# 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

> **NIH NIH U01** · BRIGHAM AND WOMEN'S HOSPITAL · 2024 · $1,020,661

## Abstract

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 preven...

## Key facts

- **NIH application ID:** 10977149
- **Project number:** 1U01AG089040-01
- **Recipient organization:** BRIGHAM AND WOMEN'S HOSPITAL
- **Principal Investigator:** SHALENDER BHASIN
- **Activity code:** U01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $1,020,661
- **Award type:** 1
- **Project period:** 2024-09-01 → 2029-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10977149

## Citation

> US National Institutes of Health, RePORTER application 10977149, 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 (1U01AG089040-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10977149. Licensed CC0.

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