Stable Isotope Variation in Pathological Bone 1 M. ANNE KATZENBERG a, * AND NANCY C. LOVELL b a Department of Archaeology, University of Calgary, Calgary, Alberta, Canada b Department of Anthropology, University of Alberta, Edmonton, Alberta, Canada ABSTRACT Bone samples taken at autopsy from seven individuals from western Canada are studied histologically and the bone protein is analysed for stable isotopes of carbon and nitrogen. The objective of the study is to determine if pathological conditions result in variations in bone protein stable isotope ratios. Of the seven individuals sampled, three are normal and four are pathological. The latter include post-paralytic atrophy, healing fracture, active periostitis and healing osteomyelitis. The normal samples are included in order to determine how much variation to expect in different segments of a bone. In the three samples analysed, the variation is 90.3 for l 13 C and 90.2 for l 15 N. Three of the four pathological specimens exceed normal variation. The greatest variation occurs in the bone with osteomyelitis from an individual who had AIDS (the diseased segment was approximately 2 greater for l 15 N than the two normal segments from the same individual). The higher nitrogen isotope ratio in the bone segment with osteomyelitis is most likely a result of the fact that the collagen was formed from the catabolism of existing proteins in the body. This finding has implications for the interpretation of nitrogen isotope ratios in individuals who may have died from wasting diseases. Copyright 1999 John Wiley & Sons, Ltd. Key words: nitrogen isotopes; bone pathology; carbon isotopes; bone chemistry Introduction In order to improve our understanding of patho- logical processes in people of the past, it is necessary to understand tissue changes in the bones of individuals with known medical his- tory. We were able to obtain bone samples from such individuals who had bequeathed their bod- ies to science. The purpose of the study is to explore carbon and nitrogen stable isotope vari- ation in bones exhibiting several different pathological responses. Histological analysis provides information on bone density and turnover, while stable isotope analysis allows the comparison of recently deposited bone tis- sue with normal adult tissue. This has implica- tions for stable isotope analysis of prehistoric bone, which is usually carried out on bone tissue from individuals who died of unknown causes. A common practice in bone chemistry studies is the rejection of bones for sampling if they exhibit pathological conditions because the effect of pathological change on isotope ratios is not well-understood. Researchers also tend to avoid destructive analyses of such specimens. Some causes of death, particularly those associ- ated with nutritional stress or diseases that affect the metabolism, may result in stable isotope ratios that are not characteristic of long-term diet. This has been demonstrated by Hobson et al. (1993), who found elevated l 15 N in nutri- tionally stressed birds. Materials and methods The bone samples used in this study were ob- tained at autopsy or as part of a broader tissue harvesting protocol at the University of Alberta * Correspondence to: Department of Archaeology, University of Calgary, 2500 University Dr. N.W., Calgary, Alberta, Canada T2N 1N4. Tel.: +1 403 2203334; fax: +1 403 2829567; e-mail: katzenbe@ucalgary.ca 1 A version of this paper was presented to the European Pale- opathology Association, Czech Republic, August 1998. CCC 1047482X/99/05031609$17.50 Copyright 1999 John Wiley & Sons, Ltd. Received 8 February 1999 Accepted 31 May 1999 Stable Isotopes in Pathological Bone 317 Table 1. Information on sex, age and cause of death for normal (i.e. no documented patholog- ical condition at time of death) and pathological specimens used in the study Age Cause of death Specimen c Sex Normal individuals 71 Male Heart attack L37 55 Automobile accident L58 Male 72 Stroke L79 Female Individuals with medically-documented pathology Pathological condition Male 62 Heart attack L6 Periostitis 34 Automobile accident L9 Fracture Female 22 Respiratory failure Female L16 Atrophy Male 46 AIDS L85 Osteomyelitis Hospital, Canada. All individuals were of Eu- ropean ancestry. After maceration and cleaning, cross-sections were taken from the proximal, middle, and distal portions of the bone diaphy- ses. One part of each section was used for stable isotope analysis while another part was prepared for histological analysis. Three samples were from individuals who had no known pathologi- cal condition of skeletal relevance and who, for the purposes of this study, were considered normal or control specimens. The causes of death for these individuals were heart attack, vehicular accident and stroke (Table 1). Four additional samples came from individuals with various pathological conditions (Table 1). The first pathological sample is a fibula from an individual who exhibited active periostitis of that bone (Figure 1). This 62-year-old male developed a localized infection from a work-re- lated injury 3 years prior to death. The injury was untreated at the time the man suffered a fatal heart attack. Periosteal new bone is clearly visible in the cross-section taken for micro- scopic analysis (Figure 2). A second fibula was obtained from a 32-year- old female who died of injuries received in a vehicular accident. Her left fibula had been fractured obliquely proximal to the malleolus approximately 5 years prior to death (Figure 3). The fracture was caused by sudden external rotation of the foot and ankle while skiing. A cross-section through the fracture callus (Figure 4) illustrates the poorly repaired bone while a thin section shows ossified woven bone and some remodelling. The third pathological specimen is a midshaft section of a femur obtained from a 22-year-old female who was a quadriplegic as a result of idiopathic polyneuritis, possibly brought on by a viral infection. Her reaction to the infection was uncommonly severe and her paralysis led to post-paralytic atrophy of her skeleton. She had contracted the infection during childhood while living in India with her missionary parents. The cause of death was complications arising from an impaired respiratory function. Her long bones all display normal length and epiphyseal union, and the external dimensions of the artic- ular surfaces are not greatly altered by the decreased mechanical loading; however, the bones exhibit generalized osteopaenia and an absence of trabecular buttressing along what would normally be functional load planes. Macroscopically, the thinned shaft and absence of muscle markings are clearly visible when Figure 1. Gross appearance of periostitis on the distal fibula of a 62-year-old male (specimen L6). Copyright 1999 John Wiley & Sons, Ltd. Int. J. Osteoarchaeol. 9: 316324 (1999) M.A. Katzenberg and N.C. Lovell 318 Figure 2. Cross-sectional appearance of the distal fibula of a 62-year-old male showing periosteal new bone on the left (specimen L6). Figure 3. Gross appearance of a healed fracture on the distal fibula of a 34-year-old female (specimen L9). was receiving medication for pneumonia and possible tuberculosis at the time of death but had not been receiving any therapy for AIDS. There is minimal evidence of remodelling over- all but there is evidence of new bone in the infected region. Normally, one would see a greater number of osteons and osteon fragments in an individual aged 46 years. The endosteal bone was probably laid down relatively near to the time of death. In preparation for stable isotope analysis, bone samples were cleaned ultrasonically, and then prepared for collagen extraction. Small chunks (approximately 1 g) were soaked in diethyl ether for 6 h in order to remove lipids. contrasted with a normal femur (Figure 5). The reduction in diaphyseal volume is consistent with the complete lack of loading of the skeletal elements. The anomalous bone structure is also displayed in a histological section where os- teocyte lacunae are clearly visible and there is a lack of Haversian bone (Figure 6). Some remod- elling is evident close to the periosteal surface, perhaps as a result of activity at muscle attach- ment sites as the woman had received daily physical therapy and manipulation of her limbs. The fourth specimen is a midshaft section of tibia from a 46-year-old male who exhibited osteomyelitis. The condition was not evident from the external surface of the bone but was clear in cross-section (Figure 7). This individual was an indigent intravenous drug user who died of AIDS. The osteomyelitis is thought to have disseminated from a primary lung infection. He Figure 4. Cross-sectional appearance of the fracture callus of the distal fibula of a 34-year-old female (specimen L9). Copyright 1999 John Wiley & Sons, Ltd. Int. J. Osteoarchaeol. 9: 316324 (1999) Stable Isotopes in Pathological Bone 319 Figure 5. Post-paralytic atrophy of the femur from a 22-year- old female (right, specimen L16), compared with a normal femur (left). Results Figure 8 shows the distribution of stable carbon and nitrogen isotope values for normal and pathological bone samples. Individual stable iso- tope values are provided in Table 2. The normal samples provide a base line for the variation in stable isotope values in different sections of the same bone. The range of variation is from 0.2 to 0.7 for l 13 C and from 0.3 to 0.4 for l 15 N among the normal individuals. Because precision of the instrument is 90.1 for l 13 C and 90.2 for l 15 N, some variation arises as a result of the sampling site along the bones. For the pathological specimens, the periostitis shows less variation than do the normal bones. One of the three segments was unaffected and the other two showed slight periosteal reaction on the bone surface. For the individual with the Figure 6. Thin section (100 magnification) from the femoral mid-shaft of a 22-year-old female with post-paralytic atrophy (specimen L16). After rinsing repeatedly, the chunks were soaked in 2% hydrochloric acid until all bone mineral was dissolved. Finally, the bone was soaked in sodium hydroxide in order to remove any remaining lipids. The remaining protein (largely collagen but with some non-collagenous proteins) was freeze-dried. For analysis by mass spectrometry, a few milligrams of collagen were weighed into tin sample holders and placed in an automated sampler that drops each sample into a Carlo Erba gas analyser. After determin- ing the percentage of carbon or nitrogen, the gas analyser introduces CO 2 or N 2 gas into the Micromass Prism mass spectrometer. Results are corrected to the international standards VPDB for carbon and atmospheric nitrogen for nitrogen stable isotopes. Precision of the instru- ment (1 S.D.) is 90.1 for l 13 C and 90.2 for l 15 N. Copyright 1999 John Wiley & Sons, Ltd. Int. J. Osteoarchaeol. 9: 316324 (1999) M.A. Katzenberg and N.C. Lovell 320 Figure 7. Cross-sectional appearance of osteomyelitis on the tibia of a 46-year-old male (right, specimen L85) clearly showing endosteal bone apposition. A normal tibia is seen in cross-section on the left. well-healed fracture, two segments from the unaffected portion of the shaft and one segment from the fracture site were used. The fracture site has a l 13 C value that is slightly lower (more negative) than the two normal segments ( 21.2 versus 20.8 and 20.9). The l 15 N value at the fracture site is also lower (8.9 versus 9.7 for the two normal seg- ments). The individual with post-paralytic atro- phy shows the greatest variation in l 13 C ( 13.8, 14.4 and 15.0 in the three sites sampled) and slightly less variation than the fracture specimen for l 15 N (6.6, 6.9 and 7.2, respectively). The greatest variation for nitrogen isotopes was seen in the individual with osteomyelitis who died of AIDS. For l 13 C, the segment with a grossly observable lesion was similar to the healed segment but these two differed from the normal segment ( 17.0, 17.0 and 17.9, respec- tively). For l 15 N, the segment with a grossly observable lesion was considerably higher at 12.9 as compared with both the healed seg- ment (11.0) and the normal segment (11.3). Discussion The null hypothesis is that there is no variation in stable carbon or nitrogen isotope values among differing sites of the same bone. Let us consider under what circumstances we might expect variation and in what direction that vari- ation could occur. In order to understand the variation in stable isotope values in pathological bone, it is necessary to understand how growth and tissue repair make use of carbon and nitro- gen that are ingested in the diet and carbon and nitrogen that are recycled in the body. Focusing on the carbon and nitrogen that make up the structural protein collagen, the nitrogen must come from either ingested protein or recycled tissues in the body. Carbon may come from dietary protein, carbohydrate or fat but the carbon found in the essential amino acids mainly comes from ingested dietary protein. As is true of nitrogen, some carbon in non-essential amino acids may come from the breakdown and recycling of tissues in the body. In the case of nitrogen balance, there are three possible conditions. Copyright 1999 John Wiley & Sons, Ltd. Int. J. Osteoarchaeol. 9: 316324 (1999) Stable Isotopes in Pathological Bone 321 1. Tissue gain during growththe individual is in positive nitrogen balance. New tissues are being produced and more nitrogen is ingested than is excreted as a result of the bodys need to manufacture muscle, bone, blood, etc. The expectation is that diet will be reflected by newly forming tissues in terms of l 15 N. Trophic enrichment relative to diet would be expected, because of the fact that more 14 N than 15 N is excreted relative to that which is ingested. 2. Tissue maintenance in healthy adultsthe individual is in nitrogen equilibrium, which means that the same amount of nitrogen is being ingested as is being excreted. Protein is being maintained. Bone collagen will largely reflect ingested protein from the diet (l 15 N of diet plus 3) averaging over sev- eral years. 3. Tissue loss during stressthe individual is in negative nitrogen balance. Less nitrogen is being ingested than is needed to maintain and replace proteins in the body. As a result, there is catabolism of existing proteins in the body where the amino group (NH 2 ) is stripped off so that the sugar can be used. In this situation, of the nitrogen that is ex- creted, there should be preferential loss of 14 N relative to 15 N as amino acids with 14 N are broken down more readily than those containing 15 N and 14 N is preferentially ex- creted (Steele & Daniel, 1978). The remain- ing tissues should be enriched in the heavier isotope relative to what those same tissues would be in a situation of tissue maintenance (Hobson et al., 1993). In response to injury or disease, newly formed tissues for repair may have as their source either ingested nitrogen from dietary protein or recy- cled nitrogen derived from the breakdown of existing proteins in the body. With reference to the specific conditions in- vestigated here: 1. Injury and repair, such as fracturevaria- tion might occur in the callus and subse- quent remodelled bone as a result of slight variations in the diet during the time of repair, as compared with the time when the healthy portions of the bone were formed. In this situation l 15 N may either decrease, as seen in the example of fracture reported here, or increase. If collagen deposited as osteoid at the fracture repair site was from recycled nitrogen in the body, we might expect elevated l 15 N values. Carbon isotope ratios would not be expected to vary under these circumstances except relative to di- etary intake. Figure 8. Graph of l 15 N and l 13 C for normal and pathological bone samples. Boxes with dashed outlines enclose segments for normal bones (three segments per bone for three different individuals). Ellipses enclose segments from the various pathological specimens, labelled as such on the graph. The asterisk (*) for the individual with osteomyelitis appears next to the point indicating the pathological segment, in comparison with unaffected segments from the same bone. All segments were similarly affected in the case of atrophy. The affected segment for the fracture is the lowest point (l 15 N=8.9). Copyright 1999 John Wiley & Sons, Ltd. Int. J. Osteoarchaeol. 9: 316324 (1999) M.A. Katzenberg and N.C. Lovell 322 Table 2. Carbon and nitrogen isotope data for normal and pathological bone samples Sample c Sample Segment l 13 C () l 15 N () L37a Normal 20.1 8.9 20.5 9.3 L37b L37c 20.5 9.2 L58a Normal 20.3 11.4 L58b 19.6 11.4 19.9 11.1 L58c L79a Normal 19.8 9.3 9.6 20.0 L79b L79c 20.0 9.3 Normal 20.9 L6a 10.9 Periostitis 10.9 20.7 Active periostitis L6b Active periostitis 20.7 L6c 11.0 Normal 20.9 L9a 9.7 Fracture 9.8 20.8 L9b Normal 8.9 21.2 Callus L9c Post-paralytic atrophy L16a 13.8 Post-paralytic atrophy 6.6 L16b Post-paralytic atrophy 15.0 7.2 L16c Post-paralytic atrophy 14.4 6.9 11.0 17.0 Normal Osteomyelitis L85a L85b Normal 17.9 11.3 L85c Osteomyelitis 17.0 12.9 2. Periostitis is a superficial reaction which may affect either a small or a large area of the bone surface. Severe periostitis may re- sult in multiple layers of periosteal bone. If the lesion is small and sampling is in cross- section (as was the case in this study), then the affected region is unlikely to be different enough to override the underlying bone which was deposited in an similar manner to the normal segments. Therefore, little or no variation would be expected. If only the periosteal reaction region had been sampled, we might have expectations similar to those in fracture repair. 3. Atrophyif we were comparing an atro- phied segment with normal segments, our expectations would be different from com- paring different segments of an atrophied limb. If atrophy was a result of general wasting that was linked to nutritional stress we would expect elevated l 15 N values. However, as the atrophy suffered by this individual was the result of nerve damage and, consequently, of disuse there is little variation in l 15 N. While we see variation in the l 13 C values in the different segments of the femur for this individual, no particular pattern of variation is expected. This indi- vidual has the greatest contribution of C 4 plants in the diet as reflected in l 13 C. (The low l 15 N suggests that the l 13 C is a result of C 4 plant consumption rather than con- sumption of marine foods.) Therefore, di- etary shifts involving varying amounts of C 3 and C 4 plants will be more apparent than in an individual with little or no C 4 plants in their diet. The l 13 C variation in this indi- vidual may have occurred in moving from India to Canada. 4. Osteomyelitisas in the case of fracture repair and periostitis, one might expect vari- ation resulting from dietary differences when the remodelled tissue is forming, or elevated l 15 N as a result of catabolism and recycling of nitrogen within the body. In this particular individual, the situation is complicated by the fact that the cause of death is AIDS and that there was overall wasting. This would further suggest that l 15 N would be elevated in newly formed tissues. In a study by Hobson et al. (1993) on stable nitrogen isotope enrichment during nutritional stress, it was found that l 15 N was enriched in Copyright 1999 John Wiley & Sons, Ltd. Int. J. Osteoarchaeol. 9: 316324 (1999) Stable Isotopes in Pathological Bone 323 the tissues of birds in negative nitrogen balance relative to those who were not stressed. Citing the work of Ambrose & DeNiro (1987) on drought tolerant animals, they provide a related explanation for their findings in birds. Specifi- cally, in situations of negative nitrogen balance, protein is synthesized from a combination of ingested protein and recycled amino acids via the breakdown of existing proteins in the body. Nitrogen isotope fractionation occurs during the deamination and the transamination of amino acids. Relatively more 14 N than 15 N will be excreted and bonds involving 14 N are broken down with less energy requirements than those involving 15 N. Therefore, more 15 N will be retained. Under conditions of fasting and nutritional stress, a greater proportion of nitrogenous compounds avail- able for protein synthesis are derived from catabolism and, since this source of nitrogen has already been enriched in 15 N relative to diet, addi- tional enrichment in the metabolic nitrogen pool must occur. A consequence of this process would be eventual enrichment in 15 N of all body tissues rela- tive to periods without stress (Hobson et al., 1993, pp. 391392). Conclusions Under normal circumstances, bone should be one of the last tissues affected by short-term dietary change, since turnover is slow com- pared with other body tissues. Bone collagen will reflect the diet over the period of normal deposition and turnover of bone. However, during times of injury, disease or nutritional stress, bone has the capacity to register such events in varying stable isotope ratios. We have presented evidence that wasting and the consequent recycling of tissue protein can re- sult in elevated l 15 N values. We have also presented evidence to show that new tissue deposited during the repair of fracture can register short-term dietary change. These find- ings caution us to avoid obvious pathological bone if our objective is to determine average diet. They also alert us to situations in which stable isotope values may not solely reflect diet. This could have implications in cases where pathology is not grossly visible, and in cases of nutritional stress, which is not always identifiable in the archaeological record. This is particularly important in interpreting l 15 N in the skeletal remains of infants and young children who may have died from nutrition-re- lated illness but who are expected to have elevated l 15 N as a result of breast-feeding (Fogel et al., 1989, 1997; Katzenberg et al., 1996). Acknowledgements The authors gratefully acknowledge a number of individuals at both the University of Calgary and the University of Alberta for their help in this research. At the University of Calgary, we thank Sharon Whitely who assisted in collagen preparation, and Nanita Lozano for technical assistance in the stable isotope laboratory. All stable isotope ratios were obtained from the stable isotope laboratory, Department of Physics, University of Calgary, under the direc- tion of H. Roy Krouse. At the University of Alberta, we thank John Marken, Department of Laboratory Medicine, for providing access to the samples and pathology reports. Dennis Carmel, Department of Oral Health Sciences, prepared the histological sections and Harvey Friebe, Department of Anthropology, assisted in the microscopic analyses. Comments from two anonymous reviewers are also gratefully acknowledged. References Ambrose, S.H. and DeNiro, M.J. (1987) Bone nitro- gen isotope composition and climate. Nature, 325: 201. Fogel, M.L., Tuross, N. and Owsley, D.W. (1989) Nitrogen isotope tracers of human lactation in modern and archaeological populations. Carnegie Institution, Annual Report of the Director; Geophysical Laboratory, 2150: 111117. Fogel, M.L., Tuross, N., Johnson, B.J. and Miller, G.H. (1997) Biogeochemical record of ancient humans. Organic Geochemistry, 27: 275287. Copyright 1999 John Wiley & Sons, Ltd. Int. J. Osteoarchaeol. 9: 316324 (1999) M.A. Katzenberg and N.C. Lovell 324 Hobson, K.A., Alisauskas, R.T. and Clark, R.G. (1993) Stable-nitrogen isotope enrichment in avian tissues due to fasting and nutritional stress: implications for isotopic analyses of diet. The Con- dor, 95: 388394. Katzenberg, M.A., Herring, D.A. and Saunders S.R. (1996) Weaning and infant mortality: evaluating the skeletal evidence. Yearbook of Physical Anthropol- ogy, 39:177199. Steele, K.W. and Daniel, R.M. (1978) Fractionation of nitrogen isotopes by animals: a further compli- cation to the use of variations in the natural abundance of 15 N for tracer studies. Journal of Agricultural Science, 90: 79. Copyright 1999 John Wiley & Sons, Ltd. Int. J. Osteoarchaeol. 9: 316324 (1999)