Study design and setting
The present analysis is based on the mortality follow-up of the German National Health Interview and Examination Survey 1998 (GNHIES98) sample. The GNHIES98 was conducted by the Robert Koch Institute between October 1997 and March 1999 and included 7124 adults representative of the 18 to 79-year-old residential, non-institutionalized population in Germany. Details of the study design, sampling procedure, response rates and data collection have been published previously [
16]. In brief, a two-stage cluster sampling procedure was applied for the selection of survey participants (response rate: 61.4%). In the first stage of sampling, sample points (i.e. study locations) reflecting community sizes and structures in Germany were drawn and in the second stage, random samples stratified by sex and age were selected from local population registries proportional to the sex and age structure of the population in Germany.
The survey included a standardized computer-assisted personal interview (CAPI) administered by specifically trained study physicians, a standardized self-administered questionnaire which was checked by trained interviewers for plausibility and completeness, and a physical examination, including standardized anthropometric measurements and blood sampling as well as a detailed medication review conducted by trained health professionals. Blood samples were drawn over the day and processed within one hour and stored at − 40 °C until analysis in the central laboratory unit at the Robert Koch Institute. Any medications taken in the past 7 days were recorded using the unique medication identifiers (“Pharmazentralnummer”, PZN) on the original medication containers brought to the study center by study participants for this purpose. The PZN was used for medication coding according to the WHO “Anatomical-Therapeutic-Chemical” classification system (ATC-Code).
The vital status could be assessed for 6979 among the 7124 GNHIES98 participants (98.0%) by the mortality follow-up as previously described in detail [
17]. Briefly, all GNHIES98 participants who had agreed to follow-up contacts were recontacted between October 2008 and October 2011 and invited to participate in the first wave of the German Health Interview and Examination Survey for Adults (DEGS1). For participants who did not respond to the invitation, vital status was obtained from local population registries including the date of death for deceased individuals. Surviving GNHIES98 participants were censored at the last date of contact, i. e. the date of contact to the population registry for non-respondents, the date of refusal for those who actively declined participation in DEGS1, or the date of DEGS1 participation for those who also took part in DEGS1.
The GNHIES98 and its mortality follow-up were approved by the Federal Office for the Protection of Data (Germany). All participants provided written informed consent before enrolment.
Study population
Among 7124 participants of the GNHIES98 study, a number of 6750 individuals reporting no history of physician-diagnosed diabetes and no use of antidiabetic medication within the last seven days preceding the interview were defined as free of known diabetes at the baseline examination and comprised the source population of the present study. After sequential exclusion of participants with no information regarding vital status (n = 138), known diabetes (n = 25), random glucose level or fasting time (n = 336) or main covariates used in the present analyses (n = 299), the final study population consisted of 5955 participants (2919 men and 3036 women) aged 18–79 years at baseline.
Assessment of random glucose and fasting time
Random glucose was determined by standardized measures of serum glucose drawn from fresh whole blood specimens randomly taken over the day using glukose-oxidase-peroxidase-4-aminophenazon-phenol by a MEGA analyzer (Merck, Darmstadt, Germany). For analyses as categorical variable, random glucose level was classified in seven categories (< 4.3, 4.3 - < 5.3, 5.3 - < 5.8, 5.8 - < 6.8, 6.8 - < 7.8, 7.8 - < 11.1 and ≥ 11.1 mmol/L) as previously suggested [
14]. The two highest categories followed classifications concerning oral glucose tolerance 2-h postload thresholds for assessing diabetes and impaired glucose tolerance [
4].
Fasting time was defined as the difference between time of drawing blood specimen and time since the last meal was taken, the latter based on self-report. For specific analyses, fasting time was classified into the categories < 2, 2 - < 4, 4 - < 8, 8 - < 12 and ≥ 12 h.
Assessment of covariates
Covariates were chosen a priori to control for potential confounding of the relationship between random glucose and all-cause mortality. Age, sex, educational level, smoking status, alcohol intake and physical activity were obtained by self-administered questionnaire, anthropometric measures by physical examination, history of chronic diseases by CAPI and HbA1c by blood sampling.
Educational level was assessed by the Comparative Analysis of Social Mobility in Industrial Nations (CASMIN) instrument, encompassing general as well as vocational training, and classified into the categories low, medium or high [
18].
Smoking status was categorized as never, former and current smoking. Alcohol intake (g/day) was obtained by a semi-quantitative food frequency questionnaire [
19] and classified into no, moderate (> 0 - < 20 g/day in men, > 0 - < 10 g/day in women) and high (≥ 20 g/day in men, ≥ 10 g/day in women) alcohol intake. Physical activity was assessed by five categories as no sport, < 1 h/week, regularly 1–2 h/week, regularly 2–4 h/week, or regularly > 4 h/week. This information was aggregated into two categories (< 2 h/week or ≥ 2 h/week) for the present analysis. Body mass index (BMI) was calculated as the ratio of body weight (kg) and height squared (m
2). History of myocardial infarction, stroke, cancer, hypertension and hyperlipidemia were chosen as chronic diseases and were each defined as no or yes.
HbA
1c was measured in fresh whole blood specimens with a Diamat high-performance liquid chromatography analyzer (Bio-Rad Laboratories, Munich, Germany) and reagents of Recipe (Recipe Chemicals and Instruments, Munich, Germany) in the Robert Koch Institute Central Epidemiological Laboratory [
20]. For specific analyses, HbA
1c level was classified into < 5.0, 5.0 - < 5.7, 5.7 - < 6.5 and ≥ 6.5% as previous studies showed a U form relation of HbA
1c and all-cause mortality [
8,
21].
Statistical analyses
Unadjusted analyses were performed by the Rao-Scott χ2 test for associations between random glucose categories and categorical variables and by the F test for mean differences for continuous and (approximately) normally distributed variables across random glucose categories. Spearman correlation was applied to assess a potential monotonic relationship between continuous random glucose and fasting time. To display distribution measures of continuous random glucose across five fasting time categories, a Box plot was created. Crude mortality rates were calculated by dividing the number of deaths by the number of person-years observed within each random glucose category.
Adjusted analyses were performed by Cox proportional hazards regression models to estimate random glucose-specific hazard ratios (HRs) including 95% confidence intervals (95% CIs) for all-cause mortality during follow-up. As basic model, model 1 was adjusted for age (continuous) and sex. Further adjustments were made for the sociodemographic factor educational level in model 2, additionally for the lifestyle factors smoking, alcohol consumption and physical activity as well as the anthropometric factors BMI (continuous) and waist circumference (continuous) in model 3, and additionally for the five chronic diseases history of myocardial infarction, stroke, cancer, hypertension and hyperlipidemia in model 4.
First, random glucose was included in the Cox regression models as categorical variable using a level of 4.3 - < 5.3 mmol/L as reference. Secondly, random glucose was included as continuous variable in the Cox regression and modelled by a spline regression approach applying restricted cubic spline functions with four knots set at the 5th, 25th, 75th and 95th percentile and choosing the median random glucose level of 5.2 mmol/L as reference.
Sensitivity analyses were performed to assess the stability of the main analyses and comprised a) an additional adjustment for fasting time (< 2, 2 - < 4, 4 - < 8, 8 - < 12, ≥ 12 h), b) an exclusion of participants with fasting time < 2 h, c) an additional adjustment for HbA
1c (< 5.0, 5.0 - < 5.7, 5.7 - < 6.5 and ≥ 6.5%), d) an exclusion of participants with HbA
1c ≥ 6.5%, e) an exclusion of participants with follow-up time ≤ 2 years and, finally, f) an analysis in the source population (
n = 6750) with multiple imputation of missing values using the “Fully Conditional Specification” method [
22]. Furthermore, interaction analyses were carried out to examine potential modifications of the random glucose-mortality association by age, BMI and waist circumference (all continuous) and sex with adding the respective interaction terms (random glucose x modifier) to the Cox regression model 4 (described above).
Statistical analyses were performed by the statistical software package SAS version 9.4 (SAS Institute, Cary, NC, USA) using the survey procedures SURVEYMEANS, SURVEYFREQ and SURVEYPHREG to account for the complex survey design, except for analyses regarding Spearman correlation and spline modelling in the Cox regression (see below). The analyses included a survey weight accounting for deviations of the study population from the population in Germany as of December 31, 1997 within strata of sex, age, education, nationality, community size, federal state, and east/west Germany which may compensate under- or over-represented groups within these strata. Spearman correlation analyses were unweighted using CORR and spline modelling in the Cox regression was performed by PHREG which permitted consideration of weights but not accounting for cluster sampling.
P values < 0.05 were considered to indicate statistical significance. The study followed the STROBE guidelines for cohort studies [
23].