Table 2.
Methods for measuring NAD+ and derivatives.
| Assay | Analyte | Advantages | Disadvantages | Ref |
|---|---|---|---|---|
| Luminometric analysis | NAD+, NADH, NADP+, and NADPH concentration | Method is reproducible and reported in tissues and cells. | Partial inactivation of luciferase system. Invasive and destructive. |
[118] |
| Colorimetric Assay using thiazolyl blue | Intracellular NAD+ concentration | Identifies biological trends that are highly reproducible in the literature. | Indirect measurement affected by minor variations in temperature and pH. Cannot detect low picomolar levels. Invasive and destructive. |
[119,120] |
| BRET-based biosensors | NAD+ concentration | Quantifies NAD+ levels in cell culture, tissue, and blood samples. The readout can be performed by a microplate reader or a simple digital camera. Minimum consumption of biological samples. |
Invasive and destructive. | [121] |
| Reverse phase HPLC | Endogenous intracellular and extracellular levels of NAD+ and related metabolites | The method uses elements to increase sensitivity. | Limited to low micromolar detection levels. Since many NAD-related metabolites can be converted to one or more metabolites the identified concentrations may be fraught with inaccuracies. Invasive and destructive detection. Static information of a population of cells. |
[122] |
| LC-MS/MS | Endogenous intracellular and extracellular levels of NAD+ and related metabolites | High specificity and sensitivity. | The assay requires time, many preparations, and materials not readily available. Static information of a population of cells. Invasive and destructive detection. |
[123,124] |
| LC-MS/MS (NAD metabolite isotopic labels) | Endogenous intracellular and extracellular levels of NAD+ and related metabolites | The method provides greater resolution and lower limit of detection. | Static information of a population cells. Invasive and destructive. |
[125,126] |
| Fluorescent imaging with metabolite sensors | NADH, NAD+ concentrations, and their ratio | Metabolite sensors may be used to profile metabolic states of living cells in real-time and with single-cell or even subcellular resolution. | Invasive (metabolite sensors are introduced into any cell or organism). With some sensors, fluorescence is sensitive to pH. Other sensors have a limited dynamic range in fluorescence. |
[127,128] |
| Novel MRI-based process | NAD+ and NADH concentrations | Non-invasive and non-destructive, measured in healthy aged human brains. |
Only measures 2 analytes. | [129] |
| Fluorescence Lifetime Imaging (FLIM) | NAD+, NADH, NADP+, and NADPH | Non-invasive and non-destructive using autofluorescence intensity. May be used to profile metabolic states of living cells in real-time. |
Requires an expensive equipment. | [99] |