Ernest Fung

Effects of Medium-Chain Triglyceride Feeding on Energy Balance in Adult Humans

In recent years, the metabolism of triglycerides has attracted much attention. Oxidation of fatty acids is an essential energy supply, especially when glucose supply is limited. In the present study, the effect of a 3-day high medium-chain triglyceride (MCT; 51% of calories), low carbohydrate intake on plasma glucose and amino acid, and urinary organic acid levels, including dicarboxylic and tricarboxylic acid cycle intermediates, was determined in eight normal adult volunteer subjects. Urine was collected at baseline and at 48 to 72 hours for amino acid and organic acid levels, and plasma collected at 0 and 72 hours for glucose and amino acid concentration. The MCT diet increased urinary levels of dicarboxylic acids (adipic 8-, suberic 65-, sebacic 284-fold) and keto acids (acetoacetate and beta-hydroxybutyrate, 67.5-fold); alanine and lactate were decreased 2.5- and 4-fold, respectively, while pyruvate, other amino acids and citric acid intermediates remained unchanged. Plasma amino acid levels were unchanged, while the plasma glucose levels decreased by 8% from baseline. The loss of calories as urinary dicarboxylic acids and keto acids, although increased during the MCT diet, was less than 1% of the daily caloric intake. The data suggest MCT sustain energy expenditure through medium-chain fatty acid (MCFA) oxidation with no decrease in citric acid cycle intermediates, while sparing protein oxidation.

Secondary loss of deoxyguanosine kinase activity in purine nucleoside phosphorylase deficient mice

The T-cell immunodeficiency associated with purine nucleoside phosphorylase (PNP) deficiency in man is believed to be due to the accumulation of dGTP which may be preferentially formed from deoxyguanosine in T-lymphocytes or their precursor cells. We found no evidence for dGTP accumulation in thymocytes or spleen leucocytes, < 1 nmol/10(9) cells, nor in erythrocytes, < 0.05 nmol/10(9) cells, of the B6-NPE- or B6-NPF PNP-deficient mice strains. There were no changes in purine or pyrimidine ribonucleotide pools. As these mice had been previously shown to excrete PNP nucleoside substrates, we examined the metabolism of deoxyguanosine. Deoxyguanosine kinase activity as compared to control mice was 6 to 52% for the B6-NPE mutant, 2 to 22% for the B6-NPF mutant. Fractionation of erythrocyte and liver lysates from the F mutation and the background strain, C57BL/6J, by anion exchange chromatography confirmed the secondary deficiency of deoxyguanosine kinase and demonstrated that this activity was distinct from adenosine kinase and two major peaks of deoxycytidine kinase activity. Mouse PNP, expressed and purified as a fusion protein, did not show evidence of being bifunctional and having deoxyguanosine kinase activity. Metabolic modelling revealed that the ratio of deoxyguanosine phosphorylation versus phosphorolysis was < 0.06 in control mice, and < or = 0.3 in lymphocytes of PNP-deficient mice. Were deoxyguanosine kinase not reduced in the PNP-deficient mice, all tissues of the B6-NPF mutant would preferentially phosphorylate deoxyguanosine at low substrate concentrations.

The relationship between d‐beta‐hydroxybutyrate blood concentrations and seizure control in children treated with the ketogenic diet for medically intractable epilepsy

The ketogenic diet (KD) is a proven treatment for drug‐resistant (DR) seizures in children and adolescents. However, the relationship between seizure control and the most commonly measured metabolite of the diet, the ketone body d‐beta‐hydroxybutyrate (D‐BHB), is controversial. This study was performed to clarify the relationship because specific ketone bodies may be useful as biomarkers of diet efficacy.

Absence of dGTP accumulation and compensatory loss of deoxyguanosine kinase in purine nucleoside phosphorylase deficient mice.

The first reported cases of purine nucleoside phosphorylase (PNP) deficiency in man were characterized by selective cellular immune dysfunction1. Patients have a pronounced decrease in T-cell numbers with normal or exaggerated B-cell function2–4. They excrete PNP substrates and have elevated plasma levels of these metabolites. Intracellular nucleotide pools of erythrocytes show an increase in deoxyguanosine triphosphate (dGTP), nicotinamide adenine dinucleotide (NAD) and a decrease in guanosine triphosphate (GTP)4–7. The metabolite thought to be responsible for the T-cell dysfunction is dGTP5.

Increased Inosinate Dehydrogenase Activity in Mycophenolic acid Resistant Neuroblastoma Cells

By incremental increase in the concentrations of inhibitors of essential biosynthetic pathways, a number of specific genes have been amplified in cultured mammalian cells. We have attempted to obtain an amplified phenotype by the progressive selection of resistance to mycophenolic acid, a potent inhibitor of the final two enzymes in guanine ribonuleotide synthesis, IMP dehydrogenase 1,2 and GMP synthetase2. In previous studies the inhibition of IMP dehydrogenase by mycophenolic acid has been shown to result in marked overproduction of purines in both human lymphoblasts 3,4 and mouse lyphoma cells5. Mycophenolic acid induced changes in ribonucleotide3,5,6 ’ and deoxyribonucleotide pools are well characterized. A mouse lymphoma line resistant to 20 μM mycophenolic acid has been obtained by sequential mutagenesis5 and single step mutagen induced resistant hamster V79 cells have also been obtained7

Mapping a Gene that Determines Erythrocytic Guanosine-5’-Triphosphate Concentration (Gtpc) on Mouse Chromosome 9

Guanosine-5’-triphosphate (GTP) is a nucleotide serving as an energy source and substrate in multiple reactions including protein synthesis, cellular signal transduction, as a precursor for pterin cofactor synthesis, and a substrate in RNA synthesis. Erythrocytes from inbred strains of mice exhibit a 10–14 fold variation in GTP concentration, but a comparison of enzymes involved in guanine metabolism did not reveal the basis for the variation in cellular GTP concentration. We describe here a genetic analysis of the erythrocytic guanine nucleotide concentration determining trait which we have designated Gtpc.

Modified GMP-affinity chromatography for the purification of mutant hypoxanthine phosphoribosyltransferase☆

Hypoxanthine-guanine phosphoribosyltransferase (HPRT) catalyzes the conversion of hypoxanthine and guanine to IMP and GMP, respectively, in the presence of 5-phosphoribosyl-1-pyrophosphate. Deficiencies of HPRT are associated with neurological abnormalities and gout. A human HPRT variant enzyme failed to bind to a GMP-affinity column under standard purification conditions. We developed a series of predictive tests for designing the affinity chromatography protocol which enabled purification of both normal and variant HPRT. The primary variable for the present variant was a difference in toleration of salt; other aspects recommended for evaluation are assessment of ligand-enzyme affinity, pH optimum, and tolerance of nonspecific ligands for washes. In addition, a method for determining the amount of GMP linked to the column material was developed and consisted of acid hydrolysis and HPLC quantitation of guanine.

146 MOUSE MODELS OF PURINE NUCLEOSIDE PHOSPHORYLASE DEFICIENCY. CHARACTERIZATION OF PARTIALLY AND SEVERELY ENZYME DEFICIENT MUTANTS

Two mutations of purine nucleoside phosphorylase (PNP) were identified in the carrier state of the first generation progeny of male mice treated with ethylnitrosourea mated to untreated females. The variants are assigned the gene symbols Np-1e and Np-1f. Both carriers have approximately half normal PNP activity in erythrocytes and Np-1a/Np-1a was distinct from the inbred strain background, Np-1a/Np-1a on isoelectric focusing, whereas Np-1a/Np-1a was not distinguishable. In the homozygous state, Np-1a and Np-1a differ and determine a more basic pattern of PNP activity than the Np-1a allele. NP-1A is stable in the presence of phosphate at 55° C whereas the half lives for the mutants were 30 and 7.5 min for NP-1E and NP-1F respectively. The substrate Michaelis constants for the variants were unchanged from controls and the maximal velocities for erythrocytes were: 16.8 ± 1.1, NP-1A; 2.16 ± 0.12, NP-1E; and 0.50 ± 0.03, NP-1F (nmole/min/mg protein). Brain, heart, kidney, liver, spleen leukocytes and thymocytes also showed reductions in activity, 4-27% of normal for NP-1E and 0.1-3.9% for NP-1F. Purine nucleoside excretion correlated with the severity of the enzyme deficiency. The substrates of PNP are not found normally ingurine < 10 uM, but inosine and guanosine were present for Np-1e/Np-1e mice, total 150 ± 84 uM; as were inosine, guanosine, deoxyinosine, and deoxyguanosine for Np-1f/Np-1f mice, total 1490 ± 190 uM. Supported by the Medical Research Council grant MT-6376.

145 MYCOPHENOLIC ACID INDUCED AMPLIFICATION AND MUTATION OF INOSINATE DEHYDROGENASE IN MOUSE NEUROBLASTOMA CELLS

Mouse neuroblastoma cells (NB) have been adapted for growth in 1 mM mycophenolic acid (NB-myco) by incremental increases in drug concentration with each passage without mutagenesis. 50% growth inhibition occurred at 0.1 uM and 1 mM for NB and NB-myco cells respectively representing a 10,000-fold increase in resistance. There were increases in IMP dehydrogenase activity, 25-fold, and a 56.7K dalton protein, 200-500 fold, in NB-myco as compared to NB cells. The resistant phenotype was also unstable. In the absence of drug there were decreases in the 56.7K dalton protein, 4-fold over 90 days, and IMP dehydrogenase activity, 3-fold over 80 days. These findings are characteristic of amplification, others are indicative of a mutation. The substrate Kms were unchanged but the Kis have increased in the NB-myco cells: 2400-fold for mycophenolic acid, and 4-fold for XMP, The kinetic studies are consistent with an ordered bi bi reaction where IMP binds first and XMP is released last; mycophenolic acid and NAD+ exhibit uncompetitive inhibition with IMP. Guanine nucleotide synthesis was also perturbed. The ATP concentration was unchanged, GTP and UTP were increased 2- and 1.5-fold respectively in NB-myco cells as compared to NB cells. Upon removal of mycophenolic acid, ATP did not change, but within hours GTP increased to 4.5-fold that of NB cells. Supported by the Medical Research Council of Canada grant MT-8665.