Endocrinology, Vol 137, 2447-2456, Copyright © 1996 by Endocrine Society
Characterization and identification of an adrenal age-related nonpolar fluorescent substance
B Cheng, KY Tserng, J Kowal, KS Buekers, S Abraham and JP Gerhart
Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA.
The adrenal cortexes of humans and rodents accumulate lipofuscin with age,
but the chemical nature of the substance that produces lipofuscin
fluorescence in the gland is not known. Analysis of rat adrenal nonpolar
lipids revealed a fluorescence profile with increased intensity in the
lipids extracted from older animals (23-24 months > 6 months > 6
weeks). The peak occurred at a wavelength of 470 +/- 5 nm(n = 26) when
excited at 340 nm. After sucrose density gradient centrifugation, the
fluorescent substance was primarily concentrated in subcellular lipid
droplets rather than supernatant or particulate. Prolonged stimulation of
rats with ACTH for 7 consecutive days caused 14-51% decreases in the
fluorescence levels, with a tendency of return to control levels
poststimulation regardless of age. In contrast, the nonpolar lipids of
mouse adrenal tumor (Y1) cells, which contain no lipofuscin, did not
display this fluorescence in the presence or absence of ACTH. The
chromatographic characteristics of the substance in a silica gel-60 column
resembled those of authentic retinyl palmitate and cholesteryl oleate.
Analysis of the substance by HPLC demonstrated at least three prominent
peaks, designated XI-3 in order. X1 and X2 were minor peaks; X3 was the
major peak. Whereas none of the peaks comigrated with cholesteryl esters,
X1 comigrated with authentic retinyl palmitate. Determination of the fatty
acid component of the major fluorescent substance X3 by gas-liquid
chromatography disclosed stearic acid. Retinyl stearate was, therefore,
synthesized. The fluorescent profiles, HPLC retention time and mass
spectrometric fragmentation of purified X3 substance were all identical to
those of the synthetic compound. In contrast, the rat liver principally
accumulated retinyl palmitate with age. Thus, we conclude that 1) the major
nonpolar fluorescent substance accumulated in the rat adrenal with age is
retinyl stearate, which may be a fluorophore of adrenal lipofuscin; 2) ACTH
action may be related to this accumulation; and 3) the type of retinyl
ester accumulated in aged animals is organ specific.
