Endovascular aneurysm repair of aortoiliac aneurysms with an iliac side-branched stent graft: studying the morphological applicability of the Cook device.

OBJECTIVE/BACKGROUND: Endovascular aneurysm (EVAR) repair of an aortic aneurysm extending to the iliac artery remains a challenge. Interventional occlusion of the internal iliac artery (IIA) intending to create a distal landing zone in the external iliac artery is a common approach with inherent morbidity (e.g., buttock claudication, impotence). Alternatively, iliac side-branched stent grafts can maintain pelvic blood supply, but the applicability is limited. The objective was to investigate the morphological applicability of the Cook Zenith branched graft (ZBIS) among patients with aorto-iliac or isolated iliac aneurysms. METHODS: This was a retrospective single centre analysis of 66 patients (60 men; median age 74 years, range 53-90 years) undergoing repair of aorto-iliac aneurysms (open repair, IIA occlusion prior to EVAR and ZBIS) between January 2008 and December 2012. All available computed tomography angiograms with post-processing imaging were compared with the criteria for morphological applicability to (i) the manufacturer's instruction for use (IFU), and (ii) to criteria published in the literature, as well as (iii) to the institutional protocol. RESULTS: In 66 patients, 88 targeted iliac aneurysms were studied. Of these, 36/88 (40.9%) were compliant with the manufacturer's IFU, 35/88 (39.8%) were compliant with the published criteria, and 51/88 (58.0%) were compliant with the in house protocol. The most common morphological exclusion criterion was an aneurysmal IIA. CONCLUSION: In the present cohort with aorto-iliac aneurysm, a maximum of 58% could have been treated with an iliac side branch based on the current experience. In particular, an aneurysmal IIA seems to be a limiting factor for the use of the iliac side-branched stent graft.

Lower limb salvage: indication and decision making for replantation, revascularisation and amputation.

Defining reproducible criteria for lower extremity salvage following severe high-energy trauma continues to be one of the most challenging and controversially discussed fields in orthopaedic surgery. At present, however, the difficult performance, limited availability and number of valid reconstructive options for complex injury types, i. e. simultaneous osteoligamentous trauma with neurovascular lesions and severe soft tissue defects ("composite/compound multilayer defects") represent the decisive prognostic injury components triggering and determining the fate of the limb. Consequently, due to the complex injury pattern of the extremity and the overall situation of multiple injured patient the treatment and decision making has to be made in a priority-adapted algorithm. In this treatment algorithm interdisciplinary cooperation with vascular and plastic surgeons is of tremendous importance. Although the number of severely injured patients remains stable in the last decade, changes in the treatment algorithms result from increased survival rates of multiple injured patients and improved modern reconstructive options leading to continuously increasing rates of salvaged limbs. This paper aimed to systematically review the current literature for lower extremity injuries in order to unravel the different surgical treatment options and provide guidelines for decision making with corresponding treatment algorithms for limb salvage. Furthermore, the experiences in the management of mangled extremities in our centre are presented and illustrated/underscored with different cases.

Refsum disease is caused by mutations in the phytanoyl-CoA hydroxylase gene.

Refsum disease is an autosomal-recessively inherited disorder characterized clinically by a tetrad of abnormalities: retinitis pigmentosa, peripheral neuropathy, cerebellar ataxia and elevated protein levels in the cerebrospinal fluid (CSF) without an increase in the number of cells in the CSF. All patients exhibit accumulation of an unusual branched-chain fatty acid, phytanic acid (3,7,11,15-tetramethylhexadecanoic acid), in blood and tissues. Biochemically, the disease is caused by the deficiency of phytanoyl-CoA hydroxylase (PhyH), a peroxisomal protein catalyzing the first step in the alpha-oxidation of phytanic acid. We have purified PhyH from rat-liver peroxisomes and determined the N-terminal amino-acid sequence, as well as an additional internal amino-acid sequence obtained after Lys-C digestion of the purified protein. A search of the EST database with these partial amino-acid sequences led to the identification of the full-length human cDNA sequence encoding PhyH: the open reading frame encodes a 41.2-kD protein of 338 amino acids, which contains a cleavable peroxisomal targeting signal type 2 (PTS2). Sequence analysis of PHYH fibroblast cDNA from five patients with Refsum disease revealed distinct mutations, including a one-nucleotide deletion, a 111-nucleotide deletion and a point mutation. This analysis confirms our finding that Refsum disease is caused by a deficiency of PhyH.

Pharmacologic rescue of lethal seizures in mice deficient in succinate semialdehyde dehydrogenase.

Succinate semialdehyde dehydrogenase (ALDH5A1, encoding SSADH deficiency is a defect of 4-aminobutyric acid (GABA) degradation that manifests in humans as 4-hydroxybutyric (gamma-hydroxybutyric, GHB) aciduria. It is characterized by a non-specific neurological disorder including psychomotor retardation, language delay, seizures, hypotonia and ataxia. The current therapy, vigabatrin (VGB), is not uniformly successful. Here we report the development of Aldh5a1-deficient mice. At postnatal day 16-22 Aldh5a1-/- mice display ataxia and develop generalized seizures leading to rapid death. We observed increased amounts of GHB and total GABA in urine, brain and liver homogenates and detected significant gliosis in the hippocampus of Aldh5a1-/- mice. We found therapeutic intervention with phenobarbital or phenytoin ineffective, whereas intervention with vigabatrin or the GABAB receptor antagonist CGP 35348 (ref. 2) prevented tonic-clonic convulsions and significantly enhanced survival of the mutant mice. Because neurologic deterioration coincided with weaning, we hypothesized the presence of a protective compound in breast milk. Indeed, treatment of mutant mice with the amino acid taurine rescued Aldh5a1-/- mice. These findings provide insight into pathomechanisms and may have therapeutic relevance for the human SSADH deficiency disease and GHB overdose and toxicity.

A lymphoblast model for IDH2 gain-of-function activity in d-2-hydroxyglutaric aciduria type II: novel avenues for biochemical and therapeutic studies.

The recent discovery of heterozygous isocitrate dehydrogenase 2 (IDH2) mutations of residue Arg(140) to Gln(140) or Gly(140) (IDH2(wt/R140Q), IDH2(wt/R140G)) in d-2-hydroxyglutaric aciduria (D-2-HGA) has defined the primary genetic lesion in 50% of D-2-HGA patients, denoted type II. Overexpression studies with IDH1(R132H) and IDH2(R172K) mutations demonstrated that the enzymes acquired a new function, converting 2-ketoglutarate (2-KG) to d-2-hydroxyglutarate (D-2-HG), in lieu of the normal IDH reaction which reversibly converts isocitrate to 2-KG. To confirm the IDH2(wt/R140Q) gain-of-function in D-2-HGA type II, and to evaluate potential therapeutic strategies, we developed a specific and sensitive IDH2(wt/R140Q) enzyme assay in lymphoblasts. This assay determines gain-of-function activity which converts 2-KG to D-2-HG in homogenates of D-2-HGA type II lymphoblasts, and uses stable-isotope-labeled 2-keto[3,3,4,4-(2)H(4)]glutarate. The specificity and sensitivity of the assay are enhanced with chiral separation and detection of stable-isotope-labeled D-2-HG by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Eleven potential inhibitors of IDH2(wt/R140Q) enzyme activity were evaluated with this procedure. The mean reaction rate in D-2-HGA type II lymphoblasts was 8-fold higher than that of controls and D-2-HGA type I cells (14.4nmolh(-1)mgprotein(-1) vs. 1.9), with a corresponding 140-fold increase in intracellular D-2-HG level. Optimal inhibition of IDH2(wt/R140Q) activity was obtained with oxaloacetate, which competitively inhibited IDH2(wt/R140Q) activity. Lymphoblast IDH2(wt/R140Q) showed long-term cell culture stability without loss of the heterozygous IDH2(wt/R140Q) mutation, underscoring the utility of the lymphoblast model for future biochemical and therapeutic studies.

Guanidinoacetate methyltransferase deficiency: first steps to newborn screening for a treatable neurometabolic disease.

BACKGROUND: GAMT deficiency is an autosomal recessive disorder of creatine biosynthesis resulting in severe neurological complications in untreated patients. Currently available treatment is only successful to stop disease progression, but is not sufficient to reverse neurological complications occurring prior to diagnosis. Normal neurodevelopmental outcome in a patient, treated in the newborn period, highlights the importance of early diagnosis. METHODS: Targeted mutation analysis (c.59G>C and c.327G>A) in the GAMT gene by the QIAxcel system and GAA measurement by a novel two-tier method were performed in 3000 anonymized newborn blood dot spot cards. RESULTS: None of the targeted mutations were detected in any newborn. Two novel heterozygous variants (c.283_285dupGTC; p.Val95dup and c.278_283delinsCTCGATGCAC; p.Asp93AlafsX35) were identified by coincidence. Carrier frequency for these insertion/deletion types of GAMT mutations was 1/1475 in this small cohort of newborns. GAA levels were at or above the 99th percentile (3.12 µmol/l) in 4 newborns. Second-tier testing showed normal results for 4 newborns revealing 0.1% false positive rate. No GAMT mutations were identified in 4 of the newborns with elevated GAA levels in the first tier testing. CONCLUSION: This is the first two-tier study to investigate carrier frequency of GAMT deficiency in the small cohort of newborn population to establish evidence base for the first steps toward newborn screening for this treatable neurometabolic disorder.

Cellular hypomethylation is associated with impaired nitric oxide production by cultured human endothelial cells.

Hyperhomocysteinemia (HHcy) is a risk factor for vascular disease, but the underlying mechanisms remain incompletely defined. Reduced bioavailability of nitric oxide (NO) is a principal manifestation of underlying endothelial dysfunction, which is an initial event in vascular disease. Inhibition of cellular methylation reactions by S-adenosylhomocysteine (AdoHcy), which accumulates during HHcy, has been suggested to contribute to vascular dysfunction. However, thus far, the effect of intracellular AdoHcy accumulation on NO bioavailability has not yet been fully substantiated by experimental evidence. The present study was carried out to evaluate whether disturbances in cellular methylation status affect NO production by cultured human endothelial cells. Here, we show that a hypomethylating environment, induced by the accumulation of AdoHcy, impairs NO production. Consistent with this finding, we observed decreased eNOS expression and activity, but, by contrast, enhanced NOS3 transcription. Taken together, our data support the existence of regulatory post-transcriptional mechanisms modulated by cellular methylation potential leading to impaired NO production by cultured human endothelial cells. As such, our conclusions may have implications for the HHcy-mediated reductions in NO bioavailability and endothelial dysfunction.

4-Hydroxybutyric aciduria associated with catheter usage: a diagnostic pitfall in the identification of SSADH deficiency.

Succinic semialdehyde dehydrogenase deficiency is a slowly progressive to static neurological disorder featuring elevated concentrations of 4-hydroxybutyric acid in body fluids. We present two patients with elevated 4-hydroxybutyric acid in urine which was later shown to be linked to catheter usage.

Elevated concentrations of sedoheptulose in bloodspots of patients with cystinosis caused by the 57-kb deletion: implications for diagnostics and neonatal screening.

Cystinosis is an autosomal recessive lysosomal storage disease caused by mutations in CTNS. The most prevalent CTNS mutation is a homozygous 57-kb deletion that also includes an adjacent gene named SHPK (CARKL), encoding sedoheptulokinase. Patients with this deletion have elevated urinary concentrations of sedoheptulose. Using derivatisation with pentafluorobenzyl hydroxylamine and liquid chromatography-tandem mass spectrometry (LC-MS/MS), we developed a new sensitive method for the quantification of sedoheptulose in dried blood spots. This method can be utilized as a quick screening test to detect cystinosis patients homozygous for the 57-kb deletion in CTNS; which is the most common mutation of cystinosis. Sedoheptulose concentrations in the deleted patients were 6 to 23 times above the upper limit for controls. The assessment of sedoheptulose in a bloodspot from a known cystinosis patient homozygous for the 57-kb deletion retrieved from the Dutch neonatal screening program showed that sedoheptulose was already elevated in the neonatal period. There was no overlap in sedoheptulose levels between cystinosis patients homozygous for the 57-kb deletion and cystinosis patients not homozygous for this deletion. Our presented method can be used prior to mutation analysis to detect cystinosis patients homozygous for the 57-kb deletion. We feel that the presented method enables fast (pre)-symptomatic detection of cystinosis patients homozygous for the 57-kb deletion, allowing early treatment.

Clinical features and X-inactivation in females heterozygous for creatine transporter defect.

The creatine transporter defect is an X-linked cause of mental retardation. We investigated the clinical features and pattern of X-inactivation in a Dutch cohort of eight female heterozygotes. We show that symptoms of the creatine transporter defect (mental retardation, learning difficulties, and constipation) can be present in female heterozygotes. We further show that the diagnosis in females is not straightforward: (i) The creatine/creatinine ratio in urine was elevated only in three of eight females. (ii) Although as a group the females had a significantly decreased cerebral creatine concentration, individual females had creatine concentrations overlapping with normal controls. (iii) Skewed X-inactivation was found in the cultured fibroblasts, in favour of either the mutated or the wild-type allele, leading to either deficient or normal results in the creatine uptake studies in fibroblasts. Thus, screening by these tests is unreliable for the diagnosis. In addition, we found no consistent skewing of the X-inactivation in peripheral tissues indicating that there is no selection against the creatine transporter defect. We conclude that testing for creatine transporter defect should be considered in females with (mild) mental retardation. Screening by DNA analysis of the SLC6A8 gene is recommended.

N-acetylaspartylglutamate in CNS hypomyelination.

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

Comparative genomics of aldehyde dehydrogenase 5a1 (succinate semialdehyde dehydrogenase) and accumulation of gamma-hydroxybutyrate associated with its deficiency.

Succinic semialdehyde dehydrogenase (SSADH; aldehyde dehydrogenase 5A1 [ALDH5A1]; locus 6p22) occupies a central position in central nervous system (CNS) neurotransmitter metabolism as one of two enzymes necessary for gamma-aminobutyric acid (GABA) recycling from the synaptic cleft. Its importance is highlighted by the neurometabolic disease associated with its inherited deficiency in humans, as well as the severe epileptic phenotype observed in Aldh5a1(-/-) knockout mice. Expanding evidence now suggests, however, that even subtle decreases in human SSADH activity, associated with rare and common single nucleotide polymorphisms, may produce subclinical pathological effects. SSADH, in conjunction with aldo-keto reductase 7A2 (AKR7A2), represent two neural enzymes responsible for further catabolism of succinic semialdehyde, producing either succinate (SSADH) or gamma-hydroxybutyrate (GHB; AKR7A2). A GABA analogue, GHB is a short-chain fatty alcohol with unusual properties in the CNS and a long pharmacological history. Moreover, SSADH occupies a further role in the CNS as the enzyme responsible for further metabolism of the lipid peroxidation aldehyde 4-hydroxy-2-nonenal (4-HNE), an intermediate known to induce oxidant stress. Accordingly, subtle decreases in SSADH activity may have the capacity to lead to regional accumulation of neurotoxic intermediates (GHB, 4-HNE). Polymorphisms in SSADH gene structure may also associate with quantitative traits, including intelligence quotient and life expectancy. Further population-based studies of human SSADH activity promise to reveal additional properties of its function and additional roles in CNS tissue.

The screening of SLC6A8 deficiency among Estonian families with X-linked mental retardation.

The urinary creatine:creatinine (Cr:Crn) ratio was measured in males from 49 families with a family history compatible with X-linked mental retardation (XLMR) in order to estimate the prevalence of SLC6A8 deficiency in Estonia. We identified 11 boys from 9 families with an increased urinary Cr:Crn ratio (18%). In three related boys, a hemizygous missense mutation (c.1271G>A; p.Gly424Asp) was identified. Their mother was heterozygous for the same mutation. Although many missense mutations have been described, the p.Gly424Asp mutation has not been previously reported. The clinical expression varied widely among affected males of this family. Patients 1 and 3 had relatively mild clinical expression (mild mental retardation (MR) and attention deficit disorder), but patient 2 had all typical clinical signs of SLC6A8 defect such as moderate MR, autistic features, expressive dysphasia and epilepsy. Among our patients, we saw significant problems in speech and language development combined with attention and behavioural difficulties. The number of false-positive biochemical results with increased urinary Cr:Crn ratio was higher (18%) in our study than in previous reports (1.8­10%). We therefore suggest that repeated biochemical testing should be performed before DNA sequencing analysis. Our study suggests that 2% (95% confidence limits: 0.05­11.1%) of this Estonian XLMR panel are due to mutations in the SLC6A8, which is similar to the prevalence reported in other populations. We therefore conclude that creatine transporter deficiency is a relatively common genetic disorder in males with sporadic or familiar MR and diagnostic screening of them should always include screening for SLC6A8 deficiency.

Use of a long-chain triglyceride-restricted/medium-chain triglyceride-supplemented diet in a case of malonyl-CoA decarboxylase deficiency with cardiomyopathy.

Malonyl coenzyme A (CoA) decarboxylase (EC 4.1.1.9, MCD) deficiency, or malonic aciduria, is a rare inborn error of metabolism characterised by a variable phenotype of developmental delay, seizures, cardiomyopathy and acidosis. There is no consensus for dietary treatment in this condition. This case describes the effect of a long-chain triglyceride (LCT)-restricted/medium-chain triglyceride (MCT)-supplemented diet upon the progress of an affected child. A full-term Asian girl of birth weight 3590 g was screened for malonic aciduria after birth due to a positive family history. She had elevated urine malonic and methylmalonic acids and was presumably homozygous for a deleterious mutation in the MLYCD gene. Her echocardiography showed mild cardiomyopathy at 0.5 months of age, but heart function was good. She was treated with carnitine 100 mg/kg per day and continued a high-energy formula feed, as her growth was slow. At 3 months of age, echocardiography showed deteriorating cardiac function with a fractional shortening of 18%. She started an angiotensin-converting enzyme (ACE) inhibitor (Captopril). Over the next few months, her diet was altered to comprise 1.9% energy from LCT, 25% from MCT and the remainder carbohydrate. Cardiac function improved and was optimal at 23 months of age, with a fractional shortening of 28% and good systolic function. During a period of low MCT intake, her cardiac function was noted to deteriorate. This reversed and stabilised following reinstatement of the diet. This case of malonic aciduria with cardiomyopathy demonstrates improvement in cardiac function attributable to LCT-restricted/MCT-supplemented diet.

Clinical, radiological and genetic aspects of leukodystrophies.

The authors summarize the pathomechanism of the myelination process, the clinical, radiological and the genetical aspects of the leukodystrophies, as in 18q deletion syndrome, adrenoleukodysrtophy, metachromatic leukodystrophy, Pelizaeus-Merzbacher leukodystrophy, Alexander disease and olivo-ponto-cerebellar atrophy (OPCA).

Low elevation of Svalbard glaciers drives high mass loss variability.

Compared to other Arctic ice masses, Svalbard glaciers are low-elevated with flat interior accumulation areas, resulting in a marked peak in their current hypsometry (area-elevation distribution) at  ~450 m above sea level. Since summer melt consistently exceeds winter snowfall, these low-lying glaciers can only survive by refreezing a considerable fraction of surface melt and rain in the porous firn layer covering their accumulation zones. We use a high-resolution climate model to show that modest atmospheric warming in the mid-1980s forced the firn zone to retreat upward by  ~100 m to coincide with the hypsometry peak. This led to a rapid areal reduction of firn cover available for refreezing, and strongly increased runoff from dark, bare ice areas, amplifying mass loss from all elevations. As the firn line fluctuates around the hypsometry peak in the current climate, Svalbard glaciers will continue to lose mass and show high sensitivity to temperature perturbations.

Measurement of D: -2-hydroxyglutarate dehydrogenase activity in cell homogenates derived from D: -2-hydroxyglutaric aciduria patients.

D: -2-Hydroxyglutaric aciduria (D: -2-HGA) is a neurometabolic disorder characterized by elevated levels of D: -2-hydroxyglutarate (D: -2-HG) in physiological fluids. Recent findings revealed that mutations in the D2HGDH gene, encoding D: -2-hydroxyglutarate dehydrogenase, cause D: -2-HGA. So far, a functionalenzyme assay to determine D: -2-hydroxyglutarate dehydrogenase activity, converting D: -2-HG into 2-ketoglutarate (2-KG), has been unavailable. We have now developed a unique enzyme assay for the determination of D: -2-hydroxyglutarate dehydrogenase activity in cells derived from D: -2-HGA patients and controls. The enzyme assay was performed using enantiomerically pure stable-isotope-labelled D: -2-hydroxy[3,3,4,4-(2)H(4)]glutarate. This substrate is convertedby D: -2-hydroxyglutarate dehydrogenase into 2-[3,3,4,4-(2)H(4)]ketoglutarate, which is subsequently converted into L: -[3,3,4,4-(2)H(4)]glutamate by L: -glutamate dehydrogenase, present in saturating amounts in cell homogenates. Enzyme activities were quantified using LC-MS/MS. The mean activities in control fibroblast and lymphoblast homogenates were 298 +/- 207 and 1670 +/- 940 pmol/h per mg protein, respectively. In fibroblast and lymphoblast cell lines derived from patients with pathogenic mutations in the D2HGDH gene, considerably decreased enzyme activities (e.g. <41 pmol/h per mg protein) were found compared with controls. This enzyme assay will have additional utility in further differentiating patients with D: -2-HGA and L: -2-HGA and in assessing the residual activities linked to pathogenic mutations in the D2HGDH gene.

Sepiapterin reductase deficiency in a 2-year-old girl with incomplete response to treatment during short-term follow-up.

Sepiapterin reductase (SR) catalyses the last step in the tetrahydrobiopterin biosynthesis pathway; it converts 6-pyruvoyl-tetrahydropterin (6-PTP) to BH(4) in an NADPH-dependent reaction. SR deficiency is a very rare autosomal recessive disorder with normal phenylalanine (Phe) concentration in blood and diagnostic abnormalities are detected in CSF. We present a 16-month-old girl with SR deficiency. From the newborn period she presented with an adaptation regulatory disorder. At the age of 3 months, abnormal eye movements with dystonic signs and at 4.5 months psychomotor retardation were noticed. Since that time axial hypotonia with limb spasticity (or rather delayed reflex development), gastro-oesophageal reflux and fatigue at the end of the day has been observed. Brain MRI was normal; EEG was without epileptiform discharges. Analysis of biogenic amine metabolites in CSF at the age of 16 months showed very low HVA and 5-HIAA concentrations. Analysis of CSF pterins revealed strongly elevated dihydrobiopterin (BH(2)), slightly elevated neopterin and elevated sepiapterin levels. Plasma and CSF amino acids concentrations were normal. A phenylalanine loading test showed increased Phe after 1 h, 2 h and 4 h and very high Phe/Tyr ratios. SR deficiency was confirmed in fibroblasts and a novel homozygous g.1330C>G (p.N127K) SPR mutation was identified. On L-dopa and then additionally 5-hydroxytryptophan, the girl showed slow but remarkable progress in motor and intellectual ability. Now, at the age of 3 years, she is able to sit; expressive speech is delayed (to 1 1/2 years), passive speech is well developed. Her visual-motor skills, eye-hand coordination and social development correspond to the age of 2 1/2 years.

Succinic semialdehyde dehydrogenase deficiency: lessons from mice and men.

Succinic semialdehyde dehydrogenase (SSADH) deficiency, a disorder of GABA degradation with subsequent elevations in brain GABA and GHB, is a neurometabolic disorder with intellectual disability, epilepsy, hypotonia, ataxia, sleep disorders, and psychiatric disturbances. Neuroimaging reveals increased T2-weighted MRI signal usually affecting the globus pallidus, cerebellar dentate nucleus, and subthalamic nucleus, and often cerebral and cerebellar atrophy. EEG abnormalities are usually generalized spike-wave, consistent with a predilection for generalized epilepsy. The murine phenotype is characterized by failure-to-thrive, progressive ataxia, and a transition from generalized absence to tonic-clonic to ultimately fatal convulsive status epilepticus. Binding and electrophysiological studies demonstrate use-dependent downregulation of GABA(A) and (B) receptors in the mutant mouse. Translational human studies similarly reveal downregulation of GABAergic activity in patients, utilizing flumazenil-PET and transcranial magnetic stimulation for GABA(A) and (B) activity, respectively. Sleep studies reveal decreased stage REM with prolonged REM latencies and diminished percentage of stage REM. An ad libitum ketogenic diet was reported as effective in the mouse model, with unclear applicability to the human condition. Acute application of SGS-742, a GABA(B) antagonist, leads to improvement in epileptiform activity on electrocorticography. Promising mouse data using compounds available for clinical use, including taurine and SGS-742, form the framework for human trials.

Development and implementation of a novel assay for L-2-hydroxyglutarate dehydrogenase (L-2-HGDH) in cell lysates: L-2-HGDH deficiency in 15 patients with L-2-hydroxyglutaric aciduria.

L-2-hydroxyglutaric aciduria (L-2-HGA) is a rare inherited autosomal recessive neurometabolic disorder caused by mutations in the gene encoding L-2-hydroxyglutarate dehydrogenase. An assay to evaluate L-2-hydroxyglutarate dehydrogenase (L-2-HGDH) activity in fibroblast, lymphoblast and/or lymphocyte lysates has hitherto been unavailable. We developed an L-2-HGDH enzyme assay in cell lysates based on the conversion of stable-isotope-labelled L-2-hydroxyglutarate to 2-ketoglutarate, which is converted into L-glutamate in situ. The formation of stable isotope labelled L-glutamate is therefore a direct measure of L-2-HGDH activity, and this product is detected by liquid chromatography-tandem mass spectrometry. A deficiency of L-2-HGDH activity was detected in cell lysates from 15 out of 15 L-2-HGA patients. Therefore, this specific assay confirmed the diagnosis unambiguously affirming the relationship between molecular and biochemical observations. Residual activity was detected in cells derived from one L-2-HGA patient. The L-2-HGDH assay will be valuable for examining in vitro riboflavin/FAD therapy to rescue L-2-HGDH activity.