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Pyridine nucleotides, NAD and NADP occur in oxidized (NAD⁺ and NADP⁺) and reduced forms (NADH and NADPH). While HPLC techniques exist for separating and quantifying NAD and NADP, both can be readily quantified through well-established and sensitive enzymatic cycling assays. It is a simple matter to distinguish between NAD and NADP either through the use of specific enzymes or by HPLC. More problematical is discrimination between the reduced and oxidized forms. Usually, this is achieved by exploiting the different stability of the two forms in acid and alkaline extraction buffers. High-throughput methods have been developed that can measure the two nucleotides at the same time as other redox compounds (Queval and Noctor 2007).

Concentrations of NAD⁺, NADH, NADP⁺ and NADPH reported in young A. thaliana are shown in Table 2.3. The NADH/NAD⁺ and NADPH/NADP⁺ ratios vary according to the physiological conditions. However, the concentration of the oxidized form (NAD⁺) in plants is higher than that of the reduced form (NADH) (Wang and Pichersky (2007).

Table 2.3 Concentrations of NAD⁺, NADH, NADP⁺, and NADH in Arabidopsis thaliana (ecotype Col-0, wild-type) plants.

Source: In part based on data of Wang and Pichersky (2007).

Tissue	NAD+	NADH	NAD+/NADH	NADP+	NADPH	NADP+/NADPH
Root	6.5	0.2	(31.4)	1.7	0.3	(5.7)
Roselle leaves	10.2	4.5	(2.8)	2.8	—	—
Stem	12.0	—	—	3.1	—	—
Cauline leaves	11.3	4.0	(3.4)	3.6	—	—
Flower	27.0	3.6	(9.1)	4.5	1.0	(4.0)
Silique	17.9	2.8	(4.5)	3.8	0.6	(6.4)
Seedlings	9.9	0.9	(11.9)	2.0	0.4	(6.0)

The values (nmol g⁻¹ f.w.) and the NAD(P)⁺/NAD(P)H ratios are obtained from different tissues.

A full understanding of pyridine nucleotide-related biological phenomena requires techniques that determine specific pool sizes in different intracellular compartments. The fluorescent properties of reduced pyridine nucleotides have been exploited in the analysis of plant mitochondrial metabolism (Kasimova et al. 2006). However, these measurements do not distinguish between the NADH and NADPH signals. The limitations of the information generated by the different methods of measuring pyridine nucleotides have been discussed by Hagedorn et al. (2004).