Quantitative profiling of endogenous retinoic acid in vivo and in vitro by tandem mass spectrometry - PubMed (original) (raw)

. 2008 Mar 1;80(5):1702-8.

doi: 10.1021/ac702030f. Epub 2008 Feb 6.

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Quantitative profiling of endogenous retinoic acid in vivo and in vitro by tandem mass spectrometry

Maureen A Kane et al. Anal Chem. 2008.

Abstract

We report an improved tandem mass spectrometric assay for retinoic acid (RA) applicable to in vitro and in vivo biological samples. This liquid chromatography tandem mass spectrometric (LC/MS/MS) assay for direct RA quantification is the most sensitive to date, with a 62.5 attomol lower limit of detection and a linear range spanning greater than 4 orders of magnitude (from 250 attomol to 10 pmol). This assay resolves all-trans-RA (atRA) from its endogenous geometric isomers, is applicable to samples of limited size (10-20 mg of tissue), and functions with complex biological matrixes. Coefficients of variation are as follows: instrumental, < or =2.6%; intraday, 5.2% +/- 0.7%; interday, 6.7% +/- 0.9%. In vitro capabilities are demonstrated by quantification of endogenous RA and RA production (from retinol) in primary cultured astrocytes. Quantification of endogenous atRA and its geometric isomers in 129SV mouse serum and tissues (liver, kidney, adipose, muscle, spleen, testis, and brain) reveals in vivo utility of the assay. The ability to discriminate spatial concentrations of RA in vivo is illustrated with C57BL/6 mouse brain loci (hippocampus, cortex, olfactory bulb, thalamus, cerebellum, and striatum), as well as with Lewis rat proximal/distal mammary gland regions during various morphological stages: virgin, early pregnancy (e7), late pregnancy (e20), lactating (day 4), involuting day 1, and involuting day 11. This assay provides the sensitivity necessary for direct, endogenous RA quantification necessary to elucidate RA function, e.g., in neurogenesis, morphogenesis, and the contribution of altered RA homeostasis to diseases, such as Alzheimer's disease, type 2 diabetes, obesity, and cancer.

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Figures

Figure 1

Figure 1

Structures of atRA, its isomers, and the internal standard 4,4-dimethyl-RA.

Figure 2

Figure 2

RA mass spectra. (A) Q1 scan showing [M + H]+ (m/z: 301.1). (B) Q3 scan showing [M + H]+ and product ions obtained after fragmentation. Both scans were obtained by infusing 200 nM RA at 10 µL/min. Note the reduction in background in (B).

Figure 3

Figure 3

SRM chromatograms of standard solutions: (A) gradient 1, cultured cell/subcellular fraction protocol; (B) gradient 2, tissue protocol. The solid line corresponds to m/z Q1:301/Q3:205, and the broken line corresponds to m/z Q1:329/Q3:151: 1, 4,4-dimethyl-RA; 2, atRA; 3, 9cRA; 4, 13cRA. 9,13-dcRA elutes between 3 and 4 (data not shown).

Figure 4

Figure 4

LOD and LOQ for (A–C) gradient 1, cultured cell/subcellular fraction protocol, and (D and F) gradient 2, tissue protocol. (A and D) 13cRA. (B and E) 9cRA. (C and F) atRA.

Figure 5

Figure 5

Representative calibration curve for atRA. Data were obtained using the cultured cell protocol (_r_2, 0.999). Similar curves were generated for the tissue protocol and for the other geometric isomers with both protocols. Note the functionality of the assay with values of <50 fmol (inset).

Figure 6

Figure 6

Quantification of endogenous atRA and atRA biosynthesis in primary cultures of hippocampus astrocytes. (A) Data were generated using gradient 1 after 5 h of incubation with retinol. Data are averages of triplicates. (B and C) Representative SRM chromatograms of RA in cells and medium, respectively, after incubation with 0.5 µM retinol: IS, internal standard.

Figure 7

Figure 7

Representative SRM chromatograms of endogenous RA. Data were generated with gradient 2. (A) Analysis of liver extract (~40 mg of tissue) from 2–4 month old, male 129SV mice fed 4 IU/g vitamin A. (B) Serum (~150 µL) from the same mice. The solid line represents RA (m/z Q1:301/Q3:205); the dashed line represents the IS 4,4-dimethyl-RA (m/z Q1:329/Q3:151). INT, nonspecific signal.

Figure 8

Figure 8

Quantification of atRA in rat mammary gland. Open bars show atRA in the proximal region; filled bars show atRA in the distal region. Also shown are serum measurements. Samples were collected from female Lewis rats fed a stock diet: V, virgin; EP, early pregnancy (e7); LP, late pregnancy (e20); L, lactation (day 4); ID1, involution day 1; ID11, involution day 11. *P < 0.05, compared to virgin (mammary) or compared to L (serum); **P < 0.05 between proximal and distal. Data are means ± SD, n = 3–13 samples.

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References

    1. Wolf G. Physiol. Rev. 1984;64:873–937. - PubMed
    1. Mark M, Ghyselinck ND, Chambon P. Annu. Rev. Pharmacol. Toxicol. 2006;46:451–480. - PubMed
    1. Maden M. Proc. Nutr. Soc. 2001;59:65–73. - PubMed
    1. Ruberte E, Friederich V, Chambon P, Moriss-Kay G. Development. 1991;111:45–60. - PubMed
    1. Ong DE. Nutr. Rev. 1994;52:S24–S31. - PubMed

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