Real-World Use of Azathioprine Metabolites Changes Clinical Management of Inflammatory Bowel Disease.

BACKGROUND: Thiopurines such as 6-mercaptopurine and azathioprine have complex metabolism, resulting in significant inter-individual differences in clinical efficacy and risk of drug toxicity, making conventional weight-based dosing inaccurate and potentially unsafe. Therapeutic drug monitoring (TDM) of thiopurine metabolites improves clinical outcomes through dose optimization and toxicity monitoring. Despite evidence for TDM, use is limited, due in part to test availability and awareness. The objectives of this study were twofold: (1) to investigate how thiopurine TDM impacts clinical management of IBD patients and (2) to evaluate proportion of patients outside therapeutic 6TGN levels or exhibiting signs of toxicity. METHODS: Patients who received thiopurine TDM as part of routine care underwent chart review of demographics, disease activity, medication dosing, metabolite levels, and adverse events. Changes in clinical management following TDM were measured. Additionally, we conducted a retrospective review of clinical decision making blinded and unblinded to TDM result. RESULTS: A total of 92 IBD patients were included. Levels of 6TGN were therapeutic in 29% of patients. 6TGN levels correlated weakly with weight-based dosing (r 2 = 0.057, P = 0.02). Adverse reactions were observed in 6.5%. TDM informed clinical management in 64%. Significantly more changes to clinical management occurred in those with active disease than in remission (73% versus 48%; P = 0.02) and in those on mono- versus combination therapy (48% versus 27.5%; P = 0.03). CONCLUSIONS: TDM informs clinical decision making in over two-thirds of patients. The demonstrated poor efficacy of weight-based dosing and impact of TDM on clinical management contributes to the evidence supporting the need for greater availability and uptake of thiopurine TDM.

Different maturities drive proteomic and metabolomic changes in Chinese black truffle.

Chinese black truffle (Tuber indicum) is rich in nutrition. However, commercial interests lead to the aroma components and nutrients of T. indicum being greatly affected by overexploitation without consideration of their maturity. This study investigated the proteomic and metabolomic profiles of truffle fruiting bodies at different maturities using a meta-proteomic approach. Among the 3007 identified proteins, the most up-expressed protein in the mature ascocarps was involved in the peptidyl-diphthamide biosynthetic process, while thiamine metabolism was the most differentially expressed pathway. Furthermore, a total of 54 metabolites identified upon LC-MS differed significantly, with 30 being up-expressed in the mature ascocarps, including organic acids, carnitine substances and polysaccharides. Additionally, the ash, protein, fat, crude fiber and total sugar contents were all higher in the mature ascocarps. Overall, our findings reveal that mature truffles have a higher nutritional value, providing a basis for further exploring protein functionality of T. indicum at different maturities.

LC-MS-Based Metabolomic Approach Revealed the Significantly Different Metabolic Profiles of Five Commercial Truffle Species.

Truffles are ascomycetous ectomycorrhizal fungi that have elevated status in the culinary field due to their unique aroma and taste as well as their nutritional value and potential biological activities. Tuber melanosporum, T. indicum, T. panzhihuanense, T. sinoaestivum, and T. pseudoexcavatum are five commercial truffle species mainly distributed in Europe or China. In this study, an untargeted metabolomics technology based on an ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was applied to analyze the metabolic profiles and variations among these five truffle species. In our results, a total of 2376 metabolites were identified under positive ion mode, of which 1282 had significantly differential amounts and covered 110 pathways or metabolisms. Principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) revealed a clear separation from each of these five truffles, indicating a significantly different metabolic profile among them, with the biggest difference between T. melanosporum and the other four truffles. The differential metabolites covered various chemical categories, and a detailed analysis was performed for nine metabolic categories, including amino acids, saccharides and nucleosides, organic acids, alkaloids, flavonoids, carnitines, phenols and alcohols, esters, and sulfur compounds. For each of the nine categories, most of metabolites predominantly accumulated in T. melanosporum compared with the other four truffles. Meanwhile, there were significant differences of the average ion intensity in each category among the five truffles, e.g., higher amounts of amino acids was detected in T. panzhihuanense and T. pseudoexcavatum; T. indicum contained significantly more carnitines, while there were more alkaloids in T. melanosporum. Additionally, some metabolites with biological activities were discussed for each category, such as acetyl-L-carnitine, adenine, neobavaisoflavone, and anandamide. Generally, this study may provide the valuable information regarding the variation of the metabolic composition of these five commercial truffle species, and the biological significance of these metabolites was uncovered to explore the metabolic mechanisms of truffles, which would be helpful for further research on the compounds and potential biological functions in truffles that have not yet been investigated.

A bifunctional cellulase-xylanase of a new Chryseobacterium strain isolated from the dung of a straw-fed cattle.

A new cellulolytic strain of Chryseobacterium genus was screened from the dung of a cattle fed with cereal straw. A putative cellulase gene (cbGH5) belonging to glycoside hydrolase family 5 subfamily 46 (GH5_46) was identified and cloned by degenerate PCR plus genome walking. The CbGH5 protein was overexpressed in Pichia pastoris, purified and characterized. It is the first bifunctional cellulase-xylanase reported in GH5_46 as well as in Chryseobacterium genus. The enzyme showed an endoglucanase activity on carboxymethylcellulose of 3237 µmol min-1  mg-1 at pH 9, 90 °C and a xylanase activity on birchwood xylan of 1793 µmol min-1  mg-1 at pH 8, 90 °C. The activity level and thermophilicity are in the front rank of all the known cellulases and xylanases. Core hydrophobicity had a positive effect on the thermophilicity of this enzyme. When similar quantity of enzymatic activity units was applied on the straws of wheat, rice, corn and oilseed rape, CbGH5 could obtain 3.5-5.0× glucose and 1.2-1.8× xylose than a mixed commercial cellulase plus xylanase of Novozymes. When applied on spent mushroom substrates made from the four straws, CbGH5 could obtain 9.2-15.7× glucose and 3.5-4.3× xylose than the mixed Novozymes cellulase+xylanase. The results suggest that CbGH5 could be a promising candidate for industrial lignocellulosic biomass conversion.

Utility of urine myoglobin for the prediction of acute renal failure in patients with suspected rhabdomyolysis: a systematic review.

BACKGROUND: Urine myoglobin continues to be used as a marker of rhabdomyolysis, particularly to assess risk of developing acute renal failure and evaluate treatment success. We sought to determine the predictive validity of urine myoglobin (uMb) for acute renal failure (ARF) in patients with suspected rhabdomyolysis. METHODS: We performed a broad systemic review of the literature from January 1980 to December 2006 using the search terms myoglobin$ AND (renal OR ARF OR kidney). Only primary studies published in English where uMb measurement was related to ARF were included. RESULTS: Of 1602 studies screened, 52 met all selection criteria. The studies covered a wide spectrum of etiologies for rhabdomyolysis, dissimilar diagnostic criteria for ARF and rhabdomyolysis, and various methods of uMb measurement and were mostly case series (n = 32). There was poor reporting on the uMb method, and 17 studies failed to provide any information about the method. The reporting of clinical criteria for ARF with respect to timing, description, performance, and interpretation also lacked adequate detail for replication. Eight studies (total 295 patients) had data for 2-by-2 tables. Sensitivity of the uMb test was 100% in 5 of the 8 studies, specificity varied widely (15% to 88%), and CIs around these measures were high. Pooling of data was not possible because of study heterogeneity. CONCLUSIONS: There is inadequate evidence evaluating the use of uMb as a predictor of ARF in patients with suspected rhabdomyolysis.

Flow-induced vascular remodeling in the mesenteric artery of spontaneously hypertensive rats.

The effect of an increased blood flow on vascular remodeling was studied in the mesenteric arteries of 11-12-week-old spontaneously hypertensive rats (SHR) and age-matched normotensive Wistar-Kyoto rats (WKY). Increased blood flow was induced by selective ligation of mesenteric arteries. Nearby arteries with normal blood flow were used as controls. 7-10 days after the ligation procedure, mesenteric arteries were fixed in situ at maximal relaxation by perfusion fixation. Morphometric measurement of vascular dimension was carried out with confocal microscopy. Apoptotic cells were detected by the TdT-mediated dUTP nick-end labelling method. Cell growth was quantified by using proliferating cell nuclear antigen (PCNA) in sections of paraffin-embedded vessels. In SHR, elevated blood flow increased the vessel wall dimension and the number of smooth muscle cell (SMC) layers and also increased the wall-to-lumen ratio and the number of PCNA-positive SMC, but did not change lumen size or number of apoptotic SMC. In WKY, on the other hand, increased blood flow resulted in an increase in lumen diameter, a reduction of apoptotic SMC, but no change in wall-to-lumen ratio, number of SMC layers, or number of PCNA-positive SMC. These results showed that mesenteric arteries from hypertensive and normotensive rats respond to an increase in blood flow differently: a lumen enlargement with reduced SMC apoptosis in WKY, but an increased wall-to-lumen ratio with enhanced SMC growth in SHR. Although it remains to be determined whether flow alteration is one of the initiating factors in the development of vascular remodeling in hypertension, we speculate that an increase in cardiac output, and therefore an increase in shear stress that occurs in young SHR, contributes to vascular remodelling in this model of hypertension.

The effects of quinapril and atorvastatin on artery structure and function in adult spontaneously hypertensive rats.

We studied the combined treatment effects of quinapril and atorvastatin on blood pressure and structure and function of resistance arteries from adult spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY rats). Apoptotic cells were identified by in situ end labeling using the terminal deoxynucleotide transferase-mediated dUTP nick end labeling method. Vascular structure was measured using a morphometric protocol and confocal microscopy and a pressurized artery system was used to study vascular functions. We found that a combined treatment with quinapril and atorvastatin lowered systolic blood pressure in both adult SHR and WKY rats and decreased medial thickness and volume and the number of smooth muscle cell layers in mesenteric arteries, as well as media-to-lumen ratio in the interlobular arteries from SHR but not in those from WKY rats. The number of apoptotic smooth muscle cells was higher in the mesenteric arteries from control WKY rats than control SHR and treatment increased the number of apoptotic smooth muscle cells in the arteries from both SHR and WKY rats. Treatment with quinapril and atorvastatin reduced ventricular weight in SHR and normalized the augmented contractile responses to norepinephrine but did not alter the contraction to electric field stimulation. Relaxation responses to acetylcholine and sodium nitroprusside were not affected by the treatment. We conclude that a combined treatment with quinapril and atorvastatin lowered blood pressure and improved cardiac and vessel hypertrophy and vessel function. An increase in apoptotic smooth muscle cells may be one of the mechanisms underlying the structural improvement.

The effect of heparin on osteoblast differentiation and activity in primary cultures of bovine aortic smooth muscle cells.

Recent studies have suggested that aortic smooth muscle cells undergo a phenotypic transition into osteoblast-like cells and mineralize when cultured in the presence of beta-glycerophosphate. Since we had previously demonstrated that heparin could inhibit osteoblast differentiation and mineralization in primary cultures of murine calvaria cells, we were interested in determining if heparin would have a similar effect when primary aortic smooth muscle cells were cultured in the presence of beta-glycerophosphate. The effect of heparin and low molecular weight heparin (LMWH) on osteoblast differentiation and activity was therefore examined in primary cultures of bovine aortic smooth muscle cells (BASMC) over a 14-day period. Here, we report that BASMC differentiate into osteoblast-like cells when cultured in the presence of beta-glycerophosphate. Moreover, we report that heparin not only inhibits this process but that it also inhibits the ability of BASMC to mineralize as well. Importantly, these effects were found not to be dependent upon heparins' anticoagulant activity since unfractionated heparin and heparins with low anti-thrombin III affinities inhibited the mineralization process equally well. Sulfation, however, was found to be a major determinant of heparins ability to inhibit BASMC mineralization since neither dermatan sulfate nor N-desulfated heparin were able to demonstrate an effect. We conclude that BASMC cultures can undergo a phenotypic transition into mature osteoblasts and that both the differentiation process and their ability to mineralize are inhibited by heparin.

Quinapril effects on resistance artery structure and function in hypertension.

The effects of chronic treatment with quinapril on blood pressure, vascular reactivity and structure of resistance arteries were examined in adult, male spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats. SHR and WKY at 15 weeks of age were treated with quinapril (10 mg/kg per day) for 10 weeks. Structural changes in the mesenteric arteries were measured by optical sectioning with confocal microscopy and in renal arteries by light microscopic measurements. Apoptotic cells in the mesenteric vessel wall were identified using the terminal deoxynucleotide transferase-mediated dUTP-nick end-labelling (TUNEL) method. The response of mesenteric arteries to noradrenaline, electrical stimulation, acetylcholine and sodium nitroprusside was studied using a pressure myograph system. Treatment with quinapril significantly lowered systolic blood pressure and ventricular weight in both SHR and WKY. It reduced wall thickness and medial volume in mesenteric arteries from SHR and WKY and media-to-lumen ratio in interlobular arteries of SHR. It also decreased the number of smooth muscle layers in SHR and increased the number of apoptotic smooth muscle cells in both SHR and WKY. In addition, treatment normalized the augmented contractile responses and improved the impaired relaxation response of SHR mesenteric arteries to the level of WKY. We conclude that treatment with quinapril lowered blood pressure and improved cardiac and vessel structure and vessel function. An increase in apoptotic process of medial smooth muscle cells is one of the mechanisms underlying the vascular structural improvement.

Differential effects of heparin and low molecular weight heparin on osteoblastogenesis and adipogenesis in vitro.

We have previously demonstrated that heparin produces cancellous bone loss in rats due in part to a decrease in the number of osteoblasts lining the trabecular bone surface. In the present study, we use a stromal-derived cell culture system together with measurements of alkaline phosphatase (ALP) activity, to compare the effects of heparin and the low molecular weight heparin (LMWH), Fragmin, on osteoblast differentiation in vitro. In addition, we examined the possibility that both heparin and LMWH can induce adipogenesis in our stromal cell culture system. Both heparin and LMWH were found to produce a statistically significant (P < 0.01) and concentration-dependent decrease in the number of osteoblasts while increasing the number of adipocytes. When the effects of gravimetrically equivalent amounts of heparin and LMWH were compared, heparin had a 4-fold greater effect than LMWH. In contrast to heparin, N-desulfated heparin was found to have minimal effects on both osteoblast and adipocyte differentiation indicating that the heparin effect is not only chain-length dependent but also charge-dependent. The observation that LMWH has less of an effect on bone formation than heparin is compatible with the results of clinical trials indicating that LMWH produces less bone loss after long-term administration.