0021-972X/91/7201-0229$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright (c) 1991 by The Endocrine Society
Vol. 72, No. 1 Printed in U.S.A.
Osteomalacia in Hereditary Hypophosphatemic Rickets with Hypercalciuria: A Correlative ClinicalHistomorphometric Study DAN GAZIT, MARTIN TIEDER, URI A. LIBERMAN, LEA PASSI-EVEN, AND ITAI A. BAB Division of Oral Pathology and Bone Laboratory, Hebrew Uniuersity-Hadassah Faculty of Dental Medicine (D.G., L.P., I.A.B.), Jerusalem; the Pediatric Nephrology Unit, Assaf Harofe Medical Center (M.T.), Zerifin; the Unit of Metabolic Diseases, Beilinson Medical Center (U.A.L.), Petah Tiqua; Sackler School of Medicine, Tel Aviv University (M.T., U.A.L.), Tel Aviv, Israel
vealed a very high inverse correlation and a tight linear relationship between serum phosphorus and osteoid parameters. Serum 1,25-dihydroxyvitamin D, which is low in other forms of hereditary hypophosphatemia and osteomalacia, is elevated in HHRH and correlated positively with osteoid parameters and the mineralization lag time. Serum alkaline phosphatase showed similar relationships. These results as well as the clinical, biochemical, and radiological remission of bone disease consequent to phosphate therapy strongly suggest that in HHRH 1) hypophosphatemia alone is sufficient to cause osteomalacia; and 2) the elevation of 1,25-dihydroxyvitamin D reflects the degree of the primary renal phosphate leak, but is not involved in the pathogenesis of the bone disease. (J Clin Endocrinol Metab 7 1 : 229235,1991)
ABSTRACT. We characterized the bone disease of transilial biopsy specimens from children with hereditary hypophosphatemic rickets with hypercalciuria (HHRH) and genetically related asymptomatic hypercalciuric subjects. All HHRH patients showed irregular mineralization fronts, markedly elevated osteoid surface and seam width, increased number of osteoid lamellae, and prolonged mineralization lag time. These findings are consistent with a mineralization defect and indicate unambiguously that the bone disease in HHRH is osteomalacia. The only abnormality seen in the asymptomatic hypercalciuric subjects was slightly extended osteoid surface. Parametric and nonparametric statistical analyses performed on a pooled sample of HHRH patients and asymptomatic hypercalciuric subjects re-
A
NEW familial syndrome, hereditary hypophosphatemic rickets with hypercalciuria (HHRH), was recently described in a group of closely related members of a Bedouine tribe (1, 2). The disease is manifested in early childhood as bone deformities, short stature, muscle weakness, bone pain, and radiological signs of rickets and osteopenia. A transilial bone biopsy from one patient implied that the osteopenia reflects a mineralization defect. The characteristic biochemical features are increased renal phosphate clearance, hypophosphatemia, elevated serum 1,25-dihydroxyvitamin D [1,25-(OH)2D] concentration, enhanced gastrointestinal absorption of calcium and phosphorus, suppressed parathyroid function, and hypercalciuria. The serum calcium concentration is normal. Of 59 members of the tribe that were evaluated, 15% express HHRH. Of the remaining asymptomatic members, 42% had hypercalciuria and mildly elevated levels
of serum 1,25-(OH)2D. The other biochemical parameters in these subjects were within normal limits, but showed a trend similar to that seen in HHRH patients. It was, therefore, suggested that 1) HHRH patients and asymptomatic subjects with hypercalciuria share a common hereditary renal phosphate leak that leads to the other biochemical abnormalities; and 2) expression of the primary defect and magnitude of the ensuing hypophosphatemia determine which subjects present bone disease and which stay asymptomatic and have only hypercalciuria (2). The present microscopic and histomorphometric study in HHRH patients and genetically related asymptomatic subjects with hypercalciuria establishes that the bone disease in HHRH is osteomalacia. A low serum phosphorus concentration appears to have a key role in determining the extent of the mineralization defect in this disease.
Received May 9, 1990. Address all correspondence and requests for reprints to: U. A. Liberman, M.D., Ph.D., Unit of Metabolic Diseases, Beilinson Medical Center, Petah Tiqva 49100, Israel.
Subjects
Subjects and Methods The study was performed with two groups of 1) HHRH patients and 2) asymptomatic hypercalciuric subjects, 3-15 229
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GAZIT ETAL.
230
(mean ± SD, 8.5 ± 3) and 7-14 (mean ± SD, 8.5 ± 1.5) yr old, respectively. Each group consisted of three females and one male. All eight individuals belonged to the same Bedouin tribe and were closely related genetically (2). The HHRH patients presented the following characteristics: short stature, long bone deformities, bone pain, muscle weakness, and radiological signs of rickets and osteopenia. The hypercalciuric subjects were asymptomatic clinically and appeared normal on skeletal x-rays. The relevant biochemical data of each group are summarized in Fig. 1. Bone biopsy Transilial bone biopsies, 5 mm in diameter, were obtained from all patients and processed as described previously (3). In short, specimens obtained after double tetracycline labeling (Ledermycin, Lederle, Germany) with a 14-day interlabel space were embedded in low viscosity resin (4) and sectioned undecalcified. Sections, 5 nm thick, were stained with modified Masson's stain (5) for general marrow histology, demonstration of bone cells, and distinction between osteoid and mineralized bone. Osteoid lamellae were differentiated from mineralized bone lamellae in 5-/mi sections stained with Von Kossa and toluidine blue and examined by polarized light microscopy. Unstained lO-^m sections were used for fluorescent microscopy of the tetracycline label. Static histomorphometric measurements were performed using a computerized digitizing system (6). Quantitation of the tetracycline labeling was performed with a Merz grid (7). The separation between double labels (dynamic measurements) was measured with a calibrated eyepiece micrometer. The following histomorphometric parameters were defined according to Parfitt et al. (8): trabecular bone volume (BV/TV), osteoid volume (OV/BV), osteoid surface (OS/BS), osteoblast surface (Ob.S/BS), osteoid thickness (O.Th), osteoclast surface (Oc.S/BS), osteoclast number (N.Oc/TA), double labeled surface (dLS/BS), single labeled surface (sLS/BS), mineral appositional rate (MAR), bone formation rate (BFR/BS), and mineralization lag time (MLT). Differences in histomorphometric and biochemical parameters between HHRH patients and asymptomatic subjects were analyzed using the Wilcoxon two-sample test. In addition, Spearman rank correlation coefficients were calculated, and Pearson linear regression analysis was carried out between pairs of biochemical and histomorphometric parameters in a pooled sample of the HHRH and asymptomatic subjects.
JCE & M • 1991 Vol 72 • No 1
revealed a considerable decrease in serum phosphorus concentration and TmP/GFR and a marked elevation in serum 1,25-(OH)2D levels and activity of alkaline phosphatase of bone origin. The serum calcium concentration was normal in both groups. Histologically, patients with HHRH presented an abundance of unmineralized matrix, to the extent that whole trabeculae appeared to be composed of osteoid (Fig. 2). The amount of osteoid in the asymptomatic subjects was normal or slightly elevated (Fig. 3). The osteomalacic nature of the hyperosteoidosis in HHRH is demonstrated by 1) the irregularity of mineralization fronts (Fig. 2), 2) the high number of osteoid lamellae (6-20 lamellae; Fig. 4) compared to that in the hypercalciuric asymptomatic subjects (5 lamellae or less; Fig. 5), and 3) the broad single fluorescent labels commonly found in biopsies from individuals with HHRH (Fig. 6). This labeling pattern was absent in bone specimens from SERUM Pi
TMP/GFR
SERUM ALK. PHOSPH.
SERUM Ca
2.50
2.25
2.00
URINARY CALCIUM
SERUM 1,25(OH)2D 2.50 2.00
Results
1.50
The biochemical comparison between HHRH patients and asymptomatic subjects is shown in Fig. 1. The data presented were obtained before the onset of any therapy. The only abnormalities in hypercalciuric asymptomatic subjects were excessive urinary calcium excretion and slightly elevated serum 1,25-(OH)2D concentration. The serum phosphorus levels and renal tubular reabsorption of phosphorus (measured as TmP/GFR), although being in the low normal range, showed a trend resembling that seen in HHRH. The HHRH patients, on the other hand,
1.00
J
)f>0 n nn
FIG. 1. Biochemical data of HHRH child patients (•) and asymptomatic hypercalciuric children (•). The normal range is indicated by shaded boxes. Serum measurements were made after overnight fasting. SD units, SD units of normal range for age; ALK. PHOSPH., alkaline phosphatase activity of bone origin. Conversion factor for serum calcium to mg/dL, 4.0; conversion factor for urinary calcium to mg/mg creatinine, 0.35. Data are the mean ± SD obtained in four subjects per group.
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OSTEOMALACIA IN HHRH
231
FIG. 2. Photomicrograph of transilial bone biopsy of HHRH patient showing broad osteoid seams (arrows), osteoid trabeculae (OT) and irregular mineralization fronts (double arrows). Modified Masson stain; magnification, X130. FIG. 3. Photomicrogaph of transilial bone biopsy of asymptomatic child with hypercalciuria. Osteoid seams are indicated by arrows. Modified Masson stain; magnification, X130. FIG. 4. Polarized light photomicrograph of transilial bone biopsy of HHRH patient with markedly increased number of osteoid lamellae (arrows). Mineralized bone (MB). Von Kossa-toluidine blue stain; magnification, X180. FIG. 5. Polarized light photomicrograph of transilial bone biopsy of an asymptomatic child with hypercalciuria, showing few osteoid lamellae (arrows). Mineralized bone (MB). Von Kossa-toluidine blue stain; magnification, X360. FlG. 6. Fluorescent photomicrograph of transilial bone biopsy of HHRH patient showing wide fluorescent bands with ground glass appearance (arrows). Unstained; magnification, X200. FlG. 7. Fluorescent photomicrograph of transilial bone biopsy of an asymptomatic child with hypercalciuria, showing single (arrows) and double (double arrows) tetracycline labels. Unstained; magnification, X200.
hypercalciuric symptom-free subjects (Fig. 7). The histomorphometric analysis of transilial bone biopsies from HHRH patients and asymptomatic hypercalciuric subjects is summarized in Tables 1 and 2. In the absence of normal child values from our laboratory,
the present findings were compared with several studies reporting normal data in children of a similar age range. Patients with HHRH exhibited hyperosteoidosis, expressed by a 3- to 8-fold increase in OV/BV, O.Th, and OS/BS over the highest mean values reported in normal
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232
GAZIT ETAL.
JCE&M-I99I Vol 72 • No 1
TABLE 1. Static bone histomorphometric parameters in HHRH, asymptomatic hypercalciuria (HC), XLH, and normal children Parameter BV/TV (%)
Normal 25.5 ± 28.6 ± 28.0 ± 26.4 ± 23.0 ± 26.4 ± 19.0 ±
2.0° 3.8C 3.5d 3.6e 4.0' 3.6s 3.0*
XLH
HHRH
HC
26.0 ± 8.0*
22.9 ± 9.1
19.6 ± 2.3
0.88
OV/BV (%)
2.5 ± 0.3° 4.0 ± 1.4C 1.2 ± 0.5d 1.2 ± 0.5e 1.1 ± 0.5' 1.2 ± 0.5* 2.4 ± 0.5*
14.4 ± S ^ * 17.0, 8.6-29' 19.0 ± 5.0*
29.8 ± 12.2
5.9 ± 2.1
0.03
OS/BS (%)
21.6 ± 2.4° 21.6 ± 8.0c 25.0 ± 3.0d 23.8 ± 6.4e 22.0 ± 2.0' 23.6 ± 6.3*
65.9 ± 7.6"
79.21 ± 12.5
41.0 ± 10.7
0.03
O.Th
11.4 ± 6.4 ± 7.4 ± 6.8 ± 10.2 ±
0.4° 1.2d 1.6* 1.5* 2.06
27.7 ± 8.3" 25.9, 12-41.5' 24.0 ± 7.0*
31.9 ± 12.2
13.3 ± 4.9
0.03
Ob.S/BS (%)
13.9 ± 1.0°
4.4 ± 4.05"
4.1 ± 6.1
8.0 ± 4.0
0.31
1.1 ± 0.3° 7.6 ± 1.7e 1.6 ± 0.05d 2.1 ± l.l e 1.8 ± 0.7* 1.0 ± 0.5*
0.2 ± 0.45h 1.8, 0.6-6.1' 0.9 ± 0.5*
0.29 ± 0.12
1.11 ± 0.42
0.03
0.1 ± 0.02° 0.8 ± 0.4c 0.6 ± 0.2" 0.8 ± 0.38e 0.3 ± 0.1' 0.65 ± 0.2*
0.6, 0.06-2.1'
0.06 ± 0.053
0.28 ± 0.14
0.06
7.0 ± 3.3C
9.7 ± 4.9e 14.0 ± 2.0' 8.4 ± 3.7*
Oc.S/BS (%)
N.Oc/TA (I/mm2)
Data are the mean ± SD obtained in four patients per group. P values for comparison between HHRH and HC were determined by Wilcoxon two-sample test; differences were considered significant at P < 0.05. 0 Baron et al. (9). * Glorieux et al. (20). c Ste-Marie et al. (10). d Glorieux et al. (11). e Marie et al. (14). ' Chen etal. (15). * Marie et al. (16). * Harrel et al. (12). ' Costa et al. (13); mean and range.
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OSTEOMALACIA IN HHRH
233
TABLE 2. Dynamic bone histomorphometric parameters in HHRH, asymptomatic hypercalciuria (HC), XLH, and normal children Normal
XLH
HHRH
HC
P
dLS/BS (%)
>60a
NA
4.09 ± 3.2
14.3 ± 12.2
0.19
sLS/BS (%)
65.4 ± 8.2a 68.8 ± 6.3"
NA
21.6 ± 20.2
9.3 ± 5.9
0.47
MAR (mm/yr)
0.33 ± 0.1c 0.47 ± 0.11°
0.16 ± 0.07d 0.10, 0.05-0.19c
0.25 ± 0.09
0.20 ± 0.05
0.24
BFR/BS (mm3/mm2-yr)
0.281 ± 0.065"
NA
0.11 ± 0.08
0.47 ± 0.59
0.19
MLT (yr)
0.023 ± 0.005°
0.27 ± 0.2d
0.13 ± 0.0
0.06 ± 0.03
0.02
Parameter
Data are the mean ± SD obtained in four patients per group. P values for comparison between HHRH and HC were determined by Wilcoxon two-sample test; differences were considered significant at P < 0.05. NA, Not available. ° Marie et al. (14). 6 Marie et al. (16). c Ste-Marie et al. (10). d Harrel et al. (12). * Costa et al. (13); mean and range.
children. These values are in a range similar to that reported for X-linked hypophosphatemia (XLH) and significantly higher in HHRH than in hypercalciuric subjects (Table 1). Osteoblast and osteoclast parameters were low in HHRH compared to normal children and similar to values reported for XLH. Compared to hypercalciuric subjects, only the Oc.S/BS was significantly lower in HHRH patients with N.Oc/TA, approaching statistical significance (Table 1). By comparison with reported normal values, both HHRH patients and hypercalciuric subjects showed considerable decreases in dLS/BS, sLS/BS, and MAR (Table 2). The BFR/BS was low only in HHRH. The MLT was prolonged in both groups; however, in HHRH this parameter was significantly higher than in hypercalciuria and within the range reported for XLH (Table 2). When values from HHRH patients and asymptomatic subjects were pooled, the Pearson linear regression analysis between histomorphometric and biochemical parameters revealed that all osteoid parameters are inversely and significantly related to the serum phosphorus concentration (Fig. 8). Spearman rank correlation coefficients were also calculated to decrease the probability that the results are a function of data clustering of the respective HHRH and hypercalciuric groups. Generally, these correlations were similar to Pearson's; namely the relationship between serum phosphorus concentration and OS/BS, O.Th., and OV/BV was -0.905 (P < 0.001), -0.88 (P < 0.01), and -0.928 (P < 0.001), respectively. The serum phosphorus levels also showed an inverse correlation with the MLT (r = -0.83; P < 0.01) and a direct correlation with Ob.S/BS, Oc.S/BS, and Oc.N./ TA (Table 3). A positive correlation was found between serum alkaline phosphatase activity and OV/BV, OS/ BS, O.Th, and MLT. In addition, the alkaline phospha-
tase activity was inversely related to resorption parameters. There was a direct correlation between serum 1,25(OH)2D and OV/BV, O.Th, and MLT and no correlation with osteoclast parameters (Table 3). Discussion Previously, the diagnosis of rickets and osteomalacia in HHRH was based mainly on the patients' clinical, radiological, and biochemical parameters (1, 2). Now we have demonstrated osteomalacia unequivocally in all biopsies from HHRH patients. This is supported by 1) the presence of irregular mineralization fronts; 2) the thickening of osteoid seams with an increase in the number of osteoid lamellae, a feature consistent with hyperosteoidosis that occurs subsequent to a mineralization defect rather than an enhanced rate of bone formation (17); 3) the occurrence of wide tetracycline labels with ground glass appearance (18); and 4) a uniform prolongation of MLT, directly indicating a substantial mineralization defect (19). In addition, HHRH patients showed a decrease in osteoclast parameters, possibly secondary to the increase in OS/BS. Most of the present histomorphometric values recorded in HHRH are similar to those found in XLH (12, 13). However, they differ considerably from results obtained previously in two sporadic cases of seemingly HHRH (20) and in the asymptomatic hypercalciuric subjects reported herein. The latter are similar to reported normal subjects except for their extended OS/BS. Likewise, extended OS/BS was noted in some patients with idiopathic hypercalciuria and was interpreted as a mild mineralization defect (21). As in other instances of impaired mineralization, the HHRH patients show an increase in serum alkaline phosphatase activity (1, 2, 12, 13, 22, 23). Furthermore,
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JCE&M«1991 Vol 72 • No 1
GAZIT ETAL.
234 \ 90
y = 125.342 - 18.878x R = -0.93 o P^O. 0009
\ D
\
a
80
Histomorphometric parameter
70 \
CO CD CO
TABLE 3. Spearman rank correlation coefficients between biochemical and histomorphometric parameters in pooled sample of HHRH patients and asymptomatic hypercalciuric subjects Biochemical parameter sPi"
sAP6
sVIT Dc
OV/BV
-0.928 P < 0.001
0.76 P < 0.048
0.71 P < 0.05
OS/BS
-0.905 P < 0.001
0.81 P < 0.02
NS d
O.Th
-0.88 P < 0.01
0.83 P < 0.02
0.79 P < 0.04
Ob.S/BS
0.67 P < 0.05
NS
NS
Oc.S/BS
0.91 P < 0.001
-0.69 P < 0.04
NS
N.Oc/TA
0.69 P < 0.05
-0.71 P < 0.05
NS
dLS
NS
NS
NS
sLS
NS
NS
NS
MAR
NS
NS
NS
BFR
NS
NS
NS
MLT
-0.83 P < 0.01
0.87 P < 0.01
0.87 P < 0.01
60 50 \
40
50
a
y = 55.821 - 9.622x R = -0.83 p
Vol. 72, No. 1 Printed in U.S.A.
Osteomalacia in Hereditary Hypophosphatemic Rickets with Hypercalciuria: A Correlative ClinicalHistomorphometric Study DAN GAZIT, MARTIN TIEDER, URI A. LIBERMAN, LEA PASSI-EVEN, AND ITAI A. BAB Division of Oral Pathology and Bone Laboratory, Hebrew Uniuersity-Hadassah Faculty of Dental Medicine (D.G., L.P., I.A.B.), Jerusalem; the Pediatric Nephrology Unit, Assaf Harofe Medical Center (M.T.), Zerifin; the Unit of Metabolic Diseases, Beilinson Medical Center (U.A.L.), Petah Tiqua; Sackler School of Medicine, Tel Aviv University (M.T., U.A.L.), Tel Aviv, Israel
vealed a very high inverse correlation and a tight linear relationship between serum phosphorus and osteoid parameters. Serum 1,25-dihydroxyvitamin D, which is low in other forms of hereditary hypophosphatemia and osteomalacia, is elevated in HHRH and correlated positively with osteoid parameters and the mineralization lag time. Serum alkaline phosphatase showed similar relationships. These results as well as the clinical, biochemical, and radiological remission of bone disease consequent to phosphate therapy strongly suggest that in HHRH 1) hypophosphatemia alone is sufficient to cause osteomalacia; and 2) the elevation of 1,25-dihydroxyvitamin D reflects the degree of the primary renal phosphate leak, but is not involved in the pathogenesis of the bone disease. (J Clin Endocrinol Metab 7 1 : 229235,1991)
ABSTRACT. We characterized the bone disease of transilial biopsy specimens from children with hereditary hypophosphatemic rickets with hypercalciuria (HHRH) and genetically related asymptomatic hypercalciuric subjects. All HHRH patients showed irregular mineralization fronts, markedly elevated osteoid surface and seam width, increased number of osteoid lamellae, and prolonged mineralization lag time. These findings are consistent with a mineralization defect and indicate unambiguously that the bone disease in HHRH is osteomalacia. The only abnormality seen in the asymptomatic hypercalciuric subjects was slightly extended osteoid surface. Parametric and nonparametric statistical analyses performed on a pooled sample of HHRH patients and asymptomatic hypercalciuric subjects re-
A
NEW familial syndrome, hereditary hypophosphatemic rickets with hypercalciuria (HHRH), was recently described in a group of closely related members of a Bedouine tribe (1, 2). The disease is manifested in early childhood as bone deformities, short stature, muscle weakness, bone pain, and radiological signs of rickets and osteopenia. A transilial bone biopsy from one patient implied that the osteopenia reflects a mineralization defect. The characteristic biochemical features are increased renal phosphate clearance, hypophosphatemia, elevated serum 1,25-dihydroxyvitamin D [1,25-(OH)2D] concentration, enhanced gastrointestinal absorption of calcium and phosphorus, suppressed parathyroid function, and hypercalciuria. The serum calcium concentration is normal. Of 59 members of the tribe that were evaluated, 15% express HHRH. Of the remaining asymptomatic members, 42% had hypercalciuria and mildly elevated levels
of serum 1,25-(OH)2D. The other biochemical parameters in these subjects were within normal limits, but showed a trend similar to that seen in HHRH patients. It was, therefore, suggested that 1) HHRH patients and asymptomatic subjects with hypercalciuria share a common hereditary renal phosphate leak that leads to the other biochemical abnormalities; and 2) expression of the primary defect and magnitude of the ensuing hypophosphatemia determine which subjects present bone disease and which stay asymptomatic and have only hypercalciuria (2). The present microscopic and histomorphometric study in HHRH patients and genetically related asymptomatic subjects with hypercalciuria establishes that the bone disease in HHRH is osteomalacia. A low serum phosphorus concentration appears to have a key role in determining the extent of the mineralization defect in this disease.
Received May 9, 1990. Address all correspondence and requests for reprints to: U. A. Liberman, M.D., Ph.D., Unit of Metabolic Diseases, Beilinson Medical Center, Petah Tiqva 49100, Israel.
Subjects
Subjects and Methods The study was performed with two groups of 1) HHRH patients and 2) asymptomatic hypercalciuric subjects, 3-15 229
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GAZIT ETAL.
230
(mean ± SD, 8.5 ± 3) and 7-14 (mean ± SD, 8.5 ± 1.5) yr old, respectively. Each group consisted of three females and one male. All eight individuals belonged to the same Bedouin tribe and were closely related genetically (2). The HHRH patients presented the following characteristics: short stature, long bone deformities, bone pain, muscle weakness, and radiological signs of rickets and osteopenia. The hypercalciuric subjects were asymptomatic clinically and appeared normal on skeletal x-rays. The relevant biochemical data of each group are summarized in Fig. 1. Bone biopsy Transilial bone biopsies, 5 mm in diameter, were obtained from all patients and processed as described previously (3). In short, specimens obtained after double tetracycline labeling (Ledermycin, Lederle, Germany) with a 14-day interlabel space were embedded in low viscosity resin (4) and sectioned undecalcified. Sections, 5 nm thick, were stained with modified Masson's stain (5) for general marrow histology, demonstration of bone cells, and distinction between osteoid and mineralized bone. Osteoid lamellae were differentiated from mineralized bone lamellae in 5-/mi sections stained with Von Kossa and toluidine blue and examined by polarized light microscopy. Unstained lO-^m sections were used for fluorescent microscopy of the tetracycline label. Static histomorphometric measurements were performed using a computerized digitizing system (6). Quantitation of the tetracycline labeling was performed with a Merz grid (7). The separation between double labels (dynamic measurements) was measured with a calibrated eyepiece micrometer. The following histomorphometric parameters were defined according to Parfitt et al. (8): trabecular bone volume (BV/TV), osteoid volume (OV/BV), osteoid surface (OS/BS), osteoblast surface (Ob.S/BS), osteoid thickness (O.Th), osteoclast surface (Oc.S/BS), osteoclast number (N.Oc/TA), double labeled surface (dLS/BS), single labeled surface (sLS/BS), mineral appositional rate (MAR), bone formation rate (BFR/BS), and mineralization lag time (MLT). Differences in histomorphometric and biochemical parameters between HHRH patients and asymptomatic subjects were analyzed using the Wilcoxon two-sample test. In addition, Spearman rank correlation coefficients were calculated, and Pearson linear regression analysis was carried out between pairs of biochemical and histomorphometric parameters in a pooled sample of the HHRH and asymptomatic subjects.
JCE & M • 1991 Vol 72 • No 1
revealed a considerable decrease in serum phosphorus concentration and TmP/GFR and a marked elevation in serum 1,25-(OH)2D levels and activity of alkaline phosphatase of bone origin. The serum calcium concentration was normal in both groups. Histologically, patients with HHRH presented an abundance of unmineralized matrix, to the extent that whole trabeculae appeared to be composed of osteoid (Fig. 2). The amount of osteoid in the asymptomatic subjects was normal or slightly elevated (Fig. 3). The osteomalacic nature of the hyperosteoidosis in HHRH is demonstrated by 1) the irregularity of mineralization fronts (Fig. 2), 2) the high number of osteoid lamellae (6-20 lamellae; Fig. 4) compared to that in the hypercalciuric asymptomatic subjects (5 lamellae or less; Fig. 5), and 3) the broad single fluorescent labels commonly found in biopsies from individuals with HHRH (Fig. 6). This labeling pattern was absent in bone specimens from SERUM Pi
TMP/GFR
SERUM ALK. PHOSPH.
SERUM Ca
2.50
2.25
2.00
URINARY CALCIUM
SERUM 1,25(OH)2D 2.50 2.00
Results
1.50
The biochemical comparison between HHRH patients and asymptomatic subjects is shown in Fig. 1. The data presented were obtained before the onset of any therapy. The only abnormalities in hypercalciuric asymptomatic subjects were excessive urinary calcium excretion and slightly elevated serum 1,25-(OH)2D concentration. The serum phosphorus levels and renal tubular reabsorption of phosphorus (measured as TmP/GFR), although being in the low normal range, showed a trend resembling that seen in HHRH. The HHRH patients, on the other hand,
1.00
J
)f>0 n nn
FIG. 1. Biochemical data of HHRH child patients (•) and asymptomatic hypercalciuric children (•). The normal range is indicated by shaded boxes. Serum measurements were made after overnight fasting. SD units, SD units of normal range for age; ALK. PHOSPH., alkaline phosphatase activity of bone origin. Conversion factor for serum calcium to mg/dL, 4.0; conversion factor for urinary calcium to mg/mg creatinine, 0.35. Data are the mean ± SD obtained in four subjects per group.
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OSTEOMALACIA IN HHRH
231
FIG. 2. Photomicrograph of transilial bone biopsy of HHRH patient showing broad osteoid seams (arrows), osteoid trabeculae (OT) and irregular mineralization fronts (double arrows). Modified Masson stain; magnification, X130. FIG. 3. Photomicrogaph of transilial bone biopsy of asymptomatic child with hypercalciuria. Osteoid seams are indicated by arrows. Modified Masson stain; magnification, X130. FIG. 4. Polarized light photomicrograph of transilial bone biopsy of HHRH patient with markedly increased number of osteoid lamellae (arrows). Mineralized bone (MB). Von Kossa-toluidine blue stain; magnification, X180. FIG. 5. Polarized light photomicrograph of transilial bone biopsy of an asymptomatic child with hypercalciuria, showing few osteoid lamellae (arrows). Mineralized bone (MB). Von Kossa-toluidine blue stain; magnification, X360. FlG. 6. Fluorescent photomicrograph of transilial bone biopsy of HHRH patient showing wide fluorescent bands with ground glass appearance (arrows). Unstained; magnification, X200. FlG. 7. Fluorescent photomicrograph of transilial bone biopsy of an asymptomatic child with hypercalciuria, showing single (arrows) and double (double arrows) tetracycline labels. Unstained; magnification, X200.
hypercalciuric symptom-free subjects (Fig. 7). The histomorphometric analysis of transilial bone biopsies from HHRH patients and asymptomatic hypercalciuric subjects is summarized in Tables 1 and 2. In the absence of normal child values from our laboratory,
the present findings were compared with several studies reporting normal data in children of a similar age range. Patients with HHRH exhibited hyperosteoidosis, expressed by a 3- to 8-fold increase in OV/BV, O.Th, and OS/BS over the highest mean values reported in normal
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232
GAZIT ETAL.
JCE&M-I99I Vol 72 • No 1
TABLE 1. Static bone histomorphometric parameters in HHRH, asymptomatic hypercalciuria (HC), XLH, and normal children Parameter BV/TV (%)
Normal 25.5 ± 28.6 ± 28.0 ± 26.4 ± 23.0 ± 26.4 ± 19.0 ±
2.0° 3.8C 3.5d 3.6e 4.0' 3.6s 3.0*
XLH
HHRH
HC
26.0 ± 8.0*
22.9 ± 9.1
19.6 ± 2.3
0.88
OV/BV (%)
2.5 ± 0.3° 4.0 ± 1.4C 1.2 ± 0.5d 1.2 ± 0.5e 1.1 ± 0.5' 1.2 ± 0.5* 2.4 ± 0.5*
14.4 ± S ^ * 17.0, 8.6-29' 19.0 ± 5.0*
29.8 ± 12.2
5.9 ± 2.1
0.03
OS/BS (%)
21.6 ± 2.4° 21.6 ± 8.0c 25.0 ± 3.0d 23.8 ± 6.4e 22.0 ± 2.0' 23.6 ± 6.3*
65.9 ± 7.6"
79.21 ± 12.5
41.0 ± 10.7
0.03
O.Th
11.4 ± 6.4 ± 7.4 ± 6.8 ± 10.2 ±
0.4° 1.2d 1.6* 1.5* 2.06
27.7 ± 8.3" 25.9, 12-41.5' 24.0 ± 7.0*
31.9 ± 12.2
13.3 ± 4.9
0.03
Ob.S/BS (%)
13.9 ± 1.0°
4.4 ± 4.05"
4.1 ± 6.1
8.0 ± 4.0
0.31
1.1 ± 0.3° 7.6 ± 1.7e 1.6 ± 0.05d 2.1 ± l.l e 1.8 ± 0.7* 1.0 ± 0.5*
0.2 ± 0.45h 1.8, 0.6-6.1' 0.9 ± 0.5*
0.29 ± 0.12
1.11 ± 0.42
0.03
0.1 ± 0.02° 0.8 ± 0.4c 0.6 ± 0.2" 0.8 ± 0.38e 0.3 ± 0.1' 0.65 ± 0.2*
0.6, 0.06-2.1'
0.06 ± 0.053
0.28 ± 0.14
0.06
7.0 ± 3.3C
9.7 ± 4.9e 14.0 ± 2.0' 8.4 ± 3.7*
Oc.S/BS (%)
N.Oc/TA (I/mm2)
Data are the mean ± SD obtained in four patients per group. P values for comparison between HHRH and HC were determined by Wilcoxon two-sample test; differences were considered significant at P < 0.05. 0 Baron et al. (9). * Glorieux et al. (20). c Ste-Marie et al. (10). d Glorieux et al. (11). e Marie et al. (14). ' Chen etal. (15). * Marie et al. (16). * Harrel et al. (12). ' Costa et al. (13); mean and range.
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OSTEOMALACIA IN HHRH
233
TABLE 2. Dynamic bone histomorphometric parameters in HHRH, asymptomatic hypercalciuria (HC), XLH, and normal children Normal
XLH
HHRH
HC
P
dLS/BS (%)
>60a
NA
4.09 ± 3.2
14.3 ± 12.2
0.19
sLS/BS (%)
65.4 ± 8.2a 68.8 ± 6.3"
NA
21.6 ± 20.2
9.3 ± 5.9
0.47
MAR (mm/yr)
0.33 ± 0.1c 0.47 ± 0.11°
0.16 ± 0.07d 0.10, 0.05-0.19c
0.25 ± 0.09
0.20 ± 0.05
0.24
BFR/BS (mm3/mm2-yr)
0.281 ± 0.065"
NA
0.11 ± 0.08
0.47 ± 0.59
0.19
MLT (yr)
0.023 ± 0.005°
0.27 ± 0.2d
0.13 ± 0.0
0.06 ± 0.03
0.02
Parameter
Data are the mean ± SD obtained in four patients per group. P values for comparison between HHRH and HC were determined by Wilcoxon two-sample test; differences were considered significant at P < 0.05. NA, Not available. ° Marie et al. (14). 6 Marie et al. (16). c Ste-Marie et al. (10). d Harrel et al. (12). * Costa et al. (13); mean and range.
children. These values are in a range similar to that reported for X-linked hypophosphatemia (XLH) and significantly higher in HHRH than in hypercalciuric subjects (Table 1). Osteoblast and osteoclast parameters were low in HHRH compared to normal children and similar to values reported for XLH. Compared to hypercalciuric subjects, only the Oc.S/BS was significantly lower in HHRH patients with N.Oc/TA, approaching statistical significance (Table 1). By comparison with reported normal values, both HHRH patients and hypercalciuric subjects showed considerable decreases in dLS/BS, sLS/BS, and MAR (Table 2). The BFR/BS was low only in HHRH. The MLT was prolonged in both groups; however, in HHRH this parameter was significantly higher than in hypercalciuria and within the range reported for XLH (Table 2). When values from HHRH patients and asymptomatic subjects were pooled, the Pearson linear regression analysis between histomorphometric and biochemical parameters revealed that all osteoid parameters are inversely and significantly related to the serum phosphorus concentration (Fig. 8). Spearman rank correlation coefficients were also calculated to decrease the probability that the results are a function of data clustering of the respective HHRH and hypercalciuric groups. Generally, these correlations were similar to Pearson's; namely the relationship between serum phosphorus concentration and OS/BS, O.Th., and OV/BV was -0.905 (P < 0.001), -0.88 (P < 0.01), and -0.928 (P < 0.001), respectively. The serum phosphorus levels also showed an inverse correlation with the MLT (r = -0.83; P < 0.01) and a direct correlation with Ob.S/BS, Oc.S/BS, and Oc.N./ TA (Table 3). A positive correlation was found between serum alkaline phosphatase activity and OV/BV, OS/ BS, O.Th, and MLT. In addition, the alkaline phospha-
tase activity was inversely related to resorption parameters. There was a direct correlation between serum 1,25(OH)2D and OV/BV, O.Th, and MLT and no correlation with osteoclast parameters (Table 3). Discussion Previously, the diagnosis of rickets and osteomalacia in HHRH was based mainly on the patients' clinical, radiological, and biochemical parameters (1, 2). Now we have demonstrated osteomalacia unequivocally in all biopsies from HHRH patients. This is supported by 1) the presence of irregular mineralization fronts; 2) the thickening of osteoid seams with an increase in the number of osteoid lamellae, a feature consistent with hyperosteoidosis that occurs subsequent to a mineralization defect rather than an enhanced rate of bone formation (17); 3) the occurrence of wide tetracycline labels with ground glass appearance (18); and 4) a uniform prolongation of MLT, directly indicating a substantial mineralization defect (19). In addition, HHRH patients showed a decrease in osteoclast parameters, possibly secondary to the increase in OS/BS. Most of the present histomorphometric values recorded in HHRH are similar to those found in XLH (12, 13). However, they differ considerably from results obtained previously in two sporadic cases of seemingly HHRH (20) and in the asymptomatic hypercalciuric subjects reported herein. The latter are similar to reported normal subjects except for their extended OS/BS. Likewise, extended OS/BS was noted in some patients with idiopathic hypercalciuria and was interpreted as a mild mineralization defect (21). As in other instances of impaired mineralization, the HHRH patients show an increase in serum alkaline phosphatase activity (1, 2, 12, 13, 22, 23). Furthermore,
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JCE&M«1991 Vol 72 • No 1
GAZIT ETAL.
234 \ 90
y = 125.342 - 18.878x R = -0.93 o P^O. 0009
\ D
\
a
80
Histomorphometric parameter
70 \
CO CD CO
TABLE 3. Spearman rank correlation coefficients between biochemical and histomorphometric parameters in pooled sample of HHRH patients and asymptomatic hypercalciuric subjects Biochemical parameter sPi"
sAP6
sVIT Dc
OV/BV
-0.928 P < 0.001
0.76 P < 0.048
0.71 P < 0.05
OS/BS
-0.905 P < 0.001
0.81 P < 0.02
NS d
O.Th
-0.88 P < 0.01
0.83 P < 0.02
0.79 P < 0.04
Ob.S/BS
0.67 P < 0.05
NS
NS
Oc.S/BS
0.91 P < 0.001
-0.69 P < 0.04
NS
N.Oc/TA
0.69 P < 0.05
-0.71 P < 0.05
NS
dLS
NS
NS
NS
sLS
NS
NS
NS
MAR
NS
NS
NS
BFR
NS
NS
NS
MLT
-0.83 P < 0.01
0.87 P < 0.01
0.87 P < 0.01
60 50 \
40
50
a
y = 55.821 - 9.622x R = -0.83 p
