Quantitative Determination of Ascorbate from the Green Alga Chlamydomonas reinhardtii by HPLC

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Plant, Cell & Environment
Jul 2016



Ascorbate (Asc, also called vitamin C) is of vital importance to the cellular functions of both animals and plants. During evolution, Asc has become one of the most abundant metabolites in seed plants; however, Asc contents in cyanobacteria, green algae and bryophytes are very low. Here we describe a sensitive and reliable HPLC method for the quantitative determination of cellular Asc content in the green alga Chlamydomonas reinhardtii.

Keywords: Ascorbate (抗坏血酸), Cell counter (细胞计数器), Chlamydomonas reinhardtii (莱茵衣藻), HPLC (HPLC), Dehydroascorbate (脱氢抗坏血酸盐)


Previous protocols for the determination of cellular Asc content were developed for higher plants, of which the amounts of cellular Asc are high enough for the analysis. Those protocols are not suitable for green algae, since both the amounts of the plant material and the levels of the cellular Asc are usually limited. Therefore, there was a need to develop a novel method with sensitivity improved to the µM range to measure the cellular Asc in green algae.

Materials and Reagents

  1. 15-ml conical centrifuge tubes (Corning, Falcon®)
  2. Polypropylene microcentrifuge tubes of 1.5 ml (Eppendorf)
  3. 0.3 ml polypropylene chromatographic vials (Focus Technology, model: VP91 ) with polypropylene cap and PTFE/silicone septa (Focus Technology, model: SC9291 )
  4. 4 mm hydrophilic PTFE syringe filter with a 0.22 µm pore size (Nantong Filterbio Membrane, catalog number: FBS4PTFE022L )
  5. 47 mm hydrophilic PTFE membrane with a 0.45 µm pore size (Nantong Filterbio Membrane, catalog number: FBM047PTFE045L )
  6. Glass beads 212-300 µm (Sigma-Aldrich, catalog number: G9143-250G )
  7. 1 ml disposable polypropylene syringe (B. Braun Medical, catalog number: 9166017V )
  8. Millex-GS 33 mm sterile syringe filter with a 0.22 µm pore size (EMD Millipore, catalog number: SLGS033SS )
  9. Milli-Q water
  10. Liquid nitrogen
  11. Tris-(2-carboxyethyl)-phosphine hydrochloride (TCEP) (Carl Roth, catalog number: HN95.2 )
  12. Potassium dihydrogen phosphate (KH2PO4) (Carl Roth, catalog number: 3904.1 )
  13. Orthophosphoric acid (H3PO4) (Sigma-Aldrich, catalog number: W290017 )
  14. EDTA (Sigma-Aldrich, catalog number: EDS-100G )
  15. Sodium chloride (NaCl) (Duchefa Biochemie, catalog number: S0520.1000 )
  16. Potassium chloride (KCl) (Duchefa Biochemie, catalog number: P0515.1000 )
  17. Di-sodium hydrogen phosphate (Na2HPO4) (Carl Roth, catalog number: P030.2 )
  18. Magnesium chloride hexahydrate (MgCl2·6H2O) (Sigma-Aldrich, catalog number: M2393-100g )
  19. Calcium chloride dihydrate (CaCl2·2H2O) (Duchefa Biochemie, catalog number: C0504.1000 )
  20. Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: 258148-2.5L-D )
  21. Sodium L-Asc (Sigma-Aldrich, catalog number: 4034-100G )
  22. Acetonitrile HPLC gradient grade (VWR, HiPerSolv Chromanorm®, catalog number: 83639.320 )
  23. Phosphate-buffered saline (PBS) (see Recipes)
  24. Extraction buffer (see Recipes)
  25. HPLC mobile phase A (see Recipes)
  26. HPLC mobile phase B (see Recipes)
  27. Sodium L-Asc standard solutions (see Recipes)


  1. ScepterTM handheld automated cell counter (EMD Millipore, catalog number: PHCC00000 )
  2. ScepterTM Sensors – 40 µm (EMD Millipore, catalog number: PHCC40050 )
  3. Refrigerated table centrifuge and microcentrifuge
  4. HPLC system (Shimadzu Scientific Instruments) equipped with:
    1. LC pump (Shimadzu Scientific Instruments, model: LC-20AD )
    2. Thermostated autosampler (Shimadzu Scientific Instruments, model: SIL-20AC )
    3. Photo Diode Array detector (Shimadzu Scientific Instruments, model: SPD-M30A )
    4. Column oven (Shimadzu Scientific Instruments, model: CTO-20AC )
    5. Synergy hydro 4u Hydro – RP 80 Å 250 x 4.6 mm (Phenomenex, catalog number: 00G-4375-E0 ) directly mounted with a guard column AQ C18 4.0 x 3.0 mm (Phenomenex, catalog number: KJ0-4282 )


  1. LabSolution (Shimadzu Scientific Instruments)


  1. Sample preparation
    1. Prepare liquid Chlamydomonas cultures and collect 1 ml sample when the cultures are in the exponential growth phase. Mix the sample with 19 ml of phosphate-buffered saline (PBS). Use a Scepter cell counter to determine cell number and cell volume by following the instructions displayed on the screen. Briefly, depress the plunger, submerge the tip, and release the plunger to soak up 50 μl liquid culture into the tip. The Scepter cell counter detects each cell passing through the orifice of the tip, calculates cell density, and displays a histogram of cell diameter or volume on the screen (Johnston, 2010).
    2. Based on the calculated cell density, harvest 2 x 106 cells, place it in a 15-ml conical centrifuge tube and centrifuge it at 12,000 x g for 1 min at room temperature.
    3. Gently re-suspend the pelleted cells in 1 ml Milli-Q water and repeat the centrifugation step.
    4. Discard the supernatant and freeze the pellet in liquid nitrogen. The samples can be stored at -80 °C for several months.
    5. Add 200 µl of extraction buffer to the frozen pellet and mix it by vigorous shaking. The extraction buffer contains TCEP to keep Asc in the reduced form.
    6. Transfer the resuspended cells into a 1.5 ml microcentrifuge tube containing 100 µl glass beads and vortex at maximum speed for 30 sec. Incubate the sample at room temperature for two minutes (during which the sample becomes brownish), then place it on ice.
    7. Centrifuge the sample at 19,000 x g at 4 °C for 30 min.
    8. Collect 120 µl of the supernatant (without stirring up the pellet) and filter it into chromatographic vials using 4 mm hydrophilic PTFE syringe filters with a pore size of 0.22 µm. The samples are stable for 24 h at 4 °C and can be stored at -80 °C for several months; however, freeze-thaw cycles should be avoided.

  2. Determination of Asc concentration by HPLC (see Nováková et al., 2008a for theoretical basics)
    1. Ascorbate can be efficiently separated chromatographically using a Synergy hydro 4u Hydro – RP 80 Å 250 x 4.6 mm column. Attach the column fitted with a suitable guard column (AQ C18 4.0 x 3.0 mm) to the HPLC. Equilibrate the column with mobile phase A for at least 30 min at a flow rate of 1 ml/min.
    2. Set the tray temperature of the autosampler at 4 °C and the column oven temperature at 20 °C.
    3. While waiting for the temperature and baseline to stabilize, prepare the sample sequence table. Insert the chromatographic vials containing the samples into the tray of the autosampler.
    4. Inject 20 µl sample. For the elution of Asc, use an isocratic mobile phase of 50 mM KH2PO4 (pH 2.5, adjusted with orthophosphoric acid) for 5 min at a flow rate of 1 ml/min then apply a short acetonitrile (mobile phase B) gradient from 0% to 30% between 3.5 and 6 min to elute the less polar components from the column. Between 8 and 9 min decrease the percentage of mobile phase B to 0% and equilibrate the column to the mobile phase A for an additional 9 min. The total running time is 18 min.
    5. Record the signal between 190 and 500 nm at a sampling frequency of 4 Hz and 1,024 spectral resolution with a slit set to 8 nm. The Asc peak appears at approximately 5.1 min with an absorption maximum at around 244 nm.

Data analysis

  1. Prepare a two-fold serial dilution between 0.2 and 50 µM Asc dissolved in the extraction buffer. Determine the peak areas of Asc in the HPLC chromatograms by the LabSolution software (Shimadzu Scientific Instruments) and use linear fitting to obtain a calibration curve (Figure 1).

    Figure 1. Typical HPLC chromatograms recorded at 244 nm and a calibration curve for the determination of L-Asc concentration. A. Chromatographic profiles of (a) 62.5 µM L-Asc dissolved in extraction buffer (95% orthophosphoric acid, 2 mM EDTA, 10 mM TCEP, pH 2.5) and (b) C. reinhardtii cell extract. The identified peaks are (1) orthophosphoric acid; (2) EDTA; (3) L-Asc. B. Calibration curve for the determination of L-Asc concentration calculated based on linear fitting of the peak areas and using 1/area weighting. The LOD and LOQ values are 0.2 and 1.5 µM, respectively.

  2. Calculate the cellular Asc concentration using the following formula:


  1. The average cell volume for C. reinhardtii is about 200 fl. The typical Asc concentration of CC124 C. reinhardtii cells grown in liquid Tris-acetate-phosphate medium for three days at approximately 80 µmole photons m-2 s-1 is about 40 µM. The cellular Asc content, however, strongly depends on the algal strain, the growth medium, the age of the cultures, growth light and stress conditions (Nagy et al., 2016; Vidal-Meireles et al., unpublished).
  2. The extraction buffer contains TCEP, which keeps Asc in the more stable reduced form; therefore, by the method described above, the total cellular Asc concentration (dehydroascorbate + Asc) is determined. However, if there is a need to determine the amount of dehydroascorbate within the cell, omit TCEP from the extraction buffer. In this case, however, the samples should be processed and measured within less than 2 h, and cannot be frozen. Dehydroascorbate does not absorb at 244 nm, thus the ratio of Asc to dehydroascorbate is calculated based on the difference in absorption at 244 nm in the presence or absence of TCEP.


  1. Phosphate-buffered saline (PBS, 1 L; based on Chazotte, 2012)
    8 g NaCl
    0.2 g KCl
    1.44 g Na2HPO4
    0.24 g KH2PO4
    0.1 g MgCl2·6H2O
    Milli-Q water
    Dissolve the salts in Milli-Q water and adjust the pH to 7.2 with HCl
    Complete the volume to 990 ml by adding Milli-Q water and dispense the solution into aliquots Sterilize the solution by autoclaving for 20 min at 121 °C, 15 psi on liquid cycle
    Dissolve 0.133 g CaCl2·2H2O in 10 ml of Milli-Q water, filter-sterilize it and add it to the solution after autoclaving
    Store the sterile PBS at room temperature
  2. Extraction buffer (100 ml; based on Nováková et al., 2008b)
    74.448 mg EDTA
    286.65 mg TCEP
    Milli-Q water
    Dissolve the salts completely in Milli-Q water
    Add 5 ml of 98% orthophosphoric acid
    Dispense the solution into aliquots, and store at -80 °C until use
  3. HPLC mobile phase A (1 L)
    6.8045 g KH2PO4
    Milli-Q water
    Adjust the pH to 2.5 with 98% orthophosphoric acid
    Filter the solution using 0.45 µm hydrophilic PTFE membrane
    Note: This solution must be freshly prepared.
  4. HPLC mobile phase B (1 L)
    100% acetonitrile
    Degas by sonication for 20 min at room temperature
  5. Sodium L-Asc standard solutions
    Dissolve 49.5 mg sodium L-Asc in 1 ml of extraction buffer to obtain a 50 mM sodium L-Asc solution
    Dilute 10 µl aliquot in 10 ml extraction buffer
    Prepare a two-fold dilution series from this 50 µM stock solution down to 0.2 µM
    Note: This solution must be freshly prepared.


This work was supported by the Lendület/Momentum Programme of the Hungarian Academy of Sciences (LP-2014/19) and the National, Research and Development Office (research grant NN114524).


  1. Chazotte, B. (2012). Labeling Golgi with fluorescent ceramides. Cold Spring Harb Protoc 2012(8).
  2. Johnston, G. (2010). Automated handheld instrument improves counting precision across multiple cell lines. BioTechniques 48: 325-327
  3. Nagy, V., Vidal-Meireles, A., Tengölics, R., Rákhely, G., Garab, G., Kovács, L. and Tóth, S. Z. (2016). Ascorbate accumulation during sulphur deprivation and its effects on photosystem II activity and H2 production of the green alga Chlamydomonas reinhardtii. Plant Cell Environ 39(7): 1460-1472.
  4. Nováková, L., Solich, P. and Solichová, D. (2008a). HPLC methods for simultaneous determination of ascorbic and dehydroascorbic acids. Trends Anal Chem 27: 942-958.
  5. Nováková, L., Solichová, D., Pavlovičová, S. and Solich, P. (2008b). Hydrophilic interaction liquid chromatography method for the determination of ascorbic acid. J Sep Sci 31(9): 1634-1644.


抗坏血酸(Asc,也称为维生素C)对动物和植物的细胞功能至关重要。 在进化过程中,Asc已经成为种子植物中最丰富的代谢物之一; 然而,蓝藻,绿藻和苔藓植物中的Asc含量非常低。 在这里,我们描述了一种灵敏可靠的HPLC方法,用于定量测定绿藻衣藻中细胞Asc含量。

背景 针对高等植物开发了用于确定细胞Asc含量的先前方案,其中细胞Asc的量足够高以用于分析。 这些方案不适用于绿色藻类,因为植物材料的量和细胞Asc的水平通常都受到限制。 因此,需要开发一种灵敏度提高到μM范围来测量绿藻中细胞Asc的新方法。

关键字:抗坏血酸, 细胞计数器, 莱茵衣藻, HPLC, 脱氢抗坏血酸盐

p class ="pptt"> 材料和试剂

  1. 15 ml锥形离心管(Corning,Falcon ®
  2. 聚丙烯微量离心管1.5 ml(Eppendorf)
  3. 0.3ml聚丙烯色谱瓶(Focus Technology,型号:VP91),聚丙烯盖和PTFE /硅隔片(Focus Technology,型号:SC9291)
  4. 4mm亲水性PTFE注射器过滤器,孔径0.22μm(南通滤膜,目录号:FBS4PTFE022L)
  5. 47mm孔径为0.45μm的亲水PTFE膜(南通滤膜,目录号:FBM047PTFE045L)
  6. 玻璃珠212-300μm(Sigma-Aldrich,目录号:G9143-250G)
  7. 1ml一次性聚丙烯注射器(B.Braun Medical,目录号:9166017V)
  8. Millex-GS 33毫米孔径为0.22微米的无菌注射器过滤器(EMD Millipore,目录号:SLGS033SS)
  9. Milli-Q水
  10. 液氮
  11. 三(2-羧乙基)膦盐酸盐(TCEP)(Carl Roth,目录号:HN95.2)
  12. 磷酸二氢钾(KH 2 PO 4)(Carl Roth,目录号:3904.1)
  13. 正磷酸(H 3 O 3 PO 4)(Sigma-Aldrich,目录号:W290017)
  14. EDTA(Sigma-Aldrich,目录号:EDS-100G)
  15. 氯化钠(NaCl)(Duchefa Biochemie,目录号:S0520.1000)
  16. 氯化钾(KCl)(Duchefa Biochemie,目录号:P0515.1000)
  17. 磷酸氢二钠(Na 2 HPO 4)(Carl Roth,目录号:P030.2)
  18. 氯化镁六水合物(MgCl 2·6H 2 O)(Sigma-Aldrich,目录号:M2393-100g)
  19. 氯化钙二水合物(CaCl 2·2H 2 O)(Duchefa Biochemie,目录号:C0504.1000)
  20. 盐酸(HCl)(Sigma-Aldrich,目录号:258148-2.5L-D)
  21. L-Asc钠(Sigma-Aldrich,目录号:4034-100G)
  22. 乙腈HPLC梯度等级(VWR,HiPerSolv Chromanorm ,目录号:83639.320)
  23. 磷酸盐缓冲盐水(PBS)(见食谱)
  24. 提取缓冲液(见配方)
  25. HPLC流动相A(参见食谱)
  26. HPLC流动相B(参见食谱)
  27. 钠L-Asc标准溶液(见配方)


  1. 权杖 TM手持式自动细胞计数器(EMD Millipore,目录号:PHCC00000)
  2. Scepter TM 传感器 - 40μm(EMD Millipore,目录号:PHCC40050)
  3. 冷藏台式离心机和微量离心机
  4. HPLC系统(Shimadzu Scientific Instruments)装备有:
    1. LC泵(Shimadzu Scientific Instruments,型号:LC-20AD)
    2. 恒温自动进样器(Shimadzu Scientific Instruments,型号:SIL-20AC)
    3. 光电二极管阵列检测器(Shimadzu Scientific Instruments,型号:SPD-M30A)
    4. 柱式炉(Shimadzu Scientific Instruments,型号:CTO-20AC)
    5. 直接安装有保护柱AQ C18 4.0×3.0mm(Phenomenex,目录号:KJ0-4282)的Synergy hydro 4u Hydro-RP 80Å250×4.6mm(Phenomenex,目录号:00G-4375-E0)


  1. LabSolution(Shimadzu Scientific Instruments)


  1. 样品制备
    1. 准备液体衣原体培养物,并在培养物处于指数生长阶段时收集1ml样品。将样品与19ml磷酸盐缓冲盐水(PBS)混合。使用权杖细胞计数器按照屏幕上显示的说明来确定细胞数量和细胞体积。简单地说,压下柱塞,淹没尖端,释放柱塞,将50μl的液体培养液吸入顶端。权杖细胞计数器检测通过尖端的孔的每个细胞,计算细胞密度,并在屏幕上显示细胞直径或体积的直方图(Johnston,2010)。
    2. 基于计算的细胞密度,收获2×10 6细胞,将其置于15ml圆锥形离心管中,并在室温下以12,000xg离心1分钟。
    3. 轻轻地将沉淀的细胞重新悬浮在1毫升Milli-Q水中,重复离心步骤
    4. 弃去上清液,并在液氮中冷冻沉淀。样品可以在-80°C下储存数月。
    5. 向冷冻的沉淀中加入200μl提取缓冲液,并通过剧烈摇动进行混合。提取缓冲液含有TCEP,以减少形式的Asc
    6. 将再悬浮的细胞转移到含有100μl玻璃珠的1.5ml微量离心管中,并以最大速度涡旋30秒。在室温下孵育样品2分钟(样品变成棕色),然后放在冰上。
    7. 在4℃下将样品以4,000 x g离心30分钟
    8. 收集120μl上清液(不搅拌沉淀),并使用孔径为0.22μm的4 mm亲水性PTFE注射器过滤器将其过滤到色谱瓶中。样品在4℃下稳定24小时,可以在-80℃下储存几个月;但是,应避免冻融周期。

  2. 通过HPLC测定Asc浓度(参见Nováková等人,2008a理论基础)
    1. 使用Synergy hydro 4u Hydro-RP 80Å250 x 4.6 mm色谱柱可以有效地色谱分离抗坏血酸。将配有合适的保护柱(AQ C18 4.0 x 3.0 mm)的色谱柱连接到HPLC。用流动相A平衡柱至少30分钟,流速为1 ml/min。
    2. 将自动进样器的纸盘温度设置为4°C,柱温箱温度设置在20°C。
    3. 在等待温度和基线稳定的同时,准备样品序列表。将含有样品的色谱柱插入自动进样器的纸盘。
    4. 注入20μl样品。对于Asc的洗脱,使用50mM KH 2 PO 4的等度流动相(pH2.5,用正磷酸调节)5分钟,流速1 ml/min,然后在3.5至6分钟之间施用0%至30%的短乙腈(流动相B)梯度洗脱柱中较不极性的组分。 8至9分钟后,流动相B的百分比降低至0%,并将柱平衡至流动相A另外9分钟。总运行时间为18分钟。
    5. 以4 Hz的采样频率和1,024光谱分辨率将信号记录在190和500 nm之间,狭缝设置为8 nm。 Asc峰出现在约5.1分钟,吸收最大值在244nm附近。


  1. 准备在0.2和50μMAsc溶解在提取缓冲液中的两倍连续稀释。通过LabSolution软件(Shimadzu Scientific Instruments)在HPLC色谱图中确定Asc的峰面积,并使用线性拟合获得校准曲线(图1)。

    图1.在244nm处记录的典型HPLC色谱图和用于测定L-Asc浓度的校准曲线A.(a)62.5μML-Asc溶解在提取缓冲液(95%正磷酸,2mM EDTA,10mM TCEP,pH 2.5)和(b)C。细胞提取物。鉴定的峰是(1)正磷酸; (2)EDTA; (3)L-Asc。 B.基于峰面积的线性拟合计算的L-Asc浓度的校准曲线,并使用1 /面积加权。 LOD和LOQ值分别为0.2和1.5μM
  2. 使用以下公式计算细胞Asc浓度:


  1. C的平均单元格容积。 reinhardtii 约为200 fl。 CC124C的典型Asc浓度。在大约80μmol的光子中,在液体Tris-乙酸盐 - 磷酸盐介质中生长三天的细胞膜细胞约为40μM。然而,细胞Asc含量强烈地取决于藻类菌株,生长培养基,培养物的年龄,生长光和胁迫条件(Nagy等人,2016; Vidal-Meireles 等,未发布)。
  2. 提取缓冲液含有TCEP,保持Asc更稳定的还原形式;因此,通过上述方法,测定总细胞Asc浓度(脱氢抗坏血酸+ Asc)。然而,如果需要确定细胞内脱氢抗坏血酸的量,则从提取缓冲液中省略TCEP。在这种情况下,样品应在不到2小时内进行处理和测量,不能冻结。脱氢抗坏血酸盐在244nm处不吸收,因此基于在存在或不存在TCEP的244nm处的吸收差异来计算Asc与脱氢抗坏血酸的比例。


  1. 磷酸盐缓冲盐水(PBS,1升;基于Chazotte,2012)
    1.44g Na 2 HPO 4
    0.24g KH 2 PO 4
    0.1g MgCl 2·6H 2 O
    溶解在Milli-Q水中的盐,并用HCl将pH调节至7.2 通过加入Milli-Q水将体积达到990ml,并将溶液分配至等分试样通过在121℃高压灭菌20分钟,液体循环15psi来消毒溶液
    将0.133g CaCl 2·2H 2 O在10ml Milli-Q水中溶解,对其进行过滤灭菌并在高压灭菌后将其加入溶液中
  2. 提取缓冲液(100ml;基于Nováková等人,,2008b)
    74.448mg EDTA
    286.65 mg TCEP
    将盐完全溶解于Milli-Q水中 加入5ml的98%正磷酸 将溶液分配到等分试样中,并储存在-80°C直到使用
  3. HPLC流动相A(1L)
    6.8045g KH 2 PO 4
    用98%正磷酸将pH调节至2.5 使用0.45μm亲水PTFE膜过滤溶液
  4. HPLC流动相B(1L)
  5. 钠L-Asc标准溶液
    将49.5mg L-Asc溶于1ml提取缓冲液中,得到50mM L-Asc溶液, 稀释10μl等分试样在10ml提取缓冲液中 准备从该50μM储备溶液的两倍稀释系列至0.2μM




  1. Chazotte,B。(2012)。用荧光标记高尔基体神经酰胺。冷泉哈勃原菌 2012(8)。
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引用:Kovács, L., Vidal-Meireles, A., Nagy, V. and Tóth, S. Z. (2016). Quantitative Determination of Ascorbate from the Green Alga Chlamydomonas reinhardtii by HPLC. Bio-protocol 6(24): e2067. DOI: 10.21769/BioProtoc.2067.