样品前处理是代谢组分析的重要环节，而冷冻干燥是样品前处理过程中去除溶剂的重要方法，但冷冻干燥对代谢组分析结果的影响还有待进一步研究.本文以人类尿液和血清样本为研究对象，采用核磁共振（NMR）技术分析了冷冻干燥对这两种重要体液代谢组的影响，发现冷冻干燥会引起体液样本中部分有机羧酸和氨基酸含量的改变；而且，冷冻干燥对尿液和血清样品代谢组的影响不同，这可能与两种体液的生物基质差异有关.本研究结果表明，在代谢组学研究中要格外关注冷冻干燥对样品前处理及后续数据归一化所产生的影响.
Sample pretreatment is essential for metabonomic analysis. Lyophilization (freeze-drying) is widely used for sample pretreatment to remove solvent, especially water, prior to nuclear magnetic resonance (NMR) and/or mass spectrum (MS) analysis. However, the effects of lyophilization on metabonomic phenotypes remain to be fully investigated. In this study, NMR analysis was carried out to reveal the lyophilization-induced changes in metabonomic phenotypes of human urinary and serum samples. It was found that lyophilization resulted in large changes in the levels of some carboxylic acids and amino acids. Such effects were different between the urine and serum samples, perhaps indicating a biological matrix-dependent effect. The results of this study suggest that extra care and considerations should be taken when lyophilization is used for sample pretreatment and normalization in metabonomic analysis.
[1] NICHOLSON J K, CONNELLY J, LINDON J C, et al. Metabonomics:a platform for studying drug toxicity and gene function[J]. Nat Rev Drug Discov, 2002, 1(2):153-161.
[2] NICHOLSON J K, LINDON J C. Systems biology-metabonomics[J]. Nature, 2008, 455(7216):1054-1056.
[3] AN Y P, XU W X, LI H H, et al. High-fat diet induces dynamic metabolic alterations in multiple biological matrices of rats[J]. J Proteome Res, 2013, 12(8):3755-3768.
[4] HOLMES E, WILSON I D, NICHOLSON J K. Metabolic phenotyping in health and disease[J]. Cell, 2008, 134(5):714-717.
[5] HE Q H, TANG H R, REN P P, et al. Dietary supplementation with L-arginine partially counteracts serum metabonome induced by weaning stress in piglets[J]. J Proteome Res, 2011, 10(11):5214-5221.
[6] HUANG C Y, LEI H H, ZHAO X J, et al. Metabolic influence of acute cyadox exposure on kunming mice[J]. J Proteome Res, 2013, 12(1):537-538.
[7] WU X Y, LI N, TANG H R. Quantitative analysis of metabolites in mungbean (Vigna Radiata) extracts using NMR techniques[J]. Chinese J Magn Reson, 2014, 31(4):548-563. 吴香玉, 李宁, 唐惠儒. 绿豆(Vigna Radiata)代谢物组成的核磁共振定量分析[J]. 波谱学杂志, 2014, 31(4):548-563.
[8] HU Y L, HAO F H, WANG Y L. NMR-based metabonomic analyses on spleen tissues of 4T1 tumor-bearing mice subjected to chemotherapies with different drug delivery strategies[J]. Chinese J Magn Reson, 2018, 35(1):8-21. 胡依黎, 豪富华, 王玉兰. 基于NMR的4T1荷瘤小鼠脾脏受不同给药方式影响的代谢组学[J]. 波谱学杂志, 2018, 35(1):8-21.
[9] ZHANG C C, WU J F, WANG Y L. Gender difference in intestinal bile acid profiles in C57BL/6 mice[J]. Chinese J Magn Reson, 2018, 35(3):328-337.张聪聪, 吴俊芳, 王玉兰. 不同性别C57BL/6小鼠肠道胆汁酸组成的差异性研究[J]. 波谱学杂志, 2018, 35(3):328-337.
[10] CHAN E C Y, PASIKANTI K K, NICHOLSON J K. Global urinary metabolic profiling procedures using gas chromatography-mass spectrometry[J]. Nat Protoc, 2011, 6(10):1483-1499.
[11] ZHOU R K, GUO K V, LI L. 5-diethylamino-naphthalene-1-sulfonyl chloride (DensCl):a novel triplex isotope labeling reagent for quantitative metabolome analysis by liquid chromatography mass spectrometry[J]. Anal Chem, 2013, 85(23):11532-11539.
[12] ABASCAL K, GANORA L, YARNELL E. The effect of freeze-drying and its implications for botanical medicine:A review[J]. Phytother Res, 2005, 19(8):655-660.
[13] RINDLER V, LUNEBERGER S, SCHWINDKE P, et al. Freeze-drying of red blood cells at ultra-low temperatures[J]. Cryobiology, 1999, 38(1):2-15.
[14] TANG X L, PIKAL M J. Design of freeze-drying processes for pharmaceuticals:Practical advice[J]. Pharm Res, 2004, 21(2):191-200.
[15] OIKAWA A, OTSUKA T, JIKUMARU Y, et al. Effects of freeze-drying of samples on metabolite levels in metabolome analyses[J]. J Sep Sci, 2011, 34(24):3561-3567.
[16] IBRAHIM A H, KHALIFA S A. Effect of freeze-drying on camel's milk nutritional properties[J]. Int Food Res J, 2015, 22(4):1438-1445.
[17] RANIERI A, BENELLI G, CASTAGNA A, et al. Freeze-drying duration influences the amino acid and rutin content in honeybee-collected chestnut pollen[J]. Saudi J Biol Sci, 2019, 26(2):252-255.
[18] DIETERLE F, ROSS A, SCHLOTTERBECK G, et al. Probabilistic quotient normalization as robust method to account for dilution of complex biological mixtures. Application in H-1 NMR metabonomics[J]. Anal Chem, 2006, 78(13):4281-4290.
[19] ERIKSSON L, TRYGG J, WOLD S. CV-ANOVA for significance testing of PLS and OPLS (R) models[J]. J Chemometr, 2008, 22(11,12):594-600.
[20] CLOAREC O, DUMAS M E, TRYGG J, et al. Evaluation of the orthogonal projection on latent structure model limitations caused by chemical shift variability and improved visualization of biomarker changes in H-1 NMR spectroscopic metabonomic studies[J]. Anal Chem, 2005, 77(2):517-526.
[21] JIMENEZ C, VENTURA R, WILLIAMS J, et al. Reference materials for analytical toxicology including doping control:freeze-dried urine samples[J]. Analyst, 2004, 129(5):449-455.
[22] RAISTRICK H. On a new type of chemical change produced by bacteria. The conversion of histidine into urocanic acid by bacteria of the Coli-Typhosus group[J]. Biochem J, 1917, 11(1):71-77.
