Battle Casualties in Korea, Studies of the Surgical Research Team, Volume I

A Study of Plasma and Erythrocyte Cholinesterase Activity in Combat Casualties

First Lieutenant John P. Frawley, MSC, USAR
Lieutenant Colonel Curtis P. Artz, MC, USA
and
Captain John M. Howard, MC, USAR

with the technical assistance of
Corporal Charles H. Adams, AMEDS
Corporal John W. Bohley, AMEDS

The work of a great number of investigators, starting with that of Loewi,¹has fully established the fact that autonomic nervous system effects are associated with the liberation of acetyl choline at synapses and neuromuscular junctions and the concurrent hydrolysis of acetyl choline by cholinesterase. The discovery of cholinesterase enzymes in the plasma and erythrocytes of animals has made it possible for clinical investigators to study the activity of these enzymes in relation to autonomic activity and other physiological functions. In the course of these studies a relationship between blood pressure and plasma cholinesterase activity was noted by Antipol²and confirmed by Morelli.³ Hall and Lucas,⁴ however, were unable to repeat the finding. A decreased plasma and erythrocyte cholinesterase activity has been observed to be a consistent and sensitive index of neuromuscular cholinesterase inhibition by such drugs as physostigmin,⁵ di-isopropylfluorophosphate,^{6, 7} and tetraethyl pyrophosphate.⁷ Other correlations have been reported with liver function by Antipol² and confirmed by Snyder,⁸ with prolonged barbiturate therapy by Schurtz,⁹ with malnutrition by McCance.¹⁰

In the present study, an attempt has been made to investigate the response of the autonomic nervous system to injury as might be reflected by the plasma and erythrocyte cholinesterase activity of combat casualties. It must be noted that an indirect approach of this nature to the measurement of autonomic activity is undesirable, but unavoidable except in experimental animals. Nevertheless, with careful interpretation, certain indications of autonomic activity are possible.

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Method

All patients studied were young, male, United Nations combat casualties, sustaining injuries from artillery, mortar, grenade, land mine or small arms fire in Korea during the summer of 1953. Samples of whole, heparinized blood were collected from 14 such patients at several intervals following admission to the forward surgical hospital. Sampling time was frequently influenced by patient condition, requirement for evacuation and military situation. All samples were centrifuged for 10 minutes at 2,000 rpm. and plasma was withdrawn. The plasma was diluted 1/50 with distilled water and maintained up to 10 days in the deep freeze until analyzed. The erythrocytes were washed with isotonic saline, centrifuged for 10 minutes at 2,000 rpm., rewashed and centrifuged for 20 minutes at 2,000 rpm. Duplicate 1 ml. aliquots of the packed cells were withdrawn with separate serological pipettes and laked in 20 ml. of distilled water for 30 minutes. The laked cell solution was then maintained in the deep freeze until analyzed. The effect of freezing and storing on the enzyme`s activity was checked and found to be negligible.

Actual measurement of the enzyme`s activity was accomplished by the electrometric method of Michel.¹¹ Although this procedure employs a temperature of 25° C for the reaction, this author has previously used a 35° C reaction temperature and obtained comparable results.⁷ The studies reported herein were conducted at 35° C for obvious technical reasons. Control values on nine normal individuals were checked and ranged for plasma from 0.80 to 1.63 and for erythrocytes from 0.50 to 0.80 unit (DpH per hour).

Clinical Summaries of Patients

Patient No. 1. This soldier was wounded at 0600on 4 June 1953, suffering shell fragment wounds, penetrating the lung, diaphragm, liver, both arms and back. He received a minimum of blood and his course was uneventful.

Patient No. 2. This 22-year-old soldier was wounded at 1450 on 24 June 1953 by shell fragments. He suffered multiple penetrating wounds of the neck, face, chest and all extremities. He was evacuated by helicopter and arrived at 46th Army Surgical Hospital at 1540 hours in very poor condition. He received 11 pints of blood and 2 bottles of dextran, preoperatively, and a total of 29 pints of blood and 3 of dextran within the first 24 hours.

Patient No. 3. This 22-year-old soldier was wounded at 1145 on 26 June, suffering shell fragment wounds of the abdomen, with lacerations of the liver and penetrating wounds of the right hip and leg. He arrived at the hospital in very poor condition after 11 pints of blood at forward medical units. He received 8 pints of blood in the operating room. His treatment required a total of 48 pints of blood during the first 24 hours because of persistent hypotension.

Patient No. 4. This 21-year-old soldier sustained shell fragment wounds from artillery fire at 2230 hours on 10 June. He received penetrating wounds of the

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right side of the chest, forearm, diaphragm and liver. He arrived at the hospital about 0100, 11 June, in good condition. He required minimum blood during his whole course, which was uneventful.. He was evacuated on 13 June 1953.

Patient No. 5. This 24-year-old soldier sustained mortar fragment wounds at 0800 hours on 6 June 1953. He arrived at the hospital at approximately 1100 hours with blood pressure 70/40 and a compound fracture of the right tibia. He received 3 pints of blood, suffered a mild transfusion reaction, and was evacuated the following day.

Patient No. 6. This 20-year-old soldier sustained shell fragment wounds at 0300 hours on 15 June 1953. He arrived at the hospital at 1105 hours with penetrating wounds of the chest and abdomen, involving the diaphragm and stomach. His blood pressure was 134/60 on arrival. He received 3 pints of blood. His course was uneventful.

Patient No. 7. This 38-year-old soldier was wounded by shell fragments at 0500 on 16 June 1953. He suffered penetrating wounds of the flank, abdomen and diaphragm. He arrived at the hospital at 1230 in good condition. He received 1 pint of blood preoperatively. His course was uneventful.

Patient No. 8. This 21-year-old soldier was wounded by mortar fragments about 1800 on 16 June 1953. He suffered a penetrating wound of the left hip. He received 2 units of dextran and 2 pints of blood prior to arrival at the hospital. His pressure upon arrival at 2130 hours was 100/00. He received an additional 2 pints of blood during surgery. His course was uneventful.

Patient No. 9. This 22-year-old soldier was wounded at 0630 on 18 June 1953 by shell fragments. He sustained a traumatic amputation of the right foot, penetrating wound of the chest, compound fracture of the left leg, and laceration of the right thigh, face and forearm. He arrived at the hospital at 0800 by helicopter with his pulse and blood pressure imperceptible. He received 2 pints of blood preoperatively (blood pressure 94/60). His recovery was sluggish.

Patient No. 10. This 20-year-old soldier was wounded at 0705, 19 June 1953, by mortar fragments. He suffered lacerations of the head and face, and penetrating wounds of both thighs and right arm. He arrived at the hospital at 0845 in moderate shock, blood pressure 90/60. He received 4 pints of blood and 2 units of dextran preoperatively and an additional 4 pints of blood in the operating room. His course was uneventful.

Patient No. 11. This 20-year-old soldier was wounded at 0445 on 7 July 1953 by small arms fire. He sustained a perforation of the abdomen, with penetration of the inferior vena cava, right ureter and duodenum. He arrived at the hospital at 1000 hours, blood pressure 100/64. He received 6 pints of blood preoperatively, 17 pints in the operating room, and 16 pints postoperatively. In addition, he received 4 units of dextran and levophed. His course was sluggish, because of a persistent hypotension unassociated with external hemorrhage.

Patient No. 12. This 26-year-old soldier sustained a penetrating wound of the abdomen at 1155 on 12 June 1953. He arrived at the hospital with a pressure of 130/70. He received 3 pints of blood preoperatively, 2 pints in the operating room and 1 pint postoperatively. In addition, he received 1 unit of dextran postoperatively. His course was uneventful.

Patient No. 13. This 23-year-old soldier sustained shell fragment wounds of the back and abdomen at 0950, on 27 June 1953. He suffered severe intra-abdominal hemorrhage and arrived at the hospital at 1210, 27 June, by helicopter. His blood pressure was 112/76. He received 5 pints of blood preoperatively. His subsequent course was uneventful.

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Patient No. 14. This 22-year-old soldier sustained shell fragment wounds of both hands, right arm, chest and abdomen including perforation of the stomach. The time of wounding was 2000 hours on 15 June1953. He required a minimum of blood preoperatively and operatively. He was evacuated the following day.

Results

Table 1 shows the plasma cholinesterase activity for these casualties and Table 2 shows the erythrocyte activity. It will be noted that all patients except Nos. 3 and 9 showed a low plasma cholinesterase activity at one interval of measurement or another. Erythrocyte activity was normal, or only slightly below normal, in all patients except No. 12; however, no particular significance can be attached to this patient since his treatment and recovery were without special incidents.

Table 1. Plasma Cholinesterase Activity* (DpH per Hour) of 14 Combat Casualties

Patient	Preop.	Days Postoperative
		1	2	3	4	5	6
1	---	0.68	---	0.65	0.57	0.55	0.48
2	---	0.56	0.60	0.60	0.58	0.41	0.46
3	---	---	0.76	0.70	0.81	0.69	---
4	0.52	0.59	---	---	---	---	---
5	0.55	0.56	---	---	---	---	---
6	---	---	0.64	0.54	0.52	---	---
7	0.86	0.81	0.32	---	---	---	---
8	---	0.38	0.32	0.35	---	---	---
9	1.13	0.82	0.79	---	---	---	---
10	0.84	1.06	0.87	0.65	---	---	---
11	---	0.85	0.79	---	---	---	---
12	---	---	---	---	0.43	0.45	0.31
13	1.01	1.12	0.81	---	---	---	---
14	0.58	0.62	---	---	---	---	---

*Normal range 0.80 to 1.63

Several samples of whole blood from the blood bank were sampled for cholinesterase activity by identical technic to that used on the patients. Table 3 shows the enzyme activity of the plasma and erythrocytes of these samples. The most significant observation on these samples was the very low activity of the erythrocytes after 11 days of storage. Since many patients received massive blood transfusions, the activity of the transfused blood must be considered in evaluating the cholinesterase activity of the patient.

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Table 2. Erythrocyte Cholinesterase Activity*(DpH per Hour) of 14 Combat Casualties

Patient	Preop.	Days Postoperative
		1	2	3	4	5	6
1	---	0.57	---	0.59	0.55	0.57	0.61
2	---	0.61	0.50	0.30	0.38	0.41	0.51
3	---	---	0.61	0.52	0.52	0.40	---
4	0.59	0.55	---	---	---	---	---
5	0.75	0.62	---	---	---	---	---
6	---	---	0.46	0.39	0.42	---	---
7	0.44	0.56	0.68	---	---	---	---
8	---	0.72	0.50	0.39	---	---	---
9	0.62	0.41	0.40	---	---	---	---
10	0.50	0.68	0.53	0.62	---	---	---
11	---	0.53	0.60	---	---	---	---
12	---	---	---	---	0.22	0.22	0.30
13	0.66	0.62	0.52	---	---	---	---
14	0.57	0.73	---	---	---	---	---

*Normal range 0.50 to 0.80

Table 3. Plasma and Erythrocyte Cholinesterase Activity (DpH per Hour) of Stored Whole Blood in Korea

Age in Days	Plasma	Erythrocytes
11	0.56	0.63
15	0.64	0.11
16	0.68	0.11
20	1.17	0.30
20	1.08	0.11
20	0.79	0.09
20	1.04	0.10
20	1.32	0.17

Discussion

The lowered plasma cholinesterase activity observed in these patients is slight and probably of little significance to autonomic activity. Such plasma activity is found commonly in patients with metabolic disorders,^2,10 especially decreased liver function.⁸ The decrease observed in these traumatized patients may be a function of decreased or altered hepatic function, commonly observed in such patients.¹²

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The slight decrease in erythrocyte cholinesterase observed in some of these patients is also less than that accompanying impaired autonomic activity.^6,7 Since the erthrocytes of stored whole blood have been shown to be low in activity, much of the patients` decreased enzyme activity maybe due to transfused blood.

An attempt to correlate plasma or erythrocyte activity with severity of injury, type of injury, liver wounds, blood pressure, and quantity of transfused blood, has been fruitless. The patients studied were too few to provide adequate sampling for any of these refinements. However, it must be noted that decreased cholinesterase activity was frequently observed, and although no conclusion of autonomic impairment is justified, an abnormal condition exists. The interpretation of these findings is made difficult by the lack of an established function for either plasma or erythrocyte cholinesterase. The ability of these proteins to hydrolyze acetyl choline is apparently incidental to their true and undiscovered physiological function. When this function is elaborated, these findings may assume added significance.

Conclusion

Serial plasma and erythrocyte cholinesterase determinations were performed on 14 Korean combat casualties at varying intervals post wounding. In general, decreased plasma cholinesterase activity was common, but the degree was not sufficient to indicate autonomic changes from inactivation of motor end plate cholinesterase. The plasma activity was more suggestive of the changes observed with decreased synthesis of this enzyme in liver disease. Erythrocyte cholinesterase was slightly below normal but this appeared to be a function of decreased enzyme activity of whole stored blood.

References

1. Loewi, O.: Transmission of Nervous Impulses. Pflüger`s Arch. 189: 239, 1921.

2. Antipol, W., Tuckman, L., and Schifrin, A.: Cholinesterase Activity of Human Sera, with Special Reference to Hyperthyroidism. Proc. Soc. Exper. Biol. & Med. 36: 46, 1937

3. Morelli, A.: Cholinesterase del siero ed impertenione arteriosa. Progresso Medico Napoli 3: 628, 1947.

4. Hall, S. E., and Lucas, C. C.: Cholinesterase Activity of Normal and Pathological Sera. J. Pharm. and Exp. Therapy 59:34, 1937.

5. Loewi, O.: Uber den mechanismus der vaguswirkung von physostigmin. Pflüger`s Arch. 214: 689, 1926.

6. Mazur, A., and Bodansky, O.: Mechanism of in viro and in vitro Inhibition of Cholinesterase by DFP. J. Biol. Chem.163: 261, 1946.

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7. Frawley, J. P., Hagen, E. C., and Fitzhugh, O. C.: Comparative Pharmocological Study of Six Anticholinesterase Compounds. J. Pharm. and Exper. Ther. 105: 156, 1952.

8. Snyder, H. E., Snyder, C. D., and Bunch, L. D.: Liver Function as Determined by Serum Cholinesterase Activity. AMA Arch. Surg.66: 426, 1953.

9. Schurtz, F.: Effect of Barbiturates on Serum Cholinesterase. J. Physiol. 102: 259, 1943.

10. McCance, R. A.: Serum Cholinesterase in Undernutrition. Proc. Roy. Soc. Med. 43: 272, 1953.

11. Michel, H. O.: Electrometric Method for the Determination of Plasma and Erythrocyte Cholinesterase. J. Lab. and Clin. Med. 34:1564, 1949.

12. Scott, R., Howard, J. M., and Olney, J. M.: Hepatic Function in the Battle Casualty (Chapter 8, this volume).