NADP+/NADPH Analysis Service

What is NADP+/NADPH?

NADP+ is the abbreviation of nicotinamide adenine dinucleotide phosphate, and is a cofactor used in anabolic reactions, such as lipid and nucleic acid synthesis. NADPH is the reduced form of NADP+. NADPH is produced in the last step of photosynthesis ferredoxin-NADP+ reductase in the photosynthetic organisms. In animals and other non-photosynthetic organisms, the major source of NADPH is the pentose phosphate pathway, which, generates NADPH and pentoses (5-carbon sugars) as well as Ribose 5-phosphate.

The regeneration of reduced glutathione, which is abbreviated as GSH in oxidation-reduction requires reducing equivalents provided by NADPH. The biochemical process can protect the cells from the toxicity of reactive oxygen species, which is abbreviated as ROS. NADPH is also used for lipid synthesis, cholesterol synthesis, and fatty acid chain elongation. The NADPH system is also involved in the generating free radicals in immune cells. These radicals are involved in the biological process named respiratory burst, which is the rapid release of reactive oxygen species (superoxide radical and hydrogen peroxide) from different types of cells in order to destroy pathogens in the immune system. NADPH also can be used as reducing equivalents for cytochrome P450 hydroxylation of aromatic compounds, steroids, alcohols, and drugs.

NADP+/NADPH Analysis Services

Creative Proteomics offers comprehensive NADP+/NADPH analysis services to support your research needs. Our specialized services include:

Quantitative Analysis

• Absolute Quantification: Accurate measurement of NADP+ and NADPH levels in various biological samples.
• Ratio Determination: Precise calculation of the NADP+/NADPH ratio, crucial for understanding cellular redox states.

Sample Types

• Tissue Samples: Analysis of NADP+/NADPH in different tissues, providing insights into organ-specific metabolic states.
• Cell Cultures: Evaluation of NADP+/NADPH levels in various cell lines to study metabolic responses and pathways.
• Body Fluids: Measurement of NADP+/NADPH in blood, serum, and other body fluids for clinical and research applications.

High-Performance Techniques

• LC-MS/MS: Utilization of Liquid Chromatography-Mass Spectrometry for high sensitivity and specificity in NADP+/NADPH detection.
• Enzymatic Assays: Employment of enzyme-based methods for reliable and reproducible NADP+/NADPH quantification.

Data Interpretation

• Comprehensive Reporting: Detailed reports including raw data, analysis results, and interpretation to facilitate your research conclusions.
• Statistical Analysis: In-depth statistical analysis to ensure data reliability and to highlight significant findings.

Customized Solutions

• Tailored Protocols: Development of customized protocols to meet specific research requirements.
• Consultation Services: Expert consultation to design experiments and interpret results, ensuring the best outcomes for your studies.

Quality Assurance

• Stringent Quality Control: Implementation of rigorous quality control measures to ensure the highest data accuracy and consistency.
• Certified Standards: Use of certified reference standards and calibration to maintain the reliability of the analysis.

Analytical Techniques for NADP+/NADPH Analysis

Liquid Chromatography-Mass Spectrometry (LC-MS/MS)

LC-MS/MS provides highly sensitive and specific detection of NADP+ and NADPH, ensuring accurate quantification even at low concentrations. This technique minimizes interference from other compounds and is ideal for various biological samples.

High-Performance Liquid Chromatography (HPLC)

HPLC offers excellent separation of NADP+ and NADPH from other cellular components. Known for its robustness and reproducibility, HPLC is suitable for tissues, cell cultures, and body fluids, providing reliable and precise measurements.

Enzymatic Cycling Assays

Enzymatic assays utilize specific enzymes to react with NADP+ and NADPH, ensuring high specificity. These assays offer both colorimetric and fluorometric detection options, making them versatile and cost-effective for routine analysis.

Spectrophotometric Assays

Spectrophotometric assays use UV-Vis detection to measure changes in absorbance, correlating to NADP+/NADPH levels. This method provides quick and efficient measurements, ideal for high-throughput screening while preserving sample integrity.

Sample Requirements for NADP+/NADPH Analysis

To ensure the highest quality of results, it is crucial to provide samples that meet our specific requirements. Below is a detailed guide on the types of samples we accept and the recommended sample volumes.

Sample Preparation Guidelines

• Animal Tissues: Fresh or frozen tissues should be provided, ideally flash-frozen in liquid nitrogen immediately after collection.
• Plant Extracts: Fresh plant material should be homogenized and extracted in appropriate solvents.
• Microbial Cultures: Cultures should be grown to the desired phase and collected via centrifugation.
• Cell Lines: Cells should be harvested and washed with phosphate-buffered saline (PBS) before submission

Deliverables for NADP+/NADPH analysis

Comprehensive Analysis Report:

• Detailed methods used for sample preparation and analysis.
• Quantitative results of NADP+ and NADPH levels.
• Calculation of NADP+/NADPH ratios if requested.
• Data interpretation and discussion of findings.

Raw Data:

• Complete records of raw data obtained during the analysis.
• Ensures transparency and traceability of results.

Quality Assurance:

• Documentation of quality control measures applied.
• Use of certified reference standards and calibration for accuracy.

The Plasma NAD+ Metabolome Is Dysregulated in "Normal" Aging

Journal: Rejuvenation research

Published: 2019

Background

Nicotinamide adenine dinucleotide (NAD+) is a crucial coenzyme involved in various biological processes such as energy metabolism, DNA repair, and cell signaling. Its levels and related metabolites have been implicated in aging and age-related diseases, making them a focus of significant research interest.

Technical Methods

Sample Collection and Preparation: Plasma samples were collected from fasting subjects to minimize potential variations due to food intake. Sample processing involved centrifugation to separate plasma from cellular components, followed by storage at -80°C to maintain sample integrity until analysis.

Analytical Methodology: LC-MS analysis was performed using a modified protocol optimized for the simultaneous quantification of multiple NAD+ metabolites. This included:

• Chromatographic Separation: Utilizing a reverse-phase chromatography column to separate metabolites based on hydrophobicity and molecular size.
• Mass Spectrometry Detection: Employing high-resolution mass spectrometry to detect and quantify specific metabolites based on their mass-to-charge ratio (m/z).

Quantification of NAD+ Metabolites: The LC-MS method allowed for the quantification of a panel of metabolites including NAD+, NADH, NADP+, NADPH, nicotinamide (NAM), nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), adenosine diphosphate ribose (ADPR), and methylated derivatives such as methyl-nicotinamide (MeNAM).

Statistical Analysis: Statistical analyses were conducted to assess age-related changes and associations between metabolite levels, age, gender, and BMI. This included:

• Correlation Studies: Calculating Pearson correlation coefficients to evaluate the strength and direction of relationships between metabolite levels.
• Regression Models: Utilizing linear regression models to quantify the impact of age, gender, and BMI on metabolite concentrations.

Quality Control and Data Interpretation: Quality control measures were implemented throughout the analytical process to ensure accuracy and reproducibility. Calibration curves using standard solutions of each metabolite were employed for quantification. Data were interpreted in the context of known biological pathways and previous research findings related to NAD+ metabolism and aging.

The study employed a cross-sectional design involving 30 subjects aged between 20 and 87 years. Plasma samples were collected and analyzed using a modified LC-MS approach to quantify various NAD+ metabolites. Statistical analyses, including correlation studies and regression models, were conducted to assess the relationship between age and metabolite levels.

Results

Age-Related Changes:

• Negative Correlations: NAD+ and NADP+ levels showed significant declines with increasing age, while NAM, MeNAM, ADPR, and NADPH levels increased.
• No Significant Change: NADH, NMN, NAMN, and NA levels did not exhibit substantial variation with age.

NAD Ratios:

• The ratios of NAD+, NAD+, and NAD+ decreased significantly with age.
• Conversely, the NADPH+ ratio increased significantly with age.

Gender and BMI Associations:

• Gender analysis indicated higher NAAD levels in men compared to women, suggesting a potential gender-specific metabolic regulation.
• BMI showed a significant correlation only with MeNAM levels, implying a link between obesity-related metabolism and NAD+ precursors.

Correlations and Associations:

• Positive correlations were observed among NAD+, NADH, and other NAD+ precursors, highlighting interconnected metabolic pathways.
• ADPR showed varied associations with different metabolites, indicating complex regulatory mechanisms within the NAD+ metabolic network.

Boxplots of NAD+ metabolite abundances across age groups. Concentrations are in nmol/L.

Correlation matrix of NAD+ metabolites with each other ordered by hierarchical clustering to group together the correlated NAD+ metabolite. Heatmap scale represents correlation strength, with no dash and dash for positive and negative correlations respectively.

Reference

1. Clement, James, et al. "The plasma NAD+ metabolome is dysregulated in "normal" aging." Rejuvenation research 22.2 (2019): 121-130.

Is there a difference between NADP and NADP+?

Yes, there is a difference between NADP and NADP+.

NADP (nicotinamide adenine dinucleotide phosphate) is the coenzyme form, which can exist in either an oxidized state (NADP+) or a reduced state (NADPH).

NADP+ is the oxidized form, which acts as an electron acceptor in various metabolic reactions. It is used in anabolic pathways that require reducing power, such as the Calvin cycle of photosynthesis and lipid/nucleic acid synthesis.

NADPH is the reduced form, which acts as an electron donor and reducing agent in biosynthetic reactions and for neutralizing reactive oxygen species. NADPH is produced during the light reactions of photosynthesis and is consumed in the Calvin cycle.

What is the role of NADP +/ NADPH?

NADP+ (nicotinamide adenine dinucleotide phosphate) and its reduced form, NADPH, play crucial roles in cellular metabolism, particularly in redox reactions and biosynthetic processes. Here's a detailed explanation of their roles:

NADP+ (Oxidized Form):

Electron Acceptor: NADP+ serves as a carrier molecule for electrons (and a hydrogen ion, H+) during oxidative reactions.

Oxidative Reactions: It participates in reactions where it accepts electrons from other molecules, becoming reduced to NADPH.

NADPH (Reduced Form):

Reducing Agent: NADPH is the reduced form of NADP+, carrying high-energy electrons and a hydrogen ion (H+).

Anabolic Processes: NADPH is critical for biosynthetic reactions such as:

• Fatty Acid Synthesis: Provides reducing power for the synthesis of fatty acids, essential for membrane structure and energy storage.
• Nucleotide Synthesis: Supports the production of nucleotides required for DNA and RNA synthesis.
• Steroid Hormone Synthesis: Facilitates the biosynthesis of steroid hormones like cortisol and aldosterone.
• Detoxification: Helps in detoxifying reactive oxygen species (ROS) through the action of antioxidant enzymes like glutathione reductase.

Role in Redox Balance:

NADPH/NADP+ ratio is crucial for maintaining cellular redox balance. High NADPH levels ensure sufficient reducing power for biosynthesis and antioxidant defense, while NADP+ levels regulate oxidative reactions.

Cellular Defense:

NADPH supports the regeneration of antioxidants such as glutathione, which protect cells from oxidative stress and maintain cellular integrity.

Is NADPH reduced to NADP+?

No, NADPH is not reduced to NADP+. Instead, NADPH is the reduced form of NADP+. It acquires electrons and a hydrogen ion (H+) during metabolic reactions, converting NADP+ into NADPH.

How is NADPH converted to NADP+?

NADPH is converted back to NADP+ through oxidative reactions where it donates electrons and a hydrogen ion (H+). This conversion is essential in maintaining the balance of redox reactions within cells, allowing NADPH to serve repeatedly as a reducing agent in various biosynthetic pathways.