Biochemistry Involving Carbon-Fluorine Bonds - ACS Publications

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Downloaded by UNIV OF MASSACHUSETTS AMHERST on June 1, 2018 | https://pubs.acs.org Publication Date: June 1, 1976 | doi: 10.1021/bk-1976-0028.ch008

Intravenous Infusion of Cis-Trans Perfluorodecalin Emulsions in the Rhesus Monkey LELAND C. CLARK, JR., EUGENE P. WESSELER, SAMUEL KAPLAN, and CAROLYN EMORY Children's Hospital Research Foundation, Elland and Bethesda Aves., Cincinnati, Ohio 45229 ROBERT MOORE Sun Ventures, Inc., Marcus-Hook, Pa. 19061 DONALD DENSON Stanford Research Institute, Menlo Park, Calif. 94025 Introduction Interest in the possibility of making fluorocarbon-based artificial blood began following the discovery by Clark (1) that animals survived the breathing of oxygen-saturated FC75 (3M Company). Since that time over 150 papers have been published, a FASEB Symposium (2) and several industrial symposia have been held on the subject. Reviews by Sloviter (3) and Geyer (4) have been published. A large number of perfluorochemicals (PFC) have been screened for this purpose. Most PFC carry oxygen in biologically significant quantities. But most are ruled out for practical use because, after performing their oxygen-carrying function, they remain in the body, largely in the liver, after being taken up mainly by the mononuclear phagocytes as are all PFC. So far it seems that only those containing fluorine and carbon or fluorine, carbon and bromine in their structure leave the body. Although we have not completed our testing of the PFC on hand and expect to test new ones in the future, nonetheless, of the PFC tested so far, perfluorodecalin (PFD) emerges as the best because it leaves the liver in a reasonable time and has a vapor pressure compatible with intravenous use. As an emulsion, its acute intravenous toxicity in the mouse is very low. It is available in commercial quantities and can be partially purified by distillation. For these, and other reasons, it was selected for testing as artificial blood in a non-human primate, the rhesus monkey. We previously reported (5) on the infusion of perfluorodecalin in one awake rhesus monkey. This paper describes the first results of further tests in 19 monkeys and 10 dogs and outlines the beginning of a protocol for its possible evaluation as a blood substitute in man. Of the 19 monkeys, 10 were infused with 10% PFD, 4 with 20% PFD emulsions, and 4 with only a "hypotensive test dose". The data included here is only a part of that on hand; it has been selected to illustrate the salient problems and findings. It is to be interpreted in terms of our previous publications (6-18) on this subject. 135 Filler; Biochemistry Involving Carbon-Fluorine Bonds ACS Symposium Series; American Chemical Society: Washington, DC, 1976.

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Data on oxygen s o l u b i l i t y i n PFC presented at t h i s meeting i s being expanded and w i l l be the subject of a separate report (19).


Methods and m a t e r i a l s Nineteen monkeys were s e l e c t e d from the I n s t i t u t e of Developmental Research's r e s i d e n t p o p u l a t i o n . Many of these monkeys had been p r e v i o u s l y used f o r t e s t i n g drugs i n t e r a t o l o g i c a l research. Three were males and had not been given drugs. The monkeys were housed i n s t a i n l e s s s t e e l cages i n a i r c o n d i t i o n e d rooms and were fed a d i e t of Purina monkey chow (Code 5038) supplemented with bananas, oranges, and raw peanuts. They were given p e r i o d i c s k i n t e s t s f o r t u b e r c u l o s i s and were given 11 mg/kg i s o n i a z i d e d a i l y impregnated as a s o l u t i o n on a sugar cube. The monkeys, a l l of which presumably have lung mites, were chest x-rayed on a r r i v a l and weighed weekly. P u b l i c Health C e r t i f i c a t i o n Procedures were used f o r monkeys obtained from India and H e l s i n k i r u l e s were f o l lowed i n the l a b o r a t o r y . The dogs were beagles obtained from a commercial source. The f i r s t monkey tested and reported (_5) , was a young male. Two of the monkeys i n the present s e r i e s were a l s o males. The v e t e r i n a r i a n s were unable to a s s i g n an approximate age to any monkey but, judging by t h e i r teeth and t h e i r behavior, many were very old. The p e r f l u o r o d e c a l i n used i n these experiments was purchased from ISC Chemicals, L t d . , Avonmouth, B r i s t o l , England and p u r i f i e d by s p i n n i n g band d i s t i l l a t i o n using a Perkin-Elmer NFA-200 Autoannular s t i l l . The f r a c t i o n b o i l i n g between 91°C and 93°C at 180 mm pressure was used f o r these s t u d i e s . The P l u r o n i c (PF68) s o l u t i o n was made by d i s s o l v i n g 200 gm i n s t e r i l e water (Abbott), brought up to 1 l i t e r , and f i l t e r i n g succ e s s i v e l y through 5.0, 0.8, and 0.22 uM M i l l i p o r e f i l t e r s at 4°C. This stock was d i l u t e d j u s t before use, the i o n i c composition was adjusted so the emulsion contained h a l f the s a l t s required f o r Ringer's and the pH was adjusted with Tham (28). Emulsions were made i n a G a u l i n Model 15M l a b homogenizer. Parts which were exposed to the emulsion were autoclaved. The shear pressure was preset to 6000 l b s / i n ^ and the gauge was r e moved. Homogenization was continued u n t i l the o p t i c a l d e n s i t y plateaued. The emulsion was placed i n s t e r i l e Pyrex v i a l s or one l i t e r b o t t l e s , frozen and preserved at -70°C. S t e r i l i t y t e s t s , performed by adding 2 ml i n t o 20 ml of a supplemented peptone broth prepared by Becton-Dickinson and Co., were conducted on random samples of the emulsion. A l l t e s t s showed the emulsion to have no b a c t e r i a l growth. Chemical methods o f s t e r i l i z a t i o n i n c l u d i n g the use of prop i o l a c t o n e were avoided, except with one monkey, Melvin 05), who r e c e i v e d emulsion s t e r i l i z e d with t h i s compound.

Filler; Biochemistry Involving Carbon-Fluorine Bonds ACS Symposium Series; American Chemical Society: Washington, DC, 1976.


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Two batches of 10% by volume PFD i n 5% PF68 and two batches of 20% by volume PFD i n 10% PF68 were prepared. A l l of the blood pressure t e s t doses were from the same batch (1 l i t e r ) of 10% PFDE and were taken from small Pyrex v i a l s which were thawed j u s t before use. A l l of the monkeys i n f u s e d with 10% PFDE r e c e i v e d emulsion (4 l i t e r s ) from the second batch where i n d i v i d u a l bott l e s were thawed j u s t before use. Two of the monkeys, C2 and C3, r e c e i v e d 20% PFDE from a t h i r d batch (1.5 l i t e r ) . Two, 74 and 113, r e c e i v e d 20% PFDE from a f o u r t h batch (4 l i t e r ) . The f i r s t three batches were prepared from pooled PFD (ISC a n a l y t i c a l r e f erence S173/A) while the f o u r t h batch was prepared from PFD (ISC a n a l y t i c a l references S172/Aand S148/B). A l l the 10% PFDE and the f i r s t two 20% PFDE emulsions were s t o r e d i n i c e water u n t i l used but the 20% PFDE f o r 74 was warmed before a d m i n i s t r a t i o n and that used f o r 113 was bubbled with oxygen at room temperature f o r three hours before i n f u s i o n . Blood samples were c o l l e c t e d from the a n e s t h e t i z e d monkeys through i n d w e l l i n g catheters and cannulae using s t e r i l e p l a s t i c syringes having the dead space f i l l e d with heparin (1000 USP u n i t s / m l ) . The samples from awake r e s t r a i n e d monkeys were obtained by venipuncture. Precautions were taken to be sure that the blood samples were not d i l u t e d with the i s o t o n i c s o l u t i o n s used to r i n s e the sampling l i n e s . Samples f o r blood gases, pH, and packed c e l l volume were analyzed immediately. The remaining blood was c e n t r i f u g e d and the plasma analyzed by Autotechnicon SMA 12 procedures (21)• Venous blood samples were taken at random from L. C l a r k , to determine the v a r i a b i l i t y of the same blood f a c t o r s being measured i n the monkey. Most of the samples from L. C l a r k were taken during the f a s t i n g s t a t e , a l l those reported here were taken while L. C l a r k was i n a s t a t e of w e l l - b e i n g , and a l l were processed l i k e monkey blood. Samples were taken p e r i o d i c a l l y from the monkey p r e v i o u s l y published (5). A l l samples not analyzed immediately were c h i l l e d i n an ice-water bath. Approximately 4800 determinations were performed as part of the research reported here. Test f o r hypotensive

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The monkeys were f a s t e d overnight and a n e s t h e t i z e d with i n travenous sodium p e n t o b a r b i t a l i n the morning. They breathed hum i d i f i e d oxygen. The a r t e r i a l pressure was measured by means of a p e d i a t r i c c u f f and a Model 802 Doppler (Parks E l e c t r o n i c s Labo r a t o r y , Oregon) using the r a d i a l a r t e r y . 0.05 ml/kg of emulsion was i n j e c t e d i n t r a v e n o u s l y and r i n s e d i n with 5 ml of Ringer's s o l u t i o n . A second t e s t dose was given w i t h i n f i v e minutes a f t e r the f i r s t i n the monkey but a f t e r the blood pressure returned to normal, 10 or 20 minutes, i n the dog. A s i m i l a r procedure was used f o r each v i a l of emulsion using the beagle w i t h i n one day of the t e s t i n the monkey. The blood pressure i n the dog was measured with a mercury manometer and d i r e c t cannulation of the femoral



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artery. Twelve monkeys were subjected to biopsy before PFDE i n f u s i o n and nine a f t e r . The o v e r n i g h t - f a s t e d monkeys were anesthetized with sodium p e n t o b a r b i t a l and a 2 or 3 gram piece of l i v e r was exc i s e d under d i r e c t v i s i o n , f i x e d , and examined by l i g h t and e l e c tron microscopy using methods p r e v i o u s l y published (18). Parts of these post i n f u s i o n samples were analyzed f o r PFD by sodium b i phenyl combustion and by GLC. C o n t r o l blood samples were taken p r i o r to making the biopsy i n c i s i o n . Post biopsy blood samples were taken one hour a f t e r the i n c i s i o n was c l o s e d and again on the f o l l o w i n g day from the r e s t r a i n e d animal. For the i n f u s i o n , the animal i s anesthetized with pentobarbit a l and kept asleep with pentothal. An endotracheal tube i s i n serted and the animal's head covered with a transparent bag being flushed with humidified oxygen. The femoral v e i n s and the r a d i a l a r t e r y are cannulated with s t e r i l e p l a s t i c tubes and the r i g h t heart i s c a t h e t e r i z e d with a Berman angiographic b a l l o o n c a t h e t e r . On the day of i n f u s i o n when the blood pressure, r e s p i r a t i o n , and ECG of the monkey have been recorded f o r s e v e r a l minutes and look s t a b l e , the f i r s t samples of blood are taken f o r blood gas analyses. A 2 ml sample of blood i s drawn into a h e p a r i n i z e d 2 ml syringe from the r i g h t heart and the r a d i a l a r t e r y . I f the p02 and pC0 readings obtained on these samples are w i t h i n normal l i m i t s , a l a r g e (12.5 ml/kg) sample of blood i s removed. This blood is c e n t r i f u g e d and analyzed as described elsewhere. At t h i s p o i n t 21 ml/kg of 57o human albumin i s infused with a Sage Instrument syringe pump at 6 ml/min. Another 12.5 ml/kg of blood i s removed c a r e f u l l y so that the blood pressure does not f a l l below 80 mm Hg. The hematocrit of t h i s blood i s determined and i f i t i s not one h a l f the o r i g i n a l hematocrit, another 4 ml/kg i s infused and another hematocrit run. To date, t h i s procedure has always reduced the hematocrit approximately 507o. In f i v e of the f i r s t s i x monkeys infused 50 ml/kg of Ringer's s o l u t i o n was given i n place of the albumin. The PFDE i s infused i n four batches and a r t e r i a l and mixed venous blood gases and pH are measured a f t e r each of the four batches are i n . A f t e r the i n f u s i o n , the a r t e r i a l cannula and a l l but one of the venous cannulae are removed and the i n c i s i o n s c l o s e d . The animal i s kept i n the operating room u n t i l i t begins to waken. Then i t i s t r a n s f e r r e d to a recovery room and c l o s e l y watched f o r the next 12 hours. F l u i d s may be given intravenously i f r e q u i r e d . U s u a l l y the monkeys are awake and d r i n k i n g l i q u i d s or even e a t i n g by l a t e afternoon. The autopsies are conducted under the auspices of the Medical School's V e t e r i n a r y Department. The organs are inspected, photographed, weighed and h a l f of each organ i s placed i n n e u t r a l 10% phosphate-buffered f o r m a l i n s o l u t i o n and h a l f i s placed i n 957o ethanol a f t e r portions of each organ are removed f o r f i x i n g , s t a i n ing and examining under l i g h t microscopy. 2


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Results and d i s c u s s i o n I n t e r l a b o r a t o r y v a r i a t i o n i n the a n a l y s i s of three p e r f l u o r i nated l i q u i d s i s given i n Table 1. I t i s apparent that the d i f f i c u l t y l i e s i n g e t t i n g q u a n t i t a t i v e recovery of f l u o r i n e and not of carbon. Some a n a l y t i c a l l a b o r a t o r i e s obtained such inaccurate r e s u l t s that they abandoned attempts to analyze the samples. Another l a b o r a t o r y i s continuing i t s e f f o r t s to develop a method. No d i f f i c u l t y was encountered, i n preparing the PFD emuls i o n s . Those preserved at -70°C remained f o r months with no d i s c e r n i b l e change. The seven day LD50 i n mice of the i n f u s e d 10% PFDE was 140 ml/kg; the LD50 of the 20% PFDE was 69 ml/kg. Measurement of p a r t i c l e s i z e d i s t r i b u t i o n i n three emulsions was performed at Sun Ventures by a technique (26) i n v o l v i n g e l e c tron microscopy and the r e s u l t s are shown i n F i g u r e 1. Tables 2 and 3 represent the changes i n blood chemistry found upon biopsy of the l i v e r . The changes i n glucose probably have l i t t l e meaning because s p e c i a l e f f o r t s were not made to prevent glycolysis. The increased l a c t i c dehydrogenase a c t i v i t y may have come from the damaged l i v e r . There i s no doubt that c r e a t i n e kinase increased as a r e s u l t of the procedure but i t i s known to increase a f t e r any kind of surgery (25). C o n t r o l values f o r blood samples from a human primate are shown i n Table 7 and those from awake monkeys i n Table 5. One reason the monkey was s e l e c t e d f o r these experiments i s that i t very o f t e n behaves l i k e the human i n i t s response to drugs. The dog sometimes shows b i z a r r e responses to emulsions and s o l u t i o n s c o n t a i n i n g s u r f a c t a n t s . In Table 4 i t can be seen that no monkey t e s t e d , but a l l dogs, s u f f e r e d a drop i n blood pressure. It sometimes takes h a l f an hour f o r the dog to recover but once i t has recovered a second dose has very l i t t l e or no e f f e c t . In Tables 10 and 11 i t can be seen that n e i t h e r the anesthes i a nor the s u r g i c a l manipulations i n v o l v e d i n g i v i n g a t e s t dose had a s i g n i f i c a n t e f f e c t upon the blood components analyzed except f o r c r e a t i n e kinase where a small but s i g n i f i c a n t increase occurred. The abnormally high values f o r twenty-four hours on monkey 80 are due to the f a c t that she was moribund and died two hours l a t e r a f t e r s e v e r a l attempts at r e s u s c i t a t i o n . The d e t a i l s concerning the replacement of withdrawn blood by Ringer's s o l u t i o n and/or 5% human albumin are given i n Table 16. The amounts of PFDE i n f u s e d are shown. For our purpose here we have r e f e r r e d to Dextran 40 and albumin as osmotic or o n c o t i c l i q quids. Albumin, however, has a longer h a l f l i f e i n the body and of course the PF68 i n PFDE has o n c o t i c a c t i v i t y . 5% human a l b u min was given p r e v i o u s l y to one monkey with no d i s c e r n i b l e e f f e c t . That we had considerable d i f f i c u l t y i n maintaining blood b a l ance a f t e r phlebotomy and PFDE i n f u s i o n i s apparent from the t a b l e . Most of the d i f f i c u l t i e s happened during the evening of the day of the i n f u s i o n and were thought to be due to the f a c t that most of the PF68 and water administered had been excreted l e a v i n g the



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Particle size distribution as determined by electron microscopy


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animals hypovolemic and although awake they were not a l e r t and were weak. When Dextran 40 was given on two occasions (C2, C3) four or f i v e hours post i n f u s i o n the animals responded to the point where they were d i f f i c u l t to r e s t r a i n . They a l s o drank Gatorade and ate. One which r e c e i v e d Dextran twenty-four hours l a t e r , f o l l o w i n g an a l l night slow b l e e d i n g from a p o o r l y t i e d i n cannula, r e v i v e d only b r i e f l y . Most of the 10% PFD monkeys that r e c e i v e d albumin during the phlebotomy r e q u i r e d l e s s f l u i d l a t e r . Two (74, 113) that r e c e i v e d 20% PFDE d i e d , p o s s i b l y because they were overloaded, but more l i k e l y because the PFDE they were given had been a t room temperature too long and coalescence of p a r t i c l e s had begun. Our experience i n maintaining blood and f l u i d balance i n thousands of dogs and human p a t i e n t s has not served us w e l l with the rhesus. I t seems to us that the rhesus monkey, p a r t i c u l a r l y the o l d monkey, i s a very d e l i c a t e and f r a g i l e animal. The main f i n d i n g i n Table 6 i s that the mixed venous oxygen tension increased i n a l l the animals r e c e i v i n g PFD emulsion. In none of those r e c e i v i n g 10% PFDE d i d the mixed venous p 0 increase beyond 100 mm, the approximate p o i n t where a l l the hemoglobin i s saturated. In three of the monkeys r e c e i v i n g 20% PFDE the mixed venous p 0 went above 100 mm. In monkey C3 the a r t e r i a l p 0 was on the low s i d e and t h i s could not be increased by chest and/or heart massage. At autopsy t h i s monkey was found to have severe emphysema. T h i s low a r t e r i a l p 0 may account, at l e a s t i n p a r t , f o r the low venous p0 . At autopsy C3 was found to have lungs h e a v i l y i n f e s t e d with mites. The heart r a t e decreased and the r e s p i r a t i o n increased as a r e s u l t of PFDE i n f u s i o n as shown i n Table 8. The Τ t e s t s shown i n t h i s t a b l e i n d i c a t e there i s no d i f f e r e n c e i n these responses to the 10% and 20% PFDE. There was no change i n a r t e r i a l pressure as a r e s u l t of PFDE i n f u s i o n as shown i n Table 9. From these and other s t u d i e s we have concluded that there i s an increase i n pulmonary a r t e r i a l pressure followed by a r e t u r n to normal i n about an hour. Because of u n c e r t a i n t y about the l o c a t i o n (RV or PA) of the catheter t i p (x-ray equipment was not a v a i l a b l e to us during these s t u d i e s ) the data i n Table 8 cannot be analyzed s t a t i s t i c a l l y . However i t can be seen that i n a l l of the cases where the pressure was low before i n f u s i o n , probably i n d i c a t i n g that pulmonary a r t e r i a l pressure was being monitored, there was a d i s t i n c t i n c r e a s e . Tables 12 and 13 give the r e s u l t s of a n a l y z i n g the blood of monkeys r e c e i v i n g 10% PFDE and Table 13 gives the average values and standard e r r o r s f o r t h i s data. Some of the apparent decreases i n c o n c e n t r a t i o n of blood components are due to the f a c t that about h a l f the blood was removed and d i l u t e d by Ringer's, albumin, and PFDE. The extent to which d i l u t i o n per se a f f e c t e d the r e s u l t s can be best judged by the extent to which the hematocrit de creased. While the t a b l e shows a decrease i n a l l components ex cept t o t a l b i l i r u b i n , i f these are " c o r r e c t e d " f o r d i l u t i o n by 2

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Figure 2. Light microscopic view of the liver of a mon key before infusion. H, hepatocyte; N, nucleus; S, sinus oid; Toluidine blue 0. χ 850.

Figure 3. Liver specimen taken at sacrifice two weeks after infusion with a 20% emulsion shows hepatocytes essentially unchanged morphologically. Cytoplasm of is completely filled with particles of PP5, and bulges into the lumen of sinusoids. H, hepatocyte; N, nucleus; M, mononuclear phagocyte. Toluidine blue 0. X 850.

Figure 4. Eleven months after infusion, the liver shows normal morphology and no structures remained which could be identified unequivocally as fluorocarbon. Other features were within normal limits. H, hepatocyte; N, nucleus; S, sinusoid. Toluidine blue 0. X 850.


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d i v i d i n g by 0.32, the hematocrit f a c t o r , the r e s u l t s i n d i c a t e an increase i n everything except c h o l e s t e r o l . The percentage change i n the components before and a f t e r c o r r e c t i o n are as f o l l o w s : TP 41,128; AB 71,222; CA 71,222; IP 90,281; GL 74,231; BN 88,275; VA 44,138; CT 80,250; TB 140,438; AP 51,159; LD 75,234; GO 45,141; CK 142,444; CH 18,56; PV 32,100. The blood chemistry changes found before and a f t e r i n f u s i o n of 20% PFDE are shown i n Table 14. Twenty-eight samples were taken from the monkey i n f u s e d with 10% PFDE about a year ago and the mean values are shown i n Table 15. The mean a l k a l i n e phosphatase i s higher than that shown i n Table 3 and Table 5 but t h i s would be expected because Melvin i s growing r a p i d l y . A l l the other data are normal according to the information we have accumulated here. Over a three month p e r i o d the average weight gain of the eight s u r v i v i n g monkeys i n f u s e d with PFDE was not s i g n i f i c a n t l y d i f f e r e n t from s i x monkeys s e l e c t e d at random as c o n t r o l s . Morphology. Three of nineteen monkeys are p i c t u r e d here which represent the p r e i n f u s i o n , post i n f u s i o n and long term r e covery of the l i v e r of a l l monkeys examined to date. The normal, or p r e i n f u s i o n morphology of the l i v e r d i f f e r e d s l i g h t l y from that of other commonly used l a b o r a t o r y mammals ( f i g . 2). V a r i a t i o n s included a marked e l e v a t i o n i n hemosiderin depos i t s i n Kupffer c e l l s , g e n e r a l l y a s s o c i a t e d with age, numerous large autophagic vacuoles (10 μ) and the presence of long tubular s t r u c t u r e s i n the mitochondria. These p e c u l i a r i t i e s have a l s o been observed by others (27). Within twenty four hours a f t e r i n f u s i o n of emulsions of PP5, p a r t i c l e s begin to appear w i t h i n the phagocytic c e l l s of the s i n u s o i d s of the l i v e r , other organs, and the c i r c u l a t i o n . Just a f t e r i n f u s i o n , while p a r t i c l e s are i n the bloodstream, the poly-' morphonuclear c e l l s show a few cytoplasmic p a r t i c l e s . Most a f fected m o r p h o l o g i c a l l y by the i n f u s i o n of emulsions are the c e l l s of the mononuclear phagocytic system. These c e l l s engulf i n d i v i dual p a r t i c l e s or clumps of p a r t i c l e s u n t i l they reach very l a r g e proportions ( f i g . 3) as they r e s t i n the l i v e r s i n u s o i d s , s p l e n i c pulp or s i m i l a r area. A f t e r a few days they may aggregate i n t o small nodules of e p i t h e l i a l - t y p e c e l l s s i m i l a r to those seen i n other f o r e i g n body responses ( f i g . 3). The hepatocytes are not changed d r a m a t i c a l l y by the i n f u s i o n of emulsions ( f i g . 4) but a small number of p a r t i c l e s do enter the cytoplasm. These d i m i n i s h , however, and u l t i m a t e l y disappear by unknown means. Residual e f f e c t s of the occupation of the l i v e r (and of course other comparable c e l l systems i n the body) by PFC i n f a c t have not been observed m o r p h o l o g i c a l l y , s i n c e mito chondria, smooth and rough endoplasmic r e t i c u l u m , lysosomes,microbodies, n u c l e i , e t c . , appear i n d i s t i n g u i s h a b l e from c o n t r o l s ( f i g s . 5,6). A l l the specimens shown were obtained at biopsy.



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Figure 5. Electron microscopic view of liver from the same monkey as figure 3. A very small percent of the cytoplasm is occupied by particles of fluorocarbon (small arrow). Organelles appear unchanged. H, hepatocyte cytoplasm; N, nucleus; M, mononuclear phagocyte with cytoplasm full of fluorocarbon particles (large arrows). Uranyl acetate, lead citrate. X 8,500.

Figure 6. Liver from the same monkey as figure 4 shows organelles which appear normal. Fluorocarbon particles, no longer identified in the cytoplasm of hepatocytes or mononuclear phagocytes, have apparently left the liver unchanged. M, mitochondria; N, nucleus; H, hepatocyte cytoplasm. Uranyl acetate, lead citrate. X 4,000.



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Post mortem f i n d i n g s . Of ten monkeys which r e c e i v e d 10% PFDE; eight are a l i v e . One, M e l v i n , has s u r v i v e d over a year. One died the day a f t e r i n f u s i o n from slow b l e e d i n g around a venous cannula. One (117) died eleven weeks a f t e r the i n f u s i o n from anesthesia preparatory to surgery f o r l i v e r biopsy. Of the four monkeys which r e c e i v e d 20% PFDE two died w i t h i n twenty four hours f o l l o w ing the i n f u s i o n and were thought to have died because the emuls i o n had begun to d e t e r i o r a t e . P o s s i b l y t h e i r c i r c u l a t i o n s were overloaded. Two s u r v i v e d i n apparent good h e a l t h but were s a c r i f i c e d at one and two weeks post i n f u s i o n because t h e i r l e g s became ischemic and p o s s i b l y gangrenous, f o l l o w i n g l i g a t i o n of t h e i r femoral a r t e r i e s as a part of the process of r e c o r d i n g t h e i r a r t e r i a l pressure. Femoral a r t e r i e s can be l i g a t e d i n cats and dogs with no v i s i b l e e f f e c t but primates are s u s c e p t i b l e to n e c r o s i s i f the femoral a r t e r i a l c i r c u l a t i o n i s compromised. One monkey's femoral a r t e r y was s u c c e s s f u l l y r e p a i r e d ; t h i s animal (117) l i v e d eleven weeks with normal l e g s . Following the problem with the femoral a r t e r y , d i r e c t pressures were measured from the r a d i a l a r tery. One monkey s u f f e r e d p a r t i a l l o s s of the f i n g e r s of the l e f t hand f o l l o w i n g l i g a t i o n of the r a d i a l . One i n j u r e d monkey, 6, was s a c r i f i c e d s h o r t l y a f t e r i t was purchased, and before i t was used as a c o n t r o l f o r organ weights. Monkey 80 b l e d to death overnight because a femoral a r t e r y was punctured during an attempt at catheterization. The organ weights obtained at autopsy are shown i n Table 17. Because of the d e a r t h of normal data i n the rhesus very l i t t l e can be s a i d about the e f f e c t s of i n f u s i o n . The l i v e r of C2 and the spleen of C3 are q u i t e p o s s i b l y enlarged. A l l the monkeys were found to have lung mites, and some were considered h e a v i l y i n f e s t e d . Many of the lungs were s t i f f and d i d not c o l l a p s e . More d e t a i l e d r e p o r t s of the f i n d i n g s with l i g h t microscopy w i l l be the s u b j e c t of a separate r e p o r t . Sixteen p i e c e s from v a r i o u s p a r t s of the l i v e r of one monkey were analyzed and were found to range from 0.61% to 2.05% with a mean of 1.45%. Information obtained by sodium biphenyl combustion of the l i v e r and GLC are shown i n Table 18. There was of course c i r c u l a t i n g PFDE i n monkeys 62, 74, and 113. Depending on the dose, i t r e q u i r e s s e v e r a l days f o r i t to completely disappear from the blood. The PFD disappears from the blood both by evaporation through the lungs and s k i n and by being engulfed by the scavenger c e l l s of the body. I n t e r e s t i n g l y , a t r a c e could be detected i n Melvin's l i v e r almost a year a f t e r the i n f u s i o n . There i s c o n s i d e r a b l e v a r i a b i l i t y between monkeys i n the way i n which they sequester the PFD i n t h e i r l i v e r s and spleen, and the r a t e at which i t leaves. The h a l f l i f e of PFD i n the l i v e r i s probably about two weeks. In the mouse, most PFD i s gone i n two weeks.


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General d i s c u s s i o n Although most of our r e s u l t s have been discussed above we think i t may be d e s i r a b l e to add some general comments about our concepts and our f i n d i n g s . Our work on a r t i f i c i a l blood i s prompted by our experience with thousands of open heart surgery p a t i e n t s and a f a m i l a r i t y with the problems surrounding plasma, plasma s u b s t i t u t e s , pare n t e r a l s o l u t i o n s and the use of donor blood. Various substances have been used over the past s e v e r a l decades as plasma s u b s t i t u t e s . C e r t a i n of these substances, such as Dextran and plasma albumin, have earned a d e f i n i t e place i n c l i n i c a l medicine f o r maintaining c i r c u l a t o r y volume. G e n e r a l l y , such s o l u t i o n s d i s s o l v e only 2 or 3 ml of oxygen per 100 ml, while whole blood d i s s o l v e s about 20 ml per 100 ml. In order to meet the oxygen demands of the body the c a r d i a c output must be increased i f the red c e l l mass decreases. In other words, the way i n which the body attempts to compensate f o r oxygen c a r r y i n g c a p a c i t y of blood i s to work harder and pump more blood. Because fluorocarbons c a r r y l a r g e q u a n t i t i e s of oxygen they can be used to increase the oxygen c a p a c i t y of the c i r c u l a t i n g blood and t h e r e f o r e decrease the work of the heart. Of course, a weak, f a i l i n g , or i n j u r e d heart may not be able to increase i t s output to meet oxygen needs and shock and death may f o l l o w . Therefore, a primary f u n c t i o n of a r t i f i c i a l blood i s to decrease the work of the heart. Oxygen c a r r y i n g l i q u i d s w i l l be most u s e f u l i n c o n d i tions where the c a r d i a c output i s low or the blood volume i s below normal. The only other way to increase the oxygen c a p a c i t y of blood i s to add stroma-free hemoglobin but t h i s has not proven to be p r a c t i c a b l e yet. Hemoglobin i s e a s i l y s t o r e d and i t may someday be p o s s i b l e to use hemoglobin from other animals, such as the cow and the p i g . One of i t s f a u l t s i s i t s high rate of e x c r e t i o n . Synthetic oxygen chelates seem to be f a r i n the f u t u r e . PFC a r t i f i c i a l blood, l i k e stroma-free hemoglobin, has no blood types. Our research on l i q u i d breathing of p e r f l u o r o c h e m i c a l l i q u i d s formed the e a r l y b a s i s of the work reported here. It indicated, f o r example, that c e r t a i n h i g h l y f l u o r i n a t e d l i q u i d s were probably biologically inert. I t has l e d to the use by Dr. David M. Long, as we hear today, of brominated p e r f l u o r i n a t e d l i q u i d as X-ray contrast agents i n the d i a g n o s i s of lung disease. Perfluorocarbon l i q u i d s may a l s o be u s e f u l some day i n the treatment of lung d i s eases, such as by washing out o b s t r u c t i v e m a t e r i a l s . I t would be h i g h l y d e s i r a b l e to have a s u i t a b l e water s o l u b l e s y n t h e t i c oxygen solvent to increase the oxygen c a p a c i t y of plasma or plasma s u b s t i t u t e s . PFC i n general are good oxygen and carbon d i o x i d e s o l v e n t s but they are completely i n s o l u b l e i n water. Therefore they can only be used as emulsions. This introduces at l e a s t three problems. (a) B i o l o g i c a l l y s u i t a b l e e m u l s i f i e r s must be found and used. (b) C e r t a i n PFC cannot be e m u l s i f i e d and



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c e r t a i n others form only unstable emulsions. (c) Because emulsions c o n s i s t of suspended p a r t i c l e s they are g r a d u a l l y removed from the c i r c u l a t i o n and deposited i n part i n the l i v e r and spleen. We have e l e c t e d to concentrate our e f f o r t s on a study of the use of p e r f l u o r o d e c a l i n p r i m a r i l y because i t g r a d u a l l y leaves the body (16), but a l s o because i t has an acceptable vapor pressure, and i s a good oxygen and carbon dioxide s o l v e n t . I t r e a d i l y forms an emulsion having a low o p t i c a l d e n s i t y but must, at present, be preserved at -70°C. For comparison, we and others have found that FC47 forms a f i n e p a r t i c l e emulsion, as judged by o p t i c a l d e n s i t y , which i s f a r more s t a b l e than that produced by PFD but once entrapped i n the l i v e r i t remains f o r years. PFD i n a d d i t i o n , unl i k e , f o r example, p e r f l u o r o m e t h y l d e c a l i n with ten isomers, has only two. A fluorocarbon having but a s i n g l e molecular c o n f i g u r a t i o n , such as adamantane would be p r e f e r a b l e to having one with two i s o mers such as PFD. PFD a f t e r p u r i f i c a t i o n by spinning band d i s t i l l a t i o n as described i n t h i s paper, was analyzed at Stanford Research I n s t i t u t e by GLC and f i e l d i o n i z a t i o n mass spectrometry and reported to be 96.4% pure with p o s s i b l y eight d i s t i n c t compounds present having molecular weights ranging from 68 to 484. A few m i l l i t e r s of the two isomers of PFD, having the publ i s h e d p r o p e r t i e s , were l a b o r i o u s l y prepared by gas chromatography. It may be that the very property of being i n e r t , which makes some of these fluorocarbons p o t e n t i a l l y u s e f u l i n medicine, makes them d i f f i c u l t to p u r i f y , c h a r a c t e r i z e , and i d e n t i f y . Of course, high p u r i t y and u n i f o r m i t y are r e q u i r e d f o r medical use. The l i s t of u s e f u l e m u l s i f i e r s i s very short indeed because they must be non-toxic, non-hemolytic and cause no undesirable p h y s i o l o g i c a l r e a c t i o n s . Only two such substances e x i s t at the present time. S p e c i a l egg p h o s p h o l i p i d (Vitrum, Sweden) has been used e x t e n s i v e l y o u t s i d e of the United States as an e m u l s i f i e r f o r intravenous f a t emulsions f o r c l i n i c a l use. This e m u l s i f i e r can only be used to formulate emulsions under very c a r e f u l l y c o n t r o l l ed c o n d i t i o n s , as to p r e p a r a t i o n , temperature, exposure to a i r and other f a c t o r s . I t i s capable of e m u l s i f y i n g PFD. The other emuls i f i e r , P l u r o n i c F68 (Wyandotte, USA), at the present time i s not used c l i n i c a l l y i n the USA as a component of intravenous emulsions of any kind. I t has found extensive use as an e m u l s i f i e r for PFC emulsions i n research with animals. I t was s e l e c t e d f o r the PFDE used here i n the rhesus because i t forms emulsions with PFD and i s r e l a t i v e l y non-toxic and s t a b l e . Most of previous work with PFDE was done using the mouse, the cat, and the dog. We decided to perform the t e s t s reported here i n the rhesus (macaca raulatta) monkey to determine i f the primate responded d i f f e r e n t l y . We found that the hypotensive e f f e c t of small doses of emuls i o n d i d not occur i n the primate. Measurements of the LD50 of plasma from the dog at the depth of the blood pressure drop



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i n d i c a t e d that no t o x i c substance was present and we tend t h e r e fore to agree with W r e t l i n d (23, 24) that t h i s i s some k i n d of a cardiovascular reflex. Emulsions prepared from PFD and PF68 are d i f f i c u l t to charact e r i z e as to p a r t i c l e s i z e and c o n c e n t r a t i o n of PF68 i n the aqueous phase a f t e r e m u l s i f i c a t i o n . There i s no one method f o r measu r i n g p a r t i c l e s i z e i n the ranges i n v o l v e d here which i s g e n e r a l l y accepted. O p t i c a l d e n s i t y measurement continues to be the best means to monitor the making of emulsions from any given PFC. I t probably cannot be used to compare p a r t i c l e s i z e when d i f f e r e n t PFC s t r u c t u r e s are i n v o l v e d . Space w i l l not permit d i s c u s s i o n of the complex f a c t o r s i n volved i n maintaining f l u i d balance by means of osmotic, o n c o t i c , and h y d r o s t a t i c f o r c e s . S u f f i c e to say that we attempt to maint a i n o n c o t i c a c t i v i t y by the s h o r t - a c t i n g P l u r o n i c and Dextran and by the long a c t i n g plasma albumin. We found that Dextran given s e v e r a l hours a f t e r the i n f u s i o n of 20% PFDE had a dramatic benef i c i a l e f f e c t ; i t has brought animals from a near unconscious s t a t e to one of near normal. The d i f f e r e n c e i n the f a t e of the two monkeys given a warmed 20% emulsion and a cooled 20% emulsion would seem to suggest that warming an emulsion i n v i t r o i s apparently completely d i f f e r e n t than warming i t i n v i v o . Not only does the emulsion appear to be much more s t a b l e i n v i v o than i n v i t r o , even though f a r above room temperature, but judging from the b l u i s h haze i n the plasma of some of these monkeys, the p a r t i c l e s i z e may even have decreased. This may be due not only to the e m u l s i f y i n g p r o p e r t i e s of blood, but to the mechanical mixing e f f e c t s i n the c a r d i o v a s c u l a r system and the unique c h a r a c t e r i s t i c s of the l i n i n g of the blood vessels. I t should be borne i n mind that most of the chemical analyses of blood reported here are done on plasma d i a l y z e d through a c e l lophane membrane and should t h e r e f o r e not be a f f e c t e d by the presence of PFD. T o t a l p r o t e i n , albumin, t o t a l b i l i r u b i n , and l a c t i c dehydrogenase were analyzed without passing through a d i a l y s i s membrane and the measurement could have been a f f e c t e d . Summary Of nineteen monkeys s e l e c t e d f o r t h i s study, fourteen were used f o r the i n f u s i o n of p u r i f i e d p e r f l u o r o d e c a l i n i n the form of 10 and 20% by volume emulsions. Four received only t e s t doses and one was s a c r i f i c e d before use as a c o n t r o l . P l u r o n i c F68 was used as the e m u l s i f y i n g agent. Previous to the i n f u s i o n of PFDE, blood was removed to decrease the hematocrit by h a l f and t h i s blood was replaced by e i t h e r Ringer's s o l u t i o n or 5% human albumin. Nine out of ten monkeys infused with 10% PFDE survived. Two of four monkeys infused with 20% PFDE survived. Mixed venous p 0 i n c r e a s ed i n a l l monkeys and exceeded 100 t o r r i n three monkeys, which r e c e i v e d 20% of PFDE. The s u r v i v i n g monkeys appear to be i n good 2



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h e a l t h . Three monkeys were s a c r i f i c e d , one as a c o n t r o l , two be cause o f compromised c i r c u l a t i o n t o the l e g . One succumbed during anesthesia preparatory to biopsy and one from a s u r g i c a l accident during cannulation. Aside from a drop i n c h o l e s t e r o l there were only questionable changes i n about 20 blood components analyzed. Small (0.05 ml/kg) doses o f PFDE given t o dogs i n v a r i a b l y caused a pronounced drop i n blood pressure but none occurred i n the mon keys. The morphologic changes i n the l i v e r of the monkeys were r e v e r s i b l e with no s i g n of damage. The major problems encountered were d i f f i c u l t y i n o b t a i n i n g pure PFD, making s t a b l e emulsions, o b t a i n i n g a n a l y s i s of compounds and t i s s u e s f o r PFD, working with the f r a g i l e c a r d i o v a s c u l a r and r e s p i r a t o r y systems of the rhesus and judging optimum f l u i d balance post phlebotomy and post i n f u sion. Abbreviations NO SP TP AB CA IP GL BN UA CT TB AP LD GO CK CH PV PF PFC PFD PFDE C GLC MK PTD M SE Τ RI BL LD50 ME PP5

Number Samples T o t a l P r o t e i n (gm%) Albumin (gm%) Calcium (mg%) Inorganic Phosphrous (mg%) Glucose (mg%) Blood Urea Nitrogen (mg%) U r i c A c i d (rag%) C r e a t i n i n e (mg%) T o t a l B i l i r u b i n (mg%) A l k a l i n e Phosphatase (mU/ml) (EC 3.1.3.1) L a c t i c Dehydrogenase (mU/ml) (EC 1.1.1.27) Glutamic-oxaloacetic Transaminase (mU/ml) (EC 2.6.1.1) Creatine Kinase (mU/ml) (EC 2.7.3.2) C h o l e s t e r o l (mg%) Packed C e l l Volume (%), hematocrit Packed PFD Volume (%), f l u o r o c r i t Perfluorochemical D i s t i l l e d Perfluorodecalin P e r f l u o r o d e c a l i n Emulsion Control Gas-Liquid Chromatography Monkey Post Test Dose Mean

Ringer's s o l u t i o n Blood Mean l e t h a l dose, i . v . i n j e c t i o n The monkey ( r e f . 5), named Melvin In the s e c t i o n on morphology t h i s i s used to designate d i s t i l l e d perfluorodecalin.


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Acknowled gemen t s The authors wish to thank Dr. George M i l l e r f o r guidance i n s u r g i c a l techniques used f o r t i s s u e biopsy. We consulted C h r i s t Taraborski concerning chemical problems. The a s s i s t a n c e of the f o l l o w i n g persons i s g r a t e f u l l y acknowledged: Dr. Marian L. M i l l e r , Dr. Steele F. M a t t i n g l y , Dr. Jag L a i , Frank Knapke, Pat Turner, L i l a m Stanley, Margaret Kelm, Steven Jones, David DeForest, Barbara Cincush, Stanley Gaines, Eleanor C l a r k , Eleanor Brinkmoeller, Sandra Hoffman, and Dotty O ' R e i l l y of Sun Ventures. The P l u r o n i c F68 was a g i f t from Dr. I r v i n g Schmolka of Wyandotte. This research i s supported i n part by grants HL17586, HL17353, GM21475, HD05221, from the N a t i o n a l I n s t i t u t e s of Health, grant 74 619 from the American Heart A s s o c i a t i o n and a grant from the Southwestern Ohio Chapter of the American Heart A s s o c i a t i o n . S h o r t l y a f t e r m a i l i n g our manuscript we r e c e i v e d , from Technicon I n d u s t r i a l Systems, Tarrytown, New York 10591, v i a Richard Carr, 1000 Crest C i r c l e , C i n c i n n a t i , Ohio 45208, a d d i t i o n a l data f o r normal values f o r c l i n i c a l blood chemistry i n the rhesus. One report (29) gives the normal values f o r blood from f o r t y - s i x female and t h i r t y - t h r e e male rhesus. Another report (30) gives the mean and range f o r eleven blood component analyses f o r f i f t y rhesus.



F

2

2

3

Comparison of a n a l y t i c a l

results.

74.22 74.09

Lab D

Table 1

68.60 68.78

66.35 77.02 76.72

Lab C

25.93 25.78

65.76 73.59 73.79

78.24 78.32

Lab Β

73.53 73.94

84.05 83.90

21.81 21.59

70.14 70.35

21.31 21.30

21.90 22.01 72.93 73.39

21.14 21.32

72.65 72.73 71.73 72.32 71.85

26.10 26.35

74.19

Lab A

73.1b

21.43

70.28

21.40

67.70

25.99

74.01

Calculated

CFo

4

CF CHF-(OCF2CF> F

%C

n=6

%F

n

%C

£o-CF-CF ]-

CF,

E4

%F

• Fa

F

Fomblin

%c

h

h

PP5

%F

Structure

Trade Name



4.6 3.9 3.9 3.9 3.5 3.3 3.6 4.0 3.8 3.7 3.7 3.8 3.9 5.7 5.2 4.5 4.9 4.5 4.2 3.9 3.7 4.1

4.8

AB gm%

10.0 9.0 9.2 10.1 9.8 10.1 10.0 9.3 9.2 9.7 8.8 9.4 10.0 10.5 10.1 9.5 9.5 9.0 9.6 8.9 8.9 9.6

9.3

CA mg%

4.1 2.6 2.4 5.9 3.0 3.5 4.6 5.4 5.0 5.7 2.4 4.0 4.2 4.6 4.3 4.6 4.7 4.5 6.0 3.8 3.4 2.9

4.7

IP mg%

93 116 97 149 105 108 122 81 78 185 119 80 109 77 72 225 75 36 122 72 70 111

198

GL mg%

16 11 14 16 20 21 12 14 15 15 15 15 21 16 16 27 18 18 19 22 24 16

14

BN mg%

0.15 0.29 0.31 0.29 0.20 0.15 0.19 0.30 0.19 0.47 0.28 0.19 0.25 0.19 0.09 0.28 0.12 0.10 0.19 0.17 0.12 0.21

0.07

UA mg%

0.9 1.0 1.0 1.3 0.9 0.9 0.9 0.8 0.8 1.1 1.1 0.8 1.1 1.1 0.8 1.5 1.1 0.9 1.1 0.8 0.7 0.8

0.8

CT mg%

0.2 0.1 0.2 0.3 0.2 0.2 0.2 0.2 0.1 0.2 0.2 0.2 0.2 0.2 0.3 0.6 0.2 0.3 0.1 0.2 0.2 0.3

0.2

TB rag%

227 152 167 167 580 610 > 350 110 107 135 128 156 170 194 195 232 138 140 186 223 234 236

187

AP mU/ml

330 226 480 336 175 439 294 660 493 381 317 569 373 583 820 > 600 195 252 224 192 109 207

—

242

LD mU/ml 40 48 18 42 36 20 38 48 48 47 51 50 66 57 86 113 > 300 38 44 40 53 60 83

—

CK mU/ml

71 >778 183 778 > 778 > 788 39 210 435 52 144 86

GO mU/ml 40 38 38 43 43 38 41 42 37 40 40 37 39 42 39 46 43 31 39 35 37 40 42 38

%

PV

Blood chemistry before and a f t e r l i v e r biopsy and before t e s t dose o r i n f u s i o n with PFDE i n the monkey.

8.0 7.7 7.6 8.2 8.2 8.3 8.4 8.1 7.4 7.6 7.2 7.4 7.8 8.8 7.5 7.5 7.2 6.8 7.6 7.3 7.2 7.6

7.3

TP gm%

continued on Table 3

Table

113

74

58

33

117

71

2

C 1H 48H C 1H 24H C 1H 24H C 1H 24H C 1H 24H C 1H 24H C 1H 48H C 1H 24H

62

86A

SP

NO



C 1H 24H

S.Ε.

0.15 0.16 0.14

9.4 9.3 9.8

9.9 10.0 9.0 9.0 9.3 9.0 8.6 9.5 9.3 9.1 10.7

CA mg%

0.36 0.29 0.93

10 7 13

103 77 143

87 176 111 97 122 92 62 110 104 66 119

4.4 12.0 1.3 2.9 2.1 4.0 5.0 3.3 2.9 3.2 3.3 3.6 3.8 4.9

GL mg%

IP mg%

0. 9 1.1 1.6

16 17 18

19 24 16 17 15 14 15 15 18 19 17

BN mg%

0.06 0.05 0.07

0.01 0.03 0.04

45 52 20

171 186 156

0.2 0.2 0.2

0.02 0.02 0.04

122 177 65 68 83 97 108 99 75 80 105

0.2 0.2 0.2 0.2 0.2 0.1 0.2 0.2 0.2 0.4 0.1

1.0 1.3 1.0 0.9 1.1 1.0 0.8 1.1 1.5 1.2 1.2 1.0 0.9 1.1

AP mU/ml

TB mg%

CT mg%

0.20 0.17 0.27

0.1 0.5 0.3 0.2 0.2 0.1 0.2 0.1 0.2 0.2 0.1

UA mg%

50 64 20

298 463 326

185 380 191 388 321 410 509 282 204 570 364

LD mU/ml

5.5 7.4 7.4

41 59 60

38 78 25 51 96 38 55 38 33 76 51

6.5 84 75

51 365 305

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Biochemistry Involving Carbon-Fluorine Bonds - ACS Publications

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