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Fibrinolytic activity and fibrinogen split products in exercise proteinuria
CLINICA CHIMICA ACTA
449
CCA 461 J
FIBRINOLYTIC EXERCISE
ACTIVITY
AND
FIBRINOGEN
SPLIT
PRODUCTS
IN
PROTEINURIA”
J. POORTMANS**,
K. H. LUKE,
A. ZIPURSKY
AND J. BIENBTSTOCK
From the Labovatoire de I’Effort, Univevsite’ Libre de Bruxelles, Bruxelles 5 (Belgium) and the Departments of Medicine and Pediatrics, McMaster University, Hamilton, Ontario (Canada.) (Received
May 18, 1971)
SUMMARY
Increased fibrinogen reactive material in the form of D and E fragments was identified in post-exercise urine from healthy individuals. No evidence for native fibrinogen in urine was found. Studies on 4 persons after severe exercise showed a striking rise in fibrinolytic activity with no significant change in fibrinogen, plasminogen or fibrin split product levels. Increased plasmin activity was found in 2 out of 4 exercise urines concentrated 30 times. A significant increase in urinary FSP can be the result of a physiological anism and does not necessarily reflect a pathological process.
In health,
fibrinogen
reactive
material
is detectable
in urine only after
mech-
very
high concentrationl. In some pathological states, however, increased urinary fibrinogen split products have been described2. The mechanism whereby these breakdown products appear in the urine has not been well studied, but it has been suggested that the presence of fibrinogen fragments in urine, in easily detectable amounts, reflects fibrin deposition in the glomeruli. After severe exercise, fibrinogen reactive material appears in urine as an accompaniment to the generalized increase in urinary protein excretion3y4. Since increased serum fibrinolytic activity occurs following exercise5r6 it was felt worthwhile to characterize the urinary fibrinogen reactive material, and study the fibrinolytic system of plasma and urine in patients undergoing strenuous exercise.
* This work was supported in part by the Fends National de la Recherche Scientifique, Belgium (J. P.), Ontario Heart Foundation (K.H.L. and A.Z.) and the Medical Research Council of Canada
(J.B.1. * * Visiting Professor to the Department
of Medicine, McMaster University. Clin. Chim. Acta, 3.5 (1971) 449-454
450
POORTMASS
MATERIALS
ft
al.
AKiD METHODS
The study consisted
of:
(a) the characterization
of urinary
fibrinogen
cise, and (b) the examination of fibrinolytic activity four normal adults after strenuous exercise.
split products and FSP
(FSP)
in serum
after exer-
and urine on
Materials A pool of post-exercise urine samples was collected in o.oI~/~ sodium azide from 27 healthy males within two hours of severe exercise. The pool was concentrated by negative pressure, lyophilized and stored at 4’. Control normal 24 h urine samples were collected from healthy male and female volunteers. The samples were stored at -20' after concentration. l’our unconditioned males (aged 20-35 years) ran cross country for 30 min to the point of exhaustion. Prior to, within I min of, and 60 min after exercise, venous blood samples were obtained and kept on ice: 9 ml whole blood in 1.0 ml 3.8% acid citrate for plasma and 2 ml blood in 0.04 M epsilon aminocaproic acid (EACA) for estimation of FSP. 24-h individual urine samples were collected in o.oI~/~ sodium azide the day before the exercise and adjusted immediately on voiding with a concentrated solution of EACA to a constant final molarity of 0.04 M. A single urine sample was obtained I h after exercise and divided into two volumes: half was then made 0.04 M with respect to EACA and half was left untreated. The pre-exercise urine samples were concentrated approximately IOOO times and the post-exercise urine samples and the normal urine samples were concentrated approximately 30 times. All urine specimens were handled at 4’. Methods Urine was concentrated by negative pressure as previously described7. Sephadex Gzoo gel filtration, sucrose density gradient ultracentrifugation, immunodiffusion and immunoelectrophoresis were carried out as outlined elsewherea. 350 mg of pooled lyophilized exercise urine were dissolved in 3 ml of 0.85% sodium chloride (saline) and applied to a Sephadex G200 column 2.5 x 105 cm and eluted with saline at a constant flow rate of 15 ml/h into 4 ml fractions. A protein elution curve was obtained by reading the fractions at 280 rnp in a Zeiss spectrophotometer. The distribution of fibrinogen reactive material was determined by an anti-fibrinogen antiserum9 in immunodiffusion against individual fractions. Six pools were made and each pool concentrated to one third of the original starting volume. Sucrose density gradient ultracentrifugation (10~4ooi;,) was performed on the exercise urine. The pool of concentrated normal urine was subjected to gel filtration on the same column. The plasmin resistant fibrinogen breakdown products D and E were prepared by a 24 hr digestion at room temperature of a ~“i; solution of fibrinogen grade A (Kabi, Stockholm) with plasmin (Kabi 0.6 casein units per ml). The digestion was stopped with an excess of soya bean trypsin inhibitor (Sigma Chem. Co., St. Louis). Fibrinolytic studies were carried out by the following methods fibrinogen assay by the method of Asrup et al. lo; fibrinolytic activity was expressed as the percentage C&n. ChiW.
A&Z, 3-j (1971) .#49-qjq
POST EXERCISE
451
PROTEINURIA
lysis of the fibrinogen
in the absence
of inhibitors
and the euglobulin
lysis time as
described by Biggs and McFarlanell. Plasminogen and plasmin assays were performed as suggested by the Committee on Thrombolytic Agents of the National Heart Institute12, and by the Hyland fibrin plate method13. FSP of serum and urine (collected in EACA) were studied by the hemagglutination Israels et a1.14.
inhibition
test as reported
The fibrinolytic activity of the urine samples after concentration both by the caseinolytic method and the fibrin plate technique.
by
was estimated
RESULTS
Characterization of urinary FSP Fig. I shows the protein elution curve of the pooled lyophilized exercise urine subjected to Sephadex G200 gel filtration. No fibrinogen reactive material was found in the high molecular fractions. Pool 5 contained the only fractions reacting with the antifibrinogen antiserum. This pool corresponded to molecules with mean effective molecular radii slightly greater than albumin.
TUBE
NO
Fig. I. Sephadex Gzoo elution of exercise urine. Reactivity indicated by horizontal black bars.
of fractions
with specific antisera is
When pool 5 was subjected to immunoelectrophoresis, two distinct anodal arcs were seen with the antifibrinogen antiserum, similar in mobility to fibrinogen split products D and E (Fig. 2). Interrupted groove immunoelectrophoresis experiments16 seen in the lower half of Fig. 2 confirmed the antigenic identity of the urinary fibrinogen reactive material with antigens D and E. D and E fibrinogen fragments were also identified in the original unfractionated pooled exercise urine samples. Sucrose density gradient ultracentrifugation experiments confirmed that the distribution of fibrinogen reactive molecules was confined to the lighter fractions, corresponding to proteins slightly larger than albumin. No fibrinogen reactive material was seen in any fractions obtained from gel filtration of the pooled normal urine. Fibrinolytic activity and FSP in serum and urine after exercise Table I summarizes the data concerning fibrinolysis in blood. The fibrinogen Clin. Chim. Acta, 35 (1971) 449-454
452
POOKTMAh’S et ak.
Fig. L. Imnlunoclectrophoresis in agar demonstrating relationships of exercise urine with plasmin digests of fibrino,qcn. A. : native fibrinogen; B.: D and E fibrinogen split product<: C.: cscrcise urine (pool 3). Antifibrinogen antiserum was placed in all troughs. These patterns show a complete reaction of identity between fragments D and E and fibrinojiell-related substances of cscrcise urine.
levels showed no significant change, however, the fibrinolytic activity was strikingly increased and the euglobulin lysis time was shortened. Plasminogen and FSP levels were within the normal range both before and after exercise. ISI? were demonstrated both by the llemagglutination inhibition and immunodiffusion techniques in each of the four samples of urine collected one hour post-exercise and concentrated 30 times. FSP were also detected in the pre-exercise urine samples but only after concentration 1000 times, indicating a quantitative increase in FSP excretion after exercise. No differences were observed beteen EACA tested and untested urine samples. Proteolytic activity of urine Increased plasmin activity was detected (0.75 and 1.0 CTA units) in z of 4 samples of post-exercise urine concentrated 30 times. The 4 normal urine samples (concentrated 30 times) also had proteolytic activity, although at a lower range of activity (0.23; 0.2; 0.3 and 0.38 CTA units respectively). A control experiment was performed in which a known amount of plasmin was added to a 24 h sample of normal urine which was then concentrated IOOO times. Full plasmin reactivity was recovered indicating no loss of plasmin during the experimental procedure and the lack of a significant plasmin inhibitor in normal urine.
POST EXERCISE
Subjects
I
Tests ~~ Flbrinogcn (1% 90) Euglobulin lysis time (min) Fibrinolysis (“0) Fibnn split products (H-41 titer) Plasminogen (CTA units) Plasmin (CT.4 untis) * BD
PROTEINURIA
2
Post
Pve ~~ 294
I,
60’
3’7
30’
‘3.5
IO
I2
IO0
1 :J
1:X
r.S_+ ND*
3.’ ND
453
70
Pre
272
>180
IO
I:L 3.1 ND
3
Post 60’
255
255
20
0
1:4 2.7 ND
II
I20
1:1 3.Oj
XD
Pre
294
>I80
0
IO0
I:+ 2.9X 0.24
4
Post 60’
255
26-t
2.5
0
2.7
I:.+ 3.0 ND
Pre
35-t
Post ~~. II
60’
3o*
313
>180
I20
IO0
1:.+
ND
I'
0
I:4 2.8 0.12
25 0
1:.f
IO0
1:4
2.7 XD
2.8
XD
-
I20
0
1:4 3.1 0.36
= not detected.
DISCUSSION
Fibrinogen has been reported to be present in trace amounts in normal urine concentrated 2000 to 3000 times ly16. It has been assumed that this is in the form of fibrinogen breakdown productslG. Fibrinogen reactive material has also been previously reported by Poortmans in exercise urine without further characterization4. We have shown in the present study that the fibrinogen reactive material found in exercise urine was made up of molecules indistinguishable antigenically or on the basis of molecular size, from the final degradation products D and E produced by digestion of the native fibrinogen molecule with plasmin. No evidence for native fibrinogen or X and Y breakdown products” was found. We have confirmed that normal urine concentrated IOOO times contained fibrinogen reactive components, and that in four individuals, exercise urine concentrated only 30 times contained detectable fibrinogen reactive material. Increased fibrinolytic activity was shown in serum. These results for the most part have confirmed previous work. Das et al.l* showed a small increase in serum FSP from 3.3 to 6.7 ,ug/ml following exercise. On the other hand we were unable to demonstrate a rise in serum FSP. One explanation to this discrepancy could be the difference in sensitivity of the techniques used. h possible mechanism for the appearance of FSP in normal highly concentrated urine and the increase of these products in exercise proteinuria is that the small D and E split products of fibrinogen are cleared by the kidney into the urine. During exercise there may be higher renal clearance of these products following increased pre-renal breakdown. The lack of rise in serum FSP in our exercised subjects could be explained by such an increased clearance as mentioned previously for some native plasma proteins (ref. 19). In the characterization of fibrinogen reactive material in the post-exercise pooled urine by gel filtration only the plasmin resistant end products D and E were identified. In the 4 normal urine samples fibrinolytic activity could be demonstrated. Increased fibrinolytic activity occurred in 2 out of 4 post-exercise urine samples. Cli?Z.Chim.
<4&a, 35
(1971)
.+.&9-1j-1_
POORT11ANS
454
et al.
Although increased fibrinolytic activity was not seen in all samples, post-renal degradation of native fibrinogen or its larger earlier breakdown products could have taken place. Therefore, the urinary fibrinolytic activity might be of significance in relation to the type of fibrin split products seen in normal or exercise urine. Since hypercoagulability occurs during severe exercise20, increased fibrinolytic activity might be regarded as a physiological compensatory mechanism. Others have measured FSP in serum and urine as an index of intrarenal disease or fibrin deposition in the kidney “; however such interpretations should perhaps be viewed with caution since a physiological process such as exercise can cause a rise in split products
in the urine.
ACKNOWLEDGMENTS
VVe would like to give our appreciation zyniak for their excellent technical assistance
to Miss M. Johnson in this study.
and Mrs. V. Wawr-
REFERENCES I 2 3 4 3 6 7
8 9 IO II
12
13
I. BERGGARD, Clin. Chim. Acta. 6 (1961) 473. W. E. BRAUIV AND J. P. MERRILL, Neze, Engl. J. Med., 278 (1968) 1366. J, I'OORTMANS AND R. W. JEANLOZ, J. Clin. Invest., 47 (1968) 386. J. R. POORTMANS, Ann. Sot. Sci. Med. Nat. Brux.. 17 (1964) 89. J. D. BILLIMORIA, J. DRYSDALE, D. C. 0. JAMES AND N. F. MACLAGEN, Lancet, ii (1959) 471. 1. D. CASH AND D. G. WOODFIELD, Nature, 215 (1967) 628. j. BIENENSTOCK, J. Immunol., IOO (1968) 280.. J. BIENENSTOCK AND T. B. TOMASI, J. C&n. Invest., 47 (1968) 1162. H. C. FERREIRA AND L. G. MURAT, Brit. J. Haemat.. 9 (1963) 299. T. ASTRUP, P. BRAKMAN ANU U. NISSEN, &and. J. C&z. Lab. Invest., 17 (1965) 57. R. BIGGS AND R. G. MCFARLANE, Human Blood Coagulation and Its Disorders, Third Edition, Blackwell, Oxford, 1962. A. J. JOHNSON, D. L. KLINE AND N. ALKJAERSIG, Thromb. Diath. Haemorrh., 21 (1969) 239. A. B. BISHOP, H. EKERT, G. S. GILCHRIST AND Z. SHANBROM. Thromb. Diath. Haemovrh., 23
(1970) 202. 14 E. D. ISRAELS, H. RAYNER, L. G. ISRAELS AXD -4. ZIPURSKY, J. Lab. Clin. Med., 71 (1968) 333. 15 E. F. OSSERMAN, J. Immunol., 84 (1960) 93. 16 H. E. SCHULTZE AND J. F. HEREMANS, Molecular Biology of Human Proteins. 1’01. I, Elsevier Publishing Company, New York, 1966, p. 701. 17 V. J. MARDER AND N. R. SHULMAN, J. Biol. Chem., 244 (1969) 211. 18 P. C. DAS, A. G. 2. ALLAN, D. G. WOODFIELD AND J. D. CASH, Brit. Med. J., 4 (1967) 718. 19 J, POORTMANS, in MANUEL et al. (Eds), Proteins in Normal and Pathological Urines, Karger ‘970, P. 229. 20 J. J. BURT, C. S. BLYTH AND H. A. RIERSON, J. Sport Med., 4 (1964) Clin. Chim. Acta, 35 (1971) 449-454
z.13
449
CCA 461 J
FIBRINOLYTIC EXERCISE
ACTIVITY
AND
FIBRINOGEN
SPLIT
PRODUCTS
IN
PROTEINURIA”
J. POORTMANS**,
K. H. LUKE,
A. ZIPURSKY
AND J. BIENBTSTOCK
From the Labovatoire de I’Effort, Univevsite’ Libre de Bruxelles, Bruxelles 5 (Belgium) and the Departments of Medicine and Pediatrics, McMaster University, Hamilton, Ontario (Canada.) (Received
May 18, 1971)
SUMMARY
Increased fibrinogen reactive material in the form of D and E fragments was identified in post-exercise urine from healthy individuals. No evidence for native fibrinogen in urine was found. Studies on 4 persons after severe exercise showed a striking rise in fibrinolytic activity with no significant change in fibrinogen, plasminogen or fibrin split product levels. Increased plasmin activity was found in 2 out of 4 exercise urines concentrated 30 times. A significant increase in urinary FSP can be the result of a physiological anism and does not necessarily reflect a pathological process.
In health,
fibrinogen
reactive
material
is detectable
in urine only after
mech-
very
high concentrationl. In some pathological states, however, increased urinary fibrinogen split products have been described2. The mechanism whereby these breakdown products appear in the urine has not been well studied, but it has been suggested that the presence of fibrinogen fragments in urine, in easily detectable amounts, reflects fibrin deposition in the glomeruli. After severe exercise, fibrinogen reactive material appears in urine as an accompaniment to the generalized increase in urinary protein excretion3y4. Since increased serum fibrinolytic activity occurs following exercise5r6 it was felt worthwhile to characterize the urinary fibrinogen reactive material, and study the fibrinolytic system of plasma and urine in patients undergoing strenuous exercise.
* This work was supported in part by the Fends National de la Recherche Scientifique, Belgium (J. P.), Ontario Heart Foundation (K.H.L. and A.Z.) and the Medical Research Council of Canada
(J.B.1. * * Visiting Professor to the Department
of Medicine, McMaster University. Clin. Chim. Acta, 3.5 (1971) 449-454
450
POORTMASS
MATERIALS
ft
al.
AKiD METHODS
The study consisted
of:
(a) the characterization
of urinary
fibrinogen
cise, and (b) the examination of fibrinolytic activity four normal adults after strenuous exercise.
split products and FSP
(FSP)
in serum
after exer-
and urine on
Materials A pool of post-exercise urine samples was collected in o.oI~/~ sodium azide from 27 healthy males within two hours of severe exercise. The pool was concentrated by negative pressure, lyophilized and stored at 4’. Control normal 24 h urine samples were collected from healthy male and female volunteers. The samples were stored at -20' after concentration. l’our unconditioned males (aged 20-35 years) ran cross country for 30 min to the point of exhaustion. Prior to, within I min of, and 60 min after exercise, venous blood samples were obtained and kept on ice: 9 ml whole blood in 1.0 ml 3.8% acid citrate for plasma and 2 ml blood in 0.04 M epsilon aminocaproic acid (EACA) for estimation of FSP. 24-h individual urine samples were collected in o.oI~/~ sodium azide the day before the exercise and adjusted immediately on voiding with a concentrated solution of EACA to a constant final molarity of 0.04 M. A single urine sample was obtained I h after exercise and divided into two volumes: half was then made 0.04 M with respect to EACA and half was left untreated. The pre-exercise urine samples were concentrated approximately IOOO times and the post-exercise urine samples and the normal urine samples were concentrated approximately 30 times. All urine specimens were handled at 4’. Methods Urine was concentrated by negative pressure as previously described7. Sephadex Gzoo gel filtration, sucrose density gradient ultracentrifugation, immunodiffusion and immunoelectrophoresis were carried out as outlined elsewherea. 350 mg of pooled lyophilized exercise urine were dissolved in 3 ml of 0.85% sodium chloride (saline) and applied to a Sephadex G200 column 2.5 x 105 cm and eluted with saline at a constant flow rate of 15 ml/h into 4 ml fractions. A protein elution curve was obtained by reading the fractions at 280 rnp in a Zeiss spectrophotometer. The distribution of fibrinogen reactive material was determined by an anti-fibrinogen antiserum9 in immunodiffusion against individual fractions. Six pools were made and each pool concentrated to one third of the original starting volume. Sucrose density gradient ultracentrifugation (10~4ooi;,) was performed on the exercise urine. The pool of concentrated normal urine was subjected to gel filtration on the same column. The plasmin resistant fibrinogen breakdown products D and E were prepared by a 24 hr digestion at room temperature of a ~“i; solution of fibrinogen grade A (Kabi, Stockholm) with plasmin (Kabi 0.6 casein units per ml). The digestion was stopped with an excess of soya bean trypsin inhibitor (Sigma Chem. Co., St. Louis). Fibrinolytic studies were carried out by the following methods fibrinogen assay by the method of Asrup et al. lo; fibrinolytic activity was expressed as the percentage C&n. ChiW.
A&Z, 3-j (1971) .#49-qjq
POST EXERCISE
451
PROTEINURIA
lysis of the fibrinogen
in the absence
of inhibitors
and the euglobulin
lysis time as
described by Biggs and McFarlanell. Plasminogen and plasmin assays were performed as suggested by the Committee on Thrombolytic Agents of the National Heart Institute12, and by the Hyland fibrin plate method13. FSP of serum and urine (collected in EACA) were studied by the hemagglutination Israels et a1.14.
inhibition
test as reported
The fibrinolytic activity of the urine samples after concentration both by the caseinolytic method and the fibrin plate technique.
by
was estimated
RESULTS
Characterization of urinary FSP Fig. I shows the protein elution curve of the pooled lyophilized exercise urine subjected to Sephadex G200 gel filtration. No fibrinogen reactive material was found in the high molecular fractions. Pool 5 contained the only fractions reacting with the antifibrinogen antiserum. This pool corresponded to molecules with mean effective molecular radii slightly greater than albumin.
TUBE
NO
Fig. I. Sephadex Gzoo elution of exercise urine. Reactivity indicated by horizontal black bars.
of fractions
with specific antisera is
When pool 5 was subjected to immunoelectrophoresis, two distinct anodal arcs were seen with the antifibrinogen antiserum, similar in mobility to fibrinogen split products D and E (Fig. 2). Interrupted groove immunoelectrophoresis experiments16 seen in the lower half of Fig. 2 confirmed the antigenic identity of the urinary fibrinogen reactive material with antigens D and E. D and E fibrinogen fragments were also identified in the original unfractionated pooled exercise urine samples. Sucrose density gradient ultracentrifugation experiments confirmed that the distribution of fibrinogen reactive molecules was confined to the lighter fractions, corresponding to proteins slightly larger than albumin. No fibrinogen reactive material was seen in any fractions obtained from gel filtration of the pooled normal urine. Fibrinolytic activity and FSP in serum and urine after exercise Table I summarizes the data concerning fibrinolysis in blood. The fibrinogen Clin. Chim. Acta, 35 (1971) 449-454
452
POOKTMAh’S et ak.
Fig. L. Imnlunoclectrophoresis in agar demonstrating relationships of exercise urine with plasmin digests of fibrino,qcn. A. : native fibrinogen; B.: D and E fibrinogen split product<: C.: cscrcise urine (pool 3). Antifibrinogen antiserum was placed in all troughs. These patterns show a complete reaction of identity between fragments D and E and fibrinojiell-related substances of cscrcise urine.
levels showed no significant change, however, the fibrinolytic activity was strikingly increased and the euglobulin lysis time was shortened. Plasminogen and FSP levels were within the normal range both before and after exercise. ISI? were demonstrated both by the llemagglutination inhibition and immunodiffusion techniques in each of the four samples of urine collected one hour post-exercise and concentrated 30 times. FSP were also detected in the pre-exercise urine samples but only after concentration 1000 times, indicating a quantitative increase in FSP excretion after exercise. No differences were observed beteen EACA tested and untested urine samples. Proteolytic activity of urine Increased plasmin activity was detected (0.75 and 1.0 CTA units) in z of 4 samples of post-exercise urine concentrated 30 times. The 4 normal urine samples (concentrated 30 times) also had proteolytic activity, although at a lower range of activity (0.23; 0.2; 0.3 and 0.38 CTA units respectively). A control experiment was performed in which a known amount of plasmin was added to a 24 h sample of normal urine which was then concentrated IOOO times. Full plasmin reactivity was recovered indicating no loss of plasmin during the experimental procedure and the lack of a significant plasmin inhibitor in normal urine.
POST EXERCISE
Subjects
I
Tests ~~ Flbrinogcn (1% 90) Euglobulin lysis time (min) Fibrinolysis (“0) Fibnn split products (H-41 titer) Plasminogen (CTA units) Plasmin (CT.4 untis) * BD
PROTEINURIA
2
Post
Pve ~~ 294
I,
60’
3’7
30’
‘3.5
IO
I2
IO0
1 :J
1:X
r.S_+ ND*
3.’ ND
453
70
Pre
272
>180
IO
I:L 3.1 ND
3
Post 60’
255
255
20
0
1:4 2.7 ND
II
I20
1:1 3.Oj
XD
Pre
294
>I80
0
IO0
I:+ 2.9X 0.24
4
Post 60’
255
26-t
2.5
0
2.7
I:.+ 3.0 ND
Pre
35-t
Post ~~. II
60’
3o*
313
>180
I20
IO0
1:.+
ND
I'
0
I:4 2.8 0.12
25 0
1:.f
IO0
1:4
2.7 XD
2.8
XD
-
I20
0
1:4 3.1 0.36
= not detected.
DISCUSSION
Fibrinogen has been reported to be present in trace amounts in normal urine concentrated 2000 to 3000 times ly16. It has been assumed that this is in the form of fibrinogen breakdown productslG. Fibrinogen reactive material has also been previously reported by Poortmans in exercise urine without further characterization4. We have shown in the present study that the fibrinogen reactive material found in exercise urine was made up of molecules indistinguishable antigenically or on the basis of molecular size, from the final degradation products D and E produced by digestion of the native fibrinogen molecule with plasmin. No evidence for native fibrinogen or X and Y breakdown products” was found. We have confirmed that normal urine concentrated IOOO times contained fibrinogen reactive components, and that in four individuals, exercise urine concentrated only 30 times contained detectable fibrinogen reactive material. Increased fibrinolytic activity was shown in serum. These results for the most part have confirmed previous work. Das et al.l* showed a small increase in serum FSP from 3.3 to 6.7 ,ug/ml following exercise. On the other hand we were unable to demonstrate a rise in serum FSP. One explanation to this discrepancy could be the difference in sensitivity of the techniques used. h possible mechanism for the appearance of FSP in normal highly concentrated urine and the increase of these products in exercise proteinuria is that the small D and E split products of fibrinogen are cleared by the kidney into the urine. During exercise there may be higher renal clearance of these products following increased pre-renal breakdown. The lack of rise in serum FSP in our exercised subjects could be explained by such an increased clearance as mentioned previously for some native plasma proteins (ref. 19). In the characterization of fibrinogen reactive material in the post-exercise pooled urine by gel filtration only the plasmin resistant end products D and E were identified. In the 4 normal urine samples fibrinolytic activity could be demonstrated. Increased fibrinolytic activity occurred in 2 out of 4 post-exercise urine samples. Cli?Z.Chim.
<4&a, 35
(1971)
.+.&9-1j-1_
POORT11ANS
454
et al.
Although increased fibrinolytic activity was not seen in all samples, post-renal degradation of native fibrinogen or its larger earlier breakdown products could have taken place. Therefore, the urinary fibrinolytic activity might be of significance in relation to the type of fibrin split products seen in normal or exercise urine. Since hypercoagulability occurs during severe exercise20, increased fibrinolytic activity might be regarded as a physiological compensatory mechanism. Others have measured FSP in serum and urine as an index of intrarenal disease or fibrin deposition in the kidney “; however such interpretations should perhaps be viewed with caution since a physiological process such as exercise can cause a rise in split products
in the urine.
ACKNOWLEDGMENTS
VVe would like to give our appreciation zyniak for their excellent technical assistance
to Miss M. Johnson in this study.
and Mrs. V. Wawr-
REFERENCES I 2 3 4 3 6 7
8 9 IO II
12
13
I. BERGGARD, Clin. Chim. Acta. 6 (1961) 473. W. E. BRAUIV AND J. P. MERRILL, Neze, Engl. J. Med., 278 (1968) 1366. J, I'OORTMANS AND R. W. JEANLOZ, J. Clin. Invest., 47 (1968) 386. J. R. POORTMANS, Ann. Sot. Sci. Med. Nat. Brux.. 17 (1964) 89. J. D. BILLIMORIA, J. DRYSDALE, D. C. 0. JAMES AND N. F. MACLAGEN, Lancet, ii (1959) 471. 1. D. CASH AND D. G. WOODFIELD, Nature, 215 (1967) 628. j. BIENENSTOCK, J. Immunol., IOO (1968) 280.. J. BIENENSTOCK AND T. B. TOMASI, J. C&n. Invest., 47 (1968) 1162. H. C. FERREIRA AND L. G. MURAT, Brit. J. Haemat.. 9 (1963) 299. T. ASTRUP, P. BRAKMAN ANU U. NISSEN, &and. J. C&z. Lab. Invest., 17 (1965) 57. R. BIGGS AND R. G. MCFARLANE, Human Blood Coagulation and Its Disorders, Third Edition, Blackwell, Oxford, 1962. A. J. JOHNSON, D. L. KLINE AND N. ALKJAERSIG, Thromb. Diath. Haemorrh., 21 (1969) 239. A. B. BISHOP, H. EKERT, G. S. GILCHRIST AND Z. SHANBROM. Thromb. Diath. Haemovrh., 23
(1970) 202. 14 E. D. ISRAELS, H. RAYNER, L. G. ISRAELS AXD -4. ZIPURSKY, J. Lab. Clin. Med., 71 (1968) 333. 15 E. F. OSSERMAN, J. Immunol., 84 (1960) 93. 16 H. E. SCHULTZE AND J. F. HEREMANS, Molecular Biology of Human Proteins. 1’01. I, Elsevier Publishing Company, New York, 1966, p. 701. 17 V. J. MARDER AND N. R. SHULMAN, J. Biol. Chem., 244 (1969) 211. 18 P. C. DAS, A. G. 2. ALLAN, D. G. WOODFIELD AND J. D. CASH, Brit. Med. J., 4 (1967) 718. 19 J, POORTMANS, in MANUEL et al. (Eds), Proteins in Normal and Pathological Urines, Karger ‘970, P. 229. 20 J. J. BURT, C. S. BLYTH AND H. A. RIERSON, J. Sport Med., 4 (1964) Clin. Chim. Acta, 35 (1971) 449-454
z.13