Plasma Changes in Endotoxin and Anaphylactic Shock (ATP, ADP and Creatine Phosphorus)
CLARENCE M. JABS, Ph .D.,* WILLIAM J. FERRELL, Ph .D .,fand HERBERT J. ROBB, M.D.*
* D epartm en t o f Surgical Research, William Beaumont Hospital,
Royal Oak, M l 48072 and
f Departm ents o f Pathology and Biological Chemistry,The University o f Michigan,
Ann Arbor, MI 48109
Decisive patterns have been dem onstrated in plasma adenosine 5 ' triphosphate (ATP) levels in both endotoxin and anaphylactic shock which correlate with periods of low platelet counts, low arterial pressures and abnormal electrocardiograms. W hen these irregularities were occurring, the plasma ATP level was low; when improvement occurred, the plasma ATP level rose. Plasma ATP levels appear to be an index to the m etabolic state of the animal.
The plasma creatine phosphate (CP) level showed a tendency to decrease w hen the ATP level dropped in anaphylactic shock, although the CP level did not recover to the same extent as the ATP level. In endotoxin shock, the plasma CP level increased on an average of six-fold. It is proposed that this rise resulted from either CP mobilization from tissues, for the purpose of replenishing the energy deficient myocardial muscle, or possible leakage from damaged cells.
Adenosine diphosphate (ADP) plasma values were m easured in both anaphylactic and endotoxin shock. High initial ADP values were prone towards a more severe anaphylactic reaction and a shorter survival time in the endotoxin shock experiments.
IntroductionA denosine 5' triphosphate (ATP) is
universally accepted as the major energy source for cellular function and creatine phosphate (CP) as the energy storehouse for muscle which intercedes to replenish ATP w hen the supply diminishes. C ellu
lar biochemical analyses in dogs during hemorrhagic and endotoxin shock have dem onstrated the existence of an aerobic state27,43 and energy failure.26,42 The high energy com pounds ATP and CP were found to be decreased during shock30 and also mechanical restriction of the blood
supply to skeletal muscle produced a reduction in the tissue content of ATP and CP.36,41 Chronic heart failure, produced in dogs by pulmonary arterial stenosis, lowered both the ATP and CP myocardial concen tra tions.14 T hese investigations have dem onstrated the apparent importance of cellular ATP and CP levels in the maintenance of an adequate cellular function.
One agent suspected to be a prime factor in platelet aggregation is adenosine diphosphate (ADP).3,17,35 W ith the abnormal loss of ATP in shock, the accum ulation of ADP is a possibility. Chen and Jorgensen7 dem onstrated the breakdown of ATP in blood with transitory accumulations of ADP as well as AMP. Ireland and M ills22 dem onstrated that when enough ADP was added to heparinized platelet rich plasma, under conditions that perm it platelet clumping, half of the added ADP was gone after 20 m inutes and AMP acc u m u la ted , th e n ADP rap id ly d isa p peared to zero after 120 minutes. This prolonged ADP breakdown time could be dangerous as a constant increasing source of ADP becomes available. The accum ulation o f ADP in the cellu lar tissues, owing to the inadequate energy restoration of ATP and the possible ability of ADP to cross the cellu lar m em brane, could lead to a dangerous level of ADP in the blood and be a stim ulation to platelet aggregation.
Success has been dem onstrated in treating dogs in experim ental hem orrhagic shock with ATP infusions with claims that this high energy nucleotide alleviated the energy im balance .29, 44,43 O th e rs6,11,13 have shown that ATP can possibly transverse the cellular membrane, which may justifiably explain its success in the treatm ent of shock. CP infusions have also been shown to increase markedly the survival rate in dogs during hem orrhagic shock.1 A knowledge of the plasma levels of these high energy compounds in the
shock state could be helpful in determ ining the m ethod or type of treatm ent. The plasm a ATP, ADP and CP level could be an index to the energy state of body tissues and possibly give some indication of the severity of the shock condition. Periodic m onitoring of these levels during their in fusion may also be important since excessively high plasma ATP and CP levels could be dangerous. ATP is known to be a p o ten t vasod ila to r48 and at excessive plasm a concentrations could lead to a dangerous drop in blood pressure, and the accumulation of plasma ADP could lead to stim ulated platelet aggregation and embolization. These reasons stim ulated our interest in the developm ent of clinically useful test to measure plasma ATP, ADP and CP concentrations.12,23,24
Previous studies from our laboratory on anaphylactic39 and endotoxin shock40 enabled one to find the time when a low cellular energy state in rabbits may exist. In the present study, these two shock models were used to determ ine if any changes occurred in the plasma ATP, ADP or CP and to co rre la te th ese ch an g es w ith changes in the p late le t count, arterial pressure and the electrocardiogram to find meaningful patterns which could indicate the arrival of a serious metabolic problem.
M aterials and MethodsNew Z ealand albino rabbits, m ain
tained on a commercial rabbit diet, were used for the studies. Anaphylactic shock was produced in eight male rabbits, sensitized with 2 ml of human serum injected slowly into the marginal ear vein on two consecutive days. Shock was induced after 12 days ofthe initial sensitizing dose. The shock dose consisted of 0.5 ml of human serum diluted to 1 ml with isotonic saline. This solution was slowly injected into the marginal ear vein over a 30 second time period. Previous to the shock dosage each rabbit was anesthetized with sodium
PLASM A CHA NGES IN EN D O TO X IN AND A N A PH Y LA CTIC SHOCK 123
p en to b a rb ita l, 30 mg p e r kg of body weight.
Endotoxin shock was produced in eight male rabbits with Difco lyophilized Escherich ia coli, (0127.B8 lipopolysac- charide). Two mg ofE. coli endotoxin per kg of rabbit w eight was dissolved in 3 ml of saline and injected into the marginal ear vein of the rabbits over a period of 90 seconds. Each rabbit was previously anesthetized w ith sodium pentobarbital (30 mg per kg of body weight) and because of the long duration of the experiments (up to 16 hours) additional sodium pen tobarbital had to be given.
Arterial pressures and blood sampling w ere a c co m p lish ed u s in g 16 gauge catheters inserted into the left carotid artery of the rabbits and attached to a three way stopcock. One lead from the stopcock w ent to an anaroid m anom eter, which m onitered the arterial pressure. The other opening of the stopcock was used to draw blood. Before a test sample was drawn, 2 ml of blood was first drawn into a syringe containing 2 ml of heparinized isotonic saline (2.5 units per ml) and the test sample was then draw n using a d ifferent syringe. The diluted saline blood was returned to the animal. This procedure in sured a fresh sample for analysis each time.
For the extraction of ATP, ADP, and CP, blood was collected in a tube containing a final concentration of 10 mM disodium ethylenediam inetetraacetic acid (Na2EDTA) to total volum e of blood. Plasma contains enzymes capable of degrading both ATP25 and CP37 and the concentration of Na2EDTA totally inhibits the breakdow n of adenine nucleotides in plasma20 as w ell as prevents blood coagulation. The whole blood was then centrifuged at 4000 x g for four minutes. The plasma layer was then decanted, to which an equal volume of 96 percent ethanol was added to denature enzymes.20 The precipitate was rem oved by centrifugation at
12000 x g for 15 m inutes and the supernatant stored at 4°C.
Electrocardiogram tracings were made.* Sm all areas on each o f the ra b b it’s legs were shaved and the four heads attach ed to th ese a reas using a lliga to r clamps. P latelet counts were then de te rm ined.f Plasma ATP, ADP, and CP determ inations were m ad e | as previously described.12,23,24
The plasma ATP level in anaphylactic shock dropped significantly five m inutes after the shocking dose of antigen (figures 1 and 2). This tim e was previously shown to be critical in the outcome of anaphylaxis. At this time the platelet count and arterial pressure are at a minimum and m ultiple p latelet emboli were observed obstructing the pulmonary blood flow.39 If the animal survives this period, platelet deaggregation and recovery occur. D uring this recovery period, the plasma ATP level gradually approached the initial level. The CP levels were found to be lower after an hour, which m ight represent its utilization to replenish the ATP study. The data also show an initial drop in the CP level at the critical time period of five m inutes.
In anaphylactic shock the plasm a ADP values indicated that the outcome was predeterm ined by the initial levels. Four animals were found to have a relatively high initial ADP level (figure 2) which dropped w ithin the critical first five m inutes by an average of 63 percent. This might be suspected if available plasma ADP was being utilized during platelet aggregation. H alf of these animals died. On the other hand, four other rabbits exhibited low initial ADP levels (figure 1)
* A Sanborn 500 Viso Cardiette was used.t A Becton-Dickinson Unopette 5855 Test Kit was
used.t A Dupont 760 Luminescence Biometer was
124 JABS, F E R R E L L AND ROBB
? F i g u r e 1. Plasm aO ATP, ADP, CP and plate-x le t coun t changes ob-
10 served in rabbits w ith£ initial low plasma ADP
lev e ls during anaphylactic shock. Each point represents the average
3 O □ of four different animals. O '—
which increased after five m inutes by an average of 78 percent, and all these animals survived the shock. The surviving animals showed a later trend to return to th e ir in itia l levels suggesting tha t both high and low levels are normally maintained.
The overall average plasma ATP level changes found in endotoxin shock were
related to three stages: initial shock, apparen t recovery and terminal shock (figure 3). In the normal and apparent recovery stage, when the pressure, platelet count and electrocardiogram were w ithin normal range, the ATP values were high (figures 4 and 5). During the shock stages, five m inutes after in jecting endotoxin and terminally, the ATP levels dropped. Ex-
F ig u r e 2. Plasma ATP, ADP, CP and platele t count changes observed in rabbits w ith initial high plasma ADP lev e ls during anaphylactic shock. Each point represents the average of four different animals except the last four points w hich are from tw o rabbits (2 died).
PLASM A C H A N G ES IN EN D O TO X IN AND A N A PHYLACTIC SHOCK 125
perim entally , the ATP level appeared again to delineate the metabolic state of the animals as it did in anaphylactic shock. The CP level in endotoxin shock showed an unexpected pattern (figures 4 and 5). In all animals, the term inal stage plasma CP level rose several fold.
The plasm a ADP pattern, which resulted during endotoxin shock, was similar to that seen during anaphylactic shock. Animals w ith initial ADP levels greater than 0.1 ¿/.M ADP (figure 5) died w ithin an average tim e of 2.8 hours, while animals w ith initial ADP levels less than 0.05 ¿iM (figure 4) survived longer to an average of14.4 hours. The plasm a ADP values of all animals which were initially high dropped five m inutes after the injection of
endotoxin, while the ADP values of animals which w ere initially low rose. This is exactly the same pattern which occurred in anaphylactic shock. The term inal ADP values were elevated an average of 2.7 fold over the initial high ADP values and10.5 fold over the initial low ADP values. The average term inal ADP value in endotoxin shock was 0,4 ¡iM.
Four animals were used as controls to determ iiie the effect of human serum on nonsensitized rabbits. The human serum was substitu ted for the endotoxin injection and these same rabbits were further used for the endotoxin controls. The results are given in table I. There was no drop in the p la te le t count or arteria l p ressu re d u r in g the 16 hour p eriod .
Stages in Endotoxin Shock
♦ In itia l ApparentNormal J Shock Recovery
4 - 4 - 4 - ; J[ j [X
J ST Wave
Terminal ShockE AR L Y
L A T E
1/\T — 'vV T ' /AV Block
FIGURE 3. Stages observed during E. coli endotoxin induced shock as ev idenced'by the’electrocardiogram, platelet count and arterial pressure.
126 JABS, F E R R E L L AND ROBB
Initial Aggregation Partia l Recovery
F i g u r e 4. Plasma ATP, ADP, CP and platele t count changes observed in rabbits w ith initial low plasma ADP levels during endotoxin shock. Each point represents the average of four different animals, how ever, the time of analysis varied depending on the severity of the shock.
Neither ATP or CP showed any significant change during this time; however, the ADP level increased in all the controls over the 16 hour period. The stress of the operative procedure may have been the cause of this ADP increase. The 16 hour
ADP control values are much lower than the term inal ADP values in endotoxin shock and, in this respect, the present authors believe the control ADP increase was insignificant. In addition, the early ADP rise or drop in anaphylactic and
A TP inIox
Initial Aggregation Partia l Recovery
F i g u r e 5. Plasm a ATP, ADP, CP and platele t count changes observed during endotoxin shock in rabbits w ith initial high plasma ADP levels. Each point represents the average of four different animals, however, the time of analysis varied depending on the severity of the shock.
A r te r ia l P re ssu r e , P l a t e l e t Count, Plasma ATP, ADP and CP L ev e ls Observed in Four C ontrol Anim als
PLASM A C H A N G ES IN EN D O TO X IN AND A N A PH Y LA CTIC SH O CK 127
Plate l e t Arterial Adenosine 5' A d e n osine CreatineControl Time Count Pressure triphosphate diphosphate phosphateAnimals Hours 1 0 3 p e r mm'3 m m H g MM/1 vM/1 \iM/l
endotoxin shock was n o t observed in either the initial high plasma ADP or initial low plasma ADP controls.
W hile the data in figures 1 ,2 ,4 and 5 are presented as the mean of a group of animals, the ATP, ADP and CP values given in table I are typical in that the range of variation for each point w ithin a group never exceeded ± 10 percent. In addition, the mean coefficient of variation for the determ ination of 1.0 /u,M per liter ATP and CP were 1.5 percent and 1.9 percent, respectively. The value for an 0.1 /u-mole per liter ADP was 1.8 percent.
I t may be presum ed from the data presented that plasma ATP m easurem ents correlate with the energy state of the tissue. W hen arterial p ressures, p la te le t counts and electrocardiograms were abnormal, in both anaphylaxis and endotoxin shock, the plasma ATP levels wesre low. W hen the anim al’s condition improved the plasma ATP levels rose. It is proposed that m onitoring ATP plasma levels could become a clinical diagnostic
tool. Past reports29,44,45 on increased survivals in hemorrhagic shock by ATP infusion add Credence to the importance of monitoring plasma ATP levels.
The plasma CP level changes during anaphylactic shock appear to be less significant w hen com pared to endotoxin shock. The overall trend in anaphylaxis (figures 1 and 2) shows a gradual CP decline after one hour. Endotoxin shock shows an explicit pattern (figures 4 and 5). The term inal high elevation in plasm a CP in endotoxin shock may rep resen t the m obilization of cellular CP to the myocardium in an attem pt to compensate for any energy deficit. This CP is not com ing from p late le t release since neither CP or creatine phosphokinase have been found in platelets.31 The rise of CP to such a high level in the plasm a leads one to assum e th a t it is cap ab le of crossing the ce llu lar m em brane. This w ould give credence to the c la im 1 of the u se fu ln e ss .o f the in travenous in fusion of CP in the treatm ent of shock.
Another possible source of plasm a CP could be the cellular destruction of tissue by the direct effect of endotoxin or anoxia
128 JABS, F E R R E L L AND ROBB
and loss of CP from these tissues. In this case the CP plasma level would be an index to cellular damage. The identification of the plasma levels of ATP and CP together appear to be an excellent index in determ ining the animals’ m etabolic state during experim ental endotoxin shock. Low plasma ATP and CP levels in endotoxin shock appear to be not as critical as a low plasm a ATP level and a high CP level. The first designates an early stage of endotoxin shock and the latter indicates the possib ility of b e in g in an irreversib le stage of shock.
Plasma ADP level evaluation appears to be more complex than ATP and CP. In the aggregometer, 0.1 to 0.5 /u,M ADP caused reversible p latelet aggregation in human platelet rich plasma.47 Some of the initial plasma values observed in the anaphylactic experim ents were greater than 0.1 /u.M. High plasm a ADP values (greater than 0.1 jU-M) do not appear to be a prerequ isite for aggregation to occur since p latelet aggregation occurred in all the se n s itiz e d rab b its . H ow ever, a h igh plasma ADP level appears to be disadvantageous since all initial low plasma level ADP (ILPL-ADP) rabbits survived, while half of the initial high plasma level ADP (IHPL-ADP) rabbits died. The drop in the plasma ADP level after fiye m inutes (figure 2) in the IHPL-ADP rabbit is puzzling w hen one takes into account that the ILPL-ADP level rose at that time (figure 1).
Platelets can be aggregated by the direct effect of antibody antigen complex- ing.32,33,34 As previously stated, platelets can also be aggregated by 0.1 /¿M ADP in an aggregometer. Since this ADP level is exceeded in the IHPL-ADP rabbits, the ADP m olecules may potentiate extra stability to the immune complexing aggregates by ADP binding on the receptor sites of the platelets. Born4 demonstrated that the num ber of platelet receptor sites for ADP is about 2 x 105 per platelet. T hus, th is ad d e d s ta b ility cou ld be adequate to h in d er deaggregation or
cause the formation of larger aggregates which may maintain the anoxic state long enough in the IHPL-ADP rabbits to cause metabolic irreversibility. Death in anaphylactic shock can occur in five minutes. Our experience has shown that five m inutes of trachea clamp off is adequate tim e to kill an anesthetized rabbit causing electrocardiogram changes similar to term inal anaphylaxis (figure 6).
The circulating ADP of the ILPL-ADP rabbits may not be initially high enough to cause any appreciable ADP bind ing to p latelet receptor sites. Once the platelets have almost totally aggregated, the ADP level may become insignificant and the rise in observed ADP in the ILPL-ADP rabbits could be due simply to the existing anoxic state. C ircu lation may still be adequate for cellular ADP leakage causing the rise in plasma ADP. In the IHPL- ADP rabbits and the ADP level may have decreased owing to ADP utilization in platelet binding. This level does not rise in the critical time of five m inutes because of severe: circulatory blockage, whereby cellular ADP leakage was not adequately picked up by the blocked capillaries. After a time, ADP levels approach pre-existing normals in both types of survivals as the circulation and metabolism improved.
A more than co inciden tal sim ilarity exists betw een the ADP levels in initial endotoxin and anaphylactic shock. Again, all the IHPL-ADP rabbits in endotoxin shock showed a plasma ADP decline in the first five m inutes while the ADP level in the ILPL-ADP rabbits rose (figures 4 and 5). Endotoxin alone has been shown to be capable of aggregating platelets.10,38 The IH PL-AD P rabbits d ied w ith in a much shorter time when compared to the ILPL-ADP rabbits. Thus, a pathway similar to that of anaphylaxis could possibly exp la in these resu lts (figure 7). The IH PL-A D P initial aggregation may be caused by both the direct effect of endotoxin and the potentiating effect of ADP, w hile in the ILPL-ADP rabbits initial
PLASM A CHA NGES IN EN D O TO X IN AND A N A PHYLACTIC SHOCK 129
plate le t aggregation may have occurred mainly from the d irect effect o f the endotoxin alone. Large aggregates could have formed in the IH PL-A D P rabbits, ow ing to greater adhesions be tw een platelets, causing the plugging of larger vessels. Isch em ia co u ld be m ore p ro n o u n c ed in these rabbits causing early severe m etabolic d isturbance leading to the earlier deaths.
In endotoxin shock, the plasma ADP level gradually increases after the first hour (figures 4 and 5). It has b een established2,28,47 with hum an plasm a that 1 to 2 /U.M ADP was necessary to obtain irreversible p latelet aggregation in an aggregome- ter w ith p latelet procoagulant liberation. Such a high ADP level was never found in any o f o u r ex p e r im en ts . C o n s ta n t in e 8 found that only 0.3 A DP in platelet rich guinea pig plasma was necessary for irreversib le p late le t aggregation to occur in the aggregometer. This plasma value was exceeded in the terminal phase of endotoxin shock in our study where irreversible p late le t aggregation has been d e te rm in ed .23,39 O th e r investigators have found that the aggregation effect of ADP can be poten tia ted by additional factors.
For example, adrenaline potentiates the effect of ADP upon platelet aggregation.46
W hen a dose of ADP was unable to cause irrevers ib le aggregation itself, an irreversible wave was produced upon the add ition of a nonaggregating amount of cepha- lin.9 Endotoxin was observed to have a direct effect on the vessel wall with disruption of the endothelial lining and exposure of collagen.16 P la telet aggregation on collagen was shown to be irreversib le .15 T h e re fo re , p lasm a A D P leve ls alone may not be the decisive p la te le t re leasing agent in irreversib le endotoxin shock, but a com bination of factors (ADP, adrenaline, lipids, exposed collagen, etc.) could be responsible for the irreversible p la te le t ag g reg a tio n th a t occurs. O u r studies, have shown that the plasma ADP level does not reach the “ so-called” critical level of 1 to 2 /u.M n eed ed for irreversible aggregation in hum ans but does exceed the value found in guinea pigs. The increased plasma ADP level in terminal endotoxin shock could play an important role in the secondary irreversible p latelet ag g rega tion . T h is e f fec t co u ld b e in creased with the synergistic help of other sources.
There are several ramifications of these experiments which may have relevance to human subjects. For example, atheroscler- otics have an in c re ased sens i t iv ity to
F igure 6. Comparison o f e lectro ca rd io g ra m s during trachea clam p-off (A) and terminal anaphylactic shock (B).
130 JA BS, F E R R E L L AND ROBB
ILPL-ADP IHPL-ADPF fin ii Fnrlntnxin In iec ted
Endotoxin Platelet Aggregation and Proposed ADP Stimulated Aggregation
Insufficient Recovery Early Death (2 -4 Hrs.)
(Platelet ADP Sensitivity\ Increased I
By Adrenaline, Lipids I(Vessel Collagen Exposed)
F ig u r e 7. P ro p o sed sc h e m e for e n d o to x in induced shock.
Plasma, ATP-i- ADP t CPT |
Irreversible Platelet Aggregation Activated Intrinsic Clotting System
Death (8 -16 Hours )
ADP.47 Thus, monitoring the plasma ATP, ADP and CP levels may be of benefit to such individuals. It may be of value to m onitor period ically the plasm a ADP level in stored blood, since high levels of ADP may develop and prove detrim ental if given in transfusions. It is known that artificial systems such as kidney dialyzers and lung oxygenators become im peded by p late le t aggregates, although the exact cause has not been established. Perhaps a contributing factor is increased levels of ADP or CP resu ltin g from hem olysis owing to mechanical shear of the red cells.
Periodic sam pling of blood levels of ATP, ADP and CP during open heart surgery may give an indication of the m etabolic state of the patient. In this respect, a 50 fold drop in plasma ATP and CP concentrations of a coronary by-pass patient just prior to death has been reported by us.24
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132 JABS, F E R R E L L AND ROBB
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