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Observations on serum and urine alkaline ribonuclease activity and urate after burn injury in man
279
Clinica
Chimica
Acta,
86 (1978)
@ Elsevier/North-Holland
279-290 Biomedical Press
CGA 9390
OBSERVATIONS ON SERUM AND URINE ALKALINE RIBONUCLEASE ACTIVITY AND URATE AFTER BURN INJURY IN MAN
E.J. COOMBES a,*, P.G. SHAKESPEARE
b and G.F. BATSTONE a
a Department of Chemical Pathology, Salisbury General Infirmary, Salisbury, Wiltshire SP2 75% and b Burns Research Unit, Odstock Wiltshire (U.K.)
Fisherton Street, Hospital, Salisbury,
(Received December 8th, 1977)
Summary In an investigation into the disturbances of body function associated with bum injury we have measured the activity of alkaline ribohuclease (EC 3.1.4.22) and the level of urate in the serum and urine of patients sustaining bum injury. Ribonuclease activity was elevated in all patients. The degree of elevation can be related to the percentage of the body surface area burned and to a predictive index of bum mortality. Increased serum ribonuclease activity was accompanied by increased urine ribonuclease output. The relationship between serum urea and ribonuclease activity has been investigated. A significant correlation between these two parameters was observed during the first week post burn. We suggest that this correlation shows as a result of increased protein catabolism and renal dysfunction. After the first week a significant correlation between serum urea and ribonuclease activity was not observed. It is possible that, at this stage, increased ribonuclease activity is perhaps a result of tissue repair. Serum urate was found to be decreased in all patients after bum injury. Serum urate decrease expressed as a percentage of initial value, correlated very strongly with the predictive index of burn mortality. In severely burned patients the decrease in serum urate was accompanied by increased urine urate output and may indicate a change in renal handling of urate after burn injury.
Introduction The morbidity associated with severe bums is the result of the massive catabolic drive following the initial injury. The elevation in catabolism is * To whom correspondence should be addressed.
280
observed as increased heat production, increased water loss, oxygen consumption, weight loss [l-3] and a negative nitrogen balance. The catabolic drive is probably the result of increased secretion of catabolic hormones such as catecholamines, glucagon and glucocorticoids [4] which leads to muscle breakdown and the use of muscle derived amino acids as a fuel source. The severity and duration of weight loss and hence a negative nitrogen balance is related to the size and extent of the burn [ 51. Assessment of the ‘catabolic state’ is difficult and hampered by the lack of simple measurement techniques for indication of severity and duration. The activity of the enzyme ribonuclease (RNAase) in the serum may be a good candidate as an indicator of tissue turnover in humans. In human nutritional experiments [6] it has been shown that serum RNAase bears an inverse relationship with nitrogen balance such that a negative nitrogen balance is associated with elevated serum RNAase activity. It is well known that a negative nitrogen balance exists for long periods after severe burn injury [7]. In malnourished children, Sigulem et al. [8] have shown that serum and urine alkaline RNAase activity can be used to differentiate the degree of malnutrition. Total urine alkaline RNAase was especially useful. Coombes et al. [9] have shown that elevated serum alkaline RNAase activity exists in females with thyrotoxicosis, a condition associated with weight loss. In this study we have evaluated the usefulness of serum and urine alkaline RNAase and urate as indicators of the degree of catabolism in the burnt patient. In an attempt to identify the tissue source of serum alkaline RNAase after burn injury, the behaviour of RNAase has been compared with the behaviour of muscle, liver and pancreas specific enzymes in the serum. In man, urate is the non metabolizable end product of purine metabolism. Purines are derived from three sources, synthesis de novo, dietary intake, and cell necrosis. Serum urate levels are dependent on these factors as well as the renal handling of this organic acid. Hormonal control may also be important as it has been shown [lo] that a positive correlation exists between serum urate and aldosterone excretion. The data in this paper attempts to distinguish between the effects of increased tissue turnover possibly due to mediating hormones and disturbances of renal function on the parameters investigated. Patients Forty one patients (27 male and 14 female) admitted to the Wessex Regional Burns Unit were studied. Patients received fluid replacement therapy, as required, according to the standard Odstock formula of Laing and Harvey [ 111. Assessment
of severity
of burn injury
Thirty five of these patients were divided into three groups based on the predictive index of burn mortality, D3, of Moores et al. [12] which is age, sex and burn area dependent. Group I, patients suffering from severe burns, was subdivided into two sections, Ia (n = 8) patients who died and Ib patients who survived (n = 9). Group II patients sustained moderate burns (n = 9) and group III mild burns (n = 9). For urine analysis patients studied were divided into two
281
groups only, group Ib (n = 5) severely burned patients who patients from groups II and III (n = 5). 24-h urines were collected a fifteen day period post burn.
survived and at times over
Methods Alkaline (pH 7.6) and acid (pH 6.5) RNAase in serum and urine were measured by the method of Biswas and Hindocha 1131 with minor modifications (Coombes et al. [9]).
Highly polymerised RNA for use as substrate was obtained from BDH Ltd. and was present in the assay at a concentration of 1 mg/ml. Phosphate buffer (0.066 M) of appropriate pH was used in the incubation. The activity of RNAase in the serum is expressed as absorbance (A) units/ml of serum and in the urine as A units X 105/24 h. Quality control requirements were fulfilled using human pooled serum {frozen in small aliquots) and bovine pancreatic RNAase (Sigma Ltd.). Patient data for each enzyme activity were grouped into 2 (or more) day intervals and the results within each period averaged. The initial result was subtracted from each average period result to produce a change in the activity of the enzyme for each time interval. Finally all the activity changes for each time period were averaged for aV members of the three groups to produce a mean change in activity. The activities of serum y-glutamyl transferase (EG 2.3.2.1), asp&ate aminotransferase (EC 2.6.1.1) and creatine kinase (EC 2.7.3.2) were measured on the L.K.B. reaction rate analyser using the automated analysis kits of Boehringer (Mannheim) kit numbers 124702, 124443 and 124184 respectively. The changes in absorbance were measured at 410 nm (for yGT) and 340 nm (for ASAT and CK) at a temperature of 37°C. Serum amylase (EC 3.2.1.1) was measured by the Phadebas@ amylase test (Pharmacia Diagnostics) at a temperature of 37°C.
Analytical deter~inat~~~s Urate in serum and urine were measured by the phosphotungstic acid method (Technicon Method N-13 b) and urea by the diacetyl monoxime method (Technicon Method N-16 b).
Statistical analysis of results Correlation coefficients have been evaluated by standard techniques and the Student’s t-test has been used to evaluate the probability (P) of significant differences between groups. Results
Changes in serum RNAase activity The thirty five patients (eight from group Ia, nine each from groups Ib, II and III) listed in Table I were studied throughout their period of stay in hospital. Alkaline (pH 7.6) RNAase activity was found to be eIevated in all patients fof-
282
lowing thermal injury. In general patients sustaining more severe burns showed greater elevation of serum RNAase activity. Serum RNAase activity is expressed as mean maximum rise (from initial activity). The mean m~imum (max.) rise in RNAase activity was found to be 187 A units/ml in group Ia, 181 in group Ib, 51.5 in group II and 27.5 in group III. There was no significant difference (P = 0.90) betweenthe max. rises in group Ia and Ib. However, there were significant differences between the max. rise in RNAase activity of group Ib and group II (P < 0,05), and group It11 (P < 0.02). There was also a significant difference (P < 0.01) between the max. rise in activity in groups II and III, Fig. 1 shows the mean change in alkaline RNAase activity in the serum post burn in the three groups of patients who survived. The patients from group Ib had a mean RNAase activity which rose rapidly reaching a peak on days lo14. This peak was followed by a slight fall in activity but elevated levels persisted for the length of time of the study (day 52). The mean change in RNAase activity in patients from groups II and III was considerably less than patients from group I, reaching a peak on day 7-9 post burn. Two patients from group II and one from group III showed increases in RNAase activity which did not
1 a 3
h
1
I
I !i \I
oh
2-3
4-6
7-9
~--I--~
10-14
TIME
I
15-20
23-27
28-32
#
38-42
I
47-52
(OAYS)
Fig. 1. Mean (C S.E.M.) change in activity of serum alkaline RNAase with time in three gyoups of patients mild burns. * indicates a signifisevere. (A) moderate and (’ -m) sustaining burns; (. -) cant difference (P G 0.05) between the group of patients with severe or moderate burns and the group sustaining mild burns. ,X indicates a significant difference (P < 0.05) between the group of patients with severe and moderate burns.
+2.27
-0.28
8
9
9
9
Ia
II
III
-2.56
+2.97
Index
Ib
Mean
Number
Group
I
GROUPS
TABLE
PATIENT
predictive
6
5
7
4
M
Sex
3
4
2
4
F
28.9
54.1
34.2
66.1
age
Mean
12.5
16.6
47.1
33.5
bum
Mean
%
max.
27.5
51.5
181
187
change
activity
RNA&w?
Mean
8
8
8
-
day
RNAase
Mean
peak
4.8
8.4
8.7
20.5
urea
SfZlUlll
Mean
peak
Mean
6
3
3
urea
peak day
0
0
0
8
Deaths
285
1-3
4:6
7-9
lo-14
15-20
23-27
28-32
38-42
45-52
Time (Days) Fig. 2. Mean (i- S.E.M.) change in activity of serum y-glutamyl patients sustaining burns. Symbols as in Fig. 1.
transferase
with time in the three groups of
urate for up to ten weeks post burn. Patients were classified into three groups, group I (severe), group II (moderate) and group III (mild) as previously described. All patients showed significant decreases in serum urate for long periods post burn. Patients from group I showed a mean max. decrease of 59.2% (S.D. = 8.8), group II a 37.0% decrease (S.D. = 13.4) and group III a 21.5% (S.D. = 9.5). There were significant differences between the mean max. decrease in group I and group II (P < 0.001) and group I and group III (P < 0.001). There was also a significant difference between group II and group III (P < 0.01). Patients from group I took much longer to return to initial urate levels and four patients had not reached this value when discharged from hospital. Fig. 3 shows the mean serum urate decrease post burn in patients from groups I, II and III. There were significant differences between the mean decrease in serum urate in patients from groups I and III on days 4% (P < O.Ol), 7-9 (P < O.OOl), lo-14 (P < O.OOl), 15-21 (P < 0.001) and 23-27 (P < 0.001). There were also significant, differences between the mean decrease in serum urate in patients from groups I and II on days 7-9 (P < O.Ol), lo-14 (P < O.Ol), 15-21 (P C 0.01) and 28-33 (P < 0.05). There was a strong correlation between max. serum urate decrease and percentage burn (r = 0.77, P < 0.001) when all patients were considered. Urine urate Ten patients, five from group I and five from groups II and III combined (referred to as group II 2 III) provided 24-h urine collections at times over the first fifteen day period post burn.
286
-6o2- 3
4-6
7-9
lo-11
15-21
I 23-27
28-33
3eJ-42
4652
TIME (DAYS) Fig. 3. Mean 9% change (+ S&M.) injury. Symbols as in Fig. 1.
of serum urate with time in the three groups of patients
sustaining burn
Urine urate excretion in group I averaged 6.1 (S.D. = 2.34, n = 5) mmol/ 24 h over this 15-day period post burn whilst urine urate excretion in group II + III averaged 2.8 (S.D. = 1.11, II = 5) mmo1/24 h. Levels of urine urate in several of the patients from the severe group reached 13.5 mmo1/24 h. Normal urate excretion can be variable but we found that the range of urate excretion in eight healthy volunteers eating a protein rich diet similar to the burn patients was 1.6-4.5 mmol/24 h. Henry [ 141 quotes a normal range of 1.5-4.5 mmol/ 24 h. The comparison of serum and urine urate levels between groups I and II + III is of interest, A fall in serum urate is accompanied by an increase in urine output (outside normal limits) in the patients from group I. There was a significant difference (P < 0.05) between urine urate output in groups I and II + III. Urine alkaline and acid ribonuclease
activity
Patients from group I showed much greater activities of both acid and alkaline RNAase in 24-h urine specimens than did patients from group II + III. The mean urine alkaline RNAase activity in patients from group I was 11.95 (S.D. = 2.84, n = 5) compared to 6.5 (S.D. = 2.91, n = 5) A units X 105/24 h in patients from group II + III. There was a significant difference between the two groups (P < 0.02). The mean acid RNAase activity in the urine of patients from group I was 13.0 (S.D. = 2.8, IZ= 5) compared to 7.00 (S.D. = 2.61, n = 5) A units X
287 Urine Alkaline RNA ase
Urine Acid RNA ase
. .
lr-. .
Patient
Group2
Fig. 4. 24-h urine output of alkaline and acid RNAase ovgr a 15 day period post burn. n, mean individual patient value in group II + III; 0, mean individual patient value in group I. Blocks represent mean group values. The mean level (? 1 S.D.) of 11 normal individuals is also represented.
105/24 h in the urine of patients from group II + III. There was a significant difference between the two groups (P < 0.01). The activity of alkaline RNAase in 24-h urine specimens from eleven normal subjects was 4.98 X 10’ (S.D. = 1.66) A units/24 h. In the same normal subjects urine acid RNAase activity was 4.78 (S.D. = 1.72) X lo5 A units/24 h. The activity of acid RNAase in 24-h urine specimens from patients in all severity groups was greater than the urine alkaline RNAase in the two groups. Urine RNAase activities are shown in Fig. 4. Clearance
of alkaline RNAase
The excretion of alkaline RNAase activity expressed as a clearance (urine/ serum) has been worked out by Sigulem et al. [8] from normal healthy subjects to be 4.1 i: 1.3 l/24 h. In a limited number of clearance studies we performed on normal subjects, we observed values approximating to the figures given by Sigulem et al. [S] . Clearances in the patients from group I were mostly within this normal range with several initial high clearances. Discussion Serum RNAase activity has been shown to be elevated in a number of disease states. Interpretation of the significance of elevated enzyme activity is rendered difficult by two complicating factors. Firstly the uncertainty of origin of the enzyme and secondly the influence of renal function in determining the serum
288
activity. It has been shown that serum RNAase activity is raised in uraemic patients [ 151 and may reflect a decreased glomerular filtration rate. It has been suggested by Ward and Lea [ 161 that increased serum RNAase activity in myelomatosis may be due to renal dysfunction and Shenkin et al. [17] have suggested that measurement of RNAase activity is only significant when serum urea is normal. The situation in patients suffering thermal injury is particularly interesting because rises in serum urea may reflect both renal dysfunction secondary to shock and increased protein catabolism. This study has shown that serum alkaline RNAase activity is elevated after thermal injury. The activity of the enzyme is elevated in the serum and the total urinary output is also increased. In the serum the alkaline RNAase is elevated whilst there may be preferential output of the acid enzyme in the urine. Higher serum alkaline RNAase activities were noted in the group of patients sustaining more severe burns and there was a strong correlation between predictive index of burn mortality and maximum increase in RNAase activity. Increased enzyme activity was shown to last up to three months post thermal injury. Our results show that there is a significant relationship between serum urea and alkaline RNAase when all results are considered. The strength of the correlation is derived from the relationship during the first week when both parameters can change rapidly. There was no significant correlation after the first week. Mean serum peak urea did not correlate with mean maximum RNAase activity in any of the groups of patients who survived. Mean differences in serum urea between group I patients who survived and group II patients were insignificant but there was a significant difference in RNAase activity. We suggest that both renal trauma and tissue damage may contribute to the observed elevation in serum urea and RNAase activity during the first week post thermal injury. However, the observation that the creatinine clearance rates are relatively unchanged during this period suggests that tissue catabolism may be the more important causative factor. This is supported by the results of measurement of the renal clearances of alkaline RNAase. Normal clearances predominate even in severely burned patients suggesting that the elevated RNAase activities are caused by relatively normal renal function with increased RNAase production in the tissues. Elevation of serum alkaline RNAase activity after the first week when serum urea levels are normal is, we suggest, an indication of tissue turnover and repair. It has been shown by a number of authors that severely burnt patients exhibit negative nitrogen balances for long periods post burn. Serum RNAase activity has been shown (Albanese et al. [6]) to bear an inverse relationship with nitrogen balance and we are led to the conclusion that this long term elevation of RNAase activit,y reflects a known negative nitrogen balance. We believe that serum and urine alkaline RNAase studies may be useful in assessing the effectiveness of diets and hormone therapy to reverse the cat,abolic drive associated with burn injury. The similarity between the mean peak activities of serum alkaline RNAase, yGT and ASAT in the group of patients with severe burns and the dissimilarity to the patterns of serum amylase and CK activities is, we suggest, an indication that the liver and not the pancreas or muscle is the source of increased serum RNAase activity. This observation that enzymes, perhaps of liver origin, may be elevated at days lo-14 post burn is suggestive of an important metabolic pro-
289
cess occurring after burn injury. The increased enzymic activity may be a reflection of late onset liver damage but it is possible that the increased yGT activity we have observed is a reflection of liver regeneration after the initial trauma of burn injury. Kataja and Gordon [ 181 have shown a significant rise in yGT activity after severe injury and have suggested that this is a reflection of post traumatic protein catabolism. One of the surprising observations in this study was the marked decrease in serum urate associated with burn injury. There was a strong correlation between 5% burn and % serum urate decrease. Decreased serum urate levels lasted for long periods post burn and a few patients had not returned t,o initial levels when discharged from hospital, Serum urate decreases were accompanied by increases in urine urate output in the severely burned patients, suggesting a change in the renal handling of urate. Mikus [19] has observed a fall in plasma urate over the first few days after myocardial infarction. This decline was attributed to increased adrenocortical activity. Weiner [ZO] found that there was a relatively high urate excretion in some infarction patients which he attributes to the uricosuric effect of cortisol. Organic acids such as lactate and urate have a common excretory pathway for which they complete. Hyperlactataemia has been shown to cause hyperuricaemia [21]. Hyperlactataemia has been shown to occur after burn injury [22]. In this paper we demonstrate that hypouricaemia occurs after burn injury together with increased urine urate output. These observations suggest that there is a change in the renal handling of urate after burn injury. The study by Batstone et al. [22] also highlighted the marked increase in the catabolic hormone, cortisol, which has been suggested as a uricosuric agent and we believe that it may have a role in the regulation of urate excretion following thermal injury. In summary we believe that the data presented in this paper indicate that severely burned patients have increased serum RNAase activity which is not the result of disturbed renal function. This implies that there is increased production of RNAase in the tissues of burned patients. It is possible that this increased production results from increased tissue turnover associated with damage and repair processes. The data suggests that measurement of serum RNAase after burn injury or other trauma may help to characterize and assess the “catabolic state” associated with trauma. Acknowledgements We are grateful to Mr. J.E. Laing for his interest in this work and permission to study patients at the Wessex Regional Burns Unit. Dr. P. Levick kindly obtained patients’ blood samples. Professor K.G.M.M. Alberti gave helpful advice throughout. Finally we acknowledge the assistance given by Dr. J.R.H. Pinkerton, Mr. D. Ely, the staff of the Pathology Department at Salisbury General Infirmary and Mrs. L. Coombes who prepared the m~uscript. References 1
Birke,
2
Harrison,
G.,
Liljedahl, H.W.,
S. and
Moncrief,
Linderholm. J.A.,
Duck&t,
H. (1958) J.C.
and
Acta Mason,
Chir. A.D.
Stand. (1964)
116.370-376 Surgery
56,
203-208
290 3 Kinney, J.M., Duke, J.H., Long, C.L. and Cump, F.E. (1970) J. Clin. Pathol. 23, Suppl. 4. 65-71 4 Wilmore. D.W., Long. J.M., Mason. A.D., Shreen, R.W. and Pruitt. B.A. (1974) Ann. Surg. 180, 653658 5 Newsome, T.W., Mason, A.D. and Pruitt, B.A. (1973) Ann. Surg. 179, 215-220 6 Albanese, A.A., Orto, L.A.. Zavattaro. D.N. and De Carlo, R. (1971) Nutr. Rep. Int. 4,151-163 7 Sunderland. A.B. (1976) Burns 2. 238-244 8 Sigulem, D.M., Brasel. J.A., Vclasco. E.C.. Ross, P. and Winick, M. (1973) Am. J. Clin. Nutr. 26. i93797 9 Coombes, E.J., Shakespeare, P.G. and Batstone. G.F. (1977) Clin. Chim. Acta 79. 271-275 10 Ramsey, L.E.. Auty, R.M., Horth, C.E., Levine, D.. Suelton, J.R. and Branch. R.A. (1975) Clin. Sci. Mol. Med. 49.613416 11 Laing. J.E. and Harvey, J. (1971) in The Management and Nursing of Burns, 2nd edn., English University Press, London 12 Moores, B., Rahman, M.M., Browning, F.S.C. and Settle, J.A.D. (1975) Burns 1,135-141 13 Biswas, S. and Hindocha, P. (1974) Clin. Chim. Acta 52, 281-289 14 Henry, R.J. (1964) in Clinical Chemistry, Principles and Techniques, P. 280, Hoeber Medical Division. Harper and Row. New York 15 Rabinovitch, M., Liberman. B. and Fausto, N. (1959) J. Lab. Clin. Med. 53, 563-567 16 Ward, D.J. and Lea, D.J. (1974) New Eng. J. Med. 291, 208 17 Shenkin, A., Citrin. D.L. and Rowan, R.M. (1975) Clin. Chim. Acta 72.223-231 18 Kataja, J. and Gordin, R. (1970) Acta Chir. Sand. 136, 277-281 19 Mikus, F. (1972) Beitr. Rheumatol. 18, 74-76 20 Weiner. K. (1976) Clin. Chim. Acta 73. 45-50 21 Sussman. K.E., Alfrey, A., Kirsch, W.. Zweig. P. and Messner. F. (1968) J. Lab. Clin. Med. 72, 10221023 22 Batstone. G.F., Alberti. K.G.M.M.. Hinks, L., Smythe. P.. Lain& J.E., Ward. C.M., Ely, D. and Bloom. S.R. (1976) Burns 2. 207-225
Clinica
Chimica
Acta,
86 (1978)
@ Elsevier/North-Holland
279-290 Biomedical Press
CGA 9390
OBSERVATIONS ON SERUM AND URINE ALKALINE RIBONUCLEASE ACTIVITY AND URATE AFTER BURN INJURY IN MAN
E.J. COOMBES a,*, P.G. SHAKESPEARE
b and G.F. BATSTONE a
a Department of Chemical Pathology, Salisbury General Infirmary, Salisbury, Wiltshire SP2 75% and b Burns Research Unit, Odstock Wiltshire (U.K.)
Fisherton Street, Hospital, Salisbury,
(Received December 8th, 1977)
Summary In an investigation into the disturbances of body function associated with bum injury we have measured the activity of alkaline ribohuclease (EC 3.1.4.22) and the level of urate in the serum and urine of patients sustaining bum injury. Ribonuclease activity was elevated in all patients. The degree of elevation can be related to the percentage of the body surface area burned and to a predictive index of bum mortality. Increased serum ribonuclease activity was accompanied by increased urine ribonuclease output. The relationship between serum urea and ribonuclease activity has been investigated. A significant correlation between these two parameters was observed during the first week post burn. We suggest that this correlation shows as a result of increased protein catabolism and renal dysfunction. After the first week a significant correlation between serum urea and ribonuclease activity was not observed. It is possible that, at this stage, increased ribonuclease activity is perhaps a result of tissue repair. Serum urate was found to be decreased in all patients after bum injury. Serum urate decrease expressed as a percentage of initial value, correlated very strongly with the predictive index of burn mortality. In severely burned patients the decrease in serum urate was accompanied by increased urine urate output and may indicate a change in renal handling of urate after burn injury.
Introduction The morbidity associated with severe bums is the result of the massive catabolic drive following the initial injury. The elevation in catabolism is * To whom correspondence should be addressed.
280
observed as increased heat production, increased water loss, oxygen consumption, weight loss [l-3] and a negative nitrogen balance. The catabolic drive is probably the result of increased secretion of catabolic hormones such as catecholamines, glucagon and glucocorticoids [4] which leads to muscle breakdown and the use of muscle derived amino acids as a fuel source. The severity and duration of weight loss and hence a negative nitrogen balance is related to the size and extent of the burn [ 51. Assessment of the ‘catabolic state’ is difficult and hampered by the lack of simple measurement techniques for indication of severity and duration. The activity of the enzyme ribonuclease (RNAase) in the serum may be a good candidate as an indicator of tissue turnover in humans. In human nutritional experiments [6] it has been shown that serum RNAase bears an inverse relationship with nitrogen balance such that a negative nitrogen balance is associated with elevated serum RNAase activity. It is well known that a negative nitrogen balance exists for long periods after severe burn injury [7]. In malnourished children, Sigulem et al. [8] have shown that serum and urine alkaline RNAase activity can be used to differentiate the degree of malnutrition. Total urine alkaline RNAase was especially useful. Coombes et al. [9] have shown that elevated serum alkaline RNAase activity exists in females with thyrotoxicosis, a condition associated with weight loss. In this study we have evaluated the usefulness of serum and urine alkaline RNAase and urate as indicators of the degree of catabolism in the burnt patient. In an attempt to identify the tissue source of serum alkaline RNAase after burn injury, the behaviour of RNAase has been compared with the behaviour of muscle, liver and pancreas specific enzymes in the serum. In man, urate is the non metabolizable end product of purine metabolism. Purines are derived from three sources, synthesis de novo, dietary intake, and cell necrosis. Serum urate levels are dependent on these factors as well as the renal handling of this organic acid. Hormonal control may also be important as it has been shown [lo] that a positive correlation exists between serum urate and aldosterone excretion. The data in this paper attempts to distinguish between the effects of increased tissue turnover possibly due to mediating hormones and disturbances of renal function on the parameters investigated. Patients Forty one patients (27 male and 14 female) admitted to the Wessex Regional Burns Unit were studied. Patients received fluid replacement therapy, as required, according to the standard Odstock formula of Laing and Harvey [ 111. Assessment
of severity
of burn injury
Thirty five of these patients were divided into three groups based on the predictive index of burn mortality, D3, of Moores et al. [12] which is age, sex and burn area dependent. Group I, patients suffering from severe burns, was subdivided into two sections, Ia (n = 8) patients who died and Ib patients who survived (n = 9). Group II patients sustained moderate burns (n = 9) and group III mild burns (n = 9). For urine analysis patients studied were divided into two
281
groups only, group Ib (n = 5) severely burned patients who patients from groups II and III (n = 5). 24-h urines were collected a fifteen day period post burn.
survived and at times over
Methods Alkaline (pH 7.6) and acid (pH 6.5) RNAase in serum and urine were measured by the method of Biswas and Hindocha 1131 with minor modifications (Coombes et al. [9]).
Highly polymerised RNA for use as substrate was obtained from BDH Ltd. and was present in the assay at a concentration of 1 mg/ml. Phosphate buffer (0.066 M) of appropriate pH was used in the incubation. The activity of RNAase in the serum is expressed as absorbance (A) units/ml of serum and in the urine as A units X 105/24 h. Quality control requirements were fulfilled using human pooled serum {frozen in small aliquots) and bovine pancreatic RNAase (Sigma Ltd.). Patient data for each enzyme activity were grouped into 2 (or more) day intervals and the results within each period averaged. The initial result was subtracted from each average period result to produce a change in the activity of the enzyme for each time interval. Finally all the activity changes for each time period were averaged for aV members of the three groups to produce a mean change in activity. The activities of serum y-glutamyl transferase (EG 2.3.2.1), asp&ate aminotransferase (EC 2.6.1.1) and creatine kinase (EC 2.7.3.2) were measured on the L.K.B. reaction rate analyser using the automated analysis kits of Boehringer (Mannheim) kit numbers 124702, 124443 and 124184 respectively. The changes in absorbance were measured at 410 nm (for yGT) and 340 nm (for ASAT and CK) at a temperature of 37°C. Serum amylase (EC 3.2.1.1) was measured by the Phadebas@ amylase test (Pharmacia Diagnostics) at a temperature of 37°C.
Analytical deter~inat~~~s Urate in serum and urine were measured by the phosphotungstic acid method (Technicon Method N-13 b) and urea by the diacetyl monoxime method (Technicon Method N-16 b).
Statistical analysis of results Correlation coefficients have been evaluated by standard techniques and the Student’s t-test has been used to evaluate the probability (P) of significant differences between groups. Results
Changes in serum RNAase activity The thirty five patients (eight from group Ia, nine each from groups Ib, II and III) listed in Table I were studied throughout their period of stay in hospital. Alkaline (pH 7.6) RNAase activity was found to be eIevated in all patients fof-
282
lowing thermal injury. In general patients sustaining more severe burns showed greater elevation of serum RNAase activity. Serum RNAase activity is expressed as mean maximum rise (from initial activity). The mean m~imum (max.) rise in RNAase activity was found to be 187 A units/ml in group Ia, 181 in group Ib, 51.5 in group II and 27.5 in group III. There was no significant difference (P = 0.90) betweenthe max. rises in group Ia and Ib. However, there were significant differences between the max. rise in RNAase activity of group Ib and group II (P < 0,05), and group It11 (P < 0.02). There was also a significant difference (P < 0.01) between the max. rise in activity in groups II and III, Fig. 1 shows the mean change in alkaline RNAase activity in the serum post burn in the three groups of patients who survived. The patients from group Ib had a mean RNAase activity which rose rapidly reaching a peak on days lo14. This peak was followed by a slight fall in activity but elevated levels persisted for the length of time of the study (day 52). The mean change in RNAase activity in patients from groups II and III was considerably less than patients from group I, reaching a peak on day 7-9 post burn. Two patients from group II and one from group III showed increases in RNAase activity which did not
1 a 3
h
1
I
I !i \I
oh
2-3
4-6
7-9
~--I--~
10-14
TIME
I
15-20
23-27
28-32
#
38-42
I
47-52
(OAYS)
Fig. 1. Mean (C S.E.M.) change in activity of serum alkaline RNAase with time in three gyoups of patients mild burns. * indicates a signifisevere. (A) moderate and (’ -m) sustaining burns; (. -) cant difference (P G 0.05) between the group of patients with severe or moderate burns and the group sustaining mild burns. ,X indicates a significant difference (P < 0.05) between the group of patients with severe and moderate burns.
+2.27
-0.28
8
9
9
9
Ia
II
III
-2.56
+2.97
Index
Ib
Mean
Number
Group
I
GROUPS
TABLE
PATIENT
predictive
6
5
7
4
M
Sex
3
4
2
4
F
28.9
54.1
34.2
66.1
age
Mean
12.5
16.6
47.1
33.5
bum
Mean
%
max.
27.5
51.5
181
187
change
activity
RNA&w?
Mean
8
8
8
-
day
RNAase
Mean
peak
4.8
8.4
8.7
20.5
urea
SfZlUlll
Mean
peak
Mean
6
3
3
urea
peak day
0
0
0
8
Deaths
285
1-3
4:6
7-9
lo-14
15-20
23-27
28-32
38-42
45-52
Time (Days) Fig. 2. Mean (i- S.E.M.) change in activity of serum y-glutamyl patients sustaining burns. Symbols as in Fig. 1.
transferase
with time in the three groups of
urate for up to ten weeks post burn. Patients were classified into three groups, group I (severe), group II (moderate) and group III (mild) as previously described. All patients showed significant decreases in serum urate for long periods post burn. Patients from group I showed a mean max. decrease of 59.2% (S.D. = 8.8), group II a 37.0% decrease (S.D. = 13.4) and group III a 21.5% (S.D. = 9.5). There were significant differences between the mean max. decrease in group I and group II (P < 0.001) and group I and group III (P < 0.001). There was also a significant difference between group II and group III (P < 0.01). Patients from group I took much longer to return to initial urate levels and four patients had not reached this value when discharged from hospital. Fig. 3 shows the mean serum urate decrease post burn in patients from groups I, II and III. There were significant differences between the mean decrease in serum urate in patients from groups I and III on days 4% (P < O.Ol), 7-9 (P < O.OOl), lo-14 (P < O.OOl), 15-21 (P < 0.001) and 23-27 (P < 0.001). There were also significant, differences between the mean decrease in serum urate in patients from groups I and II on days 7-9 (P < O.Ol), lo-14 (P < O.Ol), 15-21 (P C 0.01) and 28-33 (P < 0.05). There was a strong correlation between max. serum urate decrease and percentage burn (r = 0.77, P < 0.001) when all patients were considered. Urine urate Ten patients, five from group I and five from groups II and III combined (referred to as group II 2 III) provided 24-h urine collections at times over the first fifteen day period post burn.
286
-6o2- 3
4-6
7-9
lo-11
15-21
I 23-27
28-33
3eJ-42
4652
TIME (DAYS) Fig. 3. Mean 9% change (+ S&M.) injury. Symbols as in Fig. 1.
of serum urate with time in the three groups of patients
sustaining burn
Urine urate excretion in group I averaged 6.1 (S.D. = 2.34, n = 5) mmol/ 24 h over this 15-day period post burn whilst urine urate excretion in group II + III averaged 2.8 (S.D. = 1.11, II = 5) mmo1/24 h. Levels of urine urate in several of the patients from the severe group reached 13.5 mmo1/24 h. Normal urate excretion can be variable but we found that the range of urate excretion in eight healthy volunteers eating a protein rich diet similar to the burn patients was 1.6-4.5 mmol/24 h. Henry [ 141 quotes a normal range of 1.5-4.5 mmol/ 24 h. The comparison of serum and urine urate levels between groups I and II + III is of interest, A fall in serum urate is accompanied by an increase in urine output (outside normal limits) in the patients from group I. There was a significant difference (P < 0.05) between urine urate output in groups I and II + III. Urine alkaline and acid ribonuclease
activity
Patients from group I showed much greater activities of both acid and alkaline RNAase in 24-h urine specimens than did patients from group II + III. The mean urine alkaline RNAase activity in patients from group I was 11.95 (S.D. = 2.84, n = 5) compared to 6.5 (S.D. = 2.91, n = 5) A units X 105/24 h in patients from group II + III. There was a significant difference between the two groups (P < 0.02). The mean acid RNAase activity in the urine of patients from group I was 13.0 (S.D. = 2.8, IZ= 5) compared to 7.00 (S.D. = 2.61, n = 5) A units X
287 Urine Alkaline RNA ase
Urine Acid RNA ase
. .
lr-. .
Patient
Group2
Fig. 4. 24-h urine output of alkaline and acid RNAase ovgr a 15 day period post burn. n, mean individual patient value in group II + III; 0, mean individual patient value in group I. Blocks represent mean group values. The mean level (? 1 S.D.) of 11 normal individuals is also represented.
105/24 h in the urine of patients from group II + III. There was a significant difference between the two groups (P < 0.01). The activity of alkaline RNAase in 24-h urine specimens from eleven normal subjects was 4.98 X 10’ (S.D. = 1.66) A units/24 h. In the same normal subjects urine acid RNAase activity was 4.78 (S.D. = 1.72) X lo5 A units/24 h. The activity of acid RNAase in 24-h urine specimens from patients in all severity groups was greater than the urine alkaline RNAase in the two groups. Urine RNAase activities are shown in Fig. 4. Clearance
of alkaline RNAase
The excretion of alkaline RNAase activity expressed as a clearance (urine/ serum) has been worked out by Sigulem et al. [8] from normal healthy subjects to be 4.1 i: 1.3 l/24 h. In a limited number of clearance studies we performed on normal subjects, we observed values approximating to the figures given by Sigulem et al. [S] . Clearances in the patients from group I were mostly within this normal range with several initial high clearances. Discussion Serum RNAase activity has been shown to be elevated in a number of disease states. Interpretation of the significance of elevated enzyme activity is rendered difficult by two complicating factors. Firstly the uncertainty of origin of the enzyme and secondly the influence of renal function in determining the serum
288
activity. It has been shown that serum RNAase activity is raised in uraemic patients [ 151 and may reflect a decreased glomerular filtration rate. It has been suggested by Ward and Lea [ 161 that increased serum RNAase activity in myelomatosis may be due to renal dysfunction and Shenkin et al. [17] have suggested that measurement of RNAase activity is only significant when serum urea is normal. The situation in patients suffering thermal injury is particularly interesting because rises in serum urea may reflect both renal dysfunction secondary to shock and increased protein catabolism. This study has shown that serum alkaline RNAase activity is elevated after thermal injury. The activity of the enzyme is elevated in the serum and the total urinary output is also increased. In the serum the alkaline RNAase is elevated whilst there may be preferential output of the acid enzyme in the urine. Higher serum alkaline RNAase activities were noted in the group of patients sustaining more severe burns and there was a strong correlation between predictive index of burn mortality and maximum increase in RNAase activity. Increased enzyme activity was shown to last up to three months post thermal injury. Our results show that there is a significant relationship between serum urea and alkaline RNAase when all results are considered. The strength of the correlation is derived from the relationship during the first week when both parameters can change rapidly. There was no significant correlation after the first week. Mean serum peak urea did not correlate with mean maximum RNAase activity in any of the groups of patients who survived. Mean differences in serum urea between group I patients who survived and group II patients were insignificant but there was a significant difference in RNAase activity. We suggest that both renal trauma and tissue damage may contribute to the observed elevation in serum urea and RNAase activity during the first week post thermal injury. However, the observation that the creatinine clearance rates are relatively unchanged during this period suggests that tissue catabolism may be the more important causative factor. This is supported by the results of measurement of the renal clearances of alkaline RNAase. Normal clearances predominate even in severely burned patients suggesting that the elevated RNAase activities are caused by relatively normal renal function with increased RNAase production in the tissues. Elevation of serum alkaline RNAase activity after the first week when serum urea levels are normal is, we suggest, an indication of tissue turnover and repair. It has been shown by a number of authors that severely burnt patients exhibit negative nitrogen balances for long periods post burn. Serum RNAase activity has been shown (Albanese et al. [6]) to bear an inverse relationship with nitrogen balance and we are led to the conclusion that this long term elevation of RNAase activit,y reflects a known negative nitrogen balance. We believe that serum and urine alkaline RNAase studies may be useful in assessing the effectiveness of diets and hormone therapy to reverse the cat,abolic drive associated with burn injury. The similarity between the mean peak activities of serum alkaline RNAase, yGT and ASAT in the group of patients with severe burns and the dissimilarity to the patterns of serum amylase and CK activities is, we suggest, an indication that the liver and not the pancreas or muscle is the source of increased serum RNAase activity. This observation that enzymes, perhaps of liver origin, may be elevated at days lo-14 post burn is suggestive of an important metabolic pro-
289
cess occurring after burn injury. The increased enzymic activity may be a reflection of late onset liver damage but it is possible that the increased yGT activity we have observed is a reflection of liver regeneration after the initial trauma of burn injury. Kataja and Gordon [ 181 have shown a significant rise in yGT activity after severe injury and have suggested that this is a reflection of post traumatic protein catabolism. One of the surprising observations in this study was the marked decrease in serum urate associated with burn injury. There was a strong correlation between 5% burn and % serum urate decrease. Decreased serum urate levels lasted for long periods post burn and a few patients had not returned t,o initial levels when discharged from hospital, Serum urate decreases were accompanied by increases in urine urate output in the severely burned patients, suggesting a change in the renal handling of urate. Mikus [19] has observed a fall in plasma urate over the first few days after myocardial infarction. This decline was attributed to increased adrenocortical activity. Weiner [ZO] found that there was a relatively high urate excretion in some infarction patients which he attributes to the uricosuric effect of cortisol. Organic acids such as lactate and urate have a common excretory pathway for which they complete. Hyperlactataemia has been shown to cause hyperuricaemia [21]. Hyperlactataemia has been shown to occur after burn injury [22]. In this paper we demonstrate that hypouricaemia occurs after burn injury together with increased urine urate output. These observations suggest that there is a change in the renal handling of urate after burn injury. The study by Batstone et al. [22] also highlighted the marked increase in the catabolic hormone, cortisol, which has been suggested as a uricosuric agent and we believe that it may have a role in the regulation of urate excretion following thermal injury. In summary we believe that the data presented in this paper indicate that severely burned patients have increased serum RNAase activity which is not the result of disturbed renal function. This implies that there is increased production of RNAase in the tissues of burned patients. It is possible that this increased production results from increased tissue turnover associated with damage and repair processes. The data suggests that measurement of serum RNAase after burn injury or other trauma may help to characterize and assess the “catabolic state” associated with trauma. Acknowledgements We are grateful to Mr. J.E. Laing for his interest in this work and permission to study patients at the Wessex Regional Burns Unit. Dr. P. Levick kindly obtained patients’ blood samples. Professor K.G.M.M. Alberti gave helpful advice throughout. Finally we acknowledge the assistance given by Dr. J.R.H. Pinkerton, Mr. D. Ely, the staff of the Pathology Department at Salisbury General Infirmary and Mrs. L. Coombes who prepared the m~uscript. References 1
Birke,
2
Harrison,
G.,
Liljedahl, H.W.,
S. and
Moncrief,
Linderholm. J.A.,
Duck&t,
H. (1958) J.C.
and
Acta Mason,
Chir. A.D.
Stand. (1964)
116.370-376 Surgery
56,
203-208
290 3 Kinney, J.M., Duke, J.H., Long, C.L. and Cump, F.E. (1970) J. Clin. Pathol. 23, Suppl. 4. 65-71 4 Wilmore. D.W., Long. J.M., Mason. A.D., Shreen, R.W. and Pruitt. B.A. (1974) Ann. Surg. 180, 653658 5 Newsome, T.W., Mason, A.D. and Pruitt, B.A. (1973) Ann. Surg. 179, 215-220 6 Albanese, A.A., Orto, L.A.. Zavattaro. D.N. and De Carlo, R. (1971) Nutr. Rep. Int. 4,151-163 7 Sunderland. A.B. (1976) Burns 2. 238-244 8 Sigulem, D.M., Brasel. J.A., Vclasco. E.C.. Ross, P. and Winick, M. (1973) Am. J. Clin. Nutr. 26. i93797 9 Coombes, E.J., Shakespeare, P.G. and Batstone. G.F. (1977) Clin. Chim. Acta 79. 271-275 10 Ramsey, L.E.. Auty, R.M., Horth, C.E., Levine, D.. Suelton, J.R. and Branch. R.A. (1975) Clin. Sci. Mol. Med. 49.613416 11 Laing. J.E. and Harvey, J. (1971) in The Management and Nursing of Burns, 2nd edn., English University Press, London 12 Moores, B., Rahman, M.M., Browning, F.S.C. and Settle, J.A.D. (1975) Burns 1,135-141 13 Biswas, S. and Hindocha, P. (1974) Clin. Chim. Acta 52, 281-289 14 Henry, R.J. (1964) in Clinical Chemistry, Principles and Techniques, P. 280, Hoeber Medical Division. Harper and Row. New York 15 Rabinovitch, M., Liberman. B. and Fausto, N. (1959) J. Lab. Clin. Med. 53, 563-567 16 Ward, D.J. and Lea, D.J. (1974) New Eng. J. Med. 291, 208 17 Shenkin, A., Citrin. D.L. and Rowan, R.M. (1975) Clin. Chim. Acta 72.223-231 18 Kataja, J. and Gordin, R. (1970) Acta Chir. Sand. 136, 277-281 19 Mikus, F. (1972) Beitr. Rheumatol. 18, 74-76 20 Weiner. K. (1976) Clin. Chim. Acta 73. 45-50 21 Sussman. K.E., Alfrey, A., Kirsch, W.. Zweig. P. and Messner. F. (1968) J. Lab. Clin. Med. 72, 10221023 22 Batstone. G.F., Alberti. K.G.M.M.. Hinks, L., Smythe. P.. Lain& J.E., Ward. C.M., Ely, D. and Bloom. S.R. (1976) Burns 2. 207-225