5-Hydroxyhexanoic Acid Predicts Early Renal Functional Decline in Type 2 Diabetes Patients with Microalbuminuria.

BACKGROUND/AIMS: Diabetic nephropathy (DN) is a leading cause of end-stage renal disease. Microalbuminuria (MA) is widely used to predict early progressive renal function decline (ERFD) of DN in type 2 diabetes mellitus (T2D) patients, but the sensitivity and specificity of MA have been questioned. Here, we determined the urine metabolites differences between T2D patients with MA who maintained stable renal function and those who progressed to ERFD in order to identify specific biomarkers of the progression of renal dysfunction. METHODS: A total of 102 T2D patients with MA and normal renal function at baseline were followed up for 5-6 years. Of these, 52 patients were selected and classified into two groups according to the later renal function; 25 patients who experienced ERFD were regarded as the progressive group, while 27 patients who maintained stable renal function were considered as the stable group. In the pilot study, untargeted, broad-spectrum urine metabolomics was performed on the urine of 12 subjects from the progressive group (5 patients as "progressors") and stable group (7 patients as "non-progressors") to discover candidate markers. We then used a targeted metabolomics analysis to identify the selected markers in the urine of an additional 40 patients (20 from the progressive group as cases, and 20 from the stable group as controls) in the validation study. RESULTS: A total of 318 known metabolites were detected in the pilot study and 6 metabolites with significant difference between progressors and non-progressors were identified. The levels of 4 metabolites, including azelaic acid, adipic acid, 5-hydroxyhexanoic acid, and L-tryptophan decreased significantly, while levels of L-pyroglutamic acid and D-norvaline increased observably in the progressors compared with non-progressors. Furthermore, in the validation study, 6 metabolites were confirmed by quantitative measurements and their concentrations were consistent with the changes in the pilot study. Concentrations of L-pyroglutamic acid and D-norvaline still increased in the cases, but were not statistically significant. Of the 4 metabolites with decreased concentrations among the cases, only 5-hydroxyhexanoic acid remained statistically significant while the other 3 metabolites did not differ between cases and controls. CONCLUSION: We have identified urine metabolites and shown that 5-hydroxyhexanoic acid can be used as a predictor of progression of ERFD in T2D patients with MA. This finding provides the new perspective that 5-hydroxyhexanoic acid may be useful to identify T2D patients with MA who are at risk of ERFD.

Association between metabolic profiles in urine and bone mineral density of pre- and postmenopausal Chinese women.

OBJECTIVE: In the present study, we aimed to characterize the pathological development of menopausal osteoporosis, as well as to explore potential biomarkers and metabolic pathways involved in osteoporosis. METHODS: Urine samples from 322 female participants categorized by menopause status and different bone conditions were collected and analyzed based on a gas chromatography-mass spectrometry (GC-MS) approach. Multivariate and univariate statistical analyses were carried out for urinary metabolomic profile characterization and comparison. RESULTS: Seventeen metabolites in the low bone mineral density (BMD) groups were clearly differentiated from those in normal BMD groups. Among these 17 differentiating metabolites, taurine, ß-alanine, and 5-hydroxycaproic acid were found to be potential biomarkers of osteoporosis. The taurine metabolic pathway and the ß-alanine metabolic pathway were found to be related to menopause and bone loss. CONCLUSIONS: Based on the GC-MS metabolomic platform, four typical pathological phases during the progression of postmenopausal osteoporosis were described. Several differentiating metabolites and metabolic pathways were found to be closely related to the pathology of postmenopausal osteoporosis. Our results provided a solid foundation for further studies on early diagnosis and pathomechanistic evaluation.

A Novel Urinary Metabolite Signature for Non-invasive Post-stroke Depression Diagnosis.

Post-stroke depression (PSD) is the most common psychiatric complication in stroke survivors that has been associated with increased physical disability, distress, poor rehabilitation, and suicidal ideation. However, there are still no biomarkers available to support objective laboratory testing for this disorder. Here, a GC-MS-based urinary metabolomics approach was used to characterize the urinary metabolic profiling of PSD (stroke) subjects and non-PSD (health controls) subjects in order to identify and validate urinary metabolite biomarkers for PSD. Six metabolites, azelaic acid, glyceric acid, pseudouridine, 5-hydroxyhexanoic acid, tyrosine, and phenylalanine, were defined as biomarkers. A combined panel of these six urinary metabolites could effectively discriminate between PSD subjects and non-PSD subjects, achieving an area under the receiver-operating characteristic curve (AUC) of 0.961 in a training set (n = 72 PSD subjects and n = 146 non-PSD subjects). Moreover, this urinary biomarker panel was capable of discriminating blinded test samples (n = 58 PSD patients and n = 109 non-PSD subjects) with an AUC of 0.954. These findings suggest that a urine-based laboratory test using these biomarkers may be useful in the diagnosis of PSD.

Hydroxyglutaric aciduria and malignant brain tumor: a case report and literature review.

L -2-Hydroxyglutaric aciduria (L -2-OHGA) is a rare autosomal recessive inherited encephalopathy. This inborn error, characterized by psychomotor retardation, progressive ataxia and typical magnetic resonance imaging findings, presents in early infancy. To make a definitive diagnosis, an anomalous accumulation of L -2-hydroxyglutaric acid must be detected in body fluids. Here, we present a 17-year-old boy with L: -2-OHGA who developed an anaplastic ependymoma during the course of this disease. We also present a literature review including seven other patients who developed malignant brain tumors during the course of L -2-OHGA. This correlation may indicate a possible increased risk of brain tumors among patients with L -2-hydroxyglutaric aciduria.

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.

[L-2-hydroxyglutaric aciduria -- a rare cause of macrocephaly]. / L-2-Hydroxy-Glutarazidurie -- eine seltene Differenzialdiagnose der Makrozephalie.

We report on a 9-year-old girl who was referred to our department because of increasing macrocephaly and school problems. The neurological examination disclosed mild cerebellar dysfunction and positive pyramidal tract signs. An MRI of the brain revealed extensive signal alterations of the white matter. Biochemical investigations established the diagnosis of L-2-hydroxyglutaric aciduria which has to be kept in mind as a rare cause of macrocephaly.

Stereoselective analysis of 2-hydroxysebacic acid in urine of patients with Zellweger syndrome and of premature infants fed with medium-chain triglycerides.

The chiral metabolite 2-hydroxysebacic acid (2-HS) is considered to be an important diagnostic marker for peroxisomal disorders. The pathway of formation of 2-HS, excreted in increased amounts in patients with peroxisomal diseases, is not absolutely clear. Moreover, there is no information about the enantiomeric distribution of 2-HS in human urine. Here, we describe the stereodifferentiation of 2-HS in urine samples of nine patients with Zellweger syndrome (ZS), and for the first time in urine samples of premature infants fed a medium-chain triglyceride (MCT)-containing diet. Using enantioselective multidimensional gas chromatography-mass spectrometry, an increased excretion of 2R-HS was observed in all investigated ZS patients. 2-HS was also present in urine samples of premature infants fed MCT. Analogously to the ZS patients, a dominant 2R-HS excretion in the urine samples of the premature infants was identified. The formation of 2-HS is expected to result from the same or similar pathways as described for ZS patients. Additionally, we determined the absolute configuration of urinary 3-hydroxysebacic acid (3-HS) in the cases investigated. The enantioselective analysis provides further information for the diagnosis and treatment of patients with impaired peroxisomal fatty acid oxidation. Further insight into the metabolic origin and the biochemical pathway leading to these urinary metabolites is provided.

Medulloblastoma in a child with the metabolic disease L-2-hydroxyglutaric aciduria.

L-2-Hydroxyglutaric aciduria (LHGA) is a rare autosomal recessively inherited neurodegenerative disorder. It is characterized by psychomotor retardation, progressive ataxia and typical magnetic resonance imaging findings, and presents in early infancy. On the other hand, medulloblastomas are very common solid tumors of childhood and infancy. We present a 3-year-old boy with LHGA who developed a medulloblastoma during the course of the disease. There has been no previous report of the coexistence of medulloblastomas with LHGA. Central nervous system tumors are encountered in children with other metabolic neurodegenerative disorders. The aim of this paper is to focus on the difficulties in the diagnosis and treatment of an intracranial tumor in a child already neurologically impaired due to metabolic neurodegenerative disease.

L-2-Hydroxyglutaric aciduria presenting as status epilepticus.

L-2-Hydroxyglutaric aciduria (L-2-HGA) is a rare organic aciduria with a slowly progressive course regarding CNS involvement. We present a 13.5-year-old female patient who presented at the Emergency Department with a generalized status epilepticus, which promptly responded to intravenous phenytoin. CT and MRI demonstrated subcortical white matter alterations. The neurological examination revealed mild mental retardation, macrocephaly and ataxic gait with cerebellar signs. Repeated urinary organic acid analysis demonstrated increased excretion of 2-hydroxyglutaric acid which was of the L-configuration. The constellation of macrocephaly in a patient with mental retardation, cerebellar tract involvement and subcortical white matter signal alterations on MRI should alert the physician to the possibility of L-2-HGA. Although rare, epileptic seizures or even status epilepticus can be among the presenting symptoms in organic acidurias with a slow course, such as L-2-HGA.

High-performance liquid chromatographic measurements of urinary hydroxycarboxylic acids as an index of the metabolic control in non-insulin-dependent diabetic patients.

Hydroxycarboxylic acids in urine of patients with non-insulin-dependent diabetes mellitus and of healthy subjects are analyzed as 2-nitrophenylhydrazides by an improved high-performance liquid chromatographic method which has advantages with respect to resolution and analysis time. Variations in levels of hydroxycarboxylic acids, originated from the metabolism of valine, leucine and isoleucine, have been described in the diabetic patients who have good and poor metabolic controls. The sum of the hydroxycarboxylic acids in both groups of diabetic patients was significantly increased compared with the values of the healthy subjects. Statistically significant difference was present between the two groups. In the whole group of diabetic patients, the sum of the hydroxycarboxylic acids correlated with fasting plasma glucose or hemoglobin A1c (r = 0.548, P < 0.01 and r = 0.629, P < 0.01, respectively). These results suggest that the relevance of these abnormalities may be used as an index of metabolic control in diabetic patients.

Indirect ultraviolet detection of biologically relevant organic acids by capillary electrophoresis.

Indirect UV detection of fourteen short-chain organic acids (e.g., oxalic acid, citric acid, malonic acid, tartaric acid, methylmalonic acid, alpha-ketoglutaric acid, succinic acid, ethylmalonic acid, methylsuccinic acid, glutaric acid, apidic acid, methylglutaric acid, lactic acid and pyruvic acid) by capillary electrophoresis is described. The method used phthalate as the UV-absorbing additive in carbonate buffer and the non-absorbing analytes were detected indirectly at 230 nm. The influences of buffer pH, ionic strength, concentration of phthalate and organic modifier on indirect signal response and migration behavior of the organic acids were investigated. Comparisons of reproducibility on migration time, limit of detection and separation efficiency among three types of capillary (e.g., polyacrylamide-coated, myristyltrimethylammonium bromide-coated and uncoated capillaries) were conducted. The method developed was applied to detect succinic acid, methylmalonic acid, citric acid, glutaric acid and lactic acid in human body fluids, and preliminary results were provided.

Sensitive high-performance liquid chromatographic fluorescence assay for the quantitation of topotecan (SKF 104864-A) and its lactone ring-opened product (hydroxy acid) in human plasma and urine.

A sensitive reversed-phase high-performance liquid chromatographic fluorescence method is described for the simultaneous determination of topotecan (I) and the hydrolysed lactone ring-opened product hydroxy acid (II) in plasma and for the determination of I in urine. To 250 microliters of plasma, a 750-microliters volume of cold methanol was added to stabilize the pH-dependent conversion of I into II. In plasma, the lower limit of quantitation (LLQ) for both compounds was 0.10 ng/ml. The between-day variation for I at the LLQ was 7.1% and for II was 5.5%. Prior to injection, urine samples were acidified with orthophosphoric acid and diluted with phosphate-buffered saline (PBS). In urine, the calibration curve for I was linear in the range of 10 to 250 ng/ml and the LLQ was 10 ng/ml. The assay was developed to enable pharmacological analysis of I, in on-going phase I and II studies, in patients with solid tumors.

Metabolic origin of urinary 3-hydroxy dicarboxylic acids.

3-Hydroxy dicarboxylic acids with chain lengths ranging from 6 to 14 carbons are excreted in human urine. The urinary excretion of these acids is increased in conditions of increased mobilization of fatty acids or inhibited fatty acid oxidation. Similar urinary profiles of 3-hydroxy dicarboxylic acids were also observed in fasting rats. The metabolic genesis of these urinary 3-hydroxy dicarboxylic acids was investigated in vitro with rat liver postmitochondrial and mitochondrial fractions. 3-Hydroxy monocarboxylic acids ranging from 3-hydroxyhexanoic acid to 3-hydroxyhexadecanoic acid were synthesized. In the rat liver postmitochondrial fraction fortified with NADPH, these 3-hydroxy fatty acids with carbon chains equal to or longer than 10 were oxidized to (omega - 1)- and omega-hydroxy metabolites as well as to the corresponding 3-hydroxy dicarboxylic acids. 3-Hydroxyhexanoic (3OHMC6) and 3-hydroxyoctanoic (3OHMC8) acids were not metabolized. Upon the addition of mitochondria together with ATP, CoA, carnitine, and MgCl2, the 3-hydroxy dicarboxylic acids were converted to 3-hydroxyoctanedioic, trans-2-hexenedioic, suberic, and adipic acids. In the urine of children with elevated 3-hydroxy dicarboxylic acid levels, 3OHMC6, 3OHMC8, 3-hydroxydecanoic, 3,10-dihydroxydecanoic, 3,9-dihydroxydecanoic, and 3,11-dihydroxydodecanoic acids were identified. On the basis of these data, we propose that the urinary 3-hydroxy dicarboxylic acids are derived from the omega-oxidation of 3-hydroxy fatty acids and the subsequent beta-oxidation of longer chain 3-hydroxy dicarboxylic acids. These urinary 3-hydroxy dicarboxylic acids are not derived from the beta-oxidation of unsubstituted dicarboxylic acids.

[Clinical and biochemical studies in a case of acute encephalopathy associated with calcium hopanthenate administration].

A case with acute disturbance of consciousness associated with calcium hopanthenate (HOPA) administration was reported. He was a 3-year-old boy with autistic developmental delay, had orally taken 1.5 g of HOPA daily for 3 months. Clinical manifestations consisted of fever, vomiting and coma. Laboratory examination revealed severe hypoglycemia and metabolic acidosis, but there were no hepatic enzyme abnormalities. Analysis of urinary organic acid profile showed that very large amounts of medium and long chain dicarboxylic acids and omega-1 hydroxy-fatty acids were excreted. In particular, 2-hydroxysebacic acid, the accumulation of which has only been reported in the urine of patients with Zellweger syndrome and neonatal adrenoleukodystrophy (NALD), was observed. Analysis of urinary acylcarnitines showed that acetylcarnitine was predominant and C6-C10 dicarboxylic acylcarnitines were also excreted. He was treated with and rapidly responded to intravenous glucose and bicarbonate. After withdrawal of the drug he has had no problems and dicarboxylic aciduria disappeared. A CT scan showed symmetric, low density areas in periventricular white matter, especially around the posterior horns of the lateral ventricles. A T2-weighted MRI scan revealed high-intensity signal in the white matter corresponding to areas of low density on CT scan. We conclude that that a large amount of HOPA administration may cause encephalopathy by the inhibition of both mitochondrial and peroxisomal beta-oxidation.

Mass spectrometric identification of 2-hydroxydodecanedioic acid and its homologues in urine from patients with hopantenate therapy during clinical episode.

Urine from patients with calcium-4-(2,4-dihydroxy-3,3-dimethylbutyramido) butyrate hemihydrate (hopantenate) therapy during episodes of Reye's-like syndrome was found to contain a number of unusual dicarboxylic acids in high concentrations; odd- and even-numbered medium-chain dicarboxylic acids, alpha-hydroxydicarboxylic acids and beta-hydroxydicarboxylic acids. The abnormal excretion of dicarboxylic acids, alpha- and beta-hydroxydicarboxylic acids disappeared after discontinuance of hopantenate therapy. Besides the excretion of 2-hydroxydecandedioic acid, which has been previously described in Zellweger syndrome or neonatal adrenoleukodystrophy, a series of alpha-hydroxydicarboxylic acids was detected and identified. In this paper, we have characterized some new compounds by gas chromatography/mass spectrometry: 2-hydroxydodecanedioic acid, 2-hydroxydodecenedioic acid, 2-hydroxytetradecanedioic acid, 2-hydroxytetradecenedioic acid and 2-hydroxyoctanedioic acid.