Anna Oksuzyan
email: aoksuzyan@health.sdu.dk

Eileen Crimmins

Yasuhiko Saito

Angela O'rand

James W Vaupel

Kaare Christensen

Á K Christensen

Cross-national comparison of sex differences in health and mortality in Denmark, Japan and the US

Keywords: Sex differences, Cross-national comparison, Health, Mortality

The present study aims to compare the direction and magnitude of sex differences in mortality and major health dimensions across Denmark, Japan and the US. The Human Mortality Database was used to examine sex differences in age-specific mortality rates. The Danish twin surveys, the Danish 1905-Cohort Study, the Health and Retirement Study, and the Nihon University Japanese Longitudinal Study of Aging were used to examine sex differences in health. Men had consistently higher mortality rates at all ages in all three countries, but they also had a substantial advantage in handgrip strength compared with the same-aged women. Sex differences in activities of daily living (ADL) became pronounced among individuals aged 85? in all three countries. Depression levels tended to be higher in women, particularly, in Denmark and the HRS, and only small sex differences were observed in the immediate recall test and Mini-Mental State Exam. The present study revealed consistent sex differentials in survival and physical health, self-rated health and cognition at older ages, whereas the pattern of sex differences in depressive symptoms was country-specific.

Background

It has been well documented that women live longer than men at all ages with the female-male difference in life expectancy at birth varying from 4.4 in Sweden to 13.2 years in the Russian Federation [START_REF] Gjonca | Sex differences in mortality, a comparison of the United Kingdom and other developed countries[END_REF]. Female advantage in life expectancy grew rapidly within the first three quarters of the twentieth century in most Western countries [START_REF] Helweg-Larsen | Sex differences in mortality in Denmark during half a century, 1943-92[END_REF][START_REF] Glei | The narrowing sex differential in life expectancy in high-income populations: effects of differences in the age pattern of mortality[END_REF]. The sex gap narrowed in most EU countries and the US within the last two decades of the twentieth century, although the timing of this reversal in differential trend by gender varied across the countries [START_REF] Gjonca | Sex differences in mortality, a comparison of the United Kingdom and other developed countries[END_REF][START_REF] Glei | The narrowing sex differential in life expectancy in high-income populations: effects of differences in the age pattern of mortality[END_REF][START_REF] Waldron | Recent trends in sex mortality ratios for adults in developed countries[END_REF]. Japan was the only population among the G7 countries with a persistently widening sex differential in life expectancy from 1950 to 1999 [START_REF] Trovato | A varied pattern of change of the sex differential in survival in the G7 countries[END_REF].

While in 2006 women outlived men even in the poorest countries of the world [START_REF] Barford | Life expectancy: women now on top everywhere[END_REF] the female advantage in healthy life expectancy is less pronounced [START_REF] Jagger | Inequalities in healthy life years in the 25 countries of the European Union in 2005: a crossnational meta-regression analysis[END_REF]. Women tend to rate their general health lower in most EU countries, [START_REF] Olsen | Health differences between European countries[END_REF] have more difficulties in performing activities of daily living (ADL) [START_REF] Arber | Gender differences in health in later life: the new paradox?[END_REF][START_REF] Nybo | Functional status and self-rated health in 2, 262 nonagenarians: the Danish 1905 cohort survey[END_REF], have inferior performance on various physical tests [START_REF] Frederiksen | Age trajectories of grip strength: cross-sectional and longitudinal data among 8,342 Danes aged 46 to 102[END_REF][START_REF] Jeune | Handgrip strength among nonagenarians and centenarians in three European regions[END_REF][START_REF] Hughes | Longitudinal muscle strength changes in older adults: influence of muscle mass, physical activity, and health[END_REF], and are more likely to report a chronic condition than men [START_REF] Denton | Gender differences in structural and behavioral determinants of health: an analysis of the social production of health[END_REF]. However, literature suggests that sex differences in health depend on the selected dimension of health and age group studied. Men report more lifethreatening conditions, such as heart insufficiency, angina pectoris or thrombosis in the leg, while women suffer more frequently from non-life-threatening but disabling conditions, e.g. migraine, musculoskeletal and autoimmune diseases [START_REF] Gold | Gender and health: a study of older unlike-sex twins[END_REF][START_REF] Case | Sex differences in morbidity and mortality[END_REF][START_REF] Whitacre | Sex differences in autoimmune disease[END_REF]. Although men have a higher incidence of coronary heart disease (CHD) in middle age, the sex gap in the CHD incidence narrows after 60 and becomes very small after 80 years [START_REF] Videbaek | The Danish Heart Foundation and the Danish National Institute of Public Health[END_REF][START_REF] Wingard | Sex differentials in morbidity and mortality risk examined by age and cause in the same cohort[END_REF][START_REF] Rich-Edwards | The primary prevention of coronary heart disease in women[END_REF].

Cross-national comparison studies have been focused primarily on sex differentials in life expectancy and mortality; less research has examined sex differences in various health domains across countries. The difficulties of crossnational comparisons include the lack of comparable data and cross-cultural differences in reference levels used in answering questions about health, response categories, and in health perceptions [START_REF] Sen | Health: perception versus observation[END_REF]. However, such factors are less likely to bias comparisons of within-country male-female differences or ratios in measured health. Besides, crossnational comparison studies increase our ability to generalize research findings about sex differentials in health. The present study aims to describe sex differences in mortality and several major health dimensions across countries representing populations in Europe, Asia, and the North America. To our knowledge, no prior study has compared sex differences in health among individuals aged 45 to 90? living in Denmark, Japan, and the USA, countries with diverse cultures, health systems and phases of demographic and epidemiologic transition.

Methods

Study populations

The study is a secondary analysis of population based studies conducted in Denmark, Japan, and the US with details described elsewhere [START_REF] Nybo | The Danish 1905 Cohort: a genetic-epidemiological nationwide survey[END_REF][START_REF] Christensen | A Danish population-based twin study on general health in the elderly[END_REF][START_REF] Gaist | Strength and anthropometric measures in identical and fraternal twins: no evidence of masculinization of females with male co-twins[END_REF][START_REF] Juster | An overview of the health and retirement study[END_REF][START_REF] Saito | to Seikatsu ni Kansuru Chyosa Houkokusho (A report on a survey of health and lifestyle[END_REF][START_REF]Longitudinal Study of Aging[END_REF].

In brief, the Danish data come from the Study of Middle-Aged Danish Twins (MADT), the Longitudinal Study of Aging Danish Twins (LSADT), and the Danish 1905-Cohort Study. Eligible participants were identified through the Danish Twin Register [START_REF] Skytthe | The Danish twin registry: 127 birth cohorts of twins[END_REF] and Danish Civil Registration System [START_REF] Pedersen | The Danish civil registration system. A cohort of eight million persons[END_REF]. The MADT represented a random sample of 120 twin pairs from each birth cohort from 1931 to 1952, aged 45-68 years in 1998 when the survey was implemented. The LSADT involved Danish twins aged 75? and residing in Denmark by January 1995 when the baseline survey was carried out. The waves in 1997, 1999, and 2001 included both follow-up of participants from previous waves and new participants aged at least 70 years. Previous research in Denmark demonstrated that twins are representative of the general population in terms of health trajectories and all-cause or cardiovascular mortality and, thus, are good population models for epidemiological and demographic research [START_REF] Christensen | Mortality among twins after age 6: fetal origins hypothesis versus twin method[END_REF][START_REF] Christensen | Cardiovascular mortality in twins and the fetal origins hypothesis[END_REF].

The participants of the 1905-Cohort Study included all Danes born in 1905 and residing in Denmark in 1998, when the baseline survey was initiated (n = 2,262). The consecutive waves in 2000, 2003 and 2005 were follow-up assessments of survivors from previous waves. In all surveys the individuals residing in nursing homes or sheltered accommodation were considered eligible to participate in the study. If persons refused or were unable to participate in the face-to-face interview, a proxy respondent, usually a close relative, was sought. In total, 4,314 middle-aged twins, 4,731 old-aged twins, and 2,262 oldest-old persons completed the intake assessment in the MADT, LSADT, and the Danish 1905-Cohort Study, respectively, and were included in the present analyses (Table 1). The data for 13 twins participated in both the LSADT and the Danish 1905-Cohort Study were taken from the LSADT.

The initial response rates were 83.1% in the MADT, 77% in the LSADT, and 62.8% in the Danish 1905-Cohort Study [START_REF] Nybo | The Danish 1905 Cohort: a genetic-epidemiological nationwide survey[END_REF][START_REF] Christensen | A Danish population-based twin study on general health in the elderly[END_REF][START_REF] Gaist | Strength and anthropometric measures in identical and fraternal twins: no evidence of masculinization of females with male co-twins[END_REF]. All three Danish studies are comparable with regard to the design, implementation and data collection instruments with only minor differences, primarily reflecting the different age distributions in the three surveys. Data collection in each survey wave was carried out within approximately 3 months and at participants' homes.

The US data are from the Health and Retirement Study (HRS), a biennial survey representative of the US population 50 years of age and over [START_REF] Juster | An overview of the health and retirement study[END_REF]. The HRS began in 1992 and the sample has been resurveyed approximately every 2 years since then. Younger cohorts have been added 1). The NUJLSOA sample at the baseline included communitydwellers only, but the follow surveys included also survivors followed into the institutions and, thus, cover institutionalized population. The NUJLSOA survey was designed to be comparable to the US Longitudinal Study of Aging and the AHEAD sample of the HRS.

Total number of participants in the Danish studies, the HRS and the NUJLSOA are presented in Table 1 by gender and age groups. Sample sizes for each health measurement may vary due to missing values: 0.1 (for physical disabilities)-10.9% (for MMSE) in Danish surveys, 0.4% (for self-rated health)-8.1% (for immediate recall test) in the HRS, and 0.85% (for physical disabilities)-27.6% (for grip strength) in the NUJLSOA.

Sex-and age-specific mortality

The Human Mortality Database (HMD) was used to examine sex differences in all-cause death rates in the three countries by 5-year age group and sex in 1998 [START_REF] Hmd | Human mortality database[END_REF]. The year 1998 was selected as it is the closest year to the time when the selected studies were initiated or expanded to include additional cohorts.

Health measures

In Denmark grip strength in kilograms was measured by using a Smedley dynamometer (TTM; Tokyo, Japan) [START_REF] Frederiksen | Age trajectories of grip strength: cross-sectional and longitudinal data among 8,342 Danes aged 46 to 102[END_REF]. In the LSADT grip strength was available starting from the second follow-up survey in 1999 and was measured as the maximum of 6 attempts, 3 attempts on each hand, whereas in the 1905-Cohort Study it was the maximum of 3 attempts. In the 2006 HRS, handgrip strength was measured twice on each hand using a Smedley spring-type hand dynamometer. Two measurements were taken for each hand and the score is the mean of the two tries for the dominant hand. If both hands were equally dominant, the right hand was used [START_REF] Crimmins | Documentation of physical measures, anthropometrics and blood pressure in the Health and Retirement Study[END_REF]. In the NUJLSOA, grip strength was measured using a TANITA Hand Grip Meter Blue 6103 in a standing position with the arm straight down at the side.

In the Danish surveys physical disability score was computed as the number of 6 basic ADL with which the person had difficulty. This included ability to dress and wash oneself, walk around the house, stand up from chair or bed, eat, and go to the toilet with 3 response options: yes, yes with aids/personal help, no. Persons reporting 'no' were coded as having difficulty. The basic ADL items were administered in the 1905-Cohort Study and LSADT 1995 and 1997 waves only. In the HRS, the physical disability score was calculated as the number of six basic ADL items with which an individual had difficulty because of a health or memory problem: dressing, walking, bathing, eating, transferring and toileting. There were 4 response options: yes, no, cannot do, and do not do. Individuals reporting 'yes' or 'cannot do' were coded as having difficulty. The same six items were used to construct ADL scores in the NUJLSOA. The question wording was similar to that in the HRS, except that ''memory problem'' was not mentioned as a cause of difficulty. The response categories were: yes, do not do, and no. The individuals who responded 'yes' and who responded 'do not do' because of a 'health or physical state' were coded as having difficulty. The analysis of sex differences in ADL included also proxy respondents in all three countries.

Cognitive function was assessed using indicators of immediate word recall in the three studies and scores on the Mini-Mental State Exam (MMSE) in Denmark and the US.

Respondents were asked to recall immediately a list of 12 nouns in the Danish surveys and 10 nouns in the HRS and NUJLSOA. For our analysis, the individual score was computed as the percentage of correctly recalled words. The Mini-Mental State Exam (MMSE) is a 19-item standard cognitive screen that was conducted in the HRS and all Danish surveys except MADT. Scores range from 0 to 30 with higher score reflecting better cognitive ability [START_REF] Mcdowell | Measuring health a guide to rating scales and questionnaires[END_REF].

The global self-rated health (SRH) question asked interviewees to evaluate their health in general (How do you consider your health in general?) with 5 possible responses in all surveys. In the Danish surveys the responses were: excellent, good, acceptable, poor, and very poor and in the HRS: excellent, very good, good, fair, and poor. The same question with slightly different response categories was asked in the NUJLSOA: very healthy, healthier than average, of average health, somewhat unhealthy, very unhealthy. In all three studies the original mean scores were linearly transformed, so that the higher scores indicate better ratings of health in all three surveys.

In the Danish study populations depression was assessed using the 21 depression items from the Cambridge Mental Disorders of the Elderly Examination (CAMDEX) reflecting participants' current state and supplemented by additional 11 items for more comprehensive assessment of depression history and affective state [START_REF] Mcdowell | Measuring health a guide to rating scales and questionnaires[END_REF]. Based on factor analysis of the individual items the total depression score was computed from 17 of the 21 depression items included in the surveys [START_REF] Mcgue | The heritability of depression symptoms in elderly Danish twins: occasion-specific versus general effects[END_REF]. Several versions of depression scales have been used in the HRS. The present analysis used a Center for Epidemiologic Studies Depression (CES-D) score based on 8 items with responses of 'yes' or 'no'. In the NUJLSOA 11 of the original 20 CES-D items were administered with 3 response categories [START_REF] Kohout | Two shorter forms of the CES-D depression symptoms index[END_REF]. For all depression scales higher scores reflected greater levels of depression.

Results

All-cause sex-and age-specific mortality rates Figure 1 illustrates age-specific all-cause mortality rates, sex ratio and absolute difference in age-specific mortality rates in 1998. The age-specific death rates among women and men were the lowest in Japan and, generally, the highest in Denmark in 1998. Only Danish men aged 50-54 and 55-59 had lower all-cause mortality rates than the same-aged men in the US.

The age-specific sex ratios of mortality rates (male/ female ratio of age-standardized mortality rates) indicate that women had consistently lower death rates at all ages in all three countries (Fig. 1). The sex ratio was highest in Japan in almost all age groups, except the 100? age group. In Denmark, the sex ratio was lower than that in the US at middle and young-old ages, whereas from 75 years the opposite pattern was observed (Fig. 1).

The trajectories of the sex ratio with age were countryspecific. In Denmark, the sex ratio varied in the younger part of the age range, 45-49 to 75-79 years, and it declined steadily at the older ages. In the US, the sex ratio was highest among persons 45-49 and 50-54 years old and declined gradually afterwards. In Japan, the sex gap increased from age 45-49 to 65-69 and then rapidly declined at the older ages.

The male-female absolute difference in the age-specific death rates increased steeply with advancing age until age 90 in the three countries, but afterwards its trajectories were country-specific. In Denmark, the absolute difference continued to increase until the oldest age group when it was the largest. In the US the absolute difference increased until the 90-94 years and remained at the same level in the oldest age groups. In Japan, however, the absolute difference in the age-specific death rates increased until the 95-99 age group, but it decreased substantially among 100? years old individuals.

Grip strength

In total 8,516 Danes (57.4% women) had handgrip strength measurement at intake, 7,063 persons (57.2% women)-in the HRS, and 2,463 individuals (54.1% women)-in the NUJLSOA. In all countries grip strength was substantially higher in men compared with the same-aged women (Fig. 2). The sex gap was higher in younger age groups and diminished with increasing age in the US and Denmark. In Japan, the sex difference in grip strength was almost constant across all age groups. Although grip strength declined with increasing age in all three studies, the cross-sectional trajectories suggest that the decline was steeper among men in Denmark and the US than in women within each country, but it was similar in Japanese men and women. Cross-country comparison of grip strength suggested that Japanese men and women had the lowest grip strength at all ages, while Danes had higher than or similar to the grip strength among US men and women. However, differences in testing equipment, testing position and protocol create difficulties for within-sex comparison of grip strength across Denmark, Japan and the US and, thus, the results should be interpreted cautiously (Fig. 2).

Physical disabilities

The analysis of sex differences in physical disability score in Danish surveys was based on 5,221 (68.3% women) individuals aged at least 70 years. Valid measurements on physical disabilities were available 17,930 (58.3% women) in HRS and for 3,374 (55.6% women) in the NUJLSOA. Disability scores were lower among Danish men than women aged 70-74, 75-79, and 80-84, but women had higher disability scores at the older ages (Fig. 3). Similarly, in Japan at younger age groups men had similar or higher levels of physical disability compared with women, but the female disadvantage in physical function became apparent in the 85-89 and 90? age groups. In the HRS, men reported fewer physical disabilities compared with women at all ages. The country-specific trajectories of physical disability indicate that the female disadvantage was larger among 85? years old individuals in all three studies, especially in Japan (Fig. 3).

Within-sex cross-country comparison revealed that the level of physical disabilities was lowest among Danes, although Japan-Denmark difference was not always statistically significant in men and women. Japanese and US men had similar levels of physical disability, but Japanese women were significantly less disabled than their same-sex US counterparts (P-value \ 0.05).

Depression

In total, 10,472 (42.9% women) Danes, 16,539 persons (59.3% women) from the HRS, and 3,020 (54.2% women) NUJLSOA participants had valid depression scores. Depression scores tended to be higher in women than in men at almost all ages in Denmark, the US and Japan (Fig. 3). In Japan, men had slightly higher depression levels than women in the 75-79 and 80-84 age groups, but the direction of the sex difference in depression reversed among individuals aged 85? with women having higher depression scores than men. However, the sex difference failed to reach statistical significance at all ages. In general, depression increased with advancing age in the Danish and Japanese study populations. In the US depression decreased until 65-69 in women and 70-74 in men and further increased until the oldest age group. However, there was no apparent sex-specific pattern in the trajectories of depression over time in all three countries. Self-rated health Self-rated health was available for 10,583 Danes (57.1% women), 17,852 persons (58.4% women) in the HRS, and 3,028 individuals (54.3% women) in the NUJLSOA. Men and women rated their general health similarly in all three countries (Fig. 4). SRH declined with advanced age and the trajectories of decline were similar in men and women in the three populations.

Danes reported significantly better self-rated health compared with the same-sex HRS (P-value \ 0.01) and NUJLSOA participants (P-value \ 0.01). Within each Cross-national comparison of sex differences in health gender self-rated health was similar in Japan and the US (Fig. 4).

Cognitive function

The analysis of sex differences in immediate recall was based on 10,313 participants of the Danish surveys, 16,481 persons (59.4% women) from the HRS, and 2,869 (54.1% women) NUJLSOA participants. The Danish data showed that women had better performance on the immediate recall test compared with men in all age groups (Fig. 5).

Although the sex difference was statistically significant until 80 years, its magnitude was very small (1.02-5.48%) corresponding approximately to the recall of one word.

Similarly, in the HRS, women had significantly more words recalled at every age; except the oldest old men and women had similar immediate recall scores. The smallest sex differences were observed in the NUJLSOA (0.3-2.7%), although the 65-79 years old women had significantly better performance on immediate recall tests compared with the same-aged men. In the oldest ages Japanese men had more favorable performance than their female counterparts, which is probably due to the small numbers and selectivity of very old participants in the NUJLSOA. The performance on the immediate recall test declined with advanced age in all three countries. The trajectories of immediate recall scores were similar in men and women in Denmark, the US, and Japan (Fig. 5). Within-sex comparison across countries revealed that immediate recall score was significantly lower in Denmark than in Japan (P-value \ 0.05) and US (P-value \ 0.01) counterparts in both genders and at all ages. Men and women in the US had the best performance on the immediate recall test (P-value \ 0.01); only in the 75-79 and 90? age groups Japanese and US men had similar levels of Fig. 4 Self-rated health cognitive function. The lowest immediate recall score among Danes is likely to be partially due to methodological differences across the studies.

Valid MMSE score was obtained for 6,216 individuals (37.4% women) in Denmark and 814 persons (60.6% women) in the HRS. US women had higher MMSE scores than men at all ages, whereas the reverse pattern was observed in Denmark (Fig. 6). However, sex differences in MMSE scores in the US and among middle-aged and younger-old Danes were not statistically significant. Although 80 years and older Danish men had significantly higher MMSE scores compared with the same-aged Danish women, the magnitude was very small. Generally, US women had higher MMSE scores compared with Danish women, but statistically significant difference was indicated in the 75-59 and 80-84 age groups. Danish and US men had similar MMSE scores, except the oldest-old US men had significantly higher cognitive function compared with the same-aged Danish men.

Discussion

The present study revealed consistent sex differences in mortality, handgrip strength and physical disabilities across Denmark, Japan, and the US. It showed also that women are inclined to have higher depression scores than men at the same ages and that women and men have similar SRH and cognitive function in the three studies.

Consistent with previous research reports, the present study revealed the highest sex ratio in mortality in Japan. Other studies have documented that at the end of the twentieth century Japan had the highest sex differential in life expectancy compared with other G7 countries expect France [START_REF] Glei | The narrowing sex differential in life expectancy in high-income populations: effects of differences in the age pattern of mortality[END_REF][START_REF] Trovato | A varied pattern of change of the sex differential in survival in the G7 countries[END_REF]. Such a divergent pattern of the sex difference in life expectancy in Japan from most other industrialized countries was shown to be due to relatively small improvements in mortality from circulatory diseases and increasing mortality from cancer and external causes among Japanese men compared with their female counterparts. Sex differences in the diffusion of cigarette use within the population may underlie widening sex differential mortality in Japan. Due to an extreme scarcity of cigarettes in Japan at the time and shortly after World War II, cigarettes were rationally distributed among men aged at least 20 years, whereas smoking among women was socially unacceptable and was observed only among old aged women [START_REF] Katanoda | Population attributable fraction of mortality associated with tobacco smoking in Japan: a pooled analysis of three large-scale cohort studies[END_REF][START_REF] Marugame | Trends in smoking by birth cohorts born between 1900 and 1977 in Japan[END_REF]. With post-war economic growth the prevalence of tobacco use increased substantially from 1950s to 1980s although with lower rates and older age of initiation among women [START_REF] Satoshi | Projection of lung cancer mortality in Japan[END_REF]. Despite the adoption of healthy lifestyles by younger generations in recent years, cigarette use remains high among Japanese men and it stays at low levels among women, 54.4 versus 8.1% in men and women, respectively [START_REF] Katanoda | Population attributable fraction of mortality associated with tobacco smoking in Japan: a pooled analysis of three large-scale cohort studies[END_REF][START_REF] Marugame | Trends in smoking by birth cohorts born between 1900 and 1977 in Japan[END_REF].

Similar to other studies [START_REF] Jeune | Handgrip strength among nonagenarians and centenarians in three European regions[END_REF][START_REF] Kuh | Grip strength, postural control, and functional leg power in a representative cohort of British men and women: associations with physical activity, health status, and socioeconomic conditions[END_REF], we observed a substantial male advantage in handgrip strength across three countries. Our study suggests that the age-related decline in grip strength may be larger among men than among women in Denmark and the US, but it is similar in Japanese men and women. Investigators studying sex differences in the rate of decline in grip strength have drawn conflicting conclusions. While some studies found that grip strength declines at a faster pace among men than among women [START_REF] Hughes | Longitudinal muscle strength changes in older adults: influence of muscle mass, physical activity, and health[END_REF], others suggested that the rate of decline in women is greater than that in men [START_REF] Bassey | Longitudinal changes in selected physical capabilities: muscle strength, flexibility and body size[END_REF]. Although differences in anthropometric characteristics, testing equipment, testing position and protocol impede cross-country comparison of grip strength in the present study, our finding of highest grip strength in Denmark is consistent with better selfreported health and a tendency toward lower physical disability levels among Danes compared with the same-sex HRS and NUJLSOA participants. In addition, it has been previously reported that Danish nonagenarians and Cross-national comparison of sex differences in health centenarians had higher grip strength than French and Italian elderly even in multivariate analysis [START_REF] Jeune | Handgrip strength among nonagenarians and centenarians in three European regions[END_REF]. Substantial geographical differences in grip strength were seen in European countries with the highest scores in northern and continental (SE, DK, NL, DE, AT, CH, FR) countries and the lowest in southern countries (ES, IT and GR) [START_REF] Andersen-Ranberg | Cross-national differences in grip strength among 50? year-old Europeans: results from the SHARE study[END_REF].

Our analysis revealed only small sex differentials in ADL among young-old individuals in Denmark and Japan, but the US women tended to have more disabilities compared with men at all ages. The female disadvantage in physical function became pronounced with advancing age in the three countries. A recently published cross-country comparison study of gender differences in health at ages 50 years and older in 11 European countries, England and the US has also reported that the sex differences were less pronounced in the abilities to provide self-care, although female disadvantage was apparent in the instrumental activities of daily living [START_REF] Crimmins | Gender differences in health: results from SHARE, ELSA and HRS[END_REF]. Other studies have also indicated marked sex differences in functional disability among very old individuals and widening sex gap with increasing age [START_REF] Strauss | Women are more disabled in basic activities of daily living than men only in very advanced ages: a study on disability, morbidity, and mortality from the Kungsholmen Project[END_REF].

Our findings of no substantial sex differential in SRH and its age-related decline in all three countries run counter to the results from several cross-national comparison studies. In most EU countries, on average, women reported worse health than men [START_REF] Olsen | Health differences between European countries[END_REF][START_REF] Bambra | Gender, health inequalities and welfare state regimes: a cross-national study of thirteen European countries[END_REF]. However, some studies revealed no or negligible sex differences in general health in Japan, US, and Denmark [START_REF] Jng | Gender inequalities in US adult health: the interplay of race and ethnicity[END_REF][START_REF] Lee | A comparison of correlates of self-rated health and functional disability of older persons in the Far East: Japan and Korea[END_REF][START_REF] Andersen | Self-rated health and age: a cross-sectional and longitudinal study of 11,000 Danes aged 45 102[END_REF]. Although crossnational differences in self-rated health may reflect also cultural differences in backgrounds, response categories and health perceptions [START_REF] Sen | Health: perception versus observation[END_REF], it has been previously reported that Danes had the highest life satisfaction among the 15 Eurobarometer countries from 1975 to 2005 [START_REF] Christensen | Why Danes are smug: comparative study of life satisfaction in the European Union[END_REF].

The present study indicated that, generally, women had higher depression scores in all three countries, although the sex difference was not always statistically significant. A decline in depression observed in the oldest age group in the NUJLSOA is likely to be due to small number of participants in the oldest age groups, 35 men and 70 women. Also, there were no apparent sex-specific patterns in the trajectories of depression over time in either country. Although there is a mounting research literature pointing towards substantially higher prevalence of depression in women compared with men from young until early old age [START_REF] Piccinelli | Gender differences in depression: critical review[END_REF], the female predominance of depression at older ages is still debatable. Some studies indicated diminished sex difference in depression after menopause [START_REF] Bebbington | The influence of age and sex on the prevalence of depressive conditions: report from the National Survey of Psychiatric Morbidity[END_REF], while others reported persistent female preponderance at older ages as well [START_REF] Cairney | The influence of age on gender differences in depression further population-based evidence on the relationship between menopause and the sex difference in depression[END_REF]. Current knowledge of age-related changes in the prevalence of depression is also inconclusive [START_REF] Katona | Depression in old age[END_REF].

Our results suggested small or no sex differential in cognitive functioning as indicated by the immediate recall test in Denmark, the US and Japan. Although women, generally, did statistically better than men at younger ages, the sex difference was very small and, thus, likely to be unimportant in everyday life. The performance declined with advanced age in the three countries, but within each country the trajectories of age-related changes in the immediate recall score were similar in men and women. Cross-sectional and longitudinal studies have revealed contradictory results on sex differences in cognitive abilities. Some studies found that women performed better on verbal memory tasks, but men had higher scores on spatial orientation tasks or no sex differences were indicated in non-verbal reasoning [START_REF] Aartsen | Gender differences in level and change in cognitive functioning. Results from the longitudinal aging study Amsterdam[END_REF][START_REF] Herlitz | Sex differences in episodic memory[END_REF]. Others found statistically significant female disadvantage in episodic memory, perceptual speed, and digit span, but, like in our study, its magnitude was small [START_REF] Read | Sex differences after all those years? Heritability of cognitive abilities in old age[END_REF].

Finally, our data suggested that the sex differential in the MMSE score and its age-related decline was also very small both in Denmark and the US. There seems to be little agreement on the sex differential in cognitive decline and in incidence and prevalence rates of dementia [START_REF] Bischkopf | Mild cognitive impairment-a review of prevalence, incidence and outcome according to current approaches[END_REF]. Some studies showed no sex difference in the level and agerelated changes in cognitive function [START_REF] Jacqmin-Gadda | A 5-year longitudinal study of the mini-mental state examination in normal aging[END_REF][START_REF] Barnes | Gender, cognitive decline, and risk of AD in older persons[END_REF] and dementia [START_REF] Fitzpatrick | Incidence and prevalence of dementia in the cardiovascular health study[END_REF]. Other researchers, however, reported higher incidence rates of Alzheimer's disease among women [START_REF] Fratiglioni | Incidence of dementia and major subtypes in Europe: a collaborative study of population-based cohorts. Neurologic Diseases in the Elderly Research Group[END_REF].

Our study indicated substantially lower scores on immediate recall test in Denmark than in Japan and the US for both genders. Danish women had also lower MMSE scores compared with their US counterparts. Methodological differences in the immediate recall test across the countries may partially explain lower performance among Danes than among their US and Japanese counterparts because it is more difficult to recall a list of 12 words than a list of 10 words. In addition, the lower levels of cognitive function in Denmark than in the US and Japan may also be partially due to educational differences across three study samples. In all Danish surveys combined 7-10% of participants had higher education (11 and more years), whereas the corresponding figure in the HRS was 85%.

Prior research suggested that cross-country differences in health may be partly attributed to the differences in welfare states [START_REF] Eikemo | Health inequalities according to educational level in different welfare regimes: a comparison of 23 European countries[END_REF], socioeconomic development, [START_REF] Carlson | Self-perceived health in East and West Europe: another European health divide[END_REF] income inequality [START_REF] Huisman | Socioeconomic inequalities in morbidity among the elderly; a European overview[END_REF], as well as cultural differences in perception of health and response styles [START_REF] Ju | True health vs response styles: exploring cross-country differences in self-reported health[END_REF]. Nevertheless, within each welfare regime the prevalence of poor/fair general health and longstanding illness was higher among women than in men in all countries, except Ireland and UK with the Anglo-Saxon welfare regime, Finland with Scandinavian welfare system, and the Netherlands and Luxemburg with Bismarckian welfare [START_REF] Eikemo | Health inequalities according to educational level in different welfare regimes: a comparison of 23 European countries[END_REF].

One key concern in cross-national comparison studies is the comparability of available data. Although some health domains in the HRS, the NULSOA and Danish surveys were measured in comparable but not identical ways, intra-country comparison of sex differences in health is less sensitive to the differences in data collection instruments. Further, although no covariates other than age were considered in the present analysis, several studies demonstrated that female disadvantage in morbidity, e.g. chronic conditions and psychological distress, persisted even after adjustment for age and socio-economic status [START_REF] Luchenski | Differences between women's and men's socioeconomic inequalities in health: longitudinal analysis of the Canadian population, 1994-2003[END_REF]. Future studies should focus on sex differences in the level and rate of decline in grip strength and physical disabilities using longitudinal data collected in Denmark, Japan and the US, as well as on more detailed analyses of the cross-national differences in health.

In conclusion, the present study suggests that sex differences in the all-cause mortality, grip strength and physical disability at the middle and old ages were consistent across countries, whereas the pattern of gender differences in depression and its age-related trajectories were country-specific. Additionally, within each country women and men had similar self-rated health and cognitive function. It is remarkable that in all three countries regardless of substantial sex differences in mortality and dimensions of physical health, no sex gap was indicated in self-reported health. It suggests that the assessment of general health include the comparison of own health with that of same-sex contemporaries of the same age.

Fig. 1 Fig. 2

 12 Fig.1Age-and sex-specific death rates, sex ratios, and absolute difference in 1998

  Fig. 3 Physical disabilities and depression

Fig. 6

 6 Fig. 5 Immediate recall

Table 1

 1 Sample size in the Danish surveys, the Health and Retirements Study, and the Nihon University Japanese Longitudinal Study of Aging

	Age group Danish surveys a	HRS		NUJLSOA
		Men	Women Men	Women Men	Women
	45-49	398	394
	50-54	515	499	640	1,013
	55-59	507	468	1,051	1,472
	60-64	479	480	936	1,463
	65-69	299	275	1,537	1,879	187	217
	70-74	833	923	1,267	1,560	456	477
	75-79	610	938	906	1,128	328	340
	80-84	291	493	647	917	299	413
	85-89	168	326	344	649	179	312
	90?	621 1,777	142	379	56	139
	Total	4,721 6,573	7,470 10,460	1,505 1,898
	a Danish surveys: the Study of Middle-Aged Danish Twins, the
	Longitudinal Study of Aging Danish Twins, the Danish-1905-Cohort
	Study, the HRS-the Health and Retirement Study; the NUJLSOA-
	the Nihon University Japanese Longitudinal Study of Aging

Acknowledgments

The study was supported by the US National Institute of Health [PO1-AG08761, P01-AG031719, P30-AG17265 and U01-AG009740]; the VELUX foundation; and MEXT Academic Frontier to the Nihon University Population Research Institute.

Conflict of interest statement No conflicts of interest are declared.

Open Access This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.


Andrea Schmidt-Pokrzywniak
email: andrea.schmidt-pokrzywniak@medizin.uni-halle.de

Andreas Stang

Á A Stang

Study of return rate and return time of undeliverable postal letters

Keywords: Response, Undeliverable letters, Epidemiological studies

Postal questionnaires are widely used to collect data in health research and epidemiologic studies. One problem related to mail surveys is the availability of an up-to-date and accurate list of people in the population from which to draw the sample for the survey. For the identification of incorrect postal addresses it is important that all incorrectly addressed mails are returned as undeliverable. This study examines the proportion of unreturned postal letters that were sent to incorrect addresses. We sent 339 letters to existing addresses throughout Germany, but used fictional names name of persons. Three hundred and three letters (98.2%) were returned as undeliverable. The return rates only slightly differed by layout of the envelopes, region and deliverer.

Introduction

Postal questionnaires are widely used to collect data in health research and epidemiologic studies. They are often the only financially viable option when collecting information from a large, geographically dispersed population. However, a common weakness of mail surveys is low response, which is a potential threat to the validity of the study results [START_REF] Edwards | Increasing response rates to postal questionnaires: systematic review[END_REF][START_REF] Asch | Response rates to mail surveys published in medical journals[END_REF]. To maximize response, several strategies have been developed. These include, for instance, an attractive format of the questionnaire, the use of monetary incentives, and different modes of delivery of the postal questionnaires to the target population [START_REF] Asch | Response rates to mail surveys published in medical journals[END_REF][START_REF] Dirmaier | Incentives increased return rates but did not influence partial nonresponse or treatment outcome in a randomized trial[END_REF].

Methodological studies have examined several factors that influence nonresponse [START_REF] Edwards | Methods to increase response to postal and electronic questionnaires[END_REF]. A common cause of nonparticipation may be anxiety for to lose personal integrity [START_REF] Olsson | To answer or not answer-that is the question. Empiric studies of non-responses and quality of the answers in postal questionnaire studies[END_REF]. In an epidemiological study of respiratory illness in Norway, incorrect postal addresses were the main reasons of non-participation, 64% of the nonrespondents were not resident at the mailing address [START_REF] Bakke | Postal survey on airborne occupational exposure and respiratory disorders in Norway: causes and consequences of non-response[END_REF]. One problem related to mail surveys is the availability of an up-to-date and accurate list of people in the population from which to draw the sample for the survey. Sometimes the mailing lists are incomplete or out of date (excluding people who have recently moved into the survey area or including people who have moved away). For the identification of incorrect postal addresses it is important that all incorrectly addressed mails are returned by the post office (Deutsche Post) or other delivery services as undeliverable. If undeliverable mails are not returned, investigators may erroneously assume nonresponse for people who actually were never reached. To our knowledge, only Sandler and Holland examined the proportion of nonrespondents who never received the mail, because of incorrect addresses. They found all letters mailed to an invalid address were returned, whereas 13.3% of the letters sent to fictitious people were not [START_REF] Sandler | Fate of incorrectly addressed mailed questionnaires[END_REF]. The purpose of this study was to examine the proportion of unreturned postal letters that were sent to incorrect addresses. Furthermore, we wanted to examine the influence of layout features of the envelopes on the return rates.

Methods

To make general statement about the proportion of return letters with incorrect addresses in Germany we used the sample points (113 communities and cities) of the German National Examination Survey 1998-a representative crosssectional study of the population in unified Germany [START_REF] Bellach | The German health survey 1997/98[END_REF]. We sent 339 letters (3 in each community or city) to existing addresses (existing city, street, and street number), but we used fictional names of persons that were not registered under the corresponding address according to public phone directories. The fictional names were common German names (male and female) and surnames. Generally we used the surnames: Kluttig, Heinig and Schenk. We used these names because they are common, but not too prevalent. All letters were sent on January 26, 2009.

We sent three different types of envelopes to addresses within each sampling point. Type 1 included the addresser only above the addressee field of the envelope. Type 2 envelopes additionally included a stamp of the full institute name and address. Finally, type 3 envelopes were additionally stamped including the following text: ''if undeliverable, return to sender'' (Fig. 1). (Fig. 2).

We could not calculate the proportion of return letters stratified by deliverer as we did not know which deliverer (Federal Post, private deliverers) received the letters. All letters were sent from a post office, from where they were either delivered via Federal Post or private deliverers. Only with the return of undeliverable letters which had a postmark of the deliverer on them would we see who had delivered them. Of the letters which were not returned it would therefore not be said which deliverer was responsible.

Results

Three hundred and three letters (98.2%) were returned as undeliverable. The return rates only slightly differed by layout of the envelopes, region and deliverer. The median time difference between sending the letters and receiving the letters as undeliverable (return time) was 5 days. The median return time was somewhat modified by the layout of the envelopes, region of sampling points, and type of deliverer. For example, the median return time was 4 days for the Federal deliverer ''Deutsche Post'' and was 8 days for the ''Mitteldeutscher Zustelldienst'', a private delivery service. The median return time for letters sent to Southern Germany was 4 days as opposed to 7 days for the New Federal States and Northern Germany. Overall, after 1 week (7 days) 81% of the undeliverable letters were returned (Table 1). Stratified by layout the return rate after 7 days varied between 68 (layout 3) and 97% (layout 1).

Discussion

The success of any study depends-among other factorson achieving a high response. Non-response to contact letters within postal surveys may be due to several factors. People might have died or moved away before the letter reached them, people might refuse without informing the investigators, and people may not be reached because their postal address contained errors. If letters with incorrect addresses are returned by the deliverer, investigators will be able to discern these address errors. Our study shows that return rates of undeliverable addresses are substantial and are close to 100% under the conditions of our study setting.

If address lists for mailed surveys are out of date and therefore not correct, the investigator will have no assurance that the questionnaire has reached the invited study person unless the letter is returned as undeliverable. In our study the return rate for letters sent to fictitious persons was very high-98% of the definitely undeliverable letters were returned with only little variation by Federal States of Germany. Only the return time differs by deliverer. The median return time for letters sent by the Federal deliverer ''Deutsche Post'' was 4 days as opposed to 8 days by the private delivery service ''Mitteldeutscher Zustelldienst''. In conclusion, study investigators in Germany can be sure that incorrectly addressed letters will mostly be returned by the deliverer.

There are some factors that limit our results. Firstly, as all letters were sent on 1 day in 2009, we could not study the influence of seasons (like holiday season) that might influence return rates. For example, during summer 2009, the Federal Post reduced the number of post men considerably so that delivery times increased and maybe also return rates of undeliverable letters decreased. Secondly, this study does not reflect exactly the situation normally found in studies in epidemiology. In epidemiological studies, addresses of potential study subjects are retrieved from the population registries. It does happen that potential subjects do not live at the address any more, usually because their address was not changed when moving away, but most always a real person by that name has lived at the address which might yield more letters not returned.

Thirdly, we studied some layout features of the letters and restricted the size of the letters to DIN C5 (162 mm times 229 mm). It is difficult to speculate how much letter size may influence return proportions of undeliverable letters.

Fig. 1

 1 Fig. 1 Three types of envelope layouts

  To calculate the proportion of return of letters with incorrect addresses we grouped the sample points from the 16 Federal German States in four groups:

	1. New Federal States: Berlin, Brandenburg, Saxony
	Anhalt, Saxony, Thuringia, Mecklenburg-West Pomerania.
	2. Northern Federal States: Schleswig-Holstein, Lower
	Saxony, Hamburg, Bremen.
	3. Western Federal States: North-Rhine-Westphalia,
	Hesse, Rhineland Palatinate, Saarland.
	4. Southern Federal States: Baden Wurttemberg, Bavaria

Table 1

 1 Response proportion of letters sent to fictitious people returned as undeliverable stratified by layout, region and deliverer Layout 1: included the addresser only above the addressee field of the envelope. Layout 2: envelopes additionally included a stamp of the full institute name and address. Layout 3: envelopes were additionally stamped including the following text: ''if undeliverable, return to sender'' c Berlin, Brandenburg, Saxony Anhalt, Saxony, Thuringia, Mecklenburg-West Pomerania

		Response	Return rate after 7 days (%)	Median	Response time [days]
		N	% (SE) a			10./90. Percentile	Minimum/Maximum
	Mailed letters N = 339
	Return letters	333	98.2 (0.7)	81.4	5	3/9	2/18
	Layout b
	1	112	99.1 (0.9)	96.5	4	3/7	2/12
	2	111	98.3 (1.2)	79.7	4	2/9	2/18
	3	110	97.3 (1.5)	68.1	7	5/10	2/18
	Region
	New Federal States c	107	96.4 (1.8)	58.6	7	3/10	2/18
	Northern Federal States d	42	100 (0)	88.1	7	3/10	2/10
	Western Federal States e	111	100 (0)	93.7	5	2/7	2/10
	Southern Federal States f	73	97.3 (1.9)	93.3	4	2/7	2/12
	Deliverer
	Deutsche Post	267			4	2/7	2/12
	Mitteldeutscher Zustelldienst	66			8	7/15	4/18
	a Standard error

b d Schleswig-Holstein, Lower Saxony, Hamburg, Bremen e North Rhine-Westphalia,Hesse, Rhineland Palatinate, Saarland f Baden Wurttemberg, Bavaria

Conflict of interest statement

No conflicting relationship exists for any author.


Eberhard Fehling
email: eberhard.fehling@mri.bund.de

Klaus Bergander

Erika Klein

Nikolaus Weber

Klaus Vosmann

_____________________________________________________ E Fehling

Thiol-functionalized copolymeric polyesters by lipase-catalyzed esterification and transesterification of 1,12-dodecanedioic acid and its diethyl ester, respectively, with 1-thioglycerol

Keywords: Esterification, 1,12-Dodecanedioic acid, Immobilized lipase B from Candida antarctica, Poly

diethyl 1,12-dodecanedioate-co-1-thioglycerol); Poly(1,12-dodecanedioic acidco-1-thioglycerol); 1-Thioglycerol (3-mercaptopropane-1,2-diol

Introduction

Various compounds containing thiol groups, such as mercaptoacetic and mercaptopropionic acids as well as mercaptoethanol and 1-thioglycerol (monothioglycerol; 3-mercaptopropane-1,2diol), are used as antioxidants. Polyfunctional thioalkanols, e.g., 1-thioglycerol, dithiothreitol and others, may also serve as starting materials for the preparation of polyesters (polyoxoesters, PE) containing free thiol functions or polythioesters. There is increasing interest in such sulfur-containing functional polymers for various applications. For example, sulfur-containing polyesters may be suitable as antioxidative ingredients for polymers, coatings, paints and lubricants and as polymers with antibacterial properties [START_REF] Kúdelka | Antioxidants and stabilizers: Part IC Hydroperoxide deactivation by aliphatic sulfides. A model study[END_REF][START_REF] Uhrich | Antibiotic polymers[END_REF]. PE having high molecular weights are formed by biotransformation of hydroxy alkanoic acids in microorganisms [START_REF] Kim | Polyesters from microorganisms[END_REF][START_REF] Steinbüchel | Biochemical and molecular basis of microbial synthesis of polyhydroxyalkanoates in microorganisms[END_REF] or by lipase-catalyzed esterification and transesterification of , -alkanedioic acids and their methyl esters, respectively, with , -alkanediols in vitro [START_REF] Kazlauskas | Biotransformations with lipases[END_REF][START_REF] Kobayashi | In vitro biosynthesis of polyesters[END_REF][START_REF] Rehm | Genetics and biochemistry of polyhydroxyalkanoate granule self-assembly: The key role of polyester synthases[END_REF]. In contrast to polythioesters, polyoxoesters are more easily decomposed by microbial hydrolases such as lipases or depolymerases [START_REF] Jendrossek | Microbial degradation of polyesters[END_REF][START_REF] Tokiwa | Degradation of microbial polyesters[END_REF]. This is also expected for PE containing thiol or thioether functions, as compared to polythioesters, which may be of advantage from an environmental point of view.

Recently, we have described the preparation of such copolymeric poly(thioalkanedioates) containing thioether groups by lipase-catalyzed esterification and transesterification of 3,3'thiodipropionic acid and its dimethyl ester with , -alkanediols [START_REF] Fehling | Linear copolymeric poly(thiaalkanedioates) by lipase-catalyzed esterification and transesterification of 3,3'thiodipropionic acid and its dimethyl ester with , -alkanediols[END_REF]. The aim of the present work was to develop an enzymatic process for the preparation of thiol-functionalized polycondensates by esterification and transesterification of 1,12-dodecanedioic acid and its diethyl ester, respectively, with 1-thioglycerol using a commercial immobilized microbial lipase as biocatalyst. The final polycondensates containing methylenethiol groups are expected to have antioxidative properties and may be suitable as, e.g., non-migrating stabilizers of polymers for both food use and technical applications.

Materials and methods

Lipase-catalyzed preparation of copolymeric PE containing methylenethiol groups

As a typical example, 1,12-dodecanedioic acid (460 mg, 2 mmol) was esterified with 1-thioglycerol (216 mg, 2 mmol) in the presence of 100 mg Novozym 435 [immobilized lipase preparation from Candida antarctica (lipase B; 10,500 propyl laurate units/g; 2% w/w water); kindly provided by Novozymes] by magnetic stirring in a screw-capped tube (placed in a Schlenk vessel) in vacuo at 80°C for periods up to 480 h with water-trapping in the gas-phase using potassium hydroxide pellets. Diethyl 1,12-dodecanedioate (516 mg, 2 mmol) was transesterified with 1-thioglycerol using similar reaction conditions as given above.

Gas chromatography (GC)

Aliquots of the reaction mixtures of esterification assays were methylated using an ethereal solution of diazomethane followed by silylation with N-methyl-N-trimethylsilylheptafluorobutyramide (MSHFBA). The resulting mixtures of dimethyl 1,12-dodecanedioate, silylated 1thioglycerol as well as various other derivatized reaction products were separated by gas chromatography as described previously [START_REF] Fehling | Linear copolymeric poly(thiaalkanedioates) by lipase-catalyzed esterification and transesterification of 3,3'thiodipropionic acid and its dimethyl ester with , -alkanediols[END_REF].

Alkali-catalyzed transmethylation

Carboxy end groups of purified poly(1,12-dodecanedioic acid-co-1-thioglycerol) were converted to the methyl esters by the addition of an ethereal diazomethane solut ion. Under standard conditions, 2 ml of 0.5 M methanolic sodium methylate solution were added to ~15 mg of the thiol-functionalized PE and the transmethylation mixture was heated to 50°C for ~15 min. After cooling, the derivatization mixture was neutralized by the addition of 75 µl acetic acid followed by concentrated aqueous sodium hydrogen carbonate solution, dried in a stream of nitrogen and finally silylated using MSHFBA reagent. Silylated samples, 1-2 µl, were directly injected into gas chromatograph and separated on a J&W DB-5HT column as described previously [START_REF] Fehling | Linear copolymeric poly(thiaalkanedioates) by lipase-catalyzed esterification and transesterification of 3,3'thiodipropionic acid and its dimethyl ester with , -alkanediols[END_REF]. This way, methyl esters were formed from carboxy ester groups of PE, whereas hydroxy and thiol groups were converted to the silyl derivatives.

GC-MS analyses

Samples withdrawn from the reaction mixtures at various times or low-molecular compounds formed by alkali-catalyzed transesterification of purified thiol-functionalized PE were methylated and silylated as described above. GC-MS analyses of these derivatives were carried out on a Hewlett-Packard model 5890 series II GC/5889A apparatus as described previously [START_REF] Fehling | Linear copolymeric poly(thiaalkanedioates) by lipase-catalyzed esterification and transesterification of 3,3'thiodipropionic acid and its dimethyl ester with , -alkanediols[END_REF].

Gel Permeation Chromatography / Size Exclusion Chromatography (GPC/SEC)

The reaction products from esterification and transesterification of 1,12-dodecanedioic acid and diethyl 1,12-dodecanedioate, respectively, with 1-thioglycerol were repeatedly extracted for GPC/SEC using tetrahydrofurane (THF; stabilized with 25 mg BHT/l) at 50°C and filtered through a 1 m PTFE syringe filter. The molecular masses of copolymeric PE containing methylenethiol groups were determined by GPC/SEC using polystyrene standards for calibration as described previously [START_REF] Fehling | Linear copolymeric poly(thiaalkanedioates) by lipase-catalyzed esterification and transesterification of 3,3'thiodipropionic acid and its dimethyl ester with , -alkanediols[END_REF].

Solvent fractionation

As an example, the combined reaction products (100 mg) from various incubations of 1,12dodecanedioic acids with 1-thioglycerol were repeatedly extracted with THF at 50°C and filtered through a 1 m PTFE syringe filter. THF extract was evaporated in a stream of nitrogen at 45°C to a concentration of around 20 mg/ml, iso-hexane was added to a final ratio of 2:3 (v/v) and cooled to -20°C. Low-molecular constituents were extracted this way from the crude reaction products. The white polymer (60 mg) formed, i.e. poly(1,12-dodecanedioic acid-co-1thioglycerol), was separated by centrifugation. The iso-hexane-THF supernatant was isolated to give 40 mg of white precipitate after evaporation of the solvents. The proportions of low-(M W <600 Da), medium-(M W >600 <1,500 Da), and high-molecular (M W >1,500 Da) constituents of both fractions were determined by GPC/SEC analysis as described above. Precipitation of poly(1,12-dodecanedioic acid-co-1-thioglycerol) was also studied using other solvent mixtures such as THF-iso-hexane-dichloromethane or THF-iso-hexane-iso-propanol. Similarly, the combined reaction products of polytransesterification of diethyl 1,12-dodecanedioate with 1thioglycerol were repeatedly extracted with methyl-tert.-butyl ether (MTBE) at 50°C and filtered as described above. Solvent fractionation of poly(diethyl 1,12-dodecanedioate-co-1-thioglycerol) reaction mixtures (200 mg) with MTBE-isohexane (1:4) led to 170 mg of PE precipitate and 30 mg of supernatant.

FTIR-and NMR-Spectrometry

Fourier Transform Infrared (FTIR) spectra of PE precipitates were obtained using a Bruker IFS 28 spectrometer. Absorbance was measured at a resolution of 1 cm -1 and a total of 30 scans were collected. Solid-state polymer spectra were measured using pressed potassium bromide discs containing ~1.5% polymer.

1 H-and 13 C-NMR spectra were recorded on a Bruker AV400 spectrometer ( 1 H, 400.13 MHz;

13 C, 100.62 MHz) as well as a Varian unity plus 600 instrument ( 1 H, 599.6 MHz; 13 C, 150.8

MHz). The measurements were carried out at 298 K with samples of ~50 mg of the PE precipitates isolated from the reaction mixtures by solvent fractionation as described above and dissolved in 1 ml d 8 -THF. In addition, DEPT, gCOSY, gHMBC and gHSQC experiments were performed with the above PE precipitates.

Results and discussion

Functional polyesters with antioxidative and/or antimicrobial properties are of great current interest as components of synthetic polymers for, e.g., food packaging as well as technical, medical and cosmetic applications. Such properties can be expected for sulfur-containing polycondensates such as polythioesters, polythioethers or polymers containing thiol groups [START_REF] Kúdelka | Antioxidants and stabilizers: Part IC Hydroperoxide deactivation by aliphatic sulfides. A model study[END_REF][START_REF] Nathan | Functionalized polymers for medical applications[END_REF][START_REF] Shaglaeva | Synthesis and antimicrobial activity of butadiene sulfide copolymers[END_REF][START_REF] Uhrich | Antibiotic polymers[END_REF]). Here we present the Novozym 435-catalyzed preparation of copolymeric polyesters (PE) containing methylenethiol functions which are formed by polyesterification and polytransesterification of bifunctional 1,12dodecanedioic acid and its diethyl ester, respectively, with trifunctional 1-thioglycerol.

Fig. 1 shows the time-course of Novozym 435-catalyzed polycondensation of 1,12dodecanedioic acid (Fig. 1a) or diethyl 1,12-dodecanedioate (Fig. 1b) with 1-thioglycerol to the corresponding low-(M W >600 -<1,500 Da) and high-molecular weight (M W >1,500 Da) esterification product, i.e. poly(1,12-dodecanedioic acid-co-1-thioglycerol), and transesterification product, i.e. poly(diethyl 1,12-dodecanedioate-co-1-thioglycerol). The formation of both copolymers was studied over a period of up to 480 h using GPC/SEC analyses for the determination of the degree of conversion and polymerization (calibration using polystyrene standards). Fig. 1c shows the time course of the increase of M W of the above methylenethiol functions-containing PE. The chromatograms were analyzed using a GPC software; the intervals chosen for the separation of the reaction products are given in Fig. 2. Fig. 1d demonstrates the polydispersities (M w /M n ) of the polymeric reaction products at different times.

(Fig. 1) (Fig. 2)

It is obvious from these results that the proportions of poly(1,12-dodecanedioic acid-co-1thioglycerol) (M W >1,500 Da) in the reaction mixture increase with time. After an initial increase (24 h), the proportion of oligomers (M W >600 <1,500 Da) does not rise as is typical of reaction intermediates (Figs. 1 and2). In addition, the rapid disappearance of both starting materials and oligomeric intermediates (M W <1500 Da) indicates that Novozym 435 is highly specific for the esterification of these two groups of reactants. Generally, copolymeric thiolfunctionalized PE are formed in high to moderate yield (oligo-plus polyesters M W >600 Da, conversion: 89% for polyesterification and 51% for polytransesterification after 24 h; M W >1,500 Da, conversion: 88% for polyesterification and 31% for polytransesterification after 96 h) as is shown in Figs. 1 and2. Moreover, M W of both copolymeric PE (M W >600 Da) steadily increase with time up to ~100,000 Da for poly(1,12-dodecanedioic acid-co-1-thioglycerol) and up to ~4,400 Da for poly(diethyl 1,12-dodecanedioate-co-1-thioglycerol). However, from Fig. 1b and c, the increase of the polytransesterification product is rather low. Fig. 1c also shows that M W of poly(1,12-dodecanedioic acid-co-1-thioglycerol) decreases after 360 h. This is attributed to insoluble polymers most probably generated by the formation of small proportions of crosslinking disulfide bridges which were detected by NMR analyses (see below) .

Table 1 demonstrates that similar enzyme activities were found for the polyesterification and polytransesterification of 1,12-dodecanedioic acid and its diethyl ester, respectively, with 1thioglycerol using Novozym 435 in the absence of a solvent. The data of 24 h incubations were used to calculate the mean polyesterification and polytransesterification activities of Novozym 435. Substantially higher activity was determined, however, when polyesterification was started after dissolving the reactants in a small volume of MTBE (193.1 vs. 31.6 units/g, after MTBE treatment)the solvent was removed in vacuo before adding the enzymedemonstrating that higher solubility of 1,12-dodecanedioic acid is responsible for the increase in esterification activity. Under similar reaction conditions, the addition of MTBE only led to a slight increase of the polytransesterification activity of Novozym 435 using diethyl 1,12-dodecanedioate and 1-thioglycerol as substrates (Table 1). It is worth noting that the conversion of substrates in the absence of lipase was negligibly low (data not shown).

(Table 1) Fig. 2 shows the GPC/SEC separation of the reaction products of Novozym 435-catalyzed polyesterification of 1,12-dodecanedioic acid (Fig. 2a) and polytransesterification of diethyl 1,12-dodecanedioate (Fig. 2b) both with 1-thioglycerol after 24, 96 and 360 h and 24, 192 and 360 h, respectively. The dotted lines show the above limits set for oligomeric and polymeric fractions. Generally, copolymeric PE having maximum molecular weights of approximately 10 7

Da were formed by polyesterification of 1,12-dodecanedioic acid with 1-thioglycerol and those with maximum molecular weights of up to 8 10 4 Da by polytransesterification of diethyl 1,12dodecanedioate with 1-thioglycerol (Fig. 2a andb).

Copolymeric PE containing methylenethiol groups were purified by solvent fractionation in order to remove the remaining reactants as well as low molecular reaction products. The PE fractions of poly(1,12-dodecanedioic acid-co-1-thioglycerol) were extracted from the reaction mixtures with THF and filtered. The mainly high-molecular components were precipitated by addition of iso-hexane and cooling at -20°C. Similarly, crude fractions of poly(diethyl 1,12dodecanedioate-co-1-thioglycerol) were dissolved in MTBE and purified by solvent fractionation from MTBE-iso-hexane (1:4, v/v) at -20°C (Table 2). Using GPC/SEC analyses, a distinct increase in M W of PE precipitates (1,12-dodecanedioic acid derived polyesters: 8 10 4 vs. 1.7 10 5 Da; diethyl 1,12-dodecanedioate derived polyesters: 5.4 10 3 vs. 7.1 10 3 Da) is observed when low-molecular compounds are removed by solvent fractionation from the crude polyester fractions (Table 2). The precipitates contained largely uniform copolymeric poly(1,12dodecanedioic acid-co-1-thioglycerol) of M W ~170,000 Da or poly(diethyl 1,12-dodecanedioateco-1-thioglycerol) of M W ~7,100 Da (Table 2). It is obvious from these results that both starting materials and low-molecular esters were largely removed from the PE precipita tes, whereas substantial proportions of low-molecular material were concentrated in the supernatants.

Experiments performed to increase M W by precipitation of the polymeric fractions using other solvent mixtures did not lead to PE with higher M W (Table 2).

(Table 2)

Precipitates of poly(1,12-dodecanedioic acid-co-1-thioglycerol) and poly(diethyl 1,12dodecanedioate-co-1-thioglycerol) having highest M W (Table 2) were chosen for FTIR as well as 1 H-and 13 C-NMR analyses. FTIR spectra of the thiol-functionalized PE showed the carbonyl band of the carboxy ester groups at 1740 cm -1 (ν, C=O, strong) as well as typical thiol absorption at 2571 cm -1 (ν, S-H, weak), as is characteristic for thiol-containing compounds including polymers [START_REF] Yu | Synthesis of thiol-and carboxyl-terminated poly(pphenylene sulfide) oligomers[END_REF]. were confirmed by DEPT, gCOSY, gHSQC and gHMBC experiments (data not shown). The chemical structure of purified PE precipitatesisolated by solvent fractionation as described abovewas also analyzed by alkaline methanolysis followed by silylation. The results of methanolyses demonstrated the formation of dimethyl 1,12-dodecanedioate and silylated 1thioglycerol as confirmed by GC and GC-MS analyses. Finally, the results of FTIR, NMR and alkaline methanolysis reveal that both polymers predominantly consist of polyoxoesters containing branched-chain methylenethiol groups.

Interestingly, preferential O-acylation was also observed for the lipase-catalyzed transesterification of various ethyl alkanoates with 1-thioglycerol, dithiothreitol and other hydroxyalkanethiols [START_REF] Baldessari | Regioselective acylation of 3-mercaptopropane-1,2diol by lipase-catalyzed transesterification[END_REF][START_REF] Iglesias | Lipase-catalyzed mono-O-acylation of dithiothreitol and dithioerythritol[END_REF]. Moreover, copolymeric polyoxoesters are commonly obtained by enzymatic polyesterification or polytransesterification of various alkanedioic acids or alkanedioates with alkanediols using immobilized lipases such as lipase B from C. antarctica [START_REF] Gross | Polymer synthesis by in vitro enzyme catalysis[END_REF][START_REF] Kazlauskas | Biotransformations with lipases[END_REF], whereas ,end thiol-functionalized PE are formed by lipase-catalyzed ring-opening polymerization ofalkanelactones with various mercaptoalkanols [START_REF] Takwa | Single-step, solvent-free enzymatic route to , -functionalized polypentadecalactone macromonomers[END_REF].

Recently, we described various environmentally friendly, solvent-free lipase-catalyzed preparations of sulfur-containing antioxidants and polycondensates such as dialkyl 3,3'thiodipropionates as well as copolymeric polythioesters and polyesters containing thioether groups using immobilized lipases from C. antarctica (lipase B; Novozym 435) and Rh. miehei (Lipozyme RM IM) as biocatalysts [START_REF] Fehling | Chemo-enzymatic preparation of copolymeric polythioesters containing branched-chain thioether groups[END_REF]2008;Weber et al. 2006a;[START_REF] Gross | Polymer synthesis by in vitro enzyme catalysis[END_REF]. Similarly, enzymatic preparation of thiol-functionalized polycondensates may be of advantage over chemical synthesis because expensive and toxic reagents are mostly applied to the latter one [START_REF] Maignan | Polymers with thiol terminal function[END_REF][START_REF] Moad | Process for synthesizing thiol terminated polymers[END_REF][START_REF] Watanabe | Synthesis of polymer microspheres with mercapto groups by polycondensation of , -alkanedithiol and ,dibromoalkane in the presence of a poly[styrene-N-(hydroxymethyl)acrylamide] latex[END_REF][START_REF] Wilczewska | Method for synthesis of polymers with thiol functions[END_REF] whe reas no chemical activators are used in the polyesterification and polytransesterification reactions described here. Moreover, chemical side-reactions are limited to a minimum and only small proportions of organic solvents may be necessary in some cases to disperse and/or dissolve the reactants at the beginning of the enzymatic conversion. Polyoxoesters are widely degraded by microbial hydrolases [START_REF] Jendrossek | Microbial degradation of polyesters[END_REF][START_REF] Tokiwa | Degradation of microbial polyesters[END_REF]. This is expected for polyoxoesters containing methylenethiol groups as well, whereas polythioesters are hardly perceptable to microbial degradation [START_REF] Steinbüchel | Non-biodegradable biopolymers from renewable resources: Perspectives and impacts[END_REF].

Figure legends

Fig. 1 Fig. 2

 12 Fig. 1 Time course of the Novozym 435-catalyzed polyesterification and polytransesterification

  Fig. 1

  2.69 as well as C 44.5 point at small proportions of disulfide bridges formed by partial interconversion of the polymeric thiol functions into disulfides. This was verified by comparing

	PE precipitate from the esterification of 1,12-dodecanedioic acid with 1-thioglycerol shows characteristic resonances at: H 5.01 (m, [CH 2 ] 2 -CH-O); 4.32 (dd, 2 J=11.9 Hz, 3 J=3.7 Hz, O-CH 2 -CH); 4.15 (dd, 2 J=11.9 Hz, 3 J=5.5 Hz, O-CH 2 -CH); 2.7 (m, CH 2 -SH); 2.28 (m, CO-CH 2 ); 1.89 (tr, 3 J=9.0 Hz, CH 2 -SH); 1.58 (m, CO-CH 2 -CH 2 ); 1.30 (m, -[CH 2 -] n ); C 197.7 (-CO-S-); 173.5-172.6 (-CO-O-); 73.24 ([CH 2 ] 2 -CH-O); 63.78 (O-CH 2 -CH); 34.5 (CO-CH 2 ); 30 (-[CH 2 ] n -); 25.78 (CO-CH 2 -CH 2 ); 25.22 (CH 2 -SH). NMR data also reveal that the polyester prepared from diethyl 1,12-dodecanedioate and 1-thioglycerol additionally contains chains with terminal carboxyethyl groups [ H 4.04 (O-CH 2 -CH 3 ) and 1.19 (O-CH 2 -CH 3 ); C 60.4 (O-CH 2 -CH 3 ) and 14.58 (O-CH 2 -CH 3 )]. The intensity of the carbon resonances at C 197.7 ppm (thioester) is generally low as diisobutyl-disulfid at H 2.60 and C 48.6. In addition, the chemical structures of poly(1,12-compared to the resonances at 1 H-and 13 C-NMR resonances of the above thiol-functionalized PE with resonances of dodecanedioic acid-co-1-thioglycerol) and poly(diethyl 1,12-dodecanedioate-co-1-thioglycerol)

Typical 1 H-and 13 C-NMR resonances were observed for the two methylenethiol-functionalized PE formed by polyesterification or polytransesterification. The spectra of the two PE precipitates, i.e. poly(1,12-dodecanedioic acid-co-1-thioglycerol) and poly(diethyl 1,12-dodecanedioate-co-1-thioglycerol), demonstrate very close sets of chemical shifts and indicate only some variations with regard to the ratio of integration. For example, the C 173 ppm (oxoester) demonstrating rather small proportions of thioester linkages in comparison with both primary and secondary oxoester groups. These results are in good agreement with the chemical structures of poly(1,12-dodecanedioic acid-co-1-thioglycerol) and poly(diethyl 1,12-dodecanedioate-co-1-thioglycerol). The NMR resonances at H

Table 1

 1 Lipase activities of immobilized lipase B from Candida antarctica (Novozym 435) Determination of molecular weight by GPC/SEC in various fractions of poly(1,12-dodecanedioic acid-co-1-thioglycerol) and poly(diethyl 1,12-dodecanedioate-co-1-thioglycerol) obtained by solvent fractionation

	used for the polyesterification and polytransesterification of 1,12-dodecanedioic acid and diethyl
	1,12-dodecanedioate, respectively, with 1-thioglycerol
			Lipase activity a
	Substrates	Solvent MTBE	[units/g SEM] (n = x)
	1,12-Dodecanedioic acid
	1-thioglycerol	without	30.0 b ± 5.4 (n=4)
	1-thioglycerol	with	193.1 c ± 13.2 (n=8)
	Diethyl 1,12-dodecanedioate
	1-thioglycerol	without	18.3 b ± 1.8 (n=2)
	1-thioglycerol	with	31.6 c ± 3.6 (n=2)
	withdrawn at various intervals, extracted with tetrahydrofurane at 50°C and filtered through
	a 1 m PTFE syringe filter to separate the biocatalyst. Aliquots of the filtrate were
	analyzed by GPC/SEC
	c Same assay conditions, but before starting the reaction, ~0.5 mL methyl tert-butyl ether
	(MTBE) was added to dissolve diethyl 1,12-dodecanedioate or partially dissolve 1,12-
	dodecanedioic acid. After 10 min of incubation at 40°C, MTBE was evaporated in vacuo
	followed by drying at 1 kPa (40°C; 10 min). The reaction was started by adding the

a Esterification units were calculated from the initial rates (24 h) of polyesterification or polytransesterification reactions, as determined by GPC/SEC (see below). One esterification unit of enzyme activity was defined as the amount of enzyme (g) that produced 1 µmol of monoester/min, irrespective of the position of the esterified hydroxy group at the glycerol backbone. Small proportions of thioesters formed were also calculated as "esterification units". n=x, number of assays b Assay conditions: as given in Materials and methods. Samples of the reaction products were immobilized lipase and a moderate vacuum (80 kPa) was used to remove reaction water or ethanol. Incubations without Novozym 435 were used as blanks Table

2