Mechanisms of Ageing and Development 129 (2008) 642648

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Mechanisms of Ageing and Development

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Telomeres and frailty

J. Woo a,*, N.L.S. Tang b, E. Suen a, J.C.S. Leung c, P.C. Leung c
a

Department of Medicine & Therapeutics, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong Department of Chemical Pathology, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong c Jockey Club Centre for Osteoporosis Care and Control, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong
b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 1 February 2008 Received in revised form 11 July 2008 Accepted 15 August 2008 Available online 2 September 2008 Keywords: Telomere length Frailty Ageing

Associations between telomere length and various chronic diseases associated with ageing have led to the suggestion that telomere length may be an ageing biomarker. At the clinical level, the suggestion of using measurements of frailty as a measure of biological ageing has also been suggested. This study examines the hypothesis that telomere shortening may form the biological basis for frailty, using data obtained from a health survey of 2000 men and women aged 65 years and over, living in the community, and followed up for 4 years to determine survival. Frailty was measured using the frailty index, a summation of decits covering physical, psychological, and functional domains. Telomere length was measured in 976 men and 1030 women, using real-time quantitative polymerase chain reaction. Women were more frail than men but had longer telomere length. In men only, there was a negative association between telomere length and age and a positive association between frailty index and mortality after adjusting for age. There was no correlation between telomere length and frailty index in either sex. While telomere length may be a biomarker of cellular senescence, this relationship may not be extrapolated to the functional level represented by the frailty phenotype. 2008 Elsevier Ireland Ltd. All rights reserved.

1. Introduction In the past decade there has been growing interest in examining the role of telomeres in the balance between cancer risk and the senescence process (Monaghan and Haussmann, 2006). Telomere length is regulated by telomerase, and while this area has been more extensively studied in relation to cancer, offering a potential for therapeutic targets (Sampedro et al., 2007; Marrone et al., 2007), the role of telomere shortening and the ageing process has aroused less interest in comparison, although it has been found in premature ageing syndromes and some age-related diseases (Blasco, 2005), and mortality (Cawthon et al., 2003). Telomere shortening has been associated with coronary heart disease, hypertension, dementia, obesity, insulin resistance, cigarette smoking, and bone mineral density (Wong and Collins, 2003; Baird, 2006; Aviv, 2006; Bekaert et al., 2005). The underlying cellular basis for the associations has also been studied, in cardiac myocyte, smooth muscle and endothelial cells (Minamino and Komuro, 2007; Serrano and Andres, 2004) and potential implications for therapy in targeting telomerase have been raised (Minamino and Komuro, 2007). These

* Corresponding author at: Department of Medicine & Therapeutics, Prince of Wales Hospital, Shatin, N.T., Hong Kong. Tel.: +852 2632 3493; fax: +852 2637 3852. E-mail address: jeanwoowong@cuhk.edu.hk (J. Woo). 0047-6374/$ see front matter 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mad.2008.08.003

studies gave rise to the concept of telomere shortening as an ageing biomarker or biochronometer, in that it is a marker of cell senescence and likely represents cumulative oxidative or inammatory stress (Monaghan and Haussmann, 2006; Bekaert et al., 2005; Von Zglinicki and Martin-Ruiz, 2005) resulting in tissue functional attenuation. At the clinical level, the majority of the ageing population spends a variable period before death in a state of declining function, described as the frailty syndrome, representing an excess of decits over assets in a dynamic state of balance, covering physical, functional, psychological, nutritional and social domains (Rockwood et al., 1994; Markle-Reid, 2003; Campbell and Buchner, 1997). The concept of a summation measure of decits as a measure of frailty, the frailty index (FI), had been developed in a Canadian population (Mitniski et al., 2001, 2002) and found to be applicable to the Chinese population (Goggins et al., 2005). The validity of the FI was supported by its association with mortality (Mitniski et al., 2002), and health outcomes such as hospitalization, functional and cognitive decline (Woo et al., 2006), and was also inuenced by social determinants (Woo et al., 2005). An interesting observation was that although women were more frail than men, at the same chronological age and frailty index, men have a higher likelihood of dying than women, suggesting an underlying genetic difference to account for this phenomenon (Goggins et al., 2005). The concept of frailty and its quantitation led to discussion regarding biological

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versus chronological age, the former being a more accurate reection of an ageing individuals health status. There is little data on the biological mechanisms underlying the frailty syndrome, such as oxidative damage or telomere shortening (Walston, 2004). An association may be expected, since telomere shortening has been found to be associated with many diseases and lifestyle that predispose to frailty, including recent studies on socio-economic and psychological factors, albeit somewhat controversial (Cherkas et al., 2006; Adams et al., 2007; Kuh, 2006; Lanzdorp, 2006; Damjanovic et al., 2007). Adverse early life environment has been documented to affect health outcomes in late life (Barker, 1998), and socio-economic factors may operate via telomere attrition, since the greatest rate of attrition occurs in early life (Demerath et al., 2004). Furthermore, telomere dynamics is inuenced by estrogens, and gender differences in frailty and life expectancy exist (Bayne and Liu, 2005; Aviv et al., 2005). However, telomere shortening may be considered a determinant of longevity of cell lineage, rather than of biological ageing, there being a need to distinguish between longevity determination from ageing changes (Walston, 2004; Hayick, 2003; Hornsby, 2006). Epidemiological studies on telomere shortening and health outcomes encounter many pitfalls, due to highly variable telomere length, multiple confounders, sample size issues (which increase with decreasing age of the population studied), survival bias, temporal life course, the method of measurement of telomere length, and the need for longitudinal designs (Aviv, 2006). In a cohort study of 2000 men and 2000 women aged 65 years and over who took part in a health survey, we documented the frailty index and measured telomere length in a subsample, to test the hypothesis that telomere shortening may in part form the biological basis for frailty, and therefore there would be a correlation between telomere length and frailty index.

ABI was calculated for each leg by dividing the posterior tibial systolic pressure in each lower extremity by the upper extremity pressure. The current standard for diagnosing peripheral vascular disease is dened as an ABI of less than 0.90. An ABI of less than 0.90 is 95% sensitive and 99% specic for angiographically diagnosed peripheral arterial disease (Bernstein and Fronek, 1982). The lowest anklearm index of the two was used to determine the extent of ischemic disease. Following the principle of calculating of FI in previous studies (Mitniski et al., 2001; Goggins et al., 2005), abnormality in each parameter was assigned a score of 1, and summated. The FI was calculated as the total score divided by the maximum total score [47] (Appendix A). Subjects were followed up at 2-year intervals for measurement of bone mineral density as part of a study on fracture risk. Mortality after 4 years was ascertained from government Death Registry.

3. Laboratory methods DNA samples were available from 976 men and 1030 women. 3.1. Sample preparation and DNA extraction The genomic DNA was extracted from the peripheral blood by standard procedures using commercial kits (General Electric, USA) by the phenolchloroform method. Afterwards, puried DNA samples stored at over 100 ng/ml in Milli-Q water on the 96-well plates and stored at 20 8C. For the working samples, 20 ng/ml of DNA was prepared just before analysis and the DNA was further diluted to about 5 ng/ml promptly before the real-time PCR experiments. 3.2. Assessment of telomere length by a real-time quantitative PCR method (DDCt) Measurement of telomere length of DNA samples follows the method published by Cawthon (2002) with modication (Gil and Coetzer, 2004). The principle of this technique is to measure the factor of the ratio between the telomere repeat copy number and a single copy gene copy number in our sample with respect to a reference DNA sample (known as T/S ratio). Real-time quantitative polymerase chain reaction (RTQPCR) was performed on Roche LightCycler 480 (Roche, Mannheim, Germany). The threshold cycle numbers (Ct) of the DNA samples were recorded. This number is inversely proportional to log (amount of DNA templates) (Higuchi et al., 1993). The master mix constituents were identical for both telomere and single copy gene PCR except for the primers. One pair of primers was used following Cawthon (mentioned in Gil and Coetzer, 2004), to amplify the telomere repeat sequences: (1) tel1b, 50 -CGGTTTGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTT-30 ; (2) telb2b, 50 -GGCTTGCCTTACCCTTACCCTTACCCTTACCCTTACCCT30 . Basically, the primers were designed to hybridize to human telomere repeat sequence TTAGGG and CCCTAA without forming primer-dimers. For longer telomeres, a smaller Ct would be detected. Another two primers were designed to amplify the single copy gene (36B4) that encodes for ribosomal phosphoprotein PO: (1) 36B4u, 50 -CAGCAAGTGGGAAGGTGTAATCC-30 ; (2) 36B4d, 50 CCCATTCTATCATCAA CGGGTACAA-30 (Cawthon, 2002). About 100 ng of DNA templates were added to LightCycler FastStart DNA Master SYBR Green kit (Roche, Mannheim, Germany) with a nal volume of 20 ml, including MgCl2 added to a nal concentration of 3 mM (Gil and Coetzer, 2004). Enzyme was preheated at 95 8C for 10 min. The cycling protocol for telomere reactions was 95 8C for 5 s, 56 8C for 10 s, 72 8C for 60 s; and for 36B4 reactions, it was 95 8C for 5 s, 58 8C for 10 s, 72 8C for 40 s. The reference DNA and additional quality control samples were used to estimate efciency and quality assurance. These reactions were carried out in duplicates. A standard curve of Ct versus concentration was plotted to get the efciency for each PCR reaction. This efciency (E) can be obtained by the formula

2. Subjects and methods 4000 men and women aged 65 years and over living in the community were invited to attend a health check carried out in the School of Public Health of the Chinese University of Hong Kong, by placing recruitment notices in community centers for the elderly and housing estates. Several talks were also given at these centers explaining the purpose, procedures and investigations to be carried out. Subjects were volunteers, and the aim was to recruit a stratied sample so that approximately 33% would be in each of these age groups: 6569, 7074, 75+. The study was approved by the Clinical Research Ethics Committee of the Chinese University of Hong Kong, which requires informed consent to be obtained. The following items from the health check questionnaire were used to construct a list of decits, used for calculating the frailty index: self-reported health, history of falls in the past 12 months, history of osteoporotic fractures, presence of back pain limiting activities, clumsiness in walking, clumsiness using hands, the number of prescription medications and any difculties with performing activities of daily living (walking two to three blocks outside on level ground, climbing up 10 steps without resting, preparing own meals, doing heavy housework such as scrubbing oors or washing windows, and doing own shopping for groceries or clothes). The presence or absence of disease was based on subjects report of diagnosis by their doctors. Depressive symptoms were assessed using the Geriatric Depression Scale (Yesavage and Brink, 1983) with a score 8 representing depressive symptoms, validated in elderly Chinese subjects (Lee et al., 1993). Cognitive impairment was assessed using the Cognitive Screening Instrument for Dementia (CSID) with a cut off point of 28.4 (Chan et al., 2003). The following measurements were carried out: height, weight, time taken to walk 6 m, grip strength, blood pressure, and ankle brachial index (ABI). Body weight was measured with subjects wearing a light gown, by the Physician Balance Beam Scale (Healthometer, IL, USA). Height was measured by the Holtain Harpenden standiometer (Holtain Ltd., Crosswell, UK). Body mass index was calculated by dividing the weight in kg by the square of the height in meters. Grip strength was measured using a dynamometer JAMAR hand dynamometer 5030JI, Sammons Preston, Bolingbrook, IL. The average reading for two readings on right and left side was used. Blood pressure was measured in the supine portion using a mercury sphygmomanometer and the averages of two readings were taken. Duplicate measures of supine blood pressure in right arm and both ankles were performed using a standard mercury sphygmomanometer and an 8 MHz Doppler probe (Pocket Doppler Model 841-A, Parks Medical Electronics, Inc. ALOHA, OR, USA). The

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10(1/slope), acceptable range 1.52.2. Relative T/S ratio (second derivative of T/S ratio or DDCt) was obtained by the formula fEtel C treference tel C tsample tel g=fE36B4 C treference 36B4 C tsample 36B4 g: In addition, calibration curve of telomere length versus DDCt was plotted using four additional reference samples with known telomere length, that has been previously measured by terminal restriction fragment length polymorphism (TRFLP) and Southern blot. Thus, by comparing the raw sample DDCt to this calibration curve, the telomere length was can also be estimated for each sample. Measurement precision was determined in 4 these different samples with different telomere length. The average coefcient of variation % (CV%) of the Ct readings were 3.6% and 1.8% for telomere and 36B4 primers reactions respectively. The overall average precision of DDCt and telomere length was 11.1%. The subtelomeric DNA segment length was estimated from the calibration curve for telomere length (regressing the TRFL on the T/ S ratio of the reference samples). The y-intercepts were estimates of the subtelomeric DNA length. This analysis was performed according to Cawthon (2002). The telomere length after subtraction of the subtelomeric segment is shown in Table 1. 4. Statistical analysis Using SPSS v15 (SPSS Inc, Chicago, USA), mean values for age, log frailty index and telomere length were compared between men and women using students test. Spearmans Rank correlation was used to examine the association between frailty index and telomere length. Pearsons correlation was used to examine the association between telomere length and age. The associations between frailty index and 4-year mortality, and between telomere length and 4-year mortality, were analyzed using Cox proportional hazard model. 5. Results There was no signicant difference in mean age between men and women (Table 1). Women were more frail compared to men, but had longer telomere length. The distribution of the frailty index was skewed to the right in both men and women (Fig. 1a and b). In contrast, the distribution of telomere length followed a more normal distribution, in both sexes (Fig. 2a and b). The only signicant association was an increase in hazard ratio with age, but not telomere length. The p-value of test for linear trend was not signicant (p = 0.35). In men, telomere length was negatively correlated with age (r: 0.069, p = 0.032), while no association with age was observed in women (Fig. 3a and b). There was no correlation between frailty index and telomere length in either men or women (Fig. 4a and b). After 4 years of follow-up, 87 men (8.9%) and 28 women (2.7%) had died. There was no association between telomere length and mortality adjusting for age in men or women after 4 years of follow-up (Tables 2a and 2b). The only signicant association was an increase in hazard ratio with age, but
Table 1 Summary of age, frailty index, log frailty index and telomere length in our sample Mean S.D. Malea Age Log frailty indexb Telomere length (kb)b
a b

Fig. 1. (a) Distribution curve of frailty index (male). (b) Distribution curve of frailty index (female).

not telomere length. The p value of test for linear trend in telomere length was not signicant (p = 0.35). In men, higher frailty index was positively associated with mortality adjusting for age (Table 3a), while the association was not signicant in women (Table 3b). 6. Discussion The different distribution curve of values for telomere length and frailty index and the absence of correlation between the two measures suggest that they represent two distinct concepts: human ageing, as represented by the frailty syndrome, is multi-

95% CI of mean Femalea 72.02 5.191 3.533 1.467 9.348 2.264 Malea 72.4373.01 4.146 to 3.943 8.7128.916 Femalea 71.7072.33 3.623 to 3.444 9.2109.486

Median Malea 72.00 3.850 8.630 Femalea 71.00 3.175 9.030

72.75 5.026 4.045 1.617 8.814 1.623

N = 976 (male), N = 1030 (female). Statistically signicant differences for log frailty index and telomere length were observed between male and female; for log frailty index, p < 0.001, 95% CI of the difference: 0.3720.643; for telomere length, p < 0.001, 95% CI for the difference: 0.3620.706).

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Fig. 2. (a) Distribution curve of telomere length (male). (b) Distribution curve of telomere length (female).

Fig. 3. (a) Telomere length and age (male). (b) Telomere length and age (female).

component, while telomere length is a specic measure of cellular lifespan. While cellular dysfunction may result in tissue/organ dysfunction, and ultimately disease and death, frailty encompasses the whole person in the context of the environment. They represent two extreme ends of a wide spectrum of changes that ultimately leads to death. Telomere length is affected by telomerase function, which is to a large extent determined by genetic polymorphism (Gilson and Landono-Vallejo, 2007) and hormonal factors (Bayne and Liu, 2005), although oxidative stress and chronic inammatory process contribute to telomere shortening (Von Zglinicki and Martin-Ruiz, 2005). In contract, frailty represents a summation of many decits, which are likely to be largely affected by many environmental factors throughout the life course. Although these factors may act directly at the cellular level, they may not represent the predominant pathway. The contrasting results in men and women also suggest that frailty and telomere length represent distinct processes, in that women have longer telomeres than men, but are more frail. The nding of greater frailty in women compared to men is compatible with previous studies (Mitniski et al., 2002; Goggins et al., 2005); similarly the observation of longer telomere length in women is compatible with currently available studies (Aviv et al., 2005). Given the documented association between short telomere length

and the many chronic diseases (Wong and Collins, 2003; Baird, 2006; Aviv, 2006; Bekaert et al., 2005), and that chronic disease burden is a component of frailty, one would have expected men to be more frail than women since telomere length is shorter in men. The opposite result was observed. A possible explanation may be that the gender difference in telomere length is largely accounted for by the effect of estrogens, the level of which has been shown to be associated with telomerase activity via hTERT gene expression, while androgen appears to be a negative regulator of telomerase in normal prostate tissue (Bayne and Liu, 2005). At the same time, women and men would have been exposed to different environment factors that are associated with frailty, such as lifestyle, socio-economic, and psychosocial factors (Rockwood et al., 1994; Markle-Reid, 2003; Campbell and Buchner, 1997). Previous studies have noted that short telomere length is associated with increased mortality in people between 60 and 75 years (Cawthon et al., 2003) but not in the oldest old age groups over 80 years old (Martin-Ruiz et al., 2005; Bischoff et al., 2006). The observation of shorter telomere length in men compared to women would be compatible with the consistent observation of shorter life expectancy in men in all populations. Shorter telomere length in men may be the underlying biological mechanism for the previous observation that at any given level of frailty, men are

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J. Woo et al. / Mechanisms of Ageing and Development 129 (2008) 642648 Table 2b Telomere length and mortality, Cox proportional hazard model for female Variable Freq (%)/mean (S.D.) Alive (n = 999) Telomere length (kb) 7.78 7.799.03 9.0410.8 10.9 or above Age (per 5 years) Bold: p-value < 0.05. Died (n = 31) Hazard ratio (95% CI)

256 (25.63%) 250 (25.03%) 245 (24.52%) 248 (24.82%) 71.91 (5.11)

3 (9.68%) 8 (25.81%) 11 (35.48%) 9 (29.03%) 75.39 (6.55)

1 2.44 3.01 3.28 1.53

(0.64, 9.29) (0.83, 10.94) (0.88, 12.16) (1.14, 2.06)

Fig. 4. (a) Frailty index and telomere length (male). (b) Frailty index and telomere length (female).

more at risk of dying than women (Goggins et al., 2005). The observation a negative association between telomere length and age in men only but not women, could be a reection of the dominant inuence of estrogens on telomere length. The lack of association with mortality is compatible with the previous studies of subjects where an association was noted in those 6075 years but not older, and also with the observation of absence of association in two other studies for subjects in the oldest old age groups (Martin-Ruiz et al., 2005; Bischoff et al., 2006). There are limitations in this study. The data is cross-sectional in nature, and subjected to survival bias. Those with diseases (and likely shorter telomere length) may have died earlier, so that the survivors may have relatively longer telomere length (Aviv et al., 2006). Therefore fewer associations with diseases or lifestyle factors may be observed. Furthermore, data obtained at one time point regarding environmental factors that are related to chronic inammatory or oxidative stress may not be an accurate reection of life course factors, or cumulative stress. The age range of subjects

studied is necessarily relatively narrow, covering about 20 years, since frailty was being examined. A larger sample size is required with increasingly narrow age range. We did not include those at the extreme spectrum of frailty residing in long-term care institutions. We used a measure of frailty that had been developed predominantly for research purposes. From the clinical viewpoint, although there is a consensus that frailty is a core geriatric syndrome (Inouye et al., 2007), there is no universal agreement of how this should be measured, except that several domains should be covered. For example, a clinical measure based on appearance, healthcare utilization, medical complexity, strength, balance, nutrition, stamina, neuromotor measures, mobility, perceived health, activities of daily living, emotional status, and social status have been proposed by Studenski et al. (2004). The frailty index we used covered all these domains with the exception of appearance, healthcare utilization, stamina and social status measure. We added healthcare utilization to the index (hospitalization 10 days in the past 12 m and no. of hospitalization visits 2 m in the past 12 m), stamina (none of the time having a lot of energy in the past 4 weeks), and social status (most of the time feeling physical health or emotional problems interfered with social activities), and removed blood pressure and anklebrachial index from the construction of the score. The results were unchanged, in that no signicant association with telomere length was demonstrated. It is possible that an association may be present with some other biological frailty measures such as hormonal or inammatory markers. The strength of the study lies in the sufciently large sample size for both men and women, so that it exceeds the sample size requirement estimated for a 10-year age range for case control studies (Aviv et al., 2006), allowing observations relating to gender differences to be made. The method used for telomere length estimation may affect results of epidemiological studies (Aviv et al., 2006), and the various methods and their attributes have been summarized by Baird (2005). The PCR method used in this study has the advantage of ease of use and high throughput (Baird, 2005), which are prerequisites for large-scale epidemiological studies.

Table 2a Telomere length and mortality, Cox proportional hazard model for male Variable Freq (%)/mean (S.D.) Alive (n = 889) Telomere length (kb) 7.63 7.648.64 8.659.81 9.82 or above Age (per 5 years) Bold: p-value < 0.05. Died (n = 87) Hazard ratio (95% CI)

Table 3a Frailty index and mortality, Cox proportional hazard model for male Variable Freq (%)/mean (S.D.) Alive (n = 1830) Frailty index 0.043 0.0440.086 0.0870.15 0.151 or above Age (per 5 years) Bold: p-value < 0.05. Died (n = 170) Hazard ratio (95% CI)

224 (25.20%) 224 (25.20%) 216 (24.30%) 225 (25.31%) 72.51 (4.93)

22 (25.29%) 24 (27.59%) 22 (25.29%) 19 (21.84%) 75.17 (5.39)

1 1.13 0.87 0.67 1.50

(0.63, 2.01) (0.47, 1.62) (0.35, 1.25) (1.24, 1.81)

540 (29.51%) 476 (26.01%) 470 (25.68%) 344 (18.80%) 72.11 (4.87)

22 (12.94%) 35 (20.59%) 48 (28.24%) 65 (38.24%) 75.46 (5.41)

1 1.70 1.75 2.88 1.52

(1.00, (1.05, (1.76, (1.33,

2.91) 2.93) 4.70) 1.73)

J. Woo et al. / Mechanisms of Ageing and Development 129 (2008) 642648 Table 3b Frailty index and mortality, Cox proportional hazard model for female Variable Freq (%)/mean (S.D.) Alive (n = 1937) Frailty index 0.064 0.0650.11 0.120.18 0.181 or above Age (per 5 years) Bold: p-value < 0.05. Died (n = 63) Hazard ratio (95% CI)

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590 (30.46%) 440 (22.72%) 512 (26.43%) 395 (20.39%) 72.50 (5.31)

12 (19.05%) 11 (17.46%) 26 (41.27%) 14 (22.22%) 74.97 (6.28)

1 1.13 1.94 1.41 1.33

(0.49, 2.58) (0.96, 3.92) (0.64, 3.12) (1.07, 1.65)

In conclusion, while telomere length may be associated with mortality and other age-related diseases, which constitute a part of the frailty syndrome, it is unlikely to play a major contributory role. While telomere length may be a biomarker of cellular senescence, this relationship may not be extrapolated to the functional level represented by the frailty phenotype. Appendix A. Frailty index Items CSID abnormal GDS  8 Self-rated health (Poor or very poor) No. of disease* Past 12 m fall >1 No. of impairments of IADL No. of fracture Clumsy walking Clumsy using hands Back pain: limited activities No. of medication use BMI < 18.5 kg/m ABI < 0.9 Grip strength < 10 percentile (M < 23, F < 15) 6 m walk time > 10 percentile (M > 7.65, F > 8.63) SBP > 140 DBP > 90 Total score Frailty index = score of frailty items/47.
* Diseases include: diabetes, high thyroid, low thyroid, osteoporosis, stroke, Parkinsons disease, hypertension, MI, angina, congestive heart failure, COPD, Prostatitis, glaucoma, cataracts, gastrectomy, arthritis, kidney stone, cancer, back pain. 2

Score Yes = 1 Yes = 1 Yes = 1 Max = 18 Yes = 1 Max = 5 Max = 4 Yes = 1 Yes = 1 Yes = 1 Max = 7 Yes = 1 Yes = 1 Yes = 1 Yes = 1 Yes = 1 Yes = 1 Max = 47

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