3 0

IMPULSE COUNTlNG OF C"-CONTENTS IN VARIOUS TISSUES AFTER INJECTION OF Ci4--i-CHOLESTEROL

AND C'4-26-CHOLESTEROL

In order to get more quantitative results for some organs o f C"-cholesterol injected mice the concentration of radioactivity in some oi-gans was measured by impulse counting. The comparison of the kinetics between cholesterol. pregnenolonc and progesterone was facilitated by this investigation, since the autoradiographic studies mainly gave qualitative data.

The distribution of C"-labelled cholesterol in the animal body as well as in humans has been studied earlier by impulse counting. Gould (1952) gave blood. containing labelled cholesterol, to dogs and found that the specific activity of cholesterol in liver and spleen reached half that of plasma in a few hours and in kidney, lung, heart, intestine and diaphragm in 1 to 2 days. In the adrenal, aorta and skin half the plasma value was reached more slowly. Chevallier (1953) administered C'*-labelled cholesterol orally to rats during several days. After 9 days of feeding, intestine, liver and serum had high and essentially equal specific activity of cholesterol. Spleen, bone marrow. lung, depot fat, heart and adrenal glands had between 60 and X O O i n o f the serum value and kidney, skin and muscle about 30 ()/a and the testicle somewhat less. The brain had no detectable activity. Borgstriim et al. (1958) gave CIJ-4-chol- esterol orally to fasted rats and found that 50-70 " i n o f the labelled cholesterol was rapidly absorbed in the intestinal mucosa and was delayed there before i t was taken up in the chyle. As a consequence of this delay of the absorbed chol- esterol the blood activity curve reached its maximuni about 16 hours after feed- ing. Avigan et al. (1962) gave C'~-4-cholesterol rats and determined the ratios between specific radioactivities of cholesterol in various organs and that in serum as a function of time. Values considerably above 1 were found in kidney and muscle after 20-49 days. Values about 1.5 were found in liver, lung. heart. spleen after 3 to 49 days. In the brain values above 1 were reached after 49 days. Chobanian et al. (1962) made post mortem analyses o f tissues for specific activity of (:"-cholesterol in humans who died 1-22b days after receiving tracer doses of C'4-labelled cholesterol. Chmplete equilibration of serum and tissue cholesterol was seen within 1 month in liver, spleen, kidney. lung. adrenal, intestine, muscle, abdominal aorta and fat. In the brain the degree o f equilibration was negligible. Lossow et al. ( 1 962) injected C:''-2T- cholesterol intravenously in rats. After 24 hours they found the highest value in o/o of injected C1* per gram tissue in the adrenal and slightly less in spleen and liver. Swell and Law (1966) studied the labelling of liver and serum cholcsterol esters after injection of C;' '-4-cholesterol and found a higher value in the liver than in serum after 24 hours.

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31

In the present study the adrenals, ovaries, uterus, testes, accessory sex glands, blood and liver were selected for impulse counting. The mice were injected intravenously with C'4-4-cholesterol or C14-26-cholesterol and were killed at different intervals from 2 minutes to 20 days after injection. The organs were then immediately removed and homogenized in saline solution and extracted in chloroform. Aliquots of the extracts were counted in a Pac- kard Tri-Carb liquid scintillation spectrometer.

MATERIALS AND METHODS

C'4-4-cholesterol and C14-26-cholesterol were obtained from The Radio- chemical Centre, Amersham, England with the specific activity of 24.6 mci/ mM and 24.0 mci/mM respectively. Radiochemical purity was tested as des- cribed earlier (p. 7 ) .

Two series of NMRI-mice, 16 male and 16 female mice, were injected intravenously with either about 0.9 pci C14-4-cholesterol or about 0.9 pci C14-26-cholesterol. The substances were dissolved in 25 microliters of ethanol and injected according to the method described earlier. The animals weighed about 20 grams. From each series 2 animals of each sex were allowed to survive for 2, 20 and 60 minutes, 4 and 24 hours, 4, 10 and 20 days. The animals were sacrificed by stretching the spines. From each animal samples of blood, liver and adrenals were taken. From the male mice testicles together with the epididymides, and accessory sex glands (vesicular, coagulating and ampullary glands and dorsal and ventral prostates) and from the female mice ovaries and uterus were taken. The samples were pooled from the 2 ani- mals with the same survival time and the wet weights were registered. The tissue samples were homogenized in 1 ml 0 . 9 O / o saline solution in a Potter- Elvehjem homogenizer and the lipids were then extracted with about 10 ml chloroform in 3 to 5 ml portions. The chloroform phase was separated from the water phase by centrifugation and was sucked out with disposable pipettes and evaporated to dryness with a gentle stream of nitrogen. The residue was dissolved in 1 ml chloroform and 0.1 ml samples were transferred to liquid scintillation vials containing a solution of 3 ml ethanol and 7 ml 0.5 O/o PPO in toluene and counted in a liquid scintillation spectrometer (Packard). From the supernatant in the water phase 0.1 ml was also counted in the same way. The values were calculated as O / O gram dose per gram tissue. The remaining tissue samples in the water phase were washed and dried and counted in a GM-counter.

32

RESULTS AND COMMENTS

T h e chromatographic control of the original substances showed that > 07 O/o had an IX-value corresponding to that of inactive cholesterol. Hardly any radioactivity was found in the extracted tissue rests and will not be considered here. T h e radioactivity of the water phases was low in blood, livci- and adrenals and varied between 0 and 3 o/o of the amounts found in the chloroform extracts. In the sex organs the concentration in a few cases exceeded 3 ')/I) and values between 0 and 1 0 O / o were registered.

T h e results of the quantitation of the chloroform phase are presented in diagrams in which the values in "10 gram dosdgram tissue are plotted against time after injection (Fig. 21). T h e concentration of CI4 between the two differently labelled compounds seemed to be almost identical after the dif- ferent intervals after injection.

T h e blood concentrations were high immediately after injection hut de- creased rapidly, which might be due to the accumulation of injected Cl4-chol- esterol in the liver. After 20 minutes an increase of C 1 4 in the blood was registered and this continued until a peak was reached between 4 and 21 hours. Then a slow decrease in blood concentration was apparent. T h e livcr reached a peak between 1 hour and 4 hours and then slowly diminished and after 4 days the values slowly sank towards zero. T h e decrease o f labelled cholesterol in the liver coincided with the increase in blood concentration. T h e differences in liver concentration between C:14-4-cholesterol and C"-26- cholesterol seen in the autoradiograms from male mice 20 and 60 days after injection may be supported by the slight difference in the concentration observed in the male mice in this investigation 10 and 20 days after injection. T h e most striking uptake of CI4 was seen in the adrenal with a peak after 24 hours to 4 days and then decreasing but remaining high in comparison with other organs.

T h e C'4-concentration in the ovurirs followed the blood concentration pattei-n and reached a maximum between 4 and 21 hours but in contrast to that of the blood then remained rather high. T h e time sequence for the t ~ s t r . ~ was very similar but somewhat lower concentrations were noted. I t must, how- ever. be remembered that the quantitative data on the whole ovaries and testicles do not give justice to the high, specific concentrations seen in various parts o f these organs in the autoradiographic investigation. Thus the dif- ferences in concentration between blood and follicles and corpora lutea in ovaries and between interstitial cells in the testicles were greater than could be judged from the diagrams. However, for comparison between the different precursors, the diagrams seem to give adequate information.

These quantitative data for the adrenals, ovaries and testes confirm the findings from the autoradiograms that both the uptake and release of radio-

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activity after injection of CIJ-cholesterol were slow in comparison with labelled pregnenolone and progesterone.

In uterus and the 7nak accessory sex glands also very good agreement bet- ween the two differently labelled compounds were seen. This indicates that cholesterol as such was taken up there and may conceal a possible uptake of steroid hormones formed from ring labelled cholesterol.