Results 



EfiBciency of Extraction 



Lipofuscin was successfully extracted and 

 quantified from three different fish tissues: brain, 

 heart, and liver. Wavelengths of fluorescence ex- 

 citation and emission maxima of extracted lipo- 

 fuscin in chloroform are presented in Figure 1. 

 All maxima are within the range cited by Shel- 

 dahl and Tappel (1973); fluorescence excitation 

 maxima were between 340 and 370 nm and fluo- 

 rescence emission maxima between 420 and 470 

 nm. 



Retinol (wavelengths of maximum fluorescence 

 excitation at 325-340 nm and maximum fluores- 

 cence emission at 475 nm) was photooxidized by 

 exposing the chloroform extract to UV irradiation 

 (254 nm). As expected, retinol was found in liver 

 and sometimes it was present in brain and heart 

 tissues. For example, wavelengths of fluorescence 

 excitation and emission maxima in liver of Dover 

 sole shifted from 352 to 365 nm and from 470 to 

 440 nm, respectively, after UV irradiation (see 

 Figure IC). Mullin and Brooks (1988) also found 

 that this UV irradiation is effective in oxidizing 

 retinol although they did not find significant 

 retinol interference in fish tissue. It seems liver 

 tissues may require UV irradiation to oxidize 

 retinol while brain and heart tissue should be 

 checked for retinol presence before irradiation. 



The extractive efficiency of the three methods 

 tested are compared in Table 1. All methods ex- 

 tracted similar fluorescent compounds from the 

 brain oi Stenella sp., as the fluorescence excita- 

 tion and emission spectra were similar. The 

 MacArthur and Sohal (1982) method extracted 



Table 1. — Comparison of the three methods of lipofuscin extrac- 

 tion in brain tissue Fluorescence excitation at 360 nm and fluores- 

 cence emission at 440 nm. Results are presented as fluorescence 

 units per mg of wet tissue, where the fluorescence emission signal 

 is normalized to the intensity of emission of a standard quinine 

 sulfate solution (1 mg L Hn IN sulphuric acid). The three methods 

 were significantly different from each other, P < 0.05, Newman- 

 Keuls range test (Zar 1974). ANOVA: F ratio = 21.81, 2 df, 

 P < 0.05. 



_L 



_L 



_L 



3E0 360 400 440 480 520 



>- 



(/) 



Z 

 LiJ 



UJ 

 U 



z: 



UJ 



o 



UJ 



(r 

 o 



Z) 



320 360 400 440 480 520 



320 360 400 440 480 520 



WAVELENGTH (nm) 



Figure 1. — Spectral characteristics of lipofuscin extracted from 

 Dover sole, Microstomus pacificus, tissues (uncorrected spec- 

 tra). In chloroform. (A) Brain: (1) Lipofuscin fluorescence exci- 

 tation spectrum (emission wavelength 440 nm); (2) lipofuscin 

 fluorescence emission spectrum (excitation wavelength 365 

 nm). (B) Heart: (1) Lipofuscin excitation spectrum (emission 

 wavelength 430 nm); (2) lipofuscin fluorescence emission spec- 

 trum (excitation wavelength 365 nm ). (C ) Liver: ( 1 ) Lipofuscin 

 fluorescence excitation spectrum (emission wavelength 430 

 rmi); (2) lipofuscin emission spectrum (excitation wavelength 

 365 nm) before UV radiation; (3) Same as (2) after UV radiation 

 (254 nm). 



significantly more fluorescent pigment, than did 

 either the Tappel (1975) or the Bligh and Dyer 

 (1959) method which extracted 66% and 46% of 

 maximum extraction, respectively). Thus all 

 three methods are useful for quantitative estima- 

 tion of extractable lipofuscin in fish tissue but 



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