Clinical Chemistry 46: 612-619, 2000;
(Clinical Chemistry. 2000;46:612-619.)
© 2000 American Association for Clinical Chemistry, Inc.
Analysis of Concentration and 13C Enrichment of D-Galactose in Human Plasma
Peter Schadewaldt1,a,
Hans-Werner Hammen1,
Kamalanathan Loganathan1,
Annette Bodner-Leidecker1,2 and
Udo Wendel2
1
Deutsches Diabetes Forschungsinstitut an der Heinrich-Heine-Universität, Auf’m Hennekamp 65, D-40225 Düsseldorf, Germany.
2
Kinderklinik, Heinrich-Heine-Universität,
Moorenstrasse 5, D-40225 Düsseldorf, Germany.
a Address correspondence to this author at: Deutsches Diabetes Forschungsinstitut, Klinische Biochemie, Auf’m Hennekamp 65, D-40225 Düsseldorf, Germany. Fax 49-211-3382-603; e-mail schadewa{at}uni-duesseldorf.de
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Abstract
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Background: A stable-isotope dilution method for the
sensitive determination of D-galactose in human plasma was
established.
Methods: D-[13C]Galactose was added to
plasma, and the concentration was measured after D-glucose
was removed from the plasma by treatment with D-glucose
oxidase and the sample was purified by ion-exchange chromatography. For
gas chromatographic–mass spectrometric analysis, aldononitrile
pentaacetate derivatives were prepared. Monitoring of the
[MH-60]+ ion intensities at m/z 328, 329,
and 334 in the positive chemical ionization mode allowed the assessment
of 1-12C-, 1-13C-, and
U-13C6-labeled D-galactose,
respectively. The D-galactose concentration was quantified
on the basis of the 13C-labeled internal standard.
Results: The method was linear (range examined, 0.1–5 µmol/L)
and of good repeatability in the low and high concentration ranges
(within- and between-run CVs <15%). The limit of quantification for
plasma D-galactose was <0.02 µmol/L. Measurements in
plasma of postabsorptive subjects yielded D-galactose
concentrations (mean ± SD) of 0.12 ± 0.03 (n = 16),
0.11 ± 0.04 (n = 15), 1.44 ± 0.54 (n = 10), and
0.17 ± 0.07 (n = 5) µmol/L in healthy adults, diabetic
patients, patients with classical galactosemia, and obligate
heterozygous parents thereof, respectively. These data were
considerably lower (3- to 18-fold) than the values of a conventional
enzymatic assay. The procedure was also applied successfully in a
stable-isotope turnover study to evaluate endogenous
D-galactose formation.
Conclusions: The present findings establish that detection of
D-galactose from endogenous sources is feasible in
human plasma and show that erroneously high results may be
obtained by enzymatic methods.
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Introduction
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In patients with classical galactosemia [McKusick 2304000,
(1)], the catabolism of D-galactose
is severely impaired because of an inherited
D-galactose-1-phosphate uridyltransferase (EC
2.7.7.10) deficiency. In galactosemic infants on an unrestricted
lactose intake, a potentially lethal organ toxicity syndrome develops,
presumably because D-galactose-derived
metabolites (D-galactose-1-phosphate and
D-galactitol) accumulate within the cells.
Patients improve rapidly on cessation of lactose intake. A strict and
lifelong dietary restriction of D-galactose is
the recommended form of therapy. Even when patients are on an extremely
D-galactose-restricted diet, however, long-term
disturbances emerge, e.g., retarded development in intellectual
performance, possible neurologic symptoms, and hypergonadotropic
hypogonadism in most females [see Segal and Berry (2) for a
comprehensive review]. Gitzelmann and Steinmann (3)
supposed that these long-term complications might be attributable to
production of free D-galactose from endogenous
sources, leading to an "autointoxication" in galactosemic patients.
In fact, isotope kinetic tracer experiments performed in a limited
number of adult patients and healthy subjects indicated that
substantial and comparable amounts of D-galactose
are produced in both study groups (4).
Knowledge of the quantitative role of endogenous production of
D-galactose is essential for judgment of the significance
of lifelong dietary restrictions in patients with hereditary
galactosemia. Therefore, it seemed important to examine a possible age
dependency of endogenous galactose production rates in galactosemic
patients. For this purpose, in vivo measurement of
D-galactose turnover in postabsorptive subjects by use of
13C-labeled D-galactose infusion
represents the method of choice (4). When we were planning
an appropriate study, we found that numerous enzymatic, gas
chromatography
(GC),1
and HPLC procedures had been published, but no method has been
detailed in the literature that is sufficiently sensitive to allow
reliable estimation of the 13C enrichment or
concentration of plasma D-galactose in
postabsorptive subjects and patients. Henderson et al. (5)
applied an enzymatic fluorometric method and originally reported on
fasting concentrations in human plasma in the range of ~5–100
µmol/L. Enzymatic measurements in plasma are known to be subject to
interferences, however, and there are indications that
D-galactose may be overestimated by these
procedures, especially when analyses are performed at low plasma
concentrations (5)(6)(7)(8). Using HPLC and electrochemical
detection, Watanabe and Kawasaki (6) established the
presumably most sensitive assay (detection limit, 2.2 µmol/L) for
D-galactose in human plasma communicated to date.
These authors were actually unable to detect free
D-galactose in postabsorptive subjects and
stated, "Absence of free galactose in human plasma is usually the
case unless deliberately added or infused" (6).
Here we report on a stable-isotope dilution method for the sensitive
and reliable measurement of D-galactose in plasma by use of
1-13C- or
U-13C6-labeled
D-galactose. The major obstacle in the determination of
D-galactose by GC-mass spectrometry (MS) procedures, i.e.,
the presence of comparatively large amounts of D-glucose in
plasma samples, has been overcome by the use of an enzymatic
D-glucose removal step. The method allowed for the first
time estimation of free plasma D-galactose in
postabsorptive subjects. The usefulness of the procedure for evaluation
of 13C label enrichment in plasma
D-galactose is demonstrated by measurements in samples from
a stable-isotope study on D-galactose turnover performed in
a healthy subject.
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Materials and Methods
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subjects and patients
For the determination of fasting D-galactose
concentrations, venous EDTA blood samples were collected between 0700
and 0900 from healthy adults [4 women and 12 men; age, 33 ± 8
years (mean ± SD); weight, 72 ± 6 kg; height, 180 ± 8
cm], patients with diabetes mellitus (8 women and 8 men; age, 56
± 12 years; weight, 84 ± 14 kg; height, 167 ± 9 cm),
patients with classical galactosemia [6 females and 4 males; age,
17 ± 10 years; weight, 44 ± 19 kg; height, 147 ± 25
cm; galactose-1-phosphate uridyltransferase activity in erythrocytes
measured according to Shin (9), <2% of control], and
obligate heterozygous parents of the galactosemic patients (2 women and
3 men; age, 43 ± 16 years; weight, 78 ± 29 kg; height;
174 ± 10 cm; galactose-1-phosphate uridyltransferase, 49% ± 5%
of control). All subjects were studied in the postabsorptive state
after an overnight fast of 
10 h. Plasma was separated by
centrifugation (3000g for 10 min at 4 °C).
The study was approved by the Ethikkommission of the
Heinrich-Heine-Universität Düsseldorf, and written informed
consent was obtained from all subjects participating in the study.
galactose turnover study
A primed continuous infusion test was performed under resting
conditions essentially as described by Berry et al. (4). In
short, after an overnight fast, a healthy volunteer (male; age, 25
years) received an intravenous priming dose of
D-[1-13C]galactose (8
µmol/kg of body weight) at ~0900. Thereafter, a continuous infusion
of 0.8 µmol
D-[1-13C]galactose · kg
body weight-1 · h-1
via a cannula inserted into the basilic vein was started and continued
for 6 h. D-Glucose was infused intravenously
at a rate of 11 µmol · kg body
weight-1 · min-1 from
~0800 until the end of the experiment. Samples of venous EDTA blood
were collected from the cannula placed on the contralateral arm, and
plasma was prepared for analysis as described above.
chemicals and enzymes
Unless otherwise noted, all chemicals were obtained in the highest
available purity from Merck or Sigma Chemie. Ion-exchange resins were
from Serva. Catalase (EC 1.11.1.6, from bovine liver),
D-galactose dehydrogenase (EC 1.1.1.48, from
Pseudomonas fluorescens), D-glucose
oxidase (EC 1.1.3.4, from Aspergillus niger),
D-glucose-6-phosphate dehydrogenase (EC 1.1.1.49,
from yeast), hexokinase (EC 2.7.1.1, from yeast), and coenzymes were
purchased from Boehringer Mannheim.
D-[1-13C]Galactose (99%
1-13C, according to the manufacturer) and
D-[U-13C6]galactose
(99% U-13C6, according to
the manufacturer), produced by Cambridge Isotope Laboratories, were
obtained from Promochem. According to our GC-MS analysis (see below),
however, the 13C label enrichment in the
D-[1-13C]galactose preparation was
97.0% ± 0.1% (n = 14; duplicates analyzed on 7 different
working days). The purity of the uniformly labeled
D-galactose was 98.5% ± 0.2% (n = 6 on 2 different
working days).
enzymatic galactose and glucose assays
A modification of the procedure described by Fujimura
(10) was used: D-Galactose solutions
containing 0 (basal value), 10, or 50 µmol/L (25 µL each) were
added to three plasma aliquots (0.2 mL). After the plasma was
deproteinized by the addition of perchloric acid (0.1 mL of a 1.5 mol/L
solution) and centrifugation (13 000g for 10 min at
4 °C), the supernatant (0.25 mL) was neutralized with 50 µL of 2.5
mol/L KHCO3, and the KClO4
was then removed by centrifugation (see above). The neutralized extract
(0.25 mL) was mixed with 0.6 mL of 1 mol/L Tris-HCl buffer (pH 8.7) and
0.25 mL of 5 mmol/L NAD+. After fluorescent
blanks were measured (
ex = 340 nm;
em = 450 nm; Model 650 fluorometer;
Perkin-Elmer), the reaction was started by the addition of 10 µL of
D-galactose dehydrogenase solution (0.25 U).
After ~60 min, a stable end-point fluorescent signal was reached,
indicating that the enzymatic reaction had gone to completion. The
D-galactose concentration in the sample to which
0 µmol/L D-galactose had been added was then
calculated on the basis of the increase in fluorescent intensities of
the two samples to which 0.25 or 1.25 µmol/L
D-galactose had been added. In the recovery
studies (see Results) with plasma (pools) to which
D-galactose had been added, appropriate blanks
were prepared with authentic plasma samples containing no added
D-galactose.
D-Glucose concentrations were measured
spectrophotometrically using the
hexokinase-D-glucose-6-phosphate dehydrogenase assay
essentially as described by Kunst et al. (11).
plasma extraction and glucose removal
For internal standardization, 20 µL of a 50 µmol/L
D-[1-13C]galactose solution was
added to 1 mL of plasma.
D-[U-13C6]Galactose
solution (20 µL of a 50 µmol/L solution) was added to plasma
samples from the in vivo galactose turnover study. Water was then added
to give a final volume of 1.25 mL, and the sample was mixed with 0.25
mL of 3 mol/L perchloric acid for deproteinization. After
centrifugation (13 000g for 5 min at 4 °C), 1.2 mL of
the supernatant was mixed with 0.2 mL of 2.5 mol/L
KHCO3 and 0.1 mL of 2.5 mol/L potassium phosphate
buffer (pH 6.5). The KClO4 was removed by
centrifugation (see above). For enzymatic removal of
D-glucose, catalase (65 kU in 50 µL) and
D-glucose oxidase (10–15 U in 50 µL) were
added to 1.3 mL of buffered extract. The mixture was equilibrated with
air and incubated at 25 °C for 90 min. The reaction was stopped by
the addition of 0.15 mL of 4.5 mol/L perchloric acid and centrifuged
(see above). The supernatant (1.4 mL) was mixed with
KHCO3 (0.25 mL of a 2.5 mol/L solution), the
KClO4 removed by centrifugation, and the
glucose-depleted extract was purified by subsequent ion-exchange
chromatography.
sample purification and derivatization
A 1.5-mL aliquot of the above extract was applied onto a Dowex
1 x 8 column (200–400 mesh, acetate form; 4 mL in disposable
PolyPrep chromatography columns from Bio-Rad); 0.25 mL of water was
then applied to the column and allowed to elute. The eluate was
discarded. D-Galactose was eluted from the column with 2 mL
of H2O. The latter eluate was applied onto a
Dowex 50 WX8 column (200–400 mesh, H+ form; 4 mL
in disposable columns; see above) followed by a wash with 2 mL of
H2O. The final 2 mL of the eluate was collected,
transferred into a reaction vial, and evaporated to dryness under a
stream of gaseous N2.
For preparation of aldononitrile pentaacetates, the dry residue was
reacted with 50 µL of hydroxylamine hydrochloride (0.3 mol/L in
pyridine) at 90 °C for 30 min. Thereafter, 50 µL of acetic
anhydride was added, and the reaction mixture was kept at 90 °C for
60 min. After evaporation under a stream of gaseous
N2, the dry residue was extracted with 0.1 mL of
hexane. The hexane was evaporated as above. The final residue was
dissolved in 50 µL of ethyl acetate and then subjected to galactose
analysis by GC-MS as described below.
gc-ms procedure
A HP 6890 gas chromatograph equipped with a HP-5 MS capillary
column [5% phenylmethylpolysiloxane; 30 m x 0.25 mm (i.d.);
0.25 µm film thickness] and directly connected to a HP mass
selective detector was used (Hewlett-Packard). Helium was the carrier
gas (0.9 mL/min). Sample (1.0 µL) was injected in the splitless mode.
The injector and the transfer line to the spectrometer were held at
250 °C. The initial column temperature was 150 °C. After 0.5 min,
the temperature was increased to 250 °C at a ramp rate of
10 °C/min and then raised to 280 °C for 2 min. Positive chemical
ionization was used with methane as the reactant gas. The ion source
was operated at 170 °C. Source pressure was 60 mPa. Selected ion
monitoring of the [MH-60]+ ion intensities at
m/z 328, 329, and 334 in the galactose chromatographic peak
allowed the assessment of 1-12C-,
1-13C-, and
U-13C6-labeled
D-galactose, respectively.
calculations
To assess the natural 13C enrichment in
plasma hexoses, we measured the ion intensities at m/z 328
and 329 in the D-galactose and
D-glucose chromatographic peaks in a
representative number of native plasma samples. The ratio
R0 = (m/z
329)/(m/z 328) was 0.159 ± 0.003 (mean ± SD;
n = 20) for either hexose, and this value was used for further
calculations.
We used the ion intensity ratio, R1 =
(m/z 329)/(m/z 328), in the galactose
chromatographic peak to estimate the concentration of
D-galactose (Cgal,
in µmol/L) in the plasma samples to which
D-[1-13C]galactose (97%
1-13C, 3% naturally labeled; final
concentration, 1 µmol/L) had been added (taking into account the
amount of naturally labeled D-galactose in the
D-[1-13C]galactose preparation) according to
the equation:
 | (1) |
In plasma samples collected in the primed continuous infusion
experiment, to which
D-[U-13C6]galactose
preparation (98.5% U-13C6,
1.5% naturally labeled; final concentration, 1 µmol/L) had been
added, the ion intensity ratios RU1 =
(m/z 334)/(m/z 328) and
RU2 = (m/z
334)/(m/z 329) in the D-galactose
chromatographic peak were used to estimate the concentration of total
D-galactose (Ctot,
i.e., sum of naturally labeled and
D-[1-13C]galactose, in
µmol/L) according to the equation:
 | (2) |
The amount of naturally labeled D-galactose
(Cnat, µmol/L) in the sample was
estimated according to the equation:
 | (3) |
and the amount of 1-13C-labeled
D-galactose in the sample
(C13C, µmol/L) was estimated
using the equation:
 | (4) |
The amount of infused exogenous D-galactose (naturally
labeled plus 1-13C-labeled;
Cexo, µmol/L) is given by:
 | (5) |
Thus, the concentration of D-galactose attributable to
endogenous sources (Cendo, µmol/L) in
the primed continuous infusion experiment can be estimated applying the
equation
 | (6) |
The rate of appearance of endogenous D-galactose in
plasma under apparent steady-state conditions
(Ra, in µmol · kg body
weight-1 · min-1) was
calculated from the ratio of 13C-labeled to total
D-galactose and the infusion rate
(Inf, in µmol · kg body
weight-1 · min-1) and
corrected for the amount of natural enriched
D-galactose in the infusate as follows:
 | (7) |
The mole-percentage of enrichment of 1-13C
label in plasma D-galactose (MPE) was calculated
according to the equation:
 | (8) |
statistics
In general, results are presented as the mean ± SD with the
number of separate determinations in parentheses. Correlations were
checked by linear regression analysis (least-squares method). For
examination of differences, the Mann–Whitney U-test was
used.
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Results
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efficiency of glucose removal
D-Glucose oxidase-catalyzed conversion of
D-glucose in plasma extracts was essentially complete at
the end of the 90-min incubation period (Fig. 1
). As tested with two plasma pools containing 7.0 ±
0.1 and 18.2 ± 0.5 mmol/L D-glucose (n = 7)
to which 10 µmol/L D-galactose had been added, the
concentration of D-glucose at the end of incubation was
reduced to 0.020 ± 0.002 and to 0.058 ± 0.019 mmol/L,
respectively. The concentration of D-galactose remained
unaffected and was 10.1 ± 0.5 and 9.8 ± 0.2 µmol/L,
respectively (n = 7). The efficiency of D-glucose
removal was 99.77% ± 0.03% and 99.71% ± 0.08% (n = 7),
respectively. Thus, the excess of D-glucose over
D-galactose in the pools was reduced by more than 300-fold,
from ~700:1 and 1800:1 to 2:1 and 6:1, respectively.
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Figure 1. Time course and specificity of D-glucose
removal from plasma samples by use of D-glucose oxidase.
Human plasma containing 7 mmol/L D-glucose ( ) was
enriched with D-galactose (•; 10 µmol/L) and
deproteinized buffered extract (pH 6.5) treated at 25 °C with
D-glucose oxidase from A. niger (10 kU/L) in
the presence of catalase (50 MU/L). At the time periods indicated,
aliquots were withdrawn from the mixture, and the D-glucose
and D-galactose concentrations were measured (see
Materials and Methods for details).
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The recovery of D-galactose in the purification procedure
was checked using 1-mL plasma samples with 5 µmol
D-galactose/L added. A total of 2.3 ± 0.2 µmol
D-galactose was recovered after the ion-exchange
chromatographic sample clean-up (n = 11). This is equivalent to
81% ± 7% of the maximal theoretical yield (2.8 µmol), which can be
estimated on the basis of the inevitable losses. Some
D-galactose had obviously been lost in either
chromatographic step.
The efficacy of the procedure is demonstrated in Fig. 2
, which shows typical GC-MS chromatograms as obtained in
analyses of authentic human plasma. In postabsorptive healthy subjects,
the ratio of D-glucose to D-galactose in plasma
was ~30 000–60 000:1 (see below). Without D-glucose
oxidase treatment, D-galactose eluted as a tiny rear rider
or shoulder peak of a huge D-glucose chromatographic peak.
Therefore, reliable measurement of D-galactose was
impossible. In contrast, in the D-glucose-depleted samples,
good separation of the two peaks was achieved, thus allowing
quantification of the D-galactose chromatographic peak.
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Figure 2. GC-MS chromatograms showing the effect of enzymatic
removal of D-glucose on the separation of the aldononitrile
pentaacetate derivatives of D-glucose and
D-galactose from human plasma.
Aliquots of plasma from a postabsorptive healthy subject were worked up
in parallel as detailed in Materials and Methods but
without the addition of D-[1-13C]galactose.
Sample preparations for A and B differed
in that glucose oxidase was absent in the workup of the sample in
A. Positive chemical ionization was used for detection.
Traces for the [MH-60]+ ion intensities
(m/z 328) are shown.
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linearity and precision
Preliminary measurements in human plasma by the
D-galactose dehydrogenase assay suggested postabsorptive
D-galactose concentrations of ~1 µmol/L. Therefore, 1
nmol of D-[1-13C]galactose was
added for each milliliter of sample in the stable-isotope dilution
assay. Before performing GC-MS determinations in authentic plasma
samples, we examined the linearity and repeatability of this approach.
The results are summarized in Fig. 3
and Table 1
, respectively.
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Figure 3. Linearity of the
D-[1-13C]galactose stable-isotope dilution
assay for measurement of D-galactose concentrations.
Increasing amounts of naturally labeled D-galactose were
added to plasma from a healthy subject as indicated; the plasma was
then analyzed for total D-galactose concentrations as
detailed in Materials and Methods. Results from two
series of analyses are shown (• and larger shaded
circles). Regression line (linear regression analysis,
least-squares method): y = 0.979 (±
0.004)x + 0.104 (± 0.005); Sy|x =
0.014; r >0.9999; n = 14. The intercept represents
the estimate of the D-galactose concentration in the
original plasma sample. (Inset), matrix-free solutions
of naturally labeled and 1-13C-labeled
D-galactose were mixed to give the ratios of
13C-labeled and naturally labeled material indicated.
Samples were then analyzed by GC-MS for the ensuing ratio of the
[MH-60]+ ion intensities of the aldononitrile
pentaacetate derivatives at m/z 329 and 328. Results
from two series of analyses are shown (• and larger open
circles). Regression line (linear regression analysis,
least-squares method): y = 1.015 (±
0.004)x + 0.166 (± 0.007); Sy|x =
0.021; r >0.9999; n = 16. The intercept represents
the estimate of the ratio m/z 329/328 in the naturally
labeled D-galactose.
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In matrix-free as well as in plasma samples, we obtained excellent
linear correlation of the m/z 329/328 ratio and the ratio of
D-[1-13C]-labeled to
naturally labeled galactose (r >0.9999; see Fig. 3
). When
imprecision was examined using human plasma pools containing low
(~0.2 µmol/L) and increased (~1.3 µmol/L) concentrations of
natural D-galactose, the within- and between-run
CVs were 
5% and <15%, respectively (Table 1
).
plasma galactose in postabsorptive subjects
The stable-isotope dilution assay was then applied to the
assessment of free D-galactose in human plasma.
Postabsorptive subjects were investigated to avoid uncontrollable and
variable contributions of exogenous D-galactose from the
diet. The results are shown in Table 2
. In healthy subjects (n = 16), diabetic patients (n =
15), and obligate heterozygous parents of patients with the classical
form of galactosemia (n = 5), plasma concentrations of
D-galactose were similarly low, with mean values of ~0.1
µmol/L. Postabsorptive patients with classical galactosemia
(n = 10), however, exhibited ~10-fold increased (P
<<0.001) plasma D-galactose concentrations.
comparison of methods
Somewhat unexpected was the observation that our preliminary
measurements by the D-galactose dehydrogenase assay (see
above) indicated considerably higher concentrations of plasma
D-galactose in postabsorptive subjects than were measured
in the D-[1-13C]galactose
stable-isotope dilution assay. This prompted us to check the enzymatic
assay by performing a series of comparative measurements. Data on the
precision of the enzymatic assay are included in Table 2
. The results
obtained in the individual samples are shown in Fig. 4
, and a summary of the data is given in Table 3
. In fact, when compared with the data of the stable-isotope
dilution assay, plasma concentrations of D-galactose in
healthy adults and in diabetic patients appeared to be overestimated
~10-fold by the enzymatic assay. In galactosemic patients exhibiting
higher plasma concentrations of D-galactose, the mean
apparent overestimation was still ~3-fold. Interestingly, the
additional amount detected by the enzymatic assay [nongalactosemic
subjects, +0.91 ± 0.50 µmol/L (median, 0.75; range, 0.26–2.21
µmol/L; n = 30); galactosemic subjects, +2.54 ± 1.42
µmol/L (median, 1.94; range, 0.58–5.19 µmol/L; n = 9)] was
rather variable, and no statistically significant correlation between
the data of the GC-MS and the enzymatic method was observed in any of
the three study groups.
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Figure 4. Comparison of plasma concentrations of
D-galactose in postabsorptive healthy subjects and patients
as measured by the D-[1-13C]galactose isotope
dilution (SID) assay and the enzymatic
D-galactose dehydrogenase assay.
See Materials and Methods for details. The results
obtained by the two methods appeared to be statistically uncorrelated
(r <0.5, linear regression, least-squares method). For
convenience, shaded areas indicating high and low
concentration ranges are under the data from nongalactosemic and
galactosemic subjects.
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galactose turnover study
We examined the applicability of the present GC-MS procedure for
the evaluation of stable-isotope turnover studies. The results of a
primed continuous infusion experiment with
D-[1-13C]galactose as performed in
a healthy volunteer are shown in Fig. 5
. Within ~3 h of
D-[1-13C]galactose infusion, a
satisfactory stable steady state of 13C
enrichment in plasma D-galactose (76 ± 2 MPE; n
= 7) was reached. According to these data, the estimated rate of
appearance of endogenous D-galactose in plasma was 0.17
µmol · kg body
weight-1 · h-1 in
this adult subject. With
D-[U-13C6]galactose
for internal standardization, it could also be shown that the portion
of the plasma concentration derived from endogenous sources remained
essentially constant during the course of the experiment (Fig. 5
).
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Figure 5. D-Galactose concentration and
1-13C enrichment (MPE) in plasma of a
postabsorptive healthy subject undergoing a 6-h primed continuous
infusion test with D-[1-13C]galactose.
The priming and infusion doses were 8 µmol/kg of body weight and 0.8
µmol · kg body weight-1 · h-1,
respectively. Control samples (basal values) were
collected before application of the priming dose. 1-13C
enrichment and plasma total D-galactose
(Galtot) were analyzed using GC-MS
procedures. For concentration measurements,
D-[U-13C6]galactose
was used as internal standard. The portion of endogenous
D-galactose in a sample
(Galendo) was estimated on the basis of
MPE and the total concentration of D-galactose. See
Materials and Methods for details. •,
13C enrichment;  , D-galactose
concentration. Lines and shaded areas
indicate means and ± 2 SD, respectively.
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Discussion
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The presence of comparatively large amounts of
D-glucose is the major problem for reliable measurement of
low D-galactose concentrations in human plasma by GC-MS
methods. Our present data indicate that, under postabsorptive
conditions, the ratios of excess D-glucose over
D-galactose in the plasma of healthy adults, diabetic
patients, and patients with classical galactosemia are
30 000–60 000:1, 50 000–200 000:1, and 2 000–10 000:1,
respectively. The present results show that interferences from
D-glucose can be readily overcome by effective and specific
removal of this compound from plasma samples by the use of
D-glucose oxidase from A. niger
(12)(13). The data further show that only minor
losses of D-galactose occur during the subsequent
ion-exchange purification procedure, provided that sample clean-up is
careful.
To date, the method for plasma D-galactose with the lowest
detection limit (2.2 µmol/L) appears to be the HPLC-electrochemical
detection method described by Watanabe and Kawasaki (6).
Thus, the detection limit of the present procedure is more than two
orders of magnitude lower than in previously described
D-galactose assays (5)(6)(7)(8).
For GC-MS analysis, we chose the aldononitrile pentaacetate derivative.
This derivative has several advantages. Acetylated aldononitriles of
the hexoses represent single chemical entities, whereas other
derivatization procedures may yield two or more products, e.g.,
alkylsilylation of aldohexoses yields four cyclic tautomers in various
amounts. These are separated on the GC column, thus reducing the
overall sensitivity of MS detection (14)(15).
Furthermore, the aldononitrile pentaacetate derivatives are rather
stable, and only minor fragmentation occurs using methane chemical
ionization (16). For the derivative of
D-galactose, most of the ion current was
concentrated in the [MH-60]+ ion cluster, and
interfering peaks were absent at m/z 328, 329, and 334, thus
enhancing the specificity and sensitivity of quantification.
The present data now firmly establish that D-galactose is
generally present in plasma of postabsorptive subjects although at very
low concentrations. The concentrations measured in the in vivo study
also show that D-galactose was released at a rather
constant rate from endogenous sources into the plasma compartment. If
the plasma D-galactose had been a remainder from dietary
sources, the concentration would be expected to decline during the
course of the experiment because of the comparatively high metabolic
clearance of plasma D-galactose in the investigated subject
(~1 µmol · kg body
weight-1 · h-1). It
should be noted in this context that constancy of the isotope dilution
of infused D-[1-13C]galactose is
only an apparent indicator of stable plasma concentrations unless
gradual accumulation of naturally labeled (endogenous)
D-galactose is excluded.
In our healthy subject, the rate of appearance of endogenous
D-galactose in plasma (0.17 µmol · kg body
weight-1 · h-1) was
remarkably low when compared with the rates observed by Berry et al.
(4) in three healthy adults under quite comparable
experimental conditions (2.9–5.4 µmol · kg body
weight-1 · h-1). In a
recently conducted control experiment with the same
13C dosage as has been applied by Berry et al.
(4), we experienced a similarly low rate of
D-galactose production (0.43 µmol · kg body
weight-1 · h-1) in a
second healthy adult. The causes for this apparent discrepancy are not
obvious at present, and whether this points to a considerable
intraindividual variation of endogenous
D-galactose production remains to be elucidated.
As expected, the postabsorptive plasma concentrations of
D-galactose were significantly higher in patients with the
classical form of galactosemia than in nongalactosemic subjects.
Whether this is attributable solely to the reduced rate of
D-galactose clearance in the liver of the galactosemic
patients or is also caused by a somewhat enhanced release of
D-galactose from extrahepatic tissues remains to be
clarified.
A conventional fluorometric D-galactose dehydrogenase assay
(10) was modified to allow determination of
D-galactose in the low concentration range. The
comparative GC-MS and enzymatic measurements, however, point to the
presence of (unidentified) interfering substances in human plasma that
lead to erroneously high D-galactose estimates in
the D-galactose dehydrogenase assay. Similar
observations have been reported previously (6)(7)(8). Although
the enzymatic assay works perfectly well with pure solutions of
D-galactose, it cannot be recommended for
analysis of plasma D-galactose in the low
micromolar concentration range.
In conclusion, the present GC-MS procedure is applicable for the
sensitive and reliable determination of the concentration and
13C enrichment of D-galactose in human plasma.
It is necessary, however, to check carefully any
13C-labeled D-galactose preparation before
using it for internal standardization or infusion because different
preparations may contain different amounts of interfering compounds
(16).
|
Acknowledgments
|
|---|
This work was supported in part by a grant from the
Elterninitiative Galaktosämie e.V., Düsseldorf, Germany. We
thank Dr. U. Spiekerkötter (Kinderklinik,
Heinrich-Heine-Universtät, Düsseldorf, Germany) for
contributing to the in vivo study.
|
Footnotes
|
|---|
1 Nonstandard abbreviations: GC, gas chromatography; MS, mass spectrometry; and MPE, mole-percentage of enrichment. 
|
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Abstract
Introduction
