Metabolic studies with seleniated compounds

International Journal of AppHed Radiation and Isotopes, 1967, Vol. 18, pp, 545-556. Pergamon Press Ltd. Printed in Northern Ireland

Metabolic Studies With Seleniated Compounds* I--Kinetic Studies with Se7503 in Ratst A. I M B A C H ++ a n d J. S T E R N B E R G Laboratory of Nuclear Medicine and Radiobiology, Department of Physiology, Faculty of Medicine, University of Montreal, Montreal, Canada

(Received 28 October 1966) Trace amounts of Se75OsNa~ were injected intravenously into adult female rats and the blood dynamics, excretion pattern, body burden and distribution in the subcellular fractions of the liver were examined between 15 rain and 72 hr after injection. I n blood, selenite is very rapidly bound to the plasma proteins, which act as carriers of the trace element as well as final products of the metabolic incorporation of the isotope. The albumin-bound Se 75 prevails in the carrier phase, while during the elaboration phase the isotope is chiefly bound to the e and ~ globulins. The binding in the carrier phase seems to be of the anionic selenlte-basic endings of the serum proteins and can be reproduced in vitro, although to a lesser extent than in vivo. A significant amount of Se 75 is exhaled through the lungs during the first 30 min after injection; this is related to the transmethylation of selenite and the formation of the volatile compound Se(CHs) 2. The phenomenon takes place chiefly within the liver mierosomes, and could be taken as an index the of liver function, since experimental modification of the liver capacity result in a modification of the rate of lung excretion of di-methyl selenide. The kidney is the most important excretion route for selenite; there is little faecal excretion only during the first 24 hr after injection. The biliary excretion of selenite is also less important than the urinary elimination; the isotope does not seem to be bound to the common biliary salts. There is little resecretion of selenite through the digestive tube. In the subcellular fractions of the liver, selenite is equally distributed among nuclei, mitochondria and microsomes; the largest fraction remains in the soluble liver compounds and is rapidly excreted through the kidney; the soluble Se 75 in the liver is not bound to the proteins. ETUDES C I N ~ T I Q U E S AVE(] LE St~LI~NITE MARQUI~ AU SE 75 CHEZ LE R A T La cin~tique du s616nite a fit6 6tudi6e chez la rate adulte, apr6s administration intraveineuse de doses traceuses de SeTnO3Na2, et la d6termination de l'isotope dans les tissus et les excretions, entre 15 minutes et 72 heures apr6s injection. Le s~l~nite est li~ rapidement par les prot~ines plasmatiques, qui agissent ~ la fols comme transporteurs du s616ninm et comme produits m6taboliques de l'isotope. La phase de transport est caract6risfie par une fraction Se75-albumine, tandis que dans la phase d'61aboration, le Se 75 est li6 surtout ~ la globuline ~ et y. La liaison du s616nite ~ la fraction albumine semble 6tre dfie * Work supported partly by a Grant-in-Aid from the Medical Research Council of Canada, and by a Research Grant-in-Aid from the Abbott Company, North Chicago and Montreal. ]" Paper presented at the 14th annual meeting of the Society of Nuclear Medicine, June 23-26, 1966, Philadelphia, U.S.A. ** These studies were carried out in fulfillment of the requirements for the degree of Ph.D., Department of Physiology, University of Montreal (A. I.). 1

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A. Imbach and J. Sternberg la c o m b i n a l s o n d u radical a n i o n i q u e SeT~O~ ~- a u x r a d i c a u x basiques des prot~ines plasmatiques; elle p e u t ~tre r e p r o d u i t e in vitro, quoique ~ u n m o i n d r e degrd q u ' in vivo. U n e p r o p o r t i o n significative de Se 7s est ~liminde p a r le p o u m o n a u b o u t des premieres 30 minutes apr~s injection intravelneuse; ce p h d n o m ~ n e est p r o b a b l e m e n t lid ~ la transm~thylation d u sdl~nite et la f o r m a t i o n d u composd volatil Se75(CHs)~. L a t r a n s m d t h y l a t i o n se p r o d u i t a u n i v e a u des microsomes hdpatlques, et u n c h a n g e m e n t de la fonction h ~ p a t i q u e est refl~td d a n s u n e v a r i a t i o n d u t a u x d'excr~tion p u l m o n a i r e d u compos6 radioactif. L'excr~tion r~nale d o m i n e chez le rat, tandis que l'dlimination f~cale est i m p o r t a n t e p e n d a n t le p r e m i e r j o u r apr~s injection; il y a p e u de resdcr~tion de l'isotope dans le t u b e digestif. Aussi, t'excr~tion biliaire d u s~ldnite est moins i m p o r t a n t e q u e ceUe u r i n a i r e ; le sdldnite n e semble pas ~tre lid a u x sels billaires c o m m u n s . D a n s les fractions sous -cellulaires d u foie, le s~Idnite est distribu~ ~galement e n t r e les noyaux, les mitochondries et les microsomes; la plus forte p r o p o r t i o n d'isotope se trouve d a n s la fraction soluble, sous u n e forme n o n li~e a u x prot~ines. H3YqEHHE M E T A B O J I H 3 M A C HOMOII~t)IO C E J - I E H H T O B b I X C O E ~ H H E H H H . I. H 3 Y t I E H H E H H H E T H H H C IIOMOII~bIO SeTaO8 H A H P b I C A X . H e r o . b r a H e ROJmqeCTBa So~sOsNa 2 BB0~HJIHCI~ BHyTpIIBeHHO BSpOCJIhIM CaMKaM Hp~c H ~IIHaMIIHa Rp0BH, THH BbI~e~eHHI~, Harpy3Ha Ha opraHHSM, pacnpeneaeH~e B HO~JIeT0qH~X cTpyRTypax HaS~IO~la~Hcb ~epe8 15 MHHyT B TeqeHH~l 72 qaCOB Hoeale BBe~eHHH. ]3 HpOBH ceaeHHv OqeHh 5MCTpO CBHS~BaeTCH C HpoTeHHaMH HJIaSMH, Hovop~e Kei~cTBymT HaR HepeHocq•nH MeqeHoro aTOMa, TaHme RaH H HOHeqHMX Hpo~yHTOB MeTaeoxl~qecHHx BRJIIOqeHH~ pa~HOHSOTOHa. CBHSH Se 7s C aJIt~SyMHHOM I~peoSJ1a~amT B CTaI~HH HepeHoca, B TO BpeMH HaR B CTa~HH HepepaS0THH HBOTOH~rJIaBHb~M05pasoM, CBHSaH C ~ H ~orJIO5y~HHOM. Bepo~TH0, CBH3H B ~ a s e nepeHoca oHaShIBa~OTCH aHHOHHO-CeJIeHHTOBMMH C OCHOBHMMH HOHI~eBI,IMH rpynnaMH CMBOpOTOqH~X HpoTeHHOB H MoryT 5~T~. B0eIIpOHSBe~eHhI in vitro XOTH H C MeHI.me~i HpOTHH~eHHOCTI,~O tIeM in vlvo. ~Haq~TeJ~H])ie ~O~HqecTBa Se Ts B~7~XamTCH ~IerRHMH B TeqeHHH HepB~X 30 MHHyT Hoc~e BBe~eHII~I. OTO CBH3aHO C TpaHCMeTHJIHp0BaHHeM ce~IeHHTa H ~opMHpOBaHHeM ~ievyqero coe~HHeHtI~I Se(CH~) 2. J:IBJIeH~le HMeeT MecTo, rJIaBHbIM oepaaoM, B MIIHpocoMax IIe~leHH H MoH~eT 5hITI~ noRaBaTeJ~eM ~yHHI][HH 1]e~eHH, TaR HaH H3MeHeHHH BeJIHqHHM neqeHH B OIIMTe HpHBO~HT R H~MeHeHHIO cRopOCTH BIaI~eJIeHHH ~IeFHHMH ceJIeHIICTOrO ~HMeTHJIa. IIot/HH O~HH H8 caMMx rJIaBHhIX IIyTel~ ~JIH BhIBO~a £eJIeHHTa, 8aMeqeHi~ He60JII~HIHe {~eHaJibH~ie BH~eJIeHHH B HepBt,Ie 24 ~aca IIOCJie BBe~eHHH. CBHSaHHMe C HetleHI~IO Bt~I~eJIeHHH HBJIHIOTCH MeHee BaH-~HLIMH~~leM BM~eJIeHHe C MOqe~ H3OTOH~ BepOHTHO~ He CBH3MBaeTCH C 05hItIHMMH COJIHMHIIetleHH. HMeeTcH HeSOJII~IIIOe B~I~eHeHHe ceJIHHHTa qepeB IIHIEIeBapHTeJIt~HhI~ TpaRT. ]3 HO~HJIeTOqHMX ~paHI~HHX HetleHH ceJIeHHT paBHOMepHo pacHpe~ie~eH MeH~y H~paMH, MHTOXOH~pHHMH, MHRpOCOMaMH~ HaHSozIi~Luee ROJIHtleCTBO ocTaeTcH B paCTB0pHMI~IX cOe~HHeHHHX IIeqeHH H 5hICTpO BMBO~HTCHHOtIHaMH. PaCTBOpHMM~ ceHeHHT B HetIeHH He CBHSaH C HpOTeHHaMH. STOFFWEGHSELSTUDIEN MIT SELENIAT-VERBINDUNGEN S T U D I E N M I T Se?SO3 I N R A T T E N

I-KINETISCHE

Es w u r d e Se75OsNa2 in S p u r e n betr~igen intraven6s in ausgewachsene weibliche R a t t e n eingespritzt u n d in d e r Zeit v o n 15 M i n u t e n bis 72 S t u n d e n n a c h d e r Einspritzung w u r d e n Blutdynamik, Ausscheidungsverhalten, K 6 r p e r b t i r d e u n d V e r t e i l u n g in d e n subzellularen Teilen d e r L e b e r gepriift. I m Blut w i r d Selenit sehr schnell a n die P l a s m a p r o t e i n e g e b u n d e n , die als Tr~iger des S p u r e n e l e m e n t s u n d a u c h als Enderzeugnisse d e r Stoffwechseleinverleibung des Isotops auftreten. Das a l b u m i n g e b u n d e n e Se 75 wiegt in d e r Tr~igerphase vor, w~ihrend in d e r A u s a r b e i t u n g s p h a s e das Isotop haupts~ichlich a n die ¢¢- u n d y - G l o b u l l n e g e b u n d e n ist. Die B i n d u n g in d e r Tr~igerphase scheint eine d e r anionischen Selenit-basischen E n d e n des Serumproteins zu sein u n d k a n n wohl in vitro r e p r o d u z i e r t werden, a b e r in e i n e m kleineren U m f a n g als in vivo. E i n e betr~chtliche M e n g e yon Se ~5 wird d u r c h die L u n g e n w~ihrend der ersten 30 M i n u t e n n a c h E i n s p r i t z u n g a u s g e a t m e t ; dies h~ingt z u s a m m e n m i t d e r T r a n s m e t h y l a t i o n y o n Selenit u n d der Bildung d e r fliichtigen Se(CH3) 2 V e r b i n d u n g . Dieser V o r g a n g vollzieht sich

Metabolic studies with seleniated compounds

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hauptsiichlich innerhalb der Mikrosomen der Leber und k6nnte als Index der Leberfunktion angesehen werden, da experimentelle Ver~inderungen der Leberkapazit~it eine Ver~_uderung der Lungenausscheidegeschwindigkeit yon Dimethylselenid ergeben. Die Niere ist der wichtigst Ausscheidungsweg fiir Selenit. Nur wenig f'~tkaleAusscheidung ist zu beobachten w/ihrend der ersten 24 Stunden nach der Einspritzung. Die Gallenausscheidung ,con Selenit ist auch weniger bedeutend als die Urinausscheidung. Das Isotop scheint nicht an die gew6hnlichen Gallensalze gebunden zu werden. Es besteht nut wenig Wiederausscheidung yon Selenit durch den Verdauungskanal. In den subzellularen Teilen der Leber ist Selenit gleichm~sig verteilt zwisehen Kernen, Mitochondrien und Mikrosomen; der gr6sste Anteil verbleibt in den 16slichen Leberverbindungen und wird schnell durch die Niere ausgeschieden; das 16sliche Se~5 in der Leber ist nicht an Proteine gebunden. INTRODUCTION THE POPULARITY of seleniated compounds i n diagnostic procedures derived largely from the assumption that selenium follows the metabolic p a t h w a y of sulfur, constituting a convenient solution for in vivo studies of protein metabolism with the aid of a y-emitting tracer. Having originated in the study of synthesis of pancreatic proteins ¢1), this procedure was recently extended to the study of parathyroid physiopathology ~9~, and also as a diagnostic agent in malignant lymphomas c3). However, there is increasing evidence that the metabolic r61e of selenium is far more complex than that of a simple periodic relative of sulfur, similar to the pairs strontium/calcium or rubidium/potassium. Classified not long ago as a toxic element, selenium has undergone a drastic change in recent years; at present, it is considered if not entirely an essential trace element, at least one with a strong participation in some fundamental respiratory mechanisms of the animal cell. At the unicellular level, there is metabolic antagonism between selenium and sulfur, such as for instance in Desulfovibrio desulfuricans, where selenate ions competitively inhibit the reduction of sulphate by the microorganism ~4). T h e growth of yeast cultures is inhibited b / s e l e n a t e , but the inhibitory effect can be reversed with the addition of methionine (5~. I n mammals, SGHWARZ furnished evidence of the nutritional role of selenium compounds in the maintenance of the functional integrity of the liver cell (6). An organoseleniated compound, different from selenomethionine or selenocystine, has a highly protective effect against liver necrosis in vitamin E-deficient rats; this compound was n a m e d

factor 3, completing the list of the protective factors, namely cystine and vitamin E. I t is even thought today that the protective effect of cystine is related to its selenium impurities, since amounts of 1-2 #g/g of selenium were detected in cystine b y activation analysis. T h e interaction between selenium and ~-tocopherol is not understood, but it points to a common target effect on the respiratory chain in the mitochondrion; (7,s) in rats in selenium deficiency or in vitamin E deficiency, the liver cell shows a marked diminution of its respiratory rate, and a fatal necrosis follows within a few days. This condition is prevented by addition of 0-1 /~g factor 3, or 0.3 #g selenite to the diet; amongst the seleniated compounds tested by SCHWARTZ, the y-y'-diseleno-divaleric acid exhibited the highest protective effect in animals, and also yielded promising results in clinical trials in children with Kwashiorkor. Undoubtedly, a more detailed knowledge of the metabolic pathway of selenium is of importance for both basic science and clinical applications. T h e present p a p e r is part of a comprehensive study of the kinetics of Se 75 in the rat, designed to probe into the metabolic interrelation between this trace element and vitamin E. T h e study in normal animals will then be compared to animals in vitamin E deficiency or treated with large amounts of ot-tocopherol. MATERIAL AND METHODS Female Sprague-Dawley rats were maintained on Purina Chow diet and tap water ad libitum the daily intake averaged 10-12 g food, per animal of 180-200 g; the daily intake of sulfur, methionine and cystine were calculated at

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15, 3.6 and 2-5 mg respectively. I t was not possible to determine the a m o u n t of selenium contained in the diet. Hemodynamie studies were carried out by inserting a polythene tube into the jugular and by withdrawing samples of 0-10-0-20 ml. blood at intervals ranging between 5 and 15 rain, during the first 2 hr after injection. An equal amount of saline was introduced through the tube after each sample, and the hematocrit was maintained as closely as possible to the initial values. With this procedure, it is possible to maintain an animal for 3-4 hr under pentothal anesthesia. Bile was collected b y insertion of a fine polythene tube into the hepatic end of the main bile duct; when the restoration of the enterohepatic cycle was needed, a second rat was cannulated in the same manner, but the distal end of the tube was inserted into the duodenal end of the bile duct of the first (receiver) animal: thus the loss of bile was replaced by a similar intake of non-radioactive bile from a donor (see Fig. 6).

Radioisotope procedures Se 75 was injected as slightly alkaline solution ( p H = 7.6) in a volume of 0"5-1.0 ml into the jugular opposite to the cannulated side; the average a m o u n t was 20/~c, with a specific activity of 13 #g stable selenium/10/~c; this is equivalent to 130 #g/kg, which is far less than the toxic dose of 1,500-2,000/~g/kg for selenite. Doses as high as 100/~c (650#g/kg) were injected when the samples were analyzed for distribution in subcellular fractions or in bile compounds; no ill-effect was noted in the animals, during the experimental period. Blood and tissue samples were assayed in single channel spectrometry at 269 keV 4- 5 %. T h e p u l m o n a r y excretion was assayed in a large volume counter, which will be described in detail elsewhere~15~; the animals were plaeed into an airtight lucite cage (Fig. 1), immobilized in another lucite holder, similar to that used for basal metabolism rate determination. A light anesthesia with nembutal was performed, and the isotope was rapidly injected into the jugular vein; the airtight cage was immediately placed into the counter, and the air was continuously extracted with a circulation pump, at a rate of

450 ml/min. This rate did not induceanychange in the respiration r h y t h m of the animal, for the experimental period. T h e radioactivity of the body was measured either by direct printing every minute, or b y a strip chart recorder (Fig. 4). T h e exhaled volatile seleniated compound was recovered by passing the exhaled air through a washing flask filled with Diginelli's solution (I0~o HgClz in HC1 2 0 ~ ) . EXPERIMENTAL

RESULTS

A. Dynamics of labeled selenite in blood Figure 2 shows the concentration of Se 7~ in plasma and red ceils for 5 hr; Fig. 3 continues the analysis up to 72 hr. T h e blood content of selenite diminishes very rapidly: at 3 rain after injection, the total amount of plasma isotope averages 58-9 % of the injected dose, failing to 17.6% at 10rain and 8.9% at 15 rain. T h e plateau reached at 30 rain at 3-4 % of the injected dose means that, of the total amount of 26 # g stable selenium given with the isotope, an amount of 0.08-0.10 /~g remains in the plasma. This ratio does not seem to be related to the level of plasma sulfur, for injection of larger doses (100 #c Se ~6, or 130 #g stable Se) or smaller doses (5 #c Se 75 or 3.3 #g selenium) had the same distribution pattern as the chosen 20/~c. T h e disappearance rate of plasma selenite is a classical double exponential, with a fast component for the first 30 rain (T/2: 2"9 rain), followed by a slower phase for the next 3-4 hr ( T / 2 : 1 9 . 5 hr). T h e curve increases then slowly, reaching a value at 72 hr equivalent to that seen at 15 rain after injection. T h e total red cell uptake averages about 1.5 % of the total dose, between 15 rain and 6 hr after injection; the ratio RBC/Plasma appears to be increasing, because of the rapid diminution of plasma selenite; a real increase of red cell selenium is noted between 6 and 72 hr after injection. T h e r e is an in vitro uptake of selenite by red cells, considerably smaller than the in vivo uptake; this has been also noted by WRIOHT and BELL in sheep, and considered b y the authors to be related to an oxygen-dependent transport mechanism in the erythrocyte, in its turn linked to the dietary intake of selenite, c9~ Selenite binds to plasma proteins with a