HPLC/UV quantitation of retinal, retinol, and retinyl esters in serum and tissues

Abstract

We report robust HPLC/UV methods for quantifying retinyl esters (RE), retinol (ROL), and retinal (RAL) applicable to diverse biological samples with lower limits of detection of 0.7, 0.2, and 0.2 pmol, respectively, and linear ranges greater than 3 orders of magnitude. These assays function well with small, complex biological samples (10-20mg tissue). Coefficients of variation range from 5.9 to 10.0% (intraday) and from 5.9 to 11.0% (interday). Quantification of endogenous RE, ROL, and RAL in mouse serum and tissues (liver, kidney, adipose, muscle, spleen, testis, skin, brain, and brain regions) reveals utility. Ability to discriminate spatial concentrations of ROL and RE is illustrated with C57BL/6 mouse brain loci (hippocampus, cortex, olfactory bulb, thalamus, cerebellum, and striatum). We also developed a method to distinguish isomeric forms of ROL to investigate precursors of retinoic acid. The ROL isomer assay has limits of detection between 3.5 and 4.5 pmol and has a linear range and coefficient of variation similar to those of the ROL/RE and RAL assays. The assays described here provide for sensitive and rigorous quantification of endogenous RE, ROL, and RAL to elucidate retinoid homeostasis in disease states such as Alzheimer's disease, type 2 diabetes, obesity, and cancer.

Figure 1

Structures of analytes in the central pathway of retinoid metabolism. Typical in vivo levels of each analyte are listed. Ranges reflect variation among tissues.

Figure 2

Conversion of RAL into RAL(O-ethyl)oxime.

Figure 3

HPLC/UV chromatograms of standard solutions. (A) Total ROL/RE method: ROL (4.8 min) and RE (16.5 min). (B) RAL-oxime method: anti-RAL-(O-ethyl)oxime (6.6 min), ROL (7.2 min), and syn-RAL-(O-ethyl)oxime (10.9 min). The solid line indicates absorbance at 368 nm (left Y-axis); the dashed line indicates absorbance at 325 nm (right Y-axis). (C) ROL isomer method: RE (2.0 min), IS (retinyl acetate, 3.6 min), 13cRA (10.9 min), 9cRA (12.1 min), atRA (13.1 min), 13cROL (20.9 min), 9cROL (27.0 min), and atROL (28.9 min). The RE standard shown is retinyl palmitate. Absorbance of ROH isomers was monitored at 325 nm (left Y-axis); absorbance in the overlay showing RA isomers was monitored at 340 nm (right Y-axis).

Figure 4

Comparison of (A) methanol-based ROL/RE mobile phase with (B) acetonitrile-based ROL/RE mobile phase. (A/B) are identical mouse kidney samples with ROL (4.5min/4.8 min), IS (retinyl acetate, 7.5 min/8.9 min) and RE (11.7 min/16.6 min). The methanol-based mobile phase in (A) uses the same gradient as the acetonitrile-based mobile phase in (B) with methanol/water/1,2-dichloroethane.

Figure 5

Representative calibration curves for (A) Total ROL/RE method, (B) RAL-oxime method, (C) ROL isomer method. All r² values were >0.99.

Figure 6

HPLC/UV chromatograms of mouse tissue. (A) Total ROL/RE method measuring adipose: ROL (4.8 min), IS (retinyl acetate, 8.9 min), and RE (16.6 min). (B) RAL-oxime method measuring liver: anti-RAL-(O-ethyl)oxime (6.6 min), ROL (7.2 min), and syn-RAL-(O-ethyl)oxime (10.9 min). (C,D) ROL isomer method measuring liver: 13cROL (20.9 min), 9cROL (27.0 min), atROL (28.9 min). Top panel in (C) is a magnified view showing 13cROL and 9cROL more clearly.

Figure 7

Addition of exogenous retinoids to mouse liver before homogenization to show 9cROL is not formed artifactually. Left panel of each pair is a magnified view of the right panel to show 9cROL more clearly. (A) Addition of 9cROL increases 9cROL only. (B) Addition of atROL increases atROL only. (C, D) Addition of either 9cRAL or atRAL does not increase either 9cROL or atROL.