Bada et al. [9] proposed to use the D-Asp content in bone to detect a postmortem heating of bone (as proof of burial “more teutonico”). Since this question can also be of significance in a forensic context, we reviewed this approach. We also tested whether postmortem boiling could be proven by analysing the Pen content of bone samples (“Pen approach”).
The number of human samples analysed is relatively small. After evaluation of the data presented here, it was very clear that no further significant insights were to be expected from analysing more human samples. Therefore—and ultimately for ethical reasons as well—it was decided not to analyse further human samples.
The “Pen approach” is not suitable to answer the question of whether bones had been heated (boiled) postmortem.
Pen proved to be remarkably heat-stable. In the boiling experiments, the Pen content of bone samples did not show significant differences between boiled and non-boiled samples (Fig. 3a, b). Therefore, the analysis of Pen is not suitable for proving a burial “more teutonico”.
The approach taken by Bada et al. [9] to clarify the question of a burial “more teutonico” should not be used uncritically.
The approach proposed by Bada et al. [9] is based on the basic assumption that temperature determines the D-Asp content of bones so significantly that an increased D-Asp content in a bone sample can be considered as proof of heat exposure. Unfortunately, this basic assumption cannot be maintained in view of the recent literature and our experiments, which demonstrate a high complexity of the factors determining the postmortem D-Asp content in bone, which were not considered by Bada et al. [9].
Water is not wine: The boiling medium influences the kinetics of D-Asp accumulation in bone
Bada et al. [9] assumed that Lothar I was boiled in water. However, this may not have been the case, as historical sources also suggest that wine and vinegar were used for boiling of human remains. Our boiling experiments demonstrate a significant influence of the type of the boiling medium used (Fig. 2b, c). This is likely due to different effects on the inorganic and organic bone matrix. Different influences from boiling media may lead to different degradation processes and molecular changes [22,23,24]. This effectively changes the protein composition of the samples. Since the kinetics of the conversion of L-aspartic acid to its D-form depend on the structure of the respective “parent proteins” [24,25,26], this may result in altered kinetics of the heat-induced accumulation of D-Asp. As it is likely impossible to reconstruct the medium used for boiling in most (historic) cases, it will remain unclear to what extent the boiling process could have influenced the D-Asp content of a bone sample.
The selection of control samples is critical, but achieving this optimally is difficult
For the “Bada approach” it is of central importance to compare the D-Asp content of presumably boiled samples with the D-Asp content of non-boiled samples from individuals with a comparable postmortem interval. However, when selecting their control cases, Bada et al. [9] failed to consider some relevant findings:
Bada et al. [9] did not consider the intravital accumulation of D-Asp in bone tissue (see data for non-boiled samples in Fig. 2c and [10,11,12,13]). The age at death significantly determines the D-Asp content in bone. The intravital age-dependent accumulation of D-Asp results in different initial values at the time of death, before any further increase in D-Asp content may have been provoked by heat exposure after death. Bada et al. [9] did not state the age at death of their control individuals. If these two individuals were significantly younger than Lothar I. when they died, this could explain significantly lower D-Asp levels in their bone samples, at least to some extent. In any case, Bada et al. [9] should have considered the intravital “basal accumulation” of D-Asp and should ideally have used non-boiled control samples from individuals of a similar age at death.
Moreover, Bada et al. [9] obviously did not consider the structural and metabolic differences of bone at specific anatomical sites, resulting in different kinetics of the intravital accumulation of D-Asp [14]. When applying the approach of Bada et al. [9], the sample to be tested would need to have come from the same anatomical location as the control samples. Bada et al. [9] do not provide any information on the anatomical region of the samples they analysed.
A further point not considered by Bada et al. [9] is the possibility of increased D-Asp content in non-boiled samples due to long postmortem intervals. Depending on postmortem conditions, D-Asp may accumulate during the postmortem interval, even in the absence of heat exposure. Other conditions apart from high temperature that can lead to an unusually rapid degradation of bone proteins are also relevant, such as specific microbial influences or exposure to high humidity, and/or extreme pH conditions [15, 27,28,29,30]. Protein degradation during the postmortem interval may result in small peptides with only low steric hindrances and a significantly faster formation of D-Asp residues [24,25,26]. Mahlke et al. [19] report unusually high D-Asp contents in (non-boiled) dentine in cases with long postmortem intervals and discuss a postmortem increase of D-Asp due to degradation. Without knowledge of the environmental conditions during the entire postmortem interval, the interpretation of strikingly high postmortem D-Asp levels is difficult, especially the differentiation between the effects of heat exposure and those of other degradation-inducing influences.
The kinetics of the heat induced accumulation of D-aspartic acid in animal bones are not comparable to those in human bones
Using data from boiling experiments with deer femur bones, Bada et al. [9] came to the conclusion that Lothar’s bones were boiled for “6 h ± 30 min”. However, our boiling experiments with pig and human bones revealed that the data obtained from animal bones cannot easily be translated to human bones. The pig bone samples show a significantly faster accumulation of D-aspartic acid at identical heat exposure. This could be due to lower steric hindrances and fewer crosslinks in the collagen, especially since the pigs were very young at the time of death (6–9 months). Dobberstein et al. [31] reported very similar results from heating experiments with teeth: The dentinal collagen from young pigs degraded much faster than that from humans (16–28 years old).
All things considered, the proof of a treatment “more teutonico” based solely on the “Bada approach” and the D-aspartic acid content seems hardly possible – and if so, only under very strict conditions.
If, despite all concerns, the use of the method proposed by Bada et al. [9] should nevertheless be considered, the following prerequisites must be met: It should be examined whether there is any information (e.g., from historical sources) regarding the “boiling method” used in the specific case. The non-boiled/unheated control samples should come from individuals with similar or, ideally, identical ages at death and death dates, and they should also stem from identical anatomical locations. Furthermore, it should be ensured that all samples show no evidence of unusual influences during the postmortem interval.