Battle Casualties in Korea: Studies of the Surgical Research Team, Volume IV
Metabolic Effects of Injury; Studies of the PlasmaNonprotein 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 disturbancein the metabolism of the essential amino acids is one of the importantfactors in the pathogenesis of the malnutrition following injury.6However, there are no conclusive data regarding this viewpoint.5,13, 22, 30 That amino acids are fundamental to protein synthesisand many energy processes in all animals is well known; yet, the quantitativestudy of these compounds in man is in its comparative infancy. Lack ofappropriate methodology accounts in part for the scarcity of informationin this important field.
A practical method for the quantitative fractionation of plasma aminoacids was introduced in 1951, when Stein and Moore23 perfectedthe method of ion exchange chromatography. Until that time, the littleinformation available concerning individual amino acids of plasma had beenobtained by the use of a microbiologic technic, a method based on the extentof growth of mutant bacterial strains on a medium containing the plasmasample. This method is open to the objection that these organisms are somewhatnondiscriminating in their choice of nutrient, and metabolize certain aminoconjugates as well as single amino acids. The microbiologic method, thencan be expected, in general, to give false high values for some plasmaamino acids. By
*In press (in modified form): Surgery, Gynecology and Obstetrics.
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the ion exchange chromatographic technic on the other hand, differentiationof 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 determiningwhether any trends could be found in the plasma levels of the free aminoacids of severely wounded soldiers from whom serial specimens were obtainedbeginning early after injury. These subjects were chosen because it isprecisely this type of patient who demonstrates the greatest metabolicderangements after injury-that is, the seriously injured, but previouslyhealthy 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 SurgicalResearch Team and the Eighth Army Medical Corps.
Methods
Virtually all the samples analyzed were obtained as plasma in 2.5 percent sodium citrate (1 ml. citrate to 5 ml. blood). The plasma sampleswere 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 anapproximate maximum molecular weight species of about 15,000 to 20,000.
The individual amino acids were separated by the ion exchange chromatographictechnic devised by Stein and Moore,23 and the analyses wereaccomplished by the photometric ninhydrin method of the same workers.31About 4 ml. of the plasma filtrates was required for the analysis. Of thecommon amino acids, tryptophane and arginine were not determined; the formeris destroyed on the column, the latter requires additional hours for itsseparation.
An acid labile amino component (amino acid conjugate) was isolated inmany samples. The fraction containing this component was evaporated ineach case by a stream of air to about 2 ml. It was then hydrolyzed with6N HCl in a sealed tube at 110° for 20 hours. The resultant hydrolysatewas evaporated of its hydrochloric acid as above, made up to 4 ml. withwater and re-chromatographed in the usual manner.
Other small molecular weight, plasma nitrogenous compounds were alsodetermined. These are urea, creatine and creatinine, uric acid, purines,and total nonprotein nitrogen. Uric acid was estimated from measurementsof absorption at 293 mµ, and 305 mµ. The validityof this determination on plasma ultrafiltrates, even in the presence of
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large quantities of aromatic amino acids has been demonstrated by us.27Purines were grossly estimated from the absorption at 260 mµ.
Urea and NPN were analyzed at first by the micro method of Seligsonand Seligson,29 and later by a titrimetric modification. From0.005 to 0.02 ml. of the ultrafiltrate was required for each of these analyses.
Creatine and creatinine were determined by application of the Jaffereaction11 to about 0.05 ml. of the plasma filtrate.
Our results are presented either as µM of nitrogen per100 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. Sincethe ratio of nitrogen to total weight of one amino acid is different fromthat of any other, the expression of levels in terms of mg. of amino acidor mg. of nitrogen leads to a distorted impression of the relative quantitativeimportance of the various amino acids. By the use of the micromole unit,the amino acids are compared, in effect, molecule for molecule, regardlessof size or weight.
Absorption spectra were obtained on ultrafiltrates, diluted 20 timeswith water, with the Beckman Du spectrophotometer in 1 cm. quartz cells.We have found that ultraviolet absorption spectra of plasma ultrafiltratesare useful in estimating certain plasma constituents, viz., uric acid andother purines, and the aromatic amino acids, phenylalanine, tyrosine andtryptophane. The ultrafiltrates are ideally suited for this procedure,since they have no absorbent material added to them in vitro, and theyare 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 otherNPN components, in five critically wounded young soldiers. Four of thosemen died. Shock and renal failure were present in all in varying degrees.In four, the renal failure was persistent. Although the plasma urea concentrationsrose as much as 30 times normal, the total free plasma amino nitrogen concentrationremained near normal.
Extracorporeal dialysis by a Kolff-type artificial kidney was performedon three patients. Temporary biochemical and/or clinical improvement followedthe dialyses, but eventually, all died. The
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total free amino nitrogen concentration was little affected by the dialysisprocedures. The plasma levels of certain individual amino acids were oftennear normal in spite of the serious injury, shock, severe renal failure,near starvation, infection and extracorporeal dialysis. In contrast, thelevels of some other amino acids changed markedly. It is likely that anywide fluctuations of the individual amino acids indicate a basic upsetin the body economy.
In the following section, the observations in each individual patientwill be described.
Patient No. 1, T. M. L. This 25-year-old Koreanwas wounded by an M-1 30-caliber armor-piercing bullet. Resuscitation (includinginfusion of 1 liter of blood) was begun within an hour of injury and hewas never in severe shock preoperatively. Operation, performed under pentothal,nitrous oxide and other anesthesia, was begun 21/2hours after injury. Five liters of blood was infused during the operation,which lasted 5 hours. The missile had penetrated the right lung and pleuralcavity, 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; gelfoamwas packed into the missile tract in the liver; an intercostal Foley catheterwas inserted into the right pleural space. There was no uncontrollablehemorrhage or hypotension during surgery. Penicillin and streptomycin weregiven.
Throughout his course, he did not move his legs, and hadabsent reflexes and sensation in his lower extremities. A collapsed vertebraat D-12 was demonstrated, presumably due to damage from the missile, andit was assumed that his spinal cord had been damaged at about this point.Postoperatively, he was oliguric and was transferred on the second post-injuryday to the Renal Insufficiency Center.
During the next 2 weeks, he ran a stormy course. Severepneumonia, high lever, weight loss, persistent renal dysfunction, nitrogenretention, acidosis and hyperkalemia were important complications. He receivedaureomycin and, later, chloramphenicol. Extracorporeal dialysis on an artificialkidney of the Kolff type was carried out on the fourth post-injury daybecause of potassium toxicity. The resultant reductions in plasma K andNPN concentrations were only transient, however, and repeat dialyses wereperformed on the eighth and twelfth day after injury.
Our first analyses were done on a plasma sample obtainedon 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 aminoacids were all lower than normal (Table 5). The effect of the dialysison the nonprotein nitrogen components will be discussed later in a specialsection devoted to this matter (Effect of Extracorporeal Dialysis on thePlasma 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 againonly temporary improvement, was done on the sixteenth day.
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Tracheotomy was done to enable suction of tracheobronchialsecretions. Because of a spiking fever, right upper quadrant pain and fixationof the diaphragm on the right, extraperitoneal exploration of the rightsubdiaphragmatic space was carried out on the eighteenth day. No abscesswas found. The next day, he suddenly died of respiratory failure followingaspiration.
The postmortem diagnoses were surgically repaired venacava, liver and diaphragm, surgical absence, left kidney; atelectasis,right lower lobe and right middle lobe of lung; emphysema, left side ofchest; abscess, left lower lobe of lung; pneumothorax, left; edema, rightupper lobe of lung, bronchopneumonia (?); hematoma, liver and diaphragm;hypertrophy, right kidney and congestion of renal medulla; abscess, retroduodenalregion; splenomegaly; malaria (?); trichuriasis; decubiti, left buttockand sacrum.
Patient No. 2, F. H. This 23-year-old Americansoldier was burned and wounded when a trip flare exploded. His legs andlower trunk were burned and a flaming missile penetrated his abdomen. Hewas hypotensive for only a brief time preoperatively; during this timehe received 1,500 cc. of blood and 750 cc. of plasma. Penicillin was given.
Operation was begun 7 hours after injury and lasted 5hours. The lower abdominal wall was severely burned, and most of the smallintestine was charred and necrotic. The anterior abdominal wall was débridedwith excision of the external oblique muscle, rectus muscle and fascia,scrotum and testes. His penis was severely burned. All of the ileum andmost of the jejunum were resected, and an end-to-side jejuno-transversecolostomy was done. A small portion of proximal jejunum were resected andan end-to-end jejuno-jejunostomy was done. The terminal ileum was closedand exteriorized with the cecum. The total, circumferential deep burnsof both legs were then débrided with removal of all skin to subcutaneoustissue. 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 didrelatively well, with stable blood pressure, daily urine outputs between2,000 and 3,000 cc. and urine specific gravity up to 1.028. He ran a low-gradefever of about 100° with occasional spikes over 102°. He receiveda daily total of 1,000 to 2,000 cc. of blood plus plasma, as well as 3,000to 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 sixthday he developed icterus, which gradually increased.
On the seventh post-injury day, a homologous split-thicknessskin graft (from a cadaver) was applied to his left thigh and leg, underbrief (10-minute) N2Oanesthesia. Following operation, his urine output dropped abruptly to lessthan 10 cc. per hour. His general condition deteriorated, and he becamelethargic and began vomiting. Because of continuing oliguria, he was transferredby helicopter to the Renal Insufficiency Center on the ninth day.
On admission, he was jaundiced and his temperature was101°. The lower abdominal wall was almost all gone, except for a thinlayer of peritoneum and transversalis fascia. There was a profuse, dirtywatery and possible fecal discharge between the tissue layers at the edgeof the burned area. There were large masses of necrotic tissue in the groinarea. The deep burn of the buttocks was crusted. His penis was black andnecrotic. Penicillin and streptomycin were given.
Admission plasma chemistries: Na, 160 mEq./L.; K, 5.8mEq./L.; Cl, 110 mEq./L.; CO2,23.4 mEq./L. The hematocrit was 31 percent. The plasma nonprotein nitrogenfractions were measured for the first time on this day (Table 1).
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This plasma NPN and urea nitrogen were very high, 336and 269 mg. per 100 cc. respectively. The uric acid, other purines, andcreatine 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 21/2times normal; isoleucine, tyrosine and histidine 11/2times normal; proline, glycine, lysine and the glutamine-serine-asparaginecomplex were about half normal; threonine, alanine, valine, leucine andcystine 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-woundday, he suddenly began having respiratory difficulty and became unresponsiverather abruptly. He was flaccid, with weak deep tendon reflexes and appearedto have respiratory paralysis. He was immediately given 3.75 gm. NaHCO3intravenously, followed by 80 cc. of 3 per cent saline, with prompt improvementof respiration and revival of consciousness. A blood sample drawn justbefore this emergency treatment showed that the serum potassium was 8.3mEq./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 creatineand 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 ofthe amino acids
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remained at their previous levels, or rose. Threonine,proline, alanine and taurine were normal at this time. Glutamic acid, incontrast to the other amino acids, fell sharply. There was a correspondingrise in that function containing glutamine.
In the next few hours, he became progressively more hypotensivedespite administration of hypertonic saline bicarbonate, glucose and insulin,and noradrenalin, and died before dialysis could be performed.
Postmortem diagnoses were second and third degree burnsof abdominal wall, buttocks, perineum, penis, scrotum and legs; diffusephlegmonous inflammation and necrosis, anterior abdominal wall; ileocolicanastomosis, and cecostomy; skin grafts, left leg; bilateral castrationand surgical absence of scrotum; hepatitis, acute, toxic, renal hypertrophy,bilateral, with clinical renal insufficiency; acute hemorrhagic esophagitiswith massive bleeding; focal fat necrosis, head of pancreas, minimal; pulmonaryedema, moderate; cerebral edema, moderate; arachnoid cyst, right temporallobe of brain; icterus.
Patient No. 3, K. D. This 22-year-old Americansoldier was wounded by shell fragments. The wounds included multiple perforationsof the small bowel, multiple perforations of the sigmoid, two holes inthe urinary bladder, severe comminuted, compound fractures of the leftfemur and left tibia and multiple soft tissue wounds of the buttocks andabdominal wall.
Prior to operation, he was given 4,000 cc. of blood. Hisblood 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, threebowel resections, colostomy, cystostomy and débridement of the manywounds. With the onset of pentothal induction (later nitrous oxide-oxygen-etheranesthesia) his blood pressure fell from 106/84 to 66/42, and his pulserose from 100 to 116. Four milligrams norepinephrine dripped in with 500cc. of blood immediately raised his pressure from 90/50 to 100/60. Laterin the operation, his blood pressure was maintained with difficulty despitethe administration of considerable blood with added norepinephrine. Atoperation, peritoneal contamination was massive. He was given 3,500 cc.of blood during operation while the measured operative blood loss was 1,670cc.
A hemoclastic reaction was strongly suggested by a dropin his white blood cell count 1 hour after operation to 1,850 and abnormalclot formation. His platelet count at this time was 710,000; hematocrit,55 per cent. His plasma volume (Evans Blue) was 2,220 cc. and calculatedblood volume 4,720 cc. His blood pressure was about 90 to 100 systolicand 50 to 60 diastolic; pulse rate, around 120 per minute. One thousandcc. of dextran was then given over a period of 4 hours. No changes in bloodpressure or pulse occurred. His hematocrit was then 42.5 per cent; plasmavolume, 3,070 cc. and blood volume, 5,200 cc.
Throughout the first postoperative night, his blood pressureranged about 80/50; pulse, 130; respirations, 40. Fifteen hundred cc. ofblood and the same amount of dextran were then given over a period of 7hours, with a rise in his blood pressure to 112/80 and a drop in his pulserate to 104. His plasma volume had risen to 3,750 cc. and his blood volumeto 5,980 cc. This was 24 hours postoperatively. Five hundred cc. more bloodwas given.
At this time, his plasma urea concentration was 74 mg.per 100 cc. Plasma uric acid concentration was slightly elevated as werecreatine and creatinine; the purine fraction and total amino acids wereessentially normal. Methionine, glutamic acid, tyrosine, aspartic acidand proline were normal; histidine, taurine, alanine and phenylalaninewere somewhat elevated; leucine, isoleucine, lysine, valine, threonineand glycine were somewhat low (Table 2).
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Table 2. Ultrafilterable Nitrogen Components of Plasma (PatientK. 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 |
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During the second postoperative night his blood pressureagain fell to 94/64, and his pulse rose to 140 per minute. No bleedingwas evident. Five hundred cc. of dextran was given with no obvious improvement.
Fifty-five hours after injury, forty-eight hours afteroperation, infusion of noradrenalin in glucose in water intravenously wasbegun. His blood pressure gradually rose to 120/70, but his pulse continuedat a rapid rate (140 per minute). Oxygen, per nasal catheter, had changedthe appearance of his skin from a cyanotic to a reddish flush. Throughoutthe day his blood pressure was maintained at 115-125/65-80 and his pulserate gradually fell to 115 per minute. The infusion of noradrenalin wasgradually slowed and stopped the next day. His blood pressure was maintainedthereafter at normal levels.
Sixty-six hours after injury and fifty-nine hours afteroperation, the NPN components had all risen. The amino nitrogen was now4.4 mg. per 100 cc., while urea nitrogen was 107 mg. per 100 cc. Of thoseamino acids previously normal, all but glutamic acid had risen; alanineand 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 3,600cc. and blood volume, 6,100 cc. His blood pressure had been normal forabout 36 hours. The plasma total free amino nitrogen concentration wasnormal, while plasma urea was still elevated (99 mg. per 100 cc. uric acidnitrogen was 2.4 mg.; purine nitrogen, 0.9 mg.; creatine plus creatininenitrogen, 3.7 mg.) Methionine, aspartic acid and histidine were greatlyelevated; 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. Creatineplus creatinine and purine nitrogen remained near normal, uric acid graduallyfell to normal, while the total amino nitrogen fluctuated slightly aroundnormal. Aspartic acid continued to rise through the sixth postoperativeday, and then fell steadily to the eleventh day. Methionine fell transientlythrough 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. Incontrast, phenylalanine, glutamic acid and taurine, which were respectivelyhigh, normal and low during the first week, rose thereafter, and all werehigh on the last day of study. Histidine and lysine fluctuated widely fromday to day from above to below normal.
Patient No. 4, A. K. This 22-year-old Americansoldier received a perforating abdominal wound by an M-1 rifle bullet.His blood pressure, which was 115/85 when first recorded at 2 hours afterinjury, fell to 90/60 1 hour later. Five hundred cc. of blood was startedand he was sent to a Mobile Army Surgical Hospital by ambulance. Four andthree-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 penicillinand tetanus toxoid.
Laparotomy was begun 7 hours after injury after the patienthad received an additional 1,500 cc. of blood. Anesthesia consisted ofpentothal and nitrous oxide-oxygen-ether. The wound was a through-and-throughperforation of the abdomen with resulting perforating wounds of the liver,kidney and colon. About 1,500 cc. of blood was aspirated from the abdomenduring surgery. A colostomy and drainage of the liver and kidney woundswere performed.
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During operation, his blood pressure became imperceptiblefor about 30 minutes, but was elevated to 60 or 80 systolic as noradrenalinwas added to the blood. He was given 7,500 cc. of blood during the operativeprocedure which lasted 5 hours. Operative blood loss was measured at 2,420cc.
Our first analysis was performed on blood taken 12 hoursafter injury, immediately after operation. His hematocrit was high, about70 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 glutamicacid were elevated; tyrosine, aspartic acid, proline, threonine, glycineand 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, someof which contained terramycin. His urine output was low, blood pressurewas normal, but pulse and respirations were rapid. Plasma urea nitrogenhad risen, as had all the other NPN components. Total amino nitrogen wasdefinitely elevated (5.8 mg. per 100 cc.). Valine stayed normal, and lysine,glutamic acid and taurine fell. All the other amino acids rose. Duringthe next day, he continued oliguric; blood pressure was normal. Aureomycinwas begun. His plasma urea rose, as did the NPN components except the totalamino nitrogen, which fell slightly below normal. All the amino acids fell,except for isoleucine, which stayed normal, methionine, which remainedhigh, and taurine, which rose. Plasma Na was 125 mEq./L.; K, 5.7 mEq./L.
Because of persistent oliguria, he was transferred tothe Renal Center on the second post-injury day. On arrival, he was feverishand toxic with signs of left lower lobe pneumonia. His sputum was positivefor Staphylococcus and Proteus, the latter sensitive only to chloromycetin.The response to chloromycetin, however, was poor. The patient became clinicallyjaundiced, presumably secondary to the liver injury, the serum bilirubinreaching a peak of 14.5 direct and 25.3 total 5 days after injury.
He was on constant Wagensteen suction and lost 600 to2,200 cc. of dark fluid daily. Oral feeding was not possible. Varying quantitiesof 50 per cent glucose in water containing 2 gm. of vitamin C, 25 mg. ofvitamin K, and 10 mg. of thiamine were given daily by intravenous catheter.
Hyperkalemia was well controlled, the plasma level droppingfrom 7.6 mEq./L. on admission to 5.8 by the next morning. The toxic effectsof potassium were also counteracted later with intravenous calcium gluconate.
Throughout his course, this patient was in good fluidbalance. Diuresis occurred immediately and, thereafter, the urinary outputwas about 2,000 cc. daily, but the plasma NPN continued to rise, reachinga 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 thistime, he underwent extracorporeal dialysis by an artificial kidney of theKolff type because of uremia, drowsiness, nausea, tremulousness, occasionalperiods of disorientation, and a severe hemorrhagic diathesis. The resultsof the dialysis were satisfactory chemically, but the clinical conditionof the patient did not improve and in retrospect the symptoms probablywere due to sepsis rather than to uremia. Just prior to dialysis, plasmaNa 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 pluscreatinine, purine and uric acid nitrogen were also much elevated, whiletotal amino nitrogen was slightly high. Phenylalanine, histidine, asparticacid, methionine, tyrosine, leucine, isoleucine and lysine were high. Allthe rest were normal except taurine, which was low. Following dialysis,all the NPN components other
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Table 3. Ultrafilterable Nitrogen Components of Plasma (PatientA. 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 descriptionof the chemical findings will be found in the section on "ExtracorporealDialysis.")
During the next day the patient continued running a septiccourse. He had lost 30 pounds. Plasma Na was 146 mEq./L.; K, 6.2 mEq./L.;CO2, 20 mEq./L. Therewas 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 werelow; all the rest were normal. No subsequent plasma samples were analyzed.
Bleeding into the nasopharynx, bowel and skin occurredand persisted in spite of fresh blood transfusions and large doses of vitaminK. Frequent hypotensive episodes occurred but could be controlled withblood. Noradrenalin in increasing amounts was required to maintain bloodpressure, but finally the patient became refractory to the drug and transfusionand he died in circulatory collapse on the twelfth post-wound day. Theclinical impression was that the uremia was reasonably well controlledand that death was due to overwhelming sepsis.
Pathologic findings included pulmonary infarcts of theleft lower lobe, acute phlebitis of the inferior vena cava, septicemia(B. proteus), fibrinopurulent peritonitis, diffuse mucosal hemorrhagesof the small intestine, gunshot wound
259
of the right lobe of the liver, an abscess containingB.proteus in the liver, and central necrosis of the right lobe of theliver. There was mild lower nephron nephrosis, a few areas of focal glomerulitis,traumatic destruction of the lower pole of the right kidney, and multipleabscesses of the right kidney containing B. proteus.
Patient No. 5, C. J. This 26-year-old Americansoldier was wounded by mortar shell fragments. The wounds included lacerationof the scalp, perforations of the liver and kidney, traumatic amputationof 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 intravenousinfusion of 320 cc. of albumin and 1,500 cc. of isotonic saline his bloodpressure was 80/40. He was then evacuated by helicopter to a Mobile ArmySurgical 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 pressureremained unobtainable. His pulse was barely palpable at a rate of 110 perminute. His right leg was amputated without anesthesia in the emergencyroom. In the next 4 hours, he was given 2,500 cc. more blood. His bloodpressure 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
260
nitrogen were already elevated, while creatine plus creatininenitrogen, purine nitrogen and total amino nitrogen were normal. Glutamicacid was twice normal; alanine and phenylalanine were slightly elevated;threonine, glycine, valine and histidine were slightly low; all the restwere normal. Plasma Na was 158 mEq./L.; K, 3.8 mEq./L.
Operation was delayed for 3 more hours because he hadnot regained consciousness. During this time he received another 1,000cc. of blood. His blood pressure had remained stable at about 130/80 andhis pulse between 120 and 130 per minute.
Operation was begun 10 hours after injury, under atropine,pentothal and nitrous oxide-ether anesthesia. His blood pressure immediatelyfell from 140/80 to 90/60, but his pulse rate remained unchanged. His bloodpressure remained low throughout most of the operative procedure (whichlasted 3 hours) despite the administration of an additional 3,500 cc. ofblood. Operative blood loss, by the washed sponge technic, was 3,030 cc.Operation consisted of exploratory laparotomy, drainage of the liver andkidney perforations, re-amputation of the thigh and débridementof many soft tissue injuries. He was found to have fractures of the femurproximal to the line of amputation.
261
Towards the end of the operation, noradrenalin was addedto the infused blood. A rise in blood pressure was immediate. Plasma NPNand urea had risen still further; creatine and creatinine nitrogen, uricacid and purine nitrogen were slightly elevated, while the total aminonitrogen was still normal. Glutamic acid, phenylalanine and alanine werestill elevated; taurine and aspartic acid had risen; all the others werenormal except methionine, glycine and valine, which were low. Plasma Nawas 146 mEq./L.; K, 5.4 mEq./L.
Noradrenalin was used throughout the night after injuryto maintain his systolic pressure between 105 and 120. The rate of noradrenalinadministration was gradually decreased and successfully stopped 24 hoursafter operation. On
the morning of the first postoperative day, the variousNPN components, other than amino N, were still elevated. NPN, urea anduric acid were rising. The total amino nitrogen, on the other hand, hadfallen below normal. Phenylalanine and alanine were still elevated whilevaline, glycine, proline, threonine, methionine, leucine, isoleucine andlysine were below normal. The other amino acids were normal. Plasma Nawas 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 110to 120; respiratory rate, 24 to 32. His temperature ranged from normalto 102° orally. He had received 500 mg. of terramycin, 1,000 cc. of5 per cent glucose and water, 100
262
mg. of thiamine and 1,000 mg. of vitamin C. His 12-hoururine output was only 115 cc.
On admission at the Renal Center, he was semicomatose;blood pressure was 140/80; temperature, 101°. His serum potassium was7.1 mEq./L.; sodium, 131; CO2,30; chloride, 86; hematocrit, 37 per cent NPN, 114 mg. per 100 cc. Becauseof the probability of pulmonary complications, he was tracheotomized andput on a Stryker frame on the third day after injury. His NPN componentsrose progressively during the next few days and all except the amino nitrogenreached very high levels. The latter was only moderately elevated. Taurinewas an
exception among the amino acids, rising to 12 times normalon the fourth post-injury day. Methionine, leucine, isoleucine, lysine,phenylalanine, valine, tyrosine and alanine all rose progressively throughthe third post-injury day; all but valine fell the next day. Glutamic acid,proline, threonine and histidine had remained normal or slightly low, whileglycine was persistently low. Aspartic acid fluctuated above and belownormal.
There was an initial drop in his serum K concentration,but then a progressive increase to 8.2 mEq./L. on the fourth post-injuryday with broadening of the QRS component on EKG. The serum Na, initially131 mEq./L., rose to 139 mEq./L. Chlorides stayed constant at about 85mEq./L., and CO2 gradually
263
dropped from 29.6 to 15.7 mEq./L. After emergency infusionof calcium gluconate, he was dialyzed by the artificial kidney (Kolff-type)with an excellent chemical result, but with little change in his generalcondition. The chemical effects of the dialysis will be discussed in thesection "Extracorporeal Dialysis."
During the next 5 days he was periodically conscious.He became increasingly jaundiced. Some of his wounds were grossly infectedand bled excessively following débridement. He was still oliguric,and the plasma NPN components
other than amino nitrogen rose steadily to levels similarto those just prior to the first dialysis. In contrast, the total aminonitrogen steadily decreased and reached the very low level of 2 mg. per100 cc. on the ninth post-injury day. Valine, tyrosine, alanine, asparticacid, leucine, isoleucine, proline and threonine all fell, while taurineremained normal and glycine persistently low. Plasma K concentration whichwas 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 139mEq./L. after the first dialysis, and rose to 149 mEq./L.; chlo-
264
ride fell from 104 to 93 mEq./L., and CO2fell from 26 to 18 mEq./L. On the ninth post-injury day, because of continuinghyperkalemia, he was again dialyzed, with correction of the potassium intoxicationand transient improvement in his general condition. (Details of the chemicalresults 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. Onday 12, urea nitrogen was 234 mg. per 100 cc., other purine nitrogen, 5.7mg. 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 dialyzedfor the third time, with correction of the hyperkalemia, but with no changein his very poor general condition. He continued to deteriorate and diedon 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 infectionof the skin flaps, muscle and fascia. There was a severe bacterial pericarditis,focal edema and necrosis of the myocardium, bronchiolar pneumonia, centralnecrosis (severe) of the liver, hyperthopy of the parathyroids, acute cystitis,necrosis of the occipital poles of the cerebral cortex, and lower nephronnephrosis.
265
Plasma Amino Conjugate
Early in our work on the plasma ultrafiltrates of human subjects, wenoticed that one chromatographically distinct ninhydrin reactive componentis unstable to acid hydrolysis. We isolated this component chromatographically,from a number of plasma samples, and rechromatographed them. The resultsare shown in Table 4 and Figure 9. The striking central fact is the quantitativeand qualitative variability of the composition of this fraction among thepatients.
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 componentamino acids in greater variety. In patients C. J. and A. K., the plasmaconjugate levels of the patients rose with the plasma urea levels, thoughnot proportionately. As the
266
Table 4. Amino Acid Composition of Plasma Ultrafilterable Amino Conjugate
Patient | 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 differentamino acids were found within the component; the quantitative relationsof the amino acids within the conjugate showed an ever shifting pattern,with first one amino acid becoming predominant then another. By the eleventhpost-wound day, in patient C. J. (Fig. 9), 12 of the naturally occurringamino acids were present in the conjugate, with leucine and proline inaddition to glycine and glutamic acid occupying
quantitatively important positions. Patient A. K. showed the same sortof 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 heightsof either C. J. or A. K., the amino conjugate concentration stayed at arelatively constant (but elevated) level. The quantitative relationshipsof the component amino acids gradually decreased in
268
Table 5. Effect of 6-Hour in Vivo Dialysis on Ultrafilterable NitrogenComponents of Plasma
Patient Day Post-injury | T. L. 12 | 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 comprisedsome 85 per cent of the conjugate on the ninth day after injury.
These observations suggested that the amino conjugate fraction is nothomogeneous. Accordingly, we chromatographed the intact conjugate on paperby the "circular" technic, using butanol-acetic acid-water (6:1:1) as thesolvent. The conjugate of
C. J. 2 days post injury was thereby separated into four ninhydrin-reactivecomponents and that of K. D. 5 days post injury, into three. A specificchromatographic analysis was made in one subject (C. J.) for the peptidescarnosine and anserine, normal intracellular constituents. Neither wasfound.
The conjugate, as a whole, acted as a metabolic end product with respectto extracorporeal dialysis (Table 5). The plasma concentration of the conjugatefraction was reduced after 6-hour dialyses in approximately the same ratiosas 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 normalplasma ultrafiltrate. The peak at 290 mµ.is almost entirely due to the purine, uric acid, which has a molecularextinction coefficient, at this wavelength, of about 12,500. Absorptionin the region of 240 to 260 mµ. is dueto other prines, but also included a second peak of uric acid. The aromaticamino acids, with considerably lower extinction coefficients (around 3,000to 4,000) have their absorption maxima at about 280 mµ.Absorption below these wavelengths is termed "nonspecific," and is characteristicof many nitrogenous compounds, including most of the amino acids.
The data for patient C. J. are shown in Figures 10 and 11. There wasan insistent and progressive rise in absorption at all wavelengths, therise being only temporarily halted by extracorporeal dialysis. The spectralabsorption 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 NitrogenComponents
Extracorporeal hemodialysis of the patients with severe renal dysfunctionwas carried out to reduce hyperpotassemia or relieve uremia. We studiedthe plasma of three patients, obtained before and after
270
271
272
6-hour dialyses, with a view toward establishing the pattern of changein the nonprotein nitrogen portion of the plasma, ostensibly that partreadily diffusible through a cellophane membrane. The data set forth inTable 5 and Figure 12 show that there is a discernible pattern. With thenotable exception of the amino acids, all the small nitrogenous compounds,including the amino conjugate, were sharply reduced in concentration afterdialysis. In contrast, the plasma concentrations of the amino acids werechanged little, despite the 6-hour "washouts" on the artificial kidney.
Discussion
When 19 plasma amino acids are measured by ion exchange chromatographyin a random group of healthy individuals, it is found that the total aminoacid N measures close to 3.5 mg. per 100 cc. This figure practically nevergoes above 3.8 (except for a short time after a high-protein meal) or below3.1 mg. per 100 cc. There is considerable variation in concentration amongthe amino acids, e. g., aspartic acid is present in a concentration ofabout 2.5 µM per 100 cc. whilethe concentration of alanine is about 32 µMper 100 cc. Speculations can be indulged in to explain this, but thereis, at present, inadequate experimental evidence to support any theories.However, each amino acid is present in about the same relative concentrationin the plasma of healthy men. Among the factors which have a possible connection
273
with maintaining the plasma amino acids relatively constant in normalindividuals are: dietary intake, absorption of ingested amino acids fromthe gut; deamination, transamination, and decarboxylation primarily inthe liver; excretion and reabsorption by the kidney; and dynamic transcapillaryexchange. The plasma amino acids are in active metabolic exchange withtissue amino acids, tissue and plasma proteins, and body carbohydrate andfat.
One of the main objects of this study was to discover if any quantitativetrend in plasma amino acids manifested itself in previously healthy youngmen who had been seriously wounded. At the present time, there is littlehope of pinpointing the causes for any noted amino acid alterations, since(a) the plasma specimens were obtained from men undergoing many and variedstresses and (b) as mentioned, the basis for the differences in plasmaconcentrations of the various amino acids in normal individuals is unknown.
Among the stresses which the patients we studied underwent, the followingmight 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, renaldysfunction, fluid and electrolyte imbalance, fever, infection, antibioticsand malnutrition. Also to be considered are the paths along which the stressesmay affect the amino acid levels, i. e., humoral, neural, etc.
Before discussing our results, the work already done on the effectsof some of the stresses mentioned may be summarized. Most of the work donein this field concerned only the total plasma amino nitrogen concentrationand not individual amino acids.
A prolonged decrease in the plasma concentration of total alpha-aminonitrogen following operation was reported by Man and co-workers.21They felt that the degree and duration of the decline in plasma total alpha-aminonitrogen concentration is related to the severity of the operative procedure.Less change postoperatively was observed in malnourished patients withlow plasma amino nitrogen levels preoperatively than in those patientswith 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 of10 individual amino acids. Fourteen of the patients were stated to be ingood nutritional status at the time of operation. Two presumably were not,but the authors do not specifically characterize the nutritional statusof these two patients.
274
Statistically significant decreases in plasma concentration immediatelyafter operation were observed for all but leucine and histidine. By themorning of the first postoperative day, only the values for threonine,arginine and lysine were still significantly lower than the preoperativelevels. The plasma lysine concentration rose to the preoperative levelby the third postoperative day, while the values for arginine and threoninerose to normal somewhere between the third and seventh postoperative days.They observed no obvious relationship among the type and duration of theoperation and the plasma concentration of the 10 individual amino acidsmeasured by them. However, the number of patients studied by them is smallfor comparisons of this nature. Presumably, none of the patients in thestudy of Everson and Fritschel was in shock. No details of fluid or bloodadministration during or after operation, however, are given.
To determine whether anesthesia, per se, might influence the plasmaamino acids, 10 dogs were subjected by Everson and Fritschel to 2 hoursof ether anesthesia. A lowering of the plasma level of the individual essentialamino acids equal to or greater than the depression of plasma levels inpatients produced by anesthesia plus operation was observed. No data werereported on dogs anesthesized and subjected to operation.
It is generally held that the total plasma amino nitrogen increasesduring severe shock. Thus, an increase in plasma amino nitrogen has beenobserved in rats with severe hemorrhagic shock7, 33 and in rats15calves12 and patients17 with shock following severeburns. Engel, Winton, and Long7 observed that the rise in plasmaalpha-amino nitrogen in hemorrhagic shock in rats was accompanied by afall in red cell alpha-amino nitrogen.
The effect on plasma amino acids of ether anesthesia and severe burnsin untreated rats was studied by Rosen and Levenson28 who foundminimal rise in the total amino acids 12 hours after burn. A marked increasein 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 nitrogenduring shock may reflect rises in substances other than, but similar to,free amino acids.
Among the explanations offered for the rise of total amino nitrogenin shock is increased production of amino acids from tissues and impairmentof liver function. In this regard, it has been demonstrated by in vitromethods that the ability of liver slices from shocked ani-
275
mals to deaminate certain amino acids, viz., alanine, is impaired.1,33 In some patients with severe liver disease, Walshe32found little abnormalities in the plasma concentrations of individual aminoacids until the last stages of hepatic failure.
Another factor bearing on the interpretation of plasma concentrationdata 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 possiblyall amino acids can be concentrated to some degree by various cells; thatthis concentrative process is related to processes which control electrolytebalance; and that amino acids can displace potassium from cells and viceversa. Sodium ion fluctuations also affect amino acids; Wolf and McDowell34have observed that a 30 per cent increase in the plasma sodium concentrationby sodium chloride infusion in dogs caused a roughly twofold increase inthe concentrations of the plasma amino acids.
Decreases in plasma total alpha-amino nitrogen concentration have beenreported during certain infectious diseases.10
The influence, if any, of fever, blood transfusions, and infusions ofother fluids, and antibiotics on the plasma amino acids is unknown.
In regard to the effect of nutrition on plasma amino acids, Man etal.,21 found lower than normal concentrations of total aminonitrogen (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 forman and of histidine and arginine in surgical patients prior to operation.Thirty-eight of the patients were considered to be in poor nutritionalstate, while the other 25 were in good nutritional status. The two groupswere made of patients having approximately the same pathologic conditions.The mean value of each of the amino acids except arginine was significantlylower in the plasma of the poorly nourished patients. They found that thedecreased levels of the free amino acids in plasma of five malnourishedpatients 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 subjectthat the oral administration of a peptone solution treated with hydrogenperoxide and containing markedly reduced quantities of methionine, lysine,histidine, leucine, isoleucine, valine and threonine, was accompanied bydefinite 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 intakeand urine output in evaluating plasma concentrations.
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Christensen and his co-workers2, 3 have shown that high plasmalevels of some amino acids (e. g., proline), brought about by feeding,appeared to interfere, perhaps on a competitive basis, with the cellularactivity for concentrating other amino acids. When glutamic acid was fedin excess, however, the concentrating activity of liver and muscle forcertain amino acids appeared to be increased and there was a concomitantfall in the plasma levels of these amino acids. From this point of view,decreased plasma levels of amino acids may result from increased concentrativeassimilation by the cells. Elevated levels may occur when there is inhibitionof the concentration process.
Regarding our own data, reference to Tables 1 to 5 and Figures 1 to8 will give some idea as to the amino acid trends. It is significant thatin 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 throughoutthe course of study; (b) leucine, isoleucine, lysine, valine, tyrosineand alanine rose moderately between days 3 and 9 post injury and then fell,(c) phenylalanine, aspartic acid and methionine showed the same pictureof elevation, but to a greater degree. Taurine, the sulfonic amino acid,may not be handled by the organism as a metabolic intermediate, and borelittle or no relationship to the other amino acids.
Patient C. J. showed, in general, the same picture, with the amino acidsof group (a) normal or slightly low, group (b) becoming moderately elevatedbetween days 1 and 8 post-injury and then falling, and group (c) becomingvery elevated and then falling. In this patient, taurine reached a levelof 1,200 per cent above normal on day 4; dialysis corrected this, and taurinethereafter 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 theirsubsequent clinical course? It is obvious that the separate effects ofinjury, shock and extensive transfusions cannot very well be delineated,since they occurred within so small a time interval. However, in none ofour patients did the total of the 19 amino acids which we analyzed riseto excessive heights in the plasma. Whereas we have obtained a normal averagevalue of 3.5 mg. per 100 cc., the highest values for the patients studiedserially were 5.8 mg. (A. K., day 1 post-injury), 5.0 mg. (C. J., day 3post-injury) and 4.8 mg. (K. D., 6 days post-injury). These values, althoughnot excessively high, are significantly so. A. K. had received a penetratingabdominal 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 theoperation, however, his blood pressure became imperceptible for about ahalf hour and remained low for some time during operation. Immediatelyafter operation, his plasma total amino acids were normal. During his firstpost-wound day, he received 10,000 cc. of blood, 2,000 cc. of gelatin and500 cc. of dextran. At the end of this day his total amino nitrogen was5.8 mg. per 100 cc.
Neither C. J. nor K. D. had elevated total amino acids on day 1 afterinjury. 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 woundsof the liver and kidney; all received large quantities of blood, but A.K. had also received gelatin. Glycine, occurring in relatively large quantitiesin gelatin, might be expected to appear in the plasma if the metabolismof gelatin were contributing to the rise in the total plasma amino acidconcentration seen in this patient at the end of the day of injury. However,the rise in glycine was only slight. Other data indicate that relativelylittle gelatin is metabolized in the first post-infusion day. The risein 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 peakof 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 theamino 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 markedweight loss. He had no oral food until the last day of study. Parenterally,he received some blood, glucose, electrolytes and vitamins. His caloricintake was grossly inadequate; his only nitrogen source was his transfusions.In this regard, it will be recalled that Man et al.,21and Everson and Fritschel9 found decreased plasma amino acidsin malnourished patients.
Patient T. L., who also had renal failure and an inadequate dietaryintake, 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 parenteralcaloric intake was significantly higher than C. J.'s. A. K.'s total plasmaamino nitrogen was slightly low on only one occasion, the second post-injuryday. 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 theother patients and included moderate amounts of oral protein
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His total plasma amino nitrogen was only slightly low on the tenth andeleventh post-injury days.
Patient F. H. had a normal amino nitrogen on day 9 post-injury, andslightly elevated amino nitrogen on day 10. No data of his dietary intakeup to this time are available, but it is likely that he, too, ate moderatelyduring 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 thehypothalamic-pituitary-adrenal axis) occurs. Many of the metabolic changesseen following injury have been ascribed, in part, to the increased activityof this system. Crimson, Hanvey, and Luck4 have shown that injectionsof adrenalin will produce lowering of plasma amino nitrogen in fastingdogs. Others have made similar observations in other species.25Since increased excretion of adrenalin during anesthesia and operationis well established, Everson and Fritschel have suggested that the decreasein the levels of plasma amino acids following anesthesia or operation observedby them may be consequent to increased adrenalin secretion. On the otherhand, there is considerable evidence to indicate an increased secretionof adrenocorticotropic hormone after injury and operation. Li, Geschwind,and Evans18 have observed an increase in plasma amino nitrogenfollowing the repeated administration of adrenocorticotropic hormone torats. On the other hand, neither Li et al.,18 nor Luckand his associates14, 20 observed this increase following asingle injection of adrenocorticotropic hormone to rats. The latter investigatorsfound no change, or a lowering, of the blood amino acid concentration followingsingle injections of various commercial adrenocorticotropin preparations.Cortisone, desoxycorticosterone, norepinephrine and testosterone did notaffect the level of the blood amino acids in normal rats.
Li et al.,18 have demonstrated that the administrationof anterior pituitary growth hormone causes a significant decrease in theblood amino acid content in rats. Luck and his associates have confirmedthis and have also noted that thyrotropin has a similar effect.14,20 Although injection of insulin will lower the concentration ofamino nitrogen in the plasma, the amount of insulin required is enoughto produce signs and symptoms of hypoglycemia.19 There is noevidence to suggest this degree of insulin secretion in the postoperativeor post-injury period.
Urinary excretion of amino acids may reflect, or induce, changes inplasma amino acid concentrations. Everson and Fritschel state that theyfailed to find an increased urinary excretion of individual amino
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acids during operation in five patients sufficient to account for thelowering of the plasma levels of the amino acids observed. Nardi24has recently applied the technic of paper chromatography to study the urinaryexcretion of amino acids in surgical patients. It should be emphasizedthat this is not a quantitative technic. He noted an apparent increasein the number and quantity of amino acids excreted by patients with burnsor patients undergoing major operations. This increase began in the firstdays after injury and was roughly proportional to the severity of the burnor operation. The increased urinary output of amino acid did not necessarilycoincide with the period of greatest negative nitrogen balance, but insome 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 patientswith Cushing's syndrome. Following bilateral adrenalectomy, the amino acidexcretion 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 reflectionof an increase in the plasma concentration of the amino acids or consequentto a decrease in renal tubular reabsorption of the amino acids. (No measurementsof the plasma amino acids were made.) He suggests the possibility thata disorderly, albeit not elevated, pattern of amino acids are presentedto the kidney leading to a disturbance of tubular reabsorption. He alsosuggests the possibility of an increased secretion of an adrenocorticosteroid,probably other than cortisone, which would decrease tubular reabsorptionof amino acids.
When renal function is normal, large increases in the plasma levelsof amino acids may not occur, since a rise in their concentration, abovethe renal threshold may result in a large increase in their excretion.
We have had the opportunity to study the plasma amino acid in patientswith persistent renal failure. While the biochemical picture of these patientsis complicated by other pathologic conditions, our general impression isthat renal failure has little specific effect on plasma amino acid levels.In spite of radically high plasma levels of other NPN components (ureanitrogen concentrations up to 400 mg. per 100 cc.), there was very littlechange in total plasma amino acid levels. The probability is that other,more powerful, regulatory processes can or do control amino acid levelsin the plasma in the absence of renal function. Particularly dramatic isthe failure of 6-hour plasma "washouts" of anuric patients on the artificialkidney, noticeably to affect plasma amino acid levels. The regulatory mechanism,
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or complex of regulatory mechanisms, can obviously operate independentlyof the kidney. This does not exclude the kidney as one of the regulatoryorgans of amino acids in normal individuals, but indicates that other processescan intervene in the homeostatic task when the kidneys fail.
As a consequence of our use of the ion exchange technic for the analysisof amino acids, we have noted the presence, in all plasma samples analyzed,of an ultrafilterable, ninhydrin-reactive compound which is unstable tohot acid, and which, on hydrolysis, yields identifiable amino acids. Weinterpret these experimental findings to mean that this compound has amolecular weight of below 15,000 to 20,000, and that it is comprised ofamino acids held in peptide or peptide-like linkages. Paper chromatographyhas revealed that this component is heterogeneous, since it is readilyresolvable into three or more fractions. Further evidence of this heterogeneityis indicated by these findings: In the two normal plasmas analyzed forthis substance, only glycine and glutamic acid could be found, with a smallamount of threonine in one. Plasma ultrafiltrates from patients with renaldysfunction, however, showed larger quantities of the substance; degradationwith hydrochloric acid, moreover, revealed many more amino acids, e. g.,leucine, valine, proline, tyrosine, histidine, etc. Furthermore, the aminoacid composition varied from patient to patient, and from day to day inthe same patient. In general, those patients with the higher NPN's hadthe substance in greatest concentration. In these cases, analysis showedmaximum numbers of different constituent amino acids.
Extracorporeal dialysis reduced the plasma level of this substance orsubstances like it in roughly the same ratio as the other NPN componentsother 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 informationconcerning 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 acidabsorb in the region 240 to 260 mµ. Thearomatic amino acids absorb in the 280 mµ.region. In general, the entire ultrafiltrate absorption spectral patternfrom 220 mµ. to 320 mµ.is elevated when plasma NPN is high. The elevation of the various portionsof the spectrum, however, only roughly parallels the NPN rise. Figures10 and 11, for example, show progressive rises in spectral pattern in apatient becoming progressively more azotemic. On day 8 post-injury, thepattern is grossly elevated, at which time the NPN was 288 mg. per 100cc. On day 9, when the NPN had risen to 383 mg. per 100 cc., the elevationof
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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 spectralabsorption vary in their relative concentrations. Since by far the largestcontribution 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 changesin the pattern of the nonprotein nitrogen components in the plasma of fivecritically wounded soldiers.
The following quantitative data are presented: NPN creatine plus creatinine,uric acid, other purines and urea. In addition, 19 individual amino acidswere quantitatively analyzed by ion exchange chromatography.
Of the five patients studied, four died. Shock and renal failure werepresent in all varying degrees. No plasma amino nitrogen level over 5.8mg. per 100 cc. was found. Renal failure was persistent in four of thepatients. Although plasma urea concentrations were as much as 30 timesnormal, the total free plasma amino acid nitrogen remained near normal.
In those patients studied for periods extending from the day of injuryto 2 weeks post-injury, the plasma amino acids followed a pattern. At notime were all the amino acids depressed or elevated. Rather, they groupedthemselves as follows: (a) glycine, histidine, threonine, proline and glutamicacid stayed near normal; (b) leucine, isoleucine, lysine, valine, tyrosineand alanine rose moderately during the first week and then fell; (c) phenylalanine,aspartic acid and methionine also rose during the first week, but to agreater 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 plasmaamino 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 samepatient from day to day. In general, those patients with the highest NPN'shad the substance in greatest concentration. In these cases, acid hydrolysisrevealed maximum numbers of different constituent amino acids.
Analysis of the ultraviolet absorption spectra of the plasma ultrafiltrateof these patients indicates that there was an increase in purines, freeor bound.
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Extracorporeal dialysis with a Kolff-type artificial kidney was performedon three patients. Temporary biochemical and/or clinical improvement followedthe dialyses. With the notable exception of the amino acids, all the smallnitrogenous compounds (including the plasma amino conjugates) were sharplyreduced after dialysis. In contrast, the plasma concentrations of the aminoacids were little changed, despite the 6-hour "washouts" on the artificialkidney.
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