Double logarithmic, linear relationship between plasma chloride concentration and time since death in humans in Chandigarh Zone of North-West India

1. Introduction 

Many attempts have been made to estimate time since death which is an essential ingredient of every medicolegal postmortem examination but unfortunately there is no device by which it can be determined accurately. Madea and Henssage [1] were of the opinion that dissolution of the morphological, physical and chemical integrity of a dead body occurs due to autolytic process which commences soon after the stopage of energy metabolism in a cell. Cessation of active membrane transport and loss of selective membrane permeability occurs because of this energy breakdown resulting into diffusion of ions depending on their concentration gradient. As the extent of autolysis in a cadaver is dependent on time since death [1], number of researchers [2], [3], [4], [5], [6], [7], [8], [9], [10], [11] have attempted to correlate time since death with the biochemical changes in various body fluids viz. blood, vitreous humor, cerebro-spinal fluid, etc. In blood, Jetter [2] was perhaps first to document that chloride concentration falls through intra-cellular shift at the rate of 80–90 mEq/l per day with the advancement of postmortem interval. Later Schliyer [3] observed that rate of fall was between 0.25 and 1 mEq/l per h, whereas Coe [4], [12] was of the opinion that its fall was approximately 0.97 mEq/l per h for first 15 h of death. Camps et al. [13] were of the view that soon after death, the chloride concentration in plasma and that of erythrocytes equalize approximately at a level of whole blood and owing to extravascular diffusion drops to half in 72 h. Querido [5] demonstrated a highly significant double logarithmic relationship between plasma chloride concentration and postmortem interval in wistar rats and had concluded that because of similarities in rat and human blood, same can be applicable to humans also. Present study was carried out to substantiate this notion in human and also to observe role of factors like environmental temperature, age, gender and cause of death over the plasma chloride concentration.

2. Materials and methods 

With valid consent of legal heirs of the deceased 10 ml of blood was collected from right femoral artery of 474 subjects (333 males, 141 females), who were admitted and died because of various medicolegal causes (Trauma-292, Burn-113, Poisoning-69) at Postgraduate Institute of Medical Education and Research, Chandigarh (India) and on whom postmortems were conducted between 3 and 58 h of death by the Department of Forensic Medicine.

In the present study only those subjects were included in which exact time, cause, mode of death and other demographic profile were known and the bodies were kept at room temperature. Subjects with significant ante mortem electrolyte imbalance or on diuretics were not included in the study. In order to avoid any external factor which could influence the plasma electrolyte concentration viz. microbes, etc., blood was collected under sterilized condition with the help of 20 gauge hypodermic needle with syringe, transferred to sterile glass vials and estimation of chloride concentration was done immediately by mercurimetric titration method [14], [15].

To ascertain the relationship between postmortem levels of plasma chloride concentration and environmental temperature, blood was collected in 294 subjects in summer season (April–September, mean room temperature 38.1°C) and in 180 subjects in winter season (October–March, mean room temp 14.1°C). Youngest subject from which blood collected was an 8 year boy and oldest a 79 year female. In order to observe the role of age factor in estimation of time since death based on chloride concentration subjects were divided into four categories viz. <12 years (children), 12–18 years (adolescent), >18–60 years (adult and middle age) and >60 years (old age). The data was statistically evaluated and tested by using the least square analysis of linear regression.

3. Results 

3.1. Absolute value of plasma chloride and mean rate of fall 

Plasma chloride concentration decreased from mean 94.96±6.24 mEq/l at postmortem interval of 3–6 h to mean 63.55±4.93 mEq/l at postmortem interval of 48–58 h. Fall of chloride concentration was rapid in initial 12 h of death and mean rate of fall of chloride concentration was 0.86 mEq/l per h (Table 1).

Table 1. Plasma chloride electrolyte concentration at various postmortem intervals

	Time since death (h)	No. of subjects	Mean time since death (h)	Mean chloride concentration (mEq/l)	Mean fall of chloride concentration (mEq/l per h)
	3–6	21	5.16±0.84	94.96±6.24	1.07
	6.01–12	87	9.95±1.70	88.75±10.98	1.18
	12.01–18	94	15.44±1.66	85.90±8.57	0.94
	18.01–24	119	21.51±1.84	81.65±11.12	0.87
	24.01–30	93	26.73±1.59	79.93±9.7	0.77
	30.01–36	21	33.87±1.92	71.71±10.73	0.85
	36.01–42	16	39.09±1.72	70.86±7.65	0.75
	42.01–48	13	45.06±1.65	66.29±13.23	0.76
	48.01–58	10	52.91±3.31	63.55±4.93	0.70
	Total	474	20.94±10.24	82.45±11.94	0.86
	Correlation with time since death			−0.549a

a P<0.001.

3.3. Relationship between plasma chloride concentration and environmental temperature, age, gender and cause of death 

Plasma chloride concentration was found highly dependent on the factors like environmental temperature, age, gender and cause of death (P<0.0001). Rate of fall of plasma chloride concentration was more in cold climate (1.03 mEq/l per h) than in hot climate (0.76 mEq/l per h). Similarly, fall was more in subjects between the age of >12–18 years (0.96 mEq/l per h) followed by in the age group of >18–60 years (0.86 meq/l per h) and >60 years (0.75 mEq/l per h) and least in children (0.70 mEq/l per h) below <12 years. Rate of fall chloride concentration was more in females (0.88 mEq/l per h) than in males (0.85 mEq/l per h). Fall was more in deaths due to burn (0.93 mEq/l per h) than due to trauma and poison (0.85 mEq/l per h).

In order to establish the effect of factors like gender, cause of death, environmental temperature and age on relationship between chloride concentration and time since death, the separate prediction equations in each factor have been derived (Table 2). The multiple R coefficient, along with SE of estimate between log time since death and log plasma chloride concentration as shown in the table were found to be significant for each equation relating to the above factors.

Table 2. Regression equations for prediction of time since death from plasma chloride concentration separately for gender, environmental temperature, cause of death and age

		Variable	log y= log a+b log x	Number of subjects	Multiple R	SE of estimate	t-value for testing Ho: β=0 versus H1: β≠0	95% confidence interval for slope	F-value	d.f.
	Gender	Male	log TSD=4.293−1.587 log Cl	333	0.470b	2.1	−9.699b	−1.908 to −1.265	94.061b	1, 331
		Female	log TSD=2.661−0.722 log Cl	141	0.452b	2.0	−4.438b	−1.044 to −0.401	19.697b	1, 139
	Season	Summer	log TSD=2.862−0.832 log Cl	294	0.520b	2.2	−5.781b	−1.115 to −0.548	33.422b	1, 292
		Winter	log TSD=5.639−2.311 log Cl	180	0.656b	1.8	−11.592b	−2.705 to −1.918	134.373b	1, 178
	Cause of death	Trauma	log TSD=4.251−1.566 log Cl	292	0.466b	2.1	−8.965b	−1.910 to −1.222	80.378b	1, 290
		Burn	log TSD=2.383−0.569 log Cl	113	0.427a	1.9	−3.648a	−0.877 to −0.260	13.310b	1, 111
		Poison	log TSD=5.862−2.402 log Cl	69	0.552b	1.9	−5.414b	−3.288 to −1.517	29.316b	1, 67
	Age groups in years	<12	log TSD=6.395−2.844 log Cl	10	0.686c	1.2	−2.666c	−5.303 to −0.384	7.107b	1, 8
		12–18	log TSD=4.734−1.839 log Cl	36	0.550a	2.0	−3.844a	−2.811 to −0.867	14.775b	1, 34
		18–60	log TSD=3.167−0.991 log Cl	385	0.484b	2.0	−8.130b	−1.231 to −0.751	66.096b	1, 383
		>60	log TSD=8.895−3.988 log Cl	43	0.727b	2.0	−6.784b	−5.175 to −2.801	46.020b	1, 41

a P<0.01. b P<0.0001. c P<0.05.

It should be noticed that regression coefficient for each equation was negative which means that decrease in chloride concentration results into increase in time since death. Moreover, these coefficient have been tested by using t-statistic along with their 95% confidence limits. Since all these values were significant which means decrease in chloride concentration was playing a major role in determination of time since death.

Validity of the regression model for each factor viz. gender, cause of death, environmental temperature and age have been tested by applying analysis of variance table and their final F-value were presented in Table 2 along with respective degree of freedom. Since these values were highly significant showing that these factors had a high degree of influence over the relationship between plasma chloride concentration and time since death on a double logarithmic scale.

4. Discussion 

Observations of the present study revealed that chloride concentration decreases linearly with the advancement of time since death and it was similar to as observed by Coe [4], [12] in human's blood for the 0–24 h postmortem period and by Querido [5] in Wistar rats. Its rate of fall (0.86 mEq/l per h) was also almost similar to what was observed by other researchers [2], [3], [4], [5]. Various methods/models have been used to establish the relationship between fall of plasma chloride concentration and time since death [2], [3], [4], [5], [8]. Coe [4] with the help of mathematical models came to the conclusion that with least square regression analysis, almost accurate result could be obtained in early postmortem interval and in long postmortem interval improvement can be made by applying the square root of time or an exponential equation. However, Querido [5] used double logarithmic relationship model and found highly significant relationship between time since death and plasma chloride concentration in the wistar rats. In present study by using the double logarithmic model, a highly significant relationship (P<0.0001) between the log time since death and log plasma chloride was observed in humans but this relationship was found highly dependent on the factors like cause of death, environmental temperature, gender and age.

No literature was available to authors to discuss the role of these factors over the relationship between time since death and plasma chloride concentration. However, duration of terminal episode which was reported to be an important factor influencing electrolyte concentration especially of potassium in various body fluids [9], [10], [11] might have also played its role in rapid fall of plasma chloride concentration in deaths due to burn as observed in present study. Extensive ante mortem damage of body tissues leading to early cessation of active membrane transport and loss of selective membrane permeability of cell(s) specially during supravital and beginning of autolysis period might have resulted in rapid extravascular shift of chloride in burn deaths. Autolytic process is modulated by factors like environmental temperature and condition of a dead body viz. fluid content and subcutaneous fat of the body [16]. Observation regarding the seasonal variation in the mean fall of plasma chloride concentration may be due to the reason that mean temperature in both climates in present study was beyond the reported optimum ambient temperature favouring the process of decomposition, i.e. 21–38°C [16]. Therefore, the bodies decomposed at different rate resulting in variation in mean fall of plasma chloride concentration in hot and cold climate. Abundant subcutaneous fatty tissues as found in females retains body heat for a longer period which enhances the process of decomposition [16], thereby may be leading to more fall of plasma chloride concentration in females than in males in present study. Similarly, low fluid contents as seen in the body of elderly persons inhibit the growth of micro-organisms causing delays in the process of decomposition and possibly because of this reason mean fall of concentration was less in elderly persons, i.e. >60 years. Due to limited number of subjects (10) in below 12 years no statistical conclusion could be drawn for this age group.

In the present study equation for prediction of log time since death from postmortem plasma chloride concentration was derived with SE estimation of 2.1 h with 95% confidence interval for slope. Besides this, equation to predict time since death in each factor, i.e. age, gender, environmental temperature and cause of death which influences the plasma chloride concentration were derived.

It can be concluded that though there is significant relationship exist between plasma chloride and time since death in human blood but this relationship is equally affected by the factors like environmental temperature, age, gender and cause of death of the deceased.