Association of baseline as well as change in lipid levels with the risk of cardiovascular diseases and all-cause deaths

High baseline atherogenic lipid level has been an established risk factor for the risk of cardiovascular events. Evidence concerning the role of lipid changes in cardiovascular and death risks are inconclusive. A cohort study was conducted based on the Taiwanese Survey on Hypertension, Hyperglycemia, and Hyperlipidemia (n = 4072, mean 44.8 years, 53.5% women) assessing lipid levels of the participants repeatedly measured in 2002 and 2007. Combined baseline and changes in lipid levels were classified into four groups—stable or decreasing lipid changes and increasing lipid changes with low- and high-risk baseline lipid levels. Developing cardiovascular events (n = 225) and all-cause deaths (n = 345) were ascertained during a median follow-up of 13.3 years. Participants with increasing and higher total cholesterol level were more likely to develop cardiovascular risks. Similar patterns for cardiovascular events were observed across other lipid profile changes. However, participants with increasing total cholesterol, LDL-C, and non-high-density lipoprotein cholesterol (non-HDL-C) levels were more likely to be at a lower risk for all-cause deaths. Baseline and changes in total cholesterol, triglycerides, and LDL-C levels were positively associated with the risk of cardiovascular diseases, whereas baseline and changes in total cholesterol and LDL-C and non-HDL-C levels were inversely associated with all-cause deaths.

Data collection and measurements. Blood sampling was performed after a 12-h fasting period. The LDL-C level was measured using the Friedewald equation, i.e., "total cholesterol − HDL-C − (triglyceride/5), " in TwSHHH 2002 and by direct measurement with homogeneous assays in TwSHHH 2007. Total cholesterol and triglyceride levels were examined by colorimetry (Bucolo method), and HDL-C was examined by electrophoresis. Apo B and hsCRP levels were measured by immunoturbidimetric methods. The coefficients of variations of these laboratory measurements were approximately 5%. A standard protocol officially registered by the Taiwan Health Promotion Administration, Ministry of Health and Welfare was applied for health examinations and data collection.

Definition of exposures.
All lipid profile levels, including total cholesterol, triglyceride, LDL-C, non-HDL-C, and apo B, in TwSHHH 2002 were divided into quartiles. For the change value in lipid profiles between TwSHHH 2002 and 2007, they were divided into increasing, stable, and decreasing groups. Exposure was assessed by a two-step combination. In step 1, the baseline lipid levels were divided by quartiles. In step 2, baseline and change in lipid levels were combined. For baseline lipid levels, low-risk groups were defined as the lipid levels in the 1st and 2nd quartiles of the atherogenic (apo)lipoproteins, while the high-risk groups were defined as lipid levels in the 3rd and 4th quartiles of these lipids.
Although several variability indicators are commonly used to describe the changes in biochemistry data, such as standard deviation, variability independent of the mean, maximum-minimum difference, and average real variability, we chose standard deviation to present the change in lipid level due to following three reasons. First, standard deviation and variability independent of the mean are indicators to describe total variability of biochemistry data; however, standard deviation is generally known and easily computed. Second, standard deviation is suitable to describe data variability with different time intervals, either short term or long term, whereas average real variability is more appropriate for short-term variability and requires multiple measurement of lipid levels. Third, compared with maximum-minimum difference, standard deviation is less likely to be influenced by extreme observations [26][27][28] . For the change in lipid profiles, stable lipid change was defined as lipid change between TwSHHH 2002 and 2007 within ± 0.25 standard deviation of the baseline lipid level; decreasing lipid change was defined as lipid changes between TwSHHH 2002 and 2007 with 0.25 standard deviation of the baseline lipid profile or lower. Increasing lipid change was defined as lipid changes between TwSHHH 2002 and 2007 with 0.25 standard deviation of baseline lipid profile or higher. Then, four groups of combined lipid change were defined as follows: (1) low-risk group with stable or decreasing lipid levels, (2) low-risk group with increasing lipid levels, (3) high-risk group with stable or decreasing lipid levels, and (4) high-risk group with increasing lipid levels.

Definition of outcomes.
Each participant in the TwSHHH database was linked to the NHIRD to determine their outcomes. The primary endpoint was the incidence of CVD, including, coronary heart disease and ischemic stroke. The secondary endpoint was all-cause death. The outcomes of coronary heart disease and ischemic stroke were ascertained in NHIRD database by using the International Classification of Diseases, Ninth Definitions of covariates. Significant covariates including age, sex, BMI, parental history of CVD, menopause status, smoking status, exercise, alcohol use, educational level, income level, and marital status were collected by questionnaires. Hypertension was defined as an average systolic blood pressure (SBP) of ≥ 140 mmHg or an average diastolic blood pressure (DBP) of ≥ 90 mmHg or use of antihypertensive medications more than 84 days within 1 year before the index day (JNC-7). Diabetes was defined as a fasting plasma glucose of ≥ 126 mg/ dL or a hemoglobin A1c of ≥ 6.5% or use of antidiabetic agents more than 84 days within 1 year before the index day (Table S1) 29 .

Statistical analyses.
Relationships between baseline lipid marker levels were examined by the Pearson correlation coefficients. The correlation coefficients between the baseline lipid level and change in lipid levels was calibrated using Oldham's method 30 . The Cox proportional-hazards regression was used to compare the hazard ratios (HRs) for the outcomes after the proportionality assumption was verified 31 . Potential confounders were adjusted via three models: model 1, adjusted for age group and sex; model 2, adjusted for factors in model 1 plus BMI, smoking status, alcohol use, regular exercise, income level, and educational level; and model 3, adjusted for factors in model 2 plus diabetes, hypertension, menopause, family history of CVD, and hsCRP level. Subgroup analyses were done to explore potential effect modifiers stratified by age, sex, LDL-C targeted or not, and median hsCRP. Sensitivity analyses were also performed to judge the robustness of our

Results
The study population included 4072 participants free of CVD who had data on lipid profiles at both baseline and follow-up surveys. The population's mean (SD) age was 44.8 (14.9) years, and 53.5% were women. In total, 612 participants were excluded, among them were 109 participants who were pregnant within 1 year in TwSHHH 2002 and 2007, 341 who were < 20 years old, 10 who had missing data, and 152 who had established CVD before TwSHHH in 2007. The study flow chart is shown in Fig. 1. During a median follow-up of 13.3 years for CVD, coronary heart disease, and ischemic stroke and 13.4 years for all-cause death, we documented 252 cases of incident CVD, 155 of incident coronary heart events, 112 of incident ischemic strokes, and 345 of all-cause deaths.
Baseline characteristics of the study population in each non-HDL-C quartile are displayed in Table 1. Participants with higher non-HDL-C levels tended to be men, older, and heavier. Hypertension, diabetes mellitus, www.nature.com/scientificreports/ menopause, current smokers, high monthly income level, family history of CVD, and failure of LDL-C target achievement were more common in the higher non-HDL-C quartiles. Higher non-HDL-C quartiles were positively associated with higher levels of total cholesterol, triglycerides, LDL-C, apo B, and hsCRP. Lifestyle factors such as alcohol consumption and exercise habits did not vary across various non-HDL quartiles. The range and median value of baseline lipid level specified by combined lipid change are demonstrated in Table S2.1 and S2.2. We found strong correlations between baseline total cholesterol, triglycerides, LDL-C, non-HDL-C, and apo B www.nature.com/scientificreports/ (correlation coefficients ranging from 0.78 to 0.91). In addition to correlation among total cholesterol and LDL-C, non-HDL-C was higher than that with apo B, both in the baseline lipid levels and lipid change (Table S3). Proportional hazard assumption was verified in the Cox models. Table 2 shows the HRs and 95% confidence intervals (CIs) for CVD according to various lipid profiles at baseline. After adjustment for potential confounders, the HR for the comparison of the participants was 1.70 (95% CI, 1.00-2.89; P for trend, 0.07) in the fourth and first total cholesterol quartiles, 1.21 (95% CI, 0.70-2.10; P for trend, 0.36) in the fourth and first triglyceride quartiles, 1.51 (95% CI, 0.89-2.59; P for trend, 0.16) in the fourth and first LDL-C quartiles, 1.70 (95% CI, 0.94-3.09; P for trend, 0.17) in the fourth and first non-HDL-C quartiles, and 2.05 (95% CI, 1.03-4.08; P for trend, 0.08) in the fourth and first apo B quartiles. Similar patterns for coronary heart disease and ischemic stroke were observed. However, no relationships between baseline lipid profile and risk of all-cause deaths were observed (Table S4). Figure 2A shows the joint analyses of baseline and change in lipid levels for CVD risk. Compared with participants with decreasing and lower total cholesterol levels, those with increasing and higher total cholesterol levels were more likely to develop CVD (HR, 1.38, 95% CI, 0.81-2.35, for low total cholesterol levels; HR, 1.70, 95% CI, 1.01-2.84, for high total cholesterol levels). Similar patterns for cardiovascular events were found with Table 2. Hazard ratios (and 95% CI values) of cardiovascular disease risk during a median 13.3 years of follow-up according to baseline lipid profiles in TwSHHH 2002. Incidence rates are presented per 1000 person-years; Model 1: adjusted for age groups (20-64/ ≥ 65 years old) and sex. Model 2: as for model 1 plus body mass index (18.4/18.5-23.9/24.0-26.9/ ≥ 27 kg/m2), current smoker (yes/no), alcohol drinking (yes/no), marital status (single, divorced or separated/Living with spouse), regular exercise habit (yes/no),education level (9 years/at least 9 years), income level (monthly income level < 40,000, ≥ 40,000 New Taiwan Dollars). Model 3: as for model 2 plus baseline hypertension (yes/no), diabetes mellitus (yes/no), menopause(yes/no), family history of CVD (yes/no), and hsCRP level. We found that increasing lipid change was inversely associated with all-cause death (Fig. 2B). Compared with participants with decreasing and lower non-HDL-C levels, those with increasing non-HDL-C levels were likely to have a lower risk for all-cause death (HR, 0.59, 95% CI, 0.37-0.94, for low non-HDL-C level; HR, 0.57, 95% CI, 0.36-0.90, for high non-HDL-C level). Similar patterns for all-cause death were observed across other lipid changes.
Subgroup analyses revealed that age may be an effect modifier for the association between baseline lipid levels and the risk of CVD (interaction p as 0.033 in non-HDL-C), indicating that elevated non-HDL-C levels had more pronounced effects on the risk of CVD in the younger population than the older population (Table 3). We found the association between baseline apo B levels and the risk of coronary heart disease varied by the status of LDL-C target achievement. This suggests that elevated apo B levels potentially play an essential role in the risk of coronary heart disease in the population who achieved their LDL-C targets (interaction p as 0.023 in apo B) (Table S5.1).
The sensitivity analyses demonstrated that our results were consistent with those obtained in the main analyses (

Discussion
Our study suggested a positive association between baseline lipid levels and the incidence of CVD, including coronary heart disease and ischemic stroke in Taiwanese populations. These findings were more pronounced in populations aged < 65 years. Higher baseline levels of apo B had a stronger risk of coronary heart diseases in the target achievement of LDL-C levels. Furthermore, the increasing change in lipid levels was significantly associated with increased future risk of CVD, including coronary heart disease and ischemic stroke. No significant association between baseline lipid level and the risk of all-cause death was found, while increasing lipid change was significantly inversely associated with all-cause death.
Previous studies have demonstrated the consistency of high baseline lipid levels with the risk of CVD, coronary heart disease, and ischemic stroke 6,[9][10][11][33][34][35] . The relationship between (apo)lipoproteins with CVD may be distinct in different populations such as younger individuals, women, and populations who achieved their LDL-C target 11,22,35,36 . Our data consistently suggested that younger populations with higher baseline lipid levels www.nature.com/scientificreports/ had significantly greater risks of CVD. Furthermore, LDL-C achievement in our study consistently acted as an effect modifier between non-LDL (apo)liproteins and cardiovascular events. While many studies have evaluated the association between baseline lipid levels and the risks of CVD and all-cause death, limited evidence elucidated the relation of the change in lipid levels to the risks of CVD and allcause death [13][14][15][16][17][18][19]37,38 . In a cohort study including 2,682,045 Korean participants using category change according to the cholesterol levels between the two periods (sustained low, low-middle, low-high, middle-low, sustained middle, middle-high, high-low, high-middle, and sustained high groups) to describe cholesterol variability, the HRs for comparisons of the increasing and stable total cholesterol group were 1.21 (95% CI, 1.03-1.42, in the low-high group) for coronary heart disease and 1.24 (95% CI, 1.05-1.47, in low-high group) for cerebrovascular disease 13 . In the present study, we explored the association between cardiovascular risks and various atherogenic (apo)lipoproteins (both the baseline and the change in lipid levels), which provided considerable information of their roles in the atherogenesis in coronary heart disease and ischemic stroke.
A novel finding in our study was that increasing apo B level showed no higher cardiovascular risks, implying that apo B may play a different role in the formation and progression of atherosclerosis than did total cholesterol, LDL-C, and non-HDL-C.
The inconsistent results of studies elucidating the relationship between baseline lipid level and all-cause death risk may be attributed to the difference in age distribution. Older population tended to yield U-curve or inverse associations between baseline lipid levels and all-cause deaths [16][17][18]35 . The lack of association between baseline lipid level and all-cause death in our study may be due to a broad range of age distribution (20-93 years).
Epidemiological studies of lipid changes and all-cause death are limited and have yielded inconsistent findings 19,20,37 . In an 8.0-year follow-up cohort study of 269,391 Koreans, change in total cholesterol levels, especially in decreasing change from high baseline total cholesterol levels (3rd tertile to 1st tertile) had higher risk of all-cause death (HR, 1.47, 95% CI, 1.32-1.64) compared with stable middle total cholesterol levels 18 . However, in a meta-analysis of lipid-lowering trials, Cholesterol Treatment Trialists' Collaboration reported that decreasing LDL levels reduced all-cause death risk (RR, 0.91, 95% CI, 0.88-0.93, per 1 mmol/L reduction in LDL-C) 20 . The inconsistency may occur because the populations in lipid-lowering trials had higher baseline cardiovascular risk, and their CVD-related deaths accounted more for all-cause death compared with that in population-based studies 4,[39][40][41][42][43][44][45] .
Atherogenic (apo)lipoproteins act differently in atherogenesis 6 . Total cholesterol represents the total cholesterol mass in all lipoprotein particles. Non-HDL-C represents cholesterol mass of very-low-density lipoprotein (VLDL), intermediate density lipoprotein (IDL), and LDL particles. LDL-C is the cholesterol mass of LDL particles. Apo B reflects the total number of atherogenic lipoprotein particles because each of these lipoproteins contains one apo B molecule. Baseline apo B contributing more risk of cardiovascular events than the change in apo B level may indicate that the atherogenic risk is more closely related to the influx of cholesterol mass to the arterial wall than to the particle numbers 6,9,11,46,47 .
The positive association between decreasing lipid change and all-cause death may be explained by at least two potential mechanisms. First, the drop in lipid levels indicate reduced resistance to oxidative stress due to lipid and may play an essential role while cells are involved in tissue repair 17 . Second, a decrease in lipid levels may interfere with the binding of lipopolysaccharide from microorganisms, which increases the risks of infection 38 . Third, lipids play an essential role in cell stability. Decreasing lipid changes can disturb cell integrity and cell growth, which may increase the risk of immunological and hematological diseases and malignancy 35,38 . Lastly, the decline in lipid levels may be attributed to malnutrition or diseases in a subclinical stage, which further increases the risk of mortality 18,37 .
For primary prevention of CVD, lipid change is as important as baseline lipid levels in predicting the risks of CVD, including coronary heart disease and ischemic stroke. This finding reminds clinicians that monitoring www.nature.com/scientificreports/ baseline lipid levels and the trend of the lipid change is necessary 34,48 . To reduce these lipid profiles, early intervention should be implemented even in relatively low baseline lipid level and younger populations to prevent cardiovascular events. In addition to LDL-C, the established primary target to prevent CVD and the monitoring of other atherogenic (apo)lipoprotein levels cannot be ignored 13,34 . In populations that achieved the LDL-C target, lowering baseline apo B is beneficial and necessary to reduce the lipid-related residual cardiovascular risks 3,5 . Furthermore, our study showed that those with decreasing lipid change had increased mortality risk. In case of decreasing lipid levels, awareness of potential preclinical nonatherosclerotic diseases to prevent further elevated all-cause death risk is strongly recommended for primary care providers.

Strengths and limitations.
This study has the following strengths. First, to our knowledge, this study is the first extensive investigation of baseline lipid level as well as lipid change and the risk of CVD, including coronary heart disease and ischemic stroke among the Taiwanese population. Evidence of single lipid profile change being positively associated with CVD risk was found in Asian populations, but no study has investigated combined baseline and change in lipid profiles in predicting CVD. Our study provided robust evidence by exploring the relations of various combinations of baseline lipid and lipid change level to the risks of CVD, including coronary heart disease and ischemic stroke, and all-cause death. Second, our study is a nationwide representative cohort study with ascertained outcomes based on the TwSHHH database. The community-based populations from the TwSHHH database may reduce the selection bias. The TwSHHH database included important clinical laboratory variables and socioeconomic and lifestyle factors to adjust for potential confounding factors. Finally, our study emphasized the importance of lipid-related residual cardiovascular risk.
Our study also has several potential limitations. First, this cohort study was restricted to the Taiwanese population; thus, our results lacked external generalizability. Second, relatively few incident cases of cardiovascular events were observed for risk estimations. However, a median 13.3-year follow-up made our results robust. Third, limited information elucidating the influence of medication use on CVD was due to the primary prevention study design. Fourth, apart from standard deviation, variability independent of the mean, maximum-minimum difference and average real variability are also indicators to describe the lipid variability. However, we have limited data measurement points currently. Future longitudinal data with enough data points of lipid level may be considered to elucidate the lipid variability more thoroughly.
In conclusion, this prospective study provided suggestive evidence on how baseline and changes in lipid profile were associated with the risks of cardiovascular diseases and all-cause death. Baseline and changes in total cholesterol, triglycerides, and LDL-C levels were positively associated with the risk of cardiovascular diseases, whereas baseline and changes in total cholesterol, LDL-C, and non-HDL-C levels were inversely associated with all-cause death. Further studies about biological mechanism for the role of baseline and change in lipid levels are warranted.