Relations of Dietary Magnesium Intake to Biomarkers of Inflammation and Endothelial Dysfunction in an Ethnically Diverse Cohort of Postmenopausal Women

The WHI-OS is an ongoing national health study designed to examine demographic, lifestyle, dietary, and biological factors on health outcomes among postmenopausal women. Details on design and recruitment have been published elsewhere (9,10). In brief, a total of 93,676 postmenopausal women aged 50–79 years were enrolled in the WHI-OS between September 1994 and December 1998 from 40 clinical centers nationwide. Eligible participants completed baseline demographic and dietary questionnaires, underwent physical examinations, and provided fasting blood samples. All WHI study procedures were approved by human subject review at each clinical center, and all women provided informed consent before participating.

For the current study, we included all participants from a case-control study of type 2 diabetes nested within the WHI-OS (n = 3,713), because all were free of cardiovascular disease, type 2 diabetes, and cancers at baseline. Details of this diabetes ancillary study have been published elsewhere (11,12).

Certified WHI trained staff measured height, weight, hip circumference, and blood pressure at the baseline visit. BMI (measured as weight in kilograms divided by the square of height in meters) was calculated. Standardized questionnaires including information on age, ethnicity, education, income, occupation, medical and family history, smoking status, alcohol use, recreational physical activity, and medication and supplement use were administered. A semiquantitative food frequency questionnaire (FFQ) used previously among similar populations (13,14) was used to assess food and nutrient intake in the last 3 months. The nutrient database for the WHI FFQ was derived from the University of Minnesota Nutrition Coding Center (Minneapolis, MN) nutrient database (15). All dietary nutrient variables were adjusted for total energy intake using the residual method (16). The energy-adjusted correlation coefficient for dietary magnesium intake comparing the FFQ with 8 days of dietary intake (four 24-h recalls and a 4-day food record) among the WHI postmenopausal female population was reported to be 0.7, indicating that the WHI FFQ reasonably captures short-term intake of dietary magnesium in this population (17).

Fasting blood specimens were collected from all participants at baseline according to a standardized protocol. Aliquots of serum, plasma, and buffy coat were frozen and shipped on dry ice to a central repository and stored at −70°C for future assays in outside laboratories. hs-CRP was measured on a chemistry analyzer (Hitachi 911; Roche Diagnostics, Indianapolis, IN) using an immunoturbidimetric assay with reagents and calibrators (Denka Seiken, Niigata, Japan). IL-6 was measured by an ultrasensitive enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN). TNF-α-R2, sICAM-1, sVCAM-1, and E-selectin were measured by an enzyme-linked immunosorbent assay (R&D Systems). The coefficients of variation for each analyte were 1.61% for hs-CRP, 7.6% for IL-6, 3.5% for TNF-α-R2 (11), 6.7% for sICAM-1, 8.9% for sVCAM-1, and 6.5% for E-selectin (12).

The distributions of hs-CRP, IL-6, TNF-α-R2, sICAM-1, sVCAM-1, and E-selectin were highly skewed, and thus log transformations were performed to achieve normal distributions. Multiple linear regression models (using PROC GLM) were used to compute geometric means of inflammatory and endothelial biomarkers across quintiles of dietary magnesium after adjustment for potential confounding variables. Geometric means were calculated by regressing the natural logarithmic values of the plasma concentration of inflammatory and endothelial biomarkers on dietary magnesium intake and taking the antilog of the resulting mean logarithmic value. Tests of linear trend across increasing quintiles of dietary magnesium intake were conducted by using the median value of each quintile as a continuous variable in the model. Regression coefficients for the change in inflammatory and endothelial biomarkers for an increase of 100 mg/day dietary magnesium stratified by ethnicity were also calculated using linear regression models. All multivariable models were adjusted for matching factors (age, ethnicity [white, African American, Hispanic/Latino, and Asian/Pacific Islander], clinical center, and time of blood draw), smoking status (never, past, and current), alcohol (never, past, and current drinkers), physical activity measured by expenditure of energy from recreational activity (continuous), total energy intake (continuous), BMI (continuous), and type 2 diabetes case-control status (case and control subjects). To specifically address the independent association of magnesium intake, the final multivariate models were further adjusted for dietary fiber intake and other dietary factors such as fruit (continuous), vegetable (continuous), folate (continuous), trans fat (continuous), and saturated fat (continuous) intake. Variance inflation factors were examined for all models to check for multicollinearity issues. To further assess potential effect modification, subgroup analyses with prespecified factors including ethnicity (white, African American, Hispanic/Latino, and Asian/Pacific Islander), BMI (<25 and ≥25 kg/m²), smoking status (never smokers and ever smokers [current and past smokers]), alcohol intake (nondrinkers and current drinkers), and dietary fiber intake (low fiber intake [<21 g/day] and high fiber intake [≥21 g/day]) were conducted. The Wald test was used to assess the significance of multiplicative interaction terms. To account for measurement error, we further conducted a deattenuation analysis using a regression coefficient relating the dietary magnesium measurement from the baseline FFQ to 8 days of food records obtained from 113 women in the WHI (17,18).

All P values were two-tailed, and P < 0.05 was considered to indicate statistical significance unless otherwise specified. All statistical analyses were conducted using SAS software (version 9.1; SAS Institute, Cary, NC).

Characteristics of participants are summarized in Table 1. On average, African Americans had a higher BMI, higher smoking rate, higher dietary fat intake, and lower intake of dietary fiber and total magnesium than women in other ethnic groups. Asian/Pacific Islanders had lower concentrations of hs-CRP, IL-6, TNF-α-R2, sICAM-1, and E-selectin than did other ethnic groups, whereas African Americans had higher concentrations of hs-CRP and IL-6.

Higher dietary magnesium intake was significantly associated with lower concentrations of inflammatory biomarkers including hs-CRP (P for linear trend = 0.003), IL-6 (P for linear trend < 0.0001), and TNF-α-R2 (P for linear trend = 0.0006) (Table 2). Further adjustment for dietary fiber intake attenuated but did not alter the linear trends for hs-CRP (P for linear trend = 0.009), IL-6 (P for linear trend = 0.0005), and TNF-α-R2 (P for linear trend = 0.10) in relation to magnesium intake. After additionally controlling for fruit and vegetable, folate, and saturated and trans fat intake, dietary magnesium intake remained significantly associated with hs-CRP and IL-6. Multivariable adjusted geometric means across increasing quintiles of dietary magnesium were 3.08, 2.63, 2.31, 2.53, and 2.16 mg/l for hs-CRP (P for linear trend = 0.005) and 2.91, 2.63, 2.45, 2.27, and 2.26 pg/ml for IL-6 (P for linear trend = 0.0005).

Similarly, we observed significant trends of an inverse association of dietary magnesium intake with concentrations of sVCAM-1 (P for linear trend = 0.06) and E-selectin (P for linear trend = 0.0007) (Table 2). After further adjustment for dietary fiber intake, the inverse association with sVCAM-1 remained strong (P for linear trend = 0.05) and the association with E-selectin was diminished. After additional adjustment for dietary factors, only sVCAM-1 remained significantly associated with dietary magnesium intake; multivariable-adjusted geometric means of sVCAM-1 across increasing quintiles of dietary magnesium were 707, 681, 673, 671, and 656 ng/ml (P for linear trend = 0.04). Tests of variance inflation indicated no serious multicollinearity issues in the models (variance inflation factor <5 for all models).

Assuming a linear relation, an increment of 100 mg/day in dietary magnesium intake was inversely associated with hs-CRP (β = −0.23 ± 0.07; P = 0.002) and IL-6 (β = −0.14 ± 0.05; P = 0.004) (model 3) (Table 3). After correction for measurement error, the association was strengthened for both hs-CRP (β = −0.31 ± 0.10) and IL-6 (β = −0.19 ± 0.07) (data not shown). There was a suggestion that the association of dietary magnesium with hs-CRP concentration varied by ethnicity, with strong associations among white and African American women but no associations among Hispanic and Asian-Pacific Islander women (P_interaction = 0.05). Inverse associations of dietary magnesium with IL-6 concentration were observed in each ethnicity, without statistical evidence of heterogeneity (P_interaction = 0.77). Compared with those in normal-weight women (BMI <25 kg/m²), concentrations of hs-CRP and IL-6 were significantly higher among overweight and obese women (BMI ≥25 kg/m²), and inverse correlations with dietary magnesium were more pronounced (Fig. 1,A and B). Similarly, in subgroup analyses stratified by smoking status (Fig. 1 C and D), concentrations of hs-CRP and IL-6 were higher and demonstrated a stronger inverse association among ever smokers than never smokers (P for linear trend among ever smokers: hs-CRP P = 0.01 and IL-6 P = 0.0008). Tests of interaction were also significant for alcohol intake (IL-6 P = 0.04 and E-selectin P = 0.008) and dietary fiber intake (TNF-α-R2 P = 0.05).

In this large, ethnically diverse cohort of postmenopausal women, dietary magnesium intake was inversely associated with plasma concentrations of hs-CRP, IL-6, TNF-α-R2, sVCAM-1, and E-selectin independent of known risk factors for metabolic outcomes. Adjustment for dietary fiber intake attenuated but did not significantly alter these associations except in the case of E-selectin. After further adjustment for fruit and vegetable intake, folate intake, and saturated and trans fat intake, these inverse trends remained significant for hs-CRP, IL-6, and sVCAM-1, suggesting that dietary magnesium may influence concentrations of these biomarkers independently of other dietary factors associated with inflammation.

Our findings support the notion that magnesium intake improves systemic inflammation and endothelial dysfunction and may play a role in the prevention of type 2 diabetes and metabolic syndrome, a longstanding relation with causality yet to be confirmed. These data are consistent with a considerable body of experimental evidence in animals suggesting that acute magnesium deficiency leads to an inflammatory response (5). Observational data in humans provide additional support for this relation. To date, several cross-sectional studies have reported a link between both low dietary magnesium intake (1,6) and serum magnesium concentrations (19) and elevated hs-CRP in white populations. Fiber intake and dietary patterns high in magnesium were also inversely associated with hs-CRP concentrations in the Nurses' Health Study (8) and in NHANES 1999–2000 (20). However, the relation of magnesium intake to IL-6 and TNF-α is less clear. No association was reported between dietary magnesium intake and TNF-α-R2 or IL-6 in the Nurses' Health Study (6). However, a “western” dietary pattern, lower in magnesium-containing foods, was found to be associated with elevated IL-6 and other markers of inflammation and endothelial dysfunction (8). Our findings provide evidence that hs-CRP and IL-6 may serve as sensitive markers of inflammation that may directly benefit from increased magnesium intake through dietary sources.

CRP is an acute-phase reactant secreted by the liver in response to inflammatory cytokines including IL-6 and TNF-α and is an independent predictor of cardiovascular disease (21) and type 2 diabetes (11). IL-6 and TNF-α are proinflammatory cytokines secreted by macrophages and T-cells to stimulate an immune response to trauma. Low plasma magnesium concentrations and the subsequent disruption in intracellular magnesium homeostasis may play a role in activating the inflammatory response (22). Because hs-CRP is a more sensitive and robust marker of systemic inflammation than other inflammatory markers (23), it may be more readily detected. Our findings also suggest that IL-6 may be sensitive to fluctuations in dietary intake of magnesium. Although TNF-α-R2, a cell surface receptor believed to modulate the action of TNF-α, was inversely associated with dietary magnesium intake before adjustment for dietary fiber intake, this relation was attenuated after adjustment, suggesting that the inverse association with TNF-α-R2 is most likely partially explained by the association with dietary fiber.

With regard to endothelial dysfunction, we observed that dietary magnesium was inversely associated with plasma concentrations of sVCAM-1 independently of other dietary factors. sVCAM-1 and sICAM-1 are cellular adhesion molecules belonging to the immunoglobulin family and are primarily involved in the attachment and transendothelial migration of leukocytes in response to inflammatory cytokines (24). We observed no association between dietary magnesium intake and plasma concentrations of sICAM-1, a finding consistent with a cross-sectional study in the Nurses' Health Study (6). However, we observed a modest association with sVCAM-1 that remained significant even after adjustment for dietary factors associated with endothelial dysfunction and cardiovascular disease. Although the biological explanation for the variability in results across markers is not clear, these findings further support the link between low magnesium intake and elevated concentrations of certain markers of endothelial dysfunction. E-selectin is a cellular adhesion molecule found primarily on the surface of stimulated endothelial cells and mediates the initial rolling of leukocytes along the endothelium (25). In the current study, we observed an inverse association of dietary magnesium intake with E-selectin that disappeared after we accounted for dietary factors.

In this multiethnic cohort of women, we observed notable variation in strengths of association across ethnicity. To our knowledge, no previous work has examined potential ethnic differences in the relation of magnesium intake to systemic inflammation and endothelial dysfunction. Our findings of possible interactions in the current study should be interpreted with caution (i.e., hypothesis-generating) because ethnicity-specific sample sizes were small and the differences could partially be due to residual confounding from demographic and lifestyle factors. More importantly, the generally consistent patterns across the four ethnic groups provide additional evidence to support the notion that increased magnesium intake may have beneficial effects on alleviation of systemic inflammation and endothelial dysfunction. We also observed stronger associations of dietary magnesium intake with hs-CRP and IL-6 concentrations among subgroups with the highest concentrations of these inflammatory markers, i.e., overweight women and smokers. Similar findings among these high-risk subgroups were reported in the Women's Health Study (1), suggesting that higher magnesium intake may be most beneficial among women who are predisposed to systemic inflammation.

There are several limitations that merit consideration. First, the measurement of dietary magnesium intake may be inaccurate because of self-report inconsistencies; however, magnesium intake assessed by our FFQ has a correlation of 0.7 when validated against dietary records (17). Furthermore, in analyses that corrected for measurement error, the relation between magnesium intake and biomarkers was strengthened. Second, dietary magnesium intake is highly correlated with several nutrients including dietary fiber, potassium, and folate, and magnesium is found in high concentrations in foods such as whole grains, nuts, and fruits and vegetables. Therefore, parsing out the independent effects of dietary magnesium is a challenge. In the current study, we sought to examine the independent effects of dietary magnesium through adjustment for dietary fiber as well as fruit and vegetable intake, folate intake, and saturated and trans fat intake in multivariable-adjusted models to control for potential confounding. Our findings suggest that dietary magnesium is associated with several markers of inflammation and endothelial dysfunction independent of these dietary factors, although we could not completely exclude the possibility of residual confounding. This, coupled with the cross-sectional design, does limit our ability to make causal inferences regarding the effect of magnesium intake on markers of inflammation and endothelial dysfunction.

In summary, we found that dietary intake of magnesium was independently and inversely associated with plasma concentrations of hs-CRP, IL-6, and sVCAM-1 in postmenopausal women. These findings are consistent with those of previous studies mostly in whites and support the notion that diets high in magnesium-rich foods including whole grains, nuts, and leafy green vegetables should be encouraged for metabolic disease prevention.

The WHI program was funded by the National Heart, Lung, and Blood Institute, National Institutes of Health (NIH), and U.S. Department of Health and Human Services through contracts N01-WH-22110, 24152, 32100-2, 32105-6, 32108-9, 32111-13, 32115, 32118-32119, 32122, 42107-26, 42129-32, and 44221. This study was supported by the National Institute of Diabetes and Digestive and Kidney Diseases, NIH (R01-DK-062290) and the Burroughs Wellcome Fund Institutional Program Unifying Population and Laboratory-Based Sciences. Y.S. was supported by a grant (K01-DK-078846) from the National Institute of Diabetes and Digestive and Kidney Diseases, NIH.

No potential conflicts of interest relevant to this article were reported.

We thank the WHI investigators and staff for their dedication and the study participants for making the program possible.