L-2-hydroxyglutaric aciduria in a West Highland white terrier.
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Amino Acid Metabolism, Inborn Errors↗
Biomedical subjects
C Jakobs
Publications and source records attributed to C Jakobs.
Oral estradiol decreases plasma homocysteine, vitamin B6, and albumin in postmenopausal women but does not change the whole-body homocysteine remethylation and transmethylation flux.
Estrogens, both endogenous and exogenous, lower the fasting levels of the independent risk factor for cardiovascular disease homocysteine. The mechanism behind this observation remains unclear. In a randomized, placebo-controlled, double-blind study, 25 postmenopausal women with a screening homocysteine concentration above 10 micromol/liter were included. We investigated the influence on homocysteine levels of a 3-month treatment with a daily oral dose of 4 mg 17beta-estradiol (ET) or 4 mg ET combined with 10 mg dydrogesterone (EPT); the comparison group received placebo treatment. We performed primed continuous infusions of L-[2H3-methyl-13C]methionine to assess steady-state flux rates of transmethylation, remethylation, and transsulfuration. Homocysteine concentration relationships with S-adenosylmethionine, S-adenosylhomocysteine, creatinine, albumin, vitamins B6 and B12, and folate status were determined as well. The mean change from baseline in homocysteine concentration by both treatments compared with placebo (ET, -13%; EPT, -10%) was accompanied by a decrease in the concentration of vitamin B6 (ET, -25%; EPT, -38%) and albumin (ET, -7%; EPT, -11%). No significant changes in flux rates were observed. In a .multiltivariate analysis, changes in homocysteine concentration were related to changes in albumin concentration. No relation to other variables was observed. We conclude that the ET- and EPT-induced homocysteine changes in this study were not accompanied by a significant change in methionine-homocysteine flux rates and hypothesize that an estrogen-induced lowering of homocysteine levels is primarily part of a change in albumin metabolism.
Aged↗
Novel L2HGDH mutations in 21 patients with L-2-hydroxyglutaric aciduria of Portuguese origin.
We studied 21 patients, from 18 families, with L-2-hydroxyglutaric aciduria (L-2-HGA), a rare neurometabolic disorder with a homogeneous presentation: progressive neurodegeneration with extrapyramidal and cerebellar signs, seizures, and subcortical leukoencephalopathy. Increased levels of L-2-hydroxyglutaric acid in body fluids proved the diagnosis of L-2-HGA in all 21 patients. We analyzed the L-2-HGA gene (L2HGDH), recently found to be mutated in consanguineous families with L-2-HGA, and identified seven novel mutations in 15 families. Three mutations appeared to be particularly prevalent in this Portuguese panel: a frameshift mutation (c.529delC) was detected in 12 out of 30 mutant alleles (40%), a nonsense mutation (c.208C>T; p.Arg70X) in 7/30 alleles (23%), and a missense mutation (c.293A>G; p.His98Arg) in four out of 30 mutant alleles (13%), suggesting that common origin may exist. Furthermore, two novel missense (c.169G>A; p.Gly57Arg, c.1301A>C; p.His434Pro) and two splice error (c.257-2A>G, c.907-2A>G) mutations were found. All the mutations presumably lead to loss-of-function with no relationship between clinical signs, progression of the disease, levels of L-2-HGA and site of the mutation. In the three remaining families, no pathogenic mutations in the L-2-HGA were found, which suggests either alterations in regulatory regions of the gene or of its intervening sequences, compound heterozygosity for large genomic deletion and, or further genetic heterogeneity.
Adolescent↗
Measurement of bile acid CoA esters by high-performance liquid chromatography-electrospray ionisation tandem mass spectrometry (HPLC-ESI-MS/MS).
The novel and rapid assay presented here combines high-performance liquid chromatography and electrospray ionisation tandem mass spectrometry (HPLC-ESI-MS/MS) to directly measure and quantify the CoA esters of 3alpha,7alpha,12alpha-trihydroxy- and 3alpha,7alpha-dihydroxy-5beta-cholestan-26-oic acid (THCA and DHCA). The latter are converted inside peroxisomes to the primary bile acids, cholic and chenodeoxycholic acids, respectively. Prior to MS/MS, esters were separated by reversed-phase HPLC on a C(18) column using an isocratic mobile phase (acetonitrile/water/2-propanol) and subsequently detected by multiple reaction monitoring. For quantification, the CoA ester of deuterium-labelled 3alpha,7alpha,12alpha-trihydroxy-5beta-cholan-24-oic acid (d(4)-CA) was used as internal standard. To complete an assay took less than 8 min. To verify the validity of the assay, the effect of peroxisomal proteins on the efficacy of extraction of the CoA esters was tested. To this end, variable amounts of the CoA esters were spiked with a fixed amount of either intact peroxisomes or peroxisomal matrix proteins and then extracted using a solid-phase extraction system. The CoA esters could be reproducibly recovered in the range of 0.1-4 micromol l(-1) (linear correlation coefficient R(2) > 0.99), with a detection limit of 0.1 micromol l(-1). In summary, electrospray ionization tandem mass spectrometry combined with HPLC as described here proved to be a rapid and versatile technique for the determination of bile acid CoA esters in a mixture with peroxisomal proteins. This suggests this technique to become a valuable tool in studies dealing with the multi-step biosynthesis of bile acids and its disturbances in disorders like the Zellweger syndrome.
Acyl Coenzyme A↗
Pipecolic acid concentrations in brain tissue of nutritionally pyridoxine-deficient rats.
Elevated concentrations of pipecolic acid have been reported in plasma and CSF of patients with pyridoxine-dependent epilepsy, but its molecular background is unclear. To investigate any further association of pyridoxine and pipecolic acid metabolism, we have performed an animal trial and have measured the concentration of pipecolic acid in brain tissue of rats with nutritional pyridoxine deficiency and in control littermates. Concentrations of pyridoxal phosphate were significantly reduced in brain tissue of pyridoxine-deficient rats (p < 0.001), while concentrations of pipecolic acid were not significantly different from the normally nourished control group (p = 0.3). These data indicate that a direct association of pyridoxine and pipecolic acid metabolism is unlikely. We therefore assume that the characteristic elevation of pipecolic acid in pyridoxine-dependent epilepsy could rather be a secondary phenomenon with the primary defect of pyridoxine-dependent epilepsy being located outside the pipecolic acid pathway.
Animal Nutritional Physiological Phenomena↗
Determination of the GABA analogue succinic semialdehyde in urine and cerebrospinal fluid by dinitrophenylhydrazine derivatization and liquid chromatography-tandem mass spectrometry: application to SSADH deficiency.
Succinic semialdehyde (SSA) accumulates in the inborn error of meta- bolism succinic semialdehyde dehydrogenase deficiency owing to impaired enzymatic conversion to succinic acid. We developed a stable-isotope dilution liquid chromato- graphy-tandem mass spectrometry method for the determination of SSA in urine and cerebrospinal fluid samples. Stable-isotope-labelled [13C4]SSA, serving as internal standard, was prepared by reaction of ninhydrin with L-[13C5]glutamic acid. SSA in body fluids was converted to its dinitrophenylhydrazine (DNPH) derivative, without sample purification prior to the derivatization procedure. The DNPH derivative of SSA was injected onto a C18 analytical column and chromatography was performed by isocratic elution. Detection was accomplished by tandem mass spectrometry operating in the negative multiple-reaction monitoring mode. The limit of detection was 10 nmol/L and the calibration curves over the range 0-500 pmol of SSA showed good linearity (r2 > 0.99). The intra-day coefficient of variation (n = 10) for urine was 2.7% and inter-day coefficient of variation (n = 5) for urine was 8.5%. The average recoveries performed on two levels by enriching urine and cerebrospinal fluid samples ranged between 85 and 115%, with coefficients of variation < 8%. The method enabled the first determination of normal values for SSA in urine and pathological values of SSA in urine and cerebrospinal fluid samples derived from patients with succinic semialdehyde dehydrogenase deficiency.
Blood Chemical Analysis↗
Kinetic characterization of human hydroxyacid-oxoacid transhydrogenase: relevance to D-2-hydroxyglutaric and gamma-hydroxybutyric acidurias.
We investigated the presence of hydroxyacid-oxoacid transhydrogenase (HOT), which catalyses the cofactor-independent conversion of gamma-hydroxybutyrate (GHB) to succinic semialdehyde coupled to reduction of 2-ketoglutarate (2-KG) to D-2-hydroxyglutarate (D-2-HG), in human liver extracts employing [2H6]GHB and 2-KG as substrates. We measured incorporation of 2H in D-[2H]2-HG using GC-MS analyses, providing evidence for HOT activity in humans. Kinetic characterization of HOT was undertaken in forward and reverse directions. We employed [2H6]GHB and [2H4]2-KG as cosubstrates in order to develop a HOT activity assay in cultured human fibroblasts derived from patients with D-2-hydroxyglutaric aciduria. HOT activity was quantified in this system by the measurement of D-[2H5]2-HG production. Fibroblasts derived from patients with D-2-hydroxyglutaric aciduria showed normal HOT activities. Our results provide the first demonstration and preliminary kinetic characterization of HOT activity in human tissues.
Alcohol Oxidoreductases↗
Analysis of polyols in urine by liquid chromatography-tandem mass spectrometry: a useful tool for recognition of inborn errors affecting polyol metabolism.
Several inborn errors of metabolism with abnormal polyol concentrations in body fluids are known to date. Most of these defects can be diagnosed by the assessment of urinary concentrations of polyols. We present two methods using tandem mass spectrometry for screening for inborn errors affecting polyol metabolism. Urine samples supplemented with internal standards ([13C4]erythritol, [13C2]arabitol and [2H3]sorbitol) were desalted by a mixed-bed ion-exchange resin. Separation was achieved by two different columns. Sugar isomers could not be separated using a Prevail Carbohydrate ES 54 column (method 1), whereas with the other column (Aminex HPX-87C) separation of the isomers was achieved (method 2). Multiple reaction monitoring polyol detection was achieved by tandem mass spectrometry with an electron ion-spray source operating in the negative mode. Age-related reference ranges of polyols (erythritol, treitol, arabitol, ribitol, xylitol, galactitol, mannitol, sorbitol, sedoheptitol and perseitol) in urine were established. The applicability of the method was demonstrated by the abnormal polyol concentrations observed in patients with transaldolase deficiency, ribose-5-phosphate isomerase deficiency and classical galactosaemia. This paper describes two methods for the analysis of urinary polyols by liquid chromatography-tandem mass spectrometry. Method 1 is a fast screening method with the quantification of total isomers and method 2 is a more selective method with the separate quantification of the polyols. Both methods can be used for diagnosing inborn errors of metabolism affecting polyol metabolism.
Aldose-Ketose Isomerases↗
Severe hypoglycaemia in isolated 3-methylcrotonyl-CoA carboxylase deficiency; a rare, severe clinical presentation.
Since the introduction of neonatal screening for branched-chain organic acidurias, it has been noted that isolated 3-methylcrotonyl-CoA carboxylase deficiency is probably one of the most frequent organic acidurias. Only a few cases with severe clinical presentation have been described. Profound hypoglycaemia is an uncommon but life-threatening complication.
Carbon-Carbon Ligases↗
Clinical, biochemical, magnetic resonance imaging (MRI) and proton magnetic resonance spectroscopy (1H MRS) findings in a fourth case of combined D- and L-2 hydroxyglutaric aciduria.
We report the fourth case of combined D-and L-2-hydroxyglutaric aciduria presenting with neonatal encephalopathy and subependymal cysts.
Brain↗
A newborn with severe liver failure, cardiomyopathy and transaldolase deficiency.
This paper describes the second patient found to be affected with a deficiency of transaldolase (TALDO1; EC 2.2.1.2). Clinically, this patient presented in the neonatal period with several signs of severe liver failure: severe coagulopathy, low serum protein, elevated blood ammonia, and hypoglycaemia. She had generalized oedema, moderate muscular hypotonia, and dysmorphic signs. Liver size was decreased, and the spleen was moderately enlarged. There was severe cardiomegaly. The clinical course was characterized by intractable liver failure and progressive myocardial hypertrophy. The child died at the age of 18 days from respiratory failure. In urine, elevations of erythritol, arabitol and ribitol were found, suggesting a deficiency of transaldolase. Enzyme studies in cultured fibroblasts showed undetectable transaldolase activity. DNA sequence analysis of the TALDO1 gene showed a homozygous missense mutation (575G>A), resulting in an amino acid alteration at position 192 (arginine to histidine, R192H). This amino acid is part of the catalytic site of the transaldolase protein. Discovery of this second patient affected with transaldolase deficiency and liver failure suggests that this disorder has a heterogeneous clinical presentation with highly variable severity.
Cardiomyopathies↗
[6S]5-methyltetrahydrofolate or folic acid supplementation and absorption and initial elimination of folate in young and middle-aged adults.
OBJECTIVES: To assess the effects of supplementation with the diastereoisomer of 5-methyltetrahydrofolate ([6S]5-methylTHF), as an alternative supplement for folic acid, on folate absorption and elimination, in two age groups. DESIGN: A randomized, double-blind intervention study. SUBJECTS: A total of 12 young (<30 y) and 12 middle-aged (> or =50 y) healthy volunteers were recruited. METHODS: Volunteers were randomized to receive daily supplementation with 400 mug folic acid or equimolar amounts of [6S]5-methylTHF during 5 weeks. Before and after supplementation, absorption and initial elimination were calculated following oral [(2)H(2)]folic acid test doses using isotope kinetics in plasma. RESULTS: Folic acid absorption was lower in the middle-aged as compared to the young adults, both before (P = 0.03) and after (P = 0.05) supplementation. In the young adults, absorption decreased by 22% after [6S]5-methylTHF and increased by 21% after folic acid (P = 0.02). In the other age group, no such changes were found. The folate rate constant of elimination increased after folic acid supplementation in the young (+50%; P = 0.05) but not in the middle-aged (+18%; P = 0.5) adults. CONCLUSIONS: Young adults show increased folate turnover after folic acid supplementation relative to the effect of [6S]5-methylTHF supplementation. Similar differences are not observed in middle-aged adults, in whom folic acid absorption was found to be lower as compared to the young adults. SPONSORSHIP: Financial support was received from the European Union 5th Framework Programme (Grant QLRT-1999-00576).
Adult↗
Pipecolic acid as a diagnostic marker of pyridoxine-dependent epilepsy.
Pyridoxine-dependent epilepsy, although described some decades ago, may still be an underdiagnosed disorder. We have recently described isolated pipecolic acid elevations in the plasma and/or CSF of three patients with pyridoxine-dependent epilepsy with an intriguing inverse correlation to the oral intake of pyridoxine. We have now confirmed these findings in a further 6 unrelated patients with pyridoxine-dependent epilepsy. Pipecolic acid in plasma was 4.3- to 15.3 fold elevated compared to the upper normal range before pyridoxine and remained in the mildly elevated range while on pyridoxine. Pipecolic acid was even more markedly elevated in CSF. The extent of pipecolic acid elevation in CSF exceeded that of plasma by a factor of 2.2 to 4.8. This clearly discriminates pyridoxine-dependent epilepsy from other possible defects with elevated pipecolic acid. Determination of pipecolic acid in plasma and/or CSF should be included in the diagnostic work-up of patients with therapy-resistant seizures. It will in addition prevent patients with pyridoxine-dependent epilepsy from experiencing potentially dangerous pyridoxine-withdrawal, which until now has been necessary to prove the diagnosis.
Anticonvulsants↗
Atypical MRI findings in Canavan disease: a patient with a mild course.
Canavan disease is a severe, progressive leukodystrophy with an autosomal recessive inheritance, caused by aspartoacylase (ASPA) deficiency. The characteristic MRI features include diffuse, symmetrical white matter degeneration in the subcortical areas, with bilateral involvement of the globus pallidus. Proton magnetic resonance spectroscopy of the brain shows an increase in the concentration of N-acetylaspartic acid (NAA). The altered NAA metabolism has been traced to mutations in the gene encoding ASPA, located on chromosome 17 (17p13-ter). We present here a patient with a mild form of Canavan disease confirmed with the absent ASPA activity, atypical MRI findings, related to compound heterozygosity for a missense mutation, p.Tyr288Cys, and the known pan-European mutation, the p.Ala305Glu.
Aspartic Acid↗
Murine succinate semialdehyde dehydrogenase (SSADH) deficiency, a heritable disorder of GABA metabolism with epileptic phenotype.
Murine models of inborn errors of metabolism represent an established approach for investigating pathophysiological mechanisms associated with the corresponding human disorder. Our laboratory studies human inherited defects of GABA synthesis and degradation. One of these, succinate semialdehyde dehydrogenase (SSADH) deficiency (or gamma-hydroxybutyric aciduria; OMIM 271980; E.C. 1.2.1.24), has recently been modeled via gene targeting in the mouse. SSADH-/- mice succumb to early lethality in status epilepticus at postnatal (PN) days 20 - 26. Numerous metabolic, neurochemical and neurophysiological abnormalities have been documented using in vitro and in vivo approaches, substantially altering our thoughts about the complexity of the corresponding human condition. Moreover, novel preclinical treatment paradigms have been developed through drug trials in gene-ablated animals. The greatest utility of this animal, however, may reside in its transition from early absence seizures to generalized convulsions and eventual status epilepticus. Accurate neurochemical assessment during this transition may provide clues to the same transition process in patients, for which the underlying mechanisms remain undefined.
Animals↗
Kinetics of homocysteine metabolism after moderate alcohol consumption.
BACKGROUND: Moderate alcohol consumption is associated with a decreased risk of cardiovascular disease. Because plasma homocysteine (tHcy) is considered an independent risk factor for cardiovascular disease and associated with alcohol consumption, the authors investigated the effect of moderate alcohol consumption on kinetics of plasma tHcy concentration, vitamin B status, and other parameters involved in tHcy metabolism. METHODS: Ten healthy men and nine healthy postmenopausal women (aged 45-65 years) participated in a randomized, diet-controlled, crossover trial. They consumed beer or alcohol-free beer (men: 4 units/day; women: 3 units/day) during 3 weeks, separated by a 1-week washout. On days 5, 10, 15, and 20 of each period, fasting blood samples were taken. RESULTS: Plasma tHcy (microM) and S-adenosyl methionine/S-adenosyl homocysteine ratio were not affected by consumption of beer or alcohol-free beer (p = 0.33 and p = 0.14, respectively). Plasma pyridoxal-5-phosphate (microg/liter) increased during consumption of beer (+11.0%), whereas it decreased during consumption of alcohol-free beer (-34.0%; p = 0.042). Changes over time of plasma vitamin B6 (microg/liter) were similar to changes in plasma pyridoxal-5-phosphate (p = 0.10). Serum vitamin B12 was higher (p < 0.001) after 3 weeks consumption of alcohol-free beer (382.8 +/- 23.7 pg/liter) as compared with beer consumption (327.5 +/- 22.2 pg/liter). Changes in serum methionine, cysteine, cystathionine, and plasma folate were not different between beer-drinking and alcohol-free beer-drinking periods. CONCLUSIONS: This study shows that moderate alcohol consumption does not affect plasma tHcy concentrations or S-adenosyl methionine/S-adenosyl homocysteine ratio. However, it does increase plasma vitamin B6 and decrease serum vitamin B12.
Aged↗
Hormone replacement influences homocysteine levels in the methionine-loading test: a randomized placebo controlled trial in postmenopausal women.
OBJECTIVE: To investigate the mechanism by which exogenous oestrogen influences the homocysteine metabolism in postmenopausal women. STUDY DESIGN: A randomized placebo controlled trial in which a methionine-loading test was performed, in 25 healthy postmenopausal women, before and after a 12-week oral treatment with placebo or daily 4 mg 17beta-estradiol with (HRT) or without (ERT) 10 mg dydrogesterone. Fasting and post-load homocysteine as well as Vitamins B(6), B(12) and folate were determined. RESULTS: In both treatment groups a significant 12% decrease in fasting homocysteine was observed. This decrease was accompanied by a post-load homocysteine increase of more than 20%. Vitamin B(6) values were decreased by more than 25%. CONCLUSION: The hormone therapy induced lowering of fasting homocysteine and Vitamin B(6) levels and an increase in post-load homocysteine, supporting the hypothesis that homocysteine-methionine metabolism is modulated by hormone therapy in postmenopausal women.
Analysis of Variance↗
Inhibition of the pentose phosphate pathway decreases ischemia-reperfusion-induced creatine kinase release in the heart.
OBJECTIVE: The oxidative pentose phosphate pathway (oxPPP) produces NADPH, which can be used to maintain glutathione in its reduced state (anti-oxidant; beneficial effects) or to produce radicals or nitric oxide (NO) through NADPH oxidase/NO synthase (detrimental effects). Changes in cytosolic redox status have been implicated in ischemic preconditioning (PC). This study investigates whether (1) PC affects mitochondrial redox state, (2) the oxPPP plays a protective or detrimental role in ischemia (I)-reperfusion (R) injury in the intact heart and (3) PPP is altered with PC. METHODS: Isolated rat hearts were subjected to 40-min global I and 30-min R (CO, control). Ischemia was either preceded by three 5-min I/R periods (PC) and/or oxPPP inhibition by 6-aminonicotinamide (6AN) or NADPH oxidase/NO synthase inhibition by diphenyleneiodonium (DPI). NADH videofluorometry was used to determine mitochondrial redox state. PPP intermediates were determined in CO and PC hearts using tandem mass spectrometry. RESULTS: PC reduced ischemic damage (creatine kinase, CK, release from 337+/-64 to 147+/-41 U/R/gdw) and contracture (from 59+/-5 to 31+/-3 mm Hg) and increased recovery of contractility (from 48+/-10% to 88+/-8%), as compared to CO. PC was without effect on NADH fluorometry. Inhibition of the oxPPP reduced injury (CK release: 91+/-24 U/R/gdw) to similar levels as PC, without improving contractility. Inhibition of NADPH oxidase/NO synthase mimicked the effects of oxPPP inhibition on injury (CK release: 140+/-22 U/R/gdw). Although levels of ribose-5P and (ribulose-5P+xylulose-5P) rose several fold during ischemia with minor changes in sedoheptulose-7P, demonstrating an active PPP in the heart, PC did not affect these levels. CONCLUSIONS: (1) PC can attenuate cardiac reperfusion injury without alterations in mitochondrial redox state; (2) inhibition of the oxPPP protects the heart against I/R-induced CK release; and (3) PC does not result in altered activity of the PPP.
Animals↗