Small-Volume Analysis of Cell–Cell Signaling Molecules in the Brain

Modern science is characterized by integration and synergy between research fields. Accordingly, as technological advances allow new and more ambitious quests in scientific inquiry, numerous analytical and engineering techniques have become useful tools in biological research. The focus of this review is on cutting edge technologies that aid direct measurement of bioactive compounds in the nervous system to facilitate fundamental research, diagnostics, and drug discovery. We discuss challenges associated with measurement of cell-to-cell signaling molecules in the nervous system, and advocate for a decrease of sample volumes to the nanoliter volume regimen for improved analysis outcomes. We highlight effective approaches for the collection, separation, and detection of such small-volume samples, present strategies for targeted and discovery-oriented research, and describe the required technology advances that will empower future translational science.     

CE-MS检测胃癌患者的尿液氨基酸代谢变化

目的检测胃癌患者尿液的游离氨基酸代谢轮廓,将癌症组和正常对照组进行比较,筛选出具有诊断意义的氨基酸,探讨胃癌患者尿液中氨基酸代谢的变化规律和临床意义。方法收集胃癌患者与健康成人晨尿。采用移动反应界面富集法的毛细管电泳质谱联用技术对尿样中游离氨基酸轮廓进行检测,结合氨基酸标准品的定性定量分析,并对所得数据运用SPSS17.0进行统计学处理,得到有意义的氨基酸。结果胃癌患者尿液代谢轮廓较正常组有明显变化。与正常组相比,胃癌组尿液中的异亮氨酸、缬氨酸、精氨酸水平升高(P﹤0.05),组氨酸、蛋氨酸、天冬氨酸含量降低(P﹤0.05)。主成分胃癌患者尿液代谢较正常组有明显变化;早期或进展期胃癌患者尿液代谢轮廓亦有明显变化。结论毛细管电泳质谱检测方法可以较好的揭示正常组与胃癌的尿液代谢信息,此方法具有可行性,简便,快速。结果表明,毛细管电泳质谱检测胃癌患者尿液中氨基酸代谢对胃癌诊断与筛查具有重要的临床意义。     

Analysis of urinary metabolites for breast cancer patients receiving chemotherapy by CE-MS coupled with on-line concentration

Objective

This study aimed to explore the relationship between urinary metabolites and clinical chemotherapy response in breast cancer by CE-MS coupled with on-line concentration.

Design and methods

Urine samples were obtained from patients with advanced or locally advanced breast cancer (n = 21) before and after chemotherapy and healthy volunteers (n = 21). A rapid and sensitive hexadimethrine bromide-coating CE-MS method coupled with normal stacking is developed for the determination of organic acids in human urine. Another CE-MS method coupled with pH-mediated sample stacking is used for the analysis of amino acids and organic acids.

Results

After receiving chemotherapy, chemotherapy-sensitive patients showed 30% change in metabolite levels compared to healthy people, while chemotherapy-insensitive patients showed only 9% change in metabolite levels compared to healthy people showing recurrence. The extent of energy insufficiency for chemotherapy-insensitive patients was greater than that for chemotherapy-sensitive patients.

Conclusions

Urinary metabolic products may be new potential predictive markers for therapy efficacy. However, more studies with a larger sample size are required to confirm these conclusions.     

毛细管电泳-质谱联用法测定灵芝药材中核苷类成分

建立一种用于测定赤芝和紫芝中的核苷类成分的毛细管电泳-质谱联用法(CE-MS)。CE条件为：熔融石英毛细管柱(120 cm×50 μm),缓冲液体系100 mmol/L甲酸-10%甲醇水溶液。运行电压25 kV;毛细管温度25 ℃。MS条件为：0.3%甲酸-75%甲醇水溶液为鞘液,流速3 μL/min;干燥气体流速6 L/min,干燥温度350 ℃;采用电喷雾离子化源(ESI),正离子模式检测,全扫描范围m/z 50～350。以5-氯胞嘧啶阿拉伯糖苷为内标物。结果表明,胞嘧啶、鸟嘌呤、胞苷、腺苷、次黄嘌呤、鸟苷、肌苷和尿嘧啶在线性范围内具有较好的线性关系(除鸟苷外,其他成分R²>0.990)。加样回收率在95.3%～104.6%之间。各待测化合物的最低检测限(LOD)和最低定量限(LOQ)分别分布在0.13～9.85 μg/mL和0.42～20.83 μg/mL之间。测定结果显示赤芝的核苷类成分的含量上高于紫芝。CE-MS适合用于中药中核苷类成分的分析检测。     

A Metabolomic Approach to Clarifying the Effect of AST-120 on 5/6 Nephrectomized Rats by Capillary Electrophoresis with Mass Spectrometry (CE-MS)

The oral adsorbent AST-120 is composed of spherical carbon particles and has an adsorption ability for certain small-molecular-weight compounds that accumulate in patients with chronic kidney disease (CKD). So far, very few compounds are known to be adsorbed by AST-120 in vivo. To examine the effect of AST-120 in vivo, we comprehensively evaluated the plasma concentrations of 146 compounds (61 anions and 85 cations) in CKD model rats, with or without four weeks of treatment with AST-120. By capillary electrophoresis with mass spectrometry, we identified 6 anions and 17 cations that were significantly decreased by AST-120 treatment. In contrast, we also identified 2 cations that were significantly increased by AST-120. Among them, 4 anions, apart from indoxyl sulfate and hippurate, and 19 cations were newly identified in this study. The plasma levels of N-acetyl-neuraminate, 4-pyridoxate, 4-oxopentanoate, glycine, γ-guanidinobutyrate, N-γ-ethylglutamine, allantoin, cytosine, 5-methylcytosine and imidazole-4-acetate were significantly increased in the CKD model compared with the sham-operated group, and were significantly decreased by AST-120 treatment. Therefore, these 10 compounds could be added as uremic compounds that indicate the effect of AST-120 treatment. This study provides useful information not only for identifying the indicators of AST-120, but also for clarifying changes in the metabolic profile by AST-120 treatment in the clinical setting.     

Hypotaurine is an Energy-Saving Hepatoprotective Compound against Ischemia-Reperfusion Injury of the Rat Liver

Metabolome analyses assisted by capillary electrophoresis-mass spectrometry (CE-MS) have allowed us to systematically grasp changes in small molecular metabolites under disease conditions. We applied CE-MS to mine out biomarkers in hepatic ischemia-reperfusion. Rat livers were exposed to ischemia by clamping of the portal inlet followed by reperfusion. Metabolomic profiling revealed that l contents of taurine in liver and plasma were significantly increased. Of interest is an elevation of hypotaurine, collectively suggesting significance of hypotaurine/taurine in post-ischemic responses. Considering the anti-oxidative capacity of hypotaurine, we examined if supplementation of the compound or its precursor amino acids could affect hepatocellular viability and contents of taurine in liver and plasma. Administration of hypotaurine, N-acetylcysteine or methionine upon reperfusion comparablly attenuated the post-ischemic hepatocellular injury but with different metabolomic profiling among groups: rats treated with methionine or N-acetylcysteine but not those treated with hypotaurine, exhibited significant elevation of hepatic lactate generation without notable recovery of the energy charge. Furthermore, the group treated with hypotaurine exhibited elevation of the plasma taurine, suggesting that the exogenously administered compound was utilized as an antioxidant. These results suggest that taurine serves as a surrogate marker for ischemia-reperfusion indicating effectiveness of hypotaurine as an energy-saving hepatoprotective amino acid.     

Detection of Acute Tubulointerstitial Rejection by Proteomic Analysis of Urinary Samples in Renal Transplant Recipients

This study investigates proteomic analysis of urinary samples as a non-invasive method to detect acute rejection of renal allografts. Capillary electrophoresis coupled to mass spectrometry (CE-MS) was used to analyze urinary samples in 19 patients with different grades of subclinical or clinical acute rejection (BANFF Ia to IIb), 10 patients with urinary tract infection and 29 patients without evidence of rejection or infection. A distinct urinary polypeptide pattern identified 16 out of 17 cases of acute tubolointerstitial rejection, but was absent in two cases of vascular rejection. Urinary tract infection resulted in a different polypeptide pattern that allowed to differentiate between infection and acute rejection in all cases. Potentially confounding variables such as acute tubular lesions, tubular atrophy, tubulointerstitial fibrosis, calcineurin inhibitor toxicity, proteinuria, hematuria, allograft function and different immunosuppressive regimens did not interfere with test results. Blinded analysis of samples with and without rejection showed correct diagnosis by CE-MS in the majority of cases. Detection of acute rejection by CE-MS offers a promising non-invasive tool for the surveillance of renal allograft recipients. Further investigation is needed to establish polypeptide patterns in vascular rejection and to explore whether changes in the urinary proteome occur before the onset of histologically discernible rejection.     

CE-MS-Based Identification of Uremic Solutes Specific to Hemodialysis Patients

Uremic toxins are suggested to be involved in the pathophysiology of hemodialysis (HD) patients. However, the profile of uremic solutes in HD patients has not been fully elucidated. In this study using capillary electrophoresis mass spectrometry (CE-MS), we comprehensively quantified the serum concentrations of 122 ionic solutes before and after HD in 11 patients. In addition, we compared the results with those in non-HD patients with chronic kidney disease (CKD) to identify HD patient-specific solutes. We identified 38 solutes whose concentrations were higher in pre-HD than in CKD stage G5. Ten solutes among them did not significantly accumulate in non-HD CKD patients, suggesting that these solutes accumulate specifically in HD patients. We also identified 23 solutes whose concentrations were lower in both pre- and post-HD than in CKD stage G5. The serum levels of 14 solutes among them were not affected by renal function in non-HD patients, suggesting that these solutes tend to be lost specifically in HD patients. Our data demonstrate that HD patients have a markedly different profile of serum uremic solute levels compared to that in non-HD CKD patients. The solutes identified in our study may contribute to the pathophysiology of HD patients.     

Collagen-Derived Peptides in CKD: A Link to Fibrosis

Collagen is a major component of the extracellular matrix (ECM) and has an imminent role in fibrosis, in, among others, chronic kidney disease (CKD). Collagen alpha-1(I) (col1a1) is the most abundant collagen type and has previously been underlined for its contribution to the disease phenotype. Here, we examined 5000 urinary peptidomic datasets randomly selected from healthy participants or patients with CKD to identify urinary col1a1 fragments and study their abundance, position in the main protein, as well as their correlation with renal function. We identified 707 col1a1 peptides that differed in their amino acid sequence and/or post-translational modifications (hydroxyprolines). Well-correlated peptides with the same amino acid sequence, but a different number of hydroxyprolines, were combined into a final list of 503 peptides. These 503 col1a1 peptides covered 69% of the full col1a1 sequence. Sixty-three col1a1 peptides were significantly and highly positively associated (rho > +0.3) with the estimated glomerular filtration rate (eGFR), while only six peptides showed a significant and strong, negative association (rho < −0.3). A similar tendency was observed for col1a1 peptides associated with ageing, where the abundance of most col1a1 peptides decreased with increasing age. Collectively the results show a strong association between collagen peptides and loss of kidney function and suggest that fibrosis, potentially also of other organs, may be the main consequence of an attenuation of collagen degradation, and not increased synthesis.