An objective means of diagnosing anorexia nervosa and bulimia nervosa using15N/14N and13C/12C ratios...

RAPID COMMUNICATIONS IN MASS SPECTROMETRY

Rapid Commun. Mass Spectrom. 2006; 20: 3367–3373

) DOI: 10.1002/rcm.2740
Published online in Wiley InterScience (www.interscience.wiley.com
An objective means of diagnosing anorexia nervosa

and bulimia nervosa using 15N/14N and 13C/12C

ratios in hair

Kent A. Hatch1*, Morgan A. Crawford1, Amanda W. Kunz1, Steven R. Thomsen2,

Dennis L. Eggett3, Stephen T. Nelson4 and Beverly L. Roeder1

1Department of Integrative Biology, Brigham Young University, Provo, UT 84602, USA2Department of Communications, Brigham Young University, Provo, UT 84602, USA3Department of Statistics, Brigham Young University, Provo, UT 84602, USA4Department of Geology, Brigham Young University, Provo, UT 84602, USA

Received 10 June 2006; Revised 8 September 2006; Accepted 12 September 2006

*CorrespoBiology,84602, USE-mail: kContract/ties (ORC

An objective means based on the carbon and nitrogen stable isotope analysis of five hairs per

individual is presented for distinguishing between individuals with anorexia nervosa and/or

bulimia nervosa from non-clinical individuals (i.e. clinically normal controls). Using discriminant

analysis, an algorithm has been developed that provides both sensitivity and specificity of 80% in

making diagnoses of individuals with these eating disorders. With further refinements, the results

suggest that it may be also possible to distinguish between individuals with anorexia or bulimia.

Finally, the study shows the value of conducting blind tests and using larger sample sizes of both

control and treatment groups. Both groups are needed to validate the diagnostic value of a method

and to provide measures of sensitivity and specificity of any diagnostic test. Copyright # 2006

John Wiley & Sons, Ltd.

Conservative estimates suggest that as many as 1–5% of all

high school and college-age women in the United States, as

well as increasing numbers throughout the developed world,

are affected in some way by pathological dietary practices.1,2

The two most prevalent forms of disordered eating are

anorexia nervosa, characterized by an extreme fear of weight

gain and a severe concomitant restriction of calories, and

bulimia nervosa, manifested in excessive binging and

purging cycles.3–5

Common to both anorexia and bulimia is the element of

denial. Those who suffer from these illnesses are often

unwilling or unable to recognize and admit to the

psychopathology of their behaviors.3,6,7 This problem raises

specific concerns and challenges for researchers and medical

professionals seeking to obtain accurate dietary information

for diagnostic and evaluative purposes when working with

eating disorder patients. In most cases, doctors and

researchers must rely on potentially unreliable self-reported

data.6,7 This problem underscores the need for and the

usefulness of a clinical test to determine diet and condition

independent of subjective criteria and self-reporting, both to

accurately diagnose and to effectively treat eating disorder

patients.

The measurement of stable isotope ratios in hair has the

potential to provide a unique method for objectively

ndence to: K. A. Hatch, Department of IntegrativeBrigham Young University, 401 WIDB, Provo, UTA.

[email protected] sponsor: Office of Research and Creative Activi-A) of Brigham Young University.

diagnosing individuals with eating disorders.8 When the

body is in an anabolic state, it incorporates dietary proteins

into growing hair. Although the mechanism is unclear, it

appears that 14N is excreted by the organism at a slightly

higher rate than 15N. As a result, 15N concentrates in body

tissues9 (but see also Ref. 10). Thus, as one moves up the food

chain, d15N increases on average at 3% with each step or

trophic level11 (but see also Ref. 12). Because of these trophic

level effects,11–15 the 15N/14N ratios of herbivores (or

vegetarians and vegans) are less than those of predators

(or humans who eat more meat). Likewise, when the body

becomes catabolic, the 15N/14N ratio of the individual’s

tissues increases further due to something functionally

similar to an increase in trophic level.12,16,17 As the individual

loses weight, the individual’s body consumes its own energy

and protein stores. The remaining proteins are 15N enriched

and their amino acids are recycled. Some of these amino

acids are incorporated into new hair growth, providing a

signal of the change in diet.8,15,18,19 Research has long

suggested that the progression of anorexic pathology is

accompanied by changing patterns in dietary habits.20–22

These patterns include periods of low or no carbohydrate

intake and an avoidance of dietary fats.20 They can also

include patterns in which the primary foods consumed are

fruits and vegetables. During this period, meat is often

avoided.21 In one study of anorexic women, for example, 47%

of the participants reported they had become vegetarians.23

Copyright # 2006 John Wiley & Sons, Ltd.

3368 K. A. Hatch et al.

Changes in relative amounts of heavy to light isotopes in the

hair indicate changes in the body’s metabolic state and

dietary intake. Since hair is produced sequentially and is

inert after production by the body, the serial sections of hair

record these changes over time.8 d15N values should decrease

over time as the eating disorder progresses and the

individual eliminates animal products from the diet, thereby

reducing the amount of protein in the diet17,24 (i.e. older

segments of hair would have higher d15N values than newer

segments). Finally, the newest segments of hair would show

an increase in d15N values in individuals who have become

highly catabolic, because they are eating a high fiber diet

providing little protein, they are losing weight rapidly, and

they are metabolizing their own proteins for energy. Since

the d15N values of hairs of normal individuals should follow

no such pattern, we predicted that it would be possible to

distinguish individuals with anorexia or bulimia (or both)

from normal individuals (non-clinical controls) based on

stable isotope analysis of the hairs alone.

The focus of much stable isotope research, to date, has been

to reconstruct dietary histories. Much of this work has been

done by anthropologists, paleontologists, and archeologists

who initially focused on bone collagen.25,26 Nitrogen was

shown to correlate well with diet, but to be approximately

3% more positive than the diet.13,27–29 Carbon in well-

preserved hairs was also found to correlate well with

diet.27,30–34 Schoeninger and DeNiro25 also suggested that

d15N values might be useful in determining the degree of

carnivory in consumers.

Surprisingly, it was not until the late 1990s that researchers

provided experimental evidence suggesting hair as a

superior material for establishing the dietary history of

archeological humans.35 Meanwhile, because of its accessi-

bility and because its sampling was not invasive, the

usefulness of hair in understanding the dietary habits of

living humans was also explored.11,36–38 Stable isotope ratios

in hair were found to closely reflect the diet of living

humans,36,38,39 and to be able to indicate a diet change in 6–

12 days.36 Shampooing and graying were shown not to affect

the stable isotope ratios in hair, but dying and bleaching may

affect them.39,40 Studies of living humans also provided solid

evidence that increasing the proportion of meat in a diet

increases the d15N of the hairs of the individual.19,35,39

Several studies demonstrated the potential that stable

isotope analysis has for providing information on an

individual’s dietary history as well as her/his body

condition over time. By using stable nitrogen isotopic ratios,

researchers have been able to distinguish people eating diets

containing animal protein (such as meat, eggs, and milk)

from people eating vegan diets.19,41 Those individuals eating

diets higher in animal proteins are naturally labeled with

elevated ratios of 15N/14N.15,42,43 Animals that go through

periods of fasting have also been shown to have tissues

enriched in 15N as a result.16,44 Other studies indicated that

the stable isotope ratio analysis of hair serial sections can

determine anabolic and catabolic states in humans.8,18,45

Nevertheless, few attempts have been made to apply

stable isotope analysis of hairs to the diagnosis and treatment

of medical conditions in humans. The use of stable isotope

analysis of hairs has been explored to detect the use of illegal


drugs,46 to determine adequate zinc intake in infants,47 to

determine the diet of elderly people with senile dementia,43

and to understand the effects of pregnancy.45 A study of

Ross’s geese first suggested that stable isotope analyses

might be used to indicate nutritional stress16,48 (but see also

Ref. 49). Fuller et al.18 were the first to show that nutritional

stress, by affecting the nitrogen balance in pregnant women,

can change the d15N ratios in hairs, although not the d13C

ratios. Most recently, Mekota et al.8 presented evidence that

the dietary changes of women with eating disorders are

recorded in the d15N and d13C of the hairs of these women.

While several studies have demonstrated that nitrogen

isotope ratios of hairs in humans are influenced by diet and

nutritional stress, they were not designed to demonstrate

how to apply stable isotope analysis of hairs to the diagnosis

or treatment of these conditions.

The goal of our study was to determine if stable isotope

analysis of hairs from a sample of young adult women could

provide an objective measure of whether or not they have an

eating disorder. The d13C and d15N values were measured of

the hairs of non-clinical volunteers (i.e. clinically normal

controls) and individuals diagnosed with anorexia nervosa,

bulimia, or both anorexia and bulimia. Individuals diag-

nosed with eating disorders had had these eating disorders

for an average of 4.6 years (standard deviation (SD)¼3.1 years) before the study and first sought treatment after, on

average, 35.5 months (SD¼ 29.2 months). A discriminant

analysis of the d13C and d15N values of the hairs was then

conducted to determine how successfully these data could be

used to separate individuals with eating disorders from

those without. Simple variables were used that avoided any

need for a subjective evaluation of the hairs. Our results

strongly suggest that stable isotope analysis of hairs can

provide an effective, objective diagnosis of whether an

individual has either or both anorexia and bulimia or

whether that individual is clinically normal.

EXPERIMENTAL

Sample collectionHair samples were collected from two groups of female

subjects: inpatients diagnosed as having anorexia, anorexia

and bulimia, or bulimia (20 individuals), and non-clinical

volunteers (23 individuals). The treatment group consisted of

inpatients at an eating disorder treatment facility (The Center

for Change, Orem, UT, USA). All patients were diagnosed by

therapists at the facility as having either anorexia nervosa,

bulimia nervosa, or both anorexia and bulimia, based on the

criteria established in the Diagnostic and Statistical Manual

for Mental Disorders, fourth edition (DSM-IV). While the

diagnoses were made based on the factors listed in the

description of anorexia and bulimia in the DSM-IV, we were

not privy to these data, including the weight and height of

the individuals. The non-clinical volunteers were recruited

from communication classes at Brigham Young University.

Each subject filled out a survey indicating dietary habits and

eating disorder history, and then pulled five hairs from her

own head. The hairs were wrapped in aluminum foil for later

stable isotope analysis. The patients were asked to pull rather

than to cut hairs because cutting hairs can cause loss of


DOI: 10.1002/rcm

Diagnosing eating disorders using stable isotopes in hair 3369

information. Cutting hairs also makes it difficult to align the

hairs such that when hair sections are combined those

sections of hair were grown during the same period of time.50

The patients were also asked to extract their longest hairs, but

the location from which the hairs should come was not

specified.

The hair samples and surveys of the subjects were assigned

numbers to preserve anonymity. The surveys were kept by

one of the authors (SRT). The hair samples were again

assigned a random identification code to ensure that analysts

did not know the original categories of the subjects. All

participants were given a $10 gift certificate for a local

shopping mall in exchange for their participation in the

study. All procedures were approved by the Institutional

Review Board of Brigham Young University and were in

compliance with federal requirements for the use of human

subjects. In addition, permission was obtained from the

eating disorder treatment facility to collect data from the

inpatients there.

Sample preparation and cleaningThe hair samples were prepared as follows: the five strands

were removed from the foil and the proximal (root) ends

were lined up and glued to a piece of surgical wire with

Super GlueTM (Super Glue, Rancho Cucamonga, CA, USA).

The samples were then sonicated for 15 min in distilled water

and for an additional 15 min in petroleum ether to remove

contaminants from the hair’s surface. The samples were

allowed to dry for 1 h and then all five hairs in a sample were

cut into 1 cm segments, starting with the root end. Each 1 cm

segment (including all five hairs from that segment) was

placed in a separate tin capsule and weighed. The sample

weights ranged from 0.3 to 0.6 mg. Occasionally, a 1 cm

segment of an individual’s hairs did not weigh 0.3 mg. In that

case, a second 1 cm segment was added to the same capsule

to bring it within the proper weight range.

Mass spectrometric analysisTin capsules containing 1–2 cm of hair from five hairs were

combusted using a elemental analyzer (Costech, Valencia,

CA USA) coupled to a Deltaplus mass spectrometer

(Finnigan, Bremen, Germany) for d15N and d13C analysis

of the hair samples. All ratios were expressed as d values

relative to Pee Dee Belemnite51 in the case of carbon and

atmospheric nitrogen52 for nitrogen isotopic ratios. Samples

were corrected using UCLA Carrera (a working standard

from Ian Kaplan’s laboratory, UCLA, d13C¼ 2.52) and

LSVEC (NIST, d13C¼�47) as external standards for carbon,

and USGS 25 (NIST, d15N¼�30.4) and USGS 26 (NIST,

d15N¼ 53.5) as external standards for nitrogen. These

standards were chosen because they bounded the expected

d values of the hair allowing the d values to be corrected via

interpolation.

Statistical analysisAnalysis of variance (ANOVA) and stepwise (forward)

multiple regression analysis were used to determine which

factors would most likely be predictive of an individual

having an eating disorder. These variables were chosen

because visual inspection of the data suggested that they


might be useful in distinguishing between anorexic, bulimic,

and non-clinical individuals. The variables considered were

as follows:

1. T
he average d15N values of the whole length of hair;
2. T
he average d15N values of the length of hair grown before
the individual entered treatment (subjects had had the

eating disorder for an average of 35.5 months, with a

standard deviation of 29 months, before seeking treat-

ment);

3. T
he average d15N values of the length of hair grown by the
individual after entering treatment. (Since all the subjects

were inpatients at The Center for Change, staff in the

center were able to monitor them and to ensure they were

getting adequate nutrition.);

4. T
he d15N values of the oldest (most distal) section of hair;
5. T
he d15N values of the youngest (most proximal) section of
hair;

6. T
he difference in d15N values between the oldest and
youngest sections of hair (i.e. D15N);

7. T
he maximum and minimum d15N values found along the
length of the hair;

8. T
he slopes of the d15N values of hair sampled before and
after the subjects entered treatment; and

9. T
he standard deviation of the d15N values along an indi-
vidual’s hair.

The same variables were considered for carbon. These

values were regressed against the following factors: months

since eating disorder was diagnosed, months in treatment,

months working with a nutritionist, age, how frequently the

individual ate meat, and how frequently the individual ate

less than 1200 calories per day. The F-to-enter was 4.00 and

the F-to-remove was 3.99. The variables that were predictive

of any one of these factors were included in the stepwise

discriminant analysis.

The ability of the equations developed by discriminant

analysis to correctly categorize individuals as either having

an eating disorder or as being clinically normal was tested.

The binomial distribution of the normal approximation of the

lower 95% confidence intervals (CIs) for both sensitivity and

specificity of these results was calculated. Only the lower

95% CI was used, since we were primarily concerned with

the degree to which a test might perform worse than, not

better than, the predicted outcome.

ANOVA (type III sums of squares) was used to determine

which of the above variables appeared to be useful in

distinguishing between individuals with anorexia, bulimia,

both anorexia and bulimia, and non-clinical volunteers. Post

hoc analyses were carried out using Fisher’s probable least-

squares difference (PLSD, p< 0.05). Variables that appeared

useful in this regard were also included in the stepwise

discriminant analysis.

RESULTS

ANOVA showed that several factors could be useful in

distinguishing between non-clinical individuals, individuals

with eating disorders and between different eating disorders.

When the d15N values of the oldest sections of hair were

compared with those of the youngest sections of hairs for all


DOI: 10.1002/rcm

Figure 1. The d15N of the oldest (the 1 cm section at tip)–the

youngest section of hair (the 1 cm section at the base). ‘Both’

indicates individuals with both anorexia and bulimia. Error

bars indicate standard error. Groups that do not share letters

above the bar differ significantly at p< 0.05.

Figure 3. Comparison of the variance in d15N along the

length of hairs between those diagnosed with an eating

disorder and the controls. ‘Both’ indicates individuals with

both anorexia and bulimia. Error bars indicate standard error.


four groups (anorexia, bulimia, both anorexia and bulimia,

and normal volunteers), the differences were significant

(F3,39 ¼ 5.688, p¼ 0.0025). Patients who described themselves

as anorexic showed the greatest difference from the controls.

Patients with both anorexia and bulimia showed less

difference. There was no significant difference between

bulimic patients and controls or between anorexic patients

and patients with both anorexia and bulimia. However,

anorexic patients were significantly different from bulimic

patients (Fig. 1).

The youngest hair growth of patients with either anorexia

or both anorexia and bulimia had significantly lower d15N

values than non-clinical controls while there was no

difference between individuals with bulimia and the non-

clinical controls (F3,39¼ 4.916, p¼ 0.005; Fig. 2). However,

there was no statistical difference between the mean d15N

values of the oldest segments of hair (the 1 cm nearest the tip

of the hair) of any of the groups. Finally, the degree of

variance in d15N along the length of an individual’s hair was

examined and compared with the hair variance between the

groups. Here there was weak evidence (F3,38¼ 2.647,

Figure 2. Comparison of the d15N values of the youngest

section of hair (the 1 cm section at the base) between those

diagnosed with an eating disorder and the controls. ‘Both’

indicates individuals with both anorexia and bulimia. Error

bars indicate standard error. Groups that do not share letters

above the bar differ significantly at p< 0.05.


p¼ 0.0624) that the hairs of individuals with anorexia were

more variable in d15N along their length than were those

either from individuals with bulimia or from the normal

volunteers and that individuals with both anorexia and

bulimia showed a similar trend (Fig. 3).

Stepwise (forward) multiple regression analyses showed

that the d15N values of the newest sections of hair were

correlated with an individual’s age (t40¼ 2.124, p< 0.04) and

with the amount of meat that an individual consumed each

day (t40 ¼ 4.341, p< 0.0001; R2¼ 0.371). The standard devi-

ation of the d15N values along the length of the hair was

found to correlate with how frequently an individual ate less

than 1200 calories per day (t41¼ 2.803, p¼ 0.008; R2¼ 0.161).

The difference between the d15N values of the oldest and the

youngest sections of hair (distal and proximal ends of the

hair) was found to correlate both with the frequency of eating

less than 1200 calories per day (t40¼ 2.185, p¼ 0.035) and

with the frequency of eating meat (t40¼�1.872, p¼ 0.07;

R2¼ 0.56).

The d13C values of the hairs appeared to be less useful in

distinguishing among individuals with eating disorders and

the non-clinical controls. Nevertheless, the d13C of the oldest–

the d13C of the youngest 1 cm sections of hair differed

significantly between individuals with and without eating

disorders (F1,40¼ 11.237, p¼ 0.002; Fig. 4). Forward stepwise

Figure 4. The d13C of the oldest–the d13C of the newest

sections of hair (i.e. the D13C) was significantly different

(p< 0.05). Error bars indicate standard error.


DOI: 10.1002/rcm

Table 1. The correct and incorrect diagnosis of individuals as

either having an eating disorder or as being clinically normal

by the discriminant analysis algorithm

Subject group (n)

Subjects assignedto control group

(% of total)

Subjects assignedto eating disordergroup (% of total)

Control (22) 18 (81.8) 4 (18.2)Eating disorder (20) 4 (20) 16 (80)


multiple regression found that the d13C of the youngest 1 cm

of hair was correlated with the number of months since the

eating disorder began (p¼ 0.04; R2¼ 0.10). The average d13C

value of an individual’s hair after entering treatment was

significantly correlated with the frequency of eating meat

(t36¼ 3.161, p¼ 0.003; R2¼ 0.22).

Thus, through the use of ANOVA and stepwise multiple

regression, the list of variables to include in a stepwise

discriminant analysis was narrowed down to the following:

1. S

Co

tandard deviation in d15N values along the length of an

individuals hair;

2. T
he d15N values of the youngest (most proximal) section of
hair;

3. T
he average d15N value of an individual’s hair after
entering treatment;

4. T
he difference in d13C values between the oldest and
youngest sections of hair; and

5. T
he d13C values of the oldest sections of hair.
Other comparisons were not significant and therefore were

not included in the above list.

The forward stepwise discriminant analysis further

reduced the list of useful variables. Variables were added

if p< 0.15 and dropped if their p values increased above 0.15

after other variables were added. The difference between the

d15N values of the oldest and youngest sections of hair was

added first, followed by the standard deviation in d15N

values along the length of the hair. When, however, the

difference in the d13C values of the oldest and the youngest

sections was added, the difference in d15N values between

them was dropped. Finally, the d15N values of the youngest

section of hair were added to the model.

The discriminant analysis procedure used three of the

above variables to produce two equations: one for individ-

uals with normal eating habits (Eqn. (1)) and one for

individuals with eating disorders (Eqn. (2)):

Control ¼ �143:7 þ 14:1S þ 35:0Y � 5:57O (1)

Eating disorder ¼ �125:6 þ 20:1S þ 32:5Y � 3:42O (2)

where S is the standard deviation of the d15N values along the

length of the hairs, Y is the d15N value of the youngest hair

section, and O is the difference in the d13C value of the oldest–

the youngest hair section. The values for the individual are

entered into each equation. If the resulting value is larger for

Eqn. (1), it is inferred that the individual is clinically normal.

If, however, the larger of the two results belongs to Eqn. (2), it

is then inferred that the individual has an eating disorder.

Based on this algorithm, the computer assigned each

individual to either the control group or the eating disorder

group. The algorithm was correct approximately 80% of the

time in both cases (see Table 1).

DISCUSSION

Our study suggests that a statistically sound and objective

measure can be triangulated with the current objective

and subjective measures to improve the diagnosis of

eating disorders. While previous studies have shown that

eating habits,8,15,18,19,35,41–43,45 weight loss or gain,8,18,45 or

pyright # 2006 John Wiley & Sons, Ltd.

eating disorders8 can affect the ratios of stable isotopes in

hair, this study is the first to establish an objective and

statistically sound means, based on stable isotope ratios of

hairs, for diagnosing eating disorders. The diagnosis of an

eating disorder is a triangulation of multiple facts, including

both objective measures (e.g. weight, amenorrhea, body mass

index, change in weight over time) and subjective assess-

ments based on qualitative interviews and self-reporting by

the patient. The accuracy of the subjective assessments is

based on the honesty of the information reported by the

patient, and it can be difficult for a clinician seeing a patient

for the first time to get a clear idea of the true history of the

patient’s disorder. Researchers, clinicians and patients

would therefore greatly benefit from an objective, biological

measure that could aid in diagnosing eating disorders.

While Mekota et al.8 were the first to attempt to analyze

isotope ratios of hair as a means of diagnosing eating

disorders, their study was not designed to establish the

validity of using stable isotope analysis to diagnose eating

disorders. To establish the validity of a method or test to

diagnose a condition, three points need to be considered.

First, the study should be a blind and independent

comparison with a reference standard. Secondly, the study

should include an appropriate spectrum of individuals,

including both those with and those without the condition so

that the sensitivity and specificity of the method can be

evaluated. Thirdly, the results should not influence the

outcome.53,54

Similarly to previous authors, we initially used a visual

examination of the data to evaluate the individual’s eating

disorder. However, this new approach differed in that the

tests were blind so that previous knowledge of the

individuals could not influence the outcome. In addition,

we included non-clinical individuals in our study. This had

not been done previously when studying the effects of eating

disorders8 or nutritional stress18,45 on the carbon and

nitrogen isotope ratios in human hairs.

A blind pilot test was carried out in which one of the

authors (KAH) was given the d15N and d13C values from the

serial analyses of the hairs of six individuals. KAH was told

only that three individuals were normal and three had eating

disorders. By visually examining graphs of the stable isotope

ratios of the hairs down their lengths, KAH was able to

correctly assign all six individuals to their correct group.

This procedure was repeated with all the individuals in the

study. However, this time not only did KAH not know who

had an eating disorder and who did not, but he did not know

how many had an eating disorder and how many did not. On

this occasion, the overall success was only 70%. This

represents only a fair ability to discriminate.55


DOI: 10.1002/rcm


Typically, when establishing the diagnostic performance

of a test, one should establish the sensitivity and specificity of

the test. The sensitivity is the probability that the test is

positive for an individual who actually has the disease, while

the specificity is the probability that the test is negative for an

individual who does not have the disease.56 The sensitivity of

the subjective, visual method was 75% with a 95% lower

confidence interval (CI) of 59%. The specificity was 65% with

a 95% lower CI of 49%.

Discriminant analysis, however, offered a means by which

one could determine which measures were most important

and predictive. It is also completely objective, since the

algorithms on which the diagnoses are based cannot be

influenced by previous knowledge or biases. Both the

sensitivity and the specificity of this method were better

than that of subjective, visual analysis. The sensitivity using

the algorithms was 80% (95% lower CI¼ 65%) as was the

specificity. Thus the overall ability to discriminate was 80%

(95% lower CI¼ 66%), which indicates good discrimi-

nation.55

While these results are promising, there are several other

potential problems that should be considered. First, it is

important to recognize that the above equations were

established using a North American population. North

Americans differ in their diets from other populations

primarily in that they eat more corn.36,57 Corn is a C4 plant

that has a different carbon isotope ratio from the C3 plants

(e.g. wheat and rice) typical of other industrial societies.58–61

Thus, the above algorithms may not be appropriate for

diagnosing individuals with eating disorders who are

residents of Europe or Japan, for example. However, this

study suggests that if the above algorithms are not universal

in their applicability, algorithms can be developed specific to

the eating habits of other societies.

Secondly, variation in the stable isotope ratios of hairs can

be caused by other factors such as seasonal variations in

diets,62 travel between locations with differing diets,36,37 or

other conditions that affect metabolism.18,45 Incorporating

this kind of information into the above algorithms or

evaluating the results of the algorithms may improve their

accuracy.

Thirdly, the small number of hairs sampled may have

introduced some error. Only five hairs per individual were

sampled, and these hairs occasionally may not have been

representative, particularly if several of the hairs sampled

were in the quiescent stage.50 Previous authors have

suggested using 25 or more hairs to avoid this.8 However,

as the results were 80% correct with regard to both sensitivity

and specificity, this suggests that the small sample size of

hairs was not a major problem. While cutting 25 hairs does

not cause a patient great stress, the most recent information is

lost when hair is cut and it is difficult to properly align the cut

hairs for analysis.50 If one wants to avoid this problem by

pulling hairs, the use of a small sample size reduces the stress

experienced by the patient.

Fourthly, it may also be possible to gain greater accuracy

by sequentially analyzing segments of hair shorter than the 1

cm length used in this study and by avoiding combining

consecutive sections of hair for analysis. Occasionally, 1 cm

segments (for a total of 2 cm) had to be combined in order to


obtain sufficient mass of hair for analysis. This may also have

introduced some error. However, the study by Schwertl

et al.50 suggests that this is unlikely to cause significant errors

because of the buffering effect that the body’s amino acid

pool has. In addition, the algorithms did not include the

sequence of hairs as a factor, except for comparing the first

and the last sequences. Rather, it included the variance of the

signal along the length of the hair. Order is not important

here nor is the occasional combining of two 1 cm segments

likely to greatly affect the variance along the length of the

hair.

Despite these potential problems, Figs. 1–3 suggest that it

may be possible to develop an algorithm that distinguishes,

not just between individuals with eating disorders and non-

clinical individuals, but also among four different groups:

individuals with anorexia, individuals with bulimia, indi-

viduals with both anorexia and bulimia, and non-clinical

controls. In the above-mentioned figures, individuals with

either anorexia or both anorexia and bulimia were consist-

ently similar, while individuals with only bulimia were

consistently similar to non-clinical volunteers. By definition,

individuals with anorexia or anorexia and bulimia are losing

weight and do not get adequate nutrition.3,63 These

individuals either get their nitrogen largely from plants,

and/or do not get sufficient nitrogen in their diet and are in

nitrogen imbalance. Consequently, the individuals in our

study with either anorexia or anorexia and bulimia differed

from non-clinical volunteers. By contrast, individuals diag-

nosed with only bulimia are maintaining their weight,3,63

and therefore get adequate nutrition, and are likely not to be

in nitrogen imbalance. These individuals may get much of

their nitrogen from meat or milk products in their diets64 and

were therefore similar to normal volunteers. These results do

suggest that further distinctions may be possible.

In conclusion, the fact that students from across the USA

were being compared with patients, that the hairs of these

students included seasonal variation and variation from

travel, that only five hairs from each individual were used,

and that it was still possible to obtain 80% or better accuracy

suggest that we were able to capture the majority of the

information needed for diagnosis and that the above

problems are not overwhelming. Accounting for problems

may refine the above algorithms further, thereby improving

the accuracy of the resulting diagnoses, but our study

suggests that the method is already quite robust.

AcknowledgementsWe would like to acknowledge the help of all the individuals

who volunteered in this study. We are especially thankful to

The Center for Change in Orem, Utah, for its assistance in this

study. We appreciate Vanessa Dewey’s help in editing the

manuscript. All funding for this study was provided by the

Office of Research and Creative Activities (ORCA) of Brig-

ham Young University as part of its effort to support under-

graduate mentored research. Funding was in the form of a

Mentoring Environment Grant to KAH and an Undergradu-

ate Research Scholarship to MAC. The $10 gift certificates

were paid for by a grant from the Wendell J. Ashton Fund,

which provides support for research in the Brigham Young

University Department of Communication.


DOI: 10.1002/rcm


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DOI: 10.1002/rcm

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An objective means of diagnosing anorexia nervosa and bulimia nervosa using15N/14N and13C/12C ratios...

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