Optimized Oxidative Stress Protocols for Low-microliter Volumes of Mammalian Plasma

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Dec 2018



Small blood volumes commonly obtained from small mammals during field studies are only sufficient for a single biochemical assay. In this study, we used blood collected from a population of wild eastern chipmunks (Tamias striatus) and developed modified methods to improve analytical selectivity and sensitivity required for measuring markers of oxidative stress using small blood volumes. Specifically, we proposed a modified malondialdehyde (MDA) analysis protocol by high performance liquid chromatography (HPLC) and also optimized both the uric acid independent ferric reducing antioxidant power (FRAP) and hypochlorous acid shock capacity (HASC) assays. We present methods in which a total volume of less than 60 μl of plasma is required to obtain a comprehensive portrait of an individual’s oxidative profile.

Keywords: High-performance liquid chromatography (高效液相层析), Oxidative stress markers (氧化应激标记物), Lipid peroxidation (脂质过氧化), Ferric reducing antioxidant power (铁离子还原力), Hypochlorous acid (次氯酸), Plasma assays (血浆检测), Low blood volume tests (低血容量实验), Small mammal ecological studies (小型哺乳动物生态学研究), Antioxidants (抗氧化剂)


Measuring markers of oxidative stress has become increasingly popular and useful for the evaluation of individual’s physiological status. Measuring multiple oxidative stress markers and estimating methodological precision usually requires large blood sample volumes, making it difficult for researchers to perform comprehensive analyses of oxidative stress profile of small wild animals. We present in-house assays optimized to yield the most selective analysis for each marker using the smallest blood volumes possible (Langille et al., 2018).

MDA is a product of lipid peroxidation that in recent years has been measured by HPLC (Nussey et al., 2009). We present an HPLC method employing UV detection that uses a low volume of plasma to obtain a sensitivity and detection range appropriate for oxidative stress studies.

The ferric reducing antioxidant power (FRAP) assay has been extensively used in various disciplines ranging from agronomy and nutrition (Carlsen et al., 2010; Di Silvestro et al., 2017) to ecological studies (Griffin and Bhagooli, 2004; Ruykys et al., 2012; Eikenaar et al., 2018). Antioxidants in the samples reduce the ferric-tripyridyltriazine (Fe3+-TPTZ) complex to ferrous tripyridyltriazine (Fe2+-TPTZ), producing a blue color. The coloration intensity is proportional to the reducing capacity of the mostly non-enzymatic antioxidants in the plasma, hence providing the overall antioxidant capacity. Uric acid has been shown to interfere with the result of this assay however, Duplancic et al. (2011) proposed removing the uric acid using uricase with larger volume sample. We optimized the amount of uricase and sample needed to successfully remove all uric acid from a smaller sample, therefore improving the efficiency and accuracy of the test.

The HASC assay is commonly referred to by its kit named OXY-Adsorbent TestTM from Diacron International (Grosseto, Italy). In this test, an excess of hypochlorous acid, a strong oxidant, is added and incubated with a plasma sample. The hypochlorous acid oxidizes any reduced components in the plasma, then a chromogenic dye is added to quantify the remaining hypochlorous acid. Although the test was already optimized for 10 µl, the relatively high cost of each plate made it difficult for researchers to utilize this kit in large studies. We reduced the required volume to 1 µl and provide an in-house protocol to prepare the reagents and execute the test.

Materials and Reagents

  1. Standard flat bottomed 96-well plates (Sarstedt AG & Co., catalog number: 82.1581)
  2. Screw cap 500 µl microcentrifuge tube (BioBasic, catalog number: TC132-SN)
  3. 300 μl microinsert HPLC vial (Chromatogaphic Specialties, catalog number: C73302101232)
  4. 1.5 ml microcentrifuge tubes (BioBasic, catalog number: BT620NS)
  5. 15 ml centrifuge tube (VWR, catalog number: 89401-568)
  6. 50 ml centrifuge tube (VWR, catalog number: 76176-956)
  7. Aluminum Foil (Fisher, catalog number: 01-213-101)
  8. Acetonitrile, HPLC grade (VWR, catalog number: CABDH83639.400)
  9. Water, HPLC grade (VWR, catalog number: CABDH23595.400)
  10. Trifluoroacetic acid, HPLC grade (VWR, catalog number: CATX1276-6)
  11. 2-thiobarbituric acid (EMD Millipore, catalog number: 108180)
  12. Malondialdehyde bis (diethylacetal) (4 °C, 12 months) (Sigma-Aldrich, catalog number: T9889)
  13. Butylated hydroxytoluene (Sigma-Aldrich, catalog number: B1378)
  14. Trichloroacetic acid 6.1 N (Sigma-Aldrich, catalog number: T0699)
  15. Uricase (-20 °C, 12 months) (Sigma-Aldrich, catalog number: U0880)
  16. Ferric Chloride Hexahydrate (BioBasic, catalog number: FD0201)
  17. Ferrous Sulfate (BioBasic, catalog number: FB0461)
  18. Sodium Acetate Trihydrate (BioBasic, catalog number: SB0481)
  19. Acetic Acid, Glacial (VWR, catalog number: CA71006-424)
  20. TPTZ (Sigma-Aldrich, catalog number: T1253) 
  21. NPD (Sigma-Aldrich, catalog number: 193992)
  22. 12.5% Sodium Hypochlorite (store in the dark, 12 months) (VWR, catalog number: BDH7038-4L)
  23. Hydrochloric Acid (HCl) (VWR, catalog number: CA11020-884)
  24. Sulfuric Acid (VWR, catalog number: CA11024-170)
  25. Sodium Hydroxide (NaOH) (VWR, catalog number: BDH9292)
  26. Anhydrous Ethanol (Commercial Alcohols, catalog number: P016EAAN)
  27. 0.1 M butylated hydroxytoluene (BHT) solution (see Recipes)
  28. 15% trichloroacetic acid solution (see Recipes)
  29. 2 N NaOH solution (see Recipes)
  30. 2-thiobarbituric acid solution (see Recipes)
  31. 0.25 M HCl solution (see Recipes)
  32. Uricase stock solution (see Recipes)
  33. Uricase working solution (see Recipes)
  34. Acetate buffer solution (see Recipes)
  35. 400 mM stock HCl solution (see Recipes)
  36. 40 mM HCl solution (see Recipes)
  37. TPTZ solution (see Recipes)
  38. Ferric chloride solution (see Recipes)
  39. 30 mM Ferrous sulfate stock solution (see Recipes)
  40. FRAP reagent (see Recipes)
  41. NPD stock solution (see Recipes)
  42. 0.6 M Sulfuric acid solution (see Recipes)
  43. Oxidant solution (see Recipes)


  1. 100 ml volumetric flask, class A (VWR, catalog number: 10124-072)
  2. 500 ml volumetric flask, class A (VWR, catalog number: 10545-998)
  3. 1 L volumetric flask, class A (VWR, catalog number: 10546-000)
  4. Amber bottles (VWR, catalog number: 89221-624)
  5. Ice bath (VWR, catalog number: 10146-190)
  6. Water bath (VWR, catalog number: 89501-464)
  7. Fume hood (VWR, catalog number: 89260-062)
  8. Magnetic stirrer (VWR, catalog number: 97042-634)
  9. Analytical balance (VWR, catalog number: 10205-026)
  10. iMark Filter absorbance microplate reader (Bio-Rad, catalog number: 1681135)
  11. 515 nm Filter for microplate reader (Bio-Rad, catalog number: 1681033)
    Note: If a 515 nm filter is not available, an alternative filter in the range of 510-520 nm will suffice. 
  12. 1100 Series Binary pump (Agilent Technologies, model: G1312A)
  13. 1100 Series Autosampler (Agilent Technologies, model: G1313A)
  14. 1100 Series Column compartment (Agilent Technologies, model: G1316A)
  15. 1100 Series Variable wavelength detector (Agilent Technologies, model: G1315A)
  16. ODS2 250 x 4.6 mm 5 µm HPLC column (Waters Corp., catalog number: PSS831915) 
  17. Vortex Mixer (VWR, catalog number: 10153-842)
  18. Shaking dry-block (Eppendorf, VWR, model: Thermomixer, catalog number: CA11028-280)
  19. Barnstead E-Pure water system (Barnstead, Thermo Fisher, model: E-Pure)
  20. Refrigerated Centrifuge (Eppendorf, model: 5418R)
  21. Micropipette set (Eppendorf, catalog number: 2231000601)
  22. Spectrophotometer (Thermo Fisher, model: Genesys10S)
  23. pH meter (VWR, catalog number: 89231-662)
  24. 4 °C refrigerator (VWR, catalog number: 89239-070)
  25. -20 °C freezer (VWR, catalog number: 10819-408)
  26. -80 °C freezer (VWR, catalog number: 10160-740)


  1. OpenLab Chromatography Data System (Agilent Technologies, http://www.agilent.com/)
  2. Microplate Manager 6 (Bio-Rad Laboratories Canada Ltd., http://www.biorad.com/)


  1. MDA quantitation
    a. Field samples should be spun down in a hematocrit centrifuge directly after collection in order to store the plasma separately. We recommend transferring the separated plasma to a 0.5 ml centrifuge tube for storage. See Langille et al., 2018 for more details on field sampling.
    b. Store samples at -80 °C before analysis and test within one year of collection.
    c. A single HPLC system and two workers can expect to process a calibration curve and about 20-30 plasma samples in one working day. 

    1. Prepare the HPLC system using the parameters listed in Table 1. 

      Table 1. HPLC system parameters to be used in MDA quantification method

    2. Prepare a 1.00 mM MDA working solution by adding 100 mM 1,1,3,3-tetraethoxypropane stock solution to a 100 ml volumetric flask, diluting to the mark with ultrapure water. Mix well. 
    3. Prepare an MDA calibration by serially diluting the freshly prepared 1.00 mM 1,1,3,3-tetraethoxypropane working solution using micropipettes. Prepare fresh calibration curve before every batch of samples. See Table 2. 

      Table 2. Preparation of serially diluted 1,1,3,3-tetraethoxypropane standards for the MDA quantification method

    4. Store standards on ice until use. 
    5. Thaw plasma samples on ice and spin for 1 min in a refrigerated centrifuge at 3,000 x g and 4 °C to collect the plasma at the bottom of the tube. 
    6. Prepare standard 1.5 ml microcentrifuge tubes with labels for the samples and the standards.
      Note: We prefer to run all in triplicate, so we purchased a label printer which can make the repeated labeling of tubes much easier. 
    7. Transfer 20 µl of either standard or plasma sample to the labeled tubes. 
    8. Add 2 µl butylated hydroxytoluene (BHT) solution (see Recipes) and 5 µl of 2 N NaOH solution (see Recipes) to each tube. 
    9. Briefly vortex tubes, and then transfer to the shaking dry block incubator set at 60 °C for 30 min and 400 rpm agitation.
    10. Once this is finished, remove tubes to the ice bucket, and add 100 μl of 15% trichloroacetic acid solution to precipitate proteins. 
    11. Briefly vortex the samples, then incubate in the ice bath for 5 min.
    12. Centrifuge the tubes for 10 min at 14,000 x g and 4 °C to pellet the protein.
    13. Carefully, without disturbing the protein pellet, transfer 100 μl of supernatant to a 500 μl screw cap microcentrifuge tube.
    14. To that tube, also add 50 μl of 2-thiobarbituric acid derivatizing solution (see Recipes). 
    15. Ensure the caps on the tubes are well sealed and place in a dry block incubator at 100 °C for 60 min.
    16. Remove the tubes from the block into an ice bath, and when cooled, centrifuge for 5 min at 14,000 x g and 4 °C. 
    17. Transfer 60 μl of the derivatized solution to the microinsert HPLC vial.
    18. Transfer 5 μl of the derivatized solution to the pooled QC vial, except for standards.
      Note: The QC is a pooled sample of all the plasma you will process in one day. Injecting this QC throughout the HPLC run process (every 8-10 samples) will allow you to observe if there are any issues with the runs or decomposition of your samples.
    19. Subject the vials to HPLC analysis in triplicate within 12 h of sample preparation. 

  2. FRAP assay
    a. Samples should be stored at -80 °C before analysis and thawed on ice.
    b. FRAP reagent is light sensitive, and unstable in ambient air. The reagent must be prepared directly before use. 

    1. Prepare a working iron sulfate solution by diluting 1 ml of the 300 mM stock with 9 ml of distilled water in a 15 ml centrifuge tube. Use this 30 mM solution to prepare the calibration solutions in 15 ml centrifuge tubes (see Table 3).

      Table 3. Dilution table of iron sulfate standards for the FRAP assay

    2. Obtain a standard flat bottomed 96-well plate, and plan for where the position of the calibration curves and the samples will be positioned. We like to run 2 calibration curves per plate to account for errors. 
    3. Pipette a 5 μl aliquot of either standard or sample into each well to be measured, followed by 5 μl of ultrapure water containing uricase (1.00 U/ml, see Recipes). Be sure that the two drops mix well. 
    4. Incubate the solution for 5 min at room temperature on an orbital shaker at 100 rpm.
    5. Add 200 μl of FRAP reagent (see Recipes) to each well using a multichannel pipette. 
    6. Place the plate into the microplate reader and shake for 30 min on low speed, seen as “LO” on software. 
    7. After 30 min, the absorbance at 595 nm is read vs. reagent blank. 

  3. HASC assay
    a. Samples should be stored at -80 °C before analysis and thawed on ice.
    b. Oxidant solution is unstable in air and should be prepared with care. Adjustment of the pH should be carried out in a fume hood as small quantities of chlorine gas will be produced.

    1. Prepare the oxidant solution as in the Recipes section. Protect the solution from light by wrapping the bottle in aluminum foil, and work quickly with the solution. 
    2. From the oxidant solution, prepare a calibration curve of HClO by diluting the 1x to 2x, 4x, 8x, 16x and 32x dilutions. Prepare 1 ml of each solution. 
    3. To a standard 96-well flat bottom microplate, add 1 μl plasma or standard and 100 μl ultrapure water.
      Note: We prefer to run at least two calibration curves per plate to account for any error which may occur. 
    4. Vortex the plate on a 2D shaker at 400 rpm for 30 s. 
    5. Using a multichannel pipette, add 100 μl of oxidant solution to sample wells only.
      Note: Do not add oxidant solution to the standard wells. The standard is directly read as a serial dilution of HClO. Instead, add 100 μl ultrapure water.
    6. Shake the plate on an orbital shaker for 10 min at room temperature and 100 rpm. 
    7. Add 20 μl of NPD solution (see Recipes) to each well of the plate. 
    8. Incubate the plate on an orbital shaker for 1 min at room temperature and 100 rpm.
    9. Read the absorbance of the wells at 515 nm.

Data analysis

  1. MDA quantification
    Area of the TBA2-MDA adduct at 3.00 min is automatically integrated by the software. Export this data and use it to prepare the calibration curves and to quantify samples. Average triplicate data in its raw format then quantify using the standard linear calibration (see Langille et al., 2018).
  2. FRAP assay
    Export the absorbance data of the wells and take the average of the triplicate wells. Use this data to prepare the linear calibration and quantify samples (see Langille et al., 2018).
  3. HASC assay
    Export the absorbance data of the wells and take the average of the triplicate wells. Use this data to prepare the linear calibration and quantify samples (see Langille et al., 2018).


  1. 2-Thiobarbituric acid is controlled under the controlled drugs and substances act (CDSA) in Canada and therefore a license is required to procure and possess this compound. An application had to be filed through Health Canada to obtain authorization to procure 2-thiobarbituric acid. In the USA, 2-thiobarbituric acid is not controlled and can be purchased from Sigma-Aldrich.
  2. It is important to note that micropipettes are used throughout this experiment and therefore investigators should be trained in good pipetting technique and be comfortable with the pipettes. It is crucial to practice good technique as very small volumes of solution are handled and therefore any error can be magnified if the pipette is not delivering an accurate volume. 
  3. All solutions should be prepared using good analytical techniques. If you plan to process a large number of samples for a single study, it is advisable to prepare the stocks once for that study to minimize variability between several preparations of the same solution. 
  4. The linear range of each UV detector or microplate reader can change depending on the model, age etc. It is important to check that the system provides a linear calibration before processing plasma samples. 
  5. The figures of merit to assess the performance of the method were calculated in the following ways: relative standard deviation (%RSD) is the standard deviation divided by the mean of three separate preparations (and three injections per preparation) of the same sample. The limit of detection (LOD) and limit of quantification (LOQ) were calculated by measuring the noise of a blank injection. The LOD and LOQ are defined as 3 and 10 times the noise level of the blank, respectively. 
  6. The linearity of the MDA calibration curve from 1.25-20 μM was high using this method (%RSD = 0.32, n = 7). The LOD and LOQ were 0.06 and 0.125 μM respectively. The inter-assay variation (%RSD = 9.31, n = 48) was representative of the laboratory measurement error. We obtained no significant increase for QC samples over time (β = 0.0195 μM per hour, r2 = 0.017, n = 8). The QC quantifications compared to the mean of all the individuals assessed in each day revealed that there is little effect on the results from different days of analysis (slope = 0.99, r2 = 0.98, n = 9). Example chromatograms are shown in Langille et al., 2018. 
  7. For the developed uric acid independent FRAP assay, the linearity of the calibration curve, from 0.5-3 mM FeSO4, was high (%RSD = 2.50, n = 7). The inter-assay variation (%RSD) was 5.82 (n = 57). 
  8. The linearity of the calibration curve for the adapted HASC assay, ranging from 0.03-0.5 mM HClO, was high (%RSD = 3.64, n = 4). The inter-assay variation (%RSD) was 2.03 (n = 32). 
  9. If you are using a monochromator-based spectrophotometer or microplate reader, you can simply use the wavelengths suggested in this paper for measurement.


  1. 0.1 M butylated hydroxytoluene (BHT) solution
    1. Dissolve 2.20 g butylated hydroxytoluene in 100 ml anhydrous ethanol
    2. The solution may require brief sonication to aid in rapid dissolution
    3. Storage: RT, Shelf life: 3 months
  2. 15% trichloroacetic acid solution
    1. In a fume hood, add 15 ml 6.1 N (100% w/v) trichloroacetic acid solution slowly to 85 ml ultrapure water
    2. Briefly vortex to mix
    3. Storage: RT, Shelf life: 3 months
  3. 2 N NaOH solution
    1. Dissolve 8 g NaOH pellets in 100 ml ultrapure water
    2. Stir with a magnetic stir plate until the pellets completely dissolve
    3. Storage: RT, Shelf life: 3 months
  4. 2-thiobarbituric acid solution
    1. Dissolve 0.375 g 2-thiobarbituric acid in 100 ml 0.25 M HCl
    2. Heat in a water bath at 50 °C for 10 min, then sonicate for 5 min to aid in rapid dissolution
    3. Storage: 4 °C, Shelf life: 3 months
  5. 0.25 M HCl solution
    1. In a fume hood, slowly add 10.4 ml 12 M (conc.) HCl to a 500 ml volumetric flask, containing 300 ml of ultrapure water
    2. Swirl to mix and dilute to the mark
    3. Storage: RT, Shelf life: 3 months
  6. Uricase stock solution
    1. Incubate 15 ml of ultrapure water on ice to cool it before preparing the solution. Using the certificate of analysis provided with the uricase, calculate the mass needed to prepare a 1,000 U/ml stock
    2. Using an analytical balance, carefully weigh the powder to the nearest 0.1 mg on an analytical balance
    3. Add 1 ml pre-cooled water using a micropipette and dissolve by slowly pipetting the solution up and down. Do not vortex! Solutions of enzymes should never be vortexed
    4. Storage: -20 °C, Shelf life: 12 months
  7. Uricase working solution
    1. Dilute the Uricase stock solution by adding 1 μl of the 1,000 U/ml to 999 μl of pre-cooled ultrapure water using micropipettes
    2. Do not vortex, mix by gently inverting the tube several times 
    3. Storage: on ice, Shelf life: prepare daily
  8. Acetate buffer solution 
    1. In a fume hood, add 500 ml ultrapure water to a 1 L volumetric flask. Then slowly add 16 ml glacial acetic acid, followed by 2.70 g sodium acetate trihydrate
    2. Mix the solution using a magnetic stirrer and dilute to about 10 ml below the mark on the flask
    3. Check the pH using a benchtop pH meter that has been calibrated a pH 4 and 7. The pH should be 3.6. If the pH is higher than 3.6, acidify the solution by adding acetic acid dropwise. If the pH is lower than 3.6, raise the pH by adding sodium acetate. 
    4. Storage: 4 °C, Shelf life: 1 month
  9. 400 mM stock HCl solution 
    1. In a fume hood, slowly add 16.5 ml concentrated HCl to a 500 ml volumetric flask, containing 300 ml of ultrapure water
    2. Swirl to mix and dilute to the mark
    3. Storage: RT, Shelf life: 6 months
  10. 40 mM HCl solution 
    Combine 100 ml of 400 mM stock solution with 900 ml ultrapure water
    Storage: RT, Shelf life: 1 week
  11. TPTZ solution
    1. In a 100 ml volumetric flask, dissolve 0.3123 g TPTZ in 100 ml of 40 mM HCl as prepared above
    2. The solution may need to be placed in a 50 °C water bath to aid in dissolution
    3. Storage: 4 °C, Shelf life: 1 month
  12. Ferric chloride solution 
    1. In a fume hood, add 500 ml of 40 mM HCl to a 1 L volumetric flask, add 5.41 g of ferric chloride hexahydrate and stir on a magnetic stir plate until dissolved
    2. Dilute the solution to the mark with 40 mM HCl. The acid is required to prevent hydrolysis and precipitation of ferrous ions
    3. The solution should be stored in amber bottles, for up to two months. If you notice a red-brown precipitate forming, you must dispose of the solution and prepare it again
  13. 30 mM Ferrous sulfate stock solution 
    Dissolve 0.834 g ferrous sulfate heptahydrate in 100 ml ultrapure water
    Storage: 4 °C, Shelf life: 1 month
  14. FRAP reagent 
    1. Combine the following directly before use in a 50 ml centrifuge tube on ice:
      20 ml Acetate buffer
      2 ml TPTZ sol’n
      2 ml FeCl3 sol’n
      2.4 ml dH2O
    2. Mix reagents by vortexing. Solution should be straw in colour, if it is blue in colour, discard and prepare again
    3. Storage: ice bath, Shelf life: 1-2 h
  15. NPD stock solution
    1. Pipette 83 μl of the pure NPD into a 50 ml centrifuge tube containing 50 ml of anhydrous ethanol
    2. Wrap the tube in foil to protect from light. Mix using vortex mixer
    3. Storage: 4 °C, Shelf life: 3 months
  16. 0.6 M Sulfuric acid solution
    1. In a fume hood, add 16.6 ml conc. sulfuric acid slowly to a volumetric flask filled with 300 ml of ultrapure water
    2. Dilute to the mark and mix by inversion
    3. Storage: RT, Shelf life: 3 months
  17. Oxidant solution
    1. Turn on the spectrophotometer and allow the lamp to warm for 30 min prior to starting. In a fume hood, add 1 ml 10% sodium hypochlorite to a 500 ml volumetric flask containing 300 ml of ultrapure water
    2. Dilute to the mark and mix by inversion
    3. Transfer 100 ml of this solution to a 250 ml media bottle and insert a pH probe that has been calibrated at pH 4 and 7. With moderate magnetic stirring, carefully add 0.6 M sulfuric acid solution until the pH reaches 6.2
    4. Using a quartz cuvette with a 1 cm pathlength, take the absorbance of the resultant solution vs. the absorbance of a water blank at 292 nm
    5. Apply the Beer-Lambert Law using the formula below:

      A = ε × l × c

      where, the molar absorptivity coefficient (ε) of HClO is 350 L mol-1 cm-1, the pathlength (l) of the cell is 1 cm and the absorbance (A) is read at 292 nm. The concentration (c) of HClO can then be solved for.


I am grateful to Patrick Bergeron, Vincent Lemieux, Dany Garant and Denis Réale for the sample collection in the field. This research was funded by Natural Sciences and Engineering Research Council of Canada (NSERC) discovery grants to PB and DG and Québec Center for Biodiversity Science (QCBS) seed grants to PB and DG. VL was supported by a scholarship from Fonds de Recherche du Québec–Nature et Technologies (FRQNT).

Competing interests

The authors have declared that no competing interests exist.


Ethics approval was obtained from both The Canadian Council on Animal Care (#A2016-01–Bishop’s University) and the Ministère des Ressources naturelles et de la Faune du Québec (#2017-05-01-102-05-S-F).


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  2. Di Silvestro, R., Di Loreto, A., Bosi, S., Bregola, V., Marotti, I., Benedettelli, S., Segura-Carretero, A. and Dinelli, G. (2017). Environment and genotype effects on antioxidant properties of organically grown wheat varieties: a 3-year study. J Sci Food Agric 97(2): 641-649.
  3. Duplancic, D., Kukoc-Modun, L., Modun, D. and Radic, N. (2011). Simple and rapid method for the determination of uric acid-independent antioxidant capacity. Molecules 16(8): 7058-7068.
  4. Eikenaar, C., Isaksson, C. and Hegemann, A. (2018). A hidden cost of migration? Innate immune function versus antioxidant defense. Ecol Evol 8(5): 2721-2728.
  5. Griffin, S. and Bhagooli, R. (2004). Measuring antioxidant potential in corals using the FRAP assay. J Exp Mar Biol Ecol 302: 201-211.
  6. Langille, E., Lemieux, V., Garant, D. and Bergeron, P. (2018). Development of small blood volume assays for the measurement of oxidative stress markers in mammals. PLoS One 13(12): e0209802.
  7. Nussey, D. H., Pemberton, J. M., Pilkington, J. G. and Blount, J. D. (2009). Life history correlates of oxidative damage in a free-living mammal population. Funct Ecol 23: 809-817.
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在田间研究期间通常从小型哺乳动物获得的小血量仅足以进行单一生化测定。 在这项研究中,我们使用从一群东部野生花栗鼠( Tamias striatus >)收集的血液,并开发了改良方法,以提高使用小血量测量氧化应激标记所需的分析选择性和灵敏度。 具体而言,我们通过高效液相色谱(HPLC)提出了改进的丙二醛(MDA)分析方案,并且还优化了尿酸非依赖性铁还原抗氧化能力(FRAP)和次氯酸冲击能力(HASC)测定。 我们提出的方法是需要总体积小于60μl的血浆来获得个体氧化曲线的综合图像。
【背景】氧化应激的测量标记已经变得越来越流行并且可用于评估个体的生理状态。测量多个氧化应激标记物并估计方法学精确度通常需要大量的血液样本量,这使得研究人员难以对小型野生动物的氧化应激谱进行全面分析。我们提供内部化验,优化使用尽可能最小的血液量对每种标记物进行最具选择性的分析(Langille et al。>,2018)。

MDA是脂质过氧化的产物,近年来已经通过HPLC测量(Nussey 等人,>,2009)。我们提出了一种采用UV检测的HPLC方法,该方法使用低体积的血浆来获得适合氧化应激研究的灵敏度和检测范围。

铁还原抗氧化能力(FRAP)测定已被广泛应用于农学和营养学等各种学科(Carlsen et al。>,2010; Di Silvestro et al。>,2017 )进行生态学研究(Griffin和Bhagooli,2004; Ruykys et al。>,2012; Eikenaar et al。>,2018)。样品中的抗氧化剂将铁 - 三吡啶基三嗪(Fe 3 + -TPTZ)络合物还原为亚铁三吡啶基三嗪(Fe 2 + -TPTZ),产生蓝色。着色强度与血浆中大部分非酶抗氧化剂的还原能力成比例,因此提供总体抗氧化能力。已经显示尿酸干扰该测定的结果,然而,Duplancic 等人>(2011)提出使用具有更大体积样品的尿酸酶去除尿酸。我们优化了从较小样本中成功去除所有尿酸所需的尿酸酶和样品量,从而提高了测试的效率和准确性。

HASC测定通常由其来自Diacron International(Grosseto,Italy)的名为OXY-Adsorbent Test TM 的试剂盒提及。在该试验中,加入过量的次氯酸(强氧化剂)并与血浆样品一起温育。次氯酸氧化血浆中的任何还原组分,然后加入生色染料以定量剩余的次氯酸。尽管该测试已针对10μl进行了优化,但每个平板的成本相对较高,使得研究人员难以在大型研究中使用该试剂盒。我们将所需的体积减少至1μl,并提供内部协议来准备试剂并执行测试。

关键字:高效液相层析, 氧化应激标记物, 脂质过氧化, 铁离子还原力, 次氯酸, 血浆检测, 低血容量实验, 小型哺乳动物生态学研究, 抗氧化剂


  1. 标准平底96孔板(Sarstedt AG& Co.,目录号:82.1581)
  2. 螺帽500μl微量离心管(BioBasic,目录号:TC132-SN)
  3. 300μlmicrosinsertHPLC样品瓶(Chromatogaphic Specialties,目录号:C73302101232)
  4. 1.5 ml微量离心管(BioBasic,目录号:BT620NS)
  5. 15毫升离心管(VWR,目录号:89401-568)
  6. 50毫升离心管(VWR,目录号:76176-956)
  7. 铝箔(Fisher,目录号:01-213-101)
  8. 乙腈,HPLC级(VWR,目录号:CABDH83639.400)
  9. 水,HPLC级(VWR,目录号:CABDH23595.400)
  10. 三氟乙酸,HPLC级(VWR,目录号:CATX1276-6)
  11. 2-硫代巴比妥酸(默克密理博中国,目录号:108180)
  12. 丙二醛二(二乙基乙缩醛)(4℃,12个月)(Sigma-Aldrich,目录号:T9889)
  13. 丁基羟基甲苯(Sigma-Aldrich,目录号:B1378)
  14. 三氯乙酸6.1 N(Sigma-Aldrich,目录号:T0699)
  15. 尿酸酶(-20°C,12个月)(西格玛奥德里奇,目录号:U0880)
  16. 六水合氯化铁(BioBasic,目录号:FD0201)
  17. 硫酸亚铁(BioBasic,目录号:FB0461)
  18. 醋酸钠三水合物(BioBasic,目录号:SB0481)
  19. 乙酸,冰川(VWR,目录号:CA71006-424)
  20. TPTZ(Sigma-Aldrich,目录号:T1253) 
  21. NPD(Sigma-Aldrich,目录号:193992)
  22. 12.5%次氯酸钠(在黑暗中储存,12个月)(VWR,目录号:BDH7038-4L)
  23. 盐酸(HCl)(VWR,目录号:CA11020-884)
  24. 硫酸(VWR,目录号:CA11024-170)
  25. 氢氧化钠(NaOH)(VWR,目录号:BDH9292)
  26. 无水乙醇(商用醇,目录号:P016EAAN)
  27. 0.1 M丁基化羟基甲苯(BHT)溶液(见食谱)
  28. 15%三氯乙酸溶液(见食谱)
  29. 2 N NaOH溶液(见食谱)
  30. 2-硫代巴比妥酸溶液(见食谱)
  31. 0.25 M HCl溶液(见食谱)
  32. 尿酸酶原液(见食谱)
  33. Uricase工作解决方案(见食谱)
  34. 醋酸盐缓冲溶液(见食谱)
  35. 400 mM HCl盐溶液(参见食谱)
  36. 40 mM HCl溶液(见食谱)
  37. TPTZ解决方案(见食谱)
  38. 氯化铁溶液(见食谱)
  39. 30 mM硫酸亚铁储备液(参见食谱)
  40. FRAP试剂(见食谱)
  41. NPD储备液(见食谱)
  42. 0.6 M硫酸溶液(见食谱)
  43. 氧化剂溶液(见食谱)


  1. 100毫升容量瓶,A级(VWR,目录号:10124-072)
  2. 500毫升容量瓶,A级(VWR,目录号:10545-998)
  3. 1升容量瓶,A级(VWR,目录号:10546-000)
  4. 琥珀色瓶(VWR,目录号:89221-624)
  5. 冰浴(VWR,目录号:10146-190)
  6. 水浴(VWR,目录号:89501-464)
  7. 通风橱(VWR,目录号:89260-062)
  8. 磁力搅拌器(VWR,目录号:97042-634)
  9. 分析天平(VWR,目录号:10205-026)
  10. iMark Filter吸光度酶标仪(Bio-Rad,目录号:1681135)
  11. 用于酶标仪的515 nm滤光片(Bio-Rad,目录号:1681033)
    注意:如果没有515 nm滤光片,则510-520 nm范围内的替代滤光片就足够了。>
  12. 1100系列二元泵(Agilent Technologies,型号:G1312A)
  13. 1100系列自动进样器(Agilent Technologies,型号:G1313A)
  14. 1100系列柱温箱(Agilent Technologies,型号:G1316A)
  15. 1100系列可变波长检测器(Agilent Technologies,型号:G1315A)
  16. ODS2 250 x 4.6 mm5μmHPLC柱(Waters Corp.,目录号:PSS831915) 
  17. 涡旋混合器(VWR,目录号:10153-842)
  18. 摇晃干块(Eppendorf,VWR,型号:Thermomixer,目录号:CA11028-280)
  19. Barnstead E-Pure水系统(Barnstead,Thermo Fisher,型号:E-Pure)
  20. 冷冻离心机(Eppendorf,型号:5418R)
  21. 微量移液器(Eppendorf,目录号:2231000601)
  22. 分光光度计(Thermo Fisher,型号:Genesys10S)
  23. pH计(VWR,目录号:89231-662)
  24. 4°C冰箱(VWR,目录号:89239-070)
  25. -20°C冰柜(VWR,目录号:10819-408)
  26. -80°C冰箱(VWR,目录号:10160-740)


  1. OpenLab色谱数据系统(Agilent Technologies, http://www.agilent.com/
  2. Microplate Manager 6(Bio-Rad Laboratories Canada Ltd., http://www.biorad.com/


  1. MDA定量
    a。现场样品应在收集后直接在血细胞比容离心机中旋转,以分别储存血浆。我们建议将分离的血浆转移到0.5 ml离心管中进行储存。有关现场采样的更多详细信息,请参见Langille等,2018。 >
    b。在分析前将样品保存在-80°C,并在收集后一年内进行测试。 >
    c。一个HPLC系统和两名工作人员可以在一个工作日内处理校准曲线和大约20-30个血浆样本。  >

    1. 使用表1中列出的参数准备HPLC系统。 


    2. 通过将100mM 1,1,3,3-四乙氧基丙烷储备溶液加入100ml容量瓶中,用超纯水稀释至刻度,制备1.00mM MDA工作溶液。混合均匀。 
    3. 通过使用微量移液管连续稀释新制备的1.00mM 1,1,3,3-四乙氧基丙烷工作溶液来制备MDA校准。在每批样品之前准备新的校准曲线。见表2. 


    4. 将标准品存放在冰上直至使用。 
    5. 在冰上解冻血浆样品,并在3000 x g >和4°C的冷冻离心机中旋转1分钟,以收集管底部的血浆。 
    6. 准备标准的1.5 ml微量离心管,样品和标准品标签。
    7. 将20μl标准或血浆样品转移到标记的试管中。 
    8. 向每个试管中加入2μl丁基化羟基甲苯(BHT)溶液(参见配方)和5μl2NNaOH溶液(参见配方)。 
    9. 简单地涡旋管,然后转移到设定在60℃的摇动干燥块培养箱中30分钟并以400rpm搅拌。
    10. 完成后,将管子移到冰桶中,加入100μl15%三氯乙酸溶液沉淀蛋白质。 
    11. 将样品短暂涡旋,然后在冰浴中孵育5分钟。
    12. 将管在14,000 x g >和4°C离心10分钟以沉淀蛋白质。
    13. 小心地,在不干扰蛋白质沉淀的情况下,将100μl上清液转移到500μl螺旋盖微量离心管中。
    14. 在该试管中,还加入50μl2-硫代巴比妥酸衍生溶液(见食谱)。 
    15. 确保管上的盖子密封良好,置于100℃的干燥块培养箱中60分钟。
    16. 将试管从块中取出放入冰浴中,冷却后,以14,000 x g >和4°C离心5分钟。
    17. 将60μl衍生化溶液转移至微插入HPLC小瓶中。
    18. 除标准品外,将5μl衍生化溶液转移至合并的QC小瓶中。
    19. 在样品制备的12小时内将小瓶进行HPLC分析,一式三份。 

  2. FRAP检测
    b。 FRAP试剂对光敏感,在环境空气中不稳定。试剂必须在使用前直接制备。  >

    1. 通过在15ml离心管中用9ml蒸馏水稀释1ml 300mM原液来制备工作硫酸铁溶液。使用这种30 mM溶液在15 ml离心管中制备校准溶液(见表3)。

      表3. FRAP测定的硫酸铁标准品稀释表

    2. 获得标准的平底96孔板,并计划校准曲线的位置和样品的位置。我们希望每个板运行2条校准曲线以解决错误。 
    3. 将5μl等分试样的标准品或样品移液到每个待测孔中,然后用5μl含有尿酸酶的超纯水(1.00U / ml,参见配方)。确保两滴混合均匀。 
    4. 将该溶液在室温下在轨道振荡器上以100rpm孵育5分钟。
    5. 使用多通道移液器向每个孔中加入200μlFRAP试剂(参见食谱)。 
    6. 将平板放入酶标仪中,低速摇动30分钟,在软件上显示为“LO”。 
    7. 30分钟后,读取595nm处的吸光度与试剂空白。 

  3. HASC检测
    a。样品应在分析前储存在-80°C并在冰上融化。 >
    b。氧化剂溶液在空气中不稳定,应小心制备。 pH值的调节应在通风橱中进行,因为会产生少量的氯气。 >

    1. 如配方部分中那样准备氧化剂溶液。将瓶子用铝箔包裹,保护溶液免受光照,并迅速使用溶液。 
    2. 从氧化剂溶液中,通过稀释1x至2x,4x,8x,16x和32x稀释液来制备HClO的校准曲线。准备1毫升的每种溶液。 
    3. 向标准96孔平底微孔板中加入1μl血浆或标准和100μl超纯水。
      注意:我们希望每个板至少运行两条校准曲线,以解决可能发生的任何错误。  >
    4. 将板在400振荡器上以400rpm涡旋30秒。 
    5. 使用多通道移液器,仅在样品孔中加入100μl氧化剂溶液。
    6. 在轨道振荡器上将板在室温和100rpm下摇动10分钟。 
    7. 向板的每个孔中加入20μlNPD溶液(参见配方)。 
    8. 将板在轨道振荡器上在室温和100rpm下孵育1分钟。
    9. 读取515nm处孔的吸光度。


  1. MDA量化
    该软件自动整合3.00分钟时TBA 2 -MDA加合物的面积。导出此数据并使用它来准备校准曲线并量化样品。然后使用标准线性校准对其原始格式的平均三份重复数据进行定量(参见Langille et al。>,2018)。
  2. FRAP检测
    输出孔的吸光度数据并取一式三份孔的平均值。使用此数据准备线性校准和量化样品(参见Langille et al。>,2018)。
  3. HASC检测
    输出孔的吸光度数据并取一式三份孔的平均值。使用此数据准备线性校准和量化样品(参见Langille et al。>。,2018)。


  1. 2-硫代巴比妥酸受加拿大受控药物和物质法(CDSA)控制,因此需要获得许可才能获得并拥有该化合物。申请必须通过加拿大卫生部提交,以获得采购2-硫代巴比妥酸的授权。在美国,2-硫代巴比妥酸不受控制,可从Sigma-Aldrich购买。
  2. 重要的是要注意在整个实验过程中使用微量移液器,因此研究人员应接受良好的移液技术培训,并对移液器感到舒适。实施良好的技术至关重要,因为处理的解决方案非常少,因此如果移液器没有提供准确的体积,任何错误都会被放大。 
  3. 应使用良好的分析技术制备所有溶液。如果您计划为一项研究处理大量样本,建议为该研究准备一次库存,以尽量减少同一溶液的几种制剂之间的差异。 
  4. 每个紫外检测器或酶标仪的线性范围可根据型号,年龄等>而变化。在处理血浆样品之前,检查系统是否提供线性校准非常重要。 
  5. 用以下方式计算评估方法性能的品质因数:相对标准偏差(%RSD)是标准偏差除以相同样品的三个单独制剂(和每个制剂三次注射)的平均值。通过测量空白注射的噪声来计算检测限(LOD)和定量限(LOQ)。 LOD和LOQ分别定义为空白噪声水平的3倍和10倍。 
  6. 使用该方法,MDA校准曲线的线性度为1.25-20μM(%RSD = 0.32,n = 7)。 LOD和LOQ分别为0.06和0.125μM。批间变异(%RSD = 9.31,n = 48)代表实验室测量误差。我们没有获得QC样品随时间的显着增加(β=0.0195μM/小时,r 2 = 0.017,n = 8)。 QC量化与每天评估的所有个体的平均值相比,显示对不同分析天数的结果几乎没有影响(斜率= 0.99,r 2 = 0.98,n = 9) 。示例色谱图显示在Langille et al。>,2018中。 
  7. 对于开发的不依赖于尿酸的FRAP测定,校准曲线的线性度(0.5-3mM FeSO 4 )高(%RSD = 2.50,n = 7)。批间变异(%RSD)为5.82(n = 57)。 
  8. 适应性HASC测定的校准曲线的线性范围为0.03-0.5mM HClO,高(%RSD = 3.64,n = 4)。批间变异(%RSD)为2.03(n = 32)。 
  9. 如果您使用的是基于单色仪的分光光度计或酶标仪,您只需使用本文中建议的波长进行测量即可。


  1. 0.1M丁基化羟基甲苯(BHT)溶液
    1. 将2.20g丁基化羟基甲苯溶于100ml无水乙醇中
    2. 该解决方案可能需要短暂的超声处理以帮助快速溶解
    3. 储存:RT,保质期:3个月
  2. 15%三氯乙酸溶液
    1. 在通风橱中,将15 ml 6.1 N(100%w / v)三氯乙酸溶液缓慢加入85 ml超纯水中
    2. 简单地涡旋混合
    3. 储存:RT,保质期:3个月
  3. 2 N NaOH溶液
    1. 将8g NaOH颗粒溶解在100ml超纯水中
    2. 用磁力搅拌板搅拌直至颗粒完全溶解
    3. 储存:RT,保质期:3个月
  4. 2-硫代巴比妥酸溶液
    1. 将0.375g 2-硫代巴比妥酸溶于100ml 0.25M HCl中
    2. 在50℃的水浴中加热10分钟,然后超声处理5分钟以帮助快速溶解
    3. 储存:4°C,保质期:3个月
  5. 0.25 M HCl溶液
    1. 在通风橱中,将10.4 ml 12 M(浓)HCl缓慢加入500 ml容量瓶中,装有300 ml超纯水
    2. 旋转混合并稀释至标记
    3. 储存:RT,保质期:3个月
  6. Uricase股票解决方案
    1. 在冰上孵育15ml超纯水以冷却,然后制备溶液。使用尿酸酶提供的分析证书,计算准备1,000 U / ml储备所需的质量
    2. 使用分析天平,在分析天平上仔细称重粉末至最接近的0.1 mg
    3. 使用微量移液管加入1ml预先冷却的水,并通过缓慢地上下移液来溶解。不要涡旋!酶的溶液不应该被涡旋
    4. 储存:-20°C,保质期:12个月
  7. Uricase工作解决方案
    1. 使用微量移液管将1μl的1,000 U / ml加入到999μl预冷的超纯水中,稀释Uricase原液
    2. 不要涡旋,轻轻颠倒管子几次混合 
    3. 储存:冰上,保质期:每日准备
  8. 醋酸盐缓冲溶液 
    1. 在通风橱中,将500毫升超纯水加入1升容量瓶中。然后缓慢加入16毫升冰醋酸,接着加入2.70克三水合乙酸钠
    2. 使用磁力搅拌器混合溶液并稀释至烧瓶上标记下方约10ml
    3. 使用已校准pH值为4和7的台式pH计检查pH值.pH值应为3.6。如果pH高于3.6,则通过逐滴加入乙酸酸化溶液。如果pH值低于3.6,则加入醋酸钠提高pH值。 
    4. 储存:4°C,保质期:1个月
  9. 400 mM HCl盐溶液 
    1. 在通风橱中,向含有300ml超纯水的500ml容量瓶中缓慢加入16.5ml浓HCl
    2. 旋转混合并稀释至标记
    3. 储存:RT,保质期:6个月
  10. 40 mM HCl溶液 
  11. TPTZ解决方案
    1. 在100ml容量瓶中,将0.3123g TPTZ溶于100ml如上制备的40mM HCl中
    2. 可能需要将溶液置于50°C水浴中以帮助溶解
    3. 储存:4°C,保质期:1个月
  12. 氯化铁溶液 
    1. 在通风橱中,向1L容量瓶中加入500ml 40mM HCl,加入5.41g六水合氯化铁,在磁力搅拌板上搅拌直至溶解。
    2. 用40mM HCl将溶液稀释至刻度。需要酸来防止亚铁离子的水解和沉淀
    3. 溶液应储存在琥珀色瓶中,最长可保存两个月。如果发现形成红褐色沉淀,则必须处理溶液并再次进行准备
  13. 30 mM硫酸亚铁储备溶液 
    将0.834克硫酸亚铁七水合物溶于100毫升超纯水中 储存:4°C,保质期:1个月
  14. FRAP试剂 
    1. 使用前将以下物质直接混合在冰上的50 ml离心管中:
      2毫升TPTZ sol'n
      2毫升FeCl 3 sol'n
      2.4ml dH 2 O.
    2. 通过涡旋混合试剂。溶液应为稻草色,如果是蓝色,则丢弃并再次准备
    3. 储存:冰浴,保质期:1-2小时
  15. NPD库存解决方案
    1. 移取83μl纯NPD到含有50ml无水乙醇的50ml离心管中
    2. 用铝箔包裹管子以防止光线照射。使用涡旋混合器混合
    3. 储存:4°C,保质期:3个月
  16. 0.6 M硫酸溶液
    1. 在通风橱中,加入16.6毫升浓缩液。将硫酸缓慢加入装有300ml超纯水的容量瓶中
    2. 稀释至标记并通过反转混合
    3. 储存:RT,保质期:3个月
  17. 氧化剂溶液
    1. 打开分光光度计,让灯泡在启动前保温30分钟。在通风橱中,向含有300毫升超纯水的500毫升容量瓶中加入1毫升10%次氯酸钠
    2. 稀释至标记并通过反转混合
    3. 将100 ml此溶液转移至250 ml培养瓶中,插入已在pH 4和7下校准的pH探针。在适度磁力搅拌下,小心地加入0.6 M硫酸溶液直至pH达到6.2
    4. 使用1 cm光程的石英比色皿,测量所得溶液的吸光度与292 nm处水空白的吸光度
    5. 使用以下公式应用Beer-Lambert定律:

      A =ε×l×c > >
      其中,HClO的摩尔吸光系数(ε)为350 L mol -1 cm -1 ,细胞的光程(l)为1 cm,吸光度( A)在292nm处读数。然后可以解决HClO的浓度(c)。


我很感谢Patrick Bergeron,Vincent Lemieux,Dany Garant和DenisRéale在该领域的样品采集。这项研究由加拿大自然科学和工程研究委员会(NSERC)资助PB和DG以及魁北克生物多样性科学中心(QCBS)种子资助PB和DG。 VL得到了魁北克省自然科学基金会(FRQNT)的奖学金资助。




道德批准来自加拿大动物保护委员会(#A2016-01-Bishop's University)和魁北克省自然保护区(#2017-05-01-102-05-S-F)。


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引用:Langille, E. A. (2019). Optimized Oxidative Stress Protocols for Low-microliter Volumes of Mammalian Plasma. Bio-protocol 9(9): e3221. DOI: 10.21769/BioProtoc.3221.