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Inflammation, oxidative damage, and platelet activation are hypothesized biological mechanisms driving the disablement process. The aim of the present study is to assess whether biomarkers representing these mechanisms predicted major adverse health-related events in older persons.
Data are from 2,234 community-dwelling nondisabled older persons enrolled in the Health Aging and Body Composition study. Biomarkers of lipid peroxidation (ie, urinary levels of 8-iso-prostaglandin F2α), platelet activation (ie, urinary levels of 11-dehydro-thromboxane B2), and inflammation (serum concentrations of interleukin-6) were considered as independent variables of interest and tested in Cox proportional hazard models as predictors of (severe) mobility disability and overall mortality.
The sample’s (women 48.0%, whites 64.3%) mean age was 74.6 (SD 2.9) years. During the follow-up (median 11.4 years), 792 (35.5%), 269 (12.0%), and 942 (42.2%) events of mobility disability, severe mobility disability, and mortality occurred, respectively. Only interleukin-6 showed significant independent associations with the onset of all the study outcomes. Higher levels of urinary 8-iso-prostaglandin F2α and 11-dehydro-thromboxane B2 independently predicted increased risk of death (hazard ratio 1.10, 95% confidence interval 1.03–1.19 and hazard ratio 1.14, 95% confidence interval 1.06–1.23, respectively). No significant interactions of gender, race, cardiovascular disease, diabetes, and antiplatelet drugs were detected on the studied relationships.
The inflammatory marker interleukin-6 is confirmed to be a robust predictor for the onset of negative health-related events. Participants with higher urinary levels of 8-iso-prostaglandin F2α and 11-dehydro-thromboxane B2 presented a higher mortality risk.
MOBILITY disability is a common early stage of the disablement process in older adults. It predicts major physical disability (1) and mortality (2), and it is associated with poor quality of life (1,3) and with substantial social and health care needs (4). Consequently, prevention of mobility disability represents a high public health priority.
The oxidative damage due to the excess of free radicals is thought to play an important role in the onset of mobility limitation, disability, and mortality. Although available evidence is particularly focused on protein oxidation (5,6), previous reports (7) and the age-related increase of lipid peroxidation levels (8,9) may well justify the evaluation of this pathway as determinant of negative health-related events in older persons. Oxidative damage (especially lipid peroxidation) is closely associated with platelet activation (10) and inflammation (11), and an interaction among these three pathways might generate the vicious cycle determining the pathophysiological modifications typical of the aging process (10,12–15). Furthermore, oxidative damage, platelet activation, and inflammation are involved in subclinical (eg, sarcopenia, obesity, insulin resistance, atherogenesis) and clinical (eg, cardiovascular disease, diabetes, cancer) conditions, each of which also represents a risk factor for disability and mortality (16).
In the present study, we hypothesize that oxidative damage (in particular, lipid peroxidation), platelet activation, and inflammation are all independently associated with the onset of major health-related events in older persons. Thus, we performed longitudinal analyses in a large sample of community-dwelling older persons aimed at evaluating the predictive value of lipid peroxidation (assessed by measuring its systemic biomarker, ie, urinary excretion of 8-iso-prostaglandin [PG] F2α, 8-iso-PGF2α ), platelet activation (by measuring its systemic biomarker, ie, urinary concentrations of 11-dehydro-thromboxane [TX] B2, 11-dehydro-TXB2 ), and inflammation (ie, serum concentrations of interleukin-6 [IL-6]) biomarkers for incident mobility disability and mortality.
The Health Aging and Body Composition (Health ABC) study is an on-going prospective cohort study investigating changes in body composition and weight-related health conditions in well-functioning older persons. Health ABC study recruited 3,075 community-dwelling black and white men and women, aged 70–79 years, from April 1997 to June 1998. White participants were randomly selected from a sample of Medicare beneficiaries residing in the areas surrounding Pittsburgh, PA, and Memphis, TN. All black participants were potentially eligible. Subjects were considered eligible if they reported no difficulty walking a quarter mile, climbing 10 steps, or performing basic activities of daily living; were free of life-threatening illness; planned to remain in the geographic area for at least 3 years and were not enrolled in lifestyle intervention trials.
The key components of Health ABC include a baseline exam, annual follow-up clinical exams for 6 years with additional examinations in Years 8, 10, and 11, and phone contacts every 6 months to identify major health events and document functional status between clinic visits. In addition, the study collects and abstracts medical records of all hospitalizations (≥24 hours) and adjudicates the occurrence of targeted health events including all deaths. Interviews with proxies are conducted when the participant cannot answer for himself. The primary measures were repeated during each annual clinic visit. The Health ABC research proposal and informed consent forms have been approved by the Institutional Review Boards at the University of Tennessee and at the University of Pittsburgh.
The baseline for the present analyses is the second visit (or first annual follow-up) for the Health ABC study (1998–1999) due to the availability of the biological specimens (urine and serum samples) for the assessment of the three biomarkers of interest. From the original Health ABC study sample (n = 3,075), we excluded 32 participants who had died during the first year, 648 who developed mobility disability before our analytical baseline, and 111 who had inadequate or missing biological specimens. Thus, the present analyses are based on 2,234 Health ABC participants. Excluded participants were older, more likely to be women, and had a higher prevalence of all the clinical conditions compared with those included in these analyses.
The 8-iso-PGF2α is a member of a family of PG isomers called F2-isoprostanes. In contrast to classic PGs, formed through an enzymatic action of the PG H synthase from arachidonic acid, F2-isoprostanes result from a free radical–catalyzed mechanism (19,20). The formation of isoprostanes in lipid bilayers may contribute to alterations in fluidity and integrity of cellular membranes (17). The 11-dehydro-TXB2 is a biologically inactive major enzymatic metabolite of TXB2, the chemically stable and biologically inactive hydration product of TXA2 (a derivative of PG endoperoxide metabolism with receptor-mediated effects on platelet function) (18). Thus, urinary excretion of 8-iso-PGF2α and 11-dehydro-TXB2 provide reliable noninvasive indexes of lipid peroxidation and platelet activation in vivo, respectively.
Urinary levels of 8-iso-PGF2α and 11-dehydro-TXB2 were measured by previously described radioimmunoassay methods (18,21) at the Laboratory of Clinical Pharmacology of Eicosanoids and Pharmacodynamic located in the Center of Excellence on Aging at the “Gabriele D’Annnunzio” University Foundation (Chieti, Italy). Measurements of urinary eicosanoid metabolites by radioimmunoassay methods have been validated with different antisera and by comparison with gas chromatography-mass spectrometry, as previously described (18,21). The intraassay and interassay coefficients of variation for the 8-iso-PGF2α were 2.0% and 2.9% at the lowest level of standard (2 pg/mL) and 3.7% and 10.8% at the highest level of standard (250 pg/mL), respectively. Both urinary levels of 8-iso-PGF2α and 11-dehydro-TXB2 were adjusted for urinary creatinine in all analyses.
Serum IL-6 concentrations were measured in duplicate using a high-sensitivity Quantikine enzyme-linked immunosorbent assay kit (R&D Systems, Minneapolis, MN). The assay has a sensitivity of <0.10 pg/mL, and a detection range of 0.156–10.0 pg/mL. In the Wake Forest University laboratory, the interassay and intraassay coefficient of variation for IL-6 are 5.4% and 3.5%, respectively.
In the present analyses, the following three different outcomes were evaluated.
This was defined by two consecutive semiannual reports of having any incident difficulty either walking a quarter mile or climbing up 10 steps without resting. This definition was chosen because of better discrimination of the onset of chronic mobility disability rather than a temporary loss of physical capacity due to acute illness.
This was defined by two consecutive semiannual reports of having great difficulty or inability either walking a quarter mile or climbing up 10 steps without resting.
Dates and causes of death were obtained from the death certificate. A Health ABC Committee representing all the study units adjudicated causes of death based on the review of medical records, proxy information and autopsy report (when performed).
The days to (severe) mobility disability event was determined from the date of the baseline visit to the date of the first of two successive reports of difficulty. For participants who did not develop a functional limitation, follow-up time was censored to the last contact or death date. For the mortality outcome, the days to event was determined from the date of the baseline to the date of death. For those participants who did not die, the follow-up time was censored to the last contact date. The three outcomes were not mutually exclusive.
We examined sociodemographic variables (age, gender, race, and study site), behavioral factors (smoking status, alcohol consumption, physical activity level), clinical conditions (presence of cardiovascular disease, diabetes, osteoarthritis, peripheral artery disease, cerebrovascular disease, and cancer), biological parameters (blood pressure, body mass index), and medications (statins, corticosteroids, antihypertensive drugs, nonsteroidal anti-inflammatory drugs [NSAIDs], and antiplatelet drugs). Physical activity (in kcal/week) was estimated through a specific questionnaire mirroring the Harvard Alumni studies on the basis of reported walking and exercise activities (22,23). Clinical conditions were based on the data from the first Health ABC visit and defined by algorithms mirroring those adopted in the Cardiovascular Health Study (24). Each disease variable was then updated taking into account the new diagnoses and events reported by participants or determined by the review of clinical documentation during the year until the second visit (the present study baseline). Blood pressure was measured twice after 5 minutes of sitting quietly; the average of the two measures was calculated. Body mass index was defined as body weight (in kilograms) divided by squared height (in meters). Medications taken in the past 2 weeks were brought in, recorded, and coded according to the Iowa Drug Information System. Because smoking status and physical activity were not assessed at our analytical baseline visit, these variables are based on the data from the first Health ABC study visit.
Given the nonnormal distribution of 8-iso-PGF2α, 11-dehydro-TXB2, and IL-6, these biomarkers were log transformed to ensure equality of variances and to make the errors approximately normally distributed. Moreover, to allow fair comparisons among the three biomarkers of interest, their values were rescaled according to their own standard deviation. Unadjusted and adjusted Cox proportional hazard models were performed to evaluate the predictive value (expressed as hazard ratio [HR], and 95% confidence interval [95% CI]) of 8-iso-PGF2α, 11-dehydro-TXB2, and IL-6 concentrations for incident (severe) mobility disability and mortality events. A p value of .05 was chosen as threshold for statistical significance. Analyses were performed using SPSS for Mac 16.0 software (SPSS Inc., Chicago, IL).
The characteristics of the study sample (n = 2,234) are reported in Table 1. Mean age of the study sample was 74.6 (SD 2.9) years. Participants were more predominantly men (52.0%), white (64.3%), with a mean body mass index of 26.8 [SD 4.4] kg/m2. Among comorbid conditions, history of cancer was the most prevalent disease (21.6%), followed by cardiovascular disease (15.8%), osteoarthritis (15.5%), and diabetes (12.7%). More than half of the study sample was on antihypertensive medications (53.6%). During the follow-up (median length 11.4 years, interquartile range 8.3–11.7 years), 792 (35.5%), 269 (12.0%), and 942 (42.2%) events of mobility disability, severe mobility disability, and mortality occurred, respectively.
Spearman's correlation analyses showed modest but statistically significant relationships between 8-iso-PGF2α and 11-dehydro-TXB2 (r = .27; p < .001) and between 11-dehydro-TXB2 and IL-6 (r = .05; p = .05). No significant correlation was found between 8-iso-PGF2α and IL-6 (r = −.01; p = .59).
Results from unadjusted and adjusted Cox proportional hazard analyses predicting (severe) mobility disability and mortality by 8-iso-PGF2α, 11-dehydro-TXB2, and IL-6 (per standard deviation increments in log values) are reported in Table 2. IL-6 was the only biomarker showing significant associations with all the study outcomes, independent of potential confounders (Model 2 adjustment: mobility disability HR 1.21, 95% CI 1.12–1.30; severe mobility disability HR 1.27, 95% CI 1.12–1.43; mortality: HR 1.23, 95% CI 1.16–1.32). Although no significant relationships were reported with mobility disability outcomes, higher urinary concentrations of 8-iso-PGF2α and 11-dehydro-TXB2 were associated with an increased risk of death (Model 2 adjustment: HR 1.10, 95% CI 1.03–1.19, and HR 1.14, 95% CI 1.06–1.23, respectively). When the three biomarkers of interest were simultaneously included in the fully-adjusted models (Model 3 adjustment), IL-6 was confirmed to be the biomarker most strongly associated with all the study outcomes, although significant relationships of 8-iso-PGF2α and 11-dehydro-TXB2 with mortality were maintained (p values <.05). Exploratory analyses did not report consistent associations of the biomarkers of interest with specific causes of death (possibly due to limited statistical power).
A single significant interaction with gender was found for the relationship between 11-dehydro-TXB2 and mortality, suggesting a possible stronger association in men (Model 2 adjustment: HR 1.20, 95% CI 1.09–1.31) than in women (HR 1.06, 95% CI 0.94–1.19). No other significant interactions with (a) gender, (b) race, (c) cardiovascular disease, (d) diabetes, and (e) antiplatelet drugs were detected on the relationships between the study outcomes (ie, [severe] mobility disability, mortality) and the biomarkers of interest (ie, 8-iso-PGF2α, 11-dehydro-TXB2, and IL-6). Restricted analyses investigating the onset of a specific study outcome (eg, mobility disability) after exclusion of participants developing the other outcomes (eg, severe mobility disability and mortality) were also performed and yielded similar results.
This study explored whether three biomarkers from different biological pathways (ie, lipid peroxidation, platelet activation, and inflammation) were associated with the onset of (severe) mobility disability and mortality in a large sample of community-dwelling older persons. We found that IL-6 concentrations were consistently associated with major health-related events, even after adjustment for several potential confounders. Independent associations were also reported between urinary concentrations of 8-iso-PGF2α and 11-dehydro-TXB2 with mortality.
Overall, our study confirms the importance of IL-6 as a valuable and clinically meaningful biomarker for gerontology and geriatric medicine (25). In fact, IL-6 concentrations have shown to significantly predict subclinical (eg, sarcopenia, body composition modifications, insulin resistance) (26,27) as well as clinical (eg, physical impairment, disability, mortality) (28–30) conditions in older persons. Interestingly, the strength of the association we reported between IL-6 and mortality was not significantly modified by the inclusion of potential confounders in the adjusted models. This finding suggests this proinflammatory cytokine is related to mortality independent of the presence of common clinical conditions and disease risk factors (thus, strengthening the clinical relevance of IL-6 as prognostic indicator).
To our knowledge, this is the first study testing lipid peroxidation and platelet activation biomarkers for the prediction of incident mobility disability and mortality events in older persons. Several studies have previously reported that 8-iso-PGF2α and 11-dehydro-TXB2 are particularly elevated in selected populations, such as subjects with increased cardiovascular risk (10,11,31–35). It is noteworthy that cardiovascular disease is not only the leading cause of death in Western countries, but also detrimental to successful aging (36). Moreover, both 8-iso-PGF2α and 11-dehydro-TXB2 have been shown to be closely related to a wide spectrum of subclinical (eg, body composition modifications, insulin resistance, inflammation) (10,11,37) and clinical (eg, obesity, diabetes, pulmonary diseases, hypercholesterolemia, hypertension) (10,13,38–40) conditions potentially contributing to the onset of disability and mortality. Whereas we found statistically significant relationships between both 8-iso-PGF2α and 11-dehydro-TXB2 and mortality, no significant associations were reported for the mobility outcomes. However, given the novelty of the study, our results cannot still be considered as definitive. In fact, although we cannot exclude these biological pathways as unrelated to the disabling process, a large amount of evidence still supports the involvement of increased oxidative damage and platelet activity in several age-related modifications responsible for negative health-related events (including physical decline) (16). Therefore, it may simply be that 8-iso-PGF2α and 11-dehydro-TXB2 do not directly influence the onset of disability, although different biomarkers from the same biological pathways may still be predictive of the outcomes we tested. Our negative results for 8-iso-PGF2α and 11-dehydro-TXB2 may also be due to the older age of our sample compared with previous studies examining these biomarkers. It is possible that aging may decrease the prognostic value of these biomarkers through the increasing number of endogenous and exogenous confounders that accompany age. It is also possible, taking into account the lack of significant age interactions on the studied relationships and the Health ABC study inclusion/exclusion criteria, that our sample may be composed by successful agers and not representative of the general older population.
Some limitations of the present study need to be mentioned. It might be that our sample population may not be representative of older persons and potentially departure from normal aging. Differently from 8-iso-PGF2α and 11-dehydro-TXB2, significant results have already been obtained for IL-6 on the mobility outcomes. This statistical significance for IL-6 may suggest that the nonsignificant findings obtained by the other two biomarkers are indeed true negative results. Our analyses are based on single time-point assessments of the studied biomarkers. This might not be sufficient to adequately represent the participants’ true underlying stress state. Moreover, these are single biomarkers of extremely complex (and interacting) pathways. Thus, we cannot categorically exclude that lipid peroxidation and platelet activation may still be involved in the pathophysiological mechanisms underlying the onset of mobility disability.
In conclusion, our findings confirm the strong association of IL-6 concentrations with the onset of major negative health-related events in older persons. Increased urinary concentrations of 8-iso-PGF2α and 11-dehydro-TXB2 are associated with a higher mortality risk. Further studies confirming our findings and exploring the predictive value for health-related events of other biomarkers of oxidative damage and platelet activation in older persons are needed.
The present study is funded by the National Institutes of Health—National Institute on Aging (R01AG26556) and by the Wake Forest University (P30-AG21332) and University of Florida-Institute on Aging (P30AG028740) Claude D. Pepper Older Americans Independence Centers. The Health ABC study is supported by the National Institute on Aging (contracts N01-AG-6-2101, N01-AG-6-2103, N01-AG-6-2106, R01-AG028050, and grant R01-AG028050), the National Institute of Nursing Research (grant R01-NR012459), and partly by the Intramural Research Program of the National Institutes of Health—National Institute on Aging.