RESULTS

Under the standardized conditions of the absorption test, there was no statistically significant difference in urinary oxalate excretion between children with CaOx urolithiasis and healthy controls (0.4950.170 vs 0.4750.144 mmol per 1.73 m² per 24 h). Obviously, mean urinary oxalate excretion was highest in PH patients (2.8861.997 mmol per 1.73 m² per 24 h; Table 1). The median intestinal oxalate absorption of 15.3% in children with urolithiasis was significantly higher than that in controls (10.4% ) and in PH patients (7% ) (P<0.05 and P<0.001, respectively, Figure 1). The absorption values in the latter group were also significantly lower in comparison to the control group (P<0.05). When analyzing boys and girls with CaOx urolithiasis separately, both groups clearly had higher median intestinal oxalate absorption than the controls: 17 vs 10.6% for boys and 14.1 vs 9.6% for girls, respectively. However, only in boys, the difference was statistically significant (P<0.05). The results of the intestinal oxalate absorption in all groups are shown in Table 2. The receiver operator characteristic (ROC) analysis revealed that intestinal oxalate absorption had an area under the curve of 0.656 for diagnosis of children with CaOx urolithiasis. This result expresses a reasonable accuracy but a less reasonable sensitivity and specificity in setting a diagnostic cutoff value for oxalate absorption. In other words, every possible cutoff does not ideally discriminate the diseased children from the disease-free children. The ROC analysis for the intestinal oxalate absorption with several cutoff points is shown in Figure 2. Therefore and as a compromise, we decided to assume intestinal oxalate absorption above the 90th percentile (18.6% ) as abnormal. Above this value, the true-positive rate amounts to 0.383 and the false-positive rate to 0.114. Accordingly, increased intestinal oxalate absorption was detected in 23 (38.3% ) patients with CaOx urolithiasis and in none of the patients with PH. A statistically significant correlation between intestinal oxalate absorption and urinary oxalate excretion was found in the group of patients with CaOx urolithiasis (r=0.572, P<0.05; Figure 3). Both parameters did not significantly correlate in the control group (r=0.282).

Figure 1.

Comparison between pediatric patients with calcium oxalate urolithiasis (CaOxU) and primary hyperoxaluria (PH) and healthy controls in reference to intestinal oxalate absorption. ^*significance of P-value CaOxU vs controls; P<0.05.

Full figure and legend (47K)

Figure 2.

ROC curve for intestinal oxalate absorption (% ) determined from the results obtained in pediatric patients with CaOx urolithiasis and healthy controls. Area under the curve: 0.656.

Full figure and legend (45K)

Figure 3.

Correlation between intestinal oxalate absorption and oxalate excretion in 24-h urines in pediatric patients with CaOx urolithiasis. ^*significance of P<0.05 for correlation between intestinal oxalate absorption and urinary oxalate excretion.

Full figure and legend (61K)

Table 1 - Urinary oxalate excretion (mmol per 1.73 m² per 24 h) in pediatric patients with CaOx urolithiasis, PH, and in healthy controls under standardized dietary conditions on the second day of the oxalate absorption test.

Full table

Table 2 - Intestinal oxalate absorption (% ) in pediatric patients with CaOx urolithiasis, PH and in healthy controls.

Full table

DISCUSSION

An etiological evaluation of children with urolithiasis poses a particular challenge. In contrast to adults, an early onset of the disease is more often associated with different underlying metabolic conditions. To prevent recurrences of stone episodes and to avoid severe complications, the underlying cause of stone disease in childhood should be detected as soon as possible and hence treated adequately. At present, CaOx-containing stones are the most frequent type of urolithiasis in children at least in western countries. A routine metabolic evaluation of such cases includes the urinary excretion of promoters (calcium, oxalate, uric acid) and inhibitors (citrate, magnesium) of crystallization. Although idiopathic hypercalciuria is supposedly diagnosed more often than hyperoxaluria,¹ the latter may play a more crucial role in the pathogenesis of CaOx urolithiasis.^{14, 15} This thesis is based on the fact that in normal urine more than 10 calcium ions are available for each oxalate ion and both ingredients crystallize in a relationship of 1:1. Therefore, even small increases in urinary oxalate excretion accelerate CaOx crystalline mass production.¹⁶

It is well known that elevation of urinary oxalate excretion in most cases of secondary hyperoxaluria is 'mild' (0.5–1.0 mmol per 1.73 m² per 24 h), that is, does not reach a level found in the PHs (>1.0 mmol per 1.73 m² per 24 h). It is caused by higher intestinal absorption of oxalic acid because of either an excessive dietary intake or enteric hyperabsorption per se.^{2, 17} Evaluation of dietary oxalate intake is rather simple, whereas assessment of an effect of intestinal oxalate absorption on its urinary excretion causes methodological problems. In the past, several methods were used for this purpose but due to their inconveniencies, that is, the excessive sodium oxalate load or the ingestion of radioactive [ ¹⁴C] oxalate, they did not find way to common clinical practice.^{8, 9, 10, 18} The introduction of the safe and simple [ ¹³C₂] oxalate test now allowed studies comprising larger groups of adult patients with stone disease as well as healthy subjects.^{11, 12, 13, 19, 20, 21, 22} One of these studies showed that the median intestinal oxalate absorption was significantly higher in patients with CaOx urolithiasis when compared with healthy volunteers (9.45 vs 6.85% ).¹³ This is in agreement with the results of our study, but the median intestinal oxalate absorption in children and adolescents was considerably higher than that in adults with 15.3% for patients vs 10.5% for healthy controls. Similar to adult data, when analyzing boys and girls separately, both groups had significantly higher absorption rates than controls.¹³ Interestingly, the boys with urolithiasis showed the highest median intestinal oxalate absorption (17% ). A tendency to higher intestinal oxalate absorption in male patients was also found in adults.^{12, 13} It is well known that males of all age groups with or without urolithiasis excrete significantly higher amounts of oxalate in urine than females.^{23, 24, 25} Therefore, the higher intestinal oxalate absorption in males may partly explain this observation.

Although the pathogenesis of PH is originally associated with endogenous overproduction of oxalic acid, there is dispute about the necessity of a specifically low-oxalate diet in this condition.²⁶ From a theoretical point of view, such recommendation could keep the contribution of the exogenous oxalate source to the total urinary oxalate amount at the lowest possible level. However, our results showed that the median intestinal oxalate absorption in this group of patients is low (7% ) and even significantly lower than that in healthy controls. We speculate that the lower absorption could be caused by a downregulation of absorption or a change of shift of intestinal oxalate transporters such as SLC26A6 in case of an extremely high body oxalate burden.²⁷ Hence, oxalate would much more be intestinally secreted, than absorbed.

The reference values of intestinal oxalate absorption in healthy adults are still a matter of debate. The studies performed with [ ¹⁴C] oxalate defined the upper normal limit of oxalate absorption at 10% . However, owing to small number (maximum 20) of volunteers, this value may be questionable.^{8, 9} Recently, the results of the [ ¹³C₂] oxalate absorption test performed in 120 healthy subjects showed that the interindividual and intraindividual variance of intestinal oxalate absorption was high (3.391.68% ) and the reference range in 95% of the individuals was 2.2–18.5% . Therefore, the authors suggested that values higher than 15% are an indicator for increased intestinal oxalate absorption.²⁰ The ROC curve for intestinal oxalate absorption in our study showed that every possible cutoff does not ideally discriminate the diseased children from the disease-free children. This situation was caused by the significant overlap of oxalate absorption values in both groups, which is reflected in a relatively small area under the ROC curve (0.656). For comparison, an area of 1 represents a perfectly discriminating diagnostic test and an area under 0.5 expresses a worthless test. The results of the ROC analysis in our study are not surprising because the pathogenesis of CaOx stone disease is obviously multifactorial. Therefore, the increased intestinal oxalate absorption seems to be a contributing factor only in a part of children with urolithiasis. It is also possible that a larger group of healthy subjects could improve the results of the ROC analysis. Unfortunately, we were only able to perform the [ ¹³C₂] oxalate absorption test in 35 healthy children. Performing a 2-day test under dietary restrictions and other limitations in healthy children is extremely difficult and prone to compliance problems. Therefore and as a significant increase of numbers was not anticipated, as a compromise, we decided to assume values of oxalate intestinal absorption above 90th percentile (18.6% ) in our controls as abnormal. For this cutoff point, the true-positive rate amounts to 0.383 and false-positive rate to 0.114. Accordingly, 38.3% of children with CaOx urolithiasis showed increased intestinal oxalate absorption. This confirmed our observation that intestinal oxalate absorption in children may be physiologically higher than that of adults. In adult stone formers who underwent the same test, the percentage of hyperabsorbers was clearly lower with 20% .¹³ In the contrary, none of the PH patients showed an increase in oxalate absorption.

Only in children with urolithiasis, we found a significant correlation between the daily urinary oxalate excretion and the intestinal oxalate absorption. The same relationship was found in adult patients with urolithiasis and in much lesser degree in healthy adult volunteers.^{12, 13} However, it should be emphasized that during the standardized conditions of the test, the exogenous oxalate load is low, which has even less influence on urinary oxalate excretion in hyperabsorptive conditions. Therefore, the correlation between oxalate absorption and urinary oxalate excretion in patients was not more pronounced (r=0.572) and was absent in healthy children. This could also explain the only slightly higher 24-h urinary oxalate excretion during the test in the group of patients compared to that in the controls. In other words, under normal conditions, in individuals with a high risk of hyperabsorption, an even small increase in oxalate intake could result in dramatic consequences.

We can only speculate about possible mechanisms of intestinal oxalate hyperabsorption in our large group of pediatric CaOx stone formers. First, the process of oxalate absorption might be genetically determined and might therefore be characteristic for each individual.²⁸ Presently, mutations in the SLC26 gene family are studied as a potential cause of a disturbed oxalate transport in the small intestine.²⁹ The second possibility might be an influence of intestinal oxalate-degrading organisms, particularly Oxalobacter formigenes, on the intestinal oxalate absorption. It was found that 65–80% of healthy adults and up to 90% of children aged 3–10 years are colonized with Oxalobacter.^{30, 31} Hence, several studies showed a correlation between hyperoxaluria and the absence of intestinal colonization with O. formigenes.^{32, 33, 34} Unfortunately, we were not able to assess the colonization rate with O. formigenes in all our studied patients, but the patients with PH tested were all negative for Oxalobacter.

The identification of oxalate hyperabsorbers among patients with CaOx urolithiasis has several implications regarding their management. In this group of patients, evaluation of dietary habits to avoid foods rich in oxalate and to assess the calcium and magnesium content of food items is clearly helpful. It was shown that adequate calcium and magnesium intake may decrease intestinal oxalate absorption effectively.²¹ Our results confirmed the opinion that patients with PH would not benefit from extremely low-oxalate diets. However, they should avoid food with very high oxalate contents (for example, spinach, rhubarb, beet root, and ice tea).

In addition, the treatment with O. formigenes in case of its absence in patients with secondary hyperoxaluria as well as PH could be a potential new therapeutic tool.³⁵

CONCLUSION

Intestinal hyperabsorption of oxalate is an important lithogenic risk factor in children with CaOx urolithiasis. It was found in one-third of our patients with stone disease. Its mechanism is not yet clear but may either be due to mutations of intestinal oxalate transporter genes or due to a lack of intestinal oxalate-degrading bacteria. Oxalate absorption in children is clearly higher than that in adults, both in stone patients and in healthy subjects. However, a larger cohort of healthy children and adolescents would have to be tested to establish more reliable reference values. At present, we regard values above 18.6% as intestinal oxalate hyperabsorption in children. In contrast, patients with PH show very low intestinal oxalate absorption rates. The identification of patients with high oxalate absorption allows the introduction of a causative therapy (for example, low-oxalate diet) to prevent stone recurrences.

MATERIALS AND METHODS

The study comprised 60 children and adolescents (33 boys and 27 girls) aged 4.3–18 years (mean: 13.34.1 years) with recurrent idiopathic urolithiasis and 13 patients (8 boys and 5 girls) aged 3.5–16.4 years (mean: 10.23.9 years) with PH. Stone analysis was performed by infrared spectroscopy in 36 (60% ) of children with CaOx urolithiasis and showed CaOx (whewellite, weddellite, pure or mixed) in all cases. In the remaining children, an assessment of stone composition was impossible due to their stone loss after lithotripsy (extracorporeal shock wave lithotripsy) or an unnoticed spontaneous passage. However, CaOx urolithiasis was suspected because all calculi were radiopaque, other possible types of stones (infection-related or cystine stones) were clinically and metabolically excluded, and more than 75% of kidney stones overall are composed of CaOx. During the study, all patients had no urinary tract infection and did not receive antibiotic treatment, but remained on their normal medication such as alkaline citrate or pyridoxine in patients with PH. No patient had received antibiotic treatment for more than 3 months before the study. The patients had no history of inflammatory bowel diseases, nor did they have bowel surgery. All patients with idiopathic CaOx urolithiasis had normal renal function expressed as normal serum creatinine or cystatin C levels, or calculated glomerular filtration rate according to Schwartz.³⁶ The renal function in patients with PH was normal (>80 ml min^{- 1}) in 11, but decreased in 2 patients (>50 to <80 ml min^{- 1}).

A group of 35 healthy children (23 boys and 12 girls) aged 5.7–16.8 years (mean 11.13.6 years) without a history of urolithiasis or nephrocalcinosis, with normal kidney function, no bowel diseases, and without antibiotic treatment for the last months served as controls.

The [ ¹³C₂] oxalate absorption test was performed according to its description for adults but with minor modifications for younger children.¹¹ The test was carried out over a period of 2 consecutive days under standardized dietary conditions: 2400 ml per 1.73 m² body surface area of fluid was distributed evenly over the day. The diet supplied approximately 40 kcal per kg body weight per day, and the calcium and oxalate intake was 800 and 57 mg day^{- 1}, respectively. The first test day was designed to achieve a steady state (stable concentration of urinary solutes) for the purpose of an adequate calibration procedure. The next day was the main test period when a capsule with either 25 mg (body weight 30 kg) or 50 mg (body weight >30 kg) of the disodium salt of [ ¹³C₂] oxalate (Promochem, Wesel, Germany) was orally administered. On both study days, a 24-h urine was collected into five storage bottles containing 25% HCl as the preservative. The timed scheme of urine collection during the test is given in Table 3. After the test, aliquots of 0.1 ml urine from every study period were taken and stored at - 20 °C. The absorption measurement was subsequently performed as previously described in detail.¹¹ In brief, organic acids were extracted from the aliquots and derivatized with N-methyl-tert. -butyldimethylsilyltrifluoroacetamide. Samples were then measured by gas chromatography/mass spectrometry. The selected ion modes at m/z 261.3, 263.3, and 274.3 were used to quantify the unlabeled oxalic acid, labeled oxalic acid, and the internal standard [ ^{2- 13}C] malonic acid, respectively. Oxalate absorption was calculated from the amount of [ ¹³C₂] oxalate in the 24-h urine and expressed as percentage of the dose of [ ¹³C₂] oxalate ingested via the capsule.

Table 3 - Time and urine collection scheme during the [ ¹³C₂] oxalate absorption test.

Full table

The statistical analysis was performed by the software Statistica (StatSoft Inc., Tulsa, OK, USA) for Windows, version 7.1. The specific differences between healthy controls and patients were calculated by the non-parametric Mann–Whitney U-test for two groups and by the Kruskal–Wallis non-parametric analysis of variance for three groups. The correlations were tested with the Spearman test. Additionally, ROC analysis was used to evaluate the accuracy of the [ ¹³C₂] oxalate absorption test in separating the tested groups.³⁷ A P-value less than 0.05 was considered statistically significant.

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Acknowledgments

The research was supported by the Polish State Committee for Scientific Research KBN Grant 2P05D 117 26. We thank Mrs M Klöckner for her great technical assistance and especially all children for their cooperation during the test.