Metabolic stability of cells for extended metabolomical measurements using NMR. A comparison between lysed and additionally heat inactivated cells.

NMR measurements for metabolic characterization of biological samples like cells, biopsies or plasma, may take several hours for advanced methods. Preanalytical issues, such as sample preparation and stability over the measurement time, may have a high impact on metabolite content, and potentially lead to misinterpretation. The aim of this study was therefore to investigate by 1H HR-MAS NMR the impact of different cell handling preparation protocols on the stability of the cell metabolite content over the measurement time. For this purpose, the metabolite content of fibroblasts and adrenal cells were measured at different time points after lysis and after additional heating. Interestingly the results showed similar metabolite concentrations between lysed and lysed-heated cells at the beginning of the measurement, but increasing differences after some hours of measurement. In lysed cells, metabolism was ongoing, producing metabolite changes over time, contrary to a stable metabolite content of the lysed-heated cells. These results were confirmed in both fibroblasts and adrenal cells. Therefore, in order to minimize metabolite content modifications over the measurement time, it is suggested to use cell lysis in combination with heat inactivation for extended HR-MAS NMR measurements.

A novel mutation in BCS1L associated with deafness, tubulopathy, growth retardation and microcephaly.

UNLABELLED: We report a novel homozygous missense mutation in the ubiquinol-cytochrome c reductase synthesis-like (BCS1L) gene in two consanguineous Turkish families associated with deafness, Fanconi syndrome (tubulopathy), microcephaly, mental and growth retardation. All three patients presented with transitory metabolic acidosis in the neonatal period and development of persistent renal de Toni-Debré-Fanconi-type tubulopathy, with subsequent rachitis, short stature, microcephaly, sensorineural hearing impairment, mild mental retardation and liver dysfunction. The novel missense mutation c.142A>G (p.M48V) in BCS1L is located at a highly conserved region associated with sorting to the mitochondria. Biochemical analysis revealed an isolated complex III deficiency in skeletal muscle not detected in fibroblasts. Native polyacrylamide gel electrophoresis (PAGE) revealed normal super complex formation, but a shift in mobility of complex III most likely caused by the absence of the BCS1L-mediated insertion of Rieske Fe/S protein into complex III. These findings expand the phenotypic spectrum of BCS1L mutations, highlight the importance of biochemical analysis of different primary affected tissue and underline that neonatal lactic acidosis with multi-organ involvement may resolve after the newborn period with a relatively spared neurological outcome and survival into adulthood. CONCLUSION: Mutation screening for BCS1L should be considered in the differential diagnosis of severe (proximal) tubulopathy in the newborn period. WHAT IS KNOWN: • Mutations in BCS1L cause mitochondrial complex III deficiencies. • Phenotypic presentations of defective BCS1L range from Bjornstad to neonatal GRACILE syndrome. What is New: • Description of a novel homozygous mutation in BCS1L with transient neonatal acidosis and persistent de Toni-Debré-Fanconi-type tubulopathy. • The long survival of patients with phenotypic presentation of severe complex III deficiency is uncommon.

Inflammation and atrial remodeling after a mountain marathon.

Endurance athletes have an increased risk of atrial fibrillation. We performed a longitudinal study on elite runners of the 2010 Jungfrau Marathon, a Swiss mountain marathon, to determine acute effects of long-distance running on the atrial myocardium. Ten healthy male athletes were included and examined 9 to 1 week prior to the race, immediately after, and 1, 5, and 8 days after the race. Mean age was 34.9 ± 4.2 years, and maximum oxygen consumption was 66.8 ± 5.8 mL/kg*min. Mean race time was 243.9 ± 17.7 min. Electrocardiographic-determined signal-averaged P-wave duration (SAPWD) increased significantly after the race and returned to baseline levels during follow-up (128.7 ± 10.9 vs. 137.6 ± 9.8 vs. 131.5 ± 8.6 ms; P < 0.001). Left and right atrial volumes showed no significant differences over time, and there were no correlations of atrial volumes and SAPWD. Prolongation of the SAPWD was accompanied by a transient increase in levels of high-sensitivity C-reactive protein, proinflammatory cytokines, total leucocytes, neutrophil granulocytes, pro atrial natriuretic peptide and high-sensitivity troponin. In conclusion, marathon running was associated with a transient conduction delay in the atria, acute inflammation and increased atrial wall tension. This may reflect exercise-induced atrial myocardial edema and may contribute to atrial remodeling over time, generating a substrate for atrial arrhythmias.

Efficacy and safety of secukinumab for the treatment of severe ABCA12 deficiency-related ichthyosis in a child.

Background: Patients with severe autosomal recessive congenital ichthyosis (ARCI) show a T helper 17/interleukin 17 (Th17/IL17) skewing in their skin and serum, resembling the inflammatory profile of psoriatic patients. Secukinumab, an IL-17A inhibitor, has shown clinical efficacy in patients with moderate-to-severe plaque psoriasis. Aims: To test the clinical efficacy and safety of secukinumab in a paediatric patient with ATP-binding cassette subfamily A member 12 deficiency-related severe erythrodermic ARCI. Materials & Methods: 6-months therapeutic trial. During the first 4-weeks induction period, the patient received weekly subcutaneous injections of 150 mg secukinumab (five injections in total). During the following 20-weeks maintenance period, the patient was given a subcutaneous injection of 150 mg secukinumab every 4 weeks. Result & Discussion: After the 6-months therapy period, there was a 48% reduction from the baseline Ichthyosis-Area-Severity-Index (-Erythema/-Scaling) score. The treatment was well tolerated. Moreover, cytokine analysis revealed a reduction of keratinocyte-derived proinflammatory cytokines and an abrogation of Th17-skewing during therapy. Conclusion: Further studies are needed to evaluate the effects of the use of IL-17A inhibition in ARCI patients.

Serum acylcarnitine profile in endurance horses with and without metabolic dysfunction.

Mitochondrial ß-oxidation is essential in fat metabolism and can be monitored with blood acylcarnitine profiling, as partly degraded fatty acids accumulate as their carnitine esters. To guarantee continuous energy supply during long-distance exercise, endurance horses oxidise considerable amounts of fat in the mitochondrion. In endurance races over 80 km, glycogen depletion is evident in equine slow-twitch high oxidative muscle fibres and as a consequence, horses participating in endurance races over 80 km rely almost entirely on ß-oxidation of fatty acids. This study investigated mitochondrial fatty acid ß-oxidation in endurance horses exposed to long-distance exercise. Electrospray tandem mass spectrometry analysis of serum acylcarnitine profiles from 10 Arab horses was performed before and after a 160 km endurance race. Results were analysed statistically using ANOVA. Mean speed over the entire race in finishing horses was 16.7 ± 1.2 km/h. Endurance exercise increased mitochondrial ß-oxidation approximately eight-fold (pre-race, 5648.62 ± 1508.52 nmol/L; post-race, 44,243.17 ± 11,504.45 nmol/L; P = 0.001). In these horses, there was an approximately 17-fold increased lipolysis, as demonstrated by elevated serum concentrations of non-esterified fatty acids (NEFA; pre-race, 0.08 ± 0.08 mmol/L; post-race, 1.32 ± 0.36 mmol/L; P < 0.001). In comparison, four Arab horses with poor performance showed an approximately five-fold increase in mitochondrial ß-oxidation (pre-race, 5286.17 ± 3355.16 nmol/L; post-race, 26,660.57 ± 10,064.27 nmol/L; P = 0.009); there was a 29-fold increase in NEFA (pre-race, 0.02 ± 0.01 mmol/L; post-race, 0.58 ± 0.07 mmol/L; P = 0.006) in these horses. Similar post-exercise free carnitine:acetylcarnitine ratios in both groups suggest that the availability of carnitine in long-distance endurance horses might limit performance.

Reproducibility of cerebral phenylalanine levels in patients with phenylketonuria determined by 1H-MR spectroscopy.

The reproducibility of metabolite content determined by MR spectroscopy (MRS) is usually at best a few percent for the prominent singlets. When studying low-concentration metabolites, like phenylalanine (Phe), where tissue content can be <100 micromol/kg, better reproducibility is paramount-particularly in view of using MRS results for potential individual treatment advice. An optimized, targeted spectroscopy method was established at 1.5T and reproducibility was established in 21 patients with phenylketonuria (PKU) where three spectra were recorded in each of three independent sessions, two of which were in immediate succession to minimize physiologic variation. Intersession variation was found to be only 7 micromol/kg Phe for back-to-back repetition of sessions, in close agreement with the variation of 16 micromol/kg observed for single spectra within a session. Analysis of variance proved the individuality of the blood/brain Phe ratio-though this ratio seems to be influenced by physiologic factors that are not stable in time. The excellent reproducibility was achieved through optimization of various factors, including signal-to-noise ratio, repositioning, and prescan calibrations, but also by enforcing as much prior information as possible (e.g., lineshape and phase from reference scans, constant prior-knowledge-locked baseline). While the application of maximum general prior knowledge is a general method to reduce fluctuations, one should remember that it may introduce systematic errors.

Novel mutation in OTC gene causes neonatal death in twin brothers.

Ornithine transcarbamylase (OTC) deficiency is the most common inborn error of the urea cycle. OTC locus is located in the short arm of X-chromosome. Authors report a case of a woman who gave birth to monozygotic male twins who later died because of severe neonatal-onset hyperammonaemic encephalopathy caused by a novel mutation of OTC gene. Post-mortem liver biopsy was taken from the second twin; afterwards, blood was drawn from the mother for examination. DNA sequence data showed that the mother was a carrier of the same novel mutation that was previously detected in the case of her son. In OTC deficiency, detection of female carriers is important for genetic counselling and eventual prenatal diagnosis.

[Hypoglycaemia--diagnosis and therapy in emergencies]. / Hypoglykämien--Diagnostik und Therapie im Notfall.

Hypoglycaemia defined as blood sugar below 2.5 mmol/L, is always an important medical emergency. The primary therapy is simple and consists of iv glucose. The occurrence of hypoglycaemia is directly connected to fuel balance, determined by the availability of glucose, free fatty acids and ketone bodies. An intact fuel balance and maintenance of normal blood sugar concentration is dependent upon: (1) an adequate caloric and qualitative dietary intake; (2) afunctionally intact hepatic glucogenolytic and gluconeogenic enzyme system; (3) an adequate supply of endogenous gluconeogenic substrates (lactate, amino acids and glycerol) (4) an adequate energy supply provided by the beta-oxidation of fatty acids to synthesize glucose and ketone bodies and (5) a normal endocrine system (insulin, glucagon, catecholamines and growth hormone) for integrating and modulating these processes. Disturbances in each of these factors may lead to hypoglycaemia. Glucose, like oxygen, is of essential and fundamental importance for the brain metabolism. The major contribution of the brain to the basal metabolic rate (70% in neonates versus 20% in adults) is an important factor contributing to the frequency and severity of a hypoglycaemic syndrome in the paediatric age. If hypoglycaemia is suspected, blood should be obtained prior to treatment for serum glucose determination and an extra tube (5 ml) serum should be obtained and refrigerated for further investigations. The first voided urine has to be tested for ketones using a dipstick and also refrigerated for further investigations. Basic laboratory investigations, anamnestic informations and clinical findings allow rapid tentative diagnosis and determine the specialized investigations out of the refrigerated material. A rapid definitive diagnosis is important for the specific treatment and to avoid recurrent and prolonged hypoglycaemia.

Regulation of human GH receptor gene transcription by 20 and 22 kDa GH in a human hepatoma cell line.

The human GH gene is 1.7 kilobase pairs (kb) in length and is composed of five exons and four introns. This gene is expressed in the pituitary gland and encodes a 22 kDa protein. In addition to this predominant (75%) form, 5-10% of pituitary GH is present as a 20 kDa protein that has an amino acid (aa) sequence identical to the 22 kDa form except for a 15 aa internal deletion of residues 32-46 as a result of an alternative splicing event. Because it has been reported that non-22-kDa GH isoforms might be partly responsible for short stature and growth retardation in children, the aim of this study was to compare the impact of both 22 kDa and 20 kDa GH on GH receptor gene (GH receptor/GH binding protein (GHR/GHBP)) expression. Various concentrations of 20 kDa and 22 kDa GH (0, 2, 5, 12.5, 25, 50 and 150 ng/ml) were added to human hepatoma (HuH7) cells cultured in serum-free hormonally defined medium for 0, 1 and 2 h. Thereafter GHR/GHBP mRNA expression was measured by quantitative PCR. Addition of either 20 kDa or 22 kDa GH, at low or normal physiological concentrations (0, 2, 5, 12.5, 25 or 50 ng/ml) induced a dose-dependent increase in GHR/GHBP expression. However, a supraphysiological concentration of 20 kDa GH (150 ng/ml) resulted in a significantly lower (P<0.05) downregulation of GHR/GHBP gene transcription compared with the downregulation achieved by this concentration of 22 kDa GH. This difference might be explained by a decreased ability to form a 1 : 1 complex with GHR and/or GHBP, which normally occurs at high concentrations of GH. Nuclear run-on experiments and GHBP determinations confirmed the changes in GHR/GHBP mRNA levels. In conclusion, we report that both 20 kDa and 22 kDa GH, in low and normal physiological concentrations, have the same effect on regulation of GHR/GHBP gene transcription in a human hepatoma cell line. At a supraphysiological concentration of 150 ng/ml, however, 20 kDa GH has a less self-inhibitory effect than the 22 kDa form.

Regulation of human growth hormone receptor gene transcription by human growth hormone binding protein.

The hypothesis that growth hormone binding protein (GHBP) has an effect on its own on the regulation of the GH-receptor/GHBP transcription was tested. Three different forms of human GHBP (recombinant non-glycosylated GHBP, recombinant glycosylated GHBP and GHBP purified and extracted from serum) were added in different concentrations determined by LIFA [0 pmol/l; 50 pmol/l (low level), 200 pmol/l (average level) and 500 pmol/l (high level in circulation)] to a human hepatoma cell line (HuH7 cells) cultured in a serum free hormonally-defined medium. Following the incubation with GHBP for 0, 1 and 2 h, GH-receptor expression was quantitatively assessed by using polymerase chain reaction amplification. Treatment with a GHBP concentration of 50 pmol/l resulted in a significant increase of GH-receptor mRNA molecules given as number of molecules x 10(6)/microg total RNA. In contrast, the concentration of 500 pmol/l presented a significant decrease of GH-receptor mRNA molecules, whereas 200 pmol/l GHBP produced a GH-receptor gene expression which was in between the values of the experiments with 50 and 500 pmol/l of GHBP added. Furthermore, the three different forms of human GHBP used provided similar data and, therefore, did not effect in any variation of GH-receptor expression. In addition, nuclear run-on experiments confirmed the changes in GH-receptor expression; and cycloheximide (10 microg/ml) did not alter the transcription indicating that the up and down regulating effects of GHBP on the GH-receptor/GHBP gene transcription was dependent, at least partly, on pre-existing factors and does not require protein synthesis. In conclusion, we present data showing that GHBP on its own has an effect on GH-receptor gene expression.

Novel mitochondrial tRNA(Ile) m.4282A>G gene mutation leads to chronic progressive external ophthalmoplegia plus phenotype.

BACKGROUND/AIM: To investigate the underlying pathomechanism in a 33-year-old female Caucasian patient presenting with chronic progressive external ophthalmoplegia (CPEO) plus symptoms. METHODS: Histochemical analysis of skeletal muscle and biochemical measurements of individual oxidative phosphorylation (OXPHOS) complexes. Genetic analysis of mitochondrial DNA in various tissues with subsequent investigation of single muscle fibres for correlation of mutational load. RESULTS: The patient's skeletal muscle showed 20% of cytochrome c oxidase-negative fibres and 8% ragged-red fibres. Genetic analysis of the mitochondrial DNA revealed a novel point mutation in the mitochondrial tRNA(Ile) (MTTI) gene at position m.4282G>A. The heteroplasmy was determined in blood, buccal cells and muscle by restriction fragment length polymorphism (RFLP) combined with a last fluorescent cycle. The total mutational load was 38% in skeletal muscle, but was not detectable in blood or buccal cells of the patient. The phenotype segregated with the mutational load as determined by analysis of single cytochrome c oxidase-negative/positive fibres by laser capture microdissection and subsequent LFC-RFLP. CONCLUSIONS: We describe a novel MTTI transition mutation at nucleotide position m.4282G>A associated with a CPEO plus phenotype. The novel variant at position m.4282G>A disrupts the middle bond of the D-stem of the tRNA(Ile) and is highly conserved. The conservation and phenotype-genotype segregation strongly suggest pathogenicity and is in good agreement with the MTTI gene being frequently associated with CPEO. This novel variant broadens the spectrum of MTTI mutations causing CPEO.

Impairment of mitochondrial tRNAIle processing by a novel mutation associated with chronic progressive external ophthalmoplegia.

We report a sporadic case of chronic progressive external ophthalmoplegia associated with ragged red fibers. The patient presented with enlarged mitochondria with deranged internal architecture and crystalline inclusions. Biochemical studies showed reduced activities of complex I, III and IV in skeletal muscle. Molecular genetic analysis of all mitochondrial tRNAs revealed a G to A transition at nt 4308; the G is a highly conserved nucleotide that participates in a GC base-pair in the T-stem of mammalian mitochondrial tRNA(Ile). The mutation was detected at a high level (approx. 50%) in muscle but not in blood. The mutation co-segregated with the phenotype, as the mutation was absent from blood and muscle in the patient's healthy mother. Functional characterization of the mutation revealed a six-fold reduced rate of tRNA(Ile) precursor 3' end maturation in vitro by tRNAse Z. Furthermore, the mutated tRNA(Ile) displays local structural differences from wild-type. These results suggest that structural perturbations reduce efficiency of tRNA(Ile) precursor 3' end processing and contribute to the molecular pathomechanism of this mutation.

Neuropeptide Y modulates steroid production of human adrenal H295R cells through Y1 receptors.

Neuropeptide Y (NPY) is abundantly expressed in the nervous system and acts on target cells through NPY receptors. The human adrenal cortex and adrenal tumors express NPY receptor subtype Y1, but its function is unknown. We studied Y1-mediated signaling, steroidogenesis and cell proliferation in human adrenal NCI-H295R cells. Radioactive ligand binding studies showed that H295R cells express Y1 receptor specifically. NPY treatment of H295R cells stimulated the MEK/ERK1/2 pathway, confirming that H295R cells express functional Y1 receptors. Studies of the effect of NPY and related peptide PYY on adrenal steroidogenesis revealed a decrease in 11-deoxycortisol production. RIA measurements of cortisol from cell culture medium confirmed this finding. Co-treatment with the Y1 antagonist BIBP2336 reversed the inhibitory effect of NPY on cortisol production proving specificity of this effect. At mRNA level, NPY decreased HSD3B2 and CYP21A2 expression. However NPY revealed no effect on cell proliferation. Our data show that NPY can directly regulate human adrenal cortisol production.

[Hypoglycemia in infancy -- not always of no importance!]. / Hypoglykämie im Säuglingsalter--nicht immer eine Banalität!

A six month old boy is admitted to the children's hospital for sudden loss of consciousness. Hypoglycemia is diagnosed and corrected. Further investigations reveal the diagnosis of hyperinsulinism as underlying cause for hypoglycaemic episodes. Differential diagnosis and therapy of hypoglycemia in infancy are discussed.

[Floppy baby with macrocytic anemia and vegan mother]. / Floppy Baby mit makrozytärer Anämie und veganischer Mutter.

We report the case of a 7 month-old girl that presented with acute anemia, generalized muscular hypotonia and failure to thrive. Laboratory evaluation revealed cobalamin deficiency, due to a vegan diet of the mother. The clinical triad of an acquired floppy baby syndrome with megaloblastic anemia and failure to thrive is pathognomic for infantile cobalamin deficiency. Neurological abnormalities are often irreversible and may be associated with delayed myelinization in the MRI. A normal cobalamin level in maternal serum and absence of anemia do not exclude subclinical deficiency. If cobalamin deficiency is suspected, e.g. in pregnant women on vegan diet, urinary methylmalonic acid excretion and plasma homocysteine levels should be determined and cobalamin substitution should be started at an early stage to avoid potentially irreversible damage of the fetus.

Treatment with low-dose diazoxide in two growth-retarded prepubertal girls with glycogen storage disease type Ia resulted in catch-up growth.

Two originally prepubertal girls suffering from glycogen storage disease type Ia and short stature were treated with low-dose diazoxide (3-4.8 mg/kg per day) for 7 and 4 years, respectively. Both showed an impressive catch-up growth following this treatment. This appeared to be due to prolongation of normoglycaemia after meals and reduction of fasting lactic acidosis by diazoxide.

[Eating behavior, diabetes and weight control in girls with insulin-dependent diabetes mellitus (type 1)]. / Essverhalten, Diabetes- und Gewichtskontrolle bei Mädchen mit insulinpflichtigem Diabetes mellitus (Typ 1).

The object of the study was, first, to investigate whether girls suffering from insulin-dependent diabetes mellitus (IDDM) are more overweight than an age- and puberty-matched control group and, second, to study the impact of diet, calorie intake and pubertal stage on body mass index (BMI), body weight and fat content. We studied 43 girls with IDDM and controls, divided into two age groups: group 1 (n = 21; 10-13 years) and group 2 (n = 22; > 13 years, 13.1-20.7 years). Overweight was assessed by BMI, relative weight and body fat from skinfold thickness. Food consumption data were collected over a one week food and drink protocol. The diabetic girls, particularly those after puberty, were more overweight than the controls. Although the calorie intake was increased compared with their peers, the proportions of energy derived from protein, fat and carbohydrate were as recommended by the American and Swiss Diabetes Association. Most importantly, the recommended proportion of saturated fatty acids (< 10%) was not achieved by either the diabetic patients or the control girls. Insulin dose/unit body weight correlated with BMI and fat content. Therefore, the increased insulin dose may be responsible for the relatively increased energy intake and, in addition, increased intake of saturated fatty acid which has been related to poor metabolic control and obesity. The food intake of the control girls was identical to that reported in adults by the Swiss Government in 1991 in the Third Report on Food Consumption.

Weekly versus biweekly lipid removal and effect of statins in severe hypercholesterolemia.

Lipid removal using a continuous-flow extracorporeal system is of proven efficacy in severe hypercholesterolemia. Because of the inconveniences and expenses of extracorporeal removal of lipids, the effects of two treatment intervals (weekly versus biweekly) were assessed in two adolescents with circulating cholesterol higher than 20.0 mmol/L. In both patients, circulating levels were largely lower on a weekly lipid removal interval when compared with a biweekly interval. A rapid reaccumulation of cholesterol was noted after lipid removal. Treatment with simvastatin decreased the rapid reappearance of total cholesterol noted during the first 2 days after lipid removal but without any major effect on the subsequent reaccumulation of cholesterol. Aggressive treatment of severe hypercholesterolemia with statins and especially with extracorporeal lipid removal is now possible and of proven efficacy. The minimal practical lipid removal treatment interval should be used.