201
Clinica Chimico Acta, 95 (1979) 201-209 0 Elsevier/North-Holland Biomedical Press
CCA 1026
URINE PROTEINS
E.J. COOMBES
AFTER
BURN INJURY
a,*, P.G. SHAKESPEARE
b and G.F. BATSTONE
a
a Department of Chemical Pathology, Salisbury General Infirmary, &L-bury, Wilts., SP2 7SX (U.K.) and b Burns Research Unit, Odstock Hospital, Salisbury, Wilts. (U.K.) (Received
November
31st, 1978)
Summary Two-dimensional immunoelectrophoresis was used to examine the proteins present in urine during the first week following burn injury. Of the “serum” proteins present in the urine some glycoproteins were found to be in different relative proportions from those observed in serum. In patients sustaining severe bums the amount of protein excreted was increased compared to patients with mild bums and to controls. al-Antichymotrypsin detected in the urine of patients with severe burns was at times seen as a twin peak. This altered peak was of slower electrophoretic mobility and may represent a polymer of the protein or a complex of the protein with some other, possibly tissue-derived, protein.
Introduction Every healthy individual is known to excrete protein in their urine. The amount they excrete daily may be relatively constant [l]. However, the “normal” amount of protein excreted by individuals is uncertain. Some workers have found very small amounts whilst others [2] have found as much as 80180 mg/24 h. The differences may be due to the relative sensitivities of the methods used to determine the amounts of protein [ 31, although Hemmingsen and Shaarup [4] suggest the most characteristic feature of the excretion of plasma proteins is the great dispersion of the values and the uneven distribution. Proteinuria has been studied most following renal damage and it is still regarded [ 51 as one of the most sensitive indicators of this condition. “Stress” and heavy physical exercise can cause a moderate increase in protein excretion [6]. It is now recognised that the kidney plays an important part in the catab* To whom correspondence
should be addressed.
202
olism of low molecular weight proteins [7]. Low molecular weight proteins, but not high molecular weight proteins, readily traverse the glomerular membrane and are largely reabsorbed and catabolized in tubular cells. Plasma proteins passing through the glomerular membrane constitute a large proportion of normal urine proteins, albumin being the predominant species. Mucoproteins synthesised on the outer surface of the cells lining the renal and genito-urinary tract [ 81 constitute some of the remaining protein in normal urine. In this study, the amount of protein in the urine of patients suffering from burn injury has been quantitatively and qualitatively examined. This work was undertaken to study not only the .total excretion of protein in the urine, but also to study specifically the excretion of individual proteins over a period of time following burn injury. It is hoped that this approach may shed light on possible kidney damage after bum injury and provide a method for assessing the efficacy of fluid replacement therapy in reducing the risk of renal stress or damage. Patients
and controls
14 patients (11 M, 3 F) who were admitted to the Wessex Regional Bums Centre were studied. Urine was collected in 24-h aliquots for a period up to 9 days post injury. Preservative was not added to the urines. The volume of the urine was measured, the urine was aliquoted and frozen (-20°C) until use. The urine was thawed once and concentrated ten times using a “Minicon” macrosolute concentrator (Amicon Corporation). Mean daily urine volumes for patients sustaining mild and severe bums are shown in Table II. One patient (C.K.) produced 24-h urine volumes below 0.5 1 on days 1 and 2. Patients were divided into 2 groups (mild and severe) based on the 5%area of their bums. Patients’ data are listed in Table I. 20 controls (healthy laboratory workers, age range 19-57 years) provided 24-h urine collections. These control urines were processed and concentrated in exactly the same manner as the urines from bums patients. Methods The method of two-dimensional immunoelectrophoresis used to examine the concentrated urine was basically that of Laurel1 [ 91, following developments by Clarke and Freeman [lO,ll] and using a small plate modification [12]. Precipitin bands were visualised by staining with Coomassie Blue. Antisera specific
TABLE PATIENT Group
I DATA No.
in
group
Mean
age (yrs)
Mean
Sex
% area of bum
(range)
(range) M
F
SWl?lW
I
23.1
(19-38)
6
1
53.6
Mild
7
29.4
(11-72)
5
2
17.7
(40-65) (8-36)
203
against albumin ( ALB),(Y1-antichymotrypsin (ACT), OL , -antitrypsin (AT), prealbumin (p-Alb), Gc globulin (GC), transferrin (TFN), orosomucoid (ORO) and a*-HS glycoprotein (HS) were obtained from Behringwerke AG (Hoechst Pharmaceuticals), “Dakopatts” anti whole human serum raised in rabbits was supplied by Mercia Diagnostics. Two-dimensional immunoelectrophoresis 3 ~1 of concentrated (X10) urine was electrophoresed in the first dimension in 1% agarose (8 V/cm, 45 min). The second dimension was performed in 1% agarose containing 100 ,ul of Dakopatts polyvalent antiserum as locating agent. Precipitin arcs were identified by adding specific protein antiserum to the polyvalent antiserum in the second dimension and observing if the peak was substantially reduced in size. Alternatively where addition of the antiserum caused excessively heavy background color, 2 plates were run in parallel. One plate contained specific protein antiserum and one plate the polyvalent antiserum, peak heights and mobility were then compared. Protein determination Protein was determined by the method of Lowry et al. [ 131 but using a preliminary 5% TCA precipitation of the concentrated urine. After centrifugation the precipitate was redissolved in 1 mol/l NaOH and the protein content estimated. Quality control serum (Versatol, Warner Ltd.) was used to construct standard curves for this determination. Results Twenty control urines were analysed by two-dimensional immunoelectrophoresis as described. The maximum height of an individual protein from any concentrated control urine was measured and taken to be the upper limit of the normal range. This only applied to two proteins, orosomucoid and albumin (the protein most commonly seen) because these were the only proteins to produce measurable precipitin arcs in the concentrated urine (Fig. la). Several small precipitin arcs were uncharacterised. Any protein occurring in the con-
Fig. 1. Proteins present in urines from a control and a patient with mild burns. Urine protein profile of (a) a normal control and (b) a patient with mild burns. 1. Orosomucoid; 2, albumin; 3, aI-antichymotrypsin.
204
TABLE
II
TOTAL
24-h URINE PROTEIN AND VOLUME
IN PATIENTS
WITH SEVERE
AND MILD BURNS
Urine volume expressed as likes and urine protein as mg/24 h relative to ‘Versatol’ standards. Days postburn
1 2 3 4 5 6 7 8 9
Severe bum 24-h tine
Mild burn
protein
__I~
24-h urine volume
24-h urine protein
24-h urine volume
Meall
Range
MeatI
Range
Mean
Range
Mean
Range
42 70 179 210 166 105 90 112 51
18-- 80 20-149 34-572 65-533 50-3 15 41-243 35-l 52 52-297 3565
1.12 1.09 1.44 1.36 1.19 1.66 1.34 1.47 1.11
0.18-1.9
73 35 34 30 30 32 35
18-211 2448 1558 2052 1637 1343 2047
1.87 1.78 1.27 1.72 1.98 1.35 1.22
0.7 -3.02 0.7 -3.54 0.53-2.42 0.58-2.8 0.84-3.12 1.03-1.8 0.64-1.76
0.19-1.63 0:69-2.1 0.56-2.03 0.52-l .7& X.06-2.12 1.1 -1.82 0.5 -2.26 0.92-1.4
-
-
centrated urine from burned patients was assessed relative to the arbitrarily set upper limit of normal. The mean protein excretion in 20 controls was found to be 35 mgf24 h (range 12-75) whilst the mean urine volume was 1.49 1 (range 0.64-2.3). In patients sustaining mild burns the mean protein excretion was not significantly different from controls on any day post-burn. Mean urine protein levels in the groups of patients sustaining mild and severe burns are shown in Table II. In patients with mild burns, orosomucoid was the protein most commonly elevated above the ~bitr~ly set upper limit of normal. On Day 1 80% of patients showed an increased excretion of orosomucoid. ACT could be detected in 40% of patients on Day 1 post-burn whilst pre-albumin and a2-HS glycop~tein could be detected in 20% of patients. Table III lists the urine proteins which were detected in the group of patients with mild burns. Fig. lb shows the protein profile of a patient sustaining mild bums on Day 1 postbum, ORO, ALB and ACT can be elearly seen. In patients sustaining severe bums the mean urine protein excretion increased to a peak value of 210 mg/24 h on Day 4 post-burn falling thereafter. There were significant differences (P < 0.05) between the mean protein excretion in TABLE III INCIDENCE
OF SPECIFIC PROTEINS
IN URINES OF PATIENTS
WITH MILD BURNS
Results expressed as % of patient with detectable protein above the upper limit of normdity. Protein
Orosomucoid o(I -Antichymotrypsin Pre-albumin a~-HS glycoprotein .-____--
Days post-burn 1
2
3
4
5
6
80% 40 20 20
67 50 0 0
57 14 0 0
43 14 0 0
40 20 0 0
28 0 0 0
7 20 0 0 0 _I
205
patients with severe and mild burns on Days 4 and 5 post-bum. Fig. 2 shows the protein profile of one patient with severe bums on Days 1, 3, 5.and 7 postbum. In the early phase (Day 1) after injury this patient is excreting ORO, HS, p-ALB, ALB, AT, ACT and a twin peak of ACT. By Day 7 only ACT and OR0 could be detected by this technique. In the urine of patients with severe burns OR0 and ACT could be detected in all patients on all days. HS was the next most commonly observed protein, present in a maximum of 80% of patients on Day 2. The excretion of AT, ALB, TFN p-ALB and GC were variable over the g-day study period. Increased excretion of ALB was not detected after Day 5 and precipitin arcs were very small. The urine protein profile of the group of patients with severe bums is shown in Fig. 3. One uncharacterised protein with mobility in the LYregion, was detected in several of the urines from patients with severe bums. It is interesting to observe that whilst the mean excretion in the patients with severe burns on Days 4 and 5 is still elevated, the number of serum proteins observed in urine is decreasing. This may be an indication that tissue derived proteins may be contributing to the total urine protein excretion at this period post-bum. Twin peaks of ACT were noted in all 7 patients with severe bums on at least
Urine
Pmteins
after Burn Injury ACT
OR0
“__Lh 2
4
6
a
ACT 2
10
Fig. 2. Proteins present in urine from a patient with severe burns. Urine protein profile 2. albumin; 3, oil-antichymotrypsin; (b) Day 3. (C) Day 5. and (d) Day 7, 1, Orosomucoid; and 6, pre-albumin. antichymotrypsin: 5, a-~-HS glycoprotein
on (a) Day 1, 4. altered (Y,-
Fig. 3. Urine protein profile of 7 patients with severe burns. Results expressed as 9% number of patients with greater than normal protein excretion. Patient ages and 96 areas of bum are shown. ORO. oroso_ mucoid: ACT, (~1-antichymotrypsin: ACT 2, altered antichymotrypsin: AT. oil-antitrypsin; ALB, albumin: TFN. transferrin; HS. (YZ-HS izlwoprotein; P-ALB, pre-albumin; GC, Gc globulin; AT 2. altered antitrypsin.
206
a
Fig. 4. Form of a]-antichymotrypsin in serum and urine of patients with acute tubular necrosis following burn injury. (a) serum (Day 1 post-burn) against al-antichymotrypsin antiserum and (b) concentrated urine (Day 1) against polyvalent antiserum. 3, orl-antichymotrypsin; 4. altered antichymotrypsin.
2 days post-injury. On Day 2 post-burn all those patients tested showed this twin peak effect. An example of this twin peak phenomenon is shown in Fig. 2 (a and b). The variant peak was always of slower electrophoretic mobility and was of varying size relative to the main peak. The altered peak could not be detected in any patients’ urine after Day 7. Attempts to produce this altered peak in vitro were unsuccessful; agents used were heat, pH change, urea and mercaptoethanol. Incubation of concentrated burn and control urine with normal serum also failed to produce a second peak (apart from the quantity of the second peak originally present in the burn urine). Fig. 4 (a) shows a twodimensional immunoelectrophoretogram of serum, from a patient with severe bums (95%) on Day 1 post-burn against ACT antiserum. Only one peak is observed. This patient had developed acute tubular necrosis and Fig. 4 (b) shows a two-dimensional immunoelectrophoretogram of concentrated urine from this patient on the same day. The twin ACT effect is clearly seen. On this day the patient excreted 250 mg of protein in a urine volume of 0.23 1. Discussion This study has shown that patients sustaining severe burns excrete greater amounts of protein than patients with mild burns and normal controls. In patients with severe burns the peak protein excretion was on Day 4 post-burn, falling thereafter towards normality. The pattern of individual protein excretion in patients with mild burns showed only orosomucoid as a reasonably constant feature in the early post-bum period. In the patients with severe burns the pattern of individual protein excretion was much more varied. 8 proteins could be detected of which transferrin (mol. wt. at 80 000) was the highest molecular weight. One of the surprising features was the apparent low excretion of albumin. Serum albumin falls rapidly after severe burn injury [ 141 and this lack of increased albumin excretion probably reflects the decreased serum concentration or an increased tubular reabsorption. The constant feature in the urine of patients with severe burns was the presence of orosomucoid and (Y,-antichymotrypsin in all patients on all days studied. The presence of orosomucoid may be a reflection of increased production or loss of mucoproteins produced on the
surface of cells lining the renal tract. Jackson et al. [15] have highlighted the increased excretion of acid mucoproteins after burn injury in children. The increased production of certain proteins termed the acute phase reactants [161 in the ‘early phase of trauma is well documented. ORO, ACT, AT and HS are all acute phase reactants known to increase in the serum after trauma and burn injury. It is possible that the increased appearance of these proteins in the urine reflects an increased serum production resulting in an overflow phenomenon i.e. the reabsorption capacity has been exceeded. However, this is unlikely because ACT increases to reach a maximum serum level on days 4-6 after injury (Shakespeare, P.G. and Spur-r, unpublished observations), whilst Fig. 2 clearly shows that the excretion of ACT in the urine is maximal on Day 1 and decreases thereafter. It is of interest that while ACT is invariably observed in the urine, AT was not observed in many of these patients. In serum, AT concentration is markedly elevated in the acute phase reaction. These observations suggest the possibility of a specific kidney response to burn injury involving increased excretion of ACT and OR0 by the kidney. Whether this results from different handling by the kidney after bum injury or to an actual production of these proteins in the kidney tissue remains as a problem for further investigations. Shakespeare et al. [17] have shown a relatively higher concentration of OR0 in blister fluid than in serum of bum patients, suggesting a localised tissue response to injury. The presence of elevated concentrations of glycoproteins in wound exudate has been known for many years [ 181. One possible reason for the presence of larger numbers of proteins in the urine from patients with severe bums might have been associated with different incidence of infection in the two groups. In the severely burned group of patients (Group I) 4 patients out of the 7 studied and in the mildly burned group of patients 2 of the 7 patients received treatment for clinical evidence of infection (but not septicaemia) during the study period. Of the 4 patients in Group I who received treatment, 1 was treated on Days 4 and 5, 2 on Day 5 and the other, on Day 2 after injury. Of the patients in group 2 who received treatment, 1 was treated on Day 2 and Day 6 and the other on Day 5 after injury. 2 of the patients in Group I received treatment for septicaemia but these events (Days 23 and 12) are outside the period studied. However, the data clearly show that the OR0 and the ACT were present immediately after injury had occurred and that the relative amounts of serum protein decreased progressively from Day 1 onwards so that infection appears unlikely to be the cause for the increased numbers of serum protein species appearing in the urine. The observations that mean urine protein fell towards normality at the end of the g-day period is suggestive that this proteinuria is reversible in nature. This study has only looked at serum proteins in the urine; it is possible that there is some contribution to total protein in urine from the tissues of kidney itself. The appearance of ACT and to a much lesser extent AT, at times as a twin peak, was one of the interesting features of this study. The slower electrophoretic mobility of the second protein peak is suggestive of an increased molecular weight or a radically altered charge on the protein. This phenomenon could arise either from aggregation of protein or possibly as a result of complex
208
formation between proteinase inhibitors and other possible tissue derived proteins or enzymes. Ohlsson and Akesson [ 191 have shown a complex formation between cationic protein and ACT migrating as &globulin on agarose gel electrophoresis. Although this study has been qualitative in respect of individual proteins and the increases which have been established are in some way arbitrarily defined, we believe that the changes in the urinary level of certain of these proteins particularly ACT and OR0 may constitute a simple way of assessing renal damage after bum injury. The technique of two-dimensional immunoelectrophoresis which we have used is particularly flexible in investigating the numbers and characteristics of proteins in urine, It possesses the advantage over the Mancini technique in that both the numbers and characteristics of the serum proteins present are revealed on one slide. It is unlikely that the Mancini test would reveal the presence of a second peak such as we observe with AT and ACT. In a totally quantitative analysis, which was not the object of this study, Mancini or one-dimensional immunoelectrophoresis would almost certainly be of considerable use. Furthermore the use of appropriate antiserum may, in future studies, enable us to distinguish between proteins originating in the serum and those from the tissues of the kidney itself. We hope that this type of analysis will enable us to study in greater depth the effect of bum injury on the kidney. Analysis of protein in the serum probably would not provide such a sensitive indication of kidney damage. Total losses of protein in the urine are small and, the levels of individual proteins in the serum after bum injury almost certainly reflect wide-ranging disturbances in body systems. If the pattern of protein excretion in the urine after burn injury is the result of renal stress, then we believe that examination of the excretion of protein in the first days after bum in patients receiving fluid therapy under different regimes may indicate that treatment which puts least stress on the kidney. Acknowledgements We would like to thank Mrs. Errol Spurr for her help and Mr. J.E. Laing for kind permission to study patients in the Wessex Regional Burns Centre. References 1 2 3 4 5 6 7 8 9 10 11 12 13
Rigas, D.A. and Heller. C.G. (1951) J. Clin. Invest. 30, 853-861 Doetsch, K. and Gadsden. R.H. (1973) Clin. Chem. 19,1170-1178 Doetsch, K. and Gadsden, R.H. (1975) J. S.C. Med. Assoc. 71, 83-87 Hemmingsen, L. and Shaarup, P. (1975) Stand. J. Clin. Lab. Invest. 35, 347-353 Free, A.H. and Free, H.M. (1972) Crit. Rev. Clin. Lab. Sci. 3. 481-531 Pollak, V.E. and Resee, A.J. (1972) in Maintenance of Body Protein Homeostasis, Pathophysiology (Folicb, E.D., ed.), pp. 195-214, Lippincott, Philadelphia Strober, W. and Waldman, T.A. (1974) Nephron 13. 35-66 Maunsbach. A.B. (1973) in Ultrastructure of the Proximal Tubule, Handbook of Physiology, Sect. 8, Renal physiology (Orloff, J. and Berliner, R.W., eds.). Williams and Wilkins, Baltimore Laurel& C.B. (1965) Anal. Biochem. 10, 358-361 Clarke, H.G.M. and Freeman, T. (1966) Prot. Biol. Fluids 14, 503-509 Clarke, H.G.M. and Freeman, T. (1968) Clin. Sci. 35, 403413 Davies, D.R., Spur. E.D. and Versey, J.B. (1971) Clin. Sci. 40. 411-417 Lowry. O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275
209
14
Davies, J.W.L.,
15 Jackson, 881-899 16 17
S.H.,
Schumacher, Shakespeare.
Rick&s,
O.R. and Bull, J.P. (1962)
Farmer.
A.W..
Slater,
R.J.
Clin. Sci. 23, 411423
and Dewolfe,
M.S. (1961)
Can. J. Biochem.
G. (1960) Verh. Dtsch. Ges. Inn. Med. 66, 875-878 P.G.. Levick, P.L. and Vaitheespara, R. (1978) Burns 4, 254-260
18 White, B.N., Shetlar, M.R. and Shilling, T.A. (1961) Ann. N.Y. Acad. 19 Ohlsson. K. and Akesson, U. (1976) Clin. Chim. Acta 73. 285-291
Sci. 94. 297-307
Physiol.
39,
Clinica Chimico Acta, 95 (1979) 201-209 0 Elsevier/North-Holland Biomedical Press
CCA 1026
URINE PROTEINS
E.J. COOMBES
AFTER
BURN INJURY
a,*, P.G. SHAKESPEARE
b and G.F. BATSTONE
a
a Department of Chemical Pathology, Salisbury General Infirmary, &L-bury, Wilts., SP2 7SX (U.K.) and b Burns Research Unit, Odstock Hospital, Salisbury, Wilts. (U.K.) (Received
November
31st, 1978)
Summary Two-dimensional immunoelectrophoresis was used to examine the proteins present in urine during the first week following burn injury. Of the “serum” proteins present in the urine some glycoproteins were found to be in different relative proportions from those observed in serum. In patients sustaining severe bums the amount of protein excreted was increased compared to patients with mild bums and to controls. al-Antichymotrypsin detected in the urine of patients with severe burns was at times seen as a twin peak. This altered peak was of slower electrophoretic mobility and may represent a polymer of the protein or a complex of the protein with some other, possibly tissue-derived, protein.
Introduction Every healthy individual is known to excrete protein in their urine. The amount they excrete daily may be relatively constant [l]. However, the “normal” amount of protein excreted by individuals is uncertain. Some workers have found very small amounts whilst others [2] have found as much as 80180 mg/24 h. The differences may be due to the relative sensitivities of the methods used to determine the amounts of protein [ 31, although Hemmingsen and Shaarup [4] suggest the most characteristic feature of the excretion of plasma proteins is the great dispersion of the values and the uneven distribution. Proteinuria has been studied most following renal damage and it is still regarded [ 51 as one of the most sensitive indicators of this condition. “Stress” and heavy physical exercise can cause a moderate increase in protein excretion [6]. It is now recognised that the kidney plays an important part in the catab* To whom correspondence
should be addressed.
202
olism of low molecular weight proteins [7]. Low molecular weight proteins, but not high molecular weight proteins, readily traverse the glomerular membrane and are largely reabsorbed and catabolized in tubular cells. Plasma proteins passing through the glomerular membrane constitute a large proportion of normal urine proteins, albumin being the predominant species. Mucoproteins synthesised on the outer surface of the cells lining the renal and genito-urinary tract [ 81 constitute some of the remaining protein in normal urine. In this study, the amount of protein in the urine of patients suffering from burn injury has been quantitatively and qualitatively examined. This work was undertaken to study not only the .total excretion of protein in the urine, but also to study specifically the excretion of individual proteins over a period of time following burn injury. It is hoped that this approach may shed light on possible kidney damage after bum injury and provide a method for assessing the efficacy of fluid replacement therapy in reducing the risk of renal stress or damage. Patients
and controls
14 patients (11 M, 3 F) who were admitted to the Wessex Regional Bums Centre were studied. Urine was collected in 24-h aliquots for a period up to 9 days post injury. Preservative was not added to the urines. The volume of the urine was measured, the urine was aliquoted and frozen (-20°C) until use. The urine was thawed once and concentrated ten times using a “Minicon” macrosolute concentrator (Amicon Corporation). Mean daily urine volumes for patients sustaining mild and severe bums are shown in Table II. One patient (C.K.) produced 24-h urine volumes below 0.5 1 on days 1 and 2. Patients were divided into 2 groups (mild and severe) based on the 5%area of their bums. Patients’ data are listed in Table I. 20 controls (healthy laboratory workers, age range 19-57 years) provided 24-h urine collections. These control urines were processed and concentrated in exactly the same manner as the urines from bums patients. Methods The method of two-dimensional immunoelectrophoresis used to examine the concentrated urine was basically that of Laurel1 [ 91, following developments by Clarke and Freeman [lO,ll] and using a small plate modification [12]. Precipitin bands were visualised by staining with Coomassie Blue. Antisera specific
TABLE PATIENT Group
I DATA No.
in
group
Mean
age (yrs)
Mean
Sex
% area of bum
(range)
(range) M
F
SWl?lW
I
23.1
(19-38)
6
1
53.6
Mild
7
29.4
(11-72)
5
2
17.7
(40-65) (8-36)
203
against albumin ( ALB),(Y1-antichymotrypsin (ACT), OL , -antitrypsin (AT), prealbumin (p-Alb), Gc globulin (GC), transferrin (TFN), orosomucoid (ORO) and a*-HS glycoprotein (HS) were obtained from Behringwerke AG (Hoechst Pharmaceuticals), “Dakopatts” anti whole human serum raised in rabbits was supplied by Mercia Diagnostics. Two-dimensional immunoelectrophoresis 3 ~1 of concentrated (X10) urine was electrophoresed in the first dimension in 1% agarose (8 V/cm, 45 min). The second dimension was performed in 1% agarose containing 100 ,ul of Dakopatts polyvalent antiserum as locating agent. Precipitin arcs were identified by adding specific protein antiserum to the polyvalent antiserum in the second dimension and observing if the peak was substantially reduced in size. Alternatively where addition of the antiserum caused excessively heavy background color, 2 plates were run in parallel. One plate contained specific protein antiserum and one plate the polyvalent antiserum, peak heights and mobility were then compared. Protein determination Protein was determined by the method of Lowry et al. [ 131 but using a preliminary 5% TCA precipitation of the concentrated urine. After centrifugation the precipitate was redissolved in 1 mol/l NaOH and the protein content estimated. Quality control serum (Versatol, Warner Ltd.) was used to construct standard curves for this determination. Results Twenty control urines were analysed by two-dimensional immunoelectrophoresis as described. The maximum height of an individual protein from any concentrated control urine was measured and taken to be the upper limit of the normal range. This only applied to two proteins, orosomucoid and albumin (the protein most commonly seen) because these were the only proteins to produce measurable precipitin arcs in the concentrated urine (Fig. la). Several small precipitin arcs were uncharacterised. Any protein occurring in the con-
Fig. 1. Proteins present in urines from a control and a patient with mild burns. Urine protein profile of (a) a normal control and (b) a patient with mild burns. 1. Orosomucoid; 2, albumin; 3, aI-antichymotrypsin.
204
TABLE
II
TOTAL
24-h URINE PROTEIN AND VOLUME
IN PATIENTS
WITH SEVERE
AND MILD BURNS
Urine volume expressed as likes and urine protein as mg/24 h relative to ‘Versatol’ standards. Days postburn
1 2 3 4 5 6 7 8 9
Severe bum 24-h tine
Mild burn
protein
__I~
24-h urine volume
24-h urine protein
24-h urine volume
Meall
Range
MeatI
Range
Mean
Range
Mean
Range
42 70 179 210 166 105 90 112 51
18-- 80 20-149 34-572 65-533 50-3 15 41-243 35-l 52 52-297 3565
1.12 1.09 1.44 1.36 1.19 1.66 1.34 1.47 1.11
0.18-1.9
73 35 34 30 30 32 35
18-211 2448 1558 2052 1637 1343 2047
1.87 1.78 1.27 1.72 1.98 1.35 1.22
0.7 -3.02 0.7 -3.54 0.53-2.42 0.58-2.8 0.84-3.12 1.03-1.8 0.64-1.76
0.19-1.63 0:69-2.1 0.56-2.03 0.52-l .7& X.06-2.12 1.1 -1.82 0.5 -2.26 0.92-1.4
-
-
centrated urine from burned patients was assessed relative to the arbitrarily set upper limit of normal. The mean protein excretion in 20 controls was found to be 35 mgf24 h (range 12-75) whilst the mean urine volume was 1.49 1 (range 0.64-2.3). In patients sustaining mild burns the mean protein excretion was not significantly different from controls on any day post-burn. Mean urine protein levels in the groups of patients sustaining mild and severe burns are shown in Table II. In patients with mild burns, orosomucoid was the protein most commonly elevated above the ~bitr~ly set upper limit of normal. On Day 1 80% of patients showed an increased excretion of orosomucoid. ACT could be detected in 40% of patients on Day 1 post-burn whilst pre-albumin and a2-HS glycop~tein could be detected in 20% of patients. Table III lists the urine proteins which were detected in the group of patients with mild burns. Fig. lb shows the protein profile of a patient sustaining mild bums on Day 1 postbum, ORO, ALB and ACT can be elearly seen. In patients sustaining severe bums the mean urine protein excretion increased to a peak value of 210 mg/24 h on Day 4 post-burn falling thereafter. There were significant differences (P < 0.05) between the mean protein excretion in TABLE III INCIDENCE
OF SPECIFIC PROTEINS
IN URINES OF PATIENTS
WITH MILD BURNS
Results expressed as % of patient with detectable protein above the upper limit of normdity. Protein
Orosomucoid o(I -Antichymotrypsin Pre-albumin a~-HS glycoprotein .-____--
Days post-burn 1
2
3
4
5
6
80% 40 20 20
67 50 0 0
57 14 0 0
43 14 0 0
40 20 0 0
28 0 0 0
7 20 0 0 0 _I
205
patients with severe and mild burns on Days 4 and 5 post-bum. Fig. 2 shows the protein profile of one patient with severe bums on Days 1, 3, 5.and 7 postbum. In the early phase (Day 1) after injury this patient is excreting ORO, HS, p-ALB, ALB, AT, ACT and a twin peak of ACT. By Day 7 only ACT and OR0 could be detected by this technique. In the urine of patients with severe burns OR0 and ACT could be detected in all patients on all days. HS was the next most commonly observed protein, present in a maximum of 80% of patients on Day 2. The excretion of AT, ALB, TFN p-ALB and GC were variable over the g-day study period. Increased excretion of ALB was not detected after Day 5 and precipitin arcs were very small. The urine protein profile of the group of patients with severe bums is shown in Fig. 3. One uncharacterised protein with mobility in the LYregion, was detected in several of the urines from patients with severe bums. It is interesting to observe that whilst the mean excretion in the patients with severe burns on Days 4 and 5 is still elevated, the number of serum proteins observed in urine is decreasing. This may be an indication that tissue derived proteins may be contributing to the total urine protein excretion at this period post-bum. Twin peaks of ACT were noted in all 7 patients with severe bums on at least
Urine
Pmteins
after Burn Injury ACT
OR0
“__Lh 2
4
6
a
ACT 2
10
Fig. 2. Proteins present in urine from a patient with severe burns. Urine protein profile 2. albumin; 3, oil-antichymotrypsin; (b) Day 3. (C) Day 5. and (d) Day 7, 1, Orosomucoid; and 6, pre-albumin. antichymotrypsin: 5, a-~-HS glycoprotein
on (a) Day 1, 4. altered (Y,-
Fig. 3. Urine protein profile of 7 patients with severe burns. Results expressed as 9% number of patients with greater than normal protein excretion. Patient ages and 96 areas of bum are shown. ORO. oroso_ mucoid: ACT, (~1-antichymotrypsin: ACT 2, altered antichymotrypsin: AT. oil-antitrypsin; ALB, albumin: TFN. transferrin; HS. (YZ-HS izlwoprotein; P-ALB, pre-albumin; GC, Gc globulin; AT 2. altered antitrypsin.
206
a
Fig. 4. Form of a]-antichymotrypsin in serum and urine of patients with acute tubular necrosis following burn injury. (a) serum (Day 1 post-burn) against al-antichymotrypsin antiserum and (b) concentrated urine (Day 1) against polyvalent antiserum. 3, orl-antichymotrypsin; 4. altered antichymotrypsin.
2 days post-injury. On Day 2 post-burn all those patients tested showed this twin peak effect. An example of this twin peak phenomenon is shown in Fig. 2 (a and b). The variant peak was always of slower electrophoretic mobility and was of varying size relative to the main peak. The altered peak could not be detected in any patients’ urine after Day 7. Attempts to produce this altered peak in vitro were unsuccessful; agents used were heat, pH change, urea and mercaptoethanol. Incubation of concentrated burn and control urine with normal serum also failed to produce a second peak (apart from the quantity of the second peak originally present in the burn urine). Fig. 4 (a) shows a twodimensional immunoelectrophoretogram of serum, from a patient with severe bums (95%) on Day 1 post-burn against ACT antiserum. Only one peak is observed. This patient had developed acute tubular necrosis and Fig. 4 (b) shows a two-dimensional immunoelectrophoretogram of concentrated urine from this patient on the same day. The twin ACT effect is clearly seen. On this day the patient excreted 250 mg of protein in a urine volume of 0.23 1. Discussion This study has shown that patients sustaining severe burns excrete greater amounts of protein than patients with mild burns and normal controls. In patients with severe burns the peak protein excretion was on Day 4 post-burn, falling thereafter towards normality. The pattern of individual protein excretion in patients with mild burns showed only orosomucoid as a reasonably constant feature in the early post-bum period. In the patients with severe burns the pattern of individual protein excretion was much more varied. 8 proteins could be detected of which transferrin (mol. wt. at 80 000) was the highest molecular weight. One of the surprising features was the apparent low excretion of albumin. Serum albumin falls rapidly after severe burn injury [ 141 and this lack of increased albumin excretion probably reflects the decreased serum concentration or an increased tubular reabsorption. The constant feature in the urine of patients with severe burns was the presence of orosomucoid and (Y,-antichymotrypsin in all patients on all days studied. The presence of orosomucoid may be a reflection of increased production or loss of mucoproteins produced on the
surface of cells lining the renal tract. Jackson et al. [15] have highlighted the increased excretion of acid mucoproteins after burn injury in children. The increased production of certain proteins termed the acute phase reactants [161 in the ‘early phase of trauma is well documented. ORO, ACT, AT and HS are all acute phase reactants known to increase in the serum after trauma and burn injury. It is possible that the increased appearance of these proteins in the urine reflects an increased serum production resulting in an overflow phenomenon i.e. the reabsorption capacity has been exceeded. However, this is unlikely because ACT increases to reach a maximum serum level on days 4-6 after injury (Shakespeare, P.G. and Spur-r, unpublished observations), whilst Fig. 2 clearly shows that the excretion of ACT in the urine is maximal on Day 1 and decreases thereafter. It is of interest that while ACT is invariably observed in the urine, AT was not observed in many of these patients. In serum, AT concentration is markedly elevated in the acute phase reaction. These observations suggest the possibility of a specific kidney response to burn injury involving increased excretion of ACT and OR0 by the kidney. Whether this results from different handling by the kidney after bum injury or to an actual production of these proteins in the kidney tissue remains as a problem for further investigations. Shakespeare et al. [17] have shown a relatively higher concentration of OR0 in blister fluid than in serum of bum patients, suggesting a localised tissue response to injury. The presence of elevated concentrations of glycoproteins in wound exudate has been known for many years [ 181. One possible reason for the presence of larger numbers of proteins in the urine from patients with severe bums might have been associated with different incidence of infection in the two groups. In the severely burned group of patients (Group I) 4 patients out of the 7 studied and in the mildly burned group of patients 2 of the 7 patients received treatment for clinical evidence of infection (but not septicaemia) during the study period. Of the 4 patients in Group I who received treatment, 1 was treated on Days 4 and 5, 2 on Day 5 and the other, on Day 2 after injury. Of the patients in group 2 who received treatment, 1 was treated on Day 2 and Day 6 and the other on Day 5 after injury. 2 of the patients in Group I received treatment for septicaemia but these events (Days 23 and 12) are outside the period studied. However, the data clearly show that the OR0 and the ACT were present immediately after injury had occurred and that the relative amounts of serum protein decreased progressively from Day 1 onwards so that infection appears unlikely to be the cause for the increased numbers of serum protein species appearing in the urine. The observations that mean urine protein fell towards normality at the end of the g-day period is suggestive that this proteinuria is reversible in nature. This study has only looked at serum proteins in the urine; it is possible that there is some contribution to total protein in urine from the tissues of kidney itself. The appearance of ACT and to a much lesser extent AT, at times as a twin peak, was one of the interesting features of this study. The slower electrophoretic mobility of the second protein peak is suggestive of an increased molecular weight or a radically altered charge on the protein. This phenomenon could arise either from aggregation of protein or possibly as a result of complex
208
formation between proteinase inhibitors and other possible tissue derived proteins or enzymes. Ohlsson and Akesson [ 191 have shown a complex formation between cationic protein and ACT migrating as &globulin on agarose gel electrophoresis. Although this study has been qualitative in respect of individual proteins and the increases which have been established are in some way arbitrarily defined, we believe that the changes in the urinary level of certain of these proteins particularly ACT and OR0 may constitute a simple way of assessing renal damage after bum injury. The technique of two-dimensional immunoelectrophoresis which we have used is particularly flexible in investigating the numbers and characteristics of proteins in urine, It possesses the advantage over the Mancini technique in that both the numbers and characteristics of the serum proteins present are revealed on one slide. It is unlikely that the Mancini test would reveal the presence of a second peak such as we observe with AT and ACT. In a totally quantitative analysis, which was not the object of this study, Mancini or one-dimensional immunoelectrophoresis would almost certainly be of considerable use. Furthermore the use of appropriate antiserum may, in future studies, enable us to distinguish between proteins originating in the serum and those from the tissues of the kidney itself. We hope that this type of analysis will enable us to study in greater depth the effect of bum injury on the kidney. Analysis of protein in the serum probably would not provide such a sensitive indication of kidney damage. Total losses of protein in the urine are small and, the levels of individual proteins in the serum after bum injury almost certainly reflect wide-ranging disturbances in body systems. If the pattern of protein excretion in the urine after burn injury is the result of renal stress, then we believe that examination of the excretion of protein in the first days after bum in patients receiving fluid therapy under different regimes may indicate that treatment which puts least stress on the kidney. Acknowledgements We would like to thank Mrs. Errol Spurr for her help and Mr. J.E. Laing for kind permission to study patients in the Wessex Regional Burns Centre. References 1 2 3 4 5 6 7 8 9 10 11 12 13
Rigas, D.A. and Heller. C.G. (1951) J. Clin. Invest. 30, 853-861 Doetsch, K. and Gadsden. R.H. (1973) Clin. Chem. 19,1170-1178 Doetsch, K. and Gadsden, R.H. (1975) J. S.C. Med. Assoc. 71, 83-87 Hemmingsen, L. and Shaarup, P. (1975) Stand. J. Clin. Lab. Invest. 35, 347-353 Free, A.H. and Free, H.M. (1972) Crit. Rev. Clin. Lab. Sci. 3. 481-531 Pollak, V.E. and Resee, A.J. (1972) in Maintenance of Body Protein Homeostasis, Pathophysiology (Folicb, E.D., ed.), pp. 195-214, Lippincott, Philadelphia Strober, W. and Waldman, T.A. (1974) Nephron 13. 35-66 Maunsbach. A.B. (1973) in Ultrastructure of the Proximal Tubule, Handbook of Physiology, Sect. 8, Renal physiology (Orloff, J. and Berliner, R.W., eds.). Williams and Wilkins, Baltimore Laurel& C.B. (1965) Anal. Biochem. 10, 358-361 Clarke, H.G.M. and Freeman, T. (1966) Prot. Biol. Fluids 14, 503-509 Clarke, H.G.M. and Freeman, T. (1968) Clin. Sci. 35, 403413 Davies, D.R., Spur. E.D. and Versey, J.B. (1971) Clin. Sci. 40. 411-417 Lowry. O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275
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