参见作者原研究论文

本实验方案简略版
Jun 2016

本文章节


 

This protocol has been corrected.See the correction notice

Two Different Methods of Quantification of Oxidized Nicotinamide Adenine Dinucleotide (NAD+) and Reduced Nicotinamide Adenine Dinucleotide (NADH) Intracellular Levels: Enzymatic Coupled Cycling Assay and Ultra-performance Liquid Chromatography (UPLC)-Mass Spectrometry
定量氧化烟酰胺腺嘌呤二核苷酸(NAD+)和还原型烟酰胺腺嘌呤二核苷酸(NADH)细胞内水平的两种不同方法:酶联偶合循环测定和超高效液相色谱(UPLC)-质谱分析法   

引用 收藏 提问与回复 分享您的反馈 Cited by

Abstract

Current studies on the age-related development of metabolic dysfunction and frailty are each day in more evidence. It is known, as aging progresses, nicotinamide adenine dinucleotide (NAD+) levels decrease in an expected physiological process. Recent studies have shown that a reduction in NAD+ is a key factor for the development of age-associated metabolic decline. Increased NAD+ levels in vivo results in activation of pro-longevity and health span-related factors. Also, it improves several physiological and metabolic parameters of aging, including muscle function, exercise capacity, glucose tolerance, and cardiac function in mouse models of natural and accelerated aging.

Given the importance of monitoring cellular NAD+ and NADH levels, it is crucial to have a trustful method to do so. This protocol has the purpose of describing the NAD+ and NADH extraction from tissues and cells in an efficient and widely applicable assay as well as its graphic and quantitative analysis.

Keywords: NAD+ levels (NAD+水平), NADH (烟酰胺腺嘌呤二核苷酸), Cycling assay (循环测定), NAD+ degradation (NAD+ 降解), Fluorescence (荧光), Mass spectroscopy (质谱法), Aging (老化), Metabolism (新陈代谢), UPLC (UPLC)

Background

Oxidized Nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide (NAD+ and NADH) are important biological cofactors that donate and accept electrons in several anabolic and catabolic functions. They participate in reactions such as glycolysis, the tricarboxylic acid cycle, and oxidative phosphorylation. In addition, it serves as a substrate for several enzymes involved in DNA damage repair, such as the sirtuins and poly (ADP-ribose) polymerases (PARPs) (Imai and Guarente, 2014; Verdin, 2015; Yoshino et al., 2018).

NAD+ levels decrease during aging and are involved in age-related metabolic decline. It has been shown that the cellular NAD pool is determined by a balance between the activity of NAD-synthesizing and NAD-consuming enzymes (Aksoy et al., 2006; Barbosa et al., 2007; Yang et al., 2007; Nahimana et al., 2009; Bai et al., 2011; Yoshino et al., 2011). In previous publications, our laboratory has demonstrated that expression and activity of the NADase CD38 increases with age and that CD38 is required for the age-related NAD decline and mitochondrial dysfunction via a pathway mediated at least in part by regulation of SIRT3 activity (Camacho-Pereira et al., 2016). We also identified CD38 as the main enzyme involved in the degradation of the NAD precursor nicotinamide mononucleotide (NMN) in vivo. That indicates that CD38 has a key role in the modulation of NAD-replacement therapy for aging and metabolic diseases (Camacho-Pereira et al., 2016). CD38 was originally identified as a cell-surface enzyme that plays a key role in several physiological processes such as immune response, inflammation, cancer, and metabolic disease (Frasca et al., 2006; Barbosa et al., 2007; Guedes et al., 2008; Malavasi et al., 2008).

Several different assays and methods have been described due to the great importance of monitoring NAD+ and NADH cellular levels under various physiological conditions. Specifically two of them, ultra-performance liquid chromatography (UPLC)-mass spectroscopy assay and cycling assay have finality and both are equally sensitive and specific (Camacho-Pereira et al., 2016).

Our laboratory also further optimized and validated the cycling assay. We determined NAD+ and NADH specific and does not detect any of the other nucleotides or NAD derivatives tested, including nicotinamide adenine dinucleotide phosphate (NADP), nicotinic acid adenine dinucleotide (NAAD), nicotinic acid adenine dinucleotide phosphate (NAADP), cyclic-adenine diphosphate ribose (cADPR), adenine triphosphate (ATP), ADP, and others. The results obtained with both methods confirm that there is indeed a decrease in levels of both NAD+ and NADH in murine tissues during chronological aging (Camacho-Pereira et al., 2016). Furthermore, both techniques correlated well for both nucleotides (correlation coefficient of r = 0.95 for NAD+ and 0.97 for NADH) (Camacho-Pereira et al., 2016).

In regards to the cycling assay (pathway in which the main reaction happens), it takes NAD+ and NADH present in the samples and they are coupled to both enzymes alcohol dehydrogenase (ADH) and diaphorase in a cycling assay. Every time NAD+ or NADH cycles, it produces a molecule of resorufin, which is highly fluorescent. This fluorescence is directly captured by the multi-well fluorescence plate reader therefore indirectly the NAD+ levels can be measured. Most studies have relied on separated extractions for NAD+ and reduced nicotinamide adenine dinucleotide (NADH) determination: a basic extraction for the reduced species and a separate acidic extraction for the oxidized species (Ashrafi et al., 2000; Smith et al., 2000; Lin et al., 2001; Anderson et al., 2002; Lin et al., 2004). The extraction conditions are specific for the stabilization of either oxidized compounds, which are more stable in acid or reduced compounds, which are more stable in base.

Conversely, the ultra-performance liquid chromatography (UPLC)-mass spectrometry is an analytical chemistry technique that combines the physical separation capabilities of liquid chromatography with the mass analysis capabilities of mass spectrometry (MS). MS systems are popular in chemical analysis because the individual capabilities of each technique are enhanced synergistically. While liquid chromatography separates mixtures with multiple components, mass spectrometry provides identity of the individual components with high molecular specificity and detection sensitivity. This tandem technique can be used to analyze very accurate components such as NAD+ and NADH (Dass, 2007).


Part I: Cycling assay

Materials and Reagents

  1. Plastic tips 10 (Thermo Fisher Scientific, Molecular Bioproducts, catalog number: 3511-05 )
  2. Plastic tips 1,000 (Thermo Fisher Scientific, Molecular Bioproducts, catalog number: 3101 )
  3. Plastic tips 200 (Thermo Fisher Scientific, Molecular Bioproducts, catalog number: 3551 )
  4. 1.5 ml Microcentrifuge tubes (USA Scientific, catalog number: 1415-2508 )
  5. 2.0 ml MCT Graduated tubes (Fisher Scientific, Fisherbrand, catalog number: 05-408-146 )
  6. 96-well Microfluor 1White flat-bottom plate (Thermo Fisher Scientific, catalog number: 7705 )
  7. Combitips Advanced 10 ml (Eppendorf, catalog number: 0030089464 )
  8. Disposable cell lifter (Fisher Scientific, Fisherbrand, catalog number: 08-100-240 )
  9. Cells of interest: any cell can be used to measure NAD+/NADH levels
  10. Tissues of interest: any tissue can be used to measure NAD+/NADH levels
  11. Trichloroacetic Acid (TCA) (Sigma-Aldrich, catalog number: T4885-500G )
  12. Sodium hydroxide (NaOH) 10 N (Fisher Scientific, Fisher Chemical, catalog number: SS267 )
  13. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E5134 )
  14. Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad Laboratories, catalog number: 5000006 )
  15. 1,2,2-Trichlorotrifluoroethane (TCTFE) (Fisher Scientific, Fisher Chemical, catalog number: T1781 )
  16. Trioctylamine (Sigma-Aldrich, catalog number: T81000-500G )
  17. Tris base, Trizma base (Sigma-Aldrich, catalog number: T6066 )
  18. Diaphorase (Sigma-Aldrich, catalog number: D5540-500UN )
  19. Alcohol dehydrogenase (ADH) (Sigma-Aldrich, catalog number: A3263-75KU )
  20. Activated charcoal (Sigma-Aldrich, catalog number: C7606-125G )
  21. Sodium phosphate monobasic (NaH2PO4) (Sigma-Aldrich, catalog number: S0751-500G )
  22. Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: S0876-500G )
  23. Absolute Ethanol (Fisher Scientific, Fisher BioReagentsTM, catalog number: BP2818-500 )
  24. Bovine Serum Albumin (Sigma-Aldrich, catalog number: A7906 )
  25. β-NAD sodium salt (Sigma-Aldrich, catalog number: N0632-1G )
  26. Riboflavin 5’-monophosphate sodium salt hydrate (FMN) (Sigma-Aldrich, catalog number: F8399 )
  27. Resazurin sodium salt (Sigma-Aldrich, catalog number: 199303-5G )
  28. Phosphate-buffered saline (PBS – pH 7.4) (Thermo Fisher Scientific, GibcoTM, catalog number: 10010023 )
  29. Organic solvent (see Recipes)
  30. Sodium Phosphate Buffer (pH 8.0) (see Recipes)
  31. 20 mM Sodium Phosphate Buffer (pH 7.0) (see Recipes)
  32. 4% charcoal suspension (in 20 mM sodium phosphate, pH 7.0) (see Recipes)
  33. Bradford Dye (see Recipes)
  34. Tris Buffer (1 M, pH 8.0) (see Recipes)

Equipment

Note: The brands and models indicated are the ones used by our group, similar equipment can be used as well.

  1. 1 L volumetric flask
  2. Pipettes (10, 200, 1,000 µl)
  3. Repeat Pipette (Eppendorf, model: Repeater® M4 )
  4. Scissors
  5. Homogenizer, Tissue Tearor (Bio Spec Products, catalog number: 780CL-04 )
  6. Microcentrifuge (Eppendorf, model: 5424 )
  7. Scale (Mettler-Toledo International, model: AG104 )
  8. Sonic Dismembrator (Fisher Scientific, model: Model 100 )
  9. Vortex (Scientific Industries, model: Vortex-Genie 2 , catalog number: G560)
  10. -80 °C freezer
  11. Plate reader (Molecular Devices, model: SpectraMax® GeminiTM XPS )
  12. Spectrophotometer (BioTek Instruments, model: Epoch 2 )

Software

  1. Gen5 Microplate Reader and Imager Software (BioTek Instruments)
  2. Microsoft Excel (Microsoft Corporation)
  3. SoftMax Pro 6 (Molecular Devices, LLC)
  4. GraphPad Prism 7 (GraphPad Software, Inc)

Procedure

  1. Sample preparation
    If using cells, use at least 3 x 106 cells. Aspirate media from plate, wash with PBS, collect the cells scratching the plate using disposable cell lifter, and pellet by centrifugation (30 sec, at 2,000 x g). Aspirate supernatant, and proceed to Step A1. If measuring NAD+ from tissue samples cut approximately 20 mg of tissue and put the piece in a 2 ml tube. Then proceed to Step A1. For all the following steps, samples should be kept on ice.
    Considering either NAD+ or NADH quantification, the only step that distinguishes those two is the extraction part. From Step A2 on, the process is the same for both NAD+ and NADH.
    1. Extraction:
      1. For NAD+: start extraction of NAD+ by adding 10% TCA (diluted in water).
      2. For NADH: NADH is extracted with 500 mM NaOH and 5 mM EDTA.
      Note: If using cells, add 250 µl of TCA; if using tissue, add 300 µl of TCA.
    2. Homogenize tissue samples with scissors and mechanical homogenizer until there are no visible chunks. Keep samples always on ice during this process.
      Note: Skip this step if using cells.
    3. Sonicate samples 3 times for 5 sec each. Configure equipment on medium power.
      Note: For NADH: at this point, heat for 30 min at 60 °C.
    4. Pellet protein by centrifuging at 11,500 x g for 2 min, 4 °C. Transfer supernatant to a new 1.5 ml tube and use it for Step A6.
    5. Save pellet for later protein determination. Re-suspend pellet in NaOH:
      1. Cells: 0.2 M NaOH, volume based on pellet size; around 100 µl (always better to use a lower volume and dilute the protein sample after, than to use a larger volume and dilute it too much). Samples should be kept on ice or a rocker platform at 4 °C until it is completely soluble.
      2. Tissue: 1 M NaOH, at least 300 µl. Leave samples on a rocker at 4 °C overnight and then measure protein concentration.
      3. We measure the protein concentration by the Bradford protein assay.
    6. Remove TCA using organic solvent
      1. Organic solvent: prepare a fresh solution of TCTFE:Trioctylamine (3:1 ratio). Use only plastic tips and tubes (TCTFE = 1,1,2-trichloro-1,2,2-trifluoroethane).
      2. Add organic solvent to remove TCA (supernatant of Step A4) at a ratio of 2:1.
      3. Vortex vigorously for 15 sec allowing mixture to separate at room temperature for 3-5 min.
      4. Carefully remove the top aqueous layer containing the NAD+ (100 µl) with a plastic pipette (Figure 1), put it in a new 1.5 ml tube, and add 10 µl of 1 M Tris to adjust the pH to 8.0.
        Note: If by mistake the bottom organic layer is perturbed or aspirated, put it back, and repeat Steps A6c and A6d.
      5. Put samples on ice and proceed to enzyme preparing steps. At this point, samples can be frozen in a -80 °C freezer and procedure can be resumed at another time, although it is advised to proceed with the procedure and reading, if possible, to minimize risk of degradation in freeze-thaw cycles.


        Figure 1. Removing top aqueous layer

    7. At this step, dilute NAD+ samples in sodium phosphate buffer considering the range of the standard curve. For all the samples NAD+/NADH measurement has to be within the curve. The dilution varies depending on the type and size of tissues or cells. It is basically empirical. Usually for 20 mg tissues, the dilutions range from 1:500 to 1:1,000. For cells, the dilution usually ranges from 1:50 to 1:200. Remember this is not rigid; sometimes a lower or higher dilution is needed. Dilute NAD+ samples, to a final volume of at least 250 µl.

  2. Preparing NAD+ standard curve
    1. In order to obtain a final NAD+ 1 µM concentration, it is necessary to perform a serial dilution as indicated below:
      1. In a 1.5 ml microcentrifuge tube, weigh β-NAD sodium salt (around 1 mg).
      2. Using the β-NAD sodium salt molecular weight find the specific volume to get a solution of 10 mM.
      3. To get the intermediate concentration of 10 µM, dilute the 10 mM 1:1,000 in sodium phosphate buffer.
      4. Finally, to get the solution concentration of 1 µM, dilute the 10 µM 1:10 in sodium phosphate buffer.
    2. To obtain the standard curve, dilute the 1 µM stock of NAD+ in sodium phosphate buffer following Table 1 below.

      Table 1. NAD+ Standard Curve


  3. Preparing the enzymes
    1. Based on Table 2:
      Stock for ADH has to be 3400 U/ml.
      Stock for Diaphorase has to be 100 U/ml.
    2. Weigh the enzymes in a 1.5 ml tube for preparation of 1 ml solution. Check the amount of U/mg in the bottle being used as it might change depending on the lot number.
      Notes:
      1. Usually, we prepare 1 ml of each enzyme solution and divide in smaller aliquots in a size enough to prepare at least 1 plate (96 wells), in order to avoid thawing the whole amount each time.
      2. We suggest preparing approximately 3-4% more volume (100 wells instead of 96 wells for entire plate), considering that some volume can be lost when moving the solution to another tube (e.g., attached to the tube walls, to the pipet tips).
    3. Remove bound NAD+ from Diaphorase and ADH by diluting the enzymes in 30% (300 µl) of sodium phosphate buffer and 70% (700 µl) of 4% charcoal suspension for a total of 1 ml solution. Gently mix the solution, and incubate it for 40 min at 37 °C. Mix gently every 10 min.
    4. Remove charcoal by centrifuging for 5 min, 4 °C, and 13,500 x g. Transfer supernatant to a new tube and centrifuge for 2 min, 4 °C, and 13,500 x g.

  4. Preparing the reaction mix
    1. Thaw FMN on ice, and bring it to room temperature.
    2. Whenever possible, prepare a Resazurin stock of 20 mM in water and use it on the same day (protect it from light until use).
      Note: According to manufacturer’s instructions, small aliquots of the Resazurin stock can be stored at -20 °C for up to 1 month (stock solutions should be protected from exposure to light). When needed to be used, thaw it on ice.
    3. Prepare the reaction mix following Table 2 below, respecting the amount and the order in which each component has to be added.

      Table 2. Reaction mix preparation


  5. Setting up the plate
    1. Add 100 µl of sample/standards into wells of a 96-well opaque plate.
    2. Add 100 µl of reaction mix to each well with a repeat pipet. Put the 96-well plate into a fluorescence plate reader. Monitor for 60 min with an excitation wavelength of 544 nm and an emission wavelength of 590 nm (Video 1).

      Video 1. Setting up the plate reader

    3. On template editor, configure dilution and unit for NAD+ Standards, and dilution of samples (Figure 2).


      Figure 2. Plate layout

Data analysis

  1. Data needed: NAD+ values obtained in fluorescence plate reader (Figure 3A), protein concentration, volume of TCA 10% and NaOH used.
  2. Calculate NAD+ concentration in nmol by dividing the NAD+ value obtained (in µM) by the volume of TCA (in ml).
  3. Calculate the mass of protein (mg) present in the sample by dividing protein concentration (mg/ml) by volume of NaOH (ml) in which the pellet was re-suspended.
  4. Divide the NAD+ concentration obtained in Step 2 by the mass of protein obtained in Step 3. This is the final result (Figure 3B).
  5. Plot the results (nM/mg protein) in a graph.
  6. Perform statistical analysis as needed.


    Figure 3. Data analysis. A. Raw data from plate reader; the encircled detail shows the adjusted result based on dilution. B. Example of final graph.

Recipes

  1. Organic solvent
    Prepare a fresh solution of TCTFE:Trioctylamine (3:1 ratio)
    Use only plastic tips and tubes (TCTFE = 1,1,2-trichloro-1,2,2-trifluoroethane)
  2. 100 mM Sodium Phosphate Buffer (pH 8.0)
    In a 1 L volumetric flask:
    Add 93.2 ml of 1 M Na2HPO4 followed by 6.6 ml of 1 M NaH2PO4
    Bring the volume up to 1 L
    The buffer can be stored up to 1 month at 4 °C
  3. 20 mM Sodium Phosphate Buffer (pH 7.0)
    Use the 100 mM Sodium Phosphate Buffer (pH 8.0) to make a 1:5 dilution
    For a final volume of 500 ml, add 100 ml of 100 mM Sodium Phosphate Buffer (pH 8.0) to a beaker
    Adjust to pH 7.0 using 1 N HCl solution
    Bring the volume up to 500 ml using a graduated cylinder
    Store at room temperature
  4. 4% charcoal suspension (in 20 mM sodium phosphate, pH 7)
    Weigh 4 g of charcoal
    Transfer it to a graduated cylinder and add 100 ml of 20 mM Sodium Phosphate Buffer (pH 7.0)
    Store at room temperature
  5. Bradford Dye
    Dilute Bio-Rad Protein Assay Dye Reagent Concentrate 1:5 in water
    The diluted reagent may be used for approximately 2 weeks when kept at room temperature
  6. Tris Buffer (1 M, pH 8.0)
    Put 8 ml of distilled water in a suitable container
    Add 1.21 g of Tris base to the solution
    Adjust the solution pH to 8.0 with HCl
    Add distilled water until the volume is 10 ml

Part II: Ultra-performance liquid chromatography (UPLC)-mass spectrometry

Materials and Reagents

  1. Agilent Poroshell 120 EC-C18 pre-column (Agilent Technologies, catalog number: 693775-902 )
  2. Agilent Poroshell 120 EC-C18 analytical column (Agilent Technologies, catalog number: 697975-902T )
  3. Formic acid (Fisher Scientific, catalog number: A117-50 )
  4. Acetonitrile (Sigma-Aldrich, catalog number: 271004 )
  5. NAD (Sigma-Aldrich, catalog number: N3014 )
  6. NADH (Sigma-Aldrich, catalog number: N6005 )
  7. n-cyclohexyl benzamide (Internal Standard - IS) (Sigma-Aldrich, catalog number: R531332 )
  8. Auto-sampler vials (Chrom Tech, catalog number: CTI-9405 )
  9. Slick microfuge tubes (Fisher Scientific, FisherbrandTM, catalog number: 02-681-320 )
  10. Primary stock solutions (see Recipes)

Equipment

  1. LC-MS/MS system
    Note: The LC-MS system consists of a Waters ACQUITY H class ultra-performance liquid chromatography (UPLC) system, containing a quaternary solvent manager and sample manager-FTN coupled to a Xevo TQ-S mass spectrometer (Waters, Milford, MA) equipped with electrospray ionization (ESI) source.

Software

  1. Waters MassLynx v4.1 software

Procedure

  1. The liquid chromatographic separation of NADH and NAD+ is accomplished using an Agilent Poroshell 120 EC-C18 pre-column (2.1 x 5 mm, 2.7 μm, Chrom Tech, Apple Valley, MN) attached to an Agilent Poroshell 120 EC-C18 analytical column (2.1 x 100 mm, 2.7 μm, Chrom Tech, Apple Valley, MN) at 40 °C.
  2. Elute the column with a gradient mobile phase composed of water with 0.1% formic acid (A) and acetonitrile (ACN) with 0.1% formic acid (B) with a constant flow rate of 0.5 ml/min and a total run time of 10 min. The elution is initiated at 100% A and held for 2 min, then B is linearly increased from 0% to 60% B for 4 min, 60% B is held for 1 min, and returning to initial conditions in 2 min, finishing with 2.8 min at 100% A.
  3. Auto-sampler temperature is 4 °C and sample injection volume is 10 μl. NAD+ and NADH should be eluted for 1.6 and 3.5 min, respectively. None of the other NAD+ related metabolites (including NADP, NADPH, ATP, NAADP, NAAD, ADP, cADPR, ADPR, nicotinamide and nicotinic acid) are co-eluted with either NAD+ or NADH.
  4. Tissue samples are prepared as the same manner as described in “sample preparation” for the cycling assay. Samples should be thawed on ice and they are diluted in water containing 500 ng/ml of internal standard.
  5. Samples for NAD+ measurement diluted 1:1,000 and 1:10,000, whereas the ones for NADH samples are diluted 1:1,000 in slick microfuge tubes. After dilution, the samples should be vortexed and transferred to 2 ml auto-sampler vials for immediate analysis.

Data analysis

Data were acquired and analyzed by Waters MassLynx v4.1 software. Detection of NAD+ and NADH is accomplished using the mass spectrometer in positive ESI mode using capillary voltage 3.5 kV, source temp 150 °C, desolvation temp 500 °C, cone gas flow 150 L/h, desolvation gas flow 500 L/h, using multiple reaction monitoring (MRM) scan mode with a dwell time of 0.075 sec (Figure 4). The cone voltages and collision energies were determined by MassLynx- Intellistart, v4.1, software. Values are as follows:
NAD+–m/z 664.27 > 136.09–cone voltage 54 V–Collision 42 eV
NADH–m/z 666.28 > 514.17–cone voltage 56 V–Collision 26 eV
n-Cyclohexyl benzamide–m/z 204.1 > 122.00–Cone voltage 80 V–Collision 20 eV


Figure 4. LC-MS tracing analysis

Recipes

  1. Primary stock solutions
    The primary stock solutions were stored at -80 °C and were prepared in silanized glass vials as follows:
    NAD+ (10 mg/ml in water)
    NADH (1 mg/ml in 0.01 N NaOH, pH 11.9)
    n-cyclohexyl benzamide (IS) (100 µg/ml in EtOH)
    For working standards:
    Working standards were prepared by dilution of the stock solution into the same solutions mentioned above. Daily standard samples were prepared by diluting above stocks 1:20 in water containing 500 ng/ml internal standard

Acknowledgments

This work was supported in part by grants from the Ted Nash Long Life Foundation, the Glenn Foundation for Medical Research via the Paul F. Glenn Laboratories for the Biology of Aging at the Mayo Clinic, a grant from Calico Laboratories, the Mayo Foundation, National Institutes of Health (NIH) grants from the National Institute of Aging (NIA, grant No. AG-26094), the Pancreatic Cancer SPORE project from NIH/NCI to E.N.C (grant No. CA102701-08), the Mayo Clinic Center for Cell Signaling in Gastroenterology (NIDDK P30DK084567). Maria Auxiliadora-Martins was supported by a grant from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Brazil (#2016/20089-4).
Declaration of interests: Dr. Chini holds a patent on the use of CD38 inhibitors for metabolic diseases.

References

  1. Aksoy, P., White, T. A., Thompson, M. and Chini, E. N. (2006). Regulation of intracellular levels of NAD: a novel role for CD38. Biochem Biophys Res Commun 345(4): 1386-1392.
  2. Anderson, R. M., Bitterman, K. J., Wood, J. G., Medvedik, O., Cohen, H., Lin, S. S., Manchester, J. K., Gordon, J. I. and Sinclair, D. A. (2002). Manipulation of a nuclear NAD+ salvage pathway delays aging without altering steady-state NAD+ levels. J Biol Chem 277(21): 18881-18890.
  3. Ashrafi, K., Lin, S. S., Manchester, J. K. and Gordon, J. I. (2000). Sip2p and its partner snf1p kinase affect aging in S. cerevisiae. Genes Dev 14(15): 1872-1885.
  4. Bai, P., Canto, C., Oudart, H., Brunyanszki, A., Cen, Y., Thomas, C., Yamamoto, H., Huber, A., Kiss, B., Houtkooper, R. H., Schoonjans, K., Schreiber, V., Sauve, A. A., Menissier-de Murcia, J. and Auwerx, J. (2011). PARP-1 inhibition increases mitochondrial metabolism through SIRT1 activation. Cell Metab 13(4): 461-468.
  5. Barbosa, M. T., Soares, S. M., Novak, C. M., Sinclair, D., Levine, J. A., Aksoy, P. and Chini, E. N. (2007). The enzyme CD38 (a NAD glycohydrolase, EC 3.2.2.5) is necessary for the development of diet-induced obesity. FASEB J 21(13): 3629-3639.
  6. Camacho-Pereira, J., Tarrago, M. G., Chini, C. C. S., Nin, V., Escande, C., Warner, G. M., Puranik, A. S., Schoon, R. A., Reid, J. M., Galina, A. and Chini, E. N. (2016). CD38 dictates age-related NAD decline and mitochondrial dysfunction through an SIRT3-dependent mechanism. Cell Metab 23(6): 1127-1139.
  7. Dass, C. (2007). Chap. 5. Hyphenated separation techniques. Fundamentals of contemporary mass spectrometry, 1. John Wiley & Sons, Inc., 151-194.
  8. Frasca, L., Fedele, G., Deaglio, S., Capuano, C., Palazzo, R., Vaisitti, T., Malavasi, F. and Ausiello, C. M. (2006). CD38 orchestrates migration, survival, and Th1 immune response of human mature dendritic cells. Blood 107(6): 2392-2399.
  9. Guedes, A. G., Jude, J. A., Paulin, J., Kita, H., Lund, F. E. and Kannan, M. S. (2008). Role of CD38 in TNF-α-induced airway hyperresponsiveness. Am J Physiol Lung Cell Mol Physiol 294(2): L290-299.
  10. Imai, S. and Guarente, L. (2014). NAD+ and sirtuins in aging and disease. Trends Cell Biol 24(8): 464-471.
  11. Lin, S. J., Ford, E., Haigis, M., Liszt, G. and Guarente, L. (2004). Calorie restriction extends yeast life span by lowering the level of NADH. Genes Dev 18(1): 12-16.
  12. Lin, S. S., Manchester, J. K. and Gordon, J. I. (2001). Enhanced gluconeogenesis and increased energy storage as hallmarks of aging in Saccharomyces cerevisiae. J Biol Chem 276(38): 36000-36007.
  13. Malavasi, F., Deaglio, S., Funaro, A., Ferrero, E., Horenstein, A. L., Ortolan, E., Vaisitti, T. and Aydin, S. (2008). Evolution and function of the ADP ribosyl cyclase/CD38 gene family in physiology and pathology. Physiol Rev 88(3): 841-886.
  14. Nahimana, A., Attinger, A., Aubry, D., Greaney, P., Ireson, C., Thougaard, A. V., Tjornelund, J., Dawson, K. M., Dupuis, M. and Duchosal, M. A. (2009). The NAD biosynthesis inhibitor APO866 has potent antitumor activity against hematologic malignancies. Blood 113(14): 3276-3286.
  15. Smith, J. S., Brachmann, C. B., Celic, I., Kenna, M. A., Muhammad, S., Starai, V. J., Avalos, J. L., Escalante-Semerena, J. C., Grubmeyer, C., Wolberger, C. and Boeke, J. D. (2000). A phylogenetically conserved NAD+-dependent protein deacetylase activity in the Sir2 protein family. Proc Natl Acad Sci U S A 97(12): 6658-6663.
  16. Verdin, E. (2015). NAD+ in aging, metabolism, and neurodegeneration. Science 350(6265): 1208-1213.
  17. Yang, H., Yang, T., Baur, J. A., Perez, E., Matsui, T., Carmona, J. J., Lamming, D. W., Souza-Pinto, N. C., Bohr, V. A., Rosenzweig, A., de Cabo, R., Sauve, A. A. and Sinclair, D. A. (2007). Nutrient-sensitive mitochondrial NAD+ levels dictate cell survival. Cell 130(6): 1095-1107.
  18. Yoshino, J., Baur, J. A. and Imai, S. I. (2018). NAD+ intermediates: The biology and therapeutic potential of NMN and NR. Cell Metab 27(3): 513-528.
  19. Yoshino, J., Mills, K. F., Yoon, M. J. and Imai, S. (2011). Nicotinamide mononucleotide, a key NAD+ intermediate, treats the pathophysiology of diet- and age-induced diabetes in mice. Cell Metab 14(4): 528-536.

简介

目前关于代谢功能障碍和虚弱的年龄相关发展的研究每天都有更多的证据。众所周知,随着衰老的进展,烟酰胺腺嘌呤二核苷酸(NAD + )水平在预期的生理过程中降低。最近的研究表明,NAD + 的减少是与年龄相关的代谢衰退发展的关键因素。增加NAD + 水平体内导致激活寿命和健康跨度相关因素。此外,它改善了老化的几个生理和代谢参数,包括自然和加速老化的小鼠模型中的肌肉功能,运动能力,葡萄糖耐量和心脏功能。

鉴于监测细胞NAD + 和NADH水平的重要性,有一个值得信赖的方法是至关重要的。该方案的目的是在有效且广泛适用的测定中描述来自组织和细胞的NAD + 和NADH提取以及其图形和定量分析。

【背景】氧化烟酰胺腺嘌呤二核苷酸和还原型烟酰胺腺嘌呤二核苷酸(NAD + 和NADH)是重要的生物辅助因子,它们在几种合成代谢和分解代谢功能中提供和接受电子。它们参与诸如糖酵解,三羧酸循环和氧化磷酸化的反应。此外,它还作为DNA损伤修复中涉及的几种酶的底物,例如sirtuins和poly(ADP-核糖)聚合酶(PARP)(Imai和Guarente,2014; Verdin,2015; Yoshino et al。 ,2018)。

NAD + 水平在衰老过程中降低,并且与年龄相关的代谢下降有关。已经表明细胞NAD库是通过NAD合成活性和消耗NAD的酶之间的平衡来确定的(Aksoy et al。,2006; Barbosa et al。,2007; Yang et al。,2007; Nahimana et al。,2009; Bai et al。,2011; Yoshino 等人,2011)。在以前的出版物中,我们的实验室已经证明NADase CD38的表达和活性随着年龄的增长而增加,并且CD38是年龄相关的NAD下降和线粒体功能障碍所必需的,其通过至少部分通过调节SIRT3活性介导的途径(Camacho-) Pereira et al。,2016)。我们还将CD38鉴定为参与NAD前体烟酰胺单核苷酸(NMN)体内降解的主要酶。这表明CD38在调节NAD替代疗法治疗衰老和代谢疾病方面具有关键作用(Camacho-Pereira et al。,2016)。 CD38最初被鉴定为细胞表面酶,在许多生理过程中发挥关键作用,如免疫反应,炎症,癌症和代谢疾病(Frasca et al。,2006; Barbosa et al。,2007; Guedes et al。,2008; Malavasi et al。,2008)。

由于在各种生理条件下监测NAD + 和NADH细胞水平非常重要,已经描述了几种不同的测定和方法。具体来说,其中两个,超高效液相色谱(UPLC) - 质谱分析和循环分析具有最终性,两者同样敏感和特异(Camacho-Pereira 等,2016)。

我们的实验室还进一步优化和验证了循环测定。我们测定了NAD + 和NADH特异性,未检测到任何其他核苷酸或NAD衍生物,包括烟酰胺腺嘌呤二核苷酸磷酸(NADP),烟酸腺嘌呤二核苷酸(NAAD),烟酸腺嘌呤二核苷酸磷酸(NAADP),环腺嘌呤二磷酸核糖(cADPR),三磷酸腺嘌呤(ATP),ADP等。两种方法获得的结果证实,在时间老化过程中,小鼠组织中NAD + 和NADH的水平确实降低(Camacho-Pereira et al。,2016) )。此外,两种技术对于两种核苷酸均相关性良好(NAD + 的r = 0.95,NADH的相关系数为0.97)(Camacho-Pereira et al。,2016)。

关于循环测定(主要反应发生的途径),样品中存在NAD + 和NADH,并且它们在循环中偶联于醇脱氢酶(ADH)和心肌黄酶。检测。每当NAD + 或NADH循环时,它就会产生一种高度荧光的试卤灵分子。这种荧光直接由多孔荧光板读数器捕获,因此间接地可以测量NAD + 水平。大多数研究依赖于NAD + 的分离提取和降低的烟酰胺腺嘌呤二核苷酸(NADH)测定:还原物种的基本提取和氧化物种的单独酸性提取(Ashrafi et al 。,2000; Smith et al。,2000; Lin et al。,2001; Anderson et al。,2002; Lin et al。,2004)。萃取条件对于氧化化合物的稳定化是特定的,氧化化合物在酸或还原化合物中更稳定,其在碱中更稳定。

相反,超高效液相色谱(UPLC) - 质谱是一种分析化学技术,它结合了液相色谱的物理分离能力和质谱(MS)的质量分析能力。 MS系统在化学分析中很受欢迎,因为每种技术的个体能力都是协同增强的。虽然液相色谱法将混合物与多种组分分离,但质谱法可提供具有高分子特异性和检测灵敏度的各组分的特性。这种串联技术可用于分析非常准确的组件,如NAD + 和NADH(Dass,2007)。

关键字:NAD+水平, 烟酰胺腺嘌呤二核苷酸, 循环测定, NAD+ 降解, 荧光, 质谱法, 老化, 新陈代谢, UPLC


第一部分:循环测定

材料和试剂

  1. 塑料尖端10(Thermo Fisher Scientific,Molecular Bioproducts,目录号:3511-05)
  2. 塑料吸头1,000(Thermo Fisher Scientific,Molecular Bioproducts,目录号:3101)
  3. 塑料尖端200(Thermo Fisher Scientific,Molecular Bioproducts,目录号:3551)
  4. 1.5 ml Microcentrifuge试管(USA Scientific,目录号:1415-2508)
  5. 2.0毫升MCT刻度管(Fisher Scientific,Fisherbrand,目录号:05-408-146)
  6. 96孔Microfluor 1White平底板(赛默飞世尔科技,目录号:7705)
  7. Combitips Advanced 10 ml(Eppendorf,目录号:0030089464)
  8. 一次性细胞提升器(Fisher Scientific,Fisherbrand,目录号:08-100-240)
  9. 感兴趣的细胞:任何细胞都可用于测量NAD + / NADH水平
  10. 感兴趣的组织:任何组织都可用于测量NAD + / NADH水平
  11. 三氯乙酸(TCA)(西格玛奥德里奇,目录号:T4885-500G)
  12. 氢氧化钠(NaOH)10N(Fisher Scientific,Fisher Chemical,目录号:SS267)
  13. 乙二胺四乙酸(EDTA)(Sigma-Aldrich,目录号:E5134)
  14. Bio-Rad蛋白质分析染料试剂浓缩物(Bio-Rad Laboratories,目录号:5000006)
  15. 1,2,2-三氯三氟乙烷(TCTFE)(Fisher Scientific,Fisher Chemical,目录号:T1781)
  16. 三辛胺(Sigma-Aldrich,目录号:T81000-500G)
  17. Tris base,Trizma base(Sigma-Aldrich,目录号:T6066)
  18. Diaphorase(Sigma-Aldrich,目录号:D5540-500UN)
  19. 酒精脱氢酶(ADH)(西格玛奥德里奇,目录号:A3263-75KU)
  20. 活性炭(Sigma-Aldrich,目录号:C7606-125G)
  21. 磷酸二氢钠(NaH 2 PO 4 )(Sigma-Aldrich,目录号:S0751-500G)
  22. 磷酸氢二钠(Na 2 HPO 4 )(Sigma-Aldrich,目录号:S0876-500G)
  23. 绝对乙醇(Fisher Scientific,Fisher BioReagents TM ,目录号:BP2818-500)
  24. 牛血清白蛋白(Sigma-Aldrich,目录号:A7906)
  25. β-NAD钠盐(Sigma-Aldrich,目录号:N0632-1G)
  26. 核黄素5'-单磷酸钠盐水合物(FMN)(西格玛奥德里奇,目录号:F8399)
  27. 刃天青钠盐(Sigma-Aldrich,目录号:199303-5G)
  28. 磷酸盐缓冲盐水(PBS - pH 7.4)(Thermo Fisher Scientific,Gibco TM ,目录号:10010023)
  29. 有机溶剂(见食谱)
  30. 磷酸钠缓冲液(pH 8.0)(见食谱)
  31. 20 mM磷酸钠缓冲液(pH 7.0)(见食谱)
  32. 4%木炭悬浮液(20 mM磷酸钠,pH 7.0)(见食谱)
  33. Bradford Dye(见食谱)
  34. Tris缓冲液(1M,pH 8.0)(见食谱)

设备

注意:所示的品牌和型号是我们集团使用的品牌和型号,也可以使用类似的设备。

  1. 1升容量瓶
  2. 移液器(10,200,1,000μl)
  3. 重复移液器(Eppendorf,型号:Repeater ® M4)
  4. 剪刀
  5. 均质器,Tissue Tearor(Bio Spec Products,目录号:780CL-04)
  6. 微量离心机(Eppendorf,型号:5424)
  7. 规模(Mettler-Toledo International,型号:AG104)
  8. Sonic Dismembrator(Fisher Scientific,型号:Model 100)
  9. Vortex(科学工业,型号:Vortex-Genie 2,目录号:G560)
  10. -80°C冰柜
  11. 读板器(Molecular Devices,型号:SpectraMax ® Gemini TM XPS)
  12. 分光光度计(BioTek Instruments,型号:Epoch 2)

软件

  1. Gen5微孔板读板机和成像仪软件(BioTek仪器)
  2. Microsoft Excel(微软公司)
  3. SoftMax Pro 6(Molecular Devices,LLC)
  4. GraphPad Prism 7(GraphPad Software,Inc)

程序

  1. 样品制备
    如果使用细胞,则使用至少3 x 10 6 细胞。从平板吸出培养基,用PBS洗涤,收集细胞用一次性细胞提升器刮擦平板,并通过离心沉淀(30秒,在2,000 x g )。吸出上清液,然后进入步骤A1。如果从组织样品中测量NAD + ,则切下约20mg组织并将该片放入2ml管中。然后进入步骤A1。对于以下所有步骤,样品应保存在冰上。
    考虑NAD + 或NADH量化,区分这两者的唯一步骤是提取部分。从步骤A2开始,NAD + 和NADH的过程相同。
    1. 萃取:
      1. 对于NAD + :通过添加 10%TCA (在水中稀释)开始提取NAD + 。
      2. 对于NADH:用500mM NaOH和5mM EDTA提取NADH。
      注意:如果使用细胞,加入250μlTCA;如果使用组织,加入300μlTCA。
    2. 用剪刀和机械均化器将组织样品均化,直到没有可见的块状物。在此过程中始终将样品保存在冰上。
      注意:如果使用单元格,请跳过此步骤。
    3. 超声处理样品3次,每次5秒。配置中等功率的设备。
      注意:对于NADH:此时,在60°C加热30分钟。
    4. 通过在11,500 x g离心2分钟,4℃下离心沉淀蛋白质。将上清液转移到新的1.5 ml管中,并将其用于步骤A6。
    5. 保存颗粒以便以后测定蛋白质。将沉淀重悬于NaOH中:
      1. 细胞:0.2M NaOH,基于颗粒大小的体积;大约100μl(总是更好地使用较低的体积并稀释蛋白质样品后,而不是使用更大的体积并稀释太多)。样品应保存在冰或摇动平台上,温度为4°C,直至完全溶解。
      2. 组织:1M NaOH,至少300μl。将样品放在摇床上4°C过夜,然后测量蛋白质浓度。
      3. 我们通过Bradford蛋白质测定法测量蛋白质浓度。
    6. 使用有机溶剂去除TCA
      1. 有机溶剂:制备新鲜的TCTFE溶液:三辛胺(3:1比例)。仅使用塑料吸头和管(TCTFE = 1,1,2-三氯-1,2,2-三氟乙烷)。
      2. 加入有机溶剂以2:1的比例除去TCA(步骤A4的上清液)。
      3. 剧烈涡旋15秒,使混合物在室温下分离3-5分钟。
      4. 用塑料移液管小心地取出含有NAD + (100μl)的顶层水层(图1),将其放入新的1.5 ml管中,加入10μl1M Tris进行调整pH值为8.0。
        注意:如果错误地将底部有机层扰动或吸出,请将其放回原处,并重复步骤A6c和A6d。
      5. 将样品置于冰上并进行酶制备步骤。此时,样品可以在-80°C冰箱中冷冻,并且可以在另一时间恢复操作,但建议在可能的情况下继续进行操作和读数,以最大限度地降低冻融循环中降解的风险。


        图1.去除顶层水层

    7. 在该步骤中,考虑标准曲线的范围,在磷酸钠缓冲液中稀释NAD + 样品。对于所有样品,NAD + / NADH测量必须在曲线内。稀释度根据组织或细胞的类型和大小而变化。它基本上是经验性的。通常对于20mg组织,稀释度范围为1:500至1:1,000。对于细胞,稀释度通常为1:50至1:200。记住这不是僵硬的;有时需要更低或更高的稀释度。将NAD + 样品稀释至最终体积至少250μl。

  2. 准备NAD + 标准曲线
    1. 为了获得最终的NAD + 1μM浓度,必须进行如下所示的连续稀释:
      1. 在1.5 ml微量离心管中,称取β-NAD钠盐(约1 mg)。
      2. 使用β-NAD钠盐分子量找到比容,得到10 mM的溶液。
      3. 为了获得10μM的中间浓度,在磷酸钠缓冲液中稀释10 mM 1:1,000。
      4. 最后,为了使溶液浓度为1μM,在磷酸钠缓冲液中稀释10μM1:100。
    2. 为获得标准曲线,按照下表1稀释磷酸钠缓冲液中1μM的NAD + 原液。

      表1. NAD + 标准曲线


  3. 准备酶
    1. 基于表2:
      ADH的库存必须为3400 U / ml。
      Diaphorase的库存必须为100 U / ml。
    2. 称取1.5ml试管中的酶,制备1ml溶液。检查所用瓶子中的U / mg含量,因为它可能会根据批号而改变。
      注意:
      1. 通常,我们准备1毫升每种酶溶液,并分成足够大小的等分试样,以制备至少1个平板(96孔),以避免每次解冻整个量。
      2. 我们建议准备大约3-4%的体积(100个孔而不是整个板的96个孔),考虑到将溶液移动到另一个管(例如,连接到管壁,到移液器吸头)。
    3. 通过将酶在30%(300μl)的磷酸钠缓冲液和70%(700μl)的4%活性炭悬浮液中稀释至总共1ml溶液,从心肌黄酶和ADH中除去结合的NAD + 。轻轻混合溶液,在37°C下孵育40分钟。每10分钟轻轻混合。
    4. 通过离心5分钟,4℃和13,500 x g 除去木炭。将上清液转移到新管中,离心2分钟,4°C和13,500 x g 。

  4. 准备反应混合物
    1. 在冰上解冻FMN,并将其置于室温。
    2. 尽可能在水中制备20 mM的刃天青,并在同一天使用(保护它免受光照直至使用)。
      注意:根据制造商的说明,小剂量的刃天青原料可以在-20°C下储存长达1个月(应保护储备溶液避免暴露在光线下)。需要使用时,将其在冰上解冻。
    3. 按照下表2制备反应混合物,考虑每种组分的添加量和顺序。

      表2.反应混合物制备


  5. 设置板
    1. 将100μl样品/标准品加入96孔不透明板的孔中。
    2. 用重复移液管向每个孔中加入100μl反应混合物。将96孔板放入荧光板读数器中。监测60分钟,激发波长为544nm,发射波长为590nm(视频1)。

      视频1
    3. 在模板编辑器上,配置NAD + 标准品的稀释和单位,以及样品稀释(图2)。


      图2.板块布局

数据分析

  1. 所需数据:在荧光板读数器中获得的NAD + 值(图3A),蛋白质浓度,TCA的体积10%和使用的NaOH。
  2. 通过将获得的NAD + 值(以μM计)除以TCA的体积(以ml计),计算nmD中的NAD + 浓度。
  3. 通过将蛋白质浓度(mg / ml)除以其中重悬浮颗粒的NaOH(ml)的体积来计算样品中存在的蛋白质(mg)的质量。
  4. 将步骤2中获得的NAD + 浓度除以步骤3中获得的蛋白质质量。这是最终结果(图3B)。
  5. 将结果(nM / mg蛋白质)绘制在图表中。
  6. 根据需要进行统计分析。


    图3.数据分析。 A.来自读板器的原始数据;环绕的细节显示基于稀释的调整结果。 B.最终图表的例子。

食谱

  1. 有机溶剂
    准备新鲜的TCTFE溶液:三辛胺(3:1比例)
    仅使用塑料尖端和管(TCTFE = 1,1,2-三氯-1,2,2-三氟乙烷)
  2. 100 mM磷酸钠缓冲液(pH 8.0)
    在1升容量瓶中:
    加入93.2ml 1M Na 2 HPO 4 ,然后加入6.6ml 1M NaH 2 PO 4 < br /> 使音量达到1 L
    缓冲液可在4°C下储存长达1个月
  3. 20 mM磷酸钠缓冲液(pH 7.0)
    使用100 mM磷酸钠缓冲液(pH 8.0)进行1:5稀释 最终体积为500 ml时,向烧杯中加入100 ml 100 mM磷酸钠缓冲液(pH 8.0)
    使用1N HCl溶液调节至pH7.0 使用量筒将体积调至500毫升
    在室温下储存
  4. 4%木炭悬浮液(20mM磷酸钠,pH7)
    称重4克木炭
    将其转移至量筒并加入100ml 20mM磷酸钠缓冲液(pH7.0)
    在室温下储存
  5. 布拉德福德染料
    稀释Bio-Rad蛋白质分析染料试剂浓缩液1:5在水中
    当保持在室温下时,稀释的试剂可以使用约2周
  6. Tris缓冲液(1M,pH 8.0)
    将8毫升蒸馏水放入合适的容器中 向溶液中加入1.21克Tris碱 用HCl调节溶液的pH值至8.0 加入蒸馏水直至体积为10毫升

第二部分:超高效液相色谱(UPLC) - 质谱法

材料和试剂

  1. Agilent Poroshell 120 EC-C18预柱(Agilent Technologies,目录号:693775-902)
  2. Agilent Poroshell 120 EC-C18分析柱(Agilent Technologies,目录号:697975-902T)
  3. 甲酸(Fisher Scientific,目录号:A117-50)
  4. 乙腈(Sigma-Aldrich,目录号:271004)
  5. NAD(Sigma-Aldrich,目录号:N3014)
  6. NADH(Sigma-Aldrich,目录号:N6005)
  7. 正环己基苯甲酰胺(内标 - IS)(西格玛奥德里奇,目录号:R531332)
  8. 自动进样器样品瓶(Chrom Tech,目录号:CTI-9405)
  9. 光滑微量离心管(Fisher Scientific,Fisherbrand TM ,目录号:02-681-320)
  10. 主要原料解决方案(见食谱)

设备

  1. LC-MS / MS系统
    注意:LC-MS系统由Waters ACQUITY H级超高效液相色谱(UPLC)系统组成,包含四元溶剂管理器和样品管理器-FTN,与Xevo TQ-S质谱仪(Waters,Milford)相连,MA)配备电喷雾电离(ESI)源。

软件

  1. Waters MassLynx v4.1软件

程序

  1. NADH和NAD + 的液相色谱分离使用Agilent Poroshell 120 EC-C18预柱(2.1 x 5 mm,2.7μm,Chrom Tech,Apple Valley,MN)连接到Agilent Poroshell 120 EC-C18分析柱(2.1 x 100 mm,2.7μm,Chrom Tech,Apple Valley,MN),温度为40°C。
  2. 用含有0.1%甲酸(A)的水和含有0.1%甲酸(B)的乙腈(ACN)组成的梯度流动相洗脱色谱柱,流速恒定为0.5 ml / min,总运行时间为10 min 。洗脱在100%A下开始并保持2分钟,然后B从0%线性增加至60%B 4分钟,60%B保持1分钟,并在2分钟内恢复到初始条件,最后完成2.8分钟,100%A。
  3. 自动进样器温度为4°C,进样体积为10μl。 NAD + 和NADH应分别洗脱1.6和3.5分钟。其他NAD + 相关代谢物(包括NADP,NADPH,ATP,NAADP,NAAD,ADP,cADPR,ADPR,烟酰胺和烟酸)均未与NAD + <共洗脱。 / sup>或NADH。
  4. 以与用于循环测定的“样品制备”中所述相同的方式制备组织样品。样品应在冰上解冻,并在含有500 ng / ml内标的水中稀释。
  5. 用于NAD + 测量的样品以1:1,000和1:10,000稀释,而用于NADH样品的样品在光滑的微量离心管中以1:1,000稀释。稀释后,应将样品涡旋并转移至2 ml自动进样器样品瓶中进行立即分析。

数据分析

通过Waters MassLynx v4.1软件获取和分析数据。 NAD + 和NADH的检测使用质谱仪在正ESI模式下使用毛细管电压3.5 kV,源温度150°C,去溶剂化温度500°C,锥形气体流量150 L / h,去溶剂化来完成气体流量500 L / h,采用多反应监测(MRM)扫描模式,停留时间为0.075秒(图4)。锥电压和碰撞能量由MassLynx-Intellistart,v4.1,软件确定。价值观如下:
NAD + -m / z 664.27&gt; 136.09-锥形电压54 V-Collision 42 eV
NADH-m / z 666.28&gt; 514.17 - 锥形电压56 V-Collision 26 eV
n-环己基苯甲酰胺-m / z 204.1> 122.00-锥形电压80 V-Collision 20 eV


图4. LC-MS追踪分析

食谱

  1. 主要库存解决方案
    将初级储备溶液储存在-80℃下,并如下制备在硅烷化玻璃小瓶中:
    NAD + (10 mg / ml水溶液)
    NADH(在0.01N NaOH中1mg / ml,pH 11.9)
    n-环己基苯甲酰胺(IS)(在EtOH中100μg/ ml)
    对于工作标准:
    通过将储备溶液稀释到上述相同的溶液中来制备工作标准。通过在含有500ng / ml内标的水中1:20稀释上述储备液来制备每日标准样品

致谢

这项工作部分得到了泰德纳什长寿基金会,格伦医学研究基金会通过梅奥诊所老年生物学保罗F.格伦实验室的资助,国家梅奥基金会卡利科实验室的资助。健康研究所(NIH)从国家老龄化研究所(NIA,批准号AG-26094),NIH / NCI到ENC的胰腺癌SPORE项目(授权号CA102701-08),Mayo细胞诊所中心获得资助胃肠病学信号(NIDDK P30DK084567)。
利益声明:Chini博士拥有CD38抑制剂用于代谢疾病的专利。

参考

  1. Aksoy,P.,White,T.A.,Thompson,M。和Chini,E.N。(2006)。 调节细胞内NAD水平:CD38的新作用。 Biochem Biophys Res Commun 345(4):1386-1392。
  2. Anderson,R.M.,Bitterman,K.J.,Wood,J.G.,Medvedik,O.,Cohen,H.,Lin,S.S.,Manchester,J.K.,Gordon,J.I。和Sinclair,D.A。(2002)。 操纵核NAD + 补救途径可延迟衰老而不会改变稳定状态状态NAD + 水平。 J Biol Chem 277(21):18881-18890。
  3. Ashrafi,K.,Lin,S。S.,Manchester,J。K.和Gordon,J.I。(2000)。 Sip2p及其伴侣snf1p激酶会影响 S中的衰老。 cerevisiae 。 Genes Dev 14(15):1872-1885。
  4. Bai,P.,Canto,C.,Oudart,H.,Brunyanszki,A.,Cen,Y.,Thomas,C.,Yamamoto,H.,Huber,A.,Kiss,B.,Houtkooper,RH,Schoonjans ,K.,Schreiber,V.,Sauve,AA,Menissier-de Murcia,J。和Auwerx,J。(2011)。 PARP-1抑制通过SIRT1激活增加线粒体代谢。 Cell Metab 13(4):461-468。
  5. Barbosa,M.T.,Soares,S.M.,Novak,C.M.,Sinclair,D.,Levine,J.A.,Aksoy,P。和Chini,E.N。(2007)。 酶CD38(NAD糖水解酶,EC 3.2.2.5)是饮食发展所必需的 - 诱发肥胖。 FASEB J 21(13):3629-3639。
  6. Camacho-Pereira,J.,Tarrago,MG,Chini,CCS,Nin,V.,Escande,C.,Warner,GM,Puranik,AS,Schoon,RA,Reid,JM,Galina,A。和Chini,EN( 2016)。 CD38通过SIRT3依赖机制决定与年龄相关的NAD下降和线粒体功能障碍。 Cell Metab 23(6):1127-1139。
  7. Dass,C。(2007)。第一章。 5. 连字分离技术。现代质谱基础知识,1。 John Wiley&amp; Sons,Inc。,151-194。
  8. Frasca,L.,Fedele,G.,Deaglio,S.,Capuano,C.,Palazzo,R.,Vaisitti,T.,Malavasi,F。和Ausiello,C.M。(2006)。 CD38协调人类成熟树突状细胞的迁移,存活和Th1免疫反应。 Blood 107(6):2392-2399。
  9. Guedes,A.G.,Jude,J.A.,Paulin,J.,Kita,H.,Lund,F.E。和Kannan,M。S.(2008)。 CD38在TNF-α诱导的气道高反应性中的作用。 Am J Physiol Lung Cell Mol Physiol 294(2):L290-299。
  10. Imai,S。和Guarente,L。(2014)。 NAD + 以及衰老和疾病中的sirtuins。 Trends Cell Biol 24(8):464-471。
  11. Lin,S.J。,Ford,E.,Haigis,M.,Liszt,G。和Guarente,L。(2004)。 卡路里限制通过降低NADH水平延长酵母寿命。 基因开发 18(1):12-16。
  12. Lin,S。S.,Manchester,J。K.和Gordon,J。I.(2001)。 增强糖原生成和增加能量储存作为酿酒酵母老化的标志。 J Biol Chem 276(38):36000-36007。
  13. Malavasi,F.,Deaglio,S.,Funaro,A.,Ferrero,E.,Horenstein,A.L.,Ortolan,E.,Vaisitti,T。和Aydin,S。(2008)。 生理和病理学中ADP核糖基环化酶/ CD38基因家族的进化和功能。 Physiol Rev 88(3):841-886。
  14. Nahimana,A.,Attinger,A.,Aubry,D.,Greaney,P.,Ireson,C.,Thougaard,A.V.,Tjornelund,J.,Dawson,K.M.,Dupuis,M。和Duchosal,M.A。(2009)。 NAD生物合成抑制剂APO866对血液系统恶性肿瘤具有强大的抗肿瘤作用。 血液 113(14):3276-3286。
  15. Smith,JS,Brachmann,CB,Celic,I.,Kenna,MA,Muhammad,S.,Starai,VJ,Avalos,JL,Escalante-Semerena,JC,Grubmeyer,C.,Wolberger,C。and Boeke,JD( 2000)。 Sir2蛋白中系统发育上保守的NAD + 依赖蛋白脱乙酰酶活性家庭。 Proc Natl Acad Sci USA 97(12):6658-6663。
  16. Verdin,E。(2015)。老化,新陈代谢和神经变性的 NAD + 。 Science 350(6265):1208-1213。
  17. Yang,H.,Yang,T.,Baur,JA,Perez,E.,Matsui,T.,Carmona,JJ,Lamming,DW,Souza-Pinto,NC,Bohr,VA,Rosenzweig,A.,de Cabo, R.,Sauve,AA和Sinclair,DA(2007)。 对营养敏感的线粒体NAD + 水平决定细胞存活。 Cell 130(6):1095-1107。
  18. Yoshino,J.,Baur,J.A。和Imai,S。I.(2018)。 NAD + 中间体:NMN和NR的生物学和治疗潜力。 Cell Metab 27(3):513-528。
  19. Yoshino,J.,Mills,K.F.,Yoon,M。J.和Imai,S。(2011)。 烟酰胺单核苷酸,一种关键的NAD + 中间体,治疗饮食的病理生理学 - 和小鼠的年龄诱发糖尿病。 Cell Metab 14(4):528-536。
登录/注册账号可免费阅读全文
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Kanamori, K. S., de Oliveira, G. C., Auxiliadora-Martins, M., Schoon, R. A., Reid, J. M. and Chini, E. N. (2018). Two Different Methods of Quantification of Oxidized Nicotinamide Adenine Dinucleotide (NAD+) and Reduced Nicotinamide Adenine Dinucleotide (NADH) Intracellular Levels: Enzymatic Coupled Cycling Assay and Ultra-performance Liquid Chromatography (UPLC)-Mass Spectrometry. Bio-protocol 8(14): e2937. DOI: 10.21769/BioProtoc.2937.
提问与回复
提交问题/评论即表示您同意遵守我们的服务条款。如果您发现恶意或不符合我们的条款的言论,请联系我们:eb@bio-protocol.org。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。