Background
Vitamin E is an important fat-soluble nutrient in the maintenance of health famous for its antioxidant functions, beneficial for a variety of disorders including cancer, heart disease and even Parkinson’s disease [
1‐
4]. Meanwhile vitamin E also appears to have a variety of roles depending on its non-antioxidant properties, such as modulation of monocyte function, inhibition of platelet aggregation, inhibition of smooth muscle cell proliferation and modulation of gene expression [
5]. Up to now, the extensive implications of vitamin E deficiency are increasing evident, especially in developing countries whose risk for deficiency is higher due to limited intake of the vitamins from food sources and greater oxidative stressors [
6]. As a result, we initiate the present study to evaluate vitamin E status in our city from China. As well known, vitamin E covers a group of eight compounds (α-, β-, γ-, δ-tocopherol, and α-, β-, γ-, δ-tocotrienol) which differ in their methyl substitution and saturation. Among them, the predominant form in the human body is α-tocopherol which demonstrates the highest vitamin E activity, comprising over 90% of vitamin E [
7‐
9]. Consequently, vitamin E status is always assessed by serum α-tocopherol concentration which provides the mostly used and direct way [
10]. It is reported that vitamin E circulating in blood is transported by lipoproteins, and vitamin E partitioning out of the cellular membrane compartment would increase with the elevation of serum lipid concentrations [
11]. Consequently, vitamin E deficiency may be underestimated without consideration of the lipid concentration in the case that serum lipid concentrations pathologically elevated, while may be overestimated under the situation that serum lipid concentrations are low. Many researches thus suggest that the correction of vitamin E for lipid concentrations is preferable to assess adequacy [
12]. Oftentimes, serum α-tocopherol concentration adjusted for serum total cholesterol (TC) or the sum of serum levels of cholesterol and triglyceride (TG) (the sum of cholesterol and triglyceride, TLs) is considered as a reliable indicator in identifying vitamin E deficiency [
13,
14]. Nonetheless, the vitamin E status of people in Wuhan from central China is less-known, and an understanding of the vitamin E distribution in our city should be useful to clinicians for clinical decision-making in public health practice.
In this study, both the circulating serum a-tocopherol concentrations and serum a-tocopherol concentrations adjusted for lipids have been used to assess the nutritional status of vitamin E of urban adults in Wuhan. Wuhan is located on the banks of the Yangtze River in central China, and the food consumption has dominated by traditional Chinese food characterized by grains and vegetables, with increasing intake of red meat, fruits, nuts, eggs, milk, river fish and plant oil, most of which are rich in vitamin E [
15,
16]. Thus, we hypothesized that the likelihood of vitamin E deficiency would be low in Wuhan in consideration of the Chinese food composition.
Methods
Study design and subjects
A single-center, cross-sectional study was performed. All subjects, residing in Wuhan, were enrolled from those who underwent physical examination program in 2019 at Tongji Hospital of Tongji Medical College of Huazhong University of Science and Technology (HUST) which is a large comprehensive hospital in Wuhan with abundant patients from local residents. The basic demographic characteristics of the participants including sex, age, BMI, blood pressure and long-term residence place were collected through medical records or face-to-face interview. This work was approved by the ethics committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology (IRB Approval Number: TJ-IRB20210807).
The sample size was calculated using the formula
N = [
Z1-α/2]
2 ×
P (1-
P)/
d2 [
17]. Where
N is the sample size,
Z1-α/2 (1.96) is the certainty wanted expressed in the percentage point of normal distribution corresponding to the 2-sided level of significant (
α = 0.05);
P (13%) is the global prevalence rate of vitamin E deficiency [
18];
d (3%) is the allowable error. Therefore,
N = [(1.96)
2 × 0.13 × (1–0.13)]/(0.03)
2 = 483. A non-response rate of 40% was added, giving a total sample of 805. In view of incomplete demographic data, a total of 850 samples were ready to be included. Finally, 846 samples were selected in real word according to the actual situations, including 471 males and 375 female aged 18 to 93 years (median age 47 years).
Overnight fasting blood samples were obtained by venipuncture. Sera were obtained by centrifugation of coagulated blood samples at 3000 rpm for 5 min at room temperature. These sera were frozen and stored at − 80 °C until analysis.
Measures
The serum α-tocopherol concentrations were determined by liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) on a ABsciex Qtrap 5500 coupled to an Exion LC system (Applied Biosystems, Foster City, CA, USA) with an electrospray ionization source in positive mode, and the testing kit was gotten from Beijing Health biotech Co. Ltd. (Beijing, China). This method was well validated with linearity, precision, accuracy, analytical sensitivity and matrix effect as demonstrated in Additional file
1: Table S1. The sera were processed as follows: 0.1 ml of serum was mixed with 0.1 ml of the internal standard (α-tocopherol-d6, Sigma-Aldrich) solution in a 1.5-ml centrifuge tube; 0.6 ml of hexane was then added and mixed thoroughly for 3 min using a vortex mixer. The tube was then closed and centrifuged for 10 min at 14680 rpm. The upper hexane extracts were evaporated by nitrogen and reconstituted in acetonitrile for LC–MS/MS analysis. A symmetry C18 column (100 × 2.1 mm, 3.5 µm, Waters, USA) was used for separation. The mobile phase was consisted of solvent A (water with 0.1% formic acid) and solvent B (2 mM ammonium acetate with 0.1% formic acid in methanol). The flow rate was 0.7 ml/min and column temperature was 60 °C. The concentrations of serum lipids, including TC, TG, high-density lipoprotein cholesterol (HDLC) and low-density lipoprotein cholesterol (LDLC), were measured on a Cobas 8000 system (Roche, Diagnostics, Germany). During the analysis, samples were protected from light and those showing signs of hemolysis were discarded.
In this study, the criterion for hypercholesterolemia is defined as TC ≥ 5.2 mmol/L [
19]. The normal blood pressure recommended by WHO is < 140/90 mmHg [
20]. The standard weight status categories associated with BMI ranges for adults are: BMI below 18.5 is associated with ‘underweight’ weight status; BMI 18.5–24.9 is associated with ‘normal’ weight status; BMI 25.0–29.9 is associated with ‘overweight’ weight status; BMI 30.0 and above is associated with ‘obese’ weight status [
21]. The vitamin E status categories for healthy adults are classified as follows [
18,
22]: vitamin E serum concentrations ≤ 12 μmol/L is considered as functional deficiency; between 13 and 29 µmol/L is considered as suboptimal status; ≥ 30 μmol/L is considered as desirable status. The prevalence rate of vitamin E deficiency according to its status categories defined for healthy adults, and its comparison with other countries have been investigated.
Statistical analysis
We present the distribution of concentrations of vitamin E and the ratios of concentrations of vitamin E to serum lipids for all participants. All data were expressed as medians and interquartile ranges (IQRs). Data normality was analyzed using the Kolmogorov–Smirnov test. Student’s t test and analysis of variance (ANOVA) based on normally distributed data, or Mann–Whitney U test and Kruskal–Wallis test based on non-normally distributed data were applied to compare the means of serum concentrations of α-tocopherol across genders, age groups and strata. Pearson’s Chi-square test or Fisher’s exact test was performed to analyze the categorical data. Spearman’s correlation test was used to determine the strength of correlation between variables, such as vitamin E, age, BMI, blood pressure, TC and TG.
A multivariate logistic regression was performed to assess odds ratios (ORs) regarding Vitamin E-associated factors. Variable with a global value P < 0.10 in the univariate analysis were entered into multivariate analyses. A P-value below 0.05 was considered statistically significant. Analyses were done using SPSS (version 20.0; SPSS, Isnc., Chicago, Ill, USA). The Correlation analysis heatmap was plotted by R language.
Discussion
As the diverse role of vitamin E, especially function as a potent antioxidant in the maintenance of health and prevention of disease, the extensive implications of its deficiency are increasingly evident. As usual, serum vitamin E should be transported to tissues for exerting their functions by using lipoproteins as the major carries [
23]. In humans, vitamin E is mostly transported in LDL and HDL at similar proportions with less carried in VLDL and other lipoproteins. As a result, vitamin E homeostasis is intimately connected to lipoprotein metabolism in vivo. Assessment of vitamin E status not only depends on its concentration, but also on the concentrations of the circulating lipoproteins. In fact, vitamin E deficiency is rare in humans. When it occurs, it is a result of lipoprotein deficiencies or lipid malabsorption syndromes. It is important to identify patients who have real vitamin E deficiency or other abnormalities known to cause vitamin E deficiency [
24]. Thus, concentration of vitamin E, both unadjusted and adjusted for cholesterol or total lipids, should be used to evaluate vitamin E adequacy [
25]. This study confirmed that the prevalence rate of vitamin E deficiency is low in urban adults of Wuhan based on both unadjusted and lipid-adjusted vitamin E levels, which provided valuable information about the distribution of vitamin E in central China.
In this study, we found that the vitamin E level was not associated with sex as revealed in Table
1 (27.63 vs 27.16 μmol/L,
P = 0.683), whereas the vitamin E level slightly increased with age in Table
2. The results were consistent with the previous report [
26]. Meanwhile, the levels of BMI, SBP, DBP, TG and LDLC were significantly higher in men, mostly in consistent with Sun’s work [
27]. However, an opposite direction of association in lipoprotein between men and women was found in Qi’s study that TC was significantly lower in men with no TG difference [
28], which might be due to dietary intake or life habit in different regions. Likewise, it is well understood that aging process affect BMI, blood pressure and lipoprotein which are risk factors for cardiovascular diseases as Yao’s or Mendes’s report [
29,
30].
In order to learn about the vitamin E level in these subjects with risk factors for cardiovascular diseases potentially imposing a great threat to human health, the subjects were divided into subgroups according to TC level, blood pressure and BMI. The transport of vitamin E is closely related to lipoprotein [
24]. Therefore, hypercholesterolemia might affect the vitamin E level. As demonstrated in Fig.
1, the level of vitamin E is positively correlated with TC, TG, LDLC and TLs (
P < 0.05), and much higher level of the vitamin E is found in subjects with hypercholesterolemia. In general, the participates who have increased blood lipid concentration also have increased serum vitamin E level, accompanied by evidently decreased lipid-adjusted vitamin E levels [
25,
31]. This might be ascertained to the increased serum carriers for delivery of vitamin E in tissues [
11]. Although TC level, blood pressure and BMI are highly correlated with each other as displayed in the Spearman analysis (Fig.
1) and stratification analysis (Additional file
1: Tables S2, S3, S4) [
32], no significant difference of vitamin E, vitamin E/TC or vitamin E/TLs is found between subgroups based on blood pressure or BMI except that vitamin E/TLs is lower in subjects with hypertension. Obesity might be the most important factor associated with blood pressure, followed by hyperlipidemia [
33], which contributes to the consistent result between blood pressure and BMI.
In this study, a considerable proportion of population presents suboptimal vitamin E status (61.47%), while a very small population exhibits vitamin E deficiency (0.47%). Both age and TC level are significantly correlated with vitamin E status, which are indirectly evidenced by associations of vitamin E concentrations and variables (Table
2 and Table
3). It is found that almost equal proportions of the men and women in distribution of vitamin E. However, the young adults (18–39 years old) exhibited lower proportion of desirable vitamin E level than that of older, which was not consistent with Oldewage-Theron’s study [
34]. Perhaps, the older adults in urban China have paid more attention to vitamin/trace element supplements in daily life due to their enhanced awareness of health. However, poor food intake as well as a monotonous diet in the elderly above 70 years old made the prevalence of vitamin E deficiency suffered. In addition, the vitamin E level in different countries is summarized in Table
6 [
18,
26,
34‐
38]. Compared to the results from other countries, the serum vitamin E level in our subjects is higher than that of most countries and in parallel with that of US, even above the global level. Given that the cut-off point of vitamin E is 12 μmol/L, the prevalence of vitamin E inadequacy for urban adults in Wuhan from central China is 0.47%, much lower than that of other countries. Meanwhile, if 11.6 μmol/L was adopted as the cut-off value [
39], the deficiency rate was 0.24%. It is noted that there is a great variation in blood lipids across nations and vitamin E is transported in lipoprotein fraction in the blood which is determinant for its concentration. However, a few studies provide lipid-adjusted vitamin E, thus it is not clear that whether the prevalence defined is comparable in various countries. If vitamin E deficiency was expressed as < 2.5 mmol/mol total cholesterol or expressed as < 1.59 mmol/mmol (total cholesterol + triacylglycerols) which was too uncommon to report [
36], the prevalence of deficiency was 0.47% or 0.35% in Wuhan, respectively. Inspiringly, no matter what methods were used to adjust or evaluate for vitamin E concentration, the subjects in this study were identified as being vitamin E sufficient in comparison with other countries or regions. The low prevalence of deficiency in present study might due to the fact that the Chinese diet depends on vegetable oils, meat, green leafy vegetables, cereals, wheat germ and egg yolk, which are known to be the principal dietary sources of vitamin E [
40].
Table 6
Comparison of the vitamin E status between Wuhan in central China and other countries
This study, 2020 | Wuhan, central China | 846 | 18–93 | Serum α-tocopherol ≤ 12.0 μmol/L (I) OR < 11.6 (II) OR serum α-tocopherol: cholesterol < 2.5 mmol/mol (III) OR α-tocopherol:(cholesterol + triacylglycerols) < 1.59 mmol/mol (IV) | 0.47(I) 0.24(II) 0.47(III) 0.35(IV) | 27.40 | 6.20 |
| Global | 132 studies | NA | Serum α-tocopherol ≤ 12.0 μmol/L | 13 | 22.1 | NA |
Oldewage-Theron et al., 2009 [ 34] | Sharpeville, South Africa | 235 | 60–93 yr | Serum α-tocopherol < 2.8 μmol/L (I) OR < 3.7 μmol/L (II) | 20.9 (I) 16.2 (II) | 4.8 | NA |
Assantachai et al.,2007 [ 35] | Thailand | 2336 | ≥ 60 yr | Plasma α-tocopherol < 14 μmol/L | 55.5 | NA | NA |
| US | 4087 | ≥ 20 yr | Serum α-tocopherol < 11.6 μmol/L | 0.50 | 27.39 | 4.93 |
| Beirut, Lebanon | 857 | 25–64 yr | Plasma α-tocopherol < 5.8 μmol/L (I) OR < 11.6 (II) OR plasma α-tocopherol: cholesterol < 2.5 μmol/mmol (III) | 0.7 (I) 3.7 (II) 4.1 (III) | 24.5 | 4.67 |
| Northern Cameroon | 81 | 3–61 yr | Serum α-tocopherol < 5.8 μmol/L (I) OR < 11.6 μmol/L (II) | 12.3 (I) 33.3 (II) | 12.2 | NA |
| Taiwan | 1841 | ≥ 19 yr | Serum α-tocopherol < 11.6 μmol/L | 7.2 | 20.0 | NA |
Certain limitations should be noted when interprets the result of this study. Firstly, the results are based on cross-sectional data and the sample size is small. Secondly, this a single-center study, and thus its explanatory power is limited. Thirdly, the information for dietary supplements is deficient to know about the relationship between food sources and the serum vitamin E level.
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