Leptin, the product encoded by the obese (ob) gene, is a 16kDa non-glycosylated polypeptide synthesized mainly by white adipocyte but also by other cells.1–4 It plays a fundamental role in regulation of bodyweight and energy balance. Leptin receptors are expressed in almost all cell types, and leptin, like other members of the long-chain helical cytokine family, has a wide spectrum of central and peripheral effects and participates in a variety of cardiovascular, endocrine, and immunological actions. It plays an important role in regulation of cardiomyocyte function and structure, angiogenesis, inflammation, insulin resistance, coagulation, platelet aggregation, fibinolysis, and bone remodeling.2,3,5
Elevated circulating leptin levels have been found in a number of cardiovascular disorders, including obesity-related metabolic syndrome,6–8 as well as in coronary artery disease,9–12 hypertension,13,14 left ventricular hypertrophy,15 and heart failure,16,17 independent of the presence of obesity. Hyperleptinemia has been associated with acute myocardial infarction and stroke independently of traditional cardiovascular risk factors and obesity status.9,10,18,19
Despite extensive studies, the cardiac effects of leptin are still not completely understood and contradictory findings have been reported. Data on the role of leptin in human cardiovascular morbidity and mortality are not consistent.20–22 Some studies reported no association between elevated leptin levels and cardiovascular events,22–24 or even a protective effect.25–27
Cardiovascular complications are recognized as the main cause of morbidity and mortality in older patients undergoing hip fracture (HF) surgery.28,29 We have shown that in this population peri-operative myocardial injury, defined as an elevated level of cardiac troponin I (cTnI), is common and associated with prolonged length of hospital stay, need to be discharged to a long-term (permanent) residential care facility, and mortality.30 Given the global HF epidemic and its enormous medical and communal burden, understanding the predisposing and precipitating causes of this association is imperative.
The relationship between serum leptin levels and myocardial injury in older patients with osteoporotic HF has not been studied and the pathophysiological and prognostic implications are unknown. We hypothesized that altered leptin levels might predict an increased risk for myocardial injury in HF patients. The aim of this study was to evaluate the serum leptin concentration in patients with HF and determine whether a relationship exists between leptin levels and peri-operative myocardial injury. We also examined whether such an association, if present, is independent of traditional risk factors (age, coronary artery disease, hypertension, diabetes, cigarette smoking), comorbid dementia, anemia, hypovitaminosis D, hyper-parathyroidism, biological markers of malnutrition (albuminemia, lymphocyte count), and inflammatory indices (C-reactive protein [CRP], ferritin levels).
Patients. Full details of clinical characteristics of the study population have been previously reported.31 Briefly, the present study includes 238 consecutive patients (172 women and 66 men; mean age 81.9±7.8 years, range 60–98 years) with confirmed diagnosis of low-trauma HF admitted to our hospital between July 2004 and December 2005. Data were obtained from a prospectively maintained electronic database of all HF patients admitted to The Canberra Hospital, a university teaching tertiary care center. Of 238 patients, 47 had a prior diagnosis of coronary artery disease (CAD), including 14 with previous myocardial infarction, 127 hypertension, 31 stroke, 27 atrial fibrillation, 34 diabetes, 65 dementia, 25 chronic obstructive pulmonary disease, and 12 Parkinson’s disease; there were 29 ex-smokers and eight current smokers. In 173 patients the American Society of Anaesthesiologists (ASA) physical status was ≥3. Medications on arrival included aspirin (70 patients), clopidogrel (24), beta-blockers (46), angiotensin-converting enzyme inhibitors (43), angiotensin receptor antagonists (37), digoxin (25), diuretics (52), statins (45), and calcium-channel blockers (19), in various combinations. Usually, all cardiac medications except antiplatelet agents and warfarin were continued during hospital admission, being given pre-operatively and recommenced the day after surgery. Antiplatelet drugs or warfarin were re-started on the third to fourth day after surgery.
The study was performed in accordance with the Declaration of Helsinki. The study protocol was approved by the local ethics committee and informed consent was obtained from all patients or their carers.
Blood Sample Collection and Measurements. Venous blood samples were collected in lithium heparin tubes after a 12-hour overnight fast within 72 hours after arrival at the emergency department (89% within 48 hours). Routine hematological and biochemical parameters were analyzed immediately. In samples centrifuged at 4°C, plasma was filtered and the separated serum was stored at -70°C until assay of cardiac troponin I (cTnI) and leptin. All samples were assayed in a single batch within 1.5 years of collection in a blind fashion. cTnI and leptin have been shown to remain stable in specimens frozen for up to three32 and 29 years,33 respectively.
Serum cTnI, leptin, 25(OH) vitamin D, parathyroid hormone (PTH), thyroid-stimulating hormone (TSH), free T4, and C-reactive protein (CRP) were measured using commercially available kits according to the manufacturers’ protocols. Serum leptin was determined by an enzyme-linked immunosorbent assay (ELISA) method (Diagnostic System Laboratories Inc, Webster, Texas, US), cTnI by a two-step chemilumenescent microparticle immunoassay (Chemiflex, Abbott Labs, Mississauga, Ontario, Canada), 25(OH) vitamin D by a radioimmunoassay kit (Dia Sorin, Stillwater, Minnesota, US), intact PTH by two-site chemiluminescent enzyme-linked immunoassay on DPC Immulite 2000 (Diagnostic Products Corp, Los Angeles, California, US), TSH and T4 by using a two-step chemilumenescent microparticle immunoassay on Architect Ci8200 (Abbott Labs, IL, US), and CRP by agglutination methodology on the Architect Ci8200 (Abbott Labs, IL, US). According to the manufacturer, the low detection limit for the cTnI assay is 0.003μg/l and the upper limit of reference range is 0.06μg/l; intra- and interassay coefficients of variation are both less than 6%. In this study all values of cTnI above this level were considered elevated, indicating myocardial injury. The minimum detectable serum leptin concentration of the assay is 0.5ng/ml and the intra- and interassay precision are both less than 7%. The inter- and intra-assay CV for other biochemical variables ranged from 1.8 to 9%. Additionally, the following parameters were measured: hemoglobin, erythrocyte count, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), leukocyte, neutrophil, and lymphocyte counts, iron, ferritin, transferrin, vitamin B12, folate, total protein, albumin, alanine aminotransferase (ALT), alkaline phosphatase (ALP), γ-glutamyl transferase (γGT), bilirubin, urea, and creatinine levels by means of standard automated laboratory methods. Serum calcium concentrations were corrected for serum albumin. Glomerular filtration rate (GFR) was estimated by the formula developed by Levey.34
Statistical Analysis. Continuous variables are expressed as mean ± standard deviation (SD) and categorical data as absolute and/or frequencies. Differences among groups were compared using two-tailed unpaired Student’s t-test for continuous variables and chi-square statistics for categorical variables. Significance was defined at the p<0.05 level. Correlations between variables were calculated using the Pearson’s coefficient of linear correlation. Variables were logtransformed if non-normally distributed. Analyses were conducted with serum measures modulated both as log-transformed continuous variables and as categorical variables based on clinical cut-points. Multiple logistical regression analysis was performed with elevated cTnI concentration as a dependent variable. Independent variables included age, sex, and clinical, hematological, biochemical, and nutritional parameters, with entry/exit criteria of a p-value <0.2 in univariate analysis. To assess the interactions of risk factors for myocardial injury on a multiplicative scale, we calculated a synergy index (SI) using the following formula: SI = ORa+b/ORa x ORb, where ORa+b is the odds ratio (OR) for the complication among patients with co-existing factors a and b, ORa is OR for the complication among subjects with factor a (but not factor b), and ORb is OR for the complication among patients with factor b. An SI >1 indicates more than multiplicative effects between factors, whereas an SI <1 indicates less than multiplicative effects. All statistical analyses were performed by using Stata software (version 10, Stata Corp, College Station Texas, US).
Demography and Comorbidity. Of 238 HF patients, 69 (29%) had peri-operatively elevated cTnI. Patients with myocardial injury compared with patients without this complication were significantly older (84.4±9.8 versus 80.5±8.0 years; p=0.002), and the absolute majority were 75 years of age or older (95.7 versus 79.2%; p=0.002), whereas there was no gender difference (females 70 and 73%, respectively). The patients with elevated cTnI more often had a history of CAD (34.8 versus 14.3%; p=0.001) or stroke (22.3 versus 8.4%; p=0.007) and an ASA score ≥3 (88.2 versus 67.2%; p=0.007), and were more likely to be current smokers (9.1 versus 1.2%; p=0.008). The proportion of patients with pre-operatively diagnosed dementia was also higher, but the difference did not reach statistical significance (33.3 versus 24.4%; p=0.067). There were no differences between the two groups in terms of other comorbidities, type of HF, medication used, residential status, time from admission to the emergency department to surgery, surgical procedures, or anesthetic techniques.
Logistic regression analysis was used to estimate the predictive role of clinical characteristics with regard to the risk for developing myocardial injury. In a model including as independent variables age, sex, CAD, history of stroke, atrial fibrillation, hypertension, diabetes, dementia, and smoking, independent and significant predictors of myocardial injury were age (for each five years from 60 OR = 1.49, 95% confidence interval [CI] 1.16–1.90; p=0.002), CAD (OR = 2.23, 95% CI 1.03–4.83; p=0.042), and current smoking (OR = 9.13, 95% CI 1.65–50.65; p=0.011). When ASA score ≥3 was added to this model, only age remained an independent and significant predictor of cTnI elevation; each five-year increase in age was associated with a 1.5-fold increase of myocardial injury in HF patients.
Serum Leptin Levels and Hematological and Metabolic Variables. Mean leptin levels were approximately 1.7 times greater in women than in men (22.9±27.3 versus 13.3±19.9ng/ml; p=0.010). Table 1 reports differences between patients with and without cTnI elevation. In patients with peri-operative myocardial injury, the mean serum leptin concentration was 1.5 times lower than in patients without such complications. When patients were subdivided by gender, the difference was statistically significant only in females. When the group was divided into tertiles of serum leptin level (separately for females and males), the risk for myocardial injury was approximately two-fold higher when comparing the lowest tertile with the highest tertile (OR = 2.3, 95% CI 1.06–4.97; p=0.034). In other words, decreasing levels of serum leptin were associated with increasing ORs of cTnI elevation. The patients with elevated cTnI had also lower erythrocyte count, hematocrit, hemoglobin, MCHC, and estimated GFR and higher PTH, urea, CRP, and ferritin concentrations and leukocyte count than patients without cTnI elevation. By contrast, there was no difference in mean values of serum levels of TSH, T4, albumin, liver enzymes, 25(OH)D, iron, transferrin, vitamin B12, folate, calcium, phosphorus, magnesium, creatinine, or neutrophil and lymphocyte count by cTnI status.
Among patients with myocardial injury there was a higher proportion of subjects with lower leptin (<12ng/ml, median value for the total group 59.4 versus 43.8%; p=0.029) and haemoglobin levels (<115g/l; 40.6 versus 22.6%; p=0.009), elevated PTH (>6.5pmol/l, 52.3 versus 34.1%; p=0.011), hypoalbuminemia (<30g/l, 24.6 versus 13.0%; p=0.003), and estimated GFR <60ml/min (55.1 versus 40.1%; p=0.037). Taken together, these data may indicate that peri-operative myonecrosis is associated with anemia, malnutrition, secondary hyperparathyroidism, inflammation and renal impairment.
We further analyzed the association between serum leptin levels and clinical, hematological, metabolic, and inflammatory variables, potential risk factors for myocardial injury, and outcomes. Table 2 shows the mean serum leptin levels according to the presence or absence of selected risk factors for peri-operative myocardial injury and poor outcomes in older HF patients. The presence of dementia and anemia (Hb <115g/l) were associated with significantly lower mean leptin concentrations. Leptinemia was not associated with any other clinical or biological parameters, nor prolonged hospital stay (≤20 days), nor in-hospital death.
To examine the inter-relationships between the biomarkers, we calculated Pearson’s correlation coefficients. Leptin log-transformed levels correlated positively with hemoglobin (r=0.208; p=0.001), red cell count (r=0.191; p=0.003), hematocrit (r=0.192; p=0.003), CRP-log (r=0.132; p=0.043), albumin (r=0.173; p=0.007), and creatinine (r=0.159; p=0.014), and negatively with GFR (r=-0.173; p=0.008). cTnI-log levels correlated positively with age (r=0.221; p=0.001), concentration of PTH-log (r=0.250; p=0.001), CRP-log (r=0.162; p=0.017), and urea (r=0.27; p=0.004), and negatively with MCHC (r=-0.237; p=0.004) and GFR (r =-0.254; p=0.001).
Logistical Regression Analyses for Myocardial Injury. To assess the determinants of peri-operative myocardial injury we also performed stepwise logistic regression analyses. Three separate models are presented (see Table 3). The first model included sex, age, leptin, red cell, white cell, and lymphocyte counts, hemoglobin, haematocrit, MCHC, 25(OH)D, PTH, ferritin, CRP, albumin, urea, creatinine, and GFR as continuous variables. The results of this model revealed that only older age, lower leptin and MCHC levels, and higher CRP and ferritin concentrations are significant independent predictors of elevated serum cTnI (see Table 3, model A). The addition to the model of clinical variables such as CAD, history of stroke, hypertension, diabetes, dementia, smoking, and ASA score (see Table 3, model B) resulted in leptin’s loss as an independent predictor (OR = 0.98, 95% CI 0.95–1.00; p=0.083), indicating that the effect of leptin is captured by clinical variables when they compute in the model. However, when we carried out a logistical regression analysis with biomarkers as categorical variables (see Table 3, model C), reduced leptinemia (<12ng/ml), elevated PTH (>6.5pmol/l), advanced age, and CAD were potent and independent determinants of myocardial injury after adjusting for other confounding factors listed in Table 3. The risk for developing myocardial injury was 1.6-fold greater for each five-year increment in age, about two-fold greater if leptin level was <12ng/ml or PTH >6.5pmol/l, and 2.7-fold greater in patients with CAD. This model correctly classified 72.7% of cases and had sensitivity of 25.5%, specificity of 91.3%, positive predictive value of 53.9%, and negative predictive value of 75.6%.
Lower leptin concentrations adjusted for sex and age were associated with increased peri-operative myocardial injury risk ranging from 81% (all patients) to 103% (age >75 years) compared with higher serum leptin levels (see Table 4). When also controlled for inflammation (markedly elevated CRP and/or ferritin), anemia, hyperparathyroidism, or presence of CAD, this association did not change in patients with lower leptin levels. The OR for myocardial injury reached 113% after combined adjustments for all these variables (see Table 4).
Prognostic Effects of Co-existence of Lower Leptin Level and Other Risk Factors for Development of Myocardial Injury. To clarify the effects of two or three co-existing independent risk factors for peri-operative myocardial injury, we assessed the statistical significance of their relationship using a logistical regression model and adjusting for sex (see Table 5). As would be expected, we found an additive or even synergistic effect between lower leptin levels and advanced age, secondary hyperparathyroidism, and CAD, suggesting a pathogenic and prognostic significance of these interactions. The strongest interactions were observed for lower leptin and advanced age (>75 years). The OR for harmful synergism increases from 3.4 for the combination of lower leptin (<12ng/ml) and older age (for each five-year increment from 60 years of age) to 12.6 for patients >75 years of age with low leptin levels. Elevated PTH in association with a lower leptin level acts synergistically, with an OR of 4.4 (SI 1.10), and the combination of lower leptin level and CAD demonstrated an additive effect, with an OR of 5.8 (SI 1.00). When age was added, these ORs increased to 6.9 and 9.1, respectively.
The main novel finding of the present study is that in older HF patients lower serum leptin levels are associated with peri-operative myocardial injury as defined by conventional criteria (elevated cTnI). Cardiac injury, the major risk factor perpetuating the poor outcomes in these patients,28–31 is multifactorial in etiology and may be related to CAD, inflammation, hypoxia, or anemia. Many HF patients present with a combination of conditions that may contribute to their total risk for myocardial injury. However, the predictive potentials of traditional risk factors for CAD, as well as such common disorders as dementia, malnutrition, inflammation, anemia, vitamin D deficiency, and hyperparathyroidism, which may contribute to poorer outcomes, have not been evaluated simultaneously and systemically.
Our data, in line with previous studies, show that peri-operative myocardial injury in HF patients is significantly associated with older age, CAD, anemia, indices of inflammatory response (CRP, ferritin), and hyperparathyroidism.31,35,36 We also found that patients with myocardial injury along with lower serum leptin levels more often demonstrated marked hypoalbuminemia and renal impairment. The serum leptin concentration was significantly lower in patients with dementia and anemia (Hb <115g/l). To the best of our knowledge, this is the first study to show that leptin status does have a significant effect on the development of myocardial injury independent of all of the above factors. Following multivariate regression analysis, lower serum leptin concentration (<12ng/ml) carries a two-fold higher risk for myocardial injury and remains an independent predictor of this complication. Adjustment for age, CAD, hypertension, history of stroke, diabetes, dementia, smoking, and ASA score, as well as indices of anemia, inflammation, hyperparathyroidism, and renal failure, does not alter this association.
The reason for the link between lower leptin levels and myocardial injury is not fully understood. Conflicting reports have been published regarding the role of leptin in cardiovascular disease. While some studies reported hyperleptinemia as an independent risk factor for myocardial infarction9–12,19,20 and stroke,18 others did not observe such an association21–24 or even found a protective effect.25,27 Potential confounders were not always adequately controlled,9,10,18,23 and when inflammatory biomarkers (CRP) and other variables were included in the analysis, the association between increased leptin levels and cardiovascular morbidity and mortality did not hold.21,22
Several aspects of myocardial injury may be related to leptin-regulatory functions. Leptin has diverse cardiovascular effects mediated by complex signaling mechanisms.37 Not surprisingly, compromised cardiac function was found in both hyperleptinemic and hypoleptinemic mice models.38,39 Leptin seems to have both vasodilatory (mediated by nitric oxide) and vasoconstrictor (by induction of endothelin 1) effects on vascular endothelium. A number of studies have demonstrated that leptin has important pro-thrombotic40,41 and pro-atherosclerotic effects, including acceleration of vascular cell calcification, smooth-muscle-cell proliferation, and migration.42 On the other hand, leptin can induce vasodilatation due to endothelial release of nitric oxide, indicating antiatherosclerotic and antithrombotic effects. In humans with angiographically normal coronary arteries, leptin causes coronary artery vasodilatation.43 A beneficial effect of elevated leptin plasma levels on coronary endothelium was observed in obese persons.27
Several studies have shown that leptin can induce cardiomyocyte hypertrophy.11,44,45 However, increased cardiomyocyte size was observed in leptin-deficient or leptin-resistant animals,38,45 and leptin administration to leptindeficient obese ob/ob mice reduces cardiac hypertrophy.38 In healthy individuals who are free from cardiovascular disease and/or obesity, an inverse relationship between leptin levels and left ventricular mass index was observed,46 indicating that physiologically leptin may have an antihypertrophic effect. These contrasting effects of leptin may be related to different experimental conditions (e.g. neonatal versus adult cardyomyocytes), as well as to different metabolic conditions (obesity, metabolic syndrome versus undernutrition), especially at the whole-body level. Evidence for leptin as a cardioprotective factor is emerging. A positive cardioprotective role of leptin has been shown in both in vitro studies with hypoxic insult47 and in vivo studies in animal models such as ischemia–reperfusion injury26 and myocardial infarction induced heart failure.48 Our clinical data are consistent with the observed beneficial affects of leptin administration in myocardial injury in these models.
Several important pathophysiological disturbances other than CAD that play a key role in myocardial injury,may be linked to leptin and share common mechanisms. Among these are inflammation, anemia, and hypoxia. Leptin is structurally and functionally related to pro-inflammatory and hemotopoietic cytokines (e.g. interleukin-6, interleukin-1, tumor necrosis factor-α, granulocyte colony-stimulating factor, etc.). Significant but different associations between leptin and inflammatory markers (CRP), anemia, and hypoxemic conditions were reported.49 In our HF patients, leptin levels correlated positively with hemoglobin, red cell count, hematocrit, and CRP. Current data suggest that mycocardial injury in HF patients depends on the complex interaction of many factors, including CAD, anemia, and pro-inflammatory cytokines, and leptin is involved in regulating each of these pathogenic mechanisms. One might speculate that inadequate leptin production, as in older HF patients, when integrated at the whole-body level may contribute to myocardial injury directly and indirectly by altering multiple pathogenic mechanisms, some of which may even counterbalance one another. The latter may explain why leptin analyzed as a continuous variable was not independently associated with myocardial injury when adjusted for CAD and other clinical factors (see Table 3, model B).
From a practical point of view, our study revealed that in older HF patients a lower circulating leptin level (<12ng/ml) is an independent predictor of peri-operative myocardial injury. The clinical prognostic utility of this biomarker increases significantly when it is used in conjunction with other risk factors. Lower leptin levels together with advanced age and/or hyperparathyroidism have synergistic effects in identifying patients at risk for myocardial injury, and in combination with CAD demonstrate an additive predictive effect. Leptin can now be added to the list of risk factors for peri-operative myocardial injury. Its use might increase the predictive power of an assessment based just on traditional risk factors. Given the association between myocardial injury and adverse outcomes in HF patients, admission leptin levels appear to be a useful biomarker in the risk stratification of these patients. As decreased serum leptin concentrations may affect important pathogenic mechanisms, our findings imply that preventive and therapeutic strategies for the reduction of peri-operative cardiovascular risk should include correction of leptin status. Further investigations involving a larger number of patients are required to confirm the validity of our results and to elucidate the mechanisms underlying leptin’s effects.
There are limitations to this study. First, because of its cross-sectional nature, the study cannot prove a causal association between leptin and myocardial injury. Second, serum leptin concentrations are known to follow a circadian pattern, and we obtained only a single morning blood sample. However, it was reported that a single fasting morning measurement accurately reflects leptin levels over the day and is sufficient to characterize an individual within a population.50 Third, our cohort comprised predominantly white patients, thus the results may not be applicable to all ethnic groups. The strengths of this prospective study include the analysis of leptin by numerous co-variates in a cohort of consecutive HF patients (no selection bias) and inclusion of relevant clinical, hematological, and biochemical variables in the adjusted models.
In conclusion, this study demonstrates for the first time that older HF patients with peri-operative myocardial injury had decreased serum leptin levels and patients with lower serum leptin concentrations (<12ng/ml) had a two-fold increased risk for myocardial injury. This association was independent of clinical, hematological, metabolic, renal, and inflammatory variables. The predictive value of lower leptin levels increased significantly when used in combination with other risk factors.
Disclosures: No payments were received for the development of this manuscript. The authors do not have any conflicts of interest in connection with this paper.
Acknowledgment: The authors sincerely thank Robyn Muscat-Presti for her dedicated secretarial assistance.