Zborník príspevkov z vedeckej konferencie - Department of ...

galactose-4-epimerase [6, 7]. First described in a variant patient in 1935 by Mason and Turner, galactose-1-phosphate
uridyltransferase (GALT) deficiency is the most common enzyme deficiency that causes hypergalactosemia. Removal of
lactose largely eliminates the toxicity associated with newborn disease, but long-term complications routinely occur, as
reported by Komrower and Lee in 1970 and then delineated in a 1990 retrospective survey by Waggoner et al [8]. Inborn
errors of metabolism are categorized according to defects in these enzymes as: type I (classical, galactose-1-phosphate
uridyltransferase deficiency), type II (galactokinase deficiency) and type III (uridine diphosphate-galactose-4-epimerase
deficiency) galactosemias, respectively.
Increased galactitol concentration is a common feature in GALT deficiency and has been implicated in galactosaemic
cataract formation. As conversion of galactose to galactitol by aldose reductase represents a dead–end metabolic pathway,
galactitol removal is confined to renal excretion. The recently observed age–dependent decrease of endogenous galactose
formation in galactosaemic patients shows that correlative changes in galactitol excretion occur in galactosaemia [5].
Classical galactosemia (GALT deficiency), an autosomal recessive disorder occurs in the population with an incidence of
approximately 1:40–60 000. Galactose-1-phosphate, a metabolite derived from ingestion of galactose, is considered to be
toxic in several tissues particularly in the liver, brain and renal tubules [9].
Galactose is a monosaccharide present in many polysaccharides, where the most clinically important source is the
disaccharide lactose. Lactose is the predominant carbohydrate in human milk and milk of most animals, including cow’s
milk, therefore many commercially available infant formulas contain lactose.
Galactose
ATP
Galactokinase
ADP
Galactose-1-phosphate
Galactose 1-phosphate
uridylyl transferase
Glucose-1-phosphate
Glucose-6-phosphate
Phosphoglucomutase
UDPglucose
UDPgalactose
UDP-galactose
4-epimerase
Glycolysis
Fig. 2. Major steps in the intermediary metabolism of galactose.
There are several well established derivatization methods [10], for sugar identification and quantification either with
high–performance liquid chromatography (HPLC) [11, 12], with enzymatic [13] and with gas chromatographic (GC) [14 –
17] procedures. All these methods are time consuming and suffer numerous limitations [18].
The most important technique for carbohydrate structural determination is GC–MS. The first application of GC to
carbohydrates was reported in 1958 and it described the separation of fully methylated monosaccharides [19, 20].
Monosaccharide analysis by GC (with FID or MS detection) requires derivatization to increase their volatility and decrease
their excessive interactions with the analytical system. The simplest and most rapid method for routine analysis is silylation
to trimethylsilyl (TMS) derivates [18].
GC faced problems of multiple peaks due to the anomers of cyclic forms [21] and long reactions times and multiple
steps sample preparation [22, 23, and 24]. The multiple peak problems was solved by reduction of the C1 position of the
acyclic form to alditols or by means of oxime formation with hydroxylamine prior to the derivatization step [23, 24, 25, 26]
as published in the early studies of Sweeley et al. [27]. Reduction to alditols has the advantage that each sugar generates only
one peak, while trimethylsilyl (TMS) oximes give 2 peaks, but hydroxylamine reactions are more selective. Alditols
preparation also include cation-exchange steps and boric acid removal [22], whereas in the TMS-oxime preparation, all
reactions are consecutively carried out in the same vial.
This report describes the presence of both galactitol and galactose in urine from galactosaemic as well as normal
subjects.
Experimental
Patients
Spontaneous urine was obtained from 25 healthy subjects (classified into 5 age groups) and from 4 patients treated with
classical galactosaemia.
Zborník príspevkov
z 18. medzinárodnej vedeckej konferencie
"Analytické metódy a zdravie loveka", ISBN 978-80-969435-7-9
- 117 -
hotel Falkensteiner, Bratislava
11. - 14. 10. 2010