Human metabolism and urinary excretion kinetics of di-n-butyl adipate (DnBA) after oral and dermal administration in three volunteers.

Di-n-butyl adipate (DnBA) is used as a plasticizer and in various consumer products (e.g. personal care products) replacing, in part, the endocrine disruptor di-n-butyl phthalate (DnBP). We provide quantitative in vivo data on human DnBA metabolism and excretion after oral dose (105-185 µg/kg bw) and dermal application to three volunteers each as a tool for exposure and risk assessment. Complete and consecutive urine samples were collected for two (oral) and four days (dermal), respectively, and analyzed for the metabolites mono-n-butyl adipate (MnBA), 3- and tentative 4-hydroxy-mono-n-butyl adipate (3OH-MnBA, 4OH-MnBA), and 3-carboxy-mono-n-propyl adipate (3cx-MnPrA), as well as the hydrolysis product adipic acid (AA) using stable isotope dilution quantification. Metabolites were excreted within 24 h after oral dose with one or two concentration maxima at 0.8-3.0 h (n = 3) and 4.8-6.3 h (n = 2). AA was the major but unspecific metabolite with urinary excretion fractions (FUEs) of 14-26 %. Mean FUEs (range) of 3cx-MnPrA, MnBA, 3OH-MnBA, and tentative 4OH-MnBA were low, but consistent between volunteers (0.47 % (0.35-0.63 %), 0.079 % (0.065-0.091 %), 0.012 % (0.006-0.016 %), and 0.005 % (0.002-0.009 %), respectively). MnBA and 3OH-MnBA seem to be suitable, specific exposure biomarkers for DnBA, whereas 3cx-MnPrA and 4OH-MnBA seem to originate also from other, unknown sources not related to DnBA. Compared to the oral study, metabolite excretion in the dermal study was delayed and MnBA excretion was somewhat higher compared to the oxidized metabolites. Based on urinary concentrations and the above excretion fractions, calculated uptakes in the dermal study did not exceed the adipate ester ADI of 5 mg/(kg bw*day).

Determination of di-n-butyl adipate (DnBA) metabolites as possible biomarkers of exposure in human urine by online-SPE-LC-MS/MS.

Di-n-butyl adipate (DnBA) is an alternative to the anti-androgenic and strictly regulated di-n-butyl phthalate (DnBP) used as a cosmetic ingredient, plasticizer, and in various articles of everyday life. Hence, exposures of the general population have to be expected. Currently, biomarkers of DnBA exposure and methods for their determination are not available. Here, we describe a sensitive, rugged and precise analytical method for the determination of the DnBA monoester metabolite mono-n-butyl adipate (MnBA), as well as its potential downstream metabolites 3-hydroxy-mono-n-butyl adipate (3OH-MnBA) and 3-carboxy-mono-n-propyl adipate (3cx-MnPrA) in human urine. Glucuronic acid conjugates present in urine were deconjugated using a pure ß-glucuronidase. The metabolites were then analyzed by liquid chromatography on a C18 column with superficially porous particles coupled to electrospray ionization-triple quadrupole-tandem mass spectrometry, applying online turbulent flow chromatography for analyte enrichment and matrix depletion (online-SPE-LC-MS/MS). The metabolites were quantified using stable isotope dilution analysis with limits of quantification of 0.05 µg/L (MnBA), 0.1 µg/L (3OH-MnBA), and 0.5 µg/L (3cx-MnPrA). Method imprecision in urinary matrix was below 7% (coefficient of variation) for all analytes. Mean relative recoveries were between 93% and 107%. The suitability of the DnBA metabolites as biomarkers of exposure was demonstrated after dermal application of a commercially available sunscreen containing DnBA. Maximum concentrations were reached 6.5 h after dose (219 µg/L 3cx-MnPrA, 91 µg/L MnBA, and 3.9 µg/L 3OH-MnBA). Elimination kinetics were similar for all three metabolites. We were able to quantify 3cx-MnPrA and MnBA until 4 d after sunscreen application. In a sample set of 35 urine samples from the general German population, 3cx-MnPrA was quantified in 94% (median 2.54 µg/L, maximum 78.3 µg/L) and MnBA in 3% (median < LOQ, maximum 0.18 µg/L) of the samples. The method will be applied in future human metabolism and human biomonitoring population studies.

Metabolism and urinary excretion kinetics of di(2-ethylhexyl) adipate (DEHA) in four human volunteers after a single oral dose.

Di(2-ethylhexyl) adipate (DEHA) is used as a substitute for the reprotoxic phthalate plasticizer di(2-ethylhexyl) phthalate (DEHP). This study reports the first quantitative data on human in vivo DEHA metabolism and urinary metabolite excretion with the aim of providing tools for DEHA exposure and risk assessments. After DEHA was administered to four healthy volunteers (107-164 µg/kg body weight (bw)), urine samples were continuously and completely collected for 48 h and analyzed for the specific oxidized monoester metabolites mono-2-ethyl-5-hydroxyhexyl adipate (5OH-MEHA), mono-2-ethyl-5-oxohexyl adipate (5oxo-MEHA), and mono-5-carboxy-2-ethylpentyl adipate (5cx-MEPA), as well as for the non-specific hydrolysis product adipic acid (AA) using stable isotope dilution analysis. AA was confirmed as a major (urinary excretion fraction (FUE): 10-40%), yet non-specific DEHA metabolite. 5cx-MEPA was the major specific DEHA metabolite with an FUE of 0.20% (range: 0.17-0.24%). FUEs for 5OH-MEHA and 5oxo-MEHA were 0.07% (0.03-0.10%) and 0.05% (0.01-0.06%), respectively. The three specific metabolites were excreted with two concentration maxima (tmax1 = 1.5-2.3 h, tmax2 = 3.8-6.4 h). Elimination half-lives (t1/2, calculated after the second tmax) for 5cx-MEPA were calculated between 2.1-3.8 h. The majority (98-100%) of metabolites was excreted within 24 h. The FUE of 5cx-MEPA was applied to demonstrate its applicability for calculating daily intakes based on urinary metabolite levels from three pilot populations. Daily intakes were generally far below the tolerable daily intake (TDI) for DEHA (300 µg/kg bw/day). The highest daily intake (114 µg/kg bw/day) was calculated in individuals after consuming food that had been wrapped in DEHA containing cling film.

Severe clinical manifestation of mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase deficiency associated with two novel mutations: a case report.

BACKGROUND: Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (mHS) deficiency is an autosomal recessive inborn error of metabolism, which will give rise to failure of ketogenesis in liver during illness or fasting. It is a very rare disease with only a few patients reported worldwide, most of which had a good prognosis after proper therapies. CASE PRESENTATION: We report a 9-month-old boy with mHS deficiency presenting with unusually severe and persistent acidosis after diarrhea and reduced oral food intake. The metabolic acidosis persisted even after supplementation with sugar and alkaline solution. Blood purification and assisted respiration alleviated symptoms, but a second onset induced by respiratory infection several days later led to multiple organ failure and death. Urine organic acid analysis during the acute episode revealed a complex pattern of ketogenic dicarboxylic and 3-hydroxydicarboxylic aciduria with prominent elevation of glutaric acid and adipic acid, which seem to be specific to mHS deficiency. Plasma acylcarnitine analysis revealed elevated 3-hydroxybutyrylcarnitine and acetylcarnitine. This is the first report of elevated 3-hydroxybutyrylcarnitine in mHS deficiency. Whole exome sequencing revealed a novel compound heterozygous mutation in HMGCS2 (c.100C > T and c.1465delA). CONCLUSION: This severe case suggests the need for patients with mHS deficiency to avoid recurrent illness because it can induce severe metabolic crisis, possibly leading to death. Such patients may also require special treatment, such as blood purification. Urine organic acid profile during the acute episode may give a hint to the disease.

Determination of human urinary metabolites of the plasticizer di(2-ethylhexyl) adipate (DEHA) by online-SPE-HPLC-MS/MS.

Di(2-ethylhexyl) adipate (DEHA) is a plasticizer and phthalate substitute used in various consumer products. Relevant population exposures have to be assumed. In this study we describe the determination of three specific side chain-oxidized monoester metabolites of DEHA, mono-2-ethyl-5-hydroxyhexyl adipate (5OH-MEHA), mono-2-ethyl-5-oxohexyl adipate (5oxo-MEHA), and mono-5-carboxy-2-ethylpentyl adipate (5cx-MEPA) in human urine as potential biomarkers of DEHA exposure. After enzymatic hydrolysis, urine samples were analyzed by online turbulent flow chromatography for matrix depletion and analyte enrichment coupled to liquid chromatography-electrospray ionization-triple quadrupole-tandem mass spectrometry (online-SPE-LC-MS/MS). For quantification stable isotope dilution was applied with limits of quantification of 0.05 µg/L for 5cx-MEPA and 5OH-MEHA, and 0.1 µg/L for 5oxo-MEHA. Method accuracies (relative recoveries) were between 92 and 109%, and relative standard deviations <5%. We investigated the applicability of the method for internal DEHA exposure assessment in six volunteers who had consumed food wrapped in commercial PVC-cling film containing DEHA and in two small pilot populations without known DEHA exposure (44 pregnant Brazilian women and 32 German adults). In the cling film experiment, we could quantify all three metabolites in all post exposure urine samples, with 5cx-MEPA being most prominent (0.30-10.2 µg/L), followed by 5OH-MEHA (0.12-4.31 µg/L) and 5oxo-MEHA (0.12-2.84 µg/L). In the Brazilian and German samples we could detect DEHA exposures in 43 and 9% of all samples, again with 5cx-MEPA as the most prominent metabolite. Based on validation and pilot biomonitoring results, the method has proven appropriate for DEHA biomonitoring and will be applied in future metabolism and population studies.

Development and validation of a bioanalytical assay based on liquid chromatography-tandem mass spectrometry for measuring biomarkers of exposure of alternative plasticizers in human urine and serum.

Alternative plasticizers (APs) have been increasingly used in the last decade to replace conventional phthalate esters, in particular di(2-ethylhexyl) phthalate (DEHP), due to the toxicity of the latter. However, there is currently very little data about the toxicity of and exposure to APs. No method exists so far for the analysis of multiple exposure biomarkers. The objective of this work consisted in developing a simple bioanalytical procedure for the analysis of multiple exposure biomarkers of APs in human urine and serum. Focus was set on metabolites of di(2-propylheptyl) phthalate (DPrHpP), di(isononyl)cyclohexane-1,2-dicarboxylate (DINCH), di(2-ethylhexyl) terephthalate (DEHTP) and di-2-ethylhexyl adipate (DEHA). A sample preparation protocol was developed and optimized using Oasis HLB solid-phase extraction (SPE) cartridges. Subsequently, an instrumental method based on liquid-chromatography coupled to tandem mass spectrometry (LC-MS/MS) was optimized. Following established guidelines, the sample preparation and instrumental methods were validated in terms of recovery, matrix effects, carry-over, linearity, limits of quantification, within- and between-run precision and trueness. Obtained results were satisfactory for all compounds except for one of the metabolites of DEHA (i.e., mono(2-ethylhexyl) adipate (MEHA)). A pilot biomonitoring study was carried out to assess the method's ability to detect and quantify target analytes in human urine and serum. In urine, most analytes could be detected with frequencies ranging from 8% for mono(2-ethyl-5-hydroxyhexyl) adipate (OH-MEHA) and cyclohexane-1,2-dicarboxylic mono hydroxyisononyl ester (OH-MINCH) to 92% for mono(2-ethyl-5-oxohexyl) adipate (oxo-MEHA), whilst most compounds could not be detected in serum, except for mono(2-ethylhexyl) terephthalate (MEHTP) and mono-(2-propyl-6-hydroxyheptyl) phthalate (OH-MPrHpP) which were detected in all samples. The obtained results show that the developed method can be used to simultaneously analyse multiple exposure biomarkers to APs in human urine and serum.

Relationship between adipic acid concentration and the core symptoms of autism spectrum disorders.

Dicarboxylic acids are an important source of information about metabolism and potential physiopathological alterations in children with autism spectrum disorders (ASDs). We measured the concentration between dicarboxylic adipic and suberic acids in children with an ASD and typically-developing (TD) children and analyzed any relationships between the severity of the core symptoms of ASDs and other clinical features (drugs, supplements, drugs, or diet). The core symptoms of autism were evaluated using the DSM-IV criteria, and adipic acid and suberic acid were measured in urine samples. Overall, no increase in the concentration of adipic acid in children with ASDs compared to TD children, however when considering vitamin B supplementation in ASD there were significantly increased level of urinary adipic acid in children with an ASD not taking vitamin B supplementation compared to supplemented children or to TD children. No significant difference were observed in suberic acid. Interestingly, the increase in adipic acid concentration was significantly and indirectly correlated with the severity of the deficit in socialization and communication skills in children with an ASD. Therefore, therapeutic treatments aimed at decreasing adipic acid concentration might not be beneficial for treating the core symptoms of ASDs.

High-performance liquid chromatographic method for profiling 2-oxo acids in urine and its application in evaluating vitamin status in rats.

B-group vitamins are involved in the catabolism of 2-oxo acids. To identify the functional biomarkers of B-group vitamins, we developed a high-performance liquid chromatographic method for profiling 2-oxo acids in urine and applied this method to urine samples from rats deficient in vitamins B1 and B6 and pantothenic acid. 2-Oxo acids were reacted with 1,2-diamino-4,5-methylenebenzene to produce fluorescent derivatives, which were then separated using a TSKgel ODS-80Ts column with 30 mmol/L of KH2PO4 (pH 3.0):acetonitrile (7:3) at a flow rate of 1.0 mL/min. Vitamin B1 deficiency increased urinary levels of all 2-oxo acids, while vitamin B6 deficiency only increased levels of sum of 2-oxaloacetic acid and pyruvic acid, and pantothenic acid deficiency only increased levels of 2-oxoisovaleric acid. Profiles of 2-oxo acids in urine samples might be a non-invasive way of clarifying the functional biomarker of B-group vitamins.

Genetic basis of alpha-aminoadipic and alpha-ketoadipic aciduria.

Alpha-aminoadipic and alpha-ketoadipic aciduria is an autosomal recessive inborn error of lysine, hydroxylysine, and tryptophan degradation. To date, DHTKD1 mutations have been reported in two alpha-aminoadipic and alpha-ketoadipic aciduria patients. We have now sequenced DHTKD1 in nine patients diagnosed with alpha-aminoadipic and alpha-ketoadipic aciduria as well as one patient with isolated alpha-aminoadipic aciduria, and identified causal mutations in eight. We report nine novel mutations, including three missense mutations, two nonsense mutations, two splice donor mutations, one duplication, and one deletion and insertion. Two missense mutations, one of which was reported before, were observed in the majority of cases. The clinical presentation of this group of patients was inhomogeneous. Our results confirm that alpha-aminoadipic and alpha-ketoadipic aciduria is caused by mutations in DHTKD1, and further establish that DHTKD1 encodes the E1 subunit of the alpha-ketoadipic acid dehydrogenase complex.

In vitro metabolites of di-2-ethylhexyl adipate (DEHA) as biomarkers of exposure in human biomonitoring applications.

Di-2-ethylhexyl adipate (DEHA) is a common plasticizer used in food packaging. At high doses, DEHA can cause adverse health effects in rats. Although the potential for human exposure to DEHA is high, no DEHA specific biomarkers are identified for human biomonitoring. Using human liver microsomes, we investigated the in vitro phase I metabolism of DEHA and its hydrolytic metabolite mono-2-ethylhexyl adipate (MEHA) and, for comparison purposes, of the analogous di-2-ethylhexyl phthalate (DEHP) and its hydrolytic metabolite mono-2-ethylhexyl phthalate. We unequivocally identified MEHA, a DEHA specific biomarker, and adipic acid, a nonspecific biomarker, using authentic standards. On the basis of their mass spectrometric fragmentation patterns, we tentatively identified two other DEHA specific metabolites: mono-2-ethylhydroxyhexyl adipate (MEHHA) and mono-2-ethyloxohexyl adipate (MEOHA), analogous to the oxidative metabolites of DEHP. Interestingly, although adipic acid was the major in vitro metabolite of DEHA, the analogous phthalic acid was not the major in vitro metabolite of DEHP. Our preliminary data for 144 adults with no known exposure to DEHA suggests that adipic acid is also the main in vivo urinary metabolite, while MEHA, MEHHA, and MEOHA are only minor metabolites. Therefore, the use of these specific metabolites for assessing the exposure of DEHA may be limited to highly exposed populations.

Pantothenic acid deficiency may increase the urinary excretion of 2-oxo acids and nicotinamide catabolites in rats.

Pantothenic acid (PaA) is involved in the metabolism of amino acids as well as fatty acid. We investigated the systemic metabolism of amino acids in PaA-deficient rats. For this purpose, urine samples were collected and 2-oxo acids and L-tryptophan (L-Trp) and its metabolites including nicotinamide were measured. Group 1 was freely fed a conventional chemically-defined complete diet and used as an ad lib-fed control, which group was used for showing reference values. Group 2 was freely fed the complete diet without PaA (PaA-free diet) and used as a PaA-deficient group. Group 3 was fed the complete diet, but the daily food amount was equal to the amount of the PaA-deficient group and used as a pair-fed control group. All rats were orally administered 100 mg of L-Trp/kg body weight at 09:00 on day 34 of the experiment and the following 24-h urine samples were collected. The urinary excretion of the sum of pyruvic acid and oxaloacetic acid was higher in rats fed the PaA-free diets than in the rats fed pair-fed the complete diet. PaA deficiency elicited the increased urinary excretion of anthranilic acid and kynurenic acid, while the urinary excretion of xanthurenic acid decreased. The urinary excretion of L-Trp itself, 3-hydroxyanthranilic acid, and quinolinic acid revealed no differences between the rats fed the PaA-free and pair-fed the complete diets. PaA deficiency elicited the increased excretion of N(1)-methylnicotinamide, N(1)-methyl-2-pyridone-5-carboxamide, and N(1)-methyl-4-pyridone-3-carboxamide. These findings suggest that PaA deficiency disturbs the amino acid catabolism.

Phthalate and di-(2-ethylhexyl) adipate (DEHA) intake by German infants based on the results of a duplicate diet study and biomonitoring data (INES 2).

Phthalates as well as di-(2-ethylhexyl) adipate (DEHA) are used as plasticizers in diverse applications and are of toxicological concern. The study was conducted with a study population of 25 German subjects aged between 15 and 21 months. Overall, 16 phthalates and DEHA were measured by gas chromatography-mass spectrometry in a total of 171 duplicate diet samples collected over 7 consecutive days, and 20 phthalate metabolites were analyzed in the urine samples collected over 7 consecutive days using a liquid chromatography-tandem mass spectrometry method. The median "high" daily dietary intake based on 95th percentiles was 4.66 µg/kg b.w. for di-2-ethylhexyl phthalate (DEHP), 1.03 µg/kg b.w. for di-isobutyl phthalate (DiBP), and 0.70 µg/kg b.w. for di-n-butyl phthalate (DnBP), and 1.0 µg/kg b.w. for DEHA. The "high" daily total intake from biomonitoring data was 4.9 µg/kg b.w. for DEHP, 2.2 µg/kg b.w. for DnBP, 3.9 µg/kg b.w. for DiBP, and 2.6 µg/kg b.w. for di-isononyl phthalate. The comparison of the two intake estimates indicates that the dominant intake source of DEHP was food ingestion, whereas other sources considerably contributed to the total intake of other phthalates. Using our "high" intake scenario, none of the analyzed phthalates reached the recommended tolerable daily intake levels.

DHTKD1 mutations cause 2-aminoadipic and 2-oxoadipic aciduria.

Abnormalities in metabolite profiles are valuable indicators of underlying pathologic conditions at the molecular level. However, their interpretation relies on detailed knowledge of the pathways, enzymes, and genes involved. Identification and characterization of their physiological function are therefore crucial for our understanding of human disease: they can provide guidance for therapeutic intervention and help us to identify suitable biomarkers for monitoring associated disorders. We studied two individuals with 2-aminoadipic and 2-oxoadipic aciduria, a metabolic condition that is still unresolved at the molecular level. This disorder has been associated with varying neurological symptoms. Exome sequencing of a single affected individual revealed compound heterozygosity for an initiating methionine mutation (c.1A>G) and a missense mutation (c.2185G>A [p.Gly729Arg]) in DHTKD1. This gene codes for dehydrogenase E1 and transketolase domain-containing protein 1, which is part of a 2-oxoglutarate-dehydrogenase-complex-like protein. Sequence analysis of a second individual identified the same missense mutation together with a nonsense mutation (c.1228C>T [p.Arg410(∗)]) in DHTKD1. Increased levels of 2-oxoadipate in individual-derived fibroblasts normalized upon lentiviral expression of the wild-type DHTKD1 mRNA. Moreover, investigation of L-lysine metabolism showed an accumulation of deuterium-labeled 2-oxoadipate only in noncomplemented cells, demonstrating that DHTKD1 codes for the enzyme mediating the last unresolved step in the L-lysine-degradation pathway. All together, our results establish mutations in DHTKD1 as a cause of human 2-aminoadipic and 2-oxoadipic aciduria via impaired turnover of decarboxylation 2-oxoadipate to glutaryl-CoA.

Cannabinoid receptor antagonist-induced striated muscle toxicity and ethylmalonic-adipic aciduria in beagle dogs.

Ibipinabant (IBI), a potent cannabinoid-1 receptor (CB1R) antagonist, previously in development for the treatment of obesity, causes skeletal and cardiac myopathy in beagle dogs. This toxicity was characterized by increases in muscle-derived enzyme activity in serum and microscopic striated muscle degeneration and accumulation of lipid droplets in myofibers. Additional changes in serum chemistry included decreases in glucose and increases in non-esterified fatty acids and cholesterol, and metabolic acidosis, consistent with disturbances in lipid and carbohydrate metabolism. No evidence of CB1R expression was detected in dog striated muscle as assessed by polymerase chain reaction, immunohistochemistry, Western blot analysis, and competitive radioligand binding. Investigative studies utilized metabonomic technology and demonstrated changes in several intermediates and metabolites of fatty acid metabolism including plasma acylcarnitines and urinary ethylmalonate, methylsuccinate, adipate, suberate, hexanoylglycine, sarcosine, dimethylglycine, isovalerylglycine, and 2-hydroxyglutarate. These results indicated that the toxic effect of IBI on striated muscle in beagle dogs is consistent with an inhibition of the mitochondrial flavin-containing enzymes including dimethyl glycine, sarcosine, isovaleryl-CoA, 2-hydroxyglutarate, and multiple acyl-CoA (short, medium, long, and very long chain) dehydrogenases. All of these enzymes converge at the level of electron transfer flavoprotein (ETF) and ETF oxidoreductase. Urinary ethylmalonate was shown to be a biomarker of IBI-induced striated muscle toxicity in dogs and could provide the ability to monitor potential IBI-induced toxic myopathy in humans. We propose that IBI-induced toxic myopathy in beagle dogs is not caused by direct antagonism of CB1R and could represent a model of ethylmalonic-adipic aciduria in humans.

B vitamin supplementation reduces excretion of urinary dicarboxylic acids in autistic children.

Urinary dicarboxylic acids are an important source of information about metabolism and potential problems especially connected with energy production, intestinal dysbiosis, and nutritional individuality in autistic children. A diet rich in vitamins and macroelements is a new idea of intervention in autism. The objective of the present study was to test the hypothesis that vitamin B2, vitamin B6, and magnesium supplementation is effective in reducing the level of dicarboxylic acids in the urine of autistic children. We examined the levels of succinic, adipic, and suberic acids in the urine of autistic children before and after vitamin supplementation. Thirty children with autism received magnesium (daily dose, 200 mg), vitamin B6 (pyridoxine; daily dose, 500 mg), and vitamin B2 (riboflavin; daily dose, 20 mg). The treatment was provided for a period of 3 months. Organic acids were determined using gas chromatography/mass spectrometry. Before supplementation, the levels of succinic, adipic, and suberic acids in the urine of autistic children were 41.47 ± 50.40 µmol/mmol creatinine, 15.61 ± 15.31 µmol/mmol creatinine, 8.02 ± 6.08 µmol/mmol creatinine; and after supplementation, the levels were 9.90 ± 8.26 µmol/mmol creatinine, 2.92 ± 2.41 µmol/mmol creatinine, and 2.57 ± 3.53 µmol/mmol creatinine, respectively. The results suggest that the supplementation reduces the level of dicarboxylic acid in the urine of autistic children.

Fluorometric determination of 2-oxoadipic acid, a common metabolite of tryptophan and lysine, by high-performance liquid chromatography with pre-chemical derivatization.

2-Oxoadipic acid, a key metabolite of tryptophan and lysine, reacted with 1,2-diamino-4,5-methylenebenzene in an acidic solution to produce a fluorescent derivative. The reaction product was separated using a Tosoh ODS-80Ts column with 20 mmol/L of KH2PO4-K2HPO4 buffer (pH 7.0) containing 26% methanol at a flow rate 0.8 mL/min. The excitation wavelength of detection was 367 nm, and the emission wavelength was 446 nm. The limit of quantification was 1 pmol per injection, sufficiently sensitive for the determination of 2-oxoadipic acid in human and experimental animal urine.

Wolcott-Rallison syndrome with 3-hydroxydicarboxylic aciduria and lethal outcome.

Wolcott-Rallison syndrome (WRS) (OMIM 226980) is a rare, autosomal recessive disorder with infancy-onset diabetes mellitus, multiple epiphyseal dysplasia, osteopenia, mental retardation or developmental delay, and hepatic and renal dysfunction as main clinical findings. Patients with WRS have mutations in the EIF2AK3 gene, which encodes the pancreatic eukaryotic translation initiation factor 2-alpha kinase 3. We report a female patient who developed insulin-requiring diabetes at 2.5 months of age. Multiple epiphyseal dysplasia was diagnosed at age 2 years. At age 5.5 years she developed a Reye-like syndrome with hypoketotic hypoglycaemia and renal and hepatic insufficiency and died. A partial autopsy showed fat infiltration in the liver and kidneys. Examination of urine by gas chromatography and mass spectrometry showed large amounts of C(6)-dicarboxylic acid (adipic acid), 3-hydroxy-C(8)-dicarboxylic acid, 3-hydroxy-C(10)-dicarboxylic acid, and 3-hydroxydecenedioic acid. Acetoacetate and 3-hydroxybutyrate were absent. The findings suggested a metabolic block in mitochondrial fatty acid oxidation, but lack of material precluded enzyme analyses. The clinical diagnosis of WRS was suggested in retrospect, and confirmed by sequencing of DNA extracted from stored autopsy material. The patient was compound heterozygous for the novel EIF2AK3 mutations c.1694_1695delAT (Y565X) and c.3044T > C (F1015S). Our data suggest that disruption of the EIF2AK3 gene may lead to defective mitochondrial fatty acid oxidation and hypoglycaemia, thus adding to the heterogeneous phenotype of WRS.

Magnetic resonance imaging findings of adult-onset glutaric aciduria type I.

Glutaric aciduria or glutaric acidemia type I, an autosomal recessive disease, usually presents with an acute encephalopathic crisis in young children. We report the magnetic resonance (MR) and proton MR spectroscopy (MRS) imaging findings of a previously healthy 20-year-old man who presented with recurrent headaches. Organic acids from the patient's urine contained large amounts of adipate, glutarate, and 3-hydroxyglutarate consistent with glutaric aciduria type I.

Dilated cardiomyopathy and adipic aciduria in nutritional rickets.

Hypocalcemia secondary to nutritional rickets is a rare cause of dilated cardiomyopathy. It is also not a recognized cause of dicarboxylic aciduria. We report the first case of adipic aciduria, presenting with dilated cardiomyopathy, secondary to hypocalcemia.