Nucleation and Growth of Brushite and Calcium Oxalate in Urine of Stone-Formers Charles Y. C. Pak and Karen Holt The physicochemical factors involved in the formation of calcium-containing renal stones have been elucidated previously and some of the techniques for their quantitation are currently available. Accordingly, urinary activity product ratio (state of saturation), formation product mtio (limit of metostability), and crystal growth of brushite and calcium oxalate in 24-hr urine samples were compared between a control group without stones and stone-forming groups composed of patients with absorptive hypercalciuria, normocalciuric nephrolithiasis, and primay hyperparathyroidism.
The activity product ratios of brushite and calcium oxalate were significantly elevated in stone-forming groups, largely because of the high renal excretion of calcium. The formbtion product ratios were reduced in most stone-forming groups, and the crystal growth was increased in the group with primay hyperpamthyroidism. Thus, the physicochemicol environment of urine in stone-forming groups was favorable to the nucleation of the nidi of brushite and calcium oxalate; in primary hyperparuthyroidism, it may be conducive to the subsequent growth of nidi as well.
R
ENAL STONES probably form by nucleation of the crystal nidus,’ followed by the growth of the nidus through processes of crystal growth,24 epitaxial growth,s and crystal aggregation. 6*7According to this scheme, nucleation commences when the urinary state of saturation exceeds the limit of metastability.* It is facilitated by factors which increase the state of saturation or lower the limit of metastability. The latter may occur from lack or deficiency in urine of inhibitors of nucleation or by heterogeneous nucleation.s Once the crystal nidus has formed, its growth will require a supersaturated state, and may be accelerated by a lack or deficiency of inhibitors of crystal growth, aggregation, or of heterogeneous nucleation. The physicochemical environment of urine, which is favorable to the nucleation of the nidus and its subsequent growth, is prerequisite to the formation of renal stones. The validity of this scheme for stone formation was tested for calciumcontaining renal stones by examining the state of saturation, limit of metastability, and crystal growth of brushite (CaHPO,. 2H,O) and calcium (Ca) oxalate in urine of patients with absorptive hypercalciuria,9-1’ normocalciuric nephrolithiasis,9 and primary hyperparathyroidism.
From the Section on Mineral Metabolism, Southwestern Medical School, The University of Texas Health Science Center at Dallas, Dallas. Tex. Received for publication on October 2.1975. Supported by Grants USPHS I-ROI-AM16061 and I-MOI-RR00633. Reprint requests should be addressed to Dr. Charles Y. C. Pak. Department of Internal Medicine, University of Texas Health Science Center at Dallas, 5323 Harry Hines Boulevard, Dallas, Tex. 75235. 0 1976 by Grune & Stratton, Inc.
Metabolism, Vol. 25, No. 6 (June), 1976
665
PAK AND HOLT
666
MATERIALS
AND METHODS
Patients The control group (C) consisted of 18 healthy normal volunteers, nine men and nine women (mean age of 40 yr). Group AH-I consisted of 19 patients with absorptive hypercalciuria type 19, 14 men and five women (mean age of 43 yr). The patients in this group had normocalcemia, hypercalciuria (urinary Ca exceeding 200 mg/day on an intake of 400 mg Ca/day), intestinal hyperabsorption of calcium, an exaggerated renal excretion of calcium following an oral Ca load,9*” and a history of recurrent passage of Ca-containing renal stones. Group AH-II consisted of 19 patients with absorptive hypercalciuria type II,‘“*” I3 men and six women (mean age of 42 yr). The patients in this group had normocalcemia, urinary Ca on a restricted Ca intake within the normal range (< 200 mg/d), an excessive excretion of urinary Ca following an oral Ca load” and a history of passage of Ca-containing renal stones. Patients in groups AH-I and AH-II had normal or low values for urinary cyclic AMP and for serum concentration of immunoGroup NN consisted of nine patients with normocalcemic reactive parathyroid hormone.‘.” nephrolithiasis, eight men and one woman (mean age of 39 yr). The patients in this group had normal values for serum Ca, urinary Ca, intestinal absorption of Ca, and parathyroid function. They also had a normal response to an oral load of Ca.” In four patients, urinary uric acid was persistently > 650 mg/day on random diet." Two patients had a history of recurrent urinary tract infection (E. co/i); they were free of infection at the time of the study. Group PHPT consisted of 17 patients with primary hyperparathyroidism, five men and I2 women (mean age of 45 yr). Following this study, the diagnosis of PHPT was proven surgically. Six patients had a history of passage of Ca-containing renal stones. Studies were performed while the patients were admitted at the General Clinical Research Center. One to four 24-hr urine samples were collected under refrigeration without preservative from each patient on the third day of constant metabolic diet, with a daily composition of approximately 400 mg calcium, 800 mg phosphorus (P). 100 meq sodium (Na), 80 mg oxalate, and 3 liters of fluid. Twenty-five samples from control subjects, 30 samples from AH-I, 26 samples from AH-II, 27 samples from NN, and 18 samples from PHPT were obtained. On each specimen, most of the following tests were performed at 37’C.
Activity
Product Ratio (A PR)
The state of saturation with respect to brushite or calcium oxalate was estimated as folIows.*~‘~~‘~Ten milliters of urine sample were incubated with an excess of brushite or calcium oxalate monohydrate (100 mg) until steady state was reached (2 days) while pH was kept within 0.1 U of original pH. The preparation of Ca oxalate monohydrate’ utilized in this study gave thermodynamic solubility product of 2.53 x lOA sq m. The activity product of brushite or Ca oxalate in the original urine sample was compared with that of the filtrate obtained after incubation with brushite or Ca oxalate, respectively. The “activity products” were calculated from total concentrations of Ca (ionized and complexed), divalent phosphate and oxalate, without consideration of the formation of soluble complexes. Thus, the activity products as calculated by this method represented overestimates. The ratio of activity products (APR) yielded the state of saturation where the value of one indicated saturation, greater than one supersaturation, and less than one undersaturation. By this means, some of the errors which may be attendant in the calculation of activity products could be eliminated.” These errors included those resulting from the failure to account for the formation of soluble complexes. However, since they contributed to the activity product of original urine as well as that of the filtrate, these. errors were largely cancelled when the ratio of activity products was obtained.16 The activity products determined by the present techniqueI differed considerably from those calculated by the method of Robertson et al.” and Finlayson and Roth”’ in corresponding urine specimens. However, the values for APR were essentially the same.‘”
Formation
Product Ratio (FPR)
The limit of metastability of brushite or calcium oxalate was determined as before,‘.’ except that it was obtained 3 hr after induction rather than at steady state. The formation product repre-
CRYSTALLIZATION
OF
SRUSHITE
AND Ca OXACATE
667
Table 1. Urinary CornPositions Absorptive Hypercalcivrio CWltrCll No samples
25
Absorph Hypercolciurio Normocolciuric Primary Nephroli~hiosis Hyperparothyroidism Type 11
Type 1
30
26
27
Co (mg/doy)
121 f 56
284 f 65**’
156 f 31**
160 f 30**
253 f
P (me/day) Oxalote (mg/day)
541 *
666 f
639 f
669 + 170’”
605*194
141
22 f 8
186”
28 f 6’
Mg (mg/dey)
97 f 36
No (meq/doy)
105 f 33
97 f 44
l 21
60 zt 32
51
101 f
31
K (meq/day) Uric Acid @g/day)
465 f
159
576 i
Citrate (mg/doy)
451 f
183
419 f 230
105*’
25 f 5
20 l 5**
25+
107 + 27
90 l 33
91
94 f 44
113*32
66 f 22*
63 f 23*
137*
18
482~~96
558 i
141’
464 f 382
371 zt 161
115*** 10
l 31
124 f 39 47* 482 f
18 156
492 f 272
Sulfate (mg/day)
744 f 328
1005 f 280”
069 zk 258
064 f 430
776 f 299
PH Total volume (ml/day)
6.33 zt 0.35
6.40 f 0.34
6.57 i 0.32’
6.27 + 0.54
6.32 +z 0.65
2411 f 511
2488 f 400
2320 f 519
Significance of values from control group is indicated by: Values are presented as mean f
lp
1 suporsatumtion, and < 1 undersatumtion. C = control group; AH-I = absorptive hyporcalciuria type I; AH-II = absorptive hyporcalciuria type II; NN = normocalciuric nephmlithiasir; PHPT = primary hyporpamthymidism. Mean f SD are mprosented by horizontal ban.
3Brushite 2-
C
AH-I
AH-II
NN
PHPT
(> 200 mg/day) in every case. The mean value of 284 f 65 mg/day was significantly higher than that in the control group (p < 0.001). In AH-II and in NN, urinary Ca was < 200 mg/day; however, the mean value was significantly higher than that in the control group. In PHPT, urinary Ca was elevated in 10 of 16 cases. The mean value of 253 f 115 mg/day was significantly elevated (p < 0.001). Urinary P was slightly higher in AH-I, AH-II and NN than in the control group. Urinary oxalate and uric acid were slightly increased in AH-I and NN. There was only a small or no significant difference in urinary Mg, Na, K, citrate, sulfate, pH, or total volume among the five groups. Urinary A PR (Fig. I, Table 2)
The urine samples were invariably undersaturated (APR < 1) with respect to brushite in the control group, whereas they were usually supersaturated (APR > 1) in AH-I and PHPT. Although many samples were undersaturated, the mean values for APR of brushite in AH-II and NN were significantly higher than that in the control group (p < 0.001). The APR of brushite was signifiTable 2. Measums of Crystallization in Urine Absorptive
Absorptive
Hypercolciurio
Hypercalciurio
Normocalciuric
Type II
Nephrolithiasis
Control
Type
1
Primary Hyparporothyroidism
Brurhite APR
0.35
f
0.26
1.74
f
1.14
f
0.45***
0.92
f
0.59***
1.61
f
i.oa***
FPR
4.11
zt 1.11
3.48
zt 0.80*
3.51
f
0.87’
3.64
f
0.77+
3.17
f
0.60***
2.69
f
1.21
3.04
zk 1.36
3.09
*
1.12
2.82
zt 1.33
3.90
k
1
APR
1.45
f
0.70
2.80
f
1.43*”
1.94
f
0.62*
2.21
*
0.64***
2.40
f
1.07’
FPR
11.08
f
3.39
7.94
+
3.26*
9.29
f
5.24
9.02
f
3.63’
7.53
f
3.50**
0.95
f
f
0.39*
1.19
f
0.64’
0.94
f
0.38
1.23
f
* p
The activity product ratios of brushite and calcium oxalate were significantly elevated in stone-forming groups, largely because of the high renal excretion of calcium. The formbtion product ratios were reduced in most stone-forming groups, and the crystal growth was increased in the group with primay hyperpamthyroidism. Thus, the physicochemicol environment of urine in stone-forming groups was favorable to the nucleation of the nidi of brushite and calcium oxalate; in primary hyperparuthyroidism, it may be conducive to the subsequent growth of nidi as well.
R
ENAL STONES probably form by nucleation of the crystal nidus,’ followed by the growth of the nidus through processes of crystal growth,24 epitaxial growth,s and crystal aggregation. 6*7According to this scheme, nucleation commences when the urinary state of saturation exceeds the limit of metastability.* It is facilitated by factors which increase the state of saturation or lower the limit of metastability. The latter may occur from lack or deficiency in urine of inhibitors of nucleation or by heterogeneous nucleation.s Once the crystal nidus has formed, its growth will require a supersaturated state, and may be accelerated by a lack or deficiency of inhibitors of crystal growth, aggregation, or of heterogeneous nucleation. The physicochemical environment of urine, which is favorable to the nucleation of the nidus and its subsequent growth, is prerequisite to the formation of renal stones. The validity of this scheme for stone formation was tested for calciumcontaining renal stones by examining the state of saturation, limit of metastability, and crystal growth of brushite (CaHPO,. 2H,O) and calcium (Ca) oxalate in urine of patients with absorptive hypercalciuria,9-1’ normocalciuric nephrolithiasis,9 and primary hyperparathyroidism.
From the Section on Mineral Metabolism, Southwestern Medical School, The University of Texas Health Science Center at Dallas, Dallas. Tex. Received for publication on October 2.1975. Supported by Grants USPHS I-ROI-AM16061 and I-MOI-RR00633. Reprint requests should be addressed to Dr. Charles Y. C. Pak. Department of Internal Medicine, University of Texas Health Science Center at Dallas, 5323 Harry Hines Boulevard, Dallas, Tex. 75235. 0 1976 by Grune & Stratton, Inc.
Metabolism, Vol. 25, No. 6 (June), 1976
665
PAK AND HOLT
666
MATERIALS
AND METHODS
Patients The control group (C) consisted of 18 healthy normal volunteers, nine men and nine women (mean age of 40 yr). Group AH-I consisted of 19 patients with absorptive hypercalciuria type 19, 14 men and five women (mean age of 43 yr). The patients in this group had normocalcemia, hypercalciuria (urinary Ca exceeding 200 mg/day on an intake of 400 mg Ca/day), intestinal hyperabsorption of calcium, an exaggerated renal excretion of calcium following an oral Ca load,9*” and a history of recurrent passage of Ca-containing renal stones. Group AH-II consisted of 19 patients with absorptive hypercalciuria type II,‘“*” I3 men and six women (mean age of 42 yr). The patients in this group had normocalcemia, urinary Ca on a restricted Ca intake within the normal range (< 200 mg/d), an excessive excretion of urinary Ca following an oral Ca load” and a history of passage of Ca-containing renal stones. Patients in groups AH-I and AH-II had normal or low values for urinary cyclic AMP and for serum concentration of immunoGroup NN consisted of nine patients with normocalcemic reactive parathyroid hormone.‘.” nephrolithiasis, eight men and one woman (mean age of 39 yr). The patients in this group had normal values for serum Ca, urinary Ca, intestinal absorption of Ca, and parathyroid function. They also had a normal response to an oral load of Ca.” In four patients, urinary uric acid was persistently > 650 mg/day on random diet." Two patients had a history of recurrent urinary tract infection (E. co/i); they were free of infection at the time of the study. Group PHPT consisted of 17 patients with primary hyperparathyroidism, five men and I2 women (mean age of 45 yr). Following this study, the diagnosis of PHPT was proven surgically. Six patients had a history of passage of Ca-containing renal stones. Studies were performed while the patients were admitted at the General Clinical Research Center. One to four 24-hr urine samples were collected under refrigeration without preservative from each patient on the third day of constant metabolic diet, with a daily composition of approximately 400 mg calcium, 800 mg phosphorus (P). 100 meq sodium (Na), 80 mg oxalate, and 3 liters of fluid. Twenty-five samples from control subjects, 30 samples from AH-I, 26 samples from AH-II, 27 samples from NN, and 18 samples from PHPT were obtained. On each specimen, most of the following tests were performed at 37’C.
Activity
Product Ratio (A PR)
The state of saturation with respect to brushite or calcium oxalate was estimated as folIows.*~‘~~‘~Ten milliters of urine sample were incubated with an excess of brushite or calcium oxalate monohydrate (100 mg) until steady state was reached (2 days) while pH was kept within 0.1 U of original pH. The preparation of Ca oxalate monohydrate’ utilized in this study gave thermodynamic solubility product of 2.53 x lOA sq m. The activity product of brushite or Ca oxalate in the original urine sample was compared with that of the filtrate obtained after incubation with brushite or Ca oxalate, respectively. The “activity products” were calculated from total concentrations of Ca (ionized and complexed), divalent phosphate and oxalate, without consideration of the formation of soluble complexes. Thus, the activity products as calculated by this method represented overestimates. The ratio of activity products (APR) yielded the state of saturation where the value of one indicated saturation, greater than one supersaturation, and less than one undersaturation. By this means, some of the errors which may be attendant in the calculation of activity products could be eliminated.” These errors included those resulting from the failure to account for the formation of soluble complexes. However, since they contributed to the activity product of original urine as well as that of the filtrate, these. errors were largely cancelled when the ratio of activity products was obtained.16 The activity products determined by the present techniqueI differed considerably from those calculated by the method of Robertson et al.” and Finlayson and Roth”’ in corresponding urine specimens. However, the values for APR were essentially the same.‘”
Formation
Product Ratio (FPR)
The limit of metastability of brushite or calcium oxalate was determined as before,‘.’ except that it was obtained 3 hr after induction rather than at steady state. The formation product repre-
CRYSTALLIZATION
OF
SRUSHITE
AND Ca OXACATE
667
Table 1. Urinary CornPositions Absorptive Hypercalcivrio CWltrCll No samples
25
Absorph Hypercolciurio Normocolciuric Primary Nephroli~hiosis Hyperparothyroidism Type 11
Type 1
30
26
27
Co (mg/doy)
121 f 56
284 f 65**’
156 f 31**
160 f 30**
253 f
P (me/day) Oxalote (mg/day)
541 *
666 f
639 f
669 + 170’”
605*194
141
22 f 8
186”
28 f 6’
Mg (mg/dey)
97 f 36
No (meq/doy)
105 f 33
97 f 44
l 21
60 zt 32
51
101 f
31
K (meq/day) Uric Acid @g/day)
465 f
159
576 i
Citrate (mg/doy)
451 f
183
419 f 230
105*’
25 f 5
20 l 5**
25+
107 + 27
90 l 33
91
94 f 44
113*32
66 f 22*
63 f 23*
137*
18
482~~96
558 i
141’
464 f 382
371 zt 161
115*** 10
l 31
124 f 39 47* 482 f
18 156
492 f 272
Sulfate (mg/day)
744 f 328
1005 f 280”
069 zk 258
064 f 430
776 f 299
PH Total volume (ml/day)
6.33 zt 0.35
6.40 f 0.34
6.57 i 0.32’
6.27 + 0.54
6.32 +z 0.65
2411 f 511
2488 f 400
2320 f 519
Significance of values from control group is indicated by: Values are presented as mean f
lp
1 suporsatumtion, and < 1 undersatumtion. C = control group; AH-I = absorptive hyporcalciuria type I; AH-II = absorptive hyporcalciuria type II; NN = normocalciuric nephmlithiasir; PHPT = primary hyporpamthymidism. Mean f SD are mprosented by horizontal ban.
3Brushite 2-
C
AH-I
AH-II
NN
PHPT
(> 200 mg/day) in every case. The mean value of 284 f 65 mg/day was significantly higher than that in the control group (p < 0.001). In AH-II and in NN, urinary Ca was < 200 mg/day; however, the mean value was significantly higher than that in the control group. In PHPT, urinary Ca was elevated in 10 of 16 cases. The mean value of 253 f 115 mg/day was significantly elevated (p < 0.001). Urinary P was slightly higher in AH-I, AH-II and NN than in the control group. Urinary oxalate and uric acid were slightly increased in AH-I and NN. There was only a small or no significant difference in urinary Mg, Na, K, citrate, sulfate, pH, or total volume among the five groups. Urinary A PR (Fig. I, Table 2)
The urine samples were invariably undersaturated (APR < 1) with respect to brushite in the control group, whereas they were usually supersaturated (APR > 1) in AH-I and PHPT. Although many samples were undersaturated, the mean values for APR of brushite in AH-II and NN were significantly higher than that in the control group (p < 0.001). The APR of brushite was signifiTable 2. Measums of Crystallization in Urine Absorptive
Absorptive
Hypercolciurio
Hypercalciurio
Normocalciuric
Type II
Nephrolithiasis
Control
Type
1
Primary Hyparporothyroidism
Brurhite APR
0.35
f
0.26
1.74
f
1.14
f
0.45***
0.92
f
0.59***
1.61
f
i.oa***
FPR
4.11
zt 1.11
3.48
zt 0.80*
3.51
f
0.87’
3.64
f
0.77+
3.17
f
0.60***
2.69
f
1.21
3.04
zk 1.36
3.09
*
1.12
2.82
zt 1.33
3.90
k
1
APR
1.45
f
0.70
2.80
f
1.43*”
1.94
f
0.62*
2.21
*
0.64***
2.40
f
1.07’
FPR
11.08
f
3.39
7.94
+
3.26*
9.29
f
5.24
9.02
f
3.63’
7.53
f
3.50**
0.95
f
f
0.39*
1.19
f
0.64’
0.94
f
0.38
1.23
f
* p
