Effects of long-term treatment with loop diuretics on bone mineral density, calcitropic hormones and bone turnover

Authors


Lars Rejnmark, University Department of Endocrinology and Metabolism C, Aarhus Sygehus, Aarhus University Hospital, Tage-Hansens Gade 2, DK-8000 Aarhus C, Denmark.
(fax: +45 8949 7684; e-mail: rejnmark@post6.tele.dk).

Abstract.

Background.  Loop diuretics (LD) are widely used in the treatment of cardiovascular diseases and disorders with fluid accumulation. LD are known to increase renal calcium losses and may thereby affect calcium homeostasis and bone metabolism.

Objective.  We studied to what extent long-term treatment with LD affects calcium homeostasis and bone metabolism.

Design and subjects.  In a cross-sectional design we compared 140 postmenopausal women treated with a LD for more than 2 years with 140 age-matched women not in diuretic therapy.

Results.  Treatment with LD was associated with significantly increased urinary calcium, plasma parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D levels. Per 40 mg day−1 of furosemide, urinary calcium was increased by 17% (P < 0.05) and plasma PTH levels were increased by 28% (P = 0.04). Users of LD had a 17% higher body weight (P < 0.001) compared with nonusers. This was due to a 32% higher fat mass (P < 0.001) and a 6% higher lean tissue mass (P < 0.001). Moreover, users of LD had a higher bone mineral density (BMD) at the spine (+7.5%, P < 0.001), hip (+4.8%, P = 0.004), forearm (+3.7%, P = 0.01) and whole body (+2.5%, P = 0.06). However, after adjustment for body weight differences, BMD did not differ between groups. Nevertheless, duration of LD treatment was positively associated with BMD at the spine (P = 0.03) and whole body (P < 0.05). BMD at the spine increases by 0.3% per 1 year of treatment.

Conclusions.  The increased renal calcium losses in users of LD are compensated for by a PTH-dependent increase in 1,25(OH)2D levels. Thereby calcium balance remains neutral without major effects on bone metabolism.

Introduction

Loop diuretics (LD) are commonly used drugs [1]. In addition to their effect on the renal and cardiovascular system, treatment with LD is known to increase the renal calcium excretion. In addition, short-term treatment with LD increases plasma parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D (1,25(OH)2D) levels in a dose-dependent manner [2–5], and treatment with LD changes the diurnal rhythm of plasma PTH levels with an almost doubling in PTH levels a few hours after medication [6]. However, the potential effects of LD on bone mineral density (BMD) and fracture risk have only been investigated in a few studies with conflicting results [7–10]. In a cohort study including 348 postmenopausal women followed for 5 ± 1.7 years, use of LD was associated with an increased risk of osteoporotic fractures (RR = 2.5, 95% CI 1.1–5.7) [7]. Similarly, in a case–control study [8], current users of LD had a nearly fourfold increased risk of hip fractures (OR = 3.9, 95% CI 1.5–10.4). However, in other two case–control studies, no association was found between use of LD and risk of fracture [9, 10]. Thus, Rashiq and Logan [9] found use of LD to be associated with a nonsignificant reduced risk of fracture (OR = 0.68, 95% CI 0.28–1.7). Similarly, after adjustment for age, sex and type of residence (community versus nursing home), Cumming and Klineberg [10] found no effect of treatment with LD on fracture risk (OR = 0.93, 95% CI 0.57–1.49). So far, there has been only one published study on effects of LD on BMD. In this cross-sectional study, LD users had a 5% lower BMD at the hip compared with nonusers (P < 0.05), corrected for confounding by age, years since menopause and body weight [7].

We investigated in a cross-sectional design, whether long-term treatment with LD is associated with changes in BMD and in plasma levels of calcitropic hormones and biochemical markers of bone turnover.

Subjects and methods

We studied 280 Caucasian women aged 55–82 years, who were more than 2 years postmenopause (Table 1). Half of the studied subjects (n = 140) had been treated with a LD for at least 2 years prior to inclusion in the study.

Table 1.  Characteristics of study subjects (mean ± SEM)
 Users of loop diuretics (n = 140)Nonusers of loop diuretics (n = 140)P-value (n = 280)
  1. aMedian (range). HRT, hormone replacement therapy. bVitamin D supplements include use of multivitamin supplements, calcium/vitamin D supplements and use of cod liver oil products.

Age (years)a67 (55–82)67 (56–82)0.84
Years postmenopausala20 (2–54)19 (4–50)0.35
Height (cm)160.9 ± 0.5162.2 ± 0.50.08
Scale weight (kg)79.8 ± 1.468.1 ± 0.9<0.001
Body mass index (kg m−2)30.8 ± 0.625.9 ± 0.3<0.001
Duration of diuretic treatment (years)a7 (2–41)  
Calcium intake (mg day−1)1062 ± 361101 ± 310.42
Use of vitamin D supplementsb23 (16%)39 (28%)0.02
No. with a previous fracture64 (46%)49 (35%)0.07
Smoking43 (31%)41 (29%)0.90
Ever use of oral contraceptives42 (30%)47 (34%)0.49
 Duration of use (years)a10 (1–22)8 (1–23)0.76
Previous use of HRT16 (11%)19 (14%)0.59
 Duration of HRT use (years)a5 (0.5–11)8 (0.3–25)0.23

We used the following exclusion criteria for both groups: immobilization (more than 1 week within the last 6 months), previous or present malignant disease, hyper- or hypothyroidism within the last 2 years, granulomatous disease, a history of renal or hepatic disease with a chronic impairment of renal or hepatic function, drug or alcohol abuse of more than 14 units of alcohol a week within the last 2 years, or treatment within the last 2 years with systemic gluco-/mineralocorticoids, anticonvulsants, bisphosphonates, oestrogen and/or raloxifene. Patients treated with LD were studied from February 2002 to June 2002, and the controls were studied from March 2002 to June 2002.

Women treated with LD were recruited through a prescription database. In the database, the public health authority files data on all subjects who redeem prescriptions of drugs in Denmark in order to reimburse on medical expenses. The data are only kept on file for a period of 2 months. Thus, all women aged 55–86 years in the county of Aarhus, Denmark, who had received reimbursement for a prescription of a LD during the months of August and September 2001 were contacted by direct mailings from the health authorities (n = 3722 contacted). Amongst the 1678 women who responded the invitation to participate in the study, we had to exclude 1254 because of diseases and/or use of medication (including hormone replacement therapy), 146 as they had not yet passed the menopause, 30 as they reported use of more than 14 units of alcohol per week, and 108 as the required number of participants had been reached.

We recruited age-matched (±6 months) women who had not been treated with a LD within the last 2 years by direct mailings to a random sample of the general population. (n = 1139 contacted). Amongst the 486 women who responded, we had to exclude 279 because of diseases and/or use of medication (including hormone replacement therapy), 38 as they had not yet passed the menopause, 11 as they reported use of more than 14 units of alcohol per week, and 18 as the required number of participants had been reached.

We performed the study in accordance with the Declaration of Helsinki II and it was approved by the regional Ethical Committee (Aarhus County no. 20010275).

Measurements and biochemistry

We examined all participants in our outpatient clinic. They were asked to fill in a questionnaire concerning their state of health. Variables covered by the questionnaire are shown in Table 1. Daily total calcium intake was assessed according to reported dietary intake of milk, cheese, milk products and use of calcium supplements [11]. In order to assess dose–effect relationships, doses of bumetanide were converted into furosemide dose-equivalents according to the natriuretic potency of the two diuretics, that is 1 mg of bumetanide is equivalent to approximately 40 mg of furosemide [12].

We measured bone mineral content (BMC, g), BMD (g cm−2), and projected bone area (cm2) at the lumbar spine (L1-L4), hip and forearm by dual-energy X-ray absorptiometry using a Hologic QDR 2000 densitometer (Hologic Inc., Waltham MA, USA; software version V4.55). Additionally, we determined body composition (BMC, fat mass and lean tissue mass) employing the same scanner. Our regions of interest for body composition were whole body, arms, legs and trunk. The CV for the actual QDR scanner was 1.5% for the lumbar spine BMD, and long-term stability was high with changes <0.2% per year.

We collected 24 h urine and drew blood samples between 7.00 and 9.30 am after an overnight fast. We determined concentrations of albumin, calcium, creatinine, magnesium, phosphate, potassium, sodium, and thyrotropin in plasma and urine using standard laboratory methods. We corrected total plasma calcium for individual variations in albumin by the equation: adjusted plasma calcium (mmol L−1) = plasma calciumtotal(mmol L−1) − 0.00086 × [650 − plasma albumin (μmol L−1)].

We measured plasma intact PTH by an IMMULITE® automated analyser (Diagnostic Products Corporation, Los Angeles, CA, USA). The total coefficient of variation (CV) in our laboratory was <7%. We measured plasma 25-hydroxyvitamin D [25-(OH)D] by an equilibrium radioimmunoassay procedure (DiaSorin Inc., Stillwater, MN, USA). The inter- and intra-assay CV were 13%, and 10%, respectively. We determined plasma 1,25(OH)2D by a competitive radioreceptor assay (Nichols Institute Diagnostics, San Juan Capistrano, CA, USA). The inter- and intra-assay CV were 10%, and 8%, respectively. We measured plasma osteocalcin and C-terminal telopeptide of type I collagen (β-CrossLaps, CTx) by ELISA using an automated instrument (Elecsys, 2010 immunoassay analyzer; Roche Diagnostics, Basel, Switzerland). We used antibodies that recognize both intact- (1–49) and N-Mid-osteocalcin (1-43) [13]. For both osteocalcin and CTx the total CV was <6%. We measured alkaline phosphatase (AP) and bone-specific alkaline phosphatase (bone-ALP) spectrophotometrically using an automated instrument (Hitachi 917; Roche Diagnostics). Bone-ALP was measured after lectin-precipitation (Boehringer Mannheim, Mannheim, Germany). The total CV was <8%. To reduce analytical variation, we analysed PTH, vitamin D metabolites and bone markers from each patient in the same run.

Statistics

In each treatment group 140 subjects provide 80% power to detect a 4% difference in BMD of the lumbar spine at a 5% significance level. We assessed differences between the two study groups using the chi-square tests for categorical variables. For continuous variables, we used a two-sample t-test or Mann–Whitney U-test as appropriate, after testing for normal distributions. When indicated, we performed logarithmic transformation of data. We tested correlations between variables by bivariate correlation analysis [Pearson's correlation (r) or Spearman's rho (R), as appropriate]. Differences between groups were adjusted for using a general linear regression model. We present all results as mean ± standard error of the mean (SEM) unless otherwise stated. We used the Statistical Package for Social Sciences (spss 8.0) for Windows.

Results

Age and years since menopause were similar in the two study groups (Table 1). However, LD users had a significantly (P < 0.001) higher body weight (79.8 ± 1.4 kg) than controls (68.1 ± 0.9 kg). More subjects in the control group (28%) than in the LD group (16%) reported daily use of vitamin D supplements (P = 0.02). The total daily calcium intake and the proportion of smokers were similar in the two study groups. LD were prescribed for the treatment of cardiac oedema (n = 105), hypertension (n = 28), renal insufficiency (n = 2), dependency (n = 2), bladder dysfunction (n = 2) and unknown indication (n = 1). Table 2 shows the type of LD used, as well as median doses and duration of use. Amongst users of LD, furosemide was used more frequently than bumetanide.

Table 2.  Type of loop diuretics, dose and duration of use (n = 140)
 No. of users (%)Dose (mg per day)Duration of use (years)a
MedianRangeMedianRange
  1. aTotal years of treatment, that is some subjects may previously have been treated with other types of loop diuretics.

Furosemide127 (91)60(10–720)  7(2–41)
Bumetanide13 (9)  1(0.5–6.0)10(2–40)

Body composition and osteodensitometry

Users of LD had a 32% higher fat mass (26.5 kg vs. 35.1 kg, P < 0.001) and a 6% higher lean tissue mass (37.9 kg vs. 40.1 kg, P < 0.001) than nonusers. Furthermore, LD users had higher BMD at the lumbar spine (7.5%, P < 0.001), the total hip (4.8%, P = 0.004), the forearm (3.7%, P = 0.01) and the whole body (2.5%, P = 0.06) compared with nonusers (Fig. 1). However, after correction for differences in body weight, BMD did not differ between the groups at any of the measuring sites (Table 3).

Figure 1.

Bone mineral density in users (n = 140) and nonusers (n = 140) of loop diuretics (mean ± SEM).

Table 3.  Bone mineral density (g cm−2) adjusted for differences in body weight and previous fracture (mean ± SEM)
 Users of loop diureticsNonusers of loop diuretics P-value
Lumbar spine (L1–L4)0.937 ± 0.0130.918 ± 0.0130.30
Hip region
 Total hip0.805 ± 0.0090.815 ± 0.0090.45
 Femoral neck0.687 ± 0.0090.690 ± 0.0080.86
 Interthrochanter0.939 ± 0.0110.956 ± 0.0110.28
 Throchanter0.622 ± 0.0080.632 ± 0.0080.41
 Wards0.500 ± 0.0110.492 ± 0.0110.85
Forearm
 Total0.436 ± 0.0050.439 ± 0.0050.69
 Upper third0.541 ± 0.0060.553 ± 0.0060.19
 Mid third0.444 ± 0.0050.449 ± 0.0050.54
 Ultradistal0.331 ± 0.0050.324 ± 0.0050.33
Whole body0.982 ± 0.0080.993 ± 0.0080.37

Amongst LD users (n = 140), duration of treatment (years of treatment) correlated positively with BMD at the lumbar spine (r = 0.19, P = 0.03) and whole body (r = 0.18, P < 0.05). Similar positive effects of duration of treatment were found in multiple regressions analyses at the lumbar spine (r = 0.20, P = 0.03) and whole body (r = 0.19, P = 0.04) after correction for differences in body weight, years since menopause, previous fracture and smoking status. In this adjusted model, BMD at the spine increases by 0.3% per 1 year of treatment.

Biochemistry

Users of LD had a higher mean plasma creatinine level than nonusers (Table 4). Thirteen subjects in the LD group and two in the control group had a plasma creatinine level above the upper limit of the reference interval. However, endogenous creatinine clearance did not differ significantly between groups (Table 4).

Table 4.  Biochemical characteristics by study group (mean ± SEM)
 Users of loop diuretics (n = 140)Nonusers of loop diuretics (n = 140)P-value (n = 280)
Plasma
 Albumin (μmol L−1)598 ± 3594 ± 30.41
 Creatinine (μmol L−1)90 ± 278 ± 1<0.001
 Calcium, total (mmol L−1)2.39 ± 0.012.39 ± 0.010.63
 Calcium, adj. (mmol L−1)2.44 ± 0.012.44 ± 0.010.98
 Potassium (mmol L−1)4.18 ± 0.034.31 ± 0.020.001
 Magnesium (mmol L−1)0.91 ± 0.010.88 ± 0.010.02
 Sodium (mmol L−1)142.1 ± 0.2142.5 ± 0.20.31
 Phosphate (mmol L−1)1.03 ± 0.011.00 ± 0.010.30
 TSH (mU L−1)2.4 ± 0.22.0 ± 0.20.14
 PTH (pmol L−1)8.6 ± 0.46.1 ± 0.2<0.001
 25-hydroxyvitamin D (nmol L−1)47 ± 259 ± 2<0.001
 1,25-dihydroxyvitamin D (pmol L−1)123 ± 3127 ± 30.36
 Osteocalcin (μg L−1)38.4 ± 1.736.7 ± 1.00.85
 Alkaline phosphatase (U L−1)220 ± 5200 ± 5<0.001
 Bone-alkaline phosphatase (U L−1)82 ± 381 ± 20.81
 CTx0.54 ± 0.030.58 ± 0.020.24
 Creatinine clearance (ml min−1)75 ± 278 ± 10.07
Urine
 Diuresis (ml 24 h−1)2237 ± 732024 ± 710.03
 Creatinine (mmol 24 h−1)9.0 ± 0.28.6 ± 0.10.25
 Calcium (mmol 24 h−1)4.8 ± 0.24.3 ± 0.20.24
 Calcium/creatinine ratio (mmol mmol−1)0.54 ± 0.020.51 ± 0. 020.24
 Potassium (mmol 24 h−1)74 ± 259 ± 2<0.001
 Magnesium (mmol 24 h−1)4.1 ± 0.23.8 ± 0.10.36
 Sodium (mmol 24 h−1)138 ± 5116 ± 40.002
 Phosphate (mmol 24 h−1)21 ± 120 ± 10.07

As expected, urinary volume as well as renal potassium and sodium excretion were higher in LD users than in nonusers (Table 4). Furthermore, plasma potassium levels were lower whereas magnesium levels were higher in users compared with nonusers. Plasma calcium and urinary calcium levels did not differ between the groups. However, plasma PTH levels were 41% higher (P < 0.001) in LD-treated women compared with controls. Plasma PTH levels were above the upper reference limit (i.e. >7.6 pmol L−1) in 51% of the women in the LD group, whereas only 16% of the controls had PTH levels above 7.6 pmol L−1 (P < 0.001). Similarly, total AP levels were 10% higher (P < 0.001) in LD-treated women than in the controls, whereas 25-(OH)D levels were 20% lower (P < 0.001) in the LD group. Neither plasma 1,25(OH)2D levels nor levels of biochemical bone markers of formation (bone-ALP and osteocalcin) or resorption (CTx) differed between groups (Table 4).

Table 5 shows adjusted levels of renal calcium excretions, calcitropic hormones and biochemical bone markers. All variables have been adjusted for body weight, age, creatinine clearance, previous fracture and smoking status. This did not change the findings of higher PTH levels in LD users compared with nonusers even after further adjustment for plasma calcium, magnesium, phosphate, 25(OH)D, and 1.25(OH)2D. Inclusion of daily dose of LD in the model revealed that plasma PTH levels are increased by 28% per 40 mg furosemide per day (P = 0.04). Plasma 25(OH)D levels did no longer differ between groups after further adjustment for the use of vitamin D supplements. However, significantly higher 1,25(OH)2D levels were found in users compared with nonusers of LD, after adjustment for plasma calcium, phosphate, PTH and 25(OH)D levels (P = 0.03, Table 3). Inclusion of dose of LD in the model did not reveal any significant dose–effect relationship on plasma 1,25(OH)2D levels. Finally, the 24 h renal calcium excretion was higher in users compared with nonusers of LD, after adjustment for differences in daily calcium intake, plasma PTH, 25(OH)D and 1.25(OH)2D levels (P < 0.01, Table 5). In the adjusted model, urinary calcium levels were increased by 17% per 40 mg furosemide per day (P < 0.05).

Table 5.  Adjusted levelsa of calcitropic hormones, biochemical bone markers and renal calcium excretion (mean ± SEM)
 Users of loop diureticsNonusers of loop diureticsP-value
  1. aAll indices have been adjusted for differences in body weight, creatinine clearance, age, previous fracture and smoking status. bAdditional adjusted for differences in plasma calcium, magnesium, phosphate, 25(OH)D and1.25(OH)2D. cAdditional adjusted for differences in use of vitamin D supplements. dAdditional adjusted for differences in plasma calcium, phosphate, PTH and 25(OH)D levels. eAdditional adjusted for differences in plasma PTH, 25(OH)D and 1.25(OH)2D levels. fAdditional adjusted for differences in daily calcium intake, plasma PTH, 25(OH)D and 1.25(OH)2D levels.

PTH (pmol L−1)b7.9 ± 0.36.6 ± 0.3<0.01
25-hydroxyvitamin D (nmol L−1)c51 ± 256 ± 2  0.07
1,25-dihydroxyvitamin D (pmol L−1)d130 ± 3120 ± 3  0.03
Osteocalcin (mg L−1)e37.2 ± 1.337.9 ± 1.3  0.75
Alkaline phosphatase (U L−1)219 ± 6201 ± 6  0.03
Bone-alkaline phosphatase (U L−1)e81 ± 382 ± 3  0.83
CTx (mg L−1)e0.54 ± 0.020.58 ± 0.02  0.22
Urinary calcium (mmol 24 h−1)f5.0 ± 0.24.1 ± 0.2<0.01

Discussion

In a cross-sectional design, comparing 140 postmenopausal women who had been treated with an LD for median 7 years with 140 age- and gender-matched nonusers of LD, we found that treatment with LD was associated with an increased 24 h renal calcium excretion, as well as increased plasma PTH and 1,25(OH)2D levels. However, no between-group effects on BMD were revealed. Nevertheless, within the group of women treated with LD, duration of treatment was positively associated with BMD at the lumbar spine and whole body.

Increased body weight in users of loop diuretics

Users of LD had a higher body weight than nonusers. Measurements on BMC showed that this was due to a higher fat and lean tissue mass. Obesity is a well-known risk factor for cardiovascular diseases [14]. We find it unlikely that treatment with LD causes obesity. More likely, the higher body weight observed in users of LD has contributed to the underlying diseases, requiring diuretic treatment. As body weight is known to influence densitometric measurements and vitamin D levels, body weight and BMC has to be adjusted for during analyses.

BMD in patients treated with loop diuretics

Despite the widespread use of LD, there are only few data on effects of LD on BMD. In a observational study from Holland, Ooms et al. [7] investigated associations between BMD and risk factors for osteoporosis in 348 apparently healthy women with a mean age of 82.3 years. Amongst studied subjects, 50 women used LD. In a multiple regression analysis, corrected for confounding by age, years since menopause and body weight, the study showed a significantly lower BMD at the hip (−5%) in users of LD [7]. In contrast, in the present study analyses on raw data showed higher BMD values in women treated with LD than in controls. However, after adjustment for differences in body weight, smoking status and years since menopause, BMD did not differ between groups at any measurement sites. Nevertheless, analyses on effects of duration of treatment showed that BMD at the lumbar spine and whole body increased slightly with increased duration of treatment.

The most obvious difference between our study and the study by Ooms et al. [7] is that our participants in average were 25 years younger than the Dutch women. Calcium metabolism changes with age, for example the endogenous vitamin D production declines [15]. Thus, we cannot exclude that LD may affect bone metabolism differentially in old compared with very old women.

Calcitropic hormones and biochemical bone markers

Due to an inhibition of the Na–K–Cl cotransporter in the thick ascending limb of the loop of Henle, LD increase the renal calcium excretion [16]. Conflicting results have been reported on the long-term effects of LD on the 24 h renal calcium excretion. Some have reported an increased calcium excretion during the first days of treatment with a subsequent return to pretreatment levels during continued therapy [17–19], whereas others have found a persistent increased renal calcium excretion during long-term treatment [2, 5, 20]. Our findings support an increased renal calcium excretion in subjects on long-term treatment with LD. A persistently negative skeletal calcium balance could be the consequence of this effect if it is not compensated for by enhanced intestinal calcium absorption. Our findings of increased 1,25(OH)2D levels may imply that the intestinal calcium absorption is increased in users of LD. It is likely that the increased PTH levels stimulate the renal 1α-hydroxylase, thereby increasing the synthesis of 1,25(OH)2D. Despite increased urinary calcium losses in users of LD, the finding of unchanged BMD and similar levels of biochemical markers of bone turnover in users compared with nonusers of LD calls for such a homeostatic regulation.

The high PTH levels in subjects treated with LD may be explained as secondary to the increased renal calcium excretion. In a previous study on short-term effects of LD on calcium homeostasis, we have shown that the diurnal rhythm of plasma PTH correlated significantly with the rhythm of urinary calcium, suggesting that PTH secretion is stimulated by the hypercalciuria induced by treatment with LD [6]. Our finding of increased plasma PTH levels in subjects on long-term treatment with LD has important clinical implications. In patients, treatment with LD may have to be considered as an aetiological reason for elevated PTH levels. In our study, blood samples were collected in the fasting state in the morning. Thus, 12–24 h had elapsed since last intake of an LD. Further studies are needed to determine the exact time needed to be left before the effect of an LD on plasma PTH levels has subsided.

Analyses on raw data revealed lower 25(OH)D levels in women on treatment with LD. However, after adjustment for differences in body weight and use of vitamin D supplements, plasma 25(OH)D levels did no longer differ between groups. Our findings agree with studies showing an inverse correlation between body weight and plasma 25(OH)D levels [21], as well as studies showing increased 25(OH)D levels in response to use of vitamin D supplements [22, 23]. Our finding of increased total AP levels in users of LD agrees with previous studies [24]. This is most likely due to the underlying disease necessitating diuretic therapy, that is congestive heart failure as well as obesity has been reported to cause liver function abnormalities including elevated AP levels [24–26].

Study strengths and limitations

The major strength of our study is the recruitment procedure. Women treated with LD were recruited through a prescription database and controls were recruited from a random sample of the general population. Thus, potential selection biases that may occur if studied subjects are recruited through an outpatient hospital clinic should not apply to our sample.

The major limitation to our study is that the cross-sectional design does not allow for causal conclusions. Most of our studied subjects were treated with LD due to cardiovascular diseases. Thus, we are not able to draw any definite conclusions on whether the observed effects are due to the underlying disease or a specific effect of drug treatment. However, as previous randomized controlled studies have shown increased plasma PTH levels in response to short-term treatment with LD, the most likely explanation for increased PTH levels is drug treatment rather than cardiovascular diseases. Previously, conflicting results have been reported on the effect of cardiovascular diseases on BMD. Although arterial hypertension was associated with an increased BMD in one study [27], a reduced BMD was found by other investigators in subjects with arterial hypertension, as well as in patients with ischaemic heart diseases [28–32]. Thus, if ischaemic heart diseases cause a reduced BMD, LD may actually affect BMD positively, as we found a similar BMD in subjects treated with LD due to cardiovascular diseases as in healthy nonusers of LD. Unfortunately, we did not measure blood pressure in studied subjects and therefore we were unable to perform subanalyses on associations between blood pressure levels and BMD.

Another limitation to our study is the difference in body weight between the two study groups. We cannot exclude that factual errors in measurement introduced by weight differences may not be completely adjusted for in a linear regression model. Finally, vitamin D insufficiency was prevalent in both study groups. Whether the observed effects of LD on calcium homeostasis may be influenced by vitamin D status needs further studies.

Our finding of an increased BMD at the spine and whole body with increased duration of treatment may indicate a potential positive effect of LD on BMD. The mechanism of action might be the increased PTH levels in response to LD therapy. In a previous study, we showed a marked peak in plasma PTH levels a few hours after medication with an LD [6]. Similarly, administration of synthetic PTH (1-34) as subcutaneous injections causing intermittent high PTH concentrations increased bone mass [33].

Conclusion

In a cross-sectional design, treatment with LD was associated with an increase in urinary calcium, plasma PTH and plasma 1,25(OH)2D levels. However, BMD and plasma levels of biochemical markers of bone turnover did not differ between users and nonusers of LD. Thus, it seems that the increased renal calcium losses in users of LD are compensated for by a PTH-dependent increase in 1,25(OH)2D levels that may increase the intestinal calcium absorption. Thereby, calcium balance remains neutral without any major effects on bone metabolism.

Conflict of interest statement

No conflict of interest was declared.

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