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Objectives: The objectives were to investigate the relationship between ketogenic diet therapy and neutropenia in children with epilepsy., Methods: A retrospective chart review of children who initiated ketogenic diet at the Hospital for Sick Children between January 1, 2000, and May 1, 2018 was performed. Factors associated with the development of neutropenia during ketogenic diet therapy were evaluated and the relationship between development of a significant or suspected infection and neutrophil count was analyzed., Results: One hundred two children met inclusion criteria and were followed on the diet for up to 24 months. Thirteen of 102 (13%) children were neutropenic at diet initiation. In the remaining 89 children, 27 developed neutropenia. Developing neutropenia was significantly associated with the ketogenic diet at 6 (13%), 12 (23%), and 24 (25%) months follow-up. Developing neutropenia was associated with higher urinary ketones (OR = 4.26, 95% CI: 1.27, 14.15) and longer duration of ketogenic diet therapy (OR = 3.29, 95% CI: 1.42, 7.96). There was no significant association between development of a clinically significant infection and neutropenia., Conclusion: Ketogenic diet therapy is associated with neutropenia in children with epilepsy, however, it does not have a significant clinical impact. Concern regarding neutropenia should not discourage the use of the ketogenic diet in children.

α-Aminoadipic semialdehyde and its cyclic form (Δ 1 -piperideine-6-carboxylate) accumulate in patients with α-aminoadipic semialdehyde dehydrogenase (AASADH; antiquitin; ALDH7A1) deficiency. Δ 1 -Piperideine-6-carboxylate is known to react with pyridoxal 5'-phosphate (PLP) to form a Knoevenagel condensation product, resulting in pyridoxine-dependent epilepsy. Despite dramatic clinical improvement following pyridoxine supplementation, many patients still suffer some degree of intellectual disability due to delayed diagnosis. In order to expedite the diagnosis of patients with suspected AASADH deficiency and minimize the delay in treatment, we used gas chromatography-mass spectrometry-based metabolomics to search for potentially diagnostic biomarkers in urine from four patients with ALDH7A1 mutations, and identified Δ 2 -piperideine-6-carboxylate, 6-oxopipecolate, and pipecolate as candidate biomarkers. In a patient at postnatal day six, but before pyridoxine treatment, Δ 2 -piperideine-6-carboxylate and pipecolate were present at very high concentrations, indicating that these compounds may be good biomarkers for untreated AASADH deficiency patients. On the other hand, following pyridoxine/PLP treatment, 6-oxopipecolate was shown to be greatly elevated. We suggest that noninvasive urine metabolomics screening for Δ 2 -piperideine-6-carboxylate, 6-oxopipecolate, and pipecolate will be useful for prompt and reliable diagnosis of AASADH deficiency in patients within any age group. The most appropriate combination among Δ 2 -piperideine-6-carboxylate, 6-oxopipecolate, and pipecolate as biomarkers for AASADH deficiency patients appears to depend on the age of the patient and whether pyridoxine/PLP supplementation has been implemented. We anticipate that the present bioanalytical information will also be useful to researchers studying glutamate, proline, lysine and ornithine metabolism in mammals and other organisms., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Antiquitin (ATQ) deficiency leads to tissue, plasma, and urinary accumulation of alpha-aminoadipic semialdehyde (AASA) and its Schiff base delta-1-piperideine-6-carboxylate (P6C). Although genetic testing of ALDH7A1 is the most definitive diagnostic method, quantifications of pathognomonic metabolites are important for the diagnosis and evaluation of therapeutic and dietary interventions. Current metabolite quantification methods use laborious, technically highly complex, and expensive liquid chromatography-tandem mass spectro-metry, which is available only in selected laboratories worldwide. Incubation of ortho-aminobenzaldehyde (oABA) with P6C leads to the formation of a triple aromatic ring structure with characteristic absorption and fluorescence properties. The mean concentration of P6C in nine urine samples from seven ATQ-deficient patients under standard treatment protocols was statistically highly significantly different (P < .001) compared to the mean of 74 healthy controls aged between 2 months and 57 years. Using this limited data set the specificity and sensitivity is 100% for all tested age groups using a P6C cut-off of 2.11 μmol/mmol creatinine, which represents the 99% prediction interval of the P6C concentrations in 17 control urine samples from children below 6 years of age. Plasma P6C concentrations were only elevated in one ATQ subject, possibly because P6C is trapped by pyridoxal-5-phosphate (PLP) blocking fusing with oABA. Nevertheless, both urine and plasma samples were amenable to the quantification of exogenous P6C with high response rates. The P6C quantification method using fusion of oABA with P6C is fast, simple, and inexpensive and might be readily implemented into routine clinical diagnostic laboratories for the early diagnosis of neonatal pyridoxine-dependent epilepsy., (© 2020 The Authors. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

The measurements of lysine metabolites provide valuable information for the rapid diagnosis of pyridoxine-dependent epilepsy (PDE). Here, we aimed to develop a sensitive method to simultaneously quantify multiple lysine metabolites in PDE, including α-aminoadipic semialdehyde (a-AASA), piperideine-6-carboxylate (P6C), pipecolic acid (PA) and α-aminoadipic acid (α-AAA) in plasma, serum, dried blood spots (DBS), urine and dried urine spots (DUS). Fifteen patients with molecularly confirmed PDE were detected using liquid chromatography-mass spectrometry (LC-MS/MS) method. Compared to the control groups, the concentrations of a-AASA, P6C and the sum of a-AASA and P6C (AASA-P6C) in all types of samples from PDE patients were markedly elevated. The PA and a-AAA concentrations ranges overlapped partially between PDE patients and control groups. The concentrations of all the analytes in plasma and serum, as well as in urine and DUS were highly correlated. Our study provided more options for the diverse sample collection in the biochemical tests according to practical requirements. With treatment modality of newly triple therapy investigated, biomarker study might play important role not only on diagnosis but also on treatment monitoring and fine tuning the diet. The persistently elevated analytes with good correlation between plasma and DBS, as well as urine and DUS made neonatal screening using DBS and DUS possible.

Objectives: The modified Atkins diet (MAD) is a high fat, low carbohydrate ketogenic diet used to treat intractable seizures in children and adults. The long-term impact on fasting lipid profiles (FLPs) remains unknown. This study was designed to detect significant lipid changes in adults on MAD., Methods: Patients were observed prospectively. A FLP was obtained in all patients at the first visit then serially. Patients were started on a 20 g per day net carbohydrate limit MAD. They were screened for risk for coronary heart disease and counseled to reduce saturated fats by a registered dietitian if deemed at risk. Patients that remained on MAD for 3 or more months with one or more follow-up FLP were included., Results: Thirty-seven patients (14 male), mean age 33 years (SD 13, range 18-59) met study criteria. Median diet duration was 16 months (range 3-41). Total cholesterol and low-density lipoprotein (LDL) increased significantly over the first 3 months of MAD (P = 0.01 and 0.008, respectively), but were not significantly different from baseline after 1 year of treatment (P = 0.2 and P = 0.5, respectively). High-density lipoprotein levels trended upward in the first 3 months (P = 0.05) and triglycerides remained unchanged (P = 0.5). In 12 patients followed for 3 or more years, no cardiovascular or cerebrovascular events were reported., Discussion: Although total cholesterol and LDL increased over the first 3 months of the MAD, these values normalized within a year of treatment, including in patients treated with MAD for more than 3 years.

Purpose: Pyridoxine-dependent epilepsy (PDE) is a rare autosomal recessive disorder caused by mutations of the ALDH7A1 gene. We aimed to analyze the relations between the clinical diagnosis and treatment of PDE and ALDH7A1 gene mutations in Chinese PDE patients., Methods: The clinical manifestations, diagnosis and treatment were observed in a cohort of PDE patients with early onset of seizure. Video-electroencephalogram (VEEG) and magnetic resonance imaging (MRI) were performed. The mutation of ALDH7A1 gene was analyzed., Results: Of eight patients, six were males and two were females. Age of seizure onset ranged from 1 to 100 days and 75% patients presented with seizures in the neonatal period. All patients showed different degrees of developmental delay. EEGs showed focal or multifocal discharges, or were normal. Molecular analysis revealed 10 ALDH7A1 mutations, including 2 splice site mutations. Five patients had mutation at IVS11+1G>A site, six patients had missense mutations, one with nonsense mutation and another patient had 9-bp genomic deletion mutation. Among them, two mutations were first time reported., Conclusions: Seizure onset was in neonatal or early infantile period in our PDE patients. Early recognition and diagnosis of the disease is necessary for early intervention and improve cognitive development in the later life. In this study, on the molecular level, we also identified the splice site mutation IVS11+1G>A as a high prevalence mutation site with a frequency of 31.25% (5 of 16 alleles) in Chinese PDE patients., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Pyridoxine-dependent epilepsy (PDE) is an epileptic encephalopathy characterized by response to pharmacologic doses of pyridoxine. PDE is caused by deficiency of α-aminoadipic semialdehyde dehydrogenase resulting in impaired lysine degradation and subsequent accumulation of α-aminoadipic semialdehyde. Despite adequate seizure control with pyridoxine monotherapy, 75% of individuals with PDE have significant developmental delay and intellectual disability. We describe a new combined therapeutic approach to reduce putative toxic metabolites from impaired lysine metabolism. This approach utilizes pyridoxine, a lysine-restricted diet to limit the substrate that leads to neurotoxic metabolite accumulation and L-arginine to compete for brain lysine influx and liver mitochondrial import. We report the developmental and biochemical outcome of six subjects who were treated with this triple therapy. Triple therapy reduced CSF, plasma, and urine biomarkers associated with neurotoxicity in PDE. The addition of arginine supplementation to children already treated with dietary lysine restriction and pyridoxine further reduced toxic metabolites, and in some subjects appeared to improve neurodevelopmental outcome. Dietary lysine restriction was associated with improved seizure control in one subject, and the addition of arginine supplementation increased the objective motor outcome scale in two twin siblings, illustrating the contribution of each component of this treatment combination. Optimal results were noted in the individual treated with triple therapy early in the course of the disease. Residual disease symptoms could be related to early injury suggested by initial MR imaging prior to initiation of treatment or from severe epilepsy prior to diagnosis. This observational study reports the use of triple therapy, which combines three effective components in this rare condition, and suggests that early diagnosis and treatment with this new triple therapy may ameliorate the cognitive impairment in PDE., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Oxidative stress is a biochemical state in which reactive oxygen species are generated and it has been associated with pathological states including epilepsy. Therein, neuroprostanes (NeuroPs) and dihomo-isoprostanes (Dihomo-IsoPs)-a series of compounds formed nonenzymatically through free radical-induced DHA, n-6 DPA, and AdA peroxidation-are implicated in the pathophysiological status of various human neurological diseases. A new, robust, and selective analytical method for the determination of 10 NeuroPs/Dihomo-IsoPs in human urine, using solid-phase extraction and UHPLC-QqQ-MS/MS in the multiple reaction monitoring mode (using a negative electrospray ionization interface), was developed. Nine NeuroPs/Dihomo-IsoPs were identified in 15 epileptic patients, matched with healthy volunteers. Among them, 17-F2t-Dihomo-IsoP, Ent-7(R)-7-F2t-Dihomo-IsoP, and Ent-7-epi-7-F2t-Dihomo-IsoP, derived from adrenic acid (AdA), were significantly higher in epileptic patients than in healthy volunteers. The validated method provided a high-throughput assay with a limit of detection and limit of quantification for each analyte of 0.10-5.90ngmL(-1) and 0.15-11.81ngmL(-1), respectively. The intra- and interday variations were lower than 14%. Dihomo-IsoPs have been considered as potential markers of epilepsy for the first time and their measurement may increase the understanding of the role of oxidative stress in neurological diseases, in intra vitam studies. The present study highlights a potential role of Dihomo-IsoPs as biomarkers in persons with epilepsy, though its mechanisms and possible implications should be the subject of further investigations., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Serum and urine samples were collected from 33 NCC patients before the albendazole treatment, 3-6 and 12 months PT. At 3 months PT, 24 (72.7%) patients had no detectable CT/MRI lesions and 9 (27.2%) patients had persistent lesions. Antibody response to crude soluble extract (CSE), excretory secretory (ES), and lower molecular mass (LMM) (10-30 KDa) antigenic fraction of T. solium cysticerci was detected in serum and urine samples by ELISA. Before the treatment, out of 33 NCC children, 14 (42.4%), 22 (66.6%), and 11 (33.3%) serum samples were found positive with the use of CSE, ES, and LMM antigen, respectively. At 3-6 months PT, positivity rate was 5 (15.1%), 2 (6%), and 4 (12.1%) and at 12 months PT, positivity rate was 5 (15.1%), 0, and 3 (9%) with the use of CSE, ES, and LMM antigen, respectively. There was no significant difference in the positivity with the use of three antigens in pretreatment and PT urine samples. The study suggests that the use of ES antigen to detect antibody in serum samples may serve better purpose to evaluate the therapeutic response in patients with NCC.

As the life expectancy of people with intellectual disability (ID) increases, it is becoming necessary to understand factors affecting survival. However, predictors that are typically assessed among healthy people have not been examined. Predictors of all-cause mortality, including blood, urine, anthropometry, and nutritional indices, were examined among institutionalized people with ID. This retrospective cohort study involved 316 participants (191 males, 125 females; mean age, 36.5 ± 10.5 years) at a public facility for people with ID in Ibaraki Prefecture, Japan. During the follow-up from the examination day in 1984-1992 through December 31, 2007 (mean follow-up, 18.6 years), 44 deaths occurred. Mean age at death was 47.1 ± 10.0 years (range, 22.3-65.3 years). Early deaths within three years (n = 4) were treated as censored cases. Cox proportional hazard regression models were used to calculate hazard ratios (HRs) and 95% confidence intervals (CIs) of all-cause mortality. Sex- and age-adjusted analysis (p<0.15) revealed positive associations with mortality for high serum cholesterol, high thymol turbidity test (TTT), and glucosuria and negative associations with mortality for high serum albumin, high uric acid, high potassium, high calcium, and high systolic blood pressure. Multivariate analysis revealed that male sex (HR, 4.11; 95% CI, 1.59-10.59), high serum cholesterol (1.01; 1.00-1.02), high serum TTT (1.21; 1.03-1.41), and epilepsy significantly increased the mortality risk. The results indicate that the predictors of life expectancy for people with ID included both factors that are shared with healthy people (male sex, high serum cholesterol) and factors specific to people with disabilities (high serum TTT and epilepsy)., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

α-Amino adipic semialdehyde (α-AASA) accumulates in body fluids from patients with pyridoxine-dependent epilepsy because of mutations in antiquitin (ALDH7A1) and serves as the biomarker for this condition. We have recently found that the urinary excretion of α-AASA was also increased in molybdenum cofactor and sulfite oxidase deficiencies. The seizures in pyridoxine-dependent epilepsy are caused by lowered cerebral levels of pyridoxal-5-phosphate (PLP), the bioactive form of pyridoxine (vitamin B(6)), which can be corrected by the supplementation of pyridoxine. The nonenzymatic trapping of PLP by the cyclic form of α-AASA is causative for the lowered cerebral PLP levels. We describe 2 siblings with clinically evident pyridoxine-responsive seizures associated with increased urinary excretion of α-AASA. Subsequent metabolic investigations revealed several metabolic abnormities, all indicative for molybdenum cofactor deficiency. Molecular investigations indeed revealed a known homozygous mutation in the MOCS2 gene. Based upon the clinically evident pyridoxine-responsive seizures in these 2 siblings, we recommend considering pyridoxine supplementation to patients affected with molybdenum cofactor or sulfite oxidase deficiencies.

A simple, accurate, and sensitive microextraction by packed sorbent-gas chromatography-mass spectrometry method has been developed for the simultaneous quantification of four antiepileptic drugs; oxcarbazepine, carbamazepine, phenytoin, and alprazolam in human plasma and urine as a tool for drug monitoring. Caffeine was used as internal standards for the electron ionization mode. An original pretreatment procedure on biological samples, based on microextraction in packed syringe using C(18) as packing material gave high extraction yields (69.92-99.38%), satisfactory precision (RSD < 4.7%) and good selectivity. Linearity was found in the 0.1-500 ng/mL range for these drugs with limits of detection (LODs) between 0.0018 and 0.0036 ng/mL. Therefore, the method has been found to be suitable for the therapeutic drug monitoring of patients treated with oxcarbazepine, carbamazepine, phenytoin, and alprazolam. After validation, the method was successfully applied to some plasma samples from patients undergoing therapy with one or more of these drugs. A comparison of the detection limit with similar methods indicates high sensitivity of the present method over the earlier reported methods. The present method is applied for the analysis of these drugs in the real urine and plasma samples of the epileptic patients., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Pyridoxine-dependent epilepsy presents early in life, even in utero. It is usually refractory to conventional antiepileptic medications and responds only to lifelong pyridoxine supplementation. Seizures are usually generalized tonic clonic. We report a 3-year-old child that was born prematurely at 25 weeks of gestation. He presented with abnormal movements in the second month of life. At 10 months of age he presented with status epilepticus, which was refractory to multiple antiepileptic medications and was controlled with intravenous pyridoxine. An elevated level of a-aminoadipic semialdehyde excretion in the urine supported the diagnosis of pyridoxine-dependent epilepsy. Subsequently, a c.1195G>C homozygous mutation in the 5q31 aldehyde dehydrogenase 7A1 gene was confirmed. This case calls for considering pyridoxine-dependent epilepsy and its early management in cases with resistant seizures; even in the presence of extreme prematurity with its neurological consequences.

The assessment of urinary α-aminoadipic semialdehyde (α-AASA) has become the diagnostic laboratory test for pyridoxine dependent seizures (PDS). α-AASA is in spontaneous equilibrium with its cyclic form Δ(1)-piperideine-6-carboxylate (P6C); a molecule with a heterocyclic ring structure. Ongoing diagnostic screening and monitoring revealed that in some individuals with milder ALDH7A1 variants, and patients co-treated with a lysine restricted diet, α-AASA was only modestly increased. This prompted us to investigate the diagnostic power and added value of the assessment of urinary P6C compared to α-AASA. Urine samples were diluted to a creatinine content of 0.1 mmol/L, followed by the addition of 0.01 nmol [(2)H(9)]pipecolic acid as internal standard (IS) and 5 μL was injected onto a Waters C(18) T3 HPLC column. Chromatography was performed using water/methanol 97/3 (v/v) including 0.03 % formic acid by volume with a flow rate of 150 μL/min and detection was accomplished in the multiple reaction monitoring mode: P6C m/z 128.1 > 82.1; [(2)H(9)]pipecolic acid m/z 139.1 > 93.1. Due to the dualistic nature of α-AASA/P6C, and the lack of a proper internal standard, the method is semi quantitative. The intra-assay CVs (n = 10) for two urine samples of proven PDS patients with only modest P6C increases were 4.7% and 8.1%, whereas their inter-assay CVs (n = 10) were 16 and 18% respectively. In all 40 urine samples from 35 individuals with proven PDS, we detected increased levels of P6C. Therefore, we conclude that the diagnostic power of the assessments of urinary P6C and α-AASA is comparable.

Objective: To determine the role of serum and urine selenium, and boron levels in children with resistant epilepsy., Methods: Serum and urine boron and selenium levels were studied in 53 cases (32 boys and 21 girls) diagnosed with resistant epilepsy between April 2006 and February 2007 at the Department of Pediatric Neurology, Erciyes University, Kayseri, Turkey. Differences between groups were assessed using Student's t-test. Countable data were defined as percentage. Inter-group difference was assessed by Chi-square test. P-values less than 0.05 were considered significant., Results: When serum and urine boron and selenium levels were evaluated and compare with controls, a statistically significant difference was found in serum selenium, urine selenium, and urine boron levels (p<0.05). No significant difference was found in serum boron levels (p>0.05)., Conclusion: It was observed that there is a need for selenium supplementation in treatment of patients with resistant epilepsy, while no etiologic role is observed for boron.

The study testifies an assumption on epilepsy as an inborn error of pyridoxine metabolism and suggests non-invasive quantitative biomarkers for clarified evaluation of clinical status and monitoring an individual treatment by antiepileptic drugs. Urinary parameters of pyridoxal-phosphate (PLP)-dependent tryptophan degradation and the level of 4-pyridoxic acid, the end product of pyridoxine metabolism, were measured by HPLC method with simultaneous ultraviolet and fluorimetric detection in children with different forms of epilepsy and matched healthy controls. The concentrations of compounds formed or metabolized in the course of tryptophan degradation (kynurenines, indoxyl-sulfate) along with correlations between them turned out to be quantitative biomarkers useful for both clarifying patient's clinical state and monitoring antiepileptic treatment. In particular, the value of the ratio of 4-pyridoxic acid to kynurenine appears to be an index of an experienced seizure attack, while the ratio of 3-hydroxyanthranilic acid to 3-hydroxykynurenine reflects activity of kynureninase, the enzyme of critical sensitivity to PLP supply. Growing progressively worse, epilepsy is accompanied by aggravation of PLP-dependent disturbances of tryptophan metabolism and expanding inhibition of kynureninase. The affected pyridoxine metabolism is discussed as an inborn genetic trait in epilepsy in general, rather than a specific sign of pyridoxine-dependent epilepsy solely., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background: Pyridoxine dependent epilepsy (PDE) is characterized by neonatal epileptic encepahalopathy responsive to pharmacological doses of vitamin B6. Recently an autosomal recessive deficiency in Antiquitin (ALDH7A1), a gene involved in the catabolism of lysine has been identified as the underlying cause., Case Report: In 21 and 23 year-old sisters, who had presented with neonatal / early infantile onset seizures, PDE was confirmed by elevated urinary alpha aminoadipic- 6- semialdehyde (α-AASA) excretion and compound heterozygosity for two known ALDH7A1 missense mutations. Although epilepsy was well controlled upon treatment with pyridoxine, thiamine, phenytoin and carbamazepine since early infancy, both had developmental delay with prominent speech delay as children. As adults, despite the same genetic background and early treatment with pyridoxine, their degree of intellectual disability (ID) differed widely. While the older sister's cognitive functions were in the moderate ID range and she was not able to live unattended, the younger sister had only mild ID and was able to live independently., Conclusion: Although seizures are a defining feature of PDE, other disease manifestations can vary widely even within the same family. Adult neurologists should be aware that the diagnosis of PDE can be delayed and PDE should be considered in the differential diagnosis of adults with seizure disorders dating from childhood.

Purpose: To investigate the physiological mechanisms behind the pronounced decline of lamotrigine (LTG) serum concentrations during pregnancy., Methods: Serum and urine concentrations of LTG and its main metabolite, LTG-N2-glucuronide (LTG-GLUC), were measured monthly in 21 pregnancies of 19 women using LTG. Simultaneously, a panel of biochemical variables was monitored to evaluate liver and kidney function and possible hemodilution effects. Pharmacokinetic parameters were calculated once at baseline and once in gestational month 8., Results: Initially, LTG and LTG-GLUC serum concentrations fell simultaneously by 27% and 38%, respectively (gestational month 2). Subsequently, the ratio of the LTG-GLUC/LTG serum concentrations increased gradually, correlating strongly with rising serum estradiol concentrations. In gestational month 8, the ratio was 164% higher than at baseline. At that time, LTG total clearance had increased by 118%, and the amount of unchanged LTG in urine had dropped by 40% while the amount of LTG-GLUC had increased by a corresponding 37%., Conclusions: The simultaneous decline of LTG and LTG-GLUC serum concentrations in early pregnancy suggests that in this phase, increased renal blood flow is the major cause. After gestational month 2, estradiol-induced glucuronidation of LTG becomes more important, leading to a further fall of LTG serum concentrations and a gradual rise of the LTG-GLUC/LTG-ratio through the remaining pregnancy. An expanded volume of distribution may also contribute to reduced LTG serum concentrations in pregnancy., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Hydroxysteroid (17beta) dehydrogenase 10 (HSD10) is a mitochondrial multifunctional enzyme encoded by the HSD17B10 gene. Missense mutations in this gene result in HSD10 deficiency, whereas a silent mutation results in mental retardation, X-linked, syndromic 10 (MRXS10). Here we report a novel missense mutation found in the HSD17B10 gene, namely c.194T>C transition (rs104886492), brought about by the loss of two forked methyl groups of valine 65 in the HSD10 active site. The affected boy, who possesses mutant HSD10 (p.V65A), has a neurological syndrome with metabolic derangements, choreoathetosis, refractory epilepsy and learning disability. He has no history of acute decompensation or metabolic acidosis whereas his urine organic acid profile, showing elevated levels of 2-methyl-3-hydroxybutyrate and tiglylglycine, is characteristic of HSD10 deficiency. His HSD10 activity was much lower than the normal control level, with normal β-ketothiolase activity. The c.194T>C mutation in HSD17B10 can be identified by the restriction fragment polymorphism analysis, thereby facilitating the screening of this novel mutation in individuals with intellectual disability of unknown etiology and their family members much easier. The patient's mother is an asymptomatic carrier, and has a mixed ancestry (Hawaiian, Japanese and Chinese). This demonstrates that HSD10 deficiency patients are not confined to a particular ethnicity although previously reported cases were either Spanish or German descendants.

Pyridoxine-dependent epilepsy was recently shown to be due to mutations in the ALDH7A1 gene, which encodes antiquitin, an enzyme that catalyses the nicotinamide adenine dinucleotide-dependent dehydrogenation of l-alpha-aminoadipic semialdehyde/L-Delta1-piperideine 6-carboxylate. However, whilst this is a highly treatable disorder, there is general uncertainty about when to consider this diagnosis and how to test for it. This study aimed to evaluate the use of measurement of urine L-alpha-aminoadipic semialdehyde/creatinine ratio and mutation analysis of ALDH7A1 (antiquitin) in investigation of patients with suspected or clinically proven pyridoxine-dependent epilepsy and to characterize further the phenotypic spectrum of antiquitin deficiency. Urinary L-alpha-aminoadipic semialdehyde concentration was determined by liquid chromatography tandem mass spectrometry. When this was above the normal range, DNA sequencing of the ALDH7A1 gene was performed. Clinicians were asked to complete questionnaires on clinical, biochemical, magnetic resonance imaging and electroencephalography features of patients. The clinical spectrum of antiquitin deficiency extended from ventriculomegaly detected on foetal ultrasound, through abnormal foetal movements and a multisystem neonatal disorder, to the onset of seizures and autistic features after the first year of life. Our relatively large series suggested that clinical diagnosis of pyridoxine dependent epilepsy can be challenging because: (i) there may be some response to antiepileptic drugs; (ii) in infants with multisystem pathology, the response to pyridoxine may not be instant and obvious; and (iii) structural brain abnormalities may co-exist and be considered sufficient cause of epilepsy, whereas the fits may be a consequence of antiquitin deficiency and are then responsive to pyridoxine. These findings support the use of biochemical and DNA tests for antiquitin deficiency and a clinical trial of pyridoxine in infants and children with epilepsy across a broad range of clinical scenarios.

For the first time, the use of urine [(1)H] magnetic resonance spectroscopy has allowed the detection of 1 case of guanidinoacetate methyl transferase in a database sample of 1500 pediatric patients with a diagnosis of central nervous system impairment of unknown origin. The urine [(1)H] magnetic resonance spectroscopy of a 9-year-old child, having severe epilepsy and nonprogressive mental and motor retardation with no apparent cause, revealed a possible guanidinoacetic acid increase. The definitive assignment of guanidinoacetic acid was checked by addition of pure substance to the urine sample and by measuring [(1)H]-[(1)H] correlation spectroscopy. Diagnosis of guanidinoacetate methyl transferase deficiency was further confirmed by liquid chromatography-mass spectrometry, brain [(1)H] magnetic resonance spectroscopy, and mutational analysis of the guanidinoacetate methyl transferase gene. The replacement therapy was promptly started and, after 1 year, the child was seizure free. We conclude that for this case, urine [(1)H] magnetic resonance spectroscopy screening was able to diagnose guanidinoacetate methyl transferase deficiency.

A pilot prospective follow-up study of the role of the branched chain amino acids as additional therapy to the ketogenic diet was carried out in 17 children, aged between 2 and 7 years, with refractory epilepsy. All of these patients were on the ketogenic diet; none of them was seizure free, while only 13 had more or less benefited from the diet. The addition of branched chain amino acids induced a 100% seizure reduction in 3 patients, while a 50% to 90% reduction was noticed in 5. Moreover, in all of the patients, no reduction in ketosis was recorded despite the change in the fat-to-protein ratio from 4:1 to 2.5:1. Although our data are preliminary, we suggest that branched chain amino acids may increase the effectiveness of the ketogenic diet and the diet could be more easily tolerated by the patients because of the change in the ratio of fat to protein.

Glutaric aciduria type 1 (GA-1, OMIM 608801) is an autosomal-recessive disorder resulting from a deficiency of glutaryl-CoA dehydrogenase (GCDH). Clinical expression usually involves an acute encephalopathic episode in infancy, followed by the development of severe dystonia-dyskinesia. Other presentations include mild developmental delay, macrocephaly, and subdural haematoma. Seizures may occur with the acute encephalopathy but are unusual in the long term, unless motor or cognitive difficulties are severe. We report a 6-year-old female who was referred with recurrent epileptic seizures that proved difficult to control with first-line anticonvulsants. There was no history of encephalopathy. She had no neurological or developmental abnormalities. The electroencephalogram was profoundly abnormal with slow background and mixed multifocal and generalized spike-and-wave discharges. Seizures deteriorated on valproic acid. Cranial magnetic resonance imaging showed widened Sylvian fissures. Metabolic investigations revealed GA-1. She has improved on a low-protein diet, carnitine, levetiracetam, and lamotrigine. This is the first report of epileptic seizures as the sole presenting feature of GA-1 and it potentially adds to the clinical spectrum of this disorder. Furthermore, the case emphasizes the role of metabolic investigation when first- or second-line treatment of epilepsy is unsuccessful.

Objects: The relationship between antiepileptic drugs (AEDs) polytherapy and urinary pH was studied to demonstrate the effect and difference of AED polytherapy compared to monotherapy., Materials and Methods: A total of 271 urine samples from patients receiving AED polytherapy aged from 7 months to 35 years were enrolled. Two AEDs were co-administered to 215 patients, three AEDs to 45 patients, four AEDs to ten patients, and five AEDs to one patient., Results: The distribution of urinary pH shifted to the alkaline range with increasing numbers of co-administered AEDs (p < 0.0001). The distribution of urinary pH shifted to the alkaline side with AED polytherapy that included valproate (p < 0.05) or acetazolamide (p < 0.03). The distribution of urinary pH did not change with or without zonisamide, carbamazepine, phenobarbital, phenytoin, or clonazepam., Conclusions: Urinary pH should be monitored in patients receiving AED polytherapy, particularly those receiving valproate, acetazolamide, or various AEDs in combination.

Objective: To determine whether valproic acid (VPA) influences urinary levels of 15-F2t -isoprostane (15-F2t -IsoP), a marker of oxidative stress, in children., Study Design: Morning urine samples were collected from children with epilepsy receiving VPA (n = 25), carbamazepine (n = 16), or clobazam (n = 12) for > or = 4 weeks and from age-matched control subjects (n = 39). Urinary 15-F2t -IsoP levels were determined by enzyme-linked immunosorbent assay., Results: The mean (+/- standard deviation) urine 15-F2t -IsoP levels (nmol/mmol Cr) were: valproic acid (0.36 +/- 0.15); carbamazepine (0.24 +/- 0.10); clobazam (0.23 +/- 0.10); control group (0.20 +/- 0.09). Patients treated with VPA had significantly elevated 15-F2t -IsoP levels when compared with the control, carbamazepine, and clobazam groups (P < .05). Multiple linear regression analysis demonstrated that younger patient age and exposure to second-hand smoke were significant predictors of elevated urine 15-F2t -IsoP levels within the control group (r2 = 0.261, P = .05 and P = .01, respectively). Subjects not exposed to second-hand smoke receiving valproic acid therapy had a significantly elevated mean urine 15-F2t -IsoP level compared to subjects not exposed to second-hand smoke in the carbamazepine, clobazam and control groups (P < .05)., Conclusions: These data demonstrate that treatment of children with VPA is associated with higher urinary levels of 15-F2t -IsoP, a marker of oxidative stress.

Hypoparathyroidism occurs due to insufficient production of parathyroid hormone to maintain extracellular calcium levels within the normal range. The acute clinical symptoms and signs of hypoparathyroidism are those of hypocalcaemia, ranging from tingling and numbness of limb extremities to intractable seizures. Often seizures are mistaken for epilepsy. Though hypoparathyroidism is not uncommon, the diagnosis is often missed due to its unusual clinical manifestation. This is the first documented report with vitamin D, Parathormone levels and urinary biochemical parameters from India. We present two cases of hypoparathyroidism who presented with seizures along with a short review of literature.

L-2-Hydroxyglutaric (L-2-HG) aciduria is a rare inherited metabolic disease usually observed in children. Patients present a very slowly progressive deterioration with cerebellar ataxia, mild or severe mental retardation, and various other clinical signs including extrapyramidal and pyramidal symptoms, and seizures. The disease is characterized by increased levels of L-2-HG in body fluids such as urine and cerebrospinal fluid. We report on two sisters from consanguineous parents, in whom L-2-HG aciduria was diagnosed at an adult age. Although magnetic resonance imaging and spectroscopic findings were severely abnormal in both, they experienced a different clinical course. The older sister presented with severe mental retardation, recurrent epileptic seizures, and progressive deterioration in her ability to walk and to talk; she is now confined to a wheelchair with severe speech deficit. In contrast, the younger sister only had a few epileptic seizures in childhood and moderate mental retardation, is still able to walk, and performs manual work, and has a social life in a specialized institution for moderately mentally handicapped persons. For the two patients, a complete deletion of exon 9 was demonstrated in a gene located on chromosome 14q22.1, which most probably encodes for L-2-hydroxyglutarate dehydrogenase. The pathological findings observed in this metabolic disorder could therefore be related to a toxic effect of L-2-hydroxyglutarate on the central nervous system, although the presence of other toxic metabolites cannot be excluded.

Objects: Since alkaline urine is a risk factor for urolithiasis, the relationship between antiepileptic drugs and urinary pH was retrospectively studied in epilepsy patients treated with antiepileptic drug monotherapy for more than 1 month., Methods: A total of 913 urinary samples from antiepileptic drug-treated patients were compared with 780 age-matched control samples, and with 112 samples from epilepsy patients who had not been treated with antiepileptic drugs. The antiepileptic drugs administered were carbamazepine, valproate, phenobarbital, zonisamide, sulthiame, and phenytoin., Conclusions: The proportion of the acid urine in the valproate-treated patients was lower than that in controls. The proportion of the alkaline urine in the valproate-treated patients was higher than that in controls. This effect was independent of age, sex, and the serum valproate concentration. There was no significant difference in urinary pH among the epilepsy patients treated with other antiepileptic drugs, the epilepsy patients who had not been treated with antiepileptic drugs, and the controls.

Best practices for monitoring the adverse effects of antiepileptic drugs (AEDs) have not been carefully studied. Routine blood and urine studies do not appear to be of value in asymptomatic patients to avoid severe acute reactions. Subtle chronic AED side effects exist but algorithms for their detection and treatment are not well developed. Randomized clinical trials do not support the value of routine AED serum levels to improve compliance, seizure control, or avoid side effects. Simple screening procedures for neurotoxicity have not been developed. Some personal suggestions are offered.

The action of some anticonvulsant drugs as the causal agents of attacks of acute porphyria has been widely documented in the literature. However, little attention has been paid to the effect of these drugs on the urinary excretion of porphyrins in non-porphyric subjects. In a sample of 82 epileptic patients treated with phenobarbital (n = 54), phenytoin (n = 64), carbamazepine (n = 33), and valproate (n = 8), the daily doses were expressed according to a drug score that would reflect the capacity of these drugs as enzymatic inducers when administered in polytherapy. A significantly increased urinary excretion of D-glucaric acid (DGA) and porphyrins was found in this group of patients (P<0.001), with coproporphyrin being the major fraction in all cases (>60%). Urinary DGA had a highly significant correlation with the drug score (r = 0.783, P<0.001); however, no significant correlations were found between the urinary porphyrins and DGA (r = 0.005) or the drug score (r = 0.053). Neither was any significant relationship found between the urinary porphyrins and the serum activity of 5'-nucleotidase (r = 0.066) or the presence of a cholestasis objectivized through the presence of the isoform of gamma-glutamyltransferase with beta-globulins electrophoretic mobility. However, in a group of 10 patients a significant correlation was found between the urinary excretion of porphyrins and beta-N-acetylhexosaminidase (r = 0.790, P<0.01). Therefore, it does not appear that the liver enzyme induction, or even a subclinical cholestasis, produced by the antiepileptic drugs administered to these patients may serve to explain the increase in the urinary excretion of porphyrins. A possible renal origin is proposed for the increase of urinary porphyrins in these cases.

Toxic causes of seizures are numerous: alcohol and other substances of abuse, drugs, and industrial and household products. However, in the absence of a clearly suggestive history and/or associated symptoms and signs, identification of the toxic origin of new-onset seizures may be extremely difficult. We report here the case of a patient admitted in our hospital after a single generalized tonic-clonic seizure. The remarkable coincidence that a colleague of his, with whom he was working to clean the same workshop, had been admitted 1 week earlier for respiratory distress, coma, and de novo nonconvulsive focal status epilepticus, led us to consider a possible toxicologic etiology. Urine analysis revealed a high nickel concentration, suggestive of acute nickel poisoning.

The ratio of urinary 6 beta-hydroxycortisol/cortisol (6 beta-OHC/FC) in morning spot urine samples collected from 8:00 a.m. to 12:00 p.m. was studied using ELIZA kits (Stabiligen) in a group of healthy adult Egyptians (control group) of both sex (n=65, age range: 16-48 years). The frequency distribution of urinary 6 beta-OHC/FC ratio was widely distributed among subjects with higher values in males in comparison to females. No bimodality in either sex was observed. Another group of adult epileptic patients (n=16) was studied for the influence of chronic carbamazepine antiepileptic drug administration on urinary 6 beta-OHC/FC ratio in spot urine samples. The induction property of carbamazepine on CYP3A4 was observed through significant increase (p=0.01) in 6 beta-OHC/FC ratio among epileptic patients in comparison with control subjects. In conclusion, the frequency distribution of urinary 6 beta-OHC/FC ratio among Egyptians shows sexual dimorphism. Also, measurement of urinary 6 beta-OHC/FC ratio provides a simple non-invasive method to monitor CYP3A4 enzyme induction during administration of carbamazepine antiepileptic drug.

Urolithiasis is a rare side effect of antiepileptic drugs. To clarify the risk factors for urolithiasis induced by antiepileptic drugs, the effect of antiepileptic drug monotherapy on crystalluria was studied, and zonisamide or sulthiame therapy and alkaline urine were demonstrated to be risk factors. In the next investigation, the effect of antiepileptic drug polytherapy on crystalluria was retrospectively studied in epilepsy patients treated for more than 1 month during the last 7 years. A total of 278 urine specimens from epilepsy patients aged between 7 months and 36 years were enrolled in this study. The mean age was 12.3 years. There were 109 samples from females and 169 from males. Antiepileptic drugs administered in this study were valproate (174 urinary samples), zonisamide (139), carbamazepine (138), phenobarbital (65), phenytoin (52), acetazolamide (17), clonazepam (15), sulthiame (6), ethosuximide (6), nitrazepam (4), and clobazam (4). Epilepsy patients treated with antiepileptic drug polytherapy were frequently found to have crystalluria in patients demonstrating alkaline urine and taking acetazolamide, zonisamide (particularly with high serum levels), or many antiepileptic drugs in combination. Regular urinalysis seems to be necessary in these patients, and the evaluation for urolithiasis should be performed if persistent crystalluria is demonstrated.

A novel liquid chromatography-mass spectrometry (LC-MS) method was developed and validated for quantification of topiramate (TPM) and its metabolites 10-hydroxy topiramate (10-OH-TPM), 9-hydroxy topiramate (9-OH-TPM), and 4,5-O-desisopropylidene topiramate (4,5-diol-TPM) in plasma and urine. The method uses 0.5 mL of plasma or 1 mL of urine that is extracted with diethyl ether and analyzed by LC-MS. Positive ion mode detection enables tandem mass spectrometric (MS/MS) identification of the aforementioned four compounds. Calibration curves of TPM, 4,5-diol-TPM, 9-OH-TPM, and 10-OH-TPM in plasma and urine were prepared and validated over the concentration range of 0.625 to 40 microg/mL using TPM-d(12) as an internal standard. Calibration curves were linear over this concentration range for TPM and its metabolites. Accuracy and precision ranged in urine from 83% to 114% and 4% to 13% (%CV), respectively, and in plasma from 82% to 108% and 6% to 13%, respectively. The applicability of the assay was evaluated by analyzing plasma samples from a healthy subject who received a single oral dose of TPM (200 mg) and urine samples from 11 patients with epilepsy treated with TPM (daily dose between 100 to 600 mg) alone or with other antiepileptic drugs. Only TPM was detected and quantified in the plasma samples, and its concentration ranged between 0.7 and 4.3 microg/mL. The concentrations of TPM and 10-OH TPM were quantifiable in all urine samples and ranged from 20 to 300 microg/mL for TPM and from 1 to 50 microg/mL for 10-OH-TPM. The metabolites 4,5-diol-TPM and 9-OH-TPM were also detected in all urine samples, but their concentrations were quantifiable only in 4 patients. An unidentified peak in the chromatograms obtained from patients' urine was attributed to 2,3-O-desisopropylidene topiramate (2,3-diol-TPM). Due to a lack of reference material of 2,3-diol TPM and the similar MS/MS spectrum with 4,5-diol-TPM, the calibration curves of 4,5-diol-TPM were used for the quantification of its isomer 2,3-diol-TPM. Based on these determinations, the apparent 2,3-diol-TPM-to-TPM concentration ratio in patients' urine ranged from 0.05 to 0.51 and the 10-OH-TPM-to-TPM ratio ranged from 0.02 to 0.17. In conclusion, a novel LC-MS method for the assay of TPM and four of its metabolites in plasma and urine was developed. Its utilization for analysis of urine samples from patients with epilepsy showed that the method was suitable for analysis of TPM and its metabolites in clinical samples. Two quantitatively significant TPM metabolites (10-OH-TPM and 2,3-diol-TPM) and two quantitatively minor metabolites (9-OH-TPM and 4,5-diol-TPM) were detected and quantified in urine samples from patients with epilepsy.

Purpose: Valproic acid (VPA) is an antiepileptic drug (AED) used for generalized and absence seizures. It has a rare but potentially fatal hepatotoxicity side effect, and many researchers believe that reactive metabolites of VPA could be involved. We demonstrated here that the thiol conjugates of (E)-2,4-diene VPA were significantly elevated in a high-risk group of patients., Methods: Thirty-four patients with seizures were divided into three groups. Group A (n = 14) were being treated with VPA; group B (n = 12) received VPA as well as other AEDs that do not induce P450-VPA metabolism; and group C (n = 8) received VPA and AEDs that induce P450-VPA metabolism. The NAC conjugates of (E)-2,4-diene VPA (NAC I and NAC II) were identified in the urine of patients by gas chromatography/mass spectrography NICI analysis., Results: VPA monotherapy (group A) or VPA polytherapy with non-P450-enzyme-inducing drugs (group B), showed that patients younger than 7.5 years excreted significantly higher concentrations of the two conjugates compared with older patients (older than 7.5 years) in the same groups (p < 0.05). Patients receiving VPA polytherapy with P450-enzyme-inducing drugs were all older than 7. 5 years (group C). They excreted significantly higher concentrations of NAC I and NAC II compared with patients in groups A and B who were older than 7.5 years (p < 0.05)., Conclusions: There were no significant differences in the excretion of NAC I and NAC II between patients in group C and those who were 7.5 years or younger in groups A and B. High doses of VPA also were a significant factor associated with elevated NAC I and NAC II among young patients and in polytherapy patients.

In this study, spectroscopic and chromatographic evidence is presented for the identification and characterization of the metabolites, valproyl glutamate (2-propylpentanoyl glutamate, VPA-GLU) and valproyl glutamine (2-propylpentanoyl glutamine, VPA-GLN) in the urine, serum, and cerebrospinal fluid (CSF) of patients on valproic acid (VPA) therapy. Moreover, the identification of valproyl glycine (2-propylpentanoyl glycine, VPA-GLY) in the serum and urine of patients on VPA, albeit in trace concentrations, is also reported here. The three amino acid conjugates excreted in urine accounted for about 1% of the VPA dose in four patients who were on VPA therapy chronically and had reached steady state. VPA-GLU was quantitatively the most prominent metabolite (0.66-13.1 microg/mg creatinine) compared with VPA-GLN (0.78-9.93 microg/mg creatinine) and VPA-GLY (trace-1.0 microg/mg creatinine) in overnight urine samples of all patients studied (n = 29). The relatively low serum concentrations of the three amino acid conjugates of VPA in six patients suggest that the metabolites are readily excreted once formed. In contrast, whereas VPA GLY was absent in the CSF of one patient on VPA, the concentrations of VPA-GLU and VPA-GLN in this CSF sample were 9 and 5 times, respectively, their corresponding serum concentrations.

Oxcarbazepine, a keto-analogue of carbamazepine, was recently approved in the United States for the treatment of seizures of partial onset. Some patients treated with oxcarbazepine showed the development of hyponatremia, which in most instances was asymptomatic. Understanding the mechanisms by which oxcarbazepine can lead to a reduction of serum sodium levels could have therapeutic implications for the few patients in whom symptomatic hyponatremia develops. In this study, we evaluated sodium and water handling in patients with epilepsy and in healthy subjects titrated over 3 weeks to a maximum daily oxcarbazepine dose of 2,400mg. All subjects were evaluated in a hospital setting after an overnight fast and after an acute water-load test performed before oxcarbazepine exposure and after maintenance on the medication for 3 weeks. Before oxcarbazepine exposure, the percentage of water load excreted was normal as both groups excreted more than 80% of the administered water load. After the intake of oxcarbazepine, the water load resulted in a reduction of the serum sodium and free water clearance without a concomitant increase in the arginine vasopressin serum levels. Most subjects in both groups failed to excrete 80% or more of the water load, suggesting that the effect of oxcarbazepine is physiological. We found that, after the water load, serum sodium and free water clearance were diminished in both groups without a concomitant increase in the arginine vasopressin serum levels. These findings indicate that oxcarbazepine-induced hyponatremia is not attributable to the syndrome of inappropriate secretion of antidiuretic hormone. Possible mechanisms include a direct effect of oxcarbazepine on the renal collecting tubules or an enhancement of their responsiveness to circulating antidiuretic hormone.

We undertook a retrospective analysis of epilepsy patients referred and treated for more than 6 months with the ketogenic diet during 1994-1999 at Connecticut Children's Medical Center. Outcome measures included antiepileptic drug number, seizure frequency, electroencephalogram background/paroxysmal activity, and adverse effects at 6 months and 1 year on the ketogenic diet. The final cohort included 24 of 48 referred patients (mean age, 6.5 years; range = 1-15 years of age). The etiology of epilepsy was equally divided between idiopathic and cryptogenic epilepsy and symptomatic epilepsy. Intention to treat analysis revealed that 35% (17 of 48) achieved more than 50% reduction in seizure frequency, and 8.5% (four of 48) were seizure-free by 6 months. A sustained 50% or greater reduction at 1 year was observed in 23% (11 of 48), and the same 8.5% (four of 48) remained seizure-free. None of these improvements were statistically related to age (P = 0.97), sex (P = 0.78), or epilepsy etiology (P = 0.80). The number of antiepileptic drugs used per patient decreased. Electroencephalogram at 1 year demonstrated an improvement in background in 31% (five of 16 patients) and a reduction in paroxysmal features in 37.5% (6 of 16 patients). Most adverse effects were mild, self-limited, and occurred early. Hyperuricemia (more than 6 mg/dL) was more persistent in three patients.

Objective: To evaluate the effectiveness, tolerability, and adverse effects of the ketogenic diet in infants with refractory epilepsy., Methods: A retrospective review of 32 infants who had been treated with the ketogenic diet at a large metropolitan institution., Results: Most infants (71%) were able to maintain strong ketosis. The overall effectiveness of the diet in infants was similar to that reported in the literature for older children; 19.4% became seizure-free, and an additional 35.5% had >50% reduction in seizure frequency. The diet was particularly effective for patients with infantile spasms/myoclonic seizures. There were concomitant reductions in antiepileptic medications. The majority of parents reported improvements in seizure frequency and in their child's behavior and function, particularly with respect to attention/alertness, activity level, and socialization. The diet generally was well-tolerated, and 96.4% maintained appropriate growth parameters. Adverse events, all reversible and occurring in one patient each, included renal stone, gastritis, ulcerative colitis, alteration of mentation, and hyperlipidemia., Conclusion: The ketogenic diet should be considered safe and effective treatment for infants with intractable seizures.

Aim: To investigate the effect of prolonged use of antiepileptic drugs on renal function in children., Methods: Prospective study of 72 children (aged 3-18 years) with epilepsy, on either monotherapy (n = 44) or combined therapy (n = 28). The length of treatment varied from 1 to 13 years. Drugs used were valproic acid, carbamazepine, ethosuximide, clonazepam, clobazepam, and vigabatrin., Results: In 65 patients plasma concentrations of the drugs were in the therapeutic range. In the remaining seven, plasma concentrations were slightly high. In 33 patients urinary N-acetyl-beta-D-glucosaminidase (NAG) activity was raised. The incidence of pathological NAG indices was significantly higher in the combined therapy group than in the monotherapy group. There were also significant differences in the NAG indices of patients depending on the duration of therapy., Conclusions: Results suggest that chronic use of some antiepileptic drugs-in spite of normal blood concentrations-may alter tubular function, and the dysfunction may result in clinical symptoms. Therefore, we recommend screening of tubular function in these patients.

We report a GC/NICI-MS assay and a LC/ESI-MS/MS assay for the analysis of N-acetylcysteine (NAC) conjugates of (E)-2,4-diene VPA (NAC I and NAC II) identified in humans. The assay also includes the analysis of the NAC conjugate of 4,5-epoxy VPA (NAC III), an identified metabolite in rats treated with 4-ene VPA for its use in metabolic studies in animals. The highly sensitive GC/MS assay was designed to monitor selectively the diagnostic and most abundant [M - 181](-) fragment anion of the di-PFB derivatives of NAC I, NAC II, and NAC IV, the internal standard (IS) and the PFB derivative of NAC III. The higher selectivity of LC/MS/MS methodology was the basis for an assay which could identify and quantitate the underivatized conjugates simultaneously using MRM of the diagnostic ions m/z 130 and 123 arising from the CID of their protonated molecular ions [MH](+). The GC/MS assay employed liquid-liquid extraction whereas the LC/MS/MS assay used a solid-phase extraction procedure. Linearity ranges of the calibration curves were 0.10-5.0microg ml(-1) by GC/MS and 0.10-1.0microg ml(-1) by LC/MS/MS for NAC I, NAC II and NAC III (r(2) = 0.999 or better). Both assays were validated for NAC I and NAC II and provided good inter- and intra-assay precision and accuracy for NAC I and NAC II. The LOQ by LC/MS/MS was 0.1microg ml(-1), representing 1 ng of NAC I and NAC II. The same LOQ (0.1microg ml(-1)) was observed by GC/MS and was equivalent to 100 pg of each metabolite. NAC III was detected at concentrations as low as 0.01 microg ml(-1) by both methods. The total urinary excretion of the NAC conjugates in four patients on VPA therapy was determined to be 0.004-0.088% of a VPA dose by GC/MS and 0.004-0. 109% of a VPA dose by LC/MS/MS., (Copyright 2000 John Wiley & Sons, Ltd.)

Cross-sectional analysis of children undergoing treatment with anti-epileptic drugs has shown an increased urinary excretion of tubular enzymes and proteins. This has usually been interpreted as a consequence of subclinical renal-tubular damage or enzyme induction. We measured excretion of tubular enzymes and proteins in 29 children who suffered from epileptic seizures and in 27 control children. Investigations were undertaken at diagnosis before the start of treatment and 3-4 months later. At diagnosis we found a slightly, but statistically significant increased excretion of N-acetyl-beta-glucosaminidase and alpha1-microglobulin. There was no significant difference between patients with an idiopathic and symptomatic aetiology of seizures or between patients with different seizure types. At the second investigation, in children treated with carbamazepine or valproate, no further increase occurred. We conclude that the increased excretion of tubular enzymes and proteins in children with epilepsy is most probably not due to a side-effect of the anti-epileptic drugs, but to a physiological alteration associated with the epilepsy itself. While the cause is unknown, the influence of serotonin metabolism is discussed.

Acquired epileptic aphasia remains a poorly understood entity, which is frequently difficult to treat. Previously described treatment modalities have included antiepileptic drugs, corticosteroids, intravenous immunoglobulin, and multiple subpial transections. We describe three patients with acquired epileptic aphasia refractory to traditional treatments who were successfully treated with the ketogenic diet. All three patients had lasting improvement of their language, behavior, and seizures for 26, 24, and 12 months, respectively. This is the first reported series of patients with acquired epileptic aphasia successfully treated with the ketogenic diet, and suggests a new therapeutic alternative for patients with this often difficult-to-treat disorder.

This case-controlled study explored the relationship between bone mineral density (BMD) and long-term treatment with antiepileptic drugs (AEDs) in older adults with epilepsy. Seventy-eight patients (47 post-menopausal females, 31 males, aged 47-76 years) with epilepsy participated in the study. Each had only ever received treatment with either enzyme-inducing (n = 52) or non-inducing (n = 26) AEDs. Individuals were matched for age, sex, height and weight with a drug-naive control. All patients underwent bone densitometry at the lumbar spine and femoral neck and had blood sampling and urine collected for a range of bone markers. Male patients had lower BMD than controls at the lumbar spine (P < 0.01) and neck of the femur (P < 0.005). Female patients had significantly reduced bone density at the femoral neck (P < 0.05) only. AED usage was independently associated with an overall reduction in bone density at femoral sites and contributed to just over 5% of the variance at the femoral neck. Duration of treatment and type of AED were not independent factors for reduction in BMD. This case-controlled study supports the hypothesis that long-term AED therapy is an independent risk factor for reduced BMD in epileptic patients. Adults receiving treatment for epilepsy are at higher risk of osteoporosis and should be offered bone densitometry., (Copyright 1999 BEA Trading Ltd.)

Vigabatrin (gamma-vinyl gamma aminobutyric acid), a recently developed antiepileptic drug, has been extensively evaluated in the treatment of drug-resistant epilepsy. Several case reports demonstrated that vigabatrin affects urinary excretion of several amino and organic acids. Fourteen children were investigated for the presence of abnormal urinary amino acids before and after treatment with vigabatrin. All demonstrated increased urinary excretion of amino acids, particularly beta-alanine, gamma-aminobutyric acid, and beta-aminoisobutyric acid while on vigabatrin, which were not detected when off medication. These results emphasize the importance of obtaining urine for metabolic evaluation before the administration of vigabatrin.

Alteration of monoamine levels by some antiepileptic drugs (AEDs) was elucidated in this study. Lamotrigine (LTG) is a new AED, acting the sodium-channels. LTG was given as add-on therapy to 16 patients aged 4.5-18 yrs with intractable epilepsy and comedicated with carbamazepine or valproate. An equal group of epileptics with comparable clinical characteristics and treatment served as control. Plasma and urinary (24 h-samples) serotonin and 5-HIAA were determined before onset of LTG therapy and after 2-3 months. HPLC and electrochemical detection was used for the determination of serotonin (5-HT) and 5-hydroxy indoleacetic acid (5-HIAA). No significant effect of LTG on both urinary 5-HT and 5-HIAA levels was found, whereas plasma 5-HT concentrations significantly decreased in comparison with levels before LTG starting and relevant values in controls. This findings was noted in 7/16 children with favourable response to LTG. Increased serotonin catabolism may be result of LTG action.

D-2-Hydroxyglutaric aciduria has been observed in patients with extremely variable clinical symptoms, creating doubt about the existence of a disease entity related to the biochemical finding. An international survey of patients with D-2-hydroxyglutaric aciduria was initiated to solve this issue. The clinical history, neuroimaging, and biochemical findings of 17 patients were studied. Ten of the patients had a severe early-infantile-onset encephalopathy characterized by epilepsy, hypotonia, cerebral visual failure, and little development. Five of these patients had a cardiomyopathy. In neuroimaging, all patients had a mild ventriculomegaly, often enlarged frontal subarachnoid spaces and subdural effusions, and always signs of delayed cerebral maturation. In all patients who underwent neuroimaging before 6 months, subependymal cysts over the head or corpus of the caudate nucleus were noted. Seven patients had a much milder and variable clinical picture, most often characterized by mental retardation, hypotonia, and macrocephaly, but sometimes no related clinical problems. Neuroimaging findings in 3 patients variably showed delayed cerebral maturation, ventriculomegaly, or subependymal cysts. Biochemical findings included elevations of D-2-hydroxyglutaric acid in urine, plasma, and cerebrospinal fluid in both groups. Cerebrospinal fluid gamma-aminobutyric acid was elevated in almost all patients investigated. Urinary citric acid cycle intermediates were variably elevated. The conclusion of the study is that D-2-hydroxyglutaric aciduria is a distinct neurometabolic disorder with at least two phenotypes.