Development of quantitative assay for simultaneous measurement of purine metabolites and creatinine in biobanked urine by liquid chromatography-tandem mass spectrometry.

Purine metabolism is essential for all known living creatures, including humans in whom elevated serum concentration of purine break-down product uric acid (UA) is probably an independent risk factor for mortality, type 2 diabetes and cardiovascular events. An automated multiplex assay that measures several purine metabolites could therefore prove useful in many areas of medical, veterinary and biological research. The aim of the present work was to develop a sensitive LC-MS/MS method for simultaneous quantitation of xanthine, hypoxanthine, UA, allantoin, and creatinine in biobanked urine samples. This article describes details and performance of the new method studied in 55 samples of human urine. Archival sample preparation and effect of storage conditions on stability of the analytes are addressed. The intra-day and inter-day coefficients of variation were small for all the analytes, not exceeding 1% and 10%, respectively. Measurements of UA and creatinine in biobanked urine showed good agreement with values obtained using routine enzymatic assays on fresh urine. Spearman's correlation coefficients were 0.869 (p < .001) for creatinine and 0.964 (p < .001) for UA. Conclusion: the newly developed LC-MS/MS method allows reliable quantitative assessment of xanthine, hypoxanthine, allantoin, UA and creatinine. The proposed pre-analytical processing makes the method suitable for both fresh and biobanked urine stored frozen at -80 °C for at least 5.5 years.

1H-NMR-based urinary metabolomic analysis for the preventive effects of gushudan on glucocorticoid-induced osteoporosis rats.

Gushudan (GSD), a traditional Chinese medicine with a history of more than 15 years, has been shown to have anti-osteoporosis effects, but the specific therapeutic mechanism behind it is still unclear. To further elucidate the pathogenesis of osteoporosis and the preventive mechanism of GSD on glucocorticoid-induced osteoporosis (GIOP) rats, a rapid and comprehensive 1H NMR metabolomics method was established to detect urinary metabolic profiles in the control group, model group and GSD treatment group in this study. The orthogonal partial least squares discriminant analysis (OPLS-DA) was performed to investigate changes in the metabolites, and related metabolic pathways were discovered using MetaboAnalyst platform. As a result, a total of 27 differential metabolites were identified. Of these, 17 metabolites such as formate, allantoin and l-threonate were newly discovered as GIOP potential biomarkers. Energy metabolism, intestinal flora metabolism, amino acid metabolism and oxidative stress response were significantly changed in the urinary profiles of GIOP rats, and GSD could play an anti-osteoporosis role by regulating these metabolic pathways. This study compliments the earlier LC-MS based urine metabolomics research, and helps further understand the pathogenesis of osteoporosis and the potential preventive effects of GSD on GIOP rats.

Simultaneous determination of allantoin and adenosine in human urine using liquid chromatography - UV detection.

We report a HPLC-UV method for the quantitative determination of allantoin and adenosine in human urine, validated according to the acceptance criteria of both the USA Food and Drug Administration (FDA) guideline for bioanalytical method validation and the European Medicines Agency (EMA) validation guidelines. Both allantoins and adenosine are compounds of the purine catabolic pathway. Adenosine is situated at the top as a uric acid (UA) precursor, while allantoin is the best-known degradation product of UA. These two compounds are endogenously present in human urine. Chromatographic separation was achieved with a gradient elution at 0.6 mL/min using a Zorbax SB-Aq column coupled to a Zorbax SB-Aq guard column. Three different mobile phases were used: mobile phase A consisted of 10 mM KH2PO4 (pH 4.7) in milli-Q water, mobile phase B was 12.5 mM KH2PO4 (pH 4.7) - ACN (80:20) and mobile phase C consisted of ACN - H2O (50:50). The linear response range in human urine was 14-800 µM for allantoin and 1.25-50 µM for adenosine. The recoveries of allantoin, adenosine and the internal standard were greater than 93.8%. The intra-day accuracy ranged between 99.5 and 104.9% for allantoin and between 96.6 and 107.3% for adenosine, while the inter-day accuracy ranged respectively from 91.2 to 103.0% and from 94.5 to 107.8%. The intra-day precision range was from 0.8 to 6.2% RSD for allantoin and from 0.6 to 15.0% for adenosine. The inter-day precision ranged from 2.1-17.5% for allantoin and from 2.9-17.9% for adenosine. This method was successfully applied to analyze both compounds in urine samples of healthy volunteers. In conclusion, an accurate, precise and stable HPLC-UV method was developed and validated to quantify the endogenously present compounds allantoin and adenosine in human urine samples.

Longitudinal urinary metabolomic profiling reveals metabolites for asthma development in early childhood.

BACKGROUND: Several metabolites and altered metabolic pathways have been reported to be associated with asthma. However, longitudinal analysis of the dynamics of metabolites contributing to the development of asthma has not yet been fully clarified. METHODS: We sought to identify the metabolic mechanisms underlying asthma development in early childhood. Thirty children with asthma and paired healthy controls from a prospective birth cohort were enrolled. Time series analysis of urinary metabolites collected at ages 1, 2, 3, and 4 years was assessed using 1 H nuclear magnetic resonance (NMR) spectroscopy coupled with partial least squares discriminant analysis (PLS-DA). Metabolites identified were studied in relation to changes over time in a linear mixed model for repeated measures. RESULTS: A total of 172 urine samples collected from the enrolled children were analyzed. Urinary metabolomics identified four metabolites significantly associated with childhood asthma development, with longitudinal analysis. Among them, dimethylamine, a metabolite produced by intestinal bacteria, appeared to shift from higher to lower level during asthma development. A persistent lower level of 1-methylnicotinamide and allantoin was found in children with asthma, with a peak difference at age 3 years (P = .032 and P = .021, respectively). Furthermore, a significant inverse correlation was found between allantoin and house dust mite sensitization (Spearman's r = -.297 P = .035). CONCLUSIONS: Longitudinal urinary metabolomic profiling provides a link of microbe-environment interactions in the development of childhood asthma. 1-Methylnicotinamide and allantoin may participate in allergic reactions in response to allergen exposure, potentially serving as specific biomarkers for asthma.

Assessing Exposome Effects on Pregnancy through Urine Metabolomics of a Portuguese (Estarreja) Cohort.

This nuclear magnetic resonance metabolomics study compared the influence of two different central Portugal exposomes, one of which comprised an important source of pollutants (the Estarreja Chemical Complex, ECC), on the urinary metabolic trajectory of a cohort of healthy pregnant women (total n = 107). An exposome-independent description of pregnancy metabolism was found to comprise a set of 18 metabolites reflecting expected changes in branched-chain amino acid catabolism and hormone and lipid metabolisms. In addition, a set of small changes in some metabolites was suggested to be exposome-dependent and characteristic of pregnant subjects from the Estarreja region. These results suggested that the Estarreja exposome may impact to a very low extent pregnancy metabolism, inducing slight changes in amino acid metabolism (alanine, glycine, and 3-hydroxyisobutyrate, possibly involved in valine metabolism), tricarboxylic acid (TCA) cycle (cis-aconitate), diet, or gut microflora (furoylglycine) as well as allantoin, 2-hydroxyisobutyrate, and an unassigned resonance at δ 8.45. Furthermore, the urine of Estarreja subjects was found to generally contain higher levels of 4-hydroxyphenylacetate and lower levels of citrate. However, out of the above metabolites, only glycine and citrate seemed to correlate with the proximity to the ECC, with slightly relative higher levels of these compounds found for subjects living closer to the ECC. This suggested possible small effects of local pollutants on energy metabolism, with the remaining exposome-dependent metabolite changes most probably originating from other aspects of the local exposome such as diet and lifestyle. Despite the limitation of this study regarding the unavailability of objective environmental parameters for the period under study, our results confirm the usefulness of metabolomics of human urine to gauge exposome effects on human health and, particularly, during pregnancy.

Urine metabolomics shows an induction of fatty acids metabolism in healthy adult volunteers after supplementation with green coffee (Coffea robusta L.) bean extract.

BACKGROUND AND OBJECTIVE: Green coffee bean extract is used as herbal medicine or supplement for weight reduction and obesity. The active constituents are considered caffeine and chlorogenic acid (CGA) derivatives. The mode of action of CGA is still unclear and can be related to peroxisome proliferator-activated receptor α (PPAR-α) and liver X receptor Rα (LXR-α). Metabolomics may be an innovative tool for the description and discovery of the multiple target nature of such phytocomplex. METHODS: 24 h urine samples were collected once a week from ten healthy adult volunteers consuming daily 400 mg of dry Green coffee bean extract (GCBE, 4.9% of chlorogenic acid) each day for 30 days (5 harvesting days, considering also the first day of supplementation). Urine samples were analyzed by LC-QTOF using both untargeted and targeted approaches. The latter was used to monitor two urinary markers of oxidative stress (allantoin, 8-OHdG). RESULTS: Metabolomics analysis (PLS-DA) revealed changes in urine composition before and during the treatment with GCBE. Markers related to treatment were metabolites related to polyphenol administration as hippuric acid, benzoic acid derivatives, dihydroferulic and dihydrosinapic acid sulphate, but also carnitine derivatives and dicarboxylic acids. On the other hand, no changes in the levels of allantoin and 8-OHdG were observed. CONCLUSION: This preliminary study showed the possible usefulness of metabolomics approach in the evaluation of GCBE consumption in healthy subjects. The observed changes in urinary composition can be related to the catabolism of GCBE constituents and to induced fatty acid metabolism, mainly related to carnitine derivatives. This latter result could be considered, at least in part, as a further proof of the mode of action of green coffee extract.

Identification of Urinary Food Intake Biomarkers for Milk, Cheese, and Soy-Based Drink by Untargeted GC-MS and NMR in Healthy Humans.

The measurement of food intake biomarkers (FIBs) in biofluids represents an objective tool for dietary assessment. FIBs of milk and cheese still need more investigation due to the absence of candidate markers. Thus, an acute intervention study has been performed to sensitively and specifically identify candidate FIBs. Eleven healthy male and female volunteers participated in the randomized, controlled crossover study that tested a single intake of milk and cheese as test products, and soy-based drink as a control. Urine samples were collected at baseline and up to 24 h at distinct time intervals (0-1, 1-2, 2-4, 4-6, 6-12, and 12-24 h) and were analyzed using an untargeted multiplatform approach (GC-MS and 1H NMR). Lactose, galactose, and galactonate were identified exclusively after milk intake while for other metabolites (allantoin, hippurate, galactitol, and galactono-1,5-lactone) a significant increase has been observed. Urinary 3-phenyllactic acid was the only compound specifically reflecting cheese intake although alanine, proline, and pyroglutamic acid were found at significantly higher levels after cheese consumption. In addition, several novel candidate markers for soy drink were identified, such as pinitol and trigonelline. Together, these candidate FIBs of dairy intake could serve as a basis for future validation studies under free-living conditions.

Chemiluminescence of Lucigenin-Allantoin and Its Application for the Detection of Allantoin.

Allantoin has been reported as a promising biomarker for monitoring of oxidative stress in humans and widely utilized in a variety of topical pharmaceuticals and cosmetics. Currently, the detection of allantoin is achieved by using chromatographic coupled techniques, which needs sample pre-extraction, derivatization, complex matrixes, and expensive instrumentation. Herein we report both the intense chemiluminescence of allantoin with lucigenin and the chemiluminescent detection of allantoin for the first time. The lucigenin-allantoin system demonstrated chemiluminescence emission intensity 17 times higher than that of the classic lucigenin-hydrogen peroxide system. Based on this fascinating phenomenon, a novel chemiluminescence method has been developed for the sensitive and selective allantoin determination with the combination of flow injection analysis. This method shows a linear calibration curve in the range 0.1-3000 µM with a detection limit (3σ/s) of 0.03 µM. Moreover, it was successfully utilized for the determination of allantoin in human eye drop and real urine samples after simple dilution with water. It shows excellent recoveries in the range 94.0-101.7%, and each measurement takes a very short time. This method exhibits potential advantages in the form of simplicity, rapidity, sensitivity, selectivity, and low cost. Allantoin could be an effective candidate for constructing new chemiluminescence systems, and it may provide a broad range of sensing applications.

Urinary excretion of uric acid, allantoin, and 8-OH-Deoxyguanosine in uricase-knockout mice.

Although uricase-knockout (Uox KO) mice are reported to develop uric acid (UA) nephropathy, those that mature without severe nephropathy could be useful for research into purine metabolism in humans. In this study, we measured the urinary excretion of creatinine, UA, allantoin, and 8-hydroxy-2'-deoxyguanosine (8-OHdG) collected from Uox KO mice housed in metabolic cages. UA and allantoin were determined using liquid chromatography-mass spectrometry and creatinine and 8-OHdG were measured with a commercial kit. Uox KO mice excreted significantly higher levels of UA than wild-type mice (C57BL/6), while the excretion of allantoin was significantly lower. Urinary allantoin was detected in Uox KO mice despite a lack of uricase, which is the same as in humans. In contrast to the elevated levels of UA, the daily excretion of 8-OHdG, an oxidative stress marker, was lower in Uox KO mice. UA is thought to act as an anti-oxidizing agent in humans; thus, these results show that Uox KO mice are potential animal models for research into human purine metabolism.

Potential mechanisms for low uric acid in Parkinson disease.

Several epidemiologic studies have described an association between low serum uric acid (UA) and Parkinson disease (PD). Uric acid is a known antioxidant, and one proposed mechanism of neurodegeneration in PD is oxidative damage of dopamine neurons. However, other complex metabolic pathways may contribute. The purpose of this study is to elucidate potential mechanisms of low serum UA in PD. Subjects who met diagnostic criteria for definite or probable PD (n = 20) and controls (n = 20) aged 55-80 years were recruited. Twenty-four hour urine samples were collected from all participants, and both uric acid and allantoin were measured and corrected for body mass index (BMI). Urinary metabolites were compared using a twoway ANOVA with diagnosis and sex as the explanatory variables. There were no significant differences between PD and controls for total UA (p = 0.60), UA corrected for BMI (p = 0.37), or in the interaction of diagnosis and sex on UA (p = 0.24). Similarly, there were no significant differences between PD and controls for allantoin (p = 0.47), allantoin corrected for BMI (p = 0.57), or in the interaction of diagnosis and sex on allantoin (p = 0.78). Allantoin/UA ratios also did not significantly differ by diagnosis (p = 0.99). Our results imply that low serum UA in PD may be due to an intrinsic mechanism that alters the homeostatic set point for serum UA in PD, and may contribute to relatively lower protection against oxidative damage. These findings provide indirect support for neuroprotection trials aimed at raising serum UA.

Urinary Allantoin Is Elevated in Severe Intraventricular Hemorrhage in the Preterm Newborn.

Germinal matrix intraventricular hemorrhage (IVH) is the most common type of intracranial hemorrhage observed in preterm neonates. It is a precursor of poor neurocognitive development, cerebral palsy, and death. The pathophysiology is not well defined, but damage to the fragile germinal matrix vasculature may be due to free radicals generated during inflammation and as a consequence of ischemia followed by reperfusion. Assessment of the oxidative stress status in these infants is therefore important. Urinary allantoin concentration was measured in preterm neonates as a marker of oxidative stress associated with IVH. Urine was collected from 44 preterm neonates at four time points between 24 and 72 hours of life (HOL), and the allantoin content was determined by gas chromatography mass spectrometry (GCMS). Records were retrospectively reviewed, and the incidence and severity of IVH was categorized as follows: no IVH (n = 24), mild (grade 1-2) IVH (n = 13), and severe (grade 3-4) IVH (n = 7). Neonates with severe IVH showed significantly elevated allantoin levels vs subjects with no IVH from 36 HOL (0.098 ± 0.013 µmol and 0.043 ± 0.007 µmol, respectively, p = 0.002). The allantoin concentration remained elevated even at 72 HOL (0.079 ± 0.014 µmol and 0.033 ± 0.008 µmol, respectively, p = 0.021). There were no significant differences in allantoin levels in the no IVH and mild IVH groups. IVH was diagnosed by head imaging on average at about 11th postnatal day. Urinary allantoin levels were significantly elevated during the first 3 days of life in the neonates subsequently diagnosed with severe IVH, suggesting that oxidative stress might be a crucial factor in IVH pathogenesis. Further studies are needed to assess the usefulness of urinary allantoin in early identification of preterm infants at risk for or with severe IVH and monitoring of the response to interventions designed to prevent or treat it.

New findings on the in vivo antioxidant activity of Curcuma longa extract by an integrated (1)H NMR and HPLC-MS metabolomic approach.

Curcuminoids possess powerful antioxidant activity as demonstrated in many chemical in vitro tests and in several in vivo trials. Nevertheless, the mechanism of this activity is not completely elucidated and studies on the in vivo antioxidant effects are still needed. Metabolomics may be used as an attractive approach for such studies and in this paper, we describe the effects of oral administration of a Curcuma longa L. extract (150 mg/kg of total curcuminoids) to 12 healthy rats with particular attention to urinary markers of oxidative stress. The experiment was carried out over 33 days and changes in the 24-h urine samples metabolome were evaluated by (1)H NMR and HPLC-MS. Both techniques produced similar representations for the collected samples confirming our previous study. Modifications of the urinary metabolome lead to the observation of different variables proving the complementarity of (1)H NMR and HPLC-MS for metabolomic purposes. The urinary levels of allantoin, m-tyrosine, 8-hydroxy-2'-deoxyguanosine, and nitrotyrosine were decreased in the treated group thus supporting an in vivo antioxidant effect of the oral administration of Curcuma extract to healthy rats. On the other hand, urinary TMAO levels were higher in the treated compared to the control group suggesting a role of curcumin supplementation on microbiota or on TMAO urinary excretion. Furthermore, the urinary levels of the sulphur containing compounds taurine and cystine were also changed suggesting a role for such constituents in the biochemical pathways involved in Curcuma extract bioactivity and indicating the need for further investigation on the complex role of antioxidant curcumin effects.

Metabonomic analysis of potential biomarkers and drug targets involved in diabetic nephropathy mice.

Diabetic nephropathy (DN) is one of the lethal manifestations of diabetic systemic microvascular disease. Elucidation of characteristic metabolic alterations during diabetic progression is critical to understand its pathogenesis and identify potential biomarkers and drug targets involved in the disease. In this study, (1)H nuclear magnetic resonance ((1)H NMR)-based metabonomics with correlative analysis was performed to study the characteristic metabolites, as well as the related pathways in urine and kidney samples of db/db diabetic mice, compared with age-matched wildtype mice. The time trajectory plot of db/db mice revealed alterations, in an age-dependent manner, in urinary metabolic profiles along with progression of renal damage and dysfunction. Age-dependent and correlated metabolite analysis identified that cis-aconitate and allantoin could serve as biomarkers for the diagnosis of DN. Further correlative analysis revealed that the enzymes dimethylarginine dimethylaminohydrolase (DDAH), guanosine triphosphate cyclohydrolase I (GTPCH I), and 3-hydroxy-3-methylglutaryl-CoA lyase (HMG-CoA lyase) were involved in dimethylamine metabolism, ketogenesis and GTP metabolism pathways, respectively, and could be potential therapeutic targets for DN. Our results highlight that metabonomic analysis can be used as a tool to identify potential biomarkers and novel therapeutic targets to gain a better understanding of the mechanisms underlying the initiation and progression of diseases.

Metabonomics revealed xanthine oxidase-induced oxidative stress and inflammation in the pathogenesis of diabetic nephropathy.

Diabetic nephropathy (DN) is a serious complication of diabetes mellitus (DM), which is a major public health problem in the world. To reveal the metabolic changes associated with DN, we analyzed the serum, urine, and renal extracts obtained from control and streptozotocin (STZ)-induced DN rats by (1)H NMR-based metabonomics and multivariate data analysis. A significant difference between control and DN rats was revealed in metabolic profiles, and we identified several important DN-related metabolites including increased levels of allantoin and uric acid (UA) in the DN rats, suggesting that disturbed purine metabolism may be involved in the DN. Combined with conventional histological and biological methods, we further demonstrated that xanthine oxidase (XO), a key enzyme for purine catabolism, was abnormally activated in the kidney of diabetic rats by hyperglycemia. The highly activated XO increased the level of intracellular ROS, which caused renal injury by direct oxidative damage to renal cells, and indirect inducing inflammatory responses via activating NF-κB signaling pathway. Our study highlighted that metabonomics is a promising tool to reveal the metabolic changes and the underlying mechanism involved in the pathogenesis of DN.

Changes in urinary metabolic profile after oral administration of curcuma extract in rats.

The diffusion of phytochemicals in health promoting products is growing, but studies related to their effects on healthy subjects are still lacking despite the large consumption of natural products as nutraceuticals or food supplements. In many cases, research supports the in vitro antioxidant activity of phytochemicals, but the health claims attributed to the final marketed nutraceutical products have dubious scientific foundation. Also, studies focussed on the definition of their biological targets and mechanisms of action can be useful to assess their efficacy and safety. In this study, the effect of oral administration of 80mg/kg of Curcuma longa Linn. extract to 12 healthy rats over 25 days was evaluated by monitoring the changes of urinary composition. 24-h urine was collected during the animal experiment and the composition was analyzed by (1)H NMR and HPLC-MS. The two datasets were studied individually through a metabolomic approach and the multivariate analysis revealed significant differences between the control and the treated group. Curcumin levels were also measured in 24-h urine samples by HPLC-MS. Both the (1)H NMR and the HPLC-MS dataset showed that the administration of 80mg/kg of Curcuma longa extract to healthy animals induces changes in urinary composition. Decreased allantoin urinary levels can be considered a partial demonstration of the in vivo effect of curcumin on oxidative stress in a healthy animal model.

GC/TOFMS analysis of metabolites in serum and urine reveals metabolic perturbation of TCA cycle in db/db mice involved in diabetic nephropathy.

Early diagnosis of diabetic nephropathy (DN) is difficult although it is of crucial importance to prevent its development. To probe potential markers and the underlying mechanism of DN, an animal model of DN, the db/db mice, was used and serum and urine metabolites were profiled using gas chromatography/time-of-flight mass spectrometry. Metabolic patterns were evaluated based on serum and urine data. Principal component analysis of the data revealed an obvious metabonomic difference between db/db mice and controls, and db/db mice showed distinctly different metabolic patterns during the progression from diabetes to early, medium, and later DN. The identified metabolites discriminating between db/db mice and controls suggested that db/db mice have perturbations in the tricarboxylic acid cycle (TCA, citrate, malate, succinate, and aconitate), lipid metabolism, glycolysis, and amino acid turnover. The db/db mice were characterized by acidic urine, high TCA intermediates in serum at week 6 and a sharp decline thereafter, and gradual elevation of free fatty acids in the serum. The sharp drop of serum TCA intermediates from week 6 to 8 indicated the downregulated glycolysis and insulin resistance. However, urinary TCA intermediates did not decrease in parallel with those in the serum from week 6 to 10, and an increased portion of TCA intermediates in the serum was excreted into the urine at 8, 10, and 12 wk than at 6 wk, indicating kidney dysfunction occurred. The relative abundances of TCA intermediates in urine relative to those in serum were suggested as an index of renal damage.

2,3,4,5-Tetrakis(3',4'-dihydroxylphenyl)thiophene: a new matrix for the selective analysis of low molecular weight amines and direct determination of creatinine in urine by MALDI-TOF MS.

Small organic matrixes are still the most commonly used ones in matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) because of their advantages of high sensitivity, convenience, and cost-effectiveness. However, due to the matrix interference in the low mass region, the direct analysis of low molecular weight amines in complex surroundings with conventional organic matrixes remains a challenge. Here, a new Brønsted-Lowry acid compound 2,3,4,5-tetrakis(3',4'-dihydroxylphenyl)thiophene (DHPT) was designed, synthesized, and applied as a matrix for analysis of low molecular weight amines by MALDI-TOF MS. DHPT displays good selectivity in the analysis of amines without matrix-related interference and the low picomole/femtomole limit-of-detection was obtained in positive ion mode. With DHPT, the metabolites including creatinine, glycine, alloxan, allantoin, and 3-hydroxyhippuric acid in human urine were directly analyzed by MALDI-TOF MS. The identity of these metabolites was confirmed by tandem mass spectrometry. Furthermore, the urine creatinine was quantitatively determined using isotope-labeled internal standard. This DHPT-assisted LDI MS method provides a general approach for both qualitative and quantitative analysis of low molecular weight amines.

Systemic oxidative stress, as measured by urinary allantoin and F(2)-isoprostanes, is not increased in Down syndrome.

PURPOSE: Oxidative stress has been implicated in Down syndrome (DS) pathology. This study compares DS individuals and controls on their urinary levels of allantoin and 2,3-dinor-iPF2α-III; these biomarkers have been previously validated in a clinical model of oxidative stress. METHODS: Urine samples were collected from 48 individuals with DS and 130 controls. Biomarkers were assayed by ultraperformance liquid chromatography-tandem mass spectrometry, normalized by urinary creatinine concentration. RESULTS: After adjusting for age and gender, mean allantoin levels were lower among DS individuals versus controls (P = .04). The adjusted mean levels of 2,3-dinor-iPF2α-III were similar in DS individuals and controls (P = .7). CONCLUSIONS: Our results do not support the hypothesis that DS individuals have chronic systemic oxidative stress.

Effect of high-dose nano-selenium and selenium-yeast on feed digestibility, rumen fermentation, and purine derivatives in sheep.

The aim of this study was to evaluate the effect of nano-selenium (NS) and yeast-selenium (YS) supplementation on feed digestibility, rumen fermentation, and urinary purine derivatives in sheep. Six male ruminally cannulated sheep, average 43.32 ± 4.8 kg of BW, were used in a replicated 3 × 3 Latin square experiment. The treatments were control (without NS and YS), NS with 4 g nano-Se (provide 4 mg Se), and YS with 4 g Se-yeast (provide 4 mg Se) per kilogram of diet dry matter (DM), respectively. Experimental periods were 25 days with 15 days of adaptation and 10 days of sampling. Ruminal pH, ammonia N concentration, molar proportion of propionate, and ratio of acetate to propionate were decreased (P < 0.01), and total ruminal VFA concentration was increased with NS and YS supplementation (P < 0.01). In situ ruminal neutral detergent fiber (aNDF) degradation of Leymus chinensis (P < 0.01) and crude protein (CP) of soybean meal (P < 0.01) were significantly improved by Se supplementation. Digestibilities of DM, organic matter, crude protein, ether extract, aNDF, and ADF in the total tract and urinary excretion of purine derivatives were also affected by feeding Se supplementation diets (P < 0.01). Ruminal fermentation was improved by feeding NS, and feed conversion efficiency was also increased compared with YS (P < 0.01). We concluded that nano-Se can be used as a preferentially available selenium source in ruminant nutrition.