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1 Lower Serum Calcium Levels are Associated with Greater Calcium Hydroxyapatite Deposition in Native Aortic Valves of Male Patients with Severe Calcific Aortic Stenosis Jan R. Ortlepp 1, Manuela Pillich 1, Fabian Schmitz 1, Vera Mevissen 1, Ralf Koos 1, Stefan Weiß 2, Ludwig Stork 2, Richard Dronskowski 2, Georg Langebartels 3, Rüdiger Autschbach 3, Vincent Brandenburg 4, Seth Woodruff 5, Jens J. Kaden 6, Rainer Hoffmann MD 1 1 Clinic for Interdisciplinary Intermediate Care and Medical Clinic I, University Hospital of Aachen, 2 Institute of Inorganic Chemistry, Aachen University, 3 Clinic for Cardiac Surgery, 4 Medical Clinic II and 5 Department of Plastic Surgery and Hand Surgery, Burn Center, University Hospital of Aachen, 6 1st Department of Medicine, Faculty of Clinical Medicine Mannheim, University of Heidelberg, Germany Background and aim of the study: The study aim was to evaluate the relationship between serum calcium levels and the degree of calcification found in stenotic aortic valves. Methods: Using atomic absorption spectroscopy, the hydroxyapatite content of 228 excised human stenotic aortic valves was determined and expressed as a percentage of valve mass. Left heart catheterization preceded valve replacement. In addition, serum levels of calcium and creatinine were determined before native calcific aortic valve excision. Results: Valves from male patients contained more hydroxyapatite than those of female patients (26 ± 9 versus 22 ± 9 mass%; p <0.001). Patients presenting with lower serum calcium levels showed a slight trend towards higher levels of valve calcification (r = -0.15, p = 0.026), but this association appeared only within the subgroup of male patients. Male patients with lowest serum calcium levels displayed greatest valvular hydroxyapatite deposition (1st calcium tertiary: 29.5 ± 8.9 mass% versus 2nd calcium tertiary 26.4 ± 7.8 mass% versus 3rd calcium tertiary 21.4 ± 8.9 mass%; n = 122; p = 0.001; r = -0.25; p = 0.006). This association was even more distinct in male patients with normal serum creatinine levels. Furthermore, serum calcium was inversely and significantly associated with serum C-reactive protein in male patients (r = ; p <0.001). Conclusion: Serum calcium levels appear to be inversely related to valve calcification in patients with severe calcific aortic stenosis (AS). This finding indicates the importance of systemic calcium metabolism in calcific AS, independent of manifest disorders of calcium metabolism or renal function. Interestingly, this association was evident only in male patients, suggesting a gender-dependent pathogenesis. The Journal of Heart Valve Disease 2006;15: Calcific aortic stenosis (AS), which is one of the most important valvular heart valve diseases of older adults in the Western world (1,2), shares many similarities with atherosclerosis. Thickening and sclerosis of the aortic valve is an excellent surrogate marker for atherosclerotic diseases, as atherosclerosis is present in almost all patients with calcific AS. Nonetheless, the exact pathogenesis of AS remains incompletely understood (3,4). Previously, common cardiovascular risk factors have been associated with aortic calcification (5,6), but more recently similar risk profiles have been demonstrated in patients with and without AS, indicating that the relationship between cardiovascular Address for correspondence: Dr. Jan R. Ortlepp, Clinic for Interdisciplinary Intermediate Care, University Hospital of Aachen, Pauwelsstrasse 30, Aachen, Germany jrortlepp@ukaachen.de risk factors and AS might be more complex, and that other factors may help to determine the pathogenesis of AS (7). Although about half of all patients with AS have coronary artery disease (CAD), only a minority of those with CAD present with AS. Therefore, it seems likely that only a certain subgroup of patients with CAD, or who are at risk of developing CAD, experience AS. Atherosclerotic lesions of the aortic valve may serve as the foundation for subsequent severe calcification, but factors other than those leading to CAD might trigger calcification (8,9). Among the traditional cardiovascular risk factors, hypercholesterolemia remains especially interesting because treatment options are available (5,6,10,11). However, the positive effects of statin therapy observed in non-randomized studies could not be demonstrated in a recent randomized clinical trial, leaving the role of lipids in the pathogenesis of calcific AS unclear (12). Inflammation is a further recognized Copyright by ICR Publishers 2006

2 J Heart Valve Dis Serum calcium and valve calcification 503 factor associated with aortic valve calcification. In one study, serum levels of C-reactive protein (CRP) were higher in patients with valve calcification than in controls; in another study, inflammatory genetic polymorphisms were associated with differing amounts of valvular hydroxyapatite (13,14). Since clinical trials with an anti-inflammatory treatment have not yet been carried out, the clinical importance of inflammation in the pathogenesis of AS remains unknown. An association between calcium metabolism and calcium deposits in the aortic valve was observed in patients with straightforward disorders of calcium homeostasis, most notably renal dysfunction (15,16). This connection was deemed unimportant for the majority of AS patients, presenting without such disease. It has been shown previously that a genetic polymorphism of the vitamin D receptor was associated with AS, indicating a potential link between calcium metabolism and AS (17). Moreover, various bone-associated factors have been shown to modulate valvular calcification, indicating a further link between calcium metabolism and AS (18,19). The study aim was, therefore, to evaluate a presumed association between serum calcium levels and the precise amount of calcium deposited within excised aortic valves. Clinical material and methods Patient population Patients with symptomatic calcific AS were included in the study. The inclusion criteria have been described previously (14); the same study population plus an additional 112 patients (total 299 patients) were recruited and evaluated in the same manner. In brief, patients with an indication for aortic valve replacement (AVR) due to calcific AS (no bicuspid valves, no history of rheumatic fever or endocarditis, no concomitant other relevant valvular heart disease and coexistence of cardiomyopathies) were recruited. All patients provided their written informed consent, and the study was approved by the local ethical committee (Ethik-Kommission des Universitätsklinikums Aachen, EK712 from 1998, addenda in 1998, 2000 and 2002). Risk factors Risk factors were defined as follows: hypercholesterolemia (total fasting serum cholesterol >220 mg/dl or medically treated); arterial hypertension (blood pressure >140/90 mmhg or medically treated); diabetes mellitus (overnight fasting serum glucose >126 mg/dl on at least two occasions, or medically treated); and smoking (yes or no). Elective diagnostic left heart catheterization was performed in all patients, the gradient across the aortic valve being measured by pull back of the catheter from the left ventricle into the ascending aorta. The mean gradient was calculated using a computer-assisted program (Metek, Roetgen, Germany). Data relating to serum calcium and creatinine (glomerular filtration rate (GFR) was assessed by the MDRD formula) prior to AVR (usually on the morning prior to left heart catheterization) were available from 228 of 299 patients (76%; the other 24% underwent investigations in other laboratories, frequently without measurement of calcium), who comprised the study population. Serum calcium and CRP measurements Serum calcium was measured by complex formation with o-cresol-phthalein complex and photometric analysis at 570 nm (Roche Hitachi, Basel, Switzerland). CRP levels were measured in 219 patients; when the CRP level was below the detection limit (5 mg/l), a value of 5 mg/l was used for further calculations. Atomic absorption analysis A total of 299 valves was excised during AVR; valve tissues were frozen at -80 C, homogenized in liquid nitrogen, and eventually re-frozen after evaporation of the liquid nitrogen. The first analytical batch (187 samples) was brought to room temperature before being solubilized in concentrated sulfuric acid (14). The second batch (112 samples) was analyzed using a modified technique to improve accuracy. Defrosted samples were dried over silica gel (16 h), dissolved in a boiling 3:1 mixture of concentrated hydrochloric/nitric acid (40 ml), diluted, and spiked with 10% solutions of lanthanum/cesium chloride. Eventually, the calcium content of all solutions was determined using atomic absorption spectroscopy (Shimadzu AA-6200) at a wavelength of nm and with an air-acetylene flame. Since all calcified material has been structurally characterized previously as consisting of calcium-deficient hexagonal hydroxyapatite (approximate chemical formula Ca 5 (PO 4 ) 3 OH), the calcium mass was multiplied by a factor of (inverse Ca mass% in hydroxyapatite) to represent the true calcification mass for both batches (14). In the final statistical analysis of all samples, for reasons of convenience, the results of the first batch were rescaled so as to be comparable with the second batch (which related to dried aortic valves and was, presumably, more accurate). The data used were only from the 228 valves in which serum calcium measurements were made. Statistical analysis Continuous data were presented as mean ± SD, and categorical data as frequencies. Differences in quantities of valvular hydroxyapatite were analyzed by

3 504 Serum calcium and valve calcification ANOVA. The association between serum calcium and valvular calcium hydroxyapatite was evaluated first by dividing the calcium data into tertiaries (Ca 2.27 mmol/l; mmol/l; Ca 2.42 mmol/l) and testing the difference between tertiaries (using ANOVA); linear regression analyses were then performed. A post-hoc analysis was performed to analyze the association of calcium and valvular calcification stratified for gender and renal function (serum creatinine 1.3 mg/dl). The null hypothesis was that the amount of hydroxyapatite of the aortic valve did not differ between the different levels of serum calcium. A p- value <0.05 was considered to be statistically significant. Results J Heart Valve Dis Study population The clinical characteristics of patients are summarized in Table I. Among 228 patients, 49 patients (15 females, 34 males) had a serum creatinine level >1.3 mg/dl, and six (two females, four males) >2.0 mg/dl. Cardiovascular risk factors were not significantly associated with elevated valvular calcium hydroxyapatite (Table II). Overall, the degree of calcification was significantly higher in male patients (Table II). Serum calcium and degree of valvular calcification There was a slight inverse trend towards greater deposition of valvular calcium hydroxyapatite in patients with lower serum calcium (Table III); this association was evident only in male patients. Moreover, in a post-hoc analysis this association was strongest in the subgroup of male patients with normal serum creatinine level ( 1.3 mg/dl). Linear regression analysis showed this effect to be only weak (r = ; F = 4.99; p = 0.026) in the entire study population, but stronger among the male population (r = ; F = 7.73; p = 0.006), especially in those males with normal serum creatinine levels (r = ; F = 20.03; p <0.0001). Serum calcium, renal function and serum CRP Patients in the lower calcium tertiary had significantly higher CRP levels (Table IV), and this was more pronounced in males. Renal function, assessed by Table I: Clinical characteristics of the study population stratified by gender. Characteristic All patients Males Females (n = 228) (n = 122) (n = 106) Age (years) + 70 ± 9 68 ± 9 72 ± 8 Weight (kg) + 74 ± ± ± 13 Height (cm) ± ± ± 6 Cardiovascular risk factors Hypertension (n) 154 (68) 77 (63) 77 (73) Hypercholesterolemia (n) 117 (51) 56 (46) 61 (58) Smoking (n) 79 (35) 62 (51) 17 (16) Diabetes mellitus (n) 51 (22) 26 (21) 25 (24) Left-heart catheterization Clear coronaries or sclerosis (n) 117 (51) 50 (41) 67 (43) CAD (n) 111 (49) 72 (59) 39 (37) Mean gradient (mmhg),+ 53 ± ± ± 19 AVA (cm 2 ), ± ± ± 0.28 AVA/BSA (cm 2 /m 2 ), ± ± ± 0.15 Degree of aortic valve calcification Ca 5 (PO 4 ) 3 OH (mass%) + 24 ± 9 26 ± 9 22 ± 9 Laboratory results Calcium (mmol/l) ± ± ± 0.21 CRP (mg/l),+ 15 ± ± ± 16 Creatinine (mg/dl) ± ± ± 0.75 GFR (ml/min) + 60 ± ± ± 13 GFR <60 ml/min (n) 118 (52) 44 (36) 74 (70) + Values in parentheses are percentages., Data available from 218 patients;, data available from 162 patients;, data available from 219 patients. p <0.05. AVA/BSA: Aortic valve area corrected for body surface area; AVA: Aortic valve area; CAD: Coronary artery disease; CRP: C- reactive protein; GFR: Glomerular filtration rate assessed by MDRD formula.

4 J Heart Valve Dis Serum calcium and valve calcification Table II: Association of cardiovascular risk factors and valvular calcification. 505 Cardiovascular risk No. of Valvular calcium p-value factor patients hydroxyapatite (mass%) Hypertension Present ± 9.2 Absent ± Hypercholesterolemia Present ± 9.0 Absent ± Smoking Present ± 8.2 Absent ± Diabetes mellitus Present ± 7.5 Absent ± Gender Male ± 8.8 Female ± 9.0 <0.001 Table III: Relationship of serum calcium and aortic valve calcification in patients stratified for gender and serum creatinine level. Serum calcium Valvular CHAP p-value (mass%) + All patients (n = 228) 1st tertiary 25.9 ± nd tertiary 24.4 ± 8.8 3rd tertiary 21.4 ± Male patients (n = 122) 1st tertiary 29.5 ± 8.9 2nd tertiary 26.4 ± 7.8 3rd tertiary 21.4 ± Male patients, serum creatinine <1.3 mg/dl (n = 88) 1st tertiary 30.2 ± 9.4 2nd tertiary 27.0 ± 6.3 3rd tertiary 19.9 ± 8.1 < Female patients (n = 106) 1st tertiary 22.6 ± 9.8 2nd tertiary 20.6 ± 9.6 3rd tertiary 21.4 ± Calcium content: 1st tertiary 2.27 mmol/l; 2nd tertiary mmol/l; 3rd tertiary 2.42 mmol/l. + CHAP: Calcium hydroxyapatite. Table IV: Relationship of serum calcium with serum C- reactive protein (CRP) and renal function. Serum calcium Serum CRP GFR (mg/l) +, (ml/min) +, All patients (n = 228) 1st tertiary 24 ± ± 16 2nd tertiary 12 ± ± 14 3rd tertiary 9 ± 9 59 ± 16 p = p = Male patients (n = 122) 1st tertiary 31 ± ± 14 2nd tertiary 14 ± ± 15 3rd tertiary 10 ± ± 17 p = p = Female patients (n = 106) 1st tertiary 17 ± ± 16 2nd tertiary 7 ± 4 58 ± 11 3rd tertiary 7 ± 5 54 ± 12 p = p = Calcium content: 1st tertiary 2.27 mmol/l; 2nd tertiary mmol/l; 3rd tertiary 2.42 mmol/l. + Data available from 219 patients. Renal function assessed by GFR (glomerular filtration rate, assessed by MDRD formula).

5 506 Serum calcium and valve calcification J Heart Valve Dis Table V: Linear regression analysis: Relationship of serum calcium, valvular hydroxyapatite and serum C-reactive protein (CRP). Independent Dependent r-value F-value p-value variable variable All patients (n = 228) Serum calcium Valvular CHAP Serum calcium Serum CRP <0.001 Serum CRP Valvular CHAP Male patients (n = 122) Serum calcium Valvular CHAP Serum calcium Serum CRP <0.001 Serum CRP Valvular CHAP Male patients, serum creatinine <1.3 mg/dl (n = 88) Serum calcium Valvular CHAP <0.001 Serum calcium Serum CRP Serum CRP Valvular CHAP Female patients (n = 106) Serum calcium Valvular CHAP Serum calcium Serum CRP Serum CRP Valvular CHAP Data available from 219 patients. CHAP: Calcium hydroxyapatite. GFR, did not differ between the calcium tertiaries. In the linear regression analysis (Table V) serum calcium was inversely and significantly associated with valvular calcium hydroxyapatite and with serum CRP but, again, this was evident only in male patients. Discussion Although the exact pathogenesis of AS remains unclear, there are many similarities between AS and atherosclerosis, and cardiovascular risk factors are often present in patients with AS (5,6). However, the traditional cardiovascular risk factors do not seem to provide a sufficient explanation for the pathogenesis of AS. While hypercholesterolemia, in particular, was thought to play a major role in the progression of valve calcification, the treatment of hypercholesterolemia did not reduce progression of the disease in a large randomized trial (8,9,12). Beyond such risk factors, genetic polymorphisms of the inflammatory system and of the vitamin D receptor have been linked to AS, suggesting that genetic variations might influence valvular calcification (14,17). Disorders of systemic calcium homeostasis have been recognized as potential causes of aortic valve calcification, though systemic calcium metabolism in normal patients has not been a major focus of research (15,16). The findings of the present study showed that there is an inverse relationship between systemic serum calcium and calcium hydroxyapatite mass in the calcified aortic valves of AS patients. The lower the level of serum calcium, the greater the amount of hydroxyapatite deposited in the valves. This situation was, however, only evident in male patients, who have per se a higher degree of calcification (14). The association was, of course, not a simple linear one, and the r-value was significant, but not very large. However, this correlation was very interesting because none of the enrolled patients suffered from overt disease of calcium metabolism such as hyperparathyroidism. Moreover, the relationship was even more pronounced among the subgroup of male patients with normal serum creatinine levels; hence, frank renal disease cannot explain this observed association. Interestingly, serum calcium and serum CRP also showed an inverse and significant association, which might emphasize the interaction between inflammation and calcium homeostasis. It seems illogical to speculate that serum calcium levels might be low in severely calcified AS secondary to local deposition in the valve tissue. In fact, inflammation might interfere with calcium-controlling pathways. For example, it might reduce the amount of serum fetuin-a, an important systemic calcification

6 J Heart Valve Dis Serum calcium and valve calcification 507 inhibitor in serum. Thus, low serum calcium levels might indicate a state of disturbed ability to stabilize serum calcium levels and indicate a predisposing condition for ectopic tissue calcification. A further suggestion for the association of systemic calcium metabolism was derived from a recent small study in which patients with AS had decreased bone mineral density, a condition frequently associated with lower calcium (20). Thus, speculatively, osteoporosis and valve calcification might be two sides of the same problem. Given the hypothesis that AS is a multifactorial disease based on atherosclerosis and is promoted by genetically determined triggers of inflammation and calcification, it appears likely that small disturbances of calcium and inflammation might influence the degree of calcification sufficiently to determine the progression of the disease. Study limitations The main limitation of the present study was its cross-sectional design, undertaken without analyzing the progression of AS. Other important limitations included a lack of analysis of serum phosphate and other calcium-relevant factors such as vitamin D receptor, vitamin D, parathormone, and calcitonin. Due to the accurate method of measuring valvular calcification used herein, the observed relationship was intriguing, but to be more conclusive this should be validated in a further study with consecutive calcium determinations, during development of AS, and also in patients with AS without operation (early stages of calcification) where calcification of the valve can be evaluated with imaging. In conclusion, these study results provide further evidence supporting the potential link between calcium homeostasis and calcific AS, especially in view of recent investigations which demonstrated various mechanisms of bone development and biomineralization in stenotic valves (18,19). Changes in systemic calcium metabolism should be an important focus of future research evaluating the pathogenesis of aortic valve calcification. Acknowledgements This study formed part of the doctoral thesis of Manuela Pillich. References 1. Lindroos M, Kupari M, Heikkila J, Tilvis R. Prevalence of aortic valve abnormalities in the elderly: An echocardiographic study of a random population sample. J Am Coll Cardiol 1993;21: Freeman RV, Otto CM. Spectrum of calcific aortic valve disease. Pathogenesis, disease progression, and treatment strategies. Circulation 2005;111: Kaden JJ, Eckert JP, Poerner T, et al. Prevalence of atherosclerosis of the coronary and extracranial cerebral arteries in patients undergoing aortic valve replacement for calcified stenosis. J Heart Valve Dis 2006:15: Otto CM, Lind BK, Kitzman DW, Gersh BJ, Siscovick DS. Association of aortic-valve sclerosis with cardiovascular mortality and morbidity in the elderly. N Engl J Med 1999;341: Aronow WS, Schwartz KS, Koenigsberg M. Correlation of serum lipids, calcium, and phosphorus, diabetes mellitus and history of systemic hypertension with presence or absence of calcified or thickened aortic cusps or root in elderly patients. Am J Cardiol 1987;59: Stewart BF, Siscovick D, Lind BK, et al. Clinical factors associated with calcific aortic valve disease. J Am Coll Cardiol 1997;29: Ortlepp JR, Schmitz F, Bozoglu T, Hanrath P, Hoffmann R. Cardiovascular risk factors in patients with aortic stenosis predict prevalence of coronary artery disease, but not the prevalence of aortic stenosis. An angiographic pair matched case-control study. Heart 2003;89: O Brien KD, Reichenbach DD, Marcovina SM, Kunsisto J, Alpers CE, Otto CM. Apolipoprotein B, (a) and E accumulate in the morphological early lesion of degenerative valvular aortic stenosis. Arterioscler Thromb Vasc Biol 1996;16: Olsson M, Thyberg J, Nilsson J. Presence of oxidized low density lipoprotein in nonrheumatic stenotic aortic valves. Arterioscler Thromb Vasc Biol 1999;19: Novaro GM, Tiong IY, Pearce GL, Lauer MS, Sprecher DL, Griffin BP. Effect of hydroxymethylglutaryl coenzyme A reductase inhibitors on the progression of calcific aortic stenosis. Circulation 2001;104: Shavelle DM, Takasu J, Budoff MJ, Mao S, Zhao XQ, O Brien KD. HMG CoA reductase inhibitor (statin) and aortic valve calcium. Lancet 2002;359: Cowell SJ, Newby DE, Prescot RJ, et al. Scottish aortic stenosis and lipid lowering trial, impact on regression (SALTIRE) investigators. N Engl J Med 2005;352: Galante A, Pietroiusti A, Vellini M, et al. C-reactive protein is increased in patients with degenerative aortic valvular stenosis. J Am Coll Cardiol 2001;38: Ortlepp JR, Schmitz F, Mevissen V, et al. The amount of calcium-deficient hexagonal hydroxyap-

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