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1
Unitat de Metabolopaties, Hospital Materno-Infantil Vall d'Hebron, 08035 Barcelona, Spain.
2
Institut de Bioquímica Clínica, C/Mejia
Lequerica s/n, Edifici Helios III, Planta Baixa, Corporació
Sanitària, 08028 Barcelona, Spain.
3
Instituto de Biomedicina de Valencia (CSIC), C/Jaume
Roig 11, 46010 Valencia, Spain.
4
Dipartimento di Pediatria, Università di Padova,
Via Giustiniani 3, 35128 Padua, Italy.
a Address correspondence to this author at: Laboratori de Metabolopaties, Hospital Materno-Infantil Vall d'Hebron, Ps. Vall d'Hebron 119-129, 08035 Barcelona, Spain. Fax 34 93 2746837; e-mail msentis.hmi{at}cs.vhebron.es
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResults and DiscussionReferences |
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Methods: Urine orotate/orotidine responses to allopurinol load in 37 children (13 OCT-deficient and 24 non-OCT-deficient) and 24 women (7 at risk for carrier status and 17 not related to OCT-deficient children) were analyzed by liquid chromatography after sample purification by anion-exchange chromatography. Diagnostic accuracy was evaluated by nonparametric ROC curves.
Results: Sample purification was necessary to prevent interferences. Orotate and orotidine excretion increased with increased protein intake during the test. At a cutoff of 8 mmol orotidine/mol creatinine, sensitivity was 1.0 and specificity was 0.92 in mild forms of OCT deficiency. Results in monoplex carrier women may differ greatly from those expected because of the genetics of this deficiency.
Conclusions: Standardization of protein intake is required in the allopurinol loading test. A negative response in the face of clinical suspicion should be followed with a repeat test during a protein intake not <2.5 g · kg-1 · day-1. Measurements of orotidine provide better clinical sensitivity than measurements of orotate.© 1999 American Association for Clinical Chemistry
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResults and DiscussionReferences |
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Mild forms of OCT deficiency may present later, even during adulthood. Symptoms and signs may include coma, mental retardation, protein avoidance, headache, bizarre behavior, or episodic hyperammonemia. Heterozygous females may present diverse clinical manifestations, including postpartum hyperammonemia, depending on the pattern of X-chromosome inactivation (1). The mild forms of and carrier status for OCT deficiency are difficult to diagnose because the changes in the clinical and biochemical characteristics in patients are less marked or inconstant and because of the analytical profiles in asymptomatic female carriers are within the reference range. Enzymatic analysis is invasive and may present diagnostic difficulties because of mosaicism. In many cases, DNA analysis requires sequencing of the whole gene, because most mutations are "private". This procedure is costly and labor-intensive. Thus, the use of these analyses as diagnostic methods in suspected patients is not feasible in many cases.
In 1990, Hauser et al. (2) reported increased allopurinol-induced excretion of orotidine in the urine in female carriers, which allowed diagnosis. This observation demonstrated high diagnostic sensitivity and specificity in detecting carrier status in obligate heterozygotes. Others have used this test with orotate (3) or orotate and orotidine (4)(5) as variables. The increase in orotate and/or orotidine excretion in urine after allopurinol ingestion is caused by inhibition by oxypurinol (an allopurinol metabolite) of orotidine-5-phosphate decarboxylase, an enzyme of the pyrimidine synthesis pathway (1). A positive allopurinol test has also been reported in the hyperornithinemia, hyperammonemia, and homocitrullinuria syndrome (6). Patients with other urea cycle disorders, such as argininemia, citrullinemia, argininosuccinic aciduria, and lysinuric protein intolerance, as well as mitochondrial disorders (7), may also show a positive allopurinol test.
In addition to its use for detecting female heterozygotes, the application of the allopurinol test has been suggested as a means to ascertain diagnosis in OCT-deficient patients with mild or nonspecific clinical or biochemical presentation (8)(9). To clarify the best conditions for making these applications reliable, we studied several factors influencing the test results, such as protein intake, urine purification to eliminate analytical interferences, the determination of orotate or orotidine, and the use of cutoff values for interpretation.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResults and DiscussionReferences |
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Diagnostic criteria.
All patients had presented with
suggestive symptoms of OCT deficiency (ranging from mild to severe
headache, bizarre behavior, vomiting, lethargy, to coma) associated
with an episode of hyperammonemia and hyperglutaminemia. Argininemia;
citrullinemia; argininosuccinic aciduria; lysinuric protein intolerance
hyperornithinemia, hyperammonemia, and homocitrullinuria syndrome; and
mitochondrial disorders were ruled out. Patients who presented with
repeated hyperammonemic crises were diagnosed with OCT deficiency if
they showed, in addition, decreased plasma citrulline and increased
orotic and/or orotidine on at least one occasion. Further confirmation
of the diagnosis was obtained in some of the patients by the finding of
decreased OCT activity in the liver or intestinal mucosa, by the
finding of mutations in the OCT gene (performed only in few patients
and in none of the controls), or if a pedigree demonstrated X-linked
inheritance.
Children.
Thirty-seven children >1 year of age were
classified into two subgroups:
(a) the OCT-deficient subgroup (n = 13; patients 25–37), which included females, ages 3–8 years, whose diagnoses were established according to the criteria described above. All patients in this subgroup currently are receiving treatment with benzoate, low protein diet, and citrulline.
(b) the non-OCT-deficient subgroup (n = 24), which included 12 control children, ages 3–18 years (patients 1–12), who were tested during a visit to a regional health screening program. The remaining 12 children [patients 13–24 (ages 1–12 years); 3 males (patients 14, 16, and 23) and 9 females] were initially suspected of having OCT deficiency because of their clinical presentations. Clinical courses and laboratory tests ruled out this diagnosis.
Women.
Twenty-four women were classified into two subgroups:
(a) 17 not related to the OCT-deficient children. This
subgroup consisted of 11 (patients 38–48) healthy female volunteers
(age range, 25–45 years) and 6 women (patients 49–54) who were
relatives of patients (patients 13, 14, 15, and 17; see Table 3
) later
discarded as OCT-deficient.
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(b) seven women at risk for OCT carrier status who were
monoplex mothers (one OCT-deficient proband): one mother (patient 61)
of an OCT-deficient boy not included in this study who died from
hyperammonemic coma. This boy had been reported previously as carrying
the P225L mutation (10). The remaining six women (patients
55–60) were relatives of OCT-deficient girls (patients 30, 31, 34, and
37 and another girl not included in the study; see Tables 2
and 3
).
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methods
Allopurinol challenge.
Tested individuals were in good health
at the time of the challenge. After collection of the first morning
urine, the following amount of allopurinol was given in a single oral
dose: women, 300 mg; children 6–10 years, 200 mg; and children under 6
years, 100 mg. Urine was collected during four 6-h periods during the
following 24 h and stored frozen. The test was performed in women
7–12 days after the beginning of the menstrual cycle. Subjects were
requested not to drink alcoholic beverages or soft drinks because of
the presence of caffeine or benzoate. Female controls were requested to
ingest >2.5
g · kg-1 · day-1
of protein during the day of testing.
Analytical methods.
Orotate, orotidine, and creatinine were
measured after urine samples were heated to 70 °C to assure total
orotate solubility. Creatinine was measured by the Jaffé reaction
in a Synchron CX7 automated analyzer (Beckman). Orotate and orotidine
were analyzed by anion-exchange HPLC according to the method of
Brusilow and Hauser (11) with a Waters HPLC consisting of a
model 510 pump, a Rheodyne injector, a model 441 ultraviolet/visible
detector, and a NEC image 466es computer with the Millenium 2010
program. The sample was purified as described by Sebesta et al.
(4) with slight modifications as follows: 1 mL of urine,
adjusted to pH 8.0 with 1 mol/L NaOH, was applied to a 20
x 5 mm column of Dowex 1x8 200–400 mesh
(Cl-; Fluka). Elution was
performed in two stages with 5 mL of 0.01 mol/L HCl and 6 mL of 0.1
mol/L HCl. Orotate and orotidine were eluted in the second fraction.
After adjustment of the pH to 8.0 with 1 mol/L NaOH, the solution was
filtered through a 0.25 µm filter (LIDA) and 50 µL was
injected into the HPLC. Orotate and orotidine (0.5 nmol. Sigma) in
water were injected and used as calibrators.
For comparison and analysis, the maximum values (peaks) for orotate and orotidine were used regardless of the collection period in which they appeared.
Statistical methods.
Gaussian distribution was assessed
in control populations by normality tests [Kolmogorov–Smirnov
(Liliefors)]. When compliance with the latter tests was not achieved,
logarithmic transformation to attain normalization was attempted. The
Student t-test and Mann–Whitney U-test
(depending on compliance with appropriate parametric conditions) were
used to compare means. Diagnostic accuracy was evaluated for both
orotate and orotidine by use of nonparametric ROC curves. Areas under
curves were calculated for comparison (ROC analyzer program).
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Results and Discussion |
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TopAbstractIntroductionSubjects and MethodsResults and DiscussionReferences |
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. When the concentrations of orotate and orotidine were low, as
in controls, the identification and quantification of orotate/orotidine
became very difficult (Fig. 1
, left panel). The purification step
eliminated these interferences (Fig. 1
, right panel) and made
analytical chromatography reliable. Run time was reduced because no
additional washes or long elution times were required to clean the
column of interferences. This reduction offset the longer preparation
time. Other authors (11) did not need to purify urine;
therefore, we believe the interferences may be caused by a specific
diet. In fact, the degree of interferences varied in different subjects
(data not shown).
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The recovery and imprecision for orotate and orotidine determination
are estimated in Table 1
. The imprecision was similar to that reported by Sebesta et al.
(4).
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The peak orotate and orotidine concentrations for all groups are shown in Tables 2(2) and 3(3). Our control values are similar to those reported by other authors for women (2)(4) and for orotate in children of >1 year (3). The orotidine responses were higher than those of orotate in 27.4% of cases.
possible influence of protein intake on orotate and orotidine peak
concentrations
The results shown in Table 4
, obtained with samples from one control woman and three
children, suggest that protein intake influences intrachallenge
orotate/orotidine excretion. Basal values in women not related
to OCT-deficient children were reported to be below detection limits by
Brusilow and Hauser (11), probably because of a low-protein
diet. On the basis of our results, we would recommend a relatively high
protein intake during challenge (not <2.5 g/kg body weight) if a
first challenge yields negative results but suspicion remains. It would
be advisable to standardize protein intake to obtain greater and more
comparable responses.
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diagnostic accuracy
Detection of mild forms of OCT deficiency.
The ROC curves
obtained for orotate and orotidine peak concentrations are shown in
Fig. 2
. Visual analysis confirmed the excellent diagnostic value of
the two variables, although the graph indicates better results with
orotidine than with orotate (the areas below the curves are
significantly different; P = 0.02). When orotate and
orotidine information were used simultaneously (curve not shown), the
orotidine curve alone was practically reproduced.
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For maximum diagnostic sensitivity, the optimum cutoff value is estimated to be ~8 mmol orotidine/mol creatinine. For this cutoff value, the sensitivity was 1.0, the specificity was 0.92, the positive predictive value was 0.88, and the negative predictive value was 1.0. The overall classification accuracy (efficiency) of the test [(true positive + true negative)/total] was 0.95.
An analogous rationale applied to orotate offers the best discriminating value at a concentration of 12 mmol/mol creatinine. The sensitivity for this concentration was 0.8, the specificity was 0.92, the positive predictive value was 0.93, and the negative predictive value was 0.88. It is noteworthy that, according to these data, the specificity and predictive values are very similar between orotate and orotidine. Therefore, the parameter that truly increases orotidine efficacy over orotate is sensitivity (1.0 vs 0.8).
Hauser et al. (2) reported that the sensitivity and specificity of the allopurinol test are substantially lower with orotate than with orotidine, concluding that orotidine is the preferred metabolite. Our results concur with this finding in relation to sensitivity, although Hauser et al. applied the test to possible female carriers of the deficiency, whereas we applied the test to children susceptible to having the disease. It should be remembered that the aim of the test may influence the election of the discriminative value. The discriminative criterion used by Hauser et al. (mean + 3 SD of the orotidine values of the control adult female population) was 5.3 mmol/mol creatinine. In our ROC curve, this discriminator, albeit optimum in sensitivity, was far from being the best in specificity.
On the other hand, Burlina et al. (3) proposed using as
discriminator the mean + 3 SD of orotate values in a control
population. In our sample, this cutoff was 10.2 mmol orotate/mol
creatinine, which also failed to provide optimum sensitivity and
specificity (Fig. 2
).
A different approach is that used by Sebesta et al. (4),
whose discriminator was the mean + 2 SD after logarithmic
transformation of control orotate and/or orotidine values. Although
this method appears a priori to pool the advantages of both analytes,
our results show that in practice, the ROC curve obtained was nearly
identical to that obtained with orotidine. The cutoff value for this
combined-parameter approach in our population would be 5.8 mmol
orotidine/mol creatinine and 8.2 mmol orotate/mol creatinine. It seems
clear from Fig. 2
that there is room to correct the false-positive
rate, although the true-positive rate is well adjusted.
The positive/negative responses of the patients studied, using 8.0 mmol
orotidine/mol creatinine as the cutoff, are summarized in Table 2
.
Two positive responses (patients 16 and 23) were false positives. Patient 16 later suffered hypertropic cardiopathy, which is suggestive of a mitochondrial disorder because he resembles the cases described by Bonham et al. (7). Patient 23 was the healthy brother of a girl (patient 15) who was initially suspected of having OCT deficiency, but who did not present with new clinical episodes, giving negatives values in the laboratory assays, including the present test. Patient 23 himself had no clinical or biochemical anomalies.
Detection of carrier status for OCT deficiency in women.
No
significant differences were observed for either orotate or orotidine
between responses of risk mothers and women not related to
OCT-deficient children. Thus, the method used in the child population
to seek an appropriate cutoff cannot be extrapolated to women.
If we used the cutoff criteria of Hauser et al. (2) or of
Sebesta et al. (4), we obtained values of 6.7 mmol
orotidine/mol creatinine in the first case and 13.2 mmol orotate/mol
creatinine and 5.9 mmol orotidine/mol creatinine in the second. With
these values, only one positive response (patient 61) was obtained, and
the remaining six were negative in the group of monoplex women at risk
for carrier status (Table 3
). The mother testing positive according to
these criteria may be a carrier having a favorable lyonization pattern
because she has never presented clinical symptoms.
These results conform with the proportion of spontaneous mutations reported by Tuchman et al. (12): the a priori risk of carrier status in a mother of an affected male is 90% or higher, whereas in a mother of an affected female, it is ~20%. Thus, genetics differs according to sex: affected girls have a high de novo mutation rate, whereas the great majority of affected boys receive the mutation from their mothers. Other causes may account for negative responses in allopurinol challenge, such as gonadal mosaicism, high residual enzymatic activity, or insufficient protein intake during the test to produce accumulation of enough carbamoyl phosphate for increased orotate and orotidine excretion. Tuchman (13) points out that carriers of milder mutations will probably present a high proportion of false-negative results in the allopurinol test.
Our results do not provide direct information on the value of the test in carrier monoplex females, although they agree with the expected rate of heterozygotes among the mothers of female patients (13). A greater number of monoplex females would need to be tested and confirmed by molecular analysis to validate the results of this test, and only the positive results would be useful.
With respect to analytical aspects, we found purification of the sample before chromatography to be necessary to prevent interferences.
Given the relationship between protein intake and orotate and/or orotidine excretion that we observed, we consider it necessary to standardize protein intake in controls and patients on the day of the test. Whenever negative response to the test is obtained and clinical suspicion persists, we recommend repeating the test under strict medical supervision with higher protein intake on the test day.
The discriminator with optimum diagnostic performance in children was 8 mmol orotidine/mol creatinine. With this value, the performance parameters were very favorable: the sensitivity was 1.0, the specificity was 0.92, and the positive and negative predictive values were 0.88 and 1.0, respectively. We believe that a confirmatory test (DNA/enzyme analyses) is required in borderline responses to establish or rule out OCT deficiency.
Interpretation of results in monoplex mothers is hindered by the sex-dependent genetic behavior of the deficiency, and our results were not sufficiently conclusive to demonstrate the reliability of the test. In any case, given the simplicity and noninvasive nature of the test, its application to potential carriers would appear appropriate.
In conclusion, the allopurinol challenge test is a very reliable tool for detecting OCT deficiency in the absence of a more rapid, simple, and cost-effective DNA-based diagnostic method.
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Acknowledgments |
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References |
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TopAbstractIntroductionSubjects and MethodsResults and DiscussionReferences |
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The following articles in journals at HighWire Press have cited this article:
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