Clinical Chemistry 43: 1315-1320, 1997;
(Clinical Chemistry. 1997;43:1315-1320.)
© 1997 American Association for Clinical Chemistry, Inc.
Extraction of glyceric and glycolic acids from urine with tetrahydrofuran: utility in detection of primary hyperoxaluria
Dennis J. Dietzen1,2,5,
Timothy R. Wilhite3,
David N. Kenagy3,6,
Dawn S. Milliner4,
Carl H. Smith1,3 and
Michael Landt1,3,a
Departments of
1
Pathology,
2
Internal Medicine, and
3
Pediatrics, Washington University School of Medicine, St. Louis, MO 63110.
4
Department of Internal Medicine, Division of Nephrology,
Mayo Medical Center, Rochester, MN 55905.
5
Current address: Dade Chemistry Systems, Inc., Bldg.
700, Box 707, Newark, DE 19714-6101.
6
This author is an employee of the US Air Force: The
opinions and conclusions in this paper are those of the authors, and do
not represent the official position of the Department of Defense, the
US Air Force, or any other government agency.
7
Nonstandard abbreviations: AGT, alanine:glyoxylate aminotransferase; GDH, D-glycerate dehydrogenase; PH, primary hyperoxaluria; PH I (II), primary hyperoxaluria type I (type II); GC-MS, gas chromatography–mass spectrometry; THF, tetrahydrofuran; and BSTFA, bis(trimethylsilyl)trifluoroacetamide.
a Author for correspondence. Fax 314-454-2274; email landt{at}kids.wustl.edu
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Abstract
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Primary hyperoxaluria (PH) is an autosomal recessive metabolic
abnormality characterized by excessive oxalate excretion leading to
nephrocalcinosis and progressive renal dysfunction. Type I primary
hyperoxaluria (PH I) results from a deficiency of alanine:glyoxylate
aminotransferase, whereas type II disease has been traced to a
deficiency of D-glycerate dehydrogenase. The two syndromes
are often distinguished on the basis of organic acids that are
coexcreted with oxalate: glycolate and L-glycerate in type
I and type II disease, respectively. Routine organic acid analysis with
diethyl ether extraction followed by gas chromatographic analysis
failed to detect normal and increased concentrations of these
diagnostic metabolites. Subsequent extraction of urine with
tetrahydrofuran (THF), however, extracted 75% of added glycerate, 42%
of added glycolate, and 75% of added ethylphosphonic acid (internal
calibrator). THF extraction was analytically sensitive enough to allow
determination of normal excretion of glycolate (14–72 µg/mg
creatinine) and glycerate (0–5 years, 12–177 µg/mg creatinine and
>5 years, 19–115 µg/mg creatinine). Four of five patients with PH I
and both patients with type II disease were correctly identified. Thus,
THF extraction is a convenient adjunct to routine organic acid analysis
and facilitates the detection of PH.
Key Words: indexing terms: inborn errors of metabolism • oxalates • renal calculi • laboratory diagnosis • gas chromatography
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Introduction
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Oxalate excreted in urine arises from metabolism of amino acids
and carbohydrates in liver cytosol and peroxisomes
(1)(2). Oxalate is ordinarily a minor end
product of these pathways. In peroxisomes, glyoxylate is derived by
oxidation of glycine and glycolate, and then is largely transaminated
to glycine through the action of alanine:glyoxylate aminotransferase
(AGT, E.C. 2.6.1.44), but can alternatively be oxidized to oxalate via
L-2-hydroxyacid oxidase (E.C.
1.1.3.1).7 In the cytosol, hydroxypyruvate derived from
glucose and fructose is primarily reduced to L-glycerate or
D-glycerate by lactate dehydrogenase (E.C. 1.1.1.27) or
D-glycerate dehydrogenase (GDH, E.C. 1.1.1.29),
respectively. Under normal circumstances a small amount of
hydroxypyruvate is also oxidized to oxalate through pathways that
remain undefined. Primary hyperoxaluria (PH) results from inherited
deficiency of AGT or GDH, which ordinarily metabolize glyoxylate and
hydroxypyruvate to less toxic products. The net effect of these
deficiencies is an increased commitment of intermediates to oxalate,
resulting in increased urinary excretion of oxalate, supersaturation of
urine with calcium oxalate, and, in advanced disease, systemic
deposition of calcium oxalate. Formation of renal stones and the
resulting renal dysfunction are the presenting features of the disease
(3)(4). This disorder is distinct from
enteric hyperoxaluria, which results from hyperabsorption of oxalate
from the digestive tract (5).
Type I primary hyperoxaluria (PH I) is an autosomal recessive
deficiency of AGT with increased excretion of both oxalate and
glycolate in urine (6)(7)(8)(9)(10). Type II primary hyperoxaluria
(PH II) results from a deficiency of GDH, leading to coaccumulation of
oxalate and L-glycerate in urine
(11)(12). European literature has reported
that 1–2% of childhood cases of end-stage renal disease may be
attributable to some form of PH (13)(14). PH I
is diagnosed more commonly, has an earlier onset, and a poorer
prognosis than PH II (11)(15)(16).
Some cases of PH I are responsive to pyridoxine (an AGT cofactor), high
fluid intake, phosphate treatment, or citrate administration, yet
heroic measures such as kidney and (or) liver transplantation are
commonly required after irreversible kidney damage
(2)(17)(18)(19)(20). Patients with PH II have a much
more benign prognosis, though some do progress to end-stage renal
disease (12).
Thus, detection of PH and the distinction between PH I and PH II is
necessary for patients who might benefit most from aggressive
intervention. Currently this is accomplished by determination of AGT
activity in liver biopsy specimens (6)(7) and
measurement of glycolate and glycerate excretion. Liver biopsy allows
direct assessment of enzymatic deficiency, but has several
disadvantages: patient discomfort, the risk of an invasive procedure,
cost, long turnaround time, and (currently) no source for GDH
determination. In addition, individuals homozygous for AGT deficiency
sometimes have enzyme activities indistinguishable from heterozygotes
who are clinically normal (21)(22).
Reliable detection of L-glycerate and -glycolate in urine
may obviate the need for biopsy in many cases. Specific methods for
glycolate and glycerate determination have been developed
(23)(24), but most laboratories, even
specialized ones aimed at detecting genetic metabolic disease, do not
maintain specific testing protocols for glycolate and
L-glycerate. These compounds are only occasionally detected
in routine organic acid analysis by gas chromatography–mass
spectrometry (GC-MS) because their polar character makes them
difficult to extract from urine with diethyl ether or ethyl acetate,
the common solvents used in routine organic acid analysis
(25). With current methods, normal concentrations of
glycolate and glycerate are rarely detected; extreme increases in
excretion may be required for detection. This could partly explain the
observation by Danpure that 30% of PH I patients with marked
hyperoxaluria did not display increased glycolate excretion
(26). We reasoned that a procedure that increased the
efficiency of glycolate/glycerate extraction would provide more
accurate normal ranges and potentially improve the sensitivity of
organic acid analysis to detect and distinguish between PH I and PH II
earlier in the course of the disease. Rimoldi et al. improved the
extraction of polar compounds such as citric, hydroxybutyric, and
orotic acids from urine by using tetrahydrofuran (THF)
(27). Here, we describe the utility of THF extraction in
aiding the diagnosis of PH.
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Materials and Methods
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Materials.
Bis(trimethylsilyl)trifluoroacetamide (BSTFA)
and oxalic, hippuric, succinic, and D,L-lactic
acids were obtained from Sigma (St. Louis, MO). Ethylphosphonic acid,
glycolic acid, and THF were purchased from Aldrich (Milwaukee, WI).
D,L-Glyceric acid was obtained from ICN
(Cleveland, OH).
Specimens.
Random urine specimens were adjusted to
pH 2 to ensure complete recovery of oxalate and maintained at
-20 °C. A preliminary experiment showed that glycerate and
glycolate were stable in acidified urine for up to 3 months. Oxalate
salts precipitated in as little as 2 weeks of storage even at pH 2, and
thus oxalate was not measured on specimens stored for longer periods.
Normal ranges for glycerate, glycolate, and oxalate (normalized to
creatinine) were established with 65 specimens from children and adults
without evidence of liver or kidney disease. Specimens from children
<6 months of age were obtained from outwardly healthy children
visiting the outpatient clinic at St. Louis Children's Hospital. One
glycolate and seven oxalate values were statistically excluded from the
normal range by the outlier analysis of Reed et al. (28);
oxalate:creatinine ratios are known to be nonnormally distributed in
children (9). This study was conducted in accord with a
protocol approved by the human studies committees of Washington
University and the Mayo Clinic.
Analytical.
A volume of urine containing 500 µg of
creatinine was diluted to 5 mL. Ethylphosphonic acid (250 µg) was
added as internal calibrator. The urine was saturated with NaCl and
acidified to pH 1 with HCl before extraction. Extraction was performed
three times with 5 mL of diethyl ether and then five times with 5 mL of
THF. THF and ether extracts were pooled separately and concentrated to
dryness under nitrogen. Residual water was removed by reconstituting
the residue with 200 µL of benzene to form an azeotropic mixture and
again reducing it to dryness under nitrogen. Residues were then
derivatized by incubation in pyridine:BSTFA (1:1 by vol) for 15 min at
60 °C. Calibrators were dissolved in pyridine and derivatized with
an equal volume of BSTFA before use. Derivatives were analyzed on a
Varian 3700 gas chromatograph (Varian Instrument Group, Palo Alto, CA)
equipped with a DB-1 column (0.53 mm i.d.; PJ Colbert Assoc., St.
Louis, MO) by using a temperature program of 7 min at 80 °C followed
by a rise to 260 °C at 6 °C per minute. The injector and detector
temperatures were both 250 °C. Compounds were detected by flame
ionization and identity of the peaks was confirmed with a Finnigan ITD
mass spectrometer (Finnigan MAT, San Jose, CA). Quantification of
oxalate, glycolate, and glycerate was based on the detector response to
a known amount of each compound and corrected for recovery of the
internal calibrator. Glycolic, oxalic, and ethylphosphonic acid
calibrators were prepared by dissolving highly pure material in
pyridine and derivatizing immediately before use. Glyceric acid was
supplied as a syrup with a significant water content and was
lyophilized before dissolving in pyridine. Creatinine was determined on
the Vitros 700 XR (Johnson and Johnson, Rochester, NY).
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Results
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Extraction of glycerate and glycolate.
An aliquot of
urine containing 500 µg of creatinine and supplemented with 250 µg
each of succinate (C4H6O4),
hippurate (C9H9O3), oxalate
(C2H4O4), glycerate
(C3H7O4), and glycolate
(C2H5O3) was saturated with NaCl,
acidified, and extracted three times with 5.0 mL of ether (standard
protocol), then multiple times with 5.0 mL of THF. The first ether and
THF extracts were concentrated, derivatized, and analyzed by GC. The
more hydrophobic molecules, hippurate and succinate, were extracted
effectively by ether while the very polar C-2 and C-3 acids were not
extracted from the urine until THF was used (Fig. 1
). Lactate, with intermediate hydrophobicity, was partially
extracted with ether but predominantly recovered in THF extracts.
Several other polar compounds (phosphate, urea, and citrate) were also
efficiently extracted with THF. Ethylphosphonic acid
(C2H7PO3) was used as an internal
calibrator because of its absence in human urine and polar character
similar to glycerate and glycolate. It was extracted exclusively with
THF.
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Figure 1. THF enables extraction of oxalate, glycerate, and
glycolate from urine.
A urine specimen was supplemented with hippurate, lactate, succinate,
glycolate, glycerate, oxalate, and ethylphosphonate (internal
calibrator) and extracted three times with ether and then with THF. The
elution profiles of the first ether extraction and the first THF
extraction are shown. Oxalate, glycerate, glycolate, and
ethylphosphonate were extracted from urine exclusively with THF.
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Optimization of THF extraction.
The goal of the protocol
was to extract as much of each diagnostic compound as possible and to
match the recovery of each to the internal calibrator. To determine the
optimal number of extractions, 250 µg each of oxalate, glycerate,
glycolate, and ethylphosphonate were added to an aliquot of normal
urine (containing 500 µg creatinine) that was extracted three times
with ether and then repeatedly extracted with THF. When individual THF
extracts were analyzed, oxalate was recovered predominantly in two
extractions, whereas five extractions were required to recover a
comparable amount of glycerate, glycolate, and ethylphosphonate (Table 1
). In subsequent studies, pooling of five successive extracts
resulted in recovery of 43% ± 13%, 76% ± 7%, 43% ± 6%, and
71% ± 9% of oxalate, glycerate, glycolate, and ethylphosphonate,
respectively (mean ± SD, n = 8). Correction of values based
on the recovery of the internal calibrator, therefore, slightly
underestimates the amount of glycolate and oxalate present. Extraction
of glycolate and glycerate was linear up to 1000 µg/mg creatinine
(Sy|x = 18 µg/mg creatinine,
r = 0.9883, and
Sy|x = 47 µg/mg creatinine,
r = 0.9683, respectively). A precision study consisting
of six runs over 6 weeks was performed with a normal urine pool
containing mean glycolate and glycerate concentrations of 28.5 and 80.6
µg/mg creatinine, respectively. At these concentrations, where
imprecision is likely to be high, CVs were 17% and 28.8% for
glycolate and glycerate determination, respectively. Peaks
corresponding to 5 µg/mg creatinine were readily detectable above the
baseline signal.
Analysis of patient specimens.
Normal ranges were
determined from random urine specimens obtained from healthy children
of laboratory employees and adults with no history of renal disease and
are reported as the actual range of observed values. Specimens from
infants <6 months of age were obtained from outwardly healthy children
visiting the outpatient clinic at St. Louis Children's Hospital.
Glycerate excretion in this healthy population was dependent on age
(Fig. 2
). Children <5 years of age (n = 19) displayed higher
excretion rates of glycerate (12–177 vs 19–115 µg/mg creatinine)
than older children and adults (n = 39). Normal glycolate
excretion was 14–72 µg/mg creatinine (n = 64). No gender
differences were apparent.
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Figure 2. Excretion of oxalate, glycerate, and glycolate in urine
from healthy individuals.
Urine specimens from healthy children and adults were subjected to the
extraction protocol and analyzed. Metabolite concentration (per
milligram creatinine) for each individual is plotted vs age.
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The new extraction protocol was applied to specimens from 16 PH
patients seen at the Mayo Clinic Division of Nephrology to assess the
ability of improved glycolate/glycerate extraction to discriminate
between PH I and PH II. Patients were all 5 years of age or older and
were classified by: (a) prior history of renal dysfunction,
(b) hyperexcretion of oxalate/glycerate or oxalate/glycolate
(determined by standard organic acid analysis at reference
laboratories), (c) liver AGT activity in liver biopsy
material (when performed), and (d) response to pyridoxine.
Pyridoxine (a cofactor for AGT but not GDH) augments existing AGT
activity in some patients, resulting in normalized oxalate excretion,
and is therefore a hallmark of PH I. We examined specimens from nine
individuals whose urine oxalate excretion was responsive to pyridoxine.
Consistent with clinical response, glycolate and glycerate
concentrations were within normal limits in this population (data not
shown). Five other patients were classified as PH I, three by history
of marked hyperoxaluria and glycolate hyperexcretion (DM, LF, AJ), one
as the result of AGT deficiency by liver biopsy (NB), and one other
(AM) on the basis of glycolate hyperexcretion and biopsy-proven AGT
deficiency in an affected sibling. Four of these five patient specimens
had increased glycolate (Table 2
). The initial specimen from AM displayed high-normal excretion
of glycolate (53 µg/mg creatinine), but a subsequent specimen did
show high glycolate concentration (78 µg/mg creatinine). Two PH II
individuals were classified by a history of oxalate/glycerate
hyperexcretion at a reference laboratory. Both patients had increases
in glycerate that were at least threefold above the upper limit of
normal with our new method. In summary, then, four of five PH I
patients unresponsive to pyridoxine and both PH II patients were
detected by THF extraction.
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Discussion
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Our results show that THF extraction of urine (as an adjunct to
routine organic acid analysis) significantly improves sensitivity for
polar compounds such as glycolate and glycerate and allows even normal
excretion to be quantified. The ability to detect normal concentrations
of these compounds may improve the utility of the test in
discriminating between normal and affected individuals. The efficiency
of extraction of glycolate and glycerate, though much greater with THF
than other standard solvents, is still incomplete, a fact that
decreases precision of analysis. Nevertheless, our data indicate that
precision and accuracy with THF extraction are sufficient to readily
distinguish PH from normality. Performed after ether extraction, THF
extraction requires no additional equipment and little additional
effort in laboratories already performing organic acid analysis.
The THF extraction strategy presented here compares favorably with
other methods involving direct (no extraction) determination
(29) or alternative extraction techniques (anion-exchange
chromatography) to detect glycolate (30). Reported normal
glycerate values, however, vary widely
(12)(31). These differences are possibly due
to the range of methods used for quantifying glycerate and their
standardization. Standardization is particularly difficult with GC-MS
assays. Glyceric acid is available commercially as a syrup with
significant water content or as its calcium salt, which is insoluble in
organic solvents and in the derivatizing agents used. Both forms must
be manipulated further to provide a free acid form suitable for
standardization. Differences due to standardization do not affect the
ratio of increased to normal concentrations of glycerate and would,
therefore, not diminish the ability of THF extraction to identify those
patients with increased excretion rates of glyceric acid.
Interpretation of results must be made with some qualification. First,
the technique described here does not distinguish between the
D and L forms of glyceric acid. Further
investigation is required to confirm excretion of the L
isomer, which is specific to PH II. Second, excretion of glycerate and
glycolate will likely vary considerably within individuals. For
instance, one sample in our study contained high-normal concentrations
of glycolate; a second sample was clearly increased. Finally,
progressive glomerular damage reduces filtration of glycerate and
glycolate, so interpretation must always take into account the extent
of residual renal function. With these considerations in mind, THF
extraction of glycerate and glycolate as an adjunct to routine organic
acid analysis should enable early detection and distinction of PH and
avoid the need for liver biopsies in many patients.
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Abstract
Introduction
