Association of low vitamin C concentrations and low consumption of fresh fruit and vegetables with cardiovascular disease in type 2 diabetes

Abstract

Purpose

Vitamin C is a fundamental antioxidant with important metabolic actions in several biological processes. Patients with type 2 diabetes (T2D) are exposed to oxidative stress as a consequence of an increased production of reactive oxygen species (ROS). The aims of the present study were to estimate the prevalence of vitamin C deficiency in ambulatory patients with T2D, to study the relationship between vitamin C levels and cardiovascular diseases and to correlate the consumption of fresh fruit and vegetables with vitamin C levels and the presence of cardiovascular diseases.

Methods

Vitamin C levels, determined by high-performance liquid chromatography (HPLC), and consumption of fresh fruit and vegetables, assessed by a food frequency questionnaire, were measured in 200 outpatients with T2D. All other laboratory variables were measured by standard methods. The association between vitamin C and cardiovascular diseases was assessed by multivariable logistic regression analysis.

Results

Vitamin C deficiency was found in 12.2% of the subjects. Vitamin C levels and consumption of fresh fruit and vegetables were lower in subjects with cardiovascular diseases. Consumption of fresh fruit and vegetables was associated with higher levels of vitamin C. In the multivariable analysis, vitamin C was independently associated with cardiovascular diseases.

Conclusion

In conclusion, our study suggests that vitamin C may have an inverse relationship with cardiovascular diseases. Intake of vitamin C from fresh fruit and vegetables could play a protective role.

Introduction

It has been reported that diet and its components can have an impact on the risk of ischemic heart diseases, even in a very long follow-up study with the Mediterranean diet [1–4]. Dietary habits have also been shown to account for approximately 45% of all deaths from cardiometabolic causes in adults in the United States [5]. Micronutrients are important components of a healthy diet [6, 7], of which vitamin C is an essential component [8]. Vitamin C is a key antioxidant that acts both as co-factor for many important metabolic processes and as a non-enzymatic quencher for free radicals that are otherwise capable of modifying cellular macromolecules [9].

Patients with T2D are exposed to oxidative stress as a consequence of increased ROS production and lipid peroxidation [10]. Oxidative stress is believed to play a physiopathological role in causing cardiovascular disease, which is the main cause of morbidity and mortality in T2D [11].

The relationship between vitamin C and cardiovascular disease in diabetes is not straightforward; a large study in women reported that supplementation of more than 300 mg/day of vitamin C was positively associated with mortality outcomes in those with diabetes [12]. In contrast, vitamin C intake from food alone did not show a clear trend with mortality outcomes [12]. Based on indirect evidence [12], it is possible to hypothesise that the effect of vitamin C may differ depending on whether intake is from natural foods or supplements; however, a formal comparative study is required to test this hypothesis.

It should be emphasised that lifestyle changes and a healthy diet are included in the prevention and/or treatment of atherosclerosis in patients with or without T2D [13–15]. A high intake of fresh fruit and vegetables, whole grains, and oil seeds, as well as a low intake of sodium and alcohol is usually recommended [16]. Fruit intake is generally limited in quality and quantity by patients with diabetes due to concerns about its impact on blood glucose; this may occur especially when patients are not well informed and/or not followed by dietitians or nutrition specialists, who recommend the consumption of fresh fruit and vegetables. However, recent studies indicate that fresh fruit has no significant impact on glycemia [17] whereas it has a beneficial effect on cardiovascular risk [18].

Therefore, the aims of the present study were to estimate the prevalence of vitamin C deficiency in ambulatory patients with T2D, to investigate the relationship between vitamin C levels and cardiovascular disease, and to correlate the consumption of fresh fruit and vegetables with vitamin C levels and the presence of cardiovascular disease. In our study, we carefully excluded patients who had taken vitamin supplements in the six months prior to the study, in order to observe the relationship with diet-only vitamin C intake.

Materials and methods

In this cross-sectional observational study, 200 patients attending our diabetic clinic were recruited. Inclusion criteria were age between 18 and 80 years, both sexes and T2D diagnosed with the standard criteria at least 3 months before the enrollment; exclusion criteria were a history of pernicious anemia, autoimmune gastritis, pregnancy, vitamin supplementation within 6 months before the study.

The study was approved by the local ethics committee of the Hospital Trust of Verona (n°3853CESC), and informed consent was obtained from each participant. The study was conducted in accordance with the Declaration of Helsinki. Patients were recruited between September 2022 and March 2023 and the study was conducted at the University of Verona.

Venous blood samples were taken in the morning after an overnight fast. Serum creatinine (measured using a Jaffé rate-blanked and compensated assay) and other biochemical blood measurements were determined using standard laboratory procedures (DAX 96; Bayer Diagnostics, Milan, Italy). Low-density lipoprotein-cholesterol was calculated using the Friedewald’s equation [19]. Hemoglobin A1c (HbA1c) was measured by an automated high-performance liquid chromatography analyzer (HA-8140; Menarini Diagnostics, Florence, Italy). The glomerular filtration rate (GFR) was estimated by the CKD Epidemiology Collaboration (CKD-EPI) equation [20]. Albuminuria was measured by an immuno-nephelometric method on a morning spot urine sample and expressed as the albumin-to-creatinine ratio. The patients in the study during the scheduled blood test for the medical control underwent an additional blood test for the measurement of vitamin C. The quantitative analysis of vitamin C in human plasma is critical, as it is known that the concentration of vitamin C starts to decrease immediately after blood sampling [21]. Indeed, due to its very labile nature, under ex vivo conditions ascorbic acid is progressively oxidized to dehydroascorbic acid, which in turn is irreversibly hydrolyzed to the more stable metabolite 2,3-diketo-L-gulonic acid. In order to prevent such oxidation, the following validated procedure was carried out [22]: Li-heparin tubes were filled up to 1/10th of their vacuum volume with a sterile saline solution containing 50 mM of 1,4-Dithioerythritol (DTE, Sigma-Aldrich^®) reducing agent at 10 × the selected final concentration. To preserve the residual vacuum, a sterile insulin syringe was used, and the delivered volumes were checked gravimetrically. The procedure was performed in a sterile environment, and the tubes thus prepared were stored at + 4 °C until use, for up to 7 days. Finally, the quantification of vitamin C in treated plasma samples, was carried out using reverse phase high-performance liquid chromatography with UV detection (HPLC Prominance, Shimadzu, ) using a CE-IVD validated kit from Chrimsystems (Munich, Germany). Vitamin C final concentrations were corrected for the plasma dilution factor.

The duration of diabetes was computed from the date of diagnosis of diabetes. The body mass index (BMI) was calculated by dividing weight in kilograms by height in meters squared; height and weight were measured using a calibrated stadiometer and balance-beam scale, respectively, during the scheduled visit. Height was recorded to the nearest 0.1 cm and weight to the nearest 0.1 kg with minimal clothing. A physician measured blood pressure with a mercury sphygmomanometer after the patients had sat quietly for at least 5 min. Patients were considered to have hypertension if their blood pressure was ≥ 140/90 mmHg or if they were taking any anti-hypertensive medication. Information on smoking status and the use of medications was obtained from all patients via interviews during medical examinations. In all participants, the presence of microvascular diabetic complications was recorded. A single ophthalmologist diagnosed diabetic retinopathy using fundoscopy after pupillary dilation according to a clinical disease severity scale (no retinopathy, non-proliferative, proliferative or laser-treated retinopathy); the presence of proliferative retinopathy was confirmed by fundus fluorescein angiography. Nephropathy was defined as the presence of eGFR < 60 ml/min/1.73 m2 and/or abnormal albuminuria (i.e., an albumin-to-creatinine ratio ≥ 30 mg/g creatinine). Distal symmetric polyneuropathy (DSPN) was assessed by evaluating ankle reflex, touch sensation using the Semmes-Weinstein monofilament and vibration perception threshold using a biothesiometer. A confirmed history of myocardial infarction, angina, coronary revascularization, stroke, transitory ischemic attack, carotid revascularization, non-traumatic amputation, gangrene and/or lower limb revascularization was considered a valid proxy for prior clinical cardiovascular disease (CVD). Ultrasonography scanning of common and internal carotid arteries was performed (Esaote Wall Track System, Esaote S.p.A., Genova, Italy) and a cut-off of 60% was used to define a significant arterial stenosis. Ultrasonography scanning of lower limb arteries was performed and any detected stenosis or moderate-to-severe reduction of blood flow at proximal and/or distal level was considered as a marker of peripheral artery disease.

Nutritional data

All patients were interviewed by a register dietitian at our center, who collected information on the frequency of consumption of fresh fruit and vegetables. The intake of fresh fruit and vegetables was assessed using a food frequency questionnaire designed to analyze daily serving intake among adult and elderly patients [23]. The intake of one serving of vegetables, equivalent to one fist, and one serving of fresh fruit, an average fruit, was recorded. The response categories for fruit and vegetables intake were: less than one serving, one serving, two to three and more than three servings a day. The data were summarised as the daily consumption.

Statistical analysis

Considering a meaningful difference of Vitamin C of 8 µmol/l between the groups with and without atherosclerosis complications and a standard deviation (SD) of 18 µmol/l, we calculated that a minimum sample of 86 subjects were sufficient to reveal a significant difference with a statistical power (1-β) of 80% and a two-tailed level of significance (α) of 5%. Data were summarized as mean ± SD for continuous variables and absolute values or percentages for categorical variables. Differences in clinical/ biochemical characteristics were tested using the Student’s t-test for normally distributed variables and the Mann–Whitney test for non-normally distributed variables. ANOVA were used for variables with three or more categories. The χ2 test was used for categorical variables to study differences in proportions or percentages between the two groups. Three forced-entry logistic regression models were performed: an unadjusted model; a model adjusted for age and BMI; and a third model adjusted for age, BMI, HbA1c, eGFR and sex. Covariates for these multivariate regression models were chosen as potential confounding based on their biological plausibility. A p value of less than 0.05 was considered statistically significant. The Analyses were carried out with SPSS Statistics for Data Analysis v.20.0.1.1.

Results

We studied 200 ambulatory patients with T2D, 33.5% were women with a slightly higher BMI than men (29.1 ± 5.9 vs. 27.7 ± 3.7 Kg/m², p = 0.072) and no difference in mean age. The mean age of the whole sample was 66.7 ± 8.9 yrs (range 34–80 years) with a mean duration of diabetes of 12.1 ± 8.1 years. The mean vitamin C level was 47.4 ± 18.7 µmol/L, mean BMI was 28.1 ± 4.6 Kg/m² (range 17.0-50.4 Kg/m²), mean glycated hemoglobin was 53.1 ± 11.3 mmol/mol Hb and mean eGFR was 80.3 ± 20.2 ml/min 1.73 m². Retinopathy was found in 14.2%, nephropathy in 28.0% and DSPN in 12.5% of patients. 12.2% (24 patients, three patients had no of vitamin C determination) of patients had of vitamin C values compatible with a deficiency (vitamin C ≤ 20 µmol/l). Table 1 shows the main clinical characteristics of the patients according to the status of vitamin C levels. The eGFR was significantly lower in the group with vitamin C deficiency. Although men had lower of vitamin C levels (45.1 ± 17.8 µmol/l men vs. 51.8 ± 20.0 µmol/l women, p = 0.017), the proportion of vitamin C deficiency was not different between men and women (p = 0.146, Χ² test). Patients with vitamin C deficiency had lower levels of total and HDL-cholesterol, while triglycerides were higher. Cholesterol-lowering treatment did not influence vitamin C levels C (47.4 ± 18.0 µmol/l without treatment vs. 47.4 ± 19.0 µmol/l with treatment, p = 0.999) despite an expected significant reduction of LDL-cholesterol levels in the treated group (103.6 ± 28.8 mg/dl without treatment vs. 64.0 ± 24.0 mg/dl with treatment, p < 0.001). Furthemore, diabetes medication did not influence vitamin C levels. The vitamin C level was not different in obese patients (34.5% of patients) 46.9 ± 17.1 compared to non-obese 47.7 ± 19.6 (p = 0.777).

Table 1.

Clinical characteristic of ambulatory patients with type 2 diabetes in relation to vitamin C deficiency

Vitamin C, µmol/L	Normal concentration	Deficiency	p
Age, yr	66.8 ± 8.8	66.1 ± 9.9	0.707
Duration of diabetes, yr	11.7 ± 8.2	14.7 ± 7.8	0.093
BMI, Kg/m2	28.2 ± 4.6	27.7 ± 4.6	0.627
Systolic blood pressure, mmHg	133.5 ± 18.0	139.3 ± 21.0	0.287
Diastolic blood pressure, mmHg	80.2 ± 10.1	77.5 ± 14.1	0.386
Glycated hemoglobin, mmol/mol Hb	52.7 ± 11.4	56.4 ± 11.4	0.140
Total cholesterol, mg/dl	146.4 ± 35.1	129.4 ± 32.1	0.029
HDL-cholesterol, mg/dl	50.5 ± 16.3	40.7 ± 7.7	< 0.001
LDL-cholestrol, mg/dl	72.0 ± 28.6	62.5 ± 29.1	0.139
Triglycerides, mg/dl	120.1 ± 94.5	130.8 ± 39.7	0.021°
AST, U/L	24.4 ± 11.7	28.4 ± 19.0	0.331
ACR, mg/gr	106.7 ± 345.46	55.3 ± 75.5	0.065°
EGFR, ml/min 1.73 m2	81.5 ± 19.6	70.2 ± 22.5	0.010

° Test Mann-Whitney, otherwise t-test. Data are presented as mean ± standard deviation. BMI, body mass index; HDL, high density lipoprotein; LDL, low density lipoprotein; AST, aspartate transaminase; ACR, albumin creatinin ratio; eGFR, estimated glomerular filtration rate

Figure 1 shows that vitamin C levels are progressively and significantly lower with the worsening of carotid artery stenosis. Vitamin C levels were significantly lower in the presence of ischemic heart disease (49.0 ± 17.2 µmol/l no vs. 38.3 ± 19.6 µmol/l yes, p = 0.004), while in the presence of peripheral artery disease, there was a trend towards lower levels of vitamin C (48.0 ± 18.3 µmol/l no vs. 41.3 ± 22.7 µmol/l yes, p = 0.148). Smoking habit showed no significant differences in vitamin C levels (49.8 ± 20.0 µmol/l no smokers, 41.8 ± 15.0 µmol/l smokers and 47.5 ± 18.0 µmol/l ex-smokers, p = 0.111, ANOVA). We then compared non-smokers with current smokers, and the results were: non-smokers 49.8 ± 20.0 µmol/l vs. 41.8 ± 15.0 µmol/l smokers, p = 0.042.

Fig. 1.

The figure shows mean values ± SD of vitamin C levels in the categories of carotid artery stenosis. ANOVA was used for comparison

The consumption of fresh fruit and vegetables was associated with a significantly higher levels of vitamin C (28.7 ± 14.8 µmol/l less than one serving per day, 45.4 ± 17.9 µmol/l less than 2 servings per day and 49.8 ± 19.2 µmol/l more than 2 servings per day, p = 0.057, ANOVA). The consumption of fresh fruit and vegetables was also associated with a trend towards higher HDL-cholesterol levels.

To assess the relationship between vitamin C levels and atherosclerotic burden, we created a categorical variable with 0 equaling absence and 1 equaling the presence of ischemic heart diseases or peripheral artery disease or cerebrovascular disease or previous carotid thromboarterectomy. Vitamin C levels were significantly lower in the group with atherosclerotic conditions: 40.5 ± 19.8 µmol/l in atherosclerotic conditions vs. 49.7 ± 17.9 µmol/l without atherosclerotic conditions, p = 0.003. In addition, the consumption of fresh fruit and vegetables was low in patients with atherosclerotic conditions (33% did not consume any, 17,9% consume less than 2 servings per day and 30,4% more than 2 servings per day, Tau of Goodman and Kruskal p = 0.019 with fresh fruit and vegetables consumption as dependent variable).

Finally, in order to investigate the independent role of vitamin C levels, we ran multivariate logistic models (Table 2) with atherosclerosis burden as dependent variable. Vitamin C levels were inversely and significantly associated with the atherosclerotic load in all models. Other significant covariates were eGFR with an inverse relation and gender with a higher risk of association with atherosclerotic load in men.

Table 2.

Multivariate logistic models of the presence of atherosclerotic disease* as dependent variable in ambulatory patients with type 2 diabetes

variables	1 model		2 model		3 model
	OR (CI-95%)	p	OR (CI-95%)	p	OR (CI-95%)	p
Vitamin C, µmol/L	0.97 (0.95–0.99)	0.004	0.97 (0.95–0.99)	0.004	0.98 (0.96-1.00)	0.059
Age, yrs			1.04 (1.00-1.09)	0.051	1.01 (0.96–1.07)	0.617
BMI, kg/m2			0.99 (0.91–1.06)	0.735	0.98 (0.90–1.07)	0.611
eGFR, ml/min					0.98 (0.96-1.00)	0.032
HbA1c, mmol/mol					1.03 (1.00-1.06)	0.062
Sex, M					2.61 (1.11–6.13)	0.028

*Atherosclerotic diseases: 0 equal to the absence and 1 equal to the presence of ischemic heart diseases or peripheral artery disease or cerebrovascular disease or previous carotid thromboarterectomy

Discussion

The main findings of the present study are that 12.2% of ambulatory patients with T2D have a vitamin C deficiency and that patients with established cardiovascular diseases show a significantly lower blood level of this vitamin. The latter finding was confirmed by multivariate logistic analysis, in which vitamin C levels were a significant inverse predictor associated with cardiovascular endpoints. The study results are strengthened by the fact that we carefully excluded patients who had taken vitamin C supplements in the six months prior to the study.

According to our study, lower of vitamin C concentrations were reported in type 2 diabetes and pre-diabetes [24].

Previous studies both in diabetic and non-diabetic populations have found a positive or negative relationship between vitamin C supplementation and cardiovascular risk [25–27]. Notably, a previous result indicated that vitamin C supplementation was associated with an increased cardiovascular risk [12]. Vitamin C acts as a potent antioxidant [28] by scavenging physiologically reactive oxygen, chlorine and nitrogen species, but in-vitro experiments have also shown that it may have pro-oxidant activity [29]. Interestingly, a recent study showed a different behavior regarding the oxidation of vitamin C, C-vitamers, and other common ascorbic acid derivatives used as supplements [9]. Thus, the beneficial effect of vitamin C may vary, and sometimes be opposite, depending on whether intake is from natural sources or from supplements.

We found that vitamin C levels increased progressively with consumption of fresh fruit and vegetables, but consumption of these foods and consequently blood vitamin C levels were lower, especially in our T2D patients with cardiovascular complications. Interestingly, a recent study reported inadequate intake of fresh fruit in diabetes [30]. Therefore, considering the results of studies suggesting that vitamin C supplementation may not have a protective effect on cardiovascular outcomes, together with our results and those of other studies [31, 32], we suggest that the consumption of fresh fruit and vegetables should be preferred over vitamin C supplementation in patients with type 2 diabetes.

Thus, we found a clear association between low vitamin C levels and carotid stenosis and ischemic heart disease. In particular, we observed an inverse relationship between vitamin C levels and the progressive rate of carotid stenosis.

Low vitamin C levels may be linked to cardiovascular diseases through endothelial dysfunction caused by increased production of ROS [33]. However, vitamin C supplementation does not appear to be effective in all subjects in reducing ROS concentrations [34, 35], again indicating a difference between natural vitamin C obtained from food or from supplements.

In this regard, the results of our study may have a clinical implication, as we reported increased vitamin C levels in patients who consumed more fresh fruit and vegetables. It has been estimated that in the general population, approximately either 5 or 8 million premature deaths can be attributed to fruit and vegetables intakes of less than 500 g/d and 800 g/d, respectively [36]. Therefore, based on the results of our study we believe that the consumption of fresh fruit and vegetables in patients with T2D and cardiovascular diseases should be encouraged and evaluated regularly to maintain an adequate intake. According to our suggestion, a recent intervention study showed that a multifactorial diet, rich in fiber, polyphenols and vitamin C, reduces of triglycerides, total and non-HDL cholesterol levels in patients with T2D [37].

Important weaknesses of our study are the absence of a control group, the normal range of vitamin C levels was extracted from the literature and the study has a cross- sectional design limiting causal inferences. The strengths of the study are the number of participants, the care taken to exclude patients taking vitamin supplements, the precise laboratory method for measuring vitamin C and the completeness of the database, in which vitamin C levels were absent in only three subjects.

In conclusion, our study suggests that vitamin C may have an inverse relationship with cardiovascular disease. Vitamin C intake from fresh fruit and vegetables may play a protective role. Importantly, the consumption of fresh fruit and vegetables can elicit a favorable effect on cardiovascular health through many different mechanisms. In this respect, vitamin C levels can represent a proxy for this.

Abbreviation

BMI

Body mass index

CVD

Cardiovascular disease

DSPN

Distal Symmetric polyneuropathy

eGFR

Estimated Glomerular filtration rate

HPLC

High performance liquid chromatography

HbA1c

Hemoglobin A1c

HDL

High density lipoprotein

LDL

Low density lipoprotein

ROS

Reactive oxygen species

SD

Standard deviation

T2D

Type 2 diabetes

DTE

1,4-Dithioerythriol

Author contributions

AT contributed to the preparation of data, NV contributed to the preparation of data, NR contributed to the preparation of manuscript, EP laboratory analyses of vitamins, MG laboratory analyses of vitamins, ED laboratory analyses of vitamins and critically reviewed the manuscript, GZ analysis of data and wrote the manuscript.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

The study was approved by the local ethics committee of the Hospital Trust of Verona (n°3853CESC), and informed consent was obtained from each participant.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.