Intermediate products of purine metabolism in an experimental model of pancreatic necrosis.

BACKGROUND AND AIM: Determine the level of purines in the blood plasma of experimental animals at three stages of induced pancreatic necrosis. Find out the potential of purines as predictors of the severity of pancreatitis. METHODS: The experiment was carried out on white outbred rabbits. The pancreatic necrosis was modeled by introducing self-bile into the pancreatic parenchyma. The pancreas of rabbits, after isolation, was subjected to microscopic description. Blood was also taken from rabbits to determine the plasma levels of adenine, guanine, hypoxanthine, xanthine, and uric acid. RESULTS: 12 hours after the administration of self-bile, the level of xanthine significantly increases and the concentration of uric acid in the blood plasma increases by 3 times. 24 hours after the introduction of self-bile, there is a slight decrease in the level of adenine, xanthine and uric acid, and the indicators of purine metabolism remain elevated. 48 hours after the introduction of self-bile, the levels of guanine, hypoxanthine and xanthine are reduced. CONCLUSIONS: The concentration indices of absolute and relative intermediate products of purine metabolism were increased at the initial stage of pancreatic necrosis. The activity of enzymes and metabolites of purine metabolism involved in the formation of reactive oxygen species and free radicals increased. The hypothesis that intermediate products of purine metabolism can be predictors of pancreatic necrosis was confirmed.

The effect of Haemonchus contortus and Trichostrongylus colubriforms infection on the ruminal microbiome of lambs.

We evaluated Haemonchus contortus (HC) and Trichostrongylus colubriformis (TC) infection on the ruminal microbial community of Santa Ines lambs to better understand the pathophysiology of parasite infections and the interactions among gastrointestinal nematodes and gut resident microbiota. In this study, 18 six months of age lambs were maintained for 34 days in individual pens divided into three treatments that included animals infected with HC and TC, and control (infection-free). Haematological, ruminal parameter and microbial nitrogen absorbed by pune derivatives, as well as enteric methane emission (CH4), were analysed, and the rumen microbial taxonomic and functional profile assessed by shotgun metagenomics. The analysis showed that total protein, albumin, urea, and butyrate level were lower in animals infected by both parasites, while HC infection also decreased the haemoglobin level. Both infected groups (TC and HC) increased the enteric methane emission (CH4). TC and HC infections increased the diversity and richness of functional microbial genes. Most alterations in the rumen microbiome composition of infected groups are associated with the suppression of microbes involved in microbial homeostasis maintenance and expansion of the archaeal community in the infected animals. Infection led to an increased abundance of nitrogen, amino acid, protein, and energy metabolism genes. Overall, TC and HC infection increased the enteric methane emission, negatively affected taxon's responsible for maintenance de rumen homeostasis and modulated some important genes related to protein and energy metabolism.

Diagnostic Utility of Serum and Urinary Metabolite Analysis in Patients with Interstitial Cystitis/Painful Bladder Syndrome.

OBJECTIVE: To identify the potential biomarkers of interstitial cystitis/painful bladder syndrome (IC), a chronic syndrome of bladder-centric pain with unknown etiology that has an adverse impact on quality of life, we analyzed the urine and serum metabolomes of a cohort of IC patients and non-disease controls (NC). METHODS: Home collection of serum and urine samples was obtained from 19 IC and 20 NC females in the Veterans Affairs (VA) Health Care System. IC was diagnosed independently by thorough review of medical records using established criteria. Biostatistics and bioinformatics analyses, including univariate analysis, unsupervised clustering, random forest analysis, and metabolite set enrichment analysis (MSEA), were then utilized to identify potential IC biomarkers. RESULTS: Metabolomics profiling revealed distinct expression patterns between NC and IC. Random forest analysis of urine samples suggested discriminators specific to IC; these include phenylalanine, purine, 5-oxoproline, and 5-hydroxyindoleacetic acid. When these urinary metabolomics-based analytes were combined into a single model, the AUC was 0.92, suggesting strong potential clinical value as a diagnostic signature. Serum-based metabolomics did not provide potential IC discriminators. CONCLUSION: Analysis of serum and urine revealed that women with IC have distinct metabolomes, highlighting key metabolic pathways that may provide insight into the pathophysiology of IC. The findings from this pilot study suggest that integrated analyses of urinary metabolites, purine, phenylalanine, 5-oxoproline, and 5-HIAA, can lead to promising IC biomarkers for pathophysiology of IC. Validation of these results using a larger dataset is currently underway.

Soybean oil supplementation and starter protein content: Effects on growth performance, digestibility, ruminal fermentation, and urinary purine derivatives of Holstein dairy calves.

This study investigated the effects of feeding dairy calves starter diets containing 19% or 22% crude protein (CP) content on a dry matter basis and either supplemented or not with soybean oil (SBO, 0 vs. 3%, dry matter basis) on growth performance, digestibility, urinary nitrogen, and purine derivatives (PD) excretion. A total of 48 female Holstein dairy calves (mean 39.8 kg of body weight) were randomly distributed to experimental diets in a 2 × 2 factorial arrangement of treatments. The 4 dietary treatments were (1) starter diet without SBO supplement and 19% CP (NSBO-19CP), (2) starter diet without SBO supplement and 22% CP (NSBO-22CP), (3) starter diet with 3% SBO and 19% CP (SBO-19CP), and (4) starter diet with 3% SBO and 22% CP (SBO-22CP). Milk feeding value was similarly based on a constant protocol across experimental treatments and calves had ad libitum access to water and starter diets throughout the study. All calves were weaned on d 63 of age and remained in the study until d 83 of age. Calves supplemented with SBO had lower starter feed intake and average daily gain (ADG) and lower feed efficiency (FE) but had a higher fecal score indicating a higher likelihood of diarrhea occurrence compared with unsupplemented calves. Wither heights, digestibilities of organic matter, CP, and neutral detergent fiber were decreased, and ruminal volatile fatty acids tended to be reduced, and the molar proportion of ruminal butyrate (preweaning) and acetate (postweaning) reduced by supplemental SBO. The urinary allantoin and total PD excretion were reduced; however, urinary nitrogen excretion was increased when calves were supplemented with SBO. The CP amount did not affect starter feed intake, FE, or diarrhea occurrence rate, whereas the 22CP diets increased neutral detergent fiber digestibility, improved ADG (tendency), and increased allantoin and urinary PD excretion compared with the 19CP diets. The starter feed intake, ADG, FE, diarrhea occurrence rate, nutrient digestibility, and ruminal fermentation were not affected by the interaction between starter SBO and CP level; however, hip height and total PD in calves that received the SBO-22CP diets were higher than those fed the SBO-19CP diets. In conclusion, based on our experimental conditions, supplemental SBO could not be recommended for dairy calves. Furthermore, our findings indicate that SBO has negative effects on performance more attributed to reducing starter intake, digestibility, and ruminal volatile fatty acid concentration rather than because of a limitation of starter metabolizable protein supply and intestinal amino acid availability. Therefore, our results indicate that feeding the higher starter CP content is not a viable strategy to compensate for the negative effects of SBO supplementation on the growth performance of dairy calves.

Supplementation of nicotinic acid to prepartum Holstein cows increases colostral immunoglobulin G, excretion of urinary purine derivatives, and feed efficiency in calves.

We evaluated the effects of incremental levels of unprotected nicotinic acid (NA) supplementation prepartum (0, 16, 32, or 48 g/d; CON, 16NA, 32NA, and 48NA, respectively) on colostrum yield and composition and cow and calf performance. Previous research indicated that 48 g/d of NA prepartum increased colostrum IgG concentration. Exact mechanisms for this increase are not clear. The effects of NA supplementation to prepartum cows on growth and performance of their calves have not been studied. Thirty-six multiparous Holstein cows housed in a tie-stall barn were blocked by expected calving date and randomly assigned to treatments at 4 wk prepartum. Blood samples were collected 3 times weekly for analysis of nonesterified fatty acids, ketones, and IgG. Urine samples were also collected 3 times weekly for analysis of creatinine and purine derivatives. Colostrum was collected within 90 min after parturition. Calves were removed from their dams before suckling, weighed within 30 min of birth, and received 4 L of maternal colostrum. The 38 calves born were blocked based on treatments of dams. All calves were fed 449 g dry matter (DM) of milk replacer (19.3% crude protein, 19.5% fat, DM basis) and a textured starter (41% starch, DM basis) at 2 d of age until weaning at 42 d, with water available ad libitum. Feeding NA resulted in linear decreases in DM intake in cows, but colostrum yield was not affected. Yield of metabolizable energy (ME) tended to change cubically, decreasing from control (CON) to 16NA, increasing from 16NA to 32NA, and decreasing from 32NA to 48NA. Concentration of IgG, protein, ash, and solids increased linearly with NA. Concentration of ME showed a tendency to increase quadratically with NA. Yield of IgG, fat, protein, and solids content increased quadratically with NA, while allantoin and total purine derivatives increased linearly. Calf 24-h IgG and apparent efficiency of absorption were not affected by NA. Calf ME intake from colostrum tended to increase quadratically with NA, but calf starter intake was not affected. Feed efficiency of calves increased quadratically with NA. Calf average daily gain changed cubically with NA, decreasing from CON to 16NA, increasing from 16NA to 32NA, and decreasing from 32NA to 48NA. Hip width gain, body length gain, and final body length changed cubically with NA, decreasing from CON to 16NA, increasing from 16NA to 32NA, and decreasing from 32NA to 48NA. Calf blood concentrations of ketones increased quadratically with NA. These data suggest that increasing levels of NA can be fed prepartum to increase colostral components and 32 g/d NA can improve calf performance.

Metabolic Tools for Identification of New Mutations of Enzymes Engaged in Purine Synthesis Leading to Neurological Impairment.

The cellular pool of purines is maintained by de novo purine synthesis (DNPS), recycling and degradation. Mutations in genes encoding DNPS enzymes cause their substrates to accumulate, which has detrimental effects on cellular division and organism development, potentially leading to neurological impairments. Unspecified neurological symptoms observed in many patients could not be elucidated even by modern techniques. It is presumable that some of these problems are induced by dysfunctions in DNPS enzymes. Therefore, we determined the concentrations of dephosphorylated DNPS intermediates by LC-MS/MS as markers of yet unpublished mutations in PFAS and PAICS genes connected with dysfunctions of carboxylase/phosphoribosylaminoimidazolesuccinocarboxamide synthase (PAICS) or phosphoribosylformylglycinamidine synthase (PFAS). We determined the criteria for normal values of metabolites and investigated 1,447 samples of urine and 365 dried blood spots of patients suffering from various forms of neurological impairment. We detected slightly elevated aminoimidazole riboside (AIr) concentrations in three urine samples and a highly elevated 5-formamidoimidazole-4-carboxamide riboside (FGAr) concentration in one urine sample. The accumulation of AIr or FGAr in body fluids can indicate PAICS or PFAS deficiency, respectively, which would be new disorders of DNPS caused by mutations in the appropriate genes. Measurement of DNPS intermediates in patients with neurological symptoms can uncover the cause of serious cellular and functional impairments that are otherwise inaccessible to detection. Further genetic and molecular analysis of these patients should establish the causal mutations for prenatal diagnosis, genetic consultation, and reinforce the DNPS pathway as a therapeutic target.

Effects of sodium selenite addition on ruminal fermentation, microflora and urinary excretion of purine derivatives in Holstein dairy bulls.

Researches on sodium selenite (SS) mainly focus on production performance and rumen fermentation in ruminants, and the influence of dietary Se addition on ruminal microbial population and enzyme activity in dairy bulls is scarce. This study mainly evaluated the effects of SS on ruminal fermentation, microflora and urinary excretion of purine derivatives (PD) in dairy bulls. Eight ruminally cannulated dairy bulls were used in a replicated 4 × 4 Latin square design. Treatments were control, low SS (LSS), medium SS (MSS) and high SS (HSS) with 0, 0.1, 0.3 and 0.5 mg/kg of selenium (Se) from SS in dietary dry matter (DM), respectively. The supplement of SS (1.0 g/kg of Se) was mixed into the first third of the daily ration. Bulls were fed a total mixed ration with corn silage to concentrate ratio of 50:50 on a DM basis. Dry matter intake was not affected, average daily gain linearly increased, while feed conversion ratio quadratically decreased with increasing Se addition. The linearly increased digestibility of DM, organic matter, crude protein, ether extract, neutral detergent fibre and acid detergent fibre was observed. Both ruminal pH and ammonia-N concentration linearly decreased, whereas total volatile fatty acid concentration linearly increased. A lower acetate to propionate ratio was observed due to the unchanged acetate proportion and increased propionate proportion. Activity of cellobiase, xylanase, pectinase, α-amylase and protease, populations of total bacteria, fungi, protozoa, Ruminococcus (R.) albus, R. flavefaciens, Fibrobacter succinogenes, Butyrivibrio fibrisolvens and Ruminobacter amylophilus as well as urinary total PD excretion linearly increased, whereas populations of total methanogens and Prevotella ruminicola linearly decreased. The data indicated that dietary Se addition stimulated ruminal microbial growth and enzyme activity, and resulting in the increased nutrient digestion and growth performance, and the optimum supplementary dose of Se was 0.3 mg/kg dietary DM from SS in dairy bulls.

Simultaneous quantification of urinary purines and creatinine by ultra high performance liquid chromatography with ultraviolet spectroscopy and quadrupole time-of-flight mass spectrometry: Method development, validation, and application to gout study.

We present an ultra high performance liquid chromatography with ultraviolet spectroscopy and quadrupole time-of-flight mass spectrometry method for the simultaneous quantification of ten purines (adenine, hypoxanthine, guanine, xanthine, deoxyadenosine, adenosine, inosine, guanosine, xanthosine, and uric acid) and creatinine in human urine. After chromatographic separation on an ACE Excel 2 AQ column, high abundant creatinine and uric acid and the other low abundant purines were sequentially detected by ultraviolet and quadrupole time-of-flight mass spectrometry within a single run. Method validations including specificity (improved by accurate mass measurement), linearity (correlation coefficients ≥0.9944), limit of quantification (0.002-9.756 µg/mL), intra- and interday precision (relative standard deviations ≤9.1 and 14.0%, respectively), accuracy (relative errors ≤13.1%), extraction recovery (between 90.3 and 109.6%), matrix effect (between 85.3 and 110.5%), and stability (relative errors ≤14.3%) were fully evaluated. This approach was applied to characterize the disordered purine metabolism in acute and chronic gout as an example. Quantitative results (normalized by creatinine) showed that an overproduction of urinary purine precursors might be involved in the gout process. The developed method represents a useful tool to investigate the purine disturbances in gout and other relevant diseases.

Metabolic disorder of amino acids, fatty acids and purines reflects the decreases in oocyte quality and potential in sows.

Oocyte quality is closely related to female fertility. Nevertheless, core nutritional metabolites influencing oocyte quality are unclear. Herein, comprehensive metabolomics analysis of follicular fluid, serum, and urine from low reproductive performance (LRP) and normal reproductive performance (NRP) sows was conducted. Twenty-seven, fourteen and sixteen metabolites (involved in metabolism of amino acids, fatty acids, purine and pyrimidine) were altered in follicular fluid, serum and urine, respectively, in LRP compared with NRP sows, and could decrease oocyte quality and developmental potential, ultimately leading to low fertility. Deoxyinosine, guanidine acetate, thymidine, 5,6-epoxy-eicosatrienoic acid, carnosine, docosahexaenoic acid and carbamoyl phosphate in follicular fluid, cysteine, carnitine, serotonin, hypoxanthine, valine and arginine in serum, as well as carnitine, phenyl glycine, N-acetyl glutamine, propionyl carnitine and choline in urine could be selected as diagnostic markers to indicate oocyte quality. Consistent with metabolomics data, we confirmed changes in concentrations of fatty acids and amino acids in follicular fluid. Targeting purine metabolism, elevating levels of deoxyinosine in in-vitro maturation medium of porcine oocyte significantly promoted the blastocyst rate. Collectively, this study provided new information of potential targets for predicting oocyte quality and developmental potential, and may help with strategies for early diagnosis or therapeutic/dietary intervention in improving reproductive outcomes.

Effect of dietary energy on digestibilities, rumen fermentation, urinary purine derivatives and serum metabolites in Tibetan and small-tailed Han sheep.

Tibetan sheep are indigenous to the Qinghai-Tibetan Plateau, graze the grassland all year round without supplementation and are well-adapted to the harsh conditions. Small-tailed Han sheep were introduced to the plateau and are raised mainly in feedlots. Based on their different backgrounds, we hypothesized that the ability to cope with poor diets would be better in Tibetan than in Han sheep. To test our prediction, we examined the effect of dietary energy on apparent digestibilities, rumen fermentation, urinary purine derivatives and serum metabolites by using a 4 × 4 Latin square design in each sheep breed. Four diets were formulated to be low in crude protein (~7%) but to differ in metabolizable energy concentration. Average daily gain was greater in Tibetan than in Han sheep (p < 0.01) and increased linearly with an increase in energy intake (p < 0.001). The digestibilities of dry matter, organic matter, gross energy, and neutral and acid detergent fibres were greater in Tibetan than in Han sheep (p < 0.05). Ruminal pH was lower (p < 0.05), while volatile fatty acids (VFAs), urea-N, ammonia-N and soluble protein-N concentrations were higher (p < 0.05) in Tibetan than in Han sheep. As a molar proportion of total VFA, acetate decreased (p < 0.001) with an increase in dietary energy whereas propionate and butyrate increased (p < 0.05). Urinary purine derivative excretion was greater in Tibetan than in Han sheep (p < 0.01), as was microbial nitrogen production; both parameters increased with dietary energy (p < 0.01). Serum concentrations of glucose, insulin and insulin-like growth factor-1 increased (p < 0.05) as energy level increased, while non-esterified fatty acids and growth hormone decreased (p < 0.05). It was concluded that Tibetan sheep were better able to cope with low-protein, low-energy diets and, consequently, our prediction was supported.

Effects of folic acid on growth performance, ruminal fermentation, nutrient digestibility and urinary excretion of purine derivatives in post-weaned dairy calves.

This study evaluated the effects of folic acid (FA) supplementation on growth performance, ruminal fermentation, nutrient digestibility and urinary purine derivatives (PD) excretion in dairy calves. Forty-eight Chinese Holstein male dairy calves at 60 ± 3.2 d of age and 89 ± 5.9 kg body weight (mean ± standard error) were assigned to one of four groups in a randomised block design. Calves in control group were fed basal diet, calves in low FA, medium FA and high FA groups with 3.6, 7.2 and 10.8 mg FA per kg basal diet, respectively. The dietary corn silage to concentrate ratio was 50:50 (dry matter [DM] basis). DM intake and average daily gain (ADG) quadratically increased, and feed conversion ratio quadratically decreased with increasing FA supplementation. Ruminal pH linearly decreased, whereas total volatile fatty acids quadratically increased. The unchanged acetate-to-propionate ratio was due to the similar change in acetate and propionate concentration. Ammonia N content quadratically decreased. Digestibility of DM, organic matter, crude protein, ether extract, neutral detergent fibre and acid detergent fibre linearly increased. Activities of carboxymethyl cellulase, cellobiase, xylanase and pectinase linearly increased, but α-amylase and protease quadratically increased. Abundance of Ruminococcus albus, Ruminococcus flavefaciens and Fibrobacter succinogenes linearly increased, but Butyrivibrio fibrisolvens and Prevotella ruminicola quadratically increased. Urinary total PD excretion quadratically increased. The results indicated that FA supplementation increased ADG, ruminal fermentation and nutrient digestibility with promoted ruminal microbial growth and enzyme activity, and the optimum dose was 7.2 mg FA per kg basal diet for calves.

Total replacement of corn silage with sorghum silage improves milk fatty acid profile and antioxidant capacity of Holstein dairy cows.

Total mixed rations containing corn silage (CS) or forage sorghum silage (SS) were fed to mid-lactation Holstein cows to determine the effects on feed intake, lactation performance, milk composition and fatty acid profile, nutrient digestibility, blood metabolites, rumen microbial N synthesis, and antioxidant status. The experiment was designed as a 2-period change-over (two 28-d periods) trial with 2 diets including CS diet or SS diet and 12 cows. Total replacement of CS with SS had no significant influence on dry matter intake. Substituting CS with SS had no effect on milk production, feed efficiency, and milk concentrations of fat, protein, lactose, and solids-not-fat, whereas yields of milk fat, protein, and lactose were greater for cows fed the CS diet. Blood parameters including glucose, albumin, cholesterol, triglyceride, total protein, urea N, and fatty acids were not affected by the dietary treatments. Apparent digestibility coefficients of dry matter, organic matter, crude protein, ether extract, neutral detergent fiber, and acid detergent fiber were not significantly influenced by the diets. Replacing CS with SS had no effect on total saturated fatty acids and total monounsaturated fatty acids, whereas total polyunsaturated fatty acid percentage was greater with the SS diet. Proportions of C20:0, C18:3n-3, and C18:3n-6 were affected by feeding SS. Cows fed CS had a greater amount of urinary purine derivatives. Feeding SS had a positive effect on total antioxidant capacity of blood and milk. In conclusion, SS can be fed to lactating Holstein cows as a total replacement for CS without undesirable effects on animal performance, but with positive effects on antioxidant capacity and polyunsaturated fatty acids of milk. This forage can be an excellent choice for dairy farms in areas where cultivation of corn is difficult due to water shortage.

Identification of Novel Pathways in Idelalisib Metabolism and Bioactivation.

Idelalisib (ILB) is a selective phosphatidylinositol-3-kinase delta inhibitor approved for the treatment of hematological malignancies. However, ILB frequently causes hepatotoxicity, and the exact mechanism remains unclear. The current study profiled the metabolites of ILB in mouse liver, urine, and feces. The major metabolites found in the liver were oxidized metabolite GS-563117 (M1) and ILB-glutathione (GSH) adduct (M2). These metabolic pathways were confirmed by analysis of urine and feces from mice treated with ILB. Identification of ILB-GSH adduct (M2) suggests the formation of reactive metabolites of ILB. We also found that M1 can produce reactive metabolites and form M1-GSH adducts. The GSH-conjugates identified in mouse liver were also found in the incubations of ILB and M1 with human liver microsomes. Furthermore, we illustrated that CYP3A4 and 2C9 are the key enzymes contributing to the bioactivation pathway of ILB and M1. In summary, our work revealed that both ILB and its major metabolite M1 can undergo bioactivation to produce reactive metabolites in the liver. Further studies are required to determine whether these metabolic pathways contribute to ILB hepatotoxicity.

Bright carbon dots via inner filter effect for the sensitive determination of the purine metabolic disorder in human fluids.

In this paper, the high performance fluorescent carbon dots were synthesized with maleic acid, tris and benzoic acid as raw materials by one-step hydrothermal method. The obtained carbon dots with uniform size emitted strong blue fluorescence, which the maximum excitation and emission wavelengths at 250 nm and 415 nm, respectively. Under the optimum condition, it was meaningfully founded that the reaction between the carbon dots and uric acid resulting in the fluorescence quenching of the carbon dots at the emission spectrum of 415 nm. The reason was that they had a synergistic effect between the fluorescence internal filtering effect and the static quenching effect. The fluorescence internal filter effect sensing system was constructed by using uric acid as the absorbable material and carbon dots as the luminophore. Hence, a fluorescence quenching method for the determination of uric acid was established in the concentration range from 5.0 to 400 µM with the detection limit (3σ/S) of 2.26 µM. Thus, a fluorescent sensing assay for the determination of uric acid was founded and confirmed in human fluids.

Effects of concentrate-to-forage ratios and 2-methylbutyrate supplementation on ruminal fermentation, bacteria abundance and urinary excretion of purine derivatives in Chinese Simmental steers.

This study evaluated the effects of dietary concentrate levels and 2-methylbutyrate (2MB) supplementation on performance, ruminal fermentation, bacteria abundance, microbial enzyme activity and urinary excretion of purine derivatives (PD) in steers. Eight ruminally cannulated Simmental steers (12 months of age; 389 ± 3.7 kg of body weight) were used in a replicated 4 × 4 Latin square design with a 2 × 2 factorial arrangement. Moderate-concentrate (400 g/kg diet [MC]) or high-concentrate (600 g/kg diet [HC]) diets were fed with or without 2MB (0 g/day [2MB-] or 15.0 g/day [2MB+]). Dry matter intake and average daily gain increased, but feed conversion ratio decreased with the HC diet or 2MB supplementation. Ruminal pH decreased, but total volatile fatty acid increased with the HC diet or 2MB supplementation. Molar proportion of acetate and acetate-to-propionate ratio decreased with the HC diet, but increased with 2MB supplementation. Propionate molar proportion and ruminal NH3 -N content increased with the HC diet, but decreased with 2MB supplementation. Neutral detergent fibre degradability decreased with the HC diet, but increased with 2MB supplementation. Crude protein degradability increased with the HC diet or 2MB supplementation. Abundance of Ruminococcus albus, Ruminococcus flavefaciens, Fibrobacter succinogenes and Bufyrivibrio fibrisolvens as well as activities of carboxymethyl cellulase, cellobiase, xylanase and pectinase decreased with the HC diet, but increased with 2MB supplementation. However, abundance of Prevotella ruminicola and Ruminobacter amylophilus as well as activities of α-amylase and protease increased with the HC diet or 2MB supplementation. Total PD excretion also increased with the HC diet or 2MB supplementation. The results suggested that growth performance, ruminal fermentation, CP degradability and total PD excretion increased with increasing dietary concentrate level from 40% to 60% or 2MB supplementation. The observed diet × 2MB interaction indicated that supplementation of 2MB was more efficacious for improving growth performance, ruminal fermentation and total PD excretion with promoted ruminal bacteria abundance and enzyme activity in the MC diet than in the HC diet.

Effect of oak (Quercus persica) acorn level on apparent digestibility, ruminal fermentation, nitrogen balance and urinary purine derivatives in pregnant goats.

The aim of this experiment was to investigate the effect of dietary oak (Quercus persica) acorn (OA) level on dry matter intake (DMI), apparent nutrient digestibility, nitrogen (N) utilization, ruminal fermentation, protozoa population and urinary purine derivatives (PD) during the last 60 days of goat pregnancy. Twenty-four multiparous pregnant goats (41.7 ± 2.3 kg BW) were assigned to one of three experimental diets consisted of control diet (C, without OA) and diets containing 20 (OA20 ) or 40 g/100 g of OA (OA40 ) on a DM basis in a completely randomized block design. Goats fed OA40 had lower DMI (p < .01), DM (p < .01), OM (p < .01) and NDF (p < .05) digestibility, ruminal NH3 -N concentration (p < .01), N intake (p < .01) and N retention (p < .01). Crude protein digestibility and ruminal acetate and total volatile fatty acid (VFA) concentration were lower in animals fed OA-contained diets (p < .01), whereas ruminal propionate concentration was higher in goats fed the C diet (p < .01). Animals fed OA40 had higher faecal N excretion and lower urinary N excretion (p < .01). Urinary PD was lower in goats fed diets containing OA in relation to those fed the C diet (p < .01). Total protozoa population decreased linearly with increasing OA level in the diet (p < .05). These results suggest that feeding OA, especially high level, has negative impacts on DMI, nutrient digestibility, VFA concentration, N retention and urinary PD excretion that may have adverse effects on metabolism and performance of pregnant goats.

Simultaneous quantification of three alkylated­purine adducts in human urine using sulfonic acid poly(glycidyl methacrylate­divinylbenzene)-based microspheres as sorbent combined with LC-MS/MS.

Three alkylated DNA adducts, N3­methyladenine, N3­ethyladenine and N7­ethylguanine, have been proved to be potential biomarkers for DNA injury caused by exposure to cigarette smoke. In this study, a highly specific and sensitive method using a new mixed-mode sulfonate-functionalized poly(glycidyl methacrylate-divinylbenzene) as a solid-phase extraction sorbent was developed for the analysis of these three alkylated-purine adducts in human urine. Under optimized conditions, the prepared sorbent interacts strongly with these urinary adducts, demonstrating high clean-up efficiency and extraction recovery. The method detection limits (S/N ≥ 3) of N3-MeA, N3-EtA and N7-EtG were 1.75, 0.20, and 0.15 pg mL-1, respectively, while the method quantitation limits were found to be 5.78, 0.66, and 0.49 pg mL-1 for N3-MeA, N3-EtA and N7-EtG, respectively. The intra-day and inter-day precisions were investigated, of which were in the range of 1.6-3.8% and 3.2-5.6%, respectively. The recovery values of the alkylated DNA adducts in spiked urine sample were ranged 89.7-104.5%. Their concentrations were statistically significantly higher in smokers than in nonsmokers. These results show that the proposed method is suitable for the analysis of alkylated DNA adducts.

Effects of dietary protein levels and 2-methylbutyrate on ruminal fermentation, nutrient degradability, bacterial populations and urinary purine derivatives in Simmental steers.

The objective of this study was to evaluate the effects of dietary crude protein (CP) levels and 2-methylbutyrate (MB) supplementation on ruminal fermentation, bacterial populations, microbial enzyme activity and urinary excretion of purine derivatives (PD) in Simmental steers. Eight ruminally cannulated Simmental steers, averaging 18 months of age and 465 ± 8.6 kg of body weight (BW), were used in a replicated 4 × 4 Latin square design by a 2 × 2 factorial arrangement. Low protein (98.5 g CP/kg dry matter [LP] or high protein (128.7 g CP/kg dry matter [HP]) diets were fed with MB supplementation (0 g [MB-] or 16.8 g steer-1  day-1 [MB+]). Steers were fed a total mixed ration with dietary corn straw to concentrate ratio of 50:50 (dry matter [DM] basis). The CP × MB interaction was observed for ruminal total VFA, molar proportions of acetate and propionate, acetate to propionate ratio, ammonia-N, effective degradability of neutral detergent fibre (NDF) and CP, microbial enzyme activity, bacterial populations and total PD excretion (p < .05). Ruminal pH decreased (p < .05), but ruminal total VFA concentration increased (p < .05) with increasing dietary CP level or MB supplementation. Acetate molar proportion increased (p = .043) with MB supplementation, but was not affected by dietary CP level. Propionate molar proportion decreased (p < .05) with increasing dietary CP level or MB supplementation. Consequently, acetate-to-propionate ratio increased (p = .001) with MB supplementation, but was not affected by dietary CP level. Ruminal ammonia-N content increased (p = .034) with increasing dietary CP level, but decreased (p = .012) with MB supplementation. The effective degradability of NDF and CP increased (p < .05) with increasing dietary CP level or MB supplementation. Microbial enzyme activity, bacterial populations and total PD excretion also increased (p < .05) with increasing dietary CP level or MB supplementation. The results indicated that ruminal fermentation, nutrient degradability, microbial enzyme activity, ruminal bacterial populations and microbial protein synthesis improved with increasing dietary CP level or MB supplementation in steers.

Urinary purine derivatives as a tool to estimate dry matter intake in cattle: A meta-analysis.

The objectives of this study were to investigate the relationship between dry matter intake (DMI) and urinary purine derivative (PD) excretion, to develop equations to predict DMI and to determine the endogenous excretion of PD for beef and dairy cattle using a meta-analytical approach. To develop the models, 62 published studies for both dairy (45 studies) and beef cattle (17 studies) were compiled. Twenty models were tested using DMI (kg/d) and digestible DMI (dDMI, kg/d) as response variables and PD:creatinine (linear term: PD:C, and quadratic term: PD:C2), allantoin:creatinine (linear term: ALLA:C, and quadratic term: ALLA:C2), metabolic body weight (BW0.75, kg), milk yield (MY, kg/d), and their combination as explanatory variables for dairy and beef (except for MY) cattle. The models developed to predict DMI for dairy cattle were validated using an independent data set from 2 research trials carried out at the University of Wisconsin (trial 1: n = 45; trial 2: n = 50). A second set of models was developed to estimate the endogenous PD excretion. In all evaluated models, the effect of PD (either as PD:C or ALLA:C) was significant, supporting our hypothesis that PD are in fact correlated with DMI. Despite the BW-independent relationship between PD and DMI, the inclusion of BW0.75 in the models with PD:C and ALLA:C as predictors slightly decreased the values of root mean square error (RMSE) and Akaike information criterion for the models of DMI. Our models suggest that both DMI and dDMI can be equally well predicted by PD-related variables; however, predicting DMI seems more useful from a practical and experimental standpoint. The inclusion of MY into the dairy models substantially decreased RMSE and Akaike information criterion values, and further increased the precision of the equations. The model including PD:C, BW0.75, and MY presented greater concordance correlation coefficient (0.93 and 0.63 for trials 1 and 2, respectively) and lower RMSE of prediction (1.90 and 3.35 kg/d for trials 1 and 2, respectively) when tested in the validation data set, emerging as a potentially useful estimator of nutrient intake in dairy cows. Endogenous PD excretion was estimated by the intercept of the linear regression between DMI (g/kg of BW0.75) and PD excretion (mmol/kg of BW0.75) for beef (0.404 mmol/kg of BW0.75) and dairy cattle (0.651 mmol/kg of BW0.75). Based on the very close agreement between our results for beef cattle and the literature, the linear regression appears to be an adequate method to estimate endogenous PD excretion.

Technical note: Evaluation of urinary purine derivatives in comparison with duodenal purines for estimating rumen microbial protein supply in sheep.

A data set of individual observations was compiled from digestibility trials to examine the relationship between the duodenal purine bases (PB) flow and urinary purine derivatives (PD) excretion and the validity of different equations for estimating rumen microbial N (Nm) supply based on urinary PD in comparison with estimates based on duodenal PB. Trials (8 trials, = 185) were conducted with male sheep fitted with a duodenal T-type cannula, housed in metabolic cages, and fed forage alone or with supplements. The amount of PD excreted in urine was linearly related to the amount of PB flowing to the duodenum ( < 0.05). The intercept of the linear regression was 0.180 mmol/(d·kg), representing the endogenous excretion of PD, and the slope was lower than 1 ( < 0.05), indicating that only 0.43% of the PB in the duodenum was excreted as PD in urine. The Nm supply estimated by either approach was linearly related ( < 0.05) to the digestible OM intake. However, the Nm supply estimated through either of 3 published PD-based equations probably underestimated the Nm supply in sheep.