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Epidermal growth factor in plasma, serum and urine before and after prolonged exercise
Regulatory Peptides, 21 (1988) 197-203 Elsevier
197
RPT 00703
Epidermal growth factor in plasma, serum and urine before and after prolonged exercise L. K o n r a d s e n a n d E. N e x o Department of Clinical Chemistry, Central Hospital, Hillerod (Denmark)
(Received 15 October 1987; revised version received and accepted 28 December 1987)
Summary The substance concentration of epidermal growth factor immunoreactivity ( E G F IR) and certain other components were studied in plasma, serum and urine from 25 individuals before and after a 2 h cross-country run. The substance concentration of plasma E G F I R increased from a median of 0.10 nM (range 0.04-0.26 nM) to a median of 0.16 nM (range 0.10~.36 nM) after 2 h of exercise, while serum E G F showed no change. The values obtained for B-platelets were a median of 192 × 109/litre (range 109-282 × 109/litre) before the run, and a median of 265 × 109/litre (range 216-387 x 109/litre) after the run. N o correlation was observed between the values obtained for B-platelets and the values for plasma or serum E G F IR. The substance concentration of E G F I R in urine increased from a median of 3.2 nM (range 0.5-7.7 nM) to a median of 7.0 nM (range 1.5-15.7 nM) after the run. Expressed relative to the output of carbamide the output of urinary E G F I R increased with a median factor of 2 following the run. Expressed relative to the output of creatinine no increase was observed. Adrenergic stimulation; Epidermal growth factor; Exercise; Platelet
Introduction Epidermal growth factor (EGF) is a mitogenic polypeptide able to stimulate cellular growth and differentiation and to inhibit gastric acid secretion [1,2].
Correspondence: E. Nexo, Department of Clinical Chemistry, Central Hospital, DK-3400 Hillerod, Denmark.
0167-0115/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
198 In animals E G F is produced in a number of tissues including the salivary gland and the kidney [1,3], and it is secreted from these tissues mainly in an exocrine manner. In rats and mice adrenergic stimulation either in the form of aggressive behaviour or in the form of ~- or/~-adrenergic agonists result in an increase in the salivary and/or the urinary output of E G F [3-6]. The origin of circulating E G F is unclear. In mice the salivary glands act to a minor degree as an endocrine organ that delivers E G F to the circulation upon adrenergic stimulation [4,6]. This has not been observed for rats [3,5]. Recent work has shown the substance concentration of E G F in human serum to be considerably higher than in plasma. It is believed that E G F delivered upon coagulation of the blood is derived from the platelets [7,8]. Human E G F is produced in the same tissues as in animals, and because of that we found it of interest to study whether the release of E G F in humans is stimulated by adrenergic agonists. In a previous paper we report the salivary concentration of E G F to increase with a median factor of 1.8 after prolonged submaximal exercise [9], a condition known to involve an increase in both ~- and//-adrenergic agonists
[lO]. In the present paper we report the substance concentrations of both plasma and urinary E G F to increase after prolonged exercise. The increase is discussed in the light of other biochemical findings in the same individuals.
Materials and Methods
Participating individuals. 25 individuals (4 females and 21 males) aged 17-49 years participating in a 2 h cross-country run performed in cloudy weather at a temperature around 10°C. All gave informed consent. The participants had been active in crosscountry racing for more than 5 years. After the race they all claimed to be maximally exercised. During the run the participants had free access to water. Collection of biological samples. Urine was voided prior to the run and within the first hour after completion of the run. Blood was drawn prior to the run and within 2 min after arrival at the finish. Plasma was separated from blood anticoagulated with E D T A within 2 h after collection (1800 g, 10 min). Serum was obtained from blood collected without added anticoagulans. The blood was allowed to clot for 2 h at room temperature and at 4°C overnight. All samples were kept at - 2 0 ° C until analyzed. Quantification ofEGF. E G F was quantified as previously described [9,11]. In brief, urinary E G F purified by immunoaffinity chromatography and pure as judged by amino terminal analysis was employed for production of the antibody (serum 4554-0186, final dilution 1:30.000), the iodinated probe and the calibrators. The sensitivity of the assay was 0.04 nM. The intra-assay precision was 5.5% (n = 36, = 1.7 nM) and the interassay precision was 8.5% (n = 60, ~ = 1.65 nM, samples were run over a period of 9 months). Urinary samples were prediluted 1:4 with assay buffer, plasma and serum were analyzed undiluted. Non-specific binding (less than 10%) was substracted prior to analysis of the data.
199
Other biochemical parameters. Urine and serum creatinine and carbamide were analyzed on a SMAC automatic analyzer (Technicon, Denmark) employing routine methods. B-hemoglobin, B-platelets and B-hematocrit were analyzed on a Coulter S + 4 (Coulter Electronics, England) according to the manufacturer. Statistical methods. Pratt's test for paired differences was employed. P-values of less than 0.05 were considered significant. Correlations were examined using a Spearmann's rho test.
Results No difference was observed between serum E G F IR before and after a 2 h cross-country run, while plasma E G F IR increased following exercise (Table I). Since circulatory E G F may be derived from the platelets we compared results obtained for B-platelets with results obtained for E G F IR. The concentration for platelets increased after exercise (Table I), but no correlation was found between the platelet count or the platelet count increase on one hand, and the substance concentration of E G F IR in plasma or in serum on the other (data not shown). To evaluate a possible dehydration of the runners, B-hemoglobin and B-hematocrit were examined, but no difference in values obtained before and after the run was found (Table I). We compared the substance concentration of a number of components in plasma or serum and in urine. While the substance concentration of both E G F IR, creatinine and carbamide increased in the circulation after exercise, the urinary concentration increased only for E G F IR and creatinine (Table II, Fig. 2). The urinary concentration of E G F IR increased with a median factor of 2.2. Relative to the urinary concentration of creatinine no increase was observed, but relative to the urinary concentration of carbamide the urinary concentration of E G F IR increased with a median factor of 2.0 after exercise. The results obtained when comparing the clearance
TABLE I Substance concentration of E G F IR and other components in h u m a n plasma and serum before and after prolonged exercise Before
P - E G F (nM) S-EGF (nM) B-platelets (109/litre) B-hemoglobin (mM) B-hematocrit (fraction)
After
Median
Range
Median
Range
0.10 0.23 192 9.6 0.46
0.04-0.26 0.16-0.46 192-282 8.9 -10.7 0.414).50
0.16 0.25 265 9.7 0.46
0.10-0.36 0.13-0.50 216~387 8.5 -10.7 0.40-0.51
S n.s. S n.s. n.s.
S = significant at P < 0.05 using Pratts test, for comparison of results obtained after exercise with values obtained prior to exercise; n.s. = non-significant: n = 25.
200
A
B
30.
.30
20,
'20
10.
-10
-5.0
5.0. z o
•
~3.0,
"7
.3.0g ~2.0~
~ 2.0,
1.0
1,0,
:
T
"-.. -0.5
0.5,
P-EGF S-CREATS-CARB
EGF
CREAT CA'RB EG'F/ EG'F/ CREAT CARB URINE
PLASMA
Fig. 1. Comparison of values for E G F IR, creatinine and carbamide obtained on serum/plasma or urine before and after prolonged exercise. The figure shows results obtained on blood samples (A) or urine samples (B) collected from 25 individuals after 2 h cross-country running divided by the results obtained prior to the run. The horizontal line indicates the median. Creat., Creatinine; Carb., Carbamide; EGF/Creat., excretion of E G F relative to excretion of creatinine: EGF/Carb., excretion of E G F relative to excretion of carbamide. T A B L E II The substance concentration of E G F IR and other components in h u m a n urine and plasma/serum before and after prolonged exercise Urine Median E G F (nM) Before After Creatinine (pM) Before After Carbamide (raM) Before After n 1 2 3
3.2 7.0
Plasma/serum Range
Median
0.5 1.5~
9920 20760
3480 6200
250 260
58 56
7.7 15.73
-15680 -615003 398 370
0.10 0.16 91 128 5.4 6.4
= 25. Plasma. Serum. p < 0.05 (Pratt's test) as compared to values obtained before exercise.
Range
0.04O. lO79 105
0.26 l 0.361'3
-1272 -1482,3
3.7 4.7 -
6.82 8.3 z
201 of the different substances were similar to the results obtained for the substance concentration in urine.
Discussion
The present paper represents the first attempt to study the substance concentration of human EGF in the circulation and in urine after a stimulation known to increase the output of EGF in animals. The results show that both plasma and urinary EGF IR increase following exercise, while serum EGF IR remains unchanged. During competitive running the runner performs at about 80-90% of his maximal aerobic capacity [12]. At this rate of activity there is both an increased activity in the sympathetic nervous system with a raised serum concentration of norepinephrine [10], and an increased activity of the suprarenal glands resulting in an increased level of blood epinephrine [13]. Compared to studies in animals the release of EGF upon the adrenergic stimulation of exercise is minimal. Following aggressive behaviour circulatory EGF increases with a factor of 100 in mice [6] and following adrenergic stimulation the urinary output of EGF, relative to the output of creatinine, increases with a factor of 2 in rats [3]~ A number of factors need to be taken into consideration when interpreting the results for plasma and serum EGF IR. The origin and metabolism of plasma EGF is unknown. Animal studies have indicated the liver and the kidney to be of major importance for the clearance of EGF [14]. Under hard physical activity blood circulation to the splanchnicus area is greatly reduced. Figures concerning the liver circulation do not exist, but results from renal circulation [15] show the renal artery flow to be less than half the normal flow. It is thus likely that a decreased metabolism of EGF plays a role in the increase of plasma EGF IR after exercise. It is believed that the EGF recovered in serum after coagulation of the blood sample is derived from the platelets [7,8,16]. After exercise the platelet count increases, while serum EGF IR remains unchanged. If EGF is released from the platelets in a simple manner one would expect correlation to exist between the platelet count, and the amount of EGF recovered. Our results therefore suggest a more complex pattern for the release of EGF upon coagulation of the blood. Other potential points of interest are the factors involved in coagulation and fibrinolysis, because many of these factors contain EGF-like domains [17]. At present it is not known whether any of these domains are released in a form recognized by the radioimmunoassay for EGF. Urinary EGF may be derived from the circulation or it may originate from the kidney. The latter possibility is supported by its immunohistochemical localization in the distal tubular cells [20], and by the recent isolation of cDNA for the EGF precursor from the human kidney [21]. The results obtained in the present paper support an adrenergic release of EGF from the kidney as previously observed in rats [3]. Both the substance concentration of EGF and the concentration relative to the concentration of carbamide increase in urine after exercise. The output of EGF in urine parallel the output of creatinine. Since the production and thereby the urinary
202
output of creatinine is increased after exercise [22] this supports an increase in urinary output of EGF after exercise. At the same time the observation suggests some kind of relation between the renal output of creatinine and EGF. A similar relation has previously been reported in a study that compared the excretion of creatinine and EGF as a function of age [19]. In conclusion the present paper shows the substance concentration of EGF IR to increase in plasma and urine following prolonged exercise in humans. It further questions whether there is a simple relation between the amount of EGF recovered upon coagulation of the blood and the platelet counts.
Acknowledgements The technical assistance of Marianne Rye Hansen is warmly acknowledged. The study was supported by The Danish Cancer Society (86-034) and by a grant from The Research Council for Sports Medicine.
References I Hollenberg, M.D. and Armstrong, G.D., Epidermal growth factor urogastrone and its receptor. In B.I. Posner (Ed.), Polypeptide Hormone Receptors, Dekker, New York, 1985, pp. 201-226. 2 Gregory, H., In vivo aspects of urogastrone-epidermal growth factor, J. Cell Sci., Suppl. 3 (1985) 11-17. 3 Olsen, P.S., Kirkegaard, P., Poulsen, S.S. and Nexo, E., Adrenergic effects on renal secretion of epidermal growth factor in the rat, Regul. Pept., 11 (1985) 17-25. 4 Nexo, E., Olsen, P.S. and Poulsen, K., Exocrine and endocrine secretion of renin and epidermal growth factor from the mouse submandibular glands, Regul. Pept., 8 (1984) 327-334. 50lsen, P.S., Kirkegaard, P., Poulsen, S.S. and Nexo, E., Adrenergic effects on exocrine secretion of rat submandibular epidermal growth factor, Gut, 25 (1984) 1234-1240. 6 Nexo, E., Hollenberg, M.D. and Bing, J., Aggressive behaviour increases both plasma epidermal growth factor and renin, Acta Physiol. Scand., I I I (1981) 367-371. 7 Savage, A.P., Chatterjee, V.K., Gregory, H. and Bloom, S.R., Epidermal growth factor in blood, Regul. Pept., 16 (1986) 199-206. 8 0 k a , Y. and Orth, D.N., Human plasma epidermal growth factor/~-urogastrone is associated with blood platelets, J. Clin. Invest., 72 (1983) 249-259. 9 Nexo, E., Hansen, M.R. and Konradsen, L., Human saliva epidermal growth factor, haptocorrin and amylase before and after prolonged exercise, Scand. J. Clin. Lab. Invest., 48 (1988) 267-271. 10 Christensen, N.J., Galbo, H., Hansen, J.F., Hesse, B., Richter, E.A. and Trap-Jensen, J., Catecholamines and exercise, Diabetes, 28 (1979) 58-62. 11 Olsen, P.S. and Nexo, E., Quantitation of epidermal growth factor in the rat. Identification and partial characterization of duodenal EGF, Scand. J. Gastroenterol., 18 (1983) 771-776. 12 Saltin, B., Eklund, B., Hult~n, B., Lundin, A., Nord, L. and Silander, L., T/ivlings orientering, L. Trygg-Hansa, 1973. 13 Hartley, L.H., Growth hormone and catecholamine response to exercise in relation to physical training, Med. Sci. Sports, 7 (1975) 34-36. 14 St. Hilaire, R.J., Hradek, G.T. and Jones, A.L., Hepatic sequestration and biliary secretion of epidermal growth factor: evidence for a high-capacity uptake system, Proc. Natl. Acad. Si. U.S.A., 80 (1983) 3797-3801. 15 Grimby, G., Renal clearence during prolonged supine exercise at different loads, J. Appl. Physiol., 20 (1965) 1294-1298.
203 16 Kurobe, M., Tokida, N., Furukawa, S. and Hayashi, K., Some properties of human epidermal growth factor (hEGF)-like immunoreactive material originating from platelets during blood coagulation, Biochem. Int., 5 (1986) 729-733. 17 Kidd, S., Kelly, M.R. and Young, M.W., Sequence of the notch locus of Drosophila melanogaster: relationship of the encoded protein to mammalian clotting and growth factors, Mol. Cell. Biol., 6 (1986) 3094-3108. 18 Mattila, A.L., Human urinary epidermal growth factor: effects of age, sex and female endocrine status, Life Sci., 39 (1986) 1879-1884. 19 Uchihashi, M., Hirata, Y., Fujita, T. and Matsukura, S., Age-related decrease of urinary excretion of human epidermal growth factor (hEGF), Life Sci., 31 (1982) 679-683. 20 Poulsen, S.S., Nexo, E., Olsen, P.S., Hess, J. and Kirkegard, P., Immunhistochemical localization of epidermal growth factor in rat and man, Histochemistry, 85 (1986) 389-394. 21 Bell, G.I., Fong, N.M. Stempien, M.M., Wormsted, M.A., Caput, D., Ku, L., Urdea, M.S., Rail, LB. and Sanchez-Pescador, R., Human epidermal growth factor precursor: cDNA sequence, expression in vitro and gene organization, Nucleic Acid Res., 14 (1986) 8427-8446. 22 Stokke, O., Clinical chemical changes in relation to physical exercise, Tidskr. Nor. L~egeforen, 12B (1980) 776-782.
197
RPT 00703
Epidermal growth factor in plasma, serum and urine before and after prolonged exercise L. K o n r a d s e n a n d E. N e x o Department of Clinical Chemistry, Central Hospital, Hillerod (Denmark)
(Received 15 October 1987; revised version received and accepted 28 December 1987)
Summary The substance concentration of epidermal growth factor immunoreactivity ( E G F IR) and certain other components were studied in plasma, serum and urine from 25 individuals before and after a 2 h cross-country run. The substance concentration of plasma E G F I R increased from a median of 0.10 nM (range 0.04-0.26 nM) to a median of 0.16 nM (range 0.10~.36 nM) after 2 h of exercise, while serum E G F showed no change. The values obtained for B-platelets were a median of 192 × 109/litre (range 109-282 × 109/litre) before the run, and a median of 265 × 109/litre (range 216-387 x 109/litre) after the run. N o correlation was observed between the values obtained for B-platelets and the values for plasma or serum E G F IR. The substance concentration of E G F I R in urine increased from a median of 3.2 nM (range 0.5-7.7 nM) to a median of 7.0 nM (range 1.5-15.7 nM) after the run. Expressed relative to the output of carbamide the output of urinary E G F I R increased with a median factor of 2 following the run. Expressed relative to the output of creatinine no increase was observed. Adrenergic stimulation; Epidermal growth factor; Exercise; Platelet
Introduction Epidermal growth factor (EGF) is a mitogenic polypeptide able to stimulate cellular growth and differentiation and to inhibit gastric acid secretion [1,2].
Correspondence: E. Nexo, Department of Clinical Chemistry, Central Hospital, DK-3400 Hillerod, Denmark.
0167-0115/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
198 In animals E G F is produced in a number of tissues including the salivary gland and the kidney [1,3], and it is secreted from these tissues mainly in an exocrine manner. In rats and mice adrenergic stimulation either in the form of aggressive behaviour or in the form of ~- or/~-adrenergic agonists result in an increase in the salivary and/or the urinary output of E G F [3-6]. The origin of circulating E G F is unclear. In mice the salivary glands act to a minor degree as an endocrine organ that delivers E G F to the circulation upon adrenergic stimulation [4,6]. This has not been observed for rats [3,5]. Recent work has shown the substance concentration of E G F in human serum to be considerably higher than in plasma. It is believed that E G F delivered upon coagulation of the blood is derived from the platelets [7,8]. Human E G F is produced in the same tissues as in animals, and because of that we found it of interest to study whether the release of E G F in humans is stimulated by adrenergic agonists. In a previous paper we report the salivary concentration of E G F to increase with a median factor of 1.8 after prolonged submaximal exercise [9], a condition known to involve an increase in both ~- and//-adrenergic agonists
[lO]. In the present paper we report the substance concentrations of both plasma and urinary E G F to increase after prolonged exercise. The increase is discussed in the light of other biochemical findings in the same individuals.
Materials and Methods
Participating individuals. 25 individuals (4 females and 21 males) aged 17-49 years participating in a 2 h cross-country run performed in cloudy weather at a temperature around 10°C. All gave informed consent. The participants had been active in crosscountry racing for more than 5 years. After the race they all claimed to be maximally exercised. During the run the participants had free access to water. Collection of biological samples. Urine was voided prior to the run and within the first hour after completion of the run. Blood was drawn prior to the run and within 2 min after arrival at the finish. Plasma was separated from blood anticoagulated with E D T A within 2 h after collection (1800 g, 10 min). Serum was obtained from blood collected without added anticoagulans. The blood was allowed to clot for 2 h at room temperature and at 4°C overnight. All samples were kept at - 2 0 ° C until analyzed. Quantification ofEGF. E G F was quantified as previously described [9,11]. In brief, urinary E G F purified by immunoaffinity chromatography and pure as judged by amino terminal analysis was employed for production of the antibody (serum 4554-0186, final dilution 1:30.000), the iodinated probe and the calibrators. The sensitivity of the assay was 0.04 nM. The intra-assay precision was 5.5% (n = 36, = 1.7 nM) and the interassay precision was 8.5% (n = 60, ~ = 1.65 nM, samples were run over a period of 9 months). Urinary samples were prediluted 1:4 with assay buffer, plasma and serum were analyzed undiluted. Non-specific binding (less than 10%) was substracted prior to analysis of the data.
199
Other biochemical parameters. Urine and serum creatinine and carbamide were analyzed on a SMAC automatic analyzer (Technicon, Denmark) employing routine methods. B-hemoglobin, B-platelets and B-hematocrit were analyzed on a Coulter S + 4 (Coulter Electronics, England) according to the manufacturer. Statistical methods. Pratt's test for paired differences was employed. P-values of less than 0.05 were considered significant. Correlations were examined using a Spearmann's rho test.
Results No difference was observed between serum E G F IR before and after a 2 h cross-country run, while plasma E G F IR increased following exercise (Table I). Since circulatory E G F may be derived from the platelets we compared results obtained for B-platelets with results obtained for E G F IR. The concentration for platelets increased after exercise (Table I), but no correlation was found between the platelet count or the platelet count increase on one hand, and the substance concentration of E G F IR in plasma or in serum on the other (data not shown). To evaluate a possible dehydration of the runners, B-hemoglobin and B-hematocrit were examined, but no difference in values obtained before and after the run was found (Table I). We compared the substance concentration of a number of components in plasma or serum and in urine. While the substance concentration of both E G F IR, creatinine and carbamide increased in the circulation after exercise, the urinary concentration increased only for E G F IR and creatinine (Table II, Fig. 2). The urinary concentration of E G F IR increased with a median factor of 2.2. Relative to the urinary concentration of creatinine no increase was observed, but relative to the urinary concentration of carbamide the urinary concentration of E G F IR increased with a median factor of 2.0 after exercise. The results obtained when comparing the clearance
TABLE I Substance concentration of E G F IR and other components in h u m a n plasma and serum before and after prolonged exercise Before
P - E G F (nM) S-EGF (nM) B-platelets (109/litre) B-hemoglobin (mM) B-hematocrit (fraction)
After
Median
Range
Median
Range
0.10 0.23 192 9.6 0.46
0.04-0.26 0.16-0.46 192-282 8.9 -10.7 0.414).50
0.16 0.25 265 9.7 0.46
0.10-0.36 0.13-0.50 216~387 8.5 -10.7 0.40-0.51
S n.s. S n.s. n.s.
S = significant at P < 0.05 using Pratts test, for comparison of results obtained after exercise with values obtained prior to exercise; n.s. = non-significant: n = 25.
200
A
B
30.
.30
20,
'20
10.
-10
-5.0
5.0. z o
•
~3.0,
"7
.3.0g ~2.0~
~ 2.0,
1.0
1,0,
:
T
"-.. -0.5
0.5,
P-EGF S-CREATS-CARB
EGF
CREAT CA'RB EG'F/ EG'F/ CREAT CARB URINE
PLASMA
Fig. 1. Comparison of values for E G F IR, creatinine and carbamide obtained on serum/plasma or urine before and after prolonged exercise. The figure shows results obtained on blood samples (A) or urine samples (B) collected from 25 individuals after 2 h cross-country running divided by the results obtained prior to the run. The horizontal line indicates the median. Creat., Creatinine; Carb., Carbamide; EGF/Creat., excretion of E G F relative to excretion of creatinine: EGF/Carb., excretion of E G F relative to excretion of carbamide. T A B L E II The substance concentration of E G F IR and other components in h u m a n urine and plasma/serum before and after prolonged exercise Urine Median E G F (nM) Before After Creatinine (pM) Before After Carbamide (raM) Before After n 1 2 3
3.2 7.0
Plasma/serum Range
Median
0.5 1.5~
9920 20760
3480 6200
250 260
58 56
7.7 15.73
-15680 -615003 398 370
0.10 0.16 91 128 5.4 6.4
= 25. Plasma. Serum. p < 0.05 (Pratt's test) as compared to values obtained before exercise.
Range
0.04O. lO79 105
0.26 l 0.361'3
-1272 -1482,3
3.7 4.7 -
6.82 8.3 z
201 of the different substances were similar to the results obtained for the substance concentration in urine.
Discussion
The present paper represents the first attempt to study the substance concentration of human EGF in the circulation and in urine after a stimulation known to increase the output of EGF in animals. The results show that both plasma and urinary EGF IR increase following exercise, while serum EGF IR remains unchanged. During competitive running the runner performs at about 80-90% of his maximal aerobic capacity [12]. At this rate of activity there is both an increased activity in the sympathetic nervous system with a raised serum concentration of norepinephrine [10], and an increased activity of the suprarenal glands resulting in an increased level of blood epinephrine [13]. Compared to studies in animals the release of EGF upon the adrenergic stimulation of exercise is minimal. Following aggressive behaviour circulatory EGF increases with a factor of 100 in mice [6] and following adrenergic stimulation the urinary output of EGF, relative to the output of creatinine, increases with a factor of 2 in rats [3]~ A number of factors need to be taken into consideration when interpreting the results for plasma and serum EGF IR. The origin and metabolism of plasma EGF is unknown. Animal studies have indicated the liver and the kidney to be of major importance for the clearance of EGF [14]. Under hard physical activity blood circulation to the splanchnicus area is greatly reduced. Figures concerning the liver circulation do not exist, but results from renal circulation [15] show the renal artery flow to be less than half the normal flow. It is thus likely that a decreased metabolism of EGF plays a role in the increase of plasma EGF IR after exercise. It is believed that the EGF recovered in serum after coagulation of the blood sample is derived from the platelets [7,8,16]. After exercise the platelet count increases, while serum EGF IR remains unchanged. If EGF is released from the platelets in a simple manner one would expect correlation to exist between the platelet count, and the amount of EGF recovered. Our results therefore suggest a more complex pattern for the release of EGF upon coagulation of the blood. Other potential points of interest are the factors involved in coagulation and fibrinolysis, because many of these factors contain EGF-like domains [17]. At present it is not known whether any of these domains are released in a form recognized by the radioimmunoassay for EGF. Urinary EGF may be derived from the circulation or it may originate from the kidney. The latter possibility is supported by its immunohistochemical localization in the distal tubular cells [20], and by the recent isolation of cDNA for the EGF precursor from the human kidney [21]. The results obtained in the present paper support an adrenergic release of EGF from the kidney as previously observed in rats [3]. Both the substance concentration of EGF and the concentration relative to the concentration of carbamide increase in urine after exercise. The output of EGF in urine parallel the output of creatinine. Since the production and thereby the urinary
202
output of creatinine is increased after exercise [22] this supports an increase in urinary output of EGF after exercise. At the same time the observation suggests some kind of relation between the renal output of creatinine and EGF. A similar relation has previously been reported in a study that compared the excretion of creatinine and EGF as a function of age [19]. In conclusion the present paper shows the substance concentration of EGF IR to increase in plasma and urine following prolonged exercise in humans. It further questions whether there is a simple relation between the amount of EGF recovered upon coagulation of the blood and the platelet counts.
Acknowledgements The technical assistance of Marianne Rye Hansen is warmly acknowledged. The study was supported by The Danish Cancer Society (86-034) and by a grant from The Research Council for Sports Medicine.
References I Hollenberg, M.D. and Armstrong, G.D., Epidermal growth factor urogastrone and its receptor. In B.I. Posner (Ed.), Polypeptide Hormone Receptors, Dekker, New York, 1985, pp. 201-226. 2 Gregory, H., In vivo aspects of urogastrone-epidermal growth factor, J. Cell Sci., Suppl. 3 (1985) 11-17. 3 Olsen, P.S., Kirkegaard, P., Poulsen, S.S. and Nexo, E., Adrenergic effects on renal secretion of epidermal growth factor in the rat, Regul. Pept., 11 (1985) 17-25. 4 Nexo, E., Olsen, P.S. and Poulsen, K., Exocrine and endocrine secretion of renin and epidermal growth factor from the mouse submandibular glands, Regul. Pept., 8 (1984) 327-334. 50lsen, P.S., Kirkegaard, P., Poulsen, S.S. and Nexo, E., Adrenergic effects on exocrine secretion of rat submandibular epidermal growth factor, Gut, 25 (1984) 1234-1240. 6 Nexo, E., Hollenberg, M.D. and Bing, J., Aggressive behaviour increases both plasma epidermal growth factor and renin, Acta Physiol. Scand., I I I (1981) 367-371. 7 Savage, A.P., Chatterjee, V.K., Gregory, H. and Bloom, S.R., Epidermal growth factor in blood, Regul. Pept., 16 (1986) 199-206. 8 0 k a , Y. and Orth, D.N., Human plasma epidermal growth factor/~-urogastrone is associated with blood platelets, J. Clin. Invest., 72 (1983) 249-259. 9 Nexo, E., Hansen, M.R. and Konradsen, L., Human saliva epidermal growth factor, haptocorrin and amylase before and after prolonged exercise, Scand. J. Clin. Lab. Invest., 48 (1988) 267-271. 10 Christensen, N.J., Galbo, H., Hansen, J.F., Hesse, B., Richter, E.A. and Trap-Jensen, J., Catecholamines and exercise, Diabetes, 28 (1979) 58-62. 11 Olsen, P.S. and Nexo, E., Quantitation of epidermal growth factor in the rat. Identification and partial characterization of duodenal EGF, Scand. J. Gastroenterol., 18 (1983) 771-776. 12 Saltin, B., Eklund, B., Hult~n, B., Lundin, A., Nord, L. and Silander, L., T/ivlings orientering, L. Trygg-Hansa, 1973. 13 Hartley, L.H., Growth hormone and catecholamine response to exercise in relation to physical training, Med. Sci. Sports, 7 (1975) 34-36. 14 St. Hilaire, R.J., Hradek, G.T. and Jones, A.L., Hepatic sequestration and biliary secretion of epidermal growth factor: evidence for a high-capacity uptake system, Proc. Natl. Acad. Si. U.S.A., 80 (1983) 3797-3801. 15 Grimby, G., Renal clearence during prolonged supine exercise at different loads, J. Appl. Physiol., 20 (1965) 1294-1298.
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