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Predictive value of serum parathyroid hormone levels for bone turnover in patients on chronic maintenance dialysis
Predictive Value of Serum Parathyroid Hormone Levels for Bone Turnover in Patients on Chronic Maintenance Dialysis Quanle Qi, MD, Marie-Claude Monier-Faugere, MD, Zhaopo Geng, MD, and Hartmut H. Malluche, MD 0 Wii the increasing occurrence of adynamic bone disease, it is essential to determine the level of bone turnover in chronically dialyzed patients before instiiting vitamin D therapy. To assess the value of serum parathyroid hormone (PTH) levels for prediction of bone turnover, we determined sensitivity, specificity, and predictive value positive of serum PTH, alone or in combination with other variables, in 79 patients who underwent one or two bone biopsies. Srum PTH levels were determined by a radioimmunometric assay and were obtained at the time of bone biopsies. Patients were classified into (1) low or normal and (2) high bone turnover according to the value of activation frequency of bone. There were 57 biopsy specimens taken from hemodialysis patients and 39 specimens from continuous ambulatory peritoneal dialysis patients (CAPD). All patients with serum PTH levels within or below the normal range had low or normal bone turnover. Values of serum PTH above 450 pg/mL were 100% and 95.5% specific for high bone turnover in hemodialysis and CAPD patients, respectively. Values of serum PTH between 65 and 450 pg/mL had worse predictive value positive in CAPD patients (48.6% to 78.6%) than in hemodialysis patients (67.3% to 87.1%). When other characteristics of the patients were taken into consideration, only age in hemodialysis patients and serum ionized calcium in CAPD patients improved the predictive value of serum PTH. All hemodialysis patients younger than 45 years of age with serum PTH levels above 65 pg/mL (n = 15) had high bone turnover, and CAPD patients with low or normal bone turnover had higher serum ionized calcium. However, overall, bone turnover could not be predicted by serum PTH measurements in 30% of hemodialysis and 51.3% of CAPD patients. The data suggest that for patients with serum PTH levels between 65 and 450 pg/mL, bone biopsies are indicated to precisely assess bone turnover prior to initiation of vitamin D therapy. 0 1995 by the National Kidney Foundation, Inc. INDEX WORDS: turnover.
Parathyroid
hormone;
renal
osteodystrophy;
R
ECENTLY, the pattern of renal osteodystrophy has changed substantially.‘-4 A progressive but definite decrease in aluminumrelated bone disease has occurred with the replacement of aluminum-containing phosphate binders by calcium salts.4-5 With less aluminum intoxication, patients on chronic maintenance dialysis were expected to exhibit more signs of secondary hyperparathyroidism.6.7 However, the incidence of low turnover renal bone disease (ie, adynamic bone disease) has increased.‘.’ Several factors have been implicated in this increase: the use of calcium salts as phosphate binders, the larger number of diabetic patients requiring From the Division of Nephrology, Bone and Mineral Metabolism, Department of Internal Medicine, University of Kentucky, Lexington, KY. Received April 6, 1995; accepted in revised form June 23, 1995. Supported in part by grants from the Shriners Hospital for Crippled Children, Lexington, KY (no. 15951) Dialysis Clinics, Inc, Lexington, KY (no. 227,233), and the National Kidney Foundation of Kentucky, Louisville, KY. Address reprint requests to Hartmut H. Malluche. MD, Division of Nephrology, Bone and Mineral Metabolism, MN572, University of Kentucky Medical Center, 800 Rose St, Lexington, KY 40536-0084. 0 1995 by the National Kidney Foundation, Inc. 0272.6386/95/X04-0010$3.00/O 622
American
Journal
chronic
dialysis;
mineralized
bone
histology;
bone
chronic maintenance dialysis, the greater number of patients on peritoneal dialysis, and possibly the more widespread and/or more aggressive treatment with 1,25(OH),D3.2.” Those patients with low bone turnover have been shown to have the same decreased intestinal calcium absorption as dialysis patients with severe predominant hyperparathyroidism, but a much lower bone calcium retention than normal controls and other patients on dialysis.x Therefore, patients with adynamic bone disease might not benefit from 1,25(OH)2D3 therapy and may be at risk for developing hypercalcemia and detrimental extraosseous calcifications when subjected to enhanced intestinal absorption, which may contribute, at least in part, to the higher mortality rate of these patients compared with those exhibiting other histologic abnormalities.’ Thus, it is important to know the status of bone turnover in chronically dialyzed patients before instituting vitamin D therapy. Serum parathyroid hormone (PTH) levels are commonly used for assessment of bone turnover in renal failure. Determination of serum FTH levels by a two-site radioimmunometric assay was introduced a few years ago”’ and was found to be more sensitive than the previously used radioimmunoassays. ’ ‘-I3 However, there is no consenof Kidney
Diseases,
Vol26,
No 4 (October),
1995:
pp 622-631
PREDICTIVE
VALUE
OF SERUM
PTH
FOR
BONE
TURNOVER
SW at the present time regarding the serum level of PTH that reflects normal bone turnover in patients with end-stage renal failure. To address this issue, the present study was conducted to precisely assess the value of serum PTH concentrations, determined by radioimmunometric assay, for prediction of the level of bone turnover in uremic patients. Specifically, the study was undertaken to answer whether there is a serum level of PTH (alone or in combination with other serum parameters) which ensures that the level of bone turnover is above normal in patients undergoing chronic maintenance dialysis. MATERIALS
AND METHODS
Patients and Protocol A prospective study was carried out from January 1991 to February 1994 in two dialysis centers from the same geographic area in Kentucky. Inclusion criteria were I8 years of age or older and requirement of chronic maintenance dialysis. Exclusion criteria included gastrointestinal disease, liver disease, malignancies, sarcoidosis, tuberculosis, acquired immunodeficiency syndrome, chronic alcoholism, drug addiction, immobilization, parathyroidectomy, and antiepileptic, steroid, or cyclosporine therapy. All patients for whom a diagnostic bone biopsy was indicated and who met the selection criteria were asked to participate in the study. In addition, randomly selected patients from the dialysis centers were screened, and those who met the inclusion and exclusion criteria were asked to participate in this research study. Seventy-nine patients fulfilled the selection criteria and agreed to participate in the study. Informed consent was obtained. Thirty-nine patients were biopsied for the study only; 40 patients were biopsied for both diagnostic purposes and inclusion in the study. In the latter group of patients, the reasons for biopsies included hypercalcemia (n = 3). hypcrphosphatemia (n = 2) elevation of serum PTH (n = I I), rule out aluminum bone disease (n = 6), extraosseous calcifications (n = 2), bone pain (n = l), and evaluation of renal osteodystrophy (n = 15). Seventeen of the latter patients underwent a repeat bone biopsy for re-evaluation of their underlying bone abnormalities 13.7 +- 0.6 months after their first biopsies (range, 7 to I8 months). Thus, the number of bone biopsy specimens available for analysis was 96. There were 36 women and 43 men, with a mean age of 52.7 2 3.0 years and 46.8 ? 2.5 years, respectively. The primary kidney diseases were hypertensive nephropathy (n = 21) diabetes mellitus (n = 20) chronic glomerulonephritis (n = 12). congenital kidney disease (n = 9), tubular disease (n = 3). interstitial nephritis (n = 2), lupus nephritis (n = I), and Goodpasture’s syndrome (n = I). The underlying renal diseases in the 10 remaining patients were unknown. Forty-five patients were on chronic maintenance hemodialysis and 13 of these patients had a second biopsy. Thirty-four patients were on continuous ambulatory peritoneal dialysis (CAPD), and four of them underwent a repeat biopsy. The mean duration of dialytic therapy at time of biopsy was 34.1
623 2 6.0 months for patients on hemodialysis and 39.2 2 11.7 months for patients undergoing CAPD. All patients on hemodialysis were dialyzed three times weekly, 4 hours per session, using a Terumo hollow fiber dialyzer (Tokyo, Japan) with a 9 pm-thick cuprophane membrane and a Cobe C-2 Rx dialysis machine (Denver, CO). The dialysate calcium content was 2.5 mEq/L (n = 34) or 3.5 mEq/L (n = 23). All patients on CAPD underwent four 1.5- to 2-L exchanges per day using a dialysate containing either 2.5 mEq/L (n = 13) or 3.5 mEq/L (n = 26) of calcium (Dianeal; Baxter, Deerfield, IL). All patients received routine dialysis support medications. Phosphate binding was achieved by calcium salts in 70 patients, aluminum-containing phosphate binders in two patients, and a combination of calcium salts and aluminum hydroxide in 13 patients. Among the 96 studied biopsy specimens, 22 were obtained while patients were treated with daily oral (n = 14, including seven repeat biopsies) or intravenous (n = 8, including five second biopsies) 1,25(OHhD, therapy. Within I week prior to bone biopsies on a day when patients did not receive tetracycline labeling, blood was drawn for determinations of serum levels of total and ionized calcium, phosphorus, alkaline phosphatase, PTH, and osteocaltin. When histomorphometric results were obtained, patients were classified into (1) low or normal and (2) high bone turnover according to the value of activation frequency, the only parameter of bone turnover encompassing both formation and resorption.‘4.‘s The results were compared with values gathered from 25 normal age- and gender-matched controls analyzed using the same histologic and morphometric techniques. The value of serum levels of PTH for prediction of high versus low or normal bone turnover was assessed by calculating sensitivity, specificity, and predictive value positive of serum PTH levels for the two categories of histologically assessed bone tumover.‘h
Analytical Methods Biochemistry. Total and ionized serum calcium, phosphorus, creatinine, and alkaline phosphatase were measured by standard laboratory techniques. Concentrations of serum PIH were determined in duplicate by the two-sites immunoradiometric assay for intact PTH using the Allegro intact PTH assay kit (Nichols Institute, San Juan Capistrano, CA).‘” The intra-assay and interassay variations were 4% and 7%, respectively, with normal values ranging from 15 to 65 pg/ mL. Serum levels of osteocalcin were evaluated in duplicate using a radioimmunometric assay with monoclonal antibodies against human osteocalcin (ELSA-Osteo; Cis Bio Intemational, Gyf-sur-Yvette, France). The intra-assay and interassay coefficients of variation were 3.8% and 5.2%. respectively. Bone biopsies, mineralized bone histology and bone histomorphometry. Patients received tetracycline hydrochloride for bone labeling 20 days prior to bone biopsy for 2 days at a dose of 500 mg orally twice a day. No tetracycline antibiotic was administered for the following IO to 14 days. Tetracycline hydrochloride or demeclocychne hydrochloride (Declomycin; LederIe, Wayne, NJ) was administered for the next 4 days at 500 or 300 mg orally twice a day, respectively. Phosphate binders and other antacids were not given on days of tetracycline administration. All patients were biopsied 3 to 4 days after receiving the second label. Bone samples were taken from the anterior iliac crest with an electric drill (Strau-
QI ET AL
624 mann AG, Waldenburg, Switzerland). Bone samples were processed as previously described.” Sections were stained with the modified Masson-Goldner trichrome stain’*; the aurin tricarboxylic acid stain” was used to measure the extent of aluminum deposits at the bone-osteoid interface and solochrome azurine”’ was used as control and for detection of aluminum within bony trabeculas. Unstained sections were prepared for phase contrast and fluorescent light microscopy. Activation frequency was measured using the Osteoplan System II (Kontron, Munich, Germany) as previously described.“,” Activation frequency represents the inverse of total remodeling period, which includes formation, reversal, resorption, and quiescent periods and is the major parameter of bone tumover.‘4
tion, no differences were found between patients treated and not treated with vitamin D and those who underwent one or two biopsies. All patients were therefore analyzed together. Based on the value of activation frequency, 44 patients presented with low or normal bone turnover and 52 patients presented with high bone turnover (Figs 1 and 2, Table 1). All patients with low bone turnover had either adynamic bone disease (n = 22) or osteomalacia (n = 1). All patients with normal bone turnover (n = 21) presented with mild mixed uremic osteodystrophy, and those with elevated bone turnover had either mixed uremic osteodystrophy (n = 36) or predominant hyperparathyroid bone disease (n = 16). In the group of CAPD patients, there were somewhat more patients with low or normal bone turnover than patients with high bone turnover; this, however, did not reach statistical significance (Table 1). This is further illustrated in Fig 2, in which the relationship between serum levels of PTH and activation frequency in CAPD (r = 0.48, P < 0.01) and hemodialysis (r = 0.55, P < 0.001) patients is shown with an overall correlation coefficient of 0.54 (P < 0.001). Diabetic patients were slightly more numerous in the group of patients with low or normal turnover than in the high bone turnover group, but this did not reach statistical significance in either mode of dialysis. Patients on hemodialysis with low or normal bone turnover were older than hemodialysis patients with high bone turnover (Table 1). The number of patients taking aluminum-con-
Statistical Analysis Results are expressed as mean values 2 SEM. All statistical tests were two-sided. An assigned significance level of 0.05 was used. Differences between numerical variables were made using nonparametric tests, ie, Mann-Whitney U tests and nonparametric analysis of variance. Differences in categorical variables were done using the chi-square tests. Possible relationships between serum levels of PTH and biochemical and histologic parameters were analyzed by linear correlation. Logistic regression using forward stepwise selection was performed to estimate the probability that patients presented with high bone turnover. All computations were performed using the SPSS software package for Windows (SPSS, Chicago, IL) using an IBM PS2 computer (IBM Corp, Boca Raton, FL). RESULTS
There were no differences in clinical characteristics, biochemical parameters, and bone histology between randomly selected patients and those who underwent bone biopsies for diagnostic purposes (PTH: 443 2 54 pg/mL and 402 + 66 pg/mL, respectively; activation frequency: 1.OO2 O.l3/yr and 1.11 + 0.28/yr, respectively; Fig 1). In addi-
10.00 8:::I ‘; E 6 6 s F u. g .-:/I
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Hormone (pg/mI)
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and activation who frequency 79 patients underwent in 98 bone biopsies for diagnostic purposes (solid circles) or who were randomly selected to undergo biopsy
6 0
1. Relationship serum levels
1600
(open&c&).Normalrangea are indicated gular areas.
by the
w&an-
PREDICTIVE
VALUE
OF SERUM
Fig 2. Relationship between setum levels of PTH and activation frequency in 79 patients who underwent 99 bone biopsies and were on CAPD (solii circles) and hemodialysk (open circles). Normal ranges are indicated by the rectangular areas.
PTH
‘; E E G s 2 IL E .2
FOR
BONE
TURNOVER
625
2.00 0
1.75 1.50
0.0
1.25
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200
300
400
Parathyroid
Hormone
500
600 640
1800
(pg/ml)
differences when patient characteristics were compared between the histologic groups in CAPD, hemodialysis, or all patients. Total and ionized serum calcium was significantly higher in CAPD patients with low or nor-
and Clinical Characteristics of 79 Chronically Dialyzed Patients According Bone Turnover at the Time of Bone Biopsies (n = 96)
to Levels of
Bone Turnover
No. of biopsies Patients on hemodialysis (%) Patients on CAPD (%) Males/females Diabetic patients (%) Repeat biopsies Age WI CAPD (range) Hemodialysis (range) Duration on dialysis (mo) CAPD (range) Hemodialysis (range) Dialysate calcium 2.5 mEq/L (% of patients) 3.5 mEq/L (% of patients) Patients receiving: Calcium salts (%) Aluminum binders (%) Daily oral 1,25D (%) Intravenous 1,25D (%) Cumulative doses of aluminum-containing binders during last 12 mo (g) Cumulative dose of 1,25 vitamin D during last 12 mo (CGg) l
Different
from
low or normal
c
l
0
0 0
0
taining phosphate binders and the cumulative dose of aluminum compounds during the year prior to bone biopsies were significantly higher in the high bone turnover group than in the low or normal groups (Table 1). There were no other Table 1. Demographic
*a
0
bone
turnover,
Low or Normal
High
Total
44 36.6 56.4 26/l 6 29.5 7
52 61.4 43.6 24/28 23 10
96 60 40 50146 26 17
43 2 3.8 (20-69) 49 2 2* (20-79)
43 % 2.7 (19.81) 54 2 2.2 (20-79)
44 2 3.7 (19-81) 63 5 2.4 (36-79) 34.3 27.0
+ 7.6 (3.6-132) + 5.4 (1.2-90)
21.4 26.2
2 5.2 (1.2-60) 2 5.2 (1.2-156)
28.7 26.5
2 4.9 (1.2-132) 2 3.8 (1.2-156)
42.6 49.0
57.4 51.0
49.0 51 .o
86.4 2.3 13.6 4.5
86.9 26.9 17.3 13.5
86.5 15.6 15.6 9.4
35 2 3.5 20.7 P < 0.05.
2 9.6
80.7
+ 22.7*
44.9
2 12.8
37.4
k 12.0
29.8
2 7.8
QI ET AL
626
Table 2. Biochemical Characteristics and Stainable Aluminum of 79 Chronically Dialyzed Patients According Levels of Bone Turnover and Mode of Dialysis at Time of Bone Biopsies (n = 96)
to
Bone Turnover Low or Normal
Serum calcium (mg/dL) CAPD Range Hemodialysis Range Serum ionized calcium (mg/dL) CAPD Range Hemodialysis Range Serum phosphorus (mg/dL) CAPD Range Hemodialysis Range Serum alkaline phosphatase (U/L) CAPD Range Hemodialysis Range Serum PTH @g/ml) CAPD Range Hemodialysis Range Serum osteocalcin (ng/mL)* CAPD Range Hemodialysis Range Patients with stainable aluminum 2 30 (%) CAPD Hemodialysis
9.54 ? 0.17 6.0-11.1 9.54 + 0.22 6.0-l 1.6
(9.6)
5.04 5 0.13 4.0-5.5 4.58 2 0.25 4.2-5.3
(5.1)
6.46 t 0.32 3.7-8.7 6.05 k 0.50 2.8-11.6
(6.4)
High
8.91
? 7.4-l 9.36 2 7.7-l
(9.4)
0.23 0.9 0.17 2.8
4.32
(4.4)
(6.0)
Normal
All
(9)t (9.3)
2 0.18 4.0-4.8 4.37 2 0.20 3.4-4.7
(4.25)t
7.16 2 0.59 3.3-l 1.7 7.84 + 0.38 4.1-13.1
(6.7)
(4.55)
(7.5)$
9.3 5 0.15 7.4-l 1 .l 9.43 t 0.13 7.7-12.8
(9.4)
4.85 k 0.13 4.0-5.5 4.45 2 0.15 3.4-5.3
(4.9)
6.76 + 3.3-l 7.14 2 2.8-l
(6.7)
0.31 1.7 0.32 3.1
8.4-10.2
(9.4)
3.8-5.2
(4.45)
2.7-4.5
(7.1)
105 2 9.4 (108) 36-204 119 ? 23 (92) 57-578
174 2 38.1 (121) 51-581 162 ? 34 (108) 61-l ,222
135
2 18.1 (119) 36-581 145 ? 23 (100) 57-l ,222
20-70
195 -+ 33 (150) l-595 130 ? 32 (67) 2-439
646 t 110 (651)t 102-l ,634 650 ? 70 (517)t 79-l ,762
391
? l-l 450 k 2-l
15-65
154 + 30 (102) 38-432 76 -c 14 (71) 17-153
592
320
2 196 (395)t 86-l ,999 559 k 185 (254)t 62-2,151
O§
27.3 36.4
14.3
NOTE. Data are presented as mean values + SEM (median). *t Different from low or normal bone turnover by Mann-Whitney $ Determination of serum osteocalcin in a subset of 53 patients. 3 Different from low or normal by the Chi-square test.
ma1 turnover than in CAPD patients with high turnover (Table 2). Serum phosphorus concentrations were significantly higher in hemodialysis patients with high bone turnover than in hemodialysis patients with low or normal bone turnover (Table 2). Serum PTH and osteocalcin levels (the latter measured in a subset of 53 patients) also were significantly higher in patients with high bone turnover than in patients with low or normal bone turnover in both dialytic modalities (Table 2). In CAPD patients, none of the patients with high bone turnover showed stainable aluminum deposits at the bone-osteoid interface covering
U test:
* P < 0.05,
Values
62 (240) ,634 56 (400) ,762
+ 85 (141) 38-l ,999 317 ? 104 (138) 17-2,151
5.2-55.9
15.4 22.8
t P < 0.01.
30% or more of the bone surface (Table 2). In hemodialysis patients, there were more patients with stainable bone aluminum deposits in the low or normal group than in the high bone turnover group (Table 2); however, this did not reach statistical significance. None of the patients exhibited stainable aluminum deposits within bone. Sensitivity and specificity of serum levels of PTH in determining high bone turnover in hemodialysis and CAPD patients are given in Table 3. Sensitivity and specificity of PTH for predicting low or normal bone turnover represent the reciprocal results of those found for high bone
PREDICTIVE
VALUE
Table 3. Sensitivity, Prediction of High
OF SERUM
PTH
FOR
BONE
TURNOVER
627
Specificity, and Predictive Value Positive of Serum Levels of Parathyroid Hormone for Bone Turnover in 57 Bone Biopsies From Patients on Hemodialysis and 39 Bone Biopsies From Patients on Chronic Ambulatory Peritoneal Dialysis PTH (pghl)
Hemodialysis No. of patients Sensitivity (%) Specificity (%) PVP (%) CAPD No. of patients Sensitivity (%) Specificity (%) PVP (%) Abbreviation:
z-65
>150
>250
>350
>450
>550
>600
35 100 50.0 67.3
34 97.1 72.7 85.0
31 88.6 77.3 86.1
27 77.1 81.8 87.1
21 60.0 100 100
16 45.7 100 100
14 40 100 100
17 100 18.2 48.6
14 82.4 50.0 56.0
11 64.7 68.2 61.1
11 64.7 86.4 78.6
11 64.7 95.5 91.7
11 64.7 95.5 91.7
9 52.9 100 100
PVP, predictive
value
positive.
turnover. All patients with high bone turnover regardless of the mode of dialysis had serum PTH concentrations above the upper limit of normal range (Fig 2, Table 3). In hemodialysis patients, all those with serum PTH concentrations above 450 pg/mL presented with high bone turnover; however, for the patients with serum PTH levels between 65 and 450 pg/ mL, the predictive values positive for high bone turnover ranged from 67.3% to 87.1% (Table 3). Logistic regression analysis in hemodialysis patients for prediction of high bone turnover showed that serum PTH levels and age of the patients were involved in the equation as follows: prediction of high bone turnover = 2.87 + 0.0116 PTH - 0.103 age (P < 0.001) It is of note that all hemodialysis patients younger than 45 year old with a serum PTH level above 65 pg/mL presented with high bone tumover (sensitivity = 100%; Fig 3). Among the hemodialysis patients with serum PTH above 65 pg/mL and older than 45 years, there was a trend, although not significant, to observe relatively more men (45.5%) than women (31.6%) with low or normal bone turnover. However, overall, bone turnover could not be predicted by means of serum PTH alone or in combination with age in 30% of hemodialysis patients. In CAPD patients, all patients with serum PTH levels above 600 pg/mL presented with high bone turnover (Fig 2, Table 3). For serum PTH concentrations
between 65 and 600 pg/mL, the predictive value positive of serum PTH levels for prediction of high bone turnover varied from 18.2% to 95.5% (Table 3). Logistic regression analysis showed that serum PTH levels were part of the equation: prediction of high bone turnover = 10.3 + 0.0068 PTH - 1.41 Ca (P < 0.001) The inclusion of low serum calcium levels in the prediction of high bone turnover was useful only in a minority of patients (n = 4). Overall, prediction of bone turnover could not be done based on serum PTH levels with and without serum calcium in more than half (5 1.3%) of the CAPD patients. The combination of serum PTH results with other patient characteristics, such as gender, duration on dialysis, diabetes, treatment with vitamin D, cumulative intake of vitamin D or aluminum-containing phosphate binders, or degree of aluminum accumulation, did not enhance the diagnostic value of serum PTH for prediction of high bone turnover. It is of note that serum levels of PTH were not different between hemodialysis patients with or without stainable bone aluminum accumulation (559 2 262 pg/mL v 666 + 71 pg/ mL, respectively) at any level of bone turnover. In CAPD patients with low or normal bone tumover, again, no differences in serum PTH were found between patients with or without aluminum (220 2 5 1 pg/mL and 185 + 41 pg/ml, respectively), while none of the CAPD patients with high bone turnover exhibited stainable bone
QI ET AL
80
0
0
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00
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50
30
_----
-
0 0
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-
/. , O;O,li;B , ;,;J, iii&kligg
20 0
100
200
400
300 Parathyroid
Hormone
500
I?00 640
’ 8oo
(open circles) bone turnover. The horizontal dotted line represents age 46 years.
(pg/ml)
alkaline phosphatase levels, alone or in combination did not improve the ability to predict bone turnover. In addition, when predictive values of serum F’TH were analyzed using bone formation rate/ bone surface (mainly a parameter of bone formation that may indirectly indicate bone turnover), results were identical (Fig 4).
aluminum. When patients with aluminum accumulation (n = 19) were removed from analysis, no significant improvement could be obtained in sensitivity, specificity, and predictive value positive of serum PTH levels, alone or in combination for prediction of bone turnover. In the 17 patients who underwent two biopsies, 15 were treated with 1,25(OH)2D3 and serum PTH levels changed in parallel with activation frequency in only half of these patients. In the untreated patients, four of five showed a good agreement between changes in serum FTH levels and bone turnover. Similarly, other biochemical or hormonal parameters, including serum osteocalcin and total
DISCUSSION
The present study shows that patients biopsied for diagnostic purpose are representative of the population of patients on dialysis selected at random. This finding is in agreement with a previous study in which the addition of patients referred
35r n
0 0 0
l 0
l
a: Fig 4. Relationship between serum levels of PTH and bone formation rate/ bone surface in 79 patients who underwent g6 bone biopsies for diagnostic purpose (solid circles) or who were randomly selected to undergo biopsy (open circles). Normal ranges are indicated by the rectangular areas.
l l
!j 10 4 E k L 5 g $
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0 0 l
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0 0
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0 0
100
200
300 Parathyroid
400 Hormone
500 (pg/ml)
600 640
1800
PREDICTIVE
VALUE
OF SERUM
PTH
FOR
BONE
TURNOVER
for clinical findings suggestive of bone disease to randomly selected patients from the same geographic area did not alter the distribution of the various histologic lesions.’ This observation emphasizes the ubiquitous nature of bone abnormalities among uremic patients. Adynamic uremic osteodystrophy was found in 22.9% of the studied patients, which is close to the percentage we found in our previous extensive survey from several geographic areas.2 The present data show that all patients with a serum PTH value within or below the normal range had low or normal turnover, and those patients with serum levels above 450 pg/mL had high bone turnover. However, serum PTH concentrations between 65 and 450 pg/mL, had poor predictive values, especially in patients with CAPD in whom the probability to make the right diagnosis is lower than in hemodialysis patients. The obtained data are different from those in previous reports which advocated that serum PTH levels should be approximately 2 to 2.5 times the upper normal range to ensure optimal bone histology based on normal bone formation rate~“‘ll-l”.2’ In the present study, patients with normal bone turnover assessed by activation frequency had serum PTH levels ranging from 11 to 439 pg/ml, and 57% of them had values of serum PTH above 163 pg/ml, ie, more than 2.5 times the upper normal range. This difference could be attributed to the methods used to assess bone turnover. Previous studies used bone formation rate instead of activation frequency; the latter encompasses both bone formation and bone resorption. Bone formation rates rely only on the activity of osteoblasts. It has been shown that serum PTH levels are a better indicator of osteoblastic than osteoclastic parameters.24,25 In addition, PTH receptors are found in cells of the osteoblastic lineage and not in osteoclasts.2h.27 However, the differences in results could be explained by different methods of assessment of bone turnover if, in the previously published reports, a marked deviation in coupling of bone formation and resorption existed. In the present study, analysis of bone biopsies showed that coupling was preserved and that bone formation rate/ bone surface provided the same information as activation frequency. It is more likely that the limited number of patients,’ ‘,I2 selection bias,’ ‘.I2 or geographic differences3.“.‘2 in previous studies may account for this discrepancy.
629
The observation that serum PTH levels between 65 and 450 pg/mL are not reliable indicators of bone turnover in dialyzed patients may be due to variations in serum PTH, ie, serum PTH levels reflect parathyroid gland activity on the day of measurement, while bone mirrors the long-term effects of parathyroid gland activity. It is of note that this is particularly pertinent to patients treated with daily or pulse intravenous vitamin D, in whom transient cessation of such treatment may induce short-term changes in serum PTH levels. However, this reflects the regular daily care of patients on dialysis and the routine of blood drawings for laboratory tests, ie, before uptake or injection of vitamin D. Further studies are needed to determine whether trends in serum PTH over an extended period of time are more indicative of bone turnover than one isolated measurement. In hemodialysis patients, older patients, especially men, were more likely to have low or normal bone turnover despite elevated serum PTH levels. This is in keeping with previous observations of an association of older age with adynamic bone disease.2*’ The reasons for such an association are unclear. Data on the relationship between bone turnover and age in healthy men are somewhat controversial. Cross-sectional studies have shown that there is no relationship between age and bone tumover,2x-“’ while one longitudinal study demonstrated an increase in bone turnover with age in normal men.3o In normal women, an increase in bone turnover with age is well documented and is due mainly to the effects of menopause.“-” Most women on dialysis are amenorrheic and have lower intestinal calcium absorption (possibly related to lack of estrogen) than age-matched dialyzed men.’ Thus, it is possible that estrogen deficiency contributes to the relatively higher bone turnover in dialyzed women. However, further studies are needed to fully understand why older male patients have a tendency toward lower bone tumover. Moreover, bone turnover is not only regulated by PTH, but also by other humoral and local factors that can depress or stimulate bone tumover, bone formation, and resorption. Immune system alterations in patients on dialysis may stimulate cytokines, such as interleukins 1 and 6, and tumor necrosis factors,34 which may act directly on bone turnover. In addition, uremia
630
QI ET AL
may depress growth factors, especially osteogenic protein 1, which is synthesized by the kidney.“5.3hFurther studies are required to determine whether combinations of serum PTH levels with measurements of other bone proteins or enzymes in blood may yield better information regarding underlying bone turnover. At the present time, the poor predictive value of serum PTH levels suggests that bone biopsies are needed in patients with serum PTH values between 65 and 450 pg/mL before initiation of vitamin D therapy to rule out adynamic bone disease. Administration of oral or intravenous vitamin D in those patients with low bone turnover would not only worsen the bone abnormalities, but also increase the risk of hypercalcemia and extraosseous calcifications.’ ACKNOWLEDGMENT The authors thank Richard M. Wheaton for technical assistance in the preparation of the bone slides, Kenloch Westberry and Terri Sexton for performing determinations of the biochemical parameters, and Louise Tipton for invaluable secretarial assistance. REFERENCES 1. Moriniere P, Cohen Solal ME, Belbrik S, Boudailliez B, Marie A, Westeel PF, Renaud H, Fievet P, Lalau JD, Sebert JL, Fomnier A: Disappearance of aluminic bone disease in a long term asymptomatic dialysis population restricting Al(OH)3 intake: Emergence of an idiopathic adynamic bone disease not related to aluminum. Nephron 53:93101, 1989 2. Malluche HH, Faugere MC: Risk of adynamic bone disease in dialyzed patients. Kidney Int 42:62-67, 1992 (suppl 38) 3. Sherrard DJ, Hertz G, Pei Y, Maloney NA, Greenwood C, Manuel A, Saiphoo C, Fenton SS, Segre GV: The spectrum of bone disease in end-stage renal failure-An evolving disorder. Kidney Int 43:436-442, 1993 4. MaIluche HH, Faugere MC: Renal bone disease 1990: An unmet challenge for the nephrologist. Kidney Int 38:193211, 1990 5. Malluche HH, Faugere MC: Uremic bone disease: Current knowledge, controversial issues and new horizons. Miner Electrolyte Metab l7:281-296, 1991 6. Faugem MC, Malluche HH: Stainable aluminum and no aluminum content reflect histologic changes in bone of dialyzed patients. Kidney Int 30:717-722, 1986 7. Faugere MC, Amala IO, Ritz E, Malluche HH: Loss of bone resulting from accumulation of aluminum in bone of patients undergoing dialysis. J Lab Clin Med lO7:481-487, 1986 8. Kurz P, Monier-Faugere MC, Bognar B, Werner E, Roth P, Vlachojannis J, Malluche HH: Evidence for abnormal calcium homeostasis in patients with adynamic bone disease. Kidney Int 46:855-861, 1994 9. Hertz G, Sherrard DJ, Chan W, Pei Y: Aplastic osteo-
dystrophy: Follow-up after 5 years. J Am Sot Nephrol 5:851, 1994 (abstr) IO. Nussbaum SR, Zahradnik RJ, Lavigne JR, Brennan GL, Nazawa-Ung K, Kim LY, Kentmann HT, Wang CA, Potts JT, Segre GV: Highly sensitive two-site immunoradiometric assay of parathyrin, and its clinical utility in evaluating patients with hypercalcemia. Clin Chem 33: 1364-l 367, 1988 I I. Cohen Solal ME, Sebert JL, Boudailliez B, Marie A, Moriniere P, Gueris J, Bouillon R, Foumier A: Comparison of intact, midregion, and carboxy terminal assays of parathyroid hormone for the diagnosis of bone disease in hemodialyzed patients. J Clin Endocrinol Metab 73:516-524, 1991 12. Quarles LD, Lobaugh B, Murphy G: Intact paratbyroid hormone overestimates the presence and severity of parathyroid-mediated osseous abnormalities in uremia. J Clin Endocrinol Metab 75:145-150, 1992 13. Gallieni M, Brancaccio D, Padovese P, Rolla D, Bedani P, Colantonio G, Bronzieri C, Bagni B, Tar010 G: Low-dose intravenous calcitriol treatment of secondary hyperparathyroidism in hemodialysis patients. Kidney Int 42:1191-I 198, 1992 14. Pa&t AM, Drezner MK, Glorieux FH, Kanis JA, MaIluche HH, Meunier PJ, Gtt SM. Reeker RR: Bone histomorphometry: Standardization of nomenclature, symbols and units. J Bone Miner Res 6:595-610, 1987 15. Eriksen EF, Axelrod DW, Melsen F: Bone Histomorphometry. New York, NY, Raven, 1994 16. Sox HC: Probability theory in the use of diagnostic tests. Ann Intern Med 10460-66, 1986 17. Malluche HH, Faugere MC: Atlas of Mineralized Bone Histology. New York, NY, Karger, 1986 18. Goldner J: A modification of the Masson trichrome technique for routine laboratory purposes. Am J Path01 14:237-243, 1938 19. Lillie PD. Fullmer HM: Metals, anions, exogenous pigments, in Histopathologic Technique and Practical Histochemistry (ed 4). New York, NY, McGraw-Hill, 1976, pp 534-557 20. Denton J, Freemont AJ, Bali J: Detection of distribution of aluminum in bone. J Clin Pathol 37: 136-142, 1984 21. Malluche HH, Sherman D, Meyer W, Massry SG: A new semiautomatic method for quantitative static and dynamic bone histology. Calcif Tissue Int 34439448, 1982 22. MaIluche HH, Meyer W, Sherman D, Massry SG: Quantitative bone histology in 84 normal American subjects. Micromorphometric analysis and evaluation of variance of iliac bone. Calcif Tissue Int 34:449-455, 1982 23. Foumier A, Yvemeau PH. Hue P, Said S, Hamdini N, Eldin HM, Mohageb S, Gprisiu R, Marie A, Cohen SolaI ME, Moriniere P: Adynamic bone disease in patients with uremia. Curr Gpin Nephrol Hypertens 3:396-410, 1994 24. Andress DL, Endres DB, Maloney NA, Kopp JB, Cobum JW, Sherrard DJ: Comparison of parathyroid hormone assays with bone histomorphometry in renal osteodystrophy. J Clin Endocrinol Metab 63:1163-l 169, 1986 25. Faugere M-C, Reitz R, Endress BP, Malluche HH: Serum parathyroid hormone levels reflect preferentially osteoblastic activity in patients with renal failure. Clin Res 32:763A, 1984 (abstr) 26. McSheehy PMJ, Chambers TJ: Osteoblastic cells mediate osteoclastic responsiveness to parathyroid hormone. Endocrinology 118:824-828, 1986
PREDICTIVE
VALUE
OF
SERUM
PTH
FOR
BONE
TURNOVER
27. Lapuz MH, Koszewski NJ, Faugem MC, Jueppner H, Mahuche HH: Demonstration of human parathymid hormone receptor messenger ribonucleic acid in osteoblasts, lining cells and osteocytesof plastic-embedded bone biopsy specimens from uremic patients. J Bone Miner Res 9S232, 1994 (abstr) 28. Epstein S, Poser J, McClintock R, Johnson C, Bryce G, Hui S: Differences in serum bone gla protein with age and sex. Lancet 1:307-310, 1984 29. Catherwood BD, Marcus R, Madvig P, Cheuny AK: Determinants of bone (y-carboxyglutamic acid containing pro tein in plasma of healthy aging subjects. Bone 6:9-13, 1985 30. Orwell ES, Deftos LJ: Serum osteocalcin (BGP) levels in normal men: A longitudinal evaluation reveals an ageassociated increase. J Bone Miner Res 5:259-262, 1990 31. Garner0 P, Grimauz M, Demiaux B, Preaudat C, Seguin P, Delmas PD: Measurement of serum osteocalcin with a human-specific two-site immunoradiometric assay. J Bone Miner Res 7:1389-1398, 1992
631
32. Delmas PD, Stenner D, Wahner HW, Mann KG, Riggs BL: Increase in serum bone gamma carboxyglutamic acid protein with aging in women. J Clin Invest 71:1316-1421, 1983 33. Kelly PJ, Pocock NA, Sambrook PN, Eisman JA: Age and menopause-related changes in indices of bone turnover. J Clin Endocrinol Metab 69: 1160-l 165, 1989 34. Descamps-Latscha B: The immune system in endstage renal disease. Curr Gpin Nephrol Hypertens 2:883-891, 1993 35. Paredes A, Piqueras AI, Briscoe DM, Jones WK. Ozkaynak E, Strange K, Somers M, Harris HW: Localization of osteogenic protein- 1 (OP- 1) mRNA and protein expression in kidney. J Am Sot Nephrol4:700, 1993 (abstr) 36. Piqueras AI, Jones WK. Ozkaynak E, Somers M, Harris HW, Paredes A: Characterization of age specific expression of osteogenic protein-l (OP-I) in rats. J Am Sot Nephrol 4:700, 1993 (abstr)
Parathyroid
hormone;
renal
osteodystrophy;
R
ECENTLY, the pattern of renal osteodystrophy has changed substantially.‘-4 A progressive but definite decrease in aluminumrelated bone disease has occurred with the replacement of aluminum-containing phosphate binders by calcium salts.4-5 With less aluminum intoxication, patients on chronic maintenance dialysis were expected to exhibit more signs of secondary hyperparathyroidism.6.7 However, the incidence of low turnover renal bone disease (ie, adynamic bone disease) has increased.‘.’ Several factors have been implicated in this increase: the use of calcium salts as phosphate binders, the larger number of diabetic patients requiring From the Division of Nephrology, Bone and Mineral Metabolism, Department of Internal Medicine, University of Kentucky, Lexington, KY. Received April 6, 1995; accepted in revised form June 23, 1995. Supported in part by grants from the Shriners Hospital for Crippled Children, Lexington, KY (no. 15951) Dialysis Clinics, Inc, Lexington, KY (no. 227,233), and the National Kidney Foundation of Kentucky, Louisville, KY. Address reprint requests to Hartmut H. Malluche. MD, Division of Nephrology, Bone and Mineral Metabolism, MN572, University of Kentucky Medical Center, 800 Rose St, Lexington, KY 40536-0084. 0 1995 by the National Kidney Foundation, Inc. 0272.6386/95/X04-0010$3.00/O 622
American
Journal
chronic
dialysis;
mineralized
bone
histology;
bone
chronic maintenance dialysis, the greater number of patients on peritoneal dialysis, and possibly the more widespread and/or more aggressive treatment with 1,25(OH),D3.2.” Those patients with low bone turnover have been shown to have the same decreased intestinal calcium absorption as dialysis patients with severe predominant hyperparathyroidism, but a much lower bone calcium retention than normal controls and other patients on dialysis.x Therefore, patients with adynamic bone disease might not benefit from 1,25(OH)2D3 therapy and may be at risk for developing hypercalcemia and detrimental extraosseous calcifications when subjected to enhanced intestinal absorption, which may contribute, at least in part, to the higher mortality rate of these patients compared with those exhibiting other histologic abnormalities.’ Thus, it is important to know the status of bone turnover in chronically dialyzed patients before instituting vitamin D therapy. Serum parathyroid hormone (PTH) levels are commonly used for assessment of bone turnover in renal failure. Determination of serum FTH levels by a two-site radioimmunometric assay was introduced a few years ago”’ and was found to be more sensitive than the previously used radioimmunoassays. ’ ‘-I3 However, there is no consenof Kidney
Diseases,
Vol26,
No 4 (October),
1995:
pp 622-631
PREDICTIVE
VALUE
OF SERUM
PTH
FOR
BONE
TURNOVER
SW at the present time regarding the serum level of PTH that reflects normal bone turnover in patients with end-stage renal failure. To address this issue, the present study was conducted to precisely assess the value of serum PTH concentrations, determined by radioimmunometric assay, for prediction of the level of bone turnover in uremic patients. Specifically, the study was undertaken to answer whether there is a serum level of PTH (alone or in combination with other serum parameters) which ensures that the level of bone turnover is above normal in patients undergoing chronic maintenance dialysis. MATERIALS
AND METHODS
Patients and Protocol A prospective study was carried out from January 1991 to February 1994 in two dialysis centers from the same geographic area in Kentucky. Inclusion criteria were I8 years of age or older and requirement of chronic maintenance dialysis. Exclusion criteria included gastrointestinal disease, liver disease, malignancies, sarcoidosis, tuberculosis, acquired immunodeficiency syndrome, chronic alcoholism, drug addiction, immobilization, parathyroidectomy, and antiepileptic, steroid, or cyclosporine therapy. All patients for whom a diagnostic bone biopsy was indicated and who met the selection criteria were asked to participate in the study. In addition, randomly selected patients from the dialysis centers were screened, and those who met the inclusion and exclusion criteria were asked to participate in this research study. Seventy-nine patients fulfilled the selection criteria and agreed to participate in the study. Informed consent was obtained. Thirty-nine patients were biopsied for the study only; 40 patients were biopsied for both diagnostic purposes and inclusion in the study. In the latter group of patients, the reasons for biopsies included hypercalcemia (n = 3). hypcrphosphatemia (n = 2) elevation of serum PTH (n = I I), rule out aluminum bone disease (n = 6), extraosseous calcifications (n = 2), bone pain (n = l), and evaluation of renal osteodystrophy (n = 15). Seventeen of the latter patients underwent a repeat bone biopsy for re-evaluation of their underlying bone abnormalities 13.7 +- 0.6 months after their first biopsies (range, 7 to I8 months). Thus, the number of bone biopsy specimens available for analysis was 96. There were 36 women and 43 men, with a mean age of 52.7 2 3.0 years and 46.8 ? 2.5 years, respectively. The primary kidney diseases were hypertensive nephropathy (n = 21) diabetes mellitus (n = 20) chronic glomerulonephritis (n = 12). congenital kidney disease (n = 9), tubular disease (n = 3). interstitial nephritis (n = 2), lupus nephritis (n = I), and Goodpasture’s syndrome (n = I). The underlying renal diseases in the 10 remaining patients were unknown. Forty-five patients were on chronic maintenance hemodialysis and 13 of these patients had a second biopsy. Thirty-four patients were on continuous ambulatory peritoneal dialysis (CAPD), and four of them underwent a repeat biopsy. The mean duration of dialytic therapy at time of biopsy was 34.1
623 2 6.0 months for patients on hemodialysis and 39.2 2 11.7 months for patients undergoing CAPD. All patients on hemodialysis were dialyzed three times weekly, 4 hours per session, using a Terumo hollow fiber dialyzer (Tokyo, Japan) with a 9 pm-thick cuprophane membrane and a Cobe C-2 Rx dialysis machine (Denver, CO). The dialysate calcium content was 2.5 mEq/L (n = 34) or 3.5 mEq/L (n = 23). All patients on CAPD underwent four 1.5- to 2-L exchanges per day using a dialysate containing either 2.5 mEq/L (n = 13) or 3.5 mEq/L (n = 26) of calcium (Dianeal; Baxter, Deerfield, IL). All patients received routine dialysis support medications. Phosphate binding was achieved by calcium salts in 70 patients, aluminum-containing phosphate binders in two patients, and a combination of calcium salts and aluminum hydroxide in 13 patients. Among the 96 studied biopsy specimens, 22 were obtained while patients were treated with daily oral (n = 14, including seven repeat biopsies) or intravenous (n = 8, including five second biopsies) 1,25(OHhD, therapy. Within I week prior to bone biopsies on a day when patients did not receive tetracycline labeling, blood was drawn for determinations of serum levels of total and ionized calcium, phosphorus, alkaline phosphatase, PTH, and osteocaltin. When histomorphometric results were obtained, patients were classified into (1) low or normal and (2) high bone turnover according to the value of activation frequency, the only parameter of bone turnover encompassing both formation and resorption.‘4.‘s The results were compared with values gathered from 25 normal age- and gender-matched controls analyzed using the same histologic and morphometric techniques. The value of serum levels of PTH for prediction of high versus low or normal bone turnover was assessed by calculating sensitivity, specificity, and predictive value positive of serum PTH levels for the two categories of histologically assessed bone tumover.‘h
Analytical Methods Biochemistry. Total and ionized serum calcium, phosphorus, creatinine, and alkaline phosphatase were measured by standard laboratory techniques. Concentrations of serum PIH were determined in duplicate by the two-sites immunoradiometric assay for intact PTH using the Allegro intact PTH assay kit (Nichols Institute, San Juan Capistrano, CA).‘” The intra-assay and interassay variations were 4% and 7%, respectively, with normal values ranging from 15 to 65 pg/ mL. Serum levels of osteocalcin were evaluated in duplicate using a radioimmunometric assay with monoclonal antibodies against human osteocalcin (ELSA-Osteo; Cis Bio Intemational, Gyf-sur-Yvette, France). The intra-assay and interassay coefficients of variation were 3.8% and 5.2%. respectively. Bone biopsies, mineralized bone histology and bone histomorphometry. Patients received tetracycline hydrochloride for bone labeling 20 days prior to bone biopsy for 2 days at a dose of 500 mg orally twice a day. No tetracycline antibiotic was administered for the following IO to 14 days. Tetracycline hydrochloride or demeclocychne hydrochloride (Declomycin; LederIe, Wayne, NJ) was administered for the next 4 days at 500 or 300 mg orally twice a day, respectively. Phosphate binders and other antacids were not given on days of tetracycline administration. All patients were biopsied 3 to 4 days after receiving the second label. Bone samples were taken from the anterior iliac crest with an electric drill (Strau-
QI ET AL
624 mann AG, Waldenburg, Switzerland). Bone samples were processed as previously described.” Sections were stained with the modified Masson-Goldner trichrome stain’*; the aurin tricarboxylic acid stain” was used to measure the extent of aluminum deposits at the bone-osteoid interface and solochrome azurine”’ was used as control and for detection of aluminum within bony trabeculas. Unstained sections were prepared for phase contrast and fluorescent light microscopy. Activation frequency was measured using the Osteoplan System II (Kontron, Munich, Germany) as previously described.“,” Activation frequency represents the inverse of total remodeling period, which includes formation, reversal, resorption, and quiescent periods and is the major parameter of bone tumover.‘4
tion, no differences were found between patients treated and not treated with vitamin D and those who underwent one or two biopsies. All patients were therefore analyzed together. Based on the value of activation frequency, 44 patients presented with low or normal bone turnover and 52 patients presented with high bone turnover (Figs 1 and 2, Table 1). All patients with low bone turnover had either adynamic bone disease (n = 22) or osteomalacia (n = 1). All patients with normal bone turnover (n = 21) presented with mild mixed uremic osteodystrophy, and those with elevated bone turnover had either mixed uremic osteodystrophy (n = 36) or predominant hyperparathyroid bone disease (n = 16). In the group of CAPD patients, there were somewhat more patients with low or normal bone turnover than patients with high bone turnover; this, however, did not reach statistical significance (Table 1). This is further illustrated in Fig 2, in which the relationship between serum levels of PTH and activation frequency in CAPD (r = 0.48, P < 0.01) and hemodialysis (r = 0.55, P < 0.001) patients is shown with an overall correlation coefficient of 0.54 (P < 0.001). Diabetic patients were slightly more numerous in the group of patients with low or normal turnover than in the high bone turnover group, but this did not reach statistical significance in either mode of dialysis. Patients on hemodialysis with low or normal bone turnover were older than hemodialysis patients with high bone turnover (Table 1). The number of patients taking aluminum-con-
Statistical Analysis Results are expressed as mean values 2 SEM. All statistical tests were two-sided. An assigned significance level of 0.05 was used. Differences between numerical variables were made using nonparametric tests, ie, Mann-Whitney U tests and nonparametric analysis of variance. Differences in categorical variables were done using the chi-square tests. Possible relationships between serum levels of PTH and biochemical and histologic parameters were analyzed by linear correlation. Logistic regression using forward stepwise selection was performed to estimate the probability that patients presented with high bone turnover. All computations were performed using the SPSS software package for Windows (SPSS, Chicago, IL) using an IBM PS2 computer (IBM Corp, Boca Raton, FL). RESULTS
There were no differences in clinical characteristics, biochemical parameters, and bone histology between randomly selected patients and those who underwent bone biopsies for diagnostic purposes (PTH: 443 2 54 pg/mL and 402 + 66 pg/mL, respectively; activation frequency: 1.OO2 O.l3/yr and 1.11 + 0.28/yr, respectively; Fig 1). In addi-
10.00 8:::I ‘; E 6 6 s F u. g .-:/I
0
l
0
0
2.00
00
0 l
1.75
l l
0.0
1.25
,; 0.75 5 0.50 1,
l -
0.25 (74, &a?
0
0
-
^ y -0
_
0
lO
d
l
l
“@em
l
0
.
100
0
l
l
l
Fig tween
l
0
l
+ee
0
l
l
n
00
l
l
l
1.00
0 0
l
0
1.50
0.00
-OS l
0
0
-e
0
,n
200
I
300 Parathyroid
0
l
400
1
500
Hormone (pg/mI)
Fe--
600640
beof Plli
and activation who frequency 79 patients underwent in 98 bone biopsies for diagnostic purposes (solid circles) or who were randomly selected to undergo biopsy
6 0
1. Relationship serum levels
1600
(open&c&).Normalrangea are indicated gular areas.
by the
w&an-
PREDICTIVE
VALUE
OF SERUM
Fig 2. Relationship between setum levels of PTH and activation frequency in 79 patients who underwent 99 bone biopsies and were on CAPD (solii circles) and hemodialysk (open circles). Normal ranges are indicated by the rectangular areas.
PTH
‘; E E G s 2 IL E .2
FOR
BONE
TURNOVER
625
2.00 0
1.75 1.50
0.0
1.25
0
0
l
a 0 00
0
l
0
1.00
0
l
0.75
0 0
l
l
0
0
o*
8:
!j 0.50 0.25
OJ)O ^ 100
U
200
300
400
Parathyroid
Hormone
500
600 640
1800
(pg/ml)
differences when patient characteristics were compared between the histologic groups in CAPD, hemodialysis, or all patients. Total and ionized serum calcium was significantly higher in CAPD patients with low or nor-
and Clinical Characteristics of 79 Chronically Dialyzed Patients According Bone Turnover at the Time of Bone Biopsies (n = 96)
to Levels of
Bone Turnover
No. of biopsies Patients on hemodialysis (%) Patients on CAPD (%) Males/females Diabetic patients (%) Repeat biopsies Age WI CAPD (range) Hemodialysis (range) Duration on dialysis (mo) CAPD (range) Hemodialysis (range) Dialysate calcium 2.5 mEq/L (% of patients) 3.5 mEq/L (% of patients) Patients receiving: Calcium salts (%) Aluminum binders (%) Daily oral 1,25D (%) Intravenous 1,25D (%) Cumulative doses of aluminum-containing binders during last 12 mo (g) Cumulative dose of 1,25 vitamin D during last 12 mo (CGg) l
Different
from
low or normal
c
l
0
0 0
0
taining phosphate binders and the cumulative dose of aluminum compounds during the year prior to bone biopsies were significantly higher in the high bone turnover group than in the low or normal groups (Table 1). There were no other Table 1. Demographic
*a
0
bone
turnover,
Low or Normal
High
Total
44 36.6 56.4 26/l 6 29.5 7
52 61.4 43.6 24/28 23 10
96 60 40 50146 26 17
43 2 3.8 (20-69) 49 2 2* (20-79)
43 % 2.7 (19.81) 54 2 2.2 (20-79)
44 2 3.7 (19-81) 63 5 2.4 (36-79) 34.3 27.0
+ 7.6 (3.6-132) + 5.4 (1.2-90)
21.4 26.2
2 5.2 (1.2-60) 2 5.2 (1.2-156)
28.7 26.5
2 4.9 (1.2-132) 2 3.8 (1.2-156)
42.6 49.0
57.4 51.0
49.0 51 .o
86.4 2.3 13.6 4.5
86.9 26.9 17.3 13.5
86.5 15.6 15.6 9.4
35 2 3.5 20.7 P < 0.05.
2 9.6
80.7
+ 22.7*
44.9
2 12.8
37.4
k 12.0
29.8
2 7.8
QI ET AL
626
Table 2. Biochemical Characteristics and Stainable Aluminum of 79 Chronically Dialyzed Patients According Levels of Bone Turnover and Mode of Dialysis at Time of Bone Biopsies (n = 96)
to
Bone Turnover Low or Normal
Serum calcium (mg/dL) CAPD Range Hemodialysis Range Serum ionized calcium (mg/dL) CAPD Range Hemodialysis Range Serum phosphorus (mg/dL) CAPD Range Hemodialysis Range Serum alkaline phosphatase (U/L) CAPD Range Hemodialysis Range Serum PTH @g/ml) CAPD Range Hemodialysis Range Serum osteocalcin (ng/mL)* CAPD Range Hemodialysis Range Patients with stainable aluminum 2 30 (%) CAPD Hemodialysis
9.54 ? 0.17 6.0-11.1 9.54 + 0.22 6.0-l 1.6
(9.6)
5.04 5 0.13 4.0-5.5 4.58 2 0.25 4.2-5.3
(5.1)
6.46 t 0.32 3.7-8.7 6.05 k 0.50 2.8-11.6
(6.4)
High
8.91
? 7.4-l 9.36 2 7.7-l
(9.4)
0.23 0.9 0.17 2.8
4.32
(4.4)
(6.0)
Normal
All
(9)t (9.3)
2 0.18 4.0-4.8 4.37 2 0.20 3.4-4.7
(4.25)t
7.16 2 0.59 3.3-l 1.7 7.84 + 0.38 4.1-13.1
(6.7)
(4.55)
(7.5)$
9.3 5 0.15 7.4-l 1 .l 9.43 t 0.13 7.7-12.8
(9.4)
4.85 k 0.13 4.0-5.5 4.45 2 0.15 3.4-5.3
(4.9)
6.76 + 3.3-l 7.14 2 2.8-l
(6.7)
0.31 1.7 0.32 3.1
8.4-10.2
(9.4)
3.8-5.2
(4.45)
2.7-4.5
(7.1)
105 2 9.4 (108) 36-204 119 ? 23 (92) 57-578
174 2 38.1 (121) 51-581 162 ? 34 (108) 61-l ,222
135
2 18.1 (119) 36-581 145 ? 23 (100) 57-l ,222
20-70
195 -+ 33 (150) l-595 130 ? 32 (67) 2-439
646 t 110 (651)t 102-l ,634 650 ? 70 (517)t 79-l ,762
391
? l-l 450 k 2-l
15-65
154 + 30 (102) 38-432 76 -c 14 (71) 17-153
592
320
2 196 (395)t 86-l ,999 559 k 185 (254)t 62-2,151
O§
27.3 36.4
14.3
NOTE. Data are presented as mean values + SEM (median). *t Different from low or normal bone turnover by Mann-Whitney $ Determination of serum osteocalcin in a subset of 53 patients. 3 Different from low or normal by the Chi-square test.
ma1 turnover than in CAPD patients with high turnover (Table 2). Serum phosphorus concentrations were significantly higher in hemodialysis patients with high bone turnover than in hemodialysis patients with low or normal bone turnover (Table 2). Serum PTH and osteocalcin levels (the latter measured in a subset of 53 patients) also were significantly higher in patients with high bone turnover than in patients with low or normal bone turnover in both dialytic modalities (Table 2). In CAPD patients, none of the patients with high bone turnover showed stainable aluminum deposits at the bone-osteoid interface covering
U test:
* P < 0.05,
Values
62 (240) ,634 56 (400) ,762
+ 85 (141) 38-l ,999 317 ? 104 (138) 17-2,151
5.2-55.9
15.4 22.8
t P < 0.01.
30% or more of the bone surface (Table 2). In hemodialysis patients, there were more patients with stainable bone aluminum deposits in the low or normal group than in the high bone turnover group (Table 2); however, this did not reach statistical significance. None of the patients exhibited stainable aluminum deposits within bone. Sensitivity and specificity of serum levels of PTH in determining high bone turnover in hemodialysis and CAPD patients are given in Table 3. Sensitivity and specificity of PTH for predicting low or normal bone turnover represent the reciprocal results of those found for high bone
PREDICTIVE
VALUE
Table 3. Sensitivity, Prediction of High
OF SERUM
PTH
FOR
BONE
TURNOVER
627
Specificity, and Predictive Value Positive of Serum Levels of Parathyroid Hormone for Bone Turnover in 57 Bone Biopsies From Patients on Hemodialysis and 39 Bone Biopsies From Patients on Chronic Ambulatory Peritoneal Dialysis PTH (pghl)
Hemodialysis No. of patients Sensitivity (%) Specificity (%) PVP (%) CAPD No. of patients Sensitivity (%) Specificity (%) PVP (%) Abbreviation:
z-65
>150
>250
>350
>450
>550
>600
35 100 50.0 67.3
34 97.1 72.7 85.0
31 88.6 77.3 86.1
27 77.1 81.8 87.1
21 60.0 100 100
16 45.7 100 100
14 40 100 100
17 100 18.2 48.6
14 82.4 50.0 56.0
11 64.7 68.2 61.1
11 64.7 86.4 78.6
11 64.7 95.5 91.7
11 64.7 95.5 91.7
9 52.9 100 100
PVP, predictive
value
positive.
turnover. All patients with high bone turnover regardless of the mode of dialysis had serum PTH concentrations above the upper limit of normal range (Fig 2, Table 3). In hemodialysis patients, all those with serum PTH concentrations above 450 pg/mL presented with high bone turnover; however, for the patients with serum PTH levels between 65 and 450 pg/ mL, the predictive values positive for high bone turnover ranged from 67.3% to 87.1% (Table 3). Logistic regression analysis in hemodialysis patients for prediction of high bone turnover showed that serum PTH levels and age of the patients were involved in the equation as follows: prediction of high bone turnover = 2.87 + 0.0116 PTH - 0.103 age (P < 0.001) It is of note that all hemodialysis patients younger than 45 year old with a serum PTH level above 65 pg/mL presented with high bone tumover (sensitivity = 100%; Fig 3). Among the hemodialysis patients with serum PTH above 65 pg/mL and older than 45 years, there was a trend, although not significant, to observe relatively more men (45.5%) than women (31.6%) with low or normal bone turnover. However, overall, bone turnover could not be predicted by means of serum PTH alone or in combination with age in 30% of hemodialysis patients. In CAPD patients, all patients with serum PTH levels above 600 pg/mL presented with high bone turnover (Fig 2, Table 3). For serum PTH concentrations
between 65 and 600 pg/mL, the predictive value positive of serum PTH levels for prediction of high bone turnover varied from 18.2% to 95.5% (Table 3). Logistic regression analysis showed that serum PTH levels were part of the equation: prediction of high bone turnover = 10.3 + 0.0068 PTH - 1.41 Ca (P < 0.001) The inclusion of low serum calcium levels in the prediction of high bone turnover was useful only in a minority of patients (n = 4). Overall, prediction of bone turnover could not be done based on serum PTH levels with and without serum calcium in more than half (5 1.3%) of the CAPD patients. The combination of serum PTH results with other patient characteristics, such as gender, duration on dialysis, diabetes, treatment with vitamin D, cumulative intake of vitamin D or aluminum-containing phosphate binders, or degree of aluminum accumulation, did not enhance the diagnostic value of serum PTH for prediction of high bone turnover. It is of note that serum levels of PTH were not different between hemodialysis patients with or without stainable bone aluminum accumulation (559 2 262 pg/mL v 666 + 71 pg/ mL, respectively) at any level of bone turnover. In CAPD patients with low or normal bone tumover, again, no differences in serum PTH were found between patients with or without aluminum (220 2 5 1 pg/mL and 185 + 41 pg/ml, respectively), while none of the CAPD patients with high bone turnover exhibited stainable bone
QI ET AL
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00
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400
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(open circles) bone turnover. The horizontal dotted line represents age 46 years.
(pg/ml)
alkaline phosphatase levels, alone or in combination did not improve the ability to predict bone turnover. In addition, when predictive values of serum F’TH were analyzed using bone formation rate/ bone surface (mainly a parameter of bone formation that may indirectly indicate bone turnover), results were identical (Fig 4).
aluminum. When patients with aluminum accumulation (n = 19) were removed from analysis, no significant improvement could be obtained in sensitivity, specificity, and predictive value positive of serum PTH levels, alone or in combination for prediction of bone turnover. In the 17 patients who underwent two biopsies, 15 were treated with 1,25(OH)2D3 and serum PTH levels changed in parallel with activation frequency in only half of these patients. In the untreated patients, four of five showed a good agreement between changes in serum FTH levels and bone turnover. Similarly, other biochemical or hormonal parameters, including serum osteocalcin and total
DISCUSSION
The present study shows that patients biopsied for diagnostic purpose are representative of the population of patients on dialysis selected at random. This finding is in agreement with a previous study in which the addition of patients referred
35r n
0 0 0
l 0
l
a: Fig 4. Relationship between serum levels of PTH and bone formation rate/ bone surface in 79 patients who underwent g6 bone biopsies for diagnostic purpose (solid circles) or who were randomly selected to undergo biopsy (open circles). Normal ranges are indicated by the rectangular areas.
l l
!j 10 4 E k L 5 g $
l OeO
0 0 l
0
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‘$
0
0
l l
0 0
l
0 0
100
200
300 Parathyroid
400 Hormone
500 (pg/ml)
600 640
1800
PREDICTIVE
VALUE
OF SERUM
PTH
FOR
BONE
TURNOVER
for clinical findings suggestive of bone disease to randomly selected patients from the same geographic area did not alter the distribution of the various histologic lesions.’ This observation emphasizes the ubiquitous nature of bone abnormalities among uremic patients. Adynamic uremic osteodystrophy was found in 22.9% of the studied patients, which is close to the percentage we found in our previous extensive survey from several geographic areas.2 The present data show that all patients with a serum PTH value within or below the normal range had low or normal turnover, and those patients with serum levels above 450 pg/mL had high bone turnover. However, serum PTH concentrations between 65 and 450 pg/mL, had poor predictive values, especially in patients with CAPD in whom the probability to make the right diagnosis is lower than in hemodialysis patients. The obtained data are different from those in previous reports which advocated that serum PTH levels should be approximately 2 to 2.5 times the upper normal range to ensure optimal bone histology based on normal bone formation rate~“‘ll-l”.2’ In the present study, patients with normal bone turnover assessed by activation frequency had serum PTH levels ranging from 11 to 439 pg/ml, and 57% of them had values of serum PTH above 163 pg/ml, ie, more than 2.5 times the upper normal range. This difference could be attributed to the methods used to assess bone turnover. Previous studies used bone formation rate instead of activation frequency; the latter encompasses both bone formation and bone resorption. Bone formation rates rely only on the activity of osteoblasts. It has been shown that serum PTH levels are a better indicator of osteoblastic than osteoclastic parameters.24,25 In addition, PTH receptors are found in cells of the osteoblastic lineage and not in osteoclasts.2h.27 However, the differences in results could be explained by different methods of assessment of bone turnover if, in the previously published reports, a marked deviation in coupling of bone formation and resorption existed. In the present study, analysis of bone biopsies showed that coupling was preserved and that bone formation rate/ bone surface provided the same information as activation frequency. It is more likely that the limited number of patients,’ ‘,I2 selection bias,’ ‘.I2 or geographic differences3.“.‘2 in previous studies may account for this discrepancy.
629
The observation that serum PTH levels between 65 and 450 pg/mL are not reliable indicators of bone turnover in dialyzed patients may be due to variations in serum PTH, ie, serum PTH levels reflect parathyroid gland activity on the day of measurement, while bone mirrors the long-term effects of parathyroid gland activity. It is of note that this is particularly pertinent to patients treated with daily or pulse intravenous vitamin D, in whom transient cessation of such treatment may induce short-term changes in serum PTH levels. However, this reflects the regular daily care of patients on dialysis and the routine of blood drawings for laboratory tests, ie, before uptake or injection of vitamin D. Further studies are needed to determine whether trends in serum PTH over an extended period of time are more indicative of bone turnover than one isolated measurement. In hemodialysis patients, older patients, especially men, were more likely to have low or normal bone turnover despite elevated serum PTH levels. This is in keeping with previous observations of an association of older age with adynamic bone disease.2*’ The reasons for such an association are unclear. Data on the relationship between bone turnover and age in healthy men are somewhat controversial. Cross-sectional studies have shown that there is no relationship between age and bone tumover,2x-“’ while one longitudinal study demonstrated an increase in bone turnover with age in normal men.3o In normal women, an increase in bone turnover with age is well documented and is due mainly to the effects of menopause.“-” Most women on dialysis are amenorrheic and have lower intestinal calcium absorption (possibly related to lack of estrogen) than age-matched dialyzed men.’ Thus, it is possible that estrogen deficiency contributes to the relatively higher bone turnover in dialyzed women. However, further studies are needed to fully understand why older male patients have a tendency toward lower bone tumover. Moreover, bone turnover is not only regulated by PTH, but also by other humoral and local factors that can depress or stimulate bone tumover, bone formation, and resorption. Immune system alterations in patients on dialysis may stimulate cytokines, such as interleukins 1 and 6, and tumor necrosis factors,34 which may act directly on bone turnover. In addition, uremia
630
QI ET AL
may depress growth factors, especially osteogenic protein 1, which is synthesized by the kidney.“5.3hFurther studies are required to determine whether combinations of serum PTH levels with measurements of other bone proteins or enzymes in blood may yield better information regarding underlying bone turnover. At the present time, the poor predictive value of serum PTH levels suggests that bone biopsies are needed in patients with serum PTH values between 65 and 450 pg/mL before initiation of vitamin D therapy to rule out adynamic bone disease. Administration of oral or intravenous vitamin D in those patients with low bone turnover would not only worsen the bone abnormalities, but also increase the risk of hypercalcemia and extraosseous calcifications.’ ACKNOWLEDGMENT The authors thank Richard M. Wheaton for technical assistance in the preparation of the bone slides, Kenloch Westberry and Terri Sexton for performing determinations of the biochemical parameters, and Louise Tipton for invaluable secretarial assistance. REFERENCES 1. Moriniere P, Cohen Solal ME, Belbrik S, Boudailliez B, Marie A, Westeel PF, Renaud H, Fievet P, Lalau JD, Sebert JL, Fomnier A: Disappearance of aluminic bone disease in a long term asymptomatic dialysis population restricting Al(OH)3 intake: Emergence of an idiopathic adynamic bone disease not related to aluminum. Nephron 53:93101, 1989 2. Malluche HH, Faugere MC: Risk of adynamic bone disease in dialyzed patients. Kidney Int 42:62-67, 1992 (suppl 38) 3. Sherrard DJ, Hertz G, Pei Y, Maloney NA, Greenwood C, Manuel A, Saiphoo C, Fenton SS, Segre GV: The spectrum of bone disease in end-stage renal failure-An evolving disorder. Kidney Int 43:436-442, 1993 4. MaIluche HH, Faugere MC: Renal bone disease 1990: An unmet challenge for the nephrologist. Kidney Int 38:193211, 1990 5. Malluche HH, Faugere MC: Uremic bone disease: Current knowledge, controversial issues and new horizons. Miner Electrolyte Metab l7:281-296, 1991 6. Faugem MC, Malluche HH: Stainable aluminum and no aluminum content reflect histologic changes in bone of dialyzed patients. Kidney Int 30:717-722, 1986 7. Faugere MC, Amala IO, Ritz E, Malluche HH: Loss of bone resulting from accumulation of aluminum in bone of patients undergoing dialysis. J Lab Clin Med lO7:481-487, 1986 8. Kurz P, Monier-Faugere MC, Bognar B, Werner E, Roth P, Vlachojannis J, Malluche HH: Evidence for abnormal calcium homeostasis in patients with adynamic bone disease. Kidney Int 46:855-861, 1994 9. Hertz G, Sherrard DJ, Chan W, Pei Y: Aplastic osteo-
dystrophy: Follow-up after 5 years. J Am Sot Nephrol 5:851, 1994 (abstr) IO. Nussbaum SR, Zahradnik RJ, Lavigne JR, Brennan GL, Nazawa-Ung K, Kim LY, Kentmann HT, Wang CA, Potts JT, Segre GV: Highly sensitive two-site immunoradiometric assay of parathyrin, and its clinical utility in evaluating patients with hypercalcemia. Clin Chem 33: 1364-l 367, 1988 I I. Cohen Solal ME, Sebert JL, Boudailliez B, Marie A, Moriniere P, Gueris J, Bouillon R, Foumier A: Comparison of intact, midregion, and carboxy terminal assays of parathyroid hormone for the diagnosis of bone disease in hemodialyzed patients. J Clin Endocrinol Metab 73:516-524, 1991 12. Quarles LD, Lobaugh B, Murphy G: Intact paratbyroid hormone overestimates the presence and severity of parathyroid-mediated osseous abnormalities in uremia. J Clin Endocrinol Metab 75:145-150, 1992 13. Gallieni M, Brancaccio D, Padovese P, Rolla D, Bedani P, Colantonio G, Bronzieri C, Bagni B, Tar010 G: Low-dose intravenous calcitriol treatment of secondary hyperparathyroidism in hemodialysis patients. Kidney Int 42:1191-I 198, 1992 14. Pa&t AM, Drezner MK, Glorieux FH, Kanis JA, MaIluche HH, Meunier PJ, Gtt SM. Reeker RR: Bone histomorphometry: Standardization of nomenclature, symbols and units. J Bone Miner Res 6:595-610, 1987 15. Eriksen EF, Axelrod DW, Melsen F: Bone Histomorphometry. New York, NY, Raven, 1994 16. Sox HC: Probability theory in the use of diagnostic tests. Ann Intern Med 10460-66, 1986 17. Malluche HH, Faugere MC: Atlas of Mineralized Bone Histology. New York, NY, Karger, 1986 18. Goldner J: A modification of the Masson trichrome technique for routine laboratory purposes. Am J Path01 14:237-243, 1938 19. Lillie PD. Fullmer HM: Metals, anions, exogenous pigments, in Histopathologic Technique and Practical Histochemistry (ed 4). New York, NY, McGraw-Hill, 1976, pp 534-557 20. Denton J, Freemont AJ, Bali J: Detection of distribution of aluminum in bone. J Clin Pathol 37: 136-142, 1984 21. Malluche HH, Sherman D, Meyer W, Massry SG: A new semiautomatic method for quantitative static and dynamic bone histology. Calcif Tissue Int 34439448, 1982 22. MaIluche HH, Meyer W, Sherman D, Massry SG: Quantitative bone histology in 84 normal American subjects. Micromorphometric analysis and evaluation of variance of iliac bone. Calcif Tissue Int 34:449-455, 1982 23. Foumier A, Yvemeau PH. Hue P, Said S, Hamdini N, Eldin HM, Mohageb S, Gprisiu R, Marie A, Cohen SolaI ME, Moriniere P: Adynamic bone disease in patients with uremia. Curr Gpin Nephrol Hypertens 3:396-410, 1994 24. Andress DL, Endres DB, Maloney NA, Kopp JB, Cobum JW, Sherrard DJ: Comparison of parathyroid hormone assays with bone histomorphometry in renal osteodystrophy. J Clin Endocrinol Metab 63:1163-l 169, 1986 25. Faugere M-C, Reitz R, Endress BP, Malluche HH: Serum parathyroid hormone levels reflect preferentially osteoblastic activity in patients with renal failure. Clin Res 32:763A, 1984 (abstr) 26. McSheehy PMJ, Chambers TJ: Osteoblastic cells mediate osteoclastic responsiveness to parathyroid hormone. Endocrinology 118:824-828, 1986
PREDICTIVE
VALUE
OF
SERUM
PTH
FOR
BONE
TURNOVER
27. Lapuz MH, Koszewski NJ, Faugem MC, Jueppner H, Mahuche HH: Demonstration of human parathymid hormone receptor messenger ribonucleic acid in osteoblasts, lining cells and osteocytesof plastic-embedded bone biopsy specimens from uremic patients. J Bone Miner Res 9S232, 1994 (abstr) 28. Epstein S, Poser J, McClintock R, Johnson C, Bryce G, Hui S: Differences in serum bone gla protein with age and sex. Lancet 1:307-310, 1984 29. Catherwood BD, Marcus R, Madvig P, Cheuny AK: Determinants of bone (y-carboxyglutamic acid containing pro tein in plasma of healthy aging subjects. Bone 6:9-13, 1985 30. Orwell ES, Deftos LJ: Serum osteocalcin (BGP) levels in normal men: A longitudinal evaluation reveals an ageassociated increase. J Bone Miner Res 5:259-262, 1990 31. Garner0 P, Grimauz M, Demiaux B, Preaudat C, Seguin P, Delmas PD: Measurement of serum osteocalcin with a human-specific two-site immunoradiometric assay. J Bone Miner Res 7:1389-1398, 1992
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32. Delmas PD, Stenner D, Wahner HW, Mann KG, Riggs BL: Increase in serum bone gamma carboxyglutamic acid protein with aging in women. J Clin Invest 71:1316-1421, 1983 33. Kelly PJ, Pocock NA, Sambrook PN, Eisman JA: Age and menopause-related changes in indices of bone turnover. J Clin Endocrinol Metab 69: 1160-l 165, 1989 34. Descamps-Latscha B: The immune system in endstage renal disease. Curr Gpin Nephrol Hypertens 2:883-891, 1993 35. Paredes A, Piqueras AI, Briscoe DM, Jones WK. Ozkaynak E, Strange K, Somers M, Harris HW: Localization of osteogenic protein- 1 (OP- 1) mRNA and protein expression in kidney. J Am Sot Nephrol4:700, 1993 (abstr) 36. Piqueras AI, Jones WK. Ozkaynak E, Somers M, Harris HW, Paredes A: Characterization of age specific expression of osteogenic protein-l (OP-I) in rats. J Am Sot Nephrol 4:700, 1993 (abstr)