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Vitamin D25-OH and D1-25OH levels were compared with cardiac R2 (1/T2), left ventricular ejection fraction (LVEF), age, ferritin and liver iron in 24 thalassaemia major patients. Vitamin D25-OH levels were reduced in 13/24 patients while vitamin D1-25OH levels were often elevated. Vitamin D25-OH levels decreased with age (r2 = 0·48) and with liver iron (r2 = 0·20). Cardiac R2 was inversely related with the ratio of D25-OH to D1-25OH levels (r2 = 0·42). LVEF was also proportional to the D25-OH/D1-25OH ratio (r2 = 0·49). Vitamin D deficiency may be associated with cardiac iron uptake and ventricular dysfunction in thalassaemia major patients.
Despite supplementation in dairy products, vitamin D deficiency is epidemic in the United States, most likely representing decreased sun exposure in recreational, educational and work environments (Holick, 2007). Thirty to forty per cent of otherwise healthy adolescents and young adults have vitamin D levels below the lower limit of 50 nmol/l and an even greater proportional are below the 75 nmol/l cut-off for vitamin D ‘sufficiency’ (Dawson-Hughes et al, 2005). Vitamin D is essential for intestinal calcium absorption and plays a critical role in global calcium homeostasis and bone metabolism (Holick, 2007). However, vitamin D receptors are found in nearly all tissues and also appear to modulate cellular calcium homeostasis (Billaudel et al, 1993). Consequently, vitamin D deficiency is associated with muscular weakness (Pfeifer et al, 2002), prolonged congestive heart failure (Bhattacharya et al, 2006), and impaired insulin secretion (Chiu et al, 2004).
Given the high prevalence of vitamin D deficiency in the general population and the added burden of increased metabolic demands, chronic medical care, and iron overload, it is not surprising that vitamin D deficiency is quite common in thalassaemia major patients (Moulas et al, 1997; Napoli et al, 2006). With the recent evidence linking L-type calcium channel activity to cardiac iron loading, we investigated the possibility that vitamin D deficiency may predispose to cardiac iron loading and dysfunction in thalassaemia major (Oudit et al, 2003).
Our chronically transfused haemoglobinopathy patients are systemically screened for vitamin deficiencies and cardiac status. Patients’ medical records were reviewed after obtaining permission from the Committee on Clinical Investigation at Children’s Hospital Los Angeles. All vitamin D levels obtained within 1 year of cardiac magnetic resonance imaging (MRI) examination. Examinations were performed at 2·8 ± 3·3 month intervals (range, 0·2–9·4 months); mean serum ferritin difference between examinations was 195 ± 888 μg/l (P = NS). Other historical parameters recorded included age, gender, ferritin, liver iron (by MRI) and transferrin saturation. Folate 1 mg/d and vitamin E 400 U/d are routinely supplemented in our chronically transfused patients; none were receiving vitamin D or calcium replacement.
Cardiac T2 and ejection fraction data were derived as previously described Wood et al (2004). Liver iron was estimated from spin-echo and gradient echo images using validated techniques. Transferrin saturation measurements, calcium levels, and serum ferritin values were collected from a single fasting blood draw at the time of the vitamin D assessment. Simple and multivariate regression was performed between vitamin D levels and age, R2, left ventricular ejection fraction (LVEF), hepatic iron concentration (HIC), ferritin, and transferrin saturation (JMP5·1, SAS, Cary, NC, USA); log transformation was applied when appropriate to improve the fit. Statistically significant (P < 0·05) variables from the univariate analysis were entered into a step-wise regression analysis. Variables were manually entered and removed over all possible permutations to minimize the Akaike Information Criteria. This cost function weights fit improvement from additional variables by a penalty for increased model complexity.
Twenty-four patients [11 women and 13 men with a mean age of 14·7 ± 7·6 years (1·4–25·8)] had records suitable for review. All patients received simple transfusions with a volume of 15 ml/kg at three weekly intervals to maintain a pretransfusion haemoglobin between 90 and 100 g/l. All patients had begun chelation with deferoxamine but 19 patients were currently on deferasirox (16·1 ± 9·2 months), and one patient was on deferiprone (26 months). The study population was moderate to severely iron overloaded with ferritin values of 4693 ± 4314 pmol/l (553–18492), liver iron 13·7 ± 11·4 mg/g dry wt (2–39·5), cardiac T2 24·9 ± 12·3 (4·4–50·5), and transferrin saturation 84 ± 18% (36–106%). Hepatitis C was uncommon (one patient) and no patients were on cardiac medications, including calcium channel blockers. Folate (1 mg/d) and vitamin E (400 IU/d) were prescribed to all patients; patients on deferoxamine were also prescribed vitamin C, 250 mg QD, with infusion. No patients were taking multivitamins and only one patient was taking vitamin D supplementation.
Vitamin D25-OH levels were markedly depressed, 42·7 ± 21·2 nmol/l (2·5–82·5), with 13/24 values below the lower limit of 50 nmol/l and 23/24 below 75 nmol/l. In contrast, vitamin D1-25OH levels were normal or elevated in all patients, 155·7 ± 50·7 pmol/l (83·2–267·8) with eight patients exceeding the upper limit of normal of 156 pmol/l. There was no correlation between D25-OH and D1-25OH levels. D25-OH levels (but not D1-25OH levels) fell sharply with age (r2 = 0·48) and were negatively associated with liver iron (r2 = 0·20, P < 0·04). Calcium levels were normal in all patients, at 2·38 ± 0·1 mmol/l (2·1–2·55); phosphate and parathyroid hormone (PTH) levels were not obtained in a sufficient percentage of patients to relate them to the above findings.
These data reinforce prior evidence that serious vitamin D deficiency is common in thalassaemia major patients (Moulas et al, 1997; Napoli et al, 2006). The sharp decline in vitamin D25-OH stores with age probably reflects a combination of decreased intake (dairy products) as well as a shift toward more indoor activities. Vitamin D is 25-hydroxylated in the liver and haemosiderosis may impair vitamin D25-OH production (Chow et al, 1985). Vitamin D1-25OH levels were normal or elevated in all patients, similar to prior studies (Moulas et al, 1997; Napoli et al, 2006). Typically, vitamin D1-25OH levels are maintained in D25-OH deficiency states by reciprocal increases in parathyroid hormone (Chapuy et al, 1997). Parathyroid levels may be inappropriately low in some thalassaemia patients, possibly reflecting subclinical hypoparathyroidism from iron deposition (Napoli et al, 2006). However, in our population, the opposite appeared to be true, with D1-25OH overcompensation of in 8/24 patients. This could occur if PTH levels are inappropriately high (primary hyperparathyroidism superimposed upon secondary hyperparathyroidism) or there has been upregulation of D1-25OH hydroxylase activity in extra-renal tissues Holick, 2007). Unfortunately, the absence of PTH measurements in the present study did not allow us to distinguish the mechanism of elevated D1-25OH.
Cardiac R2 was log-linearly correlated with D25-OH level (r2 = 0·44, P = 0·0001; Fig 1A). All six patients with severe cardiac iron loading (R2 >100 Hz) had vitamin D25-OH levels below 32·5 nmol/l. Figure 1B compares cardiac R2 with the ratio of D25-OH to D1-25OH, demonstrating a sharp increase in detectable cardiac iron for ratios less than 25%. Univariate regression demonstrated significant correlations of log cardiac R2 with vitamin D25-OH (r2 = 0·44), log D25-OH/D1-25OH (r2 = 0·42), log ferritin (r2 = 0·29), HIC (r2 = 0·20) and borderline correlation with age (r2 = 0·16); there was no association with transferrin saturation or D1-25OH levels. Multivariate analysis of these variables demonstrated that the D25-OH/D1-25 ratio was the best predictor of abnormal cardiac R2 (r2 = 0·42), although serum ferritin retained a small contribution (combined r2 = 0·50).
The LVEF was positively correlated to D25-OH levels (r2 = 0·35, P = 0·002; Fig 2A). Figure 2B is a similar comparison of LVEF and the ratio of D25-OH/D1-25OH. Univariate regression demonstrated significant relationships of log LVEF with the ratio of log D25-OH/D1-25OH (r2 = 0·49), log cardiac R2 (r2 = 0·41), D25-OH (r2 = 0·37), log ferritin (r2 = 0·35), and age (r2 = 0·20); log HIC, log D1-25OH, and transferrin saturation exhibited trends (r2 = 0·10–0·14, P = 0·09–0·13) that did not reach significance. In multivariate analysis, the log ratio of D25OH/D1-25OH and log ferritin were the optimum predictors of cardiac function (combined r2 = 0·61). Although cardiac R2 was a strong univariate predictor, it did not provide additional information to these two variables. If cardiac R2 was forced to remain in the regression, both log D25-OH/D1-25OH and log ferritin were retained as terms. Thus while cardiac iron deposition will probably have a primary role in left ventricular dysfunction, further work is be necessary to decouple confounding effects from vitamin D derangements.
The association of vitamin D deficiency with left ventricular dysfunction is not surprising. Skeletal muscle weakness and chronic heart failure exacerbation have been well described with vitamin D deficiency (Pfeifer et al, 2002; Bhattacharya et al, 2006). Correction of vitamin D deficiency is well-documented to improve cardiac function in chronic renal failure patients (Lemmila et al, 1998). However, this data is the first to suggest an association between cardiac iron uptake and vitamin D25-OH deficiency. Low D25-OH levels produce reciprocal increases in serum parathyroid levels, leading to higher heart rates, cardiac intracellular calcium levels, and hypertrophy (Smogorzewski & Massry, 1997). Paradoxically, vitamin D overdose also increases cardiac intracellular calcium and has been used as a model for vascular calcinosis (Thorin et al, 1990). Both parathyroid hormone and vitamin D1-25OH appear to stimulate transmembrane calcium movement via L-type voltage-dependent calcium channels (LVDCC), although the details of this interaction remain poorly characterized (Billaudel et al, 1993; Smogorzewski & Massry, 1997). Murine data indicate that LVDCC are important in transporting non-transferrin bound iron (NTBI) into the myocardium (Oudit et al, 2003). Thus LVDCC modulation represents the logical link between vitamin D deficiency, cardiac iron, and cardiac function. According to this model, we hypothesized that secondary hyperparathyroidism (high PTH, normal or high vitamin D1-25OH levels), in the presence of elevated NTBI, may exacerbate cardiac iron uptake through LVDCC. The fact that the ratio of D25-OH to D1-25OH has greater predictive value than either variable alone supports this hypothesis. This model does not preclude cardiac iron deposition in vitamin D sufficient patients with overwhelming NTBI exposure. Similarly, secondary hyperparathyroidism would not be expected to produce cardiac iron loading in the absence of elevated circulating NTBI, but might independently impair myocardial calcium cycling and cardiac function.
Even though the multivariate regression analysis suggests otherwise, we cannot exclude the possibility that vitamin D deficiency is an epiphenomenon rather than a causative modulator of myocardial iron loading. Nonetheless, the present data are sufficiently compelling to warrant larger observational trials in addition to subsequent replacement studies should the cardiac associations be maintained in larger studies.
This work was supported by the NHLBI (1 RO1 HL075592-01A1), the General Clinical Research Center at Childrens Hospital Los Angeles (RR000043-43), Center for Disease Control (Thalassaemia Center Grant U27/CCU922106) and Novartis Pharma.