Very early increase in nitric oxide formation and oxidative cell damage associated with the reduction of tissue oxygenation is a trait of blast casualties

Background. In our previous experimental studies, we found evidence for the increase of nitric oxide (NO) formation immediately after blast injury. In the present study we investigated whether NO overproduction was a trait for the period immediately after blast injury in humans. Concomitant metabolic disturbances were also studied, and compared to the alterations in other traumatized patients. Methods. Blast casualties (group B, n = 13), surgical patients with the hip replacement or fractures, not exposed to blast effects (group S, n = 7) and healthy volunteers as controls (group C, n = 10), were examined. Both arterial and venous blood samples were taken within 6 hours, and 24 hours after blast injuries or surgical procedures, respectively. Plasma levels of nitrite/nitrate (NOx), superoxyde anion (O2), sulfhydrils (SH), malondialdehyde (MDA) as well as acid-base status and other biochemical parameters (glucose, urea, creatinine, total proteins, albumin) were measured. Results. Significant, but transient increase in plasma NOx levels occurred only in group B. It was associated with the significant increase of hemoglobin oxygen (sO2) saturation of the venous blood and the concomitant decrease of its arterial venous difference. In group S the venous sO2 decreased, its arterial venous difference increased, while NOx levels were within the control limits. In both groups, other parameters of arterial acid-base status were kept within the control limits throughout the examined period. The decrease of SH levels were similar in the examined groups, while the increase of O2 was greater in group B. Conclusion. Early NO overproduction was a trait of blast injuries in humans, contributing to the reduction of tissue the oxygenation and intensifying the oxidative cell damage that had to be considered in the therapy of casualties with blast injuries. These alterations were different from those observed in other surgical patients without blast injuries.


Introduction
Due to the increasing frequency of combat operations throughout the world, blast injuries represent more often the sources of trauma (1í5).Also, there is an increasing number of victims of terrorism who need special medical care (6í7).
Exposure to blast overpressure or to a sudden rise in atmospheric pressure after an explosion results in the dam-age mainly of the gas-filled organs (8).The organs most affected by the blast wave are those that include the structures with different specific weights (ear, lungs, intestine) (3, 9).Animal models have shown that the exposure to non-lethal blast resulted in pathological changes mostly of the hollow organs associated with pulmonary hemorrhage and edema (1).In addition, it was documented that blast waves traversing the body initiated various humoral, metabolic and neuroendocrine changes involving various mediators and modulators (10í14).However, the assessment of injury severity (12) as well as the molecular mechanism involved in blast injury pathogenesis (13,15,16) are still a challenging issue.
Our experimental studies have shown that pulmonary blast injuries increased nitric oxide (NO) production, disturbed arginine metabolism, and altered the plasma-free amino acid pool in rabbits within first posttraumatic minutes (15).Recently, the others have also observed the progressive formation of NO in blood and other tissues following blast injury (1).Some authors suggested that the increase in NO formation had the neuroprotective function after these injuries (17), while the others demonstrated the possibility of NO involvement in cognitive deficits following blast injury-induced neurotrauma (14).Moreover, in previous experimental studies we observed that an early NO overproduction was involved in oxidative cell damages following the blast injuries (16,18).There was a possibility of its association with hypotension, insufficient tissue perfusion, and free oxygen radical formation during the initial period after the injury (13,16,18).
Considering these experimental observations, we could suppose that an early increase in NO formation and oxidative cell damages occurred in injured humans following severe explosions, too.Thus, the main purpose of the present study was to examine the early alterations in NO formation, tissue oxygenation and oxidative cell damages as well as their relationships in bombing casualties.We also compared them to metabolic disturbances in the patients treated surgicaly in the similar way, but not exposed to blast overpressure, in terms of similarities in anesthesia, surgery, infusions, transfusions, etc.Thus, we examined the plasma levels of nitrites plus nitrates (also called nitrates, NOx), superoxyde anion (O 2 .-),sulfhydrils (SH), malondialdehyde (MDA), as well as the characteristics of acid-base balance in the arterial and venous circulation in the hemodynamically stable patients immediatily treated by appropriate surgery.We demonstrated, as far as we know for the first time, the increase of NO formation in human casualties during the very early period after blast injuries and its involvement in the metabolic disturbances.

Methods
Patients.Blast and non-blast surgical patients (n = 20) admitted to the Military Medical Academy, Belgrade for surgery, as well as healthy volunteers as controls (n = 10), were included in this study.They were males, aged 20í61 years.There was no significant age difference between patients (32.6 r 12.9 years) and controls (38.9 r 7.9 years).The study was approved by the Ethics Committee of the Military Medical Academy, Belgrade.In order to examine the effects of blast overpressure, the patients were divided in two groups.Casualties injured during bombing in May 1999, were admitted to hospital via the Emergency Department, following initial evaluation, adequate resuscitation and surgery and included in the group of blast casualties (group B, n = 13).Patients admitted to the hospital due to orthopaedic surgery procedures (fractures, hip replacement), and not exposed to blast overpressure, were in the surgical group (group S, n = 7).
All the patients were mechanically ventilated (IPPC+PEEP between 3 and 7 cm H 2 O; ventilators Draeger Evita 4).Volume replacements with crystalloid solution (Ringer's solution) and colloids (Haemmacell and Dextran 70) with CVP range of 5í7cm H 2 O were used.Transfusion of red blood cells was indicated in the patients with hemoglobin < 100 g/l.Antibiotic prophylaxis was achieved by cephalosporins, metronidazole and aminoglycosides.As a result of these prompt therapeutic measures, all the patients were hemodynamically stable during the investigation.
Blood sampling and analysis.Heparinized arterial and venous blood samples were used for analysis, and were taken as soon as possible after the surgery (less than 6 hours after injury), as well as 24 hours after injury.Samples taken from the control group were considered zero-time samples.
Arterial plasma NOx, O 2 .-andMDA levels were used for the assessment of NO, free oxygen radical formation and oxidative cell damage, respectively.Capillary electrophoretic procedure (19) was used for NOx measurements, while colorimetric procedures were employed for O 2 .
-and MDA determinations (20,21).Plasma albumin, total proteins, glucose, creatinine, urea, and sodium levels were measured in venous plasma using the standard procedures (SMA-12, Technicon).Acid-base parameters, including: actual pH (pH), standard and actual bicarbonates (SB and AB), standard and actual base excess (SBE and ABE), partial pressure of carbon dioxide (pCO 2 ), oxygen partial pressure (pO 2 ), hemoglobin concentration (Hb) and its saturation with oxygen (sO 2 ), were measured both in arterial and venous blood samples (ABL-520, Radiometer, Copenhagen).Moreover, in order to consider the effects of dilution and plasma volumes, we expressed the obtained values of the measured parameters according to the actual plasma creatinine, protein, or hemoglobin levels in the examined patients, respectively.
Statistics.Values were expressed as means r SD.Analysis of variance followed by Bonferroni t-test, Student's t-test, and correlation analysis, were used for statistical evaluations.Values of p < 0.05 were taken as statistically significant.

Results
The average levels of arterial plasma NOx in control group (n = 10) were 39.1 Pmol/l r 9.4, ranging from 10.2 to 61.1 Pmol/l.These values were similar to those in other reports on healthy subjects (19,22).
The obtained results indicated that NOx levels in the group of blast casualties increased within the first posttraumatic hours (marked as < 6 hours).In group B, it reached an average concentration level above 120 Pmol/l that was sig-nificantly higher (p<0.05)than that in the controls (39.1 r 9.4 Pmol/l) or the one in the group S (60.1 r 16.1 Pmol/l) (Fig. 1).This increase was transient, and following 24 hours, even in group B NOx levels were within the control limits.Similar alterations were observed while analyzing NOx/creatinine or NOx/proteins ratios (Table 1).These findings confirmed that the increased NO formation occurred only after the blast but not after the other surgical injuries.Moreover, the increased values of the ratio between NOx and hemoglobin levels were observed only in group B (Table 1).Interestingly, the increased values of NOx/hemoglobin in the group of blast casualties persisted throughout the period of examination, and were significantly higher than the corresponding values in group S.
On the other hand, both in group B and S, the levels of glucose were above the control limits, while total plasma proteins were below the control limits throughout the examined period (Table 2).Also, an early posttraumatic decrease in plasma albumin occurred in both groups (Table 2).In addition, the transient increase of plasma urea levels was observed only in group B, particularly within very early posttraumatic period (Table 2).Althought in both groups of the patients creatinine levels were within the normal range, the values obtained in group B were significantlly higher than those in group S or control group (p<0.05)within the first hours after the injury (< 6 hours).
In both groups of the patients AB, SB, ABE, SBE, and pCO 2 were kept within the control limits both in arterial and venous blood, while the transient decrease of actual pH was observed only in group S (Table 3).Also, in both groups a certain decrease of hemoglobin level was observed (Table 3), while the arterial sO 2 , was similar to the control values (Fig. 2).However, there were the opposite trends in the venous sO 2 , and concomitant arterial-venous sO 2 differences alterations in the examined groups of patients (Fig. 2).Namely, within the first posttraumatic hours, in the patients without blast injuries (group S), the transient decrease in venous sO 2 was associated with the increase of its arterialvenous difference.On the other hand, in the patients with blast injuries, (B group), a progressive increase in venous sO 2 , and a concomitant decrease of its arterial -venous difference persisted throughout the examined period (Fig. 2).
Thus, there was statistically significant difference of arterial -venous sO 2 between the examined groups of patients.Also, in the blast casualtie, arterial -venous sO 2 difference correlated with venous sO 2 (r = -0.948,t = 9.879, p < 0.001), but not with its arterial values (r = -0.216,t= 0.733, p > 0.05).Moreover, the levels of MDA in group B were significantly higher (p < 0.05) than those in the controls, while in group S they were within the control limits throughout the examined period (Fig. 3).As compared to the controls, there was an increase of plasma O 2 .-level in both groups, with the greater increase in group B than in group S.However, the similar decrease of plasma SH occurred in both groups (Fig. 3).Interestingly, in the group of blast casualties no correlation between MDA and O 2 .-(r = -0.153),SH (r = -0.240)or NOx (r = -0.105)was observed.On the other hand, in group S MDA correlated with NOx (r = 0.577, t = 2.331, p < 0.05) and SH (r = 0.740, t = 3.649, p < 0.01), but not with O 2 .-(r =-0.252, t = 0.863, p > 0.05) levels.

Discussion
The main observations of the present study indicated an early increase in NO production in the group of casualties with blast injuries, but not in the group of surgical patients.The obtained findings were in accordance with the other experimental findings (1, 16í18), and were also consistent with recently reported experimental data on the significant increase in NOx of arterial plasma after pulmonary blast injury in rabbits, indicating an early and immediate increase in NO formation after the blast injury (15).They also confirmed our hypothesis that an early increase in NO formation occurred in humans exposed to the blast overpressure action.Moreover, it was likely that an early and prompt NO overproduction was one of the main characteristics of the blast injury.Considering that a blast wave caused the shearing and streching of the various tissues, associated with the distending vascular beds, and producing blood flow disturbances, we supposed that an early NO overproduction was the consequence of an early physical activation of endothelial constitutive NO synthase.It was probably caused by the vascular shear stress already occurring during the blast overpressure action.Different NOx alterations following the examined blast and non-blast injuries were consistent with this opinion.
It is known that NO represents an endogenously generated gas with a diverse array of biological functions (23), having both beneficial and cytotoxic effects (24í26).The role of NO following blast injuries has not been elucidated yet.Thus, before considering these findings in the therapy of blast injuries, it is important to resolve whether an early NO overproduction represents a part of the defense mechanism or it imposes damaging effects.
Previously, we suggested that an increase in endogenous NO production following a blast injury had the beneficial effect during the action of blast waves (16).The other authors also suggested that its beneficial effects affeced the blast injury (17).However, as larger quantities of NO seemed to inhibit cellular respiration and proliferation (27), and to have hypotensive effects, it was likely that the longer lasting increase in NO might be involved in the reduction of oxygen utilization in the peripheral tissue, following the blast injury.The obtained results indicated that during the first posttraumatic hours only in the group of casualties with blast injuries, the levels of plasma NOx were almost trebled, while MDA levels were doubled, compared to the controls.Moreover, the simultaneous increase of NOx, O 2 .-andMDA levels observed in the blast casualties suggesed an involvement of NO in oxidative cell damage during the early period after the injury.These findings supported the opinion that NO overproduction could have had the negative effects (24).
It has to be pointed out that the proper oxygenation of arterial blood was achieved in both groups B and S throughout the examined period.Also, in the examined patients, the majority of other parameters of acid-base status in the arterial circulation was kept within the control limits.However, the significant increase of the venous sO 2 and concomitant decrease of its arterial -venous difference in the group of blast casualties, almost inside the first hours, suggested the simultaneous reduction of oxygen delivery to the peripheral tissues.These findings require further elucidation before considering them in the trentment of blast casualties.
Interestingly, in the group of non-blast surgical patients (group S), metabolic alterations were different.Namely, in the group S venous sO 2 decreased within the first hours, while arterial -venous sO 2 difference increased, suggesting the increased oxygen utilization in the peripheral tissue of non-blast surgical patients.Higher venous pCO 2 obtained in this group of patients than in the casualties with blast injuries also confirmed that.However, comparing both to controls and to the values in group B, a certain decrease of venous pH in group S was observed, suggesting the intensifications of glycolitic rather than the aerobic metabolic pathways.In our previous experimental studies, certain disturbances of acid-base balance were observed both in arterial and venous circulation, following blast or other injuries (10,13,28).It probably occurred owing to the lack of any therapy in these experimental investigations.Moreover, a decrease of the venous sO 2 was observed following injuries associated with hypovolemia (28).However, due to the proper medical treatment in the present study, the examined patients were hemodynamically stable during the whole period of investigations.In addition, the applied procedures, such as transfusion and/or infusions, kept acid-base status in arterial circulation mostly within the control limits.In fact, the observed disturbances of acid-base status occurred mainly venous circulation in both group B and S. Interestingly, their trends were different, and completely oppo-site, suggesting the different metabolic disturbance following the examined injuries.In our opinion, that might be attributed partly to the increase of NO formation observed only in the group of blast casualties.Namely, in the group of casualties without blast injuries, the plasma levels of both NOx and MDA were within the control limits, suggesting the normal NO formation, without the presence of oxidative cell damage.The other authors reported the decrease of plasma NOx levels in patients after the surgery, unless they became clinically septic (22), as well as some increase of NOx after thoracic surgery (29).
Finally, it has to be considered that we measured NOx, as the main, final, and stable oxidative products of NO metabolism in mammals (30,31).These anions are commonly used for the monitoring of changes in the concentration of NO released in physiological fluids and the rate of the whole body NO synthesis (32,33).However, they are cleared primarily by the kidneys (34), and their plasma levels could be significantly influenced by the alterations in renal function.
In the present study, the levels of plasma creatinine were within the normal ranges, not exceeding 130 Pmol/l, indicating a normal renal function in the whole group of the examined patients.However, in the group of blast casualties the shift to the higher creatinine levels occurred, and statistically higher values in group B than in S were obtained.In spite of that, in the group of blast casualties, the increased plasma NOx / creatinine ratios occurred, confirming the existence of the increased NO formation, particularly within very early posttraumatic period.In addition, an early increase of plasma NOx/total protein as well as NOx/hemoglobin ratio observed only in the group B confirmed that, as well.Moreover, in the group of blast casualties, the increased values of NOx/hemoglobin ratio, higher than the values in group S, persisted throughout the examined period.Finally, it seemed likely that the increase of NO production might cause the observed reduction in oxygen delivery to the peripheral tissues in the examined blast casualties.Recently reported opinion that NO could increase hemoglobin O 2 -binding affinity (35), has confirmed this possibility.
Conclusion.An early NO overproduction did characterized blast injury in humans, contributing to the reduction of tissue oxygenation and intensifying oxidative cell damages in the casualties with blast injuries.These alterations were different from those observed in the examined surgical patients with no blast injuries R E F E R E N C E S 1. Elsayed Fig. 1. í Arterial plasma nitrite/nitrate (NOx) levels in blast casualties (group B) and non-blast surgical (group S) patients.*** p<0.001 vs O-time (controls).x p<0.05 B vs. S group.
Fig. 2. í Arterial and venous blood hemoglobin saturation with oxygen (sO2) and its arterial -venous difference in blast casualties (group B) and non-blast surgical patients (group S) * p<0.05 vs O-time (controls).x p<0.05 B vs. S group.

Table 2 Plasma levels of glucose, urea, creatinine, total proteins and albumin in blast casualties (group B) and non-blast surgical patients (group S)
* p<0.05; † p<0.001 vs O-time (controls).‡ p<0.05 B vs. S group.

Table 3 Parameters of acid-base status in arterial (a) and venous (v) blood of blast casualties (group B) and non-blast surgical patients (group S)
* p<0.05 vs O-time (controls).† p<0.05 B vs. S group.