THE APPROACH FOR EXPRESS SPECTROMETRIC DETERMINATION OF THE REDUCED FORM OF NICOTINAMIDE ADENINE DINUCLEOTIDE (NADH) CONTENT

Niacin (vitamin B3) and its coenzyme forms NAD (nicotinamide adenine dinucleotide) and NADP (nicotinamide adenine dinucleotide phosphate) play an important role in cellular metabolism, including glysolysis, Krebs cycle, fatty acids oxidation, etc [1–3]. As well, NAD/NADH have a critical impact on the cell signaling [2, 4], redox state [1], biodegradation and detoxification [5], stress response [6], DNA reparation [7], stemness and pluripotency [8, 9], differentiation, development, and transformation [10]. Novel studies demonstrate that NAD supplementation may be used for treatment or correction of amyotrophic lateral sclerosis (ALS) [11], congenital malformations [12], and Friedreich’s ataxia (FA) [13]. Also, Ye and colleagues demonstrated the essential role of NAD salvage pathway and the enzyme nicotinamide phosphoribosyltransferase in the proliferation of colorectal cancer [14]. Nicotinamide adenine dinucleotide exists in two forms: an oxidized (NAD) and reduced (NADH) form. In cell metabolism, NAD/ NADH are involved in redox reactions, carrying electrons and protons from one compound to another [1]. Biochemical studies use various enzymatic methods with the evaluation of produced NADH concentration [15–18]. However, UDC 577.113.3:681.785.58 https://10.15407/biotech13.02.032

Nicotinamide adenine dinucleotide exists in two forms: an oxidized (NAD + ) and reduced (NADH) form. In cell metabolism, NAD + / NADH are involved in redox reactions, carrying electrons and protons from one compound to another [1].
Biochemical studies use various enzymatic methods with the evaluation of produced NADH concentration [15][16][17][18]. However, NADH may be spontaneously oxidized to NAD + during storage [19,20], so there is a strong need to develop a quick procedure for measurement the real NADH content in NADH reagent for the proper estimation of the obtained results.
Both NAD + and NADH strongly absorb ultraviolet (UV) light because of the presence of adenine. For example, absorption peak of NAD + is at a wavelength of 259 nanometers (nm), with an extinction coefficient of 16,900 M -1 cm -1 . NADH also absorbs at higher wavelengths, with a second UV absorption peak at 339 nm with an extinction coefficient of 6,220 M -1 cm -1 . This difference in the ultraviolet absorption spectra between the oxidized and reduced forms of the coenzymes at higher wavelengths makes it simple to measure the conversion of one to another forms in enzyme assays by measuring the amount of UV absorption at 340 nm using a spectrophotometer [21,22]. NAD + and NADH also differ in their fluorescence. NADH in solution has an emission peak at 445 nm ( excitation -340 nm) while the oxidized form of the coenzyme does not fluorescent [21,22]. But sometimes such fluorescent estimation is not appropriate or not available for the determination of reduced/oxidized form ratio.
The aim of this study was to elaborate a method for the rapid evaluation of NADH content in NADH reagent by the UV spectroscopic measurement and calculation.

Materials and Methods
Chemicals and reagents. NADH and NAD + were obtained from AppliChem. Resazurin sodium was obtained from Sigma Chemical Company. Phenazine methosulfate and other reagents were obtained from Alfarus Company. All used reagents and chemicals were of analytical grade.
Equipment and sofrware. Spectrophotometer mQuant Microplate Reader and fluorimeter FL800 Microplate Reader (BioTek Instruments) were used. Data was plotted using Gen5™ Data Analysis Software.
Estimation of NADH samples fluorescence. Three 0.18 mM NADH samples in 50 mM phosphate buffer (pH 7.6) were prepared from NADH-reagents with different storage time. These NADH samples were going to be used as the NADH concentration standard solution for enzymatic dehydrogenase reactions. To these samples were added 0.1 mM PMS (phenazine methosulfate) and 0.1 mM Resazurin dye and in 10 min the fluorescence data (ex 545 nm; em 600 nm) were measured.
Data analysis. Fluorescence (ex 545 nm; em 600 nm) of NADH samples with PMS/ resazurin dye was present as mean±sd. Percent of NADH in selected samples of NADH reagent was calculated in Microsoft Excel using developed formula "%NADH". Pearson correlation coefficients were determined using Microsoft Excel build-in procedure.

Results and Discussion
Development of the method. NADH evaluation may be conducted using spectrophotometric or fluorescent methods [21,22]. BioTek's kit of reagents "Determination of NADH Concentrations" demonstrates linear concentration dependence on absorbance at 340 nm and fluorescence at 445 nm especially for low NADH concentration [22]. But for applying such methods, the standard of exact NADH concentration is needed for the correct calibration. Thereby, an express approach for the NADH content correct evaluation in NADHreagents would be useful.
Spectral scan of the NADH solution is represented at Figure. It is supposed that five wavelength points reproduce such spectral feature. They are: 234 nm, 260 nm, 290 nm, 340 nm, and 400 nm. Three wavelength points (234 nm, 290 nm, 400 nm) describe the background of the spectra. Two other points (260 nm and 340 nm) are the maximum absorbance of NAD + and NADH, correspondingly.
To diminish the influence of background data, it was calculated "relative absorbance" (OD): for wavelength range from 234 nm to 290 nm for wavelength range from 290 nm to 400 nm.
The sum of the relative absorbance points ("integral") for the two bands was calculated.
Then, these two obtained integrals were normalized by the maximal relative absorbance for each range. Application of the formula "%NADH". Three samples of 0,18 mM NADH solution were used in order to demonstrate possible application of developed "%NADH" formula. After addition of PMS/resazurin dye fluorescence (ex 545 nm; em 600 nm) was measured. Obtained fluorescence intensity from three NADH samples with PMS/ resazurin were very different from one to another. The fluorescence intensity data (relative units) were for the 1-st sample -(630 ± 20); for the 2-nd sample -(400 ± 10); for the 3-rd sample -(260 ± 10). The non-coincidence of the results indicated the different concentration of NADH in these samples. During NADH reagent storage, its reduced form gradually becomes oxidized one. Therefore, in order to use NADH solution as a standard, it is necessary to control its current concentration. These NADH samples were then tested by the optical absorbance and fluorescence (ex 340 nm; em 440 nm) methods. The results are presented in the Table. Different fluorescence intensity of these samples confirms the varying of NADH concentration. Applying the formula "%NADH" to the optical absorbance data resulted in the evaluation of NADH content in these samples. The concentration of NADH varied from 39% to 19% in the samples. The correlation coefficient between the calculated concentration of NADH using the formula "%NADH" and the samples fluorescence intensity (ex 340 nm; em 440 nm) Representative spectral scan of the NADH sample. Relative absorbances (OD') are shown graphically was 0.95; and the fluorescence intensity of samples with PMS/resazurin (ex 545 nm; em 600 nm) was 0.98. Strong correlation between calculated NADH content and fluorescent data demonstrates precision of developed "%NADH" formula.

Conclusion
Taken together, this study developed an original formula for rapid and precise evaluation of NADH content in (NADH+NAD + ) reagent by the UV spectrometric measurement of the optical absorbance at the wavelengths 234 nm, 260 nm, 290 nm, 340 nm, and 400 nm. Proposed procedure is simple, quick, and require only routine laboratory equipment, such as spectrometer.

Financial support
This study was funded by budget themes "The Role of Amino Oxidases in the Development of Carbonyl-Oxidative Stress in Pathologies of Different Genesis", registration number 0115U003644 (2015-2017) and "Study of the features of extracellular matrix changes as an indicator of cancer progression and development of ways for its correction", registration number 0117U004344 (2017-2021).

Conflicts of Interest
No potential conflict of interest relevant to this article was reported.