteinization was performed with 100 µL of 10% trichloroacetic acid containing 1 mmol/L EDTA for 15 min at 0 °C. After centrifugation (2000g for 10 min at 4 °C), 50 µL of the supernatant was mixed with 100 µL of 2.5 mol/L borate buffer, pH 10.5, and 50 µL of 4.3 mmol/L ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate (SBD-F; Sigma Chemical Co., St. Louis, MO) in 2.5 mol/L borate buffer, pH 9.5. Derivatization was performed at 60 °C for 1 h. After cooling, samples were ready for HPLC analysis. We prepared 50, 20, and 10 µmol/L calibrators of homocysteine, cysteine (Sigma Chemical Co.), and cysteinylglycine (Bachem Feinchemikalien, Bubendorf, Switzerland) in MilliQ (Millipore, Bedford, MA) water.
Chromatographic conditions were as follows: Perkin-Elmer (Norwalk, CT) Integral 4000, Perkin-Elmer LC 240 fluorescence detector, Spherisorb C18 column (83 x 4.6 mm, 3-µm particle size), C18 precolumn. Eluting solvents were: A, 0.15 mol/L potassium phosphate buffer pH 1.75; B, 1000 mL/L acetonitrile. Gradient was 93% A (flow 1.5 mL/min) and 7% B, increased linearly to 20% B during the first 3 min (flow 2 mL/min), and from 3–5 min to 70% B (same flow); the mixture was maintained for 2 min (flow 1.5 mL/min) and returned to the start eluent composition in 4 min. Equilibrating time was 3 min. Fluorescence detection was excitation 385 nm, emission 515 nm. With these chromatographic conditions we measured cysteine, cysteinylglycine, homocysteine, glutathione, and mercaptopropionylglycine in 6 min, with a total chromatographic time of 14 min.
Within-run imprecision (CV; n = 18) was 3% (70.9 ± 2.2 µmol/L) and 4% (8.9 ± 0.3 µmol/L); between-day imprecision (n = 18), 6% (6.9 ± 0.4 µmol/L) and 6% (55.4 ± 3.1 µmol/L). Linearity was apparent between 1 and 150 µmol/L (r = 0.98). Recovery was 97.8 ± 6%.
Because total homocysteine values in our sample of pediatric patients
did not follow a normal distribution (assessed by the
Kolmogorov–Smirnov test), we calculated the median (6.3 µmol/L) and
the 2.5 and 97.5 percentiles (3.7–10.3). tHcy was independent of sex,
e.g., in teenagers 14–18 years old (n = 56), the medians (central
95% intervals) were 7.8 µmol/L (5.2–11.3) for boys (n = 24)
and 7.4 µmol/L (4.7–10.8) for girls (n = 32), respectively
(P = not significant, Mann–Whitney test). The tHcy
increased significantly with age (r = 0.5556; n =
195; P <0.001). After applying statistical analysis to all
age groups (Kruskal–Wallis), we established three age groups whose
tHcy concentrations were the most significantly different from one
another (Mann–Whitney; P <0.001): 2 month–10 years
(n = 105; median 5.8 µmol/L; interval 3.3–8.3), 11–15 years
(n = 59; median 6.6 µmol/L; interval 4.7–10.3), and 16–18
years (n = 31; median 8.1 µmol/L; interval 4.7–11.3) (Fig. 1
).
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Total homocysteine assay is used routinely (13), but
reference values differ among laboratories, reflecting premetrologic
and methodologic differences and, perhaps, variation in vitamin intake
(14). Our results also show differences between children
and adults (Fig. 1
). In addition, we found hyperhomocysteinemia in
three teenagers who smoked more than one pack per day (17–28
µmol/L), in agreement with the Hordaland study (15),
which concluded that tHcy is strongly influenced by life-style. With
this information, we documented subtle folate and cobalamin
deficiencies in children with anorexia nervosa (n = 40; median 9.4
µmol/L, range 6.2–30.1) (16). Moreover, our results
were the basis for the detection of heterozygotes for cystathionine
ß-synthase deficiency (n = 8; median 16 µmol/L,
range12.3–21.6), which had to be confirmed by genetic analysis, and
for further studies of the genetic basis of homocysteine-associated
vascular diseases.
We thank H.J. Blom (University Hospital Nijmegen) and L.J.M. Spaapen (Stichting Klinische Genetica Limburg, Maastricht) for their comments and technical advice.
References
The following articles in journals at HighWire Press have cited this article:
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