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Optimization of allopurinol challenge: sample purification, protein intake control, and the use of orotidine response as a discriminative variable improve performance of the test for diagnosing ornithine carbamoyltransferase deficiency.

Introduction

X-linked ornithine carbamoyltransferase (OCT; EC 2.1.3.3) deficiency (McKusick 31125) causes severe disease in male hemizygote neonates, and neonates with this deficiency present with lethargy, vomiting, coma, and hyperammonemia. Diagnosis is established by the finding of increased plasma ammonia and glutamine, decreased plasma citrulline, and increased urine orotate excretion. The demonstration of low OCT activity in the liver or small intestine and the detection of alterations in the OCT gene are confirmatory of the diagnosis.

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.

Subjects and Methods

subjects

Analytical procedures were in accordance with recommendations of the respective local ethics committees. Informed consent for the test was obtained. Two age groups were studied: children >1 year and women.

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.

(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).

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 x [kg.sup.-1] x [day.sup.-1] of protein during the day of testing.

Analytical methods.

Orotate, orotidine, and creatinine were measured after urine samples were heated to 70[degrees]C to assure total orotate solubility. Creatinine was measured by the Jaffe 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 [micro]m filter (LIDA) and 50 [micro]L 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).

Results and Discussion

analytical method evaluation

The presence of interfering ultraviolet-absorbing substances in untreated urine is illustrated in Fig. 1. 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).

[FIGURE 1 OMITTED]

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).

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.

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.

[FIGURE 2 OMITTED]

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.

Acknowledgments

The DNA analysis of patient 13 was kindly performed by Dr. M. Tuchman, Department of Pediatrics, University of Minnesota Hospitals, Minneapolis, MN. We thank the clinicians who took care of the patients: Drs. A. Baldellou, G. Pintos, L. Callis, F. Nigro, J. Vaquerizo, and L. Monreal. We thank C. Bahima, M.V. Herrero, and M. Murillo for skillful technical assistance. We especially thank Dr. C. Bachmann for valuable criticisms.

References

(1.) Brusilow SW, Horwich AL. Urea cycle enzymes. Scriver CR Beaudet AL Sly WS Valle D eds. The metabolic and molecular bases of inherited disease 7th ed. 1995:1187-1232 McGraw-Hill New York.

(2.) Hauser ER, Finkelstein JE, Valle D, Brusilow SW. Allopurinol-induced orotidinuria. A test for mutations at the ornithine carbamoyltransferase locus in women. N Engl J Med 1990;322:1641-1645. [Abstract]

(3.) Burlina AB, Ferrari V, Dionisi-Vici C, Bordugo A, Zachello F, Tuchman M. Allopurinol challenge test in children. J Inherit Metab Dis 1992;15:707-712. [Web of Science][Medline] [Order article via Infotrieve]

(4.) Sebesta I, Fairbanks LD, Davies PM, Simmonds JA, Leonard JV. The allopurinol loading test for identification of carriers of ornithine carbamoyl transferase deficiency: studies in a healthy control population and females at risk. Clin Chim Acta 1994;224:45-54. [Web of Science][Medline] [Order article via Infotrieve]

(5.) Sebesta I, Krijt J, Fairbanks LD, Simmonds HA. The allopurinol loading test in detecting obligate heterozygotes for OCT deficiency. J Inherit Metab Dis 1994;17:133-134. [Web of Science][Medline] [Order article via Infotrieve]

(6.) Tuchman M, Knopman DS, Shih VE. Episodic hyperammonemia in adult siblings with hyperornithinemia, hyperammonemia and homocitrullinuria syndrome. Arch Neurol 1990;47:1134-1137. [Abstract/Free Full Text]

(7.) Bonham JR, Downing M, Guthrie P, Olpin SE, Manning NJ, Pollit RJ, et al. Mitochondrial disorders may cause an increased excretion of orotic acid and a positive response to allopurinol loading [Abstract]. J Inherit Metab Dis 1997;20(Suppl 1):61 (P5.13)..

(8.) Spada M, Guardamagna O, Rabier D, van der Meer SB, Parvy P, Bardet J, et al. Recurrent episodes of bizarre behavior in a boy with ornithine transcarbamylase deficiency: diagnostic failure of protein loading and allopurinol challenge tests. J Pediatr 1994;125:249-251. [Web of Science][Medline] [Order article via Infotrieve]

(9.) Ahrens MJ, Berry SA, Whitley CB, Markowitz DJ, Plante RJ, Tuchman M. Clinical and biochemical heterogeneity in females of a large pedigree with ornithine transcarbamylase deficiency due to the R141Q mutation. Am J Med Genet 1996;66:311-315. [Web of Science][Medline] [Order article via Infotrieve]

(10.) Garcia-Perez MA, Climent C, Briones P, Vilaseca MA, Rodes M, Rubio V. Missense mutations in codon 225 of ornithine transcarbamylase (OTC) result in decreased amounts of OTC protein: a hypothesis on the molecular mechanism of the OTC deficiency. J Inherit Metab Dis 1997;20:769-777. [Web of Science][Medline] [Order article via Infotrieve]

(11.) Brusilow SW, Hauser E. Simple method of measurement of orotate and orotidine in urine. J Chromatogr 1989;493:388-391. [Web of Science][Medline] [Order article via Infotrieve]

(12.) Tuchman M, Matsuda I, Munnich A, Malcom S, Strantnieks S, de Brie T. Proportions of spontaneous mutations in males and females with ornithine transcarbamylase deficiency. Am J Med Genet 1995;55:67-70. [Web of Science][Medline] [Order article via Infotrieve]

(13.) Tuchman M. Inherited hyperammonemia. Chapter 12. Blau N Duran M Blaskovics ME eds. Physician's guide to the laboratory diagnosis of metabolic disease 1996:209-222 Chapman & Hall London.

Jose A. Arranz [1,a], Encarnacio Riudor [1], Margarita Rodes [2], Manuel Roig [1], Consuelo Climent [3], Vicente Rubio [3], Margarita Sentis [1] and Alberto Burlina [4]

[1] Unitat de Metabolopaties, Hospital Materno-Infantil Vall d'Hebron, 08035 Barcelona, Spain.

[2] Institut de Bioquimica Clinica, C/Mejia Lequerica s/n, Edifici Helios III, Planta Baixa, Corporacio Sanitaria, 08028 Barcelona, Spain.

[3] Instituto de Biomedicina de Valencia (CSIC), C/Jaume Roig 11, 46010 Valencia, Spain.

[4] Dipartimento di Pediatria, Universita 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@cs.vhebron.es.
Table 1. Analytical method performance.

 Orotate Orotidine

Recovery, %

From urine (mean [+ or -]
SD, n = 4 100.2 [+ or -] 6.8 106.0 [+ or -] 10.4

 Concentration (1) CV, % Concentration (1) CV, %

Imprecision
Within-run 4.6 [+ or -] 0.5 12 2.1 [+ or -] 0.3 14
 (n = 10) 58.6 [+ or -] 6.2 11 63.8 [+ or -] 6.4 10
Between-run (15 3.8 [+ or -] 0.3 9.7 1.4 [+ or -] 0.1 12
 days, five 61.6 [+ or -] 3.8 7 28.2 [+ or -] 2.8 11
 replications
 each day)

(1) Mean [+ or -] SD, mmol/mol creatinine.

Table 2. Peak values of orotate and orotidine (mmol/mol creatinine)
after allopurinol load in children.

Non-OCT-deficient children

Patient Orotate Orotidine Sex

1 5.4 2.3
2 6.6 1.1
3 5.7 2.1
4 6.5 2.5
5 3.1 0.9
6 1.9 1.9
7 0.2 1.7
8 2.1 1.0
9 4.5 4.8
10 4.9 0.2
11 2.6 2.2
12 1.2 1.0
13 9.3 5.4 Female
14 10.7 5.9 Male
15 3.3 3.6 Female
16 24.0 11.9 (1) Male
17 6.0 4.8 Female
18 14.6 7.6 Female
19 2.0 2.8 Female
20 5.8 4.9 Female
21 4.6 3.0 Female
22 0.9 4.2 Female
23 10.3 9.0 (1) Male
24 2.9 6.0 Female

Mean 9.9 8.1
Median 5.9 6.0
SD 7.1 4.6
Range 0.2-24.0 0.2-11.9

OCT-deficient children

Patient Orotate Orotidine Sex

25 4.2 13.1 (1) Female
26 15.9 10.1 (1) Female
27 22.8 12.8 (1) Female
28 4.3 9.9 (1) Female
29 66.6 103.4 (1) Female
30 22.2 16.2 (1) Female
31 236.0 27.0 (1) Female
32 1670.0 35.3 (1) Female
33 36.9 35.3 (1) Female
34 55.9 13.4 (1) Female
35 17.0 17.2 (1) Female
36 62.6 30.7 (1) Female
37 21.3 10.0 (1) Female

Mean 253.2 29.6
Median 46.4 16.7
SD 540.0 29.0
Range 4.2-1670.0 9.9-103.4

(1) Positive for criterion >8 mmol orotidine/mol creatinine.

Table 3. Peak values of orotate and orotidine (mmol/mol creatinine)
after allopurinol load in women.

Women not related to OCT-deficient children

Patient Orotate Orotidine

38 4.7 6.1
39 4.3 2.4
40 3.8 2.7
41 2.1 2.4
42 10.2 2.2
43 3.2 2.0
44 1.2 1.8
44 1.2 1.8
45 4.0 2.0
46 9.8 4.3
47 3.5 2.7
48 4.0 1.7
49 6.0 1.0 (Mother of patient 14)
50 3.6 4.6 (Grandmother of
 patient 14)
51 4.7 2.3 (Mother of patient 13)
52 11.1 3.9 (Sister of patient 13)
53 6.2 2.5 (Mother of patient 17)
54 6.1 2.9 (Mother of patient 15)
Mean 5.2 2.8
Median 4.3 2.4
SD 2.8 1.3
Range 1.2- 1.0-
 11.1 6.1

Women related to OCT-deficient children

Patient Orotate Orotidine
55 2.0 2.2 (Mother of patient 30)
56 3.0 2.3 (Grandmother of patient 30)
57 10.2 4.6 (Mother of patient 31)
58 6.3 2.9 (Mother of patient 34)
59 2.8 4.0 (Mother of patient 37)
60 9.4 4.2 (Mother of OCT-deficient
 girl not included in study)
61 4.6 8.7 (1) (Mother of OCT-deficient
 boy not included in study)
Mean 5.2 (2) 4.1 (3)
Median 4.6 4.0
SD 3.3 2.2
Range 2.0- 2.2-8.7
 10.2

(1) Positive according to cutoff criteria of Hauser et al. (2)
(> [bar.x] + 3 SD), with a value of 6.7 mmol orotidine/mol
creatinine, and the cutoff criteria of Sebesta et al. (4)
(> [bar.x] + 2 SD for orotate + orotidine after log-transformation),
with values of 13.2 mmol orotate/mol creatinine and 5.9 mmol
orotidine/mol creatinine.

(2) Not significant vs orotate for women not related to
OCT-deficient children.

(3) Not significant vs orotidine for women not related
to OCT-deficient children.

Table 4. Response of orotate/orotidine excretion, depending
on protein intake during test.

Subject Orotate (1) Orotidine (1)

ER (patient 48) 1.8 Traces
 4.0 1.7
LZ (patient 13) 5.3 4.9
 9.3 5.4
AJ (2) 17.3 20.9
 33.7 26.8
PT (patient 16) 8.7 7.4
 24.0 11.9

Subject Age Protein intake, g x
 [kg.sup.-1] x [day.sup.-1]

ER (patient 48) 45 years 1.3
 3.0
LZ (patient 13) 9 years <2.0
 3.0
AJ (2) 7 months <2.0
 9 months 3.0
PT (patient 16) 4 years <2.0
 3.0

(1) Peak value (mmol/mol creatinine).

(2) Not included in the statistical calculations.
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Author:Arranz, Jose A.; Riudor, Encarnacio; Rodes, Margarita; Roig, Manuel; Climent, Consuelo; Rubio, Vicen
Publication:Clinical Chemistry
Article Type:Clinical report
Date:Jul 1, 1999
Words:4338
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