Phenylmethanesulfonyl fluoride pretreatment stabilizes plasma lipidome in lipidomic and metabolomic analysis

Though it is standard practice to test the stability of analytes in the matrix for routine bioanalytical method, stability evaluation is always impractical and skipped in untargeted lipidomic and metabolomic analysis because analytes in these studies are enormous, diverse and sometimes unknown. Lipidome represents a major class of plasma metabolome and shows great potential to be diagnostic and prognostic biomarkers. However, lipidome also faces stability problems because plasma contains kinds of lipid degradation enzyme. Here, using liquid chromatography time of flight mass spectrometry based lipidomic methodology, plasma levels of various lipids including triglyceride (TG), diglyceride (DG), free fatty acid (FFA), phosphatidylethanolamine (PE) phosphatidylcholine (PC), lyso-phosphatidylcholine (LPC), lyso-phosphatidylethanolamine (LPE), and sphingomyelin (SM) were dynamically determined within 4 h at ambient temperature. In mouse and rat plasma, the levels of most TG, DG, PC and PE species significantly decreased with respect to time, whereas those of LPC, LPE and FFA significantly increased with respect to time. However, such changes did not occur in human plasma, thus indicating hepatic lipase and esterase might involve in the species-specified degradation of lipid classes in plasma. Phenylmethanesulfonyl fluoride (PMSF) pretreatment prevented such lipidome instability in mouse plasma. The results suggested the instability of plasma lipidome should be highly concerned, and the enhancement of ex vivo stability of plasma lipidome could enable more reliable clinical translation of lipidomic data for biomarker discovery.     

Prospective trial of a navigator setting under left hepatic lobe on magnetic resonance cholangiopancreatography using a free-breathing prospective acquisition correction technique

PURPOSE: To prospectively compare the image acquisition time and image quality obtained by navigator setting under the left hepatic lobe vs. on the right diaphragm on magnetic resonance cholangiopancreatography (MRCP) using a free-breathing navigator-triggered prospective acquisition correction technique (PACE). MATERIALS AND METHODS: Fifty consecutive patients prospectively underwent three-dimensional T2-weighted turbo spin-echo MRCP using PACE with the navigator randomly set either under the left hepatic lobe or on top of the right diaphragm. Image acquisition time and subjective image quality were compared on a five-point scale using Student's t-test and Mann-Whitney's U test, respectively. RESULTS: There was no significant difference for mean acquisition time (6.1+/-1.6 vs. 6.3+/-1.2 min, P=.689) between the left hepatic lobe group and right diaphragm group. Mean subjective image quality was significantly worse in the left hepatic lobe group than in the right diaphragm group (4.1 vs. 4.7, P=.044). CONCLUSION: Setting the navigator under the left hepatic lobe for MRCP using PACE causes the data processing to be more difficult. As well, under current circumstances, it does not contribute to reducing acquisition time or improving the image quality.     

基于点击化学反应的半乳糖糖基化马蹄金素衍生物的合成及抗HBV活性

以D-半乳糖和二缩三乙二醇为原料,经乙酰化、糖基化和叠氮化钠取代等反应合成了带叠氮连接臂的半乳糖配基,通过点击化学反应将其与炔丙基修饰的马蹄金素(MTS)衍生物进行连接,设计合成了6个具有潜在肝靶向性的半乳糖糖基化MTS衍生物.通过1H NMR,13C NMR,1H-1H COSY,HMQC,DEPT和ESI-MS对其结构进行了表征;采用Hep G2 2.2.15细胞模型初步评价了目标化合物的抗乙型肝炎病毒(HBV)活性.结果表明,所有目标化合物对HBV DNA的复制均有抑制作用,且具有一定的量效关系;化合物15f在50μg/m L浓度下对Hep G2 2.2.15细胞株的抑制率为83%,具有进一步研究的价值.     

In silico characterization of the interaction between LSKL peptide,a LAP-TGF-beta derived peptide,and ADAMTS1

Metalloproteases involved in extracellular matrix remodeling play a pivotal role in cell response by regulating the bioavailability of cytokines and growth factors. Recently, the disintegrin and metalloprotease, ADAMTS1 has been demonstrated to be able to activate the transforming growth factor TGF-β, a major factor in fibrosis and cancer. The KTFR sequence from ADAMTS1 is responsible for the interaction with the LSKL peptide from the latent form of TGF-β, leading to its activation. While the atomic details of the interaction site can be the basis of the rational design of efficient inhibitory molecules, the binding mode of interaction is totally unknown.In this study, we show that recombinant fragments of human ADAMTS1 containing KTFR sequence keep the ability to bind the latent form of TGF-β. The recombinant fragment with the best affinity is modeled to investigate the binding mode of LSKL peptide with ADAMTS1 at the atomic level. Using a combined approach with molecular docking and multiple independent molecular dynamics (MD) simulations, we provide the binding mode of LSKL peptide with ADAMTS1. The MD simulations starting with the two lowest energy model predicted by molecular docking shows stable interactions characterized by 3 salt bridges (K₃–NH₃⁺ with E₆₂₆–COO⁻; L₄–COO⁻ with K₆₁₉–NH₃⁺; L₁–NH₃⁺ with E₆₂₄–COO⁻) and 2 hydrogen bonds (S₂–OH with E₆₂₃–COO⁻; L₄–NH with E₆₂₃–COO⁻). The knowledge of this interaction mechanism paves the way to the design of more potent and more specific inhibitors against the inappropriate activation of TGF-β by ADAMTS1 in liver diseases.     

含乳糖聚电解质靶向微胶囊的层层自组装构筑

设计合成了己二酸乳糖乙烯酯/对苯乙烯磺酸钠共聚物（PLESS）,通过层层自组装技术构筑聚烯丙基胺盐酸盐（PAH）与含乳糖聚电解质PLESS的靶向微胶囊。以紫外-可见吸收光谱监测了PAH与PLESS在平面的石英片进行层层自组装过程,研究了不同实验条件（聚电解质浓度、溶液盐度、盐种类）对PAH/PLESS多层膜自组装的影响;PAH与PLESS在球形碳酸钙微球模板上层层自组装,去除模板后得到层状结构的微胶囊,用透射电镜（TEM）等方法观察其形态形貌;通过花生凝集素识别考察其潜在靶向性;通过细胞MTT活性试验评价其生物相容性。     

Optimal region-of-interest MRI R2* measurements for the assessment of hepatic iron content in thalassaemia major

Objectives

To evaluate the performance of region-of-interest (ROI)-based MRI R2* measurements by using the first-moment noise-corrected model (M¹NCM) to correct the non-central Chi noise in magnitude images from phased arrays for hepatic iron content (HIC) assessment.

Methods

R2* values were quantified using the M¹NCM model. Three approaches were employed to determine the representative R2*: fitting of the ROI-averaged signal (average-then-fit, ATF); outputting the median and mean of R2*s from the pixel-wise fitting of decay signals within the ROI (denoted as PWFmed and PWFmea, respectively). The accuracy and precision of the three approaches were evaluated on synthesized data. The agreement among these approaches and their intra- and inter-observer reproducibility were assessed on 105 thalassaemia major patients.

Results

Simulations showed that ATF consistently yielded the highest accuracy and precision at varying noise levels. By contrast, PWFmed and PWFmea slightly and significantly overestimated high R2* at poor signal-to-noise ratios, respectively. Patient study showed that ATF agreed well with PWFmed, whereas PWFmea produced high R2* measurements for patients with severe HIC. No significant difference was observed in the reproducibility of the three approaches.

Conclusions

PWFmea tends to overestimate high R2*, whereas ATF and PWFmed can produce more accurate R2* measurements for HIC assessment.     

Hepatic lipid composition analysis using 3.0-T MR spectroscopy in a steatotic rat model

Purpose

To investigate the feasibility of in vivo assessment of hepatic lipid composition using 3.0-T proton magnetic resonance spectroscopy (¹H-MRS) in a steatotic rat model and compare it to histopathological and biochemical assessment.

Materials and Methods

Hepatic steatosis was induced by feeding rats with a methionine/choline-deficient (MCD) diet for 1, 2, 3, 5 or 7 weeks (n=5 per group). At the end of the diet period, ¹H-MRS of the liver was performed, and rats were sacrificed for histopathological and biochemical assessment of the liver. Spectra were acquired in a single voxel (1.2 cc) using a point-resolved spectroscopic sequence with TE/TR=35/2000 ms and 64 signal acquisitions. From the MR spectra, peak area ratios were calculated to estimate hepatic lipid composition.

Results

During MCD diet periods, hepatic steatosis significantly increased on histopathology (P<.001). The ¹H-MRS measurements of total hepatic fat content [1.3/(1.3+4.65) ppm] correlated strongly with histological macrovesicular hepatic steatosis (r=0.93, P<.001) and with the biochemical total hepatic fatty acids (r=0.94, P<.001). Total unsaturated fatty acids [TUFA, 5.4/(1.3+4.65) ppm] estimated with ¹H-MRS strongly correlated with the biochemical unsaturated fatty acids (r=0.90, P<.001). Polyunsaturated fatty acids [PUFA, 2.8/(1.3+4.65) ppm] estimated with ¹H-MRS strongly correlated with biochemical PUFA (r=0.91, P<.001). The proportion of total unsaturated fatty acids relative to the amount of total fatty acids (rTUFA, 5.4/1.3ppm) measured with ¹H-MRS strongly correlated with the biochemical amount of unsaturated relative to total hepatic fatty acids (r=0.81, P<.001). The proportion of PUFA relative to the amount of total fatty acids (rPUFA, 2.8/1.3 ppm) measured with ¹H-MRS correlated with the biochemical amount of PUFA relative to total fatty acids (r=0.59, P=0.005,) and with the biochemical amount of omega-6 PUFA relative to total fatty acids (r=0.73, P<.001).PUFA at ¹H-MRS correlated with the histopathologically assessed degree of lobular inflammation in the liver (r=0.57, P=.001).

Conclusion

3.0T ¹H-MRS is able to measure poly- and unsaturated hepatic fatty acids and this strongly correlates with biochemical assessment. This study provides evidence that 3.0-T ¹H-MRS is a noninvasive technique to assess hepatic lipid composition.     

Determination of metabolic profile of anti-malarial trioxane CDRI 99/411 in rat liver microsomes using HPLC

CDRI 99/411 is a potent 1,2,4‐trioxane anti‐malarial candidate compound of the Central Drug Research Institute, India. This study aimed to conduct comprehensive in vitro metabolic investigations of CDRI 99/411 to corroborate its preclinical investigations. Preliminary in vitro metabolic investigations were performed to assess the metabolic stability [in vitro half‐life (t_1/2) and in vitro hepatic intrinsic clearance (Cl_int)] of CDRI 99/411 in male Sprague–Dawley rat and human liver microsomes using validated high‐performance liquid chromatography with photodiode array detector. The observed in vitro t_1/2 of the compound in rat and human liver microsomes was 13 min with in vitro Cl_int 130.7 ± 25.0 μL/min/mg and 19 min with in vitro Cl_int 89.3 ± 17.40 μL/min/mg. These observations suggested moderate metabolic degradation and in vitro Cl_int with insignificant difference (p > 0.05) in the metabolic stability profile in rat and human. Hence, in vitro metabolic investigations were performed with rat liver microsomes. It was observed that CDRI 99/411 exhibited sigmoidal kinetics. At nonlinear regression (r ≥ 0.99) EC₅₀ and Hill slope values were 17 µm and 1.50, respectively. The metabolism of CDRI 99/411 was primarily mediated by CYP3A2 and was inferred by CYP reaction phenotyping with known potent inhibitors. Two metabolites of CDRI 99/411 were detected which were undetectable on incubation with 1‐aminobenzotriazole and ketoconazole. Copyright © 2011 John Wiley & Sons, Ltd.     

Citric acid-loaded W1/O/W2 multiple emulsions efficiently remove colonic ammonia both in vitro and in vivo

Hepatic encephalopathy (HE) is a neuropsychiatric disorder caused by chronic and acute liver diseases. It is believed that ammonia played an important role on the pathogenesis of the disease. Herein, acid-loaded water-in-oil-in-water (W₁/O/W₂) multiple emulsions with over 95% encapsulation efficiency were prepared and used to absorb colonic ammonia. The study of citric acid release from W₁ to W₂ in bulk deionized water indicated that only 17% acid released in 8 hours, which proved the stability of the multiple emulsions and hence prevented the intestine from being irritated by acid burst release. In vitro, the W₁/O/W₂ emulsion could remove around 90% ammonia in 1.5 hours from either the 3 mmol/L ammonia solution or the artificial colonic fluid with 1 and 0.5 mmol/L ammonia without acidifying the artificial colonic fluid. In vivo, compared with lactulose, W₁/O/W₂ emulsions could efficiently reduce the blood ammonia to the similar level in the rat models with HE without increasing the water content in feces. All these results indicated a potential application of W₁/O/W₂ multiple emulsions for the treatment of HE.     

A new metabolite of nodakenetin by rat liver microsomes and its quantification by RP‐HPLC method

The biotransformation of nodakenetin (NANI) by rat liver microsomes in vitro was investigated. Two major polar metabolites were produced by liver microsomes from phenobarbital‐pretreated rats and detected by reversed‐phase high‐performance liquid chromatography (RP‐HPLC) analysis. The chemical structures of two metabolites were firmly identified as 3′(R)‐hydroxy‐nodakenetin‐3′‐ol and 3′(S)‐hydroxy‐nodakenetin‐3′‐ol, respectively, on the basis of their ¹H‐NMR, MS and optical rotation analysis. The latter was a new compound. A sensitive, selective and simple RP‐HPLC method has been developed for the simultaneous determination of NANI and its two major metabolites in rat liver microsomes. Chromatographic conditions comprise a C₁₈ column, a mobile phase with MeOH‐H₂O (40 : 60, v/v), a total run time of 40 min, and ultraviolet absorbance detection at 330 nm. In the rat heat‐inactivated liver microsomal supernatant, the lower limits of detection and quantification of metabolite I, metabolite II and NANI were 5.0, 2.0, 10.0 ng/mL and 20.0, 5.0, 50.0 ng/mL, respectively, and their calibration curves were linear over the concentration range 50–400, 20–120 and 150–24000 ng/mL, respectively. The results provided a firm basis for further evaluating the pharmacokinetics and clinical efficacy of NANI. Copyright © 2009 John Wiley & Sons, Ltd.