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Effect of porphyrinogenic agents on protein synthesis and bilirubin formation by the isolated perfused rat liver
52
Biochimica e t Biophysica Acta, 496 (1977) 52--64 © Elsevier/North-Holland Biomedical Press
BBA 28130
EFFECT OF PORPHYRINOGENIC AGENTS ON PROTEIN SYNTHESIS AND BILIRUBIN FORMATION BY THE ISOLATED PERFUSED RAT LIVER
H. HENG LIEM a, KATSUMI MIYAI b and URSULA MULLER-EBERHARD b a Department o f Biochemistry, Scripps Clinic and Research Foundation and the b Departments o f Pathology and Pediatrics, University o f California al San Diego, La Jolla, Calif. 92037 (U.S.A.) ,
(Received May 31st, 1976) Summary We established an isolated rat liver perfusion system for the study of heme catabolism. The liver of rats fasted for 48 h is perfused with an e r y t h r o c y t e - f r e e medium. Ultrastructural analysis shows integrity of all subcellular organelles with the e x c e p t i o n of minor alterations in the rough endoplasmic reticulum. The perfused liver synthesizes serum proteins at a constant rate for 5 h. Albumin is secreted at a mean rate of 17 -+ 2 mg/h per 100 g liver, h e m o p e x i n at 5.0 + 0.7, haptoglobin at 3.2 + 0.6 and transferrin at 5.1 -+ 0.8 mg/h per 100 g liver. The mean ratio o f ATP : ADP is 3.5 -+ 0.1, and that of lactate : pyruvate 27 + 6. The rate of conversion of heme into bilirubin is comparable to t hat reported for in vivo studies. A minimal ef f ect on protein synthesis is observed after administration of the porphyrinogenic agents, allylisopropylacetamide (AIA) and 3,5-diethoxycarb o n y l - l , 4 dihydrocollidine (DDC). Pr e t r eat m e nt of the rats with the iron chelator, Desferal, causes a 3--4-fold increase in h e m o p e x i n but n o t in albumin and transferrin synthesis. A striking 2--3-fold e n h a n c e m e n t of bile bilirubin production follows t r e a t m e n t with DDC and Desferal, but not with AIA. The a m o u n t o f bilirubin f o r m e d from heme added to the perfusate is reduced by AIA and DDC and enhanced by Desferal treatment. It is proposed that unavailability of iron in a certain hepatic tissue pool causes p r o t o p o r p h y r i n IX accumulation which may serve as an alternate source for bilirubin production. Introduction The isolated perfused rat liver has been used extensively to study protein, c a r b oh y d r ate, and lipid metabolism [1--12]. In some studies, evaluation of the Abbreviations: AIA, allylisopylacetamide; D D C ,
3,5-diethoxy c a rbonyl -l ,4-di hydroc ol l i di ne .
53 histological and ultrastructural integrity of the perfused liver was of primary concern [13--18]. Seldom were metabolic performances correlated with ultrastructural changes in the same experiment. The viability of the liver has been assessed by determining plasma protein synthesis, bile formation, oxygen consumption, CO2 production, bromosulphalein uptake and biliary excretion. Removal of particulate matter from the perfusate by the reticuloendothelial cells has served as another criterion of liver function. Recently, hepatic functions such as bile formation, secretion of K ÷and lactic acid, and carbon particle clearance were correlated with the fine structure of the perfused liver by Schmucker and Curtis [19]. These investigators found that the hepatic ultrastructure was preserved best when colloidal clearance was efficient and the rate of K ÷secretion into the perfusate was low. Perfusion is generally achieved with a semi-synthetic medium containing homologous or heterologous erythrocytes or with diluted rat blood. An erythrocyte-free medium is rarely employed [7,20,21] and, to our knowledge, has not been utilized to monitor bile pigment secretion. We are describing such a perfusion system in which bilirubin formation from endogenous and exogenous sources is studied under a variety of conditions. To facilitate the metabolism of heme *, the donor rats are fasted and erythrocytes and rat serum are omitted from the perfusion medium. This system synthesized for 5 h several serum proteins at a linear rate and converts heme into bilirubin at a rate similar to that found in vivo. We report the effect of the porphyrinogenic drugs, allylisopropylacetamide (AIA) and 3,5-diethoxycarbonyl-l,4-dihydrocollidine {DDC) as well as that of the iron chelator, Desferal, on serum protein synthesis and bilirubin formation from endogenous and exogeneous heme. Materials and Methods
Animals and drugs. Adult male Sprague-Dawley rats 250--400 g served as liver donors. They were fed a diet of Purina Laboratory Chow, containing 23% crude protein (Harrison-Riedy, Chula Vista, Calif.). Food was witheld for 40-48 h prior to the experiment. Water was allowed ad libitum. 3,5-Diethoxycarbonyl-l,4-dihydrocollidine (DDC) was kindly supplied by Dr. Robert Labbe, University of Washington, Seattle, Wash. The drug was dissolved in corn oil and administered intraperitoneally into the donor rats (300 mg/kg) 24 h before death. Allylisopropylacetamide (AIA), a gift of Hoffman La Roche, Inc., Nutley, N.J., was dissolved in phosphate-buffered saline, pH 7.4 and injected subcutaneously (300 mg/kg) 16 h before death. Desferal (desferrioxamine mesylate, Ciba Pharm. Co., Summit, N.J.) dissolved in normal saline was administered intramuscularly (125 mg/rat) once a day for 2 days. Hemin was purchased from Eastman Organic Chemicals. Perfusion methods. The perfusion technique and apparatus were designed according to Miller et al. [22], with minor modifications. The modifications are: the perfusate drains directly from the hepatic veins into the reservoir since we do not cannulate the vena cava inferior. After cannulating the portal vein and the bile duct, the liver is perfused in situ with oxygenated Krebs-Henseleit * Heme, iron-protoporphyrin
IX.
54 bicarbonate buffer, pH 7.4. Within 1 min, the excised liver, placed on a circular plexiglass disc and resting on the platform, is connected to the circulating perfusion medium. A mixture of 95% O2 and 5% CO2 passes through the glass "thin f i l m " oxygenator at a flow rate of 3.5 1/min. The perfusion medium, approx. 150 ml, contains Krebs-Henseleit bicarbonate buffer, saturated with 95% O2 and 5% CO2, 2.5% bovine serum albumin (Pentex Miles Laboratory, Inc., Kankakee, Ill.) dialyzed overnight against the buffer, 4000 units sodium heparin, 2 ml TC amino acids (Hela 100 × Difco Laboratories, Detroit, Mich.)* and 240 mg glucose. The medium is titrated with 3 M NaOH to pH 7.4. Heme is solubilized as previously described [23] and added slowly to the perfusate in the beginning of the third hour. The pH of the perfusate varies little and is maintained at pH 7.4 by monitoring once every hour, adding 1 M NaOH when necessary. The liver is perfused at 37°C, with a flow rate of 30--45 ml/min. The portal pressure varies from 13 to 14 cm of water. Each perfusion is continued for 5 h; 1-ml samples are withdrawn at time 0 (5 min after initiation of the perfusion), and at 1, 2, 3, 4 and 5 h. The bile is usually collected in two portions in graduated centrifuge tubes for the first 2 h and for the next 3 h. Analytical methods. Rat albumin concentrations in the perfusate are measured by the radial immunodiffusion technique [24], and hemopexin, transferrin, and haptoglobin by a radioimmunoassay [25]. The bile volume is recorded and its content analyzed for total bilirubin, determined as the alkaline "Azo bilirubin" [26]. Heme in the perfusate is determined by the benzidine m e t h o d [27]. At the end of the perfusion, following the technique described by Hohorst et al. [28], liver sections are instantly frozen, weighed and stored in liquid nitrogen for up to 7 days. The frozen liver pieces are pulverized and a HC104 extract is prepared. The protein-free filtrates are then assayed for ATP, ADP and AMP, as well as for lactate and pyruvate [29,30]. When protein synthesis and bilirubin formation are studied, the livers are disconnected from the canula at the end of the perfusion, blotted with filter paper and weighed. Electron microscopy. For ultrastructural studies, two livers were perfused at the end of the experiment through the portal vein with 20 ml of 1% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. The fixative was infused with a hydrostatic pressure of 15--17 cm water for 2--3 min. Liver tissue was immediately cut into approx. 1-mm cubes, rinsed with 0.1 M phosphate buffer, and post-fixed with 2% OsO4 in 0.1 M phosphate buffer, pH 7.4, for 1 h at 4°C. After dehydration with graded concentrations of acetone, tissue blocks were embedded in Epon. Ultrathin sections were stained with lead citrate and examined in a Siemens Filmiskop 101. Results
The rate of net secretion of albumin, hemopexin, haptoglobin and transferrin into the perfusion media is illustrated in Fig. 1. The synthesis rate of * T C - M e d i u m E a g l e H e l a 1 0 0 X is a 1 0 0 - f o l d c o n c e n t r a t i o n Hela medium of Eagle (Science 122, 501 (1955)).
of the original chemically
defined basal
55 Plasma protein synthesis in the isoleted perfueed liver ,
,
,
,
,
50
• - - • Albumin x m x Transferrin I00
:::
3 D
,/fl
o E
v
25
-~
.I .I ~. __
o E
•
g
~ 1 I
I
I
I
I
2
:~
4
5
Hours F i g . 1. P r o t e i n s y n t h e t i s b y t h e i s o l a t e d r a t l i v e r , p e r f u s e d w i t h a h e m o g l o b i n - f r e e m e d i u m . C u m u l a t i v e average net change in the concentrations of albumin (16) • e, transferrin (8) X X, hemopexin (4) © ©, a n d h a p t o g l o b i n ( 4 ) A - in the perfusates. Numbers in parentheses indicate the number of experiments, and the vertical bars represent S.E. The r values are: albumin, 0.999; transferrin, 0.995; hemopcxin, 0.988; haptoglobin, 0.976.
albumin and transferrin becomes linear after the first hour and that of hemopexin and haptoglobin is linear from the beginning of the experiment. The latter two proteins may be consumed during the initial phase of perfusion associated with hemolysis. Albumin and transferrin molecules, on the other hand, may be trapped in the hepatocytes and/or within the vascular system of the liver [20]. We consider the linear phase after the first hour to reflect the rate of de novo protein synthesis in the isolated liver. In Table I, the liver c o n t e n t of ATP, ADP, AMP, lactate and pyruvate is shown at the conclusion of a 5 h perfusion. The concentration of the nucleotides for the perfused liver is, in general, slightly lower than that of the liver in vivo, but is in good agreement with that found by others for perfused livers [21,31]. The average values, for the energy charge as defined by Atkinson and Walton [32] ([ATP] + 1/2[ADP])/([ATP] + [ADP] + [AMP]) for the perfused liver of non-fasted and fasted rats are 0.82 and 0.83, respec-
LACTATE AND PYRUVATE
CONTENT
OF RAT LIVER PERFUSED
WITH A HEMOGLOBIN-FREE
MEDIUM
2 . 5 -+ 0.1
2 . 4 -+ 0.1
(5)
(6)
2.8 + 0 . 2 ( 1 0 )
ATP
* In v i v o , n o n - p e r f u s e d livers.
fasted
Perfused
non-fasted
Perfused
non-fasted *
Normal
Liver
+ 0.1
0.75 + 0.03
0 . 7 2 -+ 0 . 0 6
1.1
ADP
(5)
(6)
(10)
0 . 2 1 -+ 0 . 0 2 (5)
0 . 2 6 -+ 0 . 0 3 ( 3 )
0 . 3 6 + 0 . 0 3 (2)
AMP _._
3 . 5 +- 0.1
3 . 4 -+ 0 . 3
(5)
(6)
2 . 7 -+ 0 . 3 ( 1 0 )
ATP/ADP
0 . 9 + 0.1 ( 5 )
1 . 6 -+ 0 . 2 ( 5 )
3 . 2 -+ 0 . 1 ( 3 )
Lactate
0 . 0 4 +- 0 . 0 1 ( 5 )
0 . 0 8 -+ 0 . 0 1 ( 5 )
0 . 1 2 + 0.01 (3)
___
Pyruvate
2 7 -+ 6 ( 5 )
2 1 -+ 1 ( 5 )
2 7 -+ 2 (3)
pyruvate
Lactate/
Livers from rats fasted for 40--48 hours were perfused with a medium containing 2.5% bovine serum aIbumin. Nucleotides, lactate and pyruvate of the livers were m e a s u r e d a t t h e e n d o f 5 h p e r f u s i o n . V a l u e s s h o w n r e p r e s e n t t h e m e a n _+ S . E . a n d e x p r e s s e d i n p m o l / g l i v e r w e t w e i g h t . N u m b e r o f a n i m a l s i n p a r e n t h e s e s . V a l u e s are n o t c o r r e c t e d f o r p e r f u s a t e c o n t e n t i n t h e l i v e r .
NUCLEOTIDES,
TABLE I
57 tively. These values are comparable to those obtained by Htilsmann and Kurpershoek-Davidov [11] for an isolated liver perfused with an erythrocyte-free medium. In situ, the energy charge is found to be close to 0.9 [33]. In the present study (Table I), concentrations of lactate and pyruvate are lower in the perfused than in the non-perfused liver, and they are lower in the liver of fasted than in non-fasted animals. However, the lactate : pyruvate ratio in the perfused is comparable to that in the non-perfused liver. The ultrastructure of the hepatic tissue after 5 h of perfusion is in general well-preserved (Figs. 2 and 3); only minor alterations can be noted. Parallel
Fig. 2. U l t r a s t r u c t u r e o f t h e m i d - z o n e o f a l o b u l e o f r a t l i v e r a f t e r 5 h o f p e r f u s i o n . E n d o t h e l i a l cells ( E ) l i n e t h e s i n u s o i d ( S d ) a n d are i n t a c t . T h e s t r u c t u r e s o f t h e h e p a t o c y t e s ( H ) are well p r e s e r v e d . N u c l e u s o f hepatoc~'tes
(N) × 6480.
58
arrays of the rough endoplasmic reticulum are at times dispersed and partially fragmented into small vesicles. The smooth endoplasmic reticulum appears more prominent than that of the liver in situ of fasted rats. Occasionally, some
F i g . 3. H i g h e r m a g n i f i c a t i o n o f h e p a t o c y t e s , e n d o t h e l i a l cells ( E ) , a n d s i n u s o i d ( S d ) a f t e r 5 h o f p e r f u s i o n . The ultrastructures of the hepatocytes are well preserved and closely resemble those of liver in vivo for fasted rats. Minor alterations are: cisternae of the rough endoplasmic reticulum (rer) are organized in parallel r o w s b u t a r e less p r o m i n e n t t h a n i n v i v o . G l y c o g e n p a r t i c l e s a r e f e w e r ( a r r o w s ) . T h e r e a r e n o o t h e r noteworthy changes in the other organelles. Mitochondria (m), peroxisomes (p), bile canalieuli (bc); X 12 200.
59 T A B L E II EFFECT OF HEME ON BILIRUBIN PRODUCTION WITH A HEMOGLOBIN-FREE MEDIUM
IN THE
ISOLATED
RAT
LIVER
PERFUSED
Livers from rats fasted for 40--48 h were perfused with a medium containing 2.5% bovine serum albumin. Bile a n d b i l i r u b i n w e r e m e a s u r e d w i t h a n d w i t h o u t a d d i t i o n o f h e m e t o t h e p e r f u s a t e . V a l u e s s h o w n r e p r e s e n t t h e m e a n +- S . E . N u m b e r o f e x p e r i m e n t s i n p a r e n t h e s e s . Time (h)
Perfusate
Bile v o l u m e (ml/100 g fiver)
Total bilirubin (pg/100 g liver)
0--2 2--5 2--5
no heme no heme 750 #g heine *
8 . 6 0 + 0 . 4 0 (7) 8.85-+ 0 . 8 0 ( 5 ) 1 1 . 0 5 + 0 . 7 3 (8)
136 ± 8 (12) 1 0 5 ± 1 2 (7) 1479 + 174 (8)
* H e m e is a d d e d t o t h e p e r f u s a t e a f t e r 1 2 0 r a i n .
hepatocytes show clear vacuoles in the centrilobular zone. These vacuoles are lined by a single limiting membrane and are primarily distributed near the sinusoid surface of the cytoplasm. A few mitochondria are slightly swollen with a peripheral displacement of their cristae. Again, this change is more c o m m o n in liver cells near the central vein. Glycogen stores are diminished but n o t depleted. No n o t e w o r t h y alterations occur in other subcellular organelles. The structure of the sinusoid is well-preserved; endothelial, Kupffer cells and lipocytes appear intact. Table II lists the a m o u n t of bile volume and total bilirubin for the bile collected for the first 2 and for the last 3 h of perfusion. A total of approx. 20% of the heme added to the perfusate at the beginning of the third hour is converted to bilirubin within 3 h. At conclusion of the experiment, 35% of the heme still circulates. Heme addition increases the bile volume, but n o t to a statistically significant degree. The effect of drugs on the rate of protein synthesis is shown in Table III. TABLE III PROTEIN SYNTHESIS BY THE ISOLATED RAT LIVER PERFUSED FREE MEDIA; NET CHANGE OF TOTAL PROTEINS IN 5 h Results are expressed of experiments.
-
-
-AIA AIA DDC DDC Desferal Desferal
AN ERYTHROCYTE-
i n r a g / 1 0 0 g l i v e r p e r 5 h a s m e a n +- S . E . ; f i g u r e s i n p a r e n t h e s e s i n d i c a t e t h e n u m b e r
Treatment In vivo
WITH
Albumin
Hemopexin
102.8 ± I I . 0 (16) 1 1 5 . 3 ± 11.2 (8) 69.5-+ 7.3 (5) 56.3± 7.7 ( 5 ) * * 1 0 5 . 9 + 26.7 (3) 7 2 . 6 ± 1 2 . 3 (3) 60.9 + 1 5 . 8 (4) 6 1 . 4 -+ 2 . 2 ( 4 ) **
23.1 -+ 3.7 19.3-+ 2.8 21.0-+ 2.5 24.7 ± 7.3 24.5 ± 6.2 31.1 + 7.1 85.8 + 9.8 4 4 . 8 -+ 6.7
Transferrin
In vitro -heme AIA AIA+ heine -heme -heine
* P < 0.05. ** P < 0.01. N.D., not determined.
(4) (8) (5) (5) (3) (3) ( 4 ) ** (4) **
29.1 n.d. 19.3 11.2 22.2 23.9 26.1 22.4
± 4.1 (8) +- 1.7 ± 1.4 ± 2.5 + 3.5 ± 1.9 ± 3.7
(5) (5) * (3) (3) (4) (4)
60 TABLE IV BILIRUBIN
SECRETION
BY THE
ISOLATED
LIVER
FROM
RATS
PRETREATED
WITH
DRUGS
L i v e r s w e r e p e r f u s e d w i t h a n d w i t h o u t t h e a d d i t i o n o f h e i n e i n t h e p e r f u s a t e . H e m e ( 7 5 0 p g ) is a d d e d t o t h e p e r f u s a t e a t the beginning of the e x p e r i m e n t . R e s u l t s axe e x p r e s s e d a s m e a n ± S . E . ; f i g u r e s i n p a r e n theses indicate the number of experiments. Treatment
Total bilirubin secreted
Percent heine in perfusate
Disappearance after 5 h
Conversion to bile in 5 h
8 0 . 4 ± 2.1
45.7 ± 1.9
84.4 ± 1.4
28.2 ± 3.3
79.4 ± 3.5
3 8 . 8 ± 8.1
9 3 . 0 -+ 1 . 0
7 1 . 6 -+ 2 . 5
In vivo
In vitro
(pg/100 g liver per 5 h
--A1A A1A DDC DDC Desferal Desferal
-heine AIA h e i n e + A1A -heme -heme
241 3091 265 1418 703 2002 443 4099
+ 10 -+ 2 9 2 ± 30 ± 202 ± 118 ~ 451 -+ 8 3 ± 363
(4) (4) (4) (4) (3) (3) (4) (4)
Addition of heme to the perfusate enhances the reduction in synthesis rate of albumin and transferrin to a significant level in response to pretreatment with AIA. Hemopexin synthesis is unaffected. Neither pretreatment with DDC, nor addition of heme to the perfusate after pretreatment significantly effects the rate of synthesis of any of these proteins. Desferal treatment, similar to AIA treatment, reduces albumin synthesis significantly only after addition of heme to the perfusate. It does, however, cause a 3--4-fold increase in hemopexin synthesis. This stimulatory effect on hemopexin production is inhibited by 50% upon addition of heme to the perfusate. The results of bitirubin excretion by isolated livers of rats pretreated with drugs are shown in Table IV. To show differences in bilirubin excretion over a period of 5 h, heme is added to the perfusate at the beginning of perfusion. DDC pretreatment increases bilirubin formation from endogenous sources approximately three times, but the ability of the liver to convert exogenous heme to bilirubin is impaired. Although the same a m o u n t of heme disappears from the circulation as in controls, only 2000 pg/100 g liver bilirubin is excreted into the bile, an approx. 30% reduction compared to control experiments. The conversion rate of exogenous heme to bilirubin (1400 pg/100 g liver) is even smaller following AIA. AIA treatment has no effect on bilirubin formation from endogenous sources. On the other hand, Desferal enhances bilirubin production from 240 to 440 pg/100 g liver from endogenous sources, and slightly increases heme clearance from the perfusate and bilirubin formation from exogenous heme. Discussion
Perfusion of the rat liver with an erythrocyte-free medium has been performed by several investigators. With the exception of a few very recent studies [19,33], most of these studies have dealt with liver metabolism [7,8,20,21]
61 and do not monitor concomitantly liver morphology. Schmucker and Curtis [19] compare the liver perfused for 1 h with Krebs-Ringer bicarbonate buffer containing 4.0% bovine serum albumin with that perfused with diluted rat blood. The latter perfusate preserves the subcellular organization better than the semi-synthetic medium. Nevertheless, endothelial damage and in particular vacuolar degeneration are minimal when the flow rate of the erythrocyte-free perfusate is maintained at approx. 45 ml/min. The ultrastructural changes correlate well with the potassium content of the perfusate and the extent of colloid carbon clearance. In the past few years, we have developed a rat liver perfusion system in which the rate of synthesis of four serum proteins remains constant during a 5 h perfusion with an erythrocyte-free medium. In addition, we achieve near physiological values for the energy charge and the lactate : pyruvate ratio, an indicator of the redox state of the liver cytosol [35,36]. We also show good preservation of the ultrastructural integrity of the liver tissue after a 5 h perfusion. No endothelial damage is observed. The vacuolar change of the haptocytes which is t h o u g h t to be induced by h y p o x i a seems to be less prominent than that reported by Schmucker and Curtis after perfusion for 1 h and equivalent to that of Krone et al. [34] after prolonged perfusion with erythrocyte-free medium. The rate of protein synthesis in our system of 17 mg albumin/h per 100 g liver agrees well with that reported by Matern et al. [20] of 18 mg/h per 100 g liver. Both values are approx. 50% of those reported by John and Miller [37] and Hoffenberg et al. [5] who used red cells in the perfusate. The fact that albumin, transferrin, hemopexin and haptoglobin are produced at a constant rate is a good indicator for the maintenance of viability of the liver cells during perfusion. Haptoglobin synthesis is 70% of tl~at reported by John and Miller [37] in fed rats. Hemopexin synthesis has not been studied before. The lower protein synthesis in the isolated organ compared to in vivo studies may be caused by the lack of humoral factors (e.g. hormones). John and Miller [37] showed that the addition of hormones such as insulin, growth hormones and cortisol significantly enhance the synthesis rate of many proteins in their perfusion system. We also found hormones to effectively enhance albumin and fibrinogen synthesis by isolated rat liver cells; but hemopexin production was independent of hormonal supplementation [38]. The c o n t e n t of ATP, ADP and AMP of the perfused liver from fasted and non-fasted rats is somewhat reduced in comparison to that of the non-perfused liver. Nevertheless, the ATP : ADP ratio as well as the energy charge are not lowered, indicating an adequately functioning liver. A reduced ATP generation per se is, therefore, probably n o t the cause of a decreased rate of protein synthesis, as has been suggested by Matern et al. [20]. Our values for pyruvate content of the liver 5 h after perfusion are comparable to those of Mannaerts et al. [21] who perfused for 30--90 min with red cells in Krebs-Henseleit bicarbonate buffer containing albumin (2.6%). They are also comparable to the values of Schimassek [9] who used erythrocytes in Tyrode's solution with 2.6% albumin for a 6 h perfusion. The pyruvate c o n t e n t of ours is, however, lower than that of Lueck and Miller [39] who perfused with diluted whole rat blood for a similar time period. Finding a lower a m o u n t of lactate in the perfused
62 liver of starved as compared to non-starved rats was not unexpected because depletion of glycogen stores decreases the rate of glycolysis [40]. The higher lactate content in vivo encountered by us as compared with that by others [9,28] is possibly due to lowering of tissue oxygenation during freeze-clamping the liver under ether narcosis [9]. Our values are not corrected for the blood content of the liver which does n o t cause a significant increase in the a m o u n t of lactate [28]. The lactate : pyruvate is a better indicator of the redox state than determination of either metabolite alone. A low lactate : pyruvate ratio indicates a good redox state of the cytosol [35,36], but as pointed out by Liibbers [41], a low ratio does n o t exclude heterogeneous oxygen supply to the liver tissue. In agreement with the results of others [13,42], we found a reduced bile flow during the last 3 h of perfusion. The progressive decline in bile flow is concomitantly decreased with the biliary excretion of bile salts and can be prevented by infusing taurocholate [42]. Within 3 h after the addition of heme to the perfusate, 20% is recovered as bile bilirubin. This percent of conversion of heme to bilirubin is similar to that observed by Snyder and Schmid [43] for studies in vivo, employing radioactive heme in bile-cannulated rats. In their studies, the maximal secretion of bile bilirubin occurred between 2 and 8 h after intravenous heme injection, and was completed at approx. 30 h; the total bilirubin recovery averaged 60% of the injected heme [43]. In our perfusion system, using unlabeled heme, 65% of the heme added to the perfusate is cleared from the circulation after 3 h, 20% of which has been converted to bile bilirubin. After 5 h, 80% of the heme is cleared and 45% has been converted to bile bilirubin. Extrapolating from the findings expected had all heme been cleared from the perfusate, we estimate a 60--70% recovery as bilirubin which is a value comparable with the in vivo studies [43]. Many carcinogenic and porphyrinogenic drugs as well as heme have been shown to stimulate hemopexin synthesis in vivo [44,45]. Pretreatment of the rats with AIA or DDC does not appear to enhance hemopexin synthesis but that of transferrin and albumin is decreased to a significant degree after addition of heme to the perfusate. These data support the observation of Sell et al. [46] for a fetal hepatocyte monolayer culture which indicates that the control of hemopexin synthesis differs from that o f albumin and haptoglobin. Pretreatm e n t of the donor rats with Desferal, an iron-chelating agent, stimulates the rate of hemopexin synthesis several-fold while that of albumin and transferrin remains normal or is slightly reduced. The stimulation of hemopexin synthesis is in part inhibited by the addition of heine into the perfusate. These findings suggest a close interrelationship between hemopexin synthesis and iron metabolism which has n o t been previously noted. The mechanism by which the synthesis of hemopexin is enhanced is unknown. The most likely signal for the initiation of hemopexin synthesis is an acute shift of iron or heme within critical tissue pools. Knowledge on the size and number of iron and heme pools of the liver is at present limited. The effect of AIA, DDC and Desferal on the formation of bilirubin is even more interesting. Whereas AIA has no effect on bilirubin formation, DDC and Desferal increase bilirubin production from endogenous sources. Both AIA and DDC are known to induce aminolevulinic acid synthetase (ALAS), the rate-
63 limiting enzyme in the heme biosynthetic pathway [47]. Both drugs also cause a loss of c y t o c h r o m e P-450 and heme [48]. In addition, DDC inhibits heme synthetase, the enzyme catalyzing the insertion of iron into the protoporphyrin IX ring to form heme. As a consequence, protoporphyrin IX accumulates in the liver cells [49]. For reasons stated above, it is unlikely that the bilirubin produced b y the liver is formed from endogenous heme since the heme pool in the liver is reduced by treatment of the rats with drugs like AIA and DDC. Little is known on the effect of Desferal on liver cells. This drug supposedly chelates iron b o u n d to ferritin [50] and the iron-desferrioxamine complex is excreted mainly via the bile and urine [51]. Desferal may, indeed, render certain liver pools temporarily deficient in iron which prevents heme formation. This in turn would increase the level of protoporphyrin IX. Furthermore, it is conceivable that n o t all bilirubin from endogenous sources, the so-called early bilirubin, necessarily originates from heme; it could in part be derived from protoporphyrin IX through an as yet u n k n o w n pathway. The source of this bilirubin is the subject of current research. When the rats are pretreated with AIA or DDC, their isolated livers convert less added heme into bilirubin than do the controls, which substantiate our earlier findings [52]. On the other hand, pretreatment of the rats with Desferal increases the efficiency of the liver to convert added heme into bilirubin from 45 to 70% in 5 h. At present, an interpretation of these findings cannot be given. Acknowledgements This work was supported by research grants awarded to Dr. U. Muller-Eberhard from the Institute of Child Health and Human Development (HD-04445) and from the Institute of Arthritis and Metabolic Diseases (AM-16737 and AM-18329). Dr. K. Miyai's support was awarded from U.S.P.H.S. research grant (5-RO1-AM-161110) and U.S.P.H.S. contract (SP-17-HL-14169 and HL123-73). We wish to thank Dr. Joseph Katz and Mr. George Bonorris from the Cedars-Sinai Medical Research Institute, Los Angeles for their help in setting up the isolated rat liver perfusion, Susan Wormsley for her expert technical assistance, and Dr. William Morgan, Department of Biochemistry, Scripps Clinic and Research Foundation and Dr. Steven Schenker, Vanderbilt University, Nashville, Tenn., for critical review of this manuscript. We also appreciate the generosity of Dr. A n t h o n y S. Tavill, Medical Research Centre, Harrow, Middlesex, U.K. and Dr. R. Engler, U E R Biomedicale des Saints P~res, Paris, France, in supplying rat transferrin and haptogobin. References 1 2 3 4 5 6 7 8
J o h n , D.W. a n d Miller, L . L . ( 1 9 6 9 ) J. Biol. C h e m . 2 4 4 , 6 1 3 4 - - 6 1 4 3 G r e e n , M. a n d Miller, L.L. ( 1 9 6 0 ) J. Biol. C h e m . 2 3 5 , 3 2 0 2 - - 3 2 0 8 W o o d s i d e , K . H . a n d M o r t i m o r e , G . E . ( 1 9 7 2 ) J. Biol. C h e m . 2 4 7 , 6 4 7 4 - - 6 4 8 1 K a t z , J., B o n o r r i s , G., O k u y a m a , S. a n d Sellers, A.L. ( 1 9 6 7 ) A m . J. P h y s i o l . 2 1 2 , 1 2 5 5 - - 1 2 6 0 H o f f e n b e r g , It., G o r d o n , A . H . a n d B l a c k , E . G . ( 1 9 7 1 ) B i o c h e m . J. 1 2 2 , 1 2 9 - - - 1 3 4 M o r t i m o r e , G.E. ( 1 9 6 3 ) A m . J. P h y s i o l . 2 0 4 , 6 9 9 - - 7 0 4 W i l l i a m s o n , J . R . , B r o w n i n g , E.T. a n d S c h o l z , R. ( 1 9 6 9 ) J. Biol. C h e m . 2 4 4 , 4 6 0 7 - - 4 6 1 6 W i l l i a m s o n , J . R . , S c h o l z , It. and B r o w n i n g , E.T. ( 1 9 6 9 ) J. Biol. C h e m . 2 4 4 , 4 6 1 7 - - 4 6 2 7
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Biochimica e t Biophysica Acta, 496 (1977) 52--64 © Elsevier/North-Holland Biomedical Press
BBA 28130
EFFECT OF PORPHYRINOGENIC AGENTS ON PROTEIN SYNTHESIS AND BILIRUBIN FORMATION BY THE ISOLATED PERFUSED RAT LIVER
H. HENG LIEM a, KATSUMI MIYAI b and URSULA MULLER-EBERHARD b a Department o f Biochemistry, Scripps Clinic and Research Foundation and the b Departments o f Pathology and Pediatrics, University o f California al San Diego, La Jolla, Calif. 92037 (U.S.A.) ,
(Received May 31st, 1976) Summary We established an isolated rat liver perfusion system for the study of heme catabolism. The liver of rats fasted for 48 h is perfused with an e r y t h r o c y t e - f r e e medium. Ultrastructural analysis shows integrity of all subcellular organelles with the e x c e p t i o n of minor alterations in the rough endoplasmic reticulum. The perfused liver synthesizes serum proteins at a constant rate for 5 h. Albumin is secreted at a mean rate of 17 -+ 2 mg/h per 100 g liver, h e m o p e x i n at 5.0 + 0.7, haptoglobin at 3.2 + 0.6 and transferrin at 5.1 -+ 0.8 mg/h per 100 g liver. The mean ratio o f ATP : ADP is 3.5 -+ 0.1, and that of lactate : pyruvate 27 + 6. The rate of conversion of heme into bilirubin is comparable to t hat reported for in vivo studies. A minimal ef f ect on protein synthesis is observed after administration of the porphyrinogenic agents, allylisopropylacetamide (AIA) and 3,5-diethoxycarb o n y l - l , 4 dihydrocollidine (DDC). Pr e t r eat m e nt of the rats with the iron chelator, Desferal, causes a 3--4-fold increase in h e m o p e x i n but n o t in albumin and transferrin synthesis. A striking 2--3-fold e n h a n c e m e n t of bile bilirubin production follows t r e a t m e n t with DDC and Desferal, but not with AIA. The a m o u n t o f bilirubin f o r m e d from heme added to the perfusate is reduced by AIA and DDC and enhanced by Desferal treatment. It is proposed that unavailability of iron in a certain hepatic tissue pool causes p r o t o p o r p h y r i n IX accumulation which may serve as an alternate source for bilirubin production. Introduction The isolated perfused rat liver has been used extensively to study protein, c a r b oh y d r ate, and lipid metabolism [1--12]. In some studies, evaluation of the Abbreviations: AIA, allylisopylacetamide; D D C ,
3,5-diethoxy c a rbonyl -l ,4-di hydroc ol l i di ne .
53 histological and ultrastructural integrity of the perfused liver was of primary concern [13--18]. Seldom were metabolic performances correlated with ultrastructural changes in the same experiment. The viability of the liver has been assessed by determining plasma protein synthesis, bile formation, oxygen consumption, CO2 production, bromosulphalein uptake and biliary excretion. Removal of particulate matter from the perfusate by the reticuloendothelial cells has served as another criterion of liver function. Recently, hepatic functions such as bile formation, secretion of K ÷and lactic acid, and carbon particle clearance were correlated with the fine structure of the perfused liver by Schmucker and Curtis [19]. These investigators found that the hepatic ultrastructure was preserved best when colloidal clearance was efficient and the rate of K ÷secretion into the perfusate was low. Perfusion is generally achieved with a semi-synthetic medium containing homologous or heterologous erythrocytes or with diluted rat blood. An erythrocyte-free medium is rarely employed [7,20,21] and, to our knowledge, has not been utilized to monitor bile pigment secretion. We are describing such a perfusion system in which bilirubin formation from endogenous and exogenous sources is studied under a variety of conditions. To facilitate the metabolism of heme *, the donor rats are fasted and erythrocytes and rat serum are omitted from the perfusion medium. This system synthesized for 5 h several serum proteins at a linear rate and converts heme into bilirubin at a rate similar to that found in vivo. We report the effect of the porphyrinogenic drugs, allylisopropylacetamide (AIA) and 3,5-diethoxycarbonyl-l,4-dihydrocollidine {DDC) as well as that of the iron chelator, Desferal, on serum protein synthesis and bilirubin formation from endogenous and exogeneous heme. Materials and Methods
Animals and drugs. Adult male Sprague-Dawley rats 250--400 g served as liver donors. They were fed a diet of Purina Laboratory Chow, containing 23% crude protein (Harrison-Riedy, Chula Vista, Calif.). Food was witheld for 40-48 h prior to the experiment. Water was allowed ad libitum. 3,5-Diethoxycarbonyl-l,4-dihydrocollidine (DDC) was kindly supplied by Dr. Robert Labbe, University of Washington, Seattle, Wash. The drug was dissolved in corn oil and administered intraperitoneally into the donor rats (300 mg/kg) 24 h before death. Allylisopropylacetamide (AIA), a gift of Hoffman La Roche, Inc., Nutley, N.J., was dissolved in phosphate-buffered saline, pH 7.4 and injected subcutaneously (300 mg/kg) 16 h before death. Desferal (desferrioxamine mesylate, Ciba Pharm. Co., Summit, N.J.) dissolved in normal saline was administered intramuscularly (125 mg/rat) once a day for 2 days. Hemin was purchased from Eastman Organic Chemicals. Perfusion methods. The perfusion technique and apparatus were designed according to Miller et al. [22], with minor modifications. The modifications are: the perfusate drains directly from the hepatic veins into the reservoir since we do not cannulate the vena cava inferior. After cannulating the portal vein and the bile duct, the liver is perfused in situ with oxygenated Krebs-Henseleit * Heme, iron-protoporphyrin
IX.
54 bicarbonate buffer, pH 7.4. Within 1 min, the excised liver, placed on a circular plexiglass disc and resting on the platform, is connected to the circulating perfusion medium. A mixture of 95% O2 and 5% CO2 passes through the glass "thin f i l m " oxygenator at a flow rate of 3.5 1/min. The perfusion medium, approx. 150 ml, contains Krebs-Henseleit bicarbonate buffer, saturated with 95% O2 and 5% CO2, 2.5% bovine serum albumin (Pentex Miles Laboratory, Inc., Kankakee, Ill.) dialyzed overnight against the buffer, 4000 units sodium heparin, 2 ml TC amino acids (Hela 100 × Difco Laboratories, Detroit, Mich.)* and 240 mg glucose. The medium is titrated with 3 M NaOH to pH 7.4. Heme is solubilized as previously described [23] and added slowly to the perfusate in the beginning of the third hour. The pH of the perfusate varies little and is maintained at pH 7.4 by monitoring once every hour, adding 1 M NaOH when necessary. The liver is perfused at 37°C, with a flow rate of 30--45 ml/min. The portal pressure varies from 13 to 14 cm of water. Each perfusion is continued for 5 h; 1-ml samples are withdrawn at time 0 (5 min after initiation of the perfusion), and at 1, 2, 3, 4 and 5 h. The bile is usually collected in two portions in graduated centrifuge tubes for the first 2 h and for the next 3 h. Analytical methods. Rat albumin concentrations in the perfusate are measured by the radial immunodiffusion technique [24], and hemopexin, transferrin, and haptoglobin by a radioimmunoassay [25]. The bile volume is recorded and its content analyzed for total bilirubin, determined as the alkaline "Azo bilirubin" [26]. Heme in the perfusate is determined by the benzidine m e t h o d [27]. At the end of the perfusion, following the technique described by Hohorst et al. [28], liver sections are instantly frozen, weighed and stored in liquid nitrogen for up to 7 days. The frozen liver pieces are pulverized and a HC104 extract is prepared. The protein-free filtrates are then assayed for ATP, ADP and AMP, as well as for lactate and pyruvate [29,30]. When protein synthesis and bilirubin formation are studied, the livers are disconnected from the canula at the end of the perfusion, blotted with filter paper and weighed. Electron microscopy. For ultrastructural studies, two livers were perfused at the end of the experiment through the portal vein with 20 ml of 1% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. The fixative was infused with a hydrostatic pressure of 15--17 cm water for 2--3 min. Liver tissue was immediately cut into approx. 1-mm cubes, rinsed with 0.1 M phosphate buffer, and post-fixed with 2% OsO4 in 0.1 M phosphate buffer, pH 7.4, for 1 h at 4°C. After dehydration with graded concentrations of acetone, tissue blocks were embedded in Epon. Ultrathin sections were stained with lead citrate and examined in a Siemens Filmiskop 101. Results
The rate of net secretion of albumin, hemopexin, haptoglobin and transferrin into the perfusion media is illustrated in Fig. 1. The synthesis rate of * T C - M e d i u m E a g l e H e l a 1 0 0 X is a 1 0 0 - f o l d c o n c e n t r a t i o n Hela medium of Eagle (Science 122, 501 (1955)).
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4
5
Hours F i g . 1. P r o t e i n s y n t h e t i s b y t h e i s o l a t e d r a t l i v e r , p e r f u s e d w i t h a h e m o g l o b i n - f r e e m e d i u m . C u m u l a t i v e average net change in the concentrations of albumin (16) • e, transferrin (8) X X, hemopexin (4) © ©, a n d h a p t o g l o b i n ( 4 ) A - in the perfusates. Numbers in parentheses indicate the number of experiments, and the vertical bars represent S.E. The r values are: albumin, 0.999; transferrin, 0.995; hemopcxin, 0.988; haptoglobin, 0.976.
albumin and transferrin becomes linear after the first hour and that of hemopexin and haptoglobin is linear from the beginning of the experiment. The latter two proteins may be consumed during the initial phase of perfusion associated with hemolysis. Albumin and transferrin molecules, on the other hand, may be trapped in the hepatocytes and/or within the vascular system of the liver [20]. We consider the linear phase after the first hour to reflect the rate of de novo protein synthesis in the isolated liver. In Table I, the liver c o n t e n t of ATP, ADP, AMP, lactate and pyruvate is shown at the conclusion of a 5 h perfusion. The concentration of the nucleotides for the perfused liver is, in general, slightly lower than that of the liver in vivo, but is in good agreement with that found by others for perfused livers [21,31]. The average values, for the energy charge as defined by Atkinson and Walton [32] ([ATP] + 1/2[ADP])/([ATP] + [ADP] + [AMP]) for the perfused liver of non-fasted and fasted rats are 0.82 and 0.83, respec-
LACTATE AND PYRUVATE
CONTENT
OF RAT LIVER PERFUSED
WITH A HEMOGLOBIN-FREE
MEDIUM
2 . 5 -+ 0.1
2 . 4 -+ 0.1
(5)
(6)
2.8 + 0 . 2 ( 1 0 )
ATP
* In v i v o , n o n - p e r f u s e d livers.
fasted
Perfused
non-fasted
Perfused
non-fasted *
Normal
Liver
+ 0.1
0.75 + 0.03
0 . 7 2 -+ 0 . 0 6
1.1
ADP
(5)
(6)
(10)
0 . 2 1 -+ 0 . 0 2 (5)
0 . 2 6 -+ 0 . 0 3 ( 3 )
0 . 3 6 + 0 . 0 3 (2)
AMP _._
3 . 5 +- 0.1
3 . 4 -+ 0 . 3
(5)
(6)
2 . 7 -+ 0 . 3 ( 1 0 )
ATP/ADP
0 . 9 + 0.1 ( 5 )
1 . 6 -+ 0 . 2 ( 5 )
3 . 2 -+ 0 . 1 ( 3 )
Lactate
0 . 0 4 +- 0 . 0 1 ( 5 )
0 . 0 8 -+ 0 . 0 1 ( 5 )
0 . 1 2 + 0.01 (3)
___
Pyruvate
2 7 -+ 6 ( 5 )
2 1 -+ 1 ( 5 )
2 7 -+ 2 (3)
pyruvate
Lactate/
Livers from rats fasted for 40--48 hours were perfused with a medium containing 2.5% bovine serum aIbumin. Nucleotides, lactate and pyruvate of the livers were m e a s u r e d a t t h e e n d o f 5 h p e r f u s i o n . V a l u e s s h o w n r e p r e s e n t t h e m e a n _+ S . E . a n d e x p r e s s e d i n p m o l / g l i v e r w e t w e i g h t . N u m b e r o f a n i m a l s i n p a r e n t h e s e s . V a l u e s are n o t c o r r e c t e d f o r p e r f u s a t e c o n t e n t i n t h e l i v e r .
NUCLEOTIDES,
TABLE I
57 tively. These values are comparable to those obtained by Htilsmann and Kurpershoek-Davidov [11] for an isolated liver perfused with an erythrocyte-free medium. In situ, the energy charge is found to be close to 0.9 [33]. In the present study (Table I), concentrations of lactate and pyruvate are lower in the perfused than in the non-perfused liver, and they are lower in the liver of fasted than in non-fasted animals. However, the lactate : pyruvate ratio in the perfused is comparable to that in the non-perfused liver. The ultrastructure of the hepatic tissue after 5 h of perfusion is in general well-preserved (Figs. 2 and 3); only minor alterations can be noted. Parallel
Fig. 2. U l t r a s t r u c t u r e o f t h e m i d - z o n e o f a l o b u l e o f r a t l i v e r a f t e r 5 h o f p e r f u s i o n . E n d o t h e l i a l cells ( E ) l i n e t h e s i n u s o i d ( S d ) a n d are i n t a c t . T h e s t r u c t u r e s o f t h e h e p a t o c y t e s ( H ) are well p r e s e r v e d . N u c l e u s o f hepatoc~'tes
(N) × 6480.
58
arrays of the rough endoplasmic reticulum are at times dispersed and partially fragmented into small vesicles. The smooth endoplasmic reticulum appears more prominent than that of the liver in situ of fasted rats. Occasionally, some
F i g . 3. H i g h e r m a g n i f i c a t i o n o f h e p a t o c y t e s , e n d o t h e l i a l cells ( E ) , a n d s i n u s o i d ( S d ) a f t e r 5 h o f p e r f u s i o n . The ultrastructures of the hepatocytes are well preserved and closely resemble those of liver in vivo for fasted rats. Minor alterations are: cisternae of the rough endoplasmic reticulum (rer) are organized in parallel r o w s b u t a r e less p r o m i n e n t t h a n i n v i v o . G l y c o g e n p a r t i c l e s a r e f e w e r ( a r r o w s ) . T h e r e a r e n o o t h e r noteworthy changes in the other organelles. Mitochondria (m), peroxisomes (p), bile canalieuli (bc); X 12 200.
59 T A B L E II EFFECT OF HEME ON BILIRUBIN PRODUCTION WITH A HEMOGLOBIN-FREE MEDIUM
IN THE
ISOLATED
RAT
LIVER
PERFUSED
Livers from rats fasted for 40--48 h were perfused with a medium containing 2.5% bovine serum albumin. Bile a n d b i l i r u b i n w e r e m e a s u r e d w i t h a n d w i t h o u t a d d i t i o n o f h e m e t o t h e p e r f u s a t e . V a l u e s s h o w n r e p r e s e n t t h e m e a n +- S . E . N u m b e r o f e x p e r i m e n t s i n p a r e n t h e s e s . Time (h)
Perfusate
Bile v o l u m e (ml/100 g fiver)
Total bilirubin (pg/100 g liver)
0--2 2--5 2--5
no heme no heme 750 #g heine *
8 . 6 0 + 0 . 4 0 (7) 8.85-+ 0 . 8 0 ( 5 ) 1 1 . 0 5 + 0 . 7 3 (8)
136 ± 8 (12) 1 0 5 ± 1 2 (7) 1479 + 174 (8)
* H e m e is a d d e d t o t h e p e r f u s a t e a f t e r 1 2 0 r a i n .
hepatocytes show clear vacuoles in the centrilobular zone. These vacuoles are lined by a single limiting membrane and are primarily distributed near the sinusoid surface of the cytoplasm. A few mitochondria are slightly swollen with a peripheral displacement of their cristae. Again, this change is more c o m m o n in liver cells near the central vein. Glycogen stores are diminished but n o t depleted. No n o t e w o r t h y alterations occur in other subcellular organelles. The structure of the sinusoid is well-preserved; endothelial, Kupffer cells and lipocytes appear intact. Table II lists the a m o u n t of bile volume and total bilirubin for the bile collected for the first 2 and for the last 3 h of perfusion. A total of approx. 20% of the heme added to the perfusate at the beginning of the third hour is converted to bilirubin within 3 h. At conclusion of the experiment, 35% of the heme still circulates. Heme addition increases the bile volume, but n o t to a statistically significant degree. The effect of drugs on the rate of protein synthesis is shown in Table III. TABLE III PROTEIN SYNTHESIS BY THE ISOLATED RAT LIVER PERFUSED FREE MEDIA; NET CHANGE OF TOTAL PROTEINS IN 5 h Results are expressed of experiments.
-
-
-AIA AIA DDC DDC Desferal Desferal
AN ERYTHROCYTE-
i n r a g / 1 0 0 g l i v e r p e r 5 h a s m e a n +- S . E . ; f i g u r e s i n p a r e n t h e s e s i n d i c a t e t h e n u m b e r
Treatment In vivo
WITH
Albumin
Hemopexin
102.8 ± I I . 0 (16) 1 1 5 . 3 ± 11.2 (8) 69.5-+ 7.3 (5) 56.3± 7.7 ( 5 ) * * 1 0 5 . 9 + 26.7 (3) 7 2 . 6 ± 1 2 . 3 (3) 60.9 + 1 5 . 8 (4) 6 1 . 4 -+ 2 . 2 ( 4 ) **
23.1 -+ 3.7 19.3-+ 2.8 21.0-+ 2.5 24.7 ± 7.3 24.5 ± 6.2 31.1 + 7.1 85.8 + 9.8 4 4 . 8 -+ 6.7
Transferrin
In vitro -heme AIA AIA+ heine -heme -heine
* P < 0.05. ** P < 0.01. N.D., not determined.
(4) (8) (5) (5) (3) (3) ( 4 ) ** (4) **
29.1 n.d. 19.3 11.2 22.2 23.9 26.1 22.4
± 4.1 (8) +- 1.7 ± 1.4 ± 2.5 + 3.5 ± 1.9 ± 3.7
(5) (5) * (3) (3) (4) (4)
60 TABLE IV BILIRUBIN
SECRETION
BY THE
ISOLATED
LIVER
FROM
RATS
PRETREATED
WITH
DRUGS
L i v e r s w e r e p e r f u s e d w i t h a n d w i t h o u t t h e a d d i t i o n o f h e i n e i n t h e p e r f u s a t e . H e m e ( 7 5 0 p g ) is a d d e d t o t h e p e r f u s a t e a t the beginning of the e x p e r i m e n t . R e s u l t s axe e x p r e s s e d a s m e a n ± S . E . ; f i g u r e s i n p a r e n theses indicate the number of experiments. Treatment
Total bilirubin secreted
Percent heine in perfusate
Disappearance after 5 h
Conversion to bile in 5 h
8 0 . 4 ± 2.1
45.7 ± 1.9
84.4 ± 1.4
28.2 ± 3.3
79.4 ± 3.5
3 8 . 8 ± 8.1
9 3 . 0 -+ 1 . 0
7 1 . 6 -+ 2 . 5
In vivo
In vitro
(pg/100 g liver per 5 h
--A1A A1A DDC DDC Desferal Desferal
-heine AIA h e i n e + A1A -heme -heme
241 3091 265 1418 703 2002 443 4099
+ 10 -+ 2 9 2 ± 30 ± 202 ± 118 ~ 451 -+ 8 3 ± 363
(4) (4) (4) (4) (3) (3) (4) (4)
Addition of heme to the perfusate enhances the reduction in synthesis rate of albumin and transferrin to a significant level in response to pretreatment with AIA. Hemopexin synthesis is unaffected. Neither pretreatment with DDC, nor addition of heme to the perfusate after pretreatment significantly effects the rate of synthesis of any of these proteins. Desferal treatment, similar to AIA treatment, reduces albumin synthesis significantly only after addition of heme to the perfusate. It does, however, cause a 3--4-fold increase in hemopexin synthesis. This stimulatory effect on hemopexin production is inhibited by 50% upon addition of heme to the perfusate. The results of bitirubin excretion by isolated livers of rats pretreated with drugs are shown in Table IV. To show differences in bilirubin excretion over a period of 5 h, heme is added to the perfusate at the beginning of perfusion. DDC pretreatment increases bilirubin formation from endogenous sources approximately three times, but the ability of the liver to convert exogenous heme to bilirubin is impaired. Although the same a m o u n t of heme disappears from the circulation as in controls, only 2000 pg/100 g liver bilirubin is excreted into the bile, an approx. 30% reduction compared to control experiments. The conversion rate of exogenous heme to bilirubin (1400 pg/100 g liver) is even smaller following AIA. AIA treatment has no effect on bilirubin formation from endogenous sources. On the other hand, Desferal enhances bilirubin production from 240 to 440 pg/100 g liver from endogenous sources, and slightly increases heme clearance from the perfusate and bilirubin formation from exogenous heme. Discussion
Perfusion of the rat liver with an erythrocyte-free medium has been performed by several investigators. With the exception of a few very recent studies [19,33], most of these studies have dealt with liver metabolism [7,8,20,21]
61 and do not monitor concomitantly liver morphology. Schmucker and Curtis [19] compare the liver perfused for 1 h with Krebs-Ringer bicarbonate buffer containing 4.0% bovine serum albumin with that perfused with diluted rat blood. The latter perfusate preserves the subcellular organization better than the semi-synthetic medium. Nevertheless, endothelial damage and in particular vacuolar degeneration are minimal when the flow rate of the erythrocyte-free perfusate is maintained at approx. 45 ml/min. The ultrastructural changes correlate well with the potassium content of the perfusate and the extent of colloid carbon clearance. In the past few years, we have developed a rat liver perfusion system in which the rate of synthesis of four serum proteins remains constant during a 5 h perfusion with an erythrocyte-free medium. In addition, we achieve near physiological values for the energy charge and the lactate : pyruvate ratio, an indicator of the redox state of the liver cytosol [35,36]. We also show good preservation of the ultrastructural integrity of the liver tissue after a 5 h perfusion. No endothelial damage is observed. The vacuolar change of the haptocytes which is t h o u g h t to be induced by h y p o x i a seems to be less prominent than that reported by Schmucker and Curtis after perfusion for 1 h and equivalent to that of Krone et al. [34] after prolonged perfusion with erythrocyte-free medium. The rate of protein synthesis in our system of 17 mg albumin/h per 100 g liver agrees well with that reported by Matern et al. [20] of 18 mg/h per 100 g liver. Both values are approx. 50% of those reported by John and Miller [37] and Hoffenberg et al. [5] who used red cells in the perfusate. The fact that albumin, transferrin, hemopexin and haptoglobin are produced at a constant rate is a good indicator for the maintenance of viability of the liver cells during perfusion. Haptoglobin synthesis is 70% of tl~at reported by John and Miller [37] in fed rats. Hemopexin synthesis has not been studied before. The lower protein synthesis in the isolated organ compared to in vivo studies may be caused by the lack of humoral factors (e.g. hormones). John and Miller [37] showed that the addition of hormones such as insulin, growth hormones and cortisol significantly enhance the synthesis rate of many proteins in their perfusion system. We also found hormones to effectively enhance albumin and fibrinogen synthesis by isolated rat liver cells; but hemopexin production was independent of hormonal supplementation [38]. The c o n t e n t of ATP, ADP and AMP of the perfused liver from fasted and non-fasted rats is somewhat reduced in comparison to that of the non-perfused liver. Nevertheless, the ATP : ADP ratio as well as the energy charge are not lowered, indicating an adequately functioning liver. A reduced ATP generation per se is, therefore, probably n o t the cause of a decreased rate of protein synthesis, as has been suggested by Matern et al. [20]. Our values for pyruvate content of the liver 5 h after perfusion are comparable to those of Mannaerts et al. [21] who perfused for 30--90 min with red cells in Krebs-Henseleit bicarbonate buffer containing albumin (2.6%). They are also comparable to the values of Schimassek [9] who used erythrocytes in Tyrode's solution with 2.6% albumin for a 6 h perfusion. The pyruvate c o n t e n t of ours is, however, lower than that of Lueck and Miller [39] who perfused with diluted whole rat blood for a similar time period. Finding a lower a m o u n t of lactate in the perfused
62 liver of starved as compared to non-starved rats was not unexpected because depletion of glycogen stores decreases the rate of glycolysis [40]. The higher lactate content in vivo encountered by us as compared with that by others [9,28] is possibly due to lowering of tissue oxygenation during freeze-clamping the liver under ether narcosis [9]. Our values are not corrected for the blood content of the liver which does n o t cause a significant increase in the a m o u n t of lactate [28]. The lactate : pyruvate is a better indicator of the redox state than determination of either metabolite alone. A low lactate : pyruvate ratio indicates a good redox state of the cytosol [35,36], but as pointed out by Liibbers [41], a low ratio does n o t exclude heterogeneous oxygen supply to the liver tissue. In agreement with the results of others [13,42], we found a reduced bile flow during the last 3 h of perfusion. The progressive decline in bile flow is concomitantly decreased with the biliary excretion of bile salts and can be prevented by infusing taurocholate [42]. Within 3 h after the addition of heme to the perfusate, 20% is recovered as bile bilirubin. This percent of conversion of heme to bilirubin is similar to that observed by Snyder and Schmid [43] for studies in vivo, employing radioactive heme in bile-cannulated rats. In their studies, the maximal secretion of bile bilirubin occurred between 2 and 8 h after intravenous heme injection, and was completed at approx. 30 h; the total bilirubin recovery averaged 60% of the injected heme [43]. In our perfusion system, using unlabeled heme, 65% of the heme added to the perfusate is cleared from the circulation after 3 h, 20% of which has been converted to bile bilirubin. After 5 h, 80% of the heme is cleared and 45% has been converted to bile bilirubin. Extrapolating from the findings expected had all heme been cleared from the perfusate, we estimate a 60--70% recovery as bilirubin which is a value comparable with the in vivo studies [43]. Many carcinogenic and porphyrinogenic drugs as well as heme have been shown to stimulate hemopexin synthesis in vivo [44,45]. Pretreatment of the rats with AIA or DDC does not appear to enhance hemopexin synthesis but that of transferrin and albumin is decreased to a significant degree after addition of heme to the perfusate. These data support the observation of Sell et al. [46] for a fetal hepatocyte monolayer culture which indicates that the control of hemopexin synthesis differs from that o f albumin and haptoglobin. Pretreatm e n t of the donor rats with Desferal, an iron-chelating agent, stimulates the rate of hemopexin synthesis several-fold while that of albumin and transferrin remains normal or is slightly reduced. The stimulation of hemopexin synthesis is in part inhibited by the addition of heine into the perfusate. These findings suggest a close interrelationship between hemopexin synthesis and iron metabolism which has n o t been previously noted. The mechanism by which the synthesis of hemopexin is enhanced is unknown. The most likely signal for the initiation of hemopexin synthesis is an acute shift of iron or heme within critical tissue pools. Knowledge on the size and number of iron and heme pools of the liver is at present limited. The effect of AIA, DDC and Desferal on the formation of bilirubin is even more interesting. Whereas AIA has no effect on bilirubin formation, DDC and Desferal increase bilirubin production from endogenous sources. Both AIA and DDC are known to induce aminolevulinic acid synthetase (ALAS), the rate-
63 limiting enzyme in the heme biosynthetic pathway [47]. Both drugs also cause a loss of c y t o c h r o m e P-450 and heme [48]. In addition, DDC inhibits heme synthetase, the enzyme catalyzing the insertion of iron into the protoporphyrin IX ring to form heme. As a consequence, protoporphyrin IX accumulates in the liver cells [49]. For reasons stated above, it is unlikely that the bilirubin produced b y the liver is formed from endogenous heme since the heme pool in the liver is reduced by treatment of the rats with drugs like AIA and DDC. Little is known on the effect of Desferal on liver cells. This drug supposedly chelates iron b o u n d to ferritin [50] and the iron-desferrioxamine complex is excreted mainly via the bile and urine [51]. Desferal may, indeed, render certain liver pools temporarily deficient in iron which prevents heme formation. This in turn would increase the level of protoporphyrin IX. Furthermore, it is conceivable that n o t all bilirubin from endogenous sources, the so-called early bilirubin, necessarily originates from heme; it could in part be derived from protoporphyrin IX through an as yet u n k n o w n pathway. The source of this bilirubin is the subject of current research. When the rats are pretreated with AIA or DDC, their isolated livers convert less added heme into bilirubin than do the controls, which substantiate our earlier findings [52]. On the other hand, pretreatment of the rats with Desferal increases the efficiency of the liver to convert added heme into bilirubin from 45 to 70% in 5 h. At present, an interpretation of these findings cannot be given. Acknowledgements This work was supported by research grants awarded to Dr. U. Muller-Eberhard from the Institute of Child Health and Human Development (HD-04445) and from the Institute of Arthritis and Metabolic Diseases (AM-16737 and AM-18329). Dr. K. Miyai's support was awarded from U.S.P.H.S. research grant (5-RO1-AM-161110) and U.S.P.H.S. contract (SP-17-HL-14169 and HL123-73). We wish to thank Dr. Joseph Katz and Mr. George Bonorris from the Cedars-Sinai Medical Research Institute, Los Angeles for their help in setting up the isolated rat liver perfusion, Susan Wormsley for her expert technical assistance, and Dr. William Morgan, Department of Biochemistry, Scripps Clinic and Research Foundation and Dr. Steven Schenker, Vanderbilt University, Nashville, Tenn., for critical review of this manuscript. We also appreciate the generosity of Dr. A n t h o n y S. Tavill, Medical Research Centre, Harrow, Middlesex, U.K. and Dr. R. Engler, U E R Biomedicale des Saints P~res, Paris, France, in supplying rat transferrin and haptogobin. References 1 2 3 4 5 6 7 8
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