Relationship of Critical Uptake Volume to Energy Production and Endotoxemia in Late Hemorrhagic Shock Robert S. Rhodes, MD, Cleveland, Ohio Ralph G. DePalma, MD, Cleveland, Ohio Ann V. Robinson, BA, Cleveland, Ohio
Improvements in the treatment of hemorrhagic shock refractory to volume replacement depend on identification of the factors responsible for transition to the “irreversible” state. Criteria indicative of “irreversibility” are difficult to identify experimentally, but the need for retransfusion of shed blood to maintain blood pressure is considered the major criterion of transition to the irreversible state. This was first emphasized in canine hemorrhagic shock by Wiggers [I]. The concept of critical uptake volume has recently been extended to the hemorrhagic shock model in the rat [2]. The precise factors responsible for blood uptake and subsequent lethality are not known. Current research into shock has focused on alterations in cellular metabolism and their possible relationships to irreversibility. Deterioration of hepatic energy-linked mitochondrial function in prolonged shock has been well demonstrated, and the degree of deterioration is related to prolongation of hypotension. Hepatic mitochondrial energy-linked function usually remains normal during the initial 120 minutes of hypovolemia. Animals sacrificed after longer intervals of hypovolemia show an increasing uncoupling of oxidative phosphorylation [3-61. There are, however, significant variations from animal to animal in susceptibility to the consequences of prolonged hypovolemia. Resistance to hemorrhagic shock is related to maintenance of efficient hepatic energy metabolism and blood glucose levels [6].
From the Department of Surgery, Case Western Reserve School of Medii tine and University Hospitals of Cleveland, Cleveland, Ohio. Reprint requests should be addressed to Robert S. Rhodes, MD. University Hospitals of Cleveland, 2065 Adelberl Road, Cleveland, Ohio 44106.
560
This study was carried out to further characterize the relationship between the onset of irreversibility and the deterioration of hepatic mitochondrial energy production. It was demonstrated that although depression of hepatic mitochondrial function appeared generally related to duration of shock, aberrations in energy production by hepatic mitochondria were specifically related to the uptake of blood from the reservoir and to endotoxemia. The infusion of Ringer’s lactate solution rather than blood during the uptake stage may prevent certain aspects of mitochondrial dysfunction. Material and Methods Experimental Preparation. Young adult SpragueDawley rats, weighing 225 to 300 gm, were used. They were allowed access to rat chow and water up to the time of the experiment. The animals were anesthetized with intraperitoneal pentoharbital sodium, 4 mg/lOO gm. A PE 50 polyethylene cannula was inserted into the left femoral artery and 500 units of heparin were administered intra-arterially. The cannula was connected to a three-way stopcock with a sterile syringe for bleeding and a mercury manometer for measuring blood pressure. Body temperature was monitored using a telethermometer and maintained at 35.5” to 37.5V using an external heat source. In the present study, prolongation of experimental periods of shock for longer than two hours often required artificial maintenance of the airway. Otherwise, the details of the animal preparation were similar to those previously reported [ 71. Hemorrhagic shock was induced by withdrawing blood until a mean arterial blood pressure of 30 to 35 mm Hg was obtained. Blood pressure was maintained at this level by further withdrawal or infusion of blood as was necessary. Identically prepared nonbled control animals were studied simultaneously and tissues obtained from these animals processed synchronously. The exper-
The Amerksn Journal ol Surgery
Endotoxemia and Hemorrhagic Shock
iments were terminated utes.
Biochemical
Studies
at intervals of 30 to 240 min-
and Mitochondriul
Function.
The functional capability of the hepatic mitochondria was assessed after isolation of the mitochondria using methods previously described [8,9]. The ultrastructural characteristics of the isolated preparations were similar to those previously reported [4]. Oxygen uptake was measured by a Gilson oxygraph at 23°C (model KM with a Clark electrode). Hepatic mitochondria isolated by differential centrifugation from 4 gm of liver were suspended in 8 ml of 0.24 M sucrose with 0.5 mM ethylenediaminetetracetic acid (EDTA). A 0.3 ml aliquot of this suspension was added to 2 ml of an assay system containing 5 mM of phosphate buffer (pH 7.4), 120 mM of potassium chloride, 5 mM of magnesium-chloride and 5 mM of tris buffer. The substrates, cY-ketoglutaratb (5 mM) or succinate (5 mM), were studied by successive additions of adenosine diphosphate (ADP) (133 mM). Hepatic mitochondrial protein was measured by the biuret method [IO]. State 3 and 4 respiratory rates, respiratory control ratio, and ADPIO ratios were calculated. Endotoxin Assay. At the time of sacrifice 2 to 3 ml of blood were withdrawn from the heart into heparinked syringes using aseptic technic. The blood was immediately centrifuged and the plasma stored at 0 to 4’C for later assay for endotoxin using the Limulus assay method described by Reinhold and Fine [Ill. In our experience, endotoxin concentrations greater than 0.01 Kg/ml were detectable.
Resutts Requiring Uptake. Fourteen of did not require reinfusion of shed blood during the course of hypotension. Seven of the fourteen remained hypotensive for 180 minutes, and all but two of the remainder were hypotensive for at least 120 minutes prior to termination of the experiment. The animals’ weights and amount of blood shed were similar to those of both the blood-infused and Ringer’s lactate-infused groups. None of these animals demonstrated impairment of hepatic energy metabolism to either a-ketoglutarate or succinate. There was no detectable endotoxemia. Thus, despite prolonged hypovolemia and in the absence of the uptake phenomenon, there was no demonstrable abnormality in hepatic mitochondrial function. Animals Requiring Uptake; Shed Blood Reinfused. A striking difference was seen in the animals in shock requiring reinfusion of shed blood. Ten of twenty animals whose hepatic mitochondrial functioh was assessed using a-ketoglutarate as substrate demonstrated uncoupling of oxidative Animals
fifty-six
Not
animals
Volume 130, November 1975
Nine of twenty-one animals phosphorylation. demonstrated uncoupling with succinate. The presence of uncoupled oxidative phosphorylation appeared to be directly related to the degree of uptake of shed blood. (Table I.) Endotoxemia was present in nine of eighteen animals tested and also appeared related to the degree of uptake, uncou-
TABLE I
Correlation of Blood Uptake with Hepatic Mitoizhondrial Function and Endotoxemia
-___-
Per Cent of Uptake Animals not requiring uptake
Animals requiring uptake of shed blood
0 0 0 0 0 0 0 0 0 0 0 0 0 0
4 5 8 9 10 11 16 16 20 20 27 27 31 36 36 50 55 61 78 96 100 100 100
Changein Change in Respiratory Respiratory Control Control Ratio* Ratio* (cu-ketogluta(succinate) rate) -1.18 -1.05 -0.52 -0.32 -0.63
+1.30 -0.95 -1.70 +0.55 -0.58
+1.75 +0x2 +0.43 +0.43 -0.24 -0.35 -1.03 -0.16
-0.26 -1.01 -0.34 +0.17 +0.02 +0.11 +0.02 -0.22
-0.32 uct +0.08
-1.35 -1.13 +1.88 UC -0.04 UC -1.18 -1.82 -1.50 -0.27 -3.67 UC -0.24 -1.00
UC +0.90 UC -2.31 -1.62 -1.08 +0.50 UC -1.45 -3.67 UC -1.14
Endotoxin
UC -1.17
UC UC UC
UC UC UC
UC UC
UC UC
*Average respiratory control ratio of normal animal minus the average respiratory control ratio of shock animal. tuncoupled oxidative phosphorylation
561
Rhodes, DePalma, and Robinson
TABLE II
Relationship of Mitochondrial Function to Type of infusion and to Endotoxemia Animals
Average A RC* &ketoglutarate No. uncoupled a-ketoglutarate Average A RC* succinate No. uncoupled succinate *Change
Infused
EndotoxemiaNegative
EndotoxemiaPositive
-0.70
-1.48
f .40
218 -0.49
in respiratory
* .34
5/g
l/9
control
f .83
-1.92
f .60 519
Significance NS
Infusedwith Ringer’s Lactate
EndotoxemiaNegative
EndotoxemiaPositive
-0.74
-0.41
* .15 l/14
NS t = 1.88 p < 0.05 X’ = 2.25 .20 > p > 0.10
-0.31
r .15
-0.51
t .27 O/9
NS NS
219 r .26
o/14
Significance
NS NS
Statistical Significance of Endotoxemia t = 1.34 .15 > p > 0.10 NS t = 2.24 p < 0.05 X’= 4.43 p < 0.05
ratio.
pling, or both. Endotoxemia was present in every animal in which the uptake exceeded 36 per cent of the shed volume.
Animals Requiring Uptake; Ringer’s Lactate Infused. The substitution of Ringer’s lactate solution for the shed blood when uptake was required had a protective effect on hepatic mitochondrial function. Mitochondrial respiratory control using cr-ketoglutarate as substrate was depressed when compared with that in animals not requiring uptake (t = 1.62, 0.05 < p < O.lO), but in only three of twenty-three animals was there complete uncoupling of oxidative phosphorylation. When compared with the incidence of uncoupling in animals receiving blood infusion, this difference was significant (x2 = 3.52,0.05 < p < 0.10). The use of Ringer’s lactate had an even more notable effect on succinate metabolism: none of the twenty-one animals demonstrated uncoupling (x2 = 8.66, p < 0.01). In fact, respiratory control was not significantly different from that in animals not requiring uptake. The mechanism of the protective effect of Ringer’s lactate was not immediately apparent. The incidence of endotoxemia in the heart blood was similar (nine of twenty-one animals) to that seen in the animals receiving blood infusion. A factor of possible significance was discovered, however, when the shed blood from the group of animals receiving Ringer’s lactate was assayed for endotoxin. Nineteen of twenty-five animals had positive results on assay for endotoxin in their shed blood. The endotoxin titers in this reservoir blood were several times higher than those in the infused animals. Animals that required uptake and were administered shed blood would receive this endotoxin load. The role of endotoxemia in the deterioration of hepatic energy metabolism was further assessed by
562
Animals
with Shed Blood
comparing the mitochondrial function of endotoxin-negative animals with that of endotoxin-positive animals in both animals receiving blood infusion and those receiving Ringer’s lactate. (Table II.) In the group receiving blood infusion, endotoxemia was not associated with a notable effect on the respiratory control of functioning mitochondria or on the incidence of uncoupled mitochondria when cr-ketoglutarate was used as the substrate. In contrast, with succinate as the substrate, the endotoxin-positive animals demonstrated a qarked loss of respiratory control and a high incidence of uncdupling. In animals infused with Ringer’s lactate, the respiratory control of hepatic mitochondria from endotoxin-negative versus endotoxin-positive animals was similar, regardless of the substrate. The possible protective effect of Ringer’s lactate on hepatic energy metabolism is emphasized by comparing the endotoxin-positive animals from the group infused with blood with the group receiving Ringer’s lactate (last column, Table II). AnimaIs infused with Ringer’s lactate had minimal mitochondrial dysfunction despite detectable endotoxemia. Interpretation of the results showing a protective effect of Ringer’s lactate infusion on hepatic energy metabolism must be qualified, however, by the shorter interval to uptake and shorter duration of hypotension in the group receiving Ringer’s lactate. These differences were anaiyzed if endotoxemia was detected at the time of sacrifice. (Table III.) In the animals receiving blood the presence of endotoxemia was associated with a shorter intervd from the onset of shock to the need for uptake and a slightly shorter duration of hypotension. In the animals receiving Ringer’s lactate the detection of endotoxemia was associated with a shorter interval of shock prior to the requirement for uptake. The
l?m Amarkan Journal cd Suquy
Endotoxemia and Hemorrhagic Shock
TABLE III
Comparison of Time to Uptake and Duration of Hypotension to Type of Infusion and Endotoxemia Animals
Animals Infusedwith Shed Blood
Time Average
interval
EndotoxemiaNegative 153
*
11
EndotoxemiaPositive
113 f 9
t = 1.38
167
p = 0.10 NS
to uptake (min)
Average duration of shock (min) __~___
180*
16
Significance
+ 9 --
overall duration of hypotension was significantly shorter in the presence of endotoxemia. When both animals receiving blood and those receiving Ringer’s lactate are compared with respect to endotoxemia, it can be seen that the time to uptake was significantly shorter in the animals receiving Ringer’s lactate whether or not endotoxemia was present. The overall duration of hypotension was also shorter in the animals receiving Ringer’s lactate, but any significant difference appeared to be confined to the endotoxin-positive groups. The differences in mitochondrial function between the animals infused with shed blood and those infused with Ringer’s lactate could not be explained by differences between the groups with regard to weight of the animal, amount of blood shed, or duration of hypotension. Nor could the differences between the endotoxin-positive and endotoxin-negative animals be attributed to differences in weight of the animal or the amount of blood shed, as these were similar in each group. Comments Wiggers [I] observed that dogs subjected to prolonged hemorrhagic shock reached a state in which they were refractory to restoration of normal blood volume. He coined the term, “irreversible shock,” to describe this state. Fine et al [12] proposed that endotoxemia was the principal etiologic factor in this circulatory decompensation. Recently, Bacalzo et al [2] have shown that when uptake exceeds 40 per cent of the shed blood volume, mortality approaches 100 per cent despite restoration of normovolemia. In the present study, both endotoxemia and severe mitochondrial dysfunction appeared as constant findings when uptake of shed blood exceeded 36 per cent of the shed blood volume. This level closely coincides with the 100 per cent mortality in Bacalzo’s studies. On the basis of the present study, it is probable that endotoxemia is closely related to mitochon-
Vohano 130, Novmnber 1975
EndotoxemiaNegative
with
EndotoxemiaPositive
127+- 9 168*
Infused
6
101 142
*
12
? 13
Ringer’s
Lactate
Significance
Statistical Significance of Endotoxemia
t = 1.75
t = 2.21
0.10 > p > 0.05 t = 2.15 p < 0.05
p < 0.05 t= 1.6 0.10 > p > 0.05
drial dysfunction. Although pure ischemia can induce mitochondrial dysfunction [13], endotoxin alone induces mitochondrial lesions identical to those seen in hemorrhagic shock [5]. These experiments do not establish such a cause and effect relationship, but such a relationship appears plausible. Intestinal barrier function, a safeguard against a major reservoir of endotoxin, has been demonstrated to be lost prior to the appearance of endotoxemia [6]. The role of endotoxemia as a causative factor in initiating the uptake phenomenon is not clear. Shorter intervals to the requirement for reinfusion are associated with detectable endotoxemia at sacrifice. However, the overall shorter intervals seen in the group of animals selected for subsequent Ringer’s lactate infusion can only be explained by chance biologic variation. In the animals infused with blood, the presence of endotoxemia was associated with only a shorter interval to uptake. Endotoxemia at sacrifice appeared to be most significantly related to the deterioration of mitochondrial energy metabolism, particularly with regard to succinate, rather than to time. The chance time difference between animals receiving shed blood and those infused with Ringer’s lactate helps establish, however, that the deterioration of hepatic energy metabolism does not precede the uptake phenomenon nor does it appear to be directly related to time. If a direct relationship between time in shock and deterioration of hepatic mitochondrial function existed, the animals receiving Ringer’s lactate would have demonstrated more mitochondrial dysfunction. This was not the case. The deleterious factors related to the infusion of shed blood have been noted previously by others [14,15]. Nahas, Mittelman, and Manger [15] believed that the acidotic pH of the reinfused blood was responsible. The high frequency and high titer of endotoxin in the shed blood reservoir in the present experiments was an exciting finding.
563
Rhodes,
DePalma, and Robinson
Readministration of such an additional endotoxin load might have a significant bearing on crucial metabolic functions. Since most of the blood is withdrawn early in the course of the experiment, it implies either contamination in the reservoir or early endotoxemia. Evidence for such early endotoxemia has been presented by Cuevas and Fine [16]. Further studies are needed to identify the source and significance of this reservoir of endotoxin. Our understanding of the complex’ metabolic and energy changes in late hemorrhagic shock and the therapeutic armamentarium to deal with them are as yet inadequate. The various models of shock have provided important concepts in understanding refractory clinical states. The findings of endotoxemia and disordered hepatic energy metabolism in this reservoir model of shock may be relevant to the frequent clinical findings of sequential organ failure, especially late hepatic failure. Summary The appearance of endotoxemia and uncoupling of oxidative phosphorylation coincided with progressive uptake from the reservoir in a murine model of late hemorrhagic shock. Reinfusion with Ringer’s_ lactate _..__~__ ~____~~rather ~_..~~~ than shed blood suggested that the endotoxemia was causally related to mitoThe mitochondrial dyschondrial dysfunction. function per se did not appear related to the uptake phenomenon. The shed blood in the reservoir had a high frequency of detectable endotoxin. The animals to be infused with Ringer’s lactate had a significantly more rapid appearance of the uptake phenomenon, probably due to intrinsic, variation in the biologic model. vv
564
References 1. Wiggers CJ: The Physiology of Shock. New York, Commonwealth Press. 1950. 2. Bacalzo LV, C&y AL, Miller LD, Parkins WM: Methods and critical uptake volume for hemorrhagic shock in rats. Surgery 70: 555, 1971 3. Baue AE, Sayeed MM: Atterations in the functional capacity of mitochondria in hemorrhagic shock. Surgerv 66: 40, 1970. 4. DePalma RG. Levey S, Hokfen WD: Ultrastructure and oxidative ohosohorvlation of liver mttochondria in experimental hembrrhagic shock. J Trauma 10: 122, 1970. ’ 5. MelaL, Bacalzo LV, Miller LD: Defective oxidative metabolism of rat liver mitochondria in hemorrhagic and endotoxic shock. Am JPhysioi220: 571, 1971. 6. Rhodes RS, DePalma RG, Robinson AV: Intestinal barrier function in hemorrhagic shock. J Surg Res 14: 305, 1973. 7. DePalma RG, Robinson AV, Hoiden WD: Fluid therapy in hemorrhagic shock: experimental evaluation. J Surg Oncol2: 349. 1970. 8. DePalma RG, Harano Y, Robinson AV, Holden WD: Structure and function of hepatic mitochondria in hemorrhage and endotoxemia. Surg Forum 2 1: 3, 1970. 9. Harano Y, DePalma RG, Lavine L, Miller M: Fatty acid oxidation, oxidative phosphoryhtion and uitrastructure of mitochondria in the diabetic rat liver. Diabetes 21: 257, 1972. 10. Layne E: Spectrophotometric and turbiiimetric methods for measuring proteins, p 450. Methods in Enzymology Ill (Colowick SP, Kaplan NO, ed). New York, Academic Press, 1957. 11. Reinhold RB, Fine J: A technique for quantitative measurement of endotoxin in human olasma. Proc Sot Exe Bio/ Med137: 334.1971. 12. Fine J, Frank ED, Ravin HA, Rutenburg SH, Schweinberg FB: The bacterial factor in traumatic shock. N Engl J Med 260: 214, 1959. 13. Castillo-Olivares JL, Gosalvez M, Azpeitii D, Romero EG, Blanc0 M, Figuera D: Mtochondrial-respiration and oxidative phosphorybtion during hepatic preservation. J Surg Res 13: 85. 1972. 14. Jesch F, Sunder-Ptassrnann L, Mesmer K: Die Bedeutung des Uptake im experimentellen Mmorrhagischen Schock. Res Exp Med 159: 141, 1973. 15. Nahas GG, Mittelman A, Manger WM: The effect of buffering ACD blood with THAM on the survival of dogs transfused after massive hemorrhage. FedProc 19: 54, 1960. 16. Cuevas P, Fine J: Route of absorption of endotoxin from the intestine in non-septic shock. J Reticu/oendothe/ Sot 11: 535, 1972.
The American Journal
of Surgery
Improvements in the treatment of hemorrhagic shock refractory to volume replacement depend on identification of the factors responsible for transition to the “irreversible” state. Criteria indicative of “irreversibility” are difficult to identify experimentally, but the need for retransfusion of shed blood to maintain blood pressure is considered the major criterion of transition to the irreversible state. This was first emphasized in canine hemorrhagic shock by Wiggers [I]. The concept of critical uptake volume has recently been extended to the hemorrhagic shock model in the rat [2]. The precise factors responsible for blood uptake and subsequent lethality are not known. Current research into shock has focused on alterations in cellular metabolism and their possible relationships to irreversibility. Deterioration of hepatic energy-linked mitochondrial function in prolonged shock has been well demonstrated, and the degree of deterioration is related to prolongation of hypotension. Hepatic mitochondrial energy-linked function usually remains normal during the initial 120 minutes of hypovolemia. Animals sacrificed after longer intervals of hypovolemia show an increasing uncoupling of oxidative phosphorylation [3-61. There are, however, significant variations from animal to animal in susceptibility to the consequences of prolonged hypovolemia. Resistance to hemorrhagic shock is related to maintenance of efficient hepatic energy metabolism and blood glucose levels [6].
From the Department of Surgery, Case Western Reserve School of Medii tine and University Hospitals of Cleveland, Cleveland, Ohio. Reprint requests should be addressed to Robert S. Rhodes, MD. University Hospitals of Cleveland, 2065 Adelberl Road, Cleveland, Ohio 44106.
560
This study was carried out to further characterize the relationship between the onset of irreversibility and the deterioration of hepatic mitochondrial energy production. It was demonstrated that although depression of hepatic mitochondrial function appeared generally related to duration of shock, aberrations in energy production by hepatic mitochondria were specifically related to the uptake of blood from the reservoir and to endotoxemia. The infusion of Ringer’s lactate solution rather than blood during the uptake stage may prevent certain aspects of mitochondrial dysfunction. Material and Methods Experimental Preparation. Young adult SpragueDawley rats, weighing 225 to 300 gm, were used. They were allowed access to rat chow and water up to the time of the experiment. The animals were anesthetized with intraperitoneal pentoharbital sodium, 4 mg/lOO gm. A PE 50 polyethylene cannula was inserted into the left femoral artery and 500 units of heparin were administered intra-arterially. The cannula was connected to a three-way stopcock with a sterile syringe for bleeding and a mercury manometer for measuring blood pressure. Body temperature was monitored using a telethermometer and maintained at 35.5” to 37.5V using an external heat source. In the present study, prolongation of experimental periods of shock for longer than two hours often required artificial maintenance of the airway. Otherwise, the details of the animal preparation were similar to those previously reported [ 71. Hemorrhagic shock was induced by withdrawing blood until a mean arterial blood pressure of 30 to 35 mm Hg was obtained. Blood pressure was maintained at this level by further withdrawal or infusion of blood as was necessary. Identically prepared nonbled control animals were studied simultaneously and tissues obtained from these animals processed synchronously. The exper-
The Amerksn Journal ol Surgery
Endotoxemia and Hemorrhagic Shock
iments were terminated utes.
Biochemical
Studies
at intervals of 30 to 240 min-
and Mitochondriul
Function.
The functional capability of the hepatic mitochondria was assessed after isolation of the mitochondria using methods previously described [8,9]. The ultrastructural characteristics of the isolated preparations were similar to those previously reported [4]. Oxygen uptake was measured by a Gilson oxygraph at 23°C (model KM with a Clark electrode). Hepatic mitochondria isolated by differential centrifugation from 4 gm of liver were suspended in 8 ml of 0.24 M sucrose with 0.5 mM ethylenediaminetetracetic acid (EDTA). A 0.3 ml aliquot of this suspension was added to 2 ml of an assay system containing 5 mM of phosphate buffer (pH 7.4), 120 mM of potassium chloride, 5 mM of magnesium-chloride and 5 mM of tris buffer. The substrates, cY-ketoglutaratb (5 mM) or succinate (5 mM), were studied by successive additions of adenosine diphosphate (ADP) (133 mM). Hepatic mitochondrial protein was measured by the biuret method [IO]. State 3 and 4 respiratory rates, respiratory control ratio, and ADPIO ratios were calculated. Endotoxin Assay. At the time of sacrifice 2 to 3 ml of blood were withdrawn from the heart into heparinked syringes using aseptic technic. The blood was immediately centrifuged and the plasma stored at 0 to 4’C for later assay for endotoxin using the Limulus assay method described by Reinhold and Fine [Ill. In our experience, endotoxin concentrations greater than 0.01 Kg/ml were detectable.
Resutts Requiring Uptake. Fourteen of did not require reinfusion of shed blood during the course of hypotension. Seven of the fourteen remained hypotensive for 180 minutes, and all but two of the remainder were hypotensive for at least 120 minutes prior to termination of the experiment. The animals’ weights and amount of blood shed were similar to those of both the blood-infused and Ringer’s lactate-infused groups. None of these animals demonstrated impairment of hepatic energy metabolism to either a-ketoglutarate or succinate. There was no detectable endotoxemia. Thus, despite prolonged hypovolemia and in the absence of the uptake phenomenon, there was no demonstrable abnormality in hepatic mitochondrial function. Animals Requiring Uptake; Shed Blood Reinfused. A striking difference was seen in the animals in shock requiring reinfusion of shed blood. Ten of twenty animals whose hepatic mitochondrial functioh was assessed using a-ketoglutarate as substrate demonstrated uncoupling of oxidative Animals
fifty-six
Not
animals
Volume 130, November 1975
Nine of twenty-one animals phosphorylation. demonstrated uncoupling with succinate. The presence of uncoupled oxidative phosphorylation appeared to be directly related to the degree of uptake of shed blood. (Table I.) Endotoxemia was present in nine of eighteen animals tested and also appeared related to the degree of uptake, uncou-
TABLE I
Correlation of Blood Uptake with Hepatic Mitoizhondrial Function and Endotoxemia
-___-
Per Cent of Uptake Animals not requiring uptake
Animals requiring uptake of shed blood
0 0 0 0 0 0 0 0 0 0 0 0 0 0
4 5 8 9 10 11 16 16 20 20 27 27 31 36 36 50 55 61 78 96 100 100 100
Changein Change in Respiratory Respiratory Control Control Ratio* Ratio* (cu-ketogluta(succinate) rate) -1.18 -1.05 -0.52 -0.32 -0.63
+1.30 -0.95 -1.70 +0.55 -0.58
+1.75 +0x2 +0.43 +0.43 -0.24 -0.35 -1.03 -0.16
-0.26 -1.01 -0.34 +0.17 +0.02 +0.11 +0.02 -0.22
-0.32 uct +0.08
-1.35 -1.13 +1.88 UC -0.04 UC -1.18 -1.82 -1.50 -0.27 -3.67 UC -0.24 -1.00
UC +0.90 UC -2.31 -1.62 -1.08 +0.50 UC -1.45 -3.67 UC -1.14
Endotoxin
UC -1.17
UC UC UC
UC UC UC
UC UC
UC UC
*Average respiratory control ratio of normal animal minus the average respiratory control ratio of shock animal. tuncoupled oxidative phosphorylation
561
Rhodes, DePalma, and Robinson
TABLE II
Relationship of Mitochondrial Function to Type of infusion and to Endotoxemia Animals
Average A RC* &ketoglutarate No. uncoupled a-ketoglutarate Average A RC* succinate No. uncoupled succinate *Change
Infused
EndotoxemiaNegative
EndotoxemiaPositive
-0.70
-1.48
f .40
218 -0.49
in respiratory
* .34
5/g
l/9
control
f .83
-1.92
f .60 519
Significance NS
Infusedwith Ringer’s Lactate
EndotoxemiaNegative
EndotoxemiaPositive
-0.74
-0.41
* .15 l/14
NS t = 1.88 p < 0.05 X’ = 2.25 .20 > p > 0.10
-0.31
r .15
-0.51
t .27 O/9
NS NS
219 r .26
o/14
Significance
NS NS
Statistical Significance of Endotoxemia t = 1.34 .15 > p > 0.10 NS t = 2.24 p < 0.05 X’= 4.43 p < 0.05
ratio.
pling, or both. Endotoxemia was present in every animal in which the uptake exceeded 36 per cent of the shed volume.
Animals Requiring Uptake; Ringer’s Lactate Infused. The substitution of Ringer’s lactate solution for the shed blood when uptake was required had a protective effect on hepatic mitochondrial function. Mitochondrial respiratory control using cr-ketoglutarate as substrate was depressed when compared with that in animals not requiring uptake (t = 1.62, 0.05 < p < O.lO), but in only three of twenty-three animals was there complete uncoupling of oxidative phosphorylation. When compared with the incidence of uncoupling in animals receiving blood infusion, this difference was significant (x2 = 3.52,0.05 < p < 0.10). The use of Ringer’s lactate had an even more notable effect on succinate metabolism: none of the twenty-one animals demonstrated uncoupling (x2 = 8.66, p < 0.01). In fact, respiratory control was not significantly different from that in animals not requiring uptake. The mechanism of the protective effect of Ringer’s lactate was not immediately apparent. The incidence of endotoxemia in the heart blood was similar (nine of twenty-one animals) to that seen in the animals receiving blood infusion. A factor of possible significance was discovered, however, when the shed blood from the group of animals receiving Ringer’s lactate was assayed for endotoxin. Nineteen of twenty-five animals had positive results on assay for endotoxin in their shed blood. The endotoxin titers in this reservoir blood were several times higher than those in the infused animals. Animals that required uptake and were administered shed blood would receive this endotoxin load. The role of endotoxemia in the deterioration of hepatic energy metabolism was further assessed by
562
Animals
with Shed Blood
comparing the mitochondrial function of endotoxin-negative animals with that of endotoxin-positive animals in both animals receiving blood infusion and those receiving Ringer’s lactate. (Table II.) In the group receiving blood infusion, endotoxemia was not associated with a notable effect on the respiratory control of functioning mitochondria or on the incidence of uncoupled mitochondria when cr-ketoglutarate was used as the substrate. In contrast, with succinate as the substrate, the endotoxin-positive animals demonstrated a qarked loss of respiratory control and a high incidence of uncdupling. In animals infused with Ringer’s lactate, the respiratory control of hepatic mitochondria from endotoxin-negative versus endotoxin-positive animals was similar, regardless of the substrate. The possible protective effect of Ringer’s lactate on hepatic energy metabolism is emphasized by comparing the endotoxin-positive animals from the group infused with blood with the group receiving Ringer’s lactate (last column, Table II). AnimaIs infused with Ringer’s lactate had minimal mitochondrial dysfunction despite detectable endotoxemia. Interpretation of the results showing a protective effect of Ringer’s lactate infusion on hepatic energy metabolism must be qualified, however, by the shorter interval to uptake and shorter duration of hypotension in the group receiving Ringer’s lactate. These differences were anaiyzed if endotoxemia was detected at the time of sacrifice. (Table III.) In the animals receiving blood the presence of endotoxemia was associated with a shorter intervd from the onset of shock to the need for uptake and a slightly shorter duration of hypotension. In the animals receiving Ringer’s lactate the detection of endotoxemia was associated with a shorter interval of shock prior to the requirement for uptake. The
l?m Amarkan Journal cd Suquy
Endotoxemia and Hemorrhagic Shock
TABLE III
Comparison of Time to Uptake and Duration of Hypotension to Type of Infusion and Endotoxemia Animals
Animals Infusedwith Shed Blood
Time Average
interval
EndotoxemiaNegative 153
*
11
EndotoxemiaPositive
113 f 9
t = 1.38
167
p = 0.10 NS
to uptake (min)
Average duration of shock (min) __~___
180*
16
Significance
+ 9 --
overall duration of hypotension was significantly shorter in the presence of endotoxemia. When both animals receiving blood and those receiving Ringer’s lactate are compared with respect to endotoxemia, it can be seen that the time to uptake was significantly shorter in the animals receiving Ringer’s lactate whether or not endotoxemia was present. The overall duration of hypotension was also shorter in the animals receiving Ringer’s lactate, but any significant difference appeared to be confined to the endotoxin-positive groups. The differences in mitochondrial function between the animals infused with shed blood and those infused with Ringer’s lactate could not be explained by differences between the groups with regard to weight of the animal, amount of blood shed, or duration of hypotension. Nor could the differences between the endotoxin-positive and endotoxin-negative animals be attributed to differences in weight of the animal or the amount of blood shed, as these were similar in each group. Comments Wiggers [I] observed that dogs subjected to prolonged hemorrhagic shock reached a state in which they were refractory to restoration of normal blood volume. He coined the term, “irreversible shock,” to describe this state. Fine et al [12] proposed that endotoxemia was the principal etiologic factor in this circulatory decompensation. Recently, Bacalzo et al [2] have shown that when uptake exceeds 40 per cent of the shed blood volume, mortality approaches 100 per cent despite restoration of normovolemia. In the present study, both endotoxemia and severe mitochondrial dysfunction appeared as constant findings when uptake of shed blood exceeded 36 per cent of the shed blood volume. This level closely coincides with the 100 per cent mortality in Bacalzo’s studies. On the basis of the present study, it is probable that endotoxemia is closely related to mitochon-
Vohano 130, Novmnber 1975
EndotoxemiaNegative
with
EndotoxemiaPositive
127+- 9 168*
Infused
6
101 142
*
12
? 13
Ringer’s
Lactate
Significance
Statistical Significance of Endotoxemia
t = 1.75
t = 2.21
0.10 > p > 0.05 t = 2.15 p < 0.05
p < 0.05 t= 1.6 0.10 > p > 0.05
drial dysfunction. Although pure ischemia can induce mitochondrial dysfunction [13], endotoxin alone induces mitochondrial lesions identical to those seen in hemorrhagic shock [5]. These experiments do not establish such a cause and effect relationship, but such a relationship appears plausible. Intestinal barrier function, a safeguard against a major reservoir of endotoxin, has been demonstrated to be lost prior to the appearance of endotoxemia [6]. The role of endotoxemia as a causative factor in initiating the uptake phenomenon is not clear. Shorter intervals to the requirement for reinfusion are associated with detectable endotoxemia at sacrifice. However, the overall shorter intervals seen in the group of animals selected for subsequent Ringer’s lactate infusion can only be explained by chance biologic variation. In the animals infused with blood, the presence of endotoxemia was associated with only a shorter interval to uptake. Endotoxemia at sacrifice appeared to be most significantly related to the deterioration of mitochondrial energy metabolism, particularly with regard to succinate, rather than to time. The chance time difference between animals receiving shed blood and those infused with Ringer’s lactate helps establish, however, that the deterioration of hepatic energy metabolism does not precede the uptake phenomenon nor does it appear to be directly related to time. If a direct relationship between time in shock and deterioration of hepatic mitochondrial function existed, the animals receiving Ringer’s lactate would have demonstrated more mitochondrial dysfunction. This was not the case. The deleterious factors related to the infusion of shed blood have been noted previously by others [14,15]. Nahas, Mittelman, and Manger [15] believed that the acidotic pH of the reinfused blood was responsible. The high frequency and high titer of endotoxin in the shed blood reservoir in the present experiments was an exciting finding.
563
Rhodes,
DePalma, and Robinson
Readministration of such an additional endotoxin load might have a significant bearing on crucial metabolic functions. Since most of the blood is withdrawn early in the course of the experiment, it implies either contamination in the reservoir or early endotoxemia. Evidence for such early endotoxemia has been presented by Cuevas and Fine [16]. Further studies are needed to identify the source and significance of this reservoir of endotoxin. Our understanding of the complex’ metabolic and energy changes in late hemorrhagic shock and the therapeutic armamentarium to deal with them are as yet inadequate. The various models of shock have provided important concepts in understanding refractory clinical states. The findings of endotoxemia and disordered hepatic energy metabolism in this reservoir model of shock may be relevant to the frequent clinical findings of sequential organ failure, especially late hepatic failure. Summary The appearance of endotoxemia and uncoupling of oxidative phosphorylation coincided with progressive uptake from the reservoir in a murine model of late hemorrhagic shock. Reinfusion with Ringer’s_ lactate _..__~__ ~____~~rather ~_..~~~ than shed blood suggested that the endotoxemia was causally related to mitoThe mitochondrial dyschondrial dysfunction. function per se did not appear related to the uptake phenomenon. The shed blood in the reservoir had a high frequency of detectable endotoxin. The animals to be infused with Ringer’s lactate had a significantly more rapid appearance of the uptake phenomenon, probably due to intrinsic, variation in the biologic model. vv
564
References 1. Wiggers CJ: The Physiology of Shock. New York, Commonwealth Press. 1950. 2. Bacalzo LV, C&y AL, Miller LD, Parkins WM: Methods and critical uptake volume for hemorrhagic shock in rats. Surgery 70: 555, 1971 3. Baue AE, Sayeed MM: Atterations in the functional capacity of mitochondria in hemorrhagic shock. Surgerv 66: 40, 1970. 4. DePalma RG. Levey S, Hokfen WD: Ultrastructure and oxidative ohosohorvlation of liver mttochondria in experimental hembrrhagic shock. J Trauma 10: 122, 1970. ’ 5. MelaL, Bacalzo LV, Miller LD: Defective oxidative metabolism of rat liver mitochondria in hemorrhagic and endotoxic shock. Am JPhysioi220: 571, 1971. 6. Rhodes RS, DePalma RG, Robinson AV: Intestinal barrier function in hemorrhagic shock. J Surg Res 14: 305, 1973. 7. DePalma RG, Robinson AV, Hoiden WD: Fluid therapy in hemorrhagic shock: experimental evaluation. J Surg Oncol2: 349. 1970. 8. DePalma RG, Harano Y, Robinson AV, Holden WD: Structure and function of hepatic mitochondria in hemorrhage and endotoxemia. Surg Forum 2 1: 3, 1970. 9. Harano Y, DePalma RG, Lavine L, Miller M: Fatty acid oxidation, oxidative phosphoryhtion and uitrastructure of mitochondria in the diabetic rat liver. Diabetes 21: 257, 1972. 10. Layne E: Spectrophotometric and turbiiimetric methods for measuring proteins, p 450. Methods in Enzymology Ill (Colowick SP, Kaplan NO, ed). New York, Academic Press, 1957. 11. Reinhold RB, Fine J: A technique for quantitative measurement of endotoxin in human olasma. Proc Sot Exe Bio/ Med137: 334.1971. 12. Fine J, Frank ED, Ravin HA, Rutenburg SH, Schweinberg FB: The bacterial factor in traumatic shock. N Engl J Med 260: 214, 1959. 13. Castillo-Olivares JL, Gosalvez M, Azpeitii D, Romero EG, Blanc0 M, Figuera D: Mtochondrial-respiration and oxidative phosphorybtion during hepatic preservation. J Surg Res 13: 85. 1972. 14. Jesch F, Sunder-Ptassrnann L, Mesmer K: Die Bedeutung des Uptake im experimentellen Mmorrhagischen Schock. Res Exp Med 159: 141, 1973. 15. Nahas GG, Mittelman A, Manger WM: The effect of buffering ACD blood with THAM on the survival of dogs transfused after massive hemorrhage. FedProc 19: 54, 1960. 16. Cuevas P, Fine J: Route of absorption of endotoxin from the intestine in non-septic shock. J Reticu/oendothe/ Sot 11: 535, 1972.
The American Journal
of Surgery
