Materials and Methods 



Specimens of Dover sole were collected on De- 

 cember 1986 ofTPoint Conception. Ages were esti- 

 mated by J. Butler, E. Lynn, and M. Drawbridge 

 of the Southwest Fisheries Center using otolith 

 sections. Ages ranged from 2.7 to 44.7 years (av- 

 erage of three readings). Specimens of the species 

 Salmo gairdneri were collected in July 1986 at 

 Hot Creek Hatchery. Ages ranged from 3 months 

 to 3 years. 



Samples were frozen after collection and kept 

 at -80°C until analysis. Studies by Nicol (1987) 

 showed that this form of preservation yielded the 

 lowest fluorescence when compared with ethanol 

 and formalin-preserved samples. There was no 

 indication that the level of fluorescence changed 

 as a function of time of preservation or by inter- 

 action with the extracted lipofuscin, as in the 

 case for formalin. To excise the brain, the top of 

 the skull was opened and the four brain lobes 

 were removed as a unit, without the optic nerve. 

 To excise the heart, we separated the muscle 

 from connective tissue, blood vessels, and fat de- 

 posits. 



Three methods were compared for maximum 

 lipofuscin extraction. The first two methods (Tap- 

 pel 1975; MacArthur and Sohal 1982) were specif- 

 ically developed for lipofuscin extraction and 

 employed chloroformrmethanol (2:1) as the ex- 

 tractive solvent; they differ in the optimal 

 volume-to-weight ratio (30:1 and 20:1, respec- 

 tively), temperature of extraction, and number of 

 times the chloroform phase is washed with water. 

 The third method was developed for lipid extrac- 

 tion in fishes (Bligh and Dyer 1959) and uses chlo- 

 roform:methanol:water (1:2:0.8) as the extractive 

 solvent. Subsamples (n = 3) of cerebellum of 

 Stenella sp. collected at the eastern tropical 

 Pacific and kept at -30°C were defrosted, dried 

 with lint-free paper, weighed, and extracted fol- 

 lowing the three methods as described originally. 



Tissues from Dover sole were extracted basi- 

 cally following the MacArthur and Sohal (1982) 

 technique, with two additional steps to ensure a 

 complete washing out of flavoproteins and pho- 

 tooxidation of retinol when present (Fletcher et 

 al. 1973). Tissue was freeze dried prior to analy- 

 sis. Ten mL of chloroform: methanol (2:1, v/v) was 

 added to a homogenizer containing the sample for 

 a final solvent:sample ratio of about 120:1 (vol/ 

 dry weight). The sample was ground with a teflon 

 pestle attached to an electric drill and later sub- 

 merged in a 40°C water bath for 1 minute. A 2 mL 



subsample was taken from the homogenate. 

 Three mL of deionized water was added, the sam- 

 ple was shaken, and the emulsion centrifuged 10 

 minutes at 1,912 g and 0°C. After centrifugation, 

 the hyperphase was decanted and a second rinse 

 performed. After decanting the hyperphase a sec- 

 ond time, 1 mL of the hypophase (chloroform con- 

 taining lipofuscin) was sampled and transferred 

 to a polypropylene tube. Three mL of chloroform 

 were added to the sample (for a total of 4 mL) 

 which was then exposed to UV irradiation (254 

 nm) for 1-2 minutes to photooxidize retinol. This 

 last step was routinely performed for liver tissue. 



Samples were then transferred to glass tubes, 

 sealed, and kept in the refrigerator until analysis. 

 Sample fluorescence was measured at the emis- 

 sion peak (430-440 nm) in a quartz cuvette with 

 a Perkin Elmer Fluorescence Spectrophotometer^ 

 Model MFP-44A. The sample was excited at the 

 peak of fluorescence excitation (—360 nm). The 

 intensity of the fluorescent emission (at 430 nm) 

 was normalized to the intensity of the quinine 

 solution standard (1 mg L"^ in IN sulphuric acid) 

 and expressed in fluorescence units. 



Lipofuscin in Dover sole is expressed as 1) total 

 lipofuscin content per organ and 2) weight- 

 specific lipofuscin concentration, calculated by di- 

 viding the total lipofuscin content by the dry 

 weight of the entire organ. 



Lipofuscin ft-om rainbow trout tissues was ex- 

 tracted following the Bligh and Dyer (1959) tech- 

 nique, without modifications. Whole tissues were 

 ground in water with a tissue homogenizer to give 

 a final concentration of 100 mg of tissue (wet 

 weight) in 0.8 mL of homogenate. A sample of 0.8 

 mL was taken and solvents were added to give a 

 final ratio of 1:2:0.8 (chloroform:methanol:water) 

 with a solvent to sample ratio of 20:1 (vol/w). The 

 sample was then filtered through a 2.4 cm glass 

 fiber filter (Whatman GF/C), the tissue re- 

 extracted with 1 mL of chloroform and refiltered. 

 The extract was then washed with 3 mL of water, 

 shaken, and centrifuged for 10 minutes at 1,912 g 

 and 0°C. One mL of the chloroform hypophase 

 was subsampled and fluorescence estimated as 

 described above. 



Lipofuscin in rainbow trout is expressed as 



1) total lipofuscin content per organ and 



2) weight-specific lipofuscin concentration (total 

 lipofuscin content of the organ divided by its wet 

 weight). 



iReference to trade names does not imply endorsement by the 

 National Marine Fisheries Service, NOAA. 



402 



