The impact of metabolic stressors on mitochondrial homeostasis in a renal epithelial cell model of methylmalonic aciduria.

Methylmalonic aciduria (MMA-uria) is caused by deficiency of the mitochondrial enzyme methylmalonyl-CoA mutase (MUT). MUT deficiency hampers energy generation from specific amino acids, odd-chain fatty acids and cholesterol. Chronic kidney disease (CKD) is a well-known long-term complication. We exposed human renal epithelial cells from healthy controls and MMA-uria patients to different culture conditions (normal treatment (NT), high protein (HP) and isoleucine/valine (I/V)) to test the effect of metabolic stressors on renal mitochondrial energy metabolism. Creatinine levels were increased and antioxidant stress defense was severely comprised in MMA-uria cells. Alterations in mitochondrial homeostasis were observed. Changes in tricarboxylic acid cycle metabolites and impaired energy generation from fatty acid oxidation were detected. Methylcitrate as potentially toxic, disease-specific metabolite was increased by HP and I/V load. Mitophagy was disabled in MMA-uria cells, while autophagy was highly active particularly under HP and I/V conditions. Mitochondrial dynamics were shifted towards fission. Sirtuin1, a stress-resistance protein, was down-regulated by HP and I/V exposure in MMA-uria cells. Taken together, both interventions aggravated metabolic fingerprints observed in MMA-uria cells at baseline. The results point to protein toxicity in MMA-uria and lead to a better understanding, how the accumulating, potentially toxic organic acids might trigger CKD.

Fatty acid metabolism in aggressive B-cell lymphoma is inhibited by tetraspanin CD37.

The importance of fatty acid (FA) metabolism in cancer is well-established, yet the mechanisms underlying metabolic reprogramming remain elusive. Here, we identify tetraspanin CD37, a prognostic marker for aggressive B-cell lymphoma, as essential membrane-localized inhibitor of FA metabolism. Deletion of CD37 on lymphoma cells results in increased FA oxidation shown by functional assays and metabolomics. Furthermore, CD37-negative lymphomas selectively deplete palmitate from serum in mouse studies. Mechanistically, CD37 inhibits the FA transporter FATP1 through molecular interaction. Consequently, deletion of CD37 induces uptake and processing of exogenous palmitate into energy and essential building blocks for proliferation, and inhibition of FATP1 reverses this phenotype. Large lipid deposits and intracellular lipid droplets are observed in CD37-negative lymphoma tissues of patients. Moreover, inhibition of carnitine palmitoyl transferase 1 A significantly compromises viability and proliferation of CD37-deficient lymphomas. Collectively, our results identify CD37 as a direct gatekeeper of the FA metabolic switch in aggressive B-cell lymphoma.

Analysis of S-Adenosylmethionine and S-Adenosylhomocysteine: Method Optimisation and Profiling in Healthy Adults upon Short-Term Dietary Intervention.

S-adenosylmethionine (SAM) is essential for methyl transfer reactions. All SAM is produced de novo via the methionine cycle. The demethylation of SAM produces S-adenosylhomocysteine (SAH), an inhibitor of methyltransferases and the precursor of homocysteine (Hcy). The measurement of SAM and SAH in plasma has value in the diagnosis of inborn errors of metabolism (IEM) and in research to assess methyl group homeostasis. The determination of SAM and SAH is complicated by the instability of SAM under neutral and alkaline conditions and the naturally low concentration of both SAM and SAH in plasma (nM range). Herein, we describe an optimised LC-MS/MS method for the determination of SAM and SAH in plasma, urine, and cells. The method is based on isotopic dilution and employs 20 µL of plasma or urine, or 500,000 cells, and has an instrumental running time of 5 min. The reference ranges for plasma SAM and SAH in a cohort of 33 healthy individuals (age: 19-60 years old; mean ± 2 SD) were 120 ± 36 nM and 21.5 ± 6.5 nM, respectively, in accordance with independent studies and diagnostic determinations. The method detected abnormal concentrations of SAM and SAH in patients with inborn errors of methyl group metabolism. Plasma and urinary SAM and SAH concentrations were determined for the first time in a randomised controlled trial of 53 healthy adult omnivores (age: 18-60 years old), before and after a 4 week intervention with a vegan or meat-rich diet, and revealed preserved variations of both metabolites and the SAM/SAH index.

Target Diseases for Neonatal Screening in Germany.

BACKGROUND: Neonatal screening in Germany currently comprises 19 congenital diseases, 13 of which are metabolic diseases. Approximately one in 1300 newborns suffers from one of these target diseases. Early diagnosis and treatment enable the affected children to undergo better development and even, in many cases, to have a normal life. METHODS: This review is based on pertinent publications retrieved by a selective search in the PubMed and Embase databases. RESULTS: Positive screening findings are confirmed in approximately one out of five newborns. The prompt evaluation of suspected diagnoses is essential, as treatment for some of these diseases must be initiated immediately after birth to prevent longterm sequelae. The most commonly identified diseases are primary hypothyroidism (1:3338), phenylketonuria/hyperphenylalaninemia (1 : 5262), cystic fibrosis (1 : 5400), and medium-chain acyl-CoA dehydrogenase deficiency (1 : 10 086). Patient numbers are rising as new variants of the target diseases are being identified, and treatments must be adapted to their heterogeneous manifestations. Precise diagnosis and the planning of treatment, which is generally lifelong, are best carried out in a specialized center. CONCLUSION: Improved diagnosis and treatment now prolong the lives of many patients with congenital diseases. The provision of appropriate long-term treatment extending into adulthood will be a central structural task for screening medicine in the future.

Evidence for a Genotype-Phenotype Correlation in Patients with Pathogenic GLUT2 (SLC2A2) Variants.

Fanconi-Bickel syndrome (FBS) is a very rare but distinct clinical entity with the combined features of hepatic glycogen storage disease, generalized proximal renal tubular dysfunction with disproportionately severe glucosuria, and impaired galactose tolerance. Here, we report five cases (out of 93 diagnosed in our lab) with pathogenic variants on both GLUT2 (SLC2A2) alleles. They come from 3 families and presented with an exceptionally mild clinical course. This course was correlated to data from old and most recent expression and transport studies in Xenopus oocytes. GLUT2 genotype in patients 1 and 2 was p.[153_4delLI];[P417R] with the first variant exhibiting normal membrane expression and partially retained transport activity (5.8%) for 2-deoxyglucose. In patient 3, the very first GLUT2 variant ever detected (p.V197I) was found, but for the first time it was present in a patient in the homozygous state. This variant had also shown unaffected membrane expression and remarkable residual activity (8%). The genotype in patient 4, p.[153_4delLI];[(E440A)], again included the 2-amino-acid deletion with residual transporter function, and patient 5 is the first found to be homozygous for this variant. Our results provide further evidence for a genotype-phenotype correlation in patients with GLUT2 variants; non-functional variants result in the full picture of FBS while dysfunctional variants may result in milder presentations, even glucosuria only, without other typical signs of FBS.

Biosynthesis, Quantification and Genetic Diseases of the Smallest Signaling Thiol Metabolite: Hydrogen Sulfide.

Hydrogen sulfide (H2S) is a gasotransmitter and the smallest signaling thiol metabolite with important roles in human health. The turnover of H2S in humans is mainly governed by enzymes of sulfur amino acid metabolism and also by the microbiome. As is the case with other small signaling molecules, disease-promoting effects of H2S largely depend on its concentration and compartmentalization. Genetic defects that impair the biogenesis and catabolism of H2S have been described; however, a gap in knowledge remains concerning physiological steady-state concentrations of H2S and their direct clinical implications. The small size and considerable reactivity of H2S renders its quantification in biological samples an experimental challenge. A compilation of methods currently employed to quantify H2S in biological specimens is provided in this review. Substantial discrepancy exists in the concentrations of H2S determined by different techniques. Available methodologies permit end-point measurement of H2S concentration, yet no definitive protocol exists for the continuous, real-time measurement of H2S produced by its enzymatic sources. We present a summary of available animal models, monogenic diseases that impair H2S metabolism in humans including structure-function relationships of pathogenic mutations, and discuss possible approaches to overcome current limitations of study.

Mitochondrial damage in renal epithelial cells is potentiated by protein exposure in propionic aciduria.

Propionic aciduria (PA) is caused by deficiency of the mitochondrial enzyme propionyl-CoA carboxylase (PCC). Due to inefficient propionate catabolism patients are endangered by life-threatening ketoacidotic crisis. Protein and amino acid restriction are major therapeutic pillars. However, long-term complications like neurological deterioration and cardiac abnormalities cannot be prevented. Chronic kidney disease (CKD), which is a well-known characteristic of methylmalonic aciduria two enzymatic steps downstream from PCC, has been recognized as a novel late-onset complication in PA. The pathophysiology of CKD in PA is unclear. We investigated mitochondrial structure and metabolism in human renal tubular cells of healthy controls and PA patients. The cells were exposed to either standard cell culture conditions (NT), high protein (HP) or high concentrations of isoleucine and valine (I/V). Mitochondrial morphology changed to condensed, fractured morphology in PA cells irrespective of the cell culture medium. HP and I/V exposure, however, potentiated oxidative stress in PA cells. Mitochondrial mass was enriched in PA cells, and further increased by HP and I/V exposure suggesting a need for compensation. Alterations in the tricarboxylic acid cycle intermediates and accumulation of medium- and long-chain acylcarnitines pointed to altered mitochondrial energy metabolism. Mitophagy was silenced while autophagy as cellular defense mechanisms was highly active in PA cells. The data demonstrate that PA is associated with renal mitochondrial damage which is aggravated by protein and I/V load. Preservation of mitochondrial energy homeostasis in renal cells may be a potential future therapeutic target.

Long-term safety and outcomes in hereditary tyrosinaemia type 1 with nitisinone treatment: a 15-year non-interventional, multicentre study.

BACKGROUND: Since the EU approval of nitisinone in 2005, prognosis for patients with hereditary tyrosinaemia type 1 has changed dramatically, with patients living with the disease now reaching adulthood for the first time in history. This study aimed to assess the long-term safety and outcomes of nitisinone treatment in patients with hereditary tyrosinaemia type 1. METHODS: We did a non-interventional, non-comparative, multicentre study in 77 sites across 17 countries in Europe and collected retrospective and prospective longitudinal data in patients with hereditary tyrosinaemia type 1 who were treated with oral nitisinone during the study period (Feb 21, 2005, to Sept 30, 2019). There were no specific exclusion criteria. Patients were followed-up with an investigator at least annually for as long as they were treated, or until the end of the study. The primary endpoints, occurrence of adverse events related to hepatic, renal, ophthalmic, haematological, or cognitive or developmental function, were assessed in the complete set (all patients already receiving treatment at the index date [Feb 21, 2005] or starting treatment thereafter) and the index set (the subset of patients who had their first dose on the index date or later only). FINDINGS: 315 patients were enrolled during the study period (complete set). Additionally, data from 24 patients who had liver transplantation or died during the post-marketing surveillance programme were retrieved (extended analysis set; 339 patients). Median treatment duration was 11·2 years (range 0·7-28·4); cumulative nitisinone exposure was 3172·7 patient-years. Patients who were diagnosed by neonatal screening started nitisinone treatment at median age 0·8 months versus 8·5 months in those who presented clinically. Incidences of hepatic, renal, ophthalmic, haematological, or cognitive or developmental adverse events were low. Occurrence of liver transplantation or death was more frequent the later that treatment was initiated (none of 70 patients who started treatment at age <28 days vs 35 [13%] of 268 patients who started treatment at age ≥28 days). 279 (89%) of 315 patients were assessed as having either very good or good nitisinone treatment compliance. Treatment and diet compliance declined as patients aged. Suboptimal plasma phenylalanine and tyrosine levels were observed. The majority of patients were reported to have good overall clinical condition throughout treatment; 176 (87%) of 203 during the entire study, 98% following 1 year of treatment. INTERPRETATION: Long-term nitisinone treatment was well tolerated and no new safety signals were revealed. Life-limiting hepatic disease appears to have been prevented by early treatment start. Neonatal screening was the most effective way of ensuring early treatment. Standardised monitoring of blood tyrosine, phenylalanine, and nitisinone levels has potential to guide individualised therapy. FUNDING: Swedish Orphan Biovitrum (Sobi).

Defective lysosomal storage in Fabry disease modifies mitochondrial structure, metabolism and turnover in renal epithelial cells.

Fabry disease (FD) is an X-linked lysosomal storage disorder. Deficiency of the lysosomal enzyme alpha-galactosidase (GLA) leads to accumulation of potentially toxic globotriaosylceramide (Gb3) on a multisystem level. Cardiac and cerebrovascular abnormalities as well as progressive renal failure are severe, life-threatening long-term complications. The complete pathophysiology of chronic kidney disease (CKD) in FD and the role of tubular involvement for its progression are unclear. We established human renal tubular epithelial cell lines from the urine of male FD patients and male controls. The renal tubular system is rich in mitochondria and involved in transport processes at high-energy costs. Our studies revealed fragmented mitochondria with disrupted cristae structure in FD patient cells. Oxidative stress levels were elevated and oxidative phosphorylation was upregulated in FD pointing at enhanced energetic needs. Mitochondrial homeostasis and energy metabolism revealed major changes as evidenced by differences in mitochondrial number, energy production and fuel consumption. The changes were accompanied by activation of the autophagy machinery in FD. Sirtuin1, an important sensor of (renal) metabolic stress and modifier of different defense pathways, was highly expressed in FD. Our data show that lysosomal FD impairs mitochondrial function and results in severe disturbance of mitochondrial energy metabolism in renal cells. This insight on a tissue-specific level points to new therapeutic targets which might enhance treatment efficacy.

Thiolatocobalamins repair the activity of pathogenic variants of the human cobalamin processing enzyme CblC.

Thiolatocobalamins are a class of cobalamins comprised of naturally occurring and synthetic ligands. Glutathionylcobalamin (GSCbl) occurs naturally in mammalian cells, and also as an intermediate in the glutathione-dependent dealkylation of methylcobalamin (MeCbl) to form cob(I)alamin by pure recombinant CblC from C. elegans. Glutathione-driven deglutathionylation of GSCbl was demonstrated both in mammalian as well as in C. elegans CblC. Dethiolation is orders of magnitude faster than dealkylation of Co-C bonded cobalamins, which motivated us to investigate two synthetic thiolatocobalamins as substrates to repair the enzymatic activity of pathogenic CblC variants in humans. We report the synthesis and kinetic characterization of cysteaminylcobalamin (CyaCbl) and 2-mercaptopropionylglycinocobalamin (MpgCbl). Both CyaCbl and MpgCbl were obtained in high purity (90-95%) and yield (78-85%). UV-visible spectral properties agreed with those reported for other thiolatocobalamins with absorbance maxima observed at 372 nm and 532 nm. Both CyaCbl and MpgCbl bound to wild type human recombinant CblC inducing spectral blue-shifts characteristic of the respective base-on to base-off transitions. Addition of excess glutathione (GSH) resulted in rapid elimination of the ß-ligand to give aquacobalamin (H2OCbl) as the reaction product under aerobic conditions. Further, CyaCbl and MpgCbl underwent spontaneous dethiolation thereby repairing the loss of activity of pathogenic variants of human CblC, namely R161G and R161Q. We posit that thiolatocobalamins could be exploited therapeutically for the treatment of inborn errors of metabolism that impair processing of dietary and supplemental cobalamin forms. While these disorders are targets for newborn screening in some countries, there is currently no effective treatment available to patients.

Metabolic Profiling in Human Fibroblasts Enables Subtype Clustering in Glycogen Storage Disease.

Glycogen storage disease subtypes I and III (GSD I and GSD III) are monogenic inherited disorders of metabolism that disrupt glycogen metabolism. Unavailability of glucose in GSD I and induction of gluconeogenesis in GSD III modify energy sources and possibly, mitochondrial function. Abnormal mitochondrial structure and function were described in mice with GSD Ia, yet significantly less research is available in human cells and ketotic forms of the disease. We hypothesized that impaired glycogen storage results in distinct metabolic phenotypes in the extra- and intracellular compartments that may contribute to pathogenesis. Herein, we examined mitochondrial organization in live cells by spinning-disk confocal microscopy and profiled extra- and intracellular metabolites by targeted LC-MS/MS in cultured fibroblasts from healthy controls and from patients with GSD Ia, GSD Ib, and GSD III. Results from live imaging revealed that mitochondrial content and network morphology of GSD cells are comparable to that of healthy controls. Likewise, healthy controls and GSD cells exhibited comparable basal oxygen consumption rates. Targeted metabolomics followed by principal component analysis (PCA) and hierarchical clustering (HC) uncovered metabolically distinct poises of healthy controls and GSD subtypes. Assessment of individual metabolites recapitulated dysfunctional energy production (glycolysis, Krebs cycle, succinate), reduced creatinine export in GSD Ia and GSD III, and reduced antioxidant defense of the cysteine and glutathione systems. Our study serves as proof-of-concept that extra- and intracellular metabolite profiles distinguish glycogen storage disease subtypes from healthy controls. We posit that metabolite profiles provide hints to disease mechanisms as well as to nutritional and pharmacological elements that may optimize current treatment strategies.

Assessment of cellular cobalamin metabolism in Gaucher disease.

BACKGROUND: Gaucher disease (GD) is a lysosomal disorder caused by biallelic pathogenic mutations in the GBA1 gene that encodes beta-glucosidase (GCase), and more rarely, by a deficiency in the GCase activator, saposin C. Clinically, GD manifests with heterogeneous multiorgan involvement mainly affecting hematological, hepatic and neurological axes. This disorder is divided into three types, based on the absence (type I) or presence and severity (types II and III) of involvement of the central nervous system. At the cellular level, deficiency of GBA1 disturbs lysosomal storage with buildup of glucocerebroside. The consequences of disturbed lysosomal metabolism on biochemical pathways that require lysosomal processing are unknown. Abnormal systemic markers of cobalamin (Cbl, B12) metabolism have been reported in patients with GD, suggesting impairments in lysosomal handling of Cbl or in its downstream utilization events. METHODS: Cultured skin fibroblasts from control humans (n = 3), from patients with GD types I (n = 1), II (n = 1) and III (n = 1) and an asymptomatic carrier of GD were examined for their GCase enzymatic activity and lysosomal compartment intactness. Control human and GD fibroblasts were cultured in growth medium with and without 500 nM hydroxocobalamin supplementation. Cellular cobalamin status was examined via determination of metabolomic markers in cell lysate (intracellular) and conditioned culture medium (extracellular). The presence of transcobalamin (TC) in whole cell lysates was examined by Western blot. RESULTS: Cultured skin fibroblasts from GD patients exhibited reduced GCase activity compared to healthy individuals and an asymptomatic carrier of GD, demonstrating a preserved disease phenotype in this cell type. The concentrations of total homocysteine (tHcy), methylmalonic acid (MMA), cysteine (Cys) and methionine (Met) in GD cells were comparable to control levels, except in one patient with GD III. The response of these metabolomic markers to supplementation with hydroxocobalamin (HOCbl) yielded variable results. The content of transcobalamin in whole cell lysates was comparable in control human and GD patients. CONCLUSIONS: Our results indicate that cobalamin transport and cellular processing pathways are overall protected from lysosomal storage damage in GD fibroblasts. Extending these studies to hepatocytes, macrophages and plasma will shed light on cell- and compartment-specific vitamin B12 metabolism in Gaucher disease.

Targeted Metabolic Profiling of Methionine Cycle Metabolites and Redox Thiol Pools in Mammalian Plasma, Cells and Urine.

The concentration of thiol and thioether metabolites in plasma has diagnostic value in genetic diseases of B-vitamin metabolism linked to methionine utilization. Among these, cysteine/cystine (Cys/CSSC) and glutathione/oxidized glutathione (GSH/GSSG) act as cellular redox buffers. A new LC-MS/MS method was developed for the simultaneous detection of cystathionine (Cysta), methionine (Met), methionine sulfoxide (MSO), creatinine and the reduced and oxidized pairs of homocysteine (Hcy/HSSH), cysteine (Cys/CSSC) and glutathione (GSH/GSSG). A one-step thiol-blocking protocol with minimal sample preparation was established to determine redox thiol pairs in plasma and cells. The concentrations of diagnostic biomarkers Hcy, Met, Cysta, and Cys in a cohort of healthy adults (n = 53) agreed with reference ranges and published values. Metabolite concentrations were also validated in commercial samples of human, mouse, rat and Beagle dog plasma and by the use of a standardized ERNDIM quality control. Analysis of fibroblasts, endothelial and epithelial cells, human embryonic stem cells, and cancer cell lines showed cell specificity for both the speciation and concentration of thiol and thioether metabolites. This LC-MS/MS platform permits the fast and simultaneous quantification of 10 thiol and thioether metabolites and creatinine using 40 µL plasma, urine or culture medium, or 500,000 cells. The sample preparation protocols are directly transferable to automated metabolomic platforms.

The fate of medium-chain fatty acids in very long-chain acyl­CoA dehydrogenase deficiency (VLCADD): A matter of sex?

Medium-chain-triglycerides (MCT) are widely applied in the treatment of long-chain fatty acid oxidation disorders (lcFAOD). Long-term treatment with MCT led to a sexually dimorphic response in the mouse model of very-long-chain-acyl-CoA-dehydrogenase-deficiency (VLCAD-/-) with the subsequent development of a metabolic syndrome in female mice. In order to evaluate the molecular mechanisms responsible for this sex specific response we performed a comprehensive metabolic phenotyping, SILAC-based quantitative proteomics and characterized the involved signaling pathways by western blot analysis and gene expression. WT and VLCAD-/- mice showed strong sex-dependent differences in basal metabolism and expression of proteins involved in the distinct metabolic pathways, even more prominent after treatment with octanoate. The investigation of molecular mechanisms responsible for the sexual dimorphisms delineated the selective activation of the ERK/mTORc1 signaling pathway leading to an increased biosynthesis and elongation of fatty acids in VLCAD-/- females. In contrast, octanoate induced the activation of ERK/PPARγ pathway and the subsequent upregulation of peroxisomal ߭oxidation in males. We here provide first evidence that sex has to be considered as important variable in disease phenotype. These findings may have implications on treatment strategies in the different sexes.

Implementation of a fast method for the measurement of carnitine palmitoyltransferase 2 activity in lymphocytes by tandem mass spectrometry as confirmation for newborn screening.

Carnitine palmitoyltransferase II (CPT2) is a rare autosomal recessive inherited disorder affecting mitochondrial ß-oxidation. Confirmation diagnostics are mostly based on molecular sequencing of the CPT2 gene, especially to distinguish CPT2 and carnitine:aclycarnitine translocase deficiencies, which present with identical acylcarnitine profiles on newborn screening (NBS). In the past, different enzyme tests in muscle biopsies have been developed in order to study the functional effect in one of the main target organs. In this study, we implemented a method for measurement of CPT2 enzyme activity in human lymphocytes with detection of the reaction products via liquid chromatography mass spectrometry to enable the simultaneous evaluation of the functional impairment and the clear diagnosis of the disease. CPT2 activity was measured in samples collected from CPT2 patients (n = 11), heterozygous carriers (n = 6), and healthy individuals (n = 52). Seven patients out of 11 were homozygous for the common mutation c.338T>C and showed a residual activity with median values of 19.2 ± 3.7% of healthy controls. Heterozygous carriers showed a residual activity in the range of 42% to 75%. Four individuals carrying the heterozygous mutation c.338T>C showed a 2-fold higher residual activity as compared to homozygous individuals. Our optimized method for the measurement of CPT2 activity is able to clearly discriminate between patients and healthy individuals and offers the possibility to determine CPT2 activity in human lymphocytes avoiding the need of an invasive muscle biopsy. This method can be successfully used for confirmation diagnosis in case of positive NBS and would markedly reduce the time to define diagnosis.

The diagnostic challenge in very-long chain acyl-CoA dehydrogenase deficiency (VLCADD).

Very long-chain acyl-CoA dehydrogenase deficiency (VLCADD) is the most common defect of mitochondrial ß-oxidation of long-chain fatty acids. However, the unambiguous diagnosis of true VLCADD patients may be challenging, and a high rate of false positive individuals identified by newborn screening undergo confirmation diagnostics. In this study, we show the outcome of enzyme testing in lymphocytes as a confirmatory tool in newborns identified by screening, and the correlation with molecular sequencing of the ACADVL gene. From April 2013 to March 2017, in 403 individuals with characteristic acylcarnitine profiles indicative of VLCADD, palmitoyl-CoA oxidation was measured followed by molecular genetic analysis in most of the patients with residual activity (RA) <50%. In almost 50% of the samples (209/403) the RA was >50%, one-third of the individuals (125/403) displayed a RA of 30-50% and 69/403 individuals showed a residual activity of 0-30%. Sequencing of the ACADVL gene revealed that all individuals with activities below 24% were true VLCADD patients, individuals with residual activities between 24 and 27% carried either one or two mutations. Twenty new mutations could be identified and functionally classified based on their effect on enzyme function. Finally, we observed an up-regulation of MCAD-activity in many patients. However, this did not correlate with the degree of VLCAD RA. Although the likely clinical phenotype cannot be fully foreseen by genetic and functional tests as it depends on many factors, our data demonstrate the strength of this functional enzyme test in lymphocytes as a quick and reliable method for confirmation diagnostics of VLCADD.

Mitochondrial fatty acid biosynthesis and muscle fiber plasticity in very long-chain acyl-CoA dehydrogenase-deficient mice.

The white skeletal muscle of very long-chain acyl-CoA-dehydrogenase-deficient (VLCAD-/- ) mice undergoes metabolic modification to compensate for defective ß-oxidation in a progressive and time-dependent manner by upregulating glucose oxidation. This metabolic regulation seems to be accompanied by morphologic adaptation of muscle fibers toward the glycolytic fiber type II with the concomitant upregulation of mitochondrial fatty acid biosynthesis (mFASII) and lipoic acid biosynthesis. Dietary supplementation of VLCAD-/- mice with different medium-chain triglycerides over 1 year revealed that odd-chain species has no effect on muscle fiber switch, whereas even-chain species inhibit progressive metabolic adaptation. Our study shows that muscle may undergo adaptive mechanisms that are modulated by dietary supplementation. We describe for the first time a concomitant change of mFASII in this muscular adaptation process.

Dysphagia after arteria lusoria dextra surgery: Anatomical considerations before redo-surgery.

Aberrant right subclavian artery (arteria lusoria) is the most common congenital root anomaly, remaining asymptomatic in most cases. Nevertheless, some of the 20%-40% of those affected present tracheo-esophageal symptoms. We report on a 6-year-old previously healthy girl presenting with progressive dysphagia over 4 wk. Diagnostics including barium swallow, echocardiography and magnetic resonance angiography (MRA) revealed a retro-esophageal compression by an aberrant right subclavian artery. Despite the successful, uneventful transposition of this arteria lusoria to the right common carotid via right-sided thoracotomy, the girl was suffering from persisting dysphagia. Another barium swallow showed the persistent compression of the esophagus on the level where the arteria lusoria had originated. As MRA showed no evidence of a significant re-obstruction by the transected vascular stump, we suspected a persisting ligamentum arteriosum. After a second surgical intervention via left-sided thoracotomy consisting of transecting the obviously persisting ligamentum and shortening the remaining arterial stump of the aberrant right subclavian artery, the patient recovered fully. In this case report we discuss the potential relevance of a persisting ligamentum arteriosum for patients with left aortic arch suffering from dysphagia lusoria and rational means of diagnosing, as well as the surgical options to prevent re-do surgery.

Hampered Vitamin B12 Metabolism in Gaucher Disease?

Abstract Untreated vitamin B12 deficiency manifests clinically with hematological abnormalities and combined degeneration of the spinal cord and polyneuropathy and biochemically with elevated homocysteine (Hcy) and methylmalonic acid (MMA). Vitamin B12 metabolism involves various cellular compartments including the lysosome, and a disruption in the lysosomal and endocytic pathways induces functional deficiency of this micronutrient. Gaucher disease (GD) is characterized by dysfunctional lysosomal metabolism brought about by mutations in the enzyme beta-glucocerebrosidase (Online Mendelian Inheritance in Man (OMIM): 606463; Enzyme Commission (EC) 3.2.1.45, gene: GBA1). In this study, we collected and examined available literature on the associations between GD, the second most prevalent lysosomal storage disorder in humans, and hampered vitamin B12 metabolism. Results from independent cohorts of patients show elevated circulating holotranscobalamin without changes in vitamin B12 levels in serum. Gaucher disease patients under enzyme replacement therapy present normal levels of Hcy and MMA. Although within the normal range, a significant increase in Hcy and MMA with normal serum vitamin B12 was documented in treated GD patients with polyneuropathy versus treated GD patients without polyneuropathy. Thus, a functional deficiency of vitamin B12 caused by disrupted lysosomal metabolism in GD is a plausible mechanism, contributing to the neurological form of the disorder but this awaits confirmation. Observational studies suggest that an assessment of vitamin B12 status prior to the initiation of enzyme replacement therapy may shed light on the role of vitamin B12 in the pathogenesis and progression of GD.

Clinical pattern, mutations and in vitro residual activity in 33 patients with severe 5, 10 methylenetetrahydrofolate reductase (MTHFR) deficiency.

BACKGROUND: Severe methylenetetrahydrofolate reductase (MTHFR) deficiency is a rare inborn defect disturbing the remethylation of homocysteine to methionine (<200 reported cases). This retrospective study evaluates clinical, biochemical genetic and in vitro enzymatic data in a cohort of 33 patients. METHODS: Clinical, biochemical and treatment data was obtained from physicians by using a questionnaire. MTHFR activity was measured in primary fibroblasts; genomic DNA was extracted from cultured fibroblasts. RESULTS: Thirty-three patients (mean age at follow-up 11.4 years; four deceased; median age at first presentation 5 weeks; 17 females) were included. Patients with very low (<1.5%) mean control values of enzyme activity (n = 14) presented earlier and with a pattern of feeding problems, encephalopathy, muscular hypotonia, neurocognitive impairment, apnoea, hydrocephalus, microcephaly and epilepsy. Patients with higher (>1.7-34.8%) residual enzyme activity had mainly psychiatric symptoms, mental retardation, myelopathy, ataxia and spasticity. Treatment with various combinations of betaine, methionine, folate and cobalamin improved the biochemical and clinical phenotype. During the disease course, patients with very low enzyme activity showed a progression of feeding problems, neurological symptoms, mental retardation, and psychiatric disease while in patients with higher residual enzyme activity, myelopathy, ataxia and spasticity increased. All other symptoms remained stable or improved in both groups upon treatment as did brain imaging in some cases. No clear genotype-phenotype correlation was obvious. DISCUSSION: MTHFR deficiency is a severe disease primarily affecting the central nervous system. Age at presentation and clinical pattern are correlated with residual enzyme activity. Treatment alleviates biochemical abnormalities and clinical symptoms partially.