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Effect of peritonitis on mitochondrial respiration
JOURNAL
OF
SURGICAL
EFFECT G. A.
RI’:SIhiRCH,
11,
528-532
(1971)
OF PERITONITIS
G. DECKER,
M.B.,
S. BLEVIKGS,
CH.B B.A.,
ON MITOCHONDRIAL (Cape Ah’D
7’OWT& L.
TN RECENT REPORTS OF BACTEREMIC shock in man by Siegel [II] and MacLean [5] and their associates, groups of patients were described in whom the cardiac output increased to a greater extent than did the oxygen consumption. This relative decrease in oxygen consumption, which is reflected by a narrowed arteriovenous oxygen difference, could be explained on the basis of arteriovenous shunting, increased cutaneous blood flow for heat dissipation, moderate anemia, a shift “to the left” of the oxyhemoglobin dissociation curve, caused by decreased erythrocytc levels of 2,3 diphosphoglycerate, or on the basis of a cellular defect. The entire subject of decreased oxygen consumption in sepsis has recently been reviewed by Gump et al. [4]. This study was undertaken in an attempt to show a cellular defect in the livers of rats with K. pneumoniae peritonitis. As a first step, the metabolic integrity of the mitochondria was tested. If, in sepsis, mitochondria lose their ability to oxidize substrates, such a defect could explain diminished oxygen extraction from the tissues, which in turn could cause the release of metabolites capable of opening up arteriovenous shunts. Mitochondrinl function involves not only the oxidation of substrates but also the compulsory coupling of this oxidation to phosphorFrom the Department of Surgery, Royal Victoria Hospital and McGill University, Montreal, Quebec, Canada. Supported by a grant from the Medical Research Council of Canada. The authors are grateful to Dr. H. Robson for his helpful advice and the donation of the K. pn.eumoniae culture. Submitted for publication June 14, 1971.
D.
F.R.C.S. MACLEAN,
RESPIRATION
(hg),
A.
M.D.,
F.R.C.S.
M.
DANIEL,
PH.
I).,
(C)
ylation. Intact,, tightly coupled mitochondria show a very slow rate of substrate oxidation in the absence of ADP (State 4 respiration) which increases manyfold when ADP is addrcl to the system (State 3 respiration). The ratio of the two rates, the respiratory control index, is a sensitive parameter of the couphng of oxidative phosphorylation in mitochondria. The efficiency of this process is expressed as the ADP/O ratio, the number of moles of ADP phosphorylated per atom oxygen used. The closer the ADP/O ratio is to the theoretical value (2, 3, or 4, depending on the substrate) the better the efficiency of phosphorylation. Most reported studies have dealt with the effects of endotoxins on rat liver mitochondrial respiration. This study, in which rats with K. pneu?noniae peritonitis are used as an experimental model, more closely resembles sepsis in man. METHOD Nine normal male rats (Royal Vict,oria HospitaI hooded strain) of average weight 308 t 42 grams were studied. A similar group of 10 rats (315 f 35 grams) was studied after severe generalized peritonitis with Klebsiella pnewmoniae was induced by the method described by C. J. Wright [12]. This technique requires the intraperitoneal injection of live organisms in a suspension of mucus in order to inhibit the rats vigorous macrophage activity. KLebsiella pneumoniae from an agar slope was inoculated into a glucose broth. After 6-hour culture in the broth the bacterial count was approximately 18 X 10s per milliter. The broth culture was diluted lo-fold with 4% mucus in saline and 0.5-0.8 ml. of the mixture
was injected into the peritoneal cavity to produce bacterial peritonitis. The control animals received the same volume of sterile glucose broth and mucus injected intraperitoneally. The animals were anesthetized with ether 18 hours after injection. The septic animals inclutlcd in the stucly wc~ Ic>tlJnrgic,showed pilocrcction, and had severe generalized prritoJJitis which was confirmccl on bacterial culture. .I third group consisting of four septic animals wal:: allo~d to clicx spontnncousl~ aird thrir livers were removed immediately after t11ecessation of respiration and heartbeat. The intraaortic blood prcssurc~was taken in both t.lJe sq)tic$ and cxontrol groups by means of a size18 caunula inserted below the lcvcl of the renal :rrtcrie$. Arterial blood l)rcssurc was rccordctl 1)~ :I Sanborn t,ransduccr and recording appar:~tiis. This system ~a:: calibrat~cd iinmediatc~l~ I~forc cacl\ experiment, using a mercury manometer. The livers were totally excised and immersed in ice-cold 0.25 M sucrose. Approximatcly 600 mg. tissue from each liver was ho~nogcnized in 2 ml. of 0.6 .V perclhloric acid for c&mat&n of adenosine tri-, di-, and mono1)1Joq)lJat(~s (XTP, ;ZDP, and AMP) by enzymatic methods using Bochringcr XanJJhcim (‘OII~~MII~ wagcnt kits. PREPARATION OF MITOCHONDRIA The remaining liver tissue was w-aalied, weighed, coarsely cut up, and homogenized in a chilled isolation medium (O-4”) containing 0.25 M sucrose with 0.2 m:V tliamino-cthanctctra-acetic acid (EDTA) with 0.3 M Triq I;ct to pH 7.4. The crude homogenate (tissue concentration 30% w/v) was spun at 500g foi 10 minutes. The supernatant fluid was retained, and the sedimentswas resuspended and respun at the same speed. The supernatant fraction obtained after each centrifugation was pooled and spun at 10,OOOgto separate the mitochonclria. The resulting mitorhondrial pellet was washed by resuspension in 10 ml. of the meclium and resedimented. For study, the final mitochondrial pellet was resuspended in the isolation medium to give a protein concentration of approximately 10 mg. per milliliter as
cletcrminetl by tht~ method of Lowry and his :lssocinres[S]. RESPIRATORY
MEASUREMENTS
()xygcn uptake way measurctl polarogralJhically with a (‘lark-type electrotlc iJJ a Ycllow Springs Tiistrumc~nt Co. Inc. Biological Oxygen Monitor. The assay medium contained a solution of 0.25 31 sucrose,0.01 -II 1Jotnssiurn clilori(lc, 7.5 niLl$ potassium phosphntc~.4, 1n3l Alg(‘L! 0.2 rn,V EDTA, and 0.01 JI Tris l)uf?c~r ipH 7.4). To this assay medium 0.2 1111. of the inito~l~ondrinl suspensionwas :ttltl~~l ~~lrcnsuecinatc ~vas to bc need a:: substrntc an(l 0.3 ml iJJ the C:W of other substrates. Sul)
530
JOURNAL
OF
Table
SURGICAL
1. Bffect
of Sepsis
ATP
__~_____.
___~~
2. Effect
VOL.
on rldenine
(pmoles, ‘gram liver tissue)
11,
Sucleotide
ADI’
NO.
Levels
(rmoles,‘gram liver tissue)
1.81 0.99 p < 0.4x p <
of Sepsis
and
+ 0.18 f 0.25 0.001 + 0.03 0.00%
Septic,
Death
on Oxidative
KC1
Septic At death
mean SF) mean SI) mean Sl)
131.7 f17.5 140.5 f35.9 171.2” f29.6 .~
Glut~arnate Control Septic At deat,h
meati SD meat) 541) mean SD
Septic At death
mean ,sn mean SD mean SD
AMP
1971
Tissue Total adenine nucleotides (fimoles J gram liver tissue) ’
(rmoleslgram liver tissue)
~~~~ 51.7 f13.7 53.9 f12.9 68.3 f20.0
41.6 xk8.5 46.8 f14.4 47.1 f9.6
1.00 1.55 p < 1.72
p
0.20 0.23 0.001 f 0.13 < 0.002
Phosphorylation
f f
of Rat
4.48 f 0.57 4.03 zk 0.3li p < 0.1 2.92 f 0.14 p < o.ooz2
Liver
Mitochondria
m/*atoms 02 uptake/ min./mg. protein ~. .With \Vithout ADP ADP with BSA with BSA .-,.
;\DI’/O
43.6 fS.70 47.1 f5.5 55.6 flO.O ~
3.16 *0.X7 2.97 ho.56 3.08 f0.38
1.62 SO.28 1.54 f0.13 1.33 *0.0x
146.2 41.1 k23.1 f12.2 170.6 47.9 f4X.8 l .1 179.6* 43.7 dz13.2 ck9.9 ~~. __----
3.72 drO.76 3.84 zto.35 4.21 f0.82
1.76 *to.21 1.72 dzo.23 1.50* Ito.
21.7 zt6.X 19.6 f3.7 23.6 f5.7
2.40 fO.Ti 2.78 +0.57 2.91 XIzO.61
2.26 zto.33 2.47 50.25 2.04 zfzO.16
52.4 AT.0 64 Ii h17.5 74.0** zk24.6 __-~-
19.3 f7.4 22.0 ~7.2 19.9 f4.7
2.79 fO.69 3.25 xko.43 3.69* f0.48 _~---
2.3i dzo.37 2.47 ~0.25 2.28 f0.16
2.45 +0.x4 2.30 ZkO.42 2.04 zko.25
2.20 f0.3!) 2.36 f0.48 1.93 Lto.20
42.x +13.3 42.2 f12.9 46.4 f9.6
1X.6 rk6.4 17.2 f4.9 19.9 Yk4.9
2.37 Ito. 2.58 f0.71 2.31 f0.14
of freedom
= 11,
--~ Pyruvate Control
NOVEMBER
in Rat Liver
1.67 + 0.31 1.50 f 0.24 p < 0.1 0.72 zt 0.07 p < 0.002
mratoms 02 uptake; min./mg. protein
Succinate Control
11,
~~~~~-~~ ~~-~
mean, SD Control Septic mean, SD Unpaired data df = 17 At death meair, SD elf = 11
Table
RESEARCH,
18.6 f7.1 20.5 f6.1 23.3 f5.6
2.36 f0.46 2.55 f0.46 2.08 3~0.16 ~~~- __
_
* (p >
0.01 5 0.05) ** Significant,ly different
from
control
group
(unpaired
tent were significantly lower in this group than in the livers of normal rats. Respiratory measurements. Table 2 shows a comparison of the parameters of liver mitochondrial respiration in the three groups. There were no significant differences found between the normal and the septic group. In the group examined at death the ADP/O ratio with succinate in the presence of BSA was significantly lower than in the control group, while the RCI with glutamate in the presence of BSA was higher. The rate of oxygen uptake per milligram protein content of the mitochon-
data,
degrees
p<
0.01)
drial preparation tended to be higher in the septic group. At death this parameter was consistently and in some casessignificantly higher than normal. This phenomenon could be due t,o an alteration in the protein content of the mitochondrial preparation at death or might represent an actual increase in oxidizing capacity of the mitochondria. DISCUSSION In this study, the rats included in the septic group had diffuse generalized peritonitis 18
hours after irljection of K. pneumoniue but did not, show signs of shock as judged by aortic blood pressure. Seventy percent of the rats which were given injections but not killed after 18 hours died q~ontaneously within 24 hours. indicating that) the animals in the spetic g-Oll]~ n-crcl subjcctccl to a potentially lethal form of peritonitis. ;\llalysis of tlic li\-cr tissue adeninc nuclcotitles (Table 1 I shops a significant reduct,ion in the level of ATY in the septic groul) of rats. Ttrc reduction in t,iswle ATP implies a depletion of energy reserroirs although there was no reduction in the t)otnl adenine nucleotides as OI~C would cxxpcct in an ischrmic l&r [2]. The group examined at, death had a significantly lower than normal liver adenine nucleotide content showing a frlrther deterioration in the c’nrrgy state of the tissue. This finding could IX due either to tllc more advanced stage of sepsis or to relative ischemia or anoxia during the agonal period. Despite the reduction of liver cell adenine nucleotitlrs, disturbance of mitochondrial rcsljiration was not) tlctccted in the septic animals CVVII at the time of clinical death. The mitochondria were Ilot only able to take up oxygen normally for oxidation of t)he various substrates-representative of substrate groups imprtarlt ill )JiOlOgid (JxidatiOnS-hilt their efficiency in trwnsforrnillg the energy of oxidat,ion into a mctabolicnlly useful form, namely, into high-energy l)hosphate bond, was unimIjairecl as judged by tllc normal ADP/O ratios. The respiratory control indices, a rnorc sensiti\Fe index of tight mitochondrial coupling than the hDP/O ratios were also within normal range in the livers of the sept,ica rats. The addition of bovine serum albumin to the test media did not alter the measurements considerably, indicating the absence of uncouplers bound by albumin. -1lbumin causes great improvement in ADP/O and RCI TTalues if accumulation of free fatty acids or t,he presence of bacterial endotoxins cause partial mitochondrial uncoupling. Interference of mitochondrial respiration in which the same substrates as those in the above experiment were used, has been reported after cndotoxemia in rats and the in vitro challenge of mit,ochondria with endotoxins 17-91.
Mela et nl. [7] noted signs of uncoupling in the liver mitochondria of endotoxemic rat,s. They found similar effects when endotoxin was added to rat liver mitochondria in vitro [8]. hloss et al. [9] rcportcd that liver mitochondria t,reatcd with endot’oxin in z~ifro showed n reduction of oxygen uptake with pyruvate ( + malate) as substrate but not with succinate and glutamate. These workers eml)hasized that endotoxins do not behave like clasric uncouplers because there is inhibition of both State 4 and State 3 reqiration. Schumcr ct nl. [lo] reported a reduction of RCI and hDP/O wit’h the in vitro challenge of humau liver mitochondria in which alpha-ketoglutarate i* used as substrate. Similar changes were found in their endotoxemic rats. The same authors found that the addition of BS.1 had a restorat’ive effect on mitochondrin damaged by endotoxins. Our findings suggest) that, wliatevcr the effect of sepsis may bc in viz,n. the liver mitochondria are able to respire effectively when studied ilt, ?litro. If any rhanges were present, they were reversed by the artificial rnilicu in which the mitochondria were studied. SUhlMhRT Rats were exposed to a lethal form of l
Chance, B. Quantitative aspects of utilization. Regul. Cell OXJ-gCII Por~nrl.
2.
3.
Symp.
the control of Metab. Ciba
91, 1959.
Deuticke. B.. Gerlach. E., and von Dierkesmann, R. Abbau frei Nucleotide in Herz, Skeletmuskel. Gchirn und Leber der Ratte bci Sauerstof’fmangel. Pflnegers Arch. Gesamte Pkysiol. 292:239, 1966. Estnbrook. It. W. Mitochondrial respiratory- control md the polarographic measurement of 4DP:O ratios, Metkods Enzymol. 10:41. 1967.
532
JOURNAL
OF
SURGICAL
RESEARCH,
4. Gump, F. E., Kinney, J. M., and Price, J. B., Jr. Energy metabolism in surgical patients: oxygen consumption and blood flow. 1. Surq. Res. 10:613, 1970. 5. MacLean, L. D., Mulligan, W. G., McLean. A. P. H.: and Duff, J. H. Patterns of septic shock in man-A detailed study of 56 patients. Ann. Surg. x6:543, 1967. 6. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:.%5, 1951. 7. Mela, L.. Bacalzo, L. V.> Jr., White, R. R., and Miller, L. D. Shock induced alterations of mitochondrinl energy-linked functions. Surg. Fmwva 21:6, 1970.
VOL.
11,
NO.
11,
NOVEMBER
1971
8. Mela, L., Miller, L. D., Disco, J. F., and Sugerman, H. J. Effect of E. co/i cndotoxin on mitochondrial energy-linked functions. Sttr!lery 65:541, 1970. 9. Moss, G. S., Erve, P. P., and Schumcr, W. Effect of cmdotoxin on mitochondrial respiration. Surg. Forrtm 20:24, 1969. 10. Schumer, W., Das Gupta, T. K.. Moss, G. S., and Nyhus, L. M. Effect of endotoxcmia on liver ccl1 mitochondria in man. Ann. Surg. 171:875,1970. 11. Siegel, J. H.. Greenspan, M., and Del Guercio, L. R. M. Abnormal vascular tone, defective oxygen transport and myocardial failure in human septic shock. Ann. Szug. 165:504, 1967. 12. Wright,. C. J. Personal communications. 1970.
OF
SURGICAL
EFFECT G. A.
RI’:SIhiRCH,
11,
528-532
(1971)
OF PERITONITIS
G. DECKER,
M.B.,
S. BLEVIKGS,
CH.B B.A.,
ON MITOCHONDRIAL (Cape Ah’D
7’OWT& L.
TN RECENT REPORTS OF BACTEREMIC shock in man by Siegel [II] and MacLean [5] and their associates, groups of patients were described in whom the cardiac output increased to a greater extent than did the oxygen consumption. This relative decrease in oxygen consumption, which is reflected by a narrowed arteriovenous oxygen difference, could be explained on the basis of arteriovenous shunting, increased cutaneous blood flow for heat dissipation, moderate anemia, a shift “to the left” of the oxyhemoglobin dissociation curve, caused by decreased erythrocytc levels of 2,3 diphosphoglycerate, or on the basis of a cellular defect. The entire subject of decreased oxygen consumption in sepsis has recently been reviewed by Gump et al. [4]. This study was undertaken in an attempt to show a cellular defect in the livers of rats with K. pneumoniae peritonitis. As a first step, the metabolic integrity of the mitochondria was tested. If, in sepsis, mitochondria lose their ability to oxidize substrates, such a defect could explain diminished oxygen extraction from the tissues, which in turn could cause the release of metabolites capable of opening up arteriovenous shunts. Mitochondrinl function involves not only the oxidation of substrates but also the compulsory coupling of this oxidation to phosphorFrom the Department of Surgery, Royal Victoria Hospital and McGill University, Montreal, Quebec, Canada. Supported by a grant from the Medical Research Council of Canada. The authors are grateful to Dr. H. Robson for his helpful advice and the donation of the K. pn.eumoniae culture. Submitted for publication June 14, 1971.
D.
F.R.C.S. MACLEAN,
RESPIRATION
(hg),
A.
M.D.,
F.R.C.S.
M.
DANIEL,
PH.
I).,
(C)
ylation. Intact,, tightly coupled mitochondria show a very slow rate of substrate oxidation in the absence of ADP (State 4 respiration) which increases manyfold when ADP is addrcl to the system (State 3 respiration). The ratio of the two rates, the respiratory control index, is a sensitive parameter of the couphng of oxidative phosphorylation in mitochondria. The efficiency of this process is expressed as the ADP/O ratio, the number of moles of ADP phosphorylated per atom oxygen used. The closer the ADP/O ratio is to the theoretical value (2, 3, or 4, depending on the substrate) the better the efficiency of phosphorylation. Most reported studies have dealt with the effects of endotoxins on rat liver mitochondrial respiration. This study, in which rats with K. pneu?noniae peritonitis are used as an experimental model, more closely resembles sepsis in man. METHOD Nine normal male rats (Royal Vict,oria HospitaI hooded strain) of average weight 308 t 42 grams were studied. A similar group of 10 rats (315 f 35 grams) was studied after severe generalized peritonitis with Klebsiella pnewmoniae was induced by the method described by C. J. Wright [12]. This technique requires the intraperitoneal injection of live organisms in a suspension of mucus in order to inhibit the rats vigorous macrophage activity. KLebsiella pneumoniae from an agar slope was inoculated into a glucose broth. After 6-hour culture in the broth the bacterial count was approximately 18 X 10s per milliter. The broth culture was diluted lo-fold with 4% mucus in saline and 0.5-0.8 ml. of the mixture
was injected into the peritoneal cavity to produce bacterial peritonitis. The control animals received the same volume of sterile glucose broth and mucus injected intraperitoneally. The animals were anesthetized with ether 18 hours after injection. The septic animals inclutlcd in the stucly wc~ Ic>tlJnrgic,showed pilocrcction, and had severe generalized prritoJJitis which was confirmccl on bacterial culture. .I third group consisting of four septic animals wal:: allo~d to clicx spontnncousl~ aird thrir livers were removed immediately after t11ecessation of respiration and heartbeat. The intraaortic blood prcssurc~was taken in both t.lJe sq)tic$ and cxontrol groups by means of a size18 caunula inserted below the lcvcl of the renal :rrtcrie$. Arterial blood l)rcssurc was rccordctl 1)~ :I Sanborn t,ransduccr and recording appar:~tiis. This system ~a:: calibrat~cd iinmediatc~l~ I~forc cacl\ experiment, using a mercury manometer. The livers were totally excised and immersed in ice-cold 0.25 M sucrose. Approximatcly 600 mg. tissue from each liver was ho~nogcnized in 2 ml. of 0.6 .V perclhloric acid for c&mat&n of adenosine tri-, di-, and mono1)1Joq)lJat(~s (XTP, ;ZDP, and AMP) by enzymatic methods using Bochringcr XanJJhcim (‘OII~~MII~ wagcnt kits. PREPARATION OF MITOCHONDRIA The remaining liver tissue was w-aalied, weighed, coarsely cut up, and homogenized in a chilled isolation medium (O-4”) containing 0.25 M sucrose with 0.2 m:V tliamino-cthanctctra-acetic acid (EDTA) with 0.3 M Triq I;ct to pH 7.4. The crude homogenate (tissue concentration 30% w/v) was spun at 500g foi 10 minutes. The supernatant fluid was retained, and the sedimentswas resuspended and respun at the same speed. The supernatant fraction obtained after each centrifugation was pooled and spun at 10,OOOgto separate the mitochonclria. The resulting mitorhondrial pellet was washed by resuspension in 10 ml. of the meclium and resedimented. For study, the final mitochondrial pellet was resuspended in the isolation medium to give a protein concentration of approximately 10 mg. per milliliter as
cletcrminetl by tht~ method of Lowry and his :lssocinres[S]. RESPIRATORY
MEASUREMENTS
()xygcn uptake way measurctl polarogralJhically with a (‘lark-type electrotlc iJJ a Ycllow Springs Tiistrumc~nt Co. Inc. Biological Oxygen Monitor. The assay medium contained a solution of 0.25 31 sucrose,0.01 -II 1Jotnssiurn clilori(lc, 7.5 niLl$ potassium phosphntc~.4, 1n3l Alg(‘L! 0.2 rn,V EDTA, and 0.01 JI Tris l)uf?c~r ipH 7.4). To this assay medium 0.2 1111. of the inito~l~ondrinl suspensionwas :ttltl~~l ~~lrcnsuecinatc ~vas to bc need a:: substrntc an(l 0.3 ml iJJ the C:W of other substrates. Sul)
530
JOURNAL
OF
Table
SURGICAL
1. Bffect
of Sepsis
ATP
__~_____.
___~~
2. Effect
VOL.
on rldenine
(pmoles, ‘gram liver tissue)
11,
Sucleotide
ADI’
NO.
Levels
(rmoles,‘gram liver tissue)
1.81 0.99 p < 0.4x p <
of Sepsis
and
+ 0.18 f 0.25 0.001 + 0.03 0.00%
Septic,
Death
on Oxidative
KC1
Septic At death
mean SF) mean SI) mean Sl)
131.7 f17.5 140.5 f35.9 171.2” f29.6 .~
Glut~arnate Control Septic At deat,h
meati SD meat) 541) mean SD
Septic At death
mean ,sn mean SD mean SD
AMP
1971
Tissue Total adenine nucleotides (fimoles J gram liver tissue) ’
(rmoleslgram liver tissue)
~~~~ 51.7 f13.7 53.9 f12.9 68.3 f20.0
41.6 xk8.5 46.8 f14.4 47.1 f9.6
1.00 1.55 p < 1.72
p
0.20 0.23 0.001 f 0.13 < 0.002
Phosphorylation
f f
of Rat
4.48 f 0.57 4.03 zk 0.3li p < 0.1 2.92 f 0.14 p < o.ooz2
Liver
Mitochondria
m/*atoms 02 uptake/ min./mg. protein ~. .With \Vithout ADP ADP with BSA with BSA .-,.
;\DI’/O
43.6 fS.70 47.1 f5.5 55.6 flO.O ~
3.16 *0.X7 2.97 ho.56 3.08 f0.38
1.62 SO.28 1.54 f0.13 1.33 *0.0x
146.2 41.1 k23.1 f12.2 170.6 47.9 f4X.8 l .1 179.6* 43.7 dz13.2 ck9.9 ~~. __----
3.72 drO.76 3.84 zto.35 4.21 f0.82
1.76 *to.21 1.72 dzo.23 1.50* Ito.
21.7 zt6.X 19.6 f3.7 23.6 f5.7
2.40 fO.Ti 2.78 +0.57 2.91 XIzO.61
2.26 zto.33 2.47 50.25 2.04 zfzO.16
52.4 AT.0 64 Ii h17.5 74.0** zk24.6 __-~-
19.3 f7.4 22.0 ~7.2 19.9 f4.7
2.79 fO.69 3.25 xko.43 3.69* f0.48 _~---
2.3i dzo.37 2.47 ~0.25 2.28 f0.16
2.45 +0.x4 2.30 ZkO.42 2.04 zko.25
2.20 f0.3!) 2.36 f0.48 1.93 Lto.20
42.x +13.3 42.2 f12.9 46.4 f9.6
1X.6 rk6.4 17.2 f4.9 19.9 Yk4.9
2.37 Ito. 2.58 f0.71 2.31 f0.14
of freedom
= 11,
--~ Pyruvate Control
NOVEMBER
in Rat Liver
1.67 + 0.31 1.50 f 0.24 p < 0.1 0.72 zt 0.07 p < 0.002
mratoms 02 uptake; min./mg. protein
Succinate Control
11,
~~~~~-~~ ~~-~
mean, SD Control Septic mean, SD Unpaired data df = 17 At death meair, SD elf = 11
Table
RESEARCH,
18.6 f7.1 20.5 f6.1 23.3 f5.6
2.36 f0.46 2.55 f0.46 2.08 3~0.16 ~~~- __
_
* (p >
0.01 5 0.05) ** Significant,ly different
from
control
group
(unpaired
tent were significantly lower in this group than in the livers of normal rats. Respiratory measurements. Table 2 shows a comparison of the parameters of liver mitochondrial respiration in the three groups. There were no significant differences found between the normal and the septic group. In the group examined at death the ADP/O ratio with succinate in the presence of BSA was significantly lower than in the control group, while the RCI with glutamate in the presence of BSA was higher. The rate of oxygen uptake per milligram protein content of the mitochon-
data,
degrees
p<
0.01)
drial preparation tended to be higher in the septic group. At death this parameter was consistently and in some casessignificantly higher than normal. This phenomenon could be due t,o an alteration in the protein content of the mitochondrial preparation at death or might represent an actual increase in oxidizing capacity of the mitochondria. DISCUSSION In this study, the rats included in the septic group had diffuse generalized peritonitis 18
hours after irljection of K. pneumoniue but did not, show signs of shock as judged by aortic blood pressure. Seventy percent of the rats which were given injections but not killed after 18 hours died q~ontaneously within 24 hours. indicating that) the animals in the spetic g-Oll]~ n-crcl subjcctccl to a potentially lethal form of peritonitis. ;\llalysis of tlic li\-cr tissue adeninc nuclcotitles (Table 1 I shops a significant reduct,ion in the level of ATY in the septic groul) of rats. Ttrc reduction in t,iswle ATP implies a depletion of energy reserroirs although there was no reduction in the t)otnl adenine nucleotides as OI~C would cxxpcct in an ischrmic l&r [2]. The group examined at, death had a significantly lower than normal liver adenine nucleotide content showing a frlrther deterioration in the c’nrrgy state of the tissue. This finding could IX due either to tllc more advanced stage of sepsis or to relative ischemia or anoxia during the agonal period. Despite the reduction of liver cell adenine nucleotitlrs, disturbance of mitochondrial rcsljiration was not) tlctccted in the septic animals CVVII at the time of clinical death. The mitochondria were Ilot only able to take up oxygen normally for oxidation of t)he various substrates-representative of substrate groups imprtarlt ill )JiOlOgid (JxidatiOnS-hilt their efficiency in trwnsforrnillg the energy of oxidat,ion into a mctabolicnlly useful form, namely, into high-energy l)hosphate bond, was unimIjairecl as judged by tllc normal ADP/O ratios. The respiratory control indices, a rnorc sensiti\Fe index of tight mitochondrial coupling than the hDP/O ratios were also within normal range in the livers of the sept,ica rats. The addition of bovine serum albumin to the test media did not alter the measurements considerably, indicating the absence of uncouplers bound by albumin. -1lbumin causes great improvement in ADP/O and RCI TTalues if accumulation of free fatty acids or t,he presence of bacterial endotoxins cause partial mitochondrial uncoupling. Interference of mitochondrial respiration in which the same substrates as those in the above experiment were used, has been reported after cndotoxemia in rats and the in vitro challenge of mit,ochondria with endotoxins 17-91.
Mela et nl. [7] noted signs of uncoupling in the liver mitochondria of endotoxemic rat,s. They found similar effects when endotoxin was added to rat liver mitochondria in vitro [8]. hloss et al. [9] rcportcd that liver mitochondria t,reatcd with endot’oxin in z~ifro showed n reduction of oxygen uptake with pyruvate ( + malate) as substrate but not with succinate and glutamate. These workers eml)hasized that endotoxins do not behave like clasric uncouplers because there is inhibition of both State 4 and State 3 reqiration. Schumcr ct nl. [lo] reported a reduction of RCI and hDP/O wit’h the in vitro challenge of humau liver mitochondria in which alpha-ketoglutarate i* used as substrate. Similar changes were found in their endotoxemic rats. The same authors found that the addition of BS.1 had a restorat’ive effect on mitochondrin damaged by endotoxins. Our findings suggest) that, wliatevcr the effect of sepsis may bc in viz,n. the liver mitochondria are able to respire effectively when studied ilt, ?litro. If any rhanges were present, they were reversed by the artificial rnilicu in which the mitochondria were studied. SUhlMhRT Rats were exposed to a lethal form of l
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