History

Days Post-wound

No. of Obser.

No. below 1.0

Per cent below 1.0

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

Investigations of Serum Protein Changes in Combat Casualties*

First Lieutenant John P. Frawley, MSC, USAR
Captain John M. Howard, MC, USAR
Lieutenant Colonel Curtis P. Artz, MC, USA
and
Pearl Anderson
with the technical assistance
of
Corporal Charles Adams, AMEDS

Previous studies in the disturbances of protein equilibrium following surgery and injury in man and experimental animals have shown a catabolic phase or period of negative nitrogen balance followed by an anabolic phase or period of positive nitrogen balance.^{1, 4, 5} The serum protein changes during the catabolic phase were described by Cuthbertson and Tompsett² primarily as a loss of albumin and an increase in globulin. However, the procedure used was a salt precipitation technic that did not separate the alpha¹ and alpha² globulin fractions from the albumin. More recently, Hock-Legeti, Irvine and Sprinkle,³ using paper-strip electrophoresis, studied the serum protein changes in a group of 45 surgical patients. Their results indicated a significant decrease in the albumin fraction and an increase in alpha¹ and alpha² globulin fractions in 80 per cent of the patients within 4 days following operation.

Purpose

The massive trauma and extensive surgical procedures experienced by combat casualties are relatively uncommon in civilian medical practice. A thorough understanding of the quantity as well as quality of the metabolic response is important for the advancement of the medical treatment of these patients. It is for this reason that these studies were undertaken. The specific purpose of this study was to investigate the serum protein changes following major trauma and surgery; and to determine if a qualitative or quantitative difference in the response existed between various types of wounds.

*In press: Archives of Surgery.

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Methods

The changes in serum protein fractions were investigated in 33 combat casualties. All patients studied were young, male, United Nations combat casualties, sustaining injuries from artillery, mortar, grenade, land mine, or small arms fire. Some were critically injured; sometimes with imperceptible pulse and blood pressure at the time of admission. In general, they represent the types of casualty that required immediate resuscitation including extensive surgical treatment.

Serial samples of venous blood were collected for serum from all patients beginning immediately after operation and continuing to the time of evacuation. Preoperative samples were drawn if plasma expanders or albumin had not been previously administered, and if hemorrhage was under control. Many patients received dextran or modified fluid gelatin postoperatively. The protein studies herein reported are exclusive of the period when the serum contained greater than 3 mg./ml. of the plasma expander, as evidenced by chemical analysis. This was necessitated by the interference of the expander with the fractionation procedure at concentrations above this level.

The procedure employed for protein fractionation was based on neutral salt flocculation, using 28 per cent sodium sulfite-a procedure that gives results most closely approximating those obtained by electrophoresis.⁶ In general the procedure consisted in the determination of total protein concentration spectrophotometrically with the biuret reagent, and the similar determination of the albumin fraction remaining in solution after flocculation of the globulin with 28 per cent sodium sulfite and a tween-ether solution. Conversions are made by comparison with known concentrations of human albumin solution. Because of the empirical nature of the procedure, the technic was performed under rigidly controlled conditions and always by the same investigator. As a check on the proficiency and reproducibility of the technic, a pooled sample of serum was subdivided into 24 small aliquots and stored in the frozen state. With each set of analyses, one aliquot of pooled serum was run. In our hands, the average a/g for these analyses on pooled serum was 1.50, with a variation of ±0.06.

After collection and separation of the serum, all samples were stored in the frozen state. Analysis was performed on all samples from the same patient simultaneously. Several samples were preserved with 1:10,000 merthiolate and returned to the Zone of Interior for complete electrophoretic fractionation.

A more detailed description and discussion of the procedure may be found in the Appendix.

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FIGURE1.
Albumin-globulin ratios in 16 combat casualties with abdominal wounds.

Results

Table 1 summarizes the serial total protein and albumin-globulin ratio for the 33 patients studied. Patients 1 to 6 and 17 to 25 received dextran preoperatively or postoperatively, and patients 7 to 13 and 26 to 30 received gelatin. The first 16 patients listed sustained abdominal perforations whereas the latter 17 patients sustained only extremity wounds.

Figures 1 and 2 are scattergrams of the albumin-globulin ratio for the patients with abdominal injuries and those with extremity injuries respectively. Tables 2 and 3 summarize the changes in albumin-globulin ratio following abdominal and extremity injury.

As can be seen from Figures 1 and 2, a progressive decrease in the albumin-globulin ratio occurs with nearly all patients. The degree of the response is noted to be more pronounced with patients sustaining abdominal wounds.

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FIGURE2.
Albumin-globulin ratios in 17 combat casualties with extremity wounds.

Table 1. Total Protein and Albumin-Globulin Ratios in 33 Patients

Patient No.	Wound Description	Time Postwound (Hrs.)	Total Protein (gm./100 ml.)	Albumin-Globulin Ratio
1	Abdominal Multiple perforations of small intestine, fracture of ilium	17 65 89	7.8 7.0 6.8	1.1 0.8 0.8
2	Abdominal-Extremity Multiple perforations of small and large intestines, perforation of iliac vein and artery, of both buttocks, right forearm and flank	11 42 66 89	7.7 6.0 7.3 7.3	1.1 0.8 0.6 0.7
3	Abdominal-Extremity Perforations of sigmoid colon, of sacral femoral arteries; fractures of sacrum, both tibia; fibula; innumerable soft-tissue wounds	28 61 85	6.7 5.8 5.4	1.2 0.7 0.7
4	Abdominal-Extremity Perforations of abdomen, and extremities, transfusion reaction oliguric 24 hours postoperative	55 77 101 125	5.1 5.2 5.7 5.6	0.9 0.8 0.8 0.8

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Table 1. Total Protein and Albumin-Globulin Ratios in 33 Patients-Continued

Patient No.	Wound Description	Time Postwound (Hrs.)	Total Protein (gm./100 ml.)	Albumin-Globulin Ratio
5	Abdominal Perforations of colon and injury to cauda equina, paralysis of both legs	24 52 76 100 124 148 172 196	6.0 5.2 5.4 5.1 5.1 4.8 4.6 5.2	1.3 1.0 1.0 0.9 0.8 0.8 0.8 0.7
6	Abdominal-Extremity Wounds of both legs, left thigh, scrotum, face; perforations of chest and large intestine	3 11 63 91 21	7.4 7.5 6.4 6.8 5.7	0.9 0.9 0.9 1.0 1.2
7	Abdominal Perforations of ascending colon and liver; laceration of kidney	26 40 64 89	6.4 5.3 5.8 6.9	1.1 0.9 1.0 0.8
8	Abdominal Perforations of ileum and rectum, compound fracture of sacrum	41 65 89	5.7 6.0 5.8	1.0 0.9 0.9
9	Abdominal Gunshot wound, perforations of the liver, kidney and colon. Renal insufficiency postoperatively. Died of renal abscesses	4 12 42 66 89 113 137 163	6.9 8.3 4.6 4.6 5.1 5.8 5.8 6.2	1.5 1.0 0.8 1.3 0.9 1.0 1.0 0.9
10	Abdominal-Extremity Perforations of intestine, penetrating wounds of both thighs and a fracture of right femur	12 33 45 69 93 111 141 165 189	6.3 5.7 5.7 5.9 6.1 5.3 5.5 5.5 5.8	1.5 1.6 1.5 1.3 1.3 1.4 1.0 0.9 0.9
11	Abdominal-Extremity Perforations of ascending colon and right flank; compound fracture of right ilium	6 21 27 43 67 91	5.2 4.7 4.6 4.9 5.0 5.1	1.1 1.3 1.3 1.1 0.9 0.8

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Table 1. Total Protein and Albumin-Globulin Ratios in 33 Patients-Continued

Patient No.	Wound Description	Time Postwound (Hrs.)	Total Protein (gm./100 ml.)	Albumin-Globulin Ratio
12	Abdominal Lacerations of ileum and sigmoid colon, retroperitoneal hematoma; multiple soft tissue wounds of buttocks, hip and back	9 26 38 62 86	7.1 6.5 6.5 9.4 6.2	1.4 1.2 1.2 1.1 1.0
13	Abdominal Perforations of intestine and spleen; splenectomy performed	5 9 16 22 32 56 80	7.4 6.3 6.9 7.0 7.0 5.6 5.6	1.1 1.1 1.2 1.0 1.1 0.9 0.9
14	Abdominal Perforations of intestine and bladder	1 5 13 16 22 34 58 83 107 131	7.1 6.2 6.3 6.3 6.9 6.5 6.5 6.0 6.6 5.4	1.3 1.5 1.5 1.5 1.3 1.4 1.1 1.3 1.2 1.2
15	Abdominal-Extremity Perforation of intestine and liver; penetrating wounds of right hip and leg	5 7 22 46 70 94 117 141	5.3 5.6 7.5 5.7 6.9 6.3 5.1 4.7	1.4 1.2 1.1 1.1 0.9 0.7 0.9 0.7
16	Abdominal-Extremity Perforations of lung, diaphragm, liver, both arms and back	10 26 38 62 86	7.2 7.2 6.2 7.2 6.3	1.5 1.3 1.1 0.9 1.0
17	Extremity Compound fracture of mandible and hard palate; extensive soft tissue wounds of face	38 62 86	5.9 6.5 6.8	1.1 1.1 1.0
18	Extremity Massive wound of left thigh; fracture of left femur and perforation of right thigh. High thigh amputation performed.	49 73	6.5 6.9	0.4 0.3

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Table 1. Total Protein and Albumin-Globulin Ratios in 33 Patients-Continued

Patient No.	Wound Description	Time Postwound (Hrs.)	Total Protein (gm./100 ml.)	Albumin-Globulin Ratio
19	Extremity Fracture of right leg and injury to posterior urethra.	56 80	6.6 7.6	0.9 0.9
20	Extremity Large wound of shoulder and pectoral region; fragment in cardiac musculature	12 40 64 88 112 136 160	7.3 6.8 6.8 6.7 7.1 7.1 7.1	1.2 1.3 1.2 1.2 1.1 1.1 1.1
21	Extremity Compound fracture of right tibia and contusion of left leg	4 31 44 68	5.9 5.7 5.9 6.2	1.2 1.0 1.0 0.9
22	Extremity Perforating wounds of left thigh and left arm; large laceration of brachial artery	6 24 48 72	7.4 6.4 6.4 6.5	1.4 1.1 1.2 1.2
23	Extremity Fragment wounds of left thigh, compound fracture of femur; massive muscle damage	3 52	8.1 5.9	1.2 1.0
24	Extremity Compound fracture of left mandible and left arm, perforating wound of left shoulder, right arm and right leg.	16 44 97	6.6 5.8 6.0	1.4 1.1 1.1
25	Extremity Traumatic amputation of right foot and ankle, penetrating wounds of right leg, fracture of 3 right fingers.	20 23 35 59 83	5.8 5.8 6.2 6.4 6.0	1.1 1.1 1.2 1.2 1.1
26	Extremity Perforating wound of right shoulder	9 22 28 40 64 88 112 136 160 184	6.8 5.3 5.4 5.3 5.0 4.8 5.0 5.0 5.1 6.3	1.8 2.0 1.8 1.5 1.5 1.3 1.4 1.4 1.2 0.9

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Table 1. Total Protein and Albumin-Globulin Ratios in 33 Patients-Continued

Patient No.	Wound Description	Time Postwound (Hrs.)	Total Protein (gm./100 ml.)	Albumin-Globulin Ratio
27	Extremity Perforating wound of left shoulder, severe compound fracture of head of humerus	5 16 22 33 57 81 105 129 153 177	7.0 5.4 5.2 5.8 6.3 5.8 6.2 6.6 6.2 6.6	1.6 1.8 1.8 1.6 1.5 1.4 1.2 1.4 1.2 0.7
28	Extremity Traumatic amputation of both legs and penetrating wound of right buttock	3 10 19 31 55 76	6.8 5.5 6.1 7.2 6.6 6.1	1.4 1.4 1.2 1.2 1.1 1.0
29	Extremity Traumatic amputation of right hand, multiple wounds of right thigh and compound fracture of right femur	30 40 64 88	6.0 5.5 5.7 5.5	1.4 1.1 0.9 0.9
30	Extremity Compound fracture of left ramus and body of mandible; penetrating wounds of left shoulder, arm and right forearm	7 16 19 22 32	6.5 8.0 6.0 5.5 5.3	1.6 1.5 1.3 1.5 1.2
31	Extremity Penetrating wounds of both thighs and right arm; laceration of head and face	6 26 56 76 140	6.1 6.3 5.4 5.8 5.1	1.6 1.5 1.5 1.2 1.2
32	Extremity Penetrating wound of neck; laceration of jugular vein on left side	24 48 72 96	7.5 8.6 8.0 8.2	1.0 0.9 0.9 0.9
33	Extremity Traumatic amputation of left leg at thigh; multiple fragment wounds of right leg and left arm	14 18 24 27 30 50 74 98	6.2 6.3 6.6 5.8 5.7 6.0 6.6 6.5	1.2 1.5 1.3 1.1 1.2 1.3 1.2 1.1

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Table 2. Albumin/Globulin Changes in 16 Patients With Abdominal Injuries

Table 3. Albumin/Globulin Changes in 17 Patients With Extremity Injuries

Table 4. Relative Per Cent Protein Fractions per Electrophoresis

Patient No.	Time Postwound Hrs.	Albumin	Alpha¹	Alpha²	Beta	Gamma	Total Protein gm./100 ml.
10	24 39 99	57% 46% 41%	12% 18% 25%	10% 14% 19%	11% 7% 9%	10% 15% 6%	6.0 5.1 5.1
30	26 76	62% 47%	15% 25%	11% 17%	6% 6%	6% 5%	6.3 5.8
31	10 62	59% 46%	4% 10%	14% 21%	6% 6%	17% 17%	7.2 7.2

In order to evaluate whether this shift in albumin-globulin ratio was due to a loss of albumin or an increase in globulin, several samples were preserved for electrophoretic analysis. These results are reported in Table4. These results indicate that there is a decrease in

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the relative per cent of albumin concurrent with an increase in the relative per cent of alpha¹ and alpha² globulin. No consistent change in the beta and gamma globulin levels was noted. This is in agreement with the findings of Hock-Legeti³ in general surgical patients.

Discussion

Previous investigators have studied the changes in serum protein following surgery. They have described decreases in albumin-globulin ratio, decrease in albumin and increase in alpha¹ and alpha² globulin fractions. Our results on 33 seriously injured combat casualties reflect the similar qualitative changes, but quantitatively the changes are more pronounced. Tables 2 and 3 indicate that 67 per cent of the patients with abdominal wounds and 33 per cent of the patients with wounds of the extremities possessed serum albumin concentrations which were less than 50 per cent of the total protein on the third post-wound day. Similar results were obtained on the fourth post-wound day. Previous to this time a progressive decrease in the albumin-globulin ratio had been observed.

Although the quality of the response may be similar for patients with abdominal wounds and for patients with wounds of the extremities, the degree appears more pronounced following abdominal injury. A study of Table 2 and Table 3, however, reveals that the progressive postoperative decrease in albumin-globulin ratio for both types of wounds is comparable, and that the real difference is the result of an early change reflecting itself in the albumin-globulin ratios of the first post-wound day. Since the observations made on the first post-wound day were generally performed within a few hours following operation, the low albumin-globulin ratios of the abdominal patient suggest that these changes took place before or during operation. Whether there is a specific response to abdominal injury involving localization of albumin in the peritoneal cavity cannot be answered at the present time.

Conclusions

A serial study of the changes in the serum protein of 33 combat casualties is reported. Following abdominal injury there is a rapid decrease in the albumin-globulin ratio of these patients followed by a continued progressive decrease for the next few days. Patients with extremity wounds alone demonstrate a delayed, but progressive decrease in the albumin-globulin ratio. Electrophoretic studies reveal the changes to involve primarily a decrease in the relative per cent albumin and an increase in the relative per cent alpha¹and alpha² globulin fractions.

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References

1. Browne, J. S. L., Hoffman, H. M., Schenker, V., Venning, E. H., and Weil, P.: Conference on Metabolic Aspects of Convalescence,9th Meeting. Josiah Macy, Jr. Foundation, 1945.

2. Cuthbertson, D. P., and Tompsett, S. L.: Brit. J. Exp. Path. 16: 471, 1935.

3. Hock-Legeti, C., Irvine, K., and Sprinkle, E. P.: Investigation of Serum Protein Patterns in Patients Undergoing Operation. Proc. Soc. Exper. Biol. & Med. 84: 707, 1953.

4. Peters, S. P.: Ann. N. Y. Acad. Sci. 47: 327,1946.

5. Pollack, H., and Halpern, S. L.: Advances in Protein Chemistry. 6: 383, 1951.

6. Weichselbaum, T. E.: Am. J. Clin. Path. 16:40, 1946.

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Appendix

Determination of Serum Albumin-Globulin Ratio

The following procedure was adopted at the Army Medical Service Graduate School with particular emphasis on use in the field. It was used in the field and found to be very satisfactory-all precautions being followed.

General: All established procedures for the measurement of A/G ratio are based on protein flocculation and the several methods and modifications that have been proposed are all subject to many critical limitations. It is very important to realize fully that flocculation of protein is a colloidal phenomenon and not a stoichiometric reaction; and that many conditions not usually critical or even considered for chemical determinations must be rigidly controlled to insure reproducible results. Moreover, some of the variables are yet unrecognized, so that any procedure is empirical in nature and is no better than the degree of exactness employed in the replication of each manipulation. "Short cuts" and modifications should never be attempted without careful consideration and understanding of the colloid phenomenon involved and the empirical nature of the procedures. The following procedure has been tested at AMSGS, Department of Biochemistry, and is believed to be the best available ad datum for field use. The recognized pitfalls and the precautions to be observed are indicated. The procedure is a modification of that reported by Weichselbaum.

Principle: The reddish-violet color that develops upon addition of a dilute alkaline copper solution to protein (biuret reaction) is determined spectrophotometrically. The serum albumin and globulin fractions are determined after precipitation of the globulins (alpha, beta, gamma) by a final concentration of 26.25 per cent sodium sulfite (anhydrous). The globulins represent the difference between the determinations of the albumin and total protein. Electrophoretic studies, and salt precipitation with 26.25 per cent sodium sulfite yield nearly identical values for serum albumin (60 per cent of the total protein value in normal sera).

Since the final concentration of sodium sulfite is near the saturation point, the determination should not be attempted until room temperature is above 20° C (68° F.). The room temperature must remain between20° and 30° C throughout the entire procedure.

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Reagents:

Biuret Reagent: Weigh 3.00 gm. of cupric sulfate (CuSO₄.5H₂O) and 12.0 gm. of sodium potassium tartrate (Rochelle salt), and add to a 1,000 ml. volumetric flask. Add 500-600 cc. of water and dissolve; add, with constant agitation, 300 ml. of 10 per cent sodium hydroxide that has been prepared from carbonate-free sodium hydroxide. Add 2 gm. of potassium iodide as a preservative and make to volume with distilled water. This solution keeps indefinitely in a paraffin-lined bottle but should be discarded on appearance of a black or reddish precipitate.

Sodium Sulfite, 28.0 per cent: Dissolve exactly 28.0 gm. of anhydrous sodium sulfite in distilled water at room temperature and make to 100 ml. This salt is difficult to dissolve but will go into solution with sufficient shaking.

This solution should be stored at all times at temperature between 20 and 40° C. If any crystallization occurs, complete solution must be effected before use by warming (not above 40° C) and violent shaking.

Sodium Hydroxide, 3 per cent:

Tween-Ether Reagent: Add 1 ml. of Tween 80 (Atlas Powder Co.) to 100 ml. of ethyl ether. Filter into a 100 ml. graduated cylinder, and make up to a volume of 100 ml. Store in a glass-stoppered bottle.

Calibration Curve:

Pipette 5.0 ml. of a 5 to 10 per cent solution of human albumin into a 100 ml. graduated cylinder and dilute to 80 ml. with 28.0 per cent Na₂SO₃ (1:16 dilution), mix. Prepare a series of cuvettes containing successively 2.0, 1.5, 1.0 and 0.5 ml. of above diluted solution. Then in the same order add 0, 0.5, 1.0 and. 1.5 ml. of 28.0 per cent Na₂SO₃. Pipette into each cuvette 8.0 ml. of biuret reagent, mix and read after 30 minutes in the spectrophotometer with the blank set to read 100 percent transmission at 540 mu.

Kjeldahl nitrogen determinations can be carried out on the remaining dilute solution (1:16). The total nitrogen content of 100 ml. of solution corrected for nonprotein nitrogen, which when multiplied by 6.25, is taken as the standard protein concentration. On semi-log paper plot the transmission values observed against 0, 0.75, 0.50, and 0.25 times the protein concentration of the standard solution as obtained by the micro Kjeldahl determination.

Standard Procedure:

1. All solutions and glassware must be kept at a temperature above 25°C throughout the procedure.

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2. Select three cuvettes and mark them B (blank), T (total protein), and A (albumin). Into the blank, pipette 2.0 ml. of the 28.0 per cent Na₂SO₃ solution.

3. Into a centrifuge tube with a cork stopper covered with plioform or aluminum foil or into a screw-cap culture tube add 6 ml. of 28.0 percent sodium sulfite solution, and add slowly with continuous mixing 0.4 ml. of serum.

4. Stopper and mix by inverting exactly 25 times.

5. Remove 2.0 ml. at once and transfer to cuvette T.

6. To the remaining solution add 1.0 ml. of Tween-ethyl ether solution, and shake by inverting exactly 25 times.

7. Centrifuge (Int. #2, two-thirds speed, 10 minutes), and after centrifugation, carefully insert a pipette through the ether layer and under the packed globulin layer by slanting the tube to separate the globulin precipitate from the wall of the tube.

8. Withdraw 2.0 ml. of the clear centrifugate and transfer to the cuvette marked A.

9. Into each of the cuvettes pipette 8.0 ml. of biuret reagent, and mix thoroughly.

10. Let stand at least 30 minutes, and then read in the spectrophotometer at 540 mu. with the blank set at 100 per cent transmission. Keep cuvettes covered with rubber stoppers during this period to prevent evaporation.

11. Determine the grams of protein and albumin from the calibration curve. The total protein minus the albumin gives the globulin concentration.

Standard: Run a standard solution with each set of determinations.

Serum Blank: If the serum sample contains any hemoglobin, is turbid, or excessively icteric, a serum blank must be run, otherwise high values for the total protein will be obtained.

Dilute 1.0 ml. of serum with 15.0 cc. of normal saline; mix.

Add 2.0 ml. of this diluted serum to a cuvette, SB, add 8.0 ml. of 3.0 per cent sodium hydroxide to the cuvette; mix.

Read in the spectrophotometer at 540 mu. with a blank of distilled water set at 100 per cent transmission.

Subtract the optical density reading of the serum blank from the optical density of the unknown (biuret total protein tube) to obtain the corrected optical density. Then convert the corrected optical density to transmission and determine the grams of total protein from the calibration curve.

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Discussion

The normal value for A/G ratio obtained by this procedure is approximately 1.3 to 1.6. This is as close to the value obtained by electrophoresis as can be obtained by any flocculation procedure. Other procedures for the separation of the protein fractions after flocculation (as filtration)are less suitable for field use since they require continuous use of non-standard filter paper, more restricted temperature range and other limiting conditions. For these reasons, the flocculation and separation procedure listed above should be satisfactory, if carefully followed.

Note that a critical stage in the separation is the flocculation of the "globulins" (i. e., the higher molecular weight portion of the native protein complex) and their adsorption into the interface between ether and water. This again is a very complex colloidal phenomenon involving the application of Reinder`s theorem and the relatively free surface energies existing between water-ether, ether-globulin surface, and water-globulin surface, all of which have been profoundly modified and complicated by the strongly polar "tween" molecules. These factors are not subject to quantitative measurement or control, and therefore emphasize the empirical nature of the procedure and the necessity for rigid standardization of all manipulations regardless of how insignificant they may seem to be.