GC × GC-TOFMS metabolomics analysis identifies elevated levels of plasma sugars and sugar alcohols in diabetic mellitus patients with kidney failure.

Two dimensional GC (GC × GC)-time-of-flight mass spectrometry (TOFMS) has been used to improve accurate metabolite identification in the chemical industry, but this method has not been applied as readily in biomedical research. Here, we evaluated and validated the performance of high resolution GC × GC-TOFMS against that of GC-TOFMS for metabolomics analysis of two different plasma matrices, from healthy controls (CON) and diabetes mellitus (DM) patients with kidney failure (DM with KF). We found GC × GC-TOFMS outperformed traditional GC-TOFMS in terms of separation performance and metabolite coverage. Several metabolites from both the CON and DM with KF matrices, such as carbohydrates and carbohydrate-conjugate metabolites, were exclusively detected using GC × GC-TOFMS. Additionally, we applied this method to characterize significant metabolites in the DM with KF group, with focused analysis of four metabolite groups: sugars, sugar alcohols, amino acids, and free fatty acids. Our plasma metabolomics results revealed 35 significant metabolites (12 unique and 23 concentration-dependent metabolites) in the DM with KF group, as compared with those in the CON and DM groups (N = 20 for each group). Interestingly, we determined 17 of the 35 (14/17 verified with reference standards) significant metabolites identified from both the analyses were metabolites from the sugar and sugar alcohol groups, with significantly higher concentrations in the DM with KF group than in the CON and DM groups. Enrichment analysis of these 14 metabolites also revealed that alterations in galactose metabolism and the polyol pathway are related to DM with KF. Overall, our application of GC × GC-TOFMS identified key metabolites in complex plasma matrices.

Sugar Alcohols of Polyol Pathway Serve as Alarmins to Mediate Local-Systemic Innate Immune Communication in Drosophila.

Interorgan immunological communication is critical to connect the local-systemic innate immune response and orchestrate a homeostatic host defense. However, the factors and their roles in this process remain unclear. We find Drosophila IMD response in guts can sequentially trigger a systemic IMD reaction in the fat body. Sugar alcohols of the polyol pathway are essential for the spatiotemporal regulation of gut-fat body immunological communication (GFIC). IMD activation in guts causes elevated levels of sorbitol and galactitol in hemolymph. Aldose reductase (AR) in hemocytes, the rate-limiting enzyme of the polyol pathway, is necessary and sufficient for the increase of plasma sugar alcohols. Sorbitol relays GFIC by subsequent activation of Metalloprotease 2, which cleaves PGRP-LC to activate IMD response in fat bodies. Thus, this work unveils how GFIC relies on the intermediate activation of the polyol pathway in hemolymph and demonstrates that AR provides a critical metabolic checkpoint in the global inflammatory response.

Exploratory metabolomics of biomarker identification for the internet gaming disorder in young Korean males.

The main aim of the current research is to characterize the molecular dynamics related to internet gaming disorder (IGD) using non-targeted plasma metabolite profiling based on gas-chromatography time-of-flight mass spectrometry (GC-TOF MS). IGD is a psychiatric disorder instigated by excessive and prolonged internet gaming, which shared many pathological symptoms with attention deficit hyperactivity disorder (ADHD). The prevalence of the disorder has been rapidly increased particularly in East Asia countries (5.9% in South Korea) compared to Europe or North America (0.3-1.0% in United States and 1.16% in Germany). Thus we comparably explored the correlation between plasma metabolites and internet addiction severity in IGD patients, and potential biomarker composite in combination with clinical parameters. The systematic metabolite profiling of 54 blood samples (normal user, N=28 and IGD, N=24) identified a total of 104 metabolites out of 1212 metabolic feature, and revealed unique relation of co-linearly regressed set of plasma metabolites (arabitol, myo-inositol, methionine, pyrrole-2-carboxylic acid, and aspartic acid) with internet addiction severity scale (R=0.795). In addition, orthogonal partial least squared discriminant analysis (OPLS-DA) and receiver operating characteristic (ROC) analysis identified the potential biomarker cluster that simultaneously discriminated the different types of the psychiatric status. The potential biomarker re-composite was comprehensively evaluated by a receiver operating characteristic (ROC) analysis where the AUCs were 0.890, 0.880, 1.000, and 0.935 for control, IGD, AD and IGD+AD, respectively (N=18, 19, 5, and 10) against the others. This exploratory method may provide robustness of predictive diagnosis in population screening of IGD. The identified metabolic features, the relatedness with clinical parameters, and the putative biochemical linkage will hopefully aid future pathological studies in IGD.

Invasive fungal infections: biomarkers and molecular approaches to diagnosis.

The biomarkers galactomannan and 1,3-ß-d-glucan have been well studied over the past years and are gaining a role in the diagnosis of invasive fungal infections. Although not as well studied until recently, molecular methods for the diagnosis of invasive fungal infection are also being evaluated. Outcomes data for molecular testing are expanding, but have not yet provided enough evidence for inclusion of molecular diagnostics in formal clinical guidelines. Lack of standardization and validation of the various molecular assays and platforms has hindered their widespread acceptance in the evaluation of invasive fungal infections, although the future is promising.

Detection of candidaemia in patients with and without underlying haematological disease.

Diagnosing candidaemia remains difficult despite the development of new diagnostics. We report a direct comparison of three different blood-culture systems and four indirect tests. One hundred and fourteen episodes either with haematological disease and fever despite antibacterials, or with documented invasive candidiasis, were enrolled prospectively. Clinical, para-clinical information and surveillance cultures were obtained. Blood culture was performed using conventional blood-culture bottles, mycosis bottles, and the Isolator 10 lysis centrifugation system. Serum D-arabinitol/L-arabinitol (DA/LA) ratios were determined by gas chromatography mass spectrometry. Antigen, mannan-antigen (Ag) and anti-mannan antibody (Ab) were detected by CandTec, Platelia Candida Ag ELISA and Candida AB/AC/AK kits, respectively. Episodes were classified as proven (n = 24), probable (n = 14), possible (n = 52) or unlikely (n = 24) invasive candidiasis. Candidaemia involved C. albicans (17), C. albicans + C. glabrata (3), C. tropicalis (1) and yeast (1). Mycosis bottles yielded two additional positives and the conventional blood culture yielded one positive not identified by other blood-culture methods. Considering proven and unlikely episodes, respectively, sensitivity and specificity were as follows: mannan-Ag and/or anti-mannan Ab: 83.3%, 78.3%; DA/LA ratio: 41.7%, 86.4%; and CandTec Candida Ag: 66.6%, 70.8%. Lowering the cut-off values to mannan-Ag 0.10 ng/mL and anti-mannan Ab 4 AU/mL, the values were: 100%, 73.9%. Applying the DA/LA ratio to only patients with haematological neutropenia the values were: 75%, 90.5%. Fungal blood culture allowed slightly improved detection of candidaemia. The best indirect test performance was obtained from combined mannan-Ag and anti-mannan Ab detection, especially with lower cut-offs. DA/LA ratio appears to be useful in the context of haematological neutropenia.

Plasma concentrations of carbohydrates and sugar alcohols in term newborns after milk feeding.

Nonglucose carbohydrates such as galactose, mannose, and inositol play a clinically important role in fetal and neonatal nutrition, though little is known about their metabolism in the neonate. The aim of this study was to determine whether postprandial changes in plasma carbohydrate and sugar alcohol concentrations are affected by clinical variables such as postnatal age (PNA), milk type, feeding volume, or feeding duration in term newborns. Neonates (n = 26) taking intermittent enteral feedings were enrolled. Blood samples were obtained at baseline (immediately before the start of a feeding) and at 2-3 subsequent time points up to 110 min. Postprandial rise was only observed for plasma glucose concentrations [Glu] and plasma galactose concentrations [Gal] and clinical variables did not predict this change. Despite equimolar delivery in milk, the median of [Glu] rise minus [Gal] rise from baseline to second postprandial plasma sample was 674 microM (-38, 3333 microM; p < 0.0001), reflecting efficient hepatic first-pass metabolism of galactose. A significant PNA effect on [Gal] was observed such that for each day PNA there was an 18% decrease in [Gal] (p = 0.03). [Gal] are a function of PNA, suggesting maintenance of a significant ductus venosus shunt in term infants.

Measurement of serum D-arabinitol/creatinine ratios for initial diagnosis and for predicting outcome in an unselected, population-based sample of patients with Candida fungemia.

D-Arabinitol (DA) is a useful diagnostic marker for candidiasis in patients with neutropenia and other high-risk groups, but its use in unselected patients with a broad range of underlying diseases and conditions has not been studied. We used an automated enzymatic fluorometric assay to measure serum DA/creatinine ratios (DA/cr's) in 30 healthy adults, 100 hospitalized controls without Candida fungemia, and 83 patients from a study of all Candida fungemias in Connecticut between October 1998 and September 1999. Sixty-three of 83 (76%) fungemic patients and 11 of 100 (11%) nonfungemic controls had serum DA/cr's >or=3.9 microM/mg/dl (mean + 3 standard deviations for 30 healthy adults). High serum DA/cr's were less frequent in patients with cancer or fungemia caused by the DA nonproducer Candida glabrata than in patients with cancer or fungemia caused by a DA producer, C. albicans, C. tropicalis, or C. parapsilosis. The serum DA/cr was first >or=3.9 microM/mg/dl before, on the same day as, or after the first positive blood culture was drawn for 30 (36%), 22 (27%), and 11 (13%) fungemia patients, respectively. Mortality did not differ significantly among the patients with high or normal initial or peak serum DA/cr's, but mortality was higher if any serum DA/cr value was >or=3.9 microM/mg/dl 3 or more days after the onset of fungemia (18/27 versus 4/24 patients, respectively; P < 0.001). We conclude that serum DA/cr's are useful both for the initial diagnosis of Candida fungemia and for prognostic purposes for unselected patients with a broad range of underlying diseases and conditions.

Simultaneous determination of glucose, 1,5-anhydro-d-glucitol and related sugar alcohols in serum by high-performance liquid chromatography with benzoic acid derivatization.

A new, simple and sensitive pre-column high-performance chromatographic method for the determination of diabetes marker d-glucose, 1,5-anhydro-d-glucitol and related compounds is reported. Sugars (d-glucose, d-galactose, d-mannose, sucrose and arabinose) were derivatized with benzoic acid (BA) at 80 degrees C for 60 min. l-Fucose, fructose, d-lactose, l-rhamnose, arabinose and ascorbic acid were not reacted. Sugar alcohols (xylitol, erythritol, mannitol, sorbitol myo-inositol) were also derivatized with BA at 80 degrees C for 60 min. The fluorescence derivatives were separated on a TSK amide 80 column (4.6 mm i.d. x 250 mm, 5 microm) with acetonitrile-50 mm acetate buffer (pH 5.6; 4:96, v/v) as the mobile phase. The detection wavelength of beizoic acid derivatives was lambda(ex) 275 nm and lambda(em) 315 nm. The detection limits of sugars were 10-80 microg/mL. The calibration graphs were linear up to 10 mg/mL. The relative standard deviations of 500 microg/mL sugars were 7.0-7.3%. The proposed method was compared with the enzymatic photometric glucose analysis method (Glucose B-Test II Wako). The correlation coefficient was 0.83 (n = 20) and y = 0.82x + 5.91, where y and x are concentrations in microg/mL obtained by the proposed pre-column HPLC and enzyme-photometric method, respectively. The detection limits of sugar alcohols were 100-1000 ng/mL. The calibration graphs were linear to 50 microg/mL and relative standard deviations of 10 microg/mL were 7.2-8.2%. The 1,5-AG data by the proposed method was also compared with the enzymatic photometric 1,5-AG analysis method (Rana AG 1,5-AG determination kit, Nihon Kayaku) and good correlation (r = 0.91, n = 20) was also obtained. The proposed method was applied to the simultaneous determination of d-glucose, 1,5-AG and related sugar alcohols in serum from healthy males.

Development and application of a rapid diagnostic method for invasive Candidiasis by the detection of D-/L-arabinitol using gas chromatography/mass spectrometry.

A rapid non-culture-based diagnostic method utilizing d-/l-arabinitol (DA/LA) ratios as a chemical marker of invasive candidiasis was developed and explored. The enantiomers-ratios detection was made possible by the use of gas chromatography coupled with mass spectrometry (GC/MS). The mean DA/LA ratios +/- standard deviation (range) in urine (n = 40) and serum (n = 20) were 2.08 +/- 0.78 (0.57 to 3.55) and 1.79 +/- 0.75 (0.74 to 3.54), respectively, from patients without evidence of fungal infection or colonization; in patients (n = 7) with culture-proven invasive candida infections, the figures were 9.91 +/- 3.04 (7.24 to 16.27) and 13.58 +/- 7.31 (5.57 to 25.88) in urine and serum, respectively. The differences in DA/LA ratios between the candidemic patients and the non-candidemic patients were statistically significant (p < 0.01) in both serum and urine samples. The DA/LA ratios were not significantly affected in patients with oral or vaginal candidiasis and candiduria.

Ribose-5-phosphate isomerase deficiency: new inborn error in the pentose phosphate pathway associated with a slowly progressive leukoencephalopathy.

The present article describes the first patient with a deficiency of ribose-5-phosphate isomerase (RPI) (Enzyme Commission number 5.3.1.6) who presented with leukoencephalopathy and peripheral neuropathy. Proton magnetic resonance spectroscopy of the brain revealed highly elevated levels of the polyols ribitol and D-arabitol, which were subsequently also found in high concentrations in body fluids. Deficient activity of RPI, one of the pentose-phosphate-pathway (PPP) enzymes, was demonstrated in fibroblasts. RPI gene-sequence analysis revealed a frameshift and a missense mutation. Recently, we described a patient with liver cirrhosis and abnormal polyol levels in body fluids, related to a deficiency of transaldolase, another enzyme in the PPP. RPI is the second known inborn error in the reversible phase of the PPP, confirming that defects in pentose and polyol metabolism constitute a new area of inborn metabolic disorders.

Pharmacokinetics and anti-hyperglycaemic efficacy of a novel inhibitor of glycogen phosphorylase, 1,4-dideoxy-1,4-imino-d- arabinitol, in glucagon-challenged rats and dogs and in diabetic ob/ob mice.

AIM: To further characterize the properties of 1,4-dideoxy-1,4-imino-d-arabinitol (DAB), a recently described novel and potent inhibitor of glycogen phosphorylase and potential anti-diabetic agent, we have determined its pharmacokinetic properties in rats, dogs and mice and compared these to its pharmacodynamic anti-hyperglycaemic efficacy. METHODS: Male Sprague Dawley rats, beagle dogs and diabetic Umeå ob/ob mice were administered DAB or 14C-DAB at various doses and by different routes and in either the conscious or the unconscious state and with or without glucagon, as appropriate. At different time points thereafter, blood, tissue and urine samples were withdrawn for analyses of DAB or 14C-DAB, and blood samples were taken for glucose concentration. RESULTS: DAB suppressed the blood glucose excursion in glucagon-challenged rats with an ID100 of 1-2 mg/kg per orally and intravenously and had a pharmacodynamic t50 for 1.6 mg/kg intravenously and for 1.2 mg/kg per orally of 50 and 60 min respectively. The pharmacokinetics of c. 2 mg/kg DAB in rats revealed elimination half-lives of 25 min after intravenous (i.v.) and 49 min after per oral (p.o.) administration; the oral bioavailability was 89%. In rats, DAB was distributed preferentially in liver vs. skeletal muscle and was eliminated predominantly through urine as parent compound. The pharmacokinetics of 4 mg/kg DAB in dogs showed elimination half-lives of 107 min after i.v. and 129 min after p.o. administration with an estimated oral availability of 78%. At 4 mg/kg DAB p.o., glucagon-induced hyperglycaemia in dogs was reduced in a time-dependent manner with an estimated t50 of 4 h. DAB was very rapidly cleared in mice; nevertheless, a dose-dependent reduction of blood glucose of up to 9 mmol/l was seen in diabetic ob/ob mice dosed subcutaneously, with statistically significant effects evident from 30 to 120 min. CONCLUSIONS: These data show that DAB is nearly completely orally available in rats and dogs and that it can reduce glucagon-induced and spontaneous hyperglycaemia. Inhibition of hepatic glycogen phosphorylase may benefit glycaemic control in patients with type 2 diabetes.

L-Arabinosuria: a new defect in human pentose metabolism.

A female patient, the first child of healthy non-consanguineous parents, presented at the age of 16 months with delayed motor development and facial dysmorphism. In addition she displayed a palatoschizis and multiple skeletal abnormalities as hypoplastic scapulae, hypoplastic os ilea, and an extreme cervical kyphosis. Biochemical investigation of urine revealed no abnormalities except for the presence of large amounts of reducing sugars. The sugar was identified as L-arabinose, which mainly originated from fruit formula in her diet. In addition highly elevated levels of L-arabitol were found in urine, plasma, and cerebrospinal fluid. Although little is known about human arabinose metabolism, we presume that L-arabitol dehydrogenase is deficient in our patient. As polyols are potentially toxic to the central nervous system there could be deleterious long-term effects of this disorder. Withdrawal of dietary fruit led to normalization of polyol levels. The above-mentioned clinical abnormalities are probably not related to this new inborn error of metabolism and should be considered as a separate entity.

[Advances in serological systems for diagnosis of systemic fungal infections, particularly those caused by Candida and Aspergillus].

Invasive fungal infections have emerged as important causes of morbidity and mortality in neutropenicand some other immunocompromised hosts; Candida and Aspergillus are among the major pathogens in this patient population. The clinical diagnosis of these infections is not specific and the traditional mycological methods for them not sensitive, with limits in the early detection of the pathogen. The potential additives or complements to the laboratory diagnosis of invasive candidiasis and aspergillosis are two non-culture-based methods, serodiagnostic methods and molecular ones. The former methods include the detection of pathogen-specific antigens, antibodies, metabolites and cell wall components. Several have already become standard laboratory tools and some others are under active investigation for developing new, more accurate detection systems. In this review, I will discuss the current status and future potential of serodiagnostic methods, highlighting both their technical and clinical implications.

Stereoselective pharmacokinetics and pharmacodynamics of organic nitrates in rats.

Plasma concentration and vasodilating effect after i.v. bolus injection of stereoisomeric organic nitrates were evaluated. Pharmacokinetics of mononitrates was analyzed with a linear one-compartment model. The apparent volumes of distribution were almost identical, but systemic clearances were different among stereoisomers. The concentration data after dinitrate administration could be described based on a two-compartment model with elimination only from the central compartment via metabolism to mononitrate, and then mononitrate-dependent metabolic clearance was estimated. In the vasodilation by mononitrate administered intravenously, the maximum effect was not observed. The reduction of mean arterial pressure from baseline level was related to plasma concentration with a log-linear model. The pharmacological effect following dinitrate dosing was analyzed by a sigmoidal Emax model assuming a simple additive effect of dinitrate and mononitrate. Although almost the same Hill's constant and maximum effect (Emax) values were estimated, the concentrations required to produce 50% of Emax (EC50) differed among stereoisomers. The clearance and EC50 values of stereoisomers with nitrate group at the exo position were generally higher than those with the same group at the endo position. This suggests that the stereostructure of organic nitrates controls the vasodilator potency and duration of action.

Transaldolase deficiency: liver cirrhosis associated with a new inborn error in the pentose phosphate pathway.

This article describes the first patient with a deficiency of transaldolase (TALDO1 [E.C.2.2.1.2]). Clinically, the patient presented with liver cirrhosis and hepatosplenomegaly during early infancy. In urine and plasma, elevated concentrations of ribitol, D-arabitol, and erythritol were found. By incubating the patient's lymphoblasts and erythrocytes with ribose-5-phosphate and subsequently analyzing phosphate sugar metabolites, we discovered a deficiency of transaldolase. Sequence analysis of the transaldolase gene from this patient showed a homozygous deletion of 3 bp. This deletion results in absence of serine at position 171 of the transaldolase protein. This amino acid is invariable between species and is located in a conserved region, indicating its importance for enzyme activity. The detection of this new inborn error of pentose metabolism has implications for the diagnostic workup of liver problems of unknown etiology.

[Interference by D-mannitol on serum D-arabinitol determination by enzymatic assay].

We examined a disparity in D-arabinitol values between two commercial assay kits, LABOFIT and ARABINITEC-AUTO. The determined values by the former were increased by 26%(y = 0.2643x) because of concomitant D-mannitol, whereas those by our newly developed ARABINITEC-AUTO was increased only by 2%(y = 0.0242x). Of 109 samples, 5 samples were found to contain more than 100 mumol/l of D-mannitol. A clear relation(r = 0.89) was noted between the degree of disparity between measurements by the two methods and D-mannitol concentrations in samples. Thus, we have proved that the disparity is mainly caused by D-mannitol.