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Articles |
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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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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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). 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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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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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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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.
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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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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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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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).
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Acknowledgments |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) 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).
, D-galactose
concentration. Lines and shaded areas
indicate means and ± 2 SD, respectively.