Effect of vitamins and aspirin on markers of platelet activation, oxidative stress and homocysteine in people at high risk of dementia

Authors

  • VITAL Trial Collaborative Group

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      Writing committee: Robert Clarke, Georgina Harrison and Sue Richards.
      Steering Committee: Marc Budge, Robert Clarke, John Grimley Evans, Georgina Harrison, Judy Haworth, Robin Jacoby, Elizabeth King, Sue Richards, A David Smith Gordon Wilcock.
      Coordinating Centre:Clinical Trial Service Unit, Radcliffe Infirmary, Oxford, UK:
      Robert Clarke, Georgina Harrison, Sue Richards, Savita Shah, Alan Young.
      Clinical Centres:Oxford Project to Investigate Memory and Ageing, Radcliffe Infirmary, Oxford, UK: Marc Budge, Elizabeth King, Margrit Campbell, Judith Rue, Donald Warden, Clare Bateman, Robin Jacoby, A David Smith.
      Department of Care of the Elderly, Frenchay Hospital, Bristol, UK: Judy Haworth, Emma Lander, Jeffrey Lennon, Julia Boot, and Gordon Wilcock.
      Laboratories:Department of Clinical Pharmacology, Royal College of Surgeons in Ireland, Dublin, Ireland: Desmond Fitzgerald, Brendan Hahren, Orina Belton, Aine ni Ainle. Department of Biochemistry, Trinity College, Dublin, Ireland: John Scott, Joseph McPartlin, Department of Clinical Biochemistry, AKH Aarhus University Hospital, Aarhus, Denmark: Ebba Nexo, Frode Engbaek. University Department of Pharmacology, Oxford, UK: Carole Johnston.


Dr Robert Clarke, Clinical Trial Service Unit, Radcliffe Infirmary, Oxford OX26HE, UK (fax: +44 (0) 1865 558817; e-mail: robert.clarke@ctsu.ox.ac.uk).

Abstract

Abstract. VITAL Trial Collaborative Group. Effect of vitamins and aspirin on markers of platelet activation, oxidative stress and homocysteine in people at high risk of dementia. J Intern Med 2003; 254:–.

Objectives. To examine the association of cognitive impairment with platelet activation and reactive oxygen species and total homocysteine levels; and to assess the biochemical efficacy of treatment with aspirin and vitamin supplements in people at high risk of dementia.

Subjects. People with dementia or mild cognitive impairment.

Design and intervention. In a 2 × 2 × 2 factorial design trial, 149 people at high-risk of dementia were randomized to receive either low-dose aspirin (81 mg) or placebo; and folic acid (2 mg) plus vitamin B12 (1 mg) or placebo; and vitamins E (500 mg) plus C (200 mg) or placebo. Participants were seen twice before and once after 12 weeks of treatment.

Main outcome measures. At each visit, participants had their cognitive function assessed and had blood collected for homocysteine, folate and vitamin B12 determination and urine collected for markers of platelet activation (11-dehydro-thromboxane B2) and reactive oxygen species (8-epi-PGF).

Results. Prior to treatment, cognitive function was inversely related with homocysteine and with urinary thromboxane and isoprostane, and these associations were independent of age. Aspirin was associated with a median reduction in 11-dehydrothromboxane B2 of 73% (P < 0.001). B-vitamins lowered plasma homocysteine concentration by 30% (P < 0.0001) and antioxidant vitamins lowered isoprostane excretion by 26% (P < 0.1). No effect of treatment on cognitive function was detected.

Conclusions. Aspirin and B-vitamins were effective in reducing biochemical factors associated with cognitive impairment in people at risk of dementia. Large-scale trials are now required to assess the relevance of aspirin and B-vitamins for the maintenance of cognitive function in people at risk of dementia.

Introduction

Atherosclerosis is associated with an increased risk of both Alzheimer's disease and vascular dementia [1]. Vascular pathology is frequently found in the brains of patients with Alzheimer's disease [2] and the combination of Alzheimer and vascular pathology is associated with a greater severity of cognitive impairment [3, 4]. These observations have prompted the need for trials of cardiovascular risk factor modification for Alzheimer's disease [5]. Amongst the possible candidate treatments, aspirin may be relevant for the maintenance of cognitive function in old age as it has been shown to reduce the risk of major vascular events by about one-quarter in people at high risk of vascular disease [6]. Elevated blood total homocysteine levels have also been associated with an increased risk of vascular disease, cognitive impairment and dementia [5–10]. Homocysteine levels are easily lowered by folic acid and vitamin B12 [11], but whether supplementation with B-vitamins can reduce the risk of vascular disease is still uncertain [12]. Alzheimer's disease may involve an accumulation of reactive oxygen species leading to lipid peroxidation and neuronal degeneration in the brain [13–15]. Observational studies have shown that dietary intake of antioxidants [16] or the use of anti-inflammatory drugs is associated with a reduced risk of Alzheimer's disease [17].

The aims of the present study were to examine the associations of severity of cognitive impairment with urinary markers of platelet activation (11-dehydro-thromboxane B2), reactive oxygen species (the isoprostane 8-epi-PGF) and with plasma total homocysteine concentrations in people at risk of dementia. The study assessed the biochemical efficacy on relevant biomarkers of low dose aspirin (81 mg) or placebo; and homocysteine lowering vitamins [folic acid (2 mg) plus vitamin B12 (1 mg)] or placebo; and antioxidant vitamins [vitamin E (500 mg) plus C (200 mg)] or placebo.

Patients and methods

Participants

People were eligible for the trial if they had either (i) clinical diagnosis of dementia and a Mini-mental State Examination (MMSE) score between 12 and 26 inclusive (of a maximum of 30) [18]; or (ii) mild cognitive impairment defined by symptoms of memory problems and a modified Telephone Interview of Cognitive Status (TICS-M) score below 27 of a maximum of 39 [19]. The clinical diagnosis of dementia made using DSM IV criteria [20]. Patients with a diagnosis of fronto-temporal dementia, Parkinson's disease, Huntington's disease, or normal pressure hydrocephalus were excluded. Individuals were eligible if they were not taking any of the study treatments and had no contraindications to them. Use of multivitamins containing less than 200 μg daily of folic acid or less than 100 mg daily of vitamin E was permitted. Drug therapy known to affect cognitive function (such as Donepezil, Metrifonate and Rivastigmine) was permitted provided it had been started at least 3 months before trial entry. Individuals with aspirin sensitivity or peptic ulcer, life threatening disease or cancer were excluded, as were those with concern about likely compliance or who were resident in a nursing home.

Recruitment

Medical collaborators appointed nurses or psychologists to run the study clinics and obtained local ethics committee approval. With the permission of their relevant consultant colleagues, records of hospital records were used to identify potentially eligible patients. Recruitment also involved screening general practice registers. As it proved difficult to identify potentially eligible people through hospital or general practice registers, a new recruitment procedure was implemented. People with mild cognitive impairment were sought by advertisements in the local newspapers and radio inviting those who believed that they had memory problems and were interested in participation in a trial of vitamins and aspirin, to contact the trial office by telephone. Respondents to this advertisement had their cognitive function assessed by telephone using the Telephone Interview for Cognitive Status (TICS-M) questionnaire [19], and individuals with a TICS-M score below 27 were invited to attend a screening visit.

Interventions

Eligible people who provided written informed consent were enrolled on a 4-week placebo-controlled run-in period prior to randomization. Using a 2 × 2 × 2 factorial design, patients who had proven compliant during the run-in were randomized to receive 12 weeks of treatment with either low-dose aspirin (81 mg) or placebo; and homocysteine lowering vitamins [folic acid (2 mg) plus vitamin B12 (1 mg)] or placebo; and antioxidant vitamins [vitamin E (500 mg) plus C (200 mg)] or placebo. All the medication was calendar packed in blister packs that were labelled for each day of the week using identical blister packs for each participant to ensure double-blinding of treatment allocation. A telephone randomization system used a minimization algorithm to ensure balance with respect to eligibility criteria and other prognostic factors (age, sex, smoking status and MMSE score). All staff involved in the trial were blinded to treatment allocation throughout the trial. The sample size was chosen to have adequate power be able to detect a plausible difference in the biochemical end-points in response to treatment.

Data collected at screening and follow-up visits

At the screening visit, participants were asked for details of their medical history and had their medication recorded. Blood pressure was recorded after 5 min in the seated position and height and weight were also measured. Participants had blood collected into a vacutainer containing EDTA and centrifuged within 30 min of collection and plasma aliquots stored at −70°C. Urine samples were collected into an empty container and stored at −80°C. All participants had their cognitive function assessed using the MMSE and the cognitive part of the Alzheimer's Disease Assessment Scale (ADAS-Cog) (total score: 0–70, with a high score indicating greater impairment). Activities of daily living (ADL) were recorded using the Bristol Activities of Daily Living Scale [21] at randomization and follow-up visits only.

Laboratory methods

Homocysteine levels were measured using a fluorescence polarization immunoassay (Abbott Diagnostics, Chicago, IL, USA) [22]. Vitamin B12 levels were determined by a competitive protein binding immunoassay (Bayer A/S, Terrytown, New York, USA)[23]. Plasma folate levels were determined by a microbiological method [24]. Urine samples were analysed for 11-dehydro-thromboxane B2[25] and the isoprostane 8-epi-PGF-levels [26] using liquid chromatography, tandem mass spectrometry (LC-tripleQ-MS, Applied Biosystems, Foster City, CA, USA). The results of 11-dehydro-thromboxane B2 and the isoprostane 8-epi-PGF are standardized for creatinine concentration in urine as μg mg−1 creatinine (μg mg−1 Cr).

Statistical methods

The median and interquartile range were presented at screening and randomization and the agreement between these was assessed by a Spearman correlation coefficient. The average of the two pretreatment measurements for each individual is defined as the ‘baseline’ value. Spearman correlation coefficients were used to assess the relationships between the baseline parameters and repeated with adjustment for age to allow for possible confounding. The Mann–Whitney test was used to compare the baseline biochemical or cognitive function scores with diagnosis of dementia by treatment allocation. The Mann–Whitney test was also used to compare post-treatment values of the active and placebo treated groups to assess the efficacy of treatment. In addition, the ratio of the post-treatment values to the baseline values of the biochemical outcomes were calculated to assess the proportional reduction with treatment, and the differences between treated groups were assessed by the Mann–Whitney test.

Results

Participant enrolment

Amongst the 1684 people available on local registers, most were not eligible, because they were already taking aspirin, or had an MMSE score outside the range or were resident in a nursing home (Fig. 1). A total of 112 (7%) of the 1684 people attended a screening visit. Following advertisement in the two centres, 345 people telephoned the local clinics to volunteer to participate, and 62 (18%) attended a screening visit. Amongst the 190 participants invited to attend a screening visit, 174 did so, and 170 agreed to participate. A total of 149 were randomized between November 1999 and March 2001 and 142 of these individuals attended for a follow-up visit.

Figure 1.

A flow diagram of the recruitment procedures.

Characteristics of included participants

Table 1 shows the distributions of age, sex and clinical diagnosis, cognitive scores and selected clinical and biochemical characteristics at screening and at randomization. Amongst the 149 included patients, 84 had a clinical diagnosis of Alzheimer's disease, 11 had both Alzheimer's and vascular dementia, three had vascular dementia, 47 had cognitive impairment and the diagnosis was not provided for four participants. The within-person correlation coefficients were high for cognitive function, plasma biochemical markers and somewhat lower for urinary markers. At baseline, MMSE and ADAS-Cog scores were highly and inversely correlated (−0.88). ADL score was positively associated with ADAS-Cog (0.71) and inversely correlated with MMSE (−0.71), indicating good agreement between the two measures of cognitive function and ADL.

Table 1.  Selected characteristics of included patients (n = 149)
 Values at screening visitValues at randomization visitCorrelation coefficientb
MedianInter-quartile rangeaRange of absolute valuesMedianInter-quartile rangeaRange of absolute values
  • a

    Interquartile range is the difference between the 75th and 25th percentile.

  • b

    Spearman correlation coefficients between screening and randomization values, P < 0.0001 in each case.

Clinical variables
 Age (years)75(10)56–89 
 Systolic blood pressure (mmHg)146(28)100–225 
 Diastolic blood pressure (mmHg)85(11)58–120 
 Height (cm)166(12)147–192 
 Weight (kg)72(19)40–118 
 BMI (kg m−2)25(6)17–42 
Cognitive function scores
 MMSE (Max 30)21(7)10–2621(7)12–290.86
 ADAS-Cog (Max 0)28(16)5–5527(17)4–540.91
 ADL (Max 0)8(13)0–37 
Biochemical variables
 Homocysteine (μmol L−1)12.3(4.9)5.7–130.212.4(5.9)5.0–115.60.90
 Folate (nmol L−1)7.1(7.5)1.7–30.57.9(6.4)1.6–30.90.85
 Vitamin B12 (pmol L−1)294(133)140–29 327290(136)106–21210.89
 Isoprostane (μg mg−1 Cr)515(418)95–1977589(435)131–13 3820.60
 Thromboxane (μg mg−1 Cr)922(743)23–3236846(750)109–30790.65

Baseline plasma total homocysteine level was inversely correlated with folate (−0.49) and with vitamin B12 (−0.23) and positively correlated with thromboxane (0.22), but was unrelated with isoprostane levels (0.07). Isoprostane were also associated with thromboxane levels (0.35). The relationship between homocysteine and thromboxane levels was no longer significant after adjustment for age (correlation coefficient 0.12), but all the other relationships between biochemical variables were independent of age.

Relationship of biochemical outcomes with cognitive function

Table 2 shows the distribution of characteristics at baseline by groups defined by age (less than 75 years or 75 years or greater) and severity of cognitive impairment. These analyses show significant associations of biochemical parameters with both severity of cognitive impairment and with age. Table 3 shows the associations of cognitive impairment with homocysteine, isoprostane and thromboxane (albeit not for MMSE with isoprostane) persisted after adjustment for age. These data indicate highly significant associations of cognitive function with thromboxane; compared with more moderate associations with homocysteine and with isoprostane.

Table 2.  Median levels (and interquartile range) of baseline characteristics by age and severity of cognitive impairment
 nAge (years)MMSE (Max 30)ADAS-Cog (Max 0)Homocysteine (μmol L−1)Folate (nmol L−1)B12 (pmol L−1)Isoprostane (μg mg−1 Cr)Thromboxane (μg mg−1 Cr)
  1. Although data are presented in groups, P-values are from Mann–Whitney U-test (diagnosis) and Spearman correlation (age and MMSE).

  2. The diagnosis include Alzheimer's disease (Alz), vascular dementia (vasc), the combination (mixed), mild cognitive impairment (MCI), other dementia (other), and if not recorded (unknown).

Overall14975 (10)21 (6)27 (917)12.3 (5.3)7.7 (6.9)291 (128)564 (396)880 (636)
Age (years)
 <757270 (6)22 (6)24 (17)11.4 (4.5)8.1 (6.3)284 (105)537 (302)796 (546)
  ≥757780 (5)21 (7)29 (14)13.5 (6.7)7.3 (6.6)301 (178)622 (416)1081 (908)
 (P for correlation) P  = 0.0003P  < 0.0001P  = 0.0008P  = 0.4P  = 0.07P  = 0.1P  < 0.0001
MMSE
 <162078 (12)15 (1)39 (11)12.7 (6.2)5.6 (6.1)295 (125)532 (374)943 (445)
 16–193574 (10)18 (3)36 (11)14.6 (7.8)6.9 (6.0)299 (137)583 (402)1063 (1237)
 20–246776 (9)22 (3)24 (11)12.5 (4.3)7.7 (6.3)289 (130)612 (441)880 (648)
  ≥252770 (11)26 (2)13 (8)10.4 (5.4)9.9 (10.7)284 (144)478 (253)687 (592)
 (P for correlation) P  = 0.0003P  < 0.0001P  = 0.006P  = 0.05P  = 0.2P  = 0.04P  = 0.0006
ADAS-Cog
  ≥353778 (10)16 (3)40 (9)15.1 (6.9)5.4 (5.9)311 (142)622 (354)1085 (785)
 28–343074 (12)19 (4)32 (3)12.7 (5.9)7.7 (5.5)289 (116)594 (579)945 (870)
 20–274176 (8)22 (3)24 (4)11.4 (4.4)8.2 (7.3)280 (157)558 (345)920 (667)
 <204171 (10)25 (2)15 (7)12.1 (4.6)8.9 (7.1)294 (129)484 (282)702 (541)
 (P for correlation) P  < 0.0001P  < 0.0001P  = 0.002P  = 0.008P  = 0.2P  = 0.01P  = 0.0003
Diagnosis
 Alz, Vasc or mixed9876 (9)20 (6)31 (12)12.7 (6.2)7.3 (7.0)290 (143)601 (449)1018 (825)
 MCI, other, unknown5172 (13)25 (4)16 (13)11.4 (4.8)8.7 (7.1)292 (109)518 (249)795 (510)
 (P for difference) P  = 0.003P  < 0.0001P  < 0.0001P  = 0.01P  = 0.2P  = 0.7P  = 0.08P  = 0.005
Table 3.  The relationships between baseline measurements of cognitive and biochemical variables before and after adjustment for age
 Unadjusted Spearman correlation coefficientsAge adjusted Spearman correlation coefficients
MMSEADAS-CogADLMMSEADAS-CogADL
  1. Baseline variables are the average of the screening and randomization values, except for ADL which was measured at randomization only. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Homocysteine −0.23**0.25**0.26** −0.17*0.19*0.18*
Folate0.16* −0.22** −0.20*0.15 −  0.20* −0.19*
B12 −0.100.100.19* −0.050.040.14
Isoprostane −0.17*0.20**0.14 −0.150.20*0.10
Thromboxane −0.28***0.29***0.35**** −0.22**0.23**0.25**

Effect of treatment on biochemical outcomes

Seven participants were lost to follow-up and the post-treatment biochemical data was incomplete on five people, so complete data on all biochemical outcomes was obtained for 137 people. Table 4 shows the median baseline and post-treatment concentrations of thromboxane, homocysteine, folate, vitamin B12 and isoprostane by treatment allocation. Figure 2 shows the percentage reduction of the post-treatment values compared with the baseline values that were calculated for thromboxane, homocysteine and isoprostane levels, respectively. Thromboxane was reduced by a median of 73% in the aspirin arm compared with only 18% in the placebo arm (P < 0.001). Aspirin had no significant effect on plasma total homocysteine or on urinary isoprostane levels. Folic acid plus vitamin B12 treatment was associated with a median reduction of 30% in homocysteine concentration compared with a 4% increase amongst those allocated to the placebo (P < 0.001). B-vitamins had no effect on thromboxane or isoprostane levels overall and in individuals who were allocated placebo rather than aspirin. Vitamin E plus C was associated with a nonsignificant median reduction in isoprostane levels of 26% compared with a 12% reduction in the placebo arm (P = 0.06), and had no significant effect in thromboxane or homocysteine levels overall or in the subset who were taking placebo rather than aspirin.

Table 4.  Median (and interquartile range) levels of biochemical variables before and after treatment
 Median (interquartile range) in those with data at both baseline and follow-up
Allocated aspirin (n = 74)Allocated placebo (n = 75)Allocated B12/folate (n = 74)Allocated placebo (n = 75)Allocated vitamin C/E (n = 75)Allocated placebo (n = 74)
  1. Mann–Whitney U-test. *P < 0.05, ***P < 0.001. Statistical significance assesses difference between treatment and placebo.

Homocysteine (μmol L−1)
 No. with data at both    baseline and follow-up  69  71  71  69  70  70
 Baseline  12.7 (5.6)  12.1 (5.3)  13.0 (6.0)*  11.7 (4.9)  12.6 (5.8)  12.2 (4.9)
 Follow-up  10.5 (3.9)  10.0 (3.7)    9.2 (2.3)0***  12.1 (5.3)  10.4 (4.9)  10.0 (3.5)
Folate (nmol L−1)
 No. with data at both    baseline and follow-up  69  71  71  69  70  70
 Baseline    8.2 (7.7)    7.3 (5.8)    7.1 (5.8)    8.3 (7.0)    7.7 (7.2)    7.2 (5.5)
 Follow-up  21.3 (48.6)  21.2 (53.2)  57.2 (59.4)***    7.7 (6.6)***  17.9 (62.9)  24.1 (41.7)
Vitamin B12 (pmol L−1)
 No. with data at both    baseline and follow-up  69  71  71  69  70  70
 Baseline284 (128)294 (126)284 (121)292 (130)296 (151)287 (97)
 Follow-up456 (456)479 (370)663 (336)***296 (125)***472 (524)468 (347)
Isoprostane (μg mg−1 Cr)
 No. with data at both    baseline and follow-up  68  70  70  68  69  69
 Baseline574 (402)558 (421)561 (359)566 (448)558 (403)571 (412)
 Follow-up502 (447)509 (336)484 (461)517 (331)495 (429)517 (347)
Thromboxane (μg mg−1 Cr)
 No. with data at both    baseline and follow-up  67  70  69  68  69  68
 Baseline851 (734)857 (484)789 (512)943 (720)913 (543)812 (658)
 Follow-up251 (231)***742 (782)402 (548)398 (563)432 (625)391 (536)
Figure 2.

Effects of treatment with aspirin or placebo, B-vitamins (folic acid plus vitamin B12) or placebo and vitamin C plus E or placebo on urinary thromboxane, plasma total homocysteine and urinary isoprostane. The data shown is the median (and 75th percentile) percentage reduction between baseline and follow-up for each individual by placebo (open bars) and active treatment (solid bars).

Effect of treatment on cognitive and other outcomes

Complete data on cognitive function before and after treatment was obtained from 128 people. Neither of the cognitive test scores, or ADL were found to be significantly altered by treatment (data not shown).

Discussion

This study demonstrated that cognitive impairment and impairment of ADL were significantly associated with platelet activation, elevated plasma total homocysteine and increased production of reactive oxygen species. The relationships of cognitive impairment with the biochemical markers were independent of age. Whilst platelet activation is closely correlated with elevated plasma total homocysteine, this association was attenuated after adjustment for age. Aspirin was associated with a 73% suppression in thromboxane biosynthesis in this population, which is consistent with its effect in healthy volunteers [27]. B-vitamins were associated with a 30% reduction in plasma homocysteine levels, which is consistent with the effects observed in other populations [11]. Antioxidant vitamins had a nonsignificant effect on isoprostane biosynthesis. None of the measures of cognitive function were materially altered by any of the treatments, which is to be expected given the size and duration of the pilot study.

Previous large-scale trials have shown beneficial effects on cognitive function of lowering blood pressure in people with cardiovascular disease [28, 29]. The Heart Protection Study failed to demonstrate any beneficial effects of lowering cholesterol [30] or of antioxidant vitamins [31] on cognitive function. The present pilot trial suggests that aspirin was the most promising of the treatments studied. Moreover, the pilot study demonstrated the efficacy of recruitment by self-referral and the feasibility of recruitment and monitoring the response to treatment in this population. Long-term trials involving a much larger number of people at high-risk of dementia are now required to assess the relevance of aspirin and B-vitamins for the maintenance of cognitive function in this population.

Conflict of interest statement

The Bayer Corporation, New Jersey, USA, donated aspirin and matching placebo tablets. However, the study was designed, conducted, analysed and interpreted independently of all funding sources.

Acknowledgements

This project was supported by a European Union BIOMED Demonstration Grant (BMH4-98-3549), BRACE (Bristol Research with Alzheimer Disease and Care of the Elderly), JS Cohen Foundation Trust, National Health Service, Research and Development Grant, Oxford and Anglia Health Authority CHK Charities Ltd. Bayer Corporation, New Jersey, USA, donated aspirin and matching placebo tablets. The study was designed, conducted, analysed and interpreted independently of all funding sources.

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