Journal of Oral Pathology 1983: 12: 319-329

Leukoedema: ultrastructural and histochemical

observations
C. W. VAN WYK and S. C. AMBROSIO

Research Group in Dental Epidemiology, South African Medical Research Council and Department of
Oral Pathology, University of Stellenbosch, South Africa

The ultrastructural features of 12 cases of leukoedema were investigated and com-

pared with clinically normal buecal mucosa, Histochemistry was undertaken to try
to resolve some of the observed ultrastructural changes. The characteristic "intra-
cellular edema" of the epithelial cells in leukoedema is due to vacuolation in the
cytoplasm of cells. Abnormal mitochondria were observed in these cells. The vac-
uoles contained a granular material somewhat like clumped glycogen granules, but
histochemistry failed to identify this material as glycogen. Towards the surface of
the epithelium, the vacuolated cells collapsed into a compact layer of flattened cells.
The outer cells of this layer abruptly swelled again to form the characteristic super-
ficial layer of "ballooning" cells of leukoedema. The latter cells were not vacuol-
ated but contained remnants of organelles, membraned vesicles with remnants of
organelles, keratohyalin granules and structures apparently related to keratohyalin
granules. We propose that the vacuolation represents a limited reversible form of
cellular degeneration resulting from cell damage and that impeded mitochondrial
function may be the cause ofthe vacuolation. The superficial "ballooning" cells are
degenerated cells. The flattening of the vacuolated cells into a compact layer and
the presence of keratohyalin granules and keratohyalin-like structures in the super-
ficial cells are regarded as features of an aborted form of keratinization.
Accepted for publication December 21, 1982

Leukoedema is a typically diffuse opalescent
lesion of the cheek mueosa whieh ean extend
to the lips. It is eommon and has been
observed in many parts of the world
(Sandstead & Lowe 1953, Arehard et al,
1968, Pindborg et al. 1968, Hamner et al.
1971, Durocher et al. 1972, Martin & Crump
1972, Borghelli et al, 1975, Van Wyk et al.
1979, Axell & Henriesson 1981). There ap-
pears to be a strong assoeiation with eertain
habits - the use of tobaeeo (Pindborg et al.
1968, Hamner et al. 1971, Tyldesley 1971,
Van Wyk et al. 1979, Axell & Henriesson
1981), ehewing of eoea leaves (Borghelli et al.
1975), betel nut ehewing (Pindborg et al.
1968) and eheek sueking (Van Wyk et al,
1979), On the other hand Arehard et al.
(1968) and Martin & Crump (1972) found no
elear assoeiation with habits, and the former
proposes that the eondition is a variant of the
normal.
The histologieal features of leukoedema are
320 VAN WYK & AMBROSIO

Table 1.
Smoking pattern of cases with and without teukoedema.

Tobacco
Age lYpe Durat ion Grammes/day Leukoedema

16 Cigarette s 6 yr s 17 Yes
16 Cigarette s 5 yrs 14 Yes
16 Cigarettes 7 yrs 14 Yes
17 Cigarettes 8 yr s 10 Yes
17 Zolle* k yr s 10 Yes
17 Cigarette s 5 yrs 12 Yes
17 Cigarette s 9 yr s 40 Yes
18 Cigarette s 8 yr s 22 Yes
18 Cigarett e s 6 yr s 30 Yes
19 Zolle 10 yrs 8 Yes
21 Cigarette s Yes
10 yrs 13
-t- Zolle
22 Cigarette s 11 yrs 9 Yes
X 17,8 X 7,^ X 16 ,6
17 Cigarettes 6 yr s 10 No
18 Cigarette s k yrs 2 No
18 Cigarette s 5 yrs 5 No
21 C i ga r e 11 e s 10 yrs 4 No
xl8,5 X 6,3 X 5,3

Zolle - pipe tobacco rolled in paper.

typical and have been thoroughly documented
by, among others, Sandstead & Lowe (1953),
Archard et al. (1968) and Hamner et al.
(1971). The most conspicuous characteristics
are the thickening of the epithelial layer and
broadening of rete pegs, "intracellular
edema" of the cells of the intermediate layer
and a "retention" of the superficial cells either
as a layer of parakeratotic cells (Archard et al.
1968) or as a layer of "ballooning" cells
(Pindborg et al. 1968) (See Fig. 1).
Although there is considerable evidence to
show that the condition is associated with
certain noxious habits, knowledge about its
pathogenesis is still lacking. In addition its ul-
trastructural characteristics are unknown and
there is no explanation for the "intracellular
edema" of the cells.
The purpose of the present investigation
was to determine whether a correlation exists
between the histological features of the con-
dition and the particular habit which is prac-
 LEUKOEDEMA
tised, to observe its ultrastructural features
and to try and explain the occurrence of the
intracellular edema".
Material and methods
The material for histology and electron mic-
roscopy had been collected over a period of
3 years and came from 12 volunteers who
were selected from among patients who pre-
sented for dental treatment. They were cho-
sen because they all depicted the classical
clinical features of leukoedema. No attempt
was made to grade the clinical features. All
the subjects were male, of European-African-
Malay extraction, and all smoked. Their ages
ranged from 16-22 years (Table 1). The
specimens were removed from cheek and lip
mucosa.
Four biopsy specimens from the premolar
areas of clinically normal cheek mucosa, were
studied as controls. These were donated by
male subjects who smoked, and who were of
the same age group and extraction as the
above (Table 1).
Immediately after removal, specimens were
divided into three pieces for histology, elec-
tron microscopy and histoehemistry. For the
histological investigations, tissues were fixed
in Bouin's fluid and stained with hematoxylin
and eosin, and the periodic acid-Schiff
method for glycogen. For electron micro-
scopy, specimens were fixed in a 4% cacody-
late (0.2 M) buffered glutaraldehyde solution
(pH 7.2) for 12 h at 4°C, post-fixed in 1%
osmium tetroxide for one h at 4°C, embedded
in epoxy resin according to Spurr (1969), sec-
tioned and stained with uranyl acetate and
lead citrate. The specimens for histoehemistry
were quenched in liquid nitrogen and stored
at -70°C. When required, frozen sections
were prepared and stained with hematoxylin
and eosin, and for glycogen, lipids, acid phos-
phatase and succinate dehydrogenase, ac-
321
cording to Chayen et al. (1973). The his-
tological features of each case were listed and
compared with each other.
Results
An analysis of the smoking habits of the sub-
jects, with and without leukoedema, are given
in Table 1. Using the Mann-Whitney U test it
was found that the quantity of tobacco
smoked by cases with leukoedema differed
significantly (at the level p = 0.002) from the
quantity smoked by those without
leukoedema.
Histology. All the cases of leukoedema,
whether the specimens came from the cheek
or lip, had the diagnostic features of the con-
dition: thickening of the epithelial layer,
broad rete pegs, "intracellular edema" of the
cells of the intermediate layer, flattening of
the superficial cells and the retention of an
outermost layer of "ballooning" cells (Fig. 1).
Parakeratinized cells were not seen. Glycogen
was present in the prickle cells and cells of the
intermediate and superficial layers of the
epithelium, but was peripherally distributed in
those cells which had undergone "intracellular
edema". There was no obvious correlation
between the microscopic features and the
duration and the amount smoked.
The epithelium of the clinically normal mu-
cosa was similar to the example of buccal
epithelium shown by Squier et al. (1976), viz.,
a thickened epithelial layer with a well-de-
fined basal-cell layer, prickle cells, an inter-
mediate and a superficial-cell layer. The
epithelium did not have broad rete pegs, as
seen in leukoedema, and neither were "in-
tracellular edema" or the superficial "bal-
looning" of cells observed.
Electron microscopy. Controls. The ultras-
tructural features of the epithelium were
 1

Fig. 1. The histology of leukoedema, (A) the layer of superficial "ballooning" cells, (B) the zone of compact
flat cells, (C) cells with "intracellular edema", (D) the area in the intermediate layer where "intracellular
edema" commences and (E) the basal cell layer, (H&E x350).

Fig. 2. An example of the basal cells in leukoedema which lie closely together. The basement lamina is
indicated. Note the numerous normal mitochondria in these cells, (x 18000),
Fig. 3. The early features of vacuolation (indicated) as seen in cells of zone (D) in Fig. 1. (x 112,000).

Fig. 4. Vacuolation in the outer cells of the intermediate layer (zone (C) in Fig. 1). The condensation of
tonofilaments and organelles against the cell membranes and around the nuclei, is indicated. Note how
tightly cells are joined together, (x 10,000). The inset shows the contents of the vacuoles. (x32,500).
Fig. 5. Abnormal mitochondria next to a vacuole (V). These features were seen in cells of zones C & D in
Fig. 1. (X 60,000).

Fig. 6. The layer of flat compact cells (zone B in Fig. 1). (V) are vacuoles (x 13,500).
IT

8A
Fig. 7. An example of the superficial "ballooning" cells (A) (zone A in Fig, 1), (B) is a cell of the compact
layer deep to it (x 7,000),

Fig. 8. The contents of the superficial "ballooning" cells, 8A illustrates a membraned vesicle (autophagic
vaeuoie?) with remnants of organelles. Note the loose mesh of tonofilaments, (x 112,000), 8B illustrates
remnants of rough endoplasmic reticulum and other organelles (x 112,000),
22 Oral Pathology 12:5
Fig. 9. An example of a keratohyalin granule in the superficial "ballooning" cells (x 60,000),

Fig. 10. An example of the structures in the superficial "ballooning" cells which may be related to
keratohyalin granules (x 60,000).
 LEUKOEDEMA
similar to those illustrated by Squier et al,
(1976) except that small cytoplasmic spaces,
filled with a granular material, were present in
cells of the superficial layer.
In leukoedema, we found that the basal cells
were in closer approximation than those in the
controls. I'his feature became even more
prominent in the cells of the intermediate
layer (Figs, 2, 4), Also present in this layer
were cytoplasmic spaces. The first signs of
these were seen in the deeper cells. The spaces
coalesced until all cytoplasmic organelles and
tonofilaments were displaced against the cell
membranes and around the nuclei in the sup-
erficial cells (Figs. 3, 4). The vacuoles con-
tained a granular reticulum resembling
clumped glycogen granules (Fig. 4 inset).
Associated with the appearance of these
spaces were abnormally shaped and swollen
mitochondria (Fig. 5). Such abnormal mito-
chondria were not seen in the basal cells of
leukoedema (Fig. 2) nor in the cells of the
controls.
Towards the periphery of the epithelial
layer, the vacuolated cells abruptly collapsed
into a band of compact, flat cells (Fig. 6). As
suddenly as the vacuolated cells collapsed to
form the compact layer of cells, so the latter
suddenly swelled again to form the outer layer
of "ballooning" cells (Fig. 7). These cells had
no vacuoles and the tonofilaments were
loosely dispersed in the cytoplasm. Within the
cytoplasm were membraned cavities which
contained remnants of organelles, and clumps
of organelles and fragments of rough endo-
plasmic reticulum (Figs. 1, 8). Glycogen
granules were scantily sprinkled among the
tonofilaments. The nuclei were irregular and
the chromatin condensed into clots. Also
present were keratohyalin granules (Fig. 9)
and structures of similar size but varying con-
figurations. The latter varied from almost
solid (like keratohyalin granules) to irregular
masses with a network-like texture (Fig. 10).
22*
327
Histochemistry. The characteristics of
leukoedema and normal mucosa in fresh-
frozen sections were similar to those in paraf-
fin sections. The distribution of glycogen was
the same and lipids were not seen in the
epithelium. Acid phosphatase and succinate
dehydrogenase were present with the heaviest
concentration in the basal-cell region. The
vacuoles in the cells of the intermediate layer
of leukoedema were devoid of precipitate and
the "ballooning" cells contained only a few
positive granules.
Discussion
Although the examples of leukoedema and of
normal cheek mucosa selected for this study
cannot be regarded as wholly representative,
it is of interest to note that the subjects'
smoking patterns differed significantly with
regard to the quantity of tobacco used. This
finding is in keeping with the conclusions of
Axell & Henricsson (1981). Thus, it seems
that there is a threshold for smoking and when
it is exceeded, leukoedema may develop. On
the other hand, Axell & Henricsson (1981)
also point out that ethnic factors may also be
of importance in the development of the con-
dition.
The reason why no correlation could be
observed between the histological features of
leukoedema and the smoking pattern can be
explained by the selection procedures. As
mentioned earlier, all cases were selected be-
cause of the characteristic features of the con-
dition. Therefore, it is reasonable to expect
that their histological features should be com-
parable - in other words they were all estab-
lished cases. The features of leukoedema pose
two important questions: (1) is leukoedema a
variant of the normal and (2) is the vacuola-
tion of the cells a pathological entity? We
suggest that it is a pathological entity and that
the vacuolation represents a limited form of
cellular damage. However, we are unable to
328 VAN WYK & AMBROSIO
explain the mechanism by which the noxious
habits cause this cell damage.
If it is a variant of the normal, one would
expect to find similar features in both normal
mucosa and leukoedema, however, that was
not the case, nor was there any similarity to
the examples of mucosa illustrated elsewhere
(Squier et al. 1976). On the other hand one
cannot rule out that the so-called "occlusal
line" or 'Hinea alba", often observed in
cheeks, may have some features similar to
leukoedema. In our experience this "line"
frequently has a swollen opalescent appear-
ance somewhat like a localized form of
leukoedema (Van Wyk et al. 1977). But even
this possibility is insufficient evidence to re-
gard leukoedema as part of the normal. As
mentioned earlier, leukoedema is a wide-
spread condition of the mucosal epithelium of
both cheeks and lips, and all the affected sur-
faces show typical microscopic features.
With regard to the "intracellular edema" or
the vacuolation in the cells of the intermediate
layer, we believe it has some resemblance to
reticular degeneration, a type of degeneration
described by Lever & Schaumburg-Lever
(1975). Ihis degeneration is not necessarily
irreversible and does not result in immediate
cell death. According to Dixon (1967) this can
happen when damage is insufficient to cause
disintegration of the surface membranes. If
the membrane remains intact, osmotic swell-
ing of the cell will continue and gross vacuola-
tion will eventually result.
On the other hand, such an inflow of fluid
can be the result of mitochondrial damage
too. The production of ATP is reduced, im-
peding the action of the cellular sodium pump,
and sodium and fluid accumulate in the cell
(Walter & Israel 1979). The abnormal mito-
chondria seen in association with vacuolation
lend support to the above explanation. Their
appearance is very similar to the illustration of
Trump et al. (1962) of damaged mitochon-
dria. The fact that we demonstrated succinate
dehydrogenase in the epithelium does not
preclude an hypothesis of mitochondrial dam-
age. According to Trump et al. (1965) one
cannot histochemically correlate the activity
of this enzyme with the ultrastructural altera-
tions of mitochondria.
The features of the abnormal mitochondria
present a special problem in that it can be
regarded as a result of faulty preparation. This
is difficult to accept because in several in-
stances the same section exhibited normal
mitochondria in the basal cells and abnormal
in the vacuolated cells. Also, as mentioned
earlier, abnormal mitochondria were not seen
in any basal cells nor in the examples of nor-
mal epithelium. In addition, the well-defined
double-membrane structure of the abnormal
mitochondria indicates good fixation. If a
method could be developed, such as that of
Sordahl et al. (1971), to measure the
oxidative phosphorylation of mitochondria in
small specimens, then the above problem
might be solved.
Another possible cause of the cavitation in
cells of the intermediate layer which was con-
sidered, was the effect of lysosomes. We saw
neither ultrastructural features of increased
lysosomal activity, nor an altered pattern of
distribution of acid phosphatase in the
epithelium, similar to that described by
Janigan & Santamaria (1961).
We cannot satisfactorily explain the abrupt
collapse of the vacuolated cells into a band of
compressed cells and their subsequent re-
swelling. We interpret the flattening of cells as
an active metabolic process with a superficial
resemblance to the process of keratinization,
where condensation of superficial cells pre-
cedes keratinization. Why they suddenly
should swell again is a complete enigma. From
the electron miscoscopic observations of the
"ballooning" cells one must conclude that they
are in a state of degeneration. The presence
in the cytoplasm of remnants of organelles
and structures resembling single-membraned
 LEUKOEDEMA
autophagic vacuoles containing organelles, as
described by Arstilla & Trump (1968), sup-
ports this interpretation.
Finally, we contend that the presence of
keratohyalin granules in the superficial cells,
as well as structures apparently related to
keratohyalin granules, also indicates an abor-
tive form of keratinization.
References
Archard, H. O., Carlson, K. P. & Stanley, H. R.
(1968) Leukoedema of the human oral mucosa.
Oral Surg 25, 717-728.
Arstilla, A. U. & Trump, B. F. (1968) The forma-
tion of autophagic vacuoles in the liver after
glucogen administration. Am J Pathol 53,
687-733.
Axell, T. & Henricsson, V. (1981) Leukoedema -
an epidemiologie study with special reference to
the influence of tobacco habits. Community Dent
Oral Epidemiol 9, 142-146.
Borghelli, R. F., Stirparo, M., Anrade, J., Barros,
R., Centofanti, M. & de Estevez, O. T. (1975)
Leukoedema in addicts to coca leaves in
Humahuaca, Argentina. Community Dent Oral
Epidemiol 3, 40-43.
Chayen, J., Bitensky, L. & Butcher, R. G. (1973)
Practical Histoehemistry, 1st edn., pp. 81, 72-74,
114-115, 191-192. London: John Wiley & Sons.
Dixon, K. C. (1967) Events in dying cells. Proc R
Soc Med 60, 271-275.
Durocher, R. T., Thalman, R. & Fiore-Donne, G.
(1972) Leukoedema of the oral mucosa. J Am
Dent Assoc 85, 1105-1109.
Hamner, J. E., Mehta, F. S., Pindborg, J. J. & Daf-
tary, D. K. (1971) An epidemiologie and his-
topathologie study of leukoedema among 50,915
rural Indian villagers. Oral Surg 32, 58-65.
Janigan, D. T. & Santamaria, A. (1961) A his-
tochemical study of swelling and vacuolation of
proximal tubular cells in sucrose nephrosis in the
rat. Am J Pathol 39, 175-193.
Lever, W. F. & Schaumberg-Lever, G. (1975) His-
topathology of the Skin, 5th edn., p. 340.
Philadelphia: J. B. Lippineott Company.
Martin, J. L. & Crump, E. P. (1972) Leukoedema
of the bueeal mucosa in Negro children and
youth. Oral Surg 34, 49-58.
329
Pindborg, J. J., Barmes, D. & Roed-Petersen, B.
(1968) Epidemiology and histology of oral
leukoplakia and leukoedema among Papuans
and New Guineans. Cancer 11, 379-384.
Sandstead, H. R. & Lowe, J. W. (1953)
Leukoedema and keratosis in relation to
leukoplakia of the buccal mucosa in man. J Natl
Cancer Inst 14, 423-437.
Sordahl, L. A., Johnson, C , Blailock, Z. R. &
Schwartz, A. (1971) The mitochondrion. In
Methods of Pharmacology, Vol. 1, ed. Schwartz,
A., pp. 247-286. New York: Appleton-Century-
Crofts.
Spurr, A. R. (1969) A low-viscosity epoxy resin
embedding medium for electron microscopy. J
Ultrasctuct Res 26, 31-43.
Squier, C. A., Johnson, N. W. & Hopps, R. M.
(1976) Human Oral Mucosa Development,
Structure and Function, 1st edn., pp. 22—33.
Oxford: Blackwell Scientific Publication.
Trump, B. F., Goldblatt, P. J. & Stowell, R. E.
(1962) An electron microscopic study of early
cytoplasmic alterations in hepatic parenchymal
cells of mouse liver during necrosis in vitro (au-
tolysis). Lab Invest 11, 986-1015.
Trump, B. F., Goldblatt, P. J. & Stowell, R. E.
(1965) Studies on necrosis of mouse liver m vitro.
Lab Invest 14, 343-371.
Tyldesley, W. R. (1971) Tobacco chewing in En-
glish coal miners. A preliminary report. Br J Oral
Surg 9, 21-28.
Van Wyk, C. W., Breytenbach, H. S. & Dreyer,
W. P. (1979) The epidemiology of preventable
oral mucosal lesions in the Cape Peninsula. J
Dent Assoc S Afr 34, 672-676.
Van Wyk, C. W., Staz, J. & Farman, A. G. (1977)
The prevalence of oral mucosal lesions among a
random sample of Asians resident in Cape Town.
J Dent Assoc S Afr 32, 589-592.
Walter, J. B. & Israel, M. S. (1979) General
Pathology, 5th edn., pp. 49-52. Edinburgh:
Churchill Livingstone.
Address:
Dr. C. W. van Wyk,
Private Bag XI,
Tygerberg,
7505,
South Africa.