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Chapter 13

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

Metabolic Effects of Injury; Studies of the Plasma Nonprotein Nitrogen Components in Patients With Severe Battle Wounds*

    Stanley M. Levenson, M. D.
    Captain John M. Howard, MC, USAR
    Hyman Rosen, M. A.

There have been many studies attesting to the importance of nutrition in surgical patients. Derangements of protein metabolism in the acutely or chronically ill have been particularly emphasized. Impaired wound healing, increased susceptibility to anesthesia, shock and infection, and malfunction of the liver and intestinal tract are some of the complications observed in protein-deficient patients. Under these circumstances, operative deaths are more frequent, convalescence is prolonged, and mortality increased.

The possibility was suggested a number of years ago that disturbance in the metabolism of the essential amino acids is one of the important factors in the pathogenesis of the malnutrition following injury.6 However, there are no conclusive data regarding this viewpoint.5, 13, 22, 30 That amino acids are fundamental to protein synthesis and many energy processes in all animals is well known; yet, the quantitative study of these compounds in man is in its comparative infancy. Lack of appropriate methodology accounts in part for the scarcity of information in this important field.

A practical method for the quantitative fractionation of plasma amino acids was introduced in 1951, when Stein and Moore23 perfected the method of ion exchange chromatography. Until that time, the little information available concerning individual amino acids of plasma had been obtained by the use of a microbiologic technic, a method based on the extent of growth of mutant bacterial strains on a medium containing the plasma sample. This method is open to the objection that these organisms are somewhat nondiscriminating in their choice of nutrient, and metabolize certain amino conjugates as well as single amino acids. The microbiologic method, then can be expected, in general, to give false high values for some plasma amino acids. By 


*In press (in modified form): Surgery, Gynecology and Obstetrics.


248

the ion exchange chromatographic technic on the other hand, differentiation of the free amino acids from amino conjugates is possible. Accordingly, we have used this technic.

The present study was undertaken with the end in view of determining whether any trends could be found in the plasma levels of the free amino acids of severely wounded soldiers from whom serial specimens were obtained beginning early after injury. These subjects were chosen because it is precisely this type of patient who demonstrates the greatest metabolic derangements after injury-that is, the seriously injured, but previously healthy and well nourished, young adult male.

These young men were wounded during the Korean hostilities in 1952-53. They were cared for by the Army Medical Service Graduate School Surgical Research Team and the Eighth Army Medical Corps.

Methods

Virtually all the samples analyzed were obtained as plasma in 2.5 per cent sodium citrate (1 ml. citrate to 5 ml. blood). The plasma samples were frozen immediately after separation of the cells and kept frozen thereafter. Deproteinization was accomplished by ultrafiltration in a Monel metal apparatus, previously described.26 This method yields a filtrate with an approximate maximum molecular weight species of about 15,000 to 20,000.

The individual amino acids were separated by the ion exchange chromatographic technic devised by Stein and Moore,23 and the analyses were accomplished by the photometric ninhydrin method of the same workers.31 About 4 ml. of the plasma filtrates was required for the analysis. Of the common amino acids, tryptophane and arginine were not determined; the former is destroyed on the column, the latter requires additional hours for its separation.

An acid labile amino component (amino acid conjugate) was isolated in many samples. The fraction containing this component was evaporated in each case by a stream of air to about 2 ml. It was then hydrolyzed with 6N HCl in a sealed tube at 110° for 20 hours. The resultant hydrolysate was evaporated of its hydrochloric acid as above, made up to 4 ml. with water and re-chromatographed in the usual manner.

Other small molecular weight, plasma nitrogenous compounds were also determined. These are urea, creatine and creatinine, uric acid, purines, and total nonprotein nitrogen. Uric acid was estimated from measurements of absorption at 293 mµ, and 305 mµ. The validity of this determination on plasma ultrafiltrates, even in the presence of


249

large quantities of aromatic amino acids has been demonstrated by us.27 Purines were grossly estimated from the absorption at 260 mµ.

Urea and NPN were analyzed at first by the micro method of Seligson and Seligson,29 and later by a titrimetric modification. From 0.005 to 0.02 ml. of the ultrafiltrate was required for each of these analyses.

Creatine and creatinine were determined by application of the Jaffe reaction11 to about 0.05 ml. of the plasma filtrate.

Our results are presented either as µM of nitrogen per 100 ml. ultrafiltrate (amino acids), or as mg. of nitrogen per 100 ml. ultrafiltrate (NPN, urea, creatine and creatinine, uric acid and purines). We have chosen to express the amino acids, in micromoles (µM), a unit possibly not as familiar as milligrams, but more logical. Since the ratio of nitrogen to total weight of one amino acid is different from that of any other, the expression of levels in terms of mg. of amino acid or mg. of nitrogen leads to a distorted impression of the relative quantitative importance of the various amino acids. By the use of the micromole unit, the amino acids are compared, in effect, molecule for molecule, regardless of size or weight.

Absorption spectra were obtained on ultrafiltrates, diluted 20 times with water, with the Beckman Du spectrophotometer in 1 cm. quartz cells. We have found that ultraviolet absorption spectra of plasma ultrafiltrates are useful in estimating certain plasma constituents, viz., uric acid and other purines, and the aromatic amino acids, phenylalanine, tyrosine and tryptophane. The ultrafiltrates are ideally suited for this procedure, since they have no absorbent material added to them in vitro, and they are naturally buffered near pH 7.

Results

Plasma Nonprotein Nitrogen Components (NPN, Urea, Creatine Plus Creatinine, Uric Acid, Other Purine Nitrogen, and Amino Acids)

We studied variations in the plasma amino acid levels and the other NPN components, in five critically wounded young soldiers. Four of those men died. Shock and renal failure were present in all in varying degrees. In four, the renal failure was persistent. Although the plasma urea concentrations rose as much as 30 times normal, the total free plasma amino nitrogen concentration remained near normal.

Extracorporeal dialysis by a Kolff-type artificial kidney was performed on three patients. Temporary biochemical and/or clinical improvement followed the dialyses, but eventually, all died. The


250

total free amino nitrogen concentration was little affected by the dialysis procedures. The plasma levels of certain individual amino acids were often near normal in spite of the serious injury, shock, severe renal failure, near starvation, infection and extracorporeal dialysis. In contrast, the levels of some other amino acids changed markedly. It is likely that any wide fluctuations of the individual amino acids indicate a basic upset in the body economy.

In the following section, the observations in each individual patient will be described.

Patient No. 1, T. M. L. This 25-year-old Korean was wounded by an M-1 30-caliber armor-piercing bullet. Resuscitation (including infusion of 1 liter of blood) was begun within an hour of injury and he was never in severe shock preoperatively. Operation, performed under pentothal, nitrous oxide and other anesthesia, was begun 21/2 hours after injury. Five liters of blood was infused during the operation, which lasted 5 hours. The missile had penetrated the right lung and pleural cavity, traversing the thickness of the liver, lacerating the vena cava (4 mm.), and tangentially wounding the superior pole of the left kidney. A left nephrectomy was done; the vena cava laceration was repaired; gelfoam was packed into the missile tract in the liver; an intercostal Foley catheter was inserted into the right pleural space. There was no uncontrollable hemorrhage or hypotension during surgery. Penicillin and streptomycin were given.

Throughout his course, he did not move his legs, and had absent reflexes and sensation in his lower extremities. A collapsed vertebra at D-12 was demonstrated, presumably due to damage from the missile, and it was assumed that his spinal cord had been damaged at about this point. Postoperatively, he was oliguric and was transferred on the second post-injury day to the Renal Insufficiency Center.

During the next 2 weeks, he ran a stormy course. Severe pneumonia, high lever, weight loss, persistent renal dysfunction, nitrogen retention, acidosis and hyperkalemia were important complications. He received aureomycin and, later, chloramphenicol. Extracorporeal dialysis on an artificial kidney of the Kolff type was carried out on the fourth post-injury day because of potassium toxicity. The resultant reductions in plasma K and NPN concentrations were only transient, however, and repeat dialyses were performed on the eighth and twelfth day after injury.

Our first analyses were done on a plasma sample obtained on the latter day, just prior to dialysis. The plasma K was 7.7 mEq./L., Na, 152 mEq./L.; Cl, 104 mEq./L.; CO2, 12 mEq./L.; NPN and urea were 392 and 336 mg. per 100 cc. respectively. Purine, uric acid, creatine and creatinine N were moderately elevated, while the total amino acids were slightly low. Aspartic acid, methionine, phenylalanine, and histidine were somewhat elevated, while the other amino acids were all lower than normal (Table 5). The effect of the dialysis on the nonprotein nitrogen components will be discussed later in a special section devoted to this matter (Effect of Extracorporeal Dialysis on the Plasma Nonprotein Nitrogen Components).

Following this dialysis, he continued to deteriorate, with increasing respiratory tract infection, spreading wound infections, high fever, uremia, hyperkalemia and melena. A fourth dialysis, with again only temporary improvement, was done on the sixteenth day.


251

Tracheotomy was done to enable suction of tracheobronchial secretions. Because of a spiking fever, right upper quadrant pain and fixation of the diaphragm on the right, extraperitoneal exploration of the right subdiaphragmatic space was carried out on the eighteenth day. No abscess was found. The next day, he suddenly died of respiratory failure following aspiration.

The postmortem diagnoses were surgically repaired vena cava, liver and diaphragm, surgical absence, left kidney; atelectasis, right lower lobe and right middle lobe of lung; emphysema, left side of chest; abscess, left lower lobe of lung; pneumothorax, left; edema, right upper lobe of lung, bronchopneumonia (?); hematoma, liver and diaphragm; hypertrophy, right kidney and congestion of renal medulla; abscess, retroduodenal region; splenomegaly; malaria (?); trichuriasis; decubiti, left buttock and sacrum.

Patient No. 2, F. H. This 23-year-old American soldier was burned and wounded when a trip flare exploded. His legs and lower trunk were burned and a flaming missile penetrated his abdomen. He was hypotensive for only a brief time preoperatively; during this time he received 1,500 cc. of blood and 750 cc. of plasma. Penicillin was given.

Operation was begun 7 hours after injury and lasted 5 hours. The lower abdominal wall was severely burned, and most of the small intestine was charred and necrotic. The anterior abdominal wall was débrided with excision of the external oblique muscle, rectus muscle and fascia, scrotum and testes. His penis was severely burned. All of the ileum and most of the jejunum were resected, and an end-to-side jejuno-transverse colostomy was done. A small portion of proximal jejunum were resected and an end-to-end jejuno-jejunostomy was done. The terminal ileum was closed and exteriorized with the cecum. The total, circumferential deep burns of both legs were then débrided with removal of all skin to subcutaneous tissue. Pressure dressings were applied. He remained moderately hypotensive (about 90/60) throughout the operation and received 5,500 cc. of blood.

Between the first and seventh post-wound days, he did relatively well, with stable blood pressure, daily urine outputs between 2,000 and 3,000 cc. and urine specific gravity up to 1.028. He ran a low-grade fever of about 100° with occasional spikes over 102°. He received a daily total of 1,000 to 2,000 cc. of blood plus plasma, as well as 3,000 to 5,000 cc. of about half and half glucose in saline and glucose in water. The BUN never rose above 45 mg. per 100 cc. during this time. On the sixth day he developed icterus, which gradually increased.

On the seventh post-injury day, a homologous split-thickness skin graft (from a cadaver) was applied to his left thigh and leg, under brief (10-minute) N2O anesthesia. Following operation, his urine output dropped abruptly to less than 10 cc. per hour. His general condition deteriorated, and he became lethargic and began vomiting. Because of continuing oliguria, he was transferred by helicopter to the Renal Insufficiency Center on the ninth day.

On admission, he was jaundiced and his temperature was 101°. The lower abdominal wall was almost all gone, except for a thin layer of peritoneum and transversalis fascia. There was a profuse, dirty watery and possible fecal discharge between the tissue layers at the edge of the burned area. There were large masses of necrotic tissue in the groin area. The deep burn of the buttocks was crusted. His penis was black and necrotic. Penicillin and streptomycin were given.

Admission plasma chemistries: Na, 160 mEq./L.; K, 5.8 mEq./L.; Cl, 110 mEq./L.; CO2, 23.4 mEq./L. The hematocrit was 31 percent. The plasma nonprotein nitrogen fractions were measured for the first time on this day (Table 1).


252

This plasma NPN and urea nitrogen were very high, 336 and 269 mg. per 100 cc. respectively. The uric acid, other purines, and creatine plus creatinine fractions were also elevated-2 to 3 times normal. In contrast, the total of the 19 free amino acids was normal. However, the distribution of the individual amino acids was abnormal. Glutamic acid, aspartic acid, phenylalanine and methionine were 2 to 21/2 times normal; isoleucine, tyrosine and histidine 11/2 times normal; proline, glycine, lysine and the glutamine-serine-asparagine complex were about half normal; threonine, alanine, valine, leucine and cystine were normal.

Table 1. Ultrafilterable Nitrogen Components of Plasma

(Patient F. H.)

Day Post-Injury

9

10

Normal

mg. N/100 ml. Plasma Ultrafiltrate

NPN

336.0

400.0

26.0

Urea N

269.0

364.0

15.4

Creatine + Creatinine N

7.2

9.8

2.5

Uric Acid N

5.2

9.1

1.5

Purine N

2.1

6.4

1.0

Amino Conjugate N

 

5.4

0.2

Amino N

3.6

4.3

3.5

µM./100 ml. Plasma Ultrafiltrate

Aspartic Acid

5.5

4.1

2.4±1

Threonine

11.7

11.7

13.1±2

Glutamic Acid

31.4

10.0

13.8±8

Proline

9.1

19.0

23.0±4

Glycine

19.0

19.8

24.0±2

Alanine

31.0

34.0

31.9±4

Valine

28.0

31.6

23.0±0.5

Methionine

4.3

4.5

2.2±0.5

Isoleucine

11.2

12.6

7.5±0.5

Leucine

12.6

17.6

11.6±0.5

Tyrosine

10.0

13.4

6.0±0.5

Phenylalanine

17.9

28.2

7.1±0.5

Histidine

21.4

30.6

14.1±2

Lysine

7.2

22.6

16.1±2

Taurine

5.0

4.5

4.8±1

Glutamine+Serine+Asparagine

28.6

44.6

49.8±3

He continued virtually anuric and late on the tenth post-wound day, he suddenly began having respiratory difficulty and became unresponsive rather abruptly. He was flaccid, with weak deep tendon reflexes and appeared to have respiratory paralysis. He was immediately given 3.75 gm. NaHCO3 intravenously, followed by 80 cc. of 3 per cent saline, with prompt improvement of respiration and revival of consciousness. A blood sample drawn just before this emergency treatment showed that the serum potassium was 8.3 mEq./L. (Earlier that day, plasma Na was 156 mEq./L.; K, 7.5 mEq./L.; Cl, 113 mEq./L.; CO2, 22 mEq./L.) At the same time, NPN, purine N, uric acid N, and creatine and creatinine N had also risen (table 1). Despite the extremely high NPN, amino acid N was only slightly elevated (4.3 mg. per 100 cc.). Most of the amino acids


253

remained at their previous levels, or rose. Threonine, proline, alanine and taurine were normal at this time. Glutamic acid, in contrast to the other amino acids, fell sharply. There was a corresponding rise in that function containing glutamine.

In the next few hours, he became progressively more hypotensive despite administration of hypertonic saline bicarbonate, glucose and insulin, and noradrenalin, and died before dialysis could be performed.

Postmortem diagnoses were second and third degree burns of abdominal wall, buttocks, perineum, penis, scrotum and legs; diffuse phlegmonous inflammation and necrosis, anterior abdominal wall; ileocolic anastomosis, and cecostomy; skin grafts, left leg; bilateral castration and surgical absence of scrotum; hepatitis, acute, toxic, renal hypertrophy, bilateral, with clinical renal insufficiency; acute hemorrhagic esophagitis with massive bleeding; focal fat necrosis, head of pancreas, minimal; pulmonary edema, moderate; cerebral edema, moderate; arachnoid cyst, right temporal lobe of brain; icterus.

Patient No. 3, K. D. This 22-year-old American soldier was wounded by shell fragments. The wounds included multiple perforations of the small bowel, multiple perforations of the sigmoid, two holes in the urinary bladder, severe comminuted, compound fractures of the left femur and left tibia and multiple soft tissue wounds of the buttocks and abdominal wall.

Prior to operation, he was given 4,000 cc. of blood. His blood pressure was normal.

Operation, lasting 7 hours, was begun 6 hours after injury. Operation consisted of repair of the eight holes in the small bowel, three bowel resections, colostomy, cystostomy and débridement of the many wounds. With the onset of pentothal induction (later nitrous oxide-oxygen-ether anesthesia) his blood pressure fell from 106/84 to 66/42, and his pulse rose from 100 to 116. Four milligrams norepinephrine dripped in with 500 cc. of blood immediately raised his pressure from 90/50 to 100/60. Later in the operation, his blood pressure was maintained with difficulty despite the administration of considerable blood with added norepinephrine. At operation, peritoneal contamination was massive. He was given 3,500 cc. of blood during operation while the measured operative blood loss was 1,670 cc.

A hemoclastic reaction was strongly suggested by a drop in his white blood cell count 1 hour after operation to 1,850 and abnormal clot formation. His platelet count at this time was 710,000; hematocrit, 55 per cent. His plasma volume (Evans Blue) was 2,220 cc. and calculated blood volume 4,720 cc. His blood pressure was about 90 to 100 systolic and 50 to 60 diastolic; pulse rate, around 120 per minute. One thousand cc. of dextran was then given over a period of 4 hours. No changes in blood pressure or pulse occurred. His hematocrit was then 42.5 per cent; plasma volume, 3,070 cc. and blood volume, 5,200 cc.

Throughout the first postoperative night, his blood pressure ranged about 80/50; pulse, 130; respirations, 40. Fifteen hundred cc. of blood and the same amount of dextran were then given over a period of 7 hours, with a rise in his blood pressure to 112/80 and a drop in his pulse rate to 104. His plasma volume had risen to 3,750 cc. and his blood volume to 5,980 cc. This was 24 hours postoperatively. Five hundred cc. more blood was given.

At this time, his plasma urea concentration was 74 mg. per 100 cc. Plasma uric acid concentration was slightly elevated as were creatine and creatinine; the purine fraction and total amino acids were essentially normal. Methionine, glutamic acid, tyrosine, aspartic acid and proline were normal; histidine, taurine, alanine and phenylalanine were somewhat elevated; leucine, isoleucine, lysine, valine, threonine and glycine were somewhat low (Table 2).


254

Table 2. Ultrafilterable Nitrogen Components of Plasma (Patient K. D.)

Day Post-Injury

11/2

21/2

31/2

5

6

7

8

11

12

Normal

 

mg. N/100 ml. Plasma Ultrafiltrate

Urea N

74.2

107.0

98.5

84.0

104.0

116.0

122.0

42.4

46.4

15.4

Creatine+Creatinine N

4.2

7.0

3.7

2.4

3.3

3.1

3.2

2.7

2.6

2.5

Uric Acid N

2.4

3.0

2.4

3.2

2.9

2.8

2.4

2.0

1.7

1.5

Purine N

1.1

2.2

0.9

1.7

0.5

0.5

0.5

1.2

1.4

1.0

Amino Conjugate N

 

 

 

1.48

 

 

1.42

1.2

 

0.2

Amino N

3.1

4.4

3.5

3.0

4.8

4.0

4.6

2.8

2.8

3.5

 

µM./100 ml. Plasma Ultrafiltrate

Aspartic Acid

3.0

6.9

6.6

7.0

9.2

6.9

8.2

5.2

4.1

2.4±1

Threonine

10.1

20.0

14.3

12.5

17.3

16.8

21.1

9.0

7.8

13.1±2

Glutamic Acid

11.0

14.8

8.5

12.5

11.8

10.9

10.4

20.1

26.1

13.8±8

Proline

20.0

30.6

11.8

11.7

27.7

17.6

28.0

3.0

1.9

23.0±4

Glycine

16.2

22.9

13.5

16.4

24.6

22.6

31.2

17.3

16.0

24.0±2

Alanine

38.0

66.0

28.6

33.0

55.0

47.2

60.1

19.2

18.6

31.9±4

Valine

17.2

21.3

25.7

36.8

42.1

44.1

22.0

26.4

19.2

23.0±0.5

Methionine

1.7

3.1

6.4

2.4

8.1

4.3

5.9

2.8

trace

2.2±0.5

Isoleucine

1.2

5.7

7.9

5.6

13.0

8.8

8.5

4.8

6.1

7.5±0.5

Leucine

8.2

16.1

17.3

14.6

25.7

22.5

18.3

13.8

19.9

11.6±0.5

Tyrosine

6.7

7.7

7.2

7.0

 

10.0

11.7

7.2

5.6

6.0±0.5

Phenylalanine

9.1

12.2

12.7

14.8

 

12.8

12.7

21.5

25.8

7.1±0.5

Histidine

25.5

11.7

30.7

5.1

20.4

2.0

16.5

8.6

5.5

14.1±2

Lysine

12.8

32.5

18.3

6.6

26.9

13.5

28.9

14.8

22.2

16.1±2

Taurine

7.6

4.6

4.6

trace

trace

trace

3.6

8.9

16.2

4.8±1

Glutamine+Serine+Asparagine

30.0

39.8

32.6

25.7

38.4

38.0

42.1

15.9

14.9

49.8±3


255

During the second postoperative night his blood pressure again fell to 94/64, and his pulse rose to 140 per minute. No bleeding was evident. Five hundred cc. of dextran was given with no obvious improvement.

Fifty-five hours after injury, forty-eight hours after operation, infusion of noradrenalin in glucose in water intravenously was begun. His blood pressure gradually rose to 120/70, but his pulse continued at a rapid rate (140 per minute). Oxygen, per nasal catheter, had changed the appearance of his skin from a cyanotic to a reddish flush. Throughout the day his blood pressure was maintained at 115-125/65-80 and his pulse rate gradually fell to 115 per minute. The infusion of noradrenalin was gradually slowed and stopped the next day. His blood pressure was maintained thereafter at normal levels.

Sixty-six hours after injury and fifty-nine hours after operation, the NPN components had all risen. The amino nitrogen was now 4.4 mg. per 100 cc., while urea nitrogen was 107 mg. per 100 cc. Of those amino acids previously normal, all but glutamic acid had risen; alanine and phenylalanine continued to rise, while taurine and histidine fell; the previously low amino acids all rose, some above normal.

Ninety-one hours after injury, his plasma volume was 3,600 cc. and blood volume, 6,100 cc. His blood pressure had been normal for about 36 hours. The plasma total free amino nitrogen concentration was normal, while plasma urea was still elevated (99 mg. per 100 cc. uric acid nitrogen was 2.4 mg.; purine nitrogen, 0.9 mg.; creatine plus creatinine nitrogen, 3.7 mg.) Methionine, aspartic acid and histidine were greatly elevated; leucine, tyrosine and phenylalanine were elevated slightly; isoleucine, lysine, valine, alanine, threonine, taurine and glutamic acid were normal; proline and glycine were low.

After the third postoperative day, his course was uneventful. Plasma urea continued elevated through the eighth post-injury day. Creatine plus creatinine and purine nitrogen remained near normal, uric acid gradually fell to normal, while the total amino nitrogen fluctuated slightly around normal. Aspartic acid continued to rise through the sixth postoperative day, and then fell steadily to the eleventh day. Methionine fell transiently through the fourth post-injury day, only to rise to its peak (3 times normal) on day 6 post injury. Thereafter, it too fell. Valine, tryosine, alanine, threonine, glycine, leucine and isoleucine reached their highest values (about twice normal) between days 6 and 8 post injury, and then fell. In contrast, phenylalanine, glutamic acid and taurine, which were respectively high, normal and low during the first week, rose thereafter, and all were high on the last day of study. Histidine and lysine fluctuated widely from day to day from above to below normal.

Patient No. 4, A. K. This 22-year-old American soldier received a perforating abdominal wound by an M-1 rifle bullet. His blood pressure, which was 115/85 when first recorded at 2 hours after injury, fell to 90/60 1 hour later. Five hundred cc. of blood was started and he was sent to a Mobile Army Surgical Hospital by ambulance. Four and three-quarters hours after injury he had received a total of 1,000 cc. of blood and his blood pressure was 120/80. He had been given penicillin and tetanus toxoid.

Laparotomy was begun 7 hours after injury after the patient had received an additional 1,500 cc. of blood. Anesthesia consisted of pentothal and nitrous oxide-oxygen-ether. The wound was a through-and-through perforation of the abdomen with resulting perforating wounds of the liver, kidney and colon. About 1,500 cc. of blood was aspirated from the abdomen during surgery. A colostomy and drainage of the liver and kidney wounds were performed.


256

During operation, his blood pressure became imperceptible for about 30 minutes, but was elevated to 60 or 80 systolic as noradrenalin was added to the blood. He was given 7,500 cc. of blood during the operative procedure which lasted 5 hours. Operative blood loss was measured at 2,420 cc.

Our first analysis was performed on blood taken 12 hours after injury, immediately after operation. His hematocrit was high, about 70 per cent. Plasma urea concentration was moderately elevated (36 mg. per 100 cc.) while all the other nonprotein nitrogen components were normal. Methionine, alanine, lysine, leucine, taurine, phenylalanine and glutamic acid were elevated; tyrosine, aspartic acid, proline, threonine, glycine and isoleucine were low; valine and histidine were normal (Table 3).

During the next 24 hours, he received 2,500 cc. of gelatin, 500 cc. of dextran, and 3,000 cc. of 5 per cent glucose in water, some of which contained terramycin. His urine output was low, blood pressure was normal, but pulse and respirations were rapid. Plasma urea nitrogen had risen, as had all the other NPN components. Total amino nitrogen was definitely elevated (5.8 mg. per 100 cc.). Valine stayed normal, and lysine, glutamic acid and taurine fell. All the other amino acids rose. During the next day, he continued oliguric; blood pressure was normal. Aureomycin was begun. His plasma urea rose, as did the NPN components except the total amino nitrogen, which fell slightly below normal. All the amino acids fell, except for isoleucine, which stayed normal, methionine, which remained high, and taurine, which rose. Plasma Na was 125 mEq./L.; K, 5.7 mEq./L.

Because of persistent oliguria, he was transferred to the Renal Center on the second post-injury day. On arrival, he was feverish and toxic with signs of left lower lobe pneumonia. His sputum was positive for Staphylococcus and Proteus, the latter sensitive only to chloromycetin. The response to chloromycetin, however, was poor. The patient became clinically jaundiced, presumably secondary to the liver injury, the serum bilirubin reaching a peak of 14.5 direct and 25.3 total 5 days after injury.

He was on constant Wagensteen suction and lost 600 to 2,200 cc. of dark fluid daily. Oral feeding was not possible. Varying quantities of 50 per cent glucose in water containing 2 gm. of vitamin C, 25 mg. of vitamin K, and 10 mg. of thiamine were given daily by intravenous catheter.

Hyperkalemia was well controlled, the plasma level dropping from 7.6 mEq./L. on admission to 5.8 by the next morning. The toxic effects of potassium were also counteracted later with intravenous calcium gluconate.

Throughout his course, this patient was in good fluid balance. Diuresis occurred immediately and, thereafter, the urinary output was about 2,000 cc. daily, but the plasma NPN continued to rise, reaching a peak of 430 mg. per 100 cc. on the seventh post-wound day. He was uremic, icteric and septic (pneumonia, wound infections and bacteremia). At this time, he underwent extracorporeal dialysis by an artificial kidney of the Kolff type because of uremia, drowsiness, nausea, tremulousness, occasional periods of disorientation, and a severe hemorrhagic diathesis. The results of the dialysis were satisfactory chemically, but the clinical condition of the patient did not improve and in retrospect the symptoms probably were due to sepsis rather than to uremia. Just prior to dialysis, plasma Na was 130 mEq./L.; K, 6.8 mEq./L.; Cl, 98 mEq./L.; CO2, 13 mEq./L. The plasma urea nitrogen was 377 mg. per 100 cc. Creatine plus creatinine, purine and uric acid nitrogen were also much elevated, while total amino nitrogen was slightly high. Phenylalanine, histidine, aspartic acid, methionine, tyrosine, leucine, isoleucine and lysine were high. All the rest were normal except taurine, which was low. Following dialysis, all the NPN components other


257

Table 3. Ultrafilterable Nitrogen Components of Plasma (Patient A. K.)

Day Post-injury

½

1

11/2

2

7 Pre-dialysis

7 Post-dialysis

8

Normal

 

mg. N/100 ml. Plasma Ultrafiltrate

Urea N

36.4

91.0

111.0

132.0

377.0

113.0

177.0

15.4

Creatine+Creatinine N

2.3

4.9

8.0

10.9

9.8

5.4

11.3

2.5

Uric Acid N

1.5

2.1

3.2

3.7

7.1

3.3

4.6

1.5

Purine N

0.4

0.9

1.5

2.8

4.3

1.8

3.2

1.0

Amino Conjugate N

0.33

 

 

0.64

2.10

 

1.32

0.2

Amino N

3.2

5.8

5.1

2.7

4.1

4.8

4.5

3.5

 

µM./100 ml. Plasma Ultrafiltrate

Aspartic Acid

0.8

 

4.9

2.3

5.4

5.6

9.9

2.4±1

Threonine

10.4

24.7

22.7

8.1

13.6

24.0

14.4

13.1±2

Glutamic Acid

29.5

20.6

15.0

6.1

18.0

14.1

10.4

13.8±8

Proline

10.4

28.1

31.5

15.2

29.8

33.0

29.0

23.0±4

Glycine

16.4

32.1

33.8

10.4

22.2

29.5

25.0

24.0±2

Alanine

49.0

104.0

70.0

31.0

28.2

55.1

56.5

31.9±4

Valine

25.0

20.5

28.2

16.9

22.9

27.3

21.0

23.0±0.5

Methionine

1.1

6.1

5.1

5.8

6.6

7.5

7.4

2.2±0.5

Isoleucine

1.3

8.5

7.4

7.8

11.7

7.4

6.7

7.5±0.5

Leucine

17.3

21.6

17.4

13.8

17.7

19.1

14.4

11.6±0.5

Tyrosine

4.8

11.4

9.7

6.8

8.6

11.2

8.0

6.0±0.5

Phenylalanine

9.8

28.2

20.0

10.6

12.1

14.7

7.3

7.1±0.5

Histidine

15.5

22.7

33.3

17.2

22.0

28.0

3.8

14.1±2

Lysine

 

33.3

23.4

13.2

29.6

36.2

16.6

16.1±2

Taurine

10.1

3.6

5.3

9.3

1.7

trace

0.5

4.8±1

Glutamine+Serine+Asparagine

16.0

43.4

35.3

14.8

40.0

30.3

39.5

49.8±3

 


258

than the amino acids dropped sharply. (A detailed description of the chemical findings will be found in the section on "Extracorporeal Dialysis.")

During the next day the patient continued running a septic course. He had lost 30 pounds. Plasma Na was 146 mEq./L.; K, 6.2 mEq./L.; CO2, 20 mEq./L. There was a rise in urea, uric acid, purine and creatine plus creatinine nitrogen. The total amino acids stayed slightly elevated. Aspartic acid, alanine, leucine, methionine and tyrosine were elevated; histidine and taurine were low; all the rest were normal. No subsequent plasma samples were analyzed.

Bleeding into the nasopharynx, bowel and skin occurred and persisted in spite of fresh blood transfusions and large doses of vitamin K. Frequent hypotensive episodes occurred but could be controlled with blood. Noradrenalin in increasing amounts was required to maintain blood pressure, but finally the patient became refractory to the drug and transfusion and he died in circulatory collapse on the twelfth post-wound day. The clinical impression was that the uremia was reasonably well controlled and that death was due to overwhelming sepsis.

Pathologic findings included pulmonary infarcts of the left lower lobe, acute phlebitis of the inferior vena cava, septicemia (B. proteus), fibrinopurulent peritonitis, diffuse mucosal hemorrhages of the small intestine, gunshot wound

FIGURE 1.


259

of the right lobe of the liver, an abscess containing B. proteus in the liver, and central necrosis of the right lobe of the liver. There was mild lower nephron nephrosis, a few areas of focal glomerulitis, traumatic destruction of the lower pole of the right kidney, and multiple abscesses of the right kidney containing B. proteus.

Patient No. 5, C. J. This 26-year-old American soldier was wounded by mortar shell fragments. The wounds included laceration of the scalp, perforations of the liver and kidney, traumatic amputation of the right thigh, and multiple soft tissue injuries.

On arrival at battalion aid station shortly after injury, he was unconscious and his blood pressure was unobtainable. After the intravenous infusion of 320 cc. of albumin and 1,500 cc. of isotonic saline his blood pressure was 80/40. He was then evacuated by helicopter to a Mobile Army Surgical Hospital.

On arrival (2 hours after injury) he was still unconscious (cerebral concussion) and his blood pressure and pulse were unobtainable. Within 25 minutes, he was given 2,500 cc. of blood, but his blood pressure remained unobtainable. His pulse was barely palpable at a rate of 110 per minute. His right leg was amputated without anesthesia in the emergency room. In the next 4 hours, he was given 2,500 cc. more blood. His blood pressure was 128/80; pulse rate, 120 to 130, and respiratory rate, 20. Our first analyses were done on a plasma sample obtained at this time (Figs. 1 to 8). NPN, urea nitrogen and uric acid

FIGURE 2.


260

nitrogen were already elevated, while creatine plus creatinine nitrogen, purine nitrogen and total amino nitrogen were normal. Glutamic acid was twice normal; alanine and phenylalanine were slightly elevated; threonine, glycine, valine and histidine were slightly low; all the rest were normal. Plasma Na was 158 mEq./L.; K, 3.8 mEq./L.

Operation was delayed for 3 more hours because he had not regained consciousness. During this time he received another 1,000 cc. of blood. His blood pressure had remained stable at about 130/80 and his pulse between 120 and 130 per minute.

FIGURE 3.

Operation was begun 10 hours after injury, under atropine, pentothal and nitrous oxide-ether anesthesia. His blood pressure immediately fell from 140/80 to 90/60, but his pulse rate remained unchanged. His blood pressure remained low throughout most of the operative procedure (which lasted 3 hours) despite the administration of an additional 3,500 cc. of blood. Operative blood loss, by the washed sponge technic, was 3,030 cc. Operation consisted of exploratory laparotomy, drainage of the liver and kidney perforations, re-amputation of the thigh and débridement of many soft tissue injuries. He was found to have fractures of the femur proximal to the line of amputation.


261

Towards the end of the operation, noradrenalin was added to the infused blood. A rise in blood pressure was immediate. Plasma NPN and urea had risen still further; creatine and creatinine nitrogen, uric acid and purine nitrogen were slightly elevated, while the total amino nitrogen was still normal. Glutamic acid, phenylalanine and alanine were still elevated; taurine and aspartic acid had risen; all the others were normal except methionine, glycine and valine, which were low. Plasma Na was 146 mEq./L.; K, 5.4 mEq./L.

Noradrenalin was used throughout the night after injury to maintain his systolic pressure between 105 and 120. The rate of noradrenalin administration was gradually decreased and successfully stopped 24 hours after operation. On

FIGURE 4.

the morning of the first postoperative day, the various NPN components, other than amino N, were still elevated. NPN, urea and uric acid were rising. The total amino nitrogen, on the other hand, had fallen below normal. Phenylalanine and alanine were still elevated while valine, glycine, proline, threonine, methionine, leucine, isoleucine and lysine were below normal. The other amino acids were normal. Plasma Na was 167 mEq./L.; K, 5.1 mEq./L.

By noon of the second day post injury, he was still unconscious; his blood pressure had risen to 140 to 160 systolic; pulse rate was 110 to 120; respiratory rate, 24 to 32. His temperature ranged from normal to 102° orally. He had received 500 mg. of terramycin, 1,000 cc. of 5 per cent glucose and water, 100


262

mg. of thiamine and 1,000 mg. of vitamin C. His 12-hour urine output was only 115 cc.

On admission at the Renal Center, he was semicomatose; blood pressure was 140/80; temperature, 101°. His serum potassium was 7.1 mEq./L.; sodium, 131; CO2, 30; chloride, 86; hematocrit, 37 per cent NPN, 114 mg. per 100 cc. Because of the probability of pulmonary complications, he was tracheotomized and put on a Stryker frame on the third day after injury. His NPN components rose progressively during the next few days and all except the amino nitrogen reached very high levels. The latter was only moderately elevated. Taurine was an

FIGURE 5.

exception among the amino acids, rising to 12 times normal on the fourth post-injury day. Methionine, leucine, isoleucine, lysine, phenylalanine, valine, tyrosine and alanine all rose progressively through the third post-injury day; all but valine fell the next day. Glutamic acid, proline, threonine and histidine had remained normal or slightly low, while glycine was persistently low. Aspartic acid fluctuated above and below normal.

There was an initial drop in his serum K concentration, but then a progressive increase to 8.2 mEq./L. on the fourth post-injury day with broadening of the QRS component on EKG. The serum Na, initially 131 mEq./L., rose to 139 mEq./L. Chlorides stayed constant at about 85 mEq./L., and CO2 gradually


263

dropped from 29.6 to 15.7 mEq./L. After emergency infusion of calcium gluconate, he was dialyzed by the artificial kidney (Kolff-type) with an excellent chemical result, but with little change in his general condition. The chemical effects of the dialysis will be discussed in the section "Extracorporeal Dialysis."

During the next 5 days he was periodically conscious. He became increasingly jaundiced. Some of his wounds were grossly infected and bled excessively following débridement. He was still oliguric, and the plasma NPN components

FIGURE 6.

other than amino nitrogen rose steadily to levels similar to those just prior to the first dialysis. In contrast, the total amino nitrogen steadily decreased and reached the very low level of 2 mg. per 100 cc. on the ninth post-injury day. Valine, tyrosine, alanine, aspartic acid, leucine, isoleucine, proline and threonine all fell, while taurine remained normal and glycine persistently low. Plasma K concentration which was 4.2 mEq./L. immediately after dialysis, on the fifth post-wound day, rose progressively to 8.6 mEq./L. by the ninth day. Plasma sodium was 139 mEq./L. after the first dialysis, and rose to 149 mEq./L.; chlo-


264

FIGURE 7.

ride fell from 104 to 93 mEq./L., and CO2 fell from 26 to 18 mEq./L. On the ninth post-injury day, because of continuing hyperkalemia, he was again dialyzed, with correction of the potassium intoxication and transient improvement in his general condition. (Details of the chemical results will be found in the section on "Extracorporeal Dialysis.")

He continued febrile and oliguric. The plasma NPN fractions, except for amino nitrogen, rose steadily again to very high levels. On day 12, urea nitrogen was 234 mg. per 100 cc., other purine nitrogen, 5.7 mg. per 100 cc. On the contrary, amino nitrogen again fell progressively. Leucine and isoleucine became slightly elevated; proline, threonine, glycine, lysine, tyrosine and alanine, were low; valine, aspartic acid, taurine, methionine, and histidine were normal. At this time plasma K was 6.9 mEq./L. A few days later, because of recurrent potassium intoxication, he was dialyzed for the third time, with correction of the hyperkalemia, but with no change in his very poor general condition. He continued to deteriorate and died on the nineteenth post-injury day.

At autopsy his entire body showed evidence of marked wasting. The amputation site of the right thigh showed extensive necrosis and infection of the skin flaps, muscle and fascia. There was a severe bacterial pericarditis, focal edema and necrosis of the myocardium, bronchiolar pneumonia, central necrosis (severe) of the liver, hyperthopy of the parathyroids, acute cystitis, necrosis of the occipital poles of the cerebral cortex, and lower nephron nephrosis.


265

FIGURE 8.

Plasma Amino Conjugate

Early in our work on the plasma ultrafiltrates of human subjects, we noticed that one chromatographically distinct ninhydrin reactive component is unstable to acid hydrolysis. We isolated this component chromatographically, from a number of plasma samples, and rechromatographed them. The results are shown in Table 4 and Figure 9. The striking central fact is the quantitative and qualitative variability of the composition of this fraction among the patients.

The two normal subjects were young, healthy, male laboratory technicians. In both, glutamic acid and glycine comprised almost all of the conjugate; a small amount of threonine was also found in one. The plasma of the patients, however, contained the conjugate in greater quantity, and the component amino acids in greater variety. In patients C. J. and A. K., the plasma conjugate levels of the patients rose with the plasma urea levels, though not proportionately. As the


266

Table 4. Amino Acid Composition of Plasma Ultrafilterable Amino Conjugate

Patient
Day Post-wound

½

C. J.

A. K.

K. D.

F. H.

Normals

1

2

11

12

½

2

7

8

3

8

11

10

A

B

 

µM./100 ml. Plasma Ultrafiltrate

Aspartic Acid

 

 

4.0

3.4

2.5

 

 

 

 

 

 

 

 

1.7

 

Threonine

 

 

1.8

3.2

2.0

3.6

2.3

7.9

2.3

15.8

 

0.8

13.3

 

 

Serine

 

 

2.6

1.3

2.2

1.8

2.2

11.1

3.6

17.7

 

6.0

12.1

 

 

Glutamic Acid

6.4

 

10.8

13.0

20.8

6.6

2.1

9.5

6.7

18.5

8.0

4.7

44.2

8.7

5.5

Proline

 

 

 

10.1

0.8

 

 

 

 

 

 

 

 

 

 

Glycine

18.1

5.3

18.0

26.0

10.0

15.1

8.8

29.5

14.6

37.5

23.5

74.4

145.0

6.6

8.8

Alanine

4.9

1.7

 

2.6

0.8

1.0

 

4.6

 

 

 

 

8.8

 

 

Valine

 

2.0

 

2.5

1.5

 

 

9.5

 

 

 

 

44.2

 

 

Leucine

4.8

4.0

10.0

14.5

10.0

 

21.7

45.6

34.0

5.4

45.2

 

57.2

 

 

Tyrosine

 

 

4.8

6.0

5.2

 

5.3

23.4

10.6

4.2

19.1

 

36.4

 

 

Phenylalanine

 

 

 

2.5

 

 

 

 

 

 

 

 

 

 

 

Histidine

 

 

3.1

2.6

5.1

3.6

 

8.5

13.0

6.3

5.7

 

23.6

 

 

Lysine

 

 

 

 

 

1.9

 

 

 

 

 

 

 

 

 

Unknown

 

 

12.9

 

6.0

 

3.6

 

9.2

 

 

 

 

 

 

Total

34.2

13.0

68.0

87.7

66.9

33.6

46.0

149.6

94.0

105.4

101.5

85.9

384.8

17.0

14.3

 

mg. N/100 ml. Plasma Ultrafiltrate

Conjugate N

0.5

0.3

1.0

1.2

0.9

0.3

0.6

2.1

1.3

1.5

1.4

1.2

5.4

0.2

0.2

Urea N

32

57

145

177

234

36.4

132

377

178

84

122

42

364

20

17

 


267

level of the plasma conjugate fraction rose, more and more different amino acids were found within the component; the quantitative relations of the amino acids within the conjugate showed an ever shifting pattern, with first one amino acid becoming predominant then another. By the eleventh post-wound day, in patient C. J. (Fig. 9), 12 of the naturally occurring amino acids were present in the conjugate, with leucine and proline in addition to glycine and glutamic acid occupying

FIGURE 9.

quantitatively important positions. Patient A. K. showed the same sort of shifts in amino acid composition, but with different amino acids involved; leucine became even more predominant.

In patient K. D., whose plasma urea never reached the extreme heights of either C. J. or A. K., the amino conjugate concentration stayed at a relatively constant (but elevated) level. The quantitative relationships of the component amino acids gradually decreased in


268

Table 5. Effect of 6-Hour in Vivo Dialysis on Ultrafilterable Nitrogen Components of Plasma

Patient Day Post-injury

T. L. 12 
Pre-dialysis

Post-dialysis

% Change

A. K. 7 Pre-dialysis

Post-dialysis

Change

C. J. 9 Pre-dialysis

Post-dialysis

% Change

 

mg. N/100 ml. Plasma Ultrafiltrate

NPN

392.0

118.0

-70

462.0

208.0

-55

383.0

76.0

-80

Urea N

336.0

97.5

-71

377.0

113.0

-70

311.0

54.0

-83

Creatine + Creatinine N

9.7

5.5

-43

9.8

5.4

-45

10.8

3.0

-72

Uric Acid N

6.7

2.5

-63

7.1

3.3

-54

6.9

1.6

-77

Purine N

3.8

0.9

-76

4.3

1.8

-55

5.2

1.1

-79

Amino Conjugate N

3.1

0.8

-74

2.1

1.3

-40

4.0

0.3

-92

Amino N

2.7

2.8

+3

4.1

4.8

+17

2.0

1.9

-5

 

uM./100 ml. Plasma Ultrafiltrate

Aspartic Acid

3.7

2.9

 

5.4

5.6

 

2.3

1.8

 

Threonine

6.2

6.6

 

13.6

24.0

 

4.8

6.2

 

Glutamic Acid

8.5

10.4

 

18.0

14.1

 

11.8

12.5

 

Proline

14.4

16.0

 

29.8

33.0

 

5.3

8.0

 

Glycine

15.8

16.6

 

22.2

29.5

 

10.1

14.7

 

Alanine

20.7

26.8

 

28.2

55.1

 

10.4

17.2

 

Valine

17.0

19.2

 

22.9

27.3

 

14.6

9.2

 

Methionine

3.5

2.2

 

6.6

7.5

 

Trace

Trace

 

Isoleucine

6.8

5.2

 

11.7

7.4

 

5.8

4.5

 

Leucine

9.6

12.3

 

17.7

19.1

 

8.0

8.0

 

Tyrosine

4.0

5.1

 

8.6

11.2

 

1.7

1.7

 

Phenylalanine

12.0

11.3

 

12.1

14.7

 

10.0

8.0

 

Histidine

28.2

18.7

 

22.0

28.0

 

19.5

 9.3

 

Lysine

11.4

13.0

 

29.6

36.2

 

8.3

12.6

 

Taurine

1.7

3.0

 

1.7

Trace

 

5.2

2.2

 

Glutamine+Serine+Asparagine

29.1

26.1

 

40.0

30.3

 

22.0

18.4

 

 


269

number, while glycine increased in concentration until it comprised some 85 per cent of the conjugate on the ninth day after injury.

These observations suggested that the amino conjugate fraction is not homogeneous. Accordingly, we chromatographed the intact conjugate on paper by the "circular" technic, using butanol-acetic acid-water (6:1:1) as the solvent. The conjugate of
C. J. 2 days post injury was thereby separated into four ninhydrin-reactive components and that of K. D. 5 days post injury, into three. A specific chromatographic analysis was made in one subject (C. J.) for the peptides carnosine and anserine, normal intracellular constituents. Neither was found.

The conjugate, as a whole, acted as a metabolic end product with respect to extracorporeal dialysis (Table 5). The plasma concentration of the conjugate fraction was reduced after 6-hour dialyses in approximately the same ratios as urea, uric acid, other purines and the creatine plus creatinine fraction. This is in marked contrast to the behavior of the plasma free amino acids, whose concentrations, in general, were little changed by the dialyses.

Ultraviolet Absorption of Plasma Ultrafiltrates

On Figure 11 is depicted the absorption spectral pattern of a normal plasma ultrafiltrate. The peak at 290 mµ. is almost entirely due to the purine, uric acid, which has a molecular extinction coefficient, at this wavelength, of about 12,500. Absorption in the region of 240 to 260 mµ. is due to other prines, but also included a second peak of uric acid. The aromatic amino acids, with considerably lower extinction coefficients (around 3,000 to 4,000) have their absorption maxima at about 280 mµ. Absorption below these wavelengths is termed "nonspecific," and is characteristic of many nitrogenous compounds, including most of the amino acids.

The data for patient C. J. are shown in Figures 10 and 11. There was an insistent and progressive rise in absorption at all wavelengths, the rise being only temporarily halted by extracorporeal dialysis. The spectral absorption was highest when the plasma NPN levels were highest.

Similar observations were made in patients A. K. and K. D.

Effects of Extracorporeal Dialysis on the Plasma Nonprotein Nitrogen Components

Extracorporeal hemodialysis of the patients with severe renal dysfunction was carried out to reduce hyperpotassemia or relieve uremia. We studied the plasma of three patients, obtained before and after


270

FIGURE 10.


271

FIGURE 11.


272

6-hour dialyses, with a view toward establishing the pattern of change in the nonprotein nitrogen portion of the plasma, ostensibly that part readily diffusible through a cellophane membrane. The data set forth in Table 5 and Figure 12 show that there is a discernible pattern. With the notable exception of the amino acids, all the small nitrogenous compounds, including the amino conjugate, were sharply reduced in concentration after dialysis. In contrast, the plasma concentrations of the amino acids were changed little, despite the 6-hour "washouts" on the artificial kidney.

FIGURE 12.

Discussion

When 19 plasma amino acids are measured by ion exchange chromatography in a random group of healthy individuals, it is found that the total amino acid N measures close to 3.5 mg. per 100 cc. This figure practically never goes above 3.8 (except for a short time after a high-protein meal) or below 3.1 mg. per 100 cc. There is considerable variation in concentration among the amino acids, e. g., aspartic acid is present in a concentration of about 2.5 µM per 100 cc. while the concentration of alanine is about 32 µM per 100 cc. Speculations can be indulged in to explain this, but there is, at present, inadequate experimental evidence to support any theories. However, each amino acid is present in about the same relative concentration in the plasma of healthy men. Among the factors which have a possible connection


273

with maintaining the plasma amino acids relatively constant in normal individuals are: dietary intake, absorption of ingested amino acids from the gut; deamination, transamination, and decarboxylation primarily in the liver; excretion and reabsorption by the kidney; and dynamic transcapillary exchange. The plasma amino acids are in active metabolic exchange with tissue amino acids, tissue and plasma proteins, and body carbohydrate and fat.

One of the main objects of this study was to discover if any quantitative trend in plasma amino acids manifested itself in previously healthy young men who had been seriously wounded. At the present time, there is little hope of pinpointing the causes for any noted amino acid alterations, since (a) the plasma specimens were obtained from men undergoing many and varied stresses and (b) as mentioned, the basis for the differences in plasma concentrations of the various amino acids in normal individuals is unknown.

Among the stresses which the patients we studied underwent, the following might be expected to influence the plasma amino acids: the initial injury, hemorrhage, shock, intravenous infusions (blood, albumin, dextran, gelatin, glucose, saline, etc.), anesthesia, operation, liver dysfunction, renal dysfunction, fluid and electrolyte imbalance, fever, infection, antibiotics and malnutrition. Also to be considered are the paths along which the stresses may affect the amino acid levels, i. e., humoral, neural, etc.

Before discussing our results, the work already done on the effects of some of the stresses mentioned may be summarized. Most of the work done in this field concerned only the total plasma amino nitrogen concentration and not individual amino acids.

A prolonged decrease in the plasma concentration of total alpha-amino nitrogen following operation was reported by Man and co-workers.21 They felt that the degree and duration of the decline in plasma total alpha-amino nitrogen concentration is related to the severity of the operative procedure. Less change postoperatively was observed in malnourished patients with low plasma amino nitrogen levels preoperatively than in those patients with normal preoperative levels.

Recently, Everson and Fritschel8 have studied, in 16 patients (specific diagnoses not tabulated), the effect of major surgical operations (in magnitude, cholecystectomy to gastrectomy) on the plasma levels of 10 individual amino acids. Fourteen of the patients were stated to be in good nutritional status at the time of operation. Two presumably were not, but the authors do not specifically characterize the nutritional status of these two patients.


274

Statistically significant decreases in plasma concentration immediately after operation were observed for all but leucine and histidine. By the morning of the first postoperative day, only the values for threonine, arginine and lysine were still significantly lower than the preoperative levels. The plasma lysine concentration rose to the preoperative level by the third postoperative day, while the values for arginine and threonine rose to normal somewhere between the third and seventh postoperative days. They observed no obvious relationship among the type and duration of the operation and the plasma concentration of the 10 individual amino acids measured by them. However, the number of patients studied by them is small for comparisons of this nature. Presumably, none of the patients in the study of Everson and Fritschel was in shock. No details of fluid or blood administration during or after operation, however, are given.

To determine whether anesthesia, per se, might influence the plasma amino acids, 10 dogs were subjected by Everson and Fritschel to 2 hours of ether anesthesia. A lowering of the plasma level of the individual essential amino acids equal to or greater than the depression of plasma levels in patients produced by anesthesia plus operation was observed. No data were reported on dogs anesthesized and subjected to operation.

It is generally held that the total plasma amino nitrogen increases during severe shock. Thus, an increase in plasma amino nitrogen has been observed in rats with severe hemorrhagic shock7, 33 and in rats15 calves12 and patients17 with shock following severe burns. Engel, Winton, and Long7 observed that the rise in plasma alpha-amino nitrogen in hemorrhagic shock in rats was accompanied by a fall in red cell alpha-amino nitrogen.

The effect on plasma amino acids of ether anesthesia and severe burns in untreated rats was studied by Rosen and Levenson28 who found minimal rise in the total amino acids 12 hours after burn. A marked increase in taurine and an amino conjugate was observed; alanine, histidine, phenyl-alanine, tyrosine, leucine, isoleucine, serine and asparagine were somewhat elevated; threonine, glutamic acid, proline, glycine and valine were decreased.

In view of these results, the reported rises in total amino nitrogen during shock may reflect rises in substances other than, but similar to, free amino acids.

Among the explanations offered for the rise of total amino nitrogen in shock is increased production of amino acids from tissues and impairment of liver function. In this regard, it has been demonstrated by in vitro methods that the ability of liver slices from shocked ani-


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mals to deaminate certain amino acids, viz., alanine, is impaired.1, 33 In some patients with severe liver disease, Walshe32 found little abnormalities in the plasma concentrations of individual amino acids until the last stages of hepatic failure.

Another factor bearing on the interpretation of plasma concentration data is the size of the plasma compartment; "false-high" or "false-low" levels might be obtained consequent to certain fluid and electrolyte shifts. It has recently been shown by Christensen,2 that some, and possibly all amino acids can be concentrated to some degree by various cells; that this concentrative process is related to processes which control electrolyte balance; and that amino acids can displace potassium from cells and vice versa. Sodium ion fluctuations also affect amino acids; Wolf and McDowell34 have observed that a 30 per cent increase in the plasma sodium concentration by sodium chloride infusion in dogs caused a roughly twofold increase in the concentrations of the plasma amino acids.

Decreases in plasma total alpha-amino nitrogen concentration have been reported during certain infectious diseases.10

The influence, if any, of fever, blood transfusions, and infusions of other fluids, and antibiotics on the plasma amino acids is unknown.

In regard to the effect of nutrition on plasma amino acids, Man et al.,21 found lower than normal concentrations of total amino nitrogen (measured chemically) in the plasma of malnourished patients. Everson and Fritschel9 measured, by microbiologic technics, the fasting plasma levels of the 8 amino acids considered essential for man and of histidine and arginine in surgical patients prior to operation. Thirty-eight of the patients were considered to be in poor nutritional state, while the other 25 were in good nutritional status. The two groups were made of patients having approximately the same pathologic conditions. The mean value of each of the amino acids except arginine was significantly lower in the plasma of the poorly nourished patients. They found that the decreased levels of the free amino acids in plasma of five malnourished patients were not restored to normal by only a few days of high-protein (level not stated) diets.

Kirsner, Sheffner, and Palmer16 found in one human subject that the oral administration of a peptone solution treated with hydrogen peroxide and containing markedly reduced quantities of methionine, lysine, histidine, leucine, isoleucine, valine and threonine, was accompanied by definite decreases in the free levels of these amino acids in the plasma. During the same periods, the amino acid outputs in the urine increase considerably. These findings point to the importance of considering recent dietary intake and urine output in evaluating plasma concentrations.


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Christensen and his co-workers2, 3 have shown that high plasma levels of some amino acids (e. g., proline), brought about by feeding, appeared to interfere, perhaps on a competitive basis, with the cellular activity for concentrating other amino acids. When glutamic acid was fed in excess, however, the concentrating activity of liver and muscle for certain amino acids appeared to be increased and there was a concomitant fall in the plasma levels of these amino acids. From this point of view, decreased plasma levels of amino acids may result from increased concentrative assimilation by the cells. Elevated levels may occur when there is inhibition of the concentration process.

Regarding our own data, reference to Tables 1 to 5 and Figures 1 to 8 will give some idea as to the amino acid trends. It is significant that in no patient, at any time, are all the amino acids depressed or elevated. Rather, they seem to group themselves. Thus, in patient K. D., (a) glycine, histidine, threonine, proline and glutamic acid stayed near normal throughout the course of study; (b) leucine, isoleucine, lysine, valine, tyrosine and alanine rose moderately between days 3 and 9 post injury and then fell, (c) phenylalanine, aspartic acid and methionine showed the same picture of elevation, but to a greater degree. Taurine, the sulfonic amino acid, may not be handled by the organism as a metabolic intermediate, and bore little or no relationship to the other amino acids.

Patient C. J. showed, in general, the same picture, with the amino acids of group (a) normal or slightly low, group (b) becoming moderately elevated between days 1 and 8 post-injury and then falling, and group (c) becoming very elevated and then falling. In this patient, taurine reached a level of 1,200 per cent above normal on day 4; dialysis corrected this, and taurine thereafter stayed normal. The data of patient A. K. are less complete, but the same picture of grouping can be seen.

Can these trends be related to the injuries of these men, and to their subsequent clinical course? It is obvious that the separate effects of injury, shock and extensive transfusions cannot very well be delineated, since they occurred within so small a time interval. However, in none of our patients did the total of the 19 amino acids which we analyzed rise to excessive heights in the plasma. Whereas we have obtained a normal average value of 3.5 mg. per 100 cc., the highest values for the patients studied serially were 5.8 mg. (A. K., day 1 post-injury), 5.0 mg. (C. J., day 3 post-injury) and 4.8 mg. (K. D., 6 days post-injury). These values, although not excessively high, are significantly so. A. K. had received a penetrating abdominal wound, with resultant perforations of the liver, kidney and colon. He apparently


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was not in shock prior to operation (7 hours after injury). During the operation, however, his blood pressure became imperceptible for about a half hour and remained low for some time during operation. Immediately after operation, his plasma total amino acids were normal. During his first post-wound day, he received 10,000 cc. of blood, 2,000 cc. of gelatin and 500 cc. of dextran. At the end of this day his total amino nitrogen was 5.8 mg. per 100 cc.

Neither C. J. nor K. D. had elevated total amino acids on day 1 after injury. Both K. D. and C. J. were even more severely wounded than A. K. Both had been in severe shock; C. J., like A. K., had perforating wounds of the liver and kidney; all received large quantities of blood, but A. K. had also received gelatin. Glycine, occurring in relatively large quantities in gelatin, might be expected to appear in the plasma if the metabolism of gelatin were contributing to the rise in the total plasma amino acid concentration seen in this patient at the end of the day of injury. However, the rise in glycine was only slight. Other data indicate that relatively little gelatin is metabolized in the first post-infusion day. The rise in total amino nitrogen was, in fact, due principally to alanine, phenylalanine, leucine, lysine, tyrosine, threonine and glutamic acid.

In patient C. J. the plasma amino nitrogen fell gradually from its peak of 5 mg. per 100 cc. to 2 mg. per 100 cc. by the ninth post-injury day, and stayed low to the end of study. At the lowest point (day 9), all the amino acids, other than aspartic acid, glutamic acid, histidine and phenylalanine, were low. This patient's course was complicated by cerebral concussion, renal failure, jaundice, wound infection, pneumonia, pericarditis and marked weight loss. He had no oral food until the last day of study. Parenterally, he received some blood, glucose, electrolytes and vitamins. His caloric intake was grossly inadequate; his only nitrogen source was his transfusions. In this regard, it will be recalled that Man et al.,21 and Everson and Fritschel9 found decreased plasma amino acids in malnourished patients.

Patient T. L., who also had renal failure and an inadequate dietary intake, also had a slightly low amino nitrogen on the one sample we studied (12 days post-injury).

Patient A. K.'s dietary protein intake was also very low, but his parenteral caloric intake was significantly higher than C. J.'s. A. K.'s total plasma amino nitrogen was slightly low on only one occasion, the second post-injury day. His course had also been complicated by persistent renal failure, jaundice, pneumonia, bacteremia, liver abscess and liver necrosis.

Patient K. D.'s dietary intake was probably higher than that of the other patients and included moderate amounts of oral protein


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His total plasma amino nitrogen was only slightly low on the tenth and eleventh post-injury days.

Patient F. H. had a normal amino nitrogen on day 9 post-injury, and slightly elevated amino nitrogen on day 10. No data of his dietary intake up to this time are available, but it is likely that he, too, ate moderately during the first week post-injury since his renal failure occurred late, on the eighth day after injury, and 1 day after a secondary operative procedure.

After injury, activation of various endocrine glands (particularly the hypothalamic-pituitary-adrenal axis) occurs. Many of the metabolic changes seen following injury have been ascribed, in part, to the increased activity of this system. Crimson, Hanvey, and Luck4 have shown that injections of adrenalin will produce lowering of plasma amino nitrogen in fasting dogs. Others have made similar observations in other species.25 Since increased excretion of adrenalin during anesthesia and operation is well established, Everson and Fritschel have suggested that the decrease in the levels of plasma amino acids following anesthesia or operation observed by them may be consequent to increased adrenalin secretion. On the other hand, there is considerable evidence to indicate an increased secretion of adrenocorticotropic hormone after injury and operation. Li, Geschwind, and Evans18 have observed an increase in plasma amino nitrogen following the repeated administration of adrenocorticotropic hormone to rats. On the other hand, neither Li et al.,18 nor Luck and his associates14, 20 observed this increase following a single injection of adrenocorticotropic hormone to rats. The latter investigators found no change, or a lowering, of the blood amino acid concentration following single injections of various commercial adrenocorticotropin preparations. Cortisone, desoxycorticosterone, norepinephrine and testosterone did not affect the level of the blood amino acids in normal rats.

Li et al.,18 have demonstrated that the administration of anterior pituitary growth hormone causes a significant decrease in the blood amino acid content in rats. Luck and his associates have confirmed this and have also noted that thyrotropin has a similar effect.14, 20 Although injection of insulin will lower the concentration of amino nitrogen in the plasma, the amount of insulin required is enough to produce signs and symptoms of hypoglycemia.19 There is no evidence to suggest this degree of insulin secretion in the postoperative or post-injury period.

Urinary excretion of amino acids may reflect, or induce, changes in plasma amino acid concentrations. Everson and Fritschel state that they failed to find an increased urinary excretion of individual amino


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acids during operation in five patients sufficient to account for the lowering of the plasma levels of the amino acids observed. Nardi24 has recently applied the technic of paper chromatography to study the urinary excretion of amino acids in surgical patients. It should be emphasized that this is not a quantitative technic. He noted an apparent increase in the number and quantity of amino acids excreted by patients with burns or patients undergoing major operations. This increase began in the first days after injury and was roughly proportional to the severity of the burn or operation. The increased urinary output of amino acid did not necessarily coincide with the period of greatest negative nitrogen balance, but in some cases extended beyond the period of negative nitrogen balance. However, no data of intake or total urinary nitrogen or amino acid outputs are given. He also observed an increased urinary excretion of amino acids in patients with Cushing's syndrome. Following bilateral adrenalectomy, the amino acid excretion became normal, the patient being maintained with cortisone.

In seeking an explanation for the amino aciduria observed in these patients, Nardi considers the possibility that the amino aciduria is a reflection of an increase in the plasma concentration of the amino acids or consequent to a decrease in renal tubular reabsorption of the amino acids. (No measurements of the plasma amino acids were made.) He suggests the possibility that a disorderly, albeit not elevated, pattern of amino acids are presented to the kidney leading to a disturbance of tubular reabsorption. He also suggests the possibility of an increased secretion of an adrenocorticosteroid, probably other than cortisone, which would decrease tubular reabsorption of amino acids.

When renal function is normal, large increases in the plasma levels of amino acids may not occur, since a rise in their concentration, above the renal threshold may result in a large increase in their excretion.

We have had the opportunity to study the plasma amino acid in patients with persistent renal failure. While the biochemical picture of these patients is complicated by other pathologic conditions, our general impression is that renal failure has little specific effect on plasma amino acid levels. In spite of radically high plasma levels of other NPN components (urea nitrogen concentrations up to 400 mg. per 100 cc.), there was very little change in total plasma amino acid levels. The probability is that other, more powerful, regulatory processes can or do control amino acid levels in the plasma in the absence of renal function. Particularly dramatic is the failure of 6-hour plasma "washouts" of anuric patients on the artificial kidney, noticeably to affect plasma amino acid levels. The regulatory mechanism,


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or complex of regulatory mechanisms, can obviously operate independently of the kidney. This does not exclude the kidney as one of the regulatory organs of amino acids in normal individuals, but indicates that other processes can intervene in the homeostatic task when the kidneys fail.

As a consequence of our use of the ion exchange technic for the analysis of amino acids, we have noted the presence, in all plasma samples analyzed, of an ultrafilterable, ninhydrin-reactive compound which is unstable to hot acid, and which, on hydrolysis, yields identifiable amino acids. We interpret these experimental findings to mean that this compound has a molecular weight of below 15,000 to 20,000, and that it is comprised of amino acids held in peptide or peptide-like linkages. Paper chromatography has revealed that this component is heterogeneous, since it is readily resolvable into three or more fractions. Further evidence of this heterogeneity is indicated by these findings: In the two normal plasmas analyzed for this substance, only glycine and glutamic acid could be found, with a small amount of threonine in one. Plasma ultrafiltrates from patients with renal dysfunction, however, showed larger quantities of the substance; degradation with hydrochloric acid, moreover, revealed many more amino acids, e. g., leucine, valine, proline, tyrosine, histidine, etc. Furthermore, the amino acid composition varied from patient to patient, and from day to day in the same patient. In general, those patients with the higher NPN's had the substance in greatest concentration. In these cases, analysis showed maximum numbers of different constituent amino acids.

Extracorporeal dialysis reduced the plasma level of this substance or substances like it in roughly the same ratio as the other NPN components other than the free amino acids. No amino-acid-like, homeostatic mechanism, therefore, regulates the level of the fraction.

The absorption of ultraviolet light by plasma ultrafiltrates gives information concerning certain small molecular compounds. In particular, uric acid, at pH 7, has a strong absorption peak at 290 mµ, and most of the other naturally occurring purines, including uric acid absorb in the region 240 to 260 mµ. The aromatic amino acids absorb in the 280 mµ. region. In general, the entire ultrafiltrate absorption spectral pattern from 220 mµ. to 320 mµ. is elevated when plasma NPN is high. The elevation of the various portions of the spectrum, however, only roughly parallels the NPN rise. Figures 10 and 11, for example, show progressive rises in spectral pattern in a patient becoming progressively more azotemic. On day 8 post-injury, the pattern is grossly elevated, at which time the NPN was 288 mg. per 100 cc. On day 9, when the NPN had risen to 383 mg. per 100 cc., the elevation of


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the spectrum is no greater than it was on the previous day. This observation, together with the observable differences in the shape of some of the patterns, makes it seem probable that the components contributing to the spectral absorption vary in their relative concentrations. Since by far the largest contribution to absorption in the ultraviolet region is due to purines, these compounds are implicated in the high NPN levels.

Summary

Serious metabolic derangements are common after severe injury. In particular, nitrogen metabolism may become abnormal. Accordingly, we have studied changes in the pattern of the nonprotein nitrogen components in the plasma of five critically wounded soldiers.

The following quantitative data are presented: NPN creatine plus creatinine, uric acid, other purines and urea. In addition, 19 individual amino acids were quantitatively analyzed by ion exchange chromatography.

Of the five patients studied, four died. Shock and renal failure were present in all varying degrees. No plasma amino nitrogen level over 5.8 mg. per 100 cc. was found. Renal failure was persistent in four of the patients. Although plasma urea concentrations were as much as 30 times normal, the total free plasma amino acid nitrogen remained near normal.

In those patients studied for periods extending from the day of injury to 2 weeks post-injury, the plasma amino acids followed a pattern. At no time were all the amino acids depressed or elevated. Rather, they grouped themselves as follows: (a) glycine, histidine, threonine, proline and glutamic acid stayed near normal; (b) leucine, isoleucine, lysine, valine, tyrosine and alanine rose moderately during the first week and then fell; (c) phenylalanine, aspartic acid and methionine also rose during the first week, but to a greater degree. Taurine bore little or no relation to the other amino acids. At times, it was extremely high.

In two patients, who became markedly malnourished, the total plasma amino nitrogen fell to low levels.

In all plasmas analyzed, a heterogeneous amino conjugate was found. The amino acid composition varied from patient to patient, and in the same patient from day to day. In general, those patients with the highest NPN's had the substance in greatest concentration. In these cases, acid hydrolysis revealed maximum numbers of different constituent amino acids.

Analysis of the ultraviolet absorption spectra of the plasma ultrafiltrate of these patients indicates that there was an increase in purines, free or bound.


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Extracorporeal dialysis with a Kolff-type artificial kidney was performed on three patients. Temporary biochemical and/or clinical improvement followed the dialyses. With the notable exception of the amino acids, all the small nitrogenous compounds (including the plasma amino conjugates) were sharply reduced after dialysis. In contrast, the plasma concentrations of the amino acids were little changed, despite the 6-hour "washouts" on the artificial kidney.

References

1. Bekkum, D. W., van, and Peters, R. A.: Observations upon Change in Enzymatic Process in Burns. Quart. J. Physiol. 36: 127-137, 1951.

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3. Christensen, H. N., Streicher, J. A., and Elbinger, R. L.: Effects of Feeding Individual Amino Acids upon Distribution of Other Amino Acids between Cells and Extracellular Fluid. J. Biol. Chem. 172: 515-524, 1948.

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10. Farr, L. E., McCarthy, W. C., and Francis, T.: Plasma Amino Acid Levels in Health and in Measles, Scarlet Fever, and Pneumonia. Am. J. Med. Sci. 203: 668-673, 1942.

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12. Glenn, W. W. L., Muus, J., and Drinker, C. K.: Observations on Physiology and Biochemistry of Quantitative Burns. J. Clin. Invest. 22: 451-460, 1943.

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32. Walshe, J. M.: Disturbances of Amino Acid Metabolism Following Liver Injury; A Study by Means of Paper Chromatography. Quart. J. Med. 22: 483-505, 1953.

33. Wilhelmi, A. E., Russell, J. A., Engel, F. L., and Long, C. N. H.: Effect of Hepatic Anoxia on Respiration of Liver Slices in vitro. Am. J. Physiol. 144: 669-673, 1945.

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