The effects of ezetimibe on non-alcoholic fatty liver disease and glucose metabolism: a randomised controlled trial

Aims/hypothesis

The cholesterol absorption inhibitor ezetimibe has been shown to ameliorate non-alcoholic fatty liver disease (NAFLD) pathology in a single-armed clinical study and in experimental animal models. In this study, we investigated the efficacy of ezetimibe on NAFLD pathology in an open-label randomised controlled clinical trial.

Methods

We had planned to enrol 80 patients in the trial, as we had estimated that, with this sample size, the study would have 90% power. The study intervention and enrolment were discontinued because of the higher proportion of adverse events (significant elevation in HbA_1c) in the ezetimibe group than in the control group. Thirty-two patients with NAFLD were enrolled and randomised (allocation by computer program). Ezetimibe (10 mg/day) was given to 17 patients with NAFLD for 6 months. The primary endpoint was change in serum aminotransferase level. Secondary outcomes were change in liver histology (12 control and 16 ezetimibe patients), insulin sensitivity including a hyperinsulinaemic–euglycaemic clamp study (ten control and 13 ezetimibe patients) and hepatic fatty acid composition (six control and nine ezetimibe patients). Hepatic gene expression profiling was completed in 15 patients using an Affymetrix gene chip. Patients and the physician in charge knew to which group the patient had been allocated, but people carrying out measurements or examinations were blinded to group.

Results

Serum total cholesterol was significantly decreased in the ezetimibe group. The fibrosis stage and ballooning score were also significantly improved with ezetimibe treatment. However, ezetimibe treatment significantly increased HbA_1c and was associated with a significant increase in hepatic long-chain fatty acids. Hepatic gene expression analysis showed coordinate downregulation of genes involved in skeletal muscle development and cell adhesion molecules in the ezetimibe treatment group, suggesting a suppression of stellate cell development into myofibroblasts. Genes involved in the l-carnitine pathway were coordinately downregulated by ezetimibe treatment and those in the steroid metabolism pathway upregulated, suggestive of impaired oxidation of long-chain fatty acids.

Conclusions/interpretation

Ezetimibe improved hepatic fibrosis but increased hepatic long-chain fatty acids and HbA_1c in patients with NAFLD. These findings shed light on previously unrecognised actions of ezetimibe that should be examined further in future studies. Trial registration University Hospital Medical Information Network (UMIN) Clinical Trials Registry UMIN000005250. Funding The study was funded by grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and research grants from MSD.     

Unconditioned and conditioned effects of intranasally administered insulin vs placebo in healthy men: a randomised controlled trial

Aims/hypothesis  

In humans, the intranasal route allows insulin to reach the brain while maintaining peripheral euglycaemia. Our aims were to examine acute (unconditioned) effects of central insulin on normal-range blood glucose and hormones in men, and to find out whether the effects of intranasal insulin can be learnt via classical conditioning.     

Low-dose glucocorticoid treatment affects multiple aspects of intermediary metabolism in healthy humans: a randomised controlled trial

Aim/hypothesis  

To assess whether low-dose glucocorticoid treatment induces adverse metabolic effects, as is evident for high glucocorticoid doses.     

HIV prevention with severely mentally ill men: a randomised controlled trial

We conducted a randomised clinical trial to test the efficacy of an enhanced version of an intervention previously shown to reduce HIV sexual risk behaviours among men with severe mental illness. One-hundred-and-forty-nine subjects aged 18-59 years were randomly assigned to the experimental or control conditions. Sexual risk behaviours were assessed every three months for 12-months. The primary analysis compared experimental and control groups with respect to sexual risk behaviours with casual partners as measured by the Vaginal Episodes Equivalent (VEE) score. Additional analyses included comparison of VEE scores of those men sexually active in the three months prior to baseline and the proportion of condom-protected sexual acts with casual partners. There were no significant differences in sexual risk behaviours with casual partners between experimental and control subjects. Additional analyses demonstrated a trend toward sexual risk reduction at six months post-intervention (p=0.06) but not at 12 months. These results may reflect a lack of efficacy or a true reduction in risk that the trial was underpowered to detect at the 0.05-level. If there was a true reduction in risk, it was not maintained after the initial six months.     

A randomised controlled trial of the effect of anticipation of a blood test on blood pressure

Blood pressure is affected by situational anxiety, such as the white coat effect. We hypothesised that blood pressure would also be affected by anticipation of a blood test. Volunteer subjects were recruited on the campus of Birmingham University. Subjects were randomly assigned to intervention and control groups. After a period of rest, three seated blood pressure measurements were taken at 1-min intervals using an electronic sphygmomanometer. Between the second and third measurements subjects in the intervention group were told that a blood test would be carried out after the last measurement. No blood test was carried out. Three blood pressure measurements were made in all 213 randomised subjects. Analysis was by intention-to-treat. In the control group mean systolic and diastolic blood pressure fell in successive measurements. Between the second and third measurements mean systolic blood pressure fell by 1.4 mm Hg in the control group and rose by 2.6 mm Hg in the intervention group (difference 4.0 mm Hg, P < 0.0001). A rise in diastolic blood pressure between the second and third measurements did not reach statistical significance. It was concluded that anticipation of a blood test affects measured systolic blood pressure in volunteers. The practice of taking blood tests at the same time as measuring blood pressure may potentially bias estimations of blood pressure.     

A randomised placebo controlled trial of the effects of tibolone on blood pressure and lipids in hypertensive women

The effects of hormone replacement therapy in hypertensive women are controversial. This randomised placebo controlled trial assessed the effect of tibolone 2.5 mg on blood pressure and fasting plasma lipids in 29 hypertensive postmenopausal women over 6 months using a 2:1 randomisation to tibolone. The primary clinical end-point was mean office blood pressure. At 6 months systolic blood pressure declined by 5.30 +/- 2.87% vs 4.94 +/- 3.37% whilst diastolic blood pressure declined 5.38 +/- 2.65% vs 0.85 +/- 3.69% on tibolone and placebo respectively. These differences were not statistically significant. Triglycerides decreased by 33.3 +/- 6.1% vs 7.6 +/- 7.9% (P < 0.01) and high-density lipoprotein (HDL)-cholesterol by 21.7 +/- 3.8% vs 2.4 +/- 2.6% (P < 0.01) with tibolone as opposed to placebo. No significant differences were observed in total cholesterol, low-density lipoprotein (LDL)-cholesterol and lipoprotein (a). Fibrinogen levels were reduced by 13.6 +/- 6.8% on tibolone compared to a 19.3 +/- 15.4% rise (P < 0.05) on placebo. This study suggests that tibolone has no deleterious effect on blood pressure in women with hypertension but has contrasting effects on biochemical risk factors. Large-scale studies are required to determine the overall effect of tibolone on cardiovascular morbidity and mortality.     

强化和标准血压控制对特定领域认知功能的影响:SPRINT随机对照试验的一项子研究

收缩压干预试验(systolic blood pressure intervention trial,SPRINT)结果显示,强化收缩压控制降低轻度认知功能障碍的发生率,但不降低痴呆的发生率。该研究在预先计划的SPRINT子研究参与者中探索强化降低收缩压对特定认知功能的影响。方法:SPRINT是一项开放标签、多中心、随机对照试验,在102个试验点进行,包括美国和波多黎各医学中心、退伍军人事务医学中心、医院和独立诊所。参与者为年龄≥50岁成年人,收缩压>130mm Hg(1mm Hg=0.133kPa),无糖尿病、脑卒中史或痴呆。参与者随机分配(1∶1)至收缩压控制目标在<120mm Hg(强化治疗)和<140mm Hg(标准治疗)的两组中。     

Effects of blood pressure lowering and intensive glucose control on the incidence and progression of retinopathy in patients with type 2 diabetes mellitus: a randomised controlled trial

Aims/hypothesis

The aim of the present study was to investigate the effect of blood pressure lowering and intensive glucose control on the incidence and progression of retinopathy in type 2 diabetic patients.

Methods

The Action in Diabetes and Vascular Disease: Preterax and Diamicron MR Controlled Evaluation (ADVANCE) Retinal Measurements study, a substudy of ADVANCE, is a randomised (using a central, computer-based procedure) controlled 2?×?2 factorial trial comprising a double-blind comparison of blood pressure lowering with perindopril–indapamide vs placebo, and an open comparison of standard vs intensive glucose control targeting a HbA_1c of?≤?6.5% in 1,602 diabetic patients from ADVANCE centres with access to retinal cameras conducted from 2001 to 2008. At baseline and the final visit, seven-field stereoscopic retinal photographs were taken and graded by blinded readers (gradeable baseline and final photographs from 1,241 patients). Progression of ≥2 steps in the Early Treatment of Diabetic Retinopathy Study classification (using the eye with worst grading) was the primary outcome.

Results

Retinopathy progressed in 59 (4.8%) patients and developed in 128 (10.3%) patients over 4.1 years. Fewer patients on blood pressure-lowering treatment (n?=?623) experienced incidence or progression of retinopathy compared with patients on placebo (n?=?618), but the difference was not significant (OR 0.78; 95% CI 0.57–1.06; p?=?0.12). Blood pressure-lowering treatment reduced the occurrence of macular oedema (OR 0.50; 95% CI 0.29–0.88; p?=?0.016) and arteriovenous nicking compared with placebo (OR 0.60; 95% CI 0.38–0.94; p?=?0.025). Compared with standard glucose control (n?=?611), intensive glucose control (n?=?630) did not reduce (p?=?0.27) the incidence and progression of retinopathy (OR 0.84; 95% CI 0.61–1.15). Lower, borderline significant risks of microaneurysms, hard exudates and macular oedema were observed with intensive glucose control, adjusted for baseline retinal haemorrhages. These effects of the two treatments were independent and additive. Adverse events in the ADVANCE study are reported elsewhere.

Conclusions/interpretation

Blood pressure lowering or intensive glucose control did not significantly reduce the incidence and progression of retinopathy, although consistent trends towards a benefit were observed, with significant reductions in some lesions observed with both interventions.

Trial registration:

ClinicalTrials.gov ID no. NCT00145925.

Funding:

Grants from Servier and the National Health and Medical Research Council of Australia     

Randomized controlled trial of sour milk on blood pressure in borderline hypertensive men

BACKGROUND: A double-blind randomized controlled trial was carried out to assess the effect of sour milk, containing two tripeptides (valine-proline-proline and isoleucine-proline-proline), on blood pressure (BP). METHODS: A total of 46 borderline hypertensive men aged 23 to 59 years were recruited at their workplace for this trial. Subjects were randomly allocated into two groups; sour milk drink group (S-group, n = 23) and placebo (acidified milk) drink group (P-group, n = 23) for 4 weeks. Blood pressure was measured twice at each occasion by a physician, at the health center of the company, with a mercury at baseline, 2 and 4 weeks. Statistical analysis was performed by SPSS 10.0J. RESULTS: The S-group and P-group showed no significant difference in baseline systolic BP (mean [SD], S: 147.6 [9.6], P: 145.3 [13.0]) or diastolic BP (S: 95.3 [9.9], P: 91.5 [9.6]). In the S-group, change in systolic BP at 2 and 4 weeks were -4.3 mm Hg (95% confidence interval [CI] -8.3 to -0.4; P = .032) and -5.2 mm Hg (95% CI -10.1 to -0.3; P = .039), both statistically significant. Diastolic BP showed change from -1.7 mm Hg (95% CI -5.4 to 2.0) at 2 weeks and -2.0 (95% CI -5.4 to 1.5) at 4 weeks, respectively. In the P-group, change in systolic BP were -0.5 (95% CI -5.8 to 4.8) at 2 weeks and -3.7 (95% CI -8.3 to 0.9) and change in diastolic BP were -0.6 (95% CI -4.7 to 3.6) and -0.3 (95% CI -3.9 to 3.3), which were not statistically significant. CONCLUSIONS: This trial demonstrated the beneficial effect of sour milk on BP in borderline hypertensive men who were not taking antihypertensive medication.     

Virus detection and its association with symptoms during influenza‐like illness in a sample of healthy adults enrolled in a randomised controlled vaccine trial

Background Viral respiratory infections are associated with significant morbidity and mortality. Many new aetiological agents have been described recently. Objectives We looked for respiratory viruses in a population‐based sample of healthy adults with influenza‐like illness (ILI). We investigated host and spatio‐temporal associations with virus isolation and host, spatio‐temporal and virus associations with self‐reported symptoms. Patients/Methods We recruited 586 participants experiencing 651 illness episodes from a population of healthy adults enrolled in an influenza vaccine effectiveness trial. At ILI assessment visits, a respiratory swab was collected and tested for viruses using a combination of polymerase chain reaction (PCR) assays. Participants also completed a questionnaire detailing their clinical course in 336 episodes. Results Of 643 samples analysed, a virus was identified in 44%. Half were picornaviruses, with influenza and coronaviruses the next most common. Individuals with influenza were significantly less likely to have been immunised than the reference (virus negative) population (OR = 0·52 (0·31, 0·87) P = 0·01). The mean symptom score (95% CI) reported by individuals with influenza was significantly higher than in all other episodes [Influenza: 10·2 (9·4, 10·9); Other: 7·4 (7·2, 7·7); Difference (95% CI): 2·5 (1·5, 3·5); P < 0·001]. In an analysis restricted to influenza‐positive cases, the symptom score was not attenuated by vaccination. Conclusions Our findings indicate that a greater number of symptoms are displayed by individuals presenting with influenza confirmed ILI compared with other agents that cause ILI. While influenza vaccination reduced the probability of influenza virus detection, symptom score for influenza‐positive ILI was not attenuated.     

Postprandial effects of potassium supplementation on vascular function and blood pressure: a randomised cross-over study

Background and aimsEndothelial dysfunction, as assessed by flow mediated dilatation (FMD) is an early event in atherosclerosis and an independent predictor of cardiovascular events. The effect of potassium supplementation on endothelial function and blood pressure (BP) in the postprandial state is not known. The aim of this study was to assess endothelial function using FMD in healthy volunteers.Methods and resultsThirty-two normotensive volunteers received a meal with 36 mmol potassium (High K) and a control 6 mmol potassium (Low K) meal on 2 separate occasions in a randomized order. FMD and BP were measured while participants were fasting and at 30, 60, 90 and 120 min after the meal. There was a postprandial decrease in FMD in both groups. FMD decreased overall less after the High K meal compared to the Low K meal (meal effect p < 0.05). Both meals produced a postprandial decrease in BP at 30 min which returned to baseline levels by 120 min. No significant differences in BP were observed between meals. FMD and systolic BP were negatively correlated at 90 (r = −0.54–0.55, p < 0.01) and 120 min (r = −0.42–0.56, p < 0.01) after both meals.ConclusionsA high potassium meal, which contains a similar amount of potassium as 2.5 serves of bananas, can lessen the postprandial reduction in brachial artery FMD when compared to a low potassium meal.     

A randomised,controlled trial of self-monitoring of blood glucose in patients with type 2 diabetes receiving conventional insulin treatment

Aims/hypothesis

We evaluated whether self-monitoring of blood glucose (SMBG) leads to better glycaemic control (HbA_1c) in patients with type 2 diabetes on conventional insulin regimens.

Methods

Patients with type 2 diabetes on a conventional insulin regimen (basal or premixed insulin with or without additional oral glucose-lowering agents) were recruited at study centres led by members of the German Diabetes Association. In a randomised, prospective, open 2?×?2 factorial design, the once-weekly performance of four-point glucose profiles (SMBG +; n?=?151 patients) was compared with no SMBG (SMBG ?; n?=?149), and the measuring and transmitting of HbA_1c results to the study centres (HbA_1c +; n?=?158, of these 82 SMBG ? and 76 SMBG +) was compared with HbA_1c measurement without disclosure of results (HbA_1c ?; n?=?142, of these 67 SMBG ? and 75 SMBG +). Randomised allocation was carried out by a central office, using sequentially numbered, sealed envelopes. The primary endpoint was the reduction of HbA_1c compared with baseline after 12 months. Secondary analyses were of therapy intensification in response to higher blood or urinary glucose or HbA_1c. Participants and caregivers were not blinded as to the allocation of interventions, whereas the laboratory determining HbA_1c remained blinded.

Results

Patient characteristics were balanced across groups. A total of 56 patients dropped out. In completers, HbA_1c was reduced in the SMBG + group from 7.3% to 7.0%, i.e. by 0.3% (0.1%, 0.5%) vs SMBG ? from 7.3% to 7.0% and 0.3% (0.2%, 0.5%), respectively, the difference being 0.0% (?0.2%, 0.2%) (p?=?0.93). The disclosure of HbA_1c results had no significant influence, with a difference of 0.1% (?0.1%, 0.4%) (p?=?0.28). Values above are mean (95% CI). The ORs for therapy intensification significantly rose as the following increased: proportions of urine samples testing positive for glucose, HbA_1c concentrations, and fasting or postprandial glucose concentrations. No important adverse events were associated with the interventions.

Conclusions/interpretation

SMBG profiles once weekly or the disclosure of HbA_1c results did not improve glycaemic control in patients with type 2 diabetes on conventional insulin treatment, although indicators of hyperglycaemia increased the likelihood of therapy intensification. Greater intensification may be necessary to impact on glycaemic control.

Trial registration:

www.clinicaltrials.gov (registration code NCT00688363)

Funding:

Deutsche Diabetes-Gesellschaft, Deutsche Diabetes-Stiftung, Bayer Vital GmbH     

Cutaneous microvascular effects of mid-term hormone replacement therapy in healthy postmenopausal women: a prospective placebo controlled trial

OBJECTIVE: To study the mid-term effects of Hormone Replacement Therapy (HRT) on cutaneous microcirculatory blood flow and reactivity in healthy postmenopausal women. DESIGN: In a double-blind placebo controlled randomized study, 16 healthy postmenopausal women received either placebo or HRT (micronized estradiol: 1 mg/day, day 1-28, promegestrone: 0.25 mg/day, day 14-28). This regimen was completed 6 times. Cutaneous microcirculatory blood flow was recorded by laser-Doppler velocimetry on the foot dorsum, in the supine and then dependent positions, and after post-ischemic hyperemia. RESULTS: At day 0, the two groups were similar and none of the following data differed significantly between treated and placebo group: (supine flux: 11.8 +/- 1.8 u vs. 13.2 +/- 3.9, venoarteriolar reflex: 5.6 +/- 1.3 vs. 6 +/- 3.3, and post-ischemic hyperemia: 35.2 +/- 3.9 vs.48.3+/-11). At the end of the study (day 26-28 of 6th cycle), the supine flux was 9.8 +/- 2.1 in the HRT group vs.12.9 +/- 6 in the placebo group (NS), the venoarteriolar reflex, 1.2 +/- 2 vs. 7+/-1.7 (p=0.04), and post ischemic hyperemia, 31.8 +/- 5.4 vs. 39.5 +/- 4.6 (NS). Intragroup values did not change significantly for any of the microcirculatory parameters measured, which remained stable throughout the 6 months of the study. Intergroup values for these parameters did not change either, except for the venoarteriolar reflex, which was lower at the end of the study in the HRT (EP period, cycle 6 day 26-28) than placebo group (p=0.04). CONCLUSIONS: HRT does not impair the resting supine cutaneous microcirculation blood flow or post-ischemic hyperemia.     

Folic acid enhances endothelial function and reduces blood pressure in smokers: a randomized controlled trial

OBJECTIVE: Cigarette smoking is associated with increased plasma homocysteine concentrations, endothelial dysfunction and arterial stiffening. Homocysteine per se induces endothelial dysfunction and arterial stiffening and might account, at least partly, for the vascular abnormalities observed in smokers. We sought to determine whether folic acid supplementation, by reducing plasma homocysteine concentrations, enhanced endothelial function and reduced arterial stiffness in smokers. DESIGN: Double-blind, randomized controlled, parallel-group, trial. SETTING: Academic medical centre. SUBJECTS: A consecutive sample of 24 healthy cigarette smokers (age 37.8 +/- 2.5 years, mean +/- SEM). INTERVENTION: Subjects were randomly assigned to 4-week folic acid 5 mg day-1 or placebo. MAIN OUTCOME MEASURES: The following were measured before and after treatment: (i) peripheral vasoreactivity (forearm arterial blood flow, FABF) during intra-arterial administration of endothelium-dependent (acetylcholine 1.5, 4.5 and 15 microg min-1) and endothelium-independent (sodium nitroprusside 1, 2 and 4 microg min-1) vasodilators; (ii) carotid-femoral pulse-wave velocity (PWV); (iii) blood pressure (BP). RESULTS: Folic acid reduced homocysteine concentrations (10.8 +/- 0.6 vs. 8.2 +/- 0.5 micromol L-1, P < 0.001) and enhanced endothelium-dependent vasodilatation during each acetylcholine infusion rate (ratio between the FABF in the infused and control arm during increasing infusion rates at baseline 1.09 +/- 0.03 vs. 1.41 +/- 0.09 after treatment, P < 0.01; 1.39 +/- 0.07 vs. 1.83 +/- 0.12, P < 0.01; 1.65 +/- 0.16 vs. 2.72 +/- 0.36, P < 0.05) whilst endothelium-independent vasodilatation was unaffected. A significant fall in BP was also observed (mean BP 88 +/- 2 vs. 83 +/- 1 mmHg, P < 0.01). By contrast, PWV did not significantly change (8.4 +/- 0.3 vs. 7.8 +/- 0.4 m s-1). No significant changes in plasma homocysteine concentrations, FABF, BP, and PWV were observed in the placebo group. A multiple regression analysis showed that changes in folic acid plasma concentrations independently predicted both FABF changes during maximal acetylcholine-mediated vasodilatation (P < 0.01) and BP changes (P = 0.01). CONCLUSIONS: Short-term folic acid supplementation significantly enhanced endothelial function and reduced BP in young chronic smokers. These effects were largely independent from the homocysteine lowering effects. Thus, a simple, nontoxic, and relatively inexpensive vitamin intervention might be useful in primary cardiovascular prevention in this high-risk group.     

Effects of coffee on ambulatory blood pressure in older men and women: A randomized controlled trial

This study assessed the effects of regular coffee drinking on 24-hour ambulatory blood pressure (ABP) in normotensive and hypertensive older men and women. Twenty-two normotensive and 26 hypertensive, nonsmoking men and women, with a mean age of 72.1 years (range, 54 to 89 years), took part in the study. After 2 weeks of a caffeine-free diet, subjects were randomized to continue with the caffeine-free diet and abstain from caffeine-containing drinks or drink instant coffee (5 cups per day, equivalent to 300 mg caffeine per day) in addition to the caffeine-free diet for a further 2 weeks. Change in systolic and diastolic blood pressures (SBP, DBP) determined by 24-hour ambulatory BP monitoring showed significant interactions between coffee drinking and hypertension status. In the hypertensive group, rise in mean 24-hour SBP was greater by 4.8 (SEM, 1.3) mm Hg (P=0.031) and increase in mean 24-hour DBP was higher by 3.0 (1.0) mm Hg (P=0.010) in coffee drinkers than in abstainers. There were no significant differences between abstainers and coffee drinkers in the normotensive group for 24-hour, daytime, or nighttime SBP or DBP. In older men and women with treated or untreated hypertension, ABP increased in coffee drinkers and decreased in abstainers. Restriction of coffee intake may be beneficial in older hypertensive individuals.     

Low ambulatory blood pressure is associated with lower cognitive function in healthy elderly men

INTRODUCTION: Low blood pressure (BP) has been found to be associated with cerebrovascular damage in the elderly. Studies of the relation of ambulatory BP to cognitive function in elderly persons aged 80 years or above is lacking, however. METHODS: Ninety-seven 81-year-old men from the population study 'Men born in 1914' underwent ambulatory BP monitoring and were given a cognitive test battery, 79 subjects completing all six tests. Low ambulatory systolic blood pressure (SBP) was defined as <130 mmHg and low ambulatory diastolic blood pressure (DBP) as <80 mmHg (corresponding in terms of office BP to approximately <140 and <90 mmHg, respectively). Odds ratios (OR) for lower cognitive function were calculated using a forward stepwise logistic regression model, controlling for confounding factors. RESULTS: Subjects with ambulatory SBP <130 mmHg had higher OR values for daytime (OR 2.6; P=0.037), nighttime (OR 3.6; P=0.032) and 24h (OR 2.6; P=0.038) BP measurements. A lower cognitive function was associated with lower nighttime SBP and DBP levels and lower 24-h mean SBP compared to subjects with higher cognitive function. OR values connected to low nocturnal SBP, had a tendency to be particularly high among subjects on anti-hypertensive drugs (OR 9.1; P=0.067, n.s.). CONCLUSION: Ambulatory SBP levels <130 mmHg and lower nighttime SBP and DBP were associated with lower cognitive function in healthy elderly men. Further investigation is needed to ascertain the effects of the presently recommended treatment goal of <140 mmHg for office SBP also on elderly over 80 years of age.