Metabolite Biomarkers of CKD Progression in Children.

BACKGROUND AND OBJECTIVES: Metabolomics facilitates the discovery of biomarkers and potential therapeutic targets for CKD progression. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: We evaluated an untargeted metabolomics quantification of stored plasma samples from 645 Chronic Kidney Disease in Children (CKiD) participants. Metabolites were standardized and logarithmically transformed. Cox proportional hazards regression examined the association between 825 nondrug metabolites and progression to the composite outcome of KRT or 50% reduction of eGFR, adjusting for age, sex, race, body mass index, hypertension, glomerular versus nonglomerular diagnosis, proteinuria, and baseline eGFR. Stratified analyses were performed within subgroups of glomerular/nonglomerular diagnosis and baseline eGFR. RESULTS: Baseline characteristics were 391 (61%) male; median age 12 years; median eGFR 54 ml/min per 1.73 m2; 448 (69%) nonglomerular diagnosis. Over a median follow-up of 4.8 years, 209 (32%) participants developed the composite outcome. Unique association signals were identified in subgroups of baseline eGFR. Among participants with baseline eGFR ≥60 ml/min per 1.73 m2, two-fold higher levels of seven metabolites were significantly associated with higher hazards of KRT/halving of eGFR events: three involved in purine and pyrimidine metabolism (N6-carbamoylthreonyladenosine, hazard ratio, 16; 95% confidence interval, 4 to 60; 5,6-dihydrouridine, hazard ratio, 17; 95% confidence interval, 5 to 55; pseudouridine, hazard ratio, 39; 95% confidence interval, 8 to 200); two amino acids, C-glycosyltryptophan, hazard ratio, 24; 95% confidence interval 6 to 95 and lanthionine, hazard ratio, 3; 95% confidence interval, 2 to 5; the tricarboxylic acid cycle intermediate 2-methylcitrate/homocitrate, hazard ratio, 4; 95% confidence interval, 2 to 7; and gulonate, hazard ratio, 10; 95% confidence interval, 3 to 29. Among those with baseline eGFR <60 ml/min per 1.73 m2, a higher level of tetrahydrocortisol sulfate was associated with lower risk of progression (hazard ratio, 0.8; 95% confidence interval, 0.7 to 0.9). CONCLUSIONS: Untargeted plasma metabolomic profiling facilitated discovery of novel metabolite associations with CKD progression in children that were independent of established clinical predictors and highlight the role of select biologic pathways.

Large-scale plasma-metabolome analysis identifies potential biomarkers of psoriasis and its clinical subtypes.

BACKGROUND: Psoriasis is an immune-mediated skin disease for which the crosstalk between genetic and environmental factors is responsible. To date, no definitive diagnostic criteria for psoriasis yet, and specific biomarkers are required. OBJECTIVE: We performed metabolome analysis to identify metabolite biomarkers of psoriasis and its subtypes such as psoriatic arthritis (PsA) and cutaneous psoriasis (PsC). METHODS: We constructed metabolomics profiling of 130 plasma samples (42 PsA patients, 50 PsC patients, and 38 healthy controls) using a nontargeted metabolomics approach. RESULTS: Psoriasis-control association tests showed that one metabolite (ethanolamine phosphate) was significantly increased in psoriasis samples than in the controls, whereas three metabolites decreased (false discovery rate [FDR] < 0.05; XA0019, nicotinic acid, and 20α-hydroxyprogesterone). In the association test between PsA and PsC, tyramine significantly increased in PsA than in PsC, whereas mucic acid decreased (FDR < 0.05). Molecular pathway analysis of the PsA-PsC association test identified enrichment of vitamin digestion and absorption pathway in PsC (P = 1.3 × 10-4). Correlation network analyses elucidated that a subnetwork centered on aspartate was constructed among the psoriasis-associated metabolites; meanwhile, the major subnetwork among metabolites with differences between PsA and PsC was primarily formed from saturated fatty acids. CONCLUSION: Our large-scale metabolome analysis highlights novel characteristics of plasma metabolites in psoriasis and the differences between PsA and PsC, which could be used as potential biomarkers of psoriasis and its clinical subtypes. These findings contribute to our understanding of psoriasis pathophysiology.

Assessment of lactase activity in humans by measurement of galactitol and galactonate in serum and urine after milk intake.

Background: Lactase is an enzyme that hydrolyzes lactose into glucose and galactose in the small intestine, where they are absorbed. Hypolactasia is a common condition, primarily caused by genetic programming, that leads to lactose maldigestion and, in certain cases, lactose intolerance. Galactitol and galactonate are 2 products of hepatic galactose metabolism that are candidate markers for the intake of lactose-containing foods. Objectives: The primary objective of the study was to explore the changes in serum and urine metabolomes during postprandial dairy product tests through the association between lactase persistence genotype and the postprandial dynamics of lactose-derived metabolites. Methods: We characterized the 6-h postprandial serum kinetics and urinary excretion of lactose, galactose, galactitol, and galactonate in 14 healthy men who had consumed a single dose of acidified milk (800 g) which contained 38.8 g lactose. Genotyping of LCT-13910 C/T (rs4988235) was performed to assess primary lactase persistence. Results: There were 2 distinct postprandial responses, classified as high and low metabolite responses, observed for galactose, and its metabolites galactitol and galactonate, in serum and urine. In all but 1 subject, there was a concordance between the high metabolite responses and genetic lactase persistence and between the low metabolite responses and genetic lactase nonpersistence (accuracy 0.92), galactitol and galactonate being more discriminative than galactose. Conclusions: Postprandial galactitol and galactonate after lactose overload appear to be good proxies for genetically determined lactase activity. The development of a noninvasive lactose digestion test based on the measurement of these metabolites in urine could be clinically useful. This trial was registered at clinicaltrials.gov as NCT02230345.

Effects of broccoli extract on biodistribution and labeling blood components with 99mTc-GH / Efeitos do extrato de brócolis na biodistribuição e marcação dos componentes do sangue com 99mTc-GH

PURPOSE: People consume vegetables without the knowledge of the side effects of the biological and chemical contents and interactions between radiopharmaceuticals and herbal extract. To this end, current study is focused on the effects of broccoli extract on biodistribution of radiolabeled glucoheptonate (99mTc-GH) and radiolabeling of blood components. METHODS: GH was labeled with 99mTc. Quality control studies were done utilizing TLC method. Biodistribution studies were performed on male rats which were treated via gavage with either broccoli extract or SF as control group for 15 days. Blood samples were withdrawn from rats' heart. Radiolabeling of blood constituents performed incubating with GH, SnCl2 and 99m Tc. RESULTS: Radiochemical yield of 99mTc-GH is 98.46±1.48 percent (n=8). Biodistribution studies have shown that according to the control, the treated group with broccoli has approximately 10 times less uptake in kidney. The percentage of the radioactivity ratios of the blood components is found to be same in both groups. CONCLUSIONS: Although there is no considerable effect on the radiolabeling of blood components, there is an outstanding change on the biodistribution studies especially on kidneys. The knowledge of this change on kidney uptake may contribute to reduce the risk of misdiagnosis and/or repetition of the examinations in Nuclear Medicine.

OBJETIVO: As pessoas consomem verduras sem o conhecimento dos efeitos colaterais dos conteúdos biológicos e químicos e interações entre os medicamentos radiofarmacêuticos e os extratos vegetais. Para este fim, o estudo atual é focado sobre os efeitos do extrato de brócolis na biodistribuição do fármaco glucoheptonato (99mTc-GH) e da marcação de componentes do sangue. MÉTODOS: GH foi marcado com 99mTc. Estudos de controle de qualidade foram feitos utilizando o método do TLC. Os estudos de biodistribuição foram realizados em ratos machos que foram tratados por gavagem com um extrato de brócolis ou SF como grupo controle para 15 dias. Amostras de sangue foram retiradas do coração de ratos. Marcação de constituintes sanguíneos realizados incubação com SnCl2 GH e 99mTc. RESULTADOS: Radioquímica rendimento de 99mTc-GH é 98,46 ± 1,48 por cento (n = 8). Os estudos de biodistribuição mostraram que de acordo com o controle, o grupo tratado com brócolis tem aproximadamente 10 vezes menor absorção no rim. O percentual do ratio de radioatividade dos componentes do sangue é encontrado para ser igual nos dois grupos. CONCLUSÕES: Embora não haja nenhum efeito considerável sobre a marcação dos componentes do sangue há uma mudança notável na biodistribuição especialmente nos rins. O conhecimento desta mudança na captação de rim pode contribuir para reduzir o risco de erro diagnóstico e/ou a repetição dos exames de Medicina Nuclear.

Effects of broccoli extract on biodistribution and labeling blood components with 99mTc-GH.

PURPOSE: People consume vegetables without the knowledge of the side effects of the biological and chemical contents and interactions between radiopharmaceuticals and herbal extract. To this end, current study is focused on the effects of broccoli extract on biodistribution of radiolabeled glucoheptonate ((99m)Tc-GH) and radiolabeling of blood components. METHODS: GH was labeled with (99m)Tc. Quality control studies were done utilizing TLC method. Biodistribution studies were performed on male rats which were treated via gavage with either broccoli extract or SF as control group for 15 days. Blood samples were withdrawn from rats' heart. Radiolabeling of blood constituents performed incubating with GH, SnCl2 and (99m) Tc. RESULTS: Radiochemical yield of (99m)Tc-GH is 98.46±1.48 % (n=8). Biodistribution studies have shown that according to the control, the treated group with broccoli has approximately 10 times less uptake in kidney. The percentage of the radioactivity ratios of the blood components is found to be same in both groups. CONCLUSIONS: Although there is no considerable effect on the radiolabeling of blood components, there is an outstanding change on the biodistribution studies especially on kidneys. The knowledge of this change on kidney uptake may contribute to reduce the risk of misdiagnosis and/or repetition of the examinations in Nuclear Medicine.

Monitoring of biochemical status in children with Duarte galactosemia: utility of galactose, galactitol, galactonate, and galactose 1-phosphate.

BACKGROUND: Duarte galactosemia (DG) is frequently detected in newborn-screening programs. DG patients do not manifest the symptoms of classic galactosemia, but whether they require dietary galactose restriction is controversial. We sought to assess the relationships of selected galactose metabolites (plasma galactose, plasma galactitol, erythrocyte (RBC) galactitol, RBC galactonate, and urine galactitol and galactonate) to RBC galactose 1-phosphate (Gal-1-P), dietary galactose intake, and neurodevelopmental/clinical outcomes in DG children. METHODS: We studied 30 children 1-6 years of age who had DG galactosemia and were on a regular diet. All participants underwent a physical and ophthalmologic examination and a neurodevelopmental assessment. RBC galactitol, RBC galactonate, RBC Gal-1-P, plasma galactose, plasma galactonate, and urine galactitol and galactonate concentrations were measured. RESULTS: RBC galactitol and galactonate concentrations were about 2 and 6 times higher, respectively, than control values. Plasma galactose and galactitol concentrations were also about twice the control values. The mean values for RBC Gal-1-P and urine galactitol were within the reference interval. We found a relationship between plasma and urine galactitol concentrations but no relationship between RBC galactose metabolites and urine galactitol. There was a significant relationship between galactose intake and RBC galactose metabolites, especially RBC galactitol (P < 0.0005) and RBC galactonate (P < 0.0005). Galactose intake was not related to the urine galactitol, plasma galactose, or plasma galactitol concentration. RBC galactitol, RBC galactonate, plasma galactose, plasma galactitol, and urine galactonate concentrations showed no relationship with clinical or developmental outcomes. CONCLUSIONS: DG children on a regular diet have RBC Gal-1-P concentrations within the reference interval but increased concentrations of other galactose metabolites, including RBC galactitol and RBC galactonate. These increased concentrations correlate with galactose intake and neither cause any developmental or clinical pathology during early childhood nor oblige a lactose-restricted diet.

Galactitol and galactonate in red blood cells of children with the Duarte/galactosemia genotype.

We measured galactitol, galactonate, and galactose-1-phosphate in the red blood cell (RBC) to elucidate the biochemical phenotype of infants with a Duarte/galactosemia (D/G) genotype by isotope dilution GC/MS. The RBC galactonate, galactitol and Gal-1-P were quantified in 14 D/G newborns on a lactose containing formula or breast milk, eight D/G newborns on a galactose-free formula, and 18 D/G children between 1 and 2 years of age that were on a regular diet. The results were compared with those of non-galactosemic subjects of comparable age. In the D/G newborns on regular formula/breast milk, the levels of RBC galactitol, galactonate, and Gal-1-P were significantly higher than those of D/G newborns on diet treatment and non-galactosemic newborns. There was no difference in the levels of RBC galactitol, galactonate, and Gal-1-P between D/G newborns on a lactose-restricted diet and the control group. There appears to be two different responses to dietary galactose intake in D/G children. The first group of D/G children placed on a regular diet after a year of lactose restriction had higher RBC galactitol, galactonate levels than those of non-galactosemic children. The mean level of RBC galactonate was higher and the mean value of RBC galactitol was as high as that of galactosemic (G/G) patients on diet treatment. The second group of D/G children on a regular diet had normal levels of RBC galactitol and galactonate. The levels of RBC Gal-1-P were normal in both groups of D/G patients. The alternative pathway products may reflect galactose intake better than RBC Gal-1-P in D/G children.

Age dependence of endogenous galactose formation in Q188R homozygous galactosemic patients.

The age dependence of endogenous galactose formation was investigated in Q188R homozygous galactosemic patients (n=18; 4-38 years) using the primed continuous infusion approach with D-[1-13C]galactose as a substrate. Studies were conducted under postabsorptive conditions (fasting >10h) and good metabolic control. In the patients, the release of galactose from endogenous sources into plasma (R(a)) decreased with age and ranged from 4.6 to 2.0 micromol/kg body weight per h. Galactitol and galactonate release rates paralleled the galactose R(a) but at a lower level. The mean relation of galactose, galactitol, and galactonate release was 10:5:1. Statistically, there was a highly significant (p<0.0001) inverse correlation between total galactose release (i.e., sum of R(a) plus galactitol and galactonate release) and age. The data (total galactose=y, age=t) were best fitted to the simple exponential model y=y(0)+axexp(-bt) by non-linear regression analysis. The parameter estimates were y(0)=3.0+/-0.2, a=6.5+/-0.4, and b=0.11+/-0.02. The value of y(0) provides an estimate of total galactose release in adult patients (i.e., approximately 13 mg/kg body weight per day), summation operator (y(0)+a) provides an estimate for galactosemic newborns (i.e., approximately 41 mg/kg body weight per day). The data show that significant amounts of endogenous galactose are formed in galactosemic patients with release rates being several fold higher in infants than in adults. The present findings can explain the persistently elevated galactose-1-phosphate levels in erythrocytes-and its age dependence-in galactosemic patients even when under strict dietary treatment.

Galactitol and galactonate in red blood cells of galactosemic patients.

The red blood cell (RBC) concentration of galactitol and galactonate was measured in 27 patients with galactose-1-phosphate uridyltransferase (GALT) deficiency galactosemia and 19 non-galactosemic subjects by a newly devised isotope dilution gas chromatography/mass spectrometry (GC/MS) method. The method utilizing UL[13C]galactitol and UL[13C]galactonate was reproducible with excellent precision and recovery of 99%. The RBC galactitol in galactosemic patients on galactose-restricted diets averaged 5.98+/-1.2 microM (M+/-SD) with a range of 3.54-8.81 microM. The mean in non-galactosemic patients was 0.73+/-0.31 microM with a range of 0.29-1.29 microM. The mean of RBC galactonate in the same galactosemic patients was 4.16+/-1.32 microM (M+/-SD) with a range of 0.68-6.47, while the mean in non-galactosemic subjects was 1.94+/-0.96 (M+/-SD) with a range of 0.69-3.84. In galactosemic RBC the galactitol was higher than galactonate while this was reversed in non-galactosemic cells. RBC galactose-1-phosphate (Gal-1-P) measured at the same time as galactitol and galactonate was 30 times the level of the other two metabolites. There was no relationship between RBC Gal-1-P and galactitol or galactonate. The ability to measure all three galactose metabolites in the same procedure offers the possibility of augmented monitoring of the galactose metabolic status of patients. The measurement of RBC galactitol and galactonate presents a new means of characterizing galactosemic patients and their levels monitored over time may provide new insight in the development of long-term complications observed in afflicted patients.

Physiological significance of 2-buten-4-olide (2-B4O), an endogenous satiety substance increased in the fasted state.

A sugar acid, 2-B4O, has been found to increase from 3.5 to 13 microM in rat serum at 36 h after food deprivation. Injections of 2-B4O (2.5 microM) into the rat III cerebral ventricle (III ICV) suppress food intake and single neuronal activity in the lateral hypothalamic area (LHA). 2-B4O is effective even in 72 h food-deprived rats. 2-B4O hyperpolarizes glucose-sensitive neurons in the LHA via Na+-K+ pump activation, but depolarizes glucoreceptor neurons in the ventromedial nucleus (VMH) via closure of ATP-sensitive K channels. The plasma levels of glucose, corticosterone, and catecholamines, and the firing rate in both parvocellular neurons in the paraventricular nucleus (PVN) and sympathetic efferent nerves, all increase 2-B4O intravenous (iv) injection, indicating activation of the hypothalamo-pituitary-adrenal axis. A 2-B4O iv injection facilitates emotional and spatial learning and memory, and pretreatment with anti-acidic fibroblast growth factor (aFGF) antibody ICV eliminates these effects. aFGF is released from ependymal cells in the III cerebral ventricle in response to the glucose increase in CSF induced by 2-B4O iv injection. 2-B4O also suppresses the clinical symptoms of experimental allergic encephalomyelitis (EAE) in Lewis rats [induced by immunization with a myelin basic protein (MBP)], a model for human multiple sclerosis. After immunization with MBP, the delayed-type hypersensitivity response to MBP is also reduced in 2-B4O-treated rats. 2-B4O thus suppresses autoimmune responses. These results indicate that 2-B4O is not only a powerful satiety substance, but also effective as an activator of the hypothalamo-pituitary-adrenal axis and sympathetic efferent outflow, and as a memory facilitation and a modulator of immune functions.

Diversity of the human erythrocyte membrane sialic acids in relation with blood groups.

The composition of the human erythrocyte membrane (RBC) glycoprotein- and glycolipid-bound sialic acids of A, B, AB and O type donors was studied using a new method (Zanetta et al., Glycobiology 11 (2001) 663-676). In addition to Neu5Ac as the major compound, Kdn, Neu5,9Ac(2), Neu5,7Ac(2), Neu (de-N-acetylated-Neu5Ac), Neu5Ac8Me, Neu5Ac9Lt, Neu4,5Ac(2), Neu5,8Ac(2)9Lt and Neu5Ac8S were characterised. Among these different compounds, Neu5Ac8Me, Neu5Ac9Lt, Neu4,5Ac(2), Neu5,8Ac(2)9Lt and Neu5Ac8S have never been described and quantitatively determined before in human tissues or cells. Neu5Gc and its O-alkylated or O-acylated derivatives were not detected.

Identification of galactitol and galactonate in red blood cells by gas chromatography/mass spectrometry.

BACKGROUND: Because the products of alternate pathways of galactose metabolism, galactitol and galactonate are important in galactosemia, we sought to identify these compounds in red blood cells (RBC). METHODS: RBC extracts were trimethylsilylated (TMS) and analyzed by gas chromatography/mass spectrometry (GC/MS). RESULTS: The presence of both galactitol and galactonate was identified in RBC of 15 galactosemic and 13 normal subjects by their mass spectra and chromatographic comparisons with both unlabeled and 13C labeled standards. The levels in RBC of galactosemics appear to be much higher than those of normal subjects. CONCLUSION: The determination of these compounds in RBC along with galactose-1-phosphate (gal-1-P) in the same procedure provides the potential for their use in better monitoring of diet therapy in galactosemic patients.

13C NMR evidence of the failure of human erythrocytes to metabolize ascorbate and dehydroascorbate to lactate.

13C-NMR spectroscopy was used to record time courses of the metabolism of [1-(13)C]-L-ascorbic acid (AA) and [2-(13)C]-L-ascorbic acid and their dehydro-counterparts (DHAA) by human erythrocytes. Under a range of experimental conditions, but most notably in the absence of glucose in the incubation medium, no (13)C-NMR signal for lactate emerged during any of the 5 h time courses. The NMR resonances that did emerge over time were assigned to diketogulonic (DKG) acid and CO(2). Only very minor resonances from degradation products of DKG appeared from samples that contained physiologically high concentrations of DHAA. These results are in contrast with those in a recent report that lactate is derived from AA in human erythrocytes. However, an explanation for this possible artifact is given.

Specific enzymatic assay for D-glucarate in human serum.

A sensitive and specific spectrophotometric assay was developed to determine levels of D-glucarate in human serum. This assay makes use of the Escherichia coli glucarate catabolic enzymes D-glucarate dehydrase, alpha-keto-beta-deoxy-D-glucarate aldolase, and tartronate semialdehyde (TSA) reductase, to convert D-glucarate to equimolar quantities of pyruvate and TSA. In a one-tube reaction that included NADH, lactate dehydrogenase, and the three E. coli enzymes, 1 mumol of D-glucarate was quantitatively converted to 1 mumol each of D-glycerate and L-lactate with concomitant utilization of 2 mumol of NADH. Using this method, D-glucarate in serum was measured, along with quantitative recovery of authentic D-glucarate from duplicate serum samples to which it had been added. Glucarate is a major serum organic acid, approximating blood pyruvate levels previously determined by others.

SPECT quantitation of cobalt-57 bleomycin delivery to human brain tumors.

A newly developed and validated noninvasive quantitative SPECT method was used to measure the in vivo uptake of [57Co]bleomycin (Co-bleo) in 13 human brain tumors and the uptake of [99mTc]glucoheptonate (GH) in 23 brain tumors. Significant differences in tumor uptake were found. The tumor concentration over time, the tumor to blood radio at 30 min and the tumor cumulative concentration of radioactivity showed marked differences even between tumors with the same histology. Only a weak correlation was found between tumor concentration of Co-bleo and of GH. Therefore a simple imaging agent such as GH cannot, at the present time, serve as an indicator of individual tumor uptake and further experience with other agents is still necessary. Contrary to the generally held view, no correlation was found between the concentration of drug in the blood and its tumor concentration. It is suggested therefore that the level of a drug in the blood cannot be used as a criterion of the amount that will penetrate the tumor. Direct SPECT measurement of the concentration of the drug in the tumor itself should be performed. The bioavailability of a drug is critical in order for it to exert it tumoricidal effect. The results, showing marked differences in uptake between brain tumors, suggest that before chemotherapy is administered, uptake of the chemotherapeutic drug in the individual tumor to be treated should be assessed and comparisons should be made between the uptake of a series of drugs to determine which drug would be most efficacious on the basis of its uptake as well as its tumor cell killing potential.

Short-chain polyhydroxymonocarboxylic acids as physiological signals for food intake.

In order to clarify the effects of endogenous organic acids on short and long-term feeding behavior, ingestive behavior was monitored for 2 hr before and after intra-third ventricular infusions of 3,4-dihydroxybutyric acid (2-deoxytetronic acid, 2-DTA), 2,4,5-trihydroxypentanoic acid (3-deoxypentonic acid, 3-DPA), and 3-hydroxybutyric acid (3-HBA). In addition, meal patterns were recorded for 2 days before and after the ventricular infusions. 2-DTA suppressed both short and long-term feeding by decreasing meal size (MS). 3-DPA elicited transient feeding behavior, but caused no change in long-term feeding. 3-HBA initially stimulated feeding, but subsequently suppressed long-term feeding by decreasing MS and prolonging postprandial intermeal interval (IMI). The suppressive effects of 3-HBA on feeding behavior lasted about 24 hr longer than those of 2-DTA. Based upon these observations as well as our previous reports, it appears that some of the processes affecting hunger and satiation are mediated by changes in central and peripheral concentrations of these organic acids.