ROCHESTER. The most common crystal present in calculi formed within the urinary tract is calcium oxalate as the monohydrate or dihydrate crystal phase. (1,2).
HYDROXYAPATITE-THE FORGOTTEN CRYSTAL IN CALCIUM UROLITHIASIS*.t LYNWOOD H. SMITHtt, and (by invitation) PETER G. WERNESS ROCHESTER
The most common crystal present in calculi formed within the urinary tract is calcium oxalate as the monohydrate or dihydrate crystal phase (1,2). Yet, with careful analysis and attention to all parts of the stone, other crystal types are usually present. This is especially true with hydroxyapatite. Boyce found at least some hydroxyapatite present in all stones that he examined including what had been considered "pure" calcium oxalate stones (3). This hydroxyapatite often was situated in close association with the matrix present in the stone. If one considers urine and its changing composition in terms of pH and the concentration of major ions throughout a 24-hour period, it is not surprising that the crystalline nature of a foreign body such as a calculus could vary as it grows within the urinary tract. Observations regarding the mixed crystalline nature of urinary calculi led Modlin in an address to the Royal College of Surgeons to suggest that heterogeneous nucleation may be an important etiologic factor in the initiation and growth of stones within the urinary tract (4). He suggested that the presence of a crystal of one type in urine supersaturated for another crystalline phase could induce the nucleation of this second phase. Dame Kathleen Lonsdale examined this question from a theoretical standpoint and confirmed the fact that a number of common urinary crystals had the potential for heterogeneous nucleation (5). In vitro observations of the ability to induce heterogeneous nucleation in mixed crystal systems have confirmed that this process can occur (6-12). Meyer, et al. examined the relationship between hydroxyapatite and calcium oxalate monohydrate and found that hydroxyapatite could induce the heterogeneous nucleation of calcium oxalate monohydrate (6). The rate of this nucleation was dependent upon the surface area of the seed
crystals added. To define further the possible role of heterogenous nucleation in the formation of calcium containing calculi within the urinary tract, we * Nephrology Research Unit, Mayo Clinic, Rochester, Minnesota. t This work was supported in part by grants AM 20605 and RR 585 from the National Institutes of Health. tt Requests for reprints should be addressed to Lynwood H. Smith, M.D., Mayo Clinic, W-17B, Rochester, Minnesota, 55905.
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examined patients with primary hyperparathyroidism and idiopathic calcium urolithiasis. We were especially interested in the relationship between hydroxyapatite and calcium oxalate with regard to the possible role of hydroxyapatite in the formation of calcium oxalate stones in these patients. METHODS AND MATERIALS
Study Subjects We studied 96 patients with idiopathic calcium urolithiasis receiving no treatment, 41 patients with primary hyperparathyroidism before surgery and 10 normal subjects. All subjects were admitted to a metabolic ward to allow two consecutive 24 hour urine collections to be made while they were receiving their normal diet and fluid intake. Other possible metabolic causes of urolithiasis had been eliminated during a detailed metabolic workup. All the patients had normal renal function and were free of bacteriuria at the time of study.
Crystalluria Crystalluria in each voiding during the 48 hour period was determined as previously described (13). At the time of each voiding, 2 ml of urine were filtered immediately through a 0.2 utM Nuclepore filter at 370C to separate solution from crystals present. The crystals on the surface of the filter were identified using petrographic microscopy and scanning electron microscopy with energy dispersive x-ray analysis. A gross quantitation of the crystalluria was estimated and a crystalluria score developed on the basis of the sum of crystal grades (0 = 0; 1 = 1-200 crystals/ filter; 3 = 200-500 crystals/filter; 5 = >500 crystals/filter) divided by the number of voids during the 48 hour period. Supersaturation The urinary pH and ion species including sodium, potassium, chloride, calcium, magnesium, phosphate, oxalate, sulfate, citrate, CO2, ammonia, and uric acid were measured in each 24 hour urine collection. From these data, free ion activity, activity products, ionic strength, and supersaturation for calcium oxalate hydroxyapatite and brushite could be estimated by a modification of the iterative computer program EQUIL (14). Data from the two 24-hour urine collections were averaged for each study subject. Hydroxyapatite supersaturation presented an especially difficult problem since it was dependent upon the fifth power of calcium activity and the cube of P04 activity. These calculations resulted in an extremely
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large range of values that were difficult to handle and compare. To avoid this problem, we elected to express the driving force for crystallization in terms of Gibbs-free energy of transfer from a supersaturated to a saturated solution as expressed in the equation -RT AG -T 1 SS n where R = 8.3144 joules/deg mole; T = temperature; n = number of ions in the molecule and SS = supersaturation ratio. Since the result of this calculation provides a negative number when the solution is supersaturated, we elected to use the function DG where DG = AG. Thus, DG will equal 0 for solutions at saturation; will be greater than 0 for:-solutions that are supersaturated, and will be less than 0 for undersaturated solutions. Inhibition of Crystal Growth The inhibition of calcium oxalate monohydrate and hydroxyapatite crystal growth was measured in seeded crystal growth systems (15-17). In these systems we arbitrarily assigned a value of 1 inhibitor unit to the amount of any material required to produce 50% inhibition in the standard systems. Known inhibitors including magnesium (by atomic absorption), citrate (enzymatically) (18), and pyrophosphate (19) were measured.
RESULTS
Study Subjects Twenty-four of the 41 patients with primary hyperparathyroidism had formed urinary stones. In 10 patients stones were available for analysis. In 7 of these patients the major crystal type was calcium oxalate and in 3 calcium phosphate. All the 96 patients with idiopathic calcium urolithiasis had a history of recurrent stone formation. The results of stone analysis were available in 72 patients including a total of 172 stones analyzed. Calcium oxalate was the major crystal present in the stones from 56 (78%) of the patients with calcium phosphate being the major crystal type in 16 (22%). Results of the analysis of the 172 stones from the 72 patients with idiopathic calcium urolithiasis showed the major crystal type to be calcium oxalate in 122 (71%), calcium phosphate in 48 (28%), and uric acid in 2 (1%). The reported minor crystal type was calcium oxalate in 17 (10%), calcium phosphate in 72 (42%), with none reported in 83 (48%)
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Crystalluria The results of the examination of the freshly voided urine for crystalluria in the study subjects are outlined in Table 1. In 16 of the normal subjects, crystalluria occurred in at least one voiding as hydroxyapatite. In 15 normal subjects it was the major crystal type and in 12 the only crystal type. Three normal subjects had mixed crystalluria with calcium oxalate and hydroxyapatite. All the patients with primary hyperparathyroidism had crystalluria in at least some of their voidings. Hydroxyapatite was present in these patients. It was the major crystal in 39 and the only crystal in 27. Six of the patients had mixed crystalluria with calcium oxalate and hydroxyapatite. All but one of the patients with idiopathic calcium urolithiasis had crystalluria in at least some of their voidings. Hydroxyapatite crystals were present in 92 of these patients. In 80 it was the major crystal and in 31 the only crystal present. Mixed crystalluria with calcium oxalate and hydroxyapatite was present in 43 of the patients with idiopathic calcium urolithiasis. Gross quantitation of crystalluria using the crystalluria score was significantly increased in both patient groups. In addition, large crystals and crystal aggregation were commonly present in the patient groups.
Supersaturation Outlined in Table 2 are the relative supersaturations for calcium oxalate, hydroxyapatite, and brushite in the study subjects. Urine was supersaturated for each of these crystal systems and these supersaturations were significantly increased for both patient groups as compared to the normal subjects, creating an environment within the urine where heterogeneous nucleation could occur.
Inhibition of Crystal Growth Inhibition of crystal growth expressed as inhibitor units per liter is summarized in Table 3 for the calcium oxalate and hydroxyapatite crystal growth systems. There were no differences between the three study groups with respect to the concentration of calcium oxalate inhibition. Hydroxyapatite inhibition was decreased in the patients with idiopathic calcium urolithiasis. This was due at least in part to the decrease in the known inhibitors of hydroxyapatite crystal growth including magnesium, citrate, and pyrophosphate, as shown in Table 3.
DISCUSSION In both groups of patients, the major crystal present in the stones that were available for analysis was most commonly calcium oxalate. This
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