Development, validation, and application of a surrogate analyte method for determining N-acetyl-l-aspartyl-l-glutamic acid levels in rat brain, plasma, and cerebrospinal fluid.

A bioanalytical strategy for the simple and accurate determination of endogenous substances in a variety of biological matrices using liquid chromatography-tandem mass spectrometry is described. The robust method described here uses two stable isotope-labeled compounds as a surrogate analyte and an internal standard to construct calibration curves with authentic matrices that can be applied to determine N-acetyl-l-aspartyl-l-glutamic acid (NAAG) levels in rat brain, plasma, and cerebrospinal fluid (CSF) using a simple extraction and with a short analysis time of 4min. The validated lower limits of quantification were 1.00nmol/g for brain and 0.0100nmol/mL for plasma and CSF. Using this method, regional differences in NAAG levels in the brain as well as plasma and CSF levels that were much lower than those in the brain were successfully confirmed in treatment-naïve rats. Moreover, after the rats were treated with the intraventricular administration of a NAAG peptidase inhibitor, the NAAG levels increased rapidly and dramatically in the CSF and slightly in the plasma in a time-dependent manner, while the brain levels were not affected. Thus, the procedure described here was easily applied to the determination of NAAG in different matrices in the same manner as that used for xenobiotics, and this method would also be easily applicable to the accurate measurement of endogenous substances in a variety of biological matrices.

Importance of Peptide transporter 2 on the cerebrospinal fluid efflux kinetics of glycylsarcosine characterized by nonlinear mixed effects modeling.

PURPOSE: To develop a population pharmacokinetic model to quantitate the distribution kinetics of glycylsarcosine (GlySar), a substrate of peptide transporter 2 (PEPT2), in blood, CSF and kidney in wild-type and PEPT2 knockout mice. METHODS: A stepwise compartment modeling approach was performed to describe the concentration profiles of GlySar in blood, CSF, and kidney simultaneously using nonlinear mixed effects modeling (NONMEM). The final model was selected based on the likelihood ratio test and graphical goodness-of-fit. RESULTS: The profiles of GlySar in blood, CSF, and kidney were best described by a four-compartment model. The estimated systemic elimination clearance, volume of distribution in the central and peripheral compartments were 0.236 vs 0.449 ml/min, 3.79 vs 4.75 ml, and 5.75 vs 9.18 ml for wild-type versus knockout mice. Total CSF efflux clearance was 4.3 fold higher for wild-type compared to knockout mice. NONMEM parameter estimates indicated that 77% of CSF efflux clearance was mediated by PEPT2 and the remaining 23% was mediated by the diffusional and bulk clearances. CONCLUSIONS: Due to the availability of PEPT2 knockout mice, we were able to quantitatively determine the significance of PEPT2 in the efflux kinetics of GlySar at the blood-cerebrospinal fluid barrier.

N-acetylaspartylglutamate in CNS hypomyelination.

CSF N-acetylaspartylglutamate (NAAG) has been found to be elevated in some hypomyelinating disorders. This study addressed the question whether it could be used as a marker for hypomyelination and as a means to distinguish between hypomyelinating disorders biochemically. We have measured CSF NAAG in a cohort of 28 patients with hypomyelination with known and unknown aetiology. NAAG was found to be elevated in 7 patients, but was normal in the majority, including patients with defined hypomyelinating disorders. CSF NAAG is not a universal marker of hypomyelination, and the mechanism of its elevation remains poorly understood.

Distribution of glycylsarcosine and cefadroxil among cerebrospinal fluid, choroid plexus, and brain parenchyma after intracerebroventricular injection is markedly different between wild-type and Pept2 null mice.

The purpose of this study was to define the cerebrospinal fluid (CSF) clearance kinetics, choroid plexus uptake, and parenchymal penetration of PEPT2 substrates in different regions of the brain after intracerebroventricular administration. To accomplish these objectives, we performed biodistribution studies using [(14)C]glycylsarcosine (GlySar) and [(3)H]cefadroxil, along with quantitative autoradiography of [(14)C]GlySar, in wild-type and Pept2 null mice. We found that PEPT2 deletion markedly reduced the uptake of GlySar and cefadroxil in choroid plexuses at 60 mins by 94% and 82% (P<0.001), respectively, and lowered their CSF clearances by about fourfold. Autoradiography showed that GlySar concentrations in the lateral, third, and fourth ventricle choroid plexuses were higher in wild-type as compared with Pept2 null mice (P<0.01). Uptake of GlySar by the ependymal-subependymal layer and septal region was higher in wild-type than in null mice, but the half-distance of penetration into parenchyma was significantly less in wild-type mice. The latter is probably because of the clearance of GlySar from interstitial fluid by brain cells expressing PEPT2, which stops further penetration. These studies show that PEPT2 knockout can significantly modify the spatial distribution of GlySar and cefadroxil (and presumably other peptides/mimetics and peptide-like drugs) in brain.

Elevated CSF N-acetylaspartylglutamate suggests specific molecular diagnostic abnormalities in patients with white matter diseases.

BACKGROUND: In order to identify biomarkers useful for the diagnosis of genetic white matter disorders we compared the metabolic profile of patients with leukodystrophies with a hypomyelinating or a non-hypomyelinating MRI pattern. METHODS: We used a non-a priori method of in vitro ¹H-NMR spectroscopy on CSF samples of 74 patients with leukodystrophies. RESULTS: We found an elevation of CSF N-acetylaspartylglutamate (NAAG) in patients with Pelizaeus-Merzbacher disease (PMD)-PLP1 gene, Pelizaeus-Merzbacher-like disease-GJC2 gene and Canavan disease-ASPA gene. In the PMD group, NAAG was significantly elevated in the CSF of all patients with PLP1 duplication (19/19) but was strictly normal in 6 out of 7 patients with PLP1 point mutations. Additionally, we previously reported increased CSF NAAG in patients with SLC17A5 mutations. CONCLUSIONS: Elevated CSF NAAG is a biomarker that suggests specific molecular diagnostic abnormalities in patients with white matter diseases. Our findings also point to unique pathological functions of the overexpressed PLP in PMD patients with duplication of this gene.

Bioanalysis of N-acetyl-aspartyl-glutamate as a marker of glutamate carboxypeptidase II inhibition.

We report the characterization of two methods for the analysis of N-acetyl-aspartyl-glutamate (NAAG) in biological fluids. In the first method, NAAG concentrations were calculated based on differences between glutamate concentrations before and after NAAG hydrolysis with exogenous glutamate carboxypeptidase II (GCP II) using high-performance liquid chromatography (HPLC) followed by fluorescence detection. In the second method, NAAG levels were quantified directly using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Analyses of NAAG levels in human cerebrospinal fluid samples using either method gave similar results within experimental error, confirming the validity of the two independent measurements. These methods will be useful in future clinical trials to assess drug-induced GCP II inhibition in biological matrices.

Elevated CSF N-acetylaspartylglutamate in patients with free sialic acid storage diseases.

OBJECTIVE: To investigate body fluids of patients with undiagnosed leukodystrophies using in vitro (1)H-NMR spectroscopy (H-NMRS). METHODS: We conducted a cross-sectional study using high-resolution in vitro H-NMRS on CSF and urine samples. RESULTS: We found a significant increase of free sialic acid in CSF or urine in 6 of 41 patients presenting with hypomyelination of unknown etiology. Molecular genetic testing revealed pathogenic mutations in the SLC17A5 gene in all 6 patients. H-NMRS revealed an increase of N-acetylaspartylglutamate in the CSF of all patients with SLC17A5 mutation (range 13-114 micromol/L, reference <12 micromol/L). CONCLUSION: In patients with undiagnosed leukodystrophies, increased free sialic acid in CSF or urine is a marker for free sialic acid storage disorder and facilitates the identification of the underlying genetic defect. Because increase of N-acetylaspartylglutamate in CSF has been observed in other hypomyelinating disorders, it can be viewed as a marker of a subgroup of hypomyelinating disorders.

Increased level of N-acetylaspartylglutamate (NAAG) in the CSF of a patient with Pelizaeus-Merzbacher-like disease due to mutation in the GJA12 gene.

Autosomal recessive Pelizaeus-Merzbacher-like disease 1 (PMLD1) is a hypomyelinating disorder of the central nervous system (CNS) with virtually identical phenotype to Pelizaeus-Merzbacher disease (PMD). PMLD1 is caused by mutations in GJA12 gene, PMD is due to mutations in PLP1 gene. Elevated levels of N-acetylaspartylglutamate (NAAG), the most abundant peptide neuromodulator in the human brain, have been recently reported in cerebral spinal fluid (CSF) of patients with PMD. Using capillary electrophoresis, we analyzed for the first time, the CSF from a girl with PMLD1 and detected high concentrations of NAAG. This finding confirms the hypothesis that NAAG may be involved in myelination-related processes and can be considered as a useful diagnostic marker not only for patients with the PLP1 related disorder, but also in those with Pelizaeus-Merzbacher like hypomyelinating disease due to other defined genetic causes, such as PMLD1.

Free amino acid and dipeptide changes in the body fluids from Alzheimer's disease subjects.

Our aim was to determine changes in free amino acid (FAA) and dipeptide (DP) concentrations in probable Alzheimer's disease (pAD) subjects compared with control (CT) subjects using liquid chromatography and electrospray ionization tandem mass spectrometry (LCMS2). We recruited gender- and age-matched study participants based on neurological and neuropsychological assessments. We measured FAAs and DPs in cerebrospinal fluid (CSF), plasma and urine using LCMS2 with selected reaction monitoring (SRM). Imidazole-containing FAAs (histidine, methyl-histidine), catecholamines (L-DOPA and dopamine), citrulline, ornithine, glycine and antioxidant DPs (carnosine and anserine) accounted for the major changes between CT and pAD. Carnosine levels were significantly lower in pAD (328.4 +/- 91.31 nmol/dl) than in CT plasma (654.23 +/- 100.61 nmol/dl). In contrast, L-DOPA levels were higher in pAD (1400.84 +/- 253.68) than CT (513.10 +/- 121.61 nmol/dl) plasma. These data underscore the importance of FAA and DP metabolism in the pathogenesis of AD. Since our data show changes in antioxidants, neurotransmitters and their precursors, or FAA associated with urea metabolism in pAD compared with CT, we propose that manipulation of these metabolic pathways may be important in preventing AD progression.

Quantification of free amino acids and dipeptides using isotope dilution liquid chromatography and electrospray ionization tandem mass spectrometry.

Our aim was to develop a liquid chromatography and electrospray ionization tandem mass spectrometry (LCMS2) method to measure free amino acid (FAA) and dipeptide (DP) concentrations in biological fluids. We synthesized chloroformate derivatives of FAA and DP, identified the major precursor ions and used LCMS2 to obtain the most intense product ions. Using serial dilutions of unlabeled and labeled standards ([2H3]-L-Dopa, homoarginine, homophenylalanine, [15N]-Glutamine and [2H3]-methionine), we observed linear relationships in MS response that we used to calculate the amounts of FAA and DP in biological samples. This method is sensitive with a limit of detection (LOD) for most of the FAAs and DPs tested in the 0.05-1 pmol range and is linear over 3-5 orders of magnitude when many metabolites were measured simultaneously. Reproducibility and between run or daily variations were <10% for most FAAs and DPs. We applied this method to human samples and quantitatively measured 21 FAAs and 2 DPs in 200 microl CSF, 31 FAAs and 6 DPs in 100 microl plasma, and 23 FAAs and 5 DPs in 200 microl urine. These data demonstrate the potential for using LCMS2 to discover changes in FAA and DP metabolic pathways that occur during disease pathogenesis.

Severe hypomyelination associated with increased levels of N-acetylaspartylglutamate in CSF.

BACKGROUND: Two unrelated girls had early onset of nystagmus and epilepsy, absent psychomotor development, and almost complete absence of myelin on cerebral MRI. The clinical features and MR images of both patients resembled the connatal form of Pelizaeus-Merzbacher disease (PMD), which is an X-linked recessive disorder caused by duplications or mutations of the proteolipid protein gene (PLP). OBJECTIVE: To define a unique neurometabolic disorder with failure of myelination. METHOD: S AND RESULTS: 1H-NMR of CSF in both girls was performed repeatedly, and both showed highly elevated concentrations of N-acetylaspartylglutamate (NAAG). The coding sequence of the gene coding for glutamate carboxypeptidase II, which converts NAAG to N-acetylaspartate (NAA) and glutamate, was entirely sequenced but revealed no mutations. Even though both patients are girls, the authors sequenced the PLP gene and found no abnormality. CONCLUSIONS: NAAG is an abundant peptide neurotransmitter whose exact role is unclear. NAAG is implicated in two cases of unresolved severe CNS disorder. Its elevated concentration in CSF may be the biochemical hallmark for a novel neurometabolic disorder. The cause of its accumulation is still unclear.

The gamma-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester reduces A beta levels in vivo in plasma and cerebrospinal fluid in young (plaque-free) and aged (plaque-bearing) Tg2576 mice.

Acute, s.c. administration of a gamma-secretase inhibitor, N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT), to young PDAPP mice dose dependently decreases cortical amyloid-beta (A beta). The present studies replicated these findings in Tg2576 mice and examined further whether DAPT would reduce cerebrospinal fluid (CSF) A beta comparably in young (plaque-free) and aged (plaque-bearing) mice. In the first study, vehicle or DAPT (10, 30, or 100 mg/kg s.c.) administered to young Tg2576 mice (6 months old) dose dependently reduced A beta peptide levels in the cortex as seen previously in the PDAPP mice. Additionally, a dose-dependent decrease in plasma A beta levels was evident. The same dosing regime was applied next to aged mice (17 months old) to assess A beta changes in the CSF in addition to plasma and brains. DAPT dose dependently reduced A beta levels in the CSF and plasma, but not in the brain wherein A beta levels were 400 to 500 times higher than those in young mice, consistent with a large pool of A beta extracted from amyloid deposits. In subsequent studies, effects of oral DAPT (100 or 200 mg/kg) were examined concurrently in young and aged mice. DAPT reduced A beta levels in CSF and plasma to a similar extent at both ages. In contrast, DAPT reduced brain A beta levels primarily in young mice, with minimal effects in aged mice. These results demonstrate that A beta levels in CSF and plasma decrease dose dependently after gamma-secretase inhibition, and this response is not affected by amyloid plaque burden. We conclude that CSF and plasma A beta may offer a clinically applicable, mechanism-based biomarker for inhibitors of A beta production.

N(epsilon)-(gamma-L-glutamyl)-L-lysine (GGEL) is increased in cerebrospinal fluid of patients with Huntington's disease.

Pathological-length polyglutamine (Q(n)) expansions, such as those that occur in the huntingtin protein (htt) in Huntington's disease (HD), are excellent substrates for tissue transglutaminase in vitro, and transglutaminase activity is increased in post-mortem HD brain. However, direct evidence for the participation of tissue transglutaminase (or other transglutaminases) in HD patients in vivo is scarce. We now report that levels of N(epsilon)-(gamma-L-glutamyl)-L-lysine (GGEL)--a 'marker' isodipeptide produced by the transglutaminase reaction--are elevated in the CSF of HD patients (708 +/- 41 pmol/mL, SEM, n = 36) vs. control CSF (228 +/- 36, n = 27); p < 0.0001. These data support the hypothesis that transglutaminase activity is increased in HD brain in vivo.

N(epsilon)(gamma-glutamyl)lysine in cerebrospinal fluid marks Alzheimer type and vascular dementia.

N(epsilon)(gamma-glutamyl)lysine isodipeptide is released from the breakdown of proteins cross-linked by transglutaminase enzymes. Transglutaminase activation is a marker of apoptosis and elevated isodipeptide concentrations in body fluids might correlate with the intensity of apoptotic cell turnover. The concentration of N(epsilon)(gamma-glutamyl)lysine was measured in the cerebrospinal fluid (CSF) of patients with probable Alzheimer's disease (n = 14) and vascular type dementia (n = 11) and compared with not demented surgical controls (n = 17). Baseline levels of 26-62 nM/l (mean 37.9 +/- 8.7 SD) free isodipeptide were detected in control patients. CSF isodipeptide levels showed significant elevation in vascular (mean 95.6 +/- 45.1 SD) as well as Alzheimer patients (176.6 +/- 77.1 SD). Isodipeptide concentrations above 120 nM/l were 72% specific and 77% sensitive to Alzheimer's dementia, although the difference between the two dementias was statistically insignificant (p > 0.05). Determination of CSF N(epsilon)(gamma-glutamyl)lysine isodipeptide concentration offers a novel method for measurement of neurodegeneration in primary and mixed dementias.

N-acetylaspartic acid (NAA) and N-acetylaspartylglutamic acid (NAAG) in human ventricular, subarachnoid, and lumbar cerebrospinal fluid.

N-Acetylaspartic and N-acetylaspartylglutamic acid concentrations in human ventricular, subarachnoid and lumbar cerebrospinal fluid were measured by combined gas chromatography-mass spectrometry using selected ion monitoring with deuterated internal standards. N-Acetylaspartate concentrations were in the range 55, 9, and 1 microM, respectively; N-acetylaspartylglutamate concentrations in the same fluids were in the range 8, 3 and 4 microM, respectively. There did not appear to be any difference in lumbar fluid concentrations of either compound between control subjects, schizophrenic patients, Alzheimer's disease patients and a pooled group of patients with neurological degeneration. Ventricular concentrations of both compounds were greatly increased in deceased patients suggesting that maintenance of their intracellular concentrations is probably energy dependent. The concentrations of these compounds in lumbar cerebrospinal fluid from living, and ventricular cerebrospinal fluid from deceased subjects were weakly correlated with one another. In lumbar fluid neither compound appeared to be correlated with age. Analysis of serially collected lumbar samples from two subjects showed a weak concentration gradient for both compounds. Neither antipsychotic medication nor the acid transport inhibitor probenecid had any effect on lumbar concentrations of either compound. Attempts to use anion exchange high pressure liquid chromatography with UV detection for measurement of the low concentrations of N-acetylaspartate found in cerebrospinal fluid from living subjects were unsuccessful.

N-acetylaspartylglutamate in Canavan disease: an adverse effector?

UNLABELLED: We measured N-acetylaspartate and its precursor/product N-acetylaspartylglutamate (NAAG) in urine of patients with Canavan disease using capillary zone electrophoresis. Abnormal levels of NAAG were found in 32 of 43 patients examined. Elevated NAAG was also present in the CSF of one patient. Given that NAAG may interfere with N-methyl-D-aspartate receptor function, the occurrence of high levels of NAAG in patients' urine conceivably represents a participating factor in the pathogenesis of Canavan disease. CONCLUSION: The biochemical role of N-acetylaspartylglutamate and its relationship to glutamatergic function may be relevant to the pathogenesis of Canavan disease.

Markers of glutamatergic neurotransmission and oxidative stress associated with tardive dyskinesia.

OBJECTIVE: Tardive dyskinesia is a movement disorder affecting 20%-40% of patients treated chronically with neuroleptic drugs. The dopamine supersensitivity hypothesis cannot account for the time course of tardive dyskinesia or for the persistence of tardive dyskinesia and the associated structural changes after neuroleptics are discontinued. The authors hypothesized that neuroleptics enhance striatal glutamatergic neurotransmission by blocking presynaptic dopamine receptors, which causes neuronal damage as a consequence of oxidative stress. METHOD: CSF was obtained from 20 patients with schizophrenia, 11 of whom had tardive dyskinesia. Markers for oxidative stress, including superoxide dismutase, lipid hydroperoxide, and protein carbonyl groups, and markers for excitatory neurotransmission, including N-acetylaspartate, N-acetylaspartylglutamate, aspartate, and glutamate, were measured in the CSF specimens. Patients were also rated for tardive dyskinesia symptoms with the Abnormal Involuntary Movement Scale. RESULTS: Tardive dyskinesia patients had significantly higher concentrations of N-acetylaspartate, N-acetylaspartylglutamate, and aspartate in their CSF than patients without tardive dyskinesia when age and neuroleptic dose were controlled for. The significance of the higher levels of protein-oxidized products associated with tardive dyskinesia did not pass Bonferroni correction, however. Tardive dyskinesia symptoms correlated positively with markers of excitatory neurotransmission and protein carbonyl group and negatively with CSF superoxide dismutase activity. CONCLUSIONS: These findings suggest that there are elevated levels of oxidative stress and glutamatergic neurotransmission in tardive dyskinesia, both of which may be relevant to the pathophysiology of tardive dyskinesia.