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Differences in postmortem urea nitrogen, creatinine and uric acid levels between blood and pericardial fluid in acute death
Legal Medicine 9 (2007) 115–122 www.elsevier.com/locate/legalmed
Differences in postmortem urea nitrogen, creatinine and uric acid levels between blood and pericardial fluid in acute death Bao-Li Zhu a
a,b,*
, Takaki Ishikawa a,b, Tomomi Michiue a,b, Sayaka Tanaka a, Dong Zhao a, Dong-Ri Li a, Li Quan a, Shigeki Oritani a, Hitoshi Maeda a,b
Department of Legal Medicine, Osaka City University Medical School, Asahi-machi 1-4-3, Abeno, 545-8585 Osaka, Japan b Osaka Medical Examiner’s Office, Bambacho 1-6, Chuo-ku, 540-0007 Osaka, Japan Received 20 April 2006; received in revised form 13 October 2006; accepted 16 October 2006 Available online 2 January 2007
Abstract Previous studies showed significant differences in postmortem urea nitrogen (UN), creatinine (Cr) and uric acid (UA) levels in heart blood depending on the causes of death, including acute death. In addition, the levels in pericardial fluid approximated the clinical serum reference ranges, and their elevations may be assessed based on clinical criteria. The present study investigated difference between blood and pericardial levels of these markers. Medicolegal autopsy cases (n = 556, within 48 h postmortem) of the following causes of death were examined: injury (n = 136), asphyxiation (n = 50), drowning (n = 39), fire fatalities (n = 99), hyperthermia (n = 11), hypothermia (n = 8), poisoning (n = 26), delayed traumatic death (n = 44) and natural diseases (n = 143). When serum UN, Cr and UA levels were compared with the pericardial levels, there was an equivalency for delayed traumatic death and chronic renal failure, although each level was markedly elevated. Parallel increases in serum and pericardial UA and/or Cr levels were also observed for hypothermia and gastrointestinal bleeding. However, in drowning cases, the left cardiac and pericardial UN levels were lower than the right cardiac and peripheral levels, suggesting the influence of water aspiration. Significant elevations in serum and pericardial Cr and UA levels with a higher serum/pericardial UA ratio for fatal methamphetamine intoxication suggest progressive skeletal muscle damage due to advanced hypoxia/acidosis. Similar findings were often observed for other acute and subacute deaths. These findings suggest that a comparison between blood and pericardial nitrogenous compounds would be useful for investigating the cause and process of death. Ó 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Forensic pathophysiology; Postmortem biochemistry; Urea nitrogen; Creatinine; Uric acid; Serum; Pericardial fluid; Acute death
1. Introduction Postmortem biochemistry can be one of the most productive ancillary procedures for forensic pathologists, although agonal and postmortem interference should be taken into consideration [1–9]. Among various biochemical markers, previous studies have shown that nitrogenous compounds including urea nitrogen (UN), creatinine (Cr) and uric acid (UA) are relatively stable in cadaveric blood and body fluids [1,10,11]. These markers in the blood may be especially useful for postmortem investigation of the *
Corresponding author. Tel.: +81 6 6645 3767; fax: +81 6 6634 3871. E-mail address: [email protected] (B.-L. Zhu).
causes of death, involving renal failure, hypoxic or thermal skeletal muscle damage [10]. Meanwhile, stability of the pericardial levels in acute death cases, approximating their clinical serum reference ranges, suggested a considerable delay in changes in these substances in pericardial fluid compared with those in the blood during systemic metabolic deterioration [11]. Comprehensive biochemical data analyses following multiple site sampling, involving pericardial fluid may be very important and useful for investigating the cause and process of death [6–9]. In the present study, we analyzed the difference between pericardial and cardiac blood levels of UN, Cr and UA to investigate the pathophysiology of death, involving systemic metabolic deterioration in acute or subacute death.
1344-6223/$ - see front matter Ó 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.legalmed.2006.10.002
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2. Materials and methods 2.1. Materials Serial medicolegal autopsy cases (n = 556, 3–48 h postmortem) at our institutes were examined. The cases comprised 410 males and 146 females, 18–96 (median, 60.0) years of age. Younger subjects (<18 years of age) were excluded due to an insufficient number of cases. Specimens were aseptically collected using syringes to withdraw blood from the left, right heart chamber and external iliac vein and pericardial fluid during autopsy. External iliac venous blood samples were drawn after clamping the vein at the proximal end and lifting the lower limbs for several minutes. The blood samples were immediately centrifuged to separate the sera. These specimens were subsequently stored at 20 °C until use. The causes of death were classified based on thorough autopsy, macromorphological, micropathological and toxicological examinations as follows: injury (n = 136, survival time <48 h: blunt injury, n = 112; sharp instrument injury, n = 24), mechanical asphyxiation (n = 50: hanging, n = 10; strangulation, n = 16; aspiration, n = 24), drowning (n = 39: freshwater, n = 21; saltwater, n = 18), fire fatalities (n = 99) consisting of those with blood carboxyhemoglobin (COHb) below 60% (n = 58) and above 60% (n = 41), hyperthermia (heat stroke, n = 11), hypothermia (cold exposure, n = 8), methamphetamine (MA) intoxication (n = 11), carbon monoxide (CO) poisoning (n = 4), other
poisoning (n = 11), delayed traumatic death due to multiple organ insufficiency (n = 44; survival time, 3–90 days), natural diseases (n = 143), including acute myocardial infarction/ischemia (AMI, n = 108), cerebrovascular diseases (n = 20: spontaneous cerebral hemorrhage, n = 17; subarachnoid hemorrhage, n = 3), gastrointestinal (GI) bleeding (n = 7) and chronic renal failure (uremia, n = 8) (Table 1). The AMI group consisted of cases of sudden death, which showed macro- and microscopic findings of acute ischemic heart diseases without evidence of death due to causes other than cardiac attack [8,9]. Fatal hyperthermia was determined based on pathology and circumstantial evidence, in part, considering estimated body temperature over 40 °C at the time of death (n = 10/11). Possible high body temperature (>40 °C) at the time of death was also estimated in most cases of MA fatality (n = 7/11). 2.2. Biochemical measurements UN was measured by a urease-glutamate dehydrogenase method [12], Cr by an alkali-picric acid method (modified Jaffe method) [13], and UA by a uricase peroxidase method using an L-type UA Kit (Wako, Tokyo) [14]. Hemoglobin contamination (<0.5 mg/dl) did not interfere with the measurements. The clinical reference serum ranges were: 6–20 mg/dl for UN, 0.61–1.04 (adult male) and 0.47–0.79 (adult female) mg/dl for Cr, and 3.7–7.6 (male) and 2.5–5.4 (female) mg/dl for UA. In cases showing strong
Table 1 Case profiles (n = 556) Cause of death
Injury Blunt injury Sharp injury Asphyxia Drowning Freshwater Saltwater Fire fatality COHb <60% COHb >60% Hyperthermia Hypothermia Poisoning Methamphetamine Carbon monoxide Others Delayed traumatic death Natural diseases Acute myocardial infarction Spontaneous cerebral hemorrhages Gastrointestinal bleeding Chronic renal failure
n
Male/female
Age (years)
Survival time
PMI (h)
Range
Median
Range
Median
Range
Median
1.5 <0.5
3–44 8–47 6–38
18.8 19.9 18.3
8–40 8–47
20.2 17.8
11.8 20.0 16.0
112 24 50
88/24 18/6 30/20
18–94 34–90 19–93
56.0 54.5 43.6
<0.5–24 <0.5–20 <0.5
21 18
14/7 12/6
45–84 33–73
60.0 56.0
<0.5 <0.5
58 41 11 8
44/14 30/11 6/5 7/1
23–93 25–87 51–88 44–80
67.5 68.0 77.0 64.0
<0.5 <0.5 3–14 3–6
6–36 6.0 3.0
15.5 5–42 16–40 9–44
11 4 11a 44b
10/1 3/1 7/4 41/3
20–52 27–59 20–64 28–84
39.0 54.0 29.0 56.5
2–24 <0.5–3 0.5–24 72–2,160
5.0 3.0 5.0 59.0
6–34 26–33 6–37 6–48
24.9 25.8 25.0 12.6
108 20 7 8
78/30 13/7 6/1 3/5
47–94 39–81 50–75 54–89
66.5 59.0 59.0 76.0
<0.5–72 <0.5–20 1.0–24 Unknown
<0.5 1.5 3.0
5–42 5–34 6–31 9–35
18.7 25.0 15.8 24.2
PMI, postmortem interval; COHb, carboxyhemoglobin concentration. a Sedative-hypnotics (n = 5), ethanol (n = 2) and others (n = 4). b Multiple organ insufficiency and secondary infection from head injury (n = 27) and other injury (n = 17).
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hemolysis, which may have influenced the measurements, the findings were not used in the analyses. 2.3. Toxicological analyses Blood COHb concentration was determined using a COoximeter system [15,16] for all fire fatalities. Volatile chemicals, including alcohol were analyzed by head-space gas chromatography in all cases [17]. Drug analyses were performed by gas chromatography/mass spectrometry, when preliminary screening tests were positive [18].
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2.4. Statistical analyses Regression equation analysis was used to compare two parameters, including biochemical markers, the age of victims, survival time and postmortem interval. Comparisons between groups were performed using the Mann–Whitney U test, and Scheffe test was used for analyses, involving multiple comparisons. These analyses were performed using Microsoft Excel and Statview (version 5.0, SAS Institute Inc., SAS Campus Drive Cary). A P-value less than 0.05 was considered to indicate significance. In Figs. 1–3, the results of the data analysis are shown as box-plots, for which 50% of the data are summarized in the box. The line in each box represents the median and the lines outside of each box represent the 90% confidence interval. The sensitivity and specificity in distinguishing the two groups using a cutoff value for each marker were estimated by receiver-operating characteristics (ROC) analysis [19,20]. The area under the curves were calculated and analyzed by one-tailed test. The optimal compromise between sensitivity and specificity was determined graphically.
b
b Fig. 1. Comparisons of urea nitrogen (UN) levels in the pericardial fluid and serum. (a) Pericardial, cardiac and peripheral serum UN levels and (b) the cardiac/pericardial ratios with regard to the cause of death. I, injury (n = 136); A, asphyxiation (n = 50); D, drowning (n = 39); F, fire fatalities (n = 99); H, hyperthermia (n = 11); C, hypothermia (n = 8), MA, fatal methamphetamine abuse (n = 11); P, other poisoning (n = 15); dT, delayed traumatic death (n = 44); AMI, acute myocardial infarction (n = 108); CD, cerebrovascular diseases (n = 20); GI, gastrointestinal bleeding (n = 7); U, chronic renal failure (uremia, n = 8) (details shown in Table 1). Significant high: (a) *C, dT, GI and U vs. other groups at each site, except for MA (P < 0.001, Scheffe test); **H and MA vs. I, A and F (P < 0.01, Mann–Whitney U test); in addition, P, AMI and SCH vs. I, A and F (P < 0.05, Mann–Whitney U test) at each site; (b) right cardiac serum/pericardial UN ratio, *D vs. other groups (P < 0.01, Mann– Whitney U test); in addition, external iliac vein/pericardial UN ratio, *D vs. I, A, F, C, MA, dT, AMI, CD, GI and U (P < 0.05, Mann–Whitney U test). Significant low: (a) left, right cardiac serum and pericardial fluid, D vs. other groups (P < 0.05, Mann–Whitney U test); in addition, for drowning cases, the right cardiac and iliac venous levels were significant high than the left cardiac and pericardial levels (P < 0.01, Mann–Whitney U test).
Fig. 2. Comparisons of creatinine (Cr) levels in the pericardial fluid and serum. (a) Pericardial, cardiac and peripheral serum Cr levels and (b) the cardiac serum/pericardial ratios with regard to the cause of death. Abbreviations are shown in Fig. 1. Significant high: (a) *MA, dT and U vs. other groups at each site (P < 0.05, Scheffe test); **H, P and GI vs. A. D and AMI at each site (P < 0.05, Mann–Whitney U test).
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B.-L. Zhu et al. / Legal Medicine 9 (2007) 115–122 Table 2 Postmortem urea nitrogen (UN), creatinine (Cr) and uric acid (UA) levels in pericardial fluid, both cardiac and external iliac venous blood
Pericardial fluid Left heart blood Right heart blood External iliac venous blood
n
UN (mg/dl)
Cr (mg/dl)
UA (mg/dl)
556 525 528
2.6–189 (17.1) 2.1–191 (16.5) 1.4–190 (17.4)
0.15–12.7 (1.49) 0.09–15.5 (1.40) 0.10–15.5 (1.64)
0.2–28.5 (5.5) 0.2–49.8 (5.9) 0.6–60.0 (7.6)
338
0.4–196 (17.4)
0.20–12.4 (1.85)
0.8–47.9 (5.4)
Median values are shown in parentheses.
b
Fig. 3. Comparisons of uric acid (UA) levels in the pericardial fluid and serum. (a) Pericardial, cardiac and peripheral serum UA levels and (b) the cardiac serum/pericardial ratios with regard to the cause of death. Abbreviations are shown in Fig. 1. Significant high: (a) *MA and C vs. other groups at each site, except for U (P < 0.001, Scheffe test); right cardiac serum, **H, dT and CD vs. F (P < 0.05, Scheffe test), external iliac venous serum, **CD and U vs. F (P < 0.05, Scheffe test); pericardial fluid, **dT and U vs. F (P < 0.05, Scheffe test). (b) Left cardiac serum/ pericardial UA ratio, *MA vs. I and F, and external iliac venous serum/ pericardial UA ratio, *MA and **CD, P and CD vs. I, A, F, C, dT, AMI, GI and U (P < 0.05, Scheffe test). In addition, right cardiac serum/ pericardial UA ratio, A, D, MA, P and CD vs: I, F, C, dT, AMI, GI and U (P < 0.05, Mann–Whitney U test).
3. Results 3.1. Gross comparisons between the serum and pericardial levels of nitrogenous compounds Postmortem ranges of UN, Cr and UA in the pericardial fluid, sera from both cardiac and external venous blood are shown in Table 2. There was no significant relationship of each marker to subject age (>18 years of age: UN, r = 0.032–0.110, P = 0.0756–0.5400; Cr, r = 0.116 to 0.025, P = 0.0906–0.3010; UA, r = 0.125 to 0.034, P = 0.1123–4030) or postmortem interval (UN, r = 0.020 to 0.124, P = 0.5846–0.9484; Cr, r = 0.013–0.151, P = 0.1059–0.7784; UA, r = 0.075 to 0.141, P = 0.0828– 0.1016). There was a mild survival time-dependent elevation for each marker (UN, r = 0.177–0.230, P = 0.0014–0.0130; Cr, r = 0.149–0.225, P = 0.0021–0.0349; UA, r = 0.120–0.157, P = 0.0084–0.0441). When all cases
were examined stepwise, significant difference between acute/subacute and prolonged death (survival time <6 h, n = 446 and >6 h, n = 110, respectively) were observed at each site (P = 0.0008 to <0.0001). Serum and pericardial Cr levels (mg/dl) were significantly higher for males (mean/median; 2.24/1.50 for left heart blood, 2.37/1.67 for right heart blood, 2.53/1.94 for external iliac venous blood and 2.18/1.58 for pericardial fluid) than for females (mean/median; 1.61/1.15 for left heart blood, 1.91/1.44 for right heart blood, 1.98/1.64 for external iliac venous blood and 1.61/1.18 for pericardial fluid) (P < 0.05). A significant gender-difference was also observed for the pericardial UA level (mean/median; 6.49/5.75 for males and 5.01/4.90 for females, P < 0.01). Correlation equations between pericardial and serum levels in all cases are shown in Table 3. There were high correlations for UN and Cr (correlation coefficient = 0.82–0.99, P < 0.0001), although the iliac venous Cr level was frequently higher than the pericardial level. UA showed moderate correlations (correlation coefficient = 0.34–0.57, P < 0.0001), and the right cardiac level was higher than the pericardial level in most cases. 3.2. Differences between serum and pericardial levels with regard to the cause of death 3.2.1. Urea nitrogen The ranges of UN levels in the serum and pericardial fluid showed a similar distribution in relation to the causes of death. There were significantly higher UN levels for delayed traumatic death, hypothermia, chronic renal failure (uremia) and GI bleeding compared with other groups, and mildly higher levels for hyperthermia and MA fatalities compared with injury, asphyxiation, drowning, fire fatalities, other poisoning, AMI and cerebrovascular diseases (Fig. 1a). An equivalency between serum and pericardial UN levels was observed for all groups other than drowning, including delayed traumatic death, hypothermia, chronic renal failure (uremia) and GI bleeding: the median ratio (median of the ratios) of each cardiac or iliac venous to pericardial level was 0.98–1.01 (Fig. 1b). In drowning cases, the right cardiac and iliac venous levels were significantly higher than the left cardiac and pericardial levels (P < 0.001), independently of
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Table 3 Correlation equations between the pericardial and serum levels of urea nitrogen (UN), creatinine (Cr) and uric acid (UA) n
UN
Cr
UA
y = 0.97x + 0.14 (r = 0.99, P < 0.0001) y = 0.97x + 0.85 (r = 0.99, P < 0.0001) y = 0.97x + 0.64 (r = 0.99, P < 0.0001)
y = 0.94x + 0.15 (r = 0.82, P < 0.0001) y = 0.92x + 0.37 (r = 0.87, P < 0.0001) y = 0.97x + 0.48 (r = 0.91, P < 0.0001)
y = 1.10x + 1.24 (r = 0.54, P < 0.0001) y = 1.01x + 4.69 (r = 0.34, P < 0.0001) y = 1.17x 0.13 (r = 0.57, P < 0.0001)
538
y = 0.93x + 0.61 (r = 0.96, P < 0.0001)
y = 0.97x 0.15 (r = 0.90, P < 0.0001)
y = 0.50x + 2.28 (r = 0.75, P < 0.0001)
336
y = 0.97x 0.26 (r = 0.99, P < 0.0001)
y = 0.87x 0.13 (r = 0.84, P < 0.0001)
y = 0.68x + 2.95 (r = 0.61, P < 0.0001)
338
y = 0.96x + 1.21 (r = 0.92, P < 0.0001)
y = 0.83x + 0.17 (r = 0.88, P < 0.0001)
y = 0.94x + 4.28 (r = 0.57, P < 0.0001)
Left heart (y)
491
Right heart (y)
493
External iliac vein (y)
318
Right heart (x) Left heart (y) External iliac vein (x) Left heart (y) Right heart (y)
Pericardial fluid (x)
the immersion medium (fresh- or saltwater), and the ratio of the right cardiac or iliac venous to pericardial level was significantly higher compared with that of the other groups except hyperthermia (P < 0.05). The left cardiac and pericardial levels were equivalent; the median ratio was 0.99. Similar findings were observed in some cases of injury, fire fatalities, poisoning and AMI. However, both cardiac, peripheral and pericardial UN levels were mildly lower in drowning cases compared with those in the other groups (P < 0.05). When analyzed by ROC, the sensitivity and specificity in distinguishing drowning and other groups using the serum/pericardial ratio were 0.76 and 0.79 for the right cardiac level and 0.91 and 0.79 for the iliac venous level, respectively, at a cutoff value of 1.1. For the left/right cardiac serum ratio, the sensitivity and specificity at a cutoff value of 0.75 were 0.84 and 0.95, respectively. 3.2.2. Creatinine The ranges of serum and pericardial Cr levels also showed a similar distribution in relation to the causes of death. Markedly higher Cr levels were observed for delayed traumatic death, chronic renal failure (uremia) and MA fatalities compared with the other groups, and moderately higher levels for other poisoning, hyperthermia and GI bleeding compared with injury, asphyxiation, drowning, fire fatalities, hypothermia, AMI and cerebrovascular diseases (Fig. 2a). For these groups, left, right and iliac venous Cr levels showed equivalent correlations to the pericardial level, although the serum level was markedly higher in some cases of hyperthermia: the median ratio were 0.99, 1.03 and 1.05, respectively (Fig. 2b). For all cases, the serum to pericardial Cr ratio was the highest for the iliac venous level (median, 1.30), followed by right cardiac level (median, 1.23) and left cardiac level (median, 1.05) (P < 0.001). There was no significant difference among the causes of death.
3.2.3. Uric acid Markedly higher serum and pericardial UA levels were observed for hypothermia and MA fatalities compared with the other groups, and moderately higher levels for hyperthermia, delayed traumatic death, chronic renal failure (uremia) and cerebrovascular diseases compared with injury, asphyxiation, drowning, fire fatalities, other poisoning, AMI and GI bleeding cases (Fig. 3a). UA levels in serum and pericardial fluid showed an equivalent correlation for hypothermia, delayed traumatic death and chronic renal failure (uremia): the median ratio to the pericardial level for the left, right cardiac and iliac venous levels were 0.99, 1.02 and 0.96, respectively. A significantly higher serum to pericardial UA ratio was observed for MA fatalities, other poisoning and cerebrovascular diseases (Fig. 3b). When analyzed by ROC, the sensitivity and specificity in distinguishing MA fatalities and other groups excluding other poisoning and cerebrovascular diseases using the serum/pericardial ratio were 0.78 and 0.86 at a cutoff value of 1.25 for left cardiac level, 0.93 and 0.80 at a cutoff value of 2.0 for the right cardiac level, and 0.92 and 0.85 at a cutoff value of 1.20 for the iliac venous level, respectively. The UN, Cr and UA levels did not show any significant difference between blunt and sharp instrument injury cases. There was also no significant difference between the subgroups of fire fatalities (COHb level below 60% and above 60%), or between cases with and without estimated high body temperature (>40 °C) at the time of death due to fatal hyperthermia and MA intoxication. 4. Discussion In the present study, a marked elevation in serum UN level along with elevations Cr and UA levels was observed for delayed traumatic death due to multiple organ insufficiency and chronic renal failure (uremia),
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as previously reported [10]. In these cases, their pericardial levels were usually equivalent to the serum levels, suggesting prolonged duration of uremic status, which may be assessed based on serum and pericardial levels following clinical criteria. Similar findings were observed for UN and UA in hypothermia cases, suggesting a prolonged death process involving protein catabolism [21,22]. Elevations in serum and pericardial UN levels in GI bleeding were also explained based on clinical criteria [23]. For hyperthermia and MA fatalities, a mild elevation of serum or pericardial UN level (median value, 22.1–28.5 mg/dl) was not associated with high body temperature. However, in drowning cases independently of the immersion medium (fresh- or saltwater), UN level was significantly lower for the left cardiac blood and pericardial fluid compared with the right cardiac and peripheral levels. This suggests hemodilution of the pulmonary venous blood in the left heart due to water aspiration and a subsequent influence on the components of pericardial effusion or water diffusion from the thoracic cavity, since the bilateral cardiac, peripheral and pericardial UN levels were mildly lower than those for other groups. For diagnosis of drowning, a cutoff value for the right cardiac or peripheral serum/pericardial ratio was estimated at 1.1, where about 70–90% of drowning cases showed positive findings, while false positive findings were observed in about 25% of the other cases, which included various cause of death. The use of this marker in combination with pathological and other biochemical findings may be useful [24–29]. For Cr levels, an increase serum level of fatal MA intoxication cases, associated with a mild elevation in UN level and a marked elevation in UA level, was similar to the clinical findings [30–32]. Similar elevation in the pericardial Cr level suggests considerable duration of an increased serum Cr level due to skeletal muscle damage (rhabdomyolysis) caused by MA intoxication [30–36]. However, the serum UA level showed greater elevation than the pericardial level, with an elevation in each cardiac or peripheral serum/pericardial UA ratios. Furthermore, the serum UA and Cr levels were higher for fatal MA intoxication than those for hyperthermia, showing that there was no significant elevation in UN level. This suggests that hyperthermia and/or renal failure may not be the main factor causing increased serum UA and Cr levels in MA fatality. UA precursors (purines) are mostly produced through the breakdown of nucleic acids in muscle tissues and subsequent enzymatic conversion into UA mainly in the liver [37]. Depressed ATP concentration in the muscle due to hypoxia may be related to overproduction of UA, and acidemia interferes with renal tubular secretion of UA [37–39]. Therefore, severe hypoxia and metabolic acidosis in fatal MA intoxication [31,40,41] may contribute to elevated serum UA. An elevated pericardial UA level suggests a gradually progressing metabolic deterioration
involved advanced hypoxia and subsequent acidosis, accompanied by elevations in UN and Cr levels, antecedent to the onset of terminal fatal events [10,11]. Thus, elevations in serum and pericardial Cr and UA levels with a higher serum/pericardial UA ratio (cutoff value: 1.25 for left cardiac level, 2.0 for the right cardiac level and 1.20 for the iliac venous level) are indicative of fatal MA intoxication. In addition, significant elevations in serum UA levels and higher serum/pericardial ratios for cerebrovascular diseases may indicate advanced terminal hypoxia/acidosis. Similar findings were also observed for fatal poisoning due to substances other than MA. The pericardial fluid is a transudate derived from the epicardium and contains plasma components [42,43]. Postmortem pericardial UN, Cr and UA levels were usually within the clinical serum reference ranges as previously described [11], suggesting considerable stability during the death process and postmortem period. Changes in the pericardial components during systemic metabolic deterioration may gradually occur following those in the coronary blood flow. Differences between serum and pericardial levels may appear depending on the survival period in acute or subacute death cases (survival time < 6 h). Significant changes in pericardial levels of these markers may become apparent hours later than those in the serum [11,17,42]. Thus, elevations in the pericardial UN, Cr and/or UA levels may be suggestive of prolonged survival over hours. As previously described, site-to-site difference in these markers, especially Cr and UA, was also observed for heart and peripheral blood. Higher Cr levels in the peripheral blood and UA levels in the right heart blood, which were often observed in acute and subacute death cases, may be explained as a result of hypoxic skeletal muscle damage and subsequent degradation of purine derivatives in the liver during the process of death [10]. Topographic analyses of these biochemical markers in blood and pericardial fluid may also be useful to investigate death processes, involving systemic nitrogenous metabolic deterioration in causes of acute or subacute death other than MA fatality and in the cerebrovascular diseases described above. These criteria may also be applicable to infantile fatalities when age-dependent reference values are considered [11]. In conclusion, elevations serum Cr and UA levels with a higher serum/pericardial UA ratio are suggestive of acute and progressive skeletal muscle damage due to advanced hypoxia/acidosis and/or systemic metabolic deterioration in the dying process, which was especially evident for MA fatalities. Higher right cardiac and peripheral venous to pericardial UN ratio can be used as a marker for the diagnosis of drowning. These findings suggest that pericardial fluid is useful as reference material to investigate the alteration in serum UN, Cr and UA levels for diagnosing various causes of death.
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Acknowledgments This study was in part supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science and the Ministry of Education, Culture, Sports, Science and Technology, Japan (Grant Nos. 15390217 and 15590585).
[18]
[19]
[20]
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Differences in postmortem urea nitrogen, creatinine and uric acid levels between blood and pericardial fluid in acute death Bao-Li Zhu a
a,b,*
, Takaki Ishikawa a,b, Tomomi Michiue a,b, Sayaka Tanaka a, Dong Zhao a, Dong-Ri Li a, Li Quan a, Shigeki Oritani a, Hitoshi Maeda a,b
Department of Legal Medicine, Osaka City University Medical School, Asahi-machi 1-4-3, Abeno, 545-8585 Osaka, Japan b Osaka Medical Examiner’s Office, Bambacho 1-6, Chuo-ku, 540-0007 Osaka, Japan Received 20 April 2006; received in revised form 13 October 2006; accepted 16 October 2006 Available online 2 January 2007
Abstract Previous studies showed significant differences in postmortem urea nitrogen (UN), creatinine (Cr) and uric acid (UA) levels in heart blood depending on the causes of death, including acute death. In addition, the levels in pericardial fluid approximated the clinical serum reference ranges, and their elevations may be assessed based on clinical criteria. The present study investigated difference between blood and pericardial levels of these markers. Medicolegal autopsy cases (n = 556, within 48 h postmortem) of the following causes of death were examined: injury (n = 136), asphyxiation (n = 50), drowning (n = 39), fire fatalities (n = 99), hyperthermia (n = 11), hypothermia (n = 8), poisoning (n = 26), delayed traumatic death (n = 44) and natural diseases (n = 143). When serum UN, Cr and UA levels were compared with the pericardial levels, there was an equivalency for delayed traumatic death and chronic renal failure, although each level was markedly elevated. Parallel increases in serum and pericardial UA and/or Cr levels were also observed for hypothermia and gastrointestinal bleeding. However, in drowning cases, the left cardiac and pericardial UN levels were lower than the right cardiac and peripheral levels, suggesting the influence of water aspiration. Significant elevations in serum and pericardial Cr and UA levels with a higher serum/pericardial UA ratio for fatal methamphetamine intoxication suggest progressive skeletal muscle damage due to advanced hypoxia/acidosis. Similar findings were often observed for other acute and subacute deaths. These findings suggest that a comparison between blood and pericardial nitrogenous compounds would be useful for investigating the cause and process of death. Ó 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Forensic pathophysiology; Postmortem biochemistry; Urea nitrogen; Creatinine; Uric acid; Serum; Pericardial fluid; Acute death
1. Introduction Postmortem biochemistry can be one of the most productive ancillary procedures for forensic pathologists, although agonal and postmortem interference should be taken into consideration [1–9]. Among various biochemical markers, previous studies have shown that nitrogenous compounds including urea nitrogen (UN), creatinine (Cr) and uric acid (UA) are relatively stable in cadaveric blood and body fluids [1,10,11]. These markers in the blood may be especially useful for postmortem investigation of the *
Corresponding author. Tel.: +81 6 6645 3767; fax: +81 6 6634 3871. E-mail address: [email protected] (B.-L. Zhu).
causes of death, involving renal failure, hypoxic or thermal skeletal muscle damage [10]. Meanwhile, stability of the pericardial levels in acute death cases, approximating their clinical serum reference ranges, suggested a considerable delay in changes in these substances in pericardial fluid compared with those in the blood during systemic metabolic deterioration [11]. Comprehensive biochemical data analyses following multiple site sampling, involving pericardial fluid may be very important and useful for investigating the cause and process of death [6–9]. In the present study, we analyzed the difference between pericardial and cardiac blood levels of UN, Cr and UA to investigate the pathophysiology of death, involving systemic metabolic deterioration in acute or subacute death.
1344-6223/$ - see front matter Ó 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.legalmed.2006.10.002
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B.-L. Zhu et al. / Legal Medicine 9 (2007) 115–122
2. Materials and methods 2.1. Materials Serial medicolegal autopsy cases (n = 556, 3–48 h postmortem) at our institutes were examined. The cases comprised 410 males and 146 females, 18–96 (median, 60.0) years of age. Younger subjects (<18 years of age) were excluded due to an insufficient number of cases. Specimens were aseptically collected using syringes to withdraw blood from the left, right heart chamber and external iliac vein and pericardial fluid during autopsy. External iliac venous blood samples were drawn after clamping the vein at the proximal end and lifting the lower limbs for several minutes. The blood samples were immediately centrifuged to separate the sera. These specimens were subsequently stored at 20 °C until use. The causes of death were classified based on thorough autopsy, macromorphological, micropathological and toxicological examinations as follows: injury (n = 136, survival time <48 h: blunt injury, n = 112; sharp instrument injury, n = 24), mechanical asphyxiation (n = 50: hanging, n = 10; strangulation, n = 16; aspiration, n = 24), drowning (n = 39: freshwater, n = 21; saltwater, n = 18), fire fatalities (n = 99) consisting of those with blood carboxyhemoglobin (COHb) below 60% (n = 58) and above 60% (n = 41), hyperthermia (heat stroke, n = 11), hypothermia (cold exposure, n = 8), methamphetamine (MA) intoxication (n = 11), carbon monoxide (CO) poisoning (n = 4), other
poisoning (n = 11), delayed traumatic death due to multiple organ insufficiency (n = 44; survival time, 3–90 days), natural diseases (n = 143), including acute myocardial infarction/ischemia (AMI, n = 108), cerebrovascular diseases (n = 20: spontaneous cerebral hemorrhage, n = 17; subarachnoid hemorrhage, n = 3), gastrointestinal (GI) bleeding (n = 7) and chronic renal failure (uremia, n = 8) (Table 1). The AMI group consisted of cases of sudden death, which showed macro- and microscopic findings of acute ischemic heart diseases without evidence of death due to causes other than cardiac attack [8,9]. Fatal hyperthermia was determined based on pathology and circumstantial evidence, in part, considering estimated body temperature over 40 °C at the time of death (n = 10/11). Possible high body temperature (>40 °C) at the time of death was also estimated in most cases of MA fatality (n = 7/11). 2.2. Biochemical measurements UN was measured by a urease-glutamate dehydrogenase method [12], Cr by an alkali-picric acid method (modified Jaffe method) [13], and UA by a uricase peroxidase method using an L-type UA Kit (Wako, Tokyo) [14]. Hemoglobin contamination (<0.5 mg/dl) did not interfere with the measurements. The clinical reference serum ranges were: 6–20 mg/dl for UN, 0.61–1.04 (adult male) and 0.47–0.79 (adult female) mg/dl for Cr, and 3.7–7.6 (male) and 2.5–5.4 (female) mg/dl for UA. In cases showing strong
Table 1 Case profiles (n = 556) Cause of death
Injury Blunt injury Sharp injury Asphyxia Drowning Freshwater Saltwater Fire fatality COHb <60% COHb >60% Hyperthermia Hypothermia Poisoning Methamphetamine Carbon monoxide Others Delayed traumatic death Natural diseases Acute myocardial infarction Spontaneous cerebral hemorrhages Gastrointestinal bleeding Chronic renal failure
n
Male/female
Age (years)
Survival time
PMI (h)
Range
Median
Range
Median
Range
Median
1.5 <0.5
3–44 8–47 6–38
18.8 19.9 18.3
8–40 8–47
20.2 17.8
11.8 20.0 16.0
112 24 50
88/24 18/6 30/20
18–94 34–90 19–93
56.0 54.5 43.6
<0.5–24 <0.5–20 <0.5
21 18
14/7 12/6
45–84 33–73
60.0 56.0
<0.5 <0.5
58 41 11 8
44/14 30/11 6/5 7/1
23–93 25–87 51–88 44–80
67.5 68.0 77.0 64.0
<0.5 <0.5 3–14 3–6
6–36 6.0 3.0
15.5 5–42 16–40 9–44
11 4 11a 44b
10/1 3/1 7/4 41/3
20–52 27–59 20–64 28–84
39.0 54.0 29.0 56.5
2–24 <0.5–3 0.5–24 72–2,160
5.0 3.0 5.0 59.0
6–34 26–33 6–37 6–48
24.9 25.8 25.0 12.6
108 20 7 8
78/30 13/7 6/1 3/5
47–94 39–81 50–75 54–89
66.5 59.0 59.0 76.0
<0.5–72 <0.5–20 1.0–24 Unknown
<0.5 1.5 3.0
5–42 5–34 6–31 9–35
18.7 25.0 15.8 24.2
PMI, postmortem interval; COHb, carboxyhemoglobin concentration. a Sedative-hypnotics (n = 5), ethanol (n = 2) and others (n = 4). b Multiple organ insufficiency and secondary infection from head injury (n = 27) and other injury (n = 17).
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hemolysis, which may have influenced the measurements, the findings were not used in the analyses. 2.3. Toxicological analyses Blood COHb concentration was determined using a COoximeter system [15,16] for all fire fatalities. Volatile chemicals, including alcohol were analyzed by head-space gas chromatography in all cases [17]. Drug analyses were performed by gas chromatography/mass spectrometry, when preliminary screening tests were positive [18].
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2.4. Statistical analyses Regression equation analysis was used to compare two parameters, including biochemical markers, the age of victims, survival time and postmortem interval. Comparisons between groups were performed using the Mann–Whitney U test, and Scheffe test was used for analyses, involving multiple comparisons. These analyses were performed using Microsoft Excel and Statview (version 5.0, SAS Institute Inc., SAS Campus Drive Cary). A P-value less than 0.05 was considered to indicate significance. In Figs. 1–3, the results of the data analysis are shown as box-plots, for which 50% of the data are summarized in the box. The line in each box represents the median and the lines outside of each box represent the 90% confidence interval. The sensitivity and specificity in distinguishing the two groups using a cutoff value for each marker were estimated by receiver-operating characteristics (ROC) analysis [19,20]. The area under the curves were calculated and analyzed by one-tailed test. The optimal compromise between sensitivity and specificity was determined graphically.
b
b Fig. 1. Comparisons of urea nitrogen (UN) levels in the pericardial fluid and serum. (a) Pericardial, cardiac and peripheral serum UN levels and (b) the cardiac/pericardial ratios with regard to the cause of death. I, injury (n = 136); A, asphyxiation (n = 50); D, drowning (n = 39); F, fire fatalities (n = 99); H, hyperthermia (n = 11); C, hypothermia (n = 8), MA, fatal methamphetamine abuse (n = 11); P, other poisoning (n = 15); dT, delayed traumatic death (n = 44); AMI, acute myocardial infarction (n = 108); CD, cerebrovascular diseases (n = 20); GI, gastrointestinal bleeding (n = 7); U, chronic renal failure (uremia, n = 8) (details shown in Table 1). Significant high: (a) *C, dT, GI and U vs. other groups at each site, except for MA (P < 0.001, Scheffe test); **H and MA vs. I, A and F (P < 0.01, Mann–Whitney U test); in addition, P, AMI and SCH vs. I, A and F (P < 0.05, Mann–Whitney U test) at each site; (b) right cardiac serum/pericardial UN ratio, *D vs. other groups (P < 0.01, Mann– Whitney U test); in addition, external iliac vein/pericardial UN ratio, *D vs. I, A, F, C, MA, dT, AMI, CD, GI and U (P < 0.05, Mann–Whitney U test). Significant low: (a) left, right cardiac serum and pericardial fluid, D vs. other groups (P < 0.05, Mann–Whitney U test); in addition, for drowning cases, the right cardiac and iliac venous levels were significant high than the left cardiac and pericardial levels (P < 0.01, Mann–Whitney U test).
Fig. 2. Comparisons of creatinine (Cr) levels in the pericardial fluid and serum. (a) Pericardial, cardiac and peripheral serum Cr levels and (b) the cardiac serum/pericardial ratios with regard to the cause of death. Abbreviations are shown in Fig. 1. Significant high: (a) *MA, dT and U vs. other groups at each site (P < 0.05, Scheffe test); **H, P and GI vs. A. D and AMI at each site (P < 0.05, Mann–Whitney U test).
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B.-L. Zhu et al. / Legal Medicine 9 (2007) 115–122 Table 2 Postmortem urea nitrogen (UN), creatinine (Cr) and uric acid (UA) levels in pericardial fluid, both cardiac and external iliac venous blood
Pericardial fluid Left heart blood Right heart blood External iliac venous blood
n
UN (mg/dl)
Cr (mg/dl)
UA (mg/dl)
556 525 528
2.6–189 (17.1) 2.1–191 (16.5) 1.4–190 (17.4)
0.15–12.7 (1.49) 0.09–15.5 (1.40) 0.10–15.5 (1.64)
0.2–28.5 (5.5) 0.2–49.8 (5.9) 0.6–60.0 (7.6)
338
0.4–196 (17.4)
0.20–12.4 (1.85)
0.8–47.9 (5.4)
Median values are shown in parentheses.
b
Fig. 3. Comparisons of uric acid (UA) levels in the pericardial fluid and serum. (a) Pericardial, cardiac and peripheral serum UA levels and (b) the cardiac serum/pericardial ratios with regard to the cause of death. Abbreviations are shown in Fig. 1. Significant high: (a) *MA and C vs. other groups at each site, except for U (P < 0.001, Scheffe test); right cardiac serum, **H, dT and CD vs. F (P < 0.05, Scheffe test), external iliac venous serum, **CD and U vs. F (P < 0.05, Scheffe test); pericardial fluid, **dT and U vs. F (P < 0.05, Scheffe test). (b) Left cardiac serum/ pericardial UA ratio, *MA vs. I and F, and external iliac venous serum/ pericardial UA ratio, *MA and **CD, P and CD vs. I, A, F, C, dT, AMI, GI and U (P < 0.05, Scheffe test). In addition, right cardiac serum/ pericardial UA ratio, A, D, MA, P and CD vs: I, F, C, dT, AMI, GI and U (P < 0.05, Mann–Whitney U test).
3. Results 3.1. Gross comparisons between the serum and pericardial levels of nitrogenous compounds Postmortem ranges of UN, Cr and UA in the pericardial fluid, sera from both cardiac and external venous blood are shown in Table 2. There was no significant relationship of each marker to subject age (>18 years of age: UN, r = 0.032–0.110, P = 0.0756–0.5400; Cr, r = 0.116 to 0.025, P = 0.0906–0.3010; UA, r = 0.125 to 0.034, P = 0.1123–4030) or postmortem interval (UN, r = 0.020 to 0.124, P = 0.5846–0.9484; Cr, r = 0.013–0.151, P = 0.1059–0.7784; UA, r = 0.075 to 0.141, P = 0.0828– 0.1016). There was a mild survival time-dependent elevation for each marker (UN, r = 0.177–0.230, P = 0.0014–0.0130; Cr, r = 0.149–0.225, P = 0.0021–0.0349; UA, r = 0.120–0.157, P = 0.0084–0.0441). When all cases
were examined stepwise, significant difference between acute/subacute and prolonged death (survival time <6 h, n = 446 and >6 h, n = 110, respectively) were observed at each site (P = 0.0008 to <0.0001). Serum and pericardial Cr levels (mg/dl) were significantly higher for males (mean/median; 2.24/1.50 for left heart blood, 2.37/1.67 for right heart blood, 2.53/1.94 for external iliac venous blood and 2.18/1.58 for pericardial fluid) than for females (mean/median; 1.61/1.15 for left heart blood, 1.91/1.44 for right heart blood, 1.98/1.64 for external iliac venous blood and 1.61/1.18 for pericardial fluid) (P < 0.05). A significant gender-difference was also observed for the pericardial UA level (mean/median; 6.49/5.75 for males and 5.01/4.90 for females, P < 0.01). Correlation equations between pericardial and serum levels in all cases are shown in Table 3. There were high correlations for UN and Cr (correlation coefficient = 0.82–0.99, P < 0.0001), although the iliac venous Cr level was frequently higher than the pericardial level. UA showed moderate correlations (correlation coefficient = 0.34–0.57, P < 0.0001), and the right cardiac level was higher than the pericardial level in most cases. 3.2. Differences between serum and pericardial levels with regard to the cause of death 3.2.1. Urea nitrogen The ranges of UN levels in the serum and pericardial fluid showed a similar distribution in relation to the causes of death. There were significantly higher UN levels for delayed traumatic death, hypothermia, chronic renal failure (uremia) and GI bleeding compared with other groups, and mildly higher levels for hyperthermia and MA fatalities compared with injury, asphyxiation, drowning, fire fatalities, other poisoning, AMI and cerebrovascular diseases (Fig. 1a). An equivalency between serum and pericardial UN levels was observed for all groups other than drowning, including delayed traumatic death, hypothermia, chronic renal failure (uremia) and GI bleeding: the median ratio (median of the ratios) of each cardiac or iliac venous to pericardial level was 0.98–1.01 (Fig. 1b). In drowning cases, the right cardiac and iliac venous levels were significantly higher than the left cardiac and pericardial levels (P < 0.001), independently of
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Table 3 Correlation equations between the pericardial and serum levels of urea nitrogen (UN), creatinine (Cr) and uric acid (UA) n
UN
Cr
UA
y = 0.97x + 0.14 (r = 0.99, P < 0.0001) y = 0.97x + 0.85 (r = 0.99, P < 0.0001) y = 0.97x + 0.64 (r = 0.99, P < 0.0001)
y = 0.94x + 0.15 (r = 0.82, P < 0.0001) y = 0.92x + 0.37 (r = 0.87, P < 0.0001) y = 0.97x + 0.48 (r = 0.91, P < 0.0001)
y = 1.10x + 1.24 (r = 0.54, P < 0.0001) y = 1.01x + 4.69 (r = 0.34, P < 0.0001) y = 1.17x 0.13 (r = 0.57, P < 0.0001)
538
y = 0.93x + 0.61 (r = 0.96, P < 0.0001)
y = 0.97x 0.15 (r = 0.90, P < 0.0001)
y = 0.50x + 2.28 (r = 0.75, P < 0.0001)
336
y = 0.97x 0.26 (r = 0.99, P < 0.0001)
y = 0.87x 0.13 (r = 0.84, P < 0.0001)
y = 0.68x + 2.95 (r = 0.61, P < 0.0001)
338
y = 0.96x + 1.21 (r = 0.92, P < 0.0001)
y = 0.83x + 0.17 (r = 0.88, P < 0.0001)
y = 0.94x + 4.28 (r = 0.57, P < 0.0001)
Left heart (y)
491
Right heart (y)
493
External iliac vein (y)
318
Right heart (x) Left heart (y) External iliac vein (x) Left heart (y) Right heart (y)
Pericardial fluid (x)
the immersion medium (fresh- or saltwater), and the ratio of the right cardiac or iliac venous to pericardial level was significantly higher compared with that of the other groups except hyperthermia (P < 0.05). The left cardiac and pericardial levels were equivalent; the median ratio was 0.99. Similar findings were observed in some cases of injury, fire fatalities, poisoning and AMI. However, both cardiac, peripheral and pericardial UN levels were mildly lower in drowning cases compared with those in the other groups (P < 0.05). When analyzed by ROC, the sensitivity and specificity in distinguishing drowning and other groups using the serum/pericardial ratio were 0.76 and 0.79 for the right cardiac level and 0.91 and 0.79 for the iliac venous level, respectively, at a cutoff value of 1.1. For the left/right cardiac serum ratio, the sensitivity and specificity at a cutoff value of 0.75 were 0.84 and 0.95, respectively. 3.2.2. Creatinine The ranges of serum and pericardial Cr levels also showed a similar distribution in relation to the causes of death. Markedly higher Cr levels were observed for delayed traumatic death, chronic renal failure (uremia) and MA fatalities compared with the other groups, and moderately higher levels for other poisoning, hyperthermia and GI bleeding compared with injury, asphyxiation, drowning, fire fatalities, hypothermia, AMI and cerebrovascular diseases (Fig. 2a). For these groups, left, right and iliac venous Cr levels showed equivalent correlations to the pericardial level, although the serum level was markedly higher in some cases of hyperthermia: the median ratio were 0.99, 1.03 and 1.05, respectively (Fig. 2b). For all cases, the serum to pericardial Cr ratio was the highest for the iliac venous level (median, 1.30), followed by right cardiac level (median, 1.23) and left cardiac level (median, 1.05) (P < 0.001). There was no significant difference among the causes of death.
3.2.3. Uric acid Markedly higher serum and pericardial UA levels were observed for hypothermia and MA fatalities compared with the other groups, and moderately higher levels for hyperthermia, delayed traumatic death, chronic renal failure (uremia) and cerebrovascular diseases compared with injury, asphyxiation, drowning, fire fatalities, other poisoning, AMI and GI bleeding cases (Fig. 3a). UA levels in serum and pericardial fluid showed an equivalent correlation for hypothermia, delayed traumatic death and chronic renal failure (uremia): the median ratio to the pericardial level for the left, right cardiac and iliac venous levels were 0.99, 1.02 and 0.96, respectively. A significantly higher serum to pericardial UA ratio was observed for MA fatalities, other poisoning and cerebrovascular diseases (Fig. 3b). When analyzed by ROC, the sensitivity and specificity in distinguishing MA fatalities and other groups excluding other poisoning and cerebrovascular diseases using the serum/pericardial ratio were 0.78 and 0.86 at a cutoff value of 1.25 for left cardiac level, 0.93 and 0.80 at a cutoff value of 2.0 for the right cardiac level, and 0.92 and 0.85 at a cutoff value of 1.20 for the iliac venous level, respectively. The UN, Cr and UA levels did not show any significant difference between blunt and sharp instrument injury cases. There was also no significant difference between the subgroups of fire fatalities (COHb level below 60% and above 60%), or between cases with and without estimated high body temperature (>40 °C) at the time of death due to fatal hyperthermia and MA intoxication. 4. Discussion In the present study, a marked elevation in serum UN level along with elevations Cr and UA levels was observed for delayed traumatic death due to multiple organ insufficiency and chronic renal failure (uremia),
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as previously reported [10]. In these cases, their pericardial levels were usually equivalent to the serum levels, suggesting prolonged duration of uremic status, which may be assessed based on serum and pericardial levels following clinical criteria. Similar findings were observed for UN and UA in hypothermia cases, suggesting a prolonged death process involving protein catabolism [21,22]. Elevations in serum and pericardial UN levels in GI bleeding were also explained based on clinical criteria [23]. For hyperthermia and MA fatalities, a mild elevation of serum or pericardial UN level (median value, 22.1–28.5 mg/dl) was not associated with high body temperature. However, in drowning cases independently of the immersion medium (fresh- or saltwater), UN level was significantly lower for the left cardiac blood and pericardial fluid compared with the right cardiac and peripheral levels. This suggests hemodilution of the pulmonary venous blood in the left heart due to water aspiration and a subsequent influence on the components of pericardial effusion or water diffusion from the thoracic cavity, since the bilateral cardiac, peripheral and pericardial UN levels were mildly lower than those for other groups. For diagnosis of drowning, a cutoff value for the right cardiac or peripheral serum/pericardial ratio was estimated at 1.1, where about 70–90% of drowning cases showed positive findings, while false positive findings were observed in about 25% of the other cases, which included various cause of death. The use of this marker in combination with pathological and other biochemical findings may be useful [24–29]. For Cr levels, an increase serum level of fatal MA intoxication cases, associated with a mild elevation in UN level and a marked elevation in UA level, was similar to the clinical findings [30–32]. Similar elevation in the pericardial Cr level suggests considerable duration of an increased serum Cr level due to skeletal muscle damage (rhabdomyolysis) caused by MA intoxication [30–36]. However, the serum UA level showed greater elevation than the pericardial level, with an elevation in each cardiac or peripheral serum/pericardial UA ratios. Furthermore, the serum UA and Cr levels were higher for fatal MA intoxication than those for hyperthermia, showing that there was no significant elevation in UN level. This suggests that hyperthermia and/or renal failure may not be the main factor causing increased serum UA and Cr levels in MA fatality. UA precursors (purines) are mostly produced through the breakdown of nucleic acids in muscle tissues and subsequent enzymatic conversion into UA mainly in the liver [37]. Depressed ATP concentration in the muscle due to hypoxia may be related to overproduction of UA, and acidemia interferes with renal tubular secretion of UA [37–39]. Therefore, severe hypoxia and metabolic acidosis in fatal MA intoxication [31,40,41] may contribute to elevated serum UA. An elevated pericardial UA level suggests a gradually progressing metabolic deterioration
involved advanced hypoxia and subsequent acidosis, accompanied by elevations in UN and Cr levels, antecedent to the onset of terminal fatal events [10,11]. Thus, elevations in serum and pericardial Cr and UA levels with a higher serum/pericardial UA ratio (cutoff value: 1.25 for left cardiac level, 2.0 for the right cardiac level and 1.20 for the iliac venous level) are indicative of fatal MA intoxication. In addition, significant elevations in serum UA levels and higher serum/pericardial ratios for cerebrovascular diseases may indicate advanced terminal hypoxia/acidosis. Similar findings were also observed for fatal poisoning due to substances other than MA. The pericardial fluid is a transudate derived from the epicardium and contains plasma components [42,43]. Postmortem pericardial UN, Cr and UA levels were usually within the clinical serum reference ranges as previously described [11], suggesting considerable stability during the death process and postmortem period. Changes in the pericardial components during systemic metabolic deterioration may gradually occur following those in the coronary blood flow. Differences between serum and pericardial levels may appear depending on the survival period in acute or subacute death cases (survival time < 6 h). Significant changes in pericardial levels of these markers may become apparent hours later than those in the serum [11,17,42]. Thus, elevations in the pericardial UN, Cr and/or UA levels may be suggestive of prolonged survival over hours. As previously described, site-to-site difference in these markers, especially Cr and UA, was also observed for heart and peripheral blood. Higher Cr levels in the peripheral blood and UA levels in the right heart blood, which were often observed in acute and subacute death cases, may be explained as a result of hypoxic skeletal muscle damage and subsequent degradation of purine derivatives in the liver during the process of death [10]. Topographic analyses of these biochemical markers in blood and pericardial fluid may also be useful to investigate death processes, involving systemic nitrogenous metabolic deterioration in causes of acute or subacute death other than MA fatality and in the cerebrovascular diseases described above. These criteria may also be applicable to infantile fatalities when age-dependent reference values are considered [11]. In conclusion, elevations serum Cr and UA levels with a higher serum/pericardial UA ratio are suggestive of acute and progressive skeletal muscle damage due to advanced hypoxia/acidosis and/or systemic metabolic deterioration in the dying process, which was especially evident for MA fatalities. Higher right cardiac and peripheral venous to pericardial UN ratio can be used as a marker for the diagnosis of drowning. These findings suggest that pericardial fluid is useful as reference material to investigate the alteration in serum UN, Cr and UA levels for diagnosing various causes of death.
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Acknowledgments This study was in part supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science and the Ministry of Education, Culture, Sports, Science and Technology, Japan (Grant Nos. 15390217 and 15590585).
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