Using Assays Instead of Mass Spectrometry for Cardiolipin Quantification

Cardiolipin is a phospholipid that occurs in the inner membrane of mitochondria. It is a dimeric phospholipid with a distinctive structure and comprises of two phosphatidic acid molecules linked through a central glycerol moiety. Cardiolipin has an important role in regulating mitochondrial respiration and takes part in processes such as mitochondrial biogenesis, apoptosis, protein transport, fission, and fusion.

Perturbation of cardiolipin levels may lead to chronic fatigue syndrome, cardiovascular disease, and several neurodegenerative disorders. Cardiolipin composition and levels could be altered as a result of exposure to environmental pollutants such as organophosphate pesticides and cigarette smoke, resulting in adverse effects on health. Congenital deficiency in cardiolipin synthesis (Barth’s syndrome) causes underdeveloped muscles, growth delay, and cardiomyopathy. As cardiolipin is critical to mitochondrial oxidative phosphorylation, assays for measuring the levels of cardiolipin in biological samples have been gaining increased attention.

To date, mass spectrometry has been employed for the detection of cardiolipin, which requires the use of costly, state-of-the-art instrumentation and expertise in the method. BioVision’s Cardiolipin Quantification Assay is a straightforward, adaptable, and simple plate-based assay that uses a proprietary fluorometric probe. This assay has been exclusively designed for cardiolipin, without cross-reactivity with other lipids such as sphingomyelin, phosphatidylserine, and phosphatidylcholine. The assay has the potential to detect as low as 0.2 nmol of cardiolipin in isolated mitochondria and also in cell lysates.

Cardiolipin

Key Features

  • Cardiolipin-specific (cardiolipin probe shows no cross-reactivity with other usual membrane phospholipids)
  • Highly stable (included reagents have a long shelf life)
  • Homogenous assay with simple protocol (easily adapted to high-throughput assay formats, enables direct fluorometric detection)
  • Versatile sample applications (can be employed with isolated mitochondrial samples and also cell lysates)
  • Reproducible and accurate results (correct spike recovery and low inter- and intra-assay variability)

A Plate-Based Fluorescent Assay to Measure Cardiolipin with Good Intra- and Inter-Assay Variability

(A) Cardiolipin standard curve for cardiolipin concentrations 0–3 nmol/well. (B) Intra-assay variation and coefficient of variation (n = 3). (C) Inter-assay variation and coefficient of variation (n = 3). All of the assays were performed using BioVision’s Cardiolipin Quantification Kit.

Figure 1. (A) Cardiolipin standard curve for cardiolipin concentrations 0–3 nmol/well. (B) Intra-assay variation and coefficient of variation (n = 3). (C) Inter-assay variation and coefficient of variation (n = 3). All of the assays were performed using BioVision’s Cardiolipin Quantification Kit.

Applications of Plate-Based Cardiolipin Detection Assay

Cardiolipin measurement in biological samples. Cardiolipin was measured using BioVision’s Cardiolipin Quantification Kit in Jurkat cell lysate, Jurkat cell isolated mitochondria, bovine heart mitochondria, and yeast mitochondria (S. cerevisiae). Each bar represents mean ± standard deviation of three samples.

Figure 2. Cardiolipin measurement in biological samples. Cardiolipin was measured using BioVision’s Cardiolipin Quantification Kit in Jurkat cell lysate, Jurkat cell isolated mitochondria, bovine heart mitochondria, and yeast mitochondria (S. cerevisiae). Each bar represents mean ± standard deviation of three samples.

Spike Recovery of Cardiolipin in Isolated Mitochondria and Cell Lysate

Spike and recovery of cardiolipin in isolated yeast mitochondria and Jurkat cell lysates. Samples were spiked with known amounts of cardiolipin and measured using BioVision’s Cardiolipin Quantification kit. The recovery was always >85%.

Figure 3. Spike and recovery of cardiolipin in isolated yeast mitochondria and Jurkat cell lysates. Samples were spiked with known amounts of cardiolipin and measured using BioVision’s Cardiolipin Quantification kit. The recovery was always >85%.

Cardiolipin Probe is Highly Specific and Does Not Bind to Other Phospholipids

Assay specificity for detection of cardiolipin versus other lipids. Signal from phosphatidylcholine (PC), phosphatidylserine (PS), sphingomyelin (SM), and cardiolipin (CL) demonstrates the specificity of the Cardiolipin Probe. All assays were performed according to the kit protocol.

Figure 4. Assay specificity for detection of cardiolipin versus other lipids. Signal from phosphatidylcholine (PC), phosphatidylserine (PS), sphingomyelin (SM), and cardiolipin (CL) demonstrates the specificity of the Cardiolipin Probe. All assays were performed according to the kit protocol.

Conclusion

  • BioVision’s Cardiolipin Quantification Kit is a versatile and high-throughput assay for the quantification of cardiolipin in biological samples such as cell lysates and isolated mitochondria
  • The kit has benefits over earlier techniques of cardiolipin measurement as it is extremely specific without cross-reactivity with other phospholipids such as phosphatidylserine and phosphatidylcholine
  • Applications include comparison of cardiolipin levels in various samples, as well as determination of variation in cardiolipin levels following exposure to drugs or toxicants

BioVision Incorporated

BioVision, Inc., is a privately held Life Science company headquartered in the beautiful San Francisco Bay Area.

BioVision develops and offers a wide variety of products including assay kits, antibodies, recombinant proteins & enzymes, and other innovative research tools for studying Apoptosis, Metabolism, Cell Proliferation, Cellular Stress, Cell Damage and Repair, Diabetes, Obesity and Metabolic Syndrome, Stem Cell Biology, Gene Regulation, Signal Transduction, etc. BioVision's products are currently being sold in more than 60 countries worldwide.


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Last updated: Feb 21, 2020 at 9:30 AM

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