Quantification of endogenous retinoic acid in limited biological samples by LC/MS/MS.
We report a sensitive LC (liquid chromatography)/MS/MS assay using selected reaction monitoring to quantify RA (retinoic acid), which is applicable to biological samples of limited size (10-20 mg of tissue wet weight), requires no sample derivatization, provides mass identification and resolves atRA (all-trans-RA) from its geometric isomers. The assay quantifies over a linear range of 20 fmol to 10 pmol, and has a 10 fmol limit of detection at a signal/noise ratio of 3. Coefficients of variation are: instrumental, 0.5-2.9%; intra-assay, 5.4+/-0.4%; inter-assay 8.9+/-1.0%. An internal standard (all-trans-4,4-dimethyl-RA) improves accuracy by confirming extraction efficiency and revealing handling-induced isomerization. Tissues of 2-4-month-old C57BL/6 male mice had atRA concentrations of 7-9.6 pmol/g and serum atRA of 1.9+/-0.6 pmol/ml (+/-S.E.M.). Tissue 13-cis-RA ranged from 2.9 to 4.2 pmol/g, and serum 13-cis-RA was 1.2+/-0.3 pmol/ml. CRBP (cellular retinol-binding protein)-null mouse liver had atRA approximately 30% lower than wild-type (P<0.05), but kidney, testis, brain and serum atRA were similar to wild-type. atRA in brain areas of 12-month-old female C57BL/6 mice were (+/-S.E.M.): whole brain, 5.4+/-0.4 pmol/g; cerebellum, 10.7+/-0.3 pmol/g; cortex, 2.6+/-0.4 pmol/g; hippocampus, 8.4+/-1.2 pmol/g; striatum, 15.3+/-4.7 pmol/g. These data provide the first analytically robust quantification of atRA in animal brain and in CRBP-null mice. Direct measurements of endogenous RA should have a substantial impact on investigating target tissues of RA, mechanisms of RA action, and the relationship between RA and chronic disease.
Kane
Maureen A
MA
Department of Nutritional Sciences and Toxicology, University of California Berkeley, Berkeley, CA 94720-3104, USA.
Chen
Na
N
Sparks
Susan
S
Napoli
Joseph L
JL
D000077556:Alitretinoin; D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods*; D005260:Female; D015474:Isotretinoin / Q000032:analysis*; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D015394:Molecular Structure; D015203:Reproducibility of Results; D054840:Retinol-Binding Proteins, Cellular / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity; D014018:Tissue Distribution; D014212:Tretinoin / Q000032:analysis*
D054840:Retinol-Binding Proteins, Cellular; D000077556:Alitretinoin; D014212:Tretinoin; D015474:Isotretinoin