Flux analysis of inborn errors of metabolism.

Patients with an inborn error of metabolism (IEM) are deficient of an enzyme involved in metabolism, and as a consequence metabolism reprograms itself to reach a new steady state. This new steady state underlies the clinical phenotype associated with the deficiency. Hence, we need to know the flux of metabolites through the different metabolic pathways in this new steady state of the reprogrammed metabolism. Stable isotope technology is best suited to study this. In this review the progress made in characterizing the altered metabolism will be presented. Studies done in patients to estimate the residual flux through the metabolic pathway affected by enzyme deficiencies will be discussed. After this, studies done in model systems will be reviewed. The focus will be on glycogen storage disease type I, medium-chain acyl-CoA dehydrogenase deficiency, propionic and methylmalonic aciduria, urea cycle defects, phenylketonuria, and combined D,L-2-hydroxyglutaric aciduria. Finally, new developments are discussed, which allow the tracing of metabolic reprogramming in IEM on a genome-wide scale. In conclusion, the outlook for flux analysis of metabolic derangement in IEMs looks promising.

A patient with acute liver failure and extreme hypoglycaemia with lactic acidosis who was not in a coma: causes and consequences of lactate-protected hypoglycaemia.

Lactate can substitute for glucose as a metabolic substrate. We report a patient with acute liver failure who was awake despite a glucose level of 0.7 mmol/l with very high lactate level of 25 mmol/l. The hypoglycaemia+hyperlactataemia combination may be considered paradoxical since glucose is the main precursor of lactate and lactate is reconverted into glucose by the Cori cycle. Literature relevant to the underlying mechanism of combined deep hypoglycaemia and severe hyperlactataemia was assessed. We also assessed the literature for evidence of protection against deep hypoglycaemia by hyperlactataemia. Four syndromes demonstrating hypoglycaemia+hyperlactataemia were found: 1) paracetamol-induced acute liver failure, 2) severe malaria, 3) lymphoma and 4) glucose-6-phosphatase deficiency. An impaired Cori cycle is a key component in all of these metabolic states. Apparently the liver, after exhausting its glycogen stores, loses the gluconeogenic pathway to generate glucose and thereby its ability to remove lactate as well. Several patients with lactic acidosis and glucose levels below 1.7 mmol/l who were not in a coma have been reported. These observations and other data coherently indicate that lactate-protected hypoglycaemia is, at least transiently, a viable state under experimental and clinical conditions. Severe hypoglycaemia+hyperlactataemia reflects failure of the gluconeogenic pathway of lactate metabolism. The existence of lactate-protected hypoglycaemia implies that patients who present with this metabolic state should not automatically be considered to have sustained irreversible brain damage. Moreover, therapies that aim to achieve hypoglycaemia might be feasible with concomitant hyperlactataemia.

From genome to phenome-Simple inborn errors of metabolism as complex traits.

Sporadically, patients with a proven defect in either mFAO or OXPHOS are described presenting with a metabolic profile and clinical phenotype expressing concurrent defects in both pathways. Biochemical linkages between both processes are tight. Therefore, it is striking that concurrent dysfunction of both systems occurs so infrequent. In this review, the linkages between OXPHOS and mFAO and the hypothesized processes responsible for concurrent problems in both systems are reviewed, both from the point of view of primary biochemical connections and secondary cellular responses, i.e. signaling pathways constituting nutrient-sensing networks. We propose that affected signaling pathways may play an important role in the phenomenon of concurrent defects. Recent data indicate that interference in the affected signaling pathways may resolve the pathological phenotype even though the primary enzyme deficiency persists. This offers new (unexpected) prospects for treatment of these inborn errors of metabolism. This article is part of a Special Issue entitled: From Genome to Function.

A novel approach to monitor glucose metabolism using stable isotopically labelled glucose in longitudinal studies in mice.

The aetiology of insulin resistance is still an enigma. Mouse models are frequently employed to study the underlying pathology. The most commonly used methods to monitor insulin resistance are the HOMA-IR, glucose or insulin tolerance tests and the hyperinsulinemic euglycaemic clamp (HIEC). Unfortunately, these tests disturb steady state glucose metabolism. Here we describe a method in which blood glucose kinetics can be determined in fasted mice without noticeably perturbing glucose homeostasis. The method involves an intraperitoneal injection of a trace amount of [6,6-(2)H2]glucose and can be performed repeatedly in individual mice. The validity and performance of this novel method was tested in mice fed on chow or high-fat diet for a period of five weeks. After administering the mice with [6,6-(2)H2]glucose, decay of the glucose label was followed in small volumes of blood collected by tail tip bleeding during a 90-minute period. The total amount of blood collected was less than 120 µL. This novel approach confirmed in detail the well-known increase in insulin resistance induced by a high-fat diet. The mice showed reduced glucose clearance rate, and reduced hepatic and peripheral insulin sensitivity. To compensate for this insulin resistance, ß-cell function was slightly increased. We conclude that this refinement of existing methods enables detailed information of glucose homeostasis in mice. Insulin resistance can be accurately determined while mechanistic insight is obtained in underlying pathology. In addition, this novel approach reduces the number of mice needed for longitudinal studies of insulin sensitivity and glucose metabolism.

Pathogenesis of cognitive dysfunction in phenylketonuria: review of hypotheses.

In untreated phenylketonuria (PKU), deficiency of phenylalanine hydroxylase (PAH) results in elevated blood phenylalanine (Phe) concentrations and severe mental retardation. Current dietary treatment prevents mental retardation, but cognitive outcome remains suboptimal. The mechanisms by which elevated blood Phe concentrations disturb cerebral metabolism and cognitive function have not been fully elucidated. In this review, we discuss different hypotheses on the pathogenesis of PKU, focusing on the effects of disturbed large neutral amino acid (LNAA) transport from blood to brain on cerebral neurotransmitter and protein synthesis. Although the definitive roles of these processes in PKU pathogenesis are not fully understood yet, both substantially influence clinical outcome.

Soraphen, an inhibitor of the acetyl-CoA carboxylase system, improves peripheral insulin sensitivity in mice fed a high-fat diet.

AIM: Inhibition of the acetyl-CoA carboxylase (ACC) system, consisting of the isozymes ACC1 and ACC2, may be beneficial for treatment of insulin resistance and/or obesity by interfering with de novo lipogenesis and beta-oxidation. We have evaluated effects of pharmacological inhibition of ACC by soraphen (SP) on high fat (HF) diet-induced insulin resistance in mice. METHOD: Male C57Bl6/J mice were fed control chow, a HF diet or a HF diet supplemented with SP (50 or 100 mg/kg/day). RESULTS: Body weight gain and total body fat content of SP-treated animals were significantly reduced compared with HF-fed mice. Fractional synthesis of palmitate was significantly reduced in mice treated with SP, indicative for ACC1 inhibition. Plasma beta-hydroxybutyrate levels were significantly elevated by SP, reflecting simultaneous inhibition of ACC2 activity. Mice treated with SP showed improved peripheral insulin sensitivity, as assessed by hyperinsulinaemic euglycaemic clamps. CONCLUSION: Pharmacological inhibition of the ACC system is of potential use for treatment of key components of the metabolic syndrome.

Adiponectin levels correlate with the severity of hypertriglyceridaemia in glycogen storage disease Ia.

Glycogen storage disease type Ia (GSD Ia) is characterized by severe hypercholesterolaemia and hypertriglyceridaemia. Little is known about the aetiology of the hyperlipidaemia in GSD Ia. Adipokines play an important regulatory role in lipid metabolism. We investigated whether adipokine concentrations were correlated with the degree of hyperlipidaemia in GSD Ia patients. Six patients with GSD Ia were studied in semi-fasted conditions. Adiponectin, but not leptin, correlated (r(2) = -0.79, p = 0.02) with plasma triglyceride concentrations in the GSD Ia patients. Leptin correlated well with BMI (r(2) = 0.59, p < 0.01). However, neither body mass index (BMI) nor homeostasis model assessment (HOMA), as a marker of insulin sensitivity, correlated with triglyceride concentrations. Although a small number of patients were studied, these results indicate that adiponectin concentrations are correlated with the degree of hypertriglyceridaemia in GSD Ia. Pharmacological treatment aimed at increasing adiponectin levels might improve the metabolic status of these patients.

Neonatal screening for medium-chain acyl-CoA dehydrogenase (MCAD) deficiency in The Netherlands: the importance of enzyme analysis to ascertain true MCAD deficiency.

The outcome was determined of population-wide neonatal screening for medium-chain acyl-CoA dehydrogenase (MCAD) deficiency using tandem mass spectrometry (MS/MS) in The Netherlands, between October 2003 and September 2005. Prospective population-wide neonatal screening for MCAD deficiency was performed in the northern part of The Netherlands. In newborns with blood octanoylcarnitine (C(8:0)) concentrations > or =0.3 micromol/L, clinical and laboratory follow-up was initiated, including MCAD enzymatic measurements which played a decisive role. In a 2-year period, 66 216 newborns were investigated for MCAD deficiency and follow-up was initiated in 28 newborns. True-positives (n = 14) were identified based upon MCAD enzyme activity <50%, measured with hexanoyl-CoA as substrate. The observed prevalence of MCAD deficiency was 1/6600 (95% CI: 1/4100-1/17 400). In addition to an elevated C(8:0) concentration, a C(8:0)/C(10:0) molar ratio >5.0 turned out to differentiate between false-positives and true-positives. Measurement of MCAD activity using phenylpropionyl-CoA as a substrate further discriminated between newborns with MCAD deficiency and so-called mild MCAD deficiency. To summarize, neonatal screening for MCAD deficiency in the northern part of The Netherlands resulted in the predicted number of affected newborns. Measurement of MCAD activity in leukocytes or lymphocytes using phenylpropionyl-CoA as a substrate can be regarded as the gold standard to diagnose MCAD deficiency upon initial positive screening test results.

Pyridoxine induces non-specific EEG alterations in infants with therapy resistant seizures.

PURPOSE: In infants with frequent therapy resistant seizures (TRS-infants), clinical detection of pyridoxine-dependency (PD) or -responsiveness (PR) occurs by empirical intravenous (IV) pyridoxine administration during recording of the EEG. However, in undiagnosed TRS-infants it is still unclear to what extent EEG alterations by pyridoxine-IV are attributable to PD/PR or to non-specific responses. Before EEG alterations by pyridoxine-IV can be ascribed to PD/PR, these non-specific responses should be excluded first. METHODS: In 10 TRS-infants under 1 year of age, we determined the EEG effect by pyridoxine-IV on the EEG-recording. RESULTS: After pyridoxine-IV administration, our data indicate declined (10-15%; p<0.05) EEG-amplitudes and total power (magnitude/frequency-band) at frontal, central and centro-temporal electrodes. CONCLUSION: In TRS-infants, pyridoxine-IV affects EEG-amplitude and -total power in a non-specific way, which does not identify PD/PR.

Partial hypoxanthine-guanine phosphoribosyl transferase deficiency without elevated urinary hypoxanthine excretion.

Partial hypoxanthine-guanine phosphoribosyl transferase (HGPRT) deficiency, also known as the Kelley-Seegmiller syndrome, can give rise to a wide range of neurological symptoms, and renal insufficiency. Biochemically, it is characterized by high uric acid concentrations in blood, high uric acid and hypoxanthine excretion in urine, and decreased activity of hypoxanthine-guanine phosphoribosyl transferase activity (HGPRT). However, normal uric acid concentrations in blood and uric acid excretions in urine have been reported. Here, a boy is presented with normal development and suffering from recurrent attacks of acute renal failure with slightly to clearly increased urinary uric acid excretion. Between these attacks, episodes of elevated urinary excretion of uric acid were observed with normal blood concentrations of uric acid and normal urinary excretion of hypoxanthine. HGPRT activity in erythrocytes, leukocytes, and fibroblasts was found to be strongly decreased. This case shows that not only normal blood uric acid but also normal urinary hypoxanthine concentrations do not exclude the diagnosis of partial HGPRT deficiency.

Sustained activation of the mammalian target of rapamycin nutrient sensing pathway is associated with hepatic insulin resistance, but not with steatosis, in mice.

AIMS/HYPOTHESIS: Activation of nutrient sensing through mammalian target of rapamycin (mTOR) has been linked to the pathogenesis of insulin resistance. We examined activation of mTOR-signalling in relation to insulin resistance and hepatic steatosis in mice. MATERIALS AND METHODS: Chronic hepatic steatosis and hepatic insulin resistance were induced by high-fat feeding of male C57BL/6Jico mice for 6 weeks. In addition, acute hepatic steatosis in the absence of insulin resistance was induced by pharmacological blockade of beta-oxidation using tetradecylglycidic acid (TDGA). mTOR signalling was examined in liver homogenates. RESULTS: High-fat feeding caused obesity (p<0.001), hepatic steatosis (p<0.05) and hepatic insulin resistance (p<0.05). The phosphorylation of mTOR and its downstream targets p70S6 kinase and S6 ribosomal protein was two-fold higher in mice on a high-fat diet than in mice fed standard chow (all p<0.05) and associated with enhanced rates of protein synthesis. Acute induction of hepatic steatosis with TDGA had no effect on mTOR activity. The increased activity of the mTOR pathway in livers from mice on a high-fat diet could not be ascribed to diet-induced alterations in known modulators of mTOR activity such as circulating plasma leucine levels, phosphorylation of protein kinase B and AMP-activated protein kinase, and changes in mitochondrial function. CONCLUSIONS/INTERPRETATION: High-fat diet induces increase of the mTOR nutrient sensing pathway in association with hepatic insulin resistance, but not with hepatic lipid accumulation as such.

High cerebral guanidinoacetate and variable creatine concentrations in argininosuccinate synthetase and lyase deficiency: implications for treatment?

Cerebral creatine and guanidinoacetate and blood and urine metabolites were studied in four patients with argininosuccinate synthetase (ASS) or argininosuccinate lyase (ASL) deficiency receiving large doses of arginine. Urine and blood metabolites varied largely. Cerebral guanidinoacetate was increased in all patients, while cerebral creatine was low in ASS and high in ASL deficiency. Because high cerebral guanidinoacetate might be toxic, lowering the arginine supplementation with additional creatine supplementation might be important.

Cerebral 1H MR spectroscopy showing elevation of brain guanidinoacetate in argininosuccinate lyase deficiency.

MR spectroscopy in a patient with argininosuccinate lyase deficiency revealed elevated cerebral guanidinoacetate signals, indicating that the phenomenon of increased levels of this compound in brain tissue is not limited to creatine deficiencies.

Enhanced glucose cycling and suppressed de novo synthesis of glucose-6-phosphate result in a net unchanged hepatic glucose output in ob/ob mice.

AIMS/HYPOTHESIS: Leptin-deficient ob/ob mice are hyperinsulinaemic and hyperglycaemic; however, the cause of hyperglycaemia remains largely unknown. METHODS: Glucose metabolism in vivo in 9-h fasted ob/ob mice and lean littermates was studied by infusing [U-(13)C]-glucose, [2-(13)C]-glycerol, [1-(2)H]-galactose and paracetamol for 6 h, applying mass isotopomer distribution analysis on blood glucose and urinary paracetamol-glucuronide. RESULTS: When expressed on the basis of body weight, endogenous glucose production (109+/-23 vs 152+/-27 micromol.kg(-1).min(-1), obese versus lean mice, p<0.01) and de novo synthesis of glucose-6-phosphate (122+/-13 vs 160+/-6 micromol.kg(-1).min(-1), obese versus lean mice, p<0.001) were lower in ob/ob mice than in lean littermates. In contrast, glucose cycling was greatly increased in obese mice (56+/-13 vs 26+/-4 micromol.kg(-1).min(-1), obese versus lean mice, p<0.001). As a result, total hepatic glucose output remained unaffected (165+/-31 vs 178+/-28 micromol.kg(-1).min(-1), obese vs lean mice, NS). The metabolic clearance rate of glucose was significantly lower in obese mice (8+/-2 vs 18+/-2 ml.kg(-1).min(-1), obese versus lean mice, p<0.001). Hepatic mRNA levels of genes encoding for glucokinase and pyruvate kinase were markedly increased in ob/ob mice. CONCLUSIONS/INTERPRETATION: Unaffected total hepatic glucose output in the presence of hyperinsulinaemia reflects hepatic insulin resistance in ob/ob mice, which is associated with markedly increased rates of glucose cycling. Hyperglycaemia in ob/ob mice primarily results from a decreased metabolic clearance rate of glucose.

[Deficiency of the fatty-acid oxidising enzyme medium-chain acyl-CoA dehydrogenase (MCAD) in an adult, detected during a neonatal screening programme]. / Defïciëntie van het vetzuuroxidatie-enzym middenketen-acyl-coënzym-A-dehydrogenase (MCAD) bij een volwassene, opgespoord tijdens een proefproject voor neonatale screening]

In a trial running since October 2003 in the Dutch provinces of Friesland, Groningen, Drenthe and Overijssel neonatal screening for medium-chain acyl-CoA dehydrogenase (MCAD) deficiency has been added to the regular newborn screening programme for phenylketonuria, congenital hypothyroidism and adrenogenital syndrome. One of the questions to be answered by this trial is the cause of the strong variation in clinical expression of the disorder. Underdiagnosing is an important factor in this phenomenon, as shown by the data of a family of which the case histories of the two oldest children were discussed in this journal in 1965. Both children died at a very young age. Recently, MCAD deficiency was diagnosed in the youngest child of this family, now a 34-year-old woman. This family history illustrates the variable clinical expression of MCAD deficiency, which can cause death but can also run a milder or even subclinical course. Moreover, this family history shows that the underdiagnosis of MCAD deficiency in deceased children may be a cause of the apparently limited clinical detection rate of this disease, for which a simple treatment consisting of life-style and dietary measures is available after diagnosis.

Low diagnostic value of fasting and post-methionine load homocysteine tests. A study in Dutch subjects with homocysteine test indications.

BACKGROUND: Homocysteine is a cardiovascular disease risk factor. We investigated, both in subjects with past plasma total homocysteine (tHcy) test indications and healthy adults, the diagnostic value of a fasting (tHcy) (f-tHcy) and the added value of a post-methionine-load tHcy (postload-tHcy). METHODS: Plasma homocysteine cut-off values were retrospectively used for hyperhomocysteinemia assessment in 3477 subjects with past tHcy test indications and 177 apparently healthy subjects. Cut-off values were based on reference limits (f-tHcy < or = 15.0; postload-tHcy < or = 50.0 micro mol/l), relative risk (f-tHcy < or = 12.0, postload-tHcy < or = 38.0; or f-tHcy < or = 10.0 micro mol/l) and vitamin-optimized reference limits (f-tHcy < or = 9.3; postload-tHcy < or = 35.1 micro mol/l). RESULTS: Use of the American Heart Association 10 micro mol/l f-tHcy cut-off value gave hyperhomocysteinemia prevalences of 65% in subjects with past tHcy test indications and 50% in healthy subjects. The combination of the vitamin-optimized reference limits for f-tHcy and postload-tHcy gave a hyperhomocysteinemia prevalence of 79% in subjects with tHcy test indications, of which only 5% was on account of increased postload-tHcy. Corresponding values for healthy subjects were 68% and 3%, respectively. CONCLUSIONS: Employment of a 10 micro mol/l (American Heart Association) or 9.3 micro mol/l (vitamin-optimized reference) cut-off value leaves no indications for tHcy testing from an evidence-based point-of-view.

Resting oxygen consumption and in vivo ADP are increased in myopathy due to complex I deficiency.

BACKGROUND: Patients with isolated complex I deficiency (CID) in skeletal muscle mitochondria often present with exercise intolerance as their major clinical symptom. OBJECTIVE: To study the in vivo bioenergetics in patients with complex I deficiency in skeletal muscle mitochondria. METHODS: In vivo bioenergetics were studied in three of these patients by measuring oxygen uptake at rest and during maximal exercise, together with forearm ADP concentrations ([ADP]) at rest. Whole-body oxygen consumption at rest (VO(2)) was measured with respiratory calorimetry. Maximal oxygen uptake (VO(2)max) was measured during maximal exercise on a cycle ergometer. Resting [ADP] was estimated from in vivo (31)P MRS measurements of inorganic phosphate, phosphocreatine, and ATP content of forearm muscle. RESULTS: Resting VO(2) was significantly increased in all three patients: 128 +/- 14% (SD) of values in healthy control subjects. VO(2)max in patients was on average 2.8 times their VO(2) at rest and was only 28% of VO(2)max in control subjects. Resting [ADP] in forearm muscle was significantly increased compared with healthy control subjects (patients 26 +/- 2 microM, healthy controls 9 +/- 2 microM). CONCLUSION: In patients with CID, the increased whole-body oxygen consumption rate at rest reflects increased electron transport through the respiratory chain, driven by a decreased phosphorylation potential. The increased electron transport rate may compensate for the decreased efficiency of oxidative phosphorylation (phosphorylation potential).

Acute inhibition of glucose-6-phosphate translocator activity leads to increased de novo lipogenesis and development of hepatic steatosis without affecting VLDL production in rats.

Glucose-6-phosphatase (G6Pase) is a key enzyme in hepatic glucose metabolism. Altered G6Pase activity in glycogen storage disease and diabetic states is associated with disturbances in lipid metabolism. We studied the effects of acute inhibition of G6Pase activity on hepatic lipid metabolism in nonanesthetized rats. Rats were infused with an inhibitor of the glucose-6-phosphate (G6P) translocator (S4048, 30 mg. kg(-1). h(-1)) for 8 h. Simultaneously, [1-(13)C]acetate was administered for determination of de novo lipogenesis and fractional cholesterol synthesis rates by mass isotopomer distribution analysis. In a separate group of rats, Triton WR 1339 was injected for determination of hepatic VLDL-triglyceride production. S4048 infusion significantly decreased plasma glucose (-11%) and insulin (-48%) levels and increased hepatic G6P (201%) and glycogen (182%) contents. Hepatic triglyceride contents increased from 5.8 +/- 1.4 micromol/g liver in controls to 20.6 +/- 5.5 micromol/g liver in S4048-treated animals. De novo lipogenesis was increased >10-fold in S4048-treated rats, without changes in cholesterol synthesis rates. Hepatic mRNA levels of acetyl-CoA carboxylase and fatty acid synthase were markedly induced. Plasma triglyceride levels increased fourfold, but no differences in plasma cholesterol levels were seen. Surprisingly, hepatic VLDL-triglyceride secretion was not increased in S4048-treated rats. These studies demonstrate that inhibition of the G6Pase system leads to acute stimulation of fat synthesis and development of hepatic steatosis, without affecting hepatic cholesterol synthesis and VLDL secretion. The results emphasize the strong interactions that exist between hepatic carbohydrate and fat metabolism.

Fatty acid and amino acid modulation of glucose cycling in isolated rat hepatocytes.

We studied the influence of glucose/glucose 6-phosphate cycling on glycogen deposition from glucose in fasted-rat hepatocytes using S4048 and CP320626, specific inhibitors of glucose-6-phosphate translocase and glycogen phosphorylase respectively. The effect of amino acids and oleate was also examined. The following observations were made: (1) with glucose alone, net glycogen production was low. Inhibition of glucose-6-phosphate translocase increased intracellular glucose 6-phosphate (3-fold), glycogen accumulation (5-fold) without change in active (dephosphorylated) glycogen synthase (GSa) activity, and lactate production (4-fold). With both glucose 6-phosphate translocase and glycogen phosphorylase inhibited, glycogen deposition increased 8-fold and approached reported in vivo rates of glycogen deposition during the fasted-->fed transition. Addition of a physiological mixture of amino acids in the presence of glucose increased glycogen accumulation (4-fold) through activation of GS and inhibition of glucose-6-phosphatase flux. Addition of oleate with glucose present decreased glycolytic flux and increased the flux through glucose 6-phosphatase with no change in glycogen deposition. With glucose 6-phosphate translocase inhibited by S4048, oleate increased intracellular glucose 6-phosphate (3-fold) and net glycogen production (1.5-fold), without a major change in GSa activity. It is concluded that glucose cycling in hepatocytes prevents the net accumulation of glycogen from glucose. Amino acids activate GS and inhibit flux through glucose-6-phosphatase, while oleate inhibits glycolysis and stimulates glucose-6-phosphatase flux. Variation in glucose 6-phosphate does not always result in activity changes of GSa. Activation of glucose 6-phosphatase flux by fatty acids may contribute to the increased hepatic glucose production as seen in Type 2 diabetes.