Cerebrospinal Fluid Glycine Research Articles

IMPROVING COGNITIVE FUNCTION IN RODENTS VIA INCREASING GLYCINE LEVELS IN BRAIN BY THE NOVEL GLYCINE TRANSPORTER-1 INHIBITOR BI 425809

Dysfunction of the glutamatergic system related to the NMDA receptor may play an important role in cognitive impairment in Alzheimer’s disease. One approach to counteract this is the extracellular increase of the NMDA receptor co-agonist glycine by glycine transporter-1 (GlyT1) inhibition. Thus, inhibiting GlyT1 has potential for treating memory impairment in Alzheimer’s disease via strengthening glutamatergic neurotransmission. Previous studies with GlyT1-inhibitors show memory-enhancing effects in animals. This study characterizes the potency and selectivity of the novel GlyT1 inhibitor BI 425809 and its effects on glycine increase in rat cerebrospinal fluid (CSF). BI 425809 was also evaluated in two rodent cognition tasks assessing episodic memory and working memory. The molecular potency of BI 425809 for GlyT1 was determined by inhibition of [3H]-glycine uptake in human SK-N-MC cells and rat primary cortical neurons. Selectivity was tested against GlyT2 and other off-targets by uptake and receptor binding assays, respectively. Concentrations of glycine in rat CSF samples collected from cisterna magna following oral administration of BI 425809 were determined by HPLC-MS/MS technique. BI 425809 was tested after oral application in the mouse T-maze spontaneous alternation test and in the social recognition test in naive rats. The IC50 value of BI 425809 on GlyT1 was 4.0 nM in SK-N-MC cells and 4.4 nM in rat primary neurons. BI 425809 demonstrated no relevant activity against GlyT2 (IC50 >10 μM) and 103 off-targets at 10 μM. A dose-dependent increase of glycine in rat CSF was observed. BI 425809 reversed MK-801-induced memory deficits in the mouse T-maze (working memory) and improved memory performance in the rat social recognition task (episodic memory). BI 425809 is a potent and selective GlyT1 inhibitor. Systemic administration of BI 425809 led to an increase in glycine levels in rat CSF demonstrating functional target engagement – i.e. GlyT1 inhibition in the brain – showing that glycine levels in CSF can be used to assess GlyT1 inhibition centrally. This might also be used to evaluate central target engagement in clinical trials. BI 425809 showed memory enhancing effects in animal cognition tests. GlyT1 inhibition may be a potential approach to pharmacologically improve cognition in Alzheimer’s disease.

Effects of the glycine reuptake inhibitors bitopertin and RG7118 on glycine in cerebrospinal fluid: results of two proofs of mechanism studies in healthy volunteers.

Hypofunction of NMDA receptors has been implicated in neuropsychiatric disorders including schizophrenia. NMDA receptor neurotransmission can be enhanced through inhibition of glycine reuptake by the glycine transporter type 1 (GlyT1). The primary objective of these studies was to explore the relationship between plasma exposure and glycine cerebrospinal fluid (CSF) concentrations following administration of bitopertin and RG7118 in healthy volunteers. The bitopertin study comprised four dose levels (3, 10, 30 and 60mg) administered once daily for 10days. In the RG7118 study, placebo, 15 or 30mg RG7118 was administered once daily for 28days. CSF samples were taken on day -2 and day 10, and day -1 and day 26 for bitopertin and RG7118, respectively. Twenty-two and 24 subjects participated in the bitopertin and RG7118 study, respectively. In the bitopertin study, CSF glycine concentrations showed a dose-dependent increase from baseline to day 10. The geometric mean ratios (coefficient of variation) of AUC0-12 h on day 10 over baseline were 1.3 (17%), 1.3 (49%), 1.7 (18%) and 2.3 (14%) after 3, 10, 30 and 60mg, respectively. In the RG7118 study, the geometric mean ratio of glycine concentration (CV) on day 26 at 6h post-dose over time-matched baseline was approx. 1.9 (24 and 15%) for 15 and 30mg. The mechanism of action of bitopertin and RG7118, i.e. inhibition of glycine reuptake in the brain, was confirmed. The maximal increase observed in healthy volunteers was similar to the one observed in animals showing the good translatability of this biomarker.

Bioanalytical method development and validation for the determination of glycine in human cerebrospinal fluid by ion-pair reversed-phase liquid chromatography–tandem mass spectrometry

A LC–MS/MS method has been developed and validated for the determination of glycine in human cerebrospinal fluid (CSF). The validated method used artificial cerebrospinal fluid as a surrogate matrix for calibration standards. The calibration curve range for the assay was 100–10,000ng/mL and 13C2, 15N-glycine was used as an internal standard (IS). Pre-validation experiments were performed to demonstrate parallelism with surrogate matrix and standard addition methods. The mean endogenous glycine concentration in a pooled human CSF determined on three days by using artificial CSF as a surrogate matrix and the method of standard addition was found to be 748±30.6 and 768±18.1ng/mL, respectively. A percentage difference of −2.6% indicated that artificial CSF could be used as a surrogate calibration matrix for the determination of glycine in human CSF. Quality control (QC) samples, except the lower limit of quantitation (LLOQ) QC and low QC samples, were prepared by spiking glycine into aliquots of pooled human CSF sample. The low QC sample was prepared from a separate pooled human CSF sample containing low endogenous glycine concentrations, while the LLOQ QC sample was prepared in artificial CSF. Standard addition was used extensively to evaluate matrix effects during validation. The validated method was used to determine the endogenous glycine concentrations in human CSF samples. Incurred sample reanalysis demonstrated reproducibility of the method.

Biochemical and molecular predictors for prognosis in nonketotic hyperglycinemia.

ObjectiveNonketotic hyperglycinemia is a neurometabolic disorder characterized by intellectual disability, seizures, and spasticity. Patients with attenuated nonketotic hyperglycinemia make variable developmental progress. Predictive factors have not been systematically assessed.MethodsWe reviewed 124 patients stratified by developmental outcome for biochemical and molecular predictive factors. Missense mutations were expressed to quantify residual activity using a new assay.ResultsPatients with severe nonketotic hyperglycinemia required multiple anticonvulsants, whereas patients with developmental quotient (DQ) > 30 did not require anticonvulsants. Brain malformations occurred mainly in patients with severe nonketotic hyperglycinemia (71%) but rarely in patients with attenuated nonketotic hyperglycinemia (7.5%). Neonatal presentation did not correlate with outcome, but age at onset ≥ 4 months was associated with attenuated nonketotic hyperglycinemia. Cerebrospinal fluid (CSF) glycine levels and CSF:plasma glycine ratio correlated inversely with DQ; CSF glycine > 230 μM indicated severe outcome and CSF:plasma glycine ratio ≤ 0.08 predicted attenuated outcome. The glycine index correlated strongly with outcome. Molecular analysis identified 99% of mutant alleles, including 96 novel mutations. Mutations near the active cleft of the P‐protein maintained stable protein levels. Presence of 1 mutation with residual activity was necessary but not sufficient for attenuated outcome; 2 such mutations conferred best outcome. Divergent outcomes for the same genotype indicate a contribution of other genetic or nongenetic factors.InterpretationAccurate prediction of outcome is possible in most patients. A combination of 4 factors available neonatally predicted 78% of severe and 49% of attenuated patients, and a score based on mutation severity predicted outcome with 70% sensitivity and 97% specificity. Ann Neurol 2015;78:606–618

Pre-therapy and post-therapy MRI and MRS findings in non-ketotic hyperglycinemia

Non-ketotic hyperglycinemia (NKH) is an uncommon metabolic disorder with an autosomal recessive inheritance due to abnormal glycine metabolism. We present the pre-therapy and post-therapy magnetic resonance imaging, biochemical and clinical features of a child with NKH with manifestation in the early neonatal period. The initial diagnosis of NKH was made on the biochemical evidence of elevated cerebrospinal fluid (CSF) glycine levels and CSF: plasma glycine ratio. Pre-therapy magnetic resonance imaging revealed thinned out corpus callosum, intracranial hemorrhages, prominent ventricular system, few cystic changes in bilateral gangliocapsular region and abnormal T2 hyperintensities in the white matter. Magnetic resonance spectroscopy did not reveal glycine peak in our case. Post-therapy, there was clinical improvement and reduction in CSF glycine levels. In the follow up scan, myelination had progressed with residual leukomalacic changes and volume loss of white matter.

Variant non ketotic hyperglycinemia is caused by mutations in LIAS, BOLA3 and the novel gene GLRX5

Patients with nonketotic hyperglycinemia and deficient glycine cleavage enzyme activity, but without mutations in AMT, GLDC or GCSH, the genes encoding its constituent proteins, constitute a clinical group which we call 'variant nonketotic hyperglycinemia'. We hypothesize that in some patients the aetiology involves genetic mutations that result in a deficiency of the cofactor lipoate, and sequenced genes involved in lipoate synthesis and iron-sulphur cluster biogenesis. Of 11 individuals identified with variant nonketotic hyperglycinemia, we were able to determine the genetic aetiology in eight patients and delineate the clinical and biochemical phenotypes. Mutations were identified in the genes for lipoate synthase (LIAS), BolA type 3 (BOLA3), and a novel gene glutaredoxin 5 (GLRX5). Patients with GLRX5-associated variant nonketotic hyperglycinemia had normal development with childhood-onset spastic paraplegia, spinal lesion, and optic atrophy. Clinical features of BOLA3-associated variant nonketotic hyperglycinemia include severe neurodegeneration after a period of normal development. Additional features include leukodystrophy, cardiomyopathy and optic atrophy. Patients with lipoate synthase-deficient variant nonketotic hyperglycinemia varied in severity from mild static encephalopathy to Leigh disease and cortical involvement. All patients had high serum and borderline elevated cerebrospinal fluid glycine and cerebrospinal fluid:plasma glycine ratio, and deficient glycine cleavage enzyme activity. They had low pyruvate dehydrogenase enzyme activity but most did not have lactic acidosis. Patients were deficient in lipoylation of mitochondrial proteins. There were minimal and inconsistent changes in cellular iron handling, and respiratory chain activity was unaffected. Identified mutations were phylogenetically conserved, and transfection with native genes corrected the biochemical deficiency proving pathogenicity. Treatments of cells with lipoate and with mitochondrially-targeted lipoate were unsuccessful at correcting the deficiency. The recognition of variant nonketotic hyperglycinemia is important for physicians evaluating patients with abnormalities in glycine as this will affect the genetic causation and genetic counselling, and provide prognostic information on the expected phenotypic course.

C.04.03 Relevance of study design for the effectiveness of long-acting injectable antipsychotics in schizophrenia

Serine and other amino acids that function as coagonists at the N-methyl-d-aspartate receptor (NMDAR) have been extensively investigated in schizophrenia (SCZ). However, studies comparing amino acid levels in body fluids of SCZ patients with healthy controls have yielded inconsistent results. We therefore conducted a meta-analysis (search: May 9, 2013) of serine, l-serine, d-serine, glycine, alanine, proline, and aspartate levels in blood and cerebrospinal fluid (CSF) obtained from adult SCZ patients and healthy controls. Standardized differences of means (SDMs) were computed, and heterogeneity, subgroup, sensitivity, and publication bias analyses were conducted. Blood serine levels were found to be significantly higher in SCZ patients compared to healthy controls (SDM = 0.280 (0.021–0.540), p = 0.034; N = 1671 subjects), whereas CSF serine, l-serine, d-serine, glycine, alanine, proline, and aspartate levels did not differ. Stratification by sex suggested that the case–control difference in blood serine levels particularly applies to male subjects. These results provide support for blood serine level aberrations in SCZ patients and warrant further research to disentangle the involvement of serine with SCZ in both sexes.

A novel liquid chromatography/tandem mass spectrometry method for the quantification of glycine as biomarker in brain microdialysis and cerebrospinal fluid samples within 5min

Glycine is an important amino acid neurotransmitter in the central nervous system (CNS) and a useful biomarker to indicate biological activity of drugs such as glycine reuptake inhibitors (GRI) in the brain. Here, we report how a liquid chromatography/tandem mass spectrometry (LC-MS/MS) method for the fast and reliable analysis of glycine in brain microdialysates and cerebrospinal fluid (CSF) samples has been established. Additionally, we compare this method with the conventional approach of high performance liquid chromatography (HPLC) coupled to fluorescence detection (FD). The present LC-MS/MS method did not require any derivatisation step. Fifteen microliters of sample were injected for analysis. Glycine was detected by a triple quadrupole mass spectrometer in the positive electrospray ionisation (ESI) mode. The total running time was 5min. The limit of quantitation (LOQ) was determined as 100nM, while linearity was given in the range from 100nM to 100μM. In order to demonstrate the feasibility of the LC-MS/MS method, we measured glycine levels in striatal in vivo microdialysates and CSF of rats after administration of the commercially available glycine transporter 1 (GlyT1) inhibitor LY 2365109 (10mg/kg, p.o.). LY 2365109 produced 2-fold and 3-fold elevated glycine concentrations from 1.52μM to 3.6μM in striatal microdialysates and from 10.38μM to 36μM in CSF, respectively. In conclusion, we established a fast and reliable LC-MS/MS method, which can be used for the quantification of glycine in brain microdialysis and CSF samples in biomarker studies.

Effect of Buyang Huanwu decoction on amino acid content in cerebrospinal fluid of rats during ischemic/reperfusion injury

The inhibitory effect of Buyang Huanwu decoction (BYHWD) on ischemic injury has been proven, but it is not clear how amino acid levels in cerebrospinal fluid (CSF) are associated with BYHWD treatment, nor the mechanism by which BYHWD protects the brain from ischemia/reperfusion injury. We investigated the effect of BYHWD on the amino acid content of CSF in rats during ischemia-reperfusion injury. Ischemia was imposed by right middle cerebral artery occlusion (MCAO). CSF was continuously collected from the striatum via brain microdialysis before and after ischemia/reperfusion. We used on-line derivatization combined with high-performance liquid chromatography with fluorescence detection (HPLC-FD) to determine levels of glutamate (Glu), aspartate (Asp), glycine (Gly), taurine (Tau), and γ-aminobutyric acid (GABA) in CSF. The MCAO model displayed an infarct lesion in the ipsilateral hemisphere and nerve injuries, as the left upper limb was unable to extend and turn leftward. Significant increases in excitatory and inhibitory amino acids were observed in the CSF of the ischemic rats relative to the sham-operated group (P<0.01). Treatment with BYHWD reduced the areas of cerebral infarction and improved the neurological behavior scores of rats after MCAO. BYHWD treatment was also associated with a significant decrease in excitatory amino acids and increase in inhibitory amino acids in the CSF. Only the higher dose of BYHWD (20mg/kg) affected all these levels significantly. Attenuated excitatory toxicity and reduced areas of cerebral infarction associated with BYHWD treatment might be due to a protective mechanism induced by BYHWD against ischemia/reperfusion injury.

CSF and Serum Biomarkers Focusing on Cerebral Vasospasm and Ischemia after Subarachnoid Hemorrhage

Delayed cerebral vasospasm (CVS) and delayed cerebral ischemia (DCI) remain severe complications after subarachnoid hemorrhage (SAH). Although focal changes in cerebral metabolism indicating ischemia are detectable by microdialysis, routinely used biomarkers are missing. We therefore sought to evaluate a panel of possible global markers in serum and cerebrospinal fluid (CSF) of patients after SAH. CSF and serum of SAH patients were analyzed retrospectively. In CSF, levels of inhibitory, excitatory, and structural amino acids were detected by high-performance liquid chromatography (HPLC). In serum, neuron-specific enolase (NSE) and S100B level were measured and examined in conjunction with CVS and DCI. CVS was detected by arteriography, and ischemic lesions were assessed by computed tomography (CT) scans. All CSF amino acids were altered after SAH. CSF glutamate, glutamine, glycine, and histidine were significantly correlated with arteriographic CVS. CSF glutamate and serum S100B were significantly correlated with ischemic events after SAH; however, NSE did not correlate neither with ischemia nor with vasospasm. Glutamate, glutamine, glycine, and histidine might be used in CSF as markers for CVS. Glutamate also indicates ischemia. Serum S100B, but not NSE, is a suitable marker for ischemia. These results need to be validated in larger prospective cohorts.

Neonatal Nonketotic Hyperglycinemia: A Case Study and Review of Management for the Advanced Practice Nurse

Nonketotic hyperglycinemia (NKH) is an autosomal recessive inborn error of glycine metabolism. In this article, I will present the case of baby girl S. who presented to the emergency room on Day 4 of life with severe lethargy, seizures, and respiratory depression requiring mechanical ventilation. A diagnosis of NKH was made secondary to elevated plasma and cerebrospinal fluid glycine concentrations. I will review the pathophysiology of NKH, methods of diagnosis, and the differential diagnosis. There are a variety of different pharmacologic and alternative therapies for NKH. Despite these treatments, the prognosis for infants with NKH is poor, with severe neurologic impairment, intractable seizures, and death common before 5 years of age. I will address the role of the advanced practice nurse in caring for an infant with NKH including clinical, educational, and research implications.

A rare inborn error of metabolism associated with a Dandy–Walker malformation

A baby girl, born to second-degree consanguineously married couple, was delivered by forceps, after a full-term pregnancy. Apgar scores were 9 and 10, birth weight 4,100 g, and head circumference 35 cm. The baby was hospitalized, at the age of 20 days, for recurrent myoclonic seizures. Physical examination found hypotonia and absent reflexes. Amino acid analysis of the cerebrospinal fluid (CSF) and plasma was performed, to exclude the possibility of metabolic disorder. A brain ultrasonography found hypoplastic corpus callosum. An expansion of the cisterna magna communicating with the fourth ventricle and a cerebellar vermis hypoplasia were also showed. These findings, suggested a Dandy–Walker malformation. 1 day later, brain magnetic resonance (MR) imaging and spectroscopy were performed. MRI confirmed the brain ultrasonography findings (Fig. 1). On the T1and T2-weighted images, the appearance of the white matter myelination was inappropriate for age. The axial T2-weighted images revealed increased signal intensity in the dorsal brain stem, the middle cerebellar peduncles, and the posterior limbs of the internal capsules. Diffusion-weighted MR images found hyperintense signal intensity in these areas, in combination with low apparent diffusion coefficient (ADC) values, consistent with restricted diffusion (Fig. 2). Localized proton MR spectroscopy showed a markedly increased peak intensity at 3.55 ppm, which was assigned to glycine and a lactate peak at 1.33 ppm (Fig. 3). The diagnosis of nonketotic hyperglycinemia (NKH) was made by laboratory findings of elevated CSF and plasma glycine levels, which were 250 lmol/L (normal range 1–15 lmol/L) and 1,540 lmol/L (normal range 230–740 lmol/L), respectively. The CSF-to-plasma glycine ratio was also elevated to 0.16 (normal range 0.02–0.03). 4 days later, the baby became lethargic, and the shallow respiration which began at that point progressed to intermittent apnea, requiring mechanical ventilation. 1 day later the infant died.

An interesting case of antenatal hiccups – a clue aiding a prompt diagnosis of non-ketotic hyperglycinaemia

A term baby returned for a routine neonatal discharge check on her second day of life. On examination was found to be apnoeic and hypotonic. She then had a generalised seizure, terminated by phenobarbitone. Over the course of the next 24 hours she was invasively ventilated having become completely apnoeic. She received treatment for possible sepsis including anti viral treatment. The only positive results yielded from blood testing and imaging were small ventricles on cranial ultrasound and a raised gamma glutamyl transferase. It was noted that the patient was hiccupping and on revisiting the antenatal history her mother commented she had thought her daughter had hiccupped consistently (multiple times every day) from twenty weeks gestation. With this history and her clinical presentation a neurological opinion was sought. Many plasma, urine and cerebrospinal fluid (CSF) investigations were requested. The paired plasma and CSF glycine levels were in a ratio of 0.4 (normal up to 0.06). This confirmed the clinical suspicion of non-ketotic hyperglycinaemia. This baby was referred to a tertiary intensive care unit, for neurological opinion and withdrawal of life support. This case illustrates the importance of a thorough antenatal history when reviewing babies ready for discharge as well as the danger of babies being discharged home prior to being reviewing a formal neonatal check. With the postnatal wards needing beds there is significant pressure to review the babies within the first day of life, many are being discharged before paediatric review. The case also revealed the importance of beginning discussions of possible outcomes including withdrawal of life support and palliation, at a local level before transfer to another centre. The outcome for this baby would not have been different. However, there are babies who potentially have serious pathology that would require prompt investigation, diagnosis and life saving treatment. The practice of babies being discharged before neonatal discharge check needs review. Trainees are often the doctors best positioned to begin breaking bad news but sometimes we can be afraid we are doing it inadequately and shy away from doing so.

Nonketotic Hyperglycinemia

Nonketotic hyperglycinemia is a rare metabolic disorder with severe, frequently fatal, neurologic manifestations. Reliable and accurate diagnosis depends on careful interpretation of laboratory findings. The clinical suspicion should lead to determination of glycine in plasma and cerebrospinal fluid. Amino acid analysis presents diagnostic values for classic nonketotic hyperglycinemia, but it also should be performed in suspected cases of atypical nonketotic hyperglycinemia and in children with seizures, failure to thrive, behavior problems, and uncoordinated movements. Clinical assessment should be reinforced by demonstration of elevated cerebrospinal fluid-to-plasma glycine ratio. Confirmatory diagnosis requires enzymatic and genetic investigation of glycine cleavage system. An early diagnosis, though not affecting clinical outcome, allows proper genetic counseling, with the possibility of prenatal diagnosis. We report 3 cases of nonketotic hyperglycinemia, 2 typical neonatal and 1 atypical, diagnosed in Pediatric Hospital of Coimbra, Portugal, and investigated at Laboratory of Biochemical Genetics in 2004 to 2010 (incidence 1:47 455; prevalence 1:782 951).

New Therapeutic Strategy for Amino Acid Medicine: Glycine Improves the Quality of Sleep

Glycine is a non-essential amino acid that has indispensable roles in both excitatory and inhibitory neurotransmission via N-methyl-D-aspartate type glutamate receptors and glycine receptors, respectively. We recently reported that glycine ingestion before bedtime significantly ameliorated subjective sleep quality in individuals with insomniac tendencies. Oral administration of glycine to rats was found to induce a significant increase in the plasma and cerebrospinal fluid glycine concentrations and a significant decrease in the core body temperature associated with an increase in cutaneous blood flow. The decline in the core body temperature might be a mechanism underlying glycine’s effect on sleep, as the onset of sleep is known to involve a decrease in the core body temperature. Moreover, a low core body temperature is maintained during sleep in humans. Pharmacological studies investigating the mechanisms of glycine on sleep were also performed. In this review, we will describe both our recent findings regarding how and where orally administered glycine acts and findings from our rat study and human trials.

Chronically saturating levels of endogenous glycine disrupt glutamatergic neurotransmission and enhance synaptogenesis in the CA1 region of mouse hippocampus

Glycine serves a dual role in neurotransmission. It is the primary inhibitory neurotransmitter in the spinal cord and brain stem and is also an obligatory coagonist at the excitatory glutamate, N-methyl-D-aspartate receptor (NMDAR). Therefore, the postsynaptic action of glycine should be strongly regulated to maintain a balance between its inhibitory and excitatory inputs. The glycine concentration at the synapse is tightly regulated by two types of glycine transporters, GlyT1 and GlyT2, located on nerve terminals or astrocytes. Genetic studies demonstrated that homozygous (GlyT1-/-) newborn mice display severe sensorimotor deficits characterized by lethargy, hypotonia, and hyporesponsivity to tactile stimuli and ultimately die in their first postnatal day. These symptoms are similar to those associated with the human disease glycine encephalopathy in which there is a high level of glycine in cerebrospinal fluid of affected individuals. The purpose of this investigation is to determine the impact of chronically high concentrations of endogenous glycine on glutamatergic neurotransmission during postnatal development using an in vivo mouse model (GlyT1+/-). The results of our study indicate the following; that compared with wild-type mice, CA1 pyramidal neurons from mutants display significant disruptions in hippocampal glutamatergic neurotransmission, as suggested by a faster kinetic of NMDAR excitatory postsynaptic currents, a lower reduction of the amplitude of NMDAR excitatory postsynaptic currents by ifenprodil, no difference in protein expression for NR2A and NR2B but a higher protein expression for PSD-95, an increase in their number of synapses and finally, enhanced neuronal excitability.

Pharmacokinetics and cerebral distribution of glycine administered to rats

High doses of glycine have been reported to improve negative schizophrenic symptoms, suggesting that ingested glycine activates glutamatergic transmission via N-methyl-D-aspartate (NMDA) receptors. However, the pharmacokinetics of administered glycine in the brain has not been evaluated. In the present study, the time- and dose-dependent distributions of administered glycine were investigated from a pharmacokinetic viewpoint. Whole-body autoradiography of radiolabeled glycine was performed, and time-concentration curves for glycine and serine in plasma, cerebrospinal fluid (CSF), and brain tissues were obtained. Furthermore, pharmacokinetic parameters were calculated. For a more detailed analysis, the amount of glycine uptake in the brain was evaluated using the brain uptake index method. Radiolabeled glycine was distributed among periventricular organs in the brain. Oral administration of 2 g/kg of glycine significantly elevated the CSF glycine concentration above the ED50 value for NMDA receptors. The glycine levels in CSF were 100 times lower than those in plasma. Glycine levels were elevated in brain tissue, but with a slower time-course than in CSF. Serine, a major metabolite of glycine, was elevated in plasma, CSF, and brain tissue. Glycine uptake in brain tissue increased in a dose-dependent manner. Time-concentration curves revealed that glycine was most likely transported via the blood-CSF barrier and activated NMDA receptors adjacent to the ventricles. The pharmacokinetic analysis and the brain uptake index for glycine suggested that glycine was transported into brain tissue by passive diffusion. These results provide further insight into the potential therapeutic applications of glycine.

Development of a method for the determination of glycine in human cerebrospinal fluid using pre-column derivatization and LC–MS/MS

An LC–MS/MS method using pre-column derivatization with phenylisothiocyanate (PITC) was developed to quantify glycine in human cerebrospinal fluid (CSF) and applied to the determination of glycine in human samples collected during clinical testing. The calibration curve range for the assay was 50–10,000 ng/mL and 13C 2 15N-glycine was used as an internal standard. Artificial CSF was used as a surrogate matrix for standards due to the presence of endogenous glycine in human CSF and this approach was validated with additional experiments involving either standard addition, or stable labeled glycine as an alternate calibration standard for endogenous glycine. Interday bias (% RE) and precision (% CV) were −4.2 and 12.3% at the LLOQ, and less than ±0.9 and 8.3% for higher concentrations, respectively. Glycine was stable in artificial CSF for at least 5 h at room temperature, 55 days at −70 °C (−60 to −80 °C range), and through three freeze–thaw cycles.

Pitfalls in Measuring Cerebrospinal Fluid Glycine Levels in Infants With Encephalopathy

In encephalopathic infants, cerebrospinal fluid hyperglycinemia and elevated cerebrospinal fluid to plasma glycine ratio are considered pathognomonic of nonketotic hyperglycinemia. To evaluate the significance of cerebrospinal fluid hyperglycinemia and elevated cerebrospinal fluid to plasma glycine ratio in acutely encephalopathic infants, a retrospective chart review of all cases of isolated elevation of cerebrospinal fluid glycine levels at Arkansas Children's Hospital from January 1995 to December 2000 was performed. Twenty-two patients (14 males) were included. The most common diagnosis was hypoxic ischemic encephalopathy (n = 8). Nine patients had elevated cerebrospinal fluid to plasma glycine ratio, which was transient in 7 patients. This study shows that elevated cerebrospinal fluid to plasma glycine ratio can be encountered in a variety of clinical conditions. The significance of this observation in light of the poor prognosis of nonketotic hyperglycinemia and the possible role of glycine in the mechanism of ischemic neuronal injury is addressed.

Perioperative care of a child with non-ketotic hyperglycinemia

Non-ketotic hyperglycinemia (NKGH) is an autosomal recessive disorder of glycine metabolism. Defective glycine cleavage results in elevated concentrations of glycine in plasma, urine and cerebrospinal fluid. The accumulation of glycine, an inhibitory neurotransmitter, leads to a clinical presentation of apnea, lethargy, hypotonia, seizures, and severe psychomotor retardation. There are four clinical variants of NKHG, which have been described in the medical literature. Neonatal NKHG is the most common as well as the most devastating and lethal form of the disorder. Given the multi-system involvement of the disorder, there are several perioperative concerns of such patients with delayed emergence requiring supported ventilation being a common postoperative outcome for NKHG patients. We report the perioperative management of a 4-year-old boy with NKGH who required anesthetic care during an adenoidectomy and tonsillectomy for obstructive sleep apnea.