Quantitation of lysophosphatidylcholine molecular species in rat cardiac tissue.
We have developed a rapid and sensitive procedure for isolation and measurement of 1-acyllysophosphatidylcholine (LPC) species in rat myocardial tissue. Tissues were spiked with heptadecanoyl-LPC internal standard and extracted with chloroform/methanol. The chloroform phase was dried, resuspended in chloroform/propan-2-ol (2/1, v/v), and applied to an aminopropyl-bonded phase (Bond Elut) column. Following stepwise elution with several solvent mixtures, the LPC fraction (ethyl acetate/methanol, 4/6, v/v) was separated by HPLC with direct quantitation of palmitoyl-LPC (P-LPC), oleoyl-LPC (O-LPC), and stearoyl-LPC (S-LPC), using an evaporative light scattering mass detector. Calibration curves were generated for each individual LPC species. Recoveries of added [14C]LPC and of heptadecanoyl-LPC internal standard after extraction and chromatography were 85.8 +/- 1.9% (mean +/- SE, N = 10) and 83.4 +/- 1.8% (N = 15), respectively. This assay showed satisfactory sensitivity, reproducibility, and accuracy for measurement of LPC species in rat myocardial tissue. The major molecular species of LPC in rat myocardium were found to be P-LPC and S-LPC, which were two- to sixfold as abundant as O-LPC. In isolated, crystalloid-perfused rat hearts the time of perfusion was found to significantly influence the content of P-LPC (0 min, 252 +/- 10; 15 min, 178 +/- 10, P less than 0.001, compared with 0 min; 40 min, 131 +/- 4, P less than 0.001; and 70 min, 129 +/- 4, P less than 0.001; nmol/g dry weight), but not the content of O-LPC and S-LPC. The method will be useful for studying the participation of LPC species in physiology, pathophysiology, and therapeutics.
Vesterqvist
O
O
Department of Pharmacology, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543-4000.
Sargent
C A
CA
Taylor
S C
SC
Newburger
J
J
Tymiak
A A
AA
Grover
G J
GJ
Ogletree
M L
ML
D000818:Animals; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D008027:Light; D008244:Lysophosphatidylcholines / Q000032:analysis*; D008297:Male; D009206:Myocardium / Q000737:chemistry*; D010713:Phosphatidylcholines / Q000032:analysis; D051381:Rats; D011919:Rats, Inbred Strains; D015203:Reproducibility of Results; D012542:Scattering, Radiation
D008244:Lysophosphatidylcholines; D010713:Phosphatidylcholines; C006065:We 201; C082823:1-oleoyl lysophosphatidylcholine; C026564:stearoyl alpha-lysolecithin