Coenzyme I Analysis Service

What is Coenzyme I?

Coenzyme I, more commonly known as Nicotinamide Adenine Dinucleotide (NAD+), is a crucial cofactor found in all living cells. NAD+ plays a fundamental role in various biochemical processes, acting primarily as an electron carrier in redox reactions. It exists in two primary forms within the cell: the oxidized form, NAD+, and the reduced form, NADH. Both forms are integral to numerous enzymatic reactions and metabolic pathways.

NAD+ is composed of ribosylnicotinamide 5'-diphosphate linked to adenosine 5'-phosphate through a pyrophosphate bond. This structure enables NAD+ to participate in redox reactions, where it alternates between its oxidized and reduced states. NAD+ is essential for the functionality of dehydrogenases, which catalyze the oxidation of substrates, while NADH acts as a reducing agent, donating electrons to various biochemical pathways.

In addition to its role in redox reactions, NAD+ is involved in various other cellular processes, including:

• Post-translational Modifications: NAD+ serves as a substrate for enzymes that add or remove chemical groups from proteins, influencing their function and activity.
• Cellular Signaling: NAD+ levels impact signaling pathways related to stress responses, aging, and cellular repair mechanisms.
• Metabolic Pathways: NAD+ is central to processes such as glycolysis, the citric acid cycle, and oxidative phosphorylation.

Given its wide range of functions, understanding NAD+ metabolism is critical for studying cellular health and disease mechanisms.

Coenzyme I Analysis in Creative Proteomics

Creative Proteomics offers targeted metabolomics for Coenzyme I analysis, leveraging advanced analytical techniques to provide precise and reliable results. Our services are designed to support researchers and professionals in various fields, including biochemistry, molecular biology, and pharmacology.

Nicotinamide Adenine Dinucleotide (NAD+) Analysis

NAD+ analytical services focus on the quantification and analysis of the oxidized (NAD+) and reduced (NADH) forms of nicotinamide adenine dinucleotide to help understand NAD+ metabolism, assess its role in cellular processes, and evaluate its involvement in various diseases. We use high performance liquid chromatography (HPLC) and mass spectrometry (MS) to provide accurate and reliable measurements of NAD+ levels in biological samples.

Coenzyme Q10 (CoQ10) Analysis

Coenzyme Q10, also known as ubiquinone, is essential for mitochondrial function and acts as a potent antioxidant. Our CoQ10 analysis service provides precise quantification of CoQ10 levels in various biological matrices, including blood, tissue extracts, and cell lysates. We utilize HPLC and UV-Vis Spectroscopy to ensure accurate measurement of CoQ10, aiding in studies related to energy metabolism, oxidative stress, and cardiovascular health.

Niacin (Nicotinic Acid) Analysis

Niacin, or Vitamin B3, is a critical precursor in the biosynthesis of NAD+. Our Niacin analysis service involves the measurement of niacin levels in biological samples to understand its role in NAD+ production and overall metabolic health. Using HPLC with fluorescence detection, we provide sensitive and specific quantification of niacin, supporting research on vitamin deficiencies and metabolic disorders.

Nicotinamide Analysis

Nicotinamide, another precursor in the NAD+ biosynthesis pathway, is analyzed to gain insights into its metabolic role and impact on cellular functions. Our service involves detailed measurement of nicotinamide concentrations using HPLC coupled with fluorescence or MS detection. This analysis is particularly useful for studies focused on NAD+ metabolism, cellular stress responses, and aging.

Custom Analytical Services

Creative Proteomics offers custom analytical services to meet specific research needs. Whether you require specialized protocols, additional sample types, or novel analytical techniques, our team of experts is capable of designing and implementing custom solutions to support your research goals.

Comprehensive Data Analysis and Reporting

Our services extend beyond sample analysis to include comprehensive data analysis and reporting. We provide detailed, clear, and concise reports that interpret the results of your Coenzyme I analyses, including graphical representations, statistical evaluations, and actionable insights. This ensures that you can effectively integrate our findings into your research or development projects.

Technology Platform Used for Coenzyme I Analysis

High-Performance Liquid Chromatography (HPLC): HPLC separates and quantifies Coenzyme I compounds based on their interaction with a chromatographic column. This technique is essential for analyzing NAD+ and NADH, providing high resolution and accuracy.

Mass Spectrometry (MS): Coupled with HPLC, MS measures the mass-to-charge ratio of ions to identify and quantify Coenzyme I compounds with high sensitivity and specificity.

Ultraviolet-Visible (UV-Vis) Spectroscopy: Used for analyzing Coenzyme Q10 (CoQ10), UV-Vis Spectroscopy quantifies CoQ10 based on its unique absorbance properties in the ultraviolet and visible light range.

Fluorescence Spectroscopy: This technique detects and quantifies NADH and nicotinamide by measuring their intrinsic fluorescence, offering high sensitivity for low concentrations.

Nuclear Magnetic Resonance (NMR) Spectroscopy: NMR provides structural insights into Coenzyme I compounds, offering detailed information about their molecular structure and dynamics.

Sample Requirements for Coenzyme I Analysis

Comparative metabolite profiling of salt sensitive Oryza sativa and the halophytic wild rice Oryza coarctata under salt stress.

Journal: Plant‐Environment Interactions

Published: 2024

Background

Crop yield is increasingly threatened by abiotic stresses such as salinity, which affects 20% of cultivated and 33% of irrigated land globally, with climate change worsening the situation. Rice (Oryza sativa L.), a staple for over half of the world's population, is particularly sensitive to salt stress, leading to substantial yield losses even at moderate salinity levels. Understanding how salt-tolerant rice varieties, like the halophytic wild rice Oryza coarctata, manage salt stress can inform the development of more resilient crops. O. coarctata, native to saline coastal regions of Southeast Asia, exhibits remarkable salt tolerance due to its unique physiology, including a specialized rhizomatous system and efficient Na+ regulation mechanisms. Comparative metabolomic studies between O. sativa and O. coarctata reveal differences in stress response strategies, with O. coarctata showing a more robust metabolic defense under salt stress. This study employs untargeted metabolomics to elucidate root-specific metabolic adjustments in response to salinity, aiming to enhance our understanding of salt tolerance mechanisms and improve crop resilience.

Materials & Methods

Plant Growth and Treatment:

The experiment took place at the Plant Biotechnology Laboratory, University of Dhaka, under controlled conditions (34 ± 3°C day, 27 ± 2°C night, 75 ± 5% humidity). Oryza sativa seeds were soaked, incubated, and grown in Yoshida's solution. Oryza coarctata seedlings were transplanted into the same solution. Salt stress was applied gradually from 60 mM to 120 mM NaCl, with control groups receiving no salt. Four groups of root tissues were analyzed: O. sativa control (Os.C), O. sativa salt-stressed (Os.S), O. coarctata control (Oc.C), and O. coarctata salt-stressed (Oc.S), with three biological replicates each.

Metabolite Extraction:

Root tissue (500 mg) was crushed and extracted using 80% methanol. The extracts were processed, freeze-dried, and sent to Creative Proteomics, Inc. for untargeted metabolite analysis.

LC-MS Analysis:

Metabolites were analyzed using ACQUITY UPLC coupled with Q Exactive MS (Thermo). The LC system employed gradient elution, and mass spectrometry was conducted in ESI+ and ESI− modes with specific parameters.

Data Analysis:

Data were processed with Compound Discoverer 3.1, analyzed using MetaboAnalyst 5.0 for PCA, clustering, and volcano plots, and pathway analysis was conducted with the KEGG Database. Statistical analysis used GraphPad Prism version 9.4.1.

Results

Metabolic Profiles Comparison:

Metabolite Identification: LC-MS analysis identified 1012 metabolites in the roots of Oryza coarctata and Oryza sativa under control and salt stress conditions.

Principal Component Analysis (PCA) and Hierarchical Clustering: Both PCA and hierarchical clustering revealed distinct metabolite clusters between control and salt-stressed samples in both species.

(a) Score plot from PCA analysis of metabolite profiles of Oryza sativa and Oryza coarctata without and with salt stress samples. (b) Hierarchical clustering for the top 500 metabolites of Oryza sativa and Oryza coarctata without and with salt stress samples.

Effects of Salt Stress:

General Findings: Under control conditions, O. coarctata had 380 differentially accumulated metabolites compared to O. sativa. Salt stress increased this number to 436, with 190 metabolites common across conditions. O. coarctata showed downregulation of most metabolites in response to salt, while O. sativa exhibited upregulation.

Differential Metabolite Analysis:

• Oc.C vs Os.C (Control Conditions): Higher levels of itaconate, vanillic acid, and xanthin compounds in O. coarctata compared to O. sativa.
• Oc.S vs Oc.C (Salt Stress in O. coarctata): Salt stress led to downregulation of glycerolipids and phospholipids, while amino acids like leucine, phenylalanine, and tyrosine were upregulated.
• Os.S vs Os.C (Salt Stress in O. sativa): Slight upregulation of xanthin compounds and some amino acids under salt stress. Glycerolipids and phospholipids showed minimal changes.
• Oc.S vs Os.S (Salt Stress Comparison): Itaconate, phosphatidylglycerol, and tyrosine were upregulated in O. coarctata compared to O. sativa. Notable differences in the regulation of various phospholipids were observed.

Comparison of amino acid levels in four sample groups: Oc.C, Oc.S, Os.C, and Os.S. Statistical analysis was performed using one-way ANOVA followed by Šídák multiple comparisons test. p<.05 were considered significant. * denotes .01 <p< .05, ** denotes.001 <p< .01, *** denotes .0001 <p< .001, **** denotes p< .0001.

Pathway Enrichment Analysis:

Activated Pathways: Amino acid, fatty acid, and carbohydrate metabolism pathways were significantly activated in both genotypes under salt stress.

O. coarctata: Enhanced sphingolipid metabolism, phenylpropanoid biosynthesis, and eicosanoid accumulation. Higher levels of secondary metabolites like phenylpropanoids and eicosanoids.

O. sativa: Altered biosynthesis of unsaturated fatty acids, nicotinate, nicotinamide, and beta-alanine metabolism. Increased levels of some TCA cycle intermediates and decreased activity in the pentose phosphate pathway.

Reference

1. Tamanna, Nishat, et al. "Comparative metabolite profiling of salt sensitive Oryza sativa and the halophytic wild rice Oryza coarctata under salt stress." Plant‐Environment Interactions 5.3 (2024): e10155.