Archives of Biochemistry and Biophysics 503 (2010) 84–94

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Archives of Biochemistry and Biophysics

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Review

Liver and bone

Núria Guañabens *, Albert Parés
Metabolic Bone Diseases Unit, Department of Rheumatology, Hospital Clínic, CIBERhed, University of Barcelona, Barcelona, Spain
Liver Unit, Digestive Diseases Institute, Hospital Clínic, CIBERhed, University of Barcelona, Barcelona, Spain

a r t i c l e i n f o a b s t r a c t

Article history: Osteoporosis is a frequent complication in patients with chronic liver disease, especially in end-stages
Available online 9 June 2010 and in cases with chronic cholestasis, hemochromatosis and alcohol abuse. The problem is more critical
in transplant patients when bone loss is accelerated during the period immediately after transplantation,
Keywords: leading to a greater incidence of fractures. Advanced age, low body mass index and severity of the liver
Osteoporosis disease are the main risk factors for bone disease in patients with cholestasis. Mechanisms underlying
Metabolic bone disease osteoporosis in chronic liver disease are complex and poorly understood, but osteoporosis mainly results
Cirrhosis
from low bone formation, related to the effects of retained substances of cholestasis, such as bilirubin and
Cholestasis
Liver transplantation
bile acids, or to the effects of alcohol on osteoblastic cells. Increased bone resorption has also been
described in cholestatic women with advanced disease. Although there is no specific treatment, bisphos-
phonates associated with supplements of calcium and vitamin D are effective for increasing bone mass in
patients with chronic cholestasis and after liver transplantation. The outcome in reducing the incidence
of fractures has not been adequately demonstrated essentially because of the low number of patients
included in the therapeutic trials. Randomized studies assessing bisphosphonates in larger series of
patients, the development of new drugs for osteoporosis and the improvement in the management of
liver transplant recipients may change the future.
Ó 2010 Elsevier Inc. All rights reserved.

Introduction liver disease [2–4]. In spite of this, many issues remain to be

elucidated.
For years ‘‘hepatic osteodystrophy”, including osteomalacia and This review describes the prevalence, risk factors, the current
osteoporosis, was used to describe the bone disorders observed in knowledge of the pathophysiology, and finally it focuses on the
patients with different liver diseases. However, osteomalacia is assessment and management of bone disorders in patients with li-
very uncommon in patients with liver diseases, and it has been re- ver disease.
ported only in isolated patients with advanced primary biliary cir-
rhosis (PBC) and severe intestinal malabsorption in geographical
Prevalence of osteoporosis
areas with limited sunlight exposure [1].
Osteoporosis occurs in patients with chronic liver disease. How-
The prevalence of osteoporosis in chronic liver diseases is
ever, there is certain heterogeneity regarding its prevalence, which
summarized in Table 1, which includes several relevant studies
depends on patient selection, liver disease etiology and additional
performed in patients with chronic cholestatic diseases, mainly
risk factors for osteoporosis. Actually, most studies have been fo-
primary biliary cirrhosis, and series of patients with mixed liver
cused on bone disease in chronic cholestatic liver disease, mainly
diseases. It should be taken into account, however, that because
in patients with PBC, and in bone disease before and after liver
of the different densitometric criteria for osteoporosis, the table
transplantation. By contrast, few studies have evaluated bone dis-
summarizes information on the studies with definite criteria for
orders in other chronic liver diseases such as viral hepatitis, hemo-
osteoporosis (T-score 6 2.5 in postmenopausal women, or Z-
chromatosis and alcoholic liver disease.
score 6 2 in men under 50 years old and premenopausal wo-
In recent years there have been substantial advances in the
men). Moreover, it should also be noted that in most studies
identification of the risk factors and in the understanding of the
including males the mean age of the group is higher than
pathogenetic mechanisms of osteoporosis in patients with chronic
50 years, thus indicating that the T-score 6 2.5 is applicable in
this setting.
* Corresponding author. Address: Servicio de Reumatología, Hospital Clínic, C/
In patients with chronic cholestasis, osteoporosis has been re-
Villarroel 170, Barcelona 08036, Spain. cently reported to be up to 37% in a series of 185 Spanish female
E-mail addresses: nguanabens@ub.edu (N. Guañabens), pares@ub.edu (A. Parés). patients, the lumbar spine being more severely affected than the

0003-9861/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.abb.2010.05.030
Table 1
Prevalence of osteoporosis and fractures in patients with liver diseases.

Author, year [Refs.] Cases (n) Age (years) Females Postmeno Diagnosis of Osteoporosis Vertebral Peripheral Overall Etiology of liver disease Advanced disease

N. Guañabens, A. Parés / Archives of Biochemistry and Biophysics 503 (2010) 84–94

mean ± SEM (%) pausal (%) osteoporosis (%) fractures (%) fractures (%) Fractures (%) or cirrhosis (%)
ALD VC Chol Cryp GH CH Other
(range)
(%) (%) (%) (%) (%) (%) (%)
Primary biliary cirrhosis
Guañabens et al. [42] 38 51 (37–62) 100 63 DPA 45 13 NR 13 100 94
Springer et al. [86] 72 55 (34–81) 100 68 DPA 24 100 11
Parés et al. [88] 61 54 ± 1.1 100 79 DXA 21 10 10 13 100 26
Menon et al. [6] 176 53 (29–72) 83 45 DXA 20 100 59
Newton et al. [8] 272 62 ± 0.7 94 63 DXA 31 100 54
Solerio et al. [10] 133 53 (21–81) 100 70 DXA 35 100 39
Guañabens et al. [7] 142 54 ± 0.8 100 69 DXA 31 14 11 14 100 26
Guichelaar et al. [35] 156 53 ± 0.7 86 76 DXA 44 22 NR 22 100 100
Guañabens et al. [5] 185 56 (28–79) 100 82 DXA 32 11 12 21 100 23
Mixed liver diseases
Diamond et al. [11] 115 50 (20–74) 37 30* QCT 16** 16 17 28 35 22 21 22 52
Bonkovsky et al. [12] 133 47 (18–80) 47 DPA 26 24 26 36 14 86
Chen et al. [45] 74 64 ± 1.2 0 DPA 20** 7 ND 7 12 73 14 1 100
Monegal et al. [33] 56 50 (32–60) 32 74 DXA 26** 22 NR 22 24 65 11 100
Sinigaglia et al. [13] 32 52 (31–69) 12 DXA 28 100 53
Ninkovic et al. [49] 37 51 (32–65) 46 77 DXA 39 35 NR 35 16 22 35 11 3 13 100
Ninkovic et al. [34] 243 51 ± 0.7 47 73 DXA 37 19 30 29 7 2 13 100
Carey et al. [37] 207 51 (32–68) 37 48 DXA 20 14 10 24 67 33 0 100
Sokhi et al. [36] 104 54 ± 1.3 48 70 DPA 12 15 66 8 10 100
Guggenbuhl et al. [14] 38 47 ± 1.5 0 DXA 34 100 NR
González-Calvin et al. [39] 84 65 (55–80) 100 100 DXA 43 100 100
Valenti et al. [15] 87 51 ± 1.2 20 47 DXA 25 100 NR

Diagnosis of vertebral fractures was established by spinal X-rays.

*
hypoogonadism; DPA: dual photon absorptiometry; DXA: dual X-ray absorptiometry; QCT: quantitative computed tomography. ND: not done; NR: not reported; ALD: alcoholic liver disease; Chol: chronic cholestatic diseases;
GH: genetic hemochromatosis; CH: chronic hepatitis; VC: cirrhosis of viral etiology; Cry: cryptogenic cirrhosis.
**
Criteria for osteoporosis was a T-score 6 2.5, except in these studies where osteoporosis was defined by a Z-score 6 2.0.

85
86 N. Guañabens, A. Parés / Archives of Biochemistry and Biophysics 503 (2010) 84–94
femoral neck [5]. Similarly, Menon et al. found that as many as 20%
of PBC patients in a large series from the Mayo Clinic had osteopo-
rosis [6]. In both studies, osteoporosis was more frequent in PBC
than in healthy women. Thus, the prevalence of osteoporosis was
significantly higher in PBC patients than in age-matched Spanish
population [5,7] and 32.1 times more frequent than expected in
the series from the Mayo Clinic [6]. These observations are of inter-
est, since it has been suggested that osteoporosis is not a specific
complication of PBC, its frequency being the one expected in a pop-
ulation of middle-aged postmenopausal women [8–10].
In non-cholestatic disorders like hemochromatosis and other
more prevalent liver diseases, such as viral hepatitis and alcoholic
liver disease, there is less information [11,12]. From available data,
25% to 34% of patients with hereditary hemochromatosis have
osteoporosis [13–15]. In a recent study of 87 patients (80% male)
with hemochromatosis, a quarter of them had osteoporosis and
41% had osteopenia, even in subjects without cirrhosis and hypo-
gonadism [15]. Regarding viral chronic hepatitis and its treatment
with ribavirin and interferon, there are conflicting results. Thus,
one small study found that hepatitis C virus infection did not lead
to discernable bone disease, although a possible increased risk of
fracture was found [16]. Other small studies assessing bone mass
in chronic virus hepatitis, mostly hepatitis C virus, have shown that
non-cirrhotic hepatitis C virus infected patients do not differ from
controls [17,18], or by contrast they have low bone mass [19], with
osteopenia or osteoporosis in 43% and 13%, respectively [20]. Also,
it has been reported that antiviral therapy with ribavirin and inter-
feron may induce bone loss [21], or on the contrary, an increase in
bone mineral density (BMD) which may last in those patients who
achieve a sustained serologic response [20]. Bone disease in alco-
holic liver disease has depicted more uniform results, since excess
alcohol intake is an independent risk factor for osteoporosis
[22,23]. Thus, alcoholic patients without liver cirrhosis have low
bone mass, and bone loss has been associated with persistent alco-
hol consumption [24–26]. Studies from our group found that alco-
holic males with or without mild liver disease have significantly
lower lumbar BMD than controls [27] and 29% had osteoporosis
[28]. Similar findings have been reported by others [29]. An inter-
esting observation was the improvement in bone mass after
2 years of abstinence [27].
The prevalence of osteoporosis has also been evaluated in pa-
tients with end-stage liver disease and after liver transplantation.
Osteoporosis, by densitometric criteria, has been found in 24–
38% of patients with advanced liver disease, depending on the ser-
ies [30–39]. Guichelaar et al. reported on a series of 360 patients
with advanced cholestatic liver disease (PBC and primary scleros-
ing cholangitis) that 38% of patients had osteoporosis, 39% osteope-
nia and only 23% of patients had normal bone mass. Interestingly,
they found an increase in the pre-transplant bone mass over a 16-
year study period (1985–2000) in the primary sclerosing cholangi-
tis population, although lumbar BMD remained stable in PBC pa-
tients [35].
Transplantation is an additional cause of osteoporosis and frac-
tures, particularly during the first few months after surgery, a per-
iod which is associated with a high rate of bone loss. Thus, lumbar
BMD decreases by roughly 6% at 3 months after liver transplanta-
tion, with a progressive and significant increase after 1 year of fol-
low-up. Moreover, lumbar BMD reaches baseline values 2 years
after transplantation. Femoral BMD has a different pattern of evo-
lution. After a marked decrease of up to 4% in femoral neck BMD
during the first 3–6 months, there is a delayed and incomplete
improvement at 3 years [40]. It must be kept in mind, however,
that these data were obtained in the nineties and that patients
transplanted more recently may have no significant changes in
lumbar BMD, although sustained bone loss still occurs at the fem-
oral neck [41].
Prevalence of fractures
The prevalence of fractures has been analyzed in few studies,
mostly limited to short series as indicated in Table 1, which sum-
marizes the available data on the studies where vertebral fractures
were disclosed by spinal X-rays. Overall, the prevalence of frac-
tures in patients with liver diseases ranges from 7% to 35% [5–
8,11,33,42–45]. We have recently assessed this issue in 170 pa-
tients with PBC, and the prevalence of vertebral (through X-rays
of the spine), non-vertebral and overall fractures was 11.2%,
12.2% and 20.8%, respectively. Non-vertebral fractures included
mostly wrist fractures (8 patients) followed by lower limb frac-
tures (4 patients); only one patient had hip fracture [5]. In another
series of 930 people with PBC, from the General Practice Research
Database in the United Kingdom, there was a modest increase in
both the absolute and relative fracture risks compared to the gen-
eral population [46]. There were approximately 2-fold relative in-
creases in the risk of any fracture, hip fracture and ulna/radius
fracture for the PBC cohort compared with the general population.
In this study, vertebral fractures were not specifically considered
because they are not always symptomatic. Interestingly, the in-
crease in risk of fracture was not greater in individuals with more
severe liver disease. In this series there were 25 incident hip frac-
tures and 44 ulna/radius fractures. When comparing the numbers
of hip fractures, it is interesting to consider that 23% of patients
of the UK series were older than 70 at the start of follow-up [46].
Fractures in patients with alcoholic liver disease have been
scarcely assessed, with the reported prevalence of radiological ver-
tebral fractures being up to 36% in a group of 76 males with or
without mild liver disease. In this series, previous history of non-
vertebral fractures was high, ribs being a frequent location since
they occurred in 26% of patients. The authors suggested that trau-
matic fractures may account for a great proportion of fractures in
the alcoholic population [28], although a fragility component can-
not be ruled out. In fact, most studies have been focused on alcohol
consumption without taking into account the contribution of liver
disease in the fracture risk. Thus, a recent meta-analysis assessing
the association between alcohol consumption and osteoporotic
fractures showed that compared to abstinence, consuming more
than 2 drinks per day is associated with higher hip fracture risk,
but the contributory effect of liver damage was not assessed [47].
Fractures are frequent in patients with end-stage liver disease
and after liver transplantation. Indeed, 6.6–22.4% of cirrhotic pa-
tients referred for liver transplantation show radiological vertebral
fractures [33,48,49]. After liver transplantation, a high proportion
of patients develop fractures during the first 6–12 months. Avail-
able data indicate that fracture incidence ranges from 22% to 65%
[32,50], although most series have shown an incidence of vertebral
fractures around 30%. The highest rates have been reported in the
earliest series involving patients with cholestatic diseases with
immunosuppressive approaches that were highly reliant on gluco-
corticoids [32]. By contrast, the lowest rates of fracture were de-
scribed in studies performed in patients with better bone health
prior to transplantation, together with the use of lower doses
and shorter duration of glucocorticoid therapy [41]. Interestingly,
a long-term follow-up study showed that few patients suffer their
first fracture in the third and fourth year after liver transplantation
[51].
Risk factors for osteoporosis and fractures
Apart from the incomplete knowledge on the key mechanisms
resulting in osteoporosis in PBC, some factors have been associated
with the presence of low bone mass (Table 2). Several years ago, it
was reported in a small series of PBC patients that osteoporosis
 N. Guañabens, A. Parés / Archives of Biochemistry and Biophysics 503 (2010) 84–94 87

Table 2 When risk factors for fractures were assessed in PBC population,
Risk factors for low bone mass and fractures. osteoporosis (by DXA), menopause, age and height, but not sever-
Primary biliary cirrhosis Liver transplantation ity of liver disease were the independent predictors. Furthermore,
Low bone mass: [5–7,35] Before surgery: [40,48–51] a T-score lower than 1.5 at the lumbar spine indicates a high risk
Advanced age Osteoporosis for vertebral facture in PBC patients, since 17 out of 19 patients
Menopause Vertebral fractures with vertebral fractures had a T-score lower than 1.5 (Fig. 1). Ta-
Low body mass index Etiology of liver disease ken together, the risk for fracture in PBC is associated with low
Severity of liver damage
Duration of cholestasis After surgery: [30,61]
bone mass and indirectly, through this condition, with the severity
Advanced histological stage Immunosuppressive agents and duration of liver disease [5].
Previous fractures Inmobilization When risk factors for developing fractures after liver transplan-
Fractures:[5] Other: [40,48] tation have been assessed, no pre-transplant indicator reliably pre-
Low bone mass Advanced age dicts the fracture risk in a given patient [55]. However, some
Advanced age Variable individual susceptibility conditions, such as low BMD and vertebral fractures before trans-
Menopause Re-transplantation
plantation, as well as older age, the type of liver disease and re-
Height
transplantation have been identified as major risk factors for frac-
tures [56]. In addition, the immunosuppressive regimens used in
liver transplantation play a key role in the development of bone
was associated with the duration of liver disease, calcium malab- loss and fractures (Table 2). Pre-transplant low BMD and fractures
sorption and the postmenopausal status [52]. Since most patients are the major predictors of osteoporotic fractures after liver trans-
with PBC and osteoporosis are postmenopausal, it has been ques- plantation. Thus, a long-term follow-up study pointed out that
tioned whether the development of bone loss is more related to only vertebral fractures before transplantation were independent
estrogen deficiency than to the consequences of liver damage [8]. predictors for incident fractures following liver transplantation
Although the two factors contribute to bone loss, the prevalence [51], and a short-term prospective study showed that 46% of pa-
of osteoporosis is significantly higher in patients with PBC than tients with one prevalent fracture and 62% of those with two or
in age-matched healthy controls [7]. This indicates that the liver more prevalent fractures had a new vertebral fracture in the first
damage plays a relevant role resulting in bone loss. The role of 3 months [49]. Another prospective study identified osteoporosis,
duration and severity of cholestasis in the development of osteopo- diagnosed according densitometric criteria, as an independent risk
rosis has also been proposed in two recent studies. Thus a higher factor for fractures during the first year after liver transplantation
Mayo risk score, calculating the severity of PBC, and advanced his- [40]. The underlying liver disease is another relevant risk factor for
tological stage of the liver disease, in addition to advanced age and fractures. Thus, patients with cholestatic liver disease seem to be at
lower body mass index were the independent risk factors for oste- higher risk of having fractures after transplantation [51]. In some
oporosis in a series of 142 women with PBC [7]. Similarly, Menon series, this has been observed together with a higher rate of pre-
et al. reported that age, body mass index, advanced stage and his- transplant fractures or low BMD [50,57].
tory of fractures were the only independent indicators of osteopo- Immunosuppressive therapy also contributes to the develop-
rosis. Furthermore, serum bilirubin level was the only ment of bone loss and fractures. Glucocorticoids seem to play a
independently associated variable with the rate of bone loss over decisive role because of the initial rapid bone loss and the highest
time [6]. Agents such as prednisolone [53] and budesonide used rate of fractures following liver transplantation occur along with
for treatment of the liver disease may also affect bone mass [54]. the highest doses, their withdrawal accelerates the recovery of

Lumbar T-score Femoral neck T-score

2.00 2.00

1.00

0.00

0.00

-1.5
-2.00 -1.00
-1.5

-2.00

-4.00

-3.00

-6.00 -4.00

yes no yes no

Vertebral fracture Vertebral fracture

OR: 8.5, 95% CI 1.9-38.0 OR: 6.4, 95% CI 1.5-31.6

Fig. 1. Lumbar and femoral neck T-scores of patients with and without vertebral fractures. A T-score < 1.5 (dashed red line) is associated with high risk for vertebral fracture.
88 N. Guañabens, A. Parés / Archives of Biochemistry and Biophysics 503 (2010) 84–94
lumbar BMD [58], and finally the use of low doses for a short per-
iod of time may be involved in a lower rate of fractures and less
bone loss [41]. After taking these data into consideration, most
studies have failed to demonstrate differences in the cumulative
doses of glucocorticoids between fractured and non-fractured pa-
tients [40], or a correlation between cumulative doses and the
course of bone density [38,40,58]. In addition to glucocorticoids,
other immunosuppressive agents such as cyclosporin A and tacrol-
imus, frequently used in liver transplantation, may influence bone
disease. Increased parathyroid hormone related to the decrease in
glomerular filtration rate, likely to be related to cyclosporin A and
tacrolimus, may contribute to increased bone turnover. Both drugs
induce high bone turnover and bone loss in rats [59], but their
independent effects, apart from glucocorticoids in transplant pa-
tients, are not well established. To this concern, femoral BMD
was negatively associated with serum cyclosporin levels 1 year
after transplantation [60] and discordant results had been found
in some studies regarding the fracture risk under treatment with
cyclosporin or tacrolimus [51,56]. In addition, one study assessing
changes in BMD had reported that after liver transplantation, treat-
ment with tacrolimus has a more favorable long-term effect on
bone mass than cyclosporin therapy, especially at the femoral
neck, although these differences could be associated with the low-
er dose of glucocorticoids used in the tacrolimus group [61]. Con-
sidering other immunosuppressive drugs as contributing risk
factors for bone loss, rapamycin does not induce trabecular bone
loss in rats, although it increases bone remodeling and it has even
been suggested that it may partially ameliorate osteoporosis
caused by portasystemic shunting, through an increase in bone for-
mation [59,62]. However, to our knowledge, there are no studies
assessing bone disease in liver transplant recipients under immu-
nosuppressive agents such as rapamycin, mycophenolate mofetil
or daclizumab.
Pathogenesis of osteoporosis in liver diseases
The pathogenesis of osteoporosis in chronic liver diseases has
been focused largely on PBC and on transplant recipients, and they
have been scarcely studied in other liver disorders such as chronic
hepatitis or alcoholic liver disease. However, some of these mech-
anisms are shared when cirrhosis or hyperbilirubinemia are in-
volved in the course of liver disease.
The mechanisms resulting in osteoporosis in patients with PBC
have not been completely elucidated since some studies indicate
an increased bone resorption, although most pointed towards a de-
creased bone formation as the main bone remodeling abnormality.
Indeed, some data have shown impaired osteoblast function,
resulting in lower mean wall thickness and a defect in matrix syn-
thesis [63], as well as a low bone formation rate [52,63]. This was
observed in one histomorphometric analysis of transiliac bone
biopsies in 20 patients with PBC [52]. The bone formation rate
was depressed in all but four patients. Another more recent histo-
morphometric analysis detected decreased bone formation in pa-
tients with advanced cholestatic liver disease [64]. These
morphometric data are consistent with other reports showing de-
creased serum levels of osteocalcin [65]. Osteoblast dysfunction
may result from reduced trophic factors such as insulin growth fac-
tor-1 (IGF-1) or from retained substances of cholestasis, including
bilirubin and bile acids. In this regard, serum IGF-1 levels are de-
creased in patients with cirrhosis [66] and experimental evidence
has indicated that low doses of IGF-1 increase bone mass and bone
density in cirrhotic rats [67]. Also, other alterations such as in-
creased production of a fibronectin isoform containing the oncofe-
tal domain during liver disease may participate in the decrease of
bone formation [68].
Both bilirubin and bile acids have been involved in the low bone
formation associated with cholestasis, through an alteration in the
proliferation and survival of osteoblasts in in vitro studies. In this
respect, it was observed that unconjugated bilirubin decreased
osteoblast proliferation in a dose-dependent fashion without
affecting osteoblast viability, supporting the hypothesis that
hyperbilirubinemia or possibly other substances impair osteoblast
proliferative capacity, and thus may play a major role in the path-
ogenesis of the osteoporosis associated with chronic liver diseases
[69]. Recently, we have confirmed the harmful effects of unconju-
gated bilirubin, which decreases survival in cultured primary hu-
man osteoblasts [70]. Moreover, sera from jaundiced patients
significantly impair osteoblast differentiation, as measured by
alkaline phosphatase activity, effect which was not observed with
sera from patients with normal bilirubin levels [70]. Although the
potential detrimental role of bilirubin seems reasonable, some re-
ports do not support this hypothesis since in one study serum bil-
irubin levels do not correlate with reduced BMD in patients with
end-stage liver disease, and chronic unconjugated hyperbilirubine-
mia does not lead to alterations in bone mineralization in Gunn
rats [71].
In other recent experiments we have also observed that litho-
cholic acid (LCA), a monohydroxylated secondary bile acid synthe-
sized mainly from the intestinal bacterial 7-dehydroxylation of
chenodeoxycholic acid, has deleterious effects on human osteo-
blasts, not only in relation to their viability, but also regarding
the potential damaging effects of LCA on the vitamin D pathways,
through the vitamin D receptor (VDR). Actually, adding LCA to the
culture media resulted in a significant decreased osteoblast viabil-
ity, which was observed with concentrations as low as 100 lM in
the culture media. Thus, under these conditions and with no FBS
or albumin in the media, the cells decreased their viability by more
than 50% [70] (Fig. 2). Another interesting result emerging from
this experiment is the fact that the presence of proteins in the cul-
ture media, that is 10% FBS or human albumin (25 or 40 g/L), de-
creased or completely abolished the detrimental effect of LCA on
cell viability. These results can be explained by the binding capac-
ity of FBS or albumin which may have, partially or completely, ab-
sorbed or sequestered the free LCA [72], and therefore, decrease its
detrimental effect on cell viability. This observation is supported
by the dose–response results with respect to the human albumin
concentration in the culture media, and is in accordance with the
well-known capacity of albumin as a bile acid transporter
[72,73]. Thus, different studies provided evidence for the great
affinity of human albumin for LCA [73]. From these results it could
be proposed that the amount of circulating albumin is one of the
critical factors for explaining the harmful effects on bone of circu-
lating bile acids retained in patients with chronic cholestasis, data
which are in agreement with the higher prevalence of osteoporosis
and bone fractures observed in patients with PBC and severe cho-
lestasis [7]. Indeed, PBC patients with osteoporosis have lower ser-
um albumin concentration than patients without osteoporosis.
Because LCA is a VDR ligand [74], and high serum and hepatic
concentrations of this bile acid have been described in patients
with cholestasis and end-stage liver disease, we analyzed the effect
of LCA on the expression of VDR-mediated genes such as vitamin D
24-hydroxylase (CYP24A), bone gamma-carboxyglutamate protein
(BGLAP), receptor activator for NFjB ligand – RANKL – (TNFSF11)
used as indicators of vitamin D metabolism, osteoblast maturation,
and regulation of osteoclast differentiation and activation, respec-
tively. As expected, vitamin D (10 7 M) in the media dramatically
increased the expression of CYP24A1, a gene which encodes the
hydroxylase involved in the catabolism of vitamin D. LCA 10 lM
was also able to induce CYP24A1 gene expression, but the LCA
capacity was about 100 times lower than vitamin D alone. More-
over, the addition of LCA at concentrations without effects on cell
 N. Guañabens, A. Parés / Archives of Biochemistry and Biophysics 503 (2010) 84–94
200 Basal
150
Viability (%)
100
50
0 0
200 10% FBS
150
Viability (%)
100
50
0 0
LCA (M) - 10-6 10-5
6 hrs
Fig. 2. Effect of lithocholic acid on the viability of primary human osteoblasts. Cells were incubated for 6 (grey), 24 (orange), 48 (green) and 72 (yellow) hours with increasing
concentrations of lithocholic acid (10–6 M, 10–5 M, 10–4 M and 10–3 M) in different medium conditions (basal, 10% FBS, albumin 25 g/L, and albumin 40 g/L). Results are
expressed as means ± SD. Culture experiments were performed in triplicate for each pool of osteoblast cells and treatments.
viability, and vitamin D in the media significantly decreased the
CYP24A1 gene expression by 72%. Apart from this capacity of
LCA on this gene involved in the catabolism of vitamin D, LCA also
decreased the expression of two other genes such as osteocalcin
and RANKL [75]. By contrast, no effect of vitamin D or LCA, alone
or combined, was observed on the osteoprotegerin, a gene which
is not dependent on VDR. Again, the presence of FBS or albumin
in the culture media also modified the ability of inducing gene
expression, since the osteocalcin and CYP24A1 mRNA expression
were decreased in the experiments with FBS 10% or albumin
25 g/L in the culture media, and were roughly and completely abol-
ished when the concentration of albumin was of 40 g/L [76].
Some histomorphometric reports have revealed increased bone
resorption and turnover even in the absence of osteoporosis as an
early feature of bone disease in PBC [77]. Reduced wall thickness
and increased bone turnover have been found to be proportional
to the severity of hepatic dysfunction and cholestasis [78]. Thus,
overt or subtle calcium and vitamin D deficiencies leading to sec-
ondary hyperparathyroidism have been proposed as the cause of
increased bone turnover found in some patients with PBC [79].
Likewise, reduced osteoprotegerin may also contribute to the
development of osteoporosis in PBC, since a decline in liver func-
tion may hypothetically result in reduced production of osteopro-
tegerin and increased osteoclast-mediated bone resorption.
However, at present the role of osteoprotegerin remains specula-
tive and there is information showing high circulating osteoproteg-
erin levels in PBC, which are unrelated to osteoporosis [80,81].
Also, a role for proinflammatory cytokines has been suggested in
the bone loss of chronic liver diseases [82]. Thus, it has been shown
in viral cirrhosis that serum concentrations of soluble tumor necro-
sis factor receptor p55 (sTNFR-55) are significantly higher in pa-
tients with osteoporosis and are inversely correlated with BMD
at the lumbar spine and femoral neck, and positively with urinary
deoxypyridoline [83]. A further mechanism that needs confirma-
tion is the observation of a higher capacity of circulating mononu-
clear cells to become osteoclasts in patients with chronic liver
disease and osteopenia [84].
Since vitamin K is involved in bone metabolism, mediating the
carboxylation of glutamyl residues in bone proteins such as osteo-
calcin, vitamin K deficiency may be considered as another supple-
mentary factor in the pathogenesis of osteoporosis in liver disease,
89
200 Albumin (25 g/L)
150
100
* * *
* ** 50 ** *
*
200 Albumin (40 g/L)
150
100
* *
* 50
10-4 10-3 - 10-6 10-5 10-4 10-3
24 hrs 48 hrs 72 hrs *p<0.05
taking into account that this vitamin can be decreased in subjects
with severe cholestasis. A preliminary study showed that serum
undercarboxylated osteocalcin was high in a small group of pa-
tients with PBC and the values decreased after administration of
vitamin K1 [85].
Genetic susceptibility for osteoporosis in PBC has been searched
for in the quest to analyze associations with vitamin D receptor
gene (VDR) and the gene encoding collagen type IaI (COLIA1).
One study in 72 Canadian women with PBC, concluded that the
VDR genotype is an independent genetic predictor of decreased
BMD in PBC [86], whereas others failed to find this association
[87,88]. The COLIA1 Sp1 polymorphism influenced the bone mass
in a group of Spanish patients with PBC [88], since it was associ-
ated with reduced baseline BMD. Nevertheless, the impact of the
COLIA1 Sp1 polymorphism on bone mass in patients with PBC
was not directly related to the liver disease itself. The influence
of IGF-1 gene microsatellite repeat polymorphism on osteoporosis
in PBC has also been investigated, with weak results [89]. Taken to-
gether, in PBC the VDR, COLIA1 and IGF-1 polymorphisms, studied
in a short series of patients, either do not influence the develop-
ment of osteoporosis in PBC or have a very small size effect [90].
Other conditions in patients with liver disease, including low
vitamin D levels, hypogonadism and poor nutrition [2,3], may be
contributing factors to the full picture of bone disease. Thus, hypo-
gonadism which is frequent in patients with hemochromatosis
[14,91,92], cirrhosis and alcoholic liver disease [93], may result
in increased bone remodeling and contribute to bone loss. Addi-
tionally, certain facts have demonstrated the harmful effects of
alcohol and iron on bone formation. A reduction in the grade of
bone formation has been observed in alcoholic patients on bone
biopsies, with low serum levels of osteocalcin during alcohol in-
take, which normalizes with abstinence from alcohol [24,27,93].
On the other hand, in hemochromatosis the deposits of iron may
be responsible for low bone formation, due to the direct lesion-pro-
ducing effects of iron on osteoblast activity [92,94].
After liver transplantation, according to a unifying hypothesis
for cardiac, lung and liver transplantation, there seem to be two
main phases in post-transplant bone disease: the early and the late
post-transplantation periods [55,95]. Before liver transplantation
there is a low bone turnover state, supported by biochemical and
especially histomorphometric analysis of bone biopsies. Shortly
90 N. Guañabens, A. Parés / Archives of Biochemistry and Biophysics 503 (2010) 84–94
after liver transplantation, in the first 3 months, there is a signifi-
cant and quantitatively large increase in bone turnover, substanti-
ated by histomorphometric data [96] and early increase in the
biochemical markers of bone resorption that exceeds bone forma-
tion markers [40,56,97]. Moreover, during this period, there is a
trend towards a normalization of mineral metabolism disorders
associated with end-stage liver disease. An improvement in gona-
dal function is observed and 25-hydroxyvitamin D levels, which
were low before transplantation, are significantly increased at
3 months [40]. Taken together, the earliest period of rapid bone
loss and high rate of fractures is associated with a high bone turn-
over with an uncoupling of resorption and formation. The normal-
ization of liver function and a superimposed effect of
immunosuppressive therapy, reliant on fairly high doses of gluco-
corticoids combined with cyclosporin or tacrolimus, with their sys-
temic effects, give rise to the picture during the early post-
transplant period [55]. The second phase, which generally appears
6 months after transplantation, is characterized by an increase in
both the histologic parameters as well as the biochemical markers
of bone formation [40,56,97]. At this time, bone loss at the lumbar
spine has stopped and BMD has begun to increase spontaneously.
Similarly, there is an increase in the parathyroid hormone levels
comparable to those of creatinine, as well as a rise in circulating
osteocalcin [40]. This secondary hyperparathyroidism, likely re-
lated to the decline of renal function associated with cyclosporin,
may be a major contributor to the disorder of bone remodeling
[98], with special implication in cortical bone loss. These features
occur while patients are maintained on cyclosporin alone or com-
bined with a low dose of glucocorticoids. The factors involved in
bone turnover in this second phase after transplantation are the
normalization of liver function and the gradual reduction in gluco-
corticoids, which are responsible for an increase in bone formation,
as well as a secondary hyperparathyroidism related to cyclosporin
and tacrolimus administration.
Diagnosis
The presence of risk factors for the development of osteoporosis
should be evaluated in patients with chronic liver disease, includ-
ing the following: chronic alcohol intake, smoking, body mass in-
dex lower than 19 kg/m2, male hypogonadism, early menopause,
secondary amenorrhea of more than 6 months, family history of
osteoporotic fracture and treatment with glucocorticoids (5 mg/d
of prednisone or over for more than 3 months).
There has been some debate about the indications of bone den-
sitometry in patients with chronic liver disease [4]. There is con-
sensus, however, that BMD should be evaluated in patients with
previous fragility fractures, exposure to glucocorticoids and before
liver transplantation [2,3]. Also, it seems appropriate to indicate
assessment with a BMD test when the diagnosis of PBC is first
made [2] or if any of the previously described risk factors are
found, and the patient has chronic cholestasis [3] or cirrhosis [2],
as well as after liver transplantation (Table 3).
Table 3
Recomendations for bone mineral density assessment.
Previous fragility fractures
Glucocorticoid therapy (>3 months; >5 mg/d prednisone)
PBC at diagnosis
Major risk factors for osteoporosis, particularly in chronic cholestasis and
cirrhosis*
Alcohol abuse
Hemochromatosis
Before and after liver transplantation
*
Postmenopausal women, low body mass index, male hypogonadism, early men-
opause, secondary amenorrea.
The frequency of follow-up BMD measurements remains debat-
able [4]. However, it is reasonable to recommend a BMD test annu-
ally or every two years in patients with chronic cholestasis with
several additional risk factors for osteoporosis, as well as in pa-
tients receiving glucocorticoids and after liver transplantation;
and at 3-year interval in those patients within the normal range
of BMD to exclude significant bone loss in the follow-up.
Lateral X-rays of the dorsal and lumbar spine should also be car-
ried out to disclose vertebral fractures [2,3]. In addition, circulating
levels of calcium, phosphorous, 25-hydroxyvitamin D and parathy-
roid hormone should be assessed. Disturbances in thyroid and go-
nadal function should be ruled out in particular cases. Biochemical
markers of bone turnover may be assessed to monitor the individ-
ual response to anti-osteoporotic treatment. However, there is lit-
tle information on changes in bone markers in therapeutic trials in
patients with chronic liver disease and practical guidelines for their
use in the clinical management of postmenopausal osteoporosis
are still lacking [99]. The indication of undecalcified transilial bone
biopsy is advisable only if a mineralizing defect is suspected, which
is at present a very rare condition in chronic liver disease.
It is interesting to consider possible artifacts resulting in inaccu-
racy in BMD and bone markers measurements in patients with ad-
vanced liver disease. Thus, we reported that collagen-related
markers of bone turnover do not accurately reflect bone remodel-
ing in PBC, since they are influenced by liver collagen metabolism.
The close association of these markers with the histologic stage of
the liver disease and the serum amino-terminal propeptide of type
III collagen levels, an index of liver fibrogenesis, supported this
affirmation [65]. In addition, central BMD measured by DXA in pa-
tients with ascites may be falsely reduced. Both, Labio et al. and our
group have found that lumbar and total hip BMD values increase
after therapeutic drainage of ascites in end-stage cirrhotic patients
[100,101].
Prevention and treatment
Modification of risk factors and supportive measures for bone health
The factors contributing to bone loss should, as far as possible,
be reduced to a minimum by stopping alcohol intake and smoking.
As much physical activity as possible is advisable, as are exercises
aimed at improving the mechanics of spine. However, the optimal
exercise programme for osteoporosis in patients with chronic liver
disease or after liver transplantation has not yet been determined.
Specific recommendations should be given in patients with in-
creased risk for falling.
Whenever possible, a balanced diet should be prescribed since
patients with advanced liver disease frequently have little appetite
and are malnourished. Supplements of calcium (1000–1500 mg/d)
and vitamin D (260 lg of 25-hydroxyvitamin D every 2 weeks or
vitamin D3 with a dose of 800 U/D or 5000 U/wk, orally) or the dose
required to maintain normal levels should be provided. Particular
care should be taken with patients receiving resins, such as chole-
styramine, since their administration may reduce the intestinal
absorption of vitamin D. The dose of glucocorticoids should be ad-
justed to the minimum necessary. Although calcium and vitamin D
supplements are recommended, there are no unequivocal data
confirming the efficacy of these supplements for preventing bone
loss in patients with liver disease.
Specific treatments
Different drugs for osteoporosis have been proposed in patients
with liver disease, but most studies have included small numbers
of patients, and therefore it is difficult to come to any definite con-
 N. Guañabens, A. Parés / Archives of Biochemistry and Biophysics 503 (2010) 84–94
clusions. Furthermore, no clear anti-fracture effect could be dem-
onstrated and except for osteoporosis in PBC and after liver trans-
plantation, no consistent studies have been carried out.
There is no agreement concerning the appropriate time to start
treatment, but patients with established osteoporosis, and there-
fore with fragility fractures, should be treated to reduce the risk
of further fractures. On the other hand, and taking into account
that PBC patients with a lumbar or a proximal femur T-score lower
than < 1.5 have a high risk for vertebral fracture, it seems reason-
able to consider treatment in patients with chronic cholestasis and
BMD below this threshold [5], particularly if they have additional
risk factors for osteoporosis. Likewise, it seems reasonable to treat
all patients immediately after liver transplantation, and before
transplantation if they have osteoporosis.
Bisphosphonates
Bisphosphonates are anti-catabolic drugs which increase bone
mass and reduce the incidence of fractures in postmenopausal
osteoporosis. Oral bisphosphonates are poorly absorbed in the
intestine; they are not metabolized and rapidly cleared from the
circulation. Approximately 50% of the absorbed fraction concen-
trates into the skeleton, and the remainder is excreted into the ur-
ine. Their effects in patients with liver disease are not completely
conclusive, basically because of the scarce number of studies and
the few treated patients [102–111]. Nonetheless, it has been dem-
onstrated that cyclic administration of etidronate (a bisphospho-
nate with a narrow therapeutic window) is able to prevent bone
loss after two years of treatment and that alendronate increases
bone mass in PBC, comparable to what occurs in osteoporosis
due to other causes [106]. Serious adverse events were not ob-
served and potential harmful effects of alendronate such as esoph-
agitis were not detected. One placebo-controlled trial of
alendronate (70 mg per week) in patients with PBC also indicated
that alendronate is able to increase bone mass after one year with
no or with only minor adverse effects [107,108]. Our results of
once-weekly 70 mg alendronate in patients with PBC indicated
that this regimen was more effective in increasing BMD, with a
non-significant increased adherence and a better tolerability pro-
file, than daily dosing in the treatment of low bone mass in pa-
tients with PBC treated for one year. An interesting observation
was that the effects of alendronate on BMD were lower in patients
with higher features of cholestasis [108]. Preliminary results of an-
other study comparing alendronate 70 mg weekly vs. ibandronate
150 mg monthly in PBC patients with osteoporosis or low bone
mass and fragility fractures, showed that both drugs have similar
effects on BMD, but the adherence to treatment was superior for
monthly ibandronate, without adverse effects on liver tests [109].
Pamidronate, administered parenterally, has been evaluated in
patients with end-stage chronic liver disease, prior to and after li-
ver transplantation [41,112–114]. The studies have reported con-
tradictory results with respect to the efficacy of this agent on
preventing bone loss and reducing the fracture rate. One study per-
formed in patients with low bone mass, the administration of
pamidronate every 3 months prior to and during 9 months after
transplantation, demonstrated a certain reduction in the incidence
of fractures compared with a reference group of untreated patients
[41]. Another study using 30 mg of pamidronate every 3 months,
calcium (1 g/day) and vitamin D (1000 IU/day) for two years was
associated with a significant increase in lumbar and femoral
BMD [113]. A third study, including 99 patients who, prior to trans-
plantation, randomly received 60 mg of pamidronate or were con-
trol patients, did not show any favorable effect of pamidronate on
either bone mineral density or the rate of new fractures [112].
These studies with pamidronate included few patients or were
inappropriately designed to detect bone loss and the capacity for
reducing the risk of new fractures. The results of a placebo-con-
91
trolled trial performed in Spain, including 79 liver transplant pa-
tients, indicated that 90 mg of pamidronate given within the first
2 weeks and at 3 months after transplantation, preserves lumbar
BMD during the first year without significant side-effects. How-
ever, pamidronate did not reduce bone loss at the femoral neck,
nor the development of fractures [114].
Alendronate and zoledronic acid have also been evaluated in li-
ver transplant recipients. In a randomized controlled trial Atamaz
et al. found that alendronate (70 mg per week) plus daily calcium
and 0.5 lg of calcitriol significantly increased BMD during the
2 years after liver transplantation, when compared with calcium
and calcitriol alone. Alendronate was well tolerated without dele-
terious effects on liver function tests. However, alendronate did not
appear to offer protection against fractures in this study [111]. A
previous study using weekly alendronate also showed that alendr-
onate prevents bone loss associated with liver transplantation
[110]. Thirty-two patients were treated with infusions of zoledron-
ic acid (4 mg) or saline within 7 days of transplantation and at 1, 3,
6 and 9 months after transplantation. Zoledronic acid treatment
was associated with favorable effects on BMD at the lumbar spine,
femoral neck and total hip, although temporary secondary hyper-
parathyroidism and particularly hypocalcemia were observed after
infusion in some cases. No effect was observed on the incidence of
fractures. The study concluded that zoledronic acid can prevent
bone loss within the first year after liver transplantation [115]. An-
other study using zoledronic acid (4 mg) monthly for the first
6 months, and again at 9 and 12 months in patients after liver
transplantation, showed that treated patients had reduced bone
turnover and most importantly, a lower fracture rate [116].
Hormone therapy
There is little information on hormonal treatment in patients
with advanced liver disease since for years this approach was con-
sidered to be contraindicated in these patients. However, transder-
mal estrogen treatment prevents bone loss or even increases BMD
in patients with PBC [117–121] or autoimmune cirrhosis with no
adverse effects on liver disease. The efficacy and safety of short-
term hormonal treatment with estradiol in postmenopausal wo-
men after liver transplantation has also been analyzed and was
associated with an increase in lumbar and femoral neck BMD, to-
gether with a decrease in the serum levels of a marker of bone for-
mation [122]. In this group of patients it must be taken into
account that estrogen enhances hepatic metabolism of cyclospor-
ine A.
In males with hemochromatosis and hypogonadism, treatment
with testosterone and venesection is also effective [123]. One con-
cern about restoring testosterone levels in cirrhotic patients is that
this might increase the risk of hepatocellular carcinoma. Therefore,
the potential risk/benefit must be discussed with each patient be-
fore starting replacement therapy.
Despite these results, hormone therapy is not considered to be
the most appropriate treatment, as there are other efficacious non-
hormonal agents with lesser side-effects for the treatment of
osteoporosis.
Calcitonin
The effect of calcitonin as an anti-catabolic drug in patients with
liver disease is not clear. A pilot study carried out in PBC reported
that the combined administration of calcitonin, calcium and vita-
min D over 12 months was associated with lower bone loss than
in untreated patients [124], and apparently the long-term effects
were more favorable in patients with osteopenia [125]. However,
other studies have described negative results [126]. In transplant
patients, the intramuscular administration of 40 IU/day of calcito-
nin over a period of one year was associated with a significant in-
crease in BMD [127]. However, the effects of calcitonin treatment
92 N. Guañabens, A. Parés / Archives of Biochemistry and Biophysics 503 (2010) 84–94
on bone mass and fracture rate are not clear, as in another 6-month
study in patients with low baseline BMD (Z-score < 2), the drug
was unable to prevent bone loss and decrease the rate of fragility
fractures during the first year after liver transplantation [128].
Raloxifene
The safety and efficacy of raloxifene has been evaluated in a pi-
lot study including nine postmenopausal women with PBC and an
age-matched control group [129]. Compared to baseline, lumbar
BMD improved significantly after one year of therapy but not in
matched controls. No significant effects were observed in femoral
neck BMD in either group. Raloxifene was well tolerated.
Other treatments
To the best of our knowledge there are no studies assessing the
effects of anabolic drugs or strontium ranelate in patients with
osteoporosis and liver diseases. Only one study assessed intermit-
tent administration of parathyroid hormone (hPTH 1–34) in bile
duct-ligated rats, an animal model of chronic cholestasis. This
study showed that PTH restores BMD as well as trabecular thick-
ness, suggesting that PTH 1–34 is a potential therapy for osteopo-
rosis in PBC [130].
Summary and perspectives
Osteoporosis is a frequently observed complication in patients
with chronic liver disease, especially in end-stages and in cases
with chronic cholestasis, hemochromatosis and alcohol abuse.
The problem is even more critical in transplant patients when bone
loss is accelerated during the period immediately after transplan-
tation, leading to a greater incidence of fractures. The pathogenesis
of osteoporosis is mainly characterized by low bone formation,
particularly related to the effect of retained substances of cholesta-
sis, such as bilirubin and bile acids, or to the iron and alcohol on
osteoblastic cells. Increased bone resorption has also been de-
scribed, especially in cholestatic women with advanced disease.
In this disorder, osteoporosis is associated with the severity of
the disease, older age and with the duration of cholestasis. Addi-
tional alterations in hormonal homeostasis, such as low vitamin
D, hypogonadism and drug-induced effects are found in the broad
spectrum of patients with liver disease.
For the prevention and treatment of osteoporosis good nutrition
is recommended, as are the suppression of the controllable risk fac-
tors for osteoporosis and the administration of supplements of cal-
cium and vitamin D. Hormone treatment has been suggested for
prevention, although at present this indication is under debate,
even in patients without liver disease because of potential severe
long-term adverse effects. There is no specific treatment for osteo-
porosis, although it has been demonstrated that bisphosphonates,
especially weekly alendronate and probably monthly ibandronate,
are effective in increasing bone mass in patients with chronic cho-
lestasis. The efficacy of bisphosphonates in patients after liver
transplantation remains to be confirmed, although weekly alendr-
onate prevents bone loss and recent appealing effects of zoledronic
acid have been reported. The outcome in reducing the incidence of
fractures has not been adequately demonstrated essentially be-
cause of the low number of patients included in the therapeutic tri-
als. The development of larger trials with bisphosphonates and the
assessment of new drugs for osteoporosis may change the future.
Acknowledgments
Supported in part by Centro de Investigación Biomédica en Red
de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de
Salud Carlos III, and FIS-08/0105, Ministerio de Ciencia e Innova-
ción, Spain.
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