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In the diagnostics of orthopedic diseases in the horse, diagnostic imaging often plays a decisive role. Cone beam computed tomography (CBCT) imaging is used in both human and small animal medicine and becoming increasingly popular. To see whether CBCT imaging can be useful in the diagnosis of orthopedic diseases of the carpal region of the horse and to explore possible limitations we compared CBCT images with multidetector computed tomography (MDCT) images of the carpal region of equine cadaveric specimens. Twenty-eight forelimbs from fifteen horses, slaughtered for reasons unrelated to this study, were examined. Native and contrast enhanced CBCT and MDCT scans were performed. Anatomical structures were blindly evaluated by three independent experienced observers using a visual scoring system previously reported and adapted to the equine carpal region. A descriptive evaluation was carried out as well as Spearman's rank correlation and interobserver agreement was shown by percent agreement (PA). Visualization of osseous structures was excellent in both MDCT and CBCT. Articular cartilage could only be assessed in contrast enhanced scans whereby MDCT showed a slightly better visualization than CBCT. Soft tissue structures were generally difficult to assess. An exception were the medial and lateral palmar intercarpal ligament, which could not be visualized in native but were well visualized in contrast enhanced scans in both MDCT and CBCT images. For the evaluation of osseous structures and some intraarticular ligaments after contrast enhancement, CBCT serves as a reliable diagnostic imaging modality for the equine carpal region. However, soft tissue structures and cartilage are imaged more reliably using MDCT.

Background:Mutations in the DNA polymerase-γ (POLG) gene are a major cause of clinically heterogeneous mitochondrial diseases, associated with mtDNA depletion and multiple deletions.Objective:To determine the spectrum of POLG mutations in our Dutch patient cohort, to evaluate the pathogenicity of novel mutations, and to establish genotype–phenotype correlations.Results:The authors identified 64 predominantly recessive mutations in 37 patients from a total of 232 patients, consisting of 23 different mutations. The substitution p.A467T was most frequently observed (n = 23), but was as frequent in childhood cases as in adult cases. Five new pathogenic recessive mutations, p.Lys925ArgfsX42, p.R275X, p.G426S, p.A804T and p.R869Q were identified. The known dominant chronic progressive external ophthalmoplegia (CPEO) mutation p.R943H was for the first time associated with premature ovarian failure as well. In 19 patients the authors identified only a single recessive mutation, or a sequence variant with unclear clinical significance. The data substantiate earlier observations that in POLG patients a fatal status epilepticus and liver failure can be triggered by sodium valproate. It is therefore important to exclude POLG mutations before administering this treatment.Conclusion:The clinical features of the patient are the most important features to select putative POLG mutation carriers and not the presence of mtDNA deletions or OXPHOS (oxidative phosphorylation) activity. The authors conclude that POLG mutations are an important cause of heterogeneous mitochondrial pathology and that more accurate genotype–phenotype correlations allow a more rapid genetic diagnosis and improved prognosis for mutation carriers.

In this time-frame the test has evolved from a single enzyme measurement using ultra-high performance liquid chromatography (UHPLC) in peripheral blood mononuclear cells (PBMCs) to a combined enzymatic and genetic test of four variants in the DPYD gene (DPYD*2A, DPYD*13, c.2846A>T and 1129-5923C>G). [...]at this moment a combination of a...

BackgroundElevated uric acid concentrations have been associated with high blood pressure (BP) in adults and children. An increased production of uric acid by the enzyme xanthine oxidoreductase (XOR), which is accompanied by the generation of reactive oxygen species, is a putative underlying mechanism that has so far scarcely been studied. We hypothesize that a higher XOR activity leads to increased concentrations of downstream purine metabolites, resulting in higher ratios of xanthine/hypoxanthine, uric acid/xanthine, and uric acid/hypoxanthine (figure 1).ObjectivesThe objectives were therefore to study the association between (i) the purine metabolites uric acid, hypoxanthine, and xanthine and (ii) the ratios of the three different purine metabolites as proxy measures for uric acid production, with systolic (SBP) and diastolic blood pressure (DBP).MethodsCross-sectional analyses were performed in a subset of 246 children from the Dutch Koala Birth Cohort Study. This cohort originates from two recruitment groups: pregnant women with either a conventional lifestyle or recruited through alternative channels. During a home visit, a nurse collected a venous blood sample and measured BP three times after a 5-minute rest, with an automated BP monitor. Furthermore, parents were in instructed to measure their child's BP on three consecutive days, in the morning and evening, using the same monitor. Purine metabolites were determined with UPLC-MS/MS. Data were analysed using a multivariable generalized estimating equations model, including repeated BP measurements, with an exchangeable correlation structure. Analyses were adjusted for sex, age, BMI, recruitment group, place and mode of delivery (hospital vs. at home; natural vs. caesarean section), maternal smoking during pregnancy (active and passive), total physical activity, and dietary intake at four years of age (total energy intake [kcal], energy from carbohydrates and protein [%] and fibre intake [grams]).ResultsAt a mean age of 7.1 yrs, mean plasma concentrations were 202.8 μmol/L (SD 37.7) for uric acid, 4.60 μmol/L (SD 6.93) for hypoxanthine, and 0.53 μmol/L (SD 0.17) for xanthine. Average BP was 104.3 mm Hg (SD 10.0) systolic and 63.4 mm Hg (SD 10.6) diastolic. Higher ratios of uric acid/xanthine and xanthine/hypoxanthine were associated with higher DBP (adj. β 0.01 mm Hg; 95%CI 0.00 to 0.02; P=0.03; and adj. β 0.11 mm Hg; 95%CI 0.01 to 0.21; P=0.03), respectively, but not with SBP. Uric acid, xanthine, and hypoxanthine concentrations and the ratio uric acid/hypoxanthine were not associated with SBP nor DBP (P≥0.05).ConclusionsIn multivariable-adjusted analyses, DBP in children was significantly associated with the ratios of uric acid/xanthine and xanthine/hypoxanthine, and might, as hypothesized, be proxies for uric acid production. The ratio uric acid/hypoxanthine was not associated with BP, may be explained by differential excretion or autofeedback (of non XOR metabolism).Disclosure of InterestNone declared

Dietary protein digestion and amino acid absorption kinetics determine the post-prandial muscle protein synthetic response. Body position may affect gastrointestinal function and modulate the post-prandial rise in plasma amino acid availability. We aimed to assess the impact of body position on gastric emptying rate and the post-prandial rise in plasma amino acid concentrations following ingestion of a single, meal-like amount of protein. In a randomized, cross-over design, eight healthy males (25 ± 2 years, 23.9 ± 0.8 kg·m−2) ingested 22 g protein and 1.5 g paracetamol (acetaminophen) in an upright seated position (control) and in a −20° head-down tilted position (inversion). Blood samples were collected during a 240-min post-prandial period and analyzed for paracetamol and plasma amino acid concentrations to assess gastric emptying rate and post-prandial amino acid availability, respectively. Peak plasma leucine concentrations were lower in the inversion compared with the control treatment (177 ± 15 vs. 236 ± 15 mmol·L−1, p < 0.05), which was accompanied by a lower plasma essential amino acid (EAA) response over 240 min (31,956 ± 6441 vs. 50,351 ± 4015 AU; p < 0.05). Peak plasma paracetamol concentrations were lower in the inversion vs. control treatment (5.8 ± 1.1 vs. 10.0 ± 0.6 mg·L−1, p < 0.05). Gastric emptying rate and post-prandial plasma amino acid availability are significantly decreased after protein ingestion in a head-down tilted position. Therefore, upright body positioning should be considered when aiming to augment post-prandial muscle protein accretion in both health and disease.

Dihydropyrimidine dehydrogenase (DPD) deficiency is an infrequently described autosomal recessive disorder of the pyrimidine degradation pathway and can lead to mental and motor retardation and convulsions. DPD deficiency is also known to cause a potentially lethal toxicity following administration of the antineoplastic agent 5-fluorouracil. In an ongoing study of 72 DPD deficient patients, we analysed the molecular background of 5 patients in more detail in whom initial sequence analysis did not reveal pathogenic mutations. In three patients, a 13.8 kb deletion of exon 12 was found and in one patient a 122 kb deletion of exon 14-16 of DPYD. In the fifth patient, a c.299_302delTCAT mutation in exon 4 was found and also loss of heterozygosity of the entire DPD gene. Further analysis demonstrated a de novo deletion of approximately 14 Mb of chromosome 1p13.3-1p21.3, which includes DPYD. Haploinsufficiency of NTNG1, LPPR4, GPSM2, COL11A1 and VAV3 might have contributed to the severe psychomotor retardation and unusual craniofacial features in this patient. Our study showed for the first time the presence of genomic deletions affecting DPYD in 7% (5/72) of all DPD deficient patients. Therefore, screening of DPD deficient patients for genomic deletions should be considered.

The effect of the CTP synthetase inhibitor cyclopentenyl cytosine (CPEC) on the metabolism and cytotoxicity of 2',2'-difluorodeoxycytidine (dFdC, gemcitabine) and the expression and activity of deoxycytidine kinase (dCK) was studied in human neuroblastoma cell lines. The cytotoxicity of dFdC and CPEC was studied in a panel of MYCN-amplified and MYCN-single-copy neuroblastoma cell lines using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-assays. dFdC-metabolism was studied in SK-N-BE(2)c cells using [3H]-radiolabeled dFdC. dCK activity and expression were measured using enzyme assays, immunoblot and quantitative PCR, respectively. Both MYCN-amplified and MYCN-single-copy neuroblastoma cell lines were highly sensitive to dFdC, with concentration of the drug resulting in 50% effect when compared to untreated controls (ED50) values in the nanomolar range after a 3-h exposure to dFdC. There was no correlation of the observed ED50 with the dCK activity. Treatment with dFdC induced cell death in MYCN-amplified cells whereas MYCN-single-copy-cell lines underwent neuronal differentiation. Pre-incubation with CPEC significantly increased dFdC-cytotoxicity from 1.3 to 5.3-fold in 13 out of 15 cell lines. [3H]dFdC-anabolism increased 6-44 fold in SK-N-BE(2)c cells after incubation with CPEC and was paralleled by a significant increase in expression and activity of dCK. In conclusion, the combination of dFdC and CPEC is highly toxic to neuroblastoma in vitro.

The postprandial rise in essential amino acid (EAA) concentrations modulates the increase in muscle protein synthesis rates after protein ingestion. The EAA content and AA composition of the dietary protein source contribute to the differential muscle protein synthetic response to the ingestion of different proteins. Lower EAA contents and specific lack of sufficient leucine, lysine, and/or methionine may be responsible for the lower anabolic capacity of plant-based compared with animal-based proteins. We compared EAA contents and AA composition of a large selection of plant-based protein sources with animal-based proteins and human skeletal muscle protein. AA composition of oat, lupin, wheat, hemp, microalgae, soy, brown rice, pea, corn, potato, milk, whey, caseinate, casein, egg, and human skeletal muscle protein were assessed using UPLC–MS/MS. EAA contents of plant-based protein isolates such as oat (21%), lupin (21%), and wheat (22%) were lower than animal-based proteins (whey 43%, milk 39%, casein 34%, and egg 32%) and muscle protein (38%). AA profiles largely differed among plant-based proteins with leucine contents ranging from 5.1% for hemp to 13.5% for corn protein, compared to 9.0% for milk, 7.0% for egg, and 7.6% for muscle protein. Methionine and lysine were typically lower in plant-based proteins (1.0 ± 0.3 and 3.6 ± 0.6%) compared with animal-based proteins (2.5 ± 0.1 and 7.0 ± 0.6%) and muscle protein (2.0 and 7.8%, respectively). In conclusion, there are large differences in EAA contents and AA composition between various plant-based protein isolates. Combinations of various plant-based protein isolates or blends of animal and plant-based proteins can provide protein characteristics that closely reflect the typical characteristics of animal-based proteins.

Protein turnover in skeletal muscle tissue is highly responsive to nutrient intake in healthy adults. To provide a comprehensive overview of post-prandial protein handling, ranging from dietary protein digestion and amino acid absorption, the uptake of dietary protein derived amino acids over the leg, the post-prandial stimulation of muscle protein synthesis rates, to the incorporation of dietary protein derived amino acids in de novo muscle protein. 12 healthy young males ingested 20 g intrinsically [1-13C]-phenylalanine labeled protein. In addition, primed continuous L-[ring-2H5]-phenylalanine, L-[ring-2H2]-tyrosine, and L-[1-13C]-leucine infusions were applied, with frequent collection of arterial and venous blood samples, and muscle biopsies throughout a 5 h post-prandial period. Dietary protein digestion, amino acid absorption, splanchnic amino acid extraction, amino acid uptake over the leg, and subsequent muscle protein synthesis were measured within a single in vivo human experiment. 55.3±2.7% of the protein-derived phenylalanine was released in the circulation during the 5 h post-prandial period. The post-prandial rise in plasma essential amino acid availability improved leg muscle protein balance (from -291±72 to 103±66 μM·min-1·100 mL leg volume-1; P<0.001). Muscle protein synthesis rates increased significantly following protein ingestion (0.029±0.002 vs 0.044±0.004%·h-1 based upon the muscle protein bound L-[ring-2H5]-phenylalanine enrichments (P<0.01)), with substantial incorporation of dietary protein derived L-[1-13C]-phenylalanine into de novo muscle protein (from 0 to 0.0201±0.0025 MPE). Ingestion of a single meal-like amount of protein allows ~55% of the protein derived amino acids to become available in the circulation, thereby improving whole-body and leg protein balance. About 20% of the dietary protein derived amino acids released in the circulation are taken up in skeletal muscle tissue following protein ingestion, thereby stimulating muscle protein synthesis rates and providing precursors for de novo muscle protein synthesis. trialregister.nl 3638.

Background Treatment with 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) and dietary phenylalanine and tyrosine restriction improves physical health and life expectancy in Tyrosinemia type 1 (TT1). However, neurocognitive outcome is suboptimal. This study aimed to investigate behavior problems and health-related quality of life (HR-QoL) in NTBC-dietary-treated TT1 and to relate this to phenylalanine and tyrosine concentrations. Results Thirty-one TT1 patients (19 males; mean age 13.9 ± 5.3 years) were included in this study. Emotional and behavioral problems, as measured by the Achenbach System of Empirically Based Assessment, were present in almost all domains. Attention and thought problems were particularly evident. HR-QoL was assessed by the TNO AZL Children’s and Adults QoL questionnaires. Poorer HR-QoL as compared to reference populations was observed for the domains: independent daily functioning, cognitive functioning and school performance, social contacts, motor functioning, and vitality. Both internalizing and externalizing behavior problems were associated with low phenylalanine (and associated lower tyrosine) concentrations during the first year of life. In contrast, high tyrosine (and associated higher phenylalanine) concentrations during life and specifically the last year before testing were associated with more internalizing behavior and/or HR-QoL problems. Conclusions TT1 patients showed several behavior problems and a lower HR-QoL. Associations with metabolic control differed for different age periods. This suggests the need for continuous fine-tuning and monitoring of dietary treatment to keep phenylalanine and tyrosine concentrations within target ranges in NTBC-treated TT1 patients.