Quantification of endogenous retinoic acid in limited biological samples by LC/MS/MS

Abstract

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.

Quantitative Profiling of Endogenous Retinoic Acid in Vivo and in Vitro by Tandem Mass Spectrometry

“…LOD/LOQ for 9cRA and 13cRA were 125 attomol (0.125 fmol)/1 fmol and 2 fmol/6 fmol, respectively, for gradient 2. This method provides the best sensitivity to date for detection and quantification of RA, with a 20-fold lower LOD than our previous work 30 and a 13-300-fold lower LOD than other published methods. [32][33][34][35][36][37][38] A representative calibration curve for atRA using gradient 1 shows the extensive linear dynamic range ( Figure 5).…”

“…For example, our previous work, done with a different instrument (API-3000), produced optimum sensitivity with methanol/water/0.1% formic acid. 30 This mobile phase, however, did not provide optimum resolution. Therefore, we used an acetonitrile/methanol/water/0.1% formic acid mixture as a compromise between resolution and sensitivity.…”

“…Data from previous work was acquired with an Applied Biosystems API-3000 triple-quadrupole mass spectrometer equipped with APCI using the conditions described. 30 …”

“…It is important to resolve these RA isomers from atRA and each other, because all four occur either endogenously and/or in metabolically altered/challenged states in humans and/or animals. [26][27][28]30,34 Recent reports of LC/MS methods limited to serum/ plasma that separate only 13cRA from atRA would produce inflated values for one or both, because 9,13dcRA (and if present, 9cRA) comigrate with 13cRA and/or atRA. 33,35 Gradient 1 (12 min run time) provides slightly better sensitivity in half the analysis time, relative to gradient 2.…”

β-Carotene conversion products and their effects on adipose tissue

“…In fact, retinal, which can be produced via BCMO1 cleavage, has been detected in adipose tissue [85]. RAs resulting from oxidation of retinal by RALDHs are also quantifiable in adipose tissue in vivo [52], especially all-trans RA; whereas evidence of the existence of 9-cis RA in vivo is still lacking [35,52,68]. Finally, apocarotenals resulting from asymmetric cleavage of b-carotene can be generated in vivo [31] and may influence parameters of adipose tissue biology, even if no data are currently available on the presence of these molecules in adipocytes.…”