Nicotinamide adenine dinucleotide (NAD) and its reduced form (NADH) are essential pyridine nucleotides in intermediary metabolism. They function as key electron carriers in redox reactions, driving processes such as glycolysis, the tricarboxylic acid (TCA) cycle, and oxidative phosphorylation. Accurate measurement of NAD and NADH levels is crucial for understanding cellular energy status, redox balance, and metabolic regulation.

Role of NAD/NADH in Metabolism

• Redox Reactions: NAD⁺ accepts electrons to become NADH during catabolic reactions, including glycolysis and the TCA cycle. NADH then donates electrons to the electron transport chain, facilitating ATP production.
• Metabolic Regulation: The NAD⁺/NADH ratio reflects the cell’s redox state and influences various metabolic pathways, including those involving sirtuins and ADP-ribosylation.
• Compartmentalization: The malate-aspartate shuttle and other mechanisms transport reducing equivalents between cellular compartments, linking cytosolic and mitochondrial metabolism.

Principle of NAD/NADH Assay Kits

• Enzymatic Cycling: NAD⁺ is reduced to NADH, which then reacts with a colorimetric or fluorometric probe, producing a detectable signal. The intensity of this signal is proportional to the amount of NAD⁺ or NADH present.
• Differentiation of NAD⁺ and NADH: Acid or base extraction selectively degrades one form, allowing separate quantification of NAD⁺ and NADH in the same sample.
• Detection: Colorimetric assays are typically read at 450–565 nm, while fluorometric assays use appropriate excitation/emission wavelengths (e.g., 530/585 nm).

Applications

• Monitoring cellular energy status and redox state.
• Evaluating drug effects on NAD/NADH metabolism.
• Studying metabolic diseases, aging, and mitochondrial function.

NAD/NADH assay kits are indispensable tools for quantifying these central metabolic intermediates, enabling researchers to probe energy metabolism, redox biology, and disease mechanisms with high sensitivity and specificity.