- Service Details
- Demo
- Case Study
- FAQ
- Publications
Introduction to Niacin (Nicotinic Acid, Vitamin B3)
Niacin, also referred to as Vitamin B3 or nicotinic acid, is a crucial water-soluble vitamin that plays a key role in various biochemical processes within the human body. Its chemical structure is represented by the formula C₆H₅NO₂, and it was first described in 1873 by Hugo Weidel during nicotine research. As one of the 13 essential vitamins, Niacin is critical to maintaining optimal human health, specifically by aiding in lipid metabolism, tissue respiration, and the breakdown of carbohydrates. It is also a precursor to the coenzymes nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP), both of which are central to energy production and DNA repair.
Niacin is naturally present in a variety of foods, including liver, poultry, fish, cereals, peanuts, and legumes, and the human body can synthesize it from the essential amino acid tryptophan. A deficiency in Niacin can lead to pellagra, a disease characterized by symptoms such as skin lesions, diarrhea, and mental disturbances. Additionally, Niacin is widely used in pharmaceuticals for the treatment of hypercholesterolemia (high cholesterol) and other metabolic disorders, making its accurate measurement vital for both clinical and research settings.
Niacin Analysis in Creative Proteomics
At Creative Proteomics, we offer a full range of niacin analysis services designed to support research and development in both clinical and industrial contexts. Our state-of-the-art analytical platform employs advanced methodologies such as High-Performance Liquid Chromatography (HPLC) and Liquid Chromatography-Mass Spectrometry (LC-MS) to ensure precise, reliable, and reproducible quantification of Niacin in a variety of sample types.
Our niacin analysis services provide a comprehensive understanding of niacin's role in biological systems, supporting a wide range of research needs, including nutritional studies, pharmaceutical testing, and metabolic research. We cater to both in vitro and in vivo research, providing tailored solutions based on individual project requirements.
Key Niacin Analysis Services We Provide:
- Quantitative Niacin Measurement: Precise quantification of niacin concentrations in diverse biological and clinical samples.
- Pharmaceutical and Nutritional Supplement Testing: Analysis of niacin levels in commercial supplements and drug formulations.
- Customized Niacin Analysis: Tailored solutions based on the specific requirements of your research, from small-scale studies to large clinical trials.
Technology Platforms Used for Niacin Analysis
High-Performance Liquid Chromatography (HPLC) with UV Detection
HPLC with UV Detection is one of the most widely used methods for Niacin quantification. This technique is ideal for separating and detecting Niacin in various sample matrices. By utilizing UV detection at 261 nm, HPLC can accurately identify and quantify Niacin with excellent reproducibility.
Why Use HPLC for Niacin Analysis?
- High Sensitivity: Capable of detecting low concentrations of Niacin in biological samples.
- Wide Application Range: Suitable for the analysis of non-volatile, polar compounds like Niacin.
- Quantitative Accuracy: Provides precise and reproducible measurements, making it ideal for clinical and research applications.
Liquid Chromatography-Mass Spectrometry (LC-MS)
LC-MS combines the separating power of liquid chromatography with the highly sensitive detection capabilities of mass spectrometry. This technique is particularly useful for identifying and quantifying Niacin and its derivatives, even at trace levels in complex biological matrices.
Why Use LC-MS for Niacin Analysis?
- Unmatched Sensitivity: LC-MS can detect extremely low levels of Niacin, making it ideal for samples with minimal concentrations.
- High Specificity: The ability to identify and quantify multiple Niacin forms and derivatives within a single sample.
- Versatile Application: Suitable for a wide range of samples, including pharmaceutical formulations, biological tissues, and nutritional supplements.
Sample Requirements for Niacin Analysis
| Sample Type | Required Volume/Weight | Storage Conditions | Notes |
|---|---|---|---|
| Plasma/Serum | 200 μL | Store at -80°C | Use heparin or EDTA as anticoagulants. Avoid hemolysis. |
| Blood | 500 μL | Store at -20°C | Whole blood samples should be collected in EDTA tubes. |
| Tissue Samples | 50 mg | Store at -80°C | Freeze tissue samples immediately after collection. |
| Urine | 500 μL | Store at -80°C | Ensure samples are free from particulates or debris. |
| Cell Culture Samples | 1 x 10⁶ cells | Store at -80°C | Freeze cell pellets rapidly after harvesting. |
| Pharmaceuticals | 5-10 mg | Store at room temperature or 4°C | Provide detailed product information for context-specific testing. |
PCA chart
PLS-DA point cloud diagram
Plot of multiplicative change volcanoes
Metabolite variation box plot
Pearson correlation heat map
HIF‐1α Modulates NK Cell Effector Function and Homeostasis through Glycolysis and NAD Metabolism
Journal: EMBO Reports
Published: 2023
Background
Natural killer (NK) cells are crucial components of the immune system, primarily responsible for identifying and eliminating virus-infected and tumor cells. Their function is highly dependent on metabolic processes, and hypoxia-inducible factor 1-alpha (HIF-1α) plays a critical role in the regulation of metabolism under hypoxic conditions. This study investigates how HIF-1α affects NK cell metabolism and effector function. Specifically, it addresses the metabolic pathways involved in resting and activated NK cells, highlighting the dual role of HIF-1α in promoting glycolysis and modulating NAD+/NADH balance, which is vital for maintaining NK cell redox homeostasis.
There has been debate about whether HIF-1α supports or impairs NK cell activity, with previous research providing conflicting results. This study uses various tumor models and different experimental conditions to reconcile some of these discrepancies, aiming to better understand the role of HIF-1α in NK cell biology.
Materials & Methods
Mouse Models
HIF-1α and VHL (von Hippel-Lindau tumor suppressor) were conditionally deleted in NK cells using mice with targeted deletions. Both male and female mice (C57Bl/6J background, 8–12 weeks old) were used. Animal experiments were approved by the Zurich Veterinary Office.
Pulmonary Metastasis Model
The B16F10 melanoma cell line was used to create a pulmonary metastasis model. Tumor cells were injected into the tail vein of mice, and the lungs were analyzed for NK cell infiltration and metastasis after 7 and 14 days, respectively.
NK Cell Purification
NK cells were purified from spleen, bone marrow, and liver using magnetic bead-based isolation kits and a protocol adapted for each tissue.
Stimulation Assay
Splenocytes were stimulated with PMA and ionomycin for 6 hours under normoxia (20% O2) or hypoxia (1% O2). NK cell degranulation (CD107a) and intracellular granzyme B and IFN-γ production were analyzed by flow cytometry.
Killing Assay
NK cells were co-cultured with B16F10 melanoma cells (stained with CellTrace Violet) in normoxia or hypoxia for 24 hours. Cytotoxicity was measured using flow cytometry.
Flow Cytometry
NK cell markers and intracellular proteins (e.g., phospho-Histone H2A.X, Granzyme B, and IFN-γ) were assessed. Apoptosis was detected using Annexin V and 7AAD staining.
Seahorse Metabolic Flux Analysis
Real-time oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using the Seahorse analyzer to assess oxidative phosphorylation (OxPhos) and glycolysis in NK cells under normoxia and hypoxia.
ATP Quantification
ATP levels were quantified in NK cells using a luciferase-based ATP determination kit.
ROS Quantification
Mitochondrial reactive oxygen species (ROS) levels were measured using the MitoSOX™ reagent by flow cytometry.
Glutamine Oxidation Rate
Glutamine oxidation was measured by incubating NK cells with radiolabeled glutamine ([14C(U)]-glutamine) and quantifying radioactivity.
Fatty Acid Oxidation Rate
Fatty acid oxidation was measured using radiolabeled palmitic acid ([9,10-3H(N)]-palmitic acid). The amount of released radioactive water was quantified to determine oxidation rates.
NAD+ and NADH Quantification
NAD+ and NADH levels were measured using an NAD/NADH quantification kit. NK cells were lysed, and NAD+ was extracted using freeze-thaw cycles, followed by colorimetric detection at 450 nm.
Apoptosis Assay
Annexin V and 7AAD staining were used to detect apoptotic NK cells by flow cytometry.
Freshly isolated NK cells were subjected to untargeted and targeted metabolomics. Cells were snap-frozen and sent to Creative Proteomics for analysis.
Although the lipidomics experimental details were not explicitly outlined in the provided text, metabolomic profiling often includes lipid metabolism analysis to understand changes in fatty acid oxidation and lipid-related metabolic pathways in NK cells under different conditions.
RNA Isolation and Transcriptomics
RNA was extracted from freshly isolated NK cells using the RNeasy Mini Kit. RNA sequencing was performed on pooled splenic NK cells to analyze the transcriptome and identify differential gene expression related to HIF-1α and metabolic pathways.
Statistical Analysis
Data were analyzed using GraphPad Prism 9.1 software. Statistical significance was evaluated using one-sample t-tests or unpaired Student's t-tests. P-values less than 0.05 were considered significant.
Results
HIF-1α-Driven Glycolysis: NK cells with enforced HIF-1α expression exhibited significantly higher glycolysis, as evidenced by increased extracellular acidification rates (ECAR) in Seahorse metabolic flux analysis. This enhanced glycolysis led to greater ATP production, demonstrating that HIF-1α promotes energy metabolism in NK cells. Conversely, HIF-1α-deficient NK cells showed reduced ATP levels, indicating a reliance on glycolysis for energy.
NAD+ Regulation: HIF-1α was found to be essential for maintaining elevated levels of free NAD+ in resting NK cells. HIF-1α-deficient NK cells had lower NAD+ levels and higher NADH levels, disrupting the NAD+/NADH balance and affecting cellular redox status.
NK Cell Cytotoxicity: In cytotoxic assays, HIF-1α-driven NK cells displayed enhanced killing of B16F10 melanoma cells compared to control cells, both under normoxic and hypoxic conditions. The cytotoxic effect was more pronounced under hypoxia (2% O2), highlighting the role of HIF-1α in optimizing NK cell function in low oxygen environments.
ROS Production: Increased mitochondrial ROS levels were observed in HIF-1α-deficient NK cells, indicating higher oxidative stress. Despite this, HIF-1α deficiency did not enhance NK cell function, suggesting that elevated ROS does not necessarily improve NK cell activity and may contribute to cellular damage.
Oxidative Phosphorylation: HIF-1α-deficient NK cells showed higher oxygen consumption rates (OCR), indicating a shift towards oxidative phosphorylation (OxPhos). However, this shift did not translate to improved cytotoxicity, suggesting that OxPhos alone is insufficient to maintain NK cell function in the absence of glycolysis.
Tumor Models: In the pulmonary metastasis model, HIF-1α-driven NK cells demonstrated greater tumor infiltration and enhanced tumor clearance compared to controls. The study noted discrepancies with previous findings by Ni et al., which may be attributed to differences in tumor models, experimental conditions, and methodologies.
Metabolomic and Transcriptomic Analysis: Metabolomic profiling revealed alterations in metabolic pathways related to glycolysis and fatty acid oxidation in HIF-1α-modulated NK cells. Transcriptomic analysis showed differential gene expression linked to metabolic pathways and immune function, further elucidating the role of HIF-1α in NK cell metabolism.
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
Reference
- Pelletier, Abigaelle, et al. "Resting natural killer cell homeostasis relies on tryptophan/NAD+ metabolism and HIF‐1α." EMBO reports 24.6 (2023): e56156.
How sensitive is niacin analysis using HPLC and LC-MS, and how does it impact my research?
Both HPLC and LC-MS are highly sensitive techniques for Niacin (Vitamin B3) analysis, but they offer different degrees of sensitivity. HPLC can reliably detect Niacin in the low microgram range, making it suitable for most nutritional studies and standard pharmaceutical testing. However, LC-MS is more sensitive, capable of detecting Niacin and its derivatives down to the nanogram or even picogram levels. This sensitivity is crucial for studies involving trace amounts of Niacin, such as pharmacokinetic studies, where small fluctuations in concentration must be accurately tracked, or in low-concentration biological matrices like cerebrospinal fluid.
For research that requires high precision—especially in complex matrices or when measuring minor metabolites—LC-MS would be the recommended method due to its ability to differentiate between Niacin derivatives and provide detailed molecular characterization.
Can your niacin analysis distinguish between different forms of Niacin, such as nicotinic acid and nicotinamide?
Yes, our advanced methodologies, particularly LC-MS, allow us to distinguish between different forms of niacin, including nicotinic acid, nicotinamide (niacinamide), and newer forms such as nicotinamide riboside or nicotinamide mononucleotide (NMN). The ability to differentiate these forms is essential, as they play distinct roles in human metabolism. For example, nicotinamide is a key player in DNA repair, while nicotinic acid is more involved in cholesterol regulation.
This distinction is important for clients involved in nutraceuticals, pharmaceuticals, or metabolic research, as different Niacin forms have specific therapeutic and physiological effects. We can provide detailed quantification of each form to better understand their role in your samples.
How stable is niacin in biological samples, and what precautions should I take during sample collection and storage?
Niacin is generally stable under proper storage conditions, but certain factors can lead to degradation, especially in biological samples. Plasma and serum samples should be stored at -80°C immediately after collection to preserve Niacin's integrity. Exposure to heat, light, and enzymatic activity can degrade Niacin, particularly in biological fluids like blood or tissue homogenates.
For tissue samples, it's critical to freeze them rapidly after collection, ideally in liquid nitrogen, to prevent enzymatic degradation. Cell culture samples should be pelleted and stored at -80°C as quickly as possible to maintain the stability of intracellular niacin.
Clients working with pharmaceutical formulations should store samples in cool, dry conditions, as nicotinic acid can degrade at higher temperatures or in the presence of moisture. We recommend sending samples on dry ice to ensure their stability during shipping.
Can Creative Proteomics analyze niacin levels in supplements and food products?
Absolutely. We specialize in analyzing niacin in both nutritional supplements and food products, providing precise quantification of niacin content. This service is especially relevant for manufacturers and researchers involved in quality control or regulatory compliance who need to verify label claims or ensure the consistency of Niacin levels across product batches.
Additionally, we can assess the bioavailability of Niacin in fortified foods and supplements, helping clients understand how efficiently Niacin is absorbed and utilized in the body. This information is vital for optimizing product formulations in the nutraceutical or functional food industry.
What is the typical turnaround time for niacin analysis, and can it be expedited?
The typical turnaround time for niacin analysis is usually between 5 to 10 business days, depending on the sample type and the complexity of the analysis. However, we understand that research timelines can be tight, so we offer expedited services upon request. For urgent projects, we can prioritize your samples and aim to deliver results within 3 to 5 business days, depending on lab capacity and sample complexity.
Expedited services are particularly useful for clients conducting time-sensitive research, such as clinical trials, pharmaceutical product development, or urgent regulatory submissions.
Can Creative Proteomics assist in method development and validation for niacin analysis?
Yes, in addition to routine niacin analysis, we offer comprehensive method development and validation services. This is particularly valuable for clients in the pharmaceutical or biotech industries who require validated methods for regulatory approval or GMP-compliant production.
Our team of experts will work with you to develop a customized analytical method tailored to your specific sample type, matrix, and concentration range. We follow strict regulatory guidelines to ensure the method is fully validated for parameters such as accuracy, precision, linearity, sensitivity, and specificity. Once validated, the method can be used for routine analysis or submitted to regulatory agencies.
Does Creative Proteomics offer niacin metabolite analysis?
Yes, we can analyze niacin metabolites, such as NAD (nicotinamide adenine dinucleotide), NADP (nicotinamide adenine dinucleotide phosphate), and their reduced forms (NADH, NADPH). The LC-MS platform allows for the precise quantification of these metabolites, which are crucial in understanding Niacin's role in energy metabolism and redox balance.
Metabolite analysis is particularly useful for clients studying metabolic pathways, age-related diseases, and cellular energy production. By quantifying Niacin and its metabolites, we can provide deeper insights into cellular health and metabolic efficiency, which is critical for developing therapeutic strategies.
What if I have a very small sample size? Can you still perform niacin analysis?
Yes, we can perform niacin analysis even with very small sample volumes, especially when using LC-MS, which is extremely sensitive and can handle trace amounts of analytes. For small sample sizes, such as microliter volumes of plasma or small tissue biopsies, we adjust the analysis protocols to ensure accurate detection.
This capability is particularly beneficial for studies where sample availability is limited, such as clinical trials, pediatric studies, or research using rare or hard-to-obtain biological samples. Please consult with us prior to submission to discuss the best approach for small sample analysis.
Can niacin analysis be performed on genetically modified organisms (GMOs) or transgenic models?
es, we regularly perform niacin analysis on samples from genetically modified organisms (GMOs) and transgenic models. These models are often used in research to study how genetic alterations impact metabolic pathways, including those involving Niacin.
Our analytical methods are fully compatible with samples from both animal models and plant-based GMOs, providing accurate insights into the role of Niacin in genetically modified systems. Whether your research involves gene editing technologies like CRISPR or traditional transgenic approaches, we can offer Niacin quantification as part of your broader metabolic or genetic research objectives.
Resting natural killer cell homeostasis relies on tryptophan/NAD+ metabolism and HIF‐1α.
Pelletier, Abigaelle, Line M. Boulay, Maxime Beaudoin, Eric A. Champagne, Arnaud Drouin, Jean-François Côté, and Philippe Roux.
Journal: EMBO reports
Year: 2023
https://doi.org/10.15252/embr.202256156
Enhance trial: effects of NAD3® on hallmarks of aging and clinical endpoints of health in middle aged adults: a subset analysis focused on blood cell NAD+ concentrations and lipid metabolism.
Roberts, Michael D., Lauren O'Neill, Grant Tinsley, Andrew Colombo, and Jacob M. Wilson.
Journal: Physiologia
Year: 2022
https://doi.org/10.3390/physiologia2010002
Metabolites and Genes behind Cardiac Metabolic Remodeling in Mice with Type 1 Diabetes Mellitus.
Kambis, Tyler N., Hamid R. Shahshahan, and Paras K. Mishra.
Journal: International Journal of Molecular Sciences
Year: 2022
https://doi.org/10.3390/ijms23031392
