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Extraction and Quantification of Tryptophan and Kynurenine from Cultured Cells and Media Using a High Performance Liquid Chromatography (HPLC) System Equipped with an Ultra-sensitive Diode Array Detector
使用配有超灵敏二极管阵列检测器的高效液相色谱(HPLC)系统从培养细胞和培养基中提取和量化色氨酸和犬尿素   

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Abstract

Evidence of the involvement of tryptophan and its metabolite, kynurenine, in various biological processes including cancer is constantly expanding. Analysis of cell extracts and culture media can allow for quick snapshots of the metabolic fluctuations occurring in vitro. Here, we describe a method for metabolite extraction from mammalian cells and analysis of extracted metabolites and cell culture media by HPLC with detection using an ultra-sensitive diode array detector.

Keywords: Tryptophan(色氨酸), Kynurenine(犬尿氨酸), Immune surveillance(免疫监视), HPLC(高效液相色谱法)

Materials and Reagents

  1. Disposable 2 ml plastic microcentrifuge tubes
  2. 1.5 ml glass vials (Agilent Technologies, catalog number: 5182-0715 ), screw top caps (Agilent Technologies, catalog number: 5182-0717 ) and 200 μl glass inserts (Agilent Technologies, catalog number: 5182-0549 )
  3. AcroVac Filter Unit (Pall Life Sciences, catalog number: AVFP02L )
  4. Chloroform (HPLC grade) (Thermo Fisher Scientific, catalog number: C607 )
  5. L-Tryptophan (Sigma-Aldrich, catalog number: T0254 )
  6. L-Kynurenine (Sigma-Aldrich, catalog number: K8625 )
  7. 3-Nitro-L-tyrosine (nitrotyrosine) (Sigma-Aldrich, catalog number: N7389 )
  8. Methanol (HPLC grade) (Thermo Fisher Scientific, catalog number: A452 )
  9. Acetonitrile (HPLC grade) (Thermo Fisher Scientific, catalog number: A998 )
  10. Potassium phosphate monobasic anhydrous (KH2PO4) (Thermo Fisher Scientific, catalog number: P285-500 )
  11. Potassium phosphate dibasic anhydrous (K2HPO4) (Thermo Fisher Scientific, catalog number: P288-500 )
  12. PIPES (Thermo Fisher Scientific, catalog number: BP292450 )
  13. EDTA (Thermo Fisher Scientific, catalog number: S311-500 )
  14. Kynurenic acid (Sigma-Aldrich, catalog number: K3375 )
  15. Quinolinate (Sigma-Aldrich, catalog number: P63204 )
  16. Phosphate buffered saline (PBS)
  17. 50% methanol/50% 3 mM PIPES-3 mM EDTA (pH 7.4) (see Recipes)
  18. Potassium phosphate (pH 6.4) with 2.7% (v/v) acetonitrile (see Recipes)
  19. Standard mix (see Recipes)

Equipment

  1. Refrigerated tabletop centrifuge
  2. HPLC system with binary pump, autosampler and UV diode array detector (DAD) (Agilent Technologies, model: 1100 )
  3. Hypersil GOLD C18 column (50 mm with 2.1 mm diameter) (Thermo Fisher Scientific, catalog number: 25003-052130 )

Procedure

  1. Tryptophan and kynurenine extraction
    1. The protocol requires a minimum of 1 x 106 cells to accurately detect tryptophan and kynurenine levels. If tryptophan and kynurenine quantification is desired from fresh media, collect 2 ml of fresh media prior to cell addition for subsequent analysis and freeze at -80 °C. This sample will be used to measure the baseline levels of tryptophan and kynurenine in the media.
    2. If cells of interest are adherent cells, then proceed to step A2a. If cells of interest are suspension cells, then proceed to step A2b. If desired, the cell culture can be spiked with an internal standard such as 3-nitro-L-tyrosine (a final concentration of 20-50 µM is generally appropriate) at this time. The internal standard can be used to quantify metabolite recovery.
      1. At the completion of the treatment schedule, collect 2 ml media in 2 ml microcentrifuge tubes. Centrifuge at 2,000 x g at 4 °C in centrifuge to pellet cells/debris from the media. Without disturbing the pellet, transfer media into two labeled tubes of equal volume. One will be used for immediate analysis while the other will serve as a backup. At this point, cell-free, centrifuged culture media can be stored at -80 °C until analysis. Media can be analyzed by HPLC without the need for extraction. Next, aspirate off remaining media in plate/flask where cells are grown. Wash cells with cold (4 °C) phosphate buffered saline (PBS) twice. Afterwards, dislodge cells from culture plate/flask by scraping. Collect liberated cells and place into 2 ml microcentrifuge tubes. Centrifuge at 2,000 x g at 4 °C in centrifuge. Proceed to step A3.
      2. At the completion of the treatment schedule, collect all media containing cells into appropriate volume conical tubes. Centrifuge at 2,000 x g at 4 °C in centrifuge to pellet cells/debris from media. Without disturbing the pellet, transfer 1 ml of media into two labeled tubes. One will be used for immediate analysis while the other will serve as a backup. At this point, cell-free, centrifuged culture media can be stored at -80 °C until analysis. Media can be analyzed by HPLC without the need for extraction. Next, wash pelleted cells with cold (4 °C) phosphate buffered saline (PBS) twice. Proceed to step A3.
    3. Centrifuge cells at 2,000 x g at 4 °C and aspirate PBS without disturbing the cell pellet. Keep cells on ice while pipetting PBS. To quench metabolism and extract metabolites, add 200 µl ice-cold 50% methanol/50% PIPES-EDTA to the cell pellet.
    4. To the quenched cell mixture, add 200 µl ice-cold chloroform and vortex vigorously for 45 min at -20 °C. (Note: This extraction length is important when extracting metabolites from organisms with cell walls including bacteria and yeast. A shorter extraction time of 5-10 min is adequate for mammalian cells. Vigorous vortexing is most effective for metabolite extraction. Use of a multi-tube vortex mixer head is recommended.) Afterwards, centrifuge at 21,000 x g at 4 °C for 10 min.
    5. Collect upper, water/methanol phase in a clean labeled tube. This is the first metabolite extract. Place tube on ice.
    6. Re-extract the chloroform phase by adding 200 µl of ice-cold 50% methanol/ 50% PIPES-EDTA. Vortex for 1 min and centrifuge again at 21,000 x g at 4 °C for 10 min.
    7. Just as before collect the upper, water/methanol with the extract from step A5. Freeze the extract until analysis.

  2. Detection and quantification of tryptophan and kynurenine
    1. Analyze metabolites from cell culture media and from cell extracts by HPLC. To separate and quantify tryptophan and kynurenine, inject 5-20 µl (Note: The required injection volume will vary depending on the concentration of metabolite in the sample and is a recommended range.) of the cell extract obtained from step A7, spent culture media collected in step A2, or the cell-free media collected from step A1 in an HPLC equipped with an ultra-sensitive diode array detector and a Hypersil GOLD C18 column (50 x 2.1 mm, 3 µm), which is a reverse phase column packed with spherical, fully porous ultrapure silica. Set column temperature to be maintained at 30 °C. Use an isocratic mobile phase of 15 mM potassium phosphate (pH 6.4), with 2.7% (v/v) acetonitrile with a flow rate of 0.8 ml/min. The run time should be set for 10 min.
    2. Quantify the area under the peaks that display absorbance at 286 nm and 360 nm to calculate the concentration of tryptophan and kynurenine, respectively, in the samples. Generate standard curves ranging from 0 to 200 µM for both tryptophan and kynurenine and other metabolites of interest as well as any internal standard (if applicable) using authentic standards. Standards of each concentration should be prepared in triplicate and measured in triplicate. A recipe for a standard mix is included in the Recipes section.
      Note: Details of standard curve generation will vary depending on the chromatography system and data analysis software used. Consult the manufacturer’s documentation to generate a standard curve with your instrument’s software.

Representative data


Figure 1. HPLC chromatogram showing separation of tryptophan, kynurenine, kynurenic acid and nitrotyrosine standards. Quinolinate is also present in the standard mix but below the limit of quantification. The upper panel shows absorbance at 360 nm, and the lower panel shows absorbance at 286 nm. The tryptophan metabolite kynurenic acid and 3-nitro-L-tyrosine (nitrotyrosine), which can be used as an internal standard, are also separated. All analytes are present at a concentration of 12.5 µM. The standard solution was prepared as described in the Recipes section.

Notes

This method can be modified for compatibility with liquid chromatography-mass spectrometry by using a mobile phase consisting of 10 mM ammonium formate (pH 6.4), containing 2.7% acetonitrile. Other chromatographic conditions remain unchanged. If desired, a known quantity of 3-nitro-L-tyrosine can be added to samples prior to extraction for use as an internal standard.

Recipes

  1. 50% methanol / 50% 3 mM PIPES -3 mM EDTA (pH 7.4)
    1. Prepare 3 mM PIPES-3 mM EDTA (pH 7.4) (for 500 ml).
      Mix 0.453 g PIPES and 0.438 g EDTA in 400 ml of sterile water (HPLC grade). After the solution becomes completely dissolved, complete to 500 ml with sterile water. Filter the solution.
    2. Mix the above with 500 ml of Methanol (HPLC grade).
    3. Degas and store at 4 °C.
  2. 15 mM potassium phosphate, pH 6.4, with 2.7% (v/v) acetonitrile (for 1 L)
    Make 1 M stock solutions of both monobasic anhydrous (KH2PO4) and dibasic anhydrous (K2HPO4).
    1. Mix 72.2 ml of KH2PO4 and 27.8 ml of K2HPO4 dilute with 900 ml of sterile water (HPLC grade) to make 100 mM stock.
    2. Take 150 ml of the 100 mM stock and dilute with 850 ml of sterile water (HPLC grade) to make 15 mM potassium phosphate stock. Filter the solution.
    3. Add appropriate amount of acetonitrile (HPLC grade) fresh when running HPLC separation.
    4. Degas and store at room temperature.
  3. Standard mix (200 µM each of tryptophan, nitrotyrosine, kynurenine, kynurenic acid, and quinolinate)
    1. Prepare each standard separately at a concentration of 1 mM in 50 ml mobile phase [15 mM potassium phosphate (pH 6.4) with 2.7% acetonitrile]. For each solution, weigh out an appropriate quantity of each standard and place in a 50 ml volumetric flask. Record the actual mass of each standard.
    2. To each volumetric flask, add 50 ml mobile phase and mix. The standards, especially kynurenic acid, may require more than an hour to go into solution. A stir bar can be added to each flask and stirred on a stir plate to facilitate dissolution of the standard.
    3. When each solution is ready, add 2 ml of each standard 1 mM standard solution to a 15 ml tube and vortex mix. The final concentration of each standard in this standard mix will be 200 µM. Perform replicate serial dilutions as required to make a series of standards for use in generation of a standard curve.
      Note: Serial dilutions of standards are generally satisfactory but can propagate concentration errors if the stock solution concentration is not correct.

Acknowledgments

This work was supported by NIH grants 1R01CA135401-01A1, and 1R01DK082690-01A1, the Medical Service of the US Department of Veterans’ Affairs, and Dialysis Clinics, Inc. (DCI) (all to R. H. W.). This work was performed in part at the Research Resource for Biomedical Accelerator Mass Spectrometry, which is operated at LLNL under the auspices of the U. S. Department of Energy under contract DEAC52-07NA27344. The Research Resource is supported by the National Institutes of Health, National Institute of General Medical Sciences under Grant P40 RR13461. The extraction protocol was adapted from Villas-Boas et al. (2005). The HPLC method was adapted from Laich et al. (2002).

References

  1. Laich, A., Neurauter, G., Widner, B. and Fuchs, D. (2002). More rapid method for simultaneous measurement of tryptophan and kynurenine by HPLC. Clin Chem 48(3): 579-581.
  2. Villas-Boas, S. G., Hojer-Pedersen, J., Akesson, M., Smedsgaard, J. and Nielsen, J. (2005). Global metabolite analysis of yeast: evaluation of sample preparation methods. Yeast 22(14): 1155-1169.
  3. Wettersten, H. I., Hakimi, A. A., Morin, D., Bianchi, C., Johnstone, M. E., Donohoe, D. R., Trott, J. F., Aboud, O. A., Stirdivant, S., Neri, B., Wolfert, R., Stewart, B., Perego, R., Hsieh, J. J. and Weiss, R. H. (2015). Grade-dependent metabolic reprogramming in kidney cancer revealed by combined proteomics and metabolomics analysis. Cancer Res 75(12): 2541-2552.

简介

色氨酸及其代谢物,犬尿氨酸参与包括癌症在内的各种生物过程的证据不断扩大。 细胞提取物和培养基的分析可以允许在体外发生的代谢波动的快速快照。 在这里,我们描述了一种从哺乳动物细胞代谢物提取的方法,提取代谢物和细胞培养基通过HPLC检测使用超敏二极管阵列检测器分析。

关键字:色氨酸, 犬尿氨酸, 免疫监视, 高效液相色谱法

材料和试剂

  1. 一次性2 ml塑料微量离心管
  2. 1.5ml玻璃小瓶(Agilent Technologies,目录号:5182-0715),螺旋盖(Agilent Technologies,目录号:5182-0717)和200μl玻璃插入物(Agilent Technologies,目录号:5182-0549)
  3. AcroVac过滤器单元(Pall Life Sciences,目录号:AVFP02L)
  4. 氯仿(HPLC级)(Thermo Fisher Scientific,目录号:C607)
  5. L-色氨酸(Sigma-Aldrich,目录号:T0254)
  6. L-犬尿氨酸(Sigma-Aldrich,目录号:K8625)
  7. 3-硝基-L-酪氨酸(硝基酪氨酸)(Sigma-Aldrich,目录号:N7389)
  8. 甲醇(HPLC级)(Thermo Fisher Scientific,目录号:A452)
  9. 乙腈(HPLC级)(Thermo Fisher Scientific,目录号:A998)
  10. 无水磷酸二氢钾(KH 2 PO 4)(Thermo Fisher Scientific,目录号:P285-500)
  11. 无水磷酸氢钾(K 2 HPO 4)(Thermo Fisher Scientific,目录号:P288-500)
  12. PIPES(Thermo Fisher Scientific,目录号:BP292450)
  13. EDTA(Thermo Fisher Scientific,目录号:S311-500)
  14. 犬尿酸(Sigma-Aldrich,目录号:K3375)
  15. 喹啉酸盐(Sigma-Aldrich,目录号:P63204)
  16. 磷酸盐缓冲盐水(PBS)
  17. 50%甲醇/50%3mM PIPES-3mM EDTA(pH7.4)(参见配方)
  18. 含2.7%(v/v)乙腈的磷酸钾(pH6.4)(见配方)
  19. 标准混合(见配方)

设备

  1. 冷冻式台式离心机
  2. 带有二元泵,自动进样器和UV二极管阵列检测器(DAD)(Agilent Technologies,型号:1100)的HPLC系统
  3. Hypersil GOLD C18柱(50mm,直径2.1mm)(Thermo Fisher Scientific,目录号:25003-052130)

程序

  1. 色氨酸和犬尿氨酸提取
    1. 该协议需要至少1×10 6个细胞来精确检测 色氨酸和犬尿氨酸水平。如果是色氨酸和犬尿氨酸 需要从新鲜培养基中定量,收集2ml新鲜培养基 然后加入细胞用于随后的分析并在-80℃下冷冻。 该样品将用于测量色氨酸的基线水平 和犬尿氨酸。
    2. 如果感兴趣的细胞是粘附的 细胞,然后进行步骤A2a。如果感兴趣的细胞悬浮 细胞,然后进行步骤A2b。如果需要,细胞培养物可以是 掺入内标如3-硝基-L-酪氨酸(最终 浓度一般为20-50μM为宜)。的 内标可用于定量代谢物的恢复
      1. 在 ?完成治疗方案,收集2ml培养基于2ml 微量离心管。在4℃下在离心机中离心2000×g离心 颗粒细胞/来自培养基的碎片。在不干扰颗粒的情况下, 转移介质转移到两个等体积的标记管中。将使用一个 用于立即分析,而另一个将用作备份。在这 点,无细胞,离心培养基可以储存在-80℃ 直到分析。可以通过HPLC分析培养基而不需要 萃取。接下来,吸出板/烧瓶中的剩余介质 细胞生长。用冷(4℃)磷酸盐缓冲盐水洗细胞 (PBS)两次。然后,从培养板/烧瓶中取出细胞 刮。收集释放的细胞,并放入2毫升微量离心机 管。在离心机中在4℃下以2,000×g离心。继续步骤
      2. 在完成治疗计划后,收集所有 介质包含在合适体积的锥形管中。离心机 ?在离心机中在4℃下以2,000×g离心,以从培养基中沉淀细胞/碎片。 在不打扰沉淀的情况下,将1ml培养基转移到两个标记的培养基中 管。一个将用于立即分析,而另一个将 充当备份。此时,无细胞,离心培养基 可以储存在-80°C直到分析。可以通过HPLC分析培养基 无需提取。接下来,用冷的洗涤沉淀的细胞(4 ℃)磷酸盐缓冲盐水(PBS)洗涤两次。继续执行步骤A3。
    3. 在4℃下以2,000×g离心细胞并吸出PBS 干扰细胞沉淀。保持细胞在冰上,同时移液PBS。至 猝灭代谢和提取代谢物,加入200μl冰冷的50% 甲醇/50%PIPES-EDTA至细胞沉淀。
    4. 到淬火的电池 ?混合,加入200μl冰冷的氯仿,并剧烈涡旋45 min。 (注意:这个提取长度很重要 从包括细菌的细胞壁的生物体中提取代谢物 ?和酵母。更短的提取时间5-10分钟就足够了 哺乳动物细胞。剧烈涡旋对代谢物最有效 萃取。建议使用多管涡流混合器头。) 然后,在4℃下以21,000×g离心10分钟。
    5. 收集上部,水/甲醇相在干净的标记管中。这是第一种代谢物提取物。将管放在冰上。
    6. 通过加入200μl冰冷的50% 甲醇/50%PIPES-EDTA。涡旋1分钟,并再次在4℃下以21,000×g离心10分钟。
    7. 正如之前收集上部,水/甲醇与步骤A5的提取物。冻结提取物直至分析。

  2. 色氨酸和犬尿氨酸的检测和定量
    1. 分析细胞培养基和细胞提取物的代谢物 HPLC。要分离和量化色氨酸和犬尿氨酸,注射5-20μl ?(注意:所需的进样体积将根据不同而有所不同 代谢物在样品中的浓度,是推荐的范围。) 的从步骤A7获得的细胞提取物中,收集用过的培养基 ?,或在HPLC中从步骤A1收集的无细胞培养基 配有超灵敏二极管阵列检测器和Hypersil GOLD C18柱(50×2.1mm,3μm),其为反相柱 填充球形,完全多孔的超纯二氧化硅。设置列 温度保持在30℃。使用等度流动相 15mM磷酸钾(pH6.4),含2.7%(v/v)乙腈,a 流速0.8ml/min。运行时间应设置为10分钟。
    2. 定量显示在286 nm处的吸光度的峰下面积 360 nm计算色氨酸和犬尿氨酸的浓度, 分别在样品中。生成从0到的标准曲线 ?200μM对于色氨酸和犬尿氨酸以及其他代谢物 利息以及任何内部标准(如果适用)使用 正宗标准。应制备每种浓度的标准品 一式三份并一式三份测量。标准混合的食谱是 ?包括在"食谱"部分。
      注:标准曲线的详细信息 生成将根据色谱系统和数据而变化 分析软件。请参阅制造商的文档 使用您的仪器软件生成标准曲线。

代表数据


图1.显示色氨酸,犬尿氨酸,犬尿烯酸和硝基酪氨酸标准品分离的HPLC色谱图。喹啉酸盐也存在于标准混合物中,但低于定量限。上图显示360nm处的吸光度,下图显示286nm处的吸光度。也可以分离色氨酸代谢物犬尿酸和3-硝基-L-酪氨酸(硝基酪氨酸),其可以用作内标。所有分析物以12.5μM的浓度存在。标准溶液如配方部分所述制备。

笔记

可以通过使用由含有2.7%乙腈的10mM甲酸铵(pH 6.4)组成的流动相来修改该方法以与液相色谱 - 质谱法的相容性。其他色谱条件保持不变。如果需要,可以在提取之前将已知量的3-硝基-L-酪氨酸加入样品中用作内标。

食谱

  1. 50%甲醇/50%3mM PIPES-3mM EDTA(pH7.4)
    1. 制备3mM PIPES-3mM EDTA(pH7.4)(500ml) 混合0.453g PIPES和0.438g EDTA在400ml无菌水(HPLC级)中。后 溶液变得完全溶解,完全达到500ml 无菌水。筛选解决方案。
    2. 将上述物质与500ml甲醇(HPLC级)混合
    3. 脱气并储存在4°C。
  2. 15mM磷酸钾,pH 6.4,用2.7%(v/v)乙腈(对于1L) 制备1M一级无水(KH 2 PO 4 PO 4)和无水二碱价(K 2 SO 4 PO 4)的1M储备溶液, >)。
    1. 将72.2ml的KH 2 PO 4和27.8ml的K 2 HPO 4稀释液与900ml无菌的水(HPLC级)以制备100mM储备液。
    2. 取150毫升的100毫米原液,用850毫升无菌稀释 水(HPLC级),得到15mM磷酸钾储备液。过滤 解决方案
    3. 运行HPLC分离时加入适量的新鲜乙腈(HPLC级)。
    4. 脱气并在室温下储存。
  3. 标准混合物(色氨酸,硝基酪氨酸,犬尿氨酸,犬尿喹啉酸和喹啉酸各200μM)
    1. 分别制备每种标准品,浓度为1mM,溶于50ml 流动相[15mM磷酸钾(pH 6.4),含2.7% 乙腈]。对于每种溶液,称取适量的 每个标准品并置于50ml容量瓶中。记录实际 每个标准的质量
    2. 向每个容量瓶中加入50ml移动 ?相和混合。标准,特别是犬尿酸,可能需要 超过一个小时去解决。可以向每个添加搅拌棒 并在搅拌盘上搅拌以促进溶解 标准。
    3. 当每个溶液准备好,添加2毫升的每个标准1 ?mM标准溶液至15ml管和涡旋混合物。最后 该标准品中各标准品的浓度为200μM。 根据需要进行重复连续稀释以制备一系列 用于产生标准曲线的标准。
      注意:Serial 标准品的稀释度通常令人满意,但可以繁殖 如果储备溶液浓度不正确,浓度误差。

致谢

这项工作由NIH拨款1R01CA135401-01A1和美国退伍军人事务部的医疗服务1R01DK082690-01A1和Dialysis Clinics,Inc。(DCI)(全部给R.H.W。)支持。这项工作部分在生物医学加速器质谱研究资源中进行,该研究资源在美国能源部在合同DEAC52-07NA27344的主持下在LLNL操作。研究资源由国家卫生研究院,国家综合医学科学研究所支持,授予P40 RR13461。提取方案改编自Villas-Boas等人(2005)。 HPLC方法改编自Laich等人(2002)。

参考文献

  1. Laich,A.,Neurauter,G.,Widner,B.and Fuchs,D。(2002)。 通过HPLC同时测量色氨酸和犬尿氨酸的更快速方法。 Clin Chem 48(3):579-581。
  2. Villas-Boas,S.G.,Hojer-Pedersen,J.,Akesson,M.,Smedsgaard,J.and Nielsen,J。(2005)。 酵母的全球代谢物分析:样品制备方法的评估酵母 22(14):1155-1169。
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引用:Kim, J., Stewart, B. and Weiss, R. H. (2016). Extraction and Quantification of Tryptophan and Kynurenine from Cultured Cells and Media Using a High Performance Liquid Chromatography (HPLC) System Equipped with an Ultra-sensitive Diode Array Detector. Bio-protocol 6(7): e1781. DOI: 10.21769/BioProtoc.1781.
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