A small number of osteoporotic nonvertebral fractures was registered (two of the hip, seven of the forearm, and one of the homer). They all occurred in men showing an incidence of 6.4%, and in the 20% of cases, a concomitant vertebral fracture was diagnosed. A significantly higher number of vertebral fractures was present in patients with femoral neck osteoporosis (P=0.01), whereas no difference was found using lumbar spine BMD. The difference remained significant only for men (P=0.02), mainly because of the small number of women and for the few fractures diagnosed in them. We observed a significant correlation between femoral neck T-score and the number vertebral fractures (P=0.03), whereas the significance was not achieved using spine T-score. Furthermore, the prevalence of osteoporotic patients with vertebral fractures was low, 20.63% in patients with spine osteoporosis and 22.22% in those with femoral osteoporosis.
Because FX were significantly older than UN-FX, we performed a logistic regression analysis. Our model included age, BMI, lumbar spine and femoral neck BMD, bone turnover, and daily and cumulative dosage of immunosuppressant treatments. Femoral neck BMD remained significantly lower in FX with respect to UN-FX (P=0.02 for T-score and 0.04 for BMD).
In consideration of the low number of vertebral fractures identified using −2.5 standard deviation (SD) T-score, we tried to test a different T-score threshold. We used femoral neck BMD because it showed a more significant relationship with fractures. We selected a T-score of −1.5 SD, because it was the mean of femoral neck T-score in our population and because it represents the threshold proposed for GIOP (21).
Fifty-three percent of subjects had a T-score lower than −1.5 (OP1; 44.06% of men and 66.6% of women). OP1 had a higher number of vertebral fractures with respect to those with a T-score more than −1.5 SD (OP0), and the significance was achieved in men (P=0.05) but not in women, according to what we have observed with −2.5 SD threshold. More interestingly, the prevalence of fractures greatly increased: it reached 60% in both genders.
In a subanalysis performed selecting patients with less than 5 years from transplantation, prevalence of fractures was even higher in OP1 than in OP0 (P=0.03, P=0.02 in men). Positive and negative predictive values for fracture risk were 0.5 and 0.63 for −1.5 SD, T-score threshold, 0.6 and 0.63 for −2.5 T-score, respectively.
Table 3 shows the most relevant biochemical parameters measured. Almost all subjects recruited had a vitamin D (VitD) deficiency (92%; 92.3% of men and 83.3% of women); a mild deficiency was diagnosed in 55.5% of patients (58.3% of men and 37.5% of women) and a severe one in 35.5% of cases (32.9% of men and 45.8% of women).
Parathyroid hormone (PTH) level was increased in 6.1% of cases (5.1% of men and 12.5% of women); it was negatively correlated with VitD levels (r=−0.22, P=0.01), whereas it was not significantly different in patients with fractures or lower BMD. Only 12.2% of patients showed an increased bone turnover (11.5% of men and 16.6% of women). An increased bone resorption was observed in 30.5% of patients (29.4% of men and 37.5% of women), whereas bone formation was increased in 21.6% of cases (21.8% of men and 20.8% of women).
Bone turnover was not different between FX and UN-FX, whereas an inverse significant correlation was observed with spine densitometry (P=0.004 for S-C-terminal collagen peptide [CTX], r=−0.22; P=0.01 for bone-specific alkaline phosphatase [B-ALP], r=−0.18). Finally, we did not find any significant difference in sexual hormones values, mainly for their important age variability that impair a reliable analysis in our study population.
We did not find any difference in concomitant pharmacologic treatments. None received any medication that was able to interfere with bone metabolism and were supplemented with calcium/VitD. Dietary daily calcium intake was 887.5±506.2 mg. All patients received a combined immunosuppressant therapy with GCS, CYA, and azathioprine. Mean daily intake of GCS was 15.3±25.3 mg (3.28–265.5 mg daily), and in the 40% of patients, cumulative dosage exceed 10 g. Mean daily intake of CYA was 280.7±264.5 mg/day, whereas cumulative intake was 381.66 ±302.26 g. Cumulative dosage of azathioprine was 37.68±51.32 g.
We did not find any significant difference in immunosuppressant dosages neither between patients with T-score less than −2.5 SD and normal population nor between FX and UN-FX. Instead, OP1 patients received a significantly higher daily dosage of GCS than OP0 patients (P=0.02). A significant inverse correlation was also observed between mean daily GCS and BMD both at lumbar spine and at femoral neck (P=0.001, r=−0.24, both for lumbar spine and femoral neck T-score; Fig. 2A).
On the contrary, our results showed a direct and significant correlation between mean daily CYA and femoral neck BMD (P=0.001, r=0.26 for neck BMD and P=0.002, r=0.25 for neck T-score; Fig. 2B). Analyzing the population with a time from transplantation less than 5 years, daily GCS persisted significantly higher in FX (P<0.001) and CYA remained significantly higher in UN-FX (P=0.03).
Our study presents one of the largest population of heart transplanted patients, in which we focused our attention on bone fragility. The incidence of TIOP is still unclear (17–19), and the real prevalence of fractures varies from 18% to 50% (15–18). We reported a prevalence of 40% that is in agreement to that observed in the majority of studies.
Although transplanted recipients are at high risk of fractures, in our work, a minority of them were osteoporotic, and just 20% of FX had a T-score less than −2.5 SD. BMD is an important determinant of bone fragility, but it is inadequate to identify all subjects at risk. Specific algorithms have been developed to improve diagnostic criteria in PO (17), and an higher densitometric threshold was proposed for the diagnosis of GIOP (−1.5 SD) (21).
In our study population, we were not able to discriminate among FX and UN-FX, and according with the experience in GIOP (21), we modified T-score threshold to −1.5 SD. As expected, the percentage of patients with fractures increased (up to 60%), but the numbers of vertebral fractures remained significantly higher in those with a T score less than −1.5 SD.
Positive and negative predictive values of the new threshold were similar to those obtained with a T-score of −2.5 SD, but the number of fractures that they referred to is significantly higher, confirming the consistency of our hypothesis. The relationship between fractures and BMD was stronger using femoral densitometry, as observed by others (3, 20). Radiologic artifacts (osteoarthritis, aortic calcifications, vertebral fractures, and scoliosis) could impair BMD assessment of the spine. A metabolic explanation is also possible: secondary and tertiary hyperparathyroidism is a frequent complication of end-stage organ disease, and PTH is known to affect rapidly both trabecular and cortical bone. As a consequence, femoral neck better represents the whole bone mineral content (5).
In our population, we observed a severe deficiency of VitD, suggesting that osteomalacia could play an important role in TIOP. Even if we might expect such an important deficiency in kidney and liver organ diseases, it is important to stress that this is a more diffuse problem (23, 24). Many activities of VitD have been revealed on immunologic system, glucose metabolism, cardiovascular risk, and neoplastic transformation (28), and because they are strictly related with transplantation, it is necessary to guarantee an adequate supplementation to exploit both skeletal and extraskeletal effects. As a matter of fact, according to the high incidence of VitD deficiency and the absence of important contraindications associated with the use of VitD precursors (cholecalciferol and ergocalciferol), we suggest to prescribe VitD supplementation even if the laboratory dosage is not available: at least 800 to 1000 U daily (28, 29).
We observed an increase of bone turnover and an inverse correlation between S-CTX and BMD. Bone remodeling markers can be used to evaluate bone disease activity and to check for treatment efficacy in transplanted recipients (30, 31), because they are effectively inhibited by antiresorptive agents that have been tested successfully in TIOP (30, 33, 34).
Finally, we described a significant inverse correlation between mean daily dosage of GCS and BMD. This observation confirms the importance of densitometry in TIOP but supports the need to change diagnostic criteria, because a minority of patients would be treated if a T-score less than −2.5 SD was chosen as a decision threshold.
Furthermore, we observed a positive correlation between CYA bone formation markers and BMD. Many works supposed that CYA first stimulates both osteoblasts and osteoclasts, counteracting the negative effect of GCS (8). On the contrary, long time treatment seems deleterious inducing a high bone turnover osteoporosis (8). This finding suggests a different action of CYA with respect to other immunosuppressant (35).
The limitation of our study is the retrospective analysis of the data and then lack of longitudinal monitoring of BMD. However, we think that it is clinically relevant, because the number of transplanted patients will increase continuously together with bone complications, and our results might help to change current medical approach for the diagnosis and treatment of TIOP.
In conclusion, TIOP is a complex model; standard densitometric criteria are unreliable to identify patients at risk of fracture, and a different densitometric threshold (T-score ≤−1.5 SD) should be considered. From a metabolic point of view, VitD deficiency is widespread, and all transplanted patients should be adequately supplemented. Finally, the effect of immunosuppressant agents on bone must be further evaluated because CYA increases bone formation markers and might counteract, at least in part, the negative effects of GCS.
MATERIALS AND METHODS
Study Population and Questionnaire
We planned a multicenter cross-sectional study. We enrolled 180 patients, aged 53.0±13.4 years (Table 1), in 3 centers of the Northern part of Italy: 40 patients in Padova, 20 in Verona, and 120 in Montescano. All patients were selected from heart transplanted patients who visited the Day Hospital service of each center.
Inclusion criteria were heart transplantation from less than 10 years. Exclusion criteria were an age less than 15 years, multiorgan transplantation, retransplantation, more than 10 years heart transplantation from, and severe worsening of renal function with respect to the time of transplantation (defined as serum creatinine >2.5 mg/dL, dialysis, or kidney transplantation). Clinical history was collected from clinical files available in each Day Hospital service and from each recipient using a printed module.
Questions analyzed anthropometric parameters and lifestyle factors, baseline heart disease and other comorbidities, history of fractures, pharmacologic treatments during at least the 6 months before and after the graft, and pain intensity and its relationship with daily activity. Dietary calcium intake was studied through a specific questionnaire. Performance status was determined according to Barthel index at the time of transplant and at the study evaluation. Written informed consent was obtained from each patient.
Bone Densitometry and Fracture Evaluation
Each patient underwent densitometry of the lumbar spine and of the hip. Three different devices were available: two Hologic QDR 4500 (Hologic, Waltham, MA) and one Norland XR-26 (Norland, Madison, WI). All scans collected with Norland XR-26 were expressed in Hologic data using a cross calibration analysis. All densitometric scores were then analyzed using Hologic standard curve. World Health Organization criteria for the diagnosis of PO were used (36).
To establish the presence of vertebral fractures, anteroposterior and latero-lateral x-ray of dorsal and lumbar spine were taken. A qualitative analysis was made by a specialized radiology, and subsequently, a semiquantitative evaluation, according to Genant score, was conducted from T4 to L4 (38). Lumbar vertebral fractures were excluded from densitometric analysis. Almost all patients had made an x-ray of the column before the transplantation even if in the majority of them only the dorsal tract was available (lateral projection of a chest x-ray). Nonvertebral fractures were recorded from clinical history, and traumatic fractures were excluded from the statistical analysis.
Laboratory analysis was made once at the time of study evaluation. Nevertheless, all patients underwent periodic laboratory assessments since the time of transplantation. Serum for biochemical analysis was obtained in the morning under fasting conditions. Some parameters regarding bone metabolism were tested independently in each center (calcium, phosphorus, ALP, creatinine, and albumin).
Centralized evaluation of PTH, S-CTX, B-ALP, osteocalcin, VitD, luteinizing hormone, follicle-stimulating hormone, testosterone, sex hormone binding globulin, 17-β-estradiol, dehydroepiandrosterone sulfate, oestrone, and androstenedion were performed in Pisa. B-ALP and S-CTX were also expressed after adjustment for age (ZB-ALP and ZCTX; Table 3).
We defined a VitD deficiency as a serum level less than 30 ng/mL, a mild deficiency between 10 and 30 ng/mL, and a severe deficiency when the value was less than 10 ng/mL. An increased bone turnover was classified by increase of both B-ALP and S-CTX, whereas an increment of bone resorption or formation was represented by a single increase of S-CTX or B-ALP, respectively.
All data are expressed as mean±SD. Differences between groups were tested for significance using Student's t test, whereas the analysis of frequency was performed using chi-square test. Logistic regression was made to verify the role of the main determinants of fracture: age, gender, BMI, lumbar BMD, femoral neck BMD, bone turnover, and daily and cumulative dosage of immunosuppressant treatments.
For the correlation analysis, Spearmen coefficients were calculated when appropriate, and linear regression was represented. Statistical significance was considered for a P less than 0.05. Statistical analyses were performed using SPSS for windows, version 16.0 (SPSS Inc, Chicago, IL).
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Keywords:© 2011 Lippincott Williams & Wilkins, Inc.
Densitometry; Vertebral fracture; Heart transplantation; Immunosuppressant treatment; Vitamin D