Clinical Chemistry 45: 2086-2093, 1999;
(Clinical Chemistry. 1999;45:2086-2093.)
© 1999 American Association for Clinical Chemistry, Inc.
Capillary Electrophoresis for Detection of Inherited Disorders of Purine and Pyrimidine Metabolism
Tomá
Adam1,3,a,
David Friedeck
2,
Lynette D. Fairbanks4,
Juraj 
ev
ík2,3 and
Petr Barták2,3
1
Laboratory for Inherited Metabolic Disorders, Department of Clinical Biochemistry, Medical Hospital, I. P. Pavlova 6, 775 20 Olomouc, Czech Republic.
2
Department of Analytical Chemistry, Palack
University, T
ída Svobody 8, 772 00 Olomouc, Czech
Republic.
3
Laboratory of Bioanalytical Research, Palack
University, Tída Svobody 8, 771 26 Olomouc, Czech
Republic.
4
Purine Research Laboratory, Guy’s and St. Thomas’s
Medical and Dental School, London Bridge, London SE1 9RT, UK.
a Address correspondence to this author at: Laboratory for Inherited Metabolic Disorders, Medical Hospital, I. P. Pavlova 6, 775 20 Olomouc, Czech Republic. Fax 420-68-5416555; e-mail tomasadam{at}email.cz
 |
Abstract
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Background: Measurement of purine and pyrimidine metabolites
presents complex problems for separations currently performed by HPLC
and thin-layer chromatography in clinical practice. We developed a
novel capillary electrophoresis method for this purpose.
Methods: Separations were performed in 60 mmol/L
borate-2-amino-2-methyl-1-propanol-80 mmol/L sodium dodecyl sulfate (pH
9.6) at 35 °C.
Results: The conditions reported allowed separation of all
diagnostic metabolites from major urinary constituents in an analysis
time of 3 min and with a separation efficiency of 220 000 theoretical
plates/m. The clinically important metabolites were detectable at
concentrations of 0.85–4.28 µmol/L. The method was linear over the
range 5–500 µmol/L (r >0.99). The within-run and
intra- and interday imprecision (CV) was <5%. Characteristic
abnormalities were detected in the electropherograms of urine samples
from patients with purine and pyrimidine enzyme deficiencies. We
provide the electrophoretic and spectral characteristics of many
intermediates in purine and pyrimidine metabolism and describe common
artifacts from medication and ultraviolet-absorbing compounds.
Conclusion: Capillary electrophoresis is a valuable screening
tool in the detection of inborn errors of purine and pyrimidine
metabolism.
|
Introduction
|
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Inherited defects in purine and pyrimidine metabolic pathways
are associated with serious, sometimes fatal, consequences. For the
correct diagnosis, it is necessary to have precise and rapid methods
for identifying and quantifying purines and pyrimidines in body fluids.
These methods must be powerful and capable of identifying known as well
as novel disorders. Because analysis is performed on biologic fluids
(in initial screening mostly with urine), the methods should be robust
and provide excellent resolution because the matrices we are dealing
with are very complex. From a practical point of view, assays should be
easy to perform, automated, and inexpensive because laboratories
providing a diagnostic service usually deal with hundreds of samples
each year.
Purine and pyrimidine species were first measured a century ago [see
Ref. (1) for review]. The first use of anion-exchange
chromatography in 1949 (2) introduced separation techniques
into this field. Since that time, many researchers have developed HPLC
methods for research and diagnostic purposes (3).
Currently HPLC is a dominant tool in diagnostic practice,
although two-dimensional thin-layer chromatography (4) is
also widely used.
Although isotachophoretic approaches were utilized for separation of
purine and pyrimidine species in the past (5), they were
never widely used in diagnostic practice. The narrow bore capillary
electrophoretic separation technique, with its high separation
efficiency, flexibility, and high sample throughput, can provide an
excellent tool for the diagnosis of inborn errors of purine and
pyrimidine metabolism (6)(7)(8)(9).
We report here an optimized capillary electrophoretic method that
enables diagnosis of purine and pyrimidine disorders. The method was
tested on urine samples from healthy infants and patients with
inherited defects of purine and pyrimidine metabolism.
|
Materials and Methods
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chemicals
All chemicals were of analytical reagent grade. Boric acid, sodium
hydroxide, and 
-cyclodextrin
(CD)1
were purchased from Merck. Bases; nucleosides;
2-amino-2-methyl-1-propanol (AMP);
3-[cyclohexylamino]-2-hydroxy-1-propanesulfonic acid; 
-, ß-, and
-CDs; and other chemicals were obtained from Sigma. Germanium (IV)
oxide and sulfated ß-CD were from Aldrich. Deionized water (18
M
/cm) was used for preparation of all solutions.
subjects and samples
Urine samples from healthy controls were from 200 children
(Caucasian; 112 males and 88 females of the Czech Republic; age range,
1–15 years; mean, 4.8 years). The urines from patients (all from UK)
were from persons with enzymatically demonstrated purine and pyrimidine
enzyme deficiencies.
All samples were obtained as 40-µL urine spots on filter paper and
were dissolved in 150 µL of deionized water, taken to dryness under
stream of nitrogen, and redissolved in 40 µL of deionized water.
Eight spot urine specimens from healthy children (creatinine range,
1.1–15.1 mmol/L; mean, 4.7 mmol/L) were used for measuring imprecision
and reproducibility. The pooled urine sample that was used for
determination of maximal injectable amounts was prepared by mixing
equal volumes of redissolved eluates from filter papers of these
samples. All samples were centrifuged (3000g, 5 min) before
loading.
capillary electrophoresis apparatus and conditions
All experiments were performed on a P/ACE 5510 with diode array
detector (Beckman Instruments). The electrophoretic separations were
carried out in an uncoated fused-silica capillary (75 µm i.d. x 375
µm o.d.; Polymicro Technologies). The capillary had an effective
length of 40 cm (total length, 47 cm) and was operated at 25 or
35 °C. Ultraviolet (UV) detection over the range 190–300 nm
(cartridge detection window, 100 x 800 µm) was used. The data
rate of the detector was set at 2 Hz for analyses performed at 10 kV
and 8 Hz for analyses performed at 30 kV, respectively. Sample was
loaded by low-pressure injection (0.5 psi). Borate buffers were
prepared from boric acid, sodium dodecyl sulfate (SDS) was added, and
the solution was adjusted with 500 g/L NaOH or AMP to the
appropriate pH. At the beginning of each working day, the capillary was
washed with water, 0.1 mol/L NaOH, water, and separation buffer for 5
min; between runs, it was washed with 0.1 mol/L NaOH for 0.5 min and
running buffer for 1 min. The analyses were run at a constant voltage,
using a ramp for 0.5 min. These standard conditions were used for all
experiments.
Fourteen bases and nucleosides (the key diagnostic metabolites)
together with three major urinary UV-absorbing constituents (urea,
creatinine, and hippuric acid) were taken as the target group of
compounds. A mixture of target compounds was prepared by dissolving
compounds in deionized water at a concentration of 300 µmol/L. The
mixture and pooled normal urine were analyzed with background
electrolytes (BGEs) at pH 7–10, with and without the addition
of micellar additive (SDS) at 25 and 35 °C, respectively. All
experiments performed at pH <9 did not allow substantial separation of
the mixture of target compounds (data not shown). Comparison of assays
performed with and without SDS revealed that zone electrophoretic
rather than a micellar separation mechanism prevailed for the
separation of purine and pyrimidine species. However, for the
separation of the pooled urine sample, micellar systems resolved a
higher number of peaks. Higher reproducibility can be also expected in
a system utilizing surfactants because it prevents interactions of
urine proteins with the capillary wall. As well as the pH, the BGE
composition substantially affected the separation. Fig. 1
shows that the borates and germanates provided good resolution
of ribonucleosides and deoxyribonucleosides via their well-known
complexation with cis-diol species. Separations performed in 60 mmol/L
borate-Na+-80 mmol/L SDS buffer with a pH >9.5 gave
substantially better resolution of the mixture (Fig. 2
). From these assays, it can be concluded that separation
(although incomplete) improved with increasing pH. At pH >10, almost
complete resolution was achieved, but the very high electrophoretic
mobility of orotic acid (OA) prolonged the separation. Moreover, at pH
>9.7, run-to-run reproducibility was reduced (data not shown) as a
consequence of a lowering of the buffer capacity because the
pKa of boric acid is 9.23. Addition of
organic modifiers (50–150 mL/L acetonitrile and
methanol) to the BGEs did not affect the resolution and lead to
peak tailing (data not shown). CDs previously had been used
successfully for the separation of adenosine derivatives
(10). The influence of -, ß-, -, and sulfo-ß-CD at
concentrations of 25, 10, 25, and 10 mmol/L, respectively in 60 mmol/L
borate-Na+-80 mmol/L SDS, pH 9.6, was tested. Of the CDs
tried, only ß-CD slightly affected the selectivity (data not shown),
but a decrease of run-to run reproducibility was observed. -CD led
to peak distortion, and -CD worsened the separation [mean CV of
effective mobility (n = 10) was 0.84% in BGE without CD, 0.99%
with -CD, 1.6% with ß-CD, 0.87% with -CD, and 4.8%
with sulfo-ß-CD, respectively].
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Figure 1. Influence of BGE composition on migration behavior of
adenine (A), adenosine (AR), and
2'-deoxyadenosine (dAR).
The concentration of the buffering ion was 60 mmol/L.
CAPSO,
3-[cyclohexylamino]-2-hydroxy-1-propanesulfonic acid.
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Figure 2. Influence of pH of BGE on the separation of a mixture of
purines and pyrimidines and major urinary constituents.
Conditions: BGE, 60 mmol/L borate-Na+-80 mmol/L SDS, pH
9.5–10.2; voltage, 15 kV; detection at 190 nm. For other conditions,
see Materials and Methods. creat,
creatinine; dAR, 2'-deoxyadenosine;
A, adenine; T, thymine;
dGR, deoxyguanosine; dHR, deoxyinosine;
AR, adenosine; U, uracil;
hipp, hippuric acid; GR, guanosine;
HR, inosine; X, xanthine;
OA, orotic acid; mAU, milliabsorbance
units.
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To stabilize migration times and effect a better separation, different
counterions of the BGEs were sought. Excellent resolution was achieved
by the use of 60 mmol/L borate-AMP-80 mmol/L SDS buffer, pH 9.6, at
35 °C at 10–30 kV (Fig. 3
). Here the separation was influenced by the counterion (compare
assays in Figs. 2
and 3
performed at the same pH). This buffer is a
double buffering system (the pKb of
AMP is 9.72) and, therefore, higher run-to-run reproducibility of
mobilities was expected. The borate-AMP buffer had a higher buffering
capacity and also lower conductivity (lower Joule heating) in
comparison with the borate-Na+ buffer (60 mmol/L
borate-Na+: capacity, 32.2 mmol/pH; conductivity,
257.8 mS/m; 60 mmol/L borate-AMP: capacity, 66.9 mmol/pH; conductivity
154.9 mS/m at pH 9.6).
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Figure 3. Separation of a mixture of purines and pyrimidines and
major urinary constituents in 60 mmol/L borate-AMP-80 mmol/L SDS
buffer, pH 9.6.
Capillary temperature, 35 °C; voltage, 10 kV (main
panel) and 30 kV (inset). For other conditions,
see Materials and Methods. creat,
creatinine; dAR, deoxyadenosine; T,
thymine; A, adenine; dGR, deoxyguanosine;
U, uracil; dHR, deoxyinosine;
AR, adenosine; GR, guanosine;
hipp, hippuric acid; HR, inosine;
mAU, milliabsorbance units.
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diagnostic metabolites and interferences
Using the final separation conditions, we measured the migration
and spectral properties of the compounds of interest in diagnosing
inherited metabolic disorders, common artifacts from medication
(3), and several other UV-absorbing compounds (Table 1
). The pure compounds dissolved in deionized water were
analyzed, with the exception of succinylaminoimidazole carboxamide
riboside (SAICAR) and succinyladenosine (SAR), for which diagnostic
metabolites are not commercially available (see Results).
separation efficiency and injection volume
Separation efficiency in 60 mmol/L borate-AMP-80 mmol/L SDS
reached 220 000 theoretical plates/m for the mixture of target
compounds and 141 000 theoretical plates/m for the pooled urine sample
with added target compounds. Borate buffers can tolerate higher
injection volumes because of a pronounced transient isotachophoretic
phenomenon. [Borate ion can act as the terminating ion, and
fast-migrating anions from the sample, e.g., chlorides from urine, act
as leading ions (11).] The maximal injectable amount of
sample was determined. The pooled urine sample (see "SUBJECTS
AND SAMPLES") was injected in increasing amounts for up to
15 s, and the separation efficiency was calculated. The
efficiencies observed (x1000 theoretical plates/m) were 132 for 3
s (24 nL), 120 for 5 s (40 nL), 114 for 7 s (55 nL), 113 for
9 s (71 nL), 112 for 13 s (102 nL), and 104 for 15 s
(119 nL).
limit of detection and linearity
Using 15-s injections (6.7% of the total capillary volume
injected with the sample), we determined the limit of detection for
compounds of interest at 200 nm (Table 2
). We tested the linearity by analyzing 10 calibration solutions
in the concentration range 5–500 µmol/L. The method was linear
(r >0.99) for all compounds of interest (Table 2
).
imprecision
The imprecision of the method was tested by assaying eight samples
of healthy volunteers with added 2,8-dihydroxyadenine (DHA; not present
in normal urine) at three concentrations for 20 days. The within-run
CVs were 3.8% for 15 µmol/L, 2.6% for 40 µmol/L, and 1.8% for 90
µmol/L. The intraday CVs were 4.2% for 15 µmol/L, 3.1% for 40
µmol/L, and 2.0% for 90 µmol/L. The interday CVs were 4.6% for 10
µmol/L, 3.6% for 40 µmol/L, and 3.2% for 90 µmol/L.
The reproducibility of the effective mobilities was measured on the
mixture of target compounds [run-to-run CV, 0.32% (n = 20);
day-to-day CV, 2.4% (n = 20)]. Because sample composition can
affect the separation in capillary electrophoresis (CE), the
sample-to-sample reproducibility of effective mobilities was measured
on eight urine samples from healthy volunteers with added calibrators
(CV, 1.5%).
recovery and stability
The recovery and stability of the compounds was measured using
urine sample spot extracts supplemented with the mixture of target
compounds (added concentrations, 30 and 100 µmol/L). The dried urine
spot was extracted on the day of preparation and also after 2 days at
room temperature. The mean recovery (± SD) was 94.8% ± 6.9%
for 30 µmol/L and 96.8% ± 4.9% for 100 µmol/L. The mean (± SD)
stability of the compounds was 95.9% ± 3.2%.
|
Results and Discussion
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Two hundred urine samples from healthy children were analyzed by
this method. No interfering compounds that can substantially confound
the analysis were observed. A typical electropherogram of a urine
sample from a healthy infant is shown in Fig. 4
. Urine from healthy infants contained dominant peaks of uric
acid (UA), urea, creatinine, and hippuric acid and usually various
amounts of hypoxanthine (HX), xanthine (X), 7-methylguanine,
pseudouridine, and uridine. As pointed out by many authors [e.g.,
Simmonds et al. (3)], purine excretion varies considerably
because of varying dietary purine intake, and local reference
values should be determined.
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Figure 4. Electropherogram of urine from healthy infant at 10 kV
(main panel) and 30 kV (inset).
Conditions as in legend for Fig. 3
. creat, creatinine;
hipp, hippuric acid; mAU, milliabsorbance
units.
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The usefulness of the method for diagnostic purposes was demonstrated
on urine samples from patients suffering from inherited disorders of
purine and pyrimidine metabolism. Electropherograms are presented at
205 nm and the optimal wavelength for each particular disease. In all
samples analyzed, the key diagnostic metabolites were easily identified
by migration times and spectral fit.
In the sample from a patient suffering from adenylosuccinate lyase (EC
4.3.2.2) deficiency, SAICAR and SAR were identified by expected
migration behavior (species contain highly charged succinate functional
group) and spectra (Fig. 5
) (6)(12). The analysis of urine from a
patient with adenine phosphoribosyl transferase (EC 2.4.2.7) deficiency
allowed easy identification of a DHA peak (Fig. 6
). Analysis of the urine from a patient with adenosine deaminase
(EC 3.5.4.4) deficiency demonstrated other peaks with spectra similar
to adenosine (indicated by an asterisk in the electropherogram) in
addition to the diagnostic metabolite, deoxyadenosine (Fig. 7
). These compounds could be methylated and incompletely
characterized AR derivatives (13)(14)(15). The electropherogram
of urine from a patient with dihydropyrimidine dehydrogenase (EC
1.3.1.2) deficiency revealed well-separated diagnostic peaks of uracil
and thymine (Fig. 8
). Peaks of inosine, guanosine, and their deoxyribosides were
well resolved in the sample from a patient with purine nucleoside
phosphorylase (EC 2.4.2.1) deficiency (Fig. 9
). The sample from a patient with xanthine oxidase (EC 1.2.3.2)
deficiency revealed only a dominant peak of X migrating after a small
peak of UA (Fig. 10
). In the analysis of urine from patients with orotic acidurias
caused by orotate phosphoribosyltransferase (EC 2.4.2.10) and ornithine
transcarbamylase (EC 2.1.3.3) deficiency (data not shown), an easily
identifiable but relatively broad peak of OA was observed. These
patients excrete very high amounts of this compound, and the comparable
opposite mobilities of the electroosmotic flow and OA slows movement of
OA through the detection window. Patients with orotic acidurias can be
verified easily by an alternative CE diagnostic method (9).
In the sample from a patient with hypoxanthine phosphoribosyl
transferase (EC 2.4.2.8) deficiency, increases in only UA and HX were
observed (data not shown).
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Figure 5. Electropherogram of urine from patient with
adenylosuccinate lyase deficiency at 10 kV (main panel)
and 30 kV (inset).
Conditions as in legend for Fig. 3
. creat, creatinine;
hipp, hippuric acid; mAU, milliabsorbance
units.
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Figure 6. Electropherogram of urine from patient with adenine
phosphoribosyl transferase deficiency at 10 kV (main
panel) and 30 kV (inset).
Conditions as in legend for Fig. 3
. creat, creatinine;
hipp, hippuric acid; mAU, milliabsorbance
units.
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Figure 7. Electropherogram of urine from patient with adenosine
deaminase deficiency at 10 kV (main panel) and 30 kV
(inset).
Conditions as in legend for Fig. 3
. creat, creatinine;
dAR, deoxyadenosine; A, adenine;
AR, adenosine; hipp, hippuric acid;
mAU, milliabsorbance units. *, adenosine derivatives
not characterized (see Results and Discussion).
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Figure 8. Electropherogram of urine from patient with
dihydropyrimidine dehydrogenase deficiency at 10 kV (main
panel) and 30 kV (inset).
Conditions as in legend for Fig. 3
. creat, creatinine;
T, thymine; U, uracil;
hipp, hippuric acid; mAU, milliabsorbance
units.
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Figure 9. Electropherogram of urine from patient with purine
nucleoside phosphorylase deficiency at 10 kV (main
panel) and 30 kV (inset).
Conditions as in legend for Fig. 3
. creat, creatinine;
dGR, deoxyguanosine; dHR, deoxyinosine;
GR, guanosine; HR, inosine;
mAU, milliabsorbance units.
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Figure 10. Electropherogram of urine from patient with xanthine
oxidase deficiency at 10 kV (main panel) and 30 kV
(inset).
Conditions as in legend for Fig. 3
. creat, creatinine;
hipp, hippuric acid; mAU, milliabsorbance
units.
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In conclusion, the method reported enables a simple, fast, and
efficient diagnosis of inherited purine and pyrimidine enzyme
deficiencies and also is potentially applicable to the screening of
other inherited disorders connected with increased excretion of
UV-absorbing compounds. The conditions used allowed separation of all
diagnostic metabolites from major urinary constituents. The method is
both efficient (separation efficiency, 220 000 theoretical plates/m)
and sensitive enough for diagnostic purposes. The migration time
reproducibility was achieved by the use of a double-buffering 60 mmol/L
borate-AMP-80 mmol/L SDS (pH 9.6) BGE. Analyses performed by the
proposed method are faster than currently performed HPLC assays
(3) (total analysis time, 3 min for CE vs 30 min for HPLC).
Separation efficiency is also substantially higher for CE compared with
HPLC (220 000 theoretical plates/m for CE vs 5000 theoretical plates/m
for HPLC). From a practical point of view, the substantially lower cost
for CE fused-silica capillaries compared with reversed-phase HPLC
columns makes this approach advantageous.
|
Acknowledgments
|
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This work was supported by Grant 3439-3 from the Ministry of Health
of the Czech Republic and partly by Grant VS 96021 from the Ministry of
Education and Youth and Sport of the Czech Republic.
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Footnotes
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1 Nonstandard abbreviations: CD, cyclodextrin; AMP, 2-amino-2-methyl-1-propanol; UV, ultraviolet; SDS, sodium dodecyl sulfate; BGE, background electrolyte; OA, orotic acid; SAICAR, succinylaminoimidazole carboxamide riboside; SAR, succinyladenosine; DHA, 2,8-dihydroxyadenine; CE, capillary electrophoresis; UA, uric acid; HX, hypoxanthine; and X, xanthine. 
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Abstract
Introduction
