Analysis of 24-Hour Urine Parameters As It Relates to Age of Onset of Cystine Stone Formation

Introduction

C ystinuria is an autosomal recessive disorder characterized by the impaired reabsorption of the dibasic amino acid, cystine, in the proximal tubule of the nephron. This results in increased excretion of cystine in the urine. The cystine crystals precipitate because of the low solubility of cystine at normal urinary pH values, making patients susceptible to kidney stones. ¹ Cystinuria affects 6% to 8% of the pediatric patients with stone disease ² and accounts for 1% to 2% of adult renal stones in large series of stone analysis. ³ The natural history of the disease is plagued by multiple stone recurrences, surgical interventions, and at worst the loss of renal function. ⁴ The first presentation of cystine stones frequently occurs in the first through third decades of life, with a decreased incidence in old age. We sought to determine whether there were differences in 24-hour urine parameters between those patients who present with their first cystine stone early in age as opposed to patients who present with cystine stones later in life.

Patients

Data for this retrospective analysis were obtained from Litholink Corporation, a clinical laboratory specializing in the measurement of urine chemistries for patients with kidney stones. As part of the company's disease management program, a medical history is obtained from patients during a telephone interview with a patient care representative. Included in the medical history questions are use of medications for stone prevention and the date of the patient's first diagnosis with kidney stones. Data were queried from the Litholink database and stripped of patient identifiers. Urine samples used for analysis were obtained for 94 patients between August 2003 and April 2009. An institutional review board (Western IRB, Olympia, WA) classified the protocol as exempt.

Only a patient's initial urine collections performed at Litholink were used for this analysis. Subsequent urine collections for an individual were assumed to be altered by treatment and, therefore, not included in the analysis. If a subject had two urine collections performed for their initial evaluation, the results were averaged so each subject is represented only once in the analysis. Of the 94 patients, 17 had two urines performed as part of the initial evaluation. Patients were excluded from analysis if they reported being treated with alkali or thiol-binding drugs such as tiopronin, d-penicillamine, or captopril.

Laboratory analysis for cystinuria patients has been described in detail elsewhere. ⁵ Briefly, urine samples were collected at room temperature with an antimicrobial preservative added to the urine container to prevent bacterial overgrowth. An aliquot was poured off for the measurement of urine creatinine, pH, and supersaturation (SS), and then the remainder of the urine was alkalinized by adding sodium carbonate to ensure that all cystine has been solubilized. An aliquot of the alkalinized urine was collected for the measurement of urine cystine. Urine cystine was measured using a nitroprusside colorimetric assay. ⁵ Creatinine was measured by alkaline picrate reaction on a Beckman autoanalyzer and urine pH was measured with a pH electrode.

Cystine SS was measured by adding 20 mg of cystine crystals to 25 mL of urine, maintained at the original urine pH. ⁶ The urine was incubated for 48 hours at 37°C with constant stirring. At the end of 48 hours, the urine was centrifuged at 3800 rpm for 20 minutes at room temperature to separate the solid-phase cystine, and the supernatant was removed. Cystine concentration of the supernatant was measured, which is the solubility of cystine in that particular urine sample. The cystine SS is calculated as the original cystine concentration of the urine divided by the solubility cystine concentration. A value of 1 means the urine is at the saturation point, a value of >1 means the urine is supersaturated, and a value of <1 means the urine is undersaturated.

Results

There were 94 cystinuric patients included in the study. Table 1 demonstrates the age and sex distribution for the patients in this study. Thirty seven were children (age <18 years) and 57 were adults. The age range for the children was 1.4 to 17.7 years at onset of stone disease. The adults were separated into tertiles of 19 subjects each, based on the age at which the subjects presented with their initial kidney stone. The first tertile was age 18 to 32 years, the second tertile age 33 to 50.5 years, and the third tertile above age 50.5 years. The sex and age distribution did not significantly differ between the groups (p = 0.22).

All patients' 24-hour urines were evaluated at the Litholink Corporation. The parameters for analysis in the 24-hour urine were pH, urine volume, cystine excretion (mg/day), cystine SS, sodium excretion (mmol/day), and urea nitrogen excretion (g/day) (Table 2). Figure 1 (left panel) demonstrates that the young adults (18–32 years old) have a significantly higher cystine SS compared with the older adults (32–50.5 and >50.5 years) (p = 0.016). Of the main determinants of cystine SS, including cystine excretion, urine pH, and urine volume, the analysis shows that the 24-hour urine volumes were significantly higher in the older adults when compared with the young adults (p = 0.003; Fig. 1, right panel). However, there were no differences in urine pH (p = 0.21) and cystine excretion (p = 0.18) between the groups. The increased urine volume in the older adults reflects a higher fluid intake, which may have been the protective factor delaying the onset of their stone disease.

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FIG. 1.

Cystine supersaturation and urine volume for adult groups (groups 2–4).

In the analysis, excretion rates of cystine and 24-hour urine volume for children were not compared with the adults directly because they will vary with the age of the subject and body size. However, cystine SS and pH are not excretion based and thus allowed valid comparisons of children with the adults. The cystine SS for the children and young adults was significantly higher when compared with the oldest adults in group 4 (p = 0.04) and of borderline significance compared with group 3 (p = 0.07), as depicted in Figure 2. Urine pH in children (group 1) was 7.04 ± 0.07, which was significantly higher when compared with the oldest adults (group 4) (6.47 ± 0.11; p < 0.001).

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FIG. 2.

Cystine supersaturation for each age group (groups 1–4).

Discussion

Cystinuria is one of the most common inherited diseases, with an estimated prevalence of 1 in 7000. ⁷ It is an autosomal recessive disorder caused by the impaired reabsorption of the dibasic amino acid, cystine, in the proximal tubule. Historically, three types of cystinuria have been recognized: type I, type II, and type III. ⁸ However, this classification correlates poorly with the molecular findings and has been revised by the International Cystinuria Consortium to take into account the chromosomal localization of the mutation in addition to the urinary cystine excretion of the obligate heterozygotes. Type A cystinurics have mutations of the SLC3A1 gene located on chromosome 2, type B cystinurics have mutations of the SLC7A9 gene located on chromosome 19, and type AB have mutations of both chromosomes. ⁹ Mean urinary cystine levels are significantly higher in heterozygotes with type B abnormalities, but no difference in stone formation between the groups has been identified.

Twenty-four-hour urine collections of cystine stone formers commonly demonstrate low urine volume, and cystine is known to be poorly soluble in the normal pH range of human urine. Both of these factors increase the risk of stone formation. Hypocitraturia, hypercalciuria, and hyperuricosuria may accompany cystinuria, further complicating treatment of this difficult disease. ¹⁰ Patients with cystinuria have a high recurrence rate of stone formation, and if left untreated, the disease can eventually cause kidney failure. ⁴ Worcester and colleagues ¹¹ have shown that mean creatinine clearance in 52 patients with cystinuria was lower compared with 3215 routine kidney stone patients, and the creatinine clearance declined over time.

Pediatric stone formers more commonly have an identifiable metabolic disorder as opposed to the adult population, and therefore, they more often undergo a thorough metabolic work-up at their first presentation of stone disease. ¹² Thus, most cystinurics are detected at a younger age and the disease is primarily suspected in patients younger than 30 years of age. Patients with cystinuria rarely present with their first stone event later in life. However, recently, we have seen a number of patients with first presentation of cystine stones in the fifth and sixth decades of life. We hypothesized that cystinuric patients presenting at an older age would have more favorable 24-hour urine parameters, such as greater urine volume and lower cystine excretion, compared with those patients who present earlier in life. We therefore sought to determine if a protective factor may be present in the urine of these older patients, which would delay them from clinically expressing the disorder earlier in life.

In our retrospective study, we found a significant correlation between the age of onset of cystine stones and 24-hour urine parameters. Our analysis was first performed in the adult population, groups 2, 3 and 4. Cystine SS was significantly higher in the young adults (group 2, age between 18 and 32) when compared with the older adults (groups 3 and 4, age >30). Children (group 1, <18 years of age) were also shown to have significantly higher cystine SS compared with the oldest adults (group 4), likely contributing to their earlier stone formation. The lower SS of cystine in the urine of the older adult groups likely confers a protective effect on these patients and the reason they may form cystine stones later in life. Of the urine parameters that are known to affect cystine SS, including cystine excretion, urine pH, and urine volume, urine volume seems to be the main determinant. The urine volume in older adults was significantly higher than the younger adults and is explained by improved fluid intake in these patients. Although 24-hour urines were collected prior to the institution of any medical therapy, one inherent limitation of the study is that we cannot exclude the possibility that the difference we are observing is a result of a patient being told to increase fluid intake as a part of a stone prevention treatment plan prior to the 24-hour urine collection. Therefore, whether increased urine volume is due to improved compliance with physician recommendations by the older patients or if they simply have increased fluid intake independently as a group is unknown.

Only a small number of patients are actually able to adequately control their cystinuria, likely as a result of poor patient compliance and lack of insight into the pathology of the disease. ¹³ As mentioned previously, other metabolic abnormalities such as hypercalciuria, hypocitraturia, and hyperuricosuria can also be present in cystinuric patients. ¹⁰ Our study highlights the importance of aggressively counseling younger cystine stone formers on the benefits of increasing fluid intake and monitoring their compliance with these recommendations closely. There is no doubt that childhood and adolescence mark a time when compliance may be difficult to achieve. Regardless of the age at first stone presentation, a complete metabolic evaluation and aggressive medical treatment are recommended for all cystine stone formers.