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Previous Article | Next Article 
Journal of Clinical Microbiology, April 2000, p. 1439-1443, Vol. 38, No. 4
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
A Rapid, Automated Enzymatic Fluorometric Assay for
Determination of D-Arabinitol in Serum
Siew Fah
Yeo,1,2
Yeyan
Zhang,1,2
David
Schafer,3
Sheldon
Campbell,3,4 and
Brian
Wong1,2,*
Departments of Internal
Medicine1 and Laboratory
Medicine,4 Yale University School of Medicine,
New Haven, and Infectious Diseases2
and Laboratory Medicine and Pathology,3
Veterans Affairs Medical Center, West Haven, Connecticut
Received 20 September 1999/Returned for modification 23 December
1999/Accepted 2 February 2000
 |
ABSTRACT |
A rapid enzymatic fluorometric assay for measuring
D-arabinitol in serum was developed using recombinant
D-arabinitol dehydrogenase from Candida
albicans (rArDH). rArDH was produced in Escherichia coli and purified by dye-ligand affinity chromatography. rArDH was highly specific for D-arabinitol, cross-reacting only
with xylitol (4.9%) among all polyols tested. A Cobas Fara II
centrifugal autoanalyzer (Roche) was used to measure NADH
fluorometrically when rArDH and NAD were added to serum extracts, and
D-arabinitol concentrations were calculated from standard
curves derived from pooled human serum containing known amounts of
D-arabinitol. The method was precise (mean intra-assay
coefficients of variation [CVs], 0.8%, and mean interassay CVs,
1.6%) and rapid (3.5 min per assay) and showed excellent recovery of
added D-arabinitol in serum (mean recovery rate, 101%).
The mean and median D-arabinitol/creatinine ratios were
2.74 and 2.23 µM/mg/dl, respectively, for the 11 patients with
candidemia compared to 1.14 and 1.23 µM/mg/dl, respectively, for 10 healthy controls (P < 0.01). These results confirm
earlier studies showing that serum D-arabinitol measurement
may help to promptly diagnose invasive candidiasis. The technique shows
a significant improvement in terms of accuracy, cost, simplicity, specificity, and speed compared with gas chromatography, mass spectrometry, and earlier enzymatic assays.
| |
INTRODUCTION |
As the incidence of invasive
candidiasis has increased dramatically in recent years, the accurate
and early detection of this infection has become of major importance.
Unfortunately, conventional culture-based clinical methods, which may
take several days to become positive, are not very sensitive for
detecting invasive disease (1, 7, 14, 15). Alternative
approaches such as PCR assays and immunodiagnostic methods have been
described, but these methods are not yet sufficiently sensitive and
specific to have been widely adopted in clinical practice
(15).
D-Arabinitol is a metabolite of several pathogenic
Candida species, and several studies have shown that serum
D-arabinitol concentrations and serum
D-arabinitol/creatinine ratios are higher in humans and
animals with invasive candidiasis than in uninfected or colonized
controls (4, 9, 18, 19). Early studies used gas
chromatography (GC) or GC-mass spectrometry to detect and quantify
D-arabinitol in serum (2, 4, 5, 9). However, these methods require expensive equipment, and specimen processing and
analysis require considerable time and effort. Enzymatic assays that
used D-arabinitol dehydrogenase from Klebsiella
pneumoniae (10, 11) to quantify
D-arabinitol are less cumbersome, but these enzymes also
react with D-mannitol, which is sometimes present in human
serum (8). In 1994, Switchenko et al. (13)
described a colorimetric endpoint enzymatic assay that used a clinical
chemistry autoanalyzer and D-arabinitol dehydrogenase
(ArDH) from Candida tropicalis to quantify
D-arabinitol in serum (13). The ArDH utilized in
this study was extracted and purified from C. tropicalis. A
method for overproducing recombinant C. tropicalis ArDH in
Escherichia coli has since been described (6),
but recombinant ArDH (rArDH) has not yet been used in automated
D-arabinitol assays.
In this study, we describe a sensitive, specific, and rapid enzymatic
fluorometric method for measuring D-arabinitol in serum utilizing an automated analyzer. This new assay is faster and simpler
than methods employing GC, GC-mass spectrometry, or the colorimetric
endpoint enzymatic assay. Moreover, fewer reagents are required, and
the automated equipment is available in many clinical laboratories. The
key reagent is rArDH from Candida albicans, which was
overproduced in E. coli and purified to homogeneity by
dye-ligand affinity chromatography. The assay is based on oxidation of
D-arabinitol to D-ribulose by rArDH, with the
concomitant reduction of NAD to NADH. The initial rate of NADH
production, which is proportional to the amount of
D-arabinitol in serum, is measured fluorometrically. The
D-arabinitol concentration was determined by comparing the
initial rate of NADH production to that for D-arabinitol calibration curves.
| |
MATERIALS AND METHODS |
Expression, purification, and properties of rArDH. (i) Production
and purification of rArDH.
In order to produce recombinant
C. albicans ArDH (rArDH) in E. coli, we used PCR
to amplify the C. albicans ARD1 coding sequence from plasmid
pJB4 (20) and to change the CATAATGGAT
sequence at the start codon (underlined) to CACCATGGAT,
thereby introducing an NcoI restriction site. The PCR
product was digested with NcoI and XbaI and
ligated into NcoI- and XbaI-digested pET19b
(Novagen), which yielded pET19b/ArDH. Next, the portion of the
ARD1 coding sequence 3' to the SpeI restriction
site was excised from pET19b/ArDH with SpeI and
XbaI and replaced with the SpeI-XbaI
fragment from pJB4, which yielded pET19b/ArDH81. Lastly, all of the
ARD1 coding sequence 5' to the SpeI restriction
site in pET19b/ArDH81 was sequenced to verify that no errors had been
introduced by PCR.
Next, E. coli BL21(DE3) (Novagen) was transformed with
pET19b/ArDH81, and the transformants were grown to an optical density value at 600 nm of 0.6 in Circlegrow broth (Bio 101, Vista, Calif.) supplemented with 50 µg of ampicillin (Sigma, St. Louis, Mo.) per ml
at 37°C with shaking.
Isopropyl-
-D-thiogalactopyranoside (American
Bioanalytical, Natick, Mass.) was added to 1 mM, and the cells were
shaken at 37°C for 3 more h. The cells were harvested by
centrifugation and suspended in 100 mM sodium phosphate buffer (pH 7.0)
(2 ml/100-ml cultures). The cells were broken by sonication for four
periods of 10 s each, and cellular debris was pelleted by
centrifugation at 30,000 × g for 30 min. The
supernatant was cleared by ultracentrifugation at 100,000 × g for 45 min, and it was loaded onto a reactive Yellow 86 column
(2.5 by 20 cm) (Sigma) and washed with 350 ml of 100 mM sodium
phosphate buffer (pH 7.0) and with 350 ml of 100 mM sodium phosphate
buffer (pH 7.0) plus 0.5 M NaCl. rArDH was eluted with 50 mM sodium
phosphate buffer (pH 7.0)-250 mM NaCl-5 mM MgSO4-1 mM
NADH. Active fractions were pooled and concentrated by ultrafiltration
(Centriprep-30; Amicon, Danvers, Mass.). NADH was removed with a
desalting column (Econo-Pac DG; Bio-Rad, Richmond, Calif.), and the
purified rArDH was stored at 
80°C in 0.1 M sodium phosphate buffer
(pH 7.0)-200 mM NaCl-5 mM MgSO4.
(ii) Characterization of rArDH.
The purity of rArDH was
assessed by denaturing sodium dodecyl sulfate-polyacrylamide gel
electrophoresis, using a 12% polyacrylamide gel. rArDH catalytic
activity was monitored using a UV2401 PC Spectrophotometer (Shimadzu
Scientific, Inc., Braintree, Mass.). The 1-ml reaction mixture
contained 50 mM glycine (pH 9.0), 0.1 U of rArDH, and 10 µl of 50 mM
NAD; the reaction was initiated by the addition of 50 µl of 15%
(wt/vol) D-arabinitol. Specific activity is reported as
units per milligram of protein, where 1 U is defined as the amount
required to generate 1 µmol of NADH/min. For substrate and cofactor
specificity studies, the reaction mixture contained 50 mM glycine (pH
9.0), 0.5 M NAD, 0.1 U of enzyme, and 100 mM substrates. The reverse
reaction was assayed in 50 mM glycine (pH 7.0), containing 0.34 mM
NADH, 0.1 U of rArDH, and 100 mM substrates.
To test the effect of pH on the enzyme activity, buffers containing 50 mM sodium citrate, 50 mM sodium phosphate, and 100 mM Tris with pH
values ranging from 4.0 to 10.5 were used.
Fluorometric enzymatic assay for measuring serum
D-arabinitol concentration. (i) Serum treatment and
analysis.
Serum was diluted 1:1 (vol/vol) with 10 mM sodium
citrate (pH 4.0), boiled for 10 min, cooled on ice, and then
centrifuged at 10,000 × g for 10 min. The supernatant
was analyzed immediately or stored at 20°C for later analysis.
A Cobas Fara II centrifugal autoanalyzer (Roche Diagnostics) was used
to detect and quantify D-arabinitol in human serum. This
instrument is equipped with a sensitive fluorometer that can accurately
measure fluorescence changes of 0.0001 
F/min.
The Cobas Fara II was programmed to mix 85 µl of pretreated sample
and 135 µl of reagent containing 50 mM glycine (pH 9.5), 0.03 mg of
bovine serum albumin per ml, 5 mM MgSO4, 100 mM NaCl, and
0.5 U of rArDH. After incubation at 25°C for 30 s, 10 µl of reagent containing 0.5 mM NAD was added. Measurement began after 5 s. The increase of fluorescence was recorded at 30-s intervals for
150 s (excitation wavelength, 345 nm; emission wavelength, 450 nm). The slope during the first 30 s was used to calculate the
rate of NAD reduction.
(ii) Calibration curves.
Six calibrators were prepared by
supplementing a normal pooled human serum with different concentrations
of D-arabinitol prior to sample pretreatment. An endogenous
D-arabinitol concentration of approximately 1.1 µM was
determined in this normal pooled human serum by the method of
Switchenko et al. (13). The final concentrations of
D-arabinitol in six calibrators were 1.1, 2.1, 6.1, 11.1, 16.1, and 21.1 µM, respectively. Calibration curves were obtained by plotting the initial rate of NADH produced by standards versus the
D-arabinitol concentration of the standards. Concentrations of D-arabinitol in unknown samples were determined by
reference to the linear least-squares fit to the calibration curves.
(iii) Human samples.
Single serum samples were obtained from
10 healthy controls and from 11 non-human immunodeficiency
virus-infected patients with Candida fungemia and no known
immunodeficiency states. The serum specimen from each fungemic patient
was obtained on the day where the first positive blood culture was
drawn. Seven patients had C. albicans fungemia, and four had
Candida parapsilosis fungemia. D-Arabinitol was
measured by the automated enzyme fluorometric method, creatinine was
measured by an alkaline picrate (modified Jaffe) reaction on a Hitachi
717 chemistry instrument, and D-arabinitol/creatinine ratios were calculated as previously described (16).
(iv) Statistical analysis.
The significance of differences
between the D-arabinitol/creatinine ratios for
Candida fungemic patients and uninfected controls was
assessed by the Mann-Whitney test, and a P value of <0.05 was considered significant.
| |
RESULTS |
Catalytic and physical properties of the rArDH.
The overall
yield of rArDH from the purification procedure was 80%, and its
specific activity was 123.5 U/mg. Analysis of the NADH elute by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis showed a single band
at a subunit molecular mass of 31 kDa.
The enzyme was highly specific for D-arabinitol, exhibiting
4.9% cross-reactivity with xylitol and no detectable reactivity with
L-arabinitol, galactitol, glycerol, mannitol,
D-ribitol, or D-ribitol-5-phosphate. For the
reverse activity, the enzyme oxidized NADH in the presence of
D-ribulose (137.2 U/mg), with 5% cross-reactivity with
D-xylulose. No cross-reactivity was seen with glucose,
D-arabinose-5-phosphate,
D-ribulose-5-phosphate, or
D-xylulose-5-phosphate. The apparent
Km for D-arabinitol was 46.6 mM in
the presence of NAD, and the Km for
D-ribulose was 42.6 mM in the presence of NADH.
The effects of metal ions, pH, and temperature on the enzyme activity
were determined. Addition of 5 mM MgCl2 and 5 mM
MgSO4 increased the catalytic activity of rArDH by 
33%,
but 5 mM ZnSO4 and 5 mM CaCl2 decreased
its activity by 66 and 30%, respectively. Also, EDTA at 
10
µM decreased the enzyme activity by 77.5%. However, 100 mM NaCl and
0.02% (wt/vol) bovine serum albumin did not alter the catalytic
activity of rArDH. Thus, NaCl and bovine serum albumin were included in
the automated assay as stabilizers. The optimum pH for
D-arabinitol oxidation is 9.0, and catalytic activity
decreased by 50% at a pH value of 10.0. rArDH was stable at 4°C
for 1 week, at 20°C for 3 weeks, and at 80°C for at least 3 months.
Automated enzymatic fluorometric assay of D-arabinitol
in serum. (i) Calibration curves.
Calibration curves generated on
six different days were linear with correlation coefficients of
0.998 ± 0.001 (Fig. 1). Assay precision was high, with a mean interassay variance of 2.5%.
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FIG. 1.
Calibration curve for the assay of
D-arabinitol in spiked human serum. A pooled human serum
containing 1.1 µM endogenous D-arabinitol was
supplemented with six different concentrations of
D-arabinitol. The x axis gives the total
concentrations of endogenous plus added D-arabinitol,
whereas the y axis gives the mean ± SD of initial rates of
change in fluorescence per minute assayed on six different days
(R = 0.9995).
|
|
(ii) Accuracy and precision.
The interassay precision was
determined by analyzing human sera spiked with 5 and 15 µM
D-arabinitol and tested on six different days. The
coefficients of variation were 1.7% at 5 µM and 1.4% at 15 µM
(mean, 1.6%). Intra-assay precision was evaluated by determining the
concentration of human sera spiked with 0.1, 0.5, 1, 5, 10, 11, 15, and
20 µM D-arabinitol. Coefficients of variation ranged from
0.1 to 1.5% (mean, 0.8%).
Accuracy was evaluated by determining the D-arabinitol
concentrations of eight sera spiked with 0.1 to 20 µM
D-arabinitol. The D-arabinitol concentrations
of each spiked serum were predicted by the total concentrations
of endogenous and supplemented D-arabinitol. A comparison
between predicted D-arabinitol values and values measured by enzymatic fluorometric assay showed no
significant difference. The linear calibration curve was obtained with
correlation coefficients of 0.999 (Fig.
2). Recovery of D-arabinitol
was studied by adding 2 µM D-arabinitol as a supplement
to nine different serum specimens. Recoveries were measured by the
difference in measured D-arabinitol concentration between
supplemented and unsupplemented samples and divided by
D-arabinitol concentration added. The average recovery
ranged from 96 to 106% with a mean of 101% (Table
1).
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FIG. 2.
Accuracy of D-arabinitol measurement
determined by automated fluorometric assay. Eight serum samples that
were spiked with different concentrations of D-arabinitol
were assayed by the automated fluorometric method, and
D-arabinitol values are shown on the y axis
(mean ± SD). The D-arabinitol concentration on the
x axis is predicted from the total concentrations of
endogenous and added D-arabinitol (R = 0.9990).
|
|
(iii) Interference with assay by sugars and therapeutic drugs.
L-Arabinitol (50 µM), ribitol (50 µM), xylitol (50 µM), D-sorbitol (50 µM), galactitol (50 µM),
D-galactose (500 µM), D-fructose (500 µM),
D-mannose (750 µM), and glucose (5,000 µM) were tested for their ability to interfere with the assay. Only 50 µM xylitol produced a measurable response, and this represented 5.0% of the response observed with an equimolar amount of D-arabinitol.
Several drugs were also tested for possible interference with the
D-arabinitol assay. These included acetaminophen (300 µg/ml), methylprednisone (120 µg/ml), amphotericin B (20 µg/ml), itraconazole (16 µg/ml), ketoconazole (16 µg/ml),
cefaclor (230 µg/ml), ciprofloxacin (43 µg/ml), erythromycin
(200 µg/ml), gentamicin (120 µg/ml), chloramphenicol (250 µg/ml),
vancomycin (630 µg/ml), and heparin (8 U/ml). None of these drugs
produced a measurable response.
(iv) D-Arabinitol concentrations and
D-arabinitol/creatinine ratios in patients with
candidemia.
Serum D-arabinitol concentrations ranged
from 1.10 to 1.58 µM in 10 healthy controls and 1.64 to 19.11 µM in
the 11 patients with Candida fungemia. The mean and median
D-arabinitol/creatinine ratios were 1.14 and 1.23 µM/mg/dl, respectively, for the healthy subjects, and they were 2.74 and 2.23 µM/mg/dl, respectively, for the patients with candidemia
(Fig. 3). The infected patients' D-arabinitol/creatinine ratios were significantly higher
than the values for the healthy subjects (P < 0.01 by
the Mann-Whitney test).
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FIG. 3.
D-Arabinitol/creatinine ratios (micromolar
concentrations per milligram per deciliter) for 10 healthy subjects and
11 non-human immunodeficiency virus-infected patients with
Candida fungemia and no known immunodeficiency state. The
infected patients' D-arabinitol/creatinine ratios were
significantly higher than the values for the controls (P < 0.01 by the Mann-Whitney test).
|
|
| |
DISCUSSION |
Many medically important species of Candida (including
C. albicans, C. tropicalis, and C. parapsilosis) produce measurable amounts of
D-arabinitol in culture as well as in infected animals and
humans (3, 4, 5, 18). It has been shown elsewhere that
patients with invasive candidiasis had higher serum
D-arabinitol levels than did uninfected controls and that
these levels declined with effective therapy (2, 4, 19).
Based on these results, D-arabinitol has been recognized as
a potentially useful diagnostic marker for invasive candidiasis. Since
D-arabinitol is cleared primarily by glomerular filtration,
the level of D-arabinitol in serum increases in proportion
to creatinine when renal function is impaired. To correct for this
effect, we and others have used serum
D-arabinitol/creatinine ratios (4, 16, 17, 18).
Despite the usefulness of D-arabinitol as a diagnostic
marker, its routine use in the clinical laboratory has been limited due
to the unavailability of rapid and simple analytical methods. GC and
GC-mass spectrometry were used originally to measure
D-arabinitol. However, these methods are complicated,
technically demanding, and time-consuming and may allow only a small
number of samples to be tested each day (8, 9). The
necessary equipment is also very expensive and is not routinely
available in hospital diagnostic laboratories. Hence, an enzymatic
method that used ArDH from K. pneumoniae was developed
(10, 11, 12). However, this enzyme showed 20%
cross-reactivity with D-mannitol, a sugar that may be
present in normal human serum (8, 10). Thereafter, an
automated enzymatic assay, based on colorimetric endpoint, for
measuring D-arabinitol in human serum using a highly
specific ArDH from C. tropicalis was developed
(13). A large prospective clinical trial carried out by
Walsh et al. (16) showed that the automated
D-arabinitol testing was useful for diagnosing invasive candidiasis initially and for assessing response to therapy. However, no further investigations using this method have been reported since 1995.
If an automated D-arabinitol test is to be used widely in
clinical practice, a reliable source of ArDH is needed. Although a
method for overproducing recombinant C. tropicalis ArDH has been described previously (6), rArDH has not yet been used in an automated D-arabinitol assay. To ensure a steady
source of rArDH, we cloned the C. albicans structural gene,
overproduced rArDH in E. coli, and purified rArDH to
homogeneity by dye-ligand affinity chromatography. Thus, it is now
possible to generate a highly purified rArDH that is highly specific
for D-arabinitol and has no cross-reactivity with sugars
commonly found in human serum such as L-arabinitol,
mannitol, and sorbitol. With a readily obtainable recombinant enzyme,
it is technically feasible to detect and quantify
D-arabinitol using clinical chemistry equipment.
The automated fluorometric enzymatic assay described here is much
faster and simpler to use than either GC or GC-mass spectrometry. The
assay has been set to perform automatically on a Cobas Fara II
centrifugal autoanalyzer, thereby simplifying the procedure. The
automated enzymatic D-arabinitol assay developed by
Switchenko et al. (13) was based on two reactions: the first
reaction is the oxidation of D-arabinitol and the
concomitant reduction of NAD to NADH. In the second reaction, the NADH
reduces p-iodonitrotetrazolium violet to
iodonitrotetrazolium-formazan, which is measured
spectrophotometrically. In the fluorometric assay described here, the
coupling reagent and dye reagent are eliminated, saving both time and
cost in reagent preparation and reducing potential sources of error.
Our reaction system, which is based on initial reaction rate, is
approximately five times faster than the dye-coupling method using the
endpoint reaction. The dye-coupling method requires 16 min for one
assay, whereas the fluorometric assay requires only 3.5 min, thereby increasing sample throughput substantially.
We have used the enzymatic fluorometric assay to monitor the levels of
D-arabinitol in the serum of healthy controls and patients with candidemia. Although the numbers of studied subjects were small,
significantly elevated D-arabinitol/creatinine ratios were found in the serum from patients with invasive candidiasis compared to
the ratios for uninfected controls. These data, in conjunction with
other clinical indicators, may provide earlier detection of invasive
candidiasis, which in turn may facilitate the earlier treatment of
invasive candidiasis.
The enzymatic fluorometric assay that we developed is simple, highly
specific, and sensitive for measuring D-arabinitol in serum
and permits analysis of many samples within a working day. Currently,
we have an ongoing prospective project in the State of Connecticut for
retrieving serum specimens from patients diagnosed with candidemia. The
study will allow us to explore the potential role of monitoring serum
D-arabinitol concentration by the fluorometric method
described here. Although blood culture is unlikely to be replaced by
other diagnostic tools, we anticipate that fluorometric detection of
D-arabinitol will ultimately lead to improved diagnosis of
invasive candidiasis.
| |
ACKNOWLEDGMENTS |
This work was supported by grants from the U.S. Department of
Veterans' Affairs and from Pfizer Pharmaceuticals, Inc.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: VA Medical
Center, Infectious Diseases, 950 Campbell Ave., 111-I, West Haven, CT
06516. Phone: (203) 932-5711, ext. 5168. Fax: (203) 937-4851. E-mail: brian.wong{at}yale.edu.
| |
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Journal of Clinical Microbiology, April 2000, p. 1439-1443, Vol. 38, No. 4
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
