Journal
JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES
Volume 831, Issue 1-2, Pages 63-71Publisher
ELSEVIER
DOI: 10.1016/j.jchromb.2005.11.031
Keywords
cardiolipin; mitochondria; skeletal muscle; human; fluorescent derivatization; HPLC; 1-pyrenyldiazomethane
Funding
- NIDDK NIH HHS [R01 DK 49200-06] Funding Source: Medline
Ask authors/readers for more resources
Cardiolipin is a phospholipid that is specific to the inner mitochondrial membrane and essential for numerous mitochondrial functions. Accordingly, a quantitative assay for cardiolipin can be a valuable aspect of assessing mitochondrial content and functional capacity. The current study was undertaken to develop a simple and reliable method for direct analysis of the major molecular species of cardiolipin and with particular application for analysis of human skeletal muscle. The method that is presented is based on derivatization of cardiolipin in a total lipid extract with I-pyrenyldiazomethane (PDAM), to form stable, fluorescent 1-pyrenylmethyl esters. The derivatization reaction takes 30 min on ice in a two-phase system (chloroform:methanol:H2O:H2SO4) containing 0.5-1.0 mM PDAM and detergent. The contents of the major cardiolipin species in the derivatization mixture can be estimated by HPLC separation with fluorescent detection during a 20 min run on a reverse phase column and with HPLC grade ethanol/0.5 mM H3PO4 as the mobile phase. The recovery is about 80%. The method is specific and sensitive with quantitation limits of 0.5-1 pmol cardiolipin. The response of the fluorescence detector (peak area) is linear across a range 5-40 pmol. The assay is linear over the range between 0.3 and 3.0 mg of tissue (R-2 = 0.998). The assay provides good reproducibility and accuracy (within 5-10%). (c) 2005 Elsevier B.V. All rights reserved.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available