Leveraging IsoArcH for isotope paleopathology: The example of the dataset from the Jedlička collection (Central Europe, 19th century CE)

The article introduces the enhancements made to the IsoArcH database for isotope paleopathology. This includes the addition of new metadata fields, which allow for describing abnormal anatomical or physiological conditions in humans and animals at either the individual or sample level. To showcase the novel features of the database, the article features a unique dataset of carbon and nitrogen isotope values obtained on bulk bone collagen from 42 clinically-documented cases of the Jedlička pathological-anatomical reference collection, dating from the 19th century CE and curated at the National Museum in Prague, Czechia. The dataset includes 70 combined isotopic measurements from individuals who underwent anatomizations between 1841 and 1900 and had distinct bone diseases/disorders: i.e. syphilis, rickets, osteosarcoma, osteomyelitis, and healed fractures. Finally, the article highlights the value of the data in helping the isotope bioarchaeology and paleopathology communities in their understanding of disease processes.

The article introduces the enhancements made to the IsoArcH database for isotope paleopathology.This includes the addition of new metadata fields, which allow for describing abnormal anatomical or physiological conditions in humans and animals at either the individual or sample level.To showcase the novel features of the database, the article features a unique dataset of carbon and nitrogen isotope values obtained on bulk bone collagen from 42 clinicallydocumented cases of the Jedli čka pathological-anatomical reference collection, dating from the 19th century CE and curated at the National Museum in Prague, Czechia.The dataset Specifications Pre-treatments on bones were performed at the National Museum's Department of Anthropology located in Prague (Czechia).Bone powder underwent defatting using Kates' and Liden et al.'s methods [ 1 , 2 ].Bone collagen was extracted using Longin's methodology [3] , with modifications made by DeNiro and Epstein [4] , Brown et al. [5] , and Bocherens et al. [ 6 , 7 ].
Stable carbon and nitrogen isotopic compositions were determined using a Europa Scientific Roboprep elemental analyzer coupled with a Europa Scientific 20-20 isotope ratio monitoring mass spectrometer at Iso-Analytical Limited (Crewe, UK).Measurement accuracy and precision are reported following the guidelines outlined by Szpak et al. [8]

Value of the Data
• Pathological-anatomical reference collections showcasing bone diseases/disorders from the pre-antibiotic era are rare on a global scale, yet their value is tremendous, particularly in the context of isotopic studies.They offer a unique opportunity to provide insight into the extent to which bone pathologies, not influenced by modern drugs, influence the inherent stable isotope signatures in bone collagen.The present paper focuses on the Czech Jedli čka collection.• The article features carbon and nitrogen isotope data -most of which have never been published before -on five bone diseases/disorders that have received little or no attention in previous isotope studies.The potential value of this data lies in its ability to aid the isotope bioarchaeology and paleopathology communities in their understanding of disease processes.• The IsoArcH database has been suitably adjusted to fulfill the requirements of isotope paleopathology and to integrate paleopathological data.The database is now a useful resource for paleopathologists and isotope researchers to share and initiate dialogue around paleopathological and isotopic data.

Objective
Isotope paleopathology is an emerging field that shows promise in shedding light on complex matters pertaining to human health [12] .In order to cope with the increasing amount of data and publications in this field, the IsoArcH database has been updated by adding new metadata fields related to paleopathological observations.The options to input free text for describing anomalous anatomical or physiological conditions is now available for both humans and animals, either at the individual or sample level.A one-of-a-kind dataset of carbon and nitrogen isotope values, specifically related to pathological materials extracted from the 19th century Jedli čka collection curated at the National Museum in Prague, Czechia, is used as an example to showcase the novel features.

Data Description
The present data article deals with materials deriving from a pathological-anatomical reference collection, the so-called Jedli čka collection, curated at the the Horní Po černice's depositories of the Department of Anthropology in the National Museum in Prague (Czechia).Individuals with syphilis ( n = 10), rickets ( n = 7), osteosarcoma ( n = 5), osteomyelitis ( n = 10) and healed fractures ( n = 10) were selected.Individuals afflicted by systemic diseases, such as rickets and syphilis, were sampled only once, with the exception of AJ3358/AJ3384, AJ3365/AJ3366 and AJ3369/AJ3370, for whom left and right femurs were sampled for testing intra-skeletal variations.Individuals afflicted by localized bone pathological changes or trauma were sampled twice, at the lesion site and distance to the lesion site.The final dataset consists of 70 combined measurements of stable carbon and nitrogen isotope ratios on bulk collagen.All but two measurements (at the lesion site for SA3374 and distant to the lesion site for OS2950) met the collagen quality criteria.These two measures are included in the dataset submitted to the IsoArcH database.The raw data are presented in Table 1 .Means and standard deviations for each pathological condition are shown in Table 2 and Fig. 1 .A subset of the dataset featured in this data article, comprising information on individuals who suffered from syphilis and had healed fractures, was released by Salesse et al. [11] .Plots were generated and calculations were conducted using Microsoft Excel.

Table 1
Biological and isotopic data of individuals from the Jedli čka collection in Prague (19th century CE).Notes: The known age-at-death is mentioned in brackets.The given sources refer to the pages where the cases are described in Smr čka et al. [13]  * and Ortner [14] * * .%Col corresponds to the extraction yield, expressed as a weight percentage (wt.%).%C and %N indicates the carbon and nitrogen contents of samples and are expressed as percentages.δ 13 C and δ 15 N values were measured on bone collagen samples and are reported as per mil ( ‰ ) deviation relative to VPDB and AIR, respectively.

Material and sampling strategy
The Jedli čka collection comprises approximately 6,0 0 0 anatomical preparations of both soft and hard tissue diseases/disorders obtained from clinical human dissections performed between roughly 1840 and 1950.It includes unique examples of inflammatory and infectious bone diseases from the pre-antibiotic era, as well as numerous examples of skeletal abnormalities, bone disorders, and other osteological changes.Medical diagnoses were predominantly made on living patients, and clinical reports or documentations detailing the dissected cases have been preserved to this day for many of them [ 11 , 13 ].The Jedli čka collection is a rare, historical resource for the study of pathological anatomy and medicine, shedding light on population health and medical care in Czech lands and Central Europe during the 19th and 20th centuries CE.
A restricted quantity of samples was selected from the Jedli čka collection, owing to its significance as a museum collection with historical importance, and the regulations in force at the National Museum in Prague to ensure its sustainability and continued preservation.A total of 45 bones from 42 individuals, who were afflicted by five different types of bone diseases/disorders, were selected ( Table 1 ).For 33 of these instances, Ortner [14] and Smr čka et al. [13] published observations of the lesions and/or interpretations of the potential diseases ( Table 1 ).
Bones with localized pathological changes related to osteosarcoma and osteomyelitis (from 5 and 10 individuals, respectively), and bone with healed fractures (from 10 individuals) were all sampled twice ( Table 1 ).The intra-bone sampling procedure involved targeting the site of the lesion and an unaffected area located at a distance from the lesion on the same bone.This sampling strategy enables the examination of isotopic variations within the bone, providing the opportunity to investigate short-term changes in diet, physiological responses to pathological conditions and trauma, and/or the aftereffects of medical treatments.Individuals who had syphilis ( n = 10), a systemic infection, or rickets ( n = 7), a metabolic disorder, were not concerned by the multi-sampling process, and therefore, were sampled only once ( Table 1 ).Bone fragments, ranging in size from 1 to 2 cm ², were collected using a rotary tool.

Bone collagen extraction for δ
Bone powder sample was defatted using the protocol outlined by Kates [1] , and further validated by Liden et al. [2] .Powdered bone was soaked in 10 mL of a methanol-chloroform mixture (2:1, v/v) and subjected to ultrasonic treatment during 10 min.The supernatant containing lipids was discarded and the solution was renewed.This procedure was reiterated iteratively until full elimination of fatty acids and their derivatives was achieved.The sample was then rinsed thoroughly and oven-dried at 65 °C for 7 h.
Bone collagen was extracted using the methodology described by Longin [3] , with modifications made by DeNiro and Epstein [4] , Brown et al. [5] , and Bocherens et al. [ 6 , 7 ].Bone powder sample (c.200 mg) was demineralized in 20 mL of 1 M hydrochloric acid at room temperature for 20 min.Gelatin was collected using a Nalgene reusable filtration unit with Fisherbrand membranes (0.5 μm pores), washed, and then soaked in 20 mL of 0.125 M sodium hydroxide at room temperature for 20 h to eliminate fulvic and humic acids.The sample was filtered as previously described, and rinsed.Gelatin was subsequently solubilized in 15 mL of 0.01 M hydrochloric acid at 100 °C for 17 h and filtered using a 5-8 mm Ezee-filter to trap possible impurities.Finally, collagen samples were freeze-dried at −110 °C for a minimum of 48 h.The extraction yield (%Col), expressed as a weight percentage (wt.%),was determined at the conclusion of the procedure.Bone collagen samples were analyzed for δ 13 C and δ 15 N using a Europa Scientific Roboprep elemental analyzer coupled with a Europa Scientific 20-20 isotope ratio monitoring mass spectrometer at Iso-Analytical Limited (Crewe, UK).Carbon and nitrogen contents of samples are expressed as percentages (%C and %N).Measurement error for both %C and %N, calculated from 14 duplicates of the international reference material IA-R042 (NBS-1577B bovine liver), was found to be ±2.49(1 σ ) and ±0.55 (1 σ ), respectively.Weight percent carbon and nitrogen were used to calculate atomic C:N ratios.The δ 13 C and δ 15 N values were reported as per mil ( ‰ ) deviation relative to VPDB and AIR, respectively.Duplicate measurements were performed on 20 % of samples to test reproducibility ( n = 15 samples, i.e. 30 measures).Measurement uncertainty was monitored using international and in-house standards with well-characterized isotopic com-positions: IA-R042 ( δ 13 C = −21.6 ± 0.06 ‰ , δ 15 N = + 7.65 ± 0.08 ‰ ), IA-R038 (L-alanine, δ 13 C = −24.99± 0.06 ‰ , δ 15 N = −0.65 ± 0.04 ‰ ), IA-R006/IA-R046 (mixture of cane sugar and ammonium sulfate, δ 13 C = −11.64± 0.03 ‰ , δ 15 N = + 22.04 ± 0.06 ‰ ).Precision, u(Rw) , for both δ 13 C and δ 15 N, determined through repeated measurements of calibration standards, check standards, and sample replicates, was found to be ±0.08 ‰ and ±0.13 ‰ , respectively.Systematic error, u(bias) , was determined to be ±0.14 for δ 13 C and ±0.13 for δ 15 N, based on the difference between observed and known δ values of the check standards and the long-term standard deviations of these standards.Total standard uncertainty was estimated to be ±0.16‰ for δ 13 C and ±0.18 ‰ for δ 15 N (Supplementary File 1).Measurement accuracy and precision are reported according to Szpak et al. [8] , with calibration tables conveniently available on the dedicated IsoArcH landing page for the present dataset.

Collagen quality indicators
In modern bones, collagen extraction yields are found to be 20.4 ± 3.9 wt% (1 σ ) [7] , and samples with less than 1 wt.% collagen are generally considered unreliable [ 15 , 16 ].Carbon and nitrogen contents of modern bone typically range from 15.3 to 47 % and from 5.5 to 17.3 %, respectively [17] .Bone collagen samples with %C and %N below 13 % and 4.8 %, respectively, are generally regarded as significantly altered [ 11 , 16 , 17 ].Atomic C:N ratios in modern bones are commonly around 3.2-3.3[ 16 , 17 ], but can vary between 2.9 and 3.6 [18] , with values outside these thresholds indicating potential alteration or contamination [ 11 , 17 ].To maintain the high level of scientific rigor in the subsequent interpretative processes, it is strongly recommended to exclude any samples that fail to meet these criteria [ 19 , 20 ].

Ethics Statements
This work does not involve any experimentation on living or modern-day human and animal subjects.
Dataset link: Dataset of stable carbon and nitrogen isotope values from bone collagen samples of anatomized individuals from the Jedli čka pathological-anatomical reference collection (Central Europe, 19th c.CE) (Original data) a b s t r a c t

Fig. 1 .
Fig. 1.Biplot showing the mean and standard deviation of δ 13 C and δ 15 N values obtained from bone collagen samples of individuals with bone diseases/disorders from the Jedli čka collection in Prague (19th century CE).

Table 2
Summary of isotopic data for individuals from the Jedli čka collection in Prague (19th century CE).Notes: All measurements, except for data obtained at the lesion site for SA3374 and distant to the lesion site for OS2950 that did not meet the collagen quality criteria, are included in the table.