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SIMULTANOUS DETERMINATION OF GALACTITOL AND GALACTOSE IN AQUEOUS SAMPLES BY GAS
CHROMATOGRAPHY – MASS SPECTROMETRY
IVAN OSTROVSKÝ*, RÓBERT KUBINEC, JAROSLAV BLAŠKO, JOZEF VIŠOVSKÝ, RENÁTA GÓROVÁ,
GABRIELA ADDOVÁ, PETER PODOLEC, ALEXANDRA SZABÓOVÁ
Chemical Institute, Faculty of Natural Sciences, Comenius University, Mlynská dolina CH-2, SK-845 45 Bratislava, Slovakia
e-mail: ostrovsky@rec.uniba.sk
Abstract
Galactosemia, a metabolic disorder associated with the intolerance of dietary galactose due to an inherited enzymatic
deficiency, is indicated by heightened levels of galactose and galactitol in urine. Gas chromatography (GC) with mass
spectrometry (MS) detection was evaluated for its ability to screen urinary carbohydrates, particularly galactose and
galactitol. The described method uses trimethylsilyl derivates of galactitol and galactose, and needs 100 l of urine for a
single run. Spontaneous urine was obtained from 25 healthy subjects (classified into 5 age groups), from 4 treated patients
with classical galactosaemia. The age dependences of galactose and galactitol excretion in urine was found in healthy
subjects and treated galactosaemic patients by using reported method.
Recognizing the clinical need for the simultaneous measurement of galactitol and galactose in urine for galactosaemic
subjects, we developed the new GC/MS method, permits for the first time, measurement in urine by an accurate and precise
single step procedure. This method does not require urine pretreatment before the silylation.
Introduction
Metabolism is the sum of all continuous biochemical reactions of breakdown and renewal of tissues of the body.
Enzymes play an indispensable role in facilitating the process by serving as catalysts in the conversion of one chemical
(metabolite) to another, often extracting the energy required for the reaction from a suitable high energy source, such as ATP
[1].
Figure 1 shows the various possible mutation–sensitive defects affecting the compartmentalization and metabolism of
compound A. This figure shows schematically the relationship between various defect types and their pathophysiologically
and diagnostically important consequences [1].
Membrane
1
2
Holoenzyme
Aoutside Ainside B C
E
6
Fig. 1. The primary consequences of inborn errors of metabolism.
1–transporter-mediated movement of A from one compartment to another, 2–defect in the conversion of B to C, 3–increased
conversion of B to D caused by accumulation of B, 4–defect in the interaction between an apoenzyme and an obligatory
cofactor, 5–decreased feedback inhibition of the conversion of Ain to B as a result of deficiency of C, 6–secondary inhibition
of the conversion of E to F caused by accumulation of D [1].
D
5
-
F
4
Apoenzyme
+
cofactor
“Inborn metabolic diseases” (IMDs) is the term applied to genetic disorders caused by loss of function of an enzyme.
Enzyme activity may be low or lacking for variety of reasons. Some IMDs produce relatively unimportant physical features
or skeletal abnormalities, but others produce serious diseases and even death. Most inborn errors of metabolism are
monitored by blood or urine tests [2]. The phrase “inborn errors of metabolism” had been first used by A. E. Garrod in the
Croonian Lectures of 1908, and published as a monograph with this title in 1909 [3].
Traditionally the IMDs are categorized as disorders of carbohydrate metabolism, amino acid metabolism, organic acid
metabolism, or lysosomal storage diseases [4].
Galactosemia was first "discovered" in 1908, when Von Ruess reported on a breast-fed infant with failure to thrive,
enlargement of the liver and spleen, and "galactosuria" in publication entitled "Sugar Excretion in Infancy". This infant
ceased to excrete galactose through urine when milk products were removed from the diet. The toxic syndrome,
galactosemia, is associated with an intolerance to dietary galactose as a result of certain enzymatic deficiencies [5].
Malfuctions in the following three enzymes, which participate in the normal metabolism of galactose (Fig. 2), are known to
be causes of galactosemia: galactose-1-phosphate uridyltransferase (GALT), galactokinase and uridine diphosphate-
Zborník príspevkov
z 18. medzinárodnej vedeckej konferencie
"Analytické metódy a zdravie loveka", ISBN 978-80-969435-7-9
- 116 -
hotel Falkensteiner, Bratislava
11. - 14. 10. 2010