RT Journal Article
SR Electronic
T1 Development of a Highly Sensitive Method Using Liquid Chromatography-Multiple Reaction Monitoring to Quantify Membrane P-Glycoprotein in Biological Matrices and Relationship to Transport Function
JF Drug Metabolism and Disposition
JO Drug Metab Dispos
FD American Society for Pharmacology and Experimental Therapeutics
SP 2440
OP 2449
DO 10.1124/dmd.111.040774
VO 39
IS 12
A1 Tasso Miliotis
A1 Liaqat Ali
A1 Johan E. Palm
A1 Anders J. Lundqvist
A1 Martin Ahnoff
A1 Tommy B. Andersson
A1 Constanze Hilgendorf
YR 2011
UL http://dmd.aspetjournals.org/content/39/12/2440.abstract
AB The quantification of P-glycoprotein [P-gp, ABCB1, multidrug resistance 1 (MDR1)] protein in biological matrices is considered a key factor missing for useful translation of in vitro functional data to the in vivo situation and for comparison of transporter data among different in vitro models. In the present study a liquid chromatography (LC)-mass spectrometry method was developed to quantify P-gp membrane protein levels in different biological matrices. The amount of P-gp transporter protein was measured in Caco-2 cell monolayers and in inside-out human embryonic kidney (HEK)-MDR1 vesicles. From both in vitro systems, two preparations with different functionality were used. Transporter function was determined as digoxin efflux in Caco-2 cell monolayers and N-methylquinidine (NMQ) uptake in membrane vesicles, and, in addition, mRNA expression in the Caco-2 monolayers was measured. The results showed an excellent relationship between NMQ uptake functionality in inside-out HEK-MDR1 vesicles and protein contents. Similar concordance between the digoxin efflux and P-gp content in different Caco-2 cell cultures was observed, whereas mRNA levels are indicative of increased P-gp content and activity in older Caco-2 cultures, however, not yielding the same quantitative relationship. The results from both Caco-2 and HEK-MDR1 membrane vesicles confirm that the protein content is directly related to the level of activity in the respective system. The method presented here to quantify P-gp protein by LC-multiple reaction monitoring will facilitate the development of future methodologies to bridge between expression systems and cell/tissue models and to scale from in vitro models to whole organs.