Currently, liver transplantation (LTX) is the treatment of choice for patients with end-stage liver failure (1). Advancements in surgery and immunosuppressive therapy have led to improved survival rates. Therefore, complications such as transplantation bone disease are of growing importance (2).
Hypogonadism, immobilization and vitamin D deficiency are major contributors to bone loss already before LTX (3, 4). Hepatic osteodystrophy is common among patients on the waiting list for liver replacement (4–6) and results in osteoporosis thereafter.
During the early phase after LTX, bone metabolism is characterized by an uncoupling of bone turnover resulting in accelerated bone resorption and suppressed bone formation (3, 7) with fracture rates of up to 50% during the first 6 months (8).
In addition, transplantation bone disease is characterized not only by bone loss leading to low bone density but also by alterations of bone microarchitecture resulting in bone fragility and high fracture rates (4). Major risk factors for posttransplant fragility fractures are a high cumulative steroid dose, multiple rejection episodes, high cyclosporine levels, older age, female sex, poor nutritional status, and low bone mineral density (BMD) (9).
Study groups commonly focus on the early posttransplantation period, showing that bone loss and fracture incidence are highest during the first year after surgery (1). There are some data indicating that during the second and third year bone mass recovers but for later time periods after LTX, the available data are scarce (1, 10).
Although controversially discussed over years (11, 12), several reviews suggested bisphosphonate therapy as potential osteoprotective treatment especially if started early after LTX (11, 13, 14), but data on fracture reduction are still missing. Low dose ibandronate (IBN) has been used effectively for fracture prevention after heart transplantation; however, data are available only for the first year after surgery (10).
Among long-term survivors of LTX (4, 15, 16) other comorbidities such as renal impairment, vitamin D deficiency, and secondary hyperparathyroidism derive as potential confounders for impaired bone metabolism (1, 9).
Long-term follow-up of bone status in transplant patients are still lacking, but there is some evidence that bone mass recovers after the first 12 months (17). One of the reasons is the loss of follow-up of these patients as the aftercare is likely to be outsourced from the transplantation department after 1 or 2 years, a fact that makes standardized aftercare difficult.
This study aimed to evaluate bone metabolism in LTX patients who were at least in their second year after surgery. Patients with osteoporosis diagnosed according to WHO criteria (18) received a therapy with quarterly low IBN intravenously. A majority of patients revealed secondary hyperparathyroidism (Table 1) likely to decreasing renal function and therefore oral calcitriol was administered on top of IBN. Patients with normal bone metabolism, normal bone density, and without fractures were invited to participate in the study as a control group. All participants received daily 1000 mg calcium and 800 IU cholecalciferol.
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TABLE 1: All baseline characteristics of the patients that have been included in the present study according to the patient's group assignment
RESULTS
Patient Characteristics at Baseline
Thirty patients with a mean age of 57±3 years (50% women, all of them postmenopausal) were included into the treatment group, 24 patients with a mean age of 54±3 years (38% women, all of them postmenopausal) served as control group. Indications for LTX were mainly alcoholic or posthepatitis liver cirrhosis. All baseline characteristics are shown in Table 1 according to patient groups.
By definition, all patients after LTX assigned to the treatment group had osteoporosis (19) with a fracture prevalence of 53% at baseline. Laboratory analysis revealed a high prevalence of impaired renal function in 60% with a mean serum creatinine level of 1.2+0.1 (0.6–1.3 mg/dL), low vitamin D levels of 13.9±3 (9–45 ng/mL) levels accompanied by elevated intact parathyroid hormone (iPTH) 80±13 serum levels (10–65 pmol/mL) (Table 1).
Bone Turnover Markers During the Study Period
Serum cross laps (sCTX) and osteocalcin (OC) were elevated in 73%, respectively, 67% of the IBN patients at baseline, indicating an accelerated bone turnover. Elevated tartarte-resistant alkaline phosphatase 5b (TRAP Vb) and bone-specific alkaline phosphatase (bALP) serum levels, turnover markers considered largely independent of renal function (20), were indicative for high bone turnover in the study group as compared with the control patients who had turnover markers within the normal range at baseline (Table 1).
During the study period, bone turnover markers decreased significantly in the treatment group during the first treatment year, leveling off within the normal range throughout the whole follow-up period (Fig. 1a, b).
FIGURE 1: (a, b) The development of serum cross lap (sCTX) and tartarte-resistant alkaline phosphatase 5b (TRAP Vb) throughout the whole study period. Both parameters decreased significantly (sCTX: −43%; P=0.002; TRAP Vb: −51%; P=0.0001) in this study group throughout the whole study period to nearly normal values at the study end.
sCTX decreased significantly (P=0.03) by 49% during the first year compared with the values observed in the control group (Fig. 1a) and remained stable in the time period later on (P=0.03, years 2 and 3 as compared with baseline). After the first year, there was no longer any significant difference in sCTX values between the two groups (Table 2).
TABLE 2: Course of all parameters measured at the different time points
TRAP Vb decreased significantly and continuously by 35% after the first (P=0.01), 40% after the second (P=0.04) and 51% throughout the third year (P=0.03) (Fig. 1b). After the first year, TRAP Vb values were comparable between the treatment and the control group (Table 2).
After a significant (P=0.03) decrease of 66% between baseline and 12 months, OC values in the treatment group remained stable throughout the whole study period (Table 2) and were significantly higher (P<0.05) during the first and the second year as compared with values in the control group. At the study end, the markers were comparable. At 36 months after treatment, OC values in the treatment group did not show significant differences as compared with the control patients.
bALP values decreased by 49% (P=0.001) during the first treatment year in the study group. After this decrease, the values remained stable throughout the whole study period (Table 2). bALP values in the control patients decreased slightly to mean levels above those in the treatment group during study years 2 and 3 (Table 2).
iPTH levels were significantly different at baseline between the two groups (P=0.001, Table 1). During the first year, iPTH levels decreased significantly (P=0.001) by 47% in the treatment group and remained stable in the period thereafter. At 12, 24, and 36 months, iPTH levels in the IBN patients did not differ significantly as compared with those in the treatment group and remained within the normal range among both groups during the whole study period (Table 2).
BMD Changes Throughout the Study Period
Per definition BMD results at the femoral neck and the trochanteric region at baseline were significantly lower in the IBN group (Table 1) when compared with the control group.
Bone density at the femoral neck increased significantly (P=0.002) by 6% within the first study year and by further 7% in the second (P=0.001 vs. baseline) and remained stable within the third year. The control patients showed a significant BMD decrease at the femoral neck (−5%, P=0.03). Despite this, the median BMD values in the IBN group were still lower as compared with the control patients at all time points (P<0.05, Table 2). The BMD at the trochanter increased significantly (P=0.03) during the first and the second year (P=0.04) and remained stable during the third year of the observation period in the study group. Patients in the control group showed a decrease of the BMD of 4.9%, which was significant at 2 years and study end (P=0.03) as compared with baseline.
Fractures During the Study Period
At baseline, none of the control patients had a prevalent vertebral fracture, whereas in more than half of the patients (53%) spinal X-ray revealed at least one fracture. After a study period of 3 years, 23% (n=5) of the control patients had sustained a vertebral fracture, whereas only 7% of the patients (n=2) in the treatment group had developed a new vertebral fracture (P=0.06). The relative risk to sustain a new vertebral fracture in the control group as compared with the treatment group was 3.12 with an odds ratio of 3.68 (P=0.035).
Treatment of Patients With Fractures
All patients who sustained fractures were administered analgetic therapy accordingly. Three patients in the control group and one patient in the treatment group received vertebroplasties.
DISCUSSION
A main and surprising result of this study is the high fracture risk not only in osteoporotic patients but also in liver transplant (LTX) recipients with normal BMD in the first years after transplantation. LTX patients were at high fracture risk with a fracture incidence of 23%, despite prophylactic treatment with calcium and vitamin D. High fracture rates in the long term: in expression beyond 5 years after LTX have been reported after LTX (21), whereas those patients had not received any treatment. Those fracture rates might be driven by development of renal impairment, secondary hyperparathyroidism, vitamin D insufficiency, and accelerated bone turnover (19). The high fracture risk in the current population is in contrast to a recently published 10-year follow-up on fracture incidence after LTX (17). In this study, the mean follow-up was 61 months and therefore comparable with our study, whereas fracture incidence was extremely low with 3.5%. A possible explanation may be that the immunosuppressive regimen used at this center is different. Missing of laboratory parameters, dual X-ray absorptiometry (DXA) readings, and reporting of comorbidities complicates further comparisons.
A further disturbing finding is the high prevalence of osteoporosis among long-term survivors. A total of 56% (30/54) of patients had osteoporosis with a fracture prevalence of 53% (16/30). Because of the promising results achieved with low dose IBN in a cardiac transplant population (10), the high prevalence of secondary hyperparathyroidism and renal failure, we decided to treat these patients with oral calcitriol and 2 mg IBN in addition to calcium and vitamin D3 supplementation. This combination therapy turned out as a successful treatment approach with a significant BMD increase, normalization of bone turnover and a trend for a favorable effect on fracture incidence. Although fracture incidence was alarmingly high in the control group, only 7% of the patients sustained new vertebral fractures throughout the study period in the study group. This implicates an absolute fracture risk reduction of 14% by the applied treatment.
Treatment for osteoporosis after LTX has been discussed and is recommended during the early-stage postsurgery (1). However, oral medication often results in nonadherence primarily due to the high number of prescribed drugs in this patient group (22, 23). Intravenous application of bisphosphonates has been suggested to be effective after LTX (10, 24, 25) and is recommended as a first line treatment for osteoporosis or osteopenia in the first year after LTX (1). The need to adequately address this frequent comorbidity seems to be severely underrecognized due to the suspected nephrotoxicity of bisphosphonates, and an underestimation of fracture risk after LTX (3, 13, 21).
After a follow-up period of 3 years, bone turnover markers in all study group patients had returned to normal. Recently, similarly promising results were published for heart transplant recipients for the early posttransplant period receiving treatment with IBN, calcium, and vitamin D (10, 26). BMD readings measured through the DXA were still higher in the control group as compared with the study group after 3 years. Despite this higher BMD results, those patients had sustained vertebral fractures during the follow-up period likely to the fact that the BMD decreased significantly in the control group especially in the first year after he presented study (Fig. 2).
FIGURE 2: The course of the bone mineral density (BMD, g/cm2) on the femoral neck in the study group and in the control patients. The BMD increased significantly throughout the whole study period in the study group (P=0.014), whereas it decreased significantly in the control group (P=0.032).
In conclusion, LTX patients are at high risk for bone loss and vertebral fractures far beyond the first year. Calcium and vitamin D supplementation is not enough to stabilize bone mass and prevent fractures even in LTX recipients with normal bone health. Parenteral low dose IBN and calcitriol in combination with calcium and vitamin D are a safe and effective therapy and normalize bone metabolism.
MATERIALS AND METHODS
Study Design
All included patients had received a liver graft at the Department of Surgery, and Division for Transplantation, Medical University Graz, Austria. Transplantation was performed using a piggy pack technique with retrograde reperfusion (23). Patient recruitment and administered therapy is described in a study flow chart in Figure 3. Study recruitment lasted from March 2005 and ended November 2006. The last patient completed the study in December 2009.
FIGURE 3: The flow chart of the presented study.
The study was performed at the osteoporosis outpatient clinic of the Department of Internal Medicine, Medical University Graz, Austria, and recruitment was part of the routinely scheduled control visits of the Department of Transplantation Surgery. The study was approved by the Ethical committee of the Medical University of Graz (protocol number EK-06-266) and informed consent was obtained from all participants. All patients underwent BMD measurement and standardized spinal X-ray beside laboratory analysis. Osteoporosis was diagnosed according to WHO criteria (18).
Inclusion Criteria
All adult survivors (>12 months after surgery) after LTX were eligible to participate in this study.
Exclusion Criteria
Exclusion criteria were previous or current osteoporosis therapy including calcium and vitamin D, primary hyperparathyroidism, a serum creatinine concentration of more than 2.0 mg/dL, liver enzymes more than three times the normal, hyperthyroidism, hypocalcaemia, cancer, or a previous transplantation and a severe disease recurrence of viral or alcoholic hepatitis resulting in recurrent cirrhosis, cognitive impairment, or bilateral hip replacement.
In total, 75 patients were screened. Twenty-one did not participate in this study (nine no consent, five disease recurrence, and seven prestudy osteoporotic treatment). A study flow chart is given in Figure 3.
Administration of Treatment Regimens
Study group patients: Quarterly IBN 2 mg was administered intravenously as part of the routine follow-up visits. Dependent on serum calcium, phosphate, and parathormone levels, all patients received oral calcitriol ranging from 0.25 to 1.0 μg daily. All participants including control patients were treated with 1000 mg of calcium carbonate in combination with 800 IU of vitamin D3 daily.
Immunosuppressive Therapy
Immediately after transplantation, all patients received horse antithymocyte globulin ranging from 2.0 to 4.0 mg/kg/d (Thymoglobulin, Pasteur Merieux, France) and a methylprednisolone taper starting with 70 mg every 8 hr for 7 days. Calcineurin inhibitors were initiated according to renal function on days 1 to 3, and standard trough levels were reached on day 7. Mycophenolate mofetil (MMF) was introduced between days 2 and 7. Starting on day 7, prednisolone was given at 15 mg/d and was tapered after the first month. In case of biopsy-proven rejection, 15 mg of steroids were administered and tapered until normalization of liver function. Our department favors standard maintenance immunosuppression with MMF together with tacrolimus and sirolimus and 2.5 mg of prednisolone per day for the first 24 months. Thereafter, cortisone is administered if indicated. Patients suffering from hepatitis C received cyclosporine preferably to MMF. Trough levels for tacrolimus were 3 to 8 ng/mL and for cyclosporine between 60 and 150 ng/mL.
All immunosuppressive agents were introduced stepwise according to the trough levels without loading dose. Drug therapy was induced for risk factor management whenever indicated according to evidence-based guidelines. The different immunosuppressive therapeutic agents are listed in Table 1.
Laboratory Analysis
Blood sampling was performed at baseline and every 12 months as part of a routine outpatient visit between 8 and 10 A.M. after an overnight fast. All analyses were performed with kits of the same lot number, with the samples determined in random order. All samples were measured in duplicate and averaged. If the CV was greater than 10%, the sample was reassayed.
Bone Density Measurements
Bone density (BMD) at the femoral neck and the trochanteric region was measured by DXA (Hologic QDR 4500 Acclaim, Wedel, Germany) at baseline, and after 12, 24, and 36 months. The in vivo coefficient of variation at our institution for BMD at the femoral neck is 2.1% and 1.8% for the trochanteric region. BMD measurements are expressed as absolute (g/cm2) and Z-score values (±SD) that compare individual results to an age-matched normative database.
Spine Radiographs
Standardized X-rays of the (lower) thoracic and lumbar spine were performed at baseline and after 36 months. The anonymized films were analyzed observer blinded by an experienced radiologist. The severity of vertebral fractures was assessed using semiquantitative visual assessment and semiquantitative scores were assigned to each individual vertebra from T4 to L4. A reduction of 20% of the anterior, middle, or posterior vertebral height was classified as a vertebral fracture (20). Only a height reduction in a prior normal vertebra was classified as an incident, respectively, new vertebral fracture.
Statistical Analysis
Sample size was calculated based on the number of prevalent fractures of patients in the IBN group (53%) and an estimation of the fracture incidence for healthy patients at the same age in this population per year for the control patients (1.5%). With an alpha of 0.05 and a beta error of 0.2, we needed 23 patients per group. All continuous variables are expressed as mean and standard deviation. Fracture incidence and changes in BMD readings were used as outcome variables.
All statistical analyses were performed using SPSS version 16.0 (SPSS Inc., Chicago, IL). Data were checked for normality by Kolmogorov-Smirnov and Shapiro-Wilk testing for the overall population and for both groups. Potential differences between the treatment and the control group were calculated using the t test and, in the case of categorical variables, the χ2 test. P values less than 0.05 were considered significant. Relative risk ratios were calculated to determine the risk to sustain a new fracture during the study period; absolute risk reduction was calculated using the relative risk and the odds ratios of both groups.
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