Experimental adipocere formation: implications for adipocere formation on buried bone

Adipocere, or grave wax (adipo = fat, cere = wax), is a distinctive decomposition product composed primarily of fatty acids (FA) and their alkali salts. FA result from the bacterial enzymatic hydrolysis of body fats. Reactions with ammonia and alkali metals originating from body fluids and pore waters of the depositional environment produce alkali salts of FA (soap). Adipocere formation is generally associated with burial of corpses with ample adipose tissue available. No indications that adipocere can form on defleshed remains have been presented in the literature. At the termination of a long-term bone diagenesis experiment, several samples were found to possess growths of an unknown compound. Gas chromatography-mass spectrometry confirmed that the growths are adipocere. The results herein reveal that adipocere can indeed form on defleshed bones under the right conditions and that even residual adipose and lipids in defleshed bones are sufficient to produce adipocere growth on the surfaces of bone.     

Modern concepts of adipocere formation mechanisms

Biochemical parameters of transformation of fresh subcutaneous fat into adipocere have been studied in experiment. Activation of lipid peroxidation is a significant component of the mechanism of adipocere transformation, which is proven by a drastic increase in the level of Schiff's bases in adipocere in comparison with subcutaneous fat. The concentrations of myristic, palmitic, and stearic fatty acids increase during the formation of adipocere, while the content of linoleic acid decreases in comparison with the unchanged human subcutaneous fat.     

Preliminary quantitative investigation of postmortem adipocere formation

The accurate determination of postmortem interval (PMI) using the formation of adipocere presents a significant challenge to forensic scientists interested in determining the time of death. Several attempts have been made to determine the time since the occurrence of death. However, up to date, this has been difficult because previous approaches have been mainly qualitative, focusing on the later stages of degradation processes. This work presents preliminary results of an experimental model of postmortem adipocere formation using liquid chromatography. Three pig cadavers were submerged in distilled water, chlorinated water, and saline water. Fresh specimens resulting from the degradation in the subcutaneous fat were obtained from the pigs at two-week intervals for a period of ten weeks, and were subjected to chromatographic analysis. By correlating the ratio of the disappearance of hydrolyzed fatty acids with the formation of hydroxystearic and oxostearic acids after death, a simple, quantitative analytical method was developed for the determination of PMI. Experimental observation of the chemistry of adipocere formation indicated that adipocere can be formed only a few hours after an incidence of death and this continues until the saturation of oleic acid degradation after several weeks. Different time courses were obtained for cadavers immersed in distilled, chlorinated, and saline water, respectively. This work has not in any way solved the time since death problem. But it may be an approach to the problem that has not been adequately explored.     

The effect of soil type on adipocere formation

Adipocere refers to a postmortem product which forms from body fat in the later stages of decomposition. Factors present in the surrounding decomposition environment will influence adipocere formation and may accelerate or retard the process of conversion. One such factor important in burial environments is the type of grave soil in which the burial has taken place. This study was conducted to investigate the influence of various soil types on the formation of adipocere in grave soils. X-ray diffraction and particle size analysis were used to characterise the soils which were essentially chosen on the basis of grain size. Infrared spectroscopy, inductively coupled plasma-mass spectrometry, and gas chromatography-mass spectrometry were used to investigate the lipid profile and chemical composition of adipocere developed from decomposing tissue. The results suggest that adipocere is able to form in various soil types and that particular soil environments may accelerate its formation.     

A preliminary investigation of the stages of adipocere formation

Adipocere is a postmortem decomposition product which forms from a body's adipose tissue. This study aimed to chemically demonstrate the process of conversion from adipose tissue to adipocere. Samples of adipocere were collected from pig cadavers that were allowed to decompose for varying intervals. Samples of soil were collected from beneath the cadavers and analyzed to determine the leaching effect of adipocere. Gas chromatography/mass spectrometry (GC/MS) was used to quantify the fatty acid composition of pig adipocere. Fourier transform infrared spectroscopy (FTIR) was used as a confirmatory test and to identify other components such as triglycerides and calcium salts of fatty acids. The study demonstrates the process of adipocere formation and the stages of formation through which the process passes using chemical techniques.     

The effect of the burial environment on adipocere formation

Adipocere is a decomposition product comprising predominantly of saturated fatty acids which results from the hydrolysis and hydrogenation of neutral fats in the body. Adipocere formation may occur in various decomposition environments but is chiefly dependent on the surrounding conditions. In a soil burial environment these conditions may include such factors as soil pH, temperature, moisture and the oxygen content within the grave site. This study was conducted to investigate the effect of these particular burial factors on the rate and extent of adipocere formation. Controlled laboratory experiments were conducted in an attempt to form adipocere from pig adipose tissue in model burial environments. Infrared spectroscopy, inductively coupled plasma-mass spectrometry, and gas chromatography-mass spectrometry were employed to determine the lipid profile and fatty acid composition of the adipocere product which formed in the burial environments. The results suggest that adipocere can form under a variety of burial conditions. Several burial factors were identified as enhancing adipocere formation whilst others clearly inhibited its formation. This study acts as a preliminary investigation into the effect of the burial environment on the resultant preservation of decomposing tissue via adipocere formation.     

The effect of the method of burial on adipocere formation

A controlled laboratory experiment was conducted in order to investigate the effect of the method of burial (i.e. the presence of coffin and clothing) on the formation of adipocere. This study follows previous studies by the authors who have investigated the effect of physical conditions on the formation of adipocere present in a controlled burial environment. The study utilises infrared spectroscopy to provide a preliminary lipid profile of the remains following a 12 month decomposition period. Inductively coupled plasma-mass spectrometry was employed as a technique for determining the salts of fatty acids present in adipocere. Gas chromatography-mass spectrometry (GC-MS) was used as the confirmatory test for the identification and determination of the chemical composition of adipocere which formed in the controlled burial environments. The results suggest that coffins will retard the rate at which adipocere forms but that clothing enhances its formation. The results concur with previous observations on adipocere formation in burial environments.     

The effect of body coverings on the formation of adipocere in an aqueous environment

Adipocere is a postmortem decomposition product that consists of a mixture of fatty acids. The rate of formation of adipocere from pig adipose tissue in an aqueous environment has been monitored. The effect of various clothing and carpet material types on the process was investigated. The fatty acid composition of the adipocere was determined at regular intervals using gas chromatography-mass spectrometry. Examination of the changes to fatty acid concentrations allowed the degree of adipocere formation in the different environments to be estimated. The study demonstrated that the rate at which adipocere forms is particularly accelerated by the presence of coverings produced from natural materials. Elemental analysis by inductively coupled plasma-mass spectrometry revealed, for the most part, little change to the cations present in the adipocere formed. However, an increase in Ca concentration was observed for tissue wrapped in acrylic carpet, which was associated with a CaCO(3) additive used in the carpet manufacture.     

The formation of early stage adipocere in submerged remains: a preliminary experimental study

In some circumstances, the presence of adipocere may retard decomposition and complicate postmortem interval estimation. This article explores the correlation between Accumulated Degree Days (ADD) and early stage formation of adipocere. Sixty wild rabbit (Oryctolagus cuniculus) carcasses were used in this experiment; a control group (N = 30) deposited directly on the ground surface and an experimental group (N = 30) completely submersed in water in individual buckets. Data (water and inner body temperature, pH, and total body score) were collected every 100 ADD. Results indicated that early stage adipocere is correlated to ADD and that its formation on submersed remains is more likely to occur after 630 ADD. Skin sloughing promoted the formation of adipocere. No adipocere was formed on any of the control group rabbits. This study also highlights the fact that multiple factors influence adipocere formation and it is suggested that further research needs to be conducted into this area.     

Forensic toxicological study on adipocere formation in submerged cadavers of female albino rats intoxicated with cadmium

There is a dearth of information on the mutual interaction between metal intoxication and adipocere formation. Herein, 40 adult female albino rats were distributed into two equal groups, one used as control while the other orally administered single dose of cadmium chloride (CdCl₂) 225 mg/kg·bw (LD_min). Control group was killed by cervical dislocation. Half of dead rats of both groups were subjected for determination of iodine value and estimation of cadmium (Cd) residues while the other half of both groups were submerged in opened glass container previously filled with 4 L dechlorinated tap water kept in closed room with an open air access (one cadaver/container). Gross morphological changes of submerged cadavers were recorded weekly along the experiment. At the end of the experiment, after 3 months, samples were collected again for iodine value determination and estimation of Cd residues. The obtained results revealed the depressant effect of Cd toxicity on development of adipocere. Cd residues were found in different tissues of cadavers at time of death with the highest amount in the intestines followed by the liver and kidneys, then lungs, adipose tissue, muscles, and finally the bones. After 3 months of water submersion, tissues exhibited significant decrease in the amount of Cd residues but to a limit that was still detected. This study concluded the possibility of detection of Cd residues even after adipocere formation. Additionally, it shed light on the possibility of the interference of environmental pollution with the natural rate of decomposition especially adipocere formation.     

Marine taphonomy: adipocere formation in a series of bodies recovered from a single shipwreck.

Taphonomy of marine environments has been studied mostly from individual cases. The formation of adipocere, or "grave-wax," is an important indicator of the postmortem interval. In the present paper, the conditions and the timing of adipoceric formation are observed in a series of 15 cadavers recovered at different times, over a period of 433 days, from the same contained environment. Initial foci of adipocere on the subcutaneous tissue of the cadavers were detected as early as 38 days from the time of immersion in cold (10-12 degrees C) sea water. The discrepancies between our findings and previous reports on the correlation between time since death and decomposition stages in marine environments are discussed.     

The identification of adipocere in grave soils

Soil samples recovered from grave exhumations have been analysed in an attempt to identify and characterise adipocere contained in the samples. The soil samples were collected from different environments, including samples recovered from forensic grave sites. Gas chromatography-mass spectrometry (GC-MS) was employed to identify adipocere and characterise the fatty acid composition. X-ray diffraction was used to characterise the soil environments.     

Identification of 10-hydroxy-12-octadecenoic acid in adipocere

Studies are reported on separation and identification of 10-hydroxy-12-octadecenoic acid in human adipocere. The chemical structure was determined by thin-layer chromatography (TLC), gas-liquid chromatography (GLC) and gas chromatography-mass spectrometry (GC-MS).     

Microbial production of hydroxy and oxo fatty acids by several microorganisms as a model of adipocere formation

Some varieties of aerobic or anaerobic microorganisms from the human stool and adipocere were separated and identified. These separated microorganisms together with other authentic ones produced 10-hydroxystearic acid from oleic acid. Some bacteria could convert oleic acid to 10-oxostearic acid as well as 10-hydroxystearic acid. These findings indicate that the microbial enzyme(s) catalyzes the hydration of oleic acid and probably the oxidation of this hydrated product. Aerobic bacteria as well as anaerobic microorganisms were found to be involved in the formation of adipocere.     

The mechanism of experimental adipocere formation: hydration and dehydrogenation in microbial synthesis of hydroxy and oxo fatty acids

The enzyme preparations from Flavobacterium meningosepticum solubilized by sonication catalyzed not only hydration of oleic acid to produce 10-hydroxystearic acid but dehydrogenation of this product. The mechanism of the hydration and dehydrogenation was proved by gas chromatography-mass spectrometry of 10-hydroxy and 10-oxostearic acids produced in the presence of D2O or H2(18)O. The activity of these enzymes was increased by preincubating Flavobacterium meningosepticum with oleic acid.     

The mechanism of experimental adipocere formation: substrate specificity on microbial production of hydroxy and oxo fatty acids

Studies are reported on microbial conversion of various unsaturated fatty acids to 10-hydroxy and/or 10-oxo fatty acids by Micrococcus luteus. Four fatty acids possessing cis-9-unsaturation produced 10-hydroxy and 10-oxo fatty acid products, but three enoic acids possessing trans-9-unsaturation or double bond(s) in other than the 9-carbon position were inactive as substrates. 10-Hydroxy palmitic and stearic acids were converted to the corresponding 10-oxo fatty acids, but the 10-oxo compounds were inactive as substrates. This indicates that the metabolic sequence of cis-9-enoic fatty acid by the microbial enzyme(s) is first converted to 10-hydroxy fatty acid and then to its 10-oxo compound.     

Waxing grave about adipocere: soft tissue change in an aquatic context

When postmortem environmental conditions are "just right," according to the "Goldilocks Phenomenon," soft tissues (and associated fatty acids) are converted into and preserved as adipocere. To better understand this conversion process and the development of adipocere three human cadavers were immersed in outside, water-filled pits for over 3 months to observe adipocere formation in an underwater context simulating actual field conditions. Recordings of environmental conditions showed that temperatures were between 21 degrees C and 45 degrees C, a range sufficient for the growth of Clostridium perfringens. Chemical analysis of liquid and tissue samples revealed an increase in palmitic acid and decrease in oleic acid. This study tracked the remarkable gross morphological changes that can occur in human bodies subjected to an aquatic postmortem environment. The results support the "Goldilocks Phenomenon" and substantiate previous findings that the presence of bacteria and water is crucial for adipocere to form.     

Postmortem decomposition of neutral lipids. Use of modern methods of analysis (HPLC, capillary GC, GC-MS and NMR) in adipocere formation

The chemical composition of subcutaneous fats was analyzed in a corpse that had died from drowning. The skin of the cadaver examined postmortem showed different stages of adipocere. Samples from these regions were chemically compared with the fatty tissue of a person who had died recently. HPLC, GC, GC-MS, NMR (1H- and 13C-NMR), TLC and titrimetrical methods were used to evaluate the degree of decomposition. The fatty acid pattern of the triglycerides (TG) and the free fatty acids (FFA) obtained by TLC separation was also investigated. Some discrepancy was observed between the autopsy findings and the results of the chemical analysis. It is suggested that the autopsy should be supplemented by chemical analysis in order to describe the state of adipocere correctly.     

A molecular genetic approach for forensic animal species identification

This study investigated potential markers within chromosomal, mitochondrial DNA (mtDNA) and ribosomal RNA (rRNA) with the aim of developing a DNA based method to allow differentiation between animal species. Such discrimination tests may have important applications in the forensic science, agriculture, quarantine and customs fields. DNA samples from five different animal individuals within the same species for 10 species of animal (including human) were analysed. DNA extraction and quantitation followed by PCR amplification and GeneScan visualisation formed the basis of the experimental analysis. Five gene markers from three different types of genes were investigated. These included genomic markers for the beta-actin and TP53 tumor suppressor gene. Mitochondrial DNA markers, designed by Bataille et al. [Forensic Sci. Int. 99 (1999) 165], examined the Cytochrome b gene and Hypervariable Displacement Loop (D-Loop) region. Finally, a ribosomal RNA marker for the 28S rRNA gene optimised by Naito et al. [J. Forensic Sci. 37 (1992) 396] was used as a possible marker for speciation. Results showed a difference of only several base pairs between all species for the beta-actin and 28S markers, with the exception of Sus scrofa (pig) beta-actin fragment length, which produced a significantly smaller fragment. Multiplexing of Cytochrome b and D-Loop markers gave limited species information, although positive discrimination of human DNA was evident. The most specific and discriminatory results were shown using the TP53 gene since this marker produced greatest fragment size differences between animal species studied. Sample differentiation for all species was possible following TP53 amplification, suggesting that this gene could be used as a potential animal species identifier.     

The effect of conditions of putrefaction on species determination in human and animal teeth

Tooth fragments freshly extracted from humans and rats were stored at either 4 degrees C or room temperature in dry or humid conditions for periods ranging from 1 to 6 months. The fragments were reduced to powder and antigens were extracted. Comparison of these samples was carried out using Counter Current Electrophoresis. Extracted sera were tested against known specific antisera and resultant precipitin reactions stained for examination. Correct species identification was possible both from desiccated and humid fragments but there was species variation in the sensitivity of the method. All the extracts from human teeth were positive against human antisera. In the rat some test specimens were initially negative but became positive following further dilution of the extracts.