Long-term effect of calcium-vitamin D 3 fortified milk on blood pressure and serum lipid concentrations in healthy older men

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1 (2009) 63, & 2009 Macmillan Publishers Limited All rights reserved /09 $ ORIGINAL ARTICLE Long-term effect of calcium-vitamin D 3 fortified milk on blood pressure and serum lipid concentrations in healthy older men RM Daly 1 and CA Nowson 2 1 Department of Medicine (RMH/WH), Western Hospital, University of Melbourne, Melbourne, Victoria, Australia and 2 School of Exercise and Nutrition Sciences, Centre for Physical Activity and Nutrition Research, Deakin University, Melbourne, Victoria, Australia Background/Objectives: Some epidemiological and clinical studies have shown that increased dairy consumption or calcium and/or vitamin D supplementation can have a beneficial effect on blood pressure, and lipid and lipoprotein concentrations. The aim of this study was to assess the long-term effects of calcium-vitamin D 3 fortified milk on blood pressure and lipid-lipoprotein concentrations in community-dwelling older men. Subjects/Methods: This is a substudy of a 2-year randomized controlled trial in which 167 men aged 450 years were assigned to receive either 400 ml per day of reduced fat (B1%) milk fortified with approximately 1000 mg of calcium and 800 IU of vitamin D 3 or to a control group receiving no additional fortified milk. Weight, blood pressure, lipid and lipoprotein concentrations were measured every 6 months. Participants on lipid-lowering (n ¼ 32) or antihypertensive medication (n ¼ 39) were included, but those who commenced, increased or decreased their medication throughout the intervention were excluded (n ¼ 27). Results: In the 140 men included in this study (milk, n ¼ 73; control, n ¼ 67), there were no significant effects of the calciumvitamin D 3 fortified milk on weight, systolic or diastolic blood pressure, total cholesterol, high-density lipoprotein or low-density lipoprotein cholesterol or triglyceride concentrations at any time throughout the intervention. Similar results were observed after excluding men taking antihypertensive or lipid-lowering medication or limiting the analysis to those with baseline calcium intakes o1000 mg per day and/or with hypovitaminosis D (25(OH)D o75 nmol/l). Conclusions: Supplementation with reduced-fat calcium-vitamin D 3 fortified milk did not have a beneficial (nor detrimental) effect on blood pressure, lipid or lipoprotein concentrations in healthy community-dwelling older men. (2009) 63, ; doi: /ejcn ; published online 21 January 2009 Keywords: blood pressure; lipids; calcium-vitamin D 3 ; fortified milk; men Introduction High intakes of calcium and/or increased consumption of dairy foods have been reported to have beneficial effects on a number of cardiovascular and metabolic-related parameters. Several clinical trials have shown that calcium supplementation may improve serum lipid concentrations (Bell et al., 1992; Denke et al., 1993; Reid et al., 2002). Higher dietary calcium intakes, primarily from dairy products, have also been shown to be related to lower lipid concentrations, Correspondence: Professor RM Daly, Department of Medicine (RMH/WH), Western Hospital, University of Melbourne, Footscray, Melbourne, Victoria 3011, Australia. Received 6 June 2008; revised 11 November 2008; accepted 2 December 2008; published online 21 January 2009 independent of fat mass or waist circumference (Jacqmain et al., 2003). There is also substantial evidence indicating that increased calcium or dairy consumption can lower the risk of hypertension. The findings from a meta-analysis of controlled clinical trials indicate that calcium supplementation (diet and supplements) of mg per day for at least 2 weeks resulted in a small but significant mean 1.4 and 0.8 mm Hg reduction in systolic (SBP) and diastolic (DBP) blood pressure, respectively (Griffith et al., 1999). There was however, considerable heterogeneity in the blood pressure response to increased calcium (McCarron and Reusser, 1999; Zemel, 2001) and there is some evidence that dietary sources of calcium may result in a greater and more consistent response in lowering blood pressure than calcium supplements (Griffith et al., 1999). Epidemiological and clinical studies have also reported an inverse association between vitamin D status and blood

2 994 pressure (Kristal-Boneh et al., 1997; Scragg et al., 2007); serum 25-hydroxyvitamin D (25(OH)D) concentrations have been shown to inversely, albeit weakly, associated with hypertension or blood pressure in some (Kokot et al., 1981; Scragg et al., 2007), but not all studies (Snijder et al., 2007). The link between vitamin D and blood pressure may relate, in part, to the fact that vitamin D is a negative regulator of renin release, which is important in the regulation of blood pressure (Li et al., 2004). There are a number of studies that have also reported a beneficial effect of combined calcium and vitamin D supplementation. In elderly women with serum 25(OH)D o50 nmol/l and increased blood pressure, 8 weeks of calcium-vitamin D supplementation reduced SBP more than calcium supplementation alone (Pfeifer et al., 2001). In overweight/obese women, combined calciumvitamin D supplementation enhanced the beneficial effects of weight loss on plasma lipid concentrations (Major et al., 2007). However, there are no long-term clinical trials that have examined whether dairy products fortified with additional calcium-vitamin D 3 have a beneficial effect on blood pressure or lipid concentrations in older adults. Some evidence suggests that dairy foods, particularly low-fat dairy products, may be important in reducing the risk of various cardiovascular and metabolic-related diseases (Alonso et al., 2005; Ruidavets et al., 2006), but these findings are not consistent (Barr et al., 2000; Al-Delaimy et al., 2003; Barr, 2003). The aim of this study, which was part of a 2-year randomized controlled trial investigating the effects of calcium-vitamin D 3 fortified milk on bone mineral density (BMD) in older men (Daly et al., 2006a, b), was to examine the effect of the fortified milk on blood pressure and serum lipid-lipoprotein concentrations. Subjects and methods Subjects As described previously (Daly et al., 2006b), communityliving Caucasian men aged 450 years were recruited into this study from residential areas of Melbourne, Australia. Participants were initially excluded if they had taken calcium-vitamin D supplements in the preceding 12 months, participated in regular resistance training in the previous 6 months or greater than 150 min per week of weight-bearing exercise, had a body mass index (BMI) 435 kg/m 2, were lactose intolerant, consumed more than four alcoholic beverages per day or had a history of osteoporotic fracture or medical disease or medication use known to affect bone metabolism. All men also had a hip or spine BMD z-score within ±2 s.d. The study was approved by the Deakin University Human Research Ethics Committees, and written consent was obtained from all the participants. Randomization All men (n ¼ 167) were randomly assigned to either a calcium-vitamin D 3 fortified milk (n ¼ 85) or control group receiving no additional fortified milk (n ¼ 82) with stratification according to age (o65 or X65 years) and dietary calcium intake (o800 and X800 mg per day). Of the 167 men, 18 withdrew from the study (9 milk, 9 control), yielding a dropout rate of 10.8% (Daly et al., 2006b). The reasons for withdrawal have been described previously (Daly et al., 2006b). For this study, participants on lipidlowering (n ¼ 32) or antihypertensive medication (n ¼ 39) were included, but those who commenced (n ¼ 16), increased (n ¼ 6) or decreased (n ¼ 5) their number of lipidlowering or antihypertensive drugs throughout the intervention were excluded. Therefore, 140 men were included in this substudy (milk, n ¼ 73; controls, n ¼ 67). Milk supplementation Men randomized to receive the calcium-vitamin D 3 fortified milk were asked to consume ml tetra packs per day of reduced-fat (B1%) ultra-high temperature milk (Murray Goulburn Co-operative Co. Ltd, Australia). Each 200 ml tetra pack contained approximately 500 mg calcium (milk calcium salt, NatraCal) and 400 IU vitamin D 3, 418 kj energy, 6.6 g protein, 2.2 g fat, 11 g lactose, 100 mg sodium and 250 mg phosphorus. The milk was fortified with a calcium salt derived from fresh milk whey. The vitamin D 3 used to fortify the milk was obtained from DSM Nutritional Products Pty Ltd (NSW, Australia). As previously reported (Daly et al., 2006b), eight batches of milk were manufactured throughout the study, with participants receiving a new batch every 3 months. The quality of each batch was analyzed in terms of the calcium and vitamin D 3 level before being distributed. The mean (±s.d.) calcium and vitamin D 3 levels per 200 ml for the eight batches of milk manufactured throughout the study were 497±24 mg and 352±30 IU, respectively. Of the initial 85 men, 7 stopped taking the fortified milk at different time points throughout the intervention. Three of these men stopped taking the milk within the first 6 months, with the remaining men stopping between 11 and 21 months. Five of these men experienced gastrointestinal side effects, one participant was concerned about weight gain and one participant was no longer willing to consume the milk. The mean±s.d. reported milk compliance calculated as the percentage of the tetra packs consumed based on daily diaries was 85±21% (Daly et al., 2006b). Participants assigned to the control group continued with their usual diet. Blood pressure Systolic and diastolic blood pressures were measured while the participants were seated, after a 5-min rest period in a quiet room using an automated blood pressure monitor (Vital Care 506DXN; Criticare System Inc.). Four measurements were taken on the left arm with a 2-min interval

3 between readings; the mean of the final three readings was used in the analysis. Biochemical measurements Fasted, resting morning ( hours) blood samples were obtained at baseline and every 6 months throughout the study and stored at 80 1C until assayed. Serum total cholesterol, triglycerides and high-density lipoprotein (HDL) cholesterol were assessed at all time points using RX daytona automated chemistry analyzer (Furuno Electronic Co. Ltd, Japan). Low-density lipoprotein (LDL) cholesterol was calculated using the Friedewald formula. Each participant s samples were analyzed in the same batch. The interassay coefficients of variance (CVs) ranged from 1.5 to 4.8%. As previously reported (Daly et al., 2006b), serum intact parathyroid hormone (hpth 1 84) was measured by an immunoradiometric assay (IRMA) using the DiaSorin N-tact PTH IRMA kit (DiaSorin Inc., Stillwater, MN, USA). The interassay CV was 5.3%. Serum concentrations of 25(OH)D were measured by a two-step process using the DiaSorin RIA kit (DiaSorin Inc.). The interassay CV was 11.8%. Other measurements Height (cm), weight (kg) and BMI (kg/m 2 ) were assessed using standard techniques. Dietary intakes were estimated from 2 weekdays and 1 weekend day measured food diaries collected every 6 months, and analyzed using the Foodworks nutrient analysis software program (Xyris Software, Brisbane, Australia). The CHAMPS survey was used to assess physical activity (expressed as kj per week) at baseline and every 6 months. Information on medication use, smoking status and alcohol consumption was determined by questionnaire and confirmed by interview at the beginning and every 6 months throughout the study. Statistical analysis Baseline characteristics between the groups were compared by independent t-tests for continuous variables and w 2 -tests for categorical variables. Time, group and interaction effects were examined using pooled time series regression analysis for longitudinal data with random effects models. This analysis is similar to an intention-to-treat analysis in that it includes all participants who entered the study and had at least one follow-up measurement. All analyses were adjusted for use of antihypertensive or lipid-lowering medication, change in weight, and alcohol and saturated fat intake. The data were also analyzed after (1) excluding participants taking antihypertensive (n ¼ 28) and/or lipid-lowering medication (n ¼ 24), or (2) including only those men with low dietary calcium intakes (o1000 mg per day) and/or hypovitaminosis D (serum 25(OH)D concentration o75 nmol/l) at baseline (n ¼ 110). The following parameters were normalized by log transformation: serum 25(OH)D, Table 1 Characteristics of the study participants at baseline-bytreatment group Characteristic Milk (n ¼ 73) Control (n ¼ 67) Age (years) 61.3± ±7.5 Height (cm) 175.5± ±7.6 BMI (kg/m 2 ) 26.2± ±3.2 Physical activity (kj/week) 8800± ±8795 Current smokers (%) Alcohol consumption (%) Antihypertensive therapy (%) Lipid-lowering therapy (%) Abbreviation: BMI, body mass index. Values are mean±s.d. triglycerides and HDL cholesterol. All analyses were performed on the raw data or the natural logarithm transformed scale. All data are presented as means±s.d. or 95% CI unless otherwise stated. Results There were no differences in the baseline characteristics between two groups, with the exception that dietary calcium, saturated fat and dietary cholesterol intakes were marginally greater (11 16%) in the milk group (Tables 1 3). Of the 140 men included in this study, 60% had a dietary calcium intake below the current Australian recommended dietary intake (RDI) of 1000 mg per day for men years of age. One participant was classified as having vitamin D deficiency (25(OH)D o25 nmol/l) at study entry; twelve (8.6%) were insufficient (25(OH)D o50 nmol/l) and sixty (42.9%) were classified as having hypovitaminosis D (25(OH)D o75 nmol/l). In total, 110 men (78.6%) had a dietary calcium intake below 1000 mg per day and/or hypovitaminosis D. Fifteen men (milk, n ¼ 8; control, n ¼ 7) were hypertensive based on a SBP and/or DBP of X140 and X90 mm Hg, respectively. A total of 28 men were on antihypertensive medication (milk, n ¼ 12 (16.4%); control, n ¼ 16 (23.9%)), of which 19 were on a single therapy and 9 were on combination therapy either with a single tablet (n ¼ 3) or with dual tablets (n ¼ 5) and one participant was taking a combination of four drugs. A total of 64 men (47.4%) had elevated serum total cholesterol (45.5 mmol/l) and/or triglyceride (42.0 mmol/l) concentrations at baseline (milk, 50.7%; control, 43.9%), and 24 (17.1%) were taking lipid-lowering medication (milk, n ¼ 12; control, n ¼ 12). Twelve men were taking both antihypertensive and lipidlowering medication. There was a kg nonsignificant increase in weight in the milk group after 12 and 24 months, but there were no between-group differences for the change in weight, physical activity or total energy intake throughout the study (Tables 2 and 3). Dietary calcium and potassium intake increased in 995

4 996 Table 2 Mean daily dietary intakes of participants in the milk supplementation and control group throughout the study (by 3-day food diary) Dietary parameters Baseline 12 months 24 months Milk (n ¼ 73) Control (n ¼ 67) Milk (n ¼ 69) Control (n ¼ 62) Milk (n ¼ 66) Control (n ¼ 58) Energy intake (kj/day) 9171± ± ± ± ± ±1639 % energy carbohydrates 46.5± ± ± ± ± ±8.1 z % energy protein 18.5± ± ±2.8 z a 18.4± ±3.7 w 19.7±4.0* % energy fat 31.0± ± ± ± ±5.6 b 32.2±6.3 w Protein (g/day) 99±24 98±26 110±30 w c 100±25 108±25* 98±21 Total fat (g/day) 79±29 75±28 76±26 77±29 75±21 75±23 % energy saturated fat 12.6±3.7 d 11.2± ±2.7 z a 11.6± ±2.9 b 11.9±3.4 Alcohol intake (g/day) 15.4± ± ±15.3* 21.6± ±14.0* 17.5±16.5 Calcium (mg/day) 1030±417 d 889± ±413 z a 914± ±464 z a 870±309 Cholesterol (mg/day) 295±109 d 257± ± ± ± ±131 Potassium (mg/day) 3555± ± ±1102 z a 3486± ±999 z a 3384±857* Sodium (mg/day) 2701± ± ± ± ± ±1140 Magnesium (mg/day) 356± ±91 402±130 z 366± ± ±94 Values are the mean±s.d. *Pp0.05; w Po0.01; z Po0.001 within-group change from baseline. a Po0.001, b Po0.05, c Po0.01 represent the significance level for the group-by-time interaction at each time point. d Po0.05 vs control group. Alcohol intake represents the mean intake for men who reported consuming alcohol. the milk group and were greater than in the controls throughout the intervention (all Ps o0.001). Dietary protein (and the percentage of energy from protein) also increased in the milk group and was greater than in the control group at 12 months (Po0.01 and o0.001, respectively). The percentage of energy derived from fat increased in the control compared to milk group after 24 months (Po0.01). The change in the percentage of energy from saturated fat also differed between the two groups after 12 and 24 months (Po0.001 and o0.05, respectively). There were no betweengroup differences for any of the remaining dietary parameters. As reported previously (Daly et al., 2006b), there was a significant difference for the change in serum 25(OH)D in favor of the milk group after 12 months (Po0.001), which persisted after 24 months (Po0.001). Serum PTH concentrations decreased in the milk compared to control group after 12 months (Po0.01), and remained different after 24 months (Po0.05). Systolic and diastolic blood pressures decreased in the control relative to milk group after 6 months (both Ps o0.05), but thereafter there were no between-group differences and both blood pressure measures increased similarly in the two groups (Table 3). Similar results were observed in the 104 men who did not take antihypertensive medication throughout the study (data not shown). For all lipid parameters there were no between-group differences for the change relative to baseline in the entire cohort of men (n ¼ 140) (Table 3) or those not taking lipid-lowering medication (n ¼ 108). Similar results were also observed for both blood pressure and all lipid measures when the analyses were limited to those men with dietary calcium intakes below 1000 mg per day and/or hypovitaminosis D (25(OH)D o75 nmol/l) at baseline (data not shown). Discussion Our results indicate that daily consumption of reduced-fat milk fortified with approximately 1000 mg of calcium and 800 IU of vitamin D 3 for 2 years did not have a beneficial (nor detrimental) effect on blood pressure or lipid-lipoprotein concentrations in healthy community-dwelling older men. These findings are consistent with several short-term (Pan et al., 1993; Major et al., 2007) and long-term trials (Orwoll and Oviatt, 1990; Margolis et al., 2008) that reported that combined calcium-vitamin D supplementation did not lower blood pressure. To the best of our knowledge, our study is the longest trial to have assessed the blood pressure responses to additional calcium-vitamin D 3, using food (milk) as a carrier. Although the baseline dietary calcium intakes of the men in our study averaged 962 mg per day, which is similar to the current Australian RDI of 1000 mg per day for men aged years, 60% of the men had intakes below this level. It has been proposed that the bloodpressure-lowering effects of calcium supplementation alone tend to be greatest in those with the lowest dietary calcium intakes (o600 mg per day) before supplementation (Reid et al., 2005) and/or those who are hypertensive (Allender et al., 1996). Similarly, several meta-analyses have failed to detect a blood-pressure-lowering effect of calcium supplementation with intakes around mg per day in groups with a wide range of usual calcium intakes (Dickinson et al., 2006; van Mierlo et al., 2006). Furthermore, because only approximately 30% of the men in our study were classified as hypertensive at baseline, it is possible that the lack of an effect of calcium plus vitamin D 3 supplementation in our study, and in several previous trials (Orwoll and Oviatt, 1990; Pan et al., 1993; Major et al., 2007), was due to

5 Table 3 Mean baseline weight, 25(OH)D, PTH, blood pressure, lipid and lipoprotein concentrations in the milk supplementation and control group, and the mean absolute changes within each group relative to baseline after 6, 12, 18 and 24 months 997 Baseline D 6 months (baseline) D 12 months (baseline) D 18 months (baseline) D 24 months (baseline) Weight (kg) Milk 80.9± ( 0.5, 0.5) 0.5 (0.0, 1.1) 0.0 ( 0.6, 0.6) 0.6 ( 0.1, 1.4) Control 81.3± ( 0.7, 0.2) 0.1 ( 0.4, 0.7) 0.7 ( 1.3, 0.1)* 0.1 ( 0.6, 0.8) 25-Hydroxyvitamin D (nmol/l) Milk 78± ( 0.3, 9.5)* a 4.8 ( 0.2, 9.7)* a Control 76± ( 19.3, 8.7) z 14.4 ( 19.6, 9.1) z PTH (pg/ml) Milk 28± ( 6.1, 2.6) z b 1.8 ( 4.2, 0.6) c Control 30±9 0.1 ( 2.0, 1.7) 2.2 ( 0.2, 4.6) Systolic blood pressure (mm Hg) Milk 123.7± ( 1.4, 3.4) c 5.8 (3.6, 8.1) z 5.4 (2.8, 7.9) z 6.8 (4.2, 9.3) z Control 120.4± ( 6.2, 0.7)* 3.4 (0.6, 6.2)* 3.9 (1.2, 6.6) w 5.3 (2.4, 8.2) z Diastolic blood pressure (mm Hg) Milk 69.5± ( 2.8, 1.7) c 1.5 ( 0.6, 3.6) 4.0 (1.8, 6.2) z 4.2 (2.1, 6.2) z Control 71.0± ( 5.5, 1.6) z 0.2 ( 2.1, 2.5) 2.6 (0.8, 4.4) w 3.9 (2.0, 5.8) z Total cholesterol (mmol/l) Milk 5.21± ( 0.09, 0.20) 0.02 ( 0.19, 0.15) 0.02 ( 0.16, 0.21) 0.04 ( 0.23, 0.16) Control 5.28± ( 0.36, 0.01) 0.07 ( 0.25, 0.11) 0.11 ( 0.28, 0.07) 0.19 ( 0.39, 0.00) HDL cholesterol (mmol/l) Milk 1.42± ( 0.02, 0.07) 0.06 ( 0.11, 0.01)* 0.02 ( 0.08, 0.03) 0.06 ( 0.12, 0.01)* Control 1.42± ( 0.07, 0.05) 0.04 ( 0.09, 0.02) 0.02 ( 0.07, 0.04) 0.06 ( 0.11, 0.00) LDL cholesterol (mmol/l) Milk 3.19± ( 0.14, 0.11) 0.00 ( 0.13, 0.14) 0.03 ( 0.11, 0.18) 0.05 ( 0.20, 0.10) Control 3.23± ( 0.33, 0.06) w 0.06 ( 0.19, 0.08) 0.09 ( 0.22, 0.04) 0.13 ( 0.27, 0.01) Total/HDL cholesterol ratio Milk 3.81± ( 0.11, 0.05) 0.19 (0.09, 0.29) z 0.07 ( 0.01, 0.16) 0.15 (0.06, 0.23) w Control 3.87± ( 0.15, 0.02) 0.06 ( 0.03, 0.15) 0.03 ( 0.11, 0.06) 0.04 ( 0.07, 0.15) Triglycerides (mmol/l) Milk 1.34± ( 0.06, 0.24) 0.09 ( 0.03, 0.21) 0.03 ( 0.10, 0.15) 0.13 ( 0.06, 0.32) Control 1.39± ( 0.12, 0.27) 0.04 ( 0.05, 0.14) 0.00 ( 0.12, 0.12) 0.14 ( 0.12, 0.10) Values are mean±s.d. or mean changes from baseline (95% CI). *Po0.05; w Po0.01; z Po0.001 within-group change from baseline; a Po0.001, b Po0.01, c Po0.05 represent the significance level for the group-by-time interaction at each time point. the fact that the participants in our study had adequate calcium intakes (B1000 mg per day) and serum 25(OH)D concentrations (B75 nmol/l) and/or were normotensive before supplementation. There is some evidence to support the beneficial effects of combined calcium-vitamin D supplementation on blood pressure in subgroups of older adults. In elderly women with serum 25(OH)D concentrations o50 nmol/l and high SBP and DBP (B142 and 83 mm Hg, respectively), supplementation with 1200 mg per day calcium plus 800 IU per day vitamin D 3 resulted in a 9.3% reduction in SBP compared to supplementation with 1200 mg per day calcium alone (Pfeifer et al., 2001). The mechanisms by which calcium and/or vitamin D supplementation may lower blood pressure are uncertain, but may include suppression of renin formation by vitamin D, thereby acting as a negative endocrine regulator of the renin angiotensin system (Li et al., 2004), and/or their effects on reducing serum PTH and 1,25(OH) 2 D, which may reduce vascular smooth muscle tone and therefore peripheral vascular resistance (Zemel, 2001). In our study, we observed a significant but small reduction in serum PTH, which may be explained by the lower initial PTH concentrations in our cohort of men. Thus, it would appear that if calcium-vitamin D supplementation does have blood-pressure-lowering effects, the benefits are likely to limited to specific subsets of the population, which may include those with hypertension, low circulating 25(OH)D (or elevated 1,25(OH) 2 D) and/or elevated PTH concentrations. A unique feature of our study was that we used fortified milk to increase dietary intakes of calcium and vitamin D 3. Several studies have reported that increased consumption of

6 998 milk and other low-fat dairy products can reduce the risk of hypertension and lower blood pressure (Buonopane et al., 1992; Appel et al., 1997; Zemel et al., 2004; Alonso et al., 2005; Ruidavets et al., 2006), and may even lead to greater reductions than can be achieved by supplements alone (Griffith et al., 1999). However, there are no long-term controlled intervention studies that have confirmed this effect. It has been proposed that the range of components in dairy products (for example, potassium and magnesium) (Massey, 2001), or the interaction between different nutrients (Alonso et al., 2005; Jauhiainen and Korpela, 2007) or the small amounts of angiotensin-i-converting enzyme inhibitory peptides in milk (Jauhiainen and Korpela, 2007) may contribute to a blood-pressure-lowering effect. However, several controlled studies have failed to confirmed this finding (Lee et al., 2007; Engberink et al., 2008), which could be explained in part by differences in the amount of calcium and/or vitamin D present in milk. In our study, consumption of reduced-fat fortified milk led to a significant increase in dietary protein, potassium and magnesium, without a corresponding increase in total fat or the percentage of energy derived from saturated fat. Although saturated fat intakes remained marginally higher than the recommended maximum of 10% energy from saturated fat, there was no evidence of a blood-pressure-lowering effect of milk supplementation even after adjusting for dietary factors. Therefore, although our results contrast with several short-term intervention studies in both normotensive (Van Beresteijn et al., 1990; Buonopane et al., 1992; Hilary Green et al., 2000) and hypertensive individuals (Hilary Green et al., 2000), they are consistent with the results from a meta-analysis indicating that increasing dietary calcium has a negligible effect on lowering blood pressure (Dickinson et al., 2006). An unexpected finding in our study was the significant ( mm Hg) increase in SBP and DBP in both groups after 2 years. Although a rise in blood pressure appears to be an inevitable consequence of aging, the average age-related increase in SBP is reported to around mm Hg per year (Pearson et al., 1997; Izzo et al., 2000). Given that there were no marked changes in any of the well-known determinants of blood pressure in our study, we are unable to explain the relatively large increases in blood pressure in both groups, but importantly there was no difference between the supplemented and control groups. Previous intervention studies examining the effects of milk supplementation or increased dairy consumption on lipid concentrations have produced varying results, which are likely due to differences in the amount and fat composition of the milk (or dairy product) used, participant characteristics, study duration and/or whether the intervention was combined with weight loss or a low-fat diet (Buonopane et al., 1992; Steinmetz et al., 1994; Barr et al., 2000; Zemel et al., 2004). Our findings are consistent with a 12-week trial that found that advice to drink three glasses of skim or fat-reduced milk per day did not change any lipid parameter in older men and women (Barr et al., 2000). Whole milk contains a significant amount of saturated fat, which raises serum cholesterol and LDL cholesterol (Stone, 1990), but other components in milk, including possibly vitamin D, may moderate any adverse effects on serum lipids; and low-fat milk products contain less saturated fat than whole milk. There are also mixed results regarding the effects of calcium supplementation on lipid concentrations (Bostick et al., 2000; Reid et al., 2002). Although supplementary calcium phosphate has been found to increase bile acid excretion (Ditscheid et al., 2005) and an increase in fecal fat excretion was observed when calcium intake was increased by around 1200 mg per day (Jacobsen et al., 2005), this effect is unlikely to be replicated when dietary calcium is increased through the consumption of food products. There are several key limitations with our study. First, neither blood pressure nor the lipid parameters were our prespecified primary end points. Second, our cohort included predominantly well-nourished healthy men, with some taking medication for the treatment of hypertension or hyperlipidemia. Although similar results were observed when we excluded these men from the analysis, the findings from this study cannot be extrapolated to other groups. Furthermore, post hoc estimates of the sample size required to detect any potential effects of the fortified milk on blood pressure indicate that our study lacked statistical power to detect differences in blood pressure. The expected between-group differences in blood pressure and total cholesterol were estimated to be 3.5 mm Hg (s.d. 9.0 mm Hg) and 0.5 mmol/l (s.d. 1.0 mmol/l), respectively. Thus, to provide 80% power to detect this difference at the 5% significance level, a sample size of 105 and 64 per group would be required. In conclusion, consumption of 400 ml per day of reducedfat fortified milk containing approximately 1000 mg per day of calcium and 800 IU per day of vitamin D 3 for 2 years does not have a beneficial nor detrimental effect on blood pressure or lipid-lipoprotein concentrations in healthy community-living older men. Therefore, these findings do not support the role for increasing dietary calcium and vitamin D to these levels as a strategy to control blood pressure or an adverse lipid profile in healthy older men. Acknowledgements We thank Murray Goulburn Co-operative Co. Ltd for providing the calcium-vitamin D 3 fortified milk used in the study. We also thank Jeni Black, Joanne Daly and Sam Korn for their assistance with the clinical testing, and Sandra Godfrey and Nicole Fitzpatrick for performing the hormonal and biochemical analyses. Most importantly, we are indebted to the volunteers whose cooperation and dedication made this study possible. This study was financially supported by a grant from the Geoffrey Gardiner Dairy Foundation. Associate professor Robin Daly was supported by a National Health and Medical Research Council (NHMRC) Career Development Award (ID ).

7 Disclosure/Conflict of interest The authors have no conflicts of interest. References Al-Delaimy WK, Rimm E, Willett WC, Stampfer MJ, Hu FB (2003). A prospective study of calcium intake from diet and supplements and risk of ischemic heart disease among men. Am J Clin Nutr 77, Allender PS, Cutler JA, Follmann D, Cappuccio FP, Pryer J, Elliott P (1996). Dietary calcium and blood pressure: a meta-analysis of randomized clinical trials. Ann Intern Med 124, Alonso A, Beunza JJ, Delgado-Rodriguez M, Martinez JA, Martinez- Gonzalez MA (2005). Low-fat dairy consumption and reduced risk of hypertension: the Seguimiento Universidad de Navarra (SUN) cohort. Am J Clin Nutr 82, Appel LJ, Moore TJ, Obarzanek E, Vollmer WM, Svetkey LP, Sacks FM et al. (1997). A clinical trial of the effects of dietary patterns on blood pressure. DASH Collaborative Research Group. N Engl J Med 336, Barr SI, McCarron DA, Heaney RP, Dawson-Hughes B, Berga SL, Stern JS et al. (2000). 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Chin J Physiol 36, Pearson JD, Morrell CH, Brant LJ, Landis PK, Fleg JL (1997). Age-associated changes in blood pressure in a longitudinal study of healthy men and women. J Gerontol A Biol Sci Med Sci 52, M177 M183. Pfeifer M, Begerow B, Minne HW, Nachtigall D, Hansen C (2001). Effects of a short-term vitamin D(3) and calcium supplementation on blood pressure and parathyroid hormone levels in elderly women. J Clin Endocrinol Metab 86, Reid IR, Mason B, Horne A, Ames R, Clearwater J, Bava U et al. (2002). Effects of calcium supplementation on serum lipid concentrations in normal older women: a randomized controlled trial. Am J Med 112, Reid IR, Horne A, Mason B, Ames R, Bava U, Gamble GD (2005). Effects of calcium supplementation on body weight and blood pressure in normal older women: a randomized controlled trial. J Clin Endocrinol Metab 90, Ruidavets JB, Bongard V, Simon C, Dallongeville J, Ducimetiere P, Arveiler D et al. (2006). 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8 1000 Scragg R, Sowers M, Bell C (2007). Serum 25-hydroxyvitamin D, ethnicity, and blood pressure in the Third National Health and Nutrition Examination Survey. Am J Hypertens 20, Snijder MB, Lips P, Seidell JC, Visser M, Deeg DJ, Dekker JM et al. (2007). Vitamin D status and parathyroid hormone levels in relation to blood pressure: a population-based study in older men and women. J Intern Med 261, Steinmetz KA, Childs MT, Stimson C, Kushi LH, McGovern PG, Potter JD et al. (1994). Effect of consumption of whole milk and skim milk on blood lipid profiles in healthy men. Am J Clin Nutr 59, Stone NJ (1990). Diet, lipids, and coronary heart disease. Endocrinol Metab Clin North Am 19, Van Beresteijn EC, van Schaik M, Schaafsma G (1990). Milk: does it affect blood pressure? A controlled intervention study. J Intern Med 228, van Mierlo LA, Arends LR, Streppel MT, Zeegers MP, Kok FJ, Grobbee DE et al. (2006). Blood pressure response to calcium supplementation: a meta-analysis of randomized controlled trials. J Hum Hypertens 20, Zemel MB (2001). Calcium modulation of hypertension and obesity: mechanisms and implications. J Am Coll Nutr 20, 428S 435S; discussion 440S 442S. Zemel MB, Thompson W, Milstead A, Morris K, Campbell P (2004). Calcium and dairy acceleration of weight and fat loss during energy restriction in obese adults. Obes Res 12,

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