Nicotinamide Adenine Dinucleotide (NAD+) Analysis Service

What Is Nicotinamide Adenine Dinucleotide (NAD)

Nicotinamide adenine dinucleotide (NAD) is a coenzyme, which can be found in all living cells. NAD consists of two nucleotides joined through their phosphate groups and it is a dinucleotide, in which the one nucleotide contains an adenine base and the other nicotinamide. There are two different NAD forms exist naturally, the one is an oxidized form, which is abbreviated as NAD+ and the other is a reduced form, which is abbreviated as NADH.

What Is Nad Synthesis Pathway

In both prokaryotic and eukaryotic systems, NAD is synthesized through two pathways: de novo pathway and salvage pathway. In the de novo pathway, NAD is generated from tryptophan through quinolinic acid (QA) and nicotinic acid (NA). In salvage pathway, NAD is synthesized by recycling degraded NAD products such as Nam and nicotinamide. Both pathways play essential roles in cell growth. Under normal physiological condition, the salvage pathway plays a more important role than de novo pathway in NAD synthesis. In yeast, the de novo pathway is making of six enzymatic steps and one non-enzymatic reaction. In the last enzymatic reaction, quinolinate is converted to nicotinic acid mononucleotide by a quinolinate phosphoribosyl transferase encoded by the BNA6/ QPT1 gene. This last enzymatic reaction is the converge point of the de novo pathway and the salvage pathway.

Physiological Functions of NAD

NAD takes part in many physiological processes, such as energy metabolism regulation, DNA repair and transcription. Besides acting as a coenzyme, NAD also serves as a substrate such as substrate for NAD-dependent DNA ligases, NAD-dependent oxidoreductases and NAD-dependent deacetylases. At the same time, the reduced form of NAD, NADH, serves as a substrate for the NADH dehydrogenase in the mitochondrial respiratory chain to transfers electrons to coenzyme Q and generate NAD. Calorie restriction accomplished by glucose limitation in wild-type Saccharomyces cerevisiae can extend replicative lifespan of cells and this process relies on Sir2 and the NAD+ salvage enzymes, nicotinic acid phosphoribosyl transferase and nicotinamidase. Together with glutathione, the derivative of NAD, the NADPH coenzyme, maintains the intracellular redox state and is also involved in many assimilatory pathways. To maintain the proper redox state, NADH needs to be constantly re-oxidised. Mostly, NAD is converted to NADH mostly in catabolic reactions such as glycolysis and TCA cycle. However, both cytosolic and mitochondrial NADH are re-oxidised mainly by the respiratory chain. Since the inner mitochondrial membrane is impermeable for NAD and NADH, there are several shuttle systems to transport permeable redox equivalents across this barrier. Ethanol-acetaldehyde shuttle is one example.

Though the changes of NAD+ to nicotinamide ratio and the NAD+ to NADH ratio can be anticipated through models to related the effects of calorie restriction. However, the putative alterations of NAD+ metabolism require a sensitive and reliable qualitative and quantitative analysis of NAD+ metabolites.

What Are The Pertinent Small Molecules Within The NAD Metabolic Pathway?

The NAD metabolic pathway encompasses a spectrum of small molecules, prominently featuring the oxidized form of nicotinamide adenine dinucleotide (NAD+), its reduced counterpart nicotinamide adenine dinucleotide (NADH), the oxidized form of nicotinamide adenine dinucleotide phosphate (NADP+), the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH), nicotinamide mononucleotide (NMN), nicotinamide (NAM), nicotinamide riboside (NAR), and adenosine triphosphate (ATP). Notably, nicotinamide adenine dinucleotide (NAD+), alternatively referred to as coenzyme I, plays an indispensable role as a coenzyme in redox processes, actively participating in diverse physiological processes such as DNA repair, cellular substance metabolism, and energy synthesis.

NAD Metabolites Analysis Solution

Creative Proteomics presents a cutting-edge analytical solution tailored to the study of NAD metabolism. This comprehensive platform leverages state-of-the-art GC/MS technology (Agilent 7890A/5975C) and ACQUITY UPLC/TripleQuad5500 instrumentation (Waters/AB Sciex) to facilitate both quantitative and qualitative assessments of a wide spectrum of small molecules involved in the NAD metabolic pathways. Central to this platform is the incorporation of standard reference materials and isotopic standards, enabling precise quantification of nearly 95% of the analytes under investigation.

Sample Requirement

Serum, plasma, urine, bile, and bile acids;

Animal tissues such as cells, liver, brain tissues, etc., and feces;

Plants, yeast, microorganisms, etc.

Blood samples, bile, etc.: 10 microliters

Various tissues: 10 milligrams

Feces, etc.: 10 milligrams

Delivery

A detailed technical report will be provided at the end of the whole project, including the experiment procedure, MS/MS instrument parameters

Analytes are reported as uM or ug/mg (tissue), and CV's are generally<10%

The name of the analytes, abbreviation, formula, molecular weight and CAS# would also be included in the report.

With integrated set of separation, characterization, identification and quantification systems featured with excellent robustness & reproducibility, high and ultra-sensitivity, Creative Proteomics provides reliable, rapid and cost-effective NAD metabolites targeted metabolomics services.

Resting natural killer cell homeostasis relies on tryptophan/NAD+ metabolism and HIF‐1α.

Journal: EMBO reports

Published: 2023

Background

Natural Killer (NK) cells are crucial components of the immune system, capable of targeting and destroying virus-infected or malignant cells. They function effectively in low-oxygen (hypoxic) environments, largely mediated by hypoxia-inducible factors (HIFs), particularly HIF-1α. Although NK cells primarily rely on aerobic glycolysis for activation and function, the role of HIF-1α in their metabolism and homeostasis, especially in resting states, is still debated.

Materials & Methods

Mouse Models

Targeted deletions of HIF-1α and VHL in NK cells were achieved as described previously (Krzywinska et al, 2017, 2022). All mice were on a >99% C57Bl/6J background, with both male and female mice (8–12 weeks old) used equally. All procedures followed Swiss animal protection laws.

Pulmonary Metastasis Model

B16F10 melanoma cells were injected intravenously (1 × 10⁵ for HIF1α model, 2 × 10⁵ for VHL model). Lungs were collected 7 days post-injection for NK cell analysis and after 14 days for metastasis assessment.

NK Cell Purification

NK cells were purified from spleen and bone marrow using the EasySep™ Mouse NK Cell Isolation Kit, and from liver following established protocols.

Stimulation and Killing Assays

Splenocytes were stimulated with PMA and ionomycin in normoxic or hypoxic conditions for 6 hours to assess granzyme B and IFN-γ expression. For the killing assay, NK cells were co-cultured with B16F10 melanoma cells and assessed by FACS after 24 hours.

Flow Cytometry

Flow cytometry was performed with antibodies targeting NK1.1, NKp46, CD107a, Granzyme B, IFN-γ, and markers for apoptosis.

Seahorse Metabolic Flux Analysis

OCR and ECAR were measured using Seahorse technology in freshly isolated NK cells. Cells were subjected to sequential injections of metabolic inhibitors for real-time measurements.

ATP and ROS Quantification

ATP levels were determined using an ATP Determination Kit, and mitochondrial ROS were quantified using MitoSOX Red™ by flow cytometry.

Metabolic Assays

Glutamine oxidation and fatty acid oxidation were measured using radioactive substrates, followed by DPM quantification with scintillation counting.

NAD Quantification

NAD⁺/NADH levels were assessed using the NAD/NADH Quantification Kit with absorbance measurements at 450 nm.

Metabolomics

NK cells from 20 mice were pooled for untargeted metabolomics, with sample processing performed by Creative Proteomics.

RNA Isolation and Transcriptomic Analysis

RNA was isolated using the RNeasy Mini Kit, and RNA-sequencing was conducted on pooled NK cells by Novogene Europe.

Statistical Analysis

Data were analyzed using GraphPad Prism 9.1, with statistical significance set at *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Results

HIF-1α and NK Cell Metabolism

To study the impact of HIF-1α on NK cells, researchers used NK cell-specific HIF-1α knockout (KO) mice. They observed that HIF-1α deficiency led to a reduction in NK cell numbers in the spleen, which is the organ with the lowest oxygen partial pressure, but did not affect NK cell numbers in the bone marrow or liver. This decrease in splenic NK cells was not due to altered maturation or receptor repertoire but was linked to metabolic changes.

Metabolomic analyses of NK cells from HIF-1α KO mice showed decreased levels of glycolytic end products like lactic acid and a reduction in several amino acids, including tryptophan. This suggested a disrupted tryptophan/NAD metabolism. Specifically, levels of tryptophan, methyl-quinoline (a tryptophan metabolite), and NAD precursors were significantly reduced. The mRNA expression of genes involved in tryptophan and NAD metabolism was also decreased in HIF-1α-deficient NK cells. Additionally, these cells exhibited a lower NAD/NADH ratio, indicating a role for HIF-1α in maintaining NAD+ levels in resting NK cells.

The transcription factor HIF1α contributes to tryptophan/NAD metabolism and maintenance of resting NK cells

HIF1α is involved in tryptophan/NAD metabolism of splenic NK cells

Impact of HIF-1α Deficiency on NK Cell Function

HIF-1α-deficient NK cells displayed increased mitochondrial reactive oxygen species (ROS) and DNA damage, particularly in the spleen, correlating with enhanced mitochondrial respiration and proton leakage. Despite increased OxPhos, these cells generated less ATP, suggesting mitochondrial dysfunction.

Furthermore, HIF-1α deficiency led to enhanced fatty acid oxidation, contributing to ROS production. However, glutamine oxidation remained unaffected. In terms of NK cell activation, HIF-1α was dispensable for glycolysis under normoxic conditions but crucial for glycolysis under hypoxic conditions. HIF-1α-deficient NK cells showed impaired activation and effector functions, such as reduced degranulation and IFN-γ production. Supplementing with NAD+ or nicotinamide did not restore NK cell activation, likely due to an anti-glycolytic effect.

In vivo, HIF-1α KO mice exhibited increased pulmonary metastasis of B16 melanoma cells. This was associated with decreased NK cell frequency and functionality in the lungs, demonstrating HIF-1α's role in NK cell infiltration into metastatic sites. Despite this, HIF-1α did not affect NK cell-mediated killing of B16 cells in vitro.

Constitutive Activation of HIF-1α Enhances NK Cell Function

To investigate the effect of constitutive HIF activation, researchers generated NK cell-specific VHL knockout (VHL KO) mice. Loss of VHL stabilizes HIF-1α, leading to increased NK cell glycolysis and enhanced effector functions, including greater Granzyme B expression. In contrast, deletion of HIF-2α did not produce significant effects on NK cell function or numbers, highlighting HIF-1α as the main regulator of hypoxic responses in NK cells.

Reference

1. Pelletier, Abigaelle, et al. "Resting natural killer cell homeostasis relies on tryptophan/NAD+ metabolism and HIF‐1α." EMBO reports 24.6 (2023): e56156.