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von Willebrand factor in cadaveric urine for forensic investigation
Legal Medicine 11 (2009) 245–247
Contents lists available at ScienceDirect
Legal Medicine journal homepage: www.elsevier.com/locate/legalmed
Brief Communication
von Willebrand factor in cadaveric urine for forensic investigation Shogo Kasuda a,*, Risa Kudo a, Yoshifumi Morimura a, Kohei Tatsumi b, Yoshihiko Sakurai b, Midori Shima b, Katsuhiko Hatake a a b
Department of Legal Medicine, Nara Medical University School of Medicine, 840 Shijo-cho, Kashihara, Nara 634-8521, Japan Department of Pediatrics, Nara Medical University School of Medicine, 840 Shijo-cho, Kashihara, Nara 634-8522, Japan
a r t i c l e
i n f o
Article history: Received 2 March 2009 Received in revised form 13 April 2009 Accepted 14 April 2009 Available online 9 June 2009 Keywords: von Willebrand factor Urine Postmortem intervals Autopsy
a b s t r a c t Preliminary experimental study of urinary von Willebrand factor (VWF) concentration was undertaken to evaluate the utility of this parameter in forensic investigations. ELISA was used to measure VWF concentration. Correlations of urinary VWF with cause of death and postmortem interval (PMI) were ascertained. As PMI advanced, urinary VWF increased but plasma VWF did not. Cause of death was not significantly correlated with VWF. This study indicated the possibility that urinalysis would be helpful to estimate PMI. Ó 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction In forensic autopsy, cadaveric blood is an awkward sample from which to estimate antemortem status or postmortem interval (PMI) since its decomposition progresses so rapidly. Hence chemical parameters of other body fluids such as vitreous humor (VH), pericardial fluid, cerebrospinal fluid (CSF) and joint fluid have been well studied as alternatives [1–3]. However, there are few reports regarding the composition of cadaveric urine. As metabolites and waste products accumulate in urine in healthy individuals, urine is a very important sample in clinical diagnosis. Even simple urinalysis provides considerable information such as amount of protein, pH, and presence of occult blood. However, the use of cadaveric urine is generally limited to toxicological analysis in practical forensic examination. von Willebrand factor (VWF) has a crucial role in hemostasis, being involved in platelet attachment to the subendothelium and acting as a carrier protein for coagulation factor VIII. VWF is synthesized and stored as ultra-large forms in Weibel–Palade bodies in vascular endothelial cells. The stored VWF is released when endothelial cells are activated by various agonists. Upon release, ultra-large VWF is cleaved by the metalloprotease ADAMTS-13 in plasma [4]. Since there are no cleaving enzymes in urine, urinary VWF should be structurally stable. We therefore focused our attention on VWF in urine.
In order to elucidate the usefulness of urinalysis in the estimation of PMI or cause of death, we took urinary VWF concentration for instance in this preliminary experiment. 2. Materials and methods 2.1. Materials With the permission of the ethics committee of Nara Medical University, urine and blood were obtained from 44 autopsied cadavers (male: n = 31, aged 54.6 ± 25.0 years, female: n = 13, aged 54.7 ± 20.0 years). We selected the cases whose PMI could be estimated strictly based on traditional autopsy findings, final confirmation of survive or death scene investigation. PMI of the sampled specimens were as follows: 6–12 h (group A, n = 4), 13– 24 h (group B, n = 17), 25–36 h (group C, n = 11), and 37–48 h (group D, n = 12). Causes of death were as follows: asphyxiation (n = 5), drowning (n = 10), fire (n = 8), stab injury (n = 3), internal causes (n = 12), and others (n = 6). We made an incision with a clean scalpel on the bladder wall and obtained urine samples. At the time of incision, we extended bladder wall not to contaminate urine with blood. No urine samples were visibly contaminated with blood. Blood samples were obtained from right heart cavities. Urine and blood samples were centrifuged at 3000 rpm for 10 m and the supernatants were stored 80 °C until use. 2.2. Measurement of VWF antigen
* Corresponding author. Tel.: +81 744 29 8843; fax: +81 744 29 1116. E-mail address: [email protected] (S. Kasuda). 1344-6223/$ - see front matter Ó 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.legalmed.2009.04.002
VWF antigen was measured by enzyme-linked immunosorbent assay (ELISA) as previously described [5]. Blood samples were
246
S. Kasuda et al. / Legal Medicine 11 (2009) 245–247
Table 1 Cause of death and concentration of urinary and plasma VWF. p-Value*
Cause of death
Urinary VWF (%) Plasma VWF (%)
Asphyxiation (5)
Drowing (10)
Fire (8)
Stab injury (3)
Internal causes (12)
Others (6)
0.243 ± 0.270 271.8 ± 124.9
0.316 ± 0.312 374.1 ± 244.2
0.273 ± 0.310 213.1 ± 167.4
0.0532 ± 0.0921 197.5 ± 75.0
0.468 ± 0.249 267.4 ± 150.6
0.145 ± 0.133 418.2 ± 142.4
0.0894 0.179
Values are expressed as means ± SD. (n): n is the number of samples. Kruskal–Wallis test.
*
diluted 10-fold with Tris-buffered saline (10 mmol/L Tris–HCl, pH 7.5, 150 mmol/L NaCl) containing 1% bovine serum albumin (Sigma–Aldrich, St. Louis, MO, USA). Urine samples were not diluted. Normal human plasma (Coagtrol N, Sysmex, Kobe, Japan) was used as a standard. VWF concentration was expressed as a percentage of that seen in normal plasma. 2.3. Statistical analyses Data are expressed as means ± SD. Scheffe test following Kruskal–Wallis analysis was carried out using Stat View software (SAS institute, NC, USA). A p-value of less than 0.05 was regarded as significant. 3. Results and discussion First, we compared VWF concentrations with respect to cause of death (Table 1). There were no significant differences in both urinary and plasma VWF concentration according to cause of death. Second, we performed a correlation analysis between VWF concentration and PMI. Urinary VWF showed a positive correlation with PMI (Fig. 1A). The p-values for group B, C and D against group A were 0.26, 0.0078 and 0.0035, respectively (Scheffe test). On the other hand, plasma VWF was not correlated with PMI (Fig. 1B). The p-values for group B, C and D against group A were 0.99, 0.97 and 1.0, respectively (Scheffe test). VWF is excreted from the kidney into urine in healthy individuals [6]. However, in this experiment, urinary VWF concentration was not correlated with cause of death but with PMI. This suggests that VWF in urine originates not from renal excretion but from leakage from the putrefactive vessel of bladder wall. The high lev-
A
els of VWF that we observed in the plasma would be due to rapid decomposition of vessel walls and platelets which are the maximal VWF storages in human body. If these considerations are correct, there is a possibility that survival time after tissue damage would affect VWF concentration because tissue damage stimulates VWF release from endothelial cells during agonal period. We could not investigate the relationship between the length of agonal period and VWF levels. However, the causes of death which include tissue damage such as fire death or stab injury did not increased VWF concentration in both urine and plasma. Intoxication also causes tissue damage. However, we could investigate only one case of intoxication (PMI was included in group B) in this experiment. The levels of VWF concentration in urine and plasma were 0.12% and 273%, respectively. These concentrations were not extremely deviated from values of group B. Taken together, although further investigation is anticipated, we considered that tissue damage did not affect VWF levels so much. Here, there arises a question; does the volume of urine affect the VWF concentration? If there is a small amount of urine in the bladder, the area where urine is in contact with the bladder wall is limited. In this case, the VWF value would decrease. On the contrary, if urine volume reduced due to concentration, the VWF value would increase. As a precautionary measure, we therefore evaluated the correlation between VWF concentration and urine volume. As shown in Table 2, VWF concentration showed a tendency to increase as urine volume decreased, but this was not statistically significant. Although we could not adjust urine volume with serum creatinine concentration, we confirmed that urine volume is not a significant determinant of VWF concentration. Estimation of PMI has been mainly based on heuristic components of forensic observation such as rigor mortis and postmortem
B 600
1.0
0.6
VWF (%)
VWF (%)
500
*
0.8
*
0.4
400 300 200
0.2
100
0
0 A
B
C
PMI (h)
D
A
B
C
D
PMI (h)
Fig. 1. The relationship between VWF concentration and PMI. PMI was categorized as follows; 6–12 h (group A, n = 4), 13–24 h (group B, n = 17), 25–36 h (group C, n = 11), 37–48 h (group D, n = 12). (A) VWF concentration in urine. *p < 0.05 compared with group A, (B) VWF concentration in plasma.
S. Kasuda et al. / Legal Medicine 11 (2009) 245–247 Table 2 Urine volume and concentration of urinary VWF.
References p-Value*
Urine volume (ml)
Urinary VWF (%)
247
9 (7)
1099 (25)
100 (12)
0.540 ± 0.289
0.267 ± 0.0579
0.250 ± 0.0590
0.0958
Values are expressed as means ± SD. (n): n is the number of samples. Kruskal–Wallis test.
*
lividity. As an objective indicator of PMI, electrolytes (sodium and potassium ion) concentration in VH or CSF has been well discussed [3,7,8]. Other various substances such as cardiac Troponin I, platelets, pancreatic b-cells and Strontium90 have been tried to use for the same purpose [9–12]. However, these substances are not frequently used. In this report, we examined whether levels of a specific protein in the urine could be another numerical indicator of PMI. VWF is a favorable target for postmortem examination because it naturally has a large-multimeric structure and immunoreactive components can be perpetuated even when urine decomposition is advanced. In this preliminary study, we only demonstrated weak correlation between PMI and urinary VWF. However, urine is an ideal sample for forensic examination, since it is easy to obtain and protected from the external environment, and abundant clinical data are available. Further studies are anticipated to determine other factors useful for PMI estimation from urine analysis.
[1] Madea B, Musshoff F. Postmortem biochemistry. Forensic Sci Int 2007;165:165–71. [2] Singh D, Prashad R, Sharma SK, Pandey AN. Estimation of postmortem interval from human pericardial fluid electrolytes concentrations in Chandigarh zone of India: log transformed linear regression model. Legal Med (Tokyo) 2006;8:279–87. [3] Yadav J, Deshpande A, Arora A, Athawal BK, Dubey BP. Estimation of time since death from CSF electrolyte concentration in Bhopal region of central India. Legal Med (Tokyo) 2007;9:309–13. [4] Dong JF. Cleavage of ultra-large von Willebrand factor by ADAMTS-13 under flow conditions. J Thromb Haemost 2005;3:1710–6. [5] Matsui T, Shimoyama T, Matsumoto M, Fujimura Y, Takemoto Y, Sako M, et al. ABO blood group antigens on human plasma von Willebrand factor after ABOmismatched bone marrow transplantation. Blood 1999;94:2895–900. [6] Yamamoto T, Silveira AM, Hessel B, Blomback B. von Willebrand factor antigen in urine. Thromb Res 1987;45:59–74. [7] Bocaz-Beneventi G, Tagliaro F, Bortolotti F, Manetto G, Havel J. Capillary zone electrophoresis and artificial neural networks for estimation of the postmortem interval (PMI) using electrolytes measurements in human vitreous humour. Int J Legal Med 2002;116:5–11. [8] Naumann HN. Cerebrospinal fluid electrolytes after death. Proc Soc Exp Biol Med 1958;98:16–8. [9] Neis P, Hille R, Paschke M, Pilwat G, Schnabel A, Niess C, et al. Strontium90 for determination of time since death. Forensic Sci Int 1999;99:47–51. [10] Sabucedo AJ, Furton KG. Estimation of postmortem interval using the protein marker cardiac Troponin I. Forensic Sci Int 2003;134:11–6. [11] Thomsen H, Krisch B. The postmortem activation status of platelets. Int J Leg Med 1994;107:111–7. [12] Wehner F, Wehner HD, Schieffer MC, Subke J. Delimitation of the time of death by immunohistochemical detection of insulin in pancreatic beta-cells. Forensic Sci Int 1999;105:161–9.
Contents lists available at ScienceDirect
Legal Medicine journal homepage: www.elsevier.com/locate/legalmed
Brief Communication
von Willebrand factor in cadaveric urine for forensic investigation Shogo Kasuda a,*, Risa Kudo a, Yoshifumi Morimura a, Kohei Tatsumi b, Yoshihiko Sakurai b, Midori Shima b, Katsuhiko Hatake a a b
Department of Legal Medicine, Nara Medical University School of Medicine, 840 Shijo-cho, Kashihara, Nara 634-8521, Japan Department of Pediatrics, Nara Medical University School of Medicine, 840 Shijo-cho, Kashihara, Nara 634-8522, Japan
a r t i c l e
i n f o
Article history: Received 2 March 2009 Received in revised form 13 April 2009 Accepted 14 April 2009 Available online 9 June 2009 Keywords: von Willebrand factor Urine Postmortem intervals Autopsy
a b s t r a c t Preliminary experimental study of urinary von Willebrand factor (VWF) concentration was undertaken to evaluate the utility of this parameter in forensic investigations. ELISA was used to measure VWF concentration. Correlations of urinary VWF with cause of death and postmortem interval (PMI) were ascertained. As PMI advanced, urinary VWF increased but plasma VWF did not. Cause of death was not significantly correlated with VWF. This study indicated the possibility that urinalysis would be helpful to estimate PMI. Ó 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction In forensic autopsy, cadaveric blood is an awkward sample from which to estimate antemortem status or postmortem interval (PMI) since its decomposition progresses so rapidly. Hence chemical parameters of other body fluids such as vitreous humor (VH), pericardial fluid, cerebrospinal fluid (CSF) and joint fluid have been well studied as alternatives [1–3]. However, there are few reports regarding the composition of cadaveric urine. As metabolites and waste products accumulate in urine in healthy individuals, urine is a very important sample in clinical diagnosis. Even simple urinalysis provides considerable information such as amount of protein, pH, and presence of occult blood. However, the use of cadaveric urine is generally limited to toxicological analysis in practical forensic examination. von Willebrand factor (VWF) has a crucial role in hemostasis, being involved in platelet attachment to the subendothelium and acting as a carrier protein for coagulation factor VIII. VWF is synthesized and stored as ultra-large forms in Weibel–Palade bodies in vascular endothelial cells. The stored VWF is released when endothelial cells are activated by various agonists. Upon release, ultra-large VWF is cleaved by the metalloprotease ADAMTS-13 in plasma [4]. Since there are no cleaving enzymes in urine, urinary VWF should be structurally stable. We therefore focused our attention on VWF in urine.
In order to elucidate the usefulness of urinalysis in the estimation of PMI or cause of death, we took urinary VWF concentration for instance in this preliminary experiment. 2. Materials and methods 2.1. Materials With the permission of the ethics committee of Nara Medical University, urine and blood were obtained from 44 autopsied cadavers (male: n = 31, aged 54.6 ± 25.0 years, female: n = 13, aged 54.7 ± 20.0 years). We selected the cases whose PMI could be estimated strictly based on traditional autopsy findings, final confirmation of survive or death scene investigation. PMI of the sampled specimens were as follows: 6–12 h (group A, n = 4), 13– 24 h (group B, n = 17), 25–36 h (group C, n = 11), and 37–48 h (group D, n = 12). Causes of death were as follows: asphyxiation (n = 5), drowning (n = 10), fire (n = 8), stab injury (n = 3), internal causes (n = 12), and others (n = 6). We made an incision with a clean scalpel on the bladder wall and obtained urine samples. At the time of incision, we extended bladder wall not to contaminate urine with blood. No urine samples were visibly contaminated with blood. Blood samples were obtained from right heart cavities. Urine and blood samples were centrifuged at 3000 rpm for 10 m and the supernatants were stored 80 °C until use. 2.2. Measurement of VWF antigen
* Corresponding author. Tel.: +81 744 29 8843; fax: +81 744 29 1116. E-mail address: [email protected] (S. Kasuda). 1344-6223/$ - see front matter Ó 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.legalmed.2009.04.002
VWF antigen was measured by enzyme-linked immunosorbent assay (ELISA) as previously described [5]. Blood samples were
246
S. Kasuda et al. / Legal Medicine 11 (2009) 245–247
Table 1 Cause of death and concentration of urinary and plasma VWF. p-Value*
Cause of death
Urinary VWF (%) Plasma VWF (%)
Asphyxiation (5)
Drowing (10)
Fire (8)
Stab injury (3)
Internal causes (12)
Others (6)
0.243 ± 0.270 271.8 ± 124.9
0.316 ± 0.312 374.1 ± 244.2
0.273 ± 0.310 213.1 ± 167.4
0.0532 ± 0.0921 197.5 ± 75.0
0.468 ± 0.249 267.4 ± 150.6
0.145 ± 0.133 418.2 ± 142.4
0.0894 0.179
Values are expressed as means ± SD. (n): n is the number of samples. Kruskal–Wallis test.
*
diluted 10-fold with Tris-buffered saline (10 mmol/L Tris–HCl, pH 7.5, 150 mmol/L NaCl) containing 1% bovine serum albumin (Sigma–Aldrich, St. Louis, MO, USA). Urine samples were not diluted. Normal human plasma (Coagtrol N, Sysmex, Kobe, Japan) was used as a standard. VWF concentration was expressed as a percentage of that seen in normal plasma. 2.3. Statistical analyses Data are expressed as means ± SD. Scheffe test following Kruskal–Wallis analysis was carried out using Stat View software (SAS institute, NC, USA). A p-value of less than 0.05 was regarded as significant. 3. Results and discussion First, we compared VWF concentrations with respect to cause of death (Table 1). There were no significant differences in both urinary and plasma VWF concentration according to cause of death. Second, we performed a correlation analysis between VWF concentration and PMI. Urinary VWF showed a positive correlation with PMI (Fig. 1A). The p-values for group B, C and D against group A were 0.26, 0.0078 and 0.0035, respectively (Scheffe test). On the other hand, plasma VWF was not correlated with PMI (Fig. 1B). The p-values for group B, C and D against group A were 0.99, 0.97 and 1.0, respectively (Scheffe test). VWF is excreted from the kidney into urine in healthy individuals [6]. However, in this experiment, urinary VWF concentration was not correlated with cause of death but with PMI. This suggests that VWF in urine originates not from renal excretion but from leakage from the putrefactive vessel of bladder wall. The high lev-
A
els of VWF that we observed in the plasma would be due to rapid decomposition of vessel walls and platelets which are the maximal VWF storages in human body. If these considerations are correct, there is a possibility that survival time after tissue damage would affect VWF concentration because tissue damage stimulates VWF release from endothelial cells during agonal period. We could not investigate the relationship between the length of agonal period and VWF levels. However, the causes of death which include tissue damage such as fire death or stab injury did not increased VWF concentration in both urine and plasma. Intoxication also causes tissue damage. However, we could investigate only one case of intoxication (PMI was included in group B) in this experiment. The levels of VWF concentration in urine and plasma were 0.12% and 273%, respectively. These concentrations were not extremely deviated from values of group B. Taken together, although further investigation is anticipated, we considered that tissue damage did not affect VWF levels so much. Here, there arises a question; does the volume of urine affect the VWF concentration? If there is a small amount of urine in the bladder, the area where urine is in contact with the bladder wall is limited. In this case, the VWF value would decrease. On the contrary, if urine volume reduced due to concentration, the VWF value would increase. As a precautionary measure, we therefore evaluated the correlation between VWF concentration and urine volume. As shown in Table 2, VWF concentration showed a tendency to increase as urine volume decreased, but this was not statistically significant. Although we could not adjust urine volume with serum creatinine concentration, we confirmed that urine volume is not a significant determinant of VWF concentration. Estimation of PMI has been mainly based on heuristic components of forensic observation such as rigor mortis and postmortem
B 600
1.0
0.6
VWF (%)
VWF (%)
500
*
0.8
*
0.4
400 300 200
0.2
100
0
0 A
B
C
PMI (h)
D
A
B
C
D
PMI (h)
Fig. 1. The relationship between VWF concentration and PMI. PMI was categorized as follows; 6–12 h (group A, n = 4), 13–24 h (group B, n = 17), 25–36 h (group C, n = 11), 37–48 h (group D, n = 12). (A) VWF concentration in urine. *p < 0.05 compared with group A, (B) VWF concentration in plasma.
S. Kasuda et al. / Legal Medicine 11 (2009) 245–247 Table 2 Urine volume and concentration of urinary VWF.
References p-Value*
Urine volume (ml)
Urinary VWF (%)
247
9 (7)
1099 (25)
100 (12)
0.540 ± 0.289
0.267 ± 0.0579
0.250 ± 0.0590
0.0958
Values are expressed as means ± SD. (n): n is the number of samples. Kruskal–Wallis test.
*
lividity. As an objective indicator of PMI, electrolytes (sodium and potassium ion) concentration in VH or CSF has been well discussed [3,7,8]. Other various substances such as cardiac Troponin I, platelets, pancreatic b-cells and Strontium90 have been tried to use for the same purpose [9–12]. However, these substances are not frequently used. In this report, we examined whether levels of a specific protein in the urine could be another numerical indicator of PMI. VWF is a favorable target for postmortem examination because it naturally has a large-multimeric structure and immunoreactive components can be perpetuated even when urine decomposition is advanced. In this preliminary study, we only demonstrated weak correlation between PMI and urinary VWF. However, urine is an ideal sample for forensic examination, since it is easy to obtain and protected from the external environment, and abundant clinical data are available. Further studies are anticipated to determine other factors useful for PMI estimation from urine analysis.
[1] Madea B, Musshoff F. Postmortem biochemistry. Forensic Sci Int 2007;165:165–71. [2] Singh D, Prashad R, Sharma SK, Pandey AN. Estimation of postmortem interval from human pericardial fluid electrolytes concentrations in Chandigarh zone of India: log transformed linear regression model. Legal Med (Tokyo) 2006;8:279–87. [3] Yadav J, Deshpande A, Arora A, Athawal BK, Dubey BP. Estimation of time since death from CSF electrolyte concentration in Bhopal region of central India. Legal Med (Tokyo) 2007;9:309–13. [4] Dong JF. Cleavage of ultra-large von Willebrand factor by ADAMTS-13 under flow conditions. J Thromb Haemost 2005;3:1710–6. [5] Matsui T, Shimoyama T, Matsumoto M, Fujimura Y, Takemoto Y, Sako M, et al. ABO blood group antigens on human plasma von Willebrand factor after ABOmismatched bone marrow transplantation. Blood 1999;94:2895–900. [6] Yamamoto T, Silveira AM, Hessel B, Blomback B. von Willebrand factor antigen in urine. Thromb Res 1987;45:59–74. [7] Bocaz-Beneventi G, Tagliaro F, Bortolotti F, Manetto G, Havel J. Capillary zone electrophoresis and artificial neural networks for estimation of the postmortem interval (PMI) using electrolytes measurements in human vitreous humour. Int J Legal Med 2002;116:5–11. [8] Naumann HN. Cerebrospinal fluid electrolytes after death. Proc Soc Exp Biol Med 1958;98:16–8. [9] Neis P, Hille R, Paschke M, Pilwat G, Schnabel A, Niess C, et al. Strontium90 for determination of time since death. Forensic Sci Int 1999;99:47–51. [10] Sabucedo AJ, Furton KG. Estimation of postmortem interval using the protein marker cardiac Troponin I. Forensic Sci Int 2003;134:11–6. [11] Thomsen H, Krisch B. The postmortem activation status of platelets. Int J Leg Med 1994;107:111–7. [12] Wehner F, Wehner HD, Schieffer MC, Subke J. Delimitation of the time of death by immunohistochemical detection of insulin in pancreatic beta-cells. Forensic Sci Int 1999;105:161–9.