Lawrence Sweetman

Anticonvulsant effects of a triheptanoin diet in two mouse chronic seizure models

We hypothesized that in epileptic brains citric acid cycle intermediate levels may be deficient leading to hyperexcitability. Anaplerosis is the metabolic refilling of deficient metabolites. Our goal was to determine the anticonvulsant effects of feeding triheptanoin, the triglyceride of anaplerotic heptanoate. CF1 mice were fed 0-35% calories from triheptanoin. Body weights and dietary intake were similar in mice fed triheptanoin vs. standard diet. Triheptanoin feeding increased blood propionyl-carnitine levels, signifying its metabolism. 35%, but not 20%, triheptanoin delayed development of corneal kindled seizures. After pilocarpine-induced status epilepticus (SE), triheptanoin feeding increased the pentylenetetrazole tonic seizure threshold during the chronically epileptic stage. Mice in the chronically epileptic stage showed various changes in brain metabolite levels, including a reduction in malate. Triheptanoin feeding largely restored a reduction in propionyl-CoA levels and increased methylmalonyl-CoA levels in SE mice. In summary, triheptanoin was anticonvulsant in two chronic mouse models and increased levels of anaplerotic precursor metabolites in epileptic mouse brains. The mechanisms of triheptanoins effects and its efficacy in humans suffering from epilepsy remain to be determined.

Postprandial changes in plasma acylcarnitine concentrations as markers of fatty acid flux in overweight and obesity.

This study determined whether reductions in postprandial plasma nonesterified fatty acid (FFA) flux would lead to reductions in plasma acylcarnitine (AC) concentrations. Plasma AC was measured by liquid chromatography with tandem mass spectrometry in the fasting state and over 6 hours after a high-fat (50% energy) meal was fed to 16 overweight and obese subjects with a wide range of insulin sensitivities. Body composition was measured by dual-energy x-ray absorptiometry; insulin sensitivity by insulin-modified, frequently sampled intravenous glucose tolerance test; substrate oxidation by indirect calorimetry; blood metabolite and hormone concentrations biochemically; and fatty acid flux by using stable isotope tracers. Lean body mass and fasting fat oxidation correlated positively (r > 0.522, P < .05), whereas glucose oxidation correlated negatively (r < -0.551, P < .04), with fasting AC. Postprandially, plasma glucose, insulin, and triglyceride concentrations increased; and FFA concentrations decreased significantly. The responses of plasma AC species depended on chain length and saturation, with C14:0, C16:0, and C18:0 remaining unchanged, and unsaturated species (eg, C14:1, C14:2) falling significantly (21%-46%, P < .03). Postmeal nadir AC concentrations were positively associated with lean body mass, postprandial fatty acid flux, and FFA concentrations (r > 0.515, P < .05). By contrast, nadir AC correlated negatively with insulin sensitivity and spillover of meal-derived fatty acids (r < -0.528, P < .04). Conditions that impact fatty acid flux contribute to the control of postprandial plasma AC concentrations. These data underscore the need for a better understanding of postprandial fatty acid oxidation and dietary fat delivery in the setting of adipose insulin resistance to determine how postprandial lipemia contributes to chronic disease risk.

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

BACKGROUNDnGAMT 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.nnnMETHODSnTargeted 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.nnnRESULTSnNone 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.nnnCONCLUSIONnThis 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.

Rapid determination of C4-acylcarnitine and C5-acylcarnitine isomers in plasma and dried blood spots by UPLC–MS/MS as a second tier test following flow-injection MS/MS acylcarnitine profile analysis

BACKGROUNDnFlow-injection MS/MS methods for elevated acylcarnitines are routinely performed in most newborn screening and biochemical genetics laboratories; however this technique cannot distinguish between isobaric compounds; therefore, chromatographic separation is required to quantitate isomers for differential diagnosis of some inborn errors of metabolism.nnnMETHODSnA UPLC-MS/MS method has been developed for the simultaneous quantitation of isobutyrylcarnitine and butyrylcarnitine, and a second UPLC-MS/MS method for the quantitation of isovalerylcarnitine, (S) and (R) 2-methylbutyrylcarnitine, pivaloylcarnitine and valerylcarnitine. Plasma and dried blood spots samples are extracted with methanol and derivatized with butanolic HCl. Deuterium labeled internal standards are used for quantitation. Separation is obtained using a methanol/water gradient with a C18 BEH, 1x100mm, 1.7microm UPLC column, at 60 degrees C; run time is less than 10min. The isomers are detected with a Quattro Premier triple quadrupole, with electrospray ionization in positive ion mode.nnnRESULTSnIntra-day precision in plasma and dried blood spots ranged from 1.4% to 14% and accuracy from 88% to 114% respectively for butyrylcarnitine and isobutyrylcarnitine. Precision for the isomers of C5-acylcarnitine ranged from 1.3% to 15% and accuracy 87% to 119%, respectively in plasma or dried blood spots. Inter-day precision was within 20% at each concentration of isobutyrylcarnitine and butyrylcarnitine. Precision for 2-methylbutyrylcarnitine and isovalerylcarnitine at concentrations above the normal range was within 24%.nnnCONCLUSIONSnTwo diagnostic tests based on the separation of C4-acylcarnitine and C5-acylcarnitine isomers by UPLC-MS/MS provide fast differential diagnosis of SCAD deficiency versus IBCD deficiency and IVA versus 2-MBCD deficiency. The separation of C5-acylcarnitines can reveal false elevation due to pivalic acid-containing antibiotics. Abnormal newborn screen results due to pivalate-generating prodrug antibiotics of maternal origin were confirmed. This separation of isomers can resolve multiple diagnostic challenges in both newborn screening and in cases with ambiguous metabolic test results.

Triheptanoin in acute mouse seizure models.

Triheptanoin, the triglyceride of heptanoate, is used to treat certain hereditary metabolic diseases in USA because of its anaplerotic potential. In two chronic mouse seizure models this clear tasteless oil was found to be reproducibly anticonvulsant. Here we investigated the effects of triheptanoin feeding in C3H and CD1 mice using standard acute seizure models. Feeding 30-40% triheptanoin (caloric intake) consistently elevated blood propionyl-carnitines, but inconsistent anticonvulsant effects were observed in the fluorothyl, pentylenetetrazole and 6Hz seizure models. A 2mA consistent increase in the maximal electroshock threshold was found after 3 weeks of 35% triheptanoin feeding (p=0.018). In summary, triheptanoin shows a unique anticonvulsant profile in seizure models, compared to other treatments that are in the clinic. Therefore, despite small and/or inconsistent effects of triheptanoin in acute seizure models, triheptanoin remains of interest as a potential add-on treatment for patients with medically refractory epilepsy.

A Novel Mutation in the HSD17B10 Gene of a 10-Year-Old Boy with Refractory Epilepsy, Choreoathetosis and Learning Disability

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 patients 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.

Risk of death in heart disease is associated with elevated urinary globotriaosylceramide

Background Elevated urinary globotriaosylceramide (Gb3) has been considered a hallmark of Fabry disease, an X‐linked lysosomal disorder that is a risk factor for most types of heart disease. Methods and Results We screened 1421 consecutive patients with common forms of heart disease for Fabry disease by measuring urinary Gb3 in whole urine using tandem mass spectrometry, α‐galactosidase A activity in dried blood spots, and we looked for GLA mutations by parallel sequencing of the whole gene (exons and introns) in pooled genomic DNA samples followed by Sanger sequencing verification. GLA variants were found in 13 patients. In the 1408 patients without GLA mutations, urinary Gb3 levels were significantly higher in heart disease patients compared to 116 apparently healthy controls (median difference=10.0 ng/mL and P<0.001). Urinary lipid profiling showed that levels of 5 other lipids significantly distinguished between urine of patients with Fabry disease (n=7) and heart disease patients with elevated urinary Gb3 (n=6). Sphingomyelin and Gb3 levels were abnormal in the left ventricular wall of patients with ischemic heart failure. Elevated levels of urinary Gb3 were independently associated with increased risk of death in the average follow‐up of 17 months (hazard ratio=1.59 for increase in Gb3 of 200, 95% CI=1.36 and 1.87, and P<0.0001). Conclusions In heart disease patients who do not have Fabry disease or GLA gene mutations, a higher level of urinary Gb3 is positively associated with near‐term mortality. The elevation of urinary Gb3 and that of other lipids suggests that heart disease is associated with multiorgan lipid abnormalities. Clinical Trial Registration URL: clinicaltrials.gov. Unique Identifier: NCT01019629.

Quantitation of gamma-hydroxybutyric acid in dried blood spots: Feasibility assessment for newborn screening of succinic semialdehyde dehydrogenase (SSADH) deficiency

OBJECTIVEnSSADH deficiency, the most prevalent autosomal recessive disorder of GABA degradation, is characterized by elevated gamma-hydroxybutyric acid (GHB). Neurological outcomes may be improved with early intervention and anticipatory guidance. Morbidity has been compounded by complications, e.g. hypotonia, in undiagnosed infants with otherwise routine childhood illnesses. We report pilot methodology on the feasibility of newborn screening for SSADH deficiency.nnnMETHODnDried blood spot (DBS) cards from patients affected with SSADH deficiency were compared with 2831 archival DBS cards for gamma-hydroxybutyric acid content. Following extraction with methanol, GHB in DBS was separated and analyzed using ultra high-performance liquid chromatography tandem mass spectrometry.nnnRESULTSnMethodology was validated to meet satisfactory accuracy and reproducibility criteria, including intra-day and inter-day validation. Archival refrigerated dried blood spot samples of babies, infants and children (N = 2831) were screened for GHB, yielding a mean +/- S.D. of 8 ± 5 nM (99.9%-tile 63 nM) (Min 0.0 Max 78 nM). The measured mean and median concentrations in blood spots derived from seven SSADH deficient patients were 1182 nM and 699 nM respectively (Min 124, Max 4851 nM).nnnCONCLUSIONSnGHB concentration in all 2831 dried blood spot cards was well below the lowest concentration of affected children. These data provide proof-of-principle for screening methodology to detect SSADH deficiency with applicability to newborn screening and earlier diagnosis.

Falsely elevated urinary Gb3 (globotriaosylceramide, CTH, GL3).

and C17-Gb3 as internal standards. Ten microliters are injected into a UPLC-MS/MS for the simultaneous determination of creatinine and Gb3. Urine of 100 healthy subjects from newborns to 65 years old was analyzed. The concentration of creatinine in urine varied widely due to diet or age: from less then 1 lmol/mL in infants and some adults to higher than 30 lmol/mL in adults. Gb3 in urine is usually expressed as Gb3/creatinine concentration because creatinine is believed to correct for the excretion of more or less concentrated urine [4]. Low creatinine (<4 lmol/mL) led to falsely elevated ratio Gb3/creatinine, especially (but not exclusively) in samples of infants and young children (Fig. 1). High creatinine falsely lowers the ratio Gb3/creatinine. The latter is especially problematic in case of individuals with moderately elevated Gb3, such as heterozygote females. The concentration of Gb3 expressed in lg/ mL of urine, does not correlate with the amount of creatinine

Neonatal presentation of ventricular tachycardia and a Reye-like syndrome episode associated with disturbed mitochondrial energy metabolism.

BackgroundHyperammonemia, hypoglycemia, hepatopathy, and ventricular tachycardia are common presenting features of carnitine-acylcarnitine translocase deficiency (Mendelian Inheritance in Man database: *212138), a mitochondrial fatty acid oxidation disorder with a lethal prognosis. These features have not been identified as the presenting features of mitochondrial cytopathy in the neonatal period.Case presentationWe describe an atypical presentation of mitochondrial cytopathy in a 2 day-old neonate. She presented with a Reye-like syndrome episode, premature ventricular contractions and ventricular tachycardia. Initial laboratory evaluation exhibited a large amount of 3-methylglutaconic acid on urine organic acid analysis, mild orotic aciduria and a nonspecific abnormal acylcarnitine profile. The evaluation for carnitine-acylcarnitine translocase deficiency and other fatty acid oxidation disorders was negative. The patient later developed a hypertrophic cardiomyopathy and continued to be affected by recurrent Reye-like syndrome episodes triggered by infections. A muscle biopsy exhibited signs of a mitochondrial cytopathy. During the course of her disease, her Reye-like syndrome episodes have subsided; however, cardiomyopathy has persisted along with fatigue and exercise intolerance.ConclusionsThis case illustrates that, in the neonatal period, hyperammonemia and ventricular tachycardia may be the presenting features of a lethal carnitine-acylcarnitine translocase deficiency or of a mitochondrial cytopathy, associated with a milder clinical course. This association broadens the spectrum of presenting phenotypes observed in patients with disturbed mitochondrial energy metabolism. Also, the presence of 3-methylglutaconic aciduria suggests mitochondrial dysfunction and mild orotic aciduria could potentially be used as a marker of mitochondrial disease.