Estimation of glucose utilization in a type 2 diabetes mellitus patient on insulin analogs with tumor hypoglycemia induced by IGF-II.

We present a 38-year-old male patient with insulin requiring type 2 diabetes mellitus (DM) who had fasting hypoglycemia caused by a non-pancreatic-islet-cell mesenchymal tumor producing IGF-II. The evaluation was confounded in that there was pre-existing DM being treated with insulin analogs. Insulin levels were assessed with an immunoassay with cross reactivity with the insulin analogs. An 18-Fluorodeoxyglucose (FDG) positron emission tomography/computerized tomography (PET/CT) scan localized the 19.7×18.0×17.8cm retroperitoneal mass. A 3.25kg tumor was resected. Post-operatively insulin treatment was resumed and circulating IGF-II levels returned to normal. The maximum standardized uptake values of FDG (SUVmax) along with a steady state glucose infusion of 17.5g/h were used to determine the components of glucose utilization due to IGF-II induced muscle glucose uptake (utilization, 62%) whereas the tumor itself was responsible for approximately 22% of measurable glucose uptake. Whereas tumor induced hypoglycemia has been ascribed to preferential glucose utilization by the tumor, the predominant hypoglycemic effect was due to hormonal IGF-II induced total body glucose uptake.

Urinary organic acids quantitated in a healthy north Indian pediatric population.

Human urine gives evidence of the metabolism in the body and contains numerous organic acids and other compounds at a variety of concentration. The concentration of organic acids in urine varies from population to population due to genotype, food habits and other epigenetic and environmental influences. Knowledge of the reference values for urinary organic acids in a healthy pediatric population is very important for critical evaluation. This study was designed to quantify 16 organic acids in a healthy north Indian pediatric population. Early morning urine samples from healthy pediatric subjects of age 1 day to 16 years who did not have symptoms of any disease were analyzed for organic acid content. The children were not on any supplemental vitamins or drugs and were on a free and unrestricted diet. The creatinine concentration of each sample was determined before organic acid analysis. Organic acids were extracted from urine with ethyl acetate, extracted residue was air dried, converted into trimethylsilyl derivatives and analysed by gas chromatography mass spectrometry. Here we reported the age wise mean values and standard deviations for each compound, adjusted for creatinine content (mmol/mol of creatinine). We found the concentration of most of the metabolites are higher in our population in comparison to other populations. Such data may help to provide a basis for diagnosing metabolic abnormalities in patients in a specific ethnicity.

Targeted mutagenesis of mitochondrial carbonic anhydrases VA and VB implicates both enzymes in ammonia detoxification and glucose metabolism.

Prior studies with carbonic anhydrase (CA) inhibitors implicated mitochondrial CA in ureagenesis and gluconeogenesis. Subsequent studies identified two mitochondrial CAs. To distinguish the contribution of each enzyme, we studied the effects of targeted disruption of the murine CA genes, called Car5A and Car5B. The Car5A mutation had several deleterious consequences. Car5A null mice were smaller than wild-type littermates and bred poorly. However, on sodium-potassium citrate-supplemented water, they produced offspring in expected numbers. Their blood ammonia concentrations were markedly elevated, but their fasting blood sugars were normal. By contrast, Car5B null mice showed normal growth and normal blood ammonia levels. They too had normal fasting blood sugars. Car5A/B double-knockout (DKO) mice showed additional abnormalities. Impaired growth was more severe than for Car5A null mice. Hyperammonemia was even greater as well. Although fertile, DKO animals were produced in less-than-predicted numbers even when supplemented with sodium-potassium citrate in their drinking water. Survival after weaning was also reduced, especially for males. In addition, fasting blood glucose levels for DKO mice were significantly lower than for controls (153 ± 33 vs. 230 ± 24 mg/dL). The enhanced hyperammonemia and lower fasting blood sugar, which are both seen in the DKO mice, indicate that both Car5A and Car5B contribute to both ammonia detoxification (ureagenesis) and regulation of fasting blood sugar (gluconeogenesis). Car5A, which is expressed mainly in liver, clearly has the predominant role in ammonia detoxification. The contribution of Car5B to ureagenesis and gluconeogenesis was evident only on a Car5A null background.

Substrate oxidation and cardiac performance during exercise in disorders of long chain fatty acid oxidation.

BACKGROUND: The use of long-chain fatty acids (LCFAs) for energy is inhibited in inherited disorders of long-chain fatty acid oxidation (FAO). Increased energy demands during exercise can lead to cardiomyopathy and rhabdomyolysis. Medium-chain triglycerides (MCTs) bypass the block in long-chain FAO and may provide an alternative energy substrate to exercising muscle. OBJECTIVES: To determine the influence of isocaloric MCT versus carbohydrate (CHO) supplementation prior to exercise on substrate oxidation and cardiac workload in participants with carnitine palmitoyltransferase 2 (CPT2), very long-chain acyl-CoA dehydrogenase (VLCAD) and long-chain 3-hydroxyacyl CoA dehydrogenase (LCHAD) deficiencies. DESIGN: Eleven subjects completed two 45-minute, moderate intensity, treadmill exercise studies in a randomized crossover design. An isocaloric oral dose of CHO or MCT-oil was administered prior to exercise; hemodynamic and metabolic indices were assessed during exertion. RESULTS: When exercise was pretreated with MCT, respiratory exchange ratio (RER), steady state heart rate and generation of glycolytic intermediates significantly decreased while circulating ketone bodies significantly increased. CONCLUSIONS: MCT supplementation prior to exercise increases the oxidation of medium chain fats, decreases the oxidation of glucose and acutely lowers cardiac workload during exercise for the same amount of work performed when compared with CHO pre-supplementation. We propose that MCT may expand the usable energy supply, particularly in the form of ketone bodies, and improve the oxidative capacity of the heart in this population.

One-step metabolomics: carbohydrates, organic and amino acids quantified in a single procedure.

Every infant born in the US is now screened for up to 42 rare genetic disorders called "inborn errors of metabolism". The screening method is based on tandem mass spectrometry and quantifies acylcarnitines as a screen for organic acidemias and also measures amino acids. All states also perform enzymatic testing for carbohydrate disorders such as galactosemia. Because the results can be non-specific, follow-up testing of positive results is required using a more definitive method. The present report describes the "urease" method of sample preparation for inborn error screening. Crystalline urease enzyme is used to remove urea from body fluids which permits most other water-soluble metabolites to be dehydrated and derivatized for gas chromatography in a single procedure. Dehydration by evaporation in a nitrogen stream is facilitated by adding acetonitrile and methylene chloride. Then, trimethylsilylation takes place in the presence of a unique catalyst, triethylammonium trifluoroacetate. Automated injection and chromatography is followed by macro-driven custom quantification of 192 metabolites and semi-quantification of every major component using specialized libraries of mass spectra of TMS derivatized biological compounds. The analysis may be performed on the widely-used Chemstation platform using the macros and libraries available from the author. In our laboratory, over 16,000 patient samples have been analyzed using the method with a diagnostic yield of about 17%--that is, 17% of the samples results reveal findings that should be acted upon by the ordering physician. Included in these are over 180 confirmed inborn errors, of which about 38% could not have been diagnosed using previous methods.

Isocitrate dehydrogenase is important for nitrosative stress resistance in Cryptococcus neoformans, but oxidative stress resistance is not dependent on glucose-6-phosphate dehydrogenase.

The opportunistic intracellular fungal pathogen Cryptococcus neoformans depends on many antioxidant and denitrosylating proteins and pathways for virulence in the immunocompromised host. These include the glutathione and thioredoxin pathways, thiol peroxidase, cytochrome c peroxidase, and flavohemoglobin denitrosylase. All of these ultimately depend on NADPH for either catalytic activity or maintenance of a reduced, functional form. The need for NADPH during oxidative stress is well established in many systems, but a role in resistance to nitrosative stress has not been as well characterized. In this study we investigated the roles of two sources of NADPH, glucose-6-phosphate dehydrogenase (Zwf1) and NADP(+)-dependent isocitrate dehydrogenase (Idp1), in production of NADPH and resistance to oxidative and nitrosative stress. Deletion of ZWF1 in C. neoformans did not result in an oxidative stress sensitivity phenotype or changes in the amount of NADPH produced during oxidative stress compared to those for the wild type. Deletion of IDP1 resulted in greater sensitivity to nitrosative stress than to oxidative stress. The amount of NADPH increased 2-fold over that in the wild type during nitrosative stress, and yet the idp1Delta strain accumulated more mitochondrial damage than the wild type during nitrosative stress. This is the first report of the importance of Idp1 and NADPH for nitrosative stress resistance.

Organic acid concentrations in amniotic fluid found in normal and Down syndrome pregnancies.

INTRODUCTION: Organic acids were examined from normal and Down syndrome pregnancies to identify possible differences between the amniotic fluid from fetuses with Down Syndrome compared with that of normal fetuses. MATERIALS AND METHODS: Amniotic fluids were obtained from prior amniocenteses. Forty-one normal and 22 Down syndrome specimens were assayed using gas chromatography/mass spectrometry. RESULTS AND DISCUSSION: 5-hydroxycaproate, methylsuccinate, alpha-ketoglutarate, and adipate were significantly elevated in Down syndrome, suggesting riboflavin deficiency. Phenylpyruvate was also significantly elevated in fetuses with Down syndrome. Phenylpyruvate inhibits the metabolism of tetrahydrobiopterin, which is necessary for neurotransmitter metabolism. Elevated phenylpyruvate is consistent with previous research, suggesting a disturbance of tetrahydrobiopterin metabolism in Down syndrome. CONCLUSION: Organic acid markers for B2 deficiency are elevated in the amniotic fluid of fetuses with Down syndrome. Elevation of phenylpyruvate may impair neurotransmitter metabolism. Organic acid markers for B12 levels are not different between the Down syndrome and normal group.

Pyridoxine-related metabolite concentrations in normal and Down syndrome amniotic fluid.

INTRODUCTION: Some studies of children with Down syndrome have found mild abnormalities in the metabolism of pyridoxine (vitamin B(6)); therefore the present question is whether such abnormalities might also be present in the amniotic fluid of fetuses with Down syndrome. MATERIALS AND METHODS: Archived specimens of amniotic fluid were obtained from chromosomally normal and from fetuses with Down syndrome. Gas chromatography/mass spectrometry quantitized B-related metabolites, including oxalate, xanthurenate, kynurenine and 4-pyridoxic acid. RESULTS: Oxalate, a marker of pyridoxine deficiency, was elevated in the amniotic fluid of fetuses with Down syndrome. This result was statistically significant. The other marker results were not statistically significant. CONCLUSION: A marker of pyridoxine deficiency, oxalate is elevated in the amniotic fluid of fetuses with Down syndrome. These results in amniotic fluid are consistent with previous studies done in the urine of young children.

Feeding Drosophila a biotin-deficient diet for multiple generations increases stress resistance and lifespan and alters gene expression and histone biotinylation patterns.

Energy restriction increases stress resistance and lifespan in Drosophila melanogaster and other species. The roles of individual nutrients in stress resistance and longevity are largely unknown. The vitamin biotin is a potential candidate for mediating these effects, given its known roles in stress signaling and gene regulation by epigenetic mechanisms, i.e. biotinylation of histones. Here, we tested the hypothesis that prolonged culture of Drosophila on biotin-deficient (BD) medium increases stress resistance and lifespan. Flies were fed a BD diet for multiple generations; controls were fed a biotin-normal diet. In some experiments, a third group of flies was fed a BD diet for 12 generations and then switched to control diets for 2 generations to eliminate potential effects of short-term biotin deficiency. Flies fed a BD diet exhibited a 30% increase in lifespan. This increase was associated with enhanced resistance to the DNA-damaging agent hydroxyurea and heat stress. Also, fertility increased significantly compared with biotin-normal controls. Biotinylation of histones was barely detectable in biotin-deprived flies, suggesting that epigenetic events might have contributed to effects of biotin deprivation.

A folate-dependent metabolite in amniotic fluid from pregnancies with normal or trisomy 21 chromosomes.

INTRODUCTION: Previous studies have given conflicting results as to whether or not folate metabolism is altered in Down syndrome. Folate is necessary to facilitate metabolism of one-carbon units. Folate accepts one-carbon units from one-carbon unit donors, including formiminoglutamate (FIGLU). Folate deficiency leads to accumulation of FIGLU and impairment of one-carbon unit metabolism. FIGLU is a functional measure of folate deficiency. MATERIALS AND METHODS: Archived anonymized amniotic fluid specimens were obtained from normal pregnancies and those with Down syndrome. Gas liquid chromatography/mass spectrometry was used to quantitate FIGLU, which is elevated in folate deficiency. A tetra-deuterated FIGLU was used as a standard, and single-ion monitoring was performed. Nonparametric statistical analysis was performed with the Mann-Whitney U test. RESULTS: FIGLU was significantly lower in pregnancies with Down syndrome. The median FIGLU level was 0.9 micromol/l in amniotic fluid from fetuses with Down syndrome. The median FIGLU level was 1.3 in amniotic fluid from control fetuses. This difference was statistically significant (p = 0.009). No statistically significant differences were found with histidine or glutamate. DISCUSSION: There was no evidence of folate deficiency. FIGLU was decreased, not increased. Decreased FIGLU might result from accelerated activity of one or more genes on chromosome 21, by a gene dosage effect. Genes which might explain the reduced FIGLU include one which degrades FIGLU (glutamate formiminotransferase-cyclodeaminase), one which participates in purine synthesis, and one which degrades homocysteine (cystathionine-beta-synthase).

Disruption of aldehyde reductase increases group size in dictyostelium.

Developing Dictyostelium cells form structures containing approximately 20,000 cells. The size regulation mechanism involves a secreted counting factor (CF) repressing cytosolic glucose levels. Glucose or a glucose metabolite affects cell-cell adhesion and motility; these in turn affect whether a group stays together, loses cells, or even breaks up. NADPH-coupled aldehyde reductase reduces a wide variety of aldehydes to the corresponding alcohols, including converting glucose to sorbitol. The levels of this enzyme previously appeared to be regulated by CF. We find that disrupting alrA, the gene encoding aldehyde reductase, results in the loss of alrA mRNA and AlrA protein and a decrease in the ability of cell lysates to reduce both glyceraldehyde and glucose in an NADPH-coupled reaction. Counterintuitively, alrA- cells grow normally and have decreased glucose levels compared with parental cells. The alrA- cells form long unbroken streams and huge groups. Expression of AlrA in alrA- cells causes cells to form normal fruiting bodies, indicating that AlrA affects group size. alrA- cells have normal adhesion but a reduced motility, and computer simulations suggest that this could indeed result in the formation of large groups. alrA- cells secrete low levels of countin and CF50, two components of CF, and this could partially account for why alrA- cells form large groups. alrA- cells are responsive to CF and are partially responsive to recombinant countin and CF50, suggesting that disrupting alrA inhibits but does not completely block the CF signal transduction pathway. Gas chromatography/mass spectroscopy indicates that the concentrations of several metabolites are altered in alrA- cells, suggesting that the Dictyostelium aldehyde reductase affects several metabolic pathways in addition to converting glucose to sorbitol. Together, our data suggest that disrupting alrA affects CF secretion, causes many effects on cellular metabolism, and has a major effect on group size.