Medical Hypotheses 79 (2012) 219–221

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Medical Hypotheses
journal homepage: www.elsevier.com/locate/mehy

Are extrinsic black stains of teeth iron-saturated bovine lactoferrin and a sign
of iron deficient anemia or iron overload?
Ilir Mesonjesi ⇑
Private Dental Practice, Rr. ‘‘Jan Kukuzeli’’, Nr. 4, 1010 Tirana, Albania

a r t i c l e i n f o a b s t r a c t

Article history: Extrinsic black stains on teeth are shown to have a relation with a low incidence of caries and are made of
Received 21 September 2011 a ferric compound. Whole composition and why those stains are formed are not fully understood. Studies
Accepted 27 April 2012 have shown low incidence of caries in individuals eating cheese. Lactoferrin is the major iron-binding
protein, constituent of milk, stays almost intact during cheese making and has antibacterial activity
against dental cavity-inducing Streptococcus mutans. Lactoferrin has a high affinity for iron and whenever
it is present it will bind iron and release it only in values of pH < 4. In a small survey that I made in dental
practice, patients (patients did not report taking any medication; had no frequent gingival bleeding) that
had extrinsic black stains on teeth eat >50 g of cheese per day and a good number of them, in addition to
cheese, drink one cup of milk per day. Cheese stays much longer in contact with tooth surface than does’
milk and bovine lactoferrin has four glycan chains that may contribute to a better adherence. Extrinsic
black stains are made of a ferric compound, and people that eat good amounts of cheese (where lactofer-
rin plays a central role) show to have black stains. Iron must be in sufficient amounts in saliva so that
lactoferrin can bind it and as a result making the black stains appear. In iron deficient anemia and in iron
overload the concentration of iron present in saliva is much higher than in individuals with no anemia. In
conclusion, extrinsic black stains of teeth may be iron-saturated bovine lactoferrin and a sign of iron defi-
cient anemia or iron overload if no iron supplements are taken or individuals have no frequent gingival
bleeding.
Ó 2012 Elsevier Ltd. All rights reserved.

Introduction The lactoferrin concentration in cheese was shown to depend

Extrinsic black stains on teeth appear like a black line in the
cervical third on bucal and palatinal (especially palatinal) surface
of teeth, especially in molars of maxilla (Fig. 1). Investigations into
the relationship between colored extrinsic tooth stains and dental
disease have been going on for a considerable period. Most results
show that children with black extrinsic tooth stain have a lower car-
ies experience than children with other colored or with no extrinsic
tooth stain. This finding is true in both the deciduous and permanent
dentition. The stains were examined and shown to contain a black
insoluble ferric compound probably ferric sulfide [3–5].
In a research conducted by Głabska et al. was found that cheese
consumption contributes to decrease of dental caries coefficient
and was proved the need to provide children and teenagers with
a proper model of diet in systematic consumption of milk and dairy
products [8].
Lactoferrin was identified originally as the major iron-binding
constituent of milk and is an iron-binding glycoprotein of the
transferrin family [2].
⇑ Tel.: +355 682304249.
E-mail address: ilirmesonjesi@yahoo.com
on the cheese-making process, with higher values in Swiss-type
and semi-hard cheeses than in soft cheeses. Furthermore, Wes-
tern-blotting analysis of lactoferrin in cheese showed that this pro-
tein stayed intact throughout ripening in raw milk cheese, whereas
it was partially hydrolyzed in cheeses made with pasteurized milk.
Based on these observations, cheese may constitute a natural dairy
source of lactoferrin beneficial to health [7].
Lactoferrin is a protein that displays antibacterial activity
against dental cavity-inducing Streptococcus mutans through an
intracellular mechanism that could involve DNA binding [6].
Lactoferrin is common in exocrine secretions of mammals,
known for its high-affinity for iron [1]. Human lactoferrin binds
iron much more strongly than does’ human transferrin. Lactoferrin
retains iron at pH values distinctly lower than transferrin: in fact,
human transferrin releases iron at pH values below 6, whereas hu-
man lactoferrin does not release iron until the value is reduced be-
low pH 4. Bovine lactoferrin exhibits a peculiar behavior in that it
binds iron more weakly than human lactoferrin and releases iron
more readily, but it has four glycan chains, as compared to three
of human lactoferrin, with only one glycosylation site (Asp 476)
in common. Bovine lactoferrin has a high percentage of homology
with the human lactoferrin polypeptide chain; as expected bovine

0306-9877/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.mehy.2012.04.044
220 I. Mesonjesi / Medical Hypotheses 79 (2012) 219–221
Fig. 1. Extrinsic black stains; black line in the cervical part of palatal surface in
maxillary premolars.
lactoferrin inhibited binding of human lactoferrin to human lacto-
ferrin receptors [2].
The hypothesis
Extrinsic black stains of teeth might be iron-saturated bovine
lactoferrin and those stains can indicate iron deficient anemia if
no iron supplement is taken or the individual does not have fre-
quent gingival bleeding.
It is not known entirely what black stains are made of and how
and why they form. The studies [3–5] have shown they have a fer-
ric compound probably ferric sulfide.
In my dental practice I have noticed those stains for quite a long
time making me very curious how and why they form. First I began
to read the studies, but they were very scarce. Studies only stated
that those stains were made of an insoluble ferric compound. I
thought my patients might have something in common. People
that live in the same region eat and drink probably in comparable
way. My patients come mostly from a region called Myzeqe, a rural
region of Albania, were milk is abundant and cheese is mostly
home made. Water stains teeth in different way for example; fluo-
rine stains. So it might be food. I began to ask them, without col-
lecting any data at first, what was nearly always present in their
daily diet, and dairy products became the main suspect. But why
some people that consumed those products do not have extrinsic
black stains? Most of the patients that had black extrinsic stains
on teeth did not have frequent gingival bleeding which is a addi-
tional source of iron in saliva, only a few women were taking iron
supplements (these patients did not take the survey at first be-
cause I had no clear hypotheses in my mind). Then the idea of
the iron deficiency came when I read the article from the Refs.
[9,11] because in the region the prevalence of beta thalassemia
minor is from 7.3% (Lushnje) to 9.96% (Fier) in the two major cities
of the region and the prevalence of G6PD deficiency 5–25% [10].
Those are hemolytic anemia and subject to iron overload that
raises the concentration of serum iron and as a result the rise of
iron concentration in saliva [9,11]. So I began collecting the data
of the survey.
Evaluation of the hypothesis
 We know that persons who have extrinsic black stains of teeth
have a low experience of caries [3–5].
 We know that cheese consumption contributes the decrease of
dental caries coefficient [7].
 We know that lactoferrin is a protein that displays antibacterial
activity against dental cavity-inducing S. mutans [6].
 We know that extrinsic black stains of teeth contain a ferric
compound probably ferric sulfide [3–5].
 The data collected from the survey indicates that persons who
consume lots of dairy products have those stains.
 We know that lactoferrin is major iron-binding constituent of
milk [2] and analysis of lactoferrin in cheese showed that this
protein stayed intact throughout ripening in raw milk cheese
[7].
 The protein that has a high affinity for iron is lactoferrin and it
can bind iron present in saliva (even in low pH values) which is
in high concentration in persons with iron deficient anemia or
beta thalassemia minor [9,11].
 Empirical data and methods: In this survey the patients that pre-
sented extrinsic black stains on teeth asked to fill a questionnaire
about the quantity of cheese and milk they consumed in a day.
The criteria of diagnosis the extrinsic black stains were according
to Koch et al. Presence of dark lines forming a linear discoloration
parallel to the gingival margin of at least two permanent teeth. In
the survey took part 94 patients (none of patients reported tak-
ing any medications at the time of the survey, and they did not
have frequent gingival bleeding) aged 7–53 years old, 54 males
and 40 females. Ninety (95.74%, p < 0.05) of them ate >50 g of
cheese per day; 2 patients ate 30 g; 1 patient 25 g and 1 ate
<50 g per day; 39 patients of the total number, besides the
amount of cheese drink a cup or more cow milk per day. The
cheese made in Albania, is a hard cheese, a type that resembles
feta cheese and is made mostly from raw milk.
Dairy product Cheese P50 g Cheese <50 g Cheese + milk one
per day per day or more cup of
milk per day
Number of 90 4 39 (of the total 94)
patients
 Bovine lactoferrin has four glycan chains which may contribute
to a better adherence to tooth surface than human lactoferrin
and cheese stays much longer in contact with tooth surface than
does’ milk.
 The hypothesis can be tested by scratching the material of the
black stains as shown in Ref. [3] and the material tested with
ELISA method to detect iron-saturated lactoferrin (this test
was not available in my country at the time). Blood sample
should be taken to detect iron deficient anemia.
 The survey should have been with a greater number of patients
and perhaps with a control group.
Consequences of the hypothesis and discussion
If the hypothesis is true; first we have found what those extrin-
sic black stains of teeth are made of, and second and perhaps more
important; we have found a new sign to detect iron deficient ane-
mia, iron overload (like in beta thalassemia minor or intermedia).
This is more important in children and pregnant women, especially
when iron deficient anemia or heterozygote beta thalassemia is not
suspected. It is quick, less invasive and, because it can be detected
in a routine visit lowers the costs especially in developing coun-
tries. Further studies should be made to investigate those stains.
Conflict of interest statement
None declared.
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