T H E AMERICAN JOURNAL OF CLINICAL PATHOLOGY
Vol. 45, No. 1
Printed in U.S.A.
Copyright © 1906 by The Williams & Wilkins Co.
ETHYLENE GLYCOL TOXICITY
KEVIN E. BOVE, M.D.
Department of Pathology, University of Cincinnati College of Medicine, and Cincinnati
General Hospital, Cincinnati, Ohio
Renal tubular oxalosis is a striking and
regular phenomenon following the ingestion
of ethylene glycol by a variety of mammals,
including man. Doubt exists, however,
regarding the relation of renal oxalate
crystal formation to the mechanism of
ethylene glycol toxicity.4 It has been
proposed that the toxicity of ethylene glycol
may be related either to a direct effect of the
unaltered compound or to the production of
toxic intermediaries in the course of degradation.2 The identification of glycolaldehyde,
glycolic, and glyoxylic acid as probable intermediaries by Gessner and associates2
prompted the following experiment, in which
the genesis of renal tubular oxalosis following
the administration of these compounds was
sought as an indicator of the validity of their
place in the sequential degradation of
ethylene glycol.
kidneys, liver, and brain in each instance.
Occasionally sections of heart and lungs
were also prepared, although the gross
appearance of these organs was in all
instances unremarkable.
Identification of crystals was accomplished
by the method of Johnson and Pani. 3 This
2-step procedure is based on the selective
insolubility of calcium oxalate in a weak
acid, such as 2 M acetic acid, and upon the
induction by sulfuric acid of C0 2 bubbles
from the carbonate residue following microincineration of oxalate crystals. Both
procedures were performed on unstained
mounted sections of tissue. The characteristic morphology and the appearance of
calcium oxalate crystals in polarized light
were utilized as supplementary aids in
identification.
RESULTS
M A T E R I A L S AND
METHODS
Ethylene glycol. Abundant renal tubular
oxalosis was readily produced at dosage
levels of 9 and 12 Gm. per kg. Four of the 7
animals which were given 12 Gm. per kg.
became markedly lethargic within 3 hr. and
died in less than 24 hr. They had striking
oxalate crystal formation in the renal
tubules. The 3 remaining recovered from
mild initial toxicity and were sacrificed after
1, 5, and 7 days, respectively. In each, only
an occasional renal oxalate crystal was
found. None of the 7 had extrarenal oxalate
crystals, and no significant histologic alterations were present in other organs.
Of the 4 animals receiving 9 Gm. per kg.,
3 exhibited moderate lethargy prior to
sacrifice at 24 hr. Each had abundant renal
oxalate crystals. The fourth failed to
develop signs of toxicity and only a rare
crystal was present in renal sections. It is of
particular interest that the brain of 1 of the
3 animals with abundant renal crystal formation contained numerous fine oxalate crystals. These were, for the most part, em-
Female white Holtzman rats weighing 200
to 225 Gra. were utilized. The ethylene
glycol,* glycolaldehyde,f glycolic acid,| and
glyoxylic acid§ used were of high purity. The
compounds were dissolved or diluted when
necessary in isotonic saline solution and
injected by means of gastric tube, the volume
varying from 1 to 4 ml. per animal.
Approximate lethal doses for each compound were established, and, utilizing these
as a guide, the dose was manipulated so that
obvious systemic toxicity usually developed
within 12 hr. of administration. Animals
were sacrificed by exsanguination at 24
hr. (except as otherwise indicated), and
necropsy performed immediately, with fixation of selected tissues in formalin.
Microscopic sections, stained with hematoxylin and eosin, were prepared from
Received, July 21, 1965.
* Eastman Organic Chemicals, Lot No. 133.
f Calbioehem, Lot No. 3617.
t Fisher Scientific Company, Lot No. A-130.
§ Matheson Company Inc., Lot No. 8089.
46
FIG. 1 (upper). Oxalate crystals in convoluted tubules 24 hr. after administration of glycolic acid,
5 Gin. per kg. Epithelial changes are slight. Hematoxylin and eosin. X 250. Polarized light.
FIG. 2 (lower). Oxalate crystals in the convoluted tubules 24 hr. after administration of glyoxalic
acid, 1 Gm. per kg. Epithelial changes are slight. Hematoxylin and eosin. X 250. Polarized light.
47
48
BOVE
bedded in the walls of small blood vessels.
In addition, an occasional crystal appeared
within the cortical substance. The meninges
were uninvolved.
Four animals receiving 6 Gm. per kg.
failed to manifest either toxic effects or renal
tubular oxalosis.
Glycolaklehyde. Two animals receiving 6
Gm. per kg. died less than 12 hr. after
administration. Crystals identifiable as
oxalate occurred in the renal tubules, but
were not common. One of 4 animals receiving
3 Gm. per kg. died 8 hr. after administration.
Three others exhibited no systemic effects
prior to sacrifice. Occasional renal oxalate
crystals were present in 2 of these, including
the animal that died. Crystals were rare or
absent in the other 2 animals. Neither
crystals nor histologic abnormalities were
present in the other organs.
Glycolic acid. Of the 6 animals receiving
5 Gm. per kg., all manifested severe toxic
effects, and 3 died S to 12 hr. after administration. All had severe renal tubular oxalosis,
but no crystals were found in the brains. The
4 animals given 3 Gm. per kg. failed to
exhibit either toxicity or oxalosis.
Glyoxylic acid. Four animals receiving 6
Gm. per kg., and 2 animals receiving 3 Gm.
per kg. died within 1 hr. after administration.
Necropsy did not disclose an obvious cause
of death. It is noteworthy, however, that
none had renal or cerebral oxalosis. Three
animals tolerated a dose of 1 Gm. per kg.
without signs of toxicity prior to sacrifice at
24 hr. All developed an impressive degree of
renal tubular oxalosis. Again, cerebral oxalosis was absent.
DISCUSSION
Profound renal oxalosis was readily
produced within 24 hr. after administration
of toxic doses of ethylene glycol, glycolic
acid, and glyoxylic acid (Figs. 1 and 2).
Glycolaldehyde administration in toxic
amounts produced renal oxalosis which was
significantly less extensive.
Vol. 45
Gross alterations were usually absent. In a
few animals, however, there was mild swelling and congestion of the kidneys. Microscopic renal changes were similar following
administration of all compounds, with the
exception of glycolaldehyde. The most profound alterations followed administration
of ethylene glycol. There was a striking
diffuse convoluted tubular dilatation, the
severity of which paralleled the extent of
tubular oxalosis. Often, pronounced hydropic
and vacuolar changes were present in the
epithelium of nondilated tubules (Fig. 3).
Focal tubular epithelial necrosis, although
not a prominent feature, occurred on
occasion. Epithelial changes usually bore
little relation to crystal formation. Occasionally, fine, needle-like crystals appeared
within intact tubular epithelium, suggestive
of intracellular genesis. Crystals appeared
throughout the proximal and distal convoluted tubules, and were distinctly less
numerous in collecting tubules. Crystals
were never present in the glomeruli or renal
interstitium.
Microscopic alterations in the kidney
following administration of glycolaldehyde
were minimal. Crystal formation was sparse
and the crystals small. Tubular dilatation
was absent. The convoluted tubular epithelial
cells, however, were somewhat swollen and
often obliterated the tubular lumina.
The impressive appearance of oxalate
crystals in the brain of a single rat is of
considerable interest (Fig. 4). Pons and
Custer4 reported a similar finding in
meningeal vessels of several men who
succumbed to ethylene glycol poisoning.
This phenomenon is important because the
reported and personally observed signs of
early toxicity indicate a functional disturbance of the central nervous system.
In humans the appearance of renal failure,
whether it results from toxic renal tubular
damage, as our findings indicate, or from
simple physical obstruction of renal tubules
by crystals, is usually somewhat delayed.
F I G . 3 (upper). Severe swelling of convoluted tubular epithelium with vacuoles and focal necrosis
24 hr. after administration of ethylene glycol, 9 Gm. per kg. These changes were often independent
of crystal formation. Hematoxylin and eosin. X 250.
F r o . 4 (lower). Oxalate crystals within the walls of cerebral blood vessels following administration
of ethylene glycol, 9 Gm. per kg. Hematoxylin and eosin. X 250. Polarized light.
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FIGS. 3-4
49
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Vol. 46
BOVE
Moreover, the observed pattern of oxalate
crystal deposition in the kidneys and in
cerebral blood vessel walls following ethylene glycol ingestion resembles that
seen in primary oxalosis. Perhaps these
disorders, one acquired and the other an
inherited metabolic defect, have much in
common at the molecular level.
The observations in this experiment
support the findings of Gessner, indicating
that ethylene glycol is successively converted
to glycolaldehyde, glycolic acid and glyoxylic acid prior to its ultimate appearance
as respiratory C0 2 (major pathway) or
urinary oxalate (minor pathway). The
3 intermediaries are each considerably more
toxic than the parent glycol, and all are
precursors of oxalate. Regarding alternate
metabolic routes, the ready conversion of
each to glycine has been demonstrated. 1, 6
It seems likely that the limit of such conversion is exceeded in ethylene glycol
overdose, resulting in an accumulation of the
toxic intermediaries. Concomitant oxalate
production represents a minor, although
histologically important, phenomenon. Considering the small quantity of administered
ethylene glycol that is ultimately converted
to oxalate,2 it is probable that toxicity is
related to either the unaltered glycol or
alternatively to one of the intermediaries.
The latter theory seems more plausible, on
the basis of the significantly greater toxicity
of these compounds, which has been demon-
strated by others and confirmed in this
investigation.
SUMMARY
Renal tubular oxalosis followed the
administration of glycolaldehyde, glycolic
acid, and glyoxylic acid to rats. This provides
anatomic evidence supporting their role as
intermediaries in the sequential degradation
of ethylene glycol. The toxicity of each of the
3 compounds is significantly greater than
that of ethylene glycol, suggesting their
importance in the genesis of toxic phenomena. On the basis of the histologic alterations in the convoluted tubular epithelium,
we conclude that renal failure following
ethylene glycol ingestion in humans is a
consequence of cytotoxicity rather than
simple mechanical obstruction of the tubular
lumina by oxalate crystals. Cerebral vascular
oxalosis was impressive in a single rat
receiving ethylene glycol.
REFERENCES
1. Friedmann, B., Levin, H. W., and Weinhouse,
S.: Metabolism of glycolaldehyde in t h e r a t .
J . Biol. Chem., 221: 065-677, 1956.
2. Gessner, P . K., Parke, D . V., and Williams, R.
T . : The metabolism of "C-labelled ethylene
glycol. Biochem. J., 79: 482-489, 1961.
3. Johnson, F . B . , and Pani, K . : Histochemical
identification of calcium oxalate. Arch.
P a t h . , 74: 347-351, 1962.
4. Pons, C. A., and Custer, R.. P . : Acute ethylene
glycol poisoning. Am. J . M. S c , 211: 544552, 1946.
5. Weinhouse, S., and Friedmann, B . : Metabolism
of 2-carbon acids in the intact r a t . J . Biol.
Chem., 191: 707-717, 1951.