Haptoglobin genotype modifies the association between dietary vitamin C and serum ascorbic acid deficiency1–3 Leah E Cahill and Ahmed El-Sohemy ABSTRACT Background: Haptoglobin (which is encoded by the Hp gene) is a hemoglobin-binding protein that has antioxidant properties and a common polymorphism that consists of 2 structurally different alleles: Hp1 and Hp2. The capacity of Hp2 to inhibit oxidation and vitamin C depletion is less than that of Hp1, but the influence on vitamin C requirements remains unknown. Objective: This study aimed to determine whether the Hp polymorphism modifies the association between dietary vitamin C and serum ascorbic acid deficiency (,11 lmol/L). Design: Nonsmoking men and women (n = 1046) between 20 and 29 y of age participated in the Toronto Nutrigenomics and Health Study. Blood samples were collected after the subjects had fasted overnight to determine serum ascorbic acid concentrations by HPLC and for genotyping. A 196-item food-frequency questionnaire was used to estimate vitamin C intake. Results: A gene-diet interaction on serum ascorbic acid was observed (P = 0.02). The overall odds ratio (95% CI) for serum ascorbic acid deficiency was 2.84 (1.73, 4.65) for subjects who did not meet the Recommended Dietary Allowance for vitamin C compared with those who did. The corresponding odds ratios were 4.77 (2.36, 9.65) for the Hp2–2 genotype and 1.69 (0.80, 3.63) for carriers of the Hp1 allele. Conclusions: Individuals with the Hp2–2 genotype had an increased risk of deficiency if they did not meet the Recommended Dietary Allowance for vitamin C, whereas carriers of the Hp1 allele did not. The findings suggest that the greater antioxidant capacity of Hp1 might spare serum ascorbic acid. Am J Clin Nutr 2010;92:1494–500.
Vitamin C (ascorbic acid) is an essential nutrient and a strong reducing agent that inhibits oxidative damage (1). Recent studies show that deficient serum vitamin C concentrations are common, occurring in young adults living in North America (2, 3) and the United Kingdom (4, 5). These high deficiency rates are a concern because an inverse relation has been observed between serum ascorbic acid concentrations and several markers of chronic disease, including glucose homeostasis (6), blood pressure (7, 8), oxidative stress (9, 10), high-sensitivity C-reactive protein (hsCRP) (11), and indicators of obesity, such as body mass index (BMI) and waist-to-hip ratio (10, 12). Serum ascorbic acid is also inversely associated with risk of cardiovascular disease (13, 14), diabetes (15), and all-cause mortality (16). However, the findings of studies on dietary vitamin C and the prevention of these same
health outcomes remain inconsistent and controversial (17, 18), potentially because of individual variability in serum ascorbic acid response to dietary vitamin C (19). Haptoglobin is a circulating protein that binds free hemoglobin to prevent heme-driven oxidative damage in tissues (20). A common polymorphism of the human Hp gene gives rise to 2 alleles—Hp1 and Hp2—and has been associated with risk of infections, autoimmune diseases, cardiovascular diseases, and other disorders, suggesting broad clinical significance of this polymorphism (21). A duplicated DNA segment of ’1700 base pairs present in the Hp2 allele, but not in Hp1, results in structurally and functionally different proteins being formed by each of the 3 common genotypes: Hp1–1, Hp2–1, and Hp2–2 (22, 23). The small linear haptoglobin protein made by the Hp1–1 homozygotes is biologically the most effective at binding free hemoglobin and suppressing inflammatory responses associated with free hemoglobin. The heterozygote produces a mediumsized protein that is moderately active, and the Hp2–2 homozygote produces a protein that is biologically the least active because of its large size and cyclic structure. When the antioxidant function of haptoglobin is insufficient, vitamin C has been proposed to act in its place, subsequently depleting serum ascorbic acid (24). Both in vivo and in vitro experiments have shown a significantly lower concentration and decreased stability of ascorbic acid in blood from Hp2–2 subjects than in blood from those who carry the Hp1 allele (25–27). However, it is not clear whether the ascorbic acid–depleting effect of Hp2–2 can be overcome by adequate dietary vitamin C and whether the difference in serum ascorbic acid concentrations between Hp types indicates phenotype-specific dietary vitamin C requirements. The present study examines the composite effect of the Hp polymorphism and dietary vitamin C on serum ascorbic acid concentrations to assess whether the Hp polymorphism modifies the relation between dietary and serum vitamin C. 1
From the Department of Nutritional Sciences, University of Toronto, Toronto, Canada. 2 Supported by a grant from the Advanced Foods and Materials Network (305352 to AE-S). LEC is a recipient of a Natural Sciences and Engineering Research Council of Canada graduate scholarship. AE-S holds a Canada Research Chair in Nutrigenomics. 3 Address reprint requests and correspondence to A El-Sohemy, Department of Nutritional Sciences, Room 350, University of Toronto, 150 College Street, Toronto, ON, Canada, M5S 3E2. E-mail: [email protected]
Received January 31, 2010. Accepted for publication August 2, 2010. First published online October 6, 2010; doi: 10.3945/ajcn.2010.29306.
Am J Clin Nutr 2010;92:1494–500. Printed in USA. Ó 2010 American Society for Nutrition
HAPTOGLOBIN GENOTYPE AND ASCORBIC ACID DEFICIENCY SUBJECTS AND METHODS
Study design and participants Subjects (n = 1277; 886 women and 391 men) were participants of the Toronto Nutrigenomics and Health Study, which is a cross-sectional examination of free-living adults between 20 and 29 y of age recruited from the University of Toronto campus. Subjects were recruited between October 2004 and July 2009. Individuals did not participate in the study if they could not provide a venous blood sample or if they were pregnant or breastfeeding. Smokers (n = 88) were excluded because of the known ascorbic acid–depleting effects of smoking (28, 29). Individuals who may have underreported (,800 kcal/d) or overreported (.3500 kcal/d for women, .4000 kcal/d for men) their energy intakes (n = 93) were excluded, as were those who had any missing data (n = 50). After exclusions, 1046 subjects (737 women and 309 men) remained. Vitamin C supplement users (n = 383) were identified as those who took a vitamin C–containing multivitamin (n = 218), a supplement containing vitamin C exclusively (n = 76), or both (n = 89). The 2 major ethnocultural groups were whites (n = 496) and East Asians (n = 366). The “others” group (n = 184), included South Asians, those of African descent, First Nations, and those with a mix of 2 ethnocultural backgrounds. The date that each subject provided the blood sample was used to classify the subjects by the 4 seasons of spring (March, April, and May), summer (June, July, and August), autumn (September, October, and November), and winter (December, January, and February). The study protocol was approved by the Research Ethics Board at the University of Toronto, and all subjects provided written informed consent. Dietary assessment A 196-item Toronto-modified Willett food-frequency questionnaire (FFQ) was used to assess habitual food intake over the past month. Each subject was given instructions on how to complete the FFQ using visual aids of portion sizes to improve the measurement of self-reported food intake. Subject responses to the individual foods were converted to the daily number of servings for each item. A vitamin C content value was assigned to a serving of each item based on the nutrient contents of the food in the US Department of Agriculture database. Vitamin C contents were combined to compute a total daily vitamin C intake for each subject. When a categorical variable for dietary vitamin C was needed, a dichotomous variable was created that stratified subjects by whether or not they reported meeting the Recommended Dietary Allowance (RDA) for dietary vitamin C (75 mg vitamin C/d for nonsmoking women, 90 mg vitamin C/d for nonsmoking men). Similarly, a categorical variable for fruit and vegetable intake was created that separated subjects into those who reported consuming 7 servings of fruit and vegetables/d and those who did not. An intake of 7 servings was chosen because this is the minimum fruit and vegetable serving recommended for adults by Canada’s Food Guide (30). Anthropometric measures and physical activity Anthropometric measurements, including height, weight, and waist circumference, were measured and body mass index (in kg/m2) was calculated. Modifiable physical activity was measured by
questionnaire and expressed as metabolic equivalent (MET)hours per week, which represents both leisure and occupational activity, but not including sedentary hours of sleeping or sitting. One MET is equal to 1 kcal expended per kg body weight per hour sitting at rest (31).
Serum ascorbic acid and other biochemical measurements After a minimum 12-h overnight fast, blood samples were collected at LifeLabs Medical Laboratory Services (Toronto, Canada), where all of the biochemical measures were performed. Serum ascorbic acid concentrations were measured as previously described by using HPLC (2), with salicylsalicylic acid as a deproteinizing agent, metaphosphoric acid as a stabilizer, and amber tubes used to prevent photooxidation. Samples were stored at 220°C for ,6 d, and plasma ascorbic acid has been shown to be stable under these conditions (32, 33). Certified controls from the National Institute of Standards and Technology (NIST) were used to ensure validity of the method (34). A control sample from NIST was run after calibrating and after every 10th sample analyzed, and the observed CV ranged from 4.9% to 7.8%. Serum ascorbic acid concentrations were considered to be adequate if .28 lmol/L, suboptimal if between 11 and 28 lmol/L, and deficient if ,11 lmol/L (19, 35), because symptoms of scurvy have been observed just below this concentration (36).
Genotyping DNA was isolated from whole blood, and subjects were genotyped for the haptoglobin polymorphism by using a previously established allele-specific polymerase chain reaction (PCR) technique that exploits the known size difference between Hp1 and Hp2 and has been previously established and validated against Hp phenotyping with full concordance (37). Oligonucleotide primers A (5#-GAGGGGAGCTTGCCTTTCCATTG-3#) and B (5#-GAGATTTTTGAGCCCTGGCTGGT-3#) were used for amplification of the 1757-bp Hp 1 allele-specific sequence. Primers C (5#-CCTGCCTCGTATTAACTGCACCAT-3#) and D (5#-CCGAGTGCTCCACATAGCCATGT-3#) were used to amplify a 349-bp Hp 2 allele-specific sequence. Primers were synthesized by ACGT Corporation (Toronto, Canada). Each 5-lL PCR reaction contained 2.55 lL distilled water, 0.5 lL buffer, 0.1 lL dNTPs (10 mmol/L), 0.05 lL Taq, 1.0 lL DNA (12.5 ng/lL), and 0.4 lL each of 2 (10 mmol/L) primers: A and B or C and D. Each sample was amplified separately by using the different primer pairs. The PCR involving the A and B primers used a touchdown method with the following conditions: 95°C for 15 min (95°C for 30 s, 69°C –1°C with each cycle for 45 s, and 72°C for 1 min 30 s) for 15 cycles, (95°C for 30 s, 55°C for 45 s, and 72°C for 1 min 30 s) for 35 cycles, and 72°C for 7 min. The PCR involving the C and D primers used standard conditions: 95°C for 15 min (95°C for 45 s, 69°C for 2 min) for 35 cycles and 72°C for 7 min. The 2 PCR products from the different primer pairs were combined for each sample and resolved together by 1.5% agarose gel electrophoresis. The gel was run for 40 min at 125 V. Each PCR and gel contained 96 samples, including controls and a 5% random replication of samples. All subjects were successfully genotyped for the polymorphism.
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Statistical analysis All statistical analyses were performed by using Statistical Analysis Systems software (SAS version 9.1; SAS Institute Inc, Cary, NC). Significant P values are 2-sided and ,0.05. Subject characteristics were compared between genotypes by using chisquare for categorical variables and unpaired t tests for continuous variables. P values from log-transformed analyses are displayed for BMI, waist circumference, total cholesterol:HDL ratio, hs-CRP, HOMA-IR, HOMA-b, insulin, and dietary vitamin C, fruit, vegetables, and alcohol because these variables were skewed. Potential gene-diet interactions were evaluated by using a general linear model because the dependent variable (serum ascorbic acid) had a normal distribution. Polytomous logistic regression was used to compute odds ratios (ORs) and 95% CIs. Covariate-adjusted mean serum ascorbic acid concentrations were compared between genotypes in an analysis of variance stratified by dietary vitamin C adequacy. Analyses were conducted with the 3 genotypes ungrouped and also with the Hp 1–1 and 2–1 individuals combined, and results were similar. Therefore, the latter is presented to maximize sample size and because previous studies have reported significantly lower concentrations of ascorbic acid in blood from Hp2–2 subjects than in blood from those with the Hp1 allele (25–27). The adjusted model used in the analyses included sex, BMI, ethnocultural group, energy intake, hs-CRP, plasma a-tocopherol, oral contraceptive use (women only), and season, as determined by stepwise linear regression and an analysis of covariance at the 0.05 significance level. No interactions between these covariates and dietary vitamin C on serum ascorbic acid concentrations were observed. Many other covariates were considered as potential confounders, including blood pressure, physical activity, serum lipids, and intakes of carotenoids, flavonoids, iron, fiber, and alcohol. However, none was statistically significant or materially altered the results; therefore, these variables were not included in the final model.
Genotype frequencies and subject characteristics are summarized in Table 1. The only subject characteristics to differ between the genotypes were ethnocultural group, BMI, and hsCRP. The difference in BMI was likely due to population admixture because a larger proportion of whites has the Hp1 allele, and the whites in this population had a higher BMI than the East Asians. Indeed, the mean BMI values are no longer different between the genotypes when adjusted for or stratified by ethnocultural group. Similarly, the difference in hs-CRP is eliminated when adjusted for either BMI or ethnocultural group. No interactions between BMI, hs-CRP, or ethnocultural group and the polymorphism on serum ascorbic acid were observed. Genotype frequencies were in Hardy-Weinberg Equilibrium within each major ethnocultural group. Prevalence of deficient, suboptimal, and adequate serum ascorbic acid concentrations differed between Hp types, although the mean serum ascorbic acid concentrations did not (Table 1), even when adjusted for covariates (data not shown). A significant diet-gene interaction was observed with dietary vitamin C and the Hp polymorphism (P = 0.02) on serum ascorbic acid. In the subjects who met the RDA for vitamin C, serum ascorbic
acid concentrations were not different between the genotypes, and mean (6SE) serum ascorbic acid concentrations were in the adequate range (.28 lmol/L) (Table 2). Of the subjects who reported not meeting the RDA for vitamin C, those with the Hp2–2 genotype had lower average serum ascorbic acid concentrations than did those with the Hp1 allele (17.5 6 3.9 compared with 23.2 6 3.8 lmol/L; P = 0.02). A similar dietgene interaction on serum ascorbic acid was observed for fruit and vegetable intake (P = 0.01). Of the subjects who reported not meeting Health Canada’s recommendation of 7 servings of fruit and vegetables per day, those with the Hp2–2 genotype had lower average serum ascorbic acid concentrations than did Hp1 allele carriers (23.8 6 2.2 compared with 26.4 6 2.1 lmol/L; P = 0.05). The interaction between fruit and vegetable intake and Hp polymorphism on serum ascorbic acid remained significant when the subjects were grouped into the 2 main ethnocultural groups (whites: n = 496, P = 0.01; East Asians: P = 0.02, n = 366; data not shown), which indicates that the interaction effect was not due to population admixture. However, the interaction with dietary vitamin C was observed among whites (P = 0.008), but not among East Asians (P = 0.68; data not shown). Overall, the multivariate-adjusted OR for serum ascorbic acid deficiency was 2.84 (95% CI: 1.73, 4.65) for subjects who reported not meeting the RDA for vitamin C compared with those who met the requirement (Table 3). The OR (95% CI) for serum ascorbic acid deficiency was 4.77 (95% CI: 2.36, 9.65) for the Hp2–2 genotype, and 1.69 (95% CI: 0.80, 3.63) for carriers of the Hp1 allele. DISCUSSION
The present study examined the influence of the Hp polymorphism on the association between dietary vitamin C and serum ascorbic acid concentrations. A significant interaction between dietary vitamin C and the Hp polymorphism on serum ascorbic acid concentrations was observed. Subjects homozygous for the Hp2 allele had lower concentrations than did those with the Hp1 allele when reported dietary vitamin C was low, but not when reported dietary vitamin C met the RDA. A similar interaction was also observed with intake of fruit and vegetables —the major source of dietary vitamin C. Three studies of independent cohorts of men and women previously compared serum ascorbic acid concentrations between the Hp2–2 genotype and Hp1 carriers, and all 3 reported significantly lower serum ascorbic acid concentrations in Hp2–2 individuals than in Hp1 carriers (25–27), although one study observed this pattern in the male, but not in the female, subjects (27). This study did not report measuring dietary vitamin C, but assumed that the subjects were on a standard national diet that included 100 mg vitamin C/d, referencing that the average adult diet in the country contains ’110 mg vitamin C/d as reported in a population of all women (38). The male subjects may have had a less adequate vitamin C intake than the female subjects, as suggested by dietary vitamin C measures reported by other studies within the same country (39), which could partially explain why the effect of the Hp polymorphism was observed in men and not in women. Because the previous studies did not consider dietary vitamin C, the adequacy of vitamin C and fruit and vegetable intakes in the populations studied could not be
HAPTOGLOBIN GENOTYPE AND ASCORBIC ACID DEFICIENCY TABLE 1 Subject characteristics by haptoglobin genotype1 Characteristic Subjects [n (%)] Sex [n (%)] Women Men Age (y) Ethnocultural group [n (%)] White East Asian Other Season [n (%)] Spring Summer Autumn Winter Activity (MET-h/wk) BMI (kg/m2) Waist circumference (cm) Oral contraceptive use, women only [n (%)] No Yes Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) Total cholesterol (mmol/L) Total cholesterol:HDL ratio hs-CRP (mg/L) HOMA-IR HOMA-b Insulin (pmol/L) Serum a-tocopherol (lmol/L) Serum ascorbic acid (lmol/L) Adequate, .28 lmol/L [n (%)] Suboptimal, 11–28 lmol/L [n (%)] Deficient, 11 lmol/L [n (%)] Dietary vitamin C (mg/d) All subjects No supplement users Dietary vitamin C adequacy [n (%)] ,RDA RDA Supplement use [n (%)] No Yes Fruit (servings/d) Vegetables (servings/d) Fruit and vegetable intake [n (%)] ,7 servings /d 7 servings /d Alcohol (g/d) Iron (mg/d) Energy (kcal/d)
Hp1–1 + Hp2–1
392 (53) 156 (50) 22.8 6 0.13
345 (47) 153 (50) 22.6 6 0.1
301 (61) 170 (46) 77 (42)
195 (39) 196 (54) 107 (58)
149 (52) 142 (51) 150 (53) 107 (55) 7.7 6 0.1 22.9 6 0.2 74.3 6 0.4
139 (48) 136 (49) 135 (47) 88 (45) 7.5 6 0.1 22.5 6 0.2 73.3 6 0.4
419 (51) 129 (56) 113.9 6 0.5 68.9 6 0.4 4.2 6 0.03 2.8 6 0.03 1.39 6 0.12 1.4 6 0.1 109.1 6 3.5 48.0 6 1.7 28.5 6 0.5 31.1 6 0.7 303 (55) 183 (53) 62 (43)
398 (49) 100 (44) 113.6 6 0.5 69.2 6 0.4 4.2 6 0.04 2.8 6 0.04 1.19 6 0.12 1.5 6 0.1 110.9 6 3.0 49.0 6 1.4 27.5 6 0.5 29.9 6 0.8 253 (45) 162 (47) 83 (57)
237 6 10 145 6 5
248 6 13 133 6 4
90 (52) 458 (53)
84 (48) 414 (47)
348 (52) 200 (52) 2.7 6 0.1 3.4 6 0.1
315 (48) 183 (48) 2.8 6 0.1 3.5 6 0.1
370 (52) 178 (54) 5.2 6 0.3 20.1 6 0.8 1950 6 27
347 (48) 151 (46) 4.8 6 0.4 20.1 6 0.8 1951 6 29
0.26 0.03 0.07 0.18
0.70 0.47 0.84 0.65 0.04 0.43 0.32 0.43 0.26 0.26 0.04
0.82 0.18 0.85
0.92 0.96 0.45
0.13 0.98 0.98
1 Supplement use includes the use of vitamin C supplements and vitamin C–containing multivitamins. Seven servings of fruit and vegetables are recommended for adults by Canada’s Food Guide (30). HOMA-b, homeostasis model of b cell function; HOMA-IR, homeostasis model of insulin resistance; hs-CRP, high-sensitivity C-reactive protein; RDA, Recommended Dietary Allowance (75 mg vitamin C/d for nonsmoking women, 90 mg vitamin C/d for nonsmoking men); MET-h, metabolic equivalent task hours. 2 Differences between genotypes were assessed by using an unpaired t test for continuous variables and a chi-square test for categorical variables. P values from log-transformed analyses are shown for BMI, waist circumference, total cholesterol:HDL ratio, hs-CRP, serum a-tocopherol, HOMA-IR, HOMA-b, insulin, and dietary vitamin C, iron, fruit, vegetables, and alcohol because these variables were skewed. 3 Mean 6 SE (all such values).
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TABLE 2 Adjusted mean ascorbic acid concentrations by haptoglobin genotype1 n All subjects Sex Women Men Ethnocultural group White East Asian Other BMI ,25 kg/m2 25 kg/m2 Dietary vitamin C adequacy ,RDA RDA Fruit and vegetable intake ,7 servings/d 7 servings/d Supplement use No Yes
Hp1–1 + Hp2–1
P for interaction
lmol/L 30.1 6 1.8
lmol/L 29.0 6 1.9
35.6 6 2.2 25.5 6 2.5
34.2 6 2.3 25.2 6 2.6
496 366 184
28.7 6 1.2 29.1 6 2.0 28.4 6 2.7
29.0 6 1.4 25.8 6 1.9 24.4 6 2.3
0.81 0.08 0.15
28.7 6 1.3 25.5 6 2.2
27.9 6 1.3 22.5 6 2.1
23.2 6 3.8 31.1 6 1.9
17.5 6 3.9 31.0 6 1.9
26.4 6 2.1 39.0 6 3.3
23.8 6 2.2 41.6 6 3.4
25.8 6 2.8 32.1 6 2.6
23.7 6 2.8 30.7 6 2.6
1 RDA, Recommended Dietary Allowance (75 mg vitamin C/d for nonsmoking women, 90 mg vitamin C/d for nonsmoking men). Supplement use includes the use of vitamin C supplements and vitamin C–containing multivitamins. Seven servings of fruit and vegetables are recommended for adults by Canada’s Food Guide (30). A general linear model was used for this analysis, and it adjusted for sex, ethnocultural group, season, BMI, plasma a-tocopherol, oral contraceptive use, and high-sensitivity C-reactive protein.
determined. Regardless, it appears that the 3 previous studies and the present study all support the same conclusion that individuals with the Hp2–2 genotype are more susceptible to serum ascorbic acid deficiency, which suggests that the greater antioxidant capacity associated with the Hp1 allele may spare serum ascorbic acid. Genotype frequencies reported in the present study are comparable with frequencies reported in other populations, with
Hp1 alleles being more frequent in whites than in Asians, which is consistently observed and explained by a proposed selective advantage and protective effect of the Hp2 allele on risk of malaria and infection (23). The ethnocultural diversity of the population in the present study offers the opportunity to investigate within major ethnocultural groups separately to account for potential population admixture. The interaction between dietary vitamin C and the haptoglobin polymorphism
TABLE 3 Odds ratios (ORs) and 95% CIs for suboptimal and deficient serum ascorbic acid in relation to dietary vitamin C, by haptoglobin genotype1 Ascorbic acid status
All subjects ,RDA [n (%)] RDA [n (%)] Unadjusted OR (95% CI) Adjusted OR (95% CI) Hp1–1 + Hp2–1 ,RDA [n (%)] RDA [n (%)] Unadjusted OR (95% CI) Adjusted OR (95% CI) Hp2–2 ,RDA [n (%)] RDA [n (%)] Unadjusted OR (95% CI) Adjusted OR (95% CI)
Adequate (.28 lmol/L)
Suboptimal (11–28 lmol/L)
Deficient (,11 lmol/L)
70 (40) 486 (56) 1.00 1.00
61 (35) 284 (32) 1.49 (1.03, 2.17) 1.58 (1.06, 2.38)
43 102 2.93 2.84
(25) (12) (1.89, 4.53) (1.73, 4.65)
41 (46) 262 (57) 1.00 1.00
35 (39) 148 (32) 1.51 (0.92, 2.48) 1.56 (0.91, 2.66)
14 48 1.86 1.69
(15) (11) (0.94, 3.68) (0.80, 3.63)
29 (34) 224 (54) 1.00 1.00
26 (32) 136 (33) 1.48 (0.84, 2.61) 1.72 (0.91, 3.26)
29 54 4.15 4.77
(34) (13) (2.29, 7.52) (2.36, 9.65)
P for trend
1 RDA, Recommended Dietary Allowance (75 mg vitamin C/d for nonsmoking women, 90 mg vitamin C/d for nonsmoking men). P = 0.02 for Hpvitamin C interaction as determined by using a general linear model with an interaction term. ORs were determined by polytomous logistic regression. The model was adjusted for BMI, sex, energy intake, oral contraceptive use (women only), high-sensitivity C-reactive protein, plasma a-tocopherol, ethnocultural group, and season.
HAPTOGLOBIN GENOTYPE AND ASCORBIC ACID DEFICIENCY
on serum ascorbic acid that was observed among all study participants together was also significant among whites alone, but not among East Asians. However, the interaction between fruit and vegetable intake and the polymorphism was significant among both ethnocultural groups separately, which suggests that the Hp1 allele appears to have a protective effect against serum ascorbic acid deficiency when fruit and vegetable intake is low, regardless of ancestral background. The sample size of the East Asian group was smaller than the white group, which may have been a limitation in the vitamin C analysis more so than in the fruit and vegetable analysis, because fewer subjects reported vitamin C intakes that were below recommendations than fruit and vegetable intakes that were below recommendations. Alternatively, compounds other than dietary vitamin C in fruit and vegetables may have contributed additional benefit to the antioxidant status. However, very good evidence indicates that the Hp polymorphism is a genetic predictor of risk of scurvy (24), and scurvy is caused solely by vitamin C deficiency. The renal threshold and urinary excretion of ascorbic acid does not differ between Hp genotypes (25), which suggests that oxidation of ascorbic acid in the blood is a more likely explanation of differences between Hp genotypes than is the vitamin C elimination route. Saturation of haptoglobin with hemoglobin has been shown to reduce the rate of ascorbic acid depletion (25). Ascorbic acid is more stable in solutions containing a hemoglobin-haptoglobin complex than in solutions containing hemoglobin alone (25), providing further evidence that whereas haptoglobin is the preferred compound to quench free hemoglobin, when haptoglobin is not available or functional, ascorbic acid will substitute to perform the antioxidant function of haptoglobin. Several studies have reported that the Hp2–2 protein is inferior to the Hp1–1 protein in preventing the oxidation of a variety of lipid and protein substrates by free hemoglobin (40, 41). In addition, whereas both Hp1-1 and Hp2–2 proteins are able to bind free hemoglobin, the Hp1–1 protein has been shown to be superior to the Hp2–2 protein in mediating the clearance of hemoglobin via the CD163 pathway, which is the only means of clearing hemoglobin in the extravascular compartment (42). The differences in the antioxidant capacity of the Hp1–1 and Hp2–2 proteins are enhanced markedly when the hemoglobin is glycosylated (43, 44), which commonly occurs in individuals with diabetes mellitus. The glycosylated hemoglobin-Hp2–2 complex can become a proatherogenic, proinflammatory compound (45), oxidatively modifying the HDL of Hp2–2 individuals with diabetes and resulting in increased oxidative damage that increases the susceptibility to atherosclerosis. Cross-sectional and longitudinal studies performed in different ethnic groups and geographic areas have consistently shown that the Hp1–1 homozygote genotype protects against the development of vascular complications of diabetes mellitus (46). However, the results of the present study and of other studies (44, 47) do not imply that supplemental vitamin C is a suitable therapy for patients with diabetes at risk of cardiovascular disease who are not deficient in serum ascorbic acid. Although the relation between serum ascorbic acid concentrations and risk of chronic disease and associated biomarkers is well-established (16, 48), the relation between vitamin C supplementation in clinical trials and the same health outcomes remains inconsistent and controversial (49). These inconsistencies may be due to individual genetic variation in the serum
ascorbic acid response to dietary vitamin C. In addition to Hp, other genes contain common polymorphisms that were recently shown to be potential determinants of serum ascorbic acid, including the genes that code for glutathione S-transferases (50, 51) and vitamin C transporters (52, 53). An additional reason for studying these diet-gene interactions is that inconsistencies between genetic association studies, such as haptoglobin polymorphism and risk of cardiovascular outcomes in nondiabetic individuals (23), may be due, in part, to the gene-disease association being present only when a dietary factor such as vitamin C or fruit and vegetable intake is inadequate. Because vitamin C is a first line antioxidant, the Hp polymorphism and its effects on vitamin C could have important clinical consequences. Findings from the present study indicate that, for some individuals, hemoglobin binding could be a significant use of ascorbic acid in the blood, whereas for others with more functional haptoglobin available to bind hemoglobin, this function of vitamin C might be less important. Regardless of Hp genotype, obtaining the daily recommendations for dietary vitamin C and fruit and vegetables appears to protect against serum ascorbic acid deficiency and could decrease the risk of long-term adverse health effects that are associated with low serum ascorbic acid concentrations. However, individuals with the Hp2–2 genotype appear to be at increased risk of serum ascorbic acid deficiency when dietary vitamin C is insufficient. We thank Daiva Nielsen for administrative assistance and Sarah Herd and Hyeon-Joo Lee for technical assistance in the laboratory. The authors’ responsibilities were as follows—LEC: genotyping, statistical analysis, and preparation of the first draft of the manuscript; AE-S: funding and supervision; and LEC and AE-S: data interpretation and critical revision of the manuscript for important intellectual content. Neither author had a conflict of interest to declare.
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