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Background Fecal metabolomic profiles differ between pediatric inflammatory bowel disease (IBD) patients and controls and may provide new insights in the pathophysiology of IBD. The role of amino acids, however, is not fully elucidated. We aimed to assess fecal amino acid profiles in pediatric IBD. Methods In this case-control study, treatment-naïve, newly diagnosed pediatric IBD patients and a non-IBD control group, matched based on sex and age, were included in 2 tertiary centres. Fecal amino acid profiles were assessed using a targeted high-performance liquid chromatography technique. A random forest classifier method was used to develop a prediction model differentiating IBD from controls and predicting IBD phenotype. The association between IBD localization and amino acid concentrations was tested with ordinal regression models. Results We included 78 newly diagnosed IBD patients (40 Crohn’s disease [CD], 38 ulcerative colitis [UC]) and 105 controls. Patients with IBD could be differentiated from controls with an accuracy of 82% (sensitivity 63%, specificity 97%). Twenty-nine out of the 42 measured unique amino acids were included in the prediction model. Increased levels of tryptophan, taurine, alanine, ornithine, valine, histidine, and leucine were the most differentiating features. Children with CD and UC could be differentiated from the controls with an accuracy of 80% and 90%, respectively. Inflammatory bowel disease phenotype could not be predicted. Tryptophan, valine, and histidine levels were positively associated with more extended disease in UC patients (P < .05). Conclusions Fecal amino acids may enhance understanding of the role of host-microbial interactions in the pathophysiology of IBD and may evolve into biomarkers for pediatric IBD diagnostic and personalized medicine.

Sulfate is the fourth most abundant anion in human plasma but is not measured in clinical practice and little is known about the consequences of sulfate deficiency. Nevertheless, sulfation plays an essential role in the modulation of numerous compounds, including proteoglycans and steroids. We report the first patient with a homozygous loss-of-function variant in the SLC13A1 gene, encoding a renal and intestinal sulfate transporter, which is essential for maintaining plasma sulfate levels. The homozygous (Arg12Ter) variant in SLC13A1 was found by exome sequencing performed in a patient with unexplained skeletal dysplasia. The main clinical features were enlargement of joints and spondylo-epi-metaphyseal radiological abnormalities in early childhood, which improved with age. In addition, autistic features were noted. We found profound hyposulfatemia due to complete loss of renal sulfate reabsorption. Cholesterol sulfate was reduced. Intravenous N-acetylcysteine administration temporarily restored plasma sulfate levels. We conclude that loss of the SLC13A1 gene leads to profound hypersulfaturia and hyposulfatemia, which is mainly associated with abnormal skeletal development, possibly predisposing to degenerative bone and joint disease. The diagnosis might be easily missed and more frequent.

Stability of the cerebrospinal fluid (CSF) composition under different pre-analytical conditions is relevant for the diagnostic potential of biomarkers. Our aim was to examine the pre-analytical stability of promising CSF biomarkers that are currently evaluated for their discriminative use in various neurological diseases. Pooled CSF was aliquoted and experimentally exposed to delayed storage: 0, 1, 2, 4, 24, 72, or 168 h at 4 °C or room temperature (RT), or 1–4 months at −20 °C; or up to 7 freeze/thaw (f/t) cycles, before final storage at −80 °C. Eleven CSF biomarkers were screened using immunoassays, liquid chromatography, or enzymatic methods. Levels of neurogranin (truncP75), chitinase-3-like protein (YKL-40), beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), acetylcholinesterase (AChE) enzymatic activity, theobromine, secreted protein acidic and rich in cysteine-like 1 (SPARCL-1) and homovanillic acid (HVA) levels were not affected by the applied storage conditions. 3-Methoxy-4-hydroxyphenylglycol (MHPG) levels linearly and strongly decreased after 4 h at RT (−10%) or 24 h at 4 °C (−27%), and with 6% after every f/t cycle. 5-Methyltetrahydrofolate (5-MTHF) (−29% after 1 week at RT) and 5-hydroxyindoleacetic acid levels (5-HIAA) (−16% after 1 week at RT) were reduced and 3,4-dihydroxyphenylacetic acid (DOPAC) levels (+22% after 1 week at RT) increased, but only after >24 h at RT. Ten out of eleven potential CSF novel biomarkers showed very limited change under common storage and f/t conditions, suggesting that these CSF biomarkers can be trustfully tested under the pre-analytical conditions present across different cohorts., This research was financially supported by Biobanking and BioMolecular resources Research Infrastructure the Netherlands (BBMRI-NL), a research infrastructure financed by the Dutch Government (NWO 184.021.007) under project CP2013-68. This study commenced within the BIOMARKAPD program of the EU Joint Programme – Neurodegenerative Disease Research (JPND). Project supported by the European Regional Development Fund (ERDF) through the COMPETE 2020 - Operational Programme for Competitiveness and Internationalisation and Portuguese national funds via FCT – Fundação para a Ciência e a Tecnologia, I.P., under the projects POCI-01-0145-FEDER-007440, POCI-01-0145-FEDER-016428, and POCI-01-0145-FEDER-016795, and by The National Mass Spectrometry Network (RNEM) under the contract POCI-01-0145-FEDER-402-022125.

Background: Brain tumors may have cysts, whose content of nutrients could influence tumor cell microenvironment and growth. Objective: To measure nutrients in cyst fluid from glioblastoma multiforme (GBM) and metastatic brain tumors. Methods: Quantification of nutrients in cyst fluid from 12 to 18 GBMs and 4 to 10 metastatic brain tumors. Results: GBM cysts contained glucose at 2.2 mmol/L (median value; range 3 times higher than in ventricular cerebrospinal fluid: 0.35 mmol/L (0.22-0.66; P

D-2-hydroxyglutaric aciduria Type I (D-2-HGA Type I), a neurometabolic disorder with a broad clinical spectrum, is caused by recessive variants in the D2HGDH gene encoding D-2-hydroxyglutarate dehydrogenase (D-2-HGDH). We and others detected 42 potentially pathogenic variants in D2HGDH of which 31 were missense. We developed functional studies to investigate the effect of missense variants on D-2-HGDH catalytic activity. Site-directed mutagenesis was used to introduce 31 missense variants in the pCMV5-D2HGDH expression vector. The wild type and missense variants were overexpressed in HEK293 cells. D-2-HGDH enzyme activity was evaluated based on the conversion of [2H4]D-2-HG to [2H4]2-ketoglutarate, which was subsequently converted into [2H4]L-glutamate and the latter quantified by LC-MS/MS. Eighteen variants resulted in almost complete ablation of D-2-HGDH activity and thus, should be considered pathogenic. The remaining 13 variants manifested residual activities ranging between 17% and 94% of control enzymatic activity. Our functional assay evaluating the effect of novel D2HGDH variants will be beneficial for the classification of missense variants and determination of pathogenicity.

D-glycerate 2 kinase (DGK) is an enzyme that mediates the conversion of D-glycerate, an intermediate metabolite of serine and fructose metabolism, to 2-phosphoglycerate. Deficiency of DGK leads to accumulation of D-glycerate in various tissues and its massive excretion in urine. D-glyceric aciduria (DGA) is an autosomal recessive metabolic disorder caused by mutations in the GLYCTK gene. The clinical spectrum of DGA is highly variable, ranging from severe progressive infantile encephalopathy to a practically asymptomatic condition. We describe a male patient from a consanguineous Arab family with infantile onset of DGA, characterized by profound psychomotor retardation, progressive microcephaly, intractable seizures, cortical blindness and deafness. Consecutive brain MR imaging showed an evolving brain atrophy, thinning of the corpus callosum and diffuse abnormal white matter signals. Whole exome sequencing identified the homozygous missense variant in the GLYCTK gene [c.455 T > C, NM_145262.3], which affected a highly conserved leucine residue located at a domain of yet unknown function of the enzyme [p.Leu152Pro, NP_660305]. In silico analysis of the variant supported its pathogenicity. A review of the 15 previously reported patients, together with the current one, confirms a clear association between DGA and severe neurological impairment. Yet, future studies of additional patients with DGA are required to better understand the clinical phenotype and pathogenesis.

Guanidinoacetate methyltransferase (GAMT) deficiency is an inborn error of metabolism (IEM), clinically characterized by intellectual disability, developmental delay, seizures, and movement disorders. Biochemical diagnosis of GAMT deficiency is based on the measurement of creatine and guanidinoacetate in urine, plasma, or CSF and is confirmed genetically by DNA analysis or by enzyme assay in lymphoblasts or fibroblasts. To obtain enough cells, these cells need to be cultured for at least 1 month. A less time-consuming diagnostic functional test is needed, since GAMT deficiency is a candidate for newborn screening (NBS) programs, to be able to confirm or rule out this IEM after an initial positive result in the NBS.Stable-isotope-labeledWe measured GAMT enzyme activity in lymphocyte extracts of 24 controls, 3 GAMT deficient patients and of 2 parents proven to be carrier. Because GAMT activity decreases when isolation time after venipuncture increases, reference values were obtained for 2 control groups: isolation on the day of venipuncture (27-130 pmol/h/mg) and 1 day afterwards (15-146 pmol/h/mg). Deficient patients had no detectable GAMT activity. The two carriers had GAMT activity within the normal range.We designed a fast, less invasive, and valid method to measure GAMT activity in lymphocytes using LC-MS/MS analysis without the need of time-consuming and laborious cell culture.

Enzymatic side reactions can give rise to the formation of wasteful and toxic products that are removed by metabolite repair pathways. In this work, we identify and characterize a mitochondrial metabolic repair mechanism in Arabidopsis thaliana involving malate dehydrogenase (mMDH) and l-2-hydroxyglutarate dehydrogenase (l-2HGDH). We analyze the kinetic properties of both A. thaliana mMDH isoforms, and show that they produce l-2-hydroxyglutarate (l-2HG) from 2-ketoglutarate (2-KG) at low rates in side reactions. We identify A. thaliana l-2HGDH as a mitochondrial FAD-containing oxidase that converts l-2HG back to 2-KG. Using loss-of-function mutants, we show that the electrons produced in the l-2HGDH reaction are transferred to the mitochondrial electron transport chain through the electron transfer protein (ETF). Thus, plants possess the biochemical components of an l-2HG metabolic repair system identical to that found in mammals. While deficiencies in the metabolism of l-2HG result in fatal disorders in mammals, accumulation of l-2HG in plants does not adversely affect their development under a range of tested conditions. However, orthologs of l-2HGDH are found in all examined genomes of viridiplantae, indicating that the repair reaction we identified makes an essential contribution to plant fitness in as yet unidentified conditions in the wild. © 2015 The Author 2015.

We have conducted biochemical studies with commercial available pyrroline-5-carboxylate (P5C) reductase (PYCR1) to investigate whether this enzyme plays a role in L-lysine degradation. Our recent studies with antiquitin/ALDH7A1 deficient fibroblasts revealed an alternative genesis of L-pipecolic acid, and we then hypothesized that PYCR1 was responsible for the conversion of Δ(1)-piperideine-6-carboxylate (P6C) into pipecolic acid. We here present evidence that PYCR1 is indeed able to produce L-pipecolic acid from P6C preparations, and the observed K m for this conversion is of the same magnitude as the K m described for the conversion of P5C to L-proline by PYCR1. Urine samples from antiquitin deficient individuals, who accumulate P6C, were also incubated with PYCR1 which resulted in a marked decrease of P6C and a huge increase of L-pipecolic acid as measured by LC-MS/MS, confirming that indeed PYCR1 generates L-pipecolic acid from P6C.

Objective: A high percentage of grade II and III gliomas have mutations in the gene encoding isocitrate dehydrogenase (IDH1). This mutation is always a heterozygous point mutation that affects the amino acid arginine at position 132 and results in loss of its native enzymatic activity and gain of alternative enzymatic activity (producing D-2-hydroxyglutarate). The objective of this study was to investigate the cellular effects of R132H mutations in IDH1.Methods: Functional consequences of IDH1(R132H) mutations were examined among others using fluorescence-activated cell sorting, kinome and expression arrays, biochemical assays, and intracranial injections on 3 different (glioma) cell lines with stable overexpression of IDH1(R132H).Results: IDH1(R132H) overexpression in established glioma cell lines in vitro resulted in a marked decrease in proliferation, decreased Akt phosphorylation, altered morphology, and a more contact-dependent cell migration. The reduced proliferation is related to accumulation of D-2-hydroxyglutarate that is produced by IDH1(R132H). Mice injected with IDH1(R132H) U87 cells have prolonged survival compared to mice injected with IDH1(wt) or green fluorescent protein-expressing U87 cells.Interpretation: Our results demonstrate that IDH1(R132H) dominantly reduces aggressiveness of established glioma cell lines in vitro and in vivo. In addition, the IDH1R132H-IDH1(wt) heterodimer has higher enzymatic activity than the IDH1(R132H)-IDH1(R132H) homodimer. Our observations in model systems of glioma might lead to a better understanding of the biology of IDH1 mutant gliomas, which are typically low grade and often slow growing. ANN NEUROL 2011;69:455-463

Activity of carnosinase (CN1), the only dipeptidase with substrate specificity for carnosine or homocarnosine, varies greatly between individuals but increases clearly and significantly with age. Surprisingly, the lower CN1 activity in children is not reflected by differences in CN1 protein concentrations. CN1 is present in different allosteric conformations in children and adults since all sera obtained from children but not from adults were positive in ELISA and addition of DTT to the latter sera increased OD450 values. There was no quantitative difference in the amount of monomeric CN1 between children and adults. Further, CN1 activity was dose dependently inhibited by homocarnosine. Addition of 80 microM homocarnosine lowered V (max) for carnosine from 440 to 356 pmol/min/microg and increased K (m) from 175 to 210 microM. The estimated K (i) for homocarnosine was higher (240 microM). Homocarnosine inhibits carnosine degradation and high homocarnosine concentrations in cerebrospinal fluid (CSF) may explain the lower carnosine degradation in CSF compared to serum. Because CN1 is implicated in the susceptibility for diabetic nephropathy (DN), our findings may have clinical implications for the treatment of diabetic patients with a high risk to develop DN. Homocarnosine treatment can be expected to reduce CN1 activity toward carnosine, resulting in higher carnosine levels.

To evaluate the initial experience with pulmonary thromboendarterectomy for cor pulmonale due to chronic pulmonary embolism.In the period 1 April 1996 to 31 October 2001, 18 patients with right ventricular failure due to chronic thromboembolic pulmonary hypertension were operated on. Their mean age was 54 (SD: 14) years. Preoperatively, all of the patients were in functional New York Heart Association (NYHA) class III or IV. Pulmonary angiography revealed pulmonary hypertension with an increased pulmonary vascular resistance, as well as typical angiographic signs of unresolved chronic emboli. Pulmonary thromboendarterectomy was performed via median sternotomy, using extracorporeal circulation and intermittent deep hypothermic circulatory arrest. The patients were monitored via the outpatients' department.From a technical viewpoint, the procedure was performed successfully in all of the patients. Initially the pulmonary thromboendarterectomy was performed unilaterally (n = 7), which did not decrease pulmonary artery pressure significantly. The following 11 patients were treated bilaterally; in them thromboendarterectomy required an average of circulatory arrest totalling 64 (SD: 30) min with 161 (SD: 35) min of myocardial ischemia. After bilateral pulmonary thromboendarterectomy, the pulmonary artery pressure decreased from 45 (SD: 13) to 28 (SD: 9) mmHg (p = 0.001). Reperfusion pulmonary oedema, requiring prolonged ventilation, occurred in 3 patients. There was no operative or later mortality. At a mean follow-up of 28 (SD: 19) months, all but one of the patients were in functional NYHA class I or II. Echocardiography revealed reduced right ventricular dimensions and pulmonary artery pressures. One patient, with mainly distally located obstructions, exhibited no substantial improvement.The initial experience with pulmonary thromboendarterectomy for patients with cor pulmonale due to chronic pulmonary emboli demonstrated satisfactory mid-term follow-up data.

Therapeutic intervention for human succinic semialdehyde dehydrogenase (SSADH) deficiency (gamma-hydroxybutyric aciduria) has been limited to vigabatrin (VGB). Pharmacologically, VGB should be highly effective due to 4-aminobutyrate-transaminase (GABA-transaminase) inhibition, lowering succinic semialdehyde and, thereby, gamma-hydroxybutyric acid (GHB) levels. Unfortunately, clinical efficacy has been limited. Because GHB possesses a number of potential receptor interactions, we addressed the hypothesis that antagonism of these interactions in mice with SSADH deficiency could lead to the development of novel treatment strategies for human patients. SSADH-deficient mice have significantly elevated tissue GHB levels, are neurologically impaired, and die within 4 weeks postnatally. In the current report, we compared oral versus intraperitoneal administration of VGB, CGP 35348 [3-aminopropyl(diethoxymethyl)phosphinic acid, a GABA(B) receptor antagonist], and the nonprotein amino acid taurine in rescue of SSADH-deficient mice from early death. In addition, we assessed the efficacy of the specific GHB receptor antagonist NCS-382 (6,7,8,9-tetrahydro-5-[H]benzocycloheptene-5-ol-6-ylideneacetic acid) using i.p. administration. All interventions led to significant lifespan extension (22-61%), with NCS-382 being most effective (50-61% survival). To explore the limited human clinical efficacy of VGB, we measured brain GHB and gamma-aminobutyric acid (GABA) levels in SSADH-deficient mice receiving VGB. Whereas high-dose VGB led to the expected elevation of brain GABA, we found no parallel decrease in GHB levels. Our data indicate that, at a minimum, GHB and GABA(B) receptors are involved in the pathophysiology of SSADH deficiency. We conclude that taurine and NCS-382 may have therapeutic relevance in human SSADH deficiency and that the poor clinical efficacy of VGB in this disease may relate to an inability to decrease brain GHB concentrations.

The Octopus Study consists of two multicenter randomized clinical trials in which coronary artery bypass grafting on the beating heart (off-pump CABG) using the Utrecht Octopus Method is compared to intracoronary stent implantation and conventional CABG. The primary endpoint in the comparison of off-pump CABG versus stent implantation (OctoStent Trial) is medical effectiveness (i.e., absence of reintervention and major adverse cardiac and cerebrovascular events at 1 year after treatment). The primary endpoint in the comparison of off-pump CABG versus conventional CABG (OctoPump Trial) is cerebral safety (i.e., absence of cognitive deficits and cerebrovascular events at 3 months after treatment). Secondary endpoints in both trials include presence and severity of angina, quality of life, exercise capacity, and cost-effectiveness. A total of 560 patients will be enrolled. A random sample of 210 patients will undergo repeat angiography at 1 year to assess angiographic restenosis rate and graft patency. Including 1-year follow-up, the study will last for 3 years. Control Clin Trials 2000;21:595-609

To assess the sequelae of temporary coronary artery occlusion in off-pump, beating heart CABG, i.e. ischemia, hemodynamic instability and the need for conversion to cardiopulmonary bypass.In 200 patients (150 male), mean age 60 (range 35-81) years, 365 distal anastomoses were performed, i.e. 1.8 anastomoses per patient through limited and full access. One hundred seventy six LAD, 61 diagonal, 71 RCA, 7 RPD and 50 circumflex branches were grafted. Patients were pretreated with calcium antagonists, long-acting beta-blockade and had thoracic epidural blockade. The anastomosis was constructed using two microvascular clamps, preceded by ischemic preconditioning in non-occlusive disease. Myocardial ischemia was defined as1 mm S-T segment elevation. A simple aorto-coronary shunt, consisting of two intravenous catheters and a 10 cm connecting tube (flow20 ml/min), was used in critical ischemia.Ischemia occurred during 35 (10%) temporary coronary artery occlusions. Fifteen of these (43%) were RCA. In five of these 15 patients, all with non-occlusive disease, critical ischemia occurred with bradycardia, third-degree heart block and subsequently severe hypotension, which normally requires conversion to cardio-pulmonary bypass. Following introduction of the shunt (4 patients) electrocardiographic and hemodynamic parameters normalized within 30 s. The off-pump procedures could be continued uneventfully. There were no peri-operative infarctions.Temporary segmental occlusion is an effective method for anastomosis suturing in off-pump, beating heart CABG. Critical ischemia was observed rarely, only in the RCA and in non-occlusive disease. Temporary aorto-coronary shunting could avoid conversion to cardiopulmonary bypass and myocardial infarction.

False aneurysms of the internal mammary artery are extremely rare. A case of false aneurysm of a branch of the right internal mammary artery after median sternotomy is reported. A large right-sided mediastinal mass was seen on the thoracic radiogram. A false aneurysm was suspected on CT-scan and confirmed by angiography. In the same setting percutaneous embolization was performed.

Postoperative recovery after (arterial) coronary bypass grafting mostly takes several months because of the effects of the heart-lung machine and the cardioplegia which are mostly haematological, pneumological and neurological in nature. The morbidity can be reduced by operating on the beating heart and via smaller access. One possibility is the 'Octopus' method developed in Utrecht, which involves local fixation of the beating heart. The postoperative recovery of the first 45 patients was favourable.

Intraoperative angioscopy was performed in three patients who underwent pulmonary embolectomy for massive pulmonary embolism. Angioscopy followed conventional techniques such as extracting the clot by a gallstone forceps, using a Fogarty catheter in the pulmonary tree or squeezing of the lungs. The rationale for angioscopy was to assess the result of these usual "blind" techniques. In two patients residual thrombus was detected and removed under direct visual control. Our initial experience suggests that intraoperative angioscopy appears to be useful in the detection of residual thrombus material, especially in the asanguinous, arrested heart. The small size of the angioscope allows easily access to the secondary, and up to the tertiary pulmonary branches. Clots can be visualized and extracted under direct visual control.

In the period January 1968 to January 1976, 1400 patients were treated for clavicular fractures. In 74 (5 percent) of cases co-adaptation osteosynthesis was carried out and the results are reported. Conservative treatment of clavicular fractures is to preferred. Surgical treatment should be performed only on strict indication. Percutaneous intramedullary Kirschner wire fixation, performed in an atraumatic manner and with proper postoperative treatment, gives good results for simple fractures. A warning is given against over-emphasis on the radiological findings in the treatment of clavicular fractures.

Intraluminal vena caval filters, inserted via the transvenous approach, are used often in the prevention of recurrent pulmonary embolism. Until lately, such filters have been unremovable. In a recent case, however, we treated a patient who experienced acute massive pulmonary embolism after having undergone an emergency right hemicolectomy. He underwent a successful emergency pulmonary embolectomy with the help of cardio-pulmonary bypass; to prevent recurrent embolism, we inserted a removable intracaval filter through the right atriotomy with an introducer set. On the 7th postoperative day, the absence of significant residual thrombi was confirmed by means of phlebography, and the caval filter was removed percutaneously via the femoral approach. Although the value of routine use of the filter in this application has yet to be established, we feel that it warrants further investigation because it relieves the surgeon, during an emergency procedure, of the need to decide quickly whether or not to place a filter. The question becomes one of whether or not to remove the filter, and that decision can await the results of proper postoperative diagnostic studies.