Immunosuppressive and postoperative effects of orthotopic liver transplantation on bone metabolism

Authors


Abstract

Bone loss occurs early after orthotopic liver transplantation (OLT) in all liver transplant recipients and leads to postoperative fractures, especially in cholestatic patients with the lowest bone mass. Little is known about the underlying changes in bone metabolism after OLT or about the etiology of these changes. Histomorphometric analysis of bone biopsies, a method that allows assessment of bone volume, resorption, and formation, has shown improved bone metabolism at 4 months after OLT. It has further suggested that accelerated posttransplant bone loss occurs in the first 1–2 months after OLT, probably by an additional insult to bone formation. This study attempts to correlate the histomorphometric bone changes in paired bone biopsies (OLT and 4 months after OLT) of 33 patients undergoing OLT for chronic cholestatic liver disease with the many clinical and biochemical changes in these patients over the same period. Cumulative steroid dosage early after OLT is shown to be important, presumably by decreasing bone formation rates. The actual effect of calcineurin inhibitors on this early phase of bone loss is less clear, although posttransplant histomorphometric findings suggest that tacrolimus-treated patients have an earlier recovery of bone metabolism and trabecular structure compared with cyclosporine patients. Other factors important in the recovery of bone metabolism after the early phase of bone loss are recovery of liver and gonadal function and better calcium balance. (Liver Transpl 2004;10:638–647.)

Introduction

Bone loss with subsequent fracturing is a major complication in the early months after orthotopic liver transplantation (OLT) and has been most extensively studied in osteopenic patients with chronic cholestatic liver disease.1–3 Many factors integral to the early posttransplant course are potentially deleterious to bone. Cyclosporine, tacrolimus, and glucocorticoids all have known in vitro effects on bone metabolism, but clinical studies have failed to elucidate the actual contribution of immunosuppressive drugs to posttransplant bone loss. The etiologic mechanisms involved in this accelerated bone loss after OLT, as well as its eventual recovery, remain undefined.

To gain insight into the pathogenetic mechanisms of bone loss and gain after OLT, detailed histomorphometric analysis of paired bone biopsies, performed before and after OLT in patients with primary biliary cirrhosis (PBC) or primary sclerosing cholangitis (PSC), has recently been reported.4 This study shows improved bone metabolism by 4 months, suggesting that the major etiologic cause of the accelerated posttransplant bone loss is present very early after OLT and is probably related to an additional insult to bone formation.

By analysis of correlations of histomorphometric parameters with many clinical, biochemical, and radiological parameters both before OLT and during the first posttransplant year, this study tries to identify factors associated with postoperative bone loss and gain. Biochemical markers of bone formation (bone alkaline phosphatase, osteocalcin), and bone resorption (urinary hydroxyproline) also were studied to assess their usefulness in predicting changes in bone metabolism and bone turnover status.

Abbreviations:

25(OH)D, serum 25-hydroxyvitamin D; Aj.Ar, adjusted rate of bone apposition; BFR/BS, bone formation rate per trabecular bone surface area; BFR/BV, bone formation rate per osteoid-covered surface area; BGP, bone Gla-protein; BMD, bone mineral density; BV/TV, cancellous bone volume; Ct.Th, cortical thickness; DEXA, dual-energy X-ray absorptiometry; ES/BS, eroded surface; FSH, follicle-stimulating hormone ; MAR, mineralization rate; MLT, mineralization lag time; N.Oc, number of osteoclasts per 100 mm trabecular surface length; OLT, orthotopic liver transplantation; Ob.S/OS, osteoblast-osteoid interface; OS/BS, osteoid surface; OS/BV, osteoid volume; O.Th, osteoid thickness; PBC, primary biliary cirrhosis; PSC, primary sclerosing cholangitis; PTH, parathyroid hormone; SE, standard error of the means; W.Th, wall thickness; OKT3, anti-CD3 monoclonal antibody therapy.

Methods

Patient Population

Thirty-three adult patients undergoing OLT for PBC or PSC and without any other complicating illnesses or medications before OLT were included in this study. The study was approved by the Institutional Review Board of the Mayo Clinic. Diagnoses of PBC and PSC were made according to well-established criteria.5–7 The 33 patients were part of a treatment trial of calcitonin, which showed that posttransplant calcitonin had no effect on posttransplant bone loss, fracture development, or histomorphometric changes after OLT.4, 8 Patients consented to undergo 2 bone biopsies (at the time of OLT and 4 months later) and had clinical, biochemical, radiological, and bone density measurements at time of activation for OLT and at 4 and 12 months after OLT.

Clinical Course

Symptoms and signs of bone pain or fractures were sought, as well as measurements of height, weight, and functional status (Karnofsky Performance Scale rated from 0 to 100).9 From time of activation for OLT until the end of the first posttransplant year, oral calcium supplements of 1.5 g/day were prescribed for all patients. Vitamin D therapy (400 IU/day) was given if (1) serum 25-hydroxyvitamin D [25(OH)D] was less than30 ng/mL, (2) patients had hypocalcemia, (3) parathyroid hormone (PTH) level was greater than 50 pmol/L, or (4) urinary calcium was less than 75 mg/24 h. After OLT, patients received either a 2-drug immunosuppressive regimen with tacrolimus and prednisone or triple therapy with cyclosporine A, prednisone, and azathioprine. Cyclosporine and tacrolimus doses were adjusted to maintain whole-blood trough levels within the desired therapeutic range. Acute cellular rejection of the allograft was treated with intravenous Solu-Medrol on days 1, 3, and 5 after the diagnosis of rejection. Steroid-resistant rejection was treated with anti-CD3 monoclonal antibody therapy (OKT3). Karnofsky score, hospitalization days, and posttransplant complications were recorded in all patients.

Biochemical Testing

Blood was taken for biochemical assessment after an overnight fast and was tested by Mayo Medical Laboratories using standard methods. Biochemical testing included parameters of liver and kidney function (serum albumin, total alkaline phosphatase, total and direct bilirubin, prothrombin time, serum creatinine, and iothalamate clearance), as well as parameters of bone mineral metabolism [25(OH)D], total calcium, ionized calcium, 24-hour urinary calcium, serum phosphorus, PTH. Gonadal status was assessed in all female (serum follicle stimulating hormone [FSH] and estradiol) and male patients (free testosterone). In addition, serum markers of bone formation (bone alkaline phosphatase, osteocalcin) and urinary marker of bone resorption (urinary hydroxyproline) were measured. Osteocalcin (bone Gla-protein, BGP) was measured by radioimmunoassay using rabbit antibovine BGP antiserum and homogeneous bovine BGP.10 Urinary hydroxyproline excretion was measured according to the methods established by Kivirikko et al.11 and Bidlingmeyer et al.12 25(OH)D was measured according to method established by Kao and Heser.13 Immunoreactive PTH was measured by immunochemiluminometric assay.14

Assessment of Bone Disease

Measurements of bone mineral density (BMD) of the lumbar spine (L1–L4) were performed by dual-energy X-ray absorptiometry (DEXA) (coefficient of variation 2.2%), using a Hologic QDR 1000 densitometer before OLT and at 4 and 12 months after OLT. Results were expressed as T-scores (gender-adjusted to peak bone mass of normal population) and Z-scores (age- and gender-adjusted).

All patients had bone biopsies taken just before OLT and again at 4 months after OLT. To assess dynamic bone parameters, tetracycline bone labeling was done with oxytetracycline and demeclocycline as previously described.4 The initial bone biopsies were performed just before OLT by the transplant surgeon at the standard iliac crest bone biopsy site, using a 7.5-mm trephine.15 Glucocorticoids were not administered until after the bone biopsy. Bone biopsies 4 months after OLT were taken during an outpatient procedure from the contralateral iliac crest under local anesthetic. After the bone biopsies were taken, bone tissue was prepared for quantification of bone histomorphometric parameters using standardized methods. Quantification of bone histomorphometric parameters was carried out by Bioquant System IV image analysis, using a microscope and digitizing tablet (R and M Biometrics, Nashville, TN, USA). Primary and derived data were generated by the software in accordance with standardized nomenclature and formulas.16 Using bone histomorphometric values obtained at the Mayo Clinic in normal female and male patients as reference values,4, 17 bone histomorphometric parameters of the study population were expressed both as raw data and as Z-scores (gender-adjusted histomorphometric values).

Histomorphometric Parameters

The following static parameters were analyzed: Cancellous bone volume (BV/TV, trabecular bone as a percentage of the total medullary volume bone), osteoid volume (OS/BV, osteoid volume as a percentage of total bone volume), osteoid surface (OS/BS, percentage of trabecular surfaces covered with osteoid), osteoid thickness (O.Th, mean thickness of osteoid in μm), number of osteoclasts per 100 mm trabecular surface length (N.Oc), eroded surface (ES/BS, percentage of trabecular surface showing resorption cavities), osteoblast–osteoid interface (Ob.S/OS, percentage of osteoid surface covered by osteoblasts), cortical thickness (Ct.Th, mean thickness of cortical seams in μm), and wall thickness (W.Th, mean thickness of the total bone structural unit in μm, measured as the distance between the cement line and the mineralized bone surface).

The following dynamic parameters were analyzed: Bone formation rate per trabecular bone surface area (BFR/BS, amount of new bone mineralized per micrometer of trabecular bone surface area per day, expressed as mm3/mm2/y), bone formation rate per osteoid-covered surface area (BFR/BV, the average amount of new mineralized bone made per day per micrometer of osteoid-covered surface, expressed as mm3/mm2/y), adjusted rate of bone apposition (Aj.Ar, mean distance between tetracycline labels divided by the labeling interval in days, expressed as mm3/mm2/y), mineralization rate (MAR, averaged distance between the midpoints of two consecutive tetracycline labels, divided by the time of the labeling periods, μm/day), mineralization lag time (MLT, the average lag time in days between deposition of osteoid and its mineralization).

Statistical Analysis

Biochemical and histomorphometric variables are expressed as means ± standard error of the means (SE). Transformation calculations were applied to convert raw bone histomorphometric values to Z-scores; Z-scores of histomorphometric data were obtained by subtracting the actual histomorphometric measurement from the mean value of gender-matched healthy control subjects and then dividing the difference by the standard deviation of the healthy control population. Associations between the biochemical parameters, BMD measurements, and histomorphometric parameters were assessed using the Pearson correlation coefficient. Univariate t tests of mean differences for all clinical, biochemical, bone density, and bone histomorphometric parameters were performed. When assessing tacrolimus and cyclosporine effects for posttransplant histomorphometric differences, the analysis of covariance test was used to adjust for differences in prednisone dosages and pretransplant histomorphometric values. All analyses were performed using the SAS data analysis system (SAS Institute, Cary, NC).18

Results

Clinical Data Before and After OLT

The thirty-three study patients (11 PBC, 22 PSC; 12 male patients, 21 female patients; 6 of the female patients were postmenopausal) had a mean age at OLT of 47.4 ± 1.5 years and a mean Child-Pugh score of 9.2 ± 0.3. As previously reported,4 the mean lumbar spine BMD was of 0.86 ± 0.03 g/cm2 at time of OLT and decreased in the first 4 months after OLT to 0.81 ± 0.03 g/cm2 before partially recovering by 1 year to 0.83 ± 0.02 g/cm2 (Table 1). Histomorphometric changes after OLT are shown in Fig. 1A and 1B. As previously reported, before OLT, bone volume parameters (cancellous bone volume, mean wall thickness) and all bone formation parameters (i.e., osteoblast number, osteoid markers, bone formation rates) were decreased. Both direct (osteoclast number, eroded surface area), and indirect (trabecular number, separation) parameters of bone resorption showed increased bone resorption. At 4 months after OLT, bone volume parameters (mean wall thickness, cortical thickness) had decreased further, bone resorption remained unchanged, and bone formation parameters and activation frequency had increased from below normal to the normal range.

Table 1. Biochemical and Bone Density Parameters Before and After Orthotopic Liver Transplantation in 33 Patients With Chronic Cholestatic Liver Disease
 Pre-OLTPost-OLT (4 months)Post-OLT (1 year)
  • Notes: Results expressed as means ± SEM. Change from baseline to 4 mo post-OLT and 4 mo to 12 mo post-OLT.

  • Abbreviations: BMD, bone mineral density; OLT, orthotopic liver transplantation; M, male patients; F, female patients.

  • a

    Total alkaline phosphatase = M > 19 y: 98–251 U/L; F: 24–45 y: 81–231 U/L; F: 46–60 y: 84–257 U/L; F: > 60 y: 108–309 U/L.

  • b

    Serum creatinine = F: 0.6–0.9 mg/dL, M: 0.8–1.2 mg/dL.

  • c

    25(OH) vitamin D = summer: 15–80 ng/mL, winter: 14–42 ng/mL.

  • d

    Osteocalcin = normal ranges for M and F 20–50 y: 2–15 ng/L, F 50–80: 6–22 ng/L.

  • e

    Estradiol = premenopausal F < 400 pg/mL, postmenopausal F < 35 pg/mL.

  • *

    P < .05

  • **

    P < .01

  • ***

    P < .001

  • ****

    P < .0001.

Bone density parameters   
 BMD (g/cm2)0.86 ± 0.030.81 ± 0.03****0.83 ± 0.02**
 T-scores−2.1 ± 0.2−2.5 ± 0.2****−2.3 ± 0.2*
 Z-scores−1.6 ± 0.2−1.9 ± 0.2****−1.7 ± 0.2*
Liver function (normal range)   
 Albumin (3.5–5.0 g/dL)2.8 ± 0.093.6 ± 0.09****3.8 ± 0.07
 Total alkaline phosphatase (U/L)a1531 ± 273.6406 ± 80.3****347 ± 80.5
 Total bilirubin (0.1–1.1 mg/dL)9.1 ± 1.01.7 ± 0.5****1.0 ± 0.1
 Direct bilirubin (0.0–0.3 mg/dL)5.8 ± 0.70.7 ± 0.4****0.2 ± 0.3
 Prothrombin time (8.4–10.0 s)13.0 ± 0.211.5 ± 0.1****11.4 ± 0.1
Kidney function   
 Serum creatinine (mg/dL)b0.8 ± 0.031.5 ± 0.09****1.5 ± 0.07
 Iothalamate clearance (>70 mL/min/SA)97.3 ± 5.149.6 ± 4.0****54.0 ± 2.7
Mineral metabolism   
 25-hydroxyvitamin D (ng/mL)c17.1 ± 1.632.3 ± 1.3****35.0 ± 1.7
 Serum calcium (8.9–10.1 mg/dL)8.7 ± 0.079.3 ± 0.07****9.3 ± 0.07
 Ionized calcium (4.8–5.3 mg/dL)5.0 ± 0.035.0 ± 0.035.0 ± 0.03
 Urinary calcium (25–300 mg/24 h)114 ± 16.2101 ± 11.672 ± 8.5*
 Phosphorus (2.5–4.5 mg/dL)3.2 ± 0.14.1 ± 0.1****3.6 ± 0.1***
 Parathyroid hormone (1.0–5.2 pmol/L)2.0 ± 0.23.4 ± 0.3****4.0 ± 0.5
Bone metabolism   
 Bone alkaline phosphatase (24–146 U/L)146 ± 32.8113 ± 12.6138 ± 13.3*
 Osteocalcin (μg/L)d15.2 ± 2.820.2 ± 12.3*33.6 ± 3.7***
 Urinary hydroxyproline (15–45 mg/24 h)34.8 ± 1.823.0 ± 1.3*23.4 ± 2.1
Gonadal hormones   
 FSH (IU/L)
  Premenopausal F; (FSH < 36)15.1 ± 4.950.8 ± 11.5*31.2 ± 57.7
  Postmenopausal F; (FSH 30–120)60.2 ± 8.493.7 ± 10.8**111.7 ± 16.0*
 Estradiol (pg/mL)e   
  Premenopausal F63.9 ± 12.387.1 ± 13.099.5 ± 13.3
  Postmenopausal F19.0 ± 3.417.0 ± 4.421.8 ± 2.7
 Free testosterone (9–30 ng/dL)6.6 ± 0.911.7 ± 1.1*12.4 ± 0.9*
Figure 1.

(A) Histomorphometric changes, expressed as Z-scores, of bone volume and resorption. (B) Bone formation and mineralization from pretransplant to 4 months after orthotopic liver transplantation in 33 patients with PBC and PSC.

Karnofsky performance scoring at the time of OLT showed that 14 (42.4%) patients had near-normal activity (score 80–100), 18 (54.5%) patients required help with activities of daily living but were ambulatory (score 40–70), and 1 patient was hospitalized at time of OLT because of a variceal bleed. After OLT, patients stayed on average for 4.0 ± 2.2 days in the intensive care unit and for 21.9 ± 10.9 days in the hospital. All patients were ambulatory at time of hospital discharge. Tacrolimus and prednisone were used for immunosuppression in 10 patients, whereas 23 patients were on cyclosporine, azathioprine, and prednisone. Cumulative prednisone dose during the first 4 months was 5,989.3 ± 338.8 mg. Sixteen patients (6 PBC, 10 PSC; 10 female patients, 6 male patients) had biopsy-proven rejection episodes within the first 4 months after OLT; of these patients, 3 had a second rejection episode within the first 6 months. Sixteen rejection episodes were treated with Solu-Medrol, and 3 rejection episodes were treated with OKT3.

Laboratory Data Before and After OLT

Liver function improved and kidney function deteriorated after OLT (Table 1). Serum calcium, 25(OH)D, PTH, and phosphorus increased after OLT, whereas urinary calcium decreased; all changes remained within the normal ranges. Hypogonadism occurred in 9 (75%) male patients (free testosterone < 9 ng/dL) and 11 (52%) female patients (estradiol < 35 pg/mL) before OLT. Of these 11 hypogonadal women, 4 were postmenopausal before and after OLT (estradiol < 35 pg/mL, FSH > 30 IU/L); 4 female patients had low estradiol and FSH before OLT, with increases of FSH after OLT to the postmenopausal range; 3 female patients had low FSH and estradiol before OLT and had normalization of estradiol after transplantation. Serum FSH increased significantly in both premenopausal and postmenopausal women after OLT, whereas free testosterone increased after OLT in male patients. Despite histomorphometric evidence of increased bone resorption before and after OLT, urinary hydroxyproline remained within the normal range throughout the study period. Although bone alkaline phosphatase did not change significantly after OLT, osteocalcin increased significantly over the whole study period to above normal values at 1 year.

Correlations of Histomorphometric Bone Indices With Clinical and Biochemical Parameters

Correlations were assessed (1) between pretransplant clinical and biochemical parameters and histomorphometric changes from pretransplant to posttransplant (Table 2A) and (2) between histomorphometric indices at 4 months and clinical and biochemical parameters at 4 months (Table 2B). Pretransplant Karnofsky and Child scores, posttransplant rejection episodes and hospitalization days did not correlate with any bone histomorphometric parameter.

Table 2A. Correlation Factors of Pretransplant Biochemical Parameters With Histomorphometric Changes (From Pre-OLT-4 Months Post-OLT)
 Bone Volume LossBone Formation IncreaseBone Resorption Increase
Cancellous Bone VolumeCortical ThicknessMean Wall ThicknessOsteoblast NumberOsteoid MarkersaDynamic FormationbMineralizationcOsteoclast NumberIndirect ActivitydEroded Surface
  • Abbreviations: 25(OH)D, serum 25-hydroxyvitamin D; FSH, follicle-stimulating hormone; OLT, orthotopic liver transplantation; PTH, parathyroid hormone.

  • Notes: The table summarizes the correlation (r) factors. All correlations are significant.

  • (P < .05).

  • *Baseline alkaline phosphatase, prothrombin time, creatinine and iothalamate clearance, serum and ionized calcium, bone alkaline phosphatase, and FSH and free testosterone did not have any correlation with bone histomorphometric changes after OLT.

  • a

    Osteoid parameters (newly formed bone) are osteoid surface, volume, thickness.

  • b

    Dynamic bone parameters are bone formation rates, apposition rates, activation frequency.

  • c

    Mineralization parameters are mineralization lag time, mineralization rate.

  • d

    Indirect parameters of bone resorption are trabecular thickness, number, and separation.

  • e

    Correlations showed that patients with high baseline albumin and PTH had an increase of trabecular number and decrease of trabecular separation after OLT: less resorption.

  • f

    Correlations showed that patients with high baseline urinary hydroxyproline had a decrease of trabecular number after OLT.

Liver/kidney function          
 Albumin−0.40       −0.53e 
 Total bilirubin 0.43        
 Direct bilirubin 0.420.35       
Mineral metabolism          
 Urinary calcium −0.48        
 Phosphorus      0.85   
 PTH        −0.35e 
 25(OH)D −0.42        
Bone metabolism          
 Osteocalcin    −0.39     
 Urinary hydroxyproline        0.53f 
Gonadal hormones          
 Estradiol   0.54      
Table 2B. Posttransplant Correlation Factors of 4-Month Biochemical Parameters With 4-Month Histomorphometric Parameters
 Bone VolumeBone FormationBone Resorption
Cancellous Bone VolumeCortical ThicknessMean Wall ThicknessOsteoblast NumberOsteoid MarkersaDynamic FormationbMineralizationcOsteoclast NumberIndirect ActivitydEroded Surface
  • Notes: The table summarizes the correlation (r) factors, all correlations are significant.

  • (P < .05, P < .01,

  • **

    P < .001.

  • Abbreviations: 25(OH)D, serum 25-hydroxy Vitamin D; FSH, follicle-stimulating hormone; PTH, parathyroid hormone.

  • *

    Posttransplant (4 months) albumin, iothalamate clearance, 25(OH)D, urinary hydroxyproline had no correlation with 4-month histomorphometric parameters.

  • a

    Osteoid parameters are osteoid surface, volume, thickness.

  • b

    Dynamic bone parameters are bone formation rates, apposition rates, activation frequency.

  • c

    Mineralization parameters are mineralization lag time, mineralization rate.

  • d

    Indirect parameters of bone resorption are trabecular thickness, number, and separation.

  • eNegative correlations ionized calcium with trabecular separation (less indirect resorption).

  • fPositive correlation estradiol and free testosterone with trabecular number (less indirect resorption).

  • g

    Negative correlation serum calcium with activation frequency.

Liver/kidney function          
 Alkaline phosphatase    −0.43−0.44−0.49−0.42 −0.42
 Total bilirubin    −0.36     
 Direct bilirubin    −0.39     
 Prothrombin time   0.390.360.45   
 Creatinine   −0.38      
Mineral metabolism          
 Serum calcium     −0.51g   
 Ionized calcium        −0.42−0.42
 Urinary calcium   0.41      
 Phosphorus0.48* 0.46*       
 PTH    0.430.46 0.38  
Bone metabolism          
 Bone alkaline phosphatase−0.35         
 Osteocalcin   0.60**0.71**0.55*    
Gonadal hormones          
 FSH    0.500.600.53   
 Estradiol        0.59 
 Free testosterone        0.59 

Bone volume.

The loss of cortical thickness at 4 months after OLT correlated positively with pretransplant total and direct bilirubin and negatively with pretransplant 25(OH)D and urinary calcium but did not correlate with any 4-month clinical or biochemical parameter (Table 2A). The loss of mean wall thickness after OLT also correlated positively with baseline direct bilirubin, and loss of cancellous bone volume correlated negatively with serum baseline albumin. Cancellous bone volume at 4 months correlated negatively with 4-month bone alkaline phosphatase and positively with phosphorus, which also correlated with mean wall thickness (Table 2B). Intravenous prednisone doses during the first month correlated positively with bone volume losses (cancellous bone volume, osteoid thickness and trabecular thickness). A summary of the effects of prednisone on bone histomorphometry is given in Table 3.

Table 3. Correlations of Prednisone Dosage With Posttransplant Bone Histomorphometric Parameters
PrednisoneChange in Bone Metabolism (pre-OLT–4 mo post-OLT)Bone Metabolism Status (4 mo Post-OLT)
  1. Note: All correlations are significant (P value < .05).

1 mo cumulative intravenous dosageLoss of cancellous bone volume (r = 0.40)Osteoblasts (r = −0.38)
 Loss of trabecular thickness (r = 0.46) 
4 mo cumulative oral dosage Adjusted appositional rate (r = −0.41)
  Bone formation rates (r = −0.41)
  Trabecular number (r = −0.42)
  Trabecular separation (r = 0.38)
1 mo total prednisoneLoss of cancellous bone volume (r = 0.37) 
 Loss of trabecular thickness (r = 0.41) 
 Loss of osteoid thickness (r = 0.35) 
4 mo total prednisoneLoss of cancellous bone volume (r = 0.38)Activation frequency (r = −0.41)
 Loss of trabecular thickness (r = 0.39) 

Static bone formation.

The increase in osteoblast number at 4 months after OLT correlated positively with baseline estradiol. Posttransplant osteoblast number correlated negatively with posttransplant creatinine and positively with osteocalcin, prothrombin time, and urinary calcium. Increases in osteoid markers (newly formed bone) at 4 months after OLT correlated positively with baseline osteocalcin. Osteoid markers at 4 months correlated positively with prothrombin time, PTH, FSH, and osteocalcin; negative correlations were seen with alkaline phosphatase and bilirubin.

Dynamic bone formation and mineralization.

Bone formation rates at 4 months correlated negatively with total alkaline phosphatase (but not with bone alkaline phosphatase) and positively with PTH, osteocalcin, and prothrombin time. Activation frequency at 4 months correlated positively with PTH and FSH and negatively with serum calcium and alkaline phosphatase. The increase in mineralization rate from pretransplant to 4 months post-OLT correlated highly with pretransplant serum phosphorus and the actual 4-month post-OLT mineralization rate with FSH and negatively with alkaline phosphatase. Intravenous prednisone doses during the first month and oral doses during the first 4 months also correlated negatively with posttransplant bone formation (osteoblast number, adjusted apposition rate, bone formation rates).

Bone resorption.

An increase in trabecular number and a decrease in trabecular separation were seen in patients with high baseline albumin and PTH. In addition, a decrease in trabecular number was seen in patients with high baseline urinary hydroxyproline. Posttransplant trabecular number at 4 months correlated positively with 4 months testosterone and estradiol, whereas posttransplant trabecular separation correlated negatively with ionized calcium. At 4 months posttransplant, the number of osteoclasts and eroded surface both correlated negatively with total alkaline phosphatase; osteoclast number also correlated positively with PTH and eroded surface negatively with ionized calcium. Oral prednisone doses during the first 4 months correlated positively with indirect bone resorption parameters (trabecular separation, and decreased trabecular number).

Histomorphometric Comparison of Tacrolimus and Cyclosporine Patients

Comparison was made of the histomorphometric changes between patients treated with cyclosporine and those treated with tacrolimus (Table 4). Patients treated with tacrolimus received less prednisone (P < .05), but there were no other clinical or biochemical differences between the two populations. After adjustment for different prednisone doses and baseline histomorphometry, the posttransplant histomorphometric comparison showed that tacrolimus-treated patients had an improvement of cancellous bone architecture by 4 months (increase of cancellous bone volume, trabecular thickness, decreased trabecular separation), whereas this was still diminished in cyclosporine patients. In addition, adjusted apposition rate and mean wall thickness—both bone formation markers—had increased in tacrolimus patients by 4 months after transplant (P < .05).

Table 4. Comparison of Clinical and Histomorphometric Parameters in Patients Treated with Cyclosporine or Tacrolimus
VariablesCyA Patients (n = 23)FK506 Patients (n = 10) 
  • Abbreviations: BMD, bone mineral density; CyA, cyclosporine; F, female patients; FK506, tacrolimus; M, male patients; PBC, primary biliary cirrhosis; PSC, primary sclerosing cholangitis; OLT, orthotopic liver transplantation.

  • Notes: P-values for differences between FK506 and cyclosporine patients, adjusted for prednisone doses and pretransplant histomorphometric variables.

  • *

    Six patients had 22 vertebral fractures (3.7 per patient), and 4 patients had rib fractures (all bilateral).

  • ##

    Two patients had 7 vertebral fractures, and 1 patient had bilateral rib fractures.

  • P < .05, **P < .01,

  • ***

    P < .001.

Gender (F/M)15 (65%)/ 8 (35%)6 (60%)/ 4 (40%)
Disease (PBC/PSC)6 (26%)/ 17 (74%)5 (50%)/ 5 (50%)
Age at OLT (yr)46.3 ± 8.649.8 ± 8.2
Child scores9 ± 1.810 ± 1.6
Total prednisone 4 mo post-OLT (mg)6679.4 ± 369.84389.3 ± 424.0***
Patients with post-OLT fractures year 18 (35%)3 (33%)##
  Post-OLT Post-OLT
 Pre-OLT4 moPre-OLT4 mo
Bone density parameters    
 BMD (g/cm2)0.86 ± 0.040.81 ± 0.030.84 ± 0.040.81 ± 0.04
 T-scores−2.0 ± 0.3−2.5 ± 0.3−2.2 ± 0.4−2.5 ± 0.4
 Z-scores−1.6 ± 0.3−2.0 ± 0.3−1.6 ± 0.4−1.8 ± 0.3
Histomorphometric parameters (unit)    
 Bone volume    
  Cancellous bone volume (%)18.1 ± 1.315.6 ± 1.2*16.6 ± 5.221.5 ± 6.7*
 Mean wall thickness (μm)30.2 ± 2.026.3 ± 8.2*25.4 ± 1.429.1 ± 9.1*
 Cortical thickness (μm)1021.3 ± 70.7712.2 ± 56.1835.5 ± 261.1802.7 ± 250.8
 Trabecular thickness (μm)125.3 ± 7.6107.1 ± 5.8*107.7 ± 6.3132.3 ± 5.9*
 Trabecular number (mm−1)1.5 ± 0.11.4 ± 0.1*1.5 ± 0.11.6 ± 0.1*
 Trabecular separation (μm)585.2 ± 31.6639.3 ± 32.8570.6 ± 35.4490.2 ± 22.5
 Bone resorption    
 Osteoclast per surface length (/100 mm)11.1 ± 1.615.2 ± 2.212.8 ± 4.015.5 ± 4.8
 Eroded surface areas (%)11.1 ± 1.211.5 ± 1.58.3 ± 2.68.3 ± 2.6
 Bone formation    
 Osteoid thickness (μm)7.9 ± 0.68.4 ± 0.57.2 ± 2.310.0 ± 3.1
  Surface (%)8.6 ± 1.914.4 ± 2.64.3 ± 1.420.3 ± 6.4
  Volume (%)1.3 ± 0.32.2 ± 0.50.5 ± 0.23.4 ± 1.1
 Osteoblast–osteoid interface (%)8.2 ± 1.816.2 ± 2.810.8 ± 3.416.8 ± 5.3
 Bone formation/sur based (mm3/mm2/y)0.0052 ± 0.0010.012 ± 0.0030.0033 ± 0.0010.048 ± 0.015
 Bone formation/vol based (mm3/mm2/y)0.065 ± 0.010.23 ± 0.050.059 ± 0.020.69 ± 0.22
 Mineralization rate (μm/day)0.53 ± 0.180.52 ± 0.030.57 ± 0.180.70 ± 0.21
 Mineralization lag time (days)18.5 ± 1.617.8 ± 1.113.9 ± 4.317.9 ± 5.6
 Adjusted apposition time (mm3/mm2/y)0.05 ± 0.010.071 ± 0.008*0.09 ± 0.030.172 ± 0.05*
 Activation frequency (days−1)0.2 ± 0.030.4 ± 0.10.10 ± 0.021.5 ± 0.4

Discussion

Previous histomorphometric assessment of the 33 study patients has showed early improvement in bone metabolism by 4 months after transplant, with recovery of bone formation and improved functional status of bone turnover toward a more coupled balance of resorption and formation.4 Despite this improvement in bone metabolism by 4 months, a loss of bone volume, both by histomorphometry and DEXA, indicates that an additional insult to bone mass occurred during this 4-month period after OLT. Detailed histomorphometric analysis suggested that this posttransplant loss of bone mass was probably mainly related to a further transient reduction in bone formation that occurred early after OLT and that recovered to normal by 4 months. It appears, therefore, that the etiologic factors causing rapid early bone loss after OLT are most active very early after OLT. By analysis of histomorphometric changes in relation to the multiple clinical and biochemical variables during this period, this study seeks to identify these etiologic factors of bone loss as well as the mechanisms of recovery of bone metabolism.

Several factors have been implicated as potential etiologic factors of early posttransplant bone loss, with immunosuppressive agents assuming the greatest importance. Most posttransplant studies have not found any consistent relationship between immunosuppressive doses and bone loss, but histomorphometric markers of bone metabolism have not been studied. Our study also failed to show any relationship between glucocorticoids and bone density changes, but several interesting correlations with histomorphometric parameters were seen. Cumulative glucocorticoid doses at 1 and 4 months correlated positively with bone volume losses and inversely with bone formation parameters. Presumably this reduced bone formation by steroids leads to, or at least contributes to, the loss of bone volume. These findings are consistent with the known inhibitory effects of steroids of osteoblasts and bone formation.19, 20 In addition, as previously mentioned, it appears that early posttransplant bone loss is mediated through an increased but transient inhibitory effect on bone formation. The correlation of steroids with reduced bone formation and bone losses after OLT in this study suggests an etiologic connection.

The role played by calcineurin inhibitors in posttransplant bone loss remains unknown. Previous studies comparing the effects of tacrolimus and cyclosporine on bone loss have been contradictory, with some studies indicating similar effects on bone metabolism and bone loss21, 22 and others suggesting that tacrolimus is more potent in stimulating bone osteogenesis and bone formation.23–25 Both cyclosporine and tacrolimus cause increased bone turnover in vitro,21, 23, 26 but no increase in bone turnover and resorption was seen here. However, although similar bone density losses were seen in both cyclosporine-treated and tacrolimus-treated patients, the histomorphometric changes after OLT were different in the two groups. After adjusting for cumulative prednisone dose, patients treated with tacrolimus had an improvement in cancellous bone architecture and increases in adjusted apposition rate of bone and mean wall thickness by 4 months compared with cyclosporine patients. These findings suggest that tacrolimus-treated patients may have an earlier recovery of bone metabolism after the initial phase of bone loss compared with cyclosporine-treated patients. The mechanism for this difference is not obvious from our study findings.

In addition to its direct effects on bone metabolism, glucocorticoids alter calcium homeostasis, causing bone loss. Glucocorticoids inhibit calcium absorption from the gastrointestinal tract and stimulate excretion of urinary calcium by interfering with renal tubular mechanisms;27, 28 this latter mechanism may cause a compensatory increase of PTH and, therefore, more bone turnover and resorption. PTH in our study correlated with bone turnover (both resorption and formation parameters) at 4 months after OLT, but no correlation between PTH and glucocorticoid doses was seen. An increase in PTH has been shown to occur as early as the first month after OLT,29 and any relation to cumulative glucocorticoid dose may, therefore, have been missed at the 4-month evaluation in our study. Early bone loss correlated with low pretransplant serum vitamin D level and urinary calcium, suggesting that calcium balance is somehow protective against early posttransplant bone loss. In addition, better calcium balance at 4 months had beneficial effects on bone formation (increased) and bone resorption (decreased).

Loss of bone volume after OLT was greatest in patients with high pretransplant bilirubin levels. In addition, at 4 months after OLT, increased formation of new bone (osteoid) was associated with lower bilirubin. With cholestasis as a recognized risk factor for osteopenia, it is not surprising that improved histomorphometric status at 4 months is seen in patients who lack cholestasis. This provides further evidence for the etiologic association of cholestasis with osteopenia before OLT and is consistent with previous in vitro studies indicating that osteoblastic proliferation was diminished by serum of cholestatic patients.30 The increased loss of bone volume at 4 months in patients with the highest pretransplant bilirubin levels suggests that the impaired bone metabolism of cholestatic osteopenia in some way renders it more susceptible to the early posttransplant influences that effect bone loss.

Changes in gonadal status also may affect bone metabolism after OLT. The importance of hypogonadism on bone mass has been studied mainly in patients with alcoholic liver disease.31, 32 Our study shows that two thirds of male cholestatic patients were hypogonadal at time of OLT, with increasing free testosterone levels after OLT. This increased testosterone at 4 months correlated with less bone resorption (increased trabecular number), a known effect of androgenic hormones. In addition, FSH increased after OLT in both postmenopausal and premenopausal women, suggesting central suppression of the hypothalamic–pituitary function in female patients with advanced liver disease, a mechanism for which there is also some evidence in male patients with chronic liver disease.33 Pretransplant estradiol and FSH at 4 months correlated, respectively, with an increase in osteoblast numbers and bone formation parameters at 4 months but had no correlation with bone loss. These findings suggest that recovery of hormonal function mainly contributes to bone metabolism after the initial phase of bone loss; in the early phase of bone loss, it is likely that other factors overwhelm the minor effects of the still-recovering gonadal status.

Biochemical indices of bone metabolism provide a noninvasive method to measure changes in bone metabolism. Although biochemical bone markers are unreliable in identifying bone resorption and formation in pretransplant cholestatic patients,34, 35 their use in posttransplant patients has not been validated. The current analysis shows that osteocalcin measured after OLT correlated with all posttransplant bone formation parameters. Interestingly, osteocalcin continued to increase after the first 4 months to higher values by 1 year; this most likely reflects a continuing increase in bone formation during this period. Bone alkaline phosphatase and urinary hydroxyproline were ineffective in predicting bone formation or resorption.

In conclusion, this study confirms for the first time that cumulative steroid dosage after OLT contributes to early posttransplant bone loss. Its effect is most likely caused by a further reduction in bone formation, compounding the already compromised bone formation of patients with chronic cholestatic liver disease. After this first phase of bone loss, histomorphometric assessment suggests that patients with tacrolimus therapy have an earlier recovery of bone formation and trabecular structure by 4 months compared with cyclosporine patients; this may alter the long-term outcome for fracturing. The improvement in bone metabolism by 4 months after OLT is probably multifactorial with contributions from improvements in hepatic synthetic function, lack of cholestasis, gonadal recovery, and better calcium balance. Of the bone biochemical indices, only osteocalcin appears to be a reliable tool to assess changes in posttransplant bone formation.

Ancillary