Assessment of bone metabolism in cadmium-induced renal tubular dysfunction by measurements of biochemical markers

Toxicology Letters 136 (2003) 183 /192 www.elsevier.com/locate/toxlet

Assessment of bone metabolism in cadmium-induced renal tubular dysfunction by measurements of biochemical markers Keiko Aoshima
, Jianjun Fan, Yunqing Cai, Terutaka Katoh, Hidetoyo Teranishi, Minoru Kasuya Department of Public Health, Faculty of Medicine, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-0194, Japan Received 2 November 2001; received in revised form 3 September 2002; accepted 3 September 2002

Abstract Bone metabolism related to the severity of cadmium (Cd)-induced renal tubular dysfunction (RTD) was assessed by measuring several bone biochemical markers. Fifty-three female subjects with RTD aged 65 /76 years (mean 70.09/3.3 years) and who lived in the Cd-polluted Jinzu River basin in Toyama, Japan were studied. Bone alkaline phosphatase (bone-ALP), intact bone Gla-protein (intact-BGP) and carboxy-terminal propeptide of type I collagen (PICP) in serum as bone formation markers and pyridinoline (Pyr) and deoxypyridinoline (Dpyr) in urine as bone resorption markers were measured. All markers of bone turnover were increased and significantly correlated with each other, suggesting that bone formation and resorption were coupled and increased in Cd-induced RTD. Fractional excretion of b2microglobulin (b2-m, FEb2-m) as an index of severity of Cd-induced RTD was extremely varied ranging from 0.45 to 53%. There were no significant correlations between FEb2-m and each of the five bone biochemical markers. The bone turnover in Cd-induced RTD appeared to be determined by the glomerular filtration rate (GFR): in subjects with GFRs above 50 ml/min, the levels of bone-ALP or intact-BGP tended to be inversely related to the GFRs, whereas in subjects with GFRs below 40 ml/min, those levels tended to decrease. These results suggest that the bone turnover, in particular the bone formation, was influenced by renal tubular function as assessed by the levels of GFR in Cd-induced RTD. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Environmental cadmium exposure; Bone metabolism; Renal tubular dysfunction; Bone biochemical markers; Itai /itai disease

1. Introduction


Corresponding author. Tel.: /81-76-434-7278; fax: /8176-434-5023 E-mail address: [email protected] (K. Aoshima).

Prolonged exposure to cadmium (Cd) in setting of industrial exposure or environmental pollution causes multiple proximal renal tubular dysfunctions (RTD; Friberg et al., 1974; World Health Organization, 1992). Thus, in workers exposed to

0378-4274/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 4 2 7 4 ( 0 2 ) 0 0 3 5 6 - 9

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Cd and inhabitants of a Cd-polluted area, lowmolecular-weight proteinuria such as b2-microglobulinuria, amino-aciduria, glucosuria, bicarbonaturia and phosphaturia due to impaired reabsorption of these substances in the proximal tubules were reported: Cd-induced renal tubular abnormalities were not different from RTD due to other causes (Brewer, 1985). In general, when the RTD presents chronically, metabolic bone disease occurs, either as rickets in children or osteomalacia in adults (Brewer, 1985). Osteomalacia was reported in the most advanced stage of chronic Cd poisoning in workers (Adams et al., 1969; Kazantzis, 1979; Blainey et al., 1980; Marouby, 1980) as well as in inhabitants of a Cdpolluted area, such as itai /itai disease in the Jinzu River basin in Toyama prefecture, Japan (Friberg et al., 1974; Nogawa et al., 1975; Takebayashi et al., 2000). Bone loss has also been observed in the early stage of Cd-induced RTD (Scott et al., 1980; Aoshima et al., 1988; Kido et al., 1990; Tsuritani et al., 1996; Aoshima et al., 1997). The pathogenesis of bone lesions of osteomalacia and osteopenia in Cd-induced RTD was suggested to result from hypophophatemia, hyperchloremic acidosis and hypercalciuria due to impaired reabsorption of phosphorus, bicarbonate and calcium in the proximal tubules (Adams et al., 1969; Friberg et al., 1974; Nogawa et al., 1975; Aoshima et al., 1988). Consequently, the relation between the severity of RTD and bone metabolism indicating bone biochemical markers such as serum total alkaline phosphatase (total-ALP; Aoshima et al., 1988) and urinary hydroxyproline (Nishino et al., 1991) has been studied. Recently, bone Gla-protein (BGP) and bone specific ALP (bone-ALP) have been employed to assess bone metabolism in patients with itai /itai disease and in inhabitants of a Cd-polluted area (Kido et al., 1991; Aoshima et al., 1993; Tsuritani et al., 1994). These studies showed that serum levels of BGP and bone-ALP were significantly higher in Cd-exposed subjects than in controls. The present study was performed to evaluate whether these new bone biochemical markers detect more precisely the early changes in bone effects of Cd-induced RTD and whether the bone

turnover in Cd-induced RTD is related to the severity of RTD.

2. Subjects and methods 2.1. Subjects Sixty-four female subjects who were diagnosed with RTD in our previous examination (Aoshima et al., 1988) were investigated. All subjects had lived in the Cd-polluted Jinzu River basin in Toyama Prefecture, Japan for more than 45 years. Among these 64 subjects examined, 11 were excluded for the following reasons; three were over 79-years old, one showed multiple myeloma, one had gastretomy for gastric cancer, two showed a urine pH lower than 5.80 because of the possibility of degradation of b2-microglobulin (b2-m) in the urine, and four showed serum b2-m higher than 4.5 mg/l because of the possibility of overflow excretion of b2-m. Therefore, 53 subjects aged 65/76 years old (mean 70.09/3.3 years) were evaluated in the present study (Table 1). Of these 53 subjects, none had been taking vitamin D analog, but five used a calcium supplement within 2 months before the present study. Informed consent was obtained from all subjects. 2.2. Renal tubular function and bone mass measurements Blood and 2 h fasting urine samples were obtained between 8:30 and 10:30 h. Height and weight were measured, and then the values of the body surface area and body mass index (BMI) were calculated. Timed-urine volume was measured precisely following measurement of urine pH with a pH meter. Serum calcium (Ca), phosphorus, chloride and creatinine were determined using an autoanalyzer at a commercial laboratory on the day of collection. Samples not analyzed on the day of collection were frozen at /20 8C until analysis. The b2-m concentration in urine and serum were measured using a radioimmunoassay method (b2m RIA Kit, Eiken Chemical Co., Ltd., Tokyo, Japan). Other urinary constituents were determined as follows: creatinine by the Jaffe’s method

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Table 1 Renal tubular function and bone metabolism in 53 female subjects with Cd-induced RTD Characteristic

Mean

Range

Reference range

Age (years) Weight (kg) Height (cm) BMI (kg/m2)

70.0 49.1 146.7 22.7

65 /76 34.0 /73.5 132 /162 16 /34

20 /24

Bone microdensitometry D (mm) d (mm) MCI BMC (mmAl) Serum Phosphorus (mg/100 ml) Calcium (mEq/l) Chloride (mEq/l) Total-ALP (IU)a Bone-ALP (IU)a Intact-BGP (ng/ml)a PICP (ng/ml)a

8.34 5.46 0.346 1.847 3.35 4.61 106.3 218.5 129.4 10.1 130.2

7.24 /9.58 3.90 /6.66 0.230 /0.461 1.37 /2.39 2.8 /4.3 4.3 /4.9 101 /117 113 /602 57 /464 4.1 /33.4 71 /269

2.5 /4.5 4.1 /5.0 98 /108 74 /223 37 /112 4.3 /9.2 30 /182

Renal function TmP/GFR (mg/100 ml) GFR (ml/min per 1.48 m2)b FECa (%)a FEb2-m (%)a

2.758 60.5 3.0 5.1

1.75 /3.68 32 /94 0.3 /8.3 0.45 /53.0

2.1 /3.9 B/60 0.3 /5.2 0.01 /0.29

Urine b2-m (mg/g Cr)a Pyr (pM/mM Cr)a DPyr (pM/mM Cr)a Cadmium (mg/g Cr)a

11.5 52.3 10.6 17.2

0.9 /84.7 31.4 /104.0 4.9 /24.2 5.7 /37.7

0.05 /0.53 17.7 /41.9 2.2 /6.1 1.0 /11.2

a b

Geometric mean. Others are arithmetic mean. Fifty two subjects were examined.

(Creatinine-test Wako, Wako Pure Chemical Ind., Osaka, Japan), phosphorus by a calcimetric assay based on the Fe reaction between phosphate and a molybdate reagent (Phospha B-test Wako), and Ca by the OCPC method (Calcium-C-test Wako). Urinary Cd was measured using atomic absorption spectrometry (Cai et al., 2001). Measurements of creatinine clearance (Ccr), fractional excretion indices for b2-m (FEb2-m) and calcium (FECa), and maximal tubular reabsorption of phosphate corrected for the glomerular filtration rate (TmP/ GFR) were performed as previously described (Aoshima and Kasuya, 1991; Aoshima et al., 1995a). GFR was determined as endogenous Ccr, which was corrected by a standard surface area of

1.48 m2. Urinary analytes were expressed per gram of excreted creatinine (Cr). The reference ranges for these indices are shown in Table 1. Bone mass was measured at the second metacarpal bone by radiographic absorptiometry (Inoue et al., 1983; Van Kuijk and Genant, 1998). The radiographs were analyzed at a commercial laboratory (Mitsubishi Chemical Bio-Clinical Laboratories, Inc., Tokyo). On a posterior /anterior hand radiograph along with an aluminum stepwedge, bone density measurements were performed at the midpoint of the right second metacarpal using a densitometer (Fig. 1). The densitometric pattern was recorded at a ten times magnification and simultaneously the optical den-

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Fig. 1. The density of the transverse section at the mid-point of the right second metacarpal bone was measured on X-ray film. Pattern and various parameters of microradiodensitogram.

sity of each step of the aluminum wedge was measured. From the densitometric pattern shown in Fig. 1, basic measurements were performed: the outer diameter of the periosteum (D ), the inner diameter of the medullary space (d) and the value of the integrated area of bone densitometric pattern (a GS ) indicating the total bone density were obtained. From these findings the metacarpal index (MCI), which is the ratio of cortical thickness of the metacarpal to the total width of the bone (D/d )/D , and metacarpal bone mass (a GS / D ) obtained by the ratio of the densitometric pattern area to the outer width were calculated. The value of a GS /D corresponded to the bone mineral content (BMC) determined by photon absorptiometry (Hayashi et al., 1984). So, the a GS /D was expressed as BMC in this paper (Tables 1/3).

2.3. Bone biochemical markers Serum total-ALP activities were determined by the Bessay /Lowry method and ALP isoenzymes were analyzed by electrophoresis using a cellulose acetate membrane at a commercial laboratory (Biomedical Laboratories Co., Ltd.) on the day of collection. From ALP isoenzymes determination, serum bone-ALP was calculated in each case according to the equation: bone-ALP (IU/l) / total-ALP (IU/l) /relative portion of ALP3, where ALP3 is an isoenzyme form recognized as

originating from bone tissue. Serum intact bone Gla-protein (intact-BGP) was measured by EIA using a kit (Osteocalcin-Test Teijin, Osaka). Carboxy-terminal telopeptide of type I collagen (PICP) in serum was assayed by RIA at a commercial laboratory (Sumikin Bio-Science, Inc. Laboratory, Kanagawa). Urinary pyridinoline (Pyr) and deoxypyridinoline (Dpyr) were measured by high performance liquid chromatography with fluorometric detection at a commercial laboratory (Mitsubishi Chemical Bio-Clinical Laboratories, Inc.). The reference values for totalALP, bone-ALP, intact-BGP, PICP, Pyr and Dpyr are shown in Table 1.

2.4. Statistical analysis Values for serum total-ALP, bone-ALP, intactBGP and PICP, urinary Pyr, Dpyr, b2-m and Cd, and FEb2-m and FECa were log-normally distributed and were log transformed before statistical analysis. Statistical evaluation was carried out following subdivision of the subjects into three groups according to the severity of the RTD based on FEb2-m as follows; Group I, B/3%; Group II, 35/10%; Group III, ‘/10% (Table 3). Analysis of variance (ANOVA) and multiple comparisons analysis with Dunnett’s method between Group I and II or III were performed using the STATVIEW 5.0 software. A P -value of 0.05 was considered statistically significant.

Table 2 Correlation coefficients between parameters of bone metabolism and renal tubular function in 53 female subjects with Cd-induced RTD

a b c d e f

BMI

MCI

BMC

/0.178 /0.080 /0.382c /0.025 /0.025 /0.074 0.002 0.028 /0.048 /0.036 /0.258 /0.036 0.092 /0.121

0.268 0.325a /0.047 /0.096 /0.186 /0.239 /0.143 /0.169 /0.079 0.135 /0.097 /0.164 0.000

0.442d /0.144 /0.150 /0.243 /0.221 /0.156 /0.164 0.116 0.060 /0.161 /0.235 /0.104

/0.184 /0.266 0.883d /0.226 0.591d /0.245 0.516d a /0.298 0.515d /0.245 0.419c 0.192 0.040 0.058 /0.135 /0.224 0.087 /0.229 0.103 /0.174 0.057

P B/0.05. P B/0.01. P B/0.005. P B/0.001. Log-transformed data used. Fifty two subjects were examined.

0.704d 0.614d 0.592d 0.499d /0.069 /0.190 0.133 0.185 0.093

0.662d 0.596d 0.528d 0.062 /0.189 0.068 0.023 0.179

Pyre

Dpyre

TmP/GFR GFRf

0.721d 0.604d 0.818d 0.009 0.247 0.238 /0.102 0.132 0.155 0.410c b 0.366 0.210 0.220 /0.350b /0.040 /0.195 /0.132 /0.553d /0.036 0.054 0.227 0.094

FECae

FEb2-me

/0.222 /0.469d 0.136 0.159 /0.131 /0.019

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BMI MCI BMC Total-ALPe Bone-ALPe Intact-BGPe PICPe Pyre Dpyre TmP/GFR GFR  FECae FEb2-me U /Cde

Total-ALPe Bone-ALPe Intact-BGPe PICPe

Age

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Table 3 Bone metabolism according to fractional excretion of b2-m in 53 female subjects with Cd-induced RTD Characteristic

Fractional excretion of b2-m (%) B/3 Group I (n/19)

Age (years) BMI (kg/m2) Bone microdensitometry D (mm) d (mm) MCI BMC (mmAl) Serum Phosphorus (mg/100 ml) Calcium (mEq/l) Chloride (mEq/l) Total-ALP (IU)a Bone-ALP (IU)a Intact-BGP (ng/ml)a PICP (ng/ml)a

70.2 (3.1) 22.9 (3.0) 8.07 (0.49) 5.15 (0.63) 0.361 (0.067) 1.91 (0.25)

35/10 Group II (n/18) 69.0 (3.5) 22.9 (2.7)

3.29 (0.29) 4.57 (0.12) 106.0 (2.7) 211.3 (1.4) 126.4 (1.6) 9.7 (1.6) 119.1 (1.2)

Renal function TmP/GFR (mg/100 ml) GFR (ml/min per 1.48 m2) FECa (%)a FEb2-m (%)a

2.96 (0.38) 63.2 (15.1) 2.7 (1.5) 1.4 (1.7)

2.80 (0.28) 67.3 (12.7) 2.8 (1.5) 5.1 (1.4)b

Urine b2-m (mg/g Cr)a Pyr (pM/mM Cr)a DPyr (pM/mM Cr)a Cadmium (mg/g Cr)a

3.3 55.9 11.0 14.8

11.9 51.2 11.2 18.3

a b

(1.5)b (1.3) (1.4) (1.5)

‘/10 Group III (n/16) 70.7 (3.1) 22.2 (3.9)

8.52 (0.53)b 5.59 (0.49) 0.343 (0.042) 1.84 (0.18)

3.49 (0.42) 4.63 (0.16) 104.8 (2.3) 216.7 (1.2) 120.2 (1.4) 10.3 (1.4) 136.7 (1.3)

(1.7) (1.3) (1.4) (1.7)

ANOVA P -value

8.44 (0.55) 5.64 (0.71)b 0.332 (0.059) 1.77 (0.20) 3.24 (0.26) 4.63 (0.17) 108.1 (3.4)b 229.0 (1.4) 144.8 (1.6) 10.2 (1.6) 135.8 (1.3)

0.284 0.741 0.030 0.039 0.317 0.188 0.073 0.394 0.004 0.772 0.472 0.916 0.314

2.45 (0.37)b 48.7 (11.3) (15)b 3.4 (2.0) 22.8 (1.5)b

0.003 0.007 0.494 B/0.001

(1.5)b (1.2) (1.3) (2.1)

B/0.001 0.393 0.326 0.376

47.6 49.4 9.5 16.6

Geometric mean (G.S.D.). Others are arithmetic mean (S.D.). Dunnett’s test; P B/0.05, compared with Group I.

3. Results Clinical findings and the results of renal tubular function and bone metabolism including six bone biochemical markers for the 53 subjects are summarized in Table 1, together with the range of reference values. One subject was excluded from the analysis of GFR because of an incorrect 2 h urine sample. All subjects showed extremely increased values of FEb2-m ranging from 0.45 to 53% and urinary excretion of b2-m from 0.9 to 84.7 mg/ g Cr. Twenty-six subjects (50%) showed decreased values of GFR less than 60 ml/min per 1.48 m2, six subjects (11%) showed increased values of FECa

Fig. 2. Relation between serum PICP and urinary pyridinoline in 53 female subjects with Cd-induced RTD.

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Fig. 3. Relation between serum bone-ALP and intact-BGP in 53 female subjects with Cd-induced RTD.

exceeding 5.2%, and two (4%) showed decreased values of TmP/GFR less than 2.1 mg/dl. None of the 53 subjects showed higher or lower values of phosphorus and calcium in serum compared with the reference values. Twelve subjects (23%) showed hyperchloremia. Fifty subjects (94%) showed increased urinary levels of Cd above 11.2 mg/g Cr which was obtained from the inhabitants of a Cdnon-polluted area in Toyama prefecture. The prevalence of subjects with higher values of bone markers than the upper limits of the normal reference ranges were 98% in Dpyr, 76% in Pyr, 62% in bone-ALP, 47% in intact-BGP, 45% in total-ALP and 11% in PICP. Table 2 shows the correlation coefficients between the values of bone remodeling markers and renal tubular function. All correlation coefficients between each of the bone remodeling markers were significant. A high correlation coefficient (r /0.7) was obtained between Pyr and Dpyr, Pyr and PICP (Fig. 2), and bone-ALP and intact-BGP (Fig. 3), suggesting that born turnover in Cdinduced RTD was coupled with bone formation and resorption. In the relation between bone mass and bone biochemical markers, only a weak negative correlation between Pyr and BMC (r/ /0.298, P /0.030) was noted. No significant correlation coefficients were noted between each index of renal tubular function including FEb2-m and each of the six bone biochemical markers, except for a significant correlation between PICP and FECa (r/0.366, P /0.007). Urinary excretion

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of Cd was not significantly correlated with each of the six bone remodeling markers. To consider the relative influence of various independent variables (predictors) on bone mass index of MCI or BMC, multiple linear regression analysis was employed: The variables of age, BMI, serum Ca and phosphorus, GFR, TmP/GFR, FEb2-m, FECa, urinary Cd, and each of the six bone biochemical markers were entered into a stepwise (forward) multiple regression model, following logarithmic transformation of FEb2-m, FECa, urinary Cd and each of the six bone biochemical markers. There were two significant variables (age and BMI, P /0.0002) for BMC (R2 /0.30): BMC /3.10-0.026 /Age/0.025 / BMI (SE of the regression coefficients were 0.008 and 0.010 for age and BMI, respectively), and for MCI only BMI was significant (R2 /0.11, P / 0.015): MCI /0.19-0.007 /BMI. The influence of the severity of RTD based on FEb2-m on bone metabolism was evaluated by dividing the 53 subjects into three groups according to Table 3. The mean values of Group II or III were compared with those of Group I with the lowest FEb2-m categories as the reference group. Bone mass indicated by MCI and BMC tended to decrease with increasing severity of RTD. Serum phosphorus also tended to decrease with increasing FEb2-m, but the difference was not significant (P /0.073). A significant difference was noted in the serum chloride concentrations between the three groups: Group III showed significantly

Fig. 4. Relation between GFR and serum bone-ALP in 52 female subjects with (m) GFRs ‘/50 ml/min and (k) B/50 ml/ min in Cd-induced RTD.

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Fig. 5. Relation between GFR and serum intact-BGP in 52 female subjects with (m) GFRs ‘/50 ml/min and (k) B/50 ml/ min in Cd-induced RTD.

increased values compared with Group I. Group III showed significantly lower values of TmP/GFR and GFR than Group I. There were no significant differences in any bone remodeling markers between the three groups. Figs. 4 and 5 show the relations between GFR and bone-ALP and between GFR and intact-BGP. In 39 subjects with GFRs above 50 ml/min per 1.48 m2, the levels of bone-ALP or intact-BGP appeared to be inversely related to the GFRs; the correlation coefficient was /0.311 (P /0.053) in the former and /0.428 (P/0.007) in the latter. Whereas, in subjects with advanced stage of GFRs below 40 ml/min per 1.48 m2, the levels of boneALP or intact-BGP tended to decrease. These results suggest that bone turnover in Cd-induced RTD assessed by bone biochemical markers was accelerated with decreased GFRs in subjects with GFRs above 50 ml/min per 1.48 m2, while in subjects with GFR below 40 ml/min per 1.48 m2 born turnover was decreased.

4. Discussion We assessed bone turnover using a battery of biochemical markers of bone formation and resorption to clarify the influence of RTD on the bone metabolism in Cd-induced RTD. The results of the present study showed that bone biochemical markers were increased and significantly corre-

lated with each other, suggesting that bone formation and bone resorption were coupled and increased in Cd-induced RTD. Bone metabolism was then evaluated between the three groups according to FEb2-m, because FEb2-m is a useful indicator of the severity of Cd-induced RTD (Aoshima et al., 1995a). Group III with FEb2m ‘/10% showed lower values of bone mass and serum phosphorus than Group I with FEb2-m B/ 3%, although the differences were not significant. The decreased serum phosphorus found in Group III might have resulted from the disturbance to renal tubular reabsorption of phosphate suggested by the significantly lower values of TmP/GFR in Group III than in Group I. Hypophosphatemia resulting from renal tubular phosphate wasting was suggested to impair osteoid mineralization, and the duration and severity of the phosphate leak correlated well with the development of osteomalacia (Clarke et al., 1995). Another important factor that induces abnormalities in mineral and bone metabolism in RTD is chronic metabolic acidosis (Clarke et al., 1995). We reported previously that blood bicarbonate levels were decreased with increasing FEb2-m in Cd-induced RTD in both males and females, and the subjects with FEb2-m ‘/30% could be diagnosed as proximal renal tubular acidosis (Aoshima et al., 1990). We did not perform blood gas analysis in the present study, but hyperchloremia found in Group III suggested that hyperchloremic proximal renal tubular acidosis existed in Group III. Based on these findings mentioned above, Group III appeared to have more affected bone metabolism than Group I or II. However, the bone biochemical markers were not significantly different between the three groups. Bone turnover assessed by bone biochemical markers in Cd-induced RTD appeared to be determined by GFR as shown in Figs. 4 and 5. In subjects with GFRs above 50 ml/min per 1.48 m2, the bone turnover was accelerated with decreased GFRs, while in subjects with GFRs below 40 ml/min per 1.48 m2, the bone turnover appeared to be depressed with decreased GFR. The decrease in GFR in Cd-induced RTD has been reported previously (Adams et al., 1969). This decrease is considered to be secondary to the

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impairment of RTD (Aoshima et al., 1995a). A significant inverse correlation between FEb2-m and GFR (r //0.469, P B/0.001) was also detected in this study (Table 2). A stepwise regression analysis demonstrated that only FEb2-m was significant for GFR when age, BMI, TmP/GFR, FEb2-m, FECa and urinary Cd were entered as independent variables, following logarithmic transformation of FEb2-m, FECa and urinary Cd. Therefore, bone turnover was depressed only during the advanced stage of Cd-induced RTD expressed as increased FEb2-m and decreased GFR. A variety of factors such as 1a,25-dihydroxyvitamin D [1a,25(OH)2D3] and parathyroid hormone (PTH) were identified as being determined by GFR and as influencing the bone metabolism in renal disease (Malluche and Faugere, 1990). In a previous study (Aoshima et al., 1993), vitamin D metabolism was examined in 34 subjects (21 males and 13 females) with Cd-induced RTD who showed FEb2-m above 10%: The serum levels of 1a,25(OH)2D3 were normal or increased but positively related to GFR in both genders. The serum levels of intact-PTH were slightly higher than the upper limit of the normal range. However, in the present study, we did not perform the measurements of serum levels of 1a,25(OH)2D3 and PTH, although these factors might play pivotal roles in influencing the bone metabolism in Cd-induced RTD (Nogawa et al., 1987; Tsuritani et al., 1992). Therefore, further studies including polymorphisms in the vitamin D receptor gene are needed to investigate the bone metabolism in Cd-induced RTD including patients with itai /itai disease, who show typical osteomalacia. In the present study, no significant correlation was noted between urinary Cd and FEb2-m (r// 0.019, P /0.05) (Table 2). In our previous study (Aoshima et al., 1995b), however, a significant correlation was observed between them (r /0.329, P B/0.01) in 73 female subjects, who included in the present study. The reason why there was no significant correlation between urinary Cd and FEb2-m in the present study could not be fully explained. However, one reason might be that in the Jinzu River basin an intervention program consisting of soil replacement of polluted paddy

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fields, which has been continually carried out since 1980, resulted in the difference of the intake of Cd through rice between the subjects who had produced rice in their paddy fields with or without Cd-free soil replacement. (Fan et al., 1998; Cai et al., 2001). In conclusion, the findings of the present study revealed that bone turnover assessed by a battery of biochemical markers in Cd-induced RTD was influenced by renal tubular function as assessed by the levels of GFR.

Acknowledgements This study was supported by a Grant-in-Aid for Scientific Research (B) No. 09480121 from the Ministry of Education, Science, Sports and Culture, Japan. We thank Dr Shigetsugu Hagino for his valuable advice and Yumiko Kawanishi and Kiyo Watanabe for assistance throughout this study.

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