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.

Cerebrospinal fluid D-serine and glycine concentrations are unaltered and unaffected by olanzapine therapy in male schizophrenic patients.

N-Methyl D-aspartate (NMDA)-receptor hypofunction has been implicated in the pathophysiology of schizophrenia and D-serine and glycine add-on therapy to antipsychotics has shown beneficial effects in schizophrenic patients. Nevertheless, previous studies have not shown consistently altered D-serine concentrations in cerebrospinal fluid (CSF) of schizophrenic patients. To confirm and extend these results, CSF concentrations of both endogenous NMDA-receptor co-agonists d-serine and glycine and their common precursor L-serine were analyzed simultaneously in 17 healthy controls and 19 schizophrenic patients before and 6 weeks after daily olanzapine (10 mg) treatment. CSF D-serine, L-serine and glycine concentrations and their relative ratios were similar between schizophrenic patients and controls and no differences were observed before and after olanzapine therapy. Thus, the NMDA-receptor hypofunction hypothesis in schizophrenia is not explained by olanzapine therapy-dependent absolute or relative decreases in CSF D-serine and glycine concentrations in this series of male patients, thereby not providing convenient markers for the disorder.

D-serine in the developing human central nervous system.

To elucidate the role of D-serine in human central nervous system, we analyzed D-serine, L-serine, and glycine concentrations in cerebrospinal fluid of healthy children and children with a defective L-serine biosynthesis (3-phosphoglycerate dehydrogenase deficiency). Healthy children showed high D-serine concentrations immediately after birth, both absolutely and relative to glycine and L-serine, declining to low values at infancy. D-Serine concentrations were almost undetectable in untreated 3-phosphoglycerate dehydrogenase-deficient patients. In one patient treated prenatally, D-serine concentration was nearly normal at birth and the clinical phenotype was normal. These observations suggest a pivotal role for D-serine in normal and aberrant human brain development.

Identification of three patients with a very mild form of Smith-Lemli-Opitz syndrome.

Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive malformation syndrome characterized by mental retardation, congenital anomalies, and growth deficiency. The syndrome is caused by a block in cholesterol biosynthesis at the level of 7-dehydrocholesterol reductase (7-DHCR), which results in elevated levels of the cholesterol precursor 7-dehydrocholesterol (7-DHC) and its isomer 8-dehydrocholesterol (8-DHC). We report on three patients from two families with a very mild clinical presentation of SLOS. Their plasma cholesterol values were normal and their plasma levels of 7- and 8- DHC were only slightly elevated. In cultured skin fibroblasts, a significant residual 7-DHCR activity was found. All three patients were compound heterozygotes for a novel mutation affecting translation initiation (M1L). Two of them had the common IVS8-1G>C null mutation and the third patient an E448K mutation in the 7-DHCR gene. Our findings emphasize the importance of using a sensitive method for measuring precursors of cholesterol in combination with mutation analysis to analyze patients with only minimal clinical SLOS-like signs.