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Protein-sparing in Cystectomy Patients
0022-534 7/'78/1193-0355$02. 00/0 Vol. 119, March Printed in U.S.A.
THE JOURNAL Olli' UROLOGY
Copyright © 1978 by The Williams & Wilkins Co.
PROTEIN-SPARING IN CYSTECTOMY PATIENTS TERRY W. HENSLE* From the Department of Urology, Massachusetts General Hospital and the Department of Surgery, Harvard Medical School, Boston, Massachusetts
ABSTRACT
Forty-four patients undergoing radical cystectomy and urinary diversion for invasive bladder cancer were studied prospectively to evaluate the substitution of crystalline amino acids (A3W) for dextrose (D 5W) in the postoperative fluid management. Nutritional assessment revealed that 17 (39 per cent) of the 44 patients were malnourished preope:ratively, while the postoperative complication rate in group 1 (D 5W) was 33 per cent versus 17 per cent in group 2 (A 3W). Nitrogen losses in both groups were low, secondary to the large amounts of albumin used postoperatively. However, more exogenous albumin was needed postoperatively to maintain serum albumin levels in group 1 compared to group 2 (77.5 ± 3.1 gm. versus 65 ± 5.2 gm.). Cell-mediated immunity determined reactivity to a battery of recall antigens revealed that 56 per cent (5) of the patients in group 1 were able to maintain the skin test reactivity throughout the postoperative course, while 94 per cent (17) of the patients in group 2 maintained reactivity during the same period. Four nutritionally depleted patients in group 1 required conversion to a forced feeding regimen (intravenous hyperalimentation), while none of the group 2 patients required a change i.n the nutritional regimen. The operative approach to invasive bladder cancer represents a major category of injury. Both preoperative x-ray therapy and the surgical extirpation of this disease are associated with considerable nutritional depletion. Early nutritional support in the management of patients with invasive bladder cancer is mandatory and in those instances when hypocaloric feeding regimens are appropriate the use of crystalline amino acids in place of dextrose solutions would appear to be indicated. A major effort in preoperative and postoperative surgical care is the maintenance of homeostasis and the preservation of cell mass. Severe protein loss has been shown to impede tissue repair, 1 reduce resistance to infection2 and interfere with enzyme and plasma protein synthesis. 3 It is now well recognized that positive nitrogen balance and tissue repair can be achieved in the depleted patient by intravenous hyperalimentation. This approach, however, requires a central venous system, considerable ancillary support, and has occasional serious complications. 4 Recently, Blackburn and associates have described an approach to parenteral proteinsparing, using isotonic amino acids delivered through peripheral veins. 5·" Peripheral intravenous support of any kind can only aim at minimizing the erosion of body cell mass but as long as protein depletion has not already resulted in significant protein malnutrition considerable preservation can be' achieved by the infusion of isotonic crystalline amino acids together with appropriate cofactors and micronutrients. 7 • 8 This study was undertaken to evaluate the substitution of crystalline amino acids (A3W) for dextrose (D 5W) in the postoperative fluid management of patients undergoing radical cystectomy and ileal conduit diversion for invasive bladder cancer. The study in no way attempts to equate the nutritional support provided by intravenous hyperalimentation with peripheral protein-sparing but it compares the postoperative delivery of hypocaloric dextrose solutions (D 5 W) with the delivery of isotonic amino acid solutions (A3 W) in association with the beneficial effects of the mobilization of endogenous fat stores. Accepted for publication July 15, 1977. Read at annual meeting of American Urological Association, Chicago, Illinois, April 24-28, 1977. Supported in part by grant from the Medical Department, McGaw Laboratories, Irvine, California. * Requests for reprints: Columbia-Presbyterian Medical Center, Babies Hospital, 3959 Broadway, New York, New York 10032. 355
MATERIAL AND METHODS
Forty-four patients undergoing radical cystectomy and ileal conduit urinary diversion for invasive bladder cancer were studied prospectively. Three patients had extensive carcinoma in situ without evidence of invasion. Preoperatively, 30 of the 44 patients had undergone a 5-week course of adjunctive radiotherapy (4,500 rads) confined to the bladder in a measuring 14 by 15 cm. Each patient was evaluated preoperatively for nutritional depletion in terms of height and weight, recent weight loss, serum albumin levels and immune competence. Serum electrolytes, blood urea nitrogen (BUN), creatinine and blood sugar levels also were measured routinely. For the sake of simplicity preoperative nutritional depletion was defined as a recent weight loss of more than 10 pounds, a serum level of less than 3.4 gm. per cent and negative reactivity to a battery of skin test antigens (candidin, strepto· kinase/streptodornase and mumps). Preoperatively, the patients were assigned randomly to group 1, control (D 5W) and group 2, study (A3 W). However, postoperatively it was necessary to allow the primary physi· cian to request a change in the type of therapy assigned based on his perception of the individual needs of the patient. After group assignment group 1 received routine postoperative fluid management, including large volumes of dextrose and baJ .. anced electrolyte solutions, as well as whole blood and blood byproducts when necessary. Group 2 received balanced elec-trolyte solutions, blood and blood byproducts when necessary but in place of the dextrose solution isotonic crystal.line amino acid solutions (A3 W) were infused. The A 3 W solution ccm.. tained 3 per cent essential and non-essential amino acids with appropriate micronutrients and cofactors designed to deliver 0.7 to 1.0 gm./kg. per 24 hours of protein with a caloric value of approximately 240 calories per day. The average study patient received 50 to 60 gm. protein and 9 to 10 gm. nitrogen per day (grams of nitrogen = grams of protein divided by 6.25).
356
HENSLE
Postoperatively, body weight, serum electrolytes, BUN, creatinine, blood sugar and serum albumin levels were checked twice weekly. Urinary nitrogen outputs were measured biweekly and the determination of nitrogen balance was made by calculating grams protein infused divided by 6.25 minus urinary nitrogen output plus a correction factor of 4 gm. per 24 hours. 9 Corrections in nitrogen balance for elevated BUN were made (5 mg./100 cc = 2 gm. nitrogen) when necessary. 10 Daily fluid intake and output were recorded accurately and urine ketone levels were measured 4 times daily using nitroprusside formulated tablets (acetate). Repeat skin testing was done at the end of each postoperative week. Careful attention was paid to signs of intercurrent infection and prolonged stress. Ongoing severe nutritional depletion and life-threatening complications in either group prompted early intervention with intravenous hyperalimentation. Postoperatively, electrolyte solutions were added routinely to both groups but sodium and potassium supplements in the A3W group were added in the form of potassium phosphate and sodium acetate to avoid any possibility of any systemic acidosis produced by adding chloride salts to an already acidic amino acid solution (table 1). These manipulations, of course, were dictated by the particular acid-base status of the patient and serum electrolyte determinations. Calcium, phosphorus and magnesium were added, when indicated, and fat and water soluble vitamins were added to both groups routinely. Patients in group 1 were allowed to begin oral alimentation when the clinical course indicated. However, patients in group 2 were kept on crystalline amino acids (A3W) and appropriate additives with non-carbohydrate oral supplements until they were totally able to meet the caloric demand with oral alimentation, at which time the study was terminated. Patients with renal failure and/or insulin-dependent diabetes mellitus were excluded from the study. Statistical analysis was done by paired and unpaired Student's t tests and data were expressed as mean values(± standard error of mean).
plication rate was 33 per cent (5) in group 1 and 17 per cent (5) in group 2 (table 2). Although 4 patients in group 1 required conversion to intravenous hyperalimentation secondary to severe ongoing nutritional depletion and life-threatening complications the over-all operative mortality was zero (operative mortality = death within 60 days of operation). Hospital days. The number of hospital days for the patients in group 1 ranged from 17 to 66, with an average of 32 and for group 2 it ranged from 16 to 44 days, with an average of 27 (fig. 1). Glucose levels. Serum glucose levels in group 1 averaged 100 ± 5 mg. per cent and in group 2 they averaged 90 ± 5 mg. per cent (fig. 1). The average change in serum glucose levels in group 2 patients between 1 and 7 days postoperatively was minus Ii glucose = minus 17.85. There was no clinical evidence of hypoglycemia in any of the group 2 patients. Albumin levels. Large amounts of colloid and albumin were infused into patients in both groups as is common practice in this procedure to maintain adequate intravascular volume. An average of77.5 ± 3.1 gm. albumin was used in the group 1 patients postoperatively compared to an average of 65 ± 5.2 gm. in the group 2 patients (fig. 2). Serum albumin levels TABLE
3. Preoperative nutritional depletion
Definition: A recent weight loss of > 10 pounds body weight A serum albumin level <3.4 gm. per cent Negative reactivity to a battery of recall skin test antigens Incidence: 17 (39 per cent) of 44 patients were depleted nutritionally preoperatively
BUN mg%
CREATININE mg% 1.5
24
d .
.
16
-.
1.0
.
RESULTS
. .
8
Patients. The patient population, although randomized originally, became unbalanced secondary to physician preference, thus accounting for 15 patients in group 1 and 29 in group 2. (table 2). The effect of changes in protocol selection was that the patients in group 2 were older and were considered to be worse operative risks. Using the aforementioned definition for preoperative nutritional depletion, we found 6 of the 15 patients in group 1 and 11 of the 29 patients in group 2 to be depleted initially. Thus, the over-all incidence was 17 (39 per cent) of 44 highly selected bladder cancer patients who were malnourished preoperatively (table 3). The postoperative com-
'
0
0.5
'
0
•
HOSPITAL DAYS
BLOOD SUGAR mg%
GROUP I (05 W) Control
Ill GROUP II fA:,WI
Study
60
·:~
40 20 0
Fm.1 TABLE
1. Peripheral protein-sparing therapy
3.0 or 3.5 per cent crystalline amino acids Sodium, 40 mEq./1. Potassium, 25 mEq./1. Magnesium, 5 mEq./1. * Chloride, 30 mEq./1. Phosphate, 15 mEq./1. * Multiple vitamins as required
+ 15
No. Pts.
Preop. Radiotherapy (4,500r)
Group 1: control (D.W) Group 2: study
+ 5
2
Preop. Postop. Nutritional CompliOoerative Depletion cations - - - - - - - - Mortality No. No. (%) (%) Pts. Pts.
15
10
6
(40)
5
(33)
0
29
20
11
(38)
5
(17)
0
- 0 5 --,--
-1.0
-10
-2.0
-15
-3.0
ALBUMIN (in) gms 90
44
30
17
(39)
30
(23)
0
3.0 2.0 1.0
0
10
5.0
n ALBUM IN LEVEL gm/%
4.0 60
(A3W)
Totals
HzO BALANCE liters
+10
* May double amount in alternate bottles. TABLE
NITROGEN BALANCE gm/24hrs + 3.0
0
Fm.2
flll
GROUP I (05 W) Control GROUP JI (A:, WI Study
PROTEIN-SPARING IN CYSTECTOMY PATIENTS
were maintained in both groups with an average of 3.6 ± 0.6 gm. per cent in group 1 and 3.8 ± 0.15 gm. per cent in group 2 (fig. 2). Nitrogen balance. Nitrogen balance in group 1 patients averaged minus 9. 7 ± 1.3 gm. per day and in group 2 patients it averaged minus 7.6 ± 1.2 gm. per day (fig. 2). The mild-tomoderate nitrogen losses in both groups with long operations and extensive surgical injury are explained primarily by the large amounts of protein administered to both groups. BUN and creatinine levels. In group 1 the average BUN was 18 ± 2 mg. per cent compared to an average of 24 ± 2 mg. per cent in group 2 (fig. 1). The average change in BUN between 1 and 7 days postoperatively in group 2 patients was .:i BUN = plus 6 mg. per cent. Significant azotemia was only noted in 2 of the group 2 patients, both of whom had obstructive uropathy. Serum creatinine levels were identical for each group, that is 1.3 ± 0.1 mg. per cent versus 1.3 ± 0.2 mg. per cent. Fluid balance. The initial daily fluid intake for patients in group 1 was 4.2 ± 0.71. compared to 4.1 ± 0.61. for the group 2 patients. Average daily outputs for the patients in group 1 were 3.01. per 24 hours compared to 3.11. per 24 hours for the patients in group 2. Water balance data were virtually identical for both groups, that is group 1 had a positive water balance of 1.9 ± 0.11. postoperatively compared to 1.8 ± 0.21. in group 2 (fig. 2). Cell-mediated immunity. Delayed cutaneous hypersensitivity was tested in all patients preoperatively with a battery of recall antigens (streptokinase/streptodornase, candidin and mumps). Positive skin test reactivity was noted preoperatively in 9 of the 15 patients in group 1 and in 18 of the 29 patients in group 2. Conversely, 6 (40 per cent) of the 15 patients in group 1 and 11 (38 per cent) of the 29 patients in group 2 had negative skin test reactivity preoperatively. Of the 9 patients in group 1 with positive skin test reactivity preoperatively 56 per cent (5) maintained the positivity postoperatively, while 94 per cent (17) of the patients in group 2 with positive skin test reactivity maintained the positivity during the same period, that is 1 patient converted the skin test from positive to negative postoperatively (table 4). Of the 6 patients in group 1 with negative skin test reactivity preoperatively 4 received subsequently intravenous hyperalimentation postoperatively and converted the skin test from negative to positive, while 2 maintained the skin test negativity postoperatively. Of the patients in group 2 with preoperative negative skin tests all maintained the negative reactivity throughout postoperatively but none required conversion to intravenous hyperalimentation (table 4). DISCUSSION
When the ability to sustain adequate nutrient intake to meet energy requirements and protein catabolism is not practical or possible, preservation of body cell mass is made feasible by using stored energy reserves, namely body fat and muscle protein. The unique interrelationship of protein, fat and carbohydrate during fasting and semistarvation has been well described by Blackburn and associates. 11 During starvation protein stores are spared progressively while the body adapts gradually to the use of non-protein calories, namely TABLE 4.
No. Pts. Group 1: control
9
(D5 W) 6
Group 2: study
18
(A,W) 11
Preop.
Skin test Postop.
No. Pts.
(%)
(56) (44)
Pos. Pos. Neg. Neg.
Pos. Neg. Neg. Pos.
5
4
Intravenous hyperalimentation
Pos. Pos. Neg. Neg.
Pos. Neg. Neg. Pos.
17 1
(94) (6)
4
2
11 0
357
free fatty acids and ketone bodies as fuel substrates. 12 This protein-sparing adaptation may be interrupted by the use of the hypocaloric carbohydrate containing nutrients (D 5W) because of the resulting increase in circulating insulin and associated peripheral insulin resistance. Fat mobilization is inhibited by insulin and the body accordingly turns to fuels from skeletal muscle and visceral protein during periods of hypocaloric intake. In the well-nourished individual this approach may be justified because the normal response to injury prevents the effective use of exogenous nutrients in the immediate postoperative or acute phase. However, in the malnourished patient the postoperative catabolic phase must be minimized so that the outcome will not be affected unfavorably by the nutritional status. Significant amounts of protein can be spared by the parenteral infusion of isotonic amino acids with appropriate electrolyte, vitamin and mineral additives. By this technique visceral protein synthesis is maintained while body fat is mobilized as a source of needed calories. The visceral protein compartment is evaluated best by the simple determination of serum albumin and transferrin levels. 13 • 14 The findings of this study indicate that less exogenous albumin was necessary to support serum albumin levels and, therefore, support the visceral protein compartment in patients receiving A3W as opposed to D5W (fig. 2). This is consistent with Skillman and associates who have demonstrated a significantly higher rate of albumin synthesis in patients receiving amino acid infusions in comparison to glucose. 15 While albumin concentrations in both groups in their studies were similar, as they were in this study, Skillman and associates were able to show a 51 per cent (p < 0.05) increase in albumin synthesis rates in patients receiving A3 . 5W as opposed to D5W. Substrate dependency for albumin synthesis may be an important indication for the provision of isotonic amino acids postoperatively, especially in those patients who are malnourished and somewhat deficient in labile muscle protein. Nitrogen loss in both groups in the present study was minimized by the large amounts of exogenous albumin given. In retropsect, the provision of0.7 to 1.0 gm./kg. per 24 hours of amino acids is inadequate for maximal support of protein synthesis. Data recently generated by O'Keefe and associates, 16• 17 and others 18 suggest that the provision of 1. 5 to 2 gm./kg. per 24 hours of protein best meets the requirements for protein synthesis in the hypocaloric state. It is now clear that the availability of amino acid substrate is physiological and a clinical limiting factor in protein synthesis. 19 It also is clear that the effect of dextrose solutions is to raise insulin levels, inhibit amino acid release from muscle and, thus, reduce effectively the redistribution of labile protein needed for increased visceral protein synthesis. 20 These observations seem to be contrary to the classical life raft experiments of Gamble, which demonstrated the protein-sparing effect of glucose in terms of nitrogen balance alone. 21 However, a distinction must be made as to what protein is being spared and for what purpose. The reallotment of protein from muscle to visceral while using fat for fuel best meets the need of the host for preservation of body cell mass. 20 The safety of this form of therapy has been well demonstrated. 18 In the present study serum glucose levels, BUN and creatinine levels were not significantly different in patients receiving A 3 W as opposed to D5W, indicating the relative metabolic safety of these solutions. The importance of thorough preoperative nutritional assessment is well supported by our study. The finding of39 per cent of the total patient population in this study who met the simple criteria of nutritional depletion preoperatively is consistent with recent surveys that demonstrate the wide prevalence of protein-calorie malnutrition in hospitalized patients. 22 • 23 It is now generally well accepted that the presence of malnutrition affects significantly the hospital course of patients and
358
HENSLE
has a direct bearing on morbidity and mortality. 24 The prevention of malnutrition and preservation of body cell mass and visceral protein by the use of peripheral amino acid therapy in patients in whom nutritional depletion is not already present to any degree is evidenced in this study by the maintenance of immune competence and serum albumin levels. In those patients who were nutritionally depleted preoperatively the use of peripheral amino acid therapy decreased the breakdown of the visceral protein compartment and limited ongoing severe nutritional depletion. This is evidenced by the fact that none of the patients in the A 3 W group required conversion to a forced feeding schedule while 4 of the patients in the D 5W group who were nutritionally depleted preoperatively required conversion to intravenous hyperalimentation. The use of skin testing and cell-mediated immunity as markers for immunocompetence and nutritional status is now well established. 23 • 25 Along with nutritional depletion blocking antibodies in patients with various forms of cancer also are a well known cause of anergy. 26 The maintenance of positive skin test reactivity in those patients receiving amino acids (group 2) as opposed to dextrose solutions (group 1) was striking (table 4). No statement can be made with any certainty regarding the patients with negative skin tests preoperative. However, the rapid conversion of skin test reactivity to positive in the 4 patients treated with intravenous hyperalimentation in the midst of the postoperative course suggests a nutritional basis for the lack of immunocompetence. In summary, the operative approach to invasive bladder cancer represents a major category of injuring. Preoperative x-ray therapy along with the surgical extirpation of this disease are associated with considerable nutritional depletion. Nitrogen balance is not a sensitive indicator of this nutritional depletion, since it does not identify readily the tissues involved in active protein turnover and it is necessary for us to pay close attention to more sensitive indicators such as cell-mediated immunity, albumin levels and transferrin levels to determine preoperative and postoperative nutritional status. Early nutritional support in the management of patients with invasive bladder cancer is indicated and during periods in which hypocaloric feeding regimens are considered appropriate the use of crystalline amino acids in place of dextrose solutions would appear to be beneficial. REFERENCES
1. Thompson, W. D., Ravdin, I. S. and Frank, I. L.: Effect of
2. 3.
4.
5. 6.
hypoproteinemia on wound disruption. Arch. Surg., 36: 500, 1938. Cannon, P.R.: Importance of proteins in resistance to infection. J.A.M.A., 128: 360, 1945. Steiger, E., Daly, J. M., Allen, T. R., Dudrick, S. J. and Vars, H. M.: Postoperative intravenous nutrition: effects on body weight protein regeneration, wound healing and liver morphology. Surgery, 73: 686, 1973. Ryan, J. A., Jr., Abel, R. M., Abbott, W. M., Hopkins, C. C., Chesney, T. M., Calley, R., Phillips, K. and Fischer, J. E.: Catheter complications in total parenteral nutrition: a prospective study of 200 consecutive patients. New Engl. J. Med., 290: 757, 1974. Blackburn, G. L., Flatt, J.P., Clowes, G. H. A. and O'Donnell, T. E.: Peripheral intravenous feeding with isotonic amino acid solutions. Amer. J. Surg., 125: 447, 1973. Blackburn, G. L., Flatt, J.P., Clowes, G. H. A., Jr., O'Donnell, T. F. and Hensle, T. E.: Protein sparing therapy during periods of starvation with sepsis or trauma. Ann. Surg., 177:
588, 1973. 7. Miller, J. D. B., Blackburn, G. L. and Bistrian, B. R.: Failure of postoperative infection to increase nitrogen excretion in patients maintained on peripheral amino acids. Amer. J. Clin. Nutr., in press. 8. Miller, J. D. B., Blackburn, G. L., Bistrian, B. R., Rienhoff, H. Y. and Trerice, M.: Effect of deep sepsis on protein sparing therapies and nitrogen balance. Amer. J. Clin. Nutr., 30: 1528, 1977. 9. MacKenzie, T. A., Blackburn, G. L. and Flatt, J. P.: Clinical assessment of nutritional status using nitrogen balance. Fed. Proc., 33: 68:,\ 1974. 10. Bergstrom, J., Bucht, H. and Furst, P.: Intravenous nutrition with amino acid solutions in patients with chronic uremia. Acta Med. Scand., 191: 359, 1972. 11. Blackburn, G. L., Flatt, J. P. and Hensle, T. W.: Peripheral amino acid infusion. In: Total Parenteral Nutrition. Edited by J. E. Fischer. Boston: Little, Brown & Co., chapt. 19, pp. 363-394, 1976. 12. Cahill, G. F., Jr.: Starvation in man. Clin. Endocr. Metab., 5: 397, 1976. 13. Jeejeebhoy, K. N., Bruce-Robertson, A. and Ho, J.: The comparative effects of nutritional and hormonal factors on the synthesis of albumin, fibrinogen, and transferrin, in protein turnover. New York: Associated Scientific Publishers, CIBA Foundation Symposium 9 (new series), p. 217, 1973. 14. Smith, M. F., Moldawer, L. L., Bistrian, B. R., and Trerice, M.: Transferrin as a measure of the efficiency of parenteral and enteral nutrition (abstract). J. Parent. Entr. Nutr., 1: 9A, 1977. 15. Skillman, J. J., Rosenoer, V. M., Smith, P. C. and Fang, M. S.: Improved albumin synthesis in postoperative patients by amino acid infusion. New Engl. J. Med., 295: 1037, 1976. 16. O'Keefe, S. J. D., Sender, P. M. and James, W. P.: "Catabolic" loss of body nitrogen in response to surgery. Lancet, 2: 1035, 1974. 17. O'Keefe, S. J., Sender, P. M., Clark, G. C. and James, W. P. T.: The effect of varying the postoperative parenteral regimen on the dynamics of protein metabolism following operative trauma. In: Proceedings of the International Conference on Parenteral Nutrition, Montpelier, France, p. 333, 1974. 18. Freeman, J.B., Stegink, L. D., Meyer, P. D., Thompson, R. G. and Den Besten, L.: Metabolic effects of amino acid vs. dextrose infusion in surgical patients. Arch. Surg., 110: 916, 1975. 19. Benotti, P. N., Blackburn, G. L., Miller, J. D. B., Bistrian, B. R., Flatt, J-P. and Trerice, M.: Role of branched chain amino acids (BCAA) intake in preventing muscle proteolysis. Surg. Forum, 27: 7, 1976. 20. Blackburn, G. L.: Nitrogen metabolism after surgical injury, in physiology in medicine. New Engl. J. Med., in press. 21. Gamble, J. L.: Physiologic information gained from studies in the life raft ration. Harvey Lectures, 42: 247, 1946. 22. Bistrian, B. R., Blackburn, G. L., Hallowell, E. and Heddie, R.: Protein status of general surgical patients. J.A.M.A., 230: 858, 1974. 23. Bistrian, B. R., Blackburn, G. L., Scrimshaw, N. S. and Flatt, J. P.: Cellular immunity in semistarved states in hospitalized adults. Amer. J. Clin. Nutr., 28: 1148, 1975. 24. Pietsch, J. B. and Meakins, J. L.: 1976 Davis & Geck Surgical Essay: The delayed hypersensitivity response: clinical application in surgery. Canad. J. Surg., 20: 15, 1977. 25. Law, D. K., Dudrick, S. J. and Abdou, N. I.: Immunocompetence of patients with protein-calorie malnutrition. The effects of nutritional repletion. Ann. Intern. Med., 79: 545, 1973. 26. Dodd, M. C., Scheetz, M. E., 2nd and Rossio, J. L.: Immunogenic RNA in immunotherapy of cancer: the transfer of antitumor cytotoxic activity and tuberculin sensitivity to human lymphocytes using xenogeneic ribonucleic acid. Ann. N. Y. Acad. Sci., 207: 454, 1973.
THE JOURNAL Olli' UROLOGY
Copyright © 1978 by The Williams & Wilkins Co.
PROTEIN-SPARING IN CYSTECTOMY PATIENTS TERRY W. HENSLE* From the Department of Urology, Massachusetts General Hospital and the Department of Surgery, Harvard Medical School, Boston, Massachusetts
ABSTRACT
Forty-four patients undergoing radical cystectomy and urinary diversion for invasive bladder cancer were studied prospectively to evaluate the substitution of crystalline amino acids (A3W) for dextrose (D 5W) in the postoperative fluid management. Nutritional assessment revealed that 17 (39 per cent) of the 44 patients were malnourished preope:ratively, while the postoperative complication rate in group 1 (D 5W) was 33 per cent versus 17 per cent in group 2 (A 3W). Nitrogen losses in both groups were low, secondary to the large amounts of albumin used postoperatively. However, more exogenous albumin was needed postoperatively to maintain serum albumin levels in group 1 compared to group 2 (77.5 ± 3.1 gm. versus 65 ± 5.2 gm.). Cell-mediated immunity determined reactivity to a battery of recall antigens revealed that 56 per cent (5) of the patients in group 1 were able to maintain the skin test reactivity throughout the postoperative course, while 94 per cent (17) of the patients in group 2 maintained reactivity during the same period. Four nutritionally depleted patients in group 1 required conversion to a forced feeding regimen (intravenous hyperalimentation), while none of the group 2 patients required a change i.n the nutritional regimen. The operative approach to invasive bladder cancer represents a major category of injury. Both preoperative x-ray therapy and the surgical extirpation of this disease are associated with considerable nutritional depletion. Early nutritional support in the management of patients with invasive bladder cancer is mandatory and in those instances when hypocaloric feeding regimens are appropriate the use of crystalline amino acids in place of dextrose solutions would appear to be indicated. A major effort in preoperative and postoperative surgical care is the maintenance of homeostasis and the preservation of cell mass. Severe protein loss has been shown to impede tissue repair, 1 reduce resistance to infection2 and interfere with enzyme and plasma protein synthesis. 3 It is now well recognized that positive nitrogen balance and tissue repair can be achieved in the depleted patient by intravenous hyperalimentation. This approach, however, requires a central venous system, considerable ancillary support, and has occasional serious complications. 4 Recently, Blackburn and associates have described an approach to parenteral proteinsparing, using isotonic amino acids delivered through peripheral veins. 5·" Peripheral intravenous support of any kind can only aim at minimizing the erosion of body cell mass but as long as protein depletion has not already resulted in significant protein malnutrition considerable preservation can be' achieved by the infusion of isotonic crystalline amino acids together with appropriate cofactors and micronutrients. 7 • 8 This study was undertaken to evaluate the substitution of crystalline amino acids (A3W) for dextrose (D 5W) in the postoperative fluid management of patients undergoing radical cystectomy and ileal conduit diversion for invasive bladder cancer. The study in no way attempts to equate the nutritional support provided by intravenous hyperalimentation with peripheral protein-sparing but it compares the postoperative delivery of hypocaloric dextrose solutions (D 5 W) with the delivery of isotonic amino acid solutions (A3 W) in association with the beneficial effects of the mobilization of endogenous fat stores. Accepted for publication July 15, 1977. Read at annual meeting of American Urological Association, Chicago, Illinois, April 24-28, 1977. Supported in part by grant from the Medical Department, McGaw Laboratories, Irvine, California. * Requests for reprints: Columbia-Presbyterian Medical Center, Babies Hospital, 3959 Broadway, New York, New York 10032. 355
MATERIAL AND METHODS
Forty-four patients undergoing radical cystectomy and ileal conduit urinary diversion for invasive bladder cancer were studied prospectively. Three patients had extensive carcinoma in situ without evidence of invasion. Preoperatively, 30 of the 44 patients had undergone a 5-week course of adjunctive radiotherapy (4,500 rads) confined to the bladder in a measuring 14 by 15 cm. Each patient was evaluated preoperatively for nutritional depletion in terms of height and weight, recent weight loss, serum albumin levels and immune competence. Serum electrolytes, blood urea nitrogen (BUN), creatinine and blood sugar levels also were measured routinely. For the sake of simplicity preoperative nutritional depletion was defined as a recent weight loss of more than 10 pounds, a serum level of less than 3.4 gm. per cent and negative reactivity to a battery of skin test antigens (candidin, strepto· kinase/streptodornase and mumps). Preoperatively, the patients were assigned randomly to group 1, control (D 5W) and group 2, study (A3 W). However, postoperatively it was necessary to allow the primary physi· cian to request a change in the type of therapy assigned based on his perception of the individual needs of the patient. After group assignment group 1 received routine postoperative fluid management, including large volumes of dextrose and baJ .. anced electrolyte solutions, as well as whole blood and blood byproducts when necessary. Group 2 received balanced elec-trolyte solutions, blood and blood byproducts when necessary but in place of the dextrose solution isotonic crystal.line amino acid solutions (A3 W) were infused. The A 3 W solution ccm.. tained 3 per cent essential and non-essential amino acids with appropriate micronutrients and cofactors designed to deliver 0.7 to 1.0 gm./kg. per 24 hours of protein with a caloric value of approximately 240 calories per day. The average study patient received 50 to 60 gm. protein and 9 to 10 gm. nitrogen per day (grams of nitrogen = grams of protein divided by 6.25).
356
HENSLE
Postoperatively, body weight, serum electrolytes, BUN, creatinine, blood sugar and serum albumin levels were checked twice weekly. Urinary nitrogen outputs were measured biweekly and the determination of nitrogen balance was made by calculating grams protein infused divided by 6.25 minus urinary nitrogen output plus a correction factor of 4 gm. per 24 hours. 9 Corrections in nitrogen balance for elevated BUN were made (5 mg./100 cc = 2 gm. nitrogen) when necessary. 10 Daily fluid intake and output were recorded accurately and urine ketone levels were measured 4 times daily using nitroprusside formulated tablets (acetate). Repeat skin testing was done at the end of each postoperative week. Careful attention was paid to signs of intercurrent infection and prolonged stress. Ongoing severe nutritional depletion and life-threatening complications in either group prompted early intervention with intravenous hyperalimentation. Postoperatively, electrolyte solutions were added routinely to both groups but sodium and potassium supplements in the A3W group were added in the form of potassium phosphate and sodium acetate to avoid any possibility of any systemic acidosis produced by adding chloride salts to an already acidic amino acid solution (table 1). These manipulations, of course, were dictated by the particular acid-base status of the patient and serum electrolyte determinations. Calcium, phosphorus and magnesium were added, when indicated, and fat and water soluble vitamins were added to both groups routinely. Patients in group 1 were allowed to begin oral alimentation when the clinical course indicated. However, patients in group 2 were kept on crystalline amino acids (A3W) and appropriate additives with non-carbohydrate oral supplements until they were totally able to meet the caloric demand with oral alimentation, at which time the study was terminated. Patients with renal failure and/or insulin-dependent diabetes mellitus were excluded from the study. Statistical analysis was done by paired and unpaired Student's t tests and data were expressed as mean values(± standard error of mean).
plication rate was 33 per cent (5) in group 1 and 17 per cent (5) in group 2 (table 2). Although 4 patients in group 1 required conversion to intravenous hyperalimentation secondary to severe ongoing nutritional depletion and life-threatening complications the over-all operative mortality was zero (operative mortality = death within 60 days of operation). Hospital days. The number of hospital days for the patients in group 1 ranged from 17 to 66, with an average of 32 and for group 2 it ranged from 16 to 44 days, with an average of 27 (fig. 1). Glucose levels. Serum glucose levels in group 1 averaged 100 ± 5 mg. per cent and in group 2 they averaged 90 ± 5 mg. per cent (fig. 1). The average change in serum glucose levels in group 2 patients between 1 and 7 days postoperatively was minus Ii glucose = minus 17.85. There was no clinical evidence of hypoglycemia in any of the group 2 patients. Albumin levels. Large amounts of colloid and albumin were infused into patients in both groups as is common practice in this procedure to maintain adequate intravascular volume. An average of77.5 ± 3.1 gm. albumin was used in the group 1 patients postoperatively compared to an average of 65 ± 5.2 gm. in the group 2 patients (fig. 2). Serum albumin levels TABLE
3. Preoperative nutritional depletion
Definition: A recent weight loss of > 10 pounds body weight A serum albumin level <3.4 gm. per cent Negative reactivity to a battery of recall skin test antigens Incidence: 17 (39 per cent) of 44 patients were depleted nutritionally preoperatively
BUN mg%
CREATININE mg% 1.5
24
d .
.
16
-.
1.0
.
RESULTS
. .
8
Patients. The patient population, although randomized originally, became unbalanced secondary to physician preference, thus accounting for 15 patients in group 1 and 29 in group 2. (table 2). The effect of changes in protocol selection was that the patients in group 2 were older and were considered to be worse operative risks. Using the aforementioned definition for preoperative nutritional depletion, we found 6 of the 15 patients in group 1 and 11 of the 29 patients in group 2 to be depleted initially. Thus, the over-all incidence was 17 (39 per cent) of 44 highly selected bladder cancer patients who were malnourished preoperatively (table 3). The postoperative com-
'
0
0.5
'
0
•
HOSPITAL DAYS
BLOOD SUGAR mg%
GROUP I (05 W) Control
Ill GROUP II fA:,WI
Study
60
·:~
40 20 0
Fm.1 TABLE
1. Peripheral protein-sparing therapy
3.0 or 3.5 per cent crystalline amino acids Sodium, 40 mEq./1. Potassium, 25 mEq./1. Magnesium, 5 mEq./1. * Chloride, 30 mEq./1. Phosphate, 15 mEq./1. * Multiple vitamins as required
+ 15
No. Pts.
Preop. Radiotherapy (4,500r)
Group 1: control (D.W) Group 2: study
+ 5
2
Preop. Postop. Nutritional CompliOoerative Depletion cations - - - - - - - - Mortality No. No. (%) (%) Pts. Pts.
15
10
6
(40)
5
(33)
0
29
20
11
(38)
5
(17)
0
- 0 5 --,--
-1.0
-10
-2.0
-15
-3.0
ALBUMIN (in) gms 90
44
30
17
(39)
30
(23)
0
3.0 2.0 1.0
0
10
5.0
n ALBUM IN LEVEL gm/%
4.0 60
(A3W)
Totals
HzO BALANCE liters
+10
* May double amount in alternate bottles. TABLE
NITROGEN BALANCE gm/24hrs + 3.0
0
Fm.2
flll
GROUP I (05 W) Control GROUP JI (A:, WI Study
PROTEIN-SPARING IN CYSTECTOMY PATIENTS
were maintained in both groups with an average of 3.6 ± 0.6 gm. per cent in group 1 and 3.8 ± 0.15 gm. per cent in group 2 (fig. 2). Nitrogen balance. Nitrogen balance in group 1 patients averaged minus 9. 7 ± 1.3 gm. per day and in group 2 patients it averaged minus 7.6 ± 1.2 gm. per day (fig. 2). The mild-tomoderate nitrogen losses in both groups with long operations and extensive surgical injury are explained primarily by the large amounts of protein administered to both groups. BUN and creatinine levels. In group 1 the average BUN was 18 ± 2 mg. per cent compared to an average of 24 ± 2 mg. per cent in group 2 (fig. 1). The average change in BUN between 1 and 7 days postoperatively in group 2 patients was .:i BUN = plus 6 mg. per cent. Significant azotemia was only noted in 2 of the group 2 patients, both of whom had obstructive uropathy. Serum creatinine levels were identical for each group, that is 1.3 ± 0.1 mg. per cent versus 1.3 ± 0.2 mg. per cent. Fluid balance. The initial daily fluid intake for patients in group 1 was 4.2 ± 0.71. compared to 4.1 ± 0.61. for the group 2 patients. Average daily outputs for the patients in group 1 were 3.01. per 24 hours compared to 3.11. per 24 hours for the patients in group 2. Water balance data were virtually identical for both groups, that is group 1 had a positive water balance of 1.9 ± 0.11. postoperatively compared to 1.8 ± 0.21. in group 2 (fig. 2). Cell-mediated immunity. Delayed cutaneous hypersensitivity was tested in all patients preoperatively with a battery of recall antigens (streptokinase/streptodornase, candidin and mumps). Positive skin test reactivity was noted preoperatively in 9 of the 15 patients in group 1 and in 18 of the 29 patients in group 2. Conversely, 6 (40 per cent) of the 15 patients in group 1 and 11 (38 per cent) of the 29 patients in group 2 had negative skin test reactivity preoperatively. Of the 9 patients in group 1 with positive skin test reactivity preoperatively 56 per cent (5) maintained the positivity postoperatively, while 94 per cent (17) of the patients in group 2 with positive skin test reactivity maintained the positivity during the same period, that is 1 patient converted the skin test from positive to negative postoperatively (table 4). Of the 6 patients in group 1 with negative skin test reactivity preoperatively 4 received subsequently intravenous hyperalimentation postoperatively and converted the skin test from negative to positive, while 2 maintained the skin test negativity postoperatively. Of the patients in group 2 with preoperative negative skin tests all maintained the negative reactivity throughout postoperatively but none required conversion to intravenous hyperalimentation (table 4). DISCUSSION
When the ability to sustain adequate nutrient intake to meet energy requirements and protein catabolism is not practical or possible, preservation of body cell mass is made feasible by using stored energy reserves, namely body fat and muscle protein. The unique interrelationship of protein, fat and carbohydrate during fasting and semistarvation has been well described by Blackburn and associates. 11 During starvation protein stores are spared progressively while the body adapts gradually to the use of non-protein calories, namely TABLE 4.
No. Pts. Group 1: control
9
(D5 W) 6
Group 2: study
18
(A,W) 11
Preop.
Skin test Postop.
No. Pts.
(%)
(56) (44)
Pos. Pos. Neg. Neg.
Pos. Neg. Neg. Pos.
5
4
Intravenous hyperalimentation
Pos. Pos. Neg. Neg.
Pos. Neg. Neg. Pos.
17 1
(94) (6)
4
2
11 0
357
free fatty acids and ketone bodies as fuel substrates. 12 This protein-sparing adaptation may be interrupted by the use of the hypocaloric carbohydrate containing nutrients (D 5W) because of the resulting increase in circulating insulin and associated peripheral insulin resistance. Fat mobilization is inhibited by insulin and the body accordingly turns to fuels from skeletal muscle and visceral protein during periods of hypocaloric intake. In the well-nourished individual this approach may be justified because the normal response to injury prevents the effective use of exogenous nutrients in the immediate postoperative or acute phase. However, in the malnourished patient the postoperative catabolic phase must be minimized so that the outcome will not be affected unfavorably by the nutritional status. Significant amounts of protein can be spared by the parenteral infusion of isotonic amino acids with appropriate electrolyte, vitamin and mineral additives. By this technique visceral protein synthesis is maintained while body fat is mobilized as a source of needed calories. The visceral protein compartment is evaluated best by the simple determination of serum albumin and transferrin levels. 13 • 14 The findings of this study indicate that less exogenous albumin was necessary to support serum albumin levels and, therefore, support the visceral protein compartment in patients receiving A3W as opposed to D5W (fig. 2). This is consistent with Skillman and associates who have demonstrated a significantly higher rate of albumin synthesis in patients receiving amino acid infusions in comparison to glucose. 15 While albumin concentrations in both groups in their studies were similar, as they were in this study, Skillman and associates were able to show a 51 per cent (p < 0.05) increase in albumin synthesis rates in patients receiving A3 . 5W as opposed to D5W. Substrate dependency for albumin synthesis may be an important indication for the provision of isotonic amino acids postoperatively, especially in those patients who are malnourished and somewhat deficient in labile muscle protein. Nitrogen loss in both groups in the present study was minimized by the large amounts of exogenous albumin given. In retropsect, the provision of0.7 to 1.0 gm./kg. per 24 hours of amino acids is inadequate for maximal support of protein synthesis. Data recently generated by O'Keefe and associates, 16• 17 and others 18 suggest that the provision of 1. 5 to 2 gm./kg. per 24 hours of protein best meets the requirements for protein synthesis in the hypocaloric state. It is now clear that the availability of amino acid substrate is physiological and a clinical limiting factor in protein synthesis. 19 It also is clear that the effect of dextrose solutions is to raise insulin levels, inhibit amino acid release from muscle and, thus, reduce effectively the redistribution of labile protein needed for increased visceral protein synthesis. 20 These observations seem to be contrary to the classical life raft experiments of Gamble, which demonstrated the protein-sparing effect of glucose in terms of nitrogen balance alone. 21 However, a distinction must be made as to what protein is being spared and for what purpose. The reallotment of protein from muscle to visceral while using fat for fuel best meets the need of the host for preservation of body cell mass. 20 The safety of this form of therapy has been well demonstrated. 18 In the present study serum glucose levels, BUN and creatinine levels were not significantly different in patients receiving A 3 W as opposed to D5W, indicating the relative metabolic safety of these solutions. The importance of thorough preoperative nutritional assessment is well supported by our study. The finding of39 per cent of the total patient population in this study who met the simple criteria of nutritional depletion preoperatively is consistent with recent surveys that demonstrate the wide prevalence of protein-calorie malnutrition in hospitalized patients. 22 • 23 It is now generally well accepted that the presence of malnutrition affects significantly the hospital course of patients and
358
HENSLE
has a direct bearing on morbidity and mortality. 24 The prevention of malnutrition and preservation of body cell mass and visceral protein by the use of peripheral amino acid therapy in patients in whom nutritional depletion is not already present to any degree is evidenced in this study by the maintenance of immune competence and serum albumin levels. In those patients who were nutritionally depleted preoperatively the use of peripheral amino acid therapy decreased the breakdown of the visceral protein compartment and limited ongoing severe nutritional depletion. This is evidenced by the fact that none of the patients in the A 3 W group required conversion to a forced feeding schedule while 4 of the patients in the D 5W group who were nutritionally depleted preoperatively required conversion to intravenous hyperalimentation. The use of skin testing and cell-mediated immunity as markers for immunocompetence and nutritional status is now well established. 23 • 25 Along with nutritional depletion blocking antibodies in patients with various forms of cancer also are a well known cause of anergy. 26 The maintenance of positive skin test reactivity in those patients receiving amino acids (group 2) as opposed to dextrose solutions (group 1) was striking (table 4). No statement can be made with any certainty regarding the patients with negative skin tests preoperative. However, the rapid conversion of skin test reactivity to positive in the 4 patients treated with intravenous hyperalimentation in the midst of the postoperative course suggests a nutritional basis for the lack of immunocompetence. In summary, the operative approach to invasive bladder cancer represents a major category of injuring. Preoperative x-ray therapy along with the surgical extirpation of this disease are associated with considerable nutritional depletion. Nitrogen balance is not a sensitive indicator of this nutritional depletion, since it does not identify readily the tissues involved in active protein turnover and it is necessary for us to pay close attention to more sensitive indicators such as cell-mediated immunity, albumin levels and transferrin levels to determine preoperative and postoperative nutritional status. Early nutritional support in the management of patients with invasive bladder cancer is indicated and during periods in which hypocaloric feeding regimens are considered appropriate the use of crystalline amino acids in place of dextrose solutions would appear to be beneficial. REFERENCES
1. Thompson, W. D., Ravdin, I. S. and Frank, I. L.: Effect of
2. 3.
4.
5. 6.
hypoproteinemia on wound disruption. Arch. Surg., 36: 500, 1938. Cannon, P.R.: Importance of proteins in resistance to infection. J.A.M.A., 128: 360, 1945. Steiger, E., Daly, J. M., Allen, T. R., Dudrick, S. J. and Vars, H. M.: Postoperative intravenous nutrition: effects on body weight protein regeneration, wound healing and liver morphology. Surgery, 73: 686, 1973. Ryan, J. A., Jr., Abel, R. M., Abbott, W. M., Hopkins, C. C., Chesney, T. M., Calley, R., Phillips, K. and Fischer, J. E.: Catheter complications in total parenteral nutrition: a prospective study of 200 consecutive patients. New Engl. J. Med., 290: 757, 1974. Blackburn, G. L., Flatt, J.P., Clowes, G. H. A. and O'Donnell, T. E.: Peripheral intravenous feeding with isotonic amino acid solutions. Amer. J. Surg., 125: 447, 1973. Blackburn, G. L., Flatt, J.P., Clowes, G. H. A., Jr., O'Donnell, T. F. and Hensle, T. E.: Protein sparing therapy during periods of starvation with sepsis or trauma. Ann. Surg., 177:
588, 1973. 7. Miller, J. D. B., Blackburn, G. L. and Bistrian, B. R.: Failure of postoperative infection to increase nitrogen excretion in patients maintained on peripheral amino acids. Amer. J. Clin. Nutr., in press. 8. Miller, J. D. B., Blackburn, G. L., Bistrian, B. R., Rienhoff, H. Y. and Trerice, M.: Effect of deep sepsis on protein sparing therapies and nitrogen balance. Amer. J. Clin. Nutr., 30: 1528, 1977. 9. MacKenzie, T. A., Blackburn, G. L. and Flatt, J. P.: Clinical assessment of nutritional status using nitrogen balance. Fed. Proc., 33: 68:,\ 1974. 10. Bergstrom, J., Bucht, H. and Furst, P.: Intravenous nutrition with amino acid solutions in patients with chronic uremia. Acta Med. Scand., 191: 359, 1972. 11. Blackburn, G. L., Flatt, J. P. and Hensle, T. W.: Peripheral amino acid infusion. In: Total Parenteral Nutrition. Edited by J. E. Fischer. Boston: Little, Brown & Co., chapt. 19, pp. 363-394, 1976. 12. Cahill, G. F., Jr.: Starvation in man. Clin. Endocr. Metab., 5: 397, 1976. 13. Jeejeebhoy, K. N., Bruce-Robertson, A. and Ho, J.: The comparative effects of nutritional and hormonal factors on the synthesis of albumin, fibrinogen, and transferrin, in protein turnover. New York: Associated Scientific Publishers, CIBA Foundation Symposium 9 (new series), p. 217, 1973. 14. Smith, M. F., Moldawer, L. L., Bistrian, B. R., and Trerice, M.: Transferrin as a measure of the efficiency of parenteral and enteral nutrition (abstract). J. Parent. Entr. Nutr., 1: 9A, 1977. 15. Skillman, J. J., Rosenoer, V. M., Smith, P. C. and Fang, M. S.: Improved albumin synthesis in postoperative patients by amino acid infusion. New Engl. J. Med., 295: 1037, 1976. 16. O'Keefe, S. J. D., Sender, P. M. and James, W. P.: "Catabolic" loss of body nitrogen in response to surgery. Lancet, 2: 1035, 1974. 17. O'Keefe, S. J., Sender, P. M., Clark, G. C. and James, W. P. T.: The effect of varying the postoperative parenteral regimen on the dynamics of protein metabolism following operative trauma. In: Proceedings of the International Conference on Parenteral Nutrition, Montpelier, France, p. 333, 1974. 18. Freeman, J.B., Stegink, L. D., Meyer, P. D., Thompson, R. G. and Den Besten, L.: Metabolic effects of amino acid vs. dextrose infusion in surgical patients. Arch. Surg., 110: 916, 1975. 19. Benotti, P. N., Blackburn, G. L., Miller, J. D. B., Bistrian, B. R., Flatt, J-P. and Trerice, M.: Role of branched chain amino acids (BCAA) intake in preventing muscle proteolysis. Surg. Forum, 27: 7, 1976. 20. Blackburn, G. L.: Nitrogen metabolism after surgical injury, in physiology in medicine. New Engl. J. Med., in press. 21. Gamble, J. L.: Physiologic information gained from studies in the life raft ration. Harvey Lectures, 42: 247, 1946. 22. Bistrian, B. R., Blackburn, G. L., Hallowell, E. and Heddie, R.: Protein status of general surgical patients. J.A.M.A., 230: 858, 1974. 23. Bistrian, B. R., Blackburn, G. L., Scrimshaw, N. S. and Flatt, J. P.: Cellular immunity in semistarved states in hospitalized adults. Amer. J. Clin. Nutr., 28: 1148, 1975. 24. Pietsch, J. B. and Meakins, J. L.: 1976 Davis & Geck Surgical Essay: The delayed hypersensitivity response: clinical application in surgery. Canad. J. Surg., 20: 15, 1977. 25. Law, D. K., Dudrick, S. J. and Abdou, N. I.: Immunocompetence of patients with protein-calorie malnutrition. The effects of nutritional repletion. Ann. Intern. Med., 79: 545, 1973. 26. Dodd, M. C., Scheetz, M. E., 2nd and Rossio, J. L.: Immunogenic RNA in immunotherapy of cancer: the transfer of antitumor cytotoxic activity and tuberculin sensitivity to human lymphocytes using xenogeneic ribonucleic acid. Ann. N. Y. Acad. Sci., 207: 454, 1973.