Association between the plasma proteome and serum ascorbic acid concentrations in humans

Available online at www.sciencedirect.com

Journal of Nutritional Biochemistry 24 (2013) 842 – 847

Association between the plasma proteome and serum ascorbic acid concentrations in humans☆ Laura A. Da Costa a , Bibiana García-Bailo a, b , Christoph H. Borchers c , Alaa Badawi b , Ahmed El-Sohemy a,⁎ a Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, Toronto, ON, Canada M5S 3E2 Office of Biotechnology, Genomics and Population Health, Public Health Agency of Toronto, ON, Canada M5V 3L7 c University of Victoria-Genome British Columbia Proteomics Centre, University of Victoria, Victoria, BC, Canada V8Z 7X8 b

Received 12 December 2011; received in revised form 4 April 2012; accepted 1 May 2012

Abstract Vitamin C has been associated with a reduced risk of chronic diseases, but the biological pathways regulated by vitamin C are not all known. The objective was to use a proteomics approach to identify plasma proteins associated with circulating levels of ascorbic acid. Men and women (n=1022) 20–29 years of age from the Toronto Nutrigenomics and Health Study completed a general health and lifestyle questionnaire and a 196-item food frequency questionnaire and provided a fasting blood sample. Circulating ascorbic acid was analyzed by high-performance liquid chromatography, and a mass-spectrometry-based multiple reaction monitoring method was used to measure 54 proteins abundant in plasma that are involved in numerous physiologic pathways. Mean protein concentrations were compared across tertiles of serum ascorbic acid using analysis of covariance adjusted for sex, ethnocultural group, season of blood draw, hormonal contraceptive use among women, waist circumference and tertiles of plasma α-tocopherol. A Bonferroni significance level of Pb.0009 was applied, and analyses were adjusted for multiple comparisons using the Tukey–Kramer procedure. Levels of complement C9, ceruloplasmin, alpha-1-anti-trypsin, angiotensinogen, complement C3, vitamin D binding protein and plasminogen were inversely associated with levels of ascorbic acid. The inverse association between ascorbic acid and vitamin D binding protein was highest in those with higher levels of serum 25-hydroxyvitamin D. In conclusion, several plasma proteins from various physiologic pathways are significantly associated with circulating levels of ascorbic acid. These findings suggest that vitamin C may have novel physiological effects. © 2013 Elsevier Inc. All rights reserved. Keywords: Plasma proteins; Ascorbic acid; Vitamin C; Biomarkers; Proteomics

1. Introduction Oxidative stress and inflammation have been implicated in the pathogenesis and progression of a number of chronic diseases [1]. Ascorbic acid (vitamin C) is an essential nutrient and one of the most important dietary hydrophilic antioxidants. In addition to scavenging and neutralizing free radicals, vitamin C also regenerates αtocopherol from the α-tocopherol radical [2,3] and is an important cofactor in reactions that produce collagen, carnitine, norepinephrine and peptide hormones [4]. Vitamin C may also have anti-inflammatory properties by inhibiting tumor necrosis factor (TNF)-α activation of nuclear factor-kappaB [5] and subsequent production of inflammatory cytokines [6]. Circulating levels of ascorbic acid have been inversely related to several chronic diseases and their associated risk factors, including C-reactive protein [7], blood pressure [8], diabetes [9], metabolic syndrome [10], cardiovascular disease (CVD) [11] and all-cause mortality [12]. Despite the promising epidemiological ☆ Grants, sponsors and funding sources: Advanced Foods and Materials Network. ⁎ Corresponding author. E-mail address: [email protected] (A. El-Sohemy).

0955-2863/$ - see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jnutbio.2012.05.002

evidence, data from antioxidant supplementation trials show little beneficial effect of vitamin C in preventing or treating certain chronic diseases [13–15]. Various factors have been suggested to explain these discrepancies including sample size, dose, duration, genetic variation, residual confounding, subject selection and disease status [1,16]. Identifying proteins in physiologic pathways that are affected by vitamin C may help unravel the effects of this vitamin on various disease-related pathways and lead to an increased understanding of its role in prevention and progression of disease. While traditional technologies prevented the measurement of more than a few biomarkers at once, technological developments in the field of proteomics, such as multiple reaction monitoring (MRM), now provide the opportunity to rapidly measure several proteins in a way that is both sensitive and specific across a wide range of concentrations [17]. These advances allow for identification of novel biomarkers, mechanisms of disease pathogenesis and targets for intervention [17–19]. Using a novel MRM-based proteomics assay [17], the aim of the current study was to assess the relationship between circulating levels of ascorbic acid and 54 high-abundance plasma proteins from several physiologic pathways in an ethnically diverse population of healthy young adults in order to identify pathways that may be regulated by vitamin C.

L.A. Da Costa et al. / Journal of Nutritional Biochemistry 24 (2013) 842–847

2. Methods and materials 2.1. Study design Subjects were from the Toronto Nutrigenomics and Health Study, which is a crosssectional examination of an ethnically diverse cohort of males (n= 520) and females (n= 1117), aged 20–29 years, recruited from the University of Toronto campus. Subjects completed a general health and lifestyle questionnaire that included questions on sociodemographic characteristics such as age, sex and ethnocultural group. Based on their self-reported ethnocultural status, subjects were categorized into four groups, including Caucasians, East Asians, South Asians and Other. Additional questions were used to collect information on medical history and smoking status. Physical activity was also assessed by questionnaire and was expressed as metabolic equivalent task hours per week. One metabolic equivalent task (MET) hour is equal to 1 kcal expended per kilogram body weight per hour sitting at rest [20]. Subjects completed a 196-item semiquantitative food frequency questionnaire (FFQ), which assessed habitual dietary intake over the past month [21]. Resting systolic blood pressure, diastolic blood pressure and heart rate were measured using the OMRON IntelliSense Blood Pressure Monitor (Model HEM-907XL, OMRON Healthcare, Vernon Hills, IL, USA). The mean of two measurements taken 1 min apart was also recorded. All subjects provided written, informed consent and the study was approved by the Research Ethics Board at the University of Toronto. 2.2. Biochemical measurements Subjects were screened at the time of participation for potential acute inflammatory events and were asked to wait 2 weeks prior to giving a blood sample if such an event was identified. Overnight fasting blood samples were collected and analyzed for biomarkers of cardiometabolic disease at LifeLabs Laboratories (Toronto, ON, Canada). High-performance liquid chromatography (HPLC) was used to measure total serum ascorbic acid concentrations and is described in detail elsewhere [21]. Homeostasis models of insulin resistance (HOMA-IR) and beta-cell function (HOMA-β) were calculated from insulin (μU/ml) and glucose (mmol/L) measurements using the formulas insulin×glucose/22.5 and 20×insulin/(glucose−3.5), respectively. Serum samples were also analyzed for 25-hydroxyvitamin D at the Toronto General Hospital University Health Network Specialty Lab (Toronto, ON, Canada) using HPLC–tandem mass spectrometry [22]. Aliquots of plasma and red blood cells treated with sodium heparin and EDTA were shipped to the University of Toronto (Toronto, ON, Canada) from LifeLabs and stored at − 80°C. Plasma α-tocopherol concentrations were determined using a reversed-phase isocratic HPLC method with fluorescence detection at the University of Toronto as previously described [23]. Frozen plasma samples were also shipped to the Genome British Columbia Proteomics Centre at the University of Victoria (Victoria, BC, Canada) for proteomic analysis. Details of this technique have been described in detail elsewhere [17]. Briefly, a mass-spectrometry-based MRM proteomics assay was designed to measure 63 high-abundance plasma proteins, many of which have been linked to inflammation, cancer, type 2 diabetes and CVD [17]. Of the 57 proteomic proteins successfully measured in this population, 3 had a coefficient of variability greater than 20% and were thus excluded from further analysis [24]. 2.3. Statistical analyses All statistical analyses were performed using SAS, version 9.2 (SAS Institute, Inc., Cary, NC, USA). Of the total number of subjects who completed the study (n=1637), plasma samples for 1126 subjects were available for proteomics analysis at the time it was conducted. Current smokers (n=2) were removed from the analysis, as smoking has been shown to deplete ascorbic acid in the circulation [25,26]. Subjects who may have overreported (N 3500 kcal/day for women, N4000 kcal/day for men) or underreported their energy intakes (b800 kcal/day), as measured by the FFQ, were also excluded from the analysis (n= 7). Subjects were excluded if missing measurements for serum ascorbic acid (n= 50), any one of the proteomic proteins (n= 8) or any other potential covariate of interest, including plasma α-tocopherol (n= 37). The total number of subjects used in the final analyses after exclusions was 1022. The distributions of continuous variables were examined and loge or square-root transformed where necessary to improve normality. Circulating levels of serum ascorbic acid were categorized into tertiles for analysis. Subject characteristics were examined by tertiles of ascorbic acid using χ2 analysis for categorical variables and analysis of variance (ANOVA) for normalized continuous variables. Differences in means between tertiles were adjusted for multiple comparisons using the Tukey– Kramer procedure. Unless indicated otherwise, all P valuesb.05 were considered significant. Mean plasma protein concentrations from the proteomics analysis were compared between tertiles of serum ascorbic acid using analysis of covariance (ANCOVA). Covariates considered in adjusted models included sex, ethnocultural group, season of blood draw, hormonal contraceptive use among women, waist circumference, body mass index (BMI), vitamin C adequacy [b or ≥ the recommended dietary allowance (RDA)], dietary vitamin C (with and without supplements), supplement use (multivitamin and vitamin C), number of daily servings of fruit and vegetables, serum low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterol,

843

total cholesterol, triglycerides, fiber intake, systolic and diastolic blood pressure, plasma α-tocopherol and total serum 25-hydroxyvitamin D. Only those that were significant covariates in most models and altered the significance of the results for at least one protein were included in the final models. The distribution of circulating αtocopherol was skewed and was both transformed and categorized into tertiles and considered as a potential covariate in the models. Because plasma α-tocopherol, defined categorically into tertiles, was a significant covariate and significantly altered the results to a greater extent than the transformed continuous variable, α-tocopherol status was retained in the model as a categorical variable. The final adjusted model included sex, ethnocultural group, season of blood draw, hormonal contraceptive use among women, waist circumference and tertiles of plasma α-tocopherol. To adjust for the 54 tests performed to examine the association between serum ascorbic acid and the 54 plasma proteins, a Bonferroni correction was applied resulting in a critical value of Pb.0009 (critical P value=.05/54). As the Bonferroni correction has been criticized as being overly conservative, the less conservative Benjamini and Yekutieli (B–Y) false discovery rate (FDR) method was also considered [27]. The B–Y method is a modified version of the FDR with a critical P value of α/Σ(1/i). In the present study, this resulted in a B–Y cut point of Pb.01 where α= 0.05 and i= 54 tests. Further comparison of means between the ascorbic acid tertiles was conducted with adjustment for multiple comparisons using the Tukey–Kramer procedure [28]. Statistical interactions were tested between tertiles of serum ascorbic acid and sex and ethnocultural group for all proteins, and total 25-hydroxyvitamin D tertiles for vitamin D binding protein only. Interactions were tested by the addition of a multiplicative interaction term in ANCOVA models.

3. Results Serum ascorbic acid values were categorized into tertiles with cut points of b23.0 μmol/L for tertile 1, 23.0–36.0 μmol/L for tertile 2 and N36.0 μmol/L for tertile 3 (Table 1). Mean ascorbic acid concentrations were 12.4, 29.7 and 49.4 μmol/L for tertiles 1, 2 and 3, respectively (Pb.0001). Distributions of sex, season of blood draw, dietary vitamin C adequacy, multivitamin and vitamin C supplement use, and hormonal contraceptive use among women were significantly associated with serum ascorbic acid. BMI, waist circumference, and systolic and diastolic blood pressures for tertile 3 were significantly lower than those for tertiles 1 and 2. Mean protein concentrations were examined between tertiles of ascorbic acid (Table 2). At the FDR critical P value b.01, 18 of the 54 proteins had significantly different mean concentrations between tertiles of ascorbic acid in adjusted models. At the Bonferroni critical P value b.0009, 7 of the 54 proteins had significantly different mean concentrations between tertiles of ascorbic acid in adjusted models. Models were adjusted for sex, ethnocultural group, season of blood draw, hormonal contraceptive use among women, waist circumference and tertiles of plasma α-tocopherol. These findings remained statistically significant (Pb.0009) in models additionally adjusted for dietary vitamin C adequacy (b or ≥ the RDA), dietary vitamin C (with and without supplements), supplement use (multivitamin and vitamin C), number of daily servings of fruit and vegetables, serum LDL and HDL cholesterol, total cholesterol, triglycerides, fiber intake, systolic and diastolic blood pressure, and total serum 25-hydroxyvitamin D (data not shown). Mean protein concentrations in ascorbic acid tertile 1 were significantly higher than those in tertiles 2 and 3 for complement C9, ceruloplasmin, alpha-1-anti-trypsin, angiotensinogen, complement C3, vitamin D binding protein and plasminogen after adjustment for multiple comparisons. No significant interactions were noted between serum ascorbic acid tertiles and sex or ethnocultural group at the Bonferroni significance level (Pb.0009). As measures of total serum 25-hydroxyvitamin D were collected in this population, the association between ascorbic acid and vitamin D binding protein, which is the main transport protein for 25hydroxyvitamin D [29], was examined further. Total serum 25hydroxyvitamin D was categorized into tertiles. Levels of total serum 25-hydroxyvitamin D were ≤38.5 nmol/L for tertile 1, 38.6–59.4 nmol/L for tertile 2 and ≥59.5 nmol/L for tertile 3. The association between serum ascorbic acid and vitamin D binding protein was examined by tertiles of 25-hydroxyvitamin D using ANCOVA with the Tukey–Kramer adjustment for multiple comparisons between means.

844

L.A. Da Costa et al. / Journal of Nutritional Biochemistry 24 (2013) 842–847

Table 1 Subject characteristics by tertiles of serum ascorbic acid Total population

Age, years Sex Male Female Ethnocultural group Caucasian East Asian South Asian Other Physical activity, MET-h/week BMI, kg/m2 Waist circumference, cm Systolic blood pressure, mmHg Diastolic blood pressure, mmHg Hormonal contraceptive use (among women) No Yes Season of blood draw Spring Summer Autumn Winter HOMA-IR HOMA-β Total serum cholesterol, mmol/L Serum HDL cholesterol, mmol/L Serum LDL cholesterol, mmol/L Triglycerides, mmol/L Serum ascorbic acid, μmol/L Plasma α-tocopherol, μmol/L Total serum 25-hydroxyvitamin D, nmol/L Energy, cal/day Alcohol, g/day Fiber intake, g/day Dietary vitamin C, mg/day Including supplements Excluding supplements Dietary vitamin C adequacy b Less than RDA Meets RDA Multivitamin or vitamin C supplement use No Yes

Serum ascorbic acid Tertile 1

Tertile 2

Tertile 3

P value a

b 23 μmol/L

23–36 μmol/L

N36 μmol/L

n= 1022

n= 338

n= 351

n= 333

22.7±2.5

22.7±2.5

22.8±2.5

22.5±2.4

302 (30) 720 (70)

117 (35) 221 (65)

121 (34) 230 (66)

64 (19) 269 (81)

483 (47) 359 (35) 105 (10) 75 (7) 7.7±3.1 22.7±3.5 73.8±8.9 113.7±11.3 69.0±8.0

155 (46) 107 (32) 44 (13) 32 (9) 7.6±2.9 23.1±3.9 a 74.8±9.8 a 114.6±11.4 a 69.8±7.8 a

166 (47) 126 (36) 39 (11) 20 (6) 7.8±3.1 23.1±3.6 a 74.8±9.4 a 114.9±11.4 a 69.4±8.1 a

162 (49) 126 (38) 22 (7) 23 (7) 7.5±3.3 22.0±2.7 b 71.8±6.9 b 111.5±10.7 b 67.7±8.0 b

497 (69) 223 (31)

131 (59) 90 (41)

161 (70) 69 (30)

205 (76) 64 (24)

.0003

277 (27) 280 (27) 273 (27) 192 (19) 1.4±0.9 109.7±72.2 4.2±0.8 1.6±0.4 2.2±0.6 1.0±0.5 30.4±17.2 30.0±11.8 52.2±25.0 1952±631 5.0±8.4 23.5±11.8

102 (30) 101 (30) 67 (20) 68 (20) 1.5±1.0 110.9±74.1 4.2±0.7 1.6±0.4ab 2.2±0.6 1.0±0.5 12.4±6.5 a 28.7±10.6 51.5±25.5 1913±606 5.3±8.2 21.7±10.1 a

101 (29) 95 (27) 97 (28) 58 (17) 1.5±1.0 111.6±76.6 4.3±0.8 1.5±0.4 a 2.3±0.7 1.0±0.5 29.7±4.0 b 30.5±11.8 52.7±24.6 2005±685 4.6±7.6 24.5±13.3 b

74 (22) 84 (25) 109 (33) 66 (20) 1.3±0.8 106.5±65.4 4.2±0.7 1.6±0.4 b 2.2±0.6 0.9±0.6 49.4±12.7c 30.7±12.8 52.4±24.9 1935±594 5.2±9.4 24.2±11.4 b

.006

.07 .9 .3 .03 .05 .08 b.0001 .1 .7 .1 .2 .003

241.6±262.8 140.3±87.7

196.0±230.2 a 126.3±82.5 a

256.2±271.7 b 145.3±87.4 b

272.5±278.5 b 149.2±91.7 b

b.0001 .0001

169 (17) 853 (83)

80 (24) 258 (76)

54 (15) 297 (85)

35 (11) 298 (89)

b.0001

648 (63) 374 (37)

235 (70) 103 (30)

219 (62) 132 (38)

194 (58) 139 (42)

.009

.3 b.0001

.05

.5 b.0001 b.0001 b.0001 .001

All values are mean±standard deviation or n (%). Means with different superscript letters are significantly different from one another (Pb.05) after Tukey–Kramer adjustment. a P value calculated from χ2 analysis for categorical variables and ANOVA for normalized continuous variables. b The current RDA for vitamin C is 90 mg/day for nonsmoking men and 75 mg/day for nonsmoking women ≥19 years of age.

The association between serum ascorbic acid and vitamin D binding protein was only significant in the highest tertile of total serum 25hydroxyvitamin D (Fig. 1). The mean concentration of vitamin D binding protein was significantly higher in tertile 1 compared to tertiles 2 and 3 in the adjusted model (P=.007 and P=.01, respectively). However, the statistical interaction between tertiles of serum ascorbic acid and tertiles of total serum 25-hydroxyvitamin D was not significant (P=.2). 4. Discussion The goal of the present study was to use a plasma proteomics approach to identify proteins in various physiologic pathways that might be regulated by vitamin C. A novel MRM assay that provides a quantitative assessment of 54 high-abundance plasma proteins was used in an ethnically diverse population of healthy young adults. The proteins analyzed belong to numerous physiologic pathways that

could become dysregulated during disease progression. We found that serum ascorbic acid concentrations were significantly associated with several of the proteins analyzed after Bonferroni and B–Y FDR adjustments. The proteins that reached the Bonferroni critical P value of b.0009 include ceruloplasmin, alpha-1-anti-trypsin, angiotensinogen, vitamin D binding protein, plasminogen, and the complements C3 and C9. Vitamin C and other antioxidant micronutrients have been inversely associated with numerous chronic diseases, possibly due to their antioxidant and anti-inflammatory properties [1]. However, the findings have been inconsistent [13–15]. In the present study, we observed an association between serum ascorbic acid and members of the complement system. The complement system consists of approximately 30 proteins involved in innate immunity with specific roles in promoting inflammation, phagocytosis and lysis of invading pathogens. Complement C3 is an acute phase reactant whose production is stimulated by several inflammatory agents including

L.A. Da Costa et al. / Journal of Nutritional Biochemistry 24 (2013) 842–847

Plasma proteins, μmol/L

Serum ascorbic acid Tertile 1

Tertile 2

Tertile 3

b23 μmol/L

23–36 μmol/L

N36 μmol/L

P value a

2.63±0.05b 2.59±0.04b b.0001 Complement C9 2.87±0.05a Ceruloplasmin 2.60±0.07a 2.25±0.05b 2.14±0.05b b.0001 Alpha-1-anti-trypsin 11.74±0.19a 10.89±0.14b 10.65±0.15b .0002 Angiotensinogen 1.12±0.05a 0.92±0.03b 0.90±0.04b .0002 Complement C3 20.77±0.29a 19.57±0.24b 18.63±0.24b .0002 Vitamin D binding protein 2.99±0.04a 2.80±0.04b 2.74±0.04b .0005 a b b 1.22±0.01 1.19±0.02 .0006 Plasminogen 1.29±0.02 L-Selectin 0.07±0.001 0.07±0.001 0.07±0.001 .001 Vitronectin 3.91±0.05 3.69±0.05 3.58±0.05 .001 Fibronectin 0.53±0.06 0.65±0.06 0.71±0.06 .002 Hemopexin 10.45±0.13 10.10±0.11 9.79±0.11 .002 Alpha-1B-glycoprotein 1.73±0.03 1.65±0.03 1.60±0.03 .003 Kininogen-1 2.27±0.03 2.14±0.03 2.08±0.03 .003 Prothrombin 0.59±0.01 0.59±0.01 0.56±0.01 .003 Zinc-alpha-2-glycoprotein 1.10±0.02 1.05±0.02 0.97±0.02 .004 Inter-alpha-trypsin 0.64±0.01 0.61±0.01 0.60±0.01 .005 inhibitor HC Alpha-2-HS-glycoprotein 9.19±0.12 8.78±0.10 8.42±0.11 .007 Complement C4 beta chain 1.54±0.03 1.45±0.03 1.34±0.03 .007 Afamin 0.26±0.004 0.25±0.003 0.24±0.003 .01 Alpha-1-acid glycoprotein 1.85±0.04 1.74±0.03 1.68±0.03 .01 1 Apolipoprotein A-I 44.74±0.62 43.14±0.51 43.18±0.52 .01 Serum amyloid 0.48±0.01 0.44±0.01 0.41±0.01 .01 P-component Alpha-2-antiplasmin 1.98±0.03 1.91±0.02 1.85±0.02 .02 Apolipoprotein B-100 0.82±0.01 0.82±0.01 0.76±0.01 .02 Apolipoprotein L1 0.45±0.01 0.41±0.01 0.39±0.01 .02 Beta-2-glycoprotein I 2.82±0.04 2.84±0.03 2.66±0.03 .02 Complement C4 gamma 1.69±0.04 1.59±0.03 1.46±0.03 .02 chain Heparin cofactor II 0.73±0.01 0.70±0.01 0.66±0.01 .02 Complement factor B 1.53±0.02 1.44±0.02 1.38±0.02 .03 Complement factor H 0.62±0.01 0.60±0.01 0.58±0.01 .04 Retinol-binding protein 0.96±0.02 0.91±0.01 0.89±0.01 .07 Albumin 966.49±9.52 958.05±7.83 943.66±7.38 .1 Clusterin 1.55±0.02 1.50±0.02 1.50±0.02 .1 Coagulation factor XIIa HC 0.28±0.01 0.26±0.01 0.25±0.01 .1 Alpha-1-antichymotrypsin 3.45±0.05 3.35±0.04 3.29±0.04 .2 Apolipoprotein C-I 3.26±0.05 3.21±0.04 3.17±0.05 .2 Gelsolin, isoform 1 1.24±0.02 1.23±0.02 1.16±0.01 .2 Transferrin 12.90±0.19 12.37±0.15 12.30±0.16 .2 Antithrombin-III 3.59±0.04 3.55±0.03 3.53±0.03 .3 Fibrinogen gamma chain 9.30±0.25 9.84±0.25 9.58±0.24 .3 Haptoglobin beta chain 11.07±0.33 10.78±0.30 10.07±0.27 .3 Histidine-rich glycoprotein 1.32±0.02 1.33±0.02 1.28±0.02 .3 Apolipoprotein A-IV 1.44±0.03 1.45±0.02 1.39±0.02 .4 Apolipoprotein C-III 2.41±0.05 2.41±0.04 2.36±0.05 .4 Alpha-2-macroglobulin 5.90±0.11 5.86±0.09 5.84±0.08 .5 Adiponectin 0.06±0.002 0.06±0.002 0.07±0.002 .5 Apolipoprotein E 0.50±0.01 0.50±0.01 0.51±0.01 .5 Fibrinogen alpha chain 11.75±0.34 12.32±0.36 12.23±0.32 .5 Transthyretin 5.84±0.07 5.76±0.07 5.55±0.06 .5 Fibrinogen beta chain 9.41±0.23 9.71±0.22 9.71±0.22 .6 Apolipoprotein 25.63±0.33 25.14±0.30 24.71±0.32 .7 A-II precursor Complement C1 4.67±0.08 4.72±0.06 4.63±0.06 .7 inactivator Apolipoprotein D 0.34±0.005 0.34±0.004 0.34±0.005 .8 Fibrinopeptide A 7.03±0.18 7.16±0.16 7.15±0.15 .9 Data presented are crude plasma protein means±standard errors. HC, heavy chain; HS, Heremans–Schmid. P values that are bolded and italicized meet the Bonferroni significance level of Pb.0009 for 54 tests. The B–Y FDR significance level is Pb.01. Means with different superscript letters are significantly different from one another (Pb.05) after Tukey–Kramer adjustment. a P value from ANCOVA with log or square-root transformed plasma protein concentrations where necessary, adjusted for sex, ethnocultural group, season of blood draw, hormonal contraceptive use among women, waist circumference and tertiles of plasma α-tocopherol.

interleukin (IL)-1, IL-6 and TNF-α [30,31]. C3 is the most abundant complement found in the circulation; has been associated with traditional cardiometabolic risk factors, such as triglycerides, BMI, waist circumference and fasting insulin [32,33]; and has been proposed to predict CVD risk [34]. Complement C9 is a hydrophilic serum glycoprotein and terminal component of the complement pathway. Together with complement C5b, C6, C7 and C8, complement C9 forms the C5b-9 membrane attack complex on the surface of cells, which can be incorporated into the cell membrane and induce cell lysis [35]. Deficiency in complement C9 has been identified and may increase susceptibility to infection [36]. Lower levels of complement C9 have been hypothesized to indicate higher levels of complement activation and utilization [37]. In the present study population, all subjects had detectable levels of complement C9 that fell within or above what has been reported as normal clinical ranges [38], with levels of complement C9 highest in the lowest tertile of serum ascorbic acid. Based on the expected beneficial effects of vitamin C on inflammation and immunity [39], one might speculate that the higher levels of complement C9 in the lowest tertile of ascorbic acid may reflect higher levels of inflammation or immune activation. Alternatively, higher levels of complement C9 may reflect a better functioning immune system. Further research will be required to clarify the significance of these findings, but the present results suggest a role for vitamin C in the complement system. Mean plasma levels of ceruloplasmin were also highest in the lowest tertile of serum ascorbic acid. Ceruloplasmin has a variety of functions including copper transport, iron homeostasis, biogenic amine metabolism, angiogenesis and antioxidant as well as prooxidant activities [40,41]. Ceruloplasmin is also an acute phase reactant whose concentration increases in response to infection, injury or inflammation. Circulating levels of ceruloplasmin have been shown to be positively associated with CVD [42]. In one study consisting of 26 subjects with coronary-angiography-verified coronary artery disease and 26 healthy controls matched for age and sex, circulating vitamin C levels were significantly higher, while circulating ceruloplasmin levels were significantly lower, in patients compared to controls

Plasma vitamin D binding protein (µmol/L)

Table 2 Mean plasma protein concentration by tertiles of serum ascorbic acid

845

4

Ascorbic acid tertile 1 Ascorbic acid tertile 2

*

Ascorbic acid tertile 3 3

2

1

0 Tertile 1

Tertile 2

Tertile 3

Total 25-hydroxyvitamin D Fig. 1. Circulating levels of vitamin D binding protein by tertiles of serum ascorbic acid and total 25-hydroxyvitamin D. Unadjusted means and standard errors shown. P values were calculated with ANCOVA using the transformed vitamin D binding protein variable and adjusted for sex, ethnocultural group, season of blood draw, hormonal contraceptive use among women, waist circumference and tertiles of plasma αtocopherol. Differences between means were adjusted for multiple comparisons using the Tukey–Kramer procedure. *Mean vitamin D binding protein concentration in ascorbic acid tertile 1 significantly higher than tertiles 2 and 3 (P=.007 and P=.01, respectively). Ascorbic acid and total 25-hydroxyvitamin D tertiles interaction, P=.2. Serum ascorbic acid tertiles are b 23 μmol/L, 23–36 μmol/L and N 36 μmol/L for tertiles 1, 2 and 3, respectively. Total 25-hydroxyvitamin D tertiles are ≤ 38.5 nmol/L, 38.6–59.4 nmol/L and ≥ 59.5 nmol/L for tertiles 1, 2 and 3, respectively.

846

L.A. Da Costa et al. / Journal of Nutritional Biochemistry 24 (2013) 842–847

[43]. When the population was examined as a whole, serum ceruloplasmin was found to be inversely correlated to vitamin C levels (r=−0.42) [43]. Similarly, in the present study, lower levels of ascorbic acid were associated with higher levels of ceruloplasmin. Alpha-1-anti-trypsin is a serpin proteinase inhibitor and an acute phase protein. Its primary function involves protection of the tissues from proteolytic enzymes, but it also has anti-inflammatory and immunomodulatory roles [44]. Alpha-1-anti-trypsin may be sensitive to oxidants such as those found in cigarette smoking, resulting in lower levels of alpha-1-anti-trypsin in smokers compared to nonsmokers [45]. However, other studies have not observed such an association [46]. While higher levels of plasma antioxidants could protect against oxidative degradation of this protein, levels of alpha1-anti-trypsin in the present study were highest in the lowest tertile of vitamin C. In addition, no smokers were present in the current analysis, so smoking cannot explain the differences observed. Angiotensinogen is a component of the renin–angiotensin system, which regulates blood pressure. Renin and various tissue proteases produce angiotensin I from angiotensinogen, which is followed by the formation of the potent vasoconstrictor angiotensin II by angiotensinconverting enzyme [47]. Circulating levels of angiotensinogen are positively associated with blood pressure in humans [48]. We found that levels of angiotensinogen were significantly higher in ascorbic acid tertile 1 compared to tertiles 2 and 3. Dietary and circulating levels of vitamin C have been inversely associated with blood pressure [8], an observation that has been attributed to several potential mechanisms including increases in nitric oxide and prostacyclin and reduction in endothelial dysfunction [49], but not alterations in the renin–angiotensin system. Furthermore, our results were not altered after we adjusted for systolic or diastolic blood pressure. Therefore, it is possible that the observed association between ascorbic acid and angiotensinogen is related to one of the other functions of angiotensinogen. Angiotensinogen is also an acute phase protein whose production in the liver is increased in response to inflammation via the action of the proinflammatory cytokine IL-6, which, when released at sites of injury and inflammation, increases gene expression of acute phase reactants including C-reactive protein, serum amyloid alpha, fibrinogen and angiotensinogen [50,51]. Therefore, altered levels of angiotensinogen may reflect changes in the renin–angiotensin system or inflammation. The vitamin D binding protein is the main transporter of vitamin D and its metabolites in the circulation [29] with additional roles in actin scavenging, macrophage activation and chemotaxis [52]. A positive correlation between vitamin D binding protein and BMI has been identified in one study [53], but not in others [54–56]. A recent study has also identified a relationship between circulating levels of vitamin D binding protein and type 1 diabetes [57]. There were no subjects with diabetes in the present study, and differences in mean vitamin D binding protein concentrations were independent of BMI and waist circumference. In the present study, higher levels of vitamin D binding protein were found in the lowest tertile of serum ascorbic acid. This association remained significant in models adjusted for total serum 25-hydroxyvitamin D. When the population was stratified by tertiles of total serum 25-hydroxyvitamin D, the association remained only in the highest tertile of vitamin D. These results suggest an inverse association between vitamin C and vitamin D binding protein that may be most evident among those who have a high circulating level of total 25-hydroxyvitamin D. Plasminogen is an inactive glycoprotein that is converted into the active serine protease plasmin by action of tissue- and urokinase-type plasminogen activators [58]. In addition to its known role in fibrinolysis, plasminogen may also have key functions in leukocyte migration, inflammation and CVD [59,60]. A study that examined 298 community-dwelling adults aged 65–79 years found that plasma plasminogen levels were positively associated with cardiometabolic

risk factors including cholesterol, triglycerides and blood glucose [61]. Plasminogen levels were also found to be significantly higher in women with ischemic heart disease (IHD), with IHD being an independent predictor of plasminogen levels in the whole population [61]. A prospective investigation of the Atherosclerosis Risk in Communities Study compared baseline plasminogen levels in 326 subjects who developed coronary heart disease (CHD) over a mean follow-up of 4.3 years to 720 subjects from a stratified random sample of the entire study population [62]. The highest quintile of plasma plasminogen had a significantly increased relative risk of CHD incidence compared to the lowest quintile after adjustment for major CHD risk factors [62]. Plasminogen was also found to be positively associated with total cholesterol and triglycerides [62]. In the present study, plasma plasminogen levels were significantly higher in the lowest tertile of serum ascorbic acid. This association was not modified by further adjustment for markers of circulating lipids including total cholesterol and triglycerides. Vitamin C may regulate the inflammatory response through inhibition of proinflammatory cytokine production or inhibition of other signaling pathways of the immune system [39]. During the inflammatory response, cytokines stimulate the production of acute phase proteins. In the present study, the lowest tertile of serum ascorbic acid was associated with significantly higher levels of several acute phase proteins, including alpha-1-anti-trypsin, angiotensinogen and plasminogen, as well as complement components C3 and C9 of the innate immune system. This is in line with previous work in this cohort, which showed serum ascorbic acid to be inversely associated with high-sensitivity C-reactive protein [21]. These results support the proposed anti-inflammatory and immunomodulatory roles of vitamin C. Alternatively, these proteins could be indicators of a higher state of inflammation and oxidative stress, and subsequent utilization and reduction of antioxidants such as vitamin C. However, the cohort consists of generally healthy young adults, and the observed trends are unlikely to be confounded by prevalent chronic disease. We cannot rule out the possibility of residual confounding, despite adjustment for a range of covariates. Circulating ascorbic acid is affected by several factors including age, smoking, prior vitamin C depletion as well as individual genetic variation [63]. Smoking habits were collected by self-report, and there is the possibility of underreporting due to social desirability bias. The population analyzed consists of healthy young adults between the ages of 20 and 29 years, and these associations may not exist in other age groups. In addition, all serum and plasma measurements were taken at a single time point and may not be reflective of long-term status or disease risk. Finally, the cross-sectional nature of the cohort precludes determination of causality in the observed associations. Prospective and experimental studies will be required to confirm these findings and explore potential mechanisms. To our knowledge, the present study is the first to examine the association between circulating levels of ascorbic acid with a large proteomic panel of plasma proteins representing numerous physiologic pathways. Serum levels of ascorbic acid were significantly associated with several of the proteins analyzed in this population of healthy young adults. These findings suggest that vitamin C may have physiological effects that impact on disease-related pathways, and these effects may already be apparent in healthy young individuals.

References [1] García-Bailo B, El-Sohemy A, Haddad P, et al. Vitamins D, C, and E in the prevention of type 2 diabetes mellitus: modulation of inflammation and oxidative stress. Biologics 2011;5:1–13. [2] May JM, Qu ZC, Mendiratta S. Protection and recycling of α-tocopherol in human erythrocytes by intracellular ascorbic acid. Arch Biochem Biophys 1998;349: 281–9.

L.A. Da Costa et al. / Journal of Nutritional Biochemistry 24 (2013) 842–847

[3] Packer JE, Slater TF, Willson RL. Direct observation of a free radical interaction between vitamin E and vitamin C. Nature 1979;278:737–8. [4] Padayatty SJ, Katz A, Wang Y, et al. Vitamin C as an antioxidant: evaluation of its role in disease prevention. J Am Coll Nutr 2003;22:18–35. [5] Cárcamo JM, Pedraza A, Bórquez-Ojeda O, Golde DW. Vitamin C suppresses TNFαinduced NFΚB activation by inhibiting IΚBα phosphorylation. Biochemistry 2002;41:12995–3002. [6] Härtel C, Strunk T, Bucsky P, Schultz C. Effects of vitamin C on intracytoplasmic cytokine production in human whole blood monocytes and lymphocytes. Cytokine 2004;27:101–6. [7] Ford ES, Liu S, Mannino DM, Giles WH, Smith SJ. C-reactive protein concentration and concentrations of blood vitamins, carotenoids, and selenium among United States adults. Eur J Clin Nutr 2003;57:1157–63. [8] Myint PK, Luben RN, Wareham NJ, Khaw KT. Association between plasma vitamin C concentrations and blood pressure in the European Prospective Investigation into Cancer–Norfolk population-based study. Hypertension 2011;58:372–9. [9] Harding AH, Wareham NJ, Bingham SA, et al. Plasma vitamin C level, fruit and vegetable consumption, and the risk of new-onset type 2 diabetes mellitus. The European Prospective Investigation of Cancer–Norfolk prospective study. Arch Intern Med 2008;168:1493–9. [10] Beydoun MA, Shroff MR, Chen X, Beydoun HA, Wang Y, Zonderman AB. Serum antioxidant status is associated with metabolic syndrome among U.S. adults in recent national surveys. J Nutr 2011;141:903–13. [11] Boekholdt SM, Meuwese MC, Day NE, et al. Plasma concentrations of ascorbic acid and C-reactive protein, and risk of future coronary artery disease, in apparently healthy men and women: the EPIC-Norfolk prospective population study. Br J Nutr 2006;96:516–22. [12] Simon JA, Hudes ES, Tice JA. Relation of serum ascorbic acid to mortality among US adults. J Am Coll Nutr 2001;20:255–63. [13] Bjelakovic G, Nikolova D, Gluud LL, Simonetti RG, Gluud C. Mortality in randomized trials of antioxidant supplements for primary and secondary prevention: systematic review and meta-analysis. J Am Med Assoc 2007;297:842–57. [14] Bleys J, Miller III ER, Pastor-Barriuso R, Appel LJ, Guallar E. Vitamin-mineral supplementation and the progression of atherosclerosis: a meta-analysis of randomized controlled trials. Am J Clin Nutr 2006;84:880–7. [15] Coulter ID, Hardy ML, Morton SC, et al. Antioxidants vitamin C and vitamin E for the prevention and treatment of cancer. J Gen Intern Med 2006;21:735–44. [16] Lawlor DA, Smith GD, Kundu D, Bruckdorfer KR, Ebrahim S. Those confounded vitamins: what can we learn from the differences between observational versus randomised trial evidence? Lancet 2004;363:1724–7. [17] Kuzyk MA, Smith D, Yang J, et al. Multiple reaction monitoring-based, multiplexed, absolute quantitation of 45 proteins in human plasma. Mol Cell Proteomics 2009;8:1860–77. [18] Didangelos A, Simper D, Monaco C, Mayr M. Proteomics of acute coronary syndromes. Curr Atheroscler Rep 2009;11:188–95. [19] Mayr M, Mayr U, Chung YL, Yin X, Griffiths JR, Xu Q. Vascular proteomics: linking proteomic and metabolomic changes. Proteomics 2004;4:3751–61. [20] Ainsworth BE, Haskell WL, Leon AS, et al. Compendium of physical activities: classification of energy costs of human physical activities. Med Sci Sports Exerc 1993;25:71–80. [21] Cahill L, Corey PN, El-Sohemy A. Vitamin C deficiency in a population of young Canadian adults. Am J Epidemiol 2009;170:464–71. [22] Wagner D, Hanwell HE, Schnabl K, et al. The ratio of serum 24,25-dihydroxyvitamin D3 to 25-hydroxyvitamin D3 is predictive of 25-hydroxyvitamin D3 response to vitamin D3 supplementation. J Steroid Biochem Mol Biol 2011;126: 72–7. [23] Liu Z, Lee H, Garofalo F, Jenkins D, El-Sohemy A. Simultaneous measurement of three tocopherols, all-trans-retinol, and eight carotenoids in human plasma by isocratic liquid chromatography. J Chromatog Sci 2011;49:221–7. [24] García-Bailo B, Brenner D, Nielsen D, et al. Dietary patterns and ethnicity are associated with distinct plasma proteomic groups. Am J Clin Nutr 2012;95: 352–61. [25] Faure H, Preziosi P, Roussel AM, et al. Factors influencing blood concentration of retinol, α-tocopherol, vitamin C, and β-carotene in the French participants of the SU.VI.MAX trial. Eur J Clin Nutr 2006;60:706–17. [26] Galan P, Viteri FE, Bertrais S, et al. Serum concentrations of β-carotene, vitamins C and E, zinc and selenium are influenced by sex, age, diet, smoking status, alcohol consumption and corpulence in a general French adult population. Eur J Clin Nutr 2005;59:1181–90. [27] Narum SR. Beyond Bonferroni: less conservative analyses for conservation genetics. Conservation Genetics 2006;7:783–7. [28] Kramer CY. Extension of multiple range tests to group means with unequal numbers of replications. Biometrics 1956;12:307–10. [29] Daiger SP, Schanfield MS, Cavalli-Sforza LL. Group specific component (Gc) proteins bind vitamin D and 25 hydroxyvitamin D. Proc Natl Acad Sci U S A 1975;72:2076–80. [30] Falus A, Rokita H, Walcz E, Brozik M, Hidvegi T, Meretey K. Hormonal regulation of complement biosynthesis in human cell lines—II. Upregulation of the biosynthesis of complement components C3, factor B and C1 inhibitor by interleukin-6 and interleukin-1 in human hepatoma cell line. Mol Immunol 1990;27:197–201. [31] Perlmutter DH, Dinarello CA, Punsal PI, Colten HR. Cachectin/tumor necrosis factor regulates hepatic acute-phase gene expression. J Clin Invest 1986;78:1349–54.

847

[32] Onat A, Uzunlar B, Hergenç G, et al. Cross-sectional study of complement C3 as a coronary risk factor among men and women. Clin Sci (Lond) 2005;108:129–35. [33] Muscari A, Massarelli G, Bastagli L, et al. Relationship of serum C3 to fasting insulin, risk factors and previous ischaemic events in middle-aged men. Eur Heart J 2000;21:1081–90. [34] Onat A, Can G, Rezvani R, Cianflone K. Complement C3 and cleavage products in cardiometabolic risk. Clin Chim Acta 2011;412:1171–9. [35] Esser AF. The membrane attack complex of complement. Assembly, structure and cytotoxic activity. Toxicology 1994;87:229–47. [36] Nagata M, Hara T, Aoki T, et al. Inherited deficiency of ninth component of complement: an increased risk of meningococcal meningitis. J Pediatr 1989;114: 260–4. [37] Falcão DA, Reis ES, Paixão-Cavalcante D, et al. Deficiency of the human complement regulatory protein factor H associated with low levels of component C9. Scand J Immunol 2008;68:445–55. [38] Ichikawa E, Furuta J, Kawachi Y, Imakado S, Otsuka F. Hereditary complement (C9) deficiency associated with dermatomyositis. Br J Dermatol 2001;144:1080–3. [39] Wintergerst ES, Maggini S, Hornig DH. Contribution of selected vitamins and trace elements to immune function. Ann Nutr Metab 2007;51:301–23. [40] Floris G, Medda R, Padiglia A, Musci G. The physiopathological significance of ceruloplasmin: a possible therapeutic approach. Biochem Pharmacol 2000;60: 1735–41. [41] Bielli P, Calabrese L. Structure to function relationships in ceruloplasmin: a 'moonlighting' protein. Cell Mol Life Sci 2002;59:1413–27. [42] Shukla N, Maher J, Masters J, Angelini GD, Jeremy JY. Does oxidative stress change ceruloplasmin from a protective to a vasculopathic factor? Atherosclerosis 2006;187:238–50. [43] Göçmen AY, Şahin E, Semiz E, Gümuşlü S. Is elevated serum ceruloplasmin level associated with increased risk of coronary artery disease? Can J Cardiol 2008;24: 209–12. [44] Janciauskiene SM, Bals R, Koczulla R, Vogelmeier C, Köhnlein T, Welte T. The discovery of α1-antitrypsin and its role in health and disease. Respir Med 2011;105:1129–39. [45] Venkata Rao S, Toora BD, Ravi Kiran VS, Indira S. Serum α1-antitrypsin level and antioxidant status in smokers with chronic obstructive pulmonary disease. Biomedicine 2011;31:27–31. [46] Pongpaew P, Tungtrongchitr R, Phonrat B, Vudhivai N, Viroonudomphol D, Schelp FP. Tobacco smoking in relation to the phenotype of alpha-1-antitrypsin and serum vitamin C concentration. J Nutr Environ Med 2001;11:167–73. [47] Morgan L, Pipkin FB, Kalsheker N. Angiotensinogen: molecular biology, biochemistry and physiology. Int J Biochem Cell Biol 1996;28:1211–22. [48] Walker WG, Whelton PK, Saito H, Russell RP, Hermann J. Relation between blood pressure and renin, renin substrate, angiotensin II, aldosterone and urinary sodium and potassium in 574 ambulatory subjects. Hypertension 1979;1:287–91. [49] Houston MC. The role of cellular micronutrient analysis, nutraceuticals, vitamins, antioxidants and minerals in the prevention and treatment of hypertension and cardiovascular disease. Ther Adv Cardiovasc Dis 2011;4:165–83. [50] Brasier AR, Recinos III A, Eledrisi MS, Runge MS. Vascular inflammation and the renin–angiotensin system. Arterioscler Thromb Vasc Biol 2002;22:1257–66. [51] Sherman CT, Brasier AR. Role of signal transducers and activators of transcription 1 and 3 in inducible regulation of the human angiotensinogen gene by interleukin-6. Mol Endocrinol 2001;15:441–57. [52] White P, Cooke N. The multifunctional properties and characteristics of vitamin Dbinding protein. Trends Endocrinol Metab 2000;11:320–7. [53] Taes YEC, Goemaere S, Huang G, et al. Vitamin D binding protein, bone status and body composition in community-dwelling elderly men. Bone 2006;38:701–7. [54] Speeckaert MM, Taes YE, De Buyzere ML, Christophe AB, Kaufman JM, Delanghe JR. Investigation of the potential association of vitamin D binding protein with lipoproteins. Ann Clin Biochem 2010;47:143–50. [55] Winters SJ, Chennubhatla R, Wang C, Miller JJ. Influence of obesity on vitamin Dbinding protein and 25-hydroxy vitamin D levels in African American and white women. Metabolism 2009;58:438–42. [56] Bolland MJ, Grey AB, Ames RW, et al. Age-, gender-, and weight-related effects on levels of 25-hydroxyvitamin D are not mediated by vitamin D binding protein. Clin Endocrinol (Oxf) 2007;67:259–64. [57] Blanton D, Han Z, Bierschenk L, et al. Reduced serum vitamin D-binding protein levels are associated with type 1 diabetes. Diabetes 2011;60:2566–70. [58] Castellino FJ, Ploplis VA. Structure and function of the plasminogen/plasmin system. Thromb Haemost 2005;93:647–54. [59] Plow EF, Hoover-Plow J. The functions of plasminogen in cardiovascular disease. Trends Cardiovasc Med 2004;14:180–6. [60] Plow EF, Ploplis VA, Busuttil S, Carmeliet P, Collen D. A role of plasminogen in atherosclerosis and restenosis models in mice. Thromb Haemost 1999;82:4–7. [61] Kostka T, Para J, Kostka B. Cardiovascular diseases (CVD) risk factors, physical activity (PA) and plasma plasminogen (Plg) in a random sample of communitydwelling elderly. Arch Gerontol Geriatr 2009;48:300–5. [62] Folsom AR, Aleksic N, Park E, Salomaa V, Juneja H, Wu KK. Prospective study of fibrinolytic factors and incident coronary heart disease: the Atherosclerosis Risk in Communities (ARIC) study. Arterioscler Thromb Vasc Biol 2001;21:611–7. [63] Cahill LE, Fontaine-Bisson B, El-Sohemy A. Functional genetic variants of glutathione S-transferase protect against serum ascorbic acid deficiency. Am J Clin Nutr 2009;90:1411–7.