Two mass-spectrometric techniques for quantifying serine enantiomers and glycine in cerebrospinal fluid: potential confounders and age-dependent ranges.
BACKGROUND
The recent discovery and specific functions of D-amino acids in humans are bound to lead to the revelation of D-amino acid abnormalities in human disorders. Therefore, high-throughput analysis techniques are warranted to determine D-amino acids in biological fluids in a routine laboratory setting.


METHODS
We developed 2 chromatographic techniques, a nonchiral derivatization with chiral (chirasil-L-val column) separation in a GC-MS system and a chiral derivatization with Marfey's reagent and LC- MS analysis. We validated the techniques for D-serine, L-serine, and glycine determination in cerebrospinal fluid (CSF), evaluated several confounders, and determined age-dependent human concentration ranges.


RESULTS
Quantification limits for D-serine, L-serine, and glycine in cerebrospinal fluid were 0.14, 0.44, and 0.14 micromol/L, respectively, for GC-MS and 0.20, 0.41, and 0.14 micromol/L for LC-MS. Within-run imprecision was <3% for both methods, and between-run imprecision was <13%. Comparison of both techniques with Deming regression yielded coefficients of 0.90 (D-serine), 0.92 (L-serine), and 0.96 (glycine). Sample collection, handling, and transport is uncomplicated-there is no rostrocaudal CSF gradient, no effect of storage at 4 degrees C for 1 week before storage at -80 degrees C, and no effect of up to 3 freeze/thaw cycles. Conversely, contamination with erythrocytes increased D-serine, L-serine, and glycine concentrations. CSF concentrations for 145 apparently healthy controls demonstrated markedly and specifically increased (5 to 9 times) D-serine concentrations during early central nervous system development.


CONCLUSIONS
These 2 clinically applicable analysis techniques will help to unravel pathophysiologic, diagnostic, and therapeutic issues for disorders associated with central nervous system abnormalities, NMDA-receptor dysfunction, and other pathology associated with D-amino acids.
Fuchs
Sabine A
SA
Department of Metabolic and Endocrine Diseases, University Medical Center Utrecht, Utrecht, The Netherlands. S.Fuchs@umcutrecht.nl
de Sain-van der Velden
Monique G M
MG
de Barse
Martina M J
MM
Roeleveld
Martin W
MW
Hendriks
Margriet
M
Dorland
Lambertus
L
Klomp
Leo W J
LW
Berger
Ruud
R
de Koning
Tom J
TJ
D000293:Adolescent; D000328:Adult; D002555:Cerebrospinal Fluid / Q000737:chemistry*; D002648:Child; D002675:Child, Preschool; D005998:Glycine / Q000737:chemistry; D006262:Health; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D013058:Mass Spectrometry / Q000379:methods*; D012694:Serine / Q000737:chemistry; D013237:Stereoisomerism
D012694:Serine; D005998:Glycine