Hepatic injury and lipid peroxidation during hemorrhagic shock and resuscitation

Life Sciences, Vol. 53, pp. 1685-16cg) Printed in the USA

HEPATIC

Pergamon Press

INJURY AND LIPID PEROXIDATION DURING HEMORRHAGIC AND RESUSCITATION

SHOCK

Richard C. Dart, l': Daniel C. Liebler, 2 and I. Glenn Sipes 2 ISection of Emergency Medicine, Department of Surgery, and ~ e p a r t m e n t Pharmacology/Toxicology, University of Arizona, Tucson, AZ 85724

of

(Received in final form September 20, 1993) Summary Resuscitation from hemorrhagic shock causes hepatic injury that is similar to the hypoxic injury caused by reperfusion after ischemia. This study was designed to describe the relationship between severe hemorrhagic shock, hepatic injury, and lipid peroxidation. Fasted Sprague-Dawley rats underwent shock (mean arterial pressure 40 + 5 mm Hg) for two hours followed by reinfusion of shed blood. Serum ALT levels increased during shock and gradually continued to increase for 24 hours after resuscitation. Lipid peroxidation was assessed by quantification of exhaled ethane and by liver content of thiobarbituric acid reactive substances (TBARS). Expired ethane was increased both during shock and after resuscitation. Hepatic content of TBARS remained at baseline levels during shock, but increased after resuscitation. The results suggest that severe, non-fatal hemorrhagic shock and resuscitation produces a modest hepatic injury that is accompanied by lipid peroxidation in the liver. Oxygen free radicals have been implicated in the pathogenesis of ischemiareperfusion injury in tissues from many species, including the liver of the rat and the human (1,2,3). Because the pathophysiology and histopathology of hemorrhagic shock-resuscitation (HS-R) are similar to ischemia-reperfusion (I-R), they may be considered variants of the same process. During hemorrhagic shock, blood flow to the liver decreases to 25-50% of normal and returns to normal or above normal flow after resuscitation (4). Both hemorrhagic shock and ischemia involve a reduction in oxygen delivery, the depletion of energy stores, an increase anaerobic metabolism, the loss of transmembrane ionic gradients, and eventually, injury to lipids, proteins and DNA (5,6,7). Mitochondrial swelling, degradation of cell membranes, and ultimately, cell death will occur if either I-R or HS-R are prolonged (8,9). Our hypothesis was that oxygen radicals are produced upon resuscitation from hemorrhagic shock and may cause hepatic injury as well as increased hepatic lipid peroxidation. In our model, hepatic injury began during shock and continued to develop after resuscitation, whereas hepatic lipid peroxidation began only after resuscitation.

Materials

and Methods

Animals

Male Sprague-Dawley rats (Harlan Sprague-Dawley, Houston, TX) weighing 260-380 grams were fed a standard diet (Teklad 4% mouse/rat, Madison, WI). In addition, six weanling rats were fed an established vitamin E deficient diet for six weeks (i0). Experiments were started between 8:00 and 9:00 a.m. to minimize diurnal glutathione variation (ii). Animals were fasted 18 hours prior to their use. Animals were anesthetized with a fentanyl-droperidol mixture (Innovar-vet, Pitman-

Richard Denver,

C. Dart, MD, CO 80204

PhD, Rocky Mountain

Poison Center,

645 Bannock St.,

0024-3205/93 $6.00 + .00 Cop~ight © 1993 Pergamon Press Ltd Allrightsrcse~cd.

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Lipid Peroxidation During Hemorrhagic Shock

Bowes) and b o d y t e m p e r a t u r e was m a i n t a i n e d w i t h a heat lamp. a p p r o v e d by the I n s t i t u t i o n a l A n i m a l C a r e and Use C o m m i t t e e .

Shock-resuscitation

Vol. 53, No. 22, 19)3

The protocols

were

protocol

B l o o d p r e s s u r e m o n i t o r i n g and v e n o u s a c c e s s w e r e e s t a b l i s h e d t h r o u g h the f e m o r a l vessels. At 15 m i n u t e s a f t e r v a s c u l a r a c c e s s was e s t a b l i s h e d , b l o o d w a s w i t h d r a w n into a h e p a r i n i z e d s y r i n g e u n t i l a m e a n a r t e r i a l p r e s s u r e (MAP) of 40 m m Hg was achieved. The M A P w a s m a i n t a i n e d at 40 + 5 m m Hg for two h o u r s by f u r t h e r w i t h d r a w a l or r e i n f u s i o n of b l o o d f r o m t h e syringe. At t h e end of the s h o c k period, all b l o o d w a s r e i n f u s e d o v e r 15 m i n u t e s and b l o o d lost d u r i n g s u r g e r y was r e p l a c e d w i t h two t i m e s its v o l u m e of 0.9% saline. S h a m o p e r a t e d and s h a m t r e a t e d c o n t r o l s w e r e p e r f o r m e d for e a c h group.

Analysis

of

expired

ethane

M e c h a n i c a l v e n t i l a t i o n w a s r e q u i r e d for c o l l e c t i o n of e x p i r e d breath. The a n i m a l was v e n t i l a t e d w i t h low h y d r o c a r b o n air (less t h a n 0.3 ppm) in a closed c o l l e c t i o n s y s t e m to e x c l u d e a m b i e n t air ethane. A v a l v e f o r c e d all air from the v e n t i l a t o r to the lungs and all e x h a l e d b r e a t h to the c o l l e c t i o n bag. After each c o l l e c t i o n , the e x h a l e d b r e a t h w a s d r a w n o v e r heat p u r i f i e d c a r b o n ( a c t i v a t e d SK4, 8 0 / 1 0 0 mesh, A p p l i e d S c i e n c e Labs, D e e r f i e l d , IL) on ice at 30 m l / m i n to a d s o r b hydrocarbons. The c h a r c o a l was t h e n t r a n s f e r r e d to a s e p t a t e d vial, h e a t e d to 2 4 0 ° C for five m i n u t e s to d e s o r b h y d r o c a r b o n s , and 5.0 ml of h e a d s p a c e gas was w i t h d r a w n , c o m p r e s s e d to 0.5 ml and i n j e c t e d into a gas c h r o m a t o g r a p h u s i n g flame i o n i z a t i o n d e t e c t i o n ( H e w l e t t - P a c k a r d , M o d e l 5910, P o r a p a k N 8 0 / 1 0 0 mesh, A l l t e c h A s s o c i a t e s , D e e r f i e l d , IL). A s t a n d a r d c u r v e was g e n e r a t e d d a i l y w i t h a s t a n d a r d h y d r o c a r b o n gas m i x t u r e (0.1% ethane, S c o t t y S p e c i a l t y Gases, P l u m s t e a d v i l l e , PA). Biochemical

Analyses

Liver samples (one/animal) for d e t e r m i n a t i o n of t h i o b a r b i t u r i c acid reactive s u b s t a n c e s (TBARS) w e r e t a k e n b e f o r e shock, at the e n d of the shock, at t h e end of the 0-15 m i n u t e p e r i o d a f t e r r e s u s c i t a t i o n , and at the e n d of the 15-30 m i n u t e period after resuscitation. The s a m p l e s w e r e o b t a i n e d by p e r f u s i n g t h e liver in r e t r o g r a d e w i t h I00 ml of an i c e - c o l d s o l u t i o n c o n t a i n i n g E D T A (2.0%), BHT ( b u t y l a t e d h y d r o x y t o l u e n e 0.1%) and NaCl (0.9%) at 50 m l / m i n u t e . A 300 m g b i o p s y was i m m e d i a t e l y t a k e n f r o m the m e d i a n lobe, h o m o g e n i z e d in 4.0 ml of the 3°C E D T A B H T - N a C I s o l u t i o n and t r i p l i c a t e a l i q u o t s w e r e a n a l y z e d w i t h i n one hour. A m o d i f i c a t i o n of the f l u o r o m e t r i c m e t h o d d e s c r i b e d by O h k a w a (12) was u s e d to d e t e r m i n e TBARS. BHT (0.01%) was a d d e d to the r e a c t i o n m i x t u r e to r e d u c e p e r o x i d a t i o n of the s a m p l e l i p i d s d u r i n g h e a t i n g (13,14). Tetraethoxypropane (Sigma C h e m i c a l , St. Louis) was u s e d to g e n e r a t e s t a n d a r d c u r v e s daily. Plasma a n i m a l s (n = and at 3, 6, a commercial Statistical

a l a n i n e a m i n o t r a n s f e r a s e (ALT) was d e t e r m i n e d in a s e p a r a t e g r o u p of 6 p e r group). S a m p l e s w e r e t a k e n b e f o r e shock, at the end of shock, and 24 h o u r s a f t e r r e s u s c i t a t i o n . ALT determinations were made with a s s a y kit (Sigma C h e m i c a l , St. Louis).

Analysis

All d a t a are e x p r e s s e d as m e a n + SD. A log(10) t r a n s f o r m a t i o n of the A L T v a l u e s w a s p e r f o r m e d from w h i c h the m e a n s + SD w e r e c a l c u l a t e d (15). Ethane exhalation, T B A R S results, and the t r a n s f o r m e d A L T v a l u e s of s h a m and H S - R g r o u p s w e r e c o m p a r e d by a n a l y s i s of v a r i a n c e for r e p e a t e d m e a s u r e s (16).

Results The s h o c k m o d e l we d e v e l o p e d w a s s i m i l a r to p r e v i o u s l y r e p o r t e d m o d e l s . The m e a n b l o o d v o l u m e w i t h d r a w n w a s 9.4 + 2.7 ml, (39.4% + 10.3% of c a l c u l a t e d total blood volume) (17). All a n i m a l s d e v e l o p e d o b v i o u s - - b r a d y c a r d i a and b r a d y p n e a d u r i n g t h e h y p o t e n s i v e period. M A P r e t u r n e d to n e a r n o r m a l v a l u e s in all a n i m a l s f o l l o w i n g r e s u s c i t a t i o n . T h e e f f e c t of the H S - R p r o t o c o l on p l a s m a A L T l e v e l s is shown in F i g u r e 1.

Vol. 53, No. 22, 1993

Lipid Peroxidation During Hemorrhagic Shock

1687

1000

T

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Response of plasma ALT activity to shock protocol. Both the HS-R group and the sham operated group developed elevated ALT levels at the 24 hour time point compared to their own sample taken before shock (ANOVA, p < 0.05 for each). Values indicated by (*) denote statistically significant values as compared to baseline (p < 0.05 repeated measures ANOVA, followed by Duncan's multiple range test). ALT activity increased by the end of the shock period and continued to rise over the course of 24 hours. ALT activity was higher in shock animals than in sham operated animals (p<0.05, repeated measures ANOVA). we next investigated whether the hemorrhagic shock-resuscitation protocol caused lipid peroxidation. Hepatic TBARS remained constant in the samples before shock and the samples during the shock period, but then increased 18.1% during the 0-15 minute and 22.2% during the 15-30 minute period after resuscitation. The hepatic TBARS of sham animals did not increase. The measured TBARS values were 22.1 ± 0.8 pmol/g liver at baseline; 21.1 ± 1.0 pmol/g during shock; 26.2 ± 1.2 pmol/g at 0-15 m i n resuscitation, and 27.0 ± 1.2 pmol/g at 15-30 min resuscitation. Hepatic TBARS content returned to baseline by 24 hours (data not shown). Increased tissue TBARS levels did not result from introduction of exogenously-formed TBARS during resuscitation as no TBARS accumulation was detected in stored blood (data not shown). Ethane exhalation increased in the regular diet group, both during the shock phase and after resuscitation (Figure 2). The opposite occurred in the sham operated animals; ethane exhalation decreased slightly over the course of the experiment. Vitamin E deficiency caused an increase in baseline ethane exhalation. Baseline ethane exhalation was 157 + 183 pmol/kg/hour in the vitamin E deficient animals compared to 27 + 23 pmol/kgThour in the regular diet group. Hepatic vitamin E deficiency was documented in the liver by HPLC analysis (data not shown) (18,19). Baseline ethane exhalation was higher in vitamin E deficient animals than in animals on a normal diet. Ethane exhalation increased

1688

Lipid Pcroxidation During Hemorrhagic Shock

Vol. 53, No. 22, 1993

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M e a n c u m u l a t i v e e x h a l e d e t h a n e of rats on a r e g u l a r diet. Animals u n d e r w e n t t h e H S - R p r o t o c o l w i t h serial c o l l e c t i o n s of e x p i r e d e t h a n e before, during, and after shock. The c o u r s e of e t h a n e e x h a l a t i o n in H S - R g r o u p was h i g h e r (*) t h a n the sham o p e r a t e d g r o u p (repeated m e a s u r e s ANOVA, f o l l o w e d by D u n c a n ' s m u l t i p l e r a n g e test, p < 0.05). T h e r e w e r e five a n i m a l s in each group.

d u r i n g s h o c k in the v i t a m i n E d e f i c i e n t g r o u p u n d e r g o i n g h e m o r r h a g i c shock, but not the d e f i c i e n t sham o p e r a t e d group (227 ± 130 p m o l / k g / h r and 13 ± 30 pmol/kg/hr, respectively). D u r i n g the r e s u s c i t a t i o n phase ethane exhalation a p p e a r e d to i n c r e a s e in the v i t a m i n E d e f i c i e n t shock group, but t h e d i f f e r e n c e b e t w e e n s h o c k and s h a m o p e r a t e d g r o u p s was not s t a t i s t i c a l l y s i g n i f i c a n t b e c a u s e of large v a r i a t i o n of e t h a n e e x h a l a t i o n within the shock group (782 ± 747 p m o l / k g / h r and 24 ± 45 p m o l / k g / h r ) . Discussion These experiments were done to i n v e s t i g a t e the relationship between hemorrhagic-shock-resuscitation and i s c h e m i a - r e p e r f u s i o n . The s h o c k m o d e l u s e d was very similar to that used by investigators over the past 50 years (1,20,22,23). Because oxygen radical production is a h a l l m a r k of i s c h e m i a reperfusion injury, radicals also m i g h t be e x p e c t e d to be p r e s e n t during resuscitation from hemorrhagic shock. If radicals are produced, lipid p e r o x i d a t i o n s h o u l d i n c r e a s e in a f f e c t e d tissues. P e r o x i d a t i o n of m e m b r a n e lipids by o x y g e n d e r i v e d free r a d i c a l s has b e e n i m p l i c a t e d in h e p a t i c I-R injury (24,25,26,27). T h e p e r o x i d a t i o n of w-3 p o l y u n s a t u r a t e d fatty acids c a u s e s the r e l e a s e of ethane, w h i c h can be d e t e c t e d in t h e e x p i r e d breath. This m e t h o d a l l o w s r e p e a t e d s a m p l i n g w i t h o u t i n j u r i n g t h e animal. However, the m e t h o d does not i n d i c a t e the t i s s u e s o u r c e of ethane. E t h a n e e x h a l a t i o n b e f o r e shock in our m o d e l was 27 p m o l / k g / h r in r e g u l a r diet a n i m a l s and w a s f o u r - f o l d h i g h e r in the v i t a m i n E d e p l e t e d rats. These r e s u l t s are a p p r o x i m a t e l y I0 - 50% of the b a s e l i n e e t h a n e e x h a l a t i o n found in p r e v i o u s r e p o r t s (11,28,29). The lower a p p a r e n t e t h a n e e x h a l a t i o n in our m o d e l m a y be d u e to our e x p r e s s i o n of net e t h a n e e x h a l a t i o n (i.e. e t h a n e e x h a l a t i o n b e f o r e shock was s u b t r a c t e d from s u b s e q u e n t d e t e r m i n a t i o n s ) and b e c a u s e we s a m p l e d

Vol. 53, No. 22, 1993

Lipid Peroxidation During Hemorrhagic Shock

only exhaled gases. Other investigators removing baseline exhalation.

have reported cumulative

1689

ethane without

Ethane exhalation increased during both shock and resuscitation (Figure 2). Ethane exhalation indicates total lipid peroxidation occurring within the body. The marked accentuation in ethane exhalation by vitamin E depletion suggests that oxygen radicals initiated this process. Hepatic TBARS content remained unchanged during the shock phase and then increased after resuscitation. The increase was modest compared to previous reports of lipid peroxidation induced by hepatotoxic chemicals (30,31). The increase in TBARS after resuscitation is consistent with the oxygen radical hypothesis, in which radicals produced during reoxygenation of tissue may cause lipid peroxidation. Production of TBARS only during resuscitation has been reported previously using an in vitro low flow-fellow variation of a perfused rat liver model (32). In that experiment, livers from fasted female Sprague-Dawley rats were perfused at 25% of normal flow rate. An increase in TBARS and LDH was noted in the effluent when flow rate was returned to normal. The increase in TBARS only during resuscitation, while ethane exhalation rose during both shock and resusciitation, could also be explained by the superior sensitivity of ethane exhalation in detecting lipid peroxidation. The lipid peroxidation in our model was associated with mild hepatocyte injury as evidenced by the serum ALT activity. In preliminary experiments, use of more severe shock or a longer duration of shock caused death. Therefore, the normal rat liver seems resistant to non-lethal hemorrhagic shock. This finding may have important implications. Under pathological conditions, the liver may be more susceptible to injury caused directly by the radicals themselves, or indirectly by processes induced by the oxygen radicals. For example, the liver may be more susceptible to endotoxin damage when antioxidant defenses of the cell are depleted (33). In addition, oxygen radicals may induce secondary inflammatory injury that amplifies the initial injury (34,35). Therefore, oxygen radical production may only become injurious in synergism with other processes that induce the production of oxygen radicals or consumption of antioxidant defenses. This relationship has been shown for ethanol and carbon tetrachloride (36). In conclusion, resuscitation from severe hemorrhagic caused modest hepatocyte injury as evaluated by ALT levels. Hepatic lipid peroxidation as measured by TBARS was increased only after resuscitation. On the other hand, total lipid peroxidation as measured by ethane exhalation increased throughout the shock and resuscitation periods, indicating that lipid peroxidation may occur throughout the shock and resuscitation periods. Acknowledqments This w o r k has been supported by research fellowship grants from the Emergency Medicine Foundation and the National Institutes of Health (ES05437-02).

References i. 2. 3. 4. 5. 6. 7. 8. 9. i0. II.

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Lipid Peroxidation During Hemorrhagic Shock

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