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Alteration of Enamel Proteins in Hypomaturation Amelogenesis Imperfecta


Pediatric Dentistry, School of Dentistry, The University of Alabama at Birmingham, Alabama 35294; and 'Department of Biochemistry, University
of Texas Health Science Center, Houston, Texas 77225

Amelogenesis imperfecta (AI) is a diverse group of disorders that nantly of components called amelogenins, with a molecular
affects primarily the enamel of teeth through a number of develop- weight of around 25,000, derived from a single gene. Although
mental processes. The purpose of this study was to characterize the it is still a controversial issue, the proteins in mature enamel
enamel proteins in normal enamel and in hypomaturation AI enamel. apparently consist of retained degradation products of amelo-
Impacted teeth., which were at similar stages of development, were genins and higher-molecular-weight proteins (50,000-70,000
obtained for analysis from an individual with Al and from normal
healthy controls. Evaluation of the amino acid profile and quantity of daltons) that are mineral-bound (Robinson et al., 1982, 1983;
organic material collected showed that there was an excess of enamel Fincham et al., 1982, 1983). The protein profile changes dur-
protein material that had an amelogenin-like amino acid profile in ing the maturation of enamel from one rich in proline, leucine,
mature hypomaturation AI enamel. The AI enamel protein content and histidine (amelogenins) to one rich in aspartic acid, gly-
was 5%o, while the control enamel had 0.1% protein (by weight). cine, and alanine (non-amelogenins) (Fincham, 1980; Robin-
These findings indicate that the maturation process had been altered son et al., 1983). According to Robinson and Kirkham (1984),
in this type of Al, and that maturation did not progress beyond the inhibition of proteolysis leads to retention of protein through
initial stages of secondary mineralization. Since this disorder is in- interference with the maturation process and may be the etiol-
herited as an autosomal recessive condition, it seems likely that the
primary defect involves an abnormality in the mechanism for protein
ogy of several pathological conditions of enamel, such as
removal in enamel maturation. amelogenesis imperfecta and fluorosis. The purpose of this
study was to characterize and quantify the enamel protein in
J Dent Res 68(9):1328-1330, September, 1989 normal human teeth and in teeth affected with autosomal re-
cessive hypomaturation amelogenesis imperfecta.
Materials and methods.
Amelogenesis imperfecta (Al) is a heterogeneous group of ge- We obtained five impacted third molars and a canine from
netic disorders characterized by different inheritance patterns a 14-year-old Caucasian female affected with autosomal re-
and a variety of enamel defects. Three basic categories of Al cessive pigmented hypomaturation amelogenesis imperfecta and
have been delineated, including hypoplastic, hypomaturation, five impacted third molars indicated for extraction from two
and hypocalcified types. The classification system proposed unaffected adolescents, ages 15 and 17 years. Only impacted
by Witkop and Sauk (1976) lists 11 distinct Al types based on teeth were analyzed, to preclude the possibility of dental car-
the different inheritance patterns and on clinical manifesta- ies, contamination with salivary proteins, or post-eruption mat-
tions. The primary defect and specific etiology for all the Al uration changes. Teeth from both groups had complete crown
diseases remain unknown. formation and one-half or more root formation, indicating that
Hypoplastic Al results from a deficiency in the production they were at a similar developmental stage. The enamel was
of enamel and is clinically characterized by thin and/or pitted consistent in color and appearance from the coronal to the
enamel that may or may not be fully mineralized (Ooya et al., cervical areas of the teeth, with the control teeth having a
1988). Hypocalcification Al is thought to result from abnor- translucent white color and the Al enamel displaying a char-
malities in the initial deposition of the hydroxyapatite crystals, acteristic yellow-brown color seen in erupted Al teeth. The
and it is clinically manifest by a hypomineralized enamel of a teeth were placed in physiological saline immediately after
yellow-brown color. It is hypothesized that the hypomaturation extraction, cleaned of all adherent soft tissue, and then frozen
types of Al occur as a result of abnormal maturation of the until the enamel could be removed. The enamel from the Al
enamel matrix after the enamel proteins have been secreted teeth was dissected from the tooth with a scalpel and imme-
and initial mineralization has taken place. Enamel crystallites diately placed in test tubes to be lyophilized. Enamel from the
would not be as large as those of normal enamel, and there control teeth was dissected from thin sections cut with a low-
would be a retention of organic material, if the maturation speed diamond saw. The enamel was powdered, lyophilized,
process were interrupted. Witkop et al. (1973) demonstrated and weighed prior to hydrolysis in 6 mol/L HCl at 108'C for
the presence of organic material within the enamel of teeth 24 h. The enamel was directly hydrolyzed in vacuo exposed
affected with hypomaturation Al, which they postulated was to a nitrogen atmosphere.
retained enamel protein. More recent studies (Wright, 1985) After removal of the HCl, the hydrolyzed samples were
have confirmed this histological finding and have shown that placed in 0.40 mL of H20 and injected onto a Beckman 121
both the enamel prism structure and crystallite structure are M amino acid analyzer equipped with a Hewlett Packard In-
abnormal in hypomaturation Al. tegrator; samples were analyzed according to the method of
Studies of the enamel proteins (Robinson et al., 1982, 1983) Butler et al. (1977). To determine whether the ion content of
have shown that there are numerous materials of variable mo- the mineralized specimens interfered with the amino acid ana-
lecular weights, with the early proteins, consisting predomi- lyzer's resolution, we ran standard control samples with similar
amounts of enamel between the test runs. The amino acid
Received for publication December 14, 1988 analyses were statistically analyzed by means of an unpaired t
Accepted for publication February 14, 1989 test that accepted p<0.05 as significant. Quantitative assess-
This investigation was supported in part by BRS Grant S07 RR05300 ment of the protein amount was determined by calculation of
from the National Institute of Dental Research, National Institutes of the residue weight of analyzed material and expression of the
Health, Bethesda, Maryland 20892. total protein as percent yield (weight/volume).
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Amino Acid Normal+ AI* t Value p Value
Cystic Acid 0 4.2 -7.5 <0.0005
OH-proline 0 0 0 -
Aspartic Acid 93.5 38.6 15.5 <0.0005
Thrconine 48.4 36.2 6.5 <0.0005
Serine 127.2 79.2 2.4 < 0.025
Glutamic Acid 124.1 122.8 0.08 <0.4
Proline 138.8 196.8 - 2.8 < 0.025
Glycine 159.7 95.4 6.2 < 0.0005
Alanine 61.9 21.1 3.4 < 0.005
Cystine 0 0 0 -
Valine 44.8 33.1 1.9 <0.05
Methionine 0 28.3 -8.2 <0.0005
Isoleucine 22.4 29.5 -1.5 <0.1
Leucine 62.2 80.3 - 4.3 < 0.005
Tyrosine 8.6 83.2 6.5 <0.005
Phenylalanine 28.3 32.6 - 2.0 < 0.05
OH-Lys 0 0 0 -
Lysine 44.0 29.7 1.0 < 0.0005
Histidine 20.8 51.8 -5.4 <0.0005
Arginine 24.3 36.4 -0.9 <0.375
Residuesper thousand. IFive samples from two individuals. *Five sam-
ples from one individual.

Amino acid standards analyzed with amounts of enamel sim-
ilar to those run in the experimental samples showed that res-
olution and protein recovery were not affected by the ion
concentration present. Direct analyses of the mineralized enamel
specimens were, therefore, considered reliable. The absence
of detectable hydroxyproline in the specimens indicated that
they had not been grossly contaminated with dentin during
The amino acid profiles were markedly different in the Al
and control enamel. Results of the amino acid analyses are
presented as residues per thousand in the Table and Figs. 1
and 2. The control teeth demonstrated amino acid profiles that
were rich in aspartic acid, serine, glycine, and alanine, in
comparison with the Al enamel. The Al enamel protein showed
a reduction in aspartic acid and elevations in proline, methi-
onine, leucine, tyrosine, and histidine, compared with the con-
trols. With a few exceptions-such as glutamic acid, isoleucine,
and arginine-statistical analysis revealed significant differ-
ences in the percentages of all the amino acids between the
control and the Al teeth.
Quantitatively, there was appreciably more protein in the Al Leu Met
enamel, compared with the control samples. The total enamel lie
protein was expressed as ng/mL of solution (weight/volume),
and then the percent of protein per sample was calculated: (Al Figs. I and 2-The amino acid profiles of normal and Al enamel,
= 5.00 + 2.709% SD) vs. (Control = 0.102 + 0.052% respectively, are presented as residues per thousand in the form of rose
SD). The normal enamel ranged from a maximum of 0.19% diagrams, for visualization of the distinctly different enamel protein char-
to a minimum of 0.06%, while the Al enamel ranged from a acter (Robinson et al., 1975).
maximum of 8.06% total protein to a minimum of 1.64%.
Total protein varied widely from one sample to the next in in this study (Robinson et al., 1975; Weidmann and Eyre,
both the control and the Al groups. 1967; Glimcher et al., 1964). The high level of protein found
in normal teeth in this study may have resulted from the direct
Discussion. hydrolysis of mineralized specimens, which may have reduced
the loss of material. Alternatively, since previous studies were
Enamel proteins in hypomaturation pigmented amelogenesis conducted on erupted teeth, and this study was conducted on
imperfecta were markedly altered quantitatively and in their unerupted enamel, there may be post-eruption maturational
amino acid composition, compared with normal control enamel. changes that continue to reduce the enamel protein content.
The control teeth's total enamel protein content was slightly The dissection process provided samples for analysis of nearly
higher than the values published previously, which range from the entire enamel thickness, including that of material directly
0.01% to 0.09%, compared with the mean of 0.102% found adjacent to the dentin-enamel junction (DEJ). Previous studies
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1330 WRIGHT & BUTLER J Dent Res September 1989
have indicated that this is probably the area of enamel richest of amelogenesis imperfecta will result in a better understanding
in protein as a result of the presence of enamel tufts (Weath- of enamel formation and of potential defects in the complex
erell et at. , 1968). Inclusion of this area can result in dentinal developmental processes involved in enamel formation. This
contamination of the enamel specimen; however, the technique study provides biochemical support for the delineation of a
used in this study did not. hypomaturation type of amelogenesis imperfecta. Analysis of
.The protein content of the Al enamel was similar to the enamel from other forms of Al will provide a diagnostic tool
figures reported for human enamel in the very early stages of for this group of diseases that will be more reliable than the
maturation (Weatherell et al., 1968). Deutsch and Alayoff (1987) current criteria of clinical, histological, and hereditary infor-
reported that the enamel protein content during the early mat- mation.
uration stage was 6.93% and dropped to 2.14% and 1.27% as
sampling moved incisally toward more mature enamel. The REFERENCES
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