International Journal of Osteoarchaeology

Int. J. Osteoarchaeol. 9: 316324 (1999)

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)