Development, Validation, and Harmonization of Methods for Collection and Measurement of NAD+ and Related Metabolites: Application to a Randomized Trial of Patients Undergoing Coronary Artery Bypass

The methods used to measure NAD+ and NADH in human samples include the enzymatic cycling assay, high pressure liquid chromatography and liquid chromatography tandem mass spectrometry (LC-MS/MS). The LC- MS/MS method offers superior sensitivity, precision, accuracy compared to the other methods. However, all

current methods suffer from one or more limitations: 1) some sample processing methods use strong acids (e.g., trichloroacetic acid or perchloric acid) which degrade NADH and NADPH; 2) separate methods are often used to measure oxidized and reduced pyridine nucleotides which poses a barrier to measuring the full

spectrum of NAD+-related metabolites; 3) scarcity of stable isotope-labeled internal standards for NAD+-related metabolites that are imperative for precise quantification of analytes; 4) Commutable reference materials used to calibrate assays and provide accuracy, metrological traceability, and comparability of measurements by

different laboratories are not available, nor are there mechanisms to harmonize assays across laboratories. We propose two integrated aims to fill these methodological gaps. In Aim 1 we will (a) optimize and validate standardized sample collection and storage protocols that will preserve the integrity of the pools of NAD+,

NADH, NADP, NADPH and related metabolites; (b) finalize the validation of our advanced LC/MS-MS method (NADome Quant) that simultaneously measures this set of metabolites in human tissues; and (c) in collaboration with Dr. Vesper's laboratory at the CDC, generate higher order commutable reference standards

and establish processes for harmonizing assays across laboratories. In Aim 2, we will conduct a randomized, double-blind, placebo-controlled trial (n=90) to determine whether oral administration of β nicotinamide mononucleotide (NMN), an immediate NAD+ precursor, is more efficacious effective than placebo in raising

myocardial NAD+ levels and reducing myocardial injury in adults undergoing elective coronary artery bypass graft (CABG) surgery. Patients will be randomized 1:1:1 to placebo or to one of two dosing regimens of NMN (administered for seven vs. two days prior to surgery) to test whether NMN administration increases

myocardial concentrations of NAD+ and its metabolites and attenuates myocardial and extracardiac (e.g., kidney) injury. CABG surgery offers a unique opportunity to harvest a variety of human tissue that would otherwise be discarded, including myocardium, aortic endothelium, subcutaneous fat, and skin.

This research proposal will establish robust sample collection and storage protocols that preserve NAD+ as well as NADH and NADP(H); more advanced LC/MS assays for measuring NAD+ and related metabolites; and commutable reference materials and procedures for harmonizing assays across laboratories through the

auspices of the CDC. The proof-of-concept trial will provide key foundational data to guide the design of larger efficacy trials of NAD+ augmentation strategies for preventing myocardial and extracardiac injury during CABG surgery, an important unmet clinical need.