Eur J Clin Chem Clin Biochem 1996; 34:651-654 © 1996 by Walter de Gruyler · Berlin · New York
Interferences of Glycerol, Propylene Glycol, and other Diols
in the Enzymatic Assay of Ethylene Glycol1)
Henri Malandain and Yves Cano
Laboratoire de Biochimie, Centre Hospitalier Chubert, Vannes, France
Summary: As an alternative to gas chromatography, the enzymatic
UV assay of ethylene glycol is often used by emergency laborato-
ries. Many variants of this technique have been published, all based
on the reaction between NAD+ and ethylene glycol in the presence
of glycerol dehydrogenase (EC 184.108.40.206). We show that other a-
diols interfere in this reaction. Some of them, like 2,3-butanediol,
give false positive reactions; whereas other diols, e. g. glycerol and
propylene glycol, interfere only when ethylene glycol is present in
the sample and lower the ethylene glycol response; these interfer-
ents are of particular concern because some parenteral drugs used
in emergency situations contain glycerol or propylene glycol in
their vehicle. This drawback has hitherto been largely underesti-
mated, and we think that ethylene glycol results obtained with these
enzymatic techniques should be interpreted with caution, even if
the sample is pre-treated with glycerokinase (EC 220.127.116.11); this
pre-treatment effectively corrects the glycerol interference but not
that of propylene glycol.
Ethylene glycol causes severe poisoning in man. In suspected
cases, an emergency ethylene glycol assay is necessary but gas
chromatography is not available in every laboratory. Hanson &
Masson (1) and Standefer & Blackwell (2) recently popularized
simple UV enzymatic assays of ethylene glycol using glycerol de-
hydrogenase (EC 18.104.22.168) from Enterobacter aerogenes:
+ NADH + H+
These authors found that glycerol and propylene glycol reacted
very quickly and thus did not interfere if absorbance readings be-
gan after a delay sufficient for their complete conversion (fig. 1).
These authors also noted an inhibitory effect of the reaction pro-
duct, glycolaldehyde; but this inhibition was of no clinical impor-
tance because glycolaldehyde, like other aldehydes, is rapidly
cleared from the circulation and does not accumulate sufficiently
to affect the ethylene glycol measurement. Nilsson & Jones (3)
later reported 2,3-butanediol as an additional substrate. 2,3-Bu-
tanediol reacts at a rate similar to that of ethylene glycol (fig. 1),
and it interferes in the ethylene glycol measurement irrespective of
the absorbance reading times.
In view of the poor specificity of glycerol dehydrogenase, we de-
cided to test some other compounds of toxicological interest. We
also performed these tests in the presence of ethylene glycol to
reveal any competitive or inhibitory effects.
Materials and Methods
All chemicals were of analytical grade.
Experiments were done on a Boehringer Hitachi 717 at 30 °C.
Sample: 10 μΐ.
Reagent 1: 250 μΐ of 15 mmol/1 NAD+ in Tris buffer 0.1 mol/1 pH
9.0 (Sigma ref. 545-2).
Reagent 2: 50 μΐ of 45 kU/1 glycerol dehydrogenase from Entero-
bacter (Boehringer ref. 258-255) in the same buffer.
Reagent 2 was added 5 min after reagent 1.
Two 340 nm absorbance readings were taken 2 min and 5 min after
reagent 2 addition (these reading times correspond to the 35th and
50th Hitachi 717 cycles, respectively).
Concentrations were calculated from the difference between these
two absorbance readings.
Calibration curves were prepared with 0, 5, 10, 20, and 40 mmol/1
ethylene glycol standards prepared in 0.154 mol/1 NaCl.
Reagent 1 was modified by the addition of glycerokinase, ATP and
Mg2"1" to catalyse the conversion of glycerol to glycerol-3-phos-
phate before the addition of glycerol dehydrogenase:
}) This work was presented in part at the 47th A. A. C. C. Annual
Meeting, July 16-20 1995, Anaheim (CA), USA
Time after reagent 2 addition [min]
Fig. 1 Reaction kinetics of ethylene glycol and of some interfer-