A new assay method that detects only intact osteocalcin. Two-step non-invasive diagnosis to predict adynamic bone disease in haemodialysed patients
Tetsuo Morishita,
Masanori Nomura,
Masato Hanaoka,
Takayuki Saruta,
Takatoshi Matsuo and
Yusuke Tsukamoto
Division of Nephrology, Fourth Department of Medicine, Kitasato University School of Medicine, Kanagawa, Japan
 |
Abstract
|
---|
Background. We studied the usefulness of a new assay method that detects only the intact human osteocalcin molecule in haemodialysed patients.
Methods. Iliac bone biopsy specimens obtained from 62 haemodialysed patients were analysed.
Results. Bone formation rates (BFR/BS) correlated positively with serum intact osteocalcin concentrations (n=62), osteocalcin concentrations assayed by a conventional method (n=31), parathyroid hormone (PTH) concentrations (n=62), and total alkaline phosphatase concentrations (r=0.602, 0.588, 0.650, and 0.401 respectively). Based on ROC curve and Youden index analysis, the optimal cut-off value to distinguish adynamic bone disease from a mild lesion was 195 pg/ml of serum PTH concentration (Youden index=0.233) or 30 ng/ml of serum intact osteocalcin concentration (Youden index=0.628). The optimal cut-off value to distinguish between hyperparathyroid bone and a mild lesion was 455 pg/ml of serum PTH level (Youden index=0.63) or 50 ng=ml of serum intact osteocalcin concentration (Youden index=0.634). Since both ROC curve and Youden index suggested that the serum PTH concentration was not a good marker to distinguish adynamic bone from a mild lesion or hyperparathyroid bone, we designed a two-step procedure. The first step was the diagnosis of adynamic bone (cut-off: 65 pg/ml) or hyperparathyroid bone (cut-off: 455 pg/ml) according to serum PTH concentration. In a second step, we assessed serum intact osteocalcin concentration in patients with serum PTH concentrations between 65 and 455 pg/ml. The cut-off values for adynamic and hyperparathyroid bone in this diagnostic approach were 30 and 70 ng/ml respectively. As a result, 49 out of 62 patients were diagnosed properly. The Youden index of this two-step diagnosis was 0.527 and 0.661 for adynamic bone and hyperparathyroid bone respectively. Sensitivity markedly improved to 94.4% and 96.2% for adynamic bone and hyperparathyroid bone respectively, without sacrificing specificity (84.0 and 88.8% respectively).
Conclusion. Measurement of serum intact osteocalcin concentration is useful for the diagnosis of adynamic bone in haemodialysed patients. A two-step procedure involving also simultaneous measurement of serum PTH concentration further improved the sensitivity of each individual marker while maintaining specificity.
Keywords: bone formation rates; haemodialysed patients; hyperparathyroid bone; intact osteocalcin assay; mild lesions; parathyroid hormone concentrations; Youden index
 |
Introduction
|
---|
Historically, dialysis therapy has aimed at suppression of parathyroid hormone (PTH) secretion as the only goal in the treatment of renal osteodystrophy. However, the recent increase in adynamic (or aplastic) bone disease in the dialysis population have alerted us to the risk generated by oversuppression of PTH secretion [1,2]. In fact, our recent national survey of 9395 chronic dialysis patients in Japan revealed that 36.2% showed serum intact PTH concentration lower than 60 pg/ml [3]. A serum PTH concentration below 60 or 65 pg/ml strongly suggests adynamic bone. However, the sensitivity of serum PTH concentration to predict adynamic bone is not sufficient. It has been postulated that bone lesions in patients with serum PTH concentration between 65 and 460 pg/ml could not be diagnosed without bone biopsy [4]. In order to increase the diagnostic utility of non-invasive methods, various serum parameters have been examined for correlation with bone histomorphometric parameters. Of these, serum bone-type alkaline phosphatase or osteocalcin concentrations have been demonstrated to have a significant positive correlation with osteoblastic activity and bone formation rates [511]. A
-carboxyglutamic-acid-containing protein, osteocalcin, is still considered to play a key role in mineralization [1216] although a study using knock-out mice could not confirm this hypothesis [11]. Serum osteocalcin concentration may reflect bone mineralization as well as bone formation rate. Indeed, a number of studies have reported that the serum osteocalcin concentration was correlated with the bone formation rate. However, this positive correlation was not greater than that for serum intact PTH concentrations. The major problem in determining osteocalcin by conventional radioimmunoassays is distinguishing intact osteocalcin from inactive fragments that accumulate in renal failure. To overcome this problem, the assay that only detects the intact molecule of serum osteocalcin was developed [17]. In this study we examined the diagnostic value of this serum intact osteocalcin measurement for predicting bone formation rate and compared the diagnostic utility of this assay to other conventional serum bone markers. For this analysis we employed the Receiver Operating Characteristic (ROC) curve and Youden index to decide the optimal cut-off value for the diagnosis and to evaluate its diagnostic value. Finally, we further modified the cut-off value to improve its sensitivity.
 |
Subjects and methods
|
---|
Patients
Eighty patients on chronic haemodialysis consented to undergo bone biopsy between January 1992 and April 1998. The bone biopsies were performed for the following reasons: symptomatic bone disease with bone pain and or fractures, suspicion of aluminium bone disease or adynamic bone, or to evaluate the indication of parathyroidectomy, outcome of oral calcitriol pulse therapy, or aetiology of hypercalcaemia. Sixty-two patients (31 males and 31 females) with adequate bone samples as determined by tetracycline double labelling and complete biochemical data were enrolled in this study. Of the biopsies included in the study, 15 bone samples exhibited positive aluminium staining and nine out of 15 samples showed a stainable bone aluminium surface (BSA) of more than 25% [18,19]. The mean patient age was 53±9 years (range 3874) and the mean duration of haemodialysis was 108±60 months (range 3242). The presumed aetiology of chronic renal failure was non-diabetic chronic renal disease in every patient. None of the patients had undergone renal transplantation. All patients were given calcium carbonate as a phosphate binder at the time of biopsy. Daily calcitriol or alfacalcidol (0.250.5 µg/day) was also given to every patient except for 10 patients who received oral calcitriol pulse therapy at the time of biopsy. Five patients had a history of parathyroidectomy. None of the patients received any other medication that might affect calcium metabolism, such as glucocorticoid, sex hormones, iprivoflabon, calcitonin, or vitamin K. Patients were dialysed three times weekly, 4 h each, using a hollow-fibre dialyser. The dialysate contained 3.5 mEq/l calcium.
Bone biopsies
Approximately 20 days prior to bone biopsy, all patients received tetracycline hydrochloride (Achromycin®, Japan Lederle, Tokyo, Japan) at a dose of 750 mg/day for 2 days. No tetracycline was administered for the following 10 days. Tetracycline hydrochloride was administered again for the next 3 days at 750 mg/day. Calcium carbonate was not given on days of tetracycline administration. All patients were biopsied 47 days after receiving the second tetracycline dose.
Under local anaesthesia, bone samples were taken from the anterior iliac crest with a vertical approach using a specially made trephine of 5 mm inner diameter.
Mineralized bone histological studies.
Samples were fixed in absolute ethanol, dehydrated, and embedded in methylmethacrylate according to Malluche and Faugere [20]. Serial sections 5 µm thick were cut with a ReichertJung microtome, Model 2050 (Finetech Scientific Instruments, Tokyo, Japan). Four serial sections were stained by the modified Goldner trichrome stain, and by the aurin tricarboxylic acid stain for detection of aluminium [18]. In addition, two unstained sections were used for fluorescence microscopy. Histomorphometric parameters were measured in trabecular bone using the semiautomatic computer program OsteoMeasure (OsteoMetrics Inc., Atlanta, GA, USA) and were expressed according to the standard nomenclature [21]. These assessments included total trabecular bone volume (BV/TV) expressed as a percentage of total bone volume; osteoid volume (OV/BV) as a percentage of trabecular bone volume; volume of fibrosis (Fb/TV) as a percentage of cancellous space; osteoid seam thickness (O Th, µ); osteoblast surface (Ob.S/BS) defined as osteoid surface covered with plump osteoblasts and expressed as a percentage of trabecular bone surface; osteoclast surface (Oc.S/BS) defined as the fraction of trabecular bone surface with osteoclasts; eroded surface (ES/BS) as a percentage of trabecular bone surface; mineral apposition rate (MAR, in µm/day); and the bone formation rate at the tissue level (BFR/BS in µm3/µm2/day), obtained by multiplying MAR by the total labelled mineralizing surface (determined as the sum of double-labelled surfaces and half of single-labelled surfaces). Bone biopsy results were classified into four groups according to histomorphometry data: hyperparathyroid bone group (n=26) defined as an increased bone formation rate (BFR/BS>47 µm3/µm2/year) and/or increased bone marrow fibrosis (Fb.V/TV
0.5%) accompanied with an increased osteoclastic activity (Oc.S/BS>0.8%); adynamic bone group (n=18) defined as a decreased bone formation rate (BFR/BS<10 µm3/µm2/year) without increased osteoid volume (OV/BV<5%); osteomalacia group (n=3) defined as a decreased bone formation rate (BFR/BS <10 µm3/µm2/year) with an increased osteoid thickness (O.Th>11.0 µm); and mild lesion group (n=15) defined as a normal bone formation rate (BFR/BS between 10 and 47 µm3/µm2/year) with normal Fb.V/TV (<0.5%), Oc.S/BS(<0.8%), and OV/BV (<15%). In this category of the disease, mixed uraemic osteodystrophy (increased BFR/BS with mineralization defect) was included in the hyperparathyroid bone group. Since a bone dynamic parameter of healthy Japanese has never been available, the normal range of BFR/BS reported by Garcia-Carrasco was employed in this study because it represents the lowest values reported in normal individuals [22].
Biochemical
Blood samples were drawn from patients after 12-h fast on the morning of bone biopsy. Bone biopsies were performed between 10 and 11 a.m. Blood was left standing at room temperature for 30 min before centrifugation. Separated serum was frozen at -20°C until assay. Serum parathyroid hormone (PTH) concentrations were measured with an intact PTH assay kit (Allegro®, Japan Mediphysics Co., Tokyo, Japan), which only detects intact PTH (normal 1565 pg/ml). Serum osteocalcin concentrations were measured by two different assays. One method employed a kit (Teijin Co., Tokyo, Japan) that only detects intact osteocalcin (intact osteocalcin concentration, normal <7 ng/ml) [17] and the other used a kit (Yamasa Co., Tokyo, Japan) that utilizes rabbit antiserum against bovine osteocalcin (conventional osteocalcin concentration, normal <12 ng/ml). Conventional osteocalcin concentration was performed in only 36 patients because of limited availability of the assay. Serum concentrations of calcium (corrected for serum albumin concentration), phosphorus, and total alkaline phosphatase (normal: 73248 IU/l) were determined with an AutoAnalyser (Hitachi Instruments, Tokyo, Japan).
Statistical analysis and calculation of post-test probability
All results are presented as the mean±one standard deviation (SD). Comparison between groups was performed using one-way analysis of variance (ANOVA). The relationship between histomorphometric and biochemical data was assessed by linear correlation analysis. The predictive value of biochemical parameters for the diagnosis of bone disease was expressed in terms of sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). Sensitivity is defined as the ability of a biochemical parameter to identify patients with selected bone disease within a population; specificity represents the ability of a biochemical test to exclude those without a given disease. PPV defines the probability of a disease if a test result is positive, NPV defines the probability of a disease being absent if a test result is negative [23]. The optimal cut-off value was estimated from the ROC curve [24]. Youden index was calculated for the determination of the diagnostic value of each cut-off level [25].
 |
Results
|
---|
Correlation between bone histomorphometric measurements and serum parameters
Correlation coefficients are shown in Table 1
. Significant correlation was found between BFR/BS and four serum parameters. The correlation coefficient was 0.650 for PTH concentration, 0.602 for intact osteocalcin concentration, 0.588 for conventional osteocalcin concentration (n=36), and 0.401 for alkaline phosphatase concentration. Correlation coefficient for alkaline phosphatase was significantly lower than those for the other three parameters (P<0.05). Since none of the serum parameters correlated with MAR or Mlt, LS/BS was the determinant factor of correlation between BFR/BS and each of the four serum parameters. Serum intact osteocalcin concentration correlated significantly with parameters for bone formation (OV/BV, Fb.V/TV, Ob.S/BS, LS/BS and BFR/BS). Serum conventional osteocalcin concentration correlated with parameters for both bone formation (Ob.S/BS, LS/BS, Fb.V/TV) and resorption (ES/BS, Oc.S./BS). Serum PTH concentration correlated with bone marrow fibrosis and bone formation (Ob.S/BV, LS/BS). Serum alkaline phosphatase concentration had the strongest correlation with bone marrow fibrosis and osteoid volume among serum bone markers.
The effect of aluminium accumulation on these correlations was evaluated by bone surface aluminium [18,19]. Twelve patients showed BSA higher than 25% (four adynamic bone, four hyperparathyroid bone, two mild lesion, two osteomalacia). The correlation coefficients of BFR/BS and serum PTH, intact osteocalcin, and alkaline phosphatase in these patients were 0.489, 0.747, and 0.495 respectively. The correlation coefficient significantly decreased with serum PTH concentration (P<0.05), while that with serum intact osteocalcin was maintained.
Differences in serum parameters and clinical data among adynamic bone, osteomalacia, mild change, and hyperparathyroid bone (Table 2
)
Since there were only three patients with osteomalacia, statistical comparison was studied among the other three bone groups. There was no difference in age or duration of haemodialysis among the groups. Serum calcium concentration was significantly higher in the hyperparathyroid bone group than the adynamic bone group (P<0.05). There was no difference in serum phosphorus concentration between the groups. Serum PTH, intact osteocalcin, and conventional osteocalcin concentrations were significantly higher in the hyperparathyroid bone group than the mild lesion group. Serum alkaline phosphatase concentration was significantly different between the hyperparathyroid bone group and the adynamic bone group. The only significant difference between the mild lesion and the adynamic bone groups was the serum intact osteocalcin concentration. Although no significant difference was found in serum aluminium concentration among the groups, the incidence of positive bone surface aluminium (>0%) was higher in the hyperparathyroid bone group than the adynamic bone group.
Differences in bone histomorphometric data among the bone groups as shown in Table 3
Osteoid volume was significantly greater in the hyperparathyroid bone group than the adynamic bone group. There was no significant difference in either osteoid surface or osteoblastic surface (Ob.S/BS) between the adynamic bone group and the mild lesion group. The percentage of woven osteoid volume (OV(Wo)/OV) was also significantly greater in the hyperparathyroid bone group than the adynamic bone group. Eroded surface (ES/BS) and osteoclastic surface (Oc.S/BS) were significantly higher in the hyperparathyroid bone group than the mild lesion or adynamic bone groups. Bone marrow fibrosis volume (Fb.V/TV) was significantly higher in the hyperparathyroid bone group than in the mild lesion or adynamic bone groups. There was a significant difference in BFR/BS among the groups.
Prediction of bone pathology by serum parameters
ROC curves are illustrated in Figures 1
and 2
. According to the analysis by the ROC curve and Youden index, the optimal cut-off value to distinguish between a diagnosis of adynamic bone from mild lesion was 195 pg/ml of serum PTH concentration (Youden index=0.233) or 30 ng/ml of serum intact osteocalcin concentration (Youden index=0.628). The cut-off value to distinguish between a diagnosis of hyperparathyroid bone from mild lesion was 455 pg/ml of serum PTH (Youden index=0.63) or 50 ng/ml of serum intact osteocalcin concentration (Youden index=0.634).

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 1. ROC curves for serum PTH concentration in (left) adynamic bone and (right) hyperparathyroid bone. True positive rate represents sensitivity (%) and false positive rate represents [100-specificity] (%).
|
|

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 2. ROC curves for serum intact osteocalcin concentration in (left) adynamic bone and (right) hyperparathyroid bone. True positive rate represents sensitivity (%) and false positive rate represents [100-specificity] (%).
|
|
PPV was 60% and NPV was only 8.8% when the cut-off value of 195 pg/ml of serum PTH was employed for the diagnosis of adynamic bone (Table 4
). With a cut-off value of 65 pg/ml, specificity, PPV, and NPV reached 95.1, 71.4 and 25% respectively. Diagnostic specificity of serum intact PTH concentration for hyperparathyroid bone reached 94.1% at concentrations greater than 455 pg/ml. NPV was also high (82%) for serum PTH concentration above 455 pg/ml.
At intact osteocalcin concentrations below 30 ng/ml (Table 5
), the diagnostic specificity and sensitivity for adynamic bone were above 90% (90 and 94.4% respectively). However, for the diagnosis of hyperparathyroid bone, a serum intact osteocalcin concentration above 70 ng/ml was required to achieve a specificity of 90%. Both specificity and PPV reached 100% at a concentration above 80 ng/ml.
Since both the ROC curve and Youden index suggested that serum PTH concentration was not a good marker to distinguish between a diagnosis of adynamic bone from a mild lesion or hyperparathyroid bone, we designed the two-step method of diagnosis as shown in Figure 3
. The first step is the diagnosis of adynamic bone or hyperparathyroid bone according to serum PTH concentration. As a result, six patients with adynamic bone and two with mild lesion were diagnosed as adynamic bone because their PTH concentrations were below 65 pg/ml. Twenty patients with PTH concentrations above 455 pg/ml were properly diagnosed as hyperparathyroid bone with one exception of mild lesion. For the second step, we applied serum intact osteocalcin concentration to patients with their serum PTH concentrations between 65 and 455 pg/ml. Nine patients with serum PTH concentrations between 65 and 455 pg/ml also had serum intact osteocalcin concentrations between 30 and 70 ng/ml. Among these patients, every patient but two was diagnosed as mild lesion. By this diagnostic approach, 49 patients out of 62 were diagnosed properly. In the rest, there was no misdiagnosis between adynamic bone and hyperparathyroid bone and the judgement of treatment according to this criterion would not harm the present status of bone. Youden index of this two-step diagnosis was calculated as 0.527 and 0.661 for adynamic bone and hyperparathyroid bone respectively. Sensitivity improved to 94.4% and 96.2% for adynamic bone and hyperparathyroid bone respectively without sacrificing specificity (84.0 and 88.9% respectively). For a diagnosis of mild lesion, sensitivity and specificity was 41.2 and 84.4% respectively. If we employed 195 pg/ml of serum PTH as the cut-off value for adynamic bone and 50 ng/ml of intact osteocalcin for hyperparathyroid bone, specificity decreased to 68.0 and 80.6% for adynamic and hyperparathyroid bone respectively (more false-positive patients). For a diagnosis of mild lesion, sensitivity turned to 0%.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 3. Two-step diagnosis of bone pathology by serum PTH and intact osteocalcin measurements. Out of 62 patients, eight had serum PTH below 65 pg/ml and 20 had concentrations above 455 pg/ml. Out of 34 patients with serum PTH between 65 and 455 pg/ml, 16 had serum intact osteocalcin below 30 ng/ml, nine between 30 and 70 ng/ml, and nine patients had concentrations above 70 ng/ml. Forty-nine patients (79%) were diagnosed accurately by this non-invasive diagnostic approach. Abbreviations: ADB, adynamic bone; HPB, hyperparathyroid bone; MC, mild lesion; OM, osteomalacia. Numbers of patients are shown in parentheses.
|
|
Diagnosis of osteomalacia was not possible using these criteria, although the number was too low to reach any conclusions.
 |
Discussion
|
---|
Osteocalcin or bone Gla-protein is a vitamin K- dependent protein secreted from osteoblasts. This peptide has been found to have selective binding affinity for insoluble Ca2+ salt, especially hydroxyapatite crystals of bone. Although the mechanism has not been fully elucidated, it seems to play an important role in the mineralization of osteoid matrix [1216]. The serum concentration of osteocalcin has been shown to correlate with histomorphometric parameters for bone formation such as osteoblastic surface and bone formation rate [79,24]. Serum osteocalcin concentration has also been reported to correlate with parameters of bone resorption in uraemic patients. In contrast to non-uraemic serum, heterogeneous immunoreactive peptide fragments are present in uraemic serum, some of which are formed by cathepsin D secreted from osteoclasts [26,27]. Since all studies employed a radioimmunoassay (RIA) using rabbit antiserum against calf osteocalcin based on the method of Price and Nishimoto [28], this assay detects not only active intact molecules but also inactive fragments in uraemic serum [27]. To employ this parameter to predict bone histology in uraemia, an assay that detects only intact human osteocalcin was required. For this purpose, a new sandwich immunoassay method for human intact osteocalcin peptide was developed [17]. Initially we compared the results obtained with this new assay with a conventional RIA which uses a rabbit antiserum against bovine osteocalcin, a serum intact PTH assay and a total alkaline phosphatase assay. Correlation of serum intact PTH and conventional osteocalcin assay with BFR/BS) was almost identical to that for serum intact osteocalcin assay. The correlation with serum total alkaline phosphatase concentration was not as good as for other parameters. This is probably due to the fact that this assay does not measure bone-specific enzyme activity. Indeed, a recent report showed that bone-specific alkaline phosphatase is a good marker for adynamic bone [29]. Every parameter could distinguish adynamic bone from hyperparathyroid bone, although only intact osteocalcin concentration could distinguish adynamic bone from mild lesion. Both the ROC curve analysis and Youden index clearly demonstrated the advantage of serum intact osteocalcin measurement. This indicates that the assay for intact osteocalcin is very useful for predicting bone formation rate in patients with mild lesion or adynamic bone because accumulation of inactive fragments does not affect intact osteocalcin measurements, particularly at low to normal concentrations. Probability testing revealed that with intact osteocalcin concentrations below 30 ng/ml, patients were more likely to have adynamic bone disease.
In this study, three cases of aluminium-induced osteomalacia were included. Two of these had serum intact osteocalcin concentrations below 30 ng/ml, although in another patient it was as high as 70 ng/ml. It is interesting that the high correlation between BFR/BS and serum intact osteocalcin was maintained in patients with high bone surface aluminium, while the correlation with serum PTH decreased. This supports the finding that aluminium depresses BFR and favours hypercalcaemia independently of PTH concentration [30]. These patients did not manifest severe mineralization defects in this study. For detecting the risk of aluminium-bone disease, a low-dose desferrioxamine test is also a good index [31].
Intact osteocalcin concentrations did not correlate with bone resorption parameters as well as conventional osteocalcin concentrations (e.g. 0.21 vs 0.634 for ES/BS). This result is in accordance with a study that showed the presence of osteoclast-cleaved fragments of osteocalcin in uraemic serum [27]. It is thought that the conventional osteocalcin assay measures osteoclast-cleaved osteocalcin fragments and is correlated with bone resorption. Another interesting aspect of our study is that correlation between each parameter and bone formation rate resulted from correlation not only with mineral apposition rate, but also with labelled surface. This suggests that osteocalcin stimulates both recruitment of osteoblasts and osteoblastic activity for mineralization of osteoid matrix. Another interesting finding was a good correlation between serum alkaline phosphatase concentration and marrow fibrosis volume or woven osteoid volume. This suggests that the serum alkaline phosphatase concentration reflects the ability of osteoblasts to produce non-calceous bone matrix.
This study confirmed the clinical usefulness of measuring serum PTH concentration (intact PTH assay), as reported by other investigators [4,32]. The ROC curve indicates that the optimal cut-off value for serum PTH concentration is 195 pg/ml and 455 pg/ml for adynamic and hyperparathyroid bone respectively. However, the Youden index was very low (0.233) at 195 pg/ml for adynamic bone. If the cut-off value was set at 65 pg/ml, specificity and sensitivity was 95 and 27.8% respectively. Sensitivity improved to 61% with PTH concentration at 130 pg/ml, which agreed well with other studies [4,32]. For prediction of the presence of hyperparathyroid bone, 94% specificity was achieved with serum PTH concentrations above 455 pg/ml and sensitivity was also good at this range (73%). This result was also almost identical to the results obtained by Qi et al. [4] (460 pg/ml) and Wang et al. [32] (500 pg/ml). Instead of 195 pg/ml, we propose a cut-off value of 65 pg/ml for serum PTH concentration to diagnose adynamic bone in order to increase specificity. Similarly, we suggest a cut-off value of 70 ng/ml rather than 50 ng/ml serum intact osteocalcin concentration for the diagnosis of hyperparathyroid bone in order to increase specificity. By using these cut-off values, we designed the two-step diagnosis method to improve poor diagnostic utility of serum PTH concentration especially for adynamic bone. Eventually, 13 patients (21%) were diagnosed as having mild lesion because they had serum intact osteocalcin concentrations between 30 and 70 ng/ml. The summary of misinterpretation of biopsy results due to this new diagnostic approach was as follows: five biopsies with mild lesion were diagnosed as adynamic bone, three biopsies with mild lesion as hyperparathyroid bone, one biopsy with adynamic bone as mild lesion and one biopsy with hyperparathyroid bone as mild lesion. From the therapeutic point of view, none of these misinterpretations of bone pathology was harmful to the patients because none of the hyperparathyroid bone was misinterpreted as adynamic bone or vice versa.
In conclusion, serum intact osteocalcin concentration is a very useful serum bone marker, especially for the diagnosis of adynamic bone, and the two-step diagnosis method, which employed both serum PTH and intact osteocalcin concentrations, can predict bone pathology at a very high sensitivity and specificity in haemodialysed patients.
 |
Acknowledgments
|
---|
We thank Dr H. H. Malluche, Dr Marie-Claude Faugere, and Mr R. Wheaton, Department of Medicine, University of Kentucky, for teaching us techniques of bone biopsy, preparation of undecalcified bone specimens, and histomorphometry. We also thank Prof. Moritosi Itoman, Department of Orthopedics, Kitasato University, for his valuable advice regarding bone histology. We also thank our dialysis specialists, Drs N. Kobayashi, T. Nagaoka, and K. Takahashi for their valuable continuous support. We thank Ms M. Saitoh for her excellent technical assistance. This work was supported by a grant from the Japanese Ministry of Welfare.
 |
Notes
|
---|
Correspondence and offprint requests to: Yusuke Tsukamoto MD, Director, Gerontology Research Institute, Morishita Memorial Hospital 4-2-18 Higashirinkan, Sagamihara-shi, Kanagawa 228-0811, Japan. 
 |
References
|
---|
-
Malluche HH, Monier-Faugere MC. Risk of adynamic bone disease in dialyzed patients. Kidney Int1992; 38 [Suppl]: S62S67
-
Sherrard DJ, Hercz G, Pei Y et al. The spectrum of bone disease in end-stage renal failurean evolving disorder. Kidney Int1993; 43: 436442[ISI][Medline]
-
Akizawa T, Kinugasa E, Akiba T, Tsukamoto Y, Kurokawa K. Incidence and clinical characteristics of hypoparathyroidism in dialysis patients. Kidney Int1997; 52 [Suppl. 62]: S72S74[ISI]
-
Qi Q, Monier-Faugere MC, Geng Z, Malluche HH. Predictive value of serum parathyroid hormone levels for bone turnover in patients on chronic maintenance dialysis. Am J Kidney Dis1995; 26: 622631[ISI][Medline]
-
Malluche HH, Faugere M-C, Fanti P, Price PA. Plasma levels of bone Gla-protein reflect bone formation in patients on chronic maintenance dialysis. Kidney Int1984; 26: 869874[ISI][Medline]
-
Epstein S, Traberg H, Raja R, Poser J. Serum and dialysate osteocalcin levels in hemodialysis and peritoneal dialysis patients and after renal transplantation. J Clin Endocrinol Metab1985; 60: 12531256[Abstract]
-
Coen G, Mazzaferro S, Bonucci E et al. Bone GLA protein in predialysis chronic renal failure. Effects of 1,25(OH)2D3 administration in a long-term follow-up. Kidney Int1985; 28: 783790[ISI][Medline]
-
Charhon SA, Delmas PD, Malaval L et al. Serum bone Gla-protein in renal osteodystrophy. comparison with bone histomorphometry. J Clin Endocrinol Metab1986; 63: 892897[Abstract]
-
Mazzaferro S, Coen G, Ballanti P et al. Osteocalcin, iPTH, alkaline phosphatase and hand X-ray scores as predictive indices of histomorphometric parameters in renal osteodystrophy. Nephron1990; 56: 261266[ISI][Medline]
-
Martinez ME, Selgas R, Miguel JL, Balaguer G, Sanchez-Cabezudo MJ, Llach F. Osteocalcin levels in uremic patients: influence of calcitriol treatment through two different routes and type of dialysis. Nephron1991; 59: 429433[ISI][Medline]
-
Ducy P, Desbois C, Boyce B et al. Increased bone formation in osteocalcin-deficient mice. Nature1996; 382: 448452[ISI][Medline]
-
Mikuni-Takagaki Y, Kakai Y, Satoyoshi M et al. Matrix mineralization and the differentiation of osteocyte-like cells in culture. J Bone Miner Res1995; 10: 231242[ISI][Medline]
-
Koshihara Y, Hoshi K, Ishibashi H, Shiraki M. Vitamin K2 promotes 1alpha,25(OH)2 vitamin D3-induced mineralization in human periosteal osteoblasts. Calcif Tissue Int1996; 59: 466473[ISI][Medline]
-
Ohgushi H, Dohi Y, Katuda T, Tamai S, Tabata S, Suwa Y. In vitro bone formation by rat marrow cell culture. J Biomed Mater Res1996; 32: 333340[ISI][Medline]
-
Koshihara Y, Hoshi K. Vitamin K2 enhances osteocalcin accumulation in the extracellular matrix of human osteoblasts in vitro. J Bone Miner Res1997; 12: 431438[ISI][Medline]
-
Boskey AL, Gadaleta S, Gundberg C, Doty SB, Ducy P, Karsenty G. Fourier transform infrared microspectroscopic analysis of bones of osteocalcin-deficient mice provides insight into the function of osteocalcin. Bone1998; 23: 187196[ISI][Medline]
-
Hosoda K, Eguchi H, Nakamoto T et al. A sandwich immunoassay for intact human osteocalcin. Clin Chem1992; 38: 22332238[Abstract]
-
Faugere MC, Malluche HH. Stainable aluminum and not aluminum content reflects bone histology in dialyzed patients. Kidney Int1986; 30: 717722[ISI][Medline]
-
Nebeker HG, Andress DL, Milliner DS et al. Indirect methods for the diagnosis of aluminum bone disease. plasma aluminum, the desferrioxamine infusion test, and serum iPTH. Kidney Int1986; 18 [Suppl]: S96S99
-
Malluche HH, Faugere M-C. Atlas of Mineralized Bone Histology. Karger, Basel, 1986
-
Parfitt AM, Drezner MK, Glorieux FH et al. Bone histomorphometry. Standardization of nomenclature, symbols, and units. Report of the ASBMR Histomorphometry Nomenclature Committee. J Bone Miner Res1987; 2: 595610[ISI][Medline]
-
Garcia-Carrasco M, Gruson M, De Vernejoul MC, Denne MA, Miravet L. Osteocalcin and bone morphometric parameters in adults. Calcif Tissue Int1988; 42: 1317[ISI][Medline]
-
Sox HCJ. Probability theory in the use of diagnostic tests. Ann Intern Med1986; 104: 6066[ISI][Medline]
-
Hanley JA, McNeil BJ. The meaning and use of the area under a Receiver Operating Characteristic (ROC) curve. Diagn Radiol1982; 143: 2936
-
Youden WJ. Index for rating diagnostic tests. Cancer1950; 3: 3235[ISI]
-
Deftos LJ, Wolfert RL, Hill CS, Burton DW. Two-site assays of bone gla protein (osteocalcin) demonstrate immunochemical heterogeneity of the intact molecule. Clin Chem1992; 38: 23182321[Abstract]
-
Gundberg CM, Weinstein RS. Multiple immunoreactive forms of osteocalcin in uremic serum. J Clin Invest1986; 77: 17621777[ISI][Medline]
-
Price PA, Nishimoto SK. Radioimmunoassay for the vitamin K-dependent protein of bone and its discovery in plasma. Proc Natl Acad Sci USA1980; 1980: 22342238
-
Jarava C, Armas JR, Salgueira M, Palma A. Bone alkaline phosphatase isoenzyme in renal osteodystrophy. Nephrol Dial Transplant1996; 11 [Suppl 3]: 4346[ISI][Medline]
-
Sebert JL, Marie A, Gueris J et al. Assessment of the aluminum overload and of its possible toxicity in asymptomatic uremic patients. evidence for a depressive effect on bone formation. Bone1985; 6: 373375[ISI][Medline]
-
D'Haese PC, Couttenye MM, Goodman WG et al. Use of the low-dose desferrioxamine test to diagnose and differentiate between patients with aluminium-related bone disease, increased risk for aluminium toxicity, or aluminium overload. Nephrol Dial Transplant1995; 10: 18741884[Abstract]
-
Wang M, Hercz G, Sherrard DJ, Maloney NA, Segre GV, Pei Y. Relationship between intact 184 parathyroid hormone and bone histomorphometric parameters in dialysis patients without aluminum toxicity. Am J Kidney Dis1995; 26: 836844[ISI][Medline]
Received for publication: 26. 6.98
Revision received 3.12.99.