Katzenberg, Lovell

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International Journal of OsteoarchaeologyInt. J. Osteoarchaeol. 9: 316324 (1999)

Stable Isotope Variation in PathologicalBone1

M. ANNE KATZENBERGa,

*AND NANCY C. LOVELLb

a Department of Archaeology,University of Calgary,Calgary,Alberta, Canadab Department of Anthropology,University of Alberta,Edmonton,Alberta, Canada

ABSTRACT Bone samples taken at autopsy from seven individuals from western Canada are studiedhistologically and the bone protein is analysed for stable isotopes of carbon and nitrogen. Theobjective of the study is to determine if pathological conditions result in variations in bone proteinstable isotope ratios. Of the seven individuals sampled, three are normal and four arepathological. The latter include post-paralytic atrophy, healing fracture, active periostitis andhealing osteomyelitis. The normal samples are included in order to determine how much variationto expect in different segments of a bone. In the three samples analysed, the variation is0.3

for 13

C and 0.2 for 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 (thediseased segment was approximately 2 greater for 15N than the two normal segments fromthe same individual). The higher nitrogen isotope ratio in the bone segment with osteomyelitis ismost likely a result of the fact that the collagen was formed from the catabolism of existingproteins in the body. This finding has implications for the interpretation of nitrogen isotope ratiosin 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 isnecessary to understand tissue changes in thebones of individuals with known medical his-tory. We were able to obtain bone samples fromsuch 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 differentpathological responses. Histological analysisprovides information on bone density andturnover, while stable isotope analysis allowsthe 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 bonetissue from individuals who died of unknowncauses. A common practice in bone chemistrystudies is the rejection of bones for sampling ifthey exhibit pathological conditions because theeffect of pathological change on isotope ratiosis not well-understood. Researchers also tend toavoid 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 isotoperatios that are not characteristic of long-termdiet. This has been demonstrated by Hobson etal. (1993), who found elevated 15N 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 tissueharvesting protocol at the University of Alberta

* Correspondence to: Department of Archaeology, University ofCalgary, 2500 University Dr. N.W., Calgary, Alberta, CanadaT2N 1N4. Tel.: +1 403 2203334; fax: +1 403 2829567; e-mail:

[email protected] A version of this paper was presented to the European Pale-opathology Association, Czech Republic, August 1998.

CCC 1047482X/99/05031609$17.50Copyright 1999 John Wiley & Sons, Ltd.

Received 8February 1999Accepted 31 May 1999


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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 cSex

Normal individuals71Male Heart attack L3755 Automobile accident L58Male

72 Stroke L79Female

Individuals with medically-documented pathologyPathological condition

Male 62 Heart attack L6 Periostitis34 Automobile accident L9 FractureFemale22 Respiratory fai lureFemale 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 stableisotope analysis while another part was preparedfor histological analysis. Three samples werefrom individuals who had no known pathologi-cal condition of skeletal relevance and who, forthe purposes of this study, were considerednormal or control specimens. The causes ofdeath for these individuals were heart attack,

vehicular accident and stroke (Table 1). Fouradditional samples came from individuals with

various pathological conditions (Table 1).The first pathological sample is a fibula from

an individual who exhibited active periostitis ofthat bone (Figure 1). This 62-year-old maledeveloped a localized infection from a work-re-lated injury 3 years prior to death. The injurywas untreated at the time the man suffered afatal 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 beenfractured obliquely proximal to the malleolusapproximately 5 years prior to death (Figure 3).The fracture was caused by sudden externalrotation of the foot and ankle while skiing. Across-section through the fracture callus (Figure

4) illustrates the poorly repaired bone while athin section shows ossified woven bone andsome remodelling.

The third pathological specimen is a midshaft

section of a femur obtained from a 22-year-oldfemale who was a quadriplegic as a result ofidiopathic polyneuritis, possibly brought on bya viral infection. Her reaction to the infectionwas uncommonly severe and her paralysis led topost-paralytic atrophy of her skeleton. She hadcontracted the infection during childhood whileliving in India with her missionary parents. Thecause of death was complications arising froman impaired respiratory function. Her longbones all display normal length and epiphysealunion, and the external dimensions of the artic-ular surfaces are not greatly altered by thedecreased mechanical loading; however, thebones exhibit generalized osteopaenia and anabsence of trabecular buttressing along whatwould normally be functional load planes.Macroscopically, the thinned shaft and absenceof muscle markings are clearly visible when

Figure 1. Gross appearance of periostitis on the distal fibula ofa 62-year-old male (specimen L6).

Copyright 1999 John Wiley & Sons, Ltd. Int. J. Osteoarchaeol. 9: 316324 (1999)


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M.A. Katzenberg and N.C. Lovell318

Figure 2. Cross-sectional appearance of the distal fibula of a62-year-old male showing periosteal new bone on the left(specimen L6).

Figure 3. Gross appearance of a healed fracture on the distalfibula of a 34-year-old female (specimen L9).

was receiving medication for pneumonia andpossible tuberculosis at the time of death buthad not been receiving any therapy for AIDS.There is minimal evidence of remodelling over-all but there is evidence of new bone in theinfected region. Normally, one would see agreater number of osteons and osteon fragmentsin an individual aged 46 years. The endostealbone was probably laid down relatively near tothe time of death.

In preparation for stable isotope analysis,bone samples were cleaned ultrasonically, andthen prepared for collagen extraction. Smallchunks (approximately 1 g) were soaked indiethyl ether for 6 h in order to remove lipids.contrasted with a normal femur (Figure 5). The

reduction in diaphyseal volume is consistentwith the complete lack of loading of the skeletalelements. The anomalous bone structure is alsodisplayed 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 dailyphysical therapy and manipulation of her limbs.

The fourth specimen is a midshaft section oftibia from a 46-year-old male who exhibitedosteomyelitis. The condition was not evidentfrom the external surface of the bone but wasclear in cross-section (Figure 7). This individual

was an indigent intravenous drug user who diedof AIDS. The osteomyelitis is thought to havedisseminated from a primary lung infection. He

Figure 4. Cross-sectional appearance of the fracture callus ofthe distal fibula of a 34-year-old female (specimen L9).



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Figure 5. Post-paralytic atrophy of the femur from a 22-year-old female (right, specimen L16), compared with a normalfemur (left).

Results

Figure 8 shows the distribution of stable carbonand nitrogen isotope values for normal andpathological bone samples. Individual stable iso-tope values are provided in Table 2. The normalsamples provide a base line for the variation instable isotope values in different sections of thesame bone. The range of variation is from 0.2to 0.7 for 13C and from 0.3 to 0.4 for

15N among the normal individuals. Becauseprecision of the instrument is 0.1 for 13Cand 0.2 for 15N, some variation arises as aresult of the sampling site along the bones.

For the pathological specimens, the periostitisshows less variation than do the normal bones.

One of the three segments was unaffected andthe other two showed slight periosteal reactionon the bone surface. For the individual with the

Figure 6. Thin section (100 magnification) from the femoralmid-shaft of a 22-year-old female with post-paralytic atrophy(specimen L16).

After rinsing repeatedly, the chunks weresoaked in 2% hydrochloric acid until all bonemineral was dissolved. Finally, the bone wassoaked in sodium hydroxide in order to removeany remaining lipids. The remaining protein(largely collagen but with some non-collagenous

proteins) was freeze-dried. For analysis by massspectrometry, a few milligrams of collagen wereweighed into tin sample holders and placed inan automated sampler that drops each sampleinto a Carlo Erba gas analyser. After determin-ing the percentage of carbon or nitrogen, thegas analyser introduces CO2 or N2 gas into theMicromass Prism mass spectrometer. Results arecorrected to the international standards VPDB for carbon and atmospheric nitrogen for

nitrogen stable isotopes. Precision of the instru-ment (1 S.D.) is 0.1 for 13C and 0.2for 15N.



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Figure 7. Cross-sectional appearance of osteomyelitis on the tibia of a 46-year-old male (right, specimen L85) clearly showingendosteal bone apposition. A normal tibia is seen in cross-section on the left.

well-healed fracture, two segments from theunaffected portion of the shaft and one segment

from the fracture site were used. The fracturesite has a 13C value that is slightly lower (morenegative) than the two normal segments(21.2 versus 20.8 and 20.9).The 15N 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 13C(13.8, 14.4 and 15.0 in thethree sites sampled) and slightly less variation

than the fracture specimen for 15

N (6.6,6.9 and 7.2, respectively). The greatest

variation for nitrogen isotopes was seen in theindividual with osteomyelitis who died of AIDS.For 13C, the segment with a grossly observablelesion was similar to the healed segment butthese two differed from the normal segment(17.0, 17.0 and 17.9, respec-tively). For 15N, the segment with a grosslyobservable 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 variationin stable carbon or nitrogen isotope valuesamong differing sites of the same bone. Let usconsider under what circumstances we mightexpect variation and in what direction that vari-ation could occur. In order to understand the

variation in stable isotope values in pathologicalbone, it is necessary to understand how growthand 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. Focusingon the carbon and nitrogen that make up thestructural protein collagen, the nitrogen mustcome from either ingested protein or recycledtissues in the body. Carbon may come fromdietary protein, carbohydrate or fat but thecarbon found in the essential amino acidsmainly comes from ingested dietary protein. Asis true of nitrogen, some carbon in non-essentialamino acids may come from the breakdown and

recycling of tissues in the body. In the case ofnitrogen balance, there are three possibleconditions.



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1. Tissue gain during growth the individualis in positive nitrogen balance. New tissuesare being produced and more nitrogen isingested than is excreted as a result of thebodys need to manufacture muscle, bone,blood, etc. The expectation is that diet will

be reflected by newly forming tissues interms of 15N. Trophic enrichment relativeto diet would be expected, because of thefact that more 14N than 15N is excretedrelative to that which is ingested.

2. Tissue maintenance in healthy adults theindividual is in nitrogen equilibrium, whichmeans that the same amount of nitrogen isbeing ingested as is being excreted. Proteinis being maintained. Bone collagen will

largely reflect ingested protein from the diet(15N of diet plus 3) averaging over sev-eral years.

3. Tissue loss during stress the individual isin negative nitrogen balance. Less nitrogenis being ingested than is needed to maintainand replace proteins in the body. As a result,there is catabolism of existing proteins inthe body where the amino group (NH2) isstripped off so that the sugar can be used. In

this situation, of the nitrogen that is ex-creted, there should be preferential loss of14N relative to 15N as amino acids with 14N

are broken down more readily than thosecontaining 15N and 14N is preferentially ex-creted (Steele & Daniel, 1978). The remain-ing tissues should be enriched in the heavierisotope relative to what those same tissueswould be in a situation of tissue maintenance

(Hobson et al., 1993).

In response to injury or disease, newly formedtissues for repair may have as their source eitheringested nitrogen from dietary protein or recy-cled nitrogen derived from the breakdown ofexisting proteins in the body.

With reference to the specific conditions in-vestigated here:

1. Injury and repair, such as fracture varia-

tion might occur in the callus and subse-quent remodelled bone as a result of slightvariations in the diet during the time ofrepair, as compared with the time when thehealthy portions of the bone were formed.In this situation 15N may either decrease,as seen in the example of fracture reportedhere, or increase. If collagen deposited asosteoid at the fracture repair site was fromrecycled nitrogen in the body, we mightexpect elevated 15N values. Carbon isotope

ratios would not be expected to vary underthese circumstances except relative to di-etary intake.

Figure 8. Graph of 15N and 13C 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 variouspathological specimens, labelled as such on the graph. The asterisk (*) for the individual with osteomyelitis appears next to thepoint indicating the pathological segment, in comparison with unaffected segments from the same bone. All segments weresimilarly affected in the case of atrophy. The affected segment for the fracture is the lowest point ( 15N=8.9).



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Table 2. Carbon and nitrogen isotope data for normal and pathological bone samples

Sample c Sample Segment 13C () 15N ()

L37a Normal 20.1 8.920.5 9.3L37b

L37c 20.5 9.2

L58a Normal 20.3 11.4L58b 19.6 11.419.9 11.1L58c

L79a Normal 19.8 9.39.620.0L79b

L79c 20.0 9.3

Normal 20.9L6a 10.9Periostitis10.920.7Active periostitisL6b

Active periostitis 20.7L6c 11.0

Normal 20.9L9a 9.7Fracture9.820.8L9b Normal8.921.2CallusL9c

Post-paralytic atrophyL16a 13.8Post-paralytic atrophy 6.6L16b Post-paralytic atrophy 15.0 7.2L16c Post-paralytic atrophy 14.4 6.9

11.017.0NormalOsteomyelitisL85aL85b Normal 17.9 11.3L85c Osteomyelitis 17.0 12.9

2. Periostitis is a superficial reaction whichmay affect either a small or a large area ofthe 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), thenthe affected region is unlikely to be differentenough to override the underlying bonewhich was deposited in an similar manner tothe normal segments. Therefore, little or no

variation would be expected. If only theperiosteal reaction region had been sampled,we might have expectations similar to thosein fracture repair.

3. Atrophy if we were comparing an atro-phied segment with normal segments, ourexpectations would be different from com-paring different segments of an atrophiedlimb. If atrophy was a result of generalwasting that was linked to nutritional stresswe would expect elevated 15N values.However, as the atrophy suffered by thisindividual was the result of nerve damageand, consequently, of disuse there is little

variation in 15

N. While we see variation inthe 13C values in the different segments ofthe femur for this individual, no particular

pattern of variation is expected. This indi-vidual has the greatest contribution of C4plants in the diet as reflected in 13C. (Thelow 15N suggests that the 13C is a result

of C4 plant consumption rather than con-sumption of marine foods.) Therefore, di-etary shifts involving varying amounts of C3and C4 plants will be more apparent than inan individual with little or no C4 plants intheir diet. The 13C variation in this indi-

vidual may have occurred in moving fromIndia to Canada.

4. Osteomyelitis as in the case of fracturerepair and periostitis, one might expect vari-

ation resulting from dietary differenceswhen the remodelled tissue is forming, orelevated 15N as a result of catabolism andrecycling of nitrogen within the body. Inthis particular individual, the situation iscomplicated by the fact that the cause ofdeath is AIDS and that there was overallwasting. This would further suggest that

15N would be elevated in newly formedtissues.

In a study by Hobson et al. (1993) on stablenitrogen isotope enrichment during nutritionalstress, it was found that 15N was enriched in



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the tissues of birds in negative nitrogen balancerelative to those who were not stressed. Citingthe work of Ambrose & DeNiro (1987) ondrought tolerant animals, they provide a relatedexplanation for their findings in birds. Specifi-cally, in situations of negative nitrogen balance,

protein is synthesized from a combination ofingested protein and recycled amino acids viathe breakdown of existing proteins in the body.Nitrogen isotope fractionation occurs during thedeamination and the transamination of aminoacids. Relatively more 14N than 15N will beexcreted and bonds involving 14N are brokendown with less energy requirements than thoseinvolving 15N. Therefore, more 15N will beretained.

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 hasalready been enriched in 15N relative to diet, addi-

tional enrichment in the metabolic nitrogen pool

must occur. A consequence of this process would be

eventual enrichment in 15N of all body tissues rela-

tive to periods without stress (Hobson et al., 1993,

pp. 391392).

Conclusions

Under normal circumstances, bone should beone of the last tissues affected by short-termdietary change, since turnover is slow com-pared with other body tissues. Bone collagenwill reflect the diet over the period of normaldeposition and turnover of bone. However,

during times of injury, disease or nutritionalstress, bone has the capacity to register suchevents in varying stable isotope ratios. Wehave presented evidence that wasting and theconsequent recycling of tissue protein can re-sult in elevated 15N values. We have alsopresented evidence to show that new tissuedeposited during the repair of fracture canregister short-term dietary change. These find-ings caution us to avoid obvious pathological

bone if our objective is to determine averagediet. They also alert us to situations in whichstable isotope values may not solely reflect

diet. This could have implications in caseswhere pathology is not grossly visible, and incases of nutritional stress, which is not alwaysidentifiable in the archaeological record. Thisis particularly important in interpreting 15Nin the skeletal remains of infants and young

children who may have died from nutrition-re-lated illness but who are expected to haveelevated 15N 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 Calgaryand the University of Alberta for their help inthis research. At the University of Calgary, wethank Sharon Whitely who assisted in collagenpreparation, and Nanita Lozano for technicalassistance in the stable isotope laboratory. Allstable isotope ratios were obtained from thestable isotope laboratory, Department ofPhysics, University of Calgary, under the direc-tion of H. Roy Krouse. At the University of

Alberta, we thank John Marken, Department ofLaboratory Medicine, for providing access tothe samples and pathology reports. DennisCarmel, Department of Oral Health Sciences,prepared the histological sections and HarveyFriebe, Department of Anthropology, assisted inthe microscopic analyses. Comments from twoanonymous reviewers are also gratefullyacknowledged.

References

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Fogel, M.L., Tuross, N. and Owsley, D.W. (1989)Nitrogen isotope tracers of human lactation inmodern and archaeological populations. CarnegieInstitution, Annual Report of the Director; GeophysicalLaboratory, 2150: 111117.

Fogel, M.L., Tuross, N., Johnson, B.J. and Miller,G.H. (1997) Biogeochemical record of ancienthumans. Organic Geochemistry, 27: 275287.



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Hobson, K.A., Alisauskas, R.T. and Clark, R.G.(1993) Stable-nitrogen isotope enrichment inavian 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) Fractionationof nitrogen isotopes by animals: a further compli-cation to the use of variations in the naturalabundance of 15N for tracer studies. Journal of

Agricultural Science, 90: 79.


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