Late non-fasting plasma glucose predicts cardiovascular mortality independent of hemoglobin A1c

Definition of fasting status

Fasting status was defined according to the American Diabetes Association (ADA) guidelines as a period of ≥ 8 h since last calorie intake^25,26,27,28 and non-fasting as a period of < 8 h since last calorie intake^25,26,27,28. The non-fasting period was further divided into two sub-periods: early non-fasting (a period of < 3 h since last calorie intake)^29,30 and late non-fasting (a period of 3–7.9 h since last calorie intake).

Study participants

This study used data from NHANES III (1988–1994) and the subsequent eight cycles of NHANES from 1999 to 2014. These cycles of NHANES were chosen because only these NHANES cycles had mortality data available³¹. A total of 35,070 adults aged ≥ 20 years had PG data. After exclusion of those without HbA1c (N = 112), without a fasting time (N = 19), or without a follow-up time (N = 32), 34,907 participants were included in the final analysis.

Ethical considerations

The National Center for Health Statistics Research Ethics Review Board (ERB) approved all study protocols (ERB Numbers: NHANES III, NHANES Protocol #98-12, NHANES Protocol #2005-06, and NHANES Protocol #2011-17)²¹. All procedures were performed following the guidelines of the Declaration of Helsinki. Written informed consent was obtained from all participants. The participants’ records were anonymized before being accessed by the authors.

Measurement of plasma glucose

PG was measured using the hexokinase-mediated reaction method³². In brief, hexokinase catalyzed the phosphorylation of glucose to glucose-6-phosphate, and the latter compound was then oxidized to gluconate-6-phosphate by glucose-6-phosphate dehydrogenase in the presence of nicotinamide adenine dinucleotide phosphate (NADP). The rate of formation of the reduced form of NADP (NADPH) during the second reaction was directly proportional to the glucose concentration and was measured photometrically.

Measurement of blood HbA1c

HbA1c in the whole blood was measured by an Automated Glycohemoglobin Analyzer³³. In brief, various forms of glycohemoglobin including HbA1c were separated by high-performance liquid chromatography (HPLC), and then be detected, and quantified by an analyzer at the absorbance of 415 nm. HbA1c was calculated as a percentage of the total amount of hemoglobin present in the sample.

Mortality from all causes and CVD

Data on mortality from all causes, heart diseases (I00-I09, I11, I13, I20-I51), and cerebrovascular diseases (I60-I69) were directly retrieved from NHANES-linked mortality files³¹. To evaluate mortality status and the cause of death, the National Center for Health Statistics conducted probabilistic matching to link the NHANES data with death certificate records from National Death Index records. The NHANES-linked mortality files used the Underlying Cause of Death 113 (UCOD_113) code to recode all deaths according to the International Classification of Diseases, 9th Revision (ICD-9) or the International Classification of Diseases, 10th Revision (ICD–10) for the underlying cause of death. Heart diseases included ischaemic heart disease (angina pectoris and myocardial infarction), heart failure, cardiac arrhythmias, cardiomyopathy, myocarditis, endocarditis, pericarditis, and valve disorders. Cerebrovascular diseases included hemorrhage stroke, ischemic stroke, occlusion and stenosis of precerebral or cerebral arteries without resulting in stroke, and other cerebrovascular diseases (e.g., cerebral aneurysm). CVD mortality included mortality from heart or cerebrovascular diseases. Follow-up time was the duration from the time when the participant was examined at the Mobile Examination Center until death, or until the end of follow-up (December 31, 2015), whichever occurred first³¹.

Covariates

Confounding covariates included age (continuous)³⁴, sex (male or female), ethnicity (non-Hispanic white, non-Hispanic black, Mexican–American, or other)³², obesity (underweight, normal, overweight, obese, or unknown)³⁵, education (< high school, high school, > high school, or unknown), poverty-income ratio (< 130%, 130%-349%, ≥ 350%, or unknown)³⁶, and survey periods (1988–1991, 1991–1994, 1999–2000, 2001–2002, 2003–2004, 2005–2006, 2007–2008, 2009–2010, 2011–2012, or 2013–2014). NHANES III was conducted in two stages, i.e., from 1988 to 1991 and then from 1991 to 1994, and the subsequent cycles of NHANES were conducted once every two years. Lifestyle confounders included physical activity (inactive, insufficiently active, or active)²¹, alcohol consumption (never, < 1 drink per week, 1–6 drinks per week, ≥ 7 drinks per week, or unknown)³⁷, and smoking status (past smoker, current smoker, or other). Clinical confounders included self-reported physician diagnosis of hypertension (yes, no, or unknown), self-reported physician diagnosis of hypercholesterolemia (yes, no, or unknown)³⁸, and HbA1c (continuous, natural log-transformed).

Statistical analyses

Data were presented as mean and standard deviation for continuous variables or percentages for categorical variables. Cox proportional hazards models were used to calculate hazard ratios and 95% confidence intervals of PG for CVD mortality and all-cause mortality. PG was treated as a continuous variable (natural log-transformed) or categorical variable (dichotomous, ≥ versus < a cut-off value). Sub-analyses were conducted in subgroups stratified according to HbA1c values: < 5.7% (normal), 5.7%-6.4% (prediabetes), or ≥ 6.5% (diabetes)⁵. Sensitivity analyses of the associations between PG and CVD mortality were conducted after exclusion of those who died of non-CVD causes.

The null hypothesis was rejected for two-sided values of P < 0.05. All analyses were performed using SPSS version 27.0 (IBM SPSS Statistics for Windows, Armonk, NY, IBM Corporation).