Xylitol is prothrombotic and associated with cardiovascular risk

Abstract

Background and aims: The pathways and metabolites that contribute to residual cardiovascular disease risks are unclear. Low-calorie sweeteners are widely used sugar substitutes in processed foods with presumed health benefits. Many low-calorie sweeteners are sugar alcohols that also are produced endogenously, albeit at levels over 1000-fold lower than observed following consumption as a sugar substitute.

Methods: Untargeted metabolomics studies were performed on overnight fasting plasma samples in a discovery cohort (n = 1157) of sequential stable subjects undergoing elective diagnostic cardiac evaluations; subsequent stable isotope dilution liquid chromatography tandem mass spectrometry (LC-MS/MS) analyses were performed on an independent, non-overlapping validation cohort (n = 2149). Complementary isolated human platelet, platelet-rich plasma, whole blood, and animal model studies examined the effect of xylitol on platelet responsiveness and thrombus formation in vivo. Finally, an intervention study was performed to assess the effects of xylitol consumption on platelet function in healthy volunteers (n = 10).

Results: In initial untargeted metabolomics studies (discovery cohort), circulating levels of a polyol tentatively assigned as xylitol were associated with incident (3-year) major adverse cardiovascular event (MACE) risk. Subsequent stable isotope dilution LC-MS/MS analyses (validation cohort) specific for xylitol (and not its structural isomers) confirmed its association with incident MACE risk [third vs. first tertile adjusted hazard ratio (95% confidence interval), 1.57 (1.12-2.21), P < .01]. Complementary mechanistic studies showed xylitol-enhanced multiple indices of platelet reactivity and in vivo thrombosis formation at levels observed in fasting plasma. In interventional studies, consumption of a xylitol-sweetened drink markedly raised plasma levels and enhanced multiple functional measures of platelet responsiveness in all subjects.

Conclusions: Xylitol is associated with incident MACE risk. Moreover, xylitol both enhanced platelet reactivity and thrombosis potential in vivo. Further studies examining the cardiovascular safety of xylitol are warranted.

Keywords: Artificial sweetener; Cardiovascular disease; Heart attack; Low-calorie sweetener; Nutrition; Platelet; Polyol; Stroke; Sugar alcohol; Thrombosis.

Structured Graphical Abstract

Role of the artificial sweetener xylitol in cardiovascular event risk. In initial untargeted metabolomics studies (discovery cohort) and subsequent stable isotope dilution liquid chromatography tandem mass spectrometry (LC-MS/MS) studies (validation cohort), fasting levels of xylitol are associated with incident major adverse cardiovascular events (MACE). Using human whole blood, platelet-rich plasma, and washed platelets, xylitol enhances multiple indices of platelet reactivity in vitro. Xylitol also was shown to enhance thrombosis formation in a murine arterial injury model in vivo. In human intervention studies, when subjects ingested a typical dietary amount of xylitol in an artificially sweetened food, multiple functional measures of platelet responsiveness were significantly increased. Xylitol is both clinically associated with cardiovascular event risks and mechanistically linked to enhanced platelet responsiveness and thrombosis potential in vivo. ADP, adenosine diphosphate; MI, myocardial infarction.

Figure 1

Xylitol levels are associated with higher risks of major adverse cardiovascular events (MACE) in the discovery and validation cohorts. (A, left) Circulating levels of a polyol tentatively assigned as xylitol (from untargeted metabolomics) in discovery cohort subjects. Boxes represent IQR with the notch indicating the median. Lower whiskers represent the smallest observation (≥25% quantile − 1.5 × IQR) and upper whiskers the largest observation (≤75% quantile + 1.5 × IQR). Two-tailed Mann–Whitney P-values are indicated. (Middle) Kaplan–Meier plot for 3-year MACE stratified by tertiles (T) of relative levels of xylitol in discovery cohort subjects. P-values were calculated with log rank test. (Right) Hazard ratios (HR) for incident 3-year MACE based on univariable and multivariable Cox proportional hazards regression analysis. Data points indicate HR, and 95% confidence intervals are represented by line length. Multivariable adjustments include age, sex, smoking, diabetes, systolic blood pressure, LDL cholesterol, HDL cholesterol, triglycerides, and hsCRP. (B, left) Circulating xylitol levels (from quantitative stable isotope dilution LC-MS/MS analysis) in discovery cohort subjects. Boxes represent IQR with the notch indicating the median. Lower whiskers represent the smallest observation (≥25% quantile − 1.5 × IQR) and upper whiskers the largest observation (≤75% quantile + 1.5 × IQR). Two-tailed Mann–Whitney P-values are indicated. (Middle) Kaplan–Meier plot for 3-year MACE stratified by tertiles (T) of plasma xylitol levels in the validation cohort. P-values were calculated with log rank test. (Right) HR for incident 3-year MACE based on univariable and multivariable Cox proportional hazards regression analysis. Data points indicate HR, and 95% confidence intervals are represented by line length. Multivariable adjustments include age, sex, smoking, diabetes, systolic blood pressure, LDL cholesterol, HDL cholesterol, triglycerides, and hsCRP

Figure 2

Xylitol levels following oral challenge and effect of xylitol platelet responsiveness. (A) Study participants (n = 10) were given 30 g of xylitol dissolved in water to ingest. Xylitol levels were quantified via LC-MS/MS in the blood before and at the indicated times after the xylitol challenge in the first four subjects. In the remainder of subjects, xylitol levels were measured before and 30 min after xylitol challenge. Values listed above data points at each time point represent median (IQR). The distribution of fasting (≥12 h) plasma xylitol levels observed in the validation cohort is also shown: the dashed lines represent the upper and lower range, and the dotted lines indicate the bottom boundaries at quartiles (Q) 2, 3, and 4 in the validation cohort. (B and C) PRP was isolated from healthy volunteers and used to study the effects of varying levels of xylitol on agonist-induced platelet aggregometry. Scatter plots show aggregometry responses for fixed concentrations of xylitol (30 μM, red circles) vs. vehicle (blue circles) with different concentrations of ADP (A) or thrombin receptor activator peptide (TRAP6, B) with line representing medians. Global P-values (for xylitol effect) were calculated with two-way ANOVA and Šídák’s multiple comparisons test to compare groups. *P < .05, **P < .01, ***P < .001. Bar graphs (magnified areas) show submaximal ADP-stimulated (2 μM, A) and TRAP6-stimulated (5 μM, B) platelet aggregometry responses of human PRP following incubation with xylitol (30 μM, red) vs. normal saline (vehicle, blue), with line and whiskers representing means (±SD). P-values were calculated by two-tailed Mann–Whitney test. (D and E) Aggregometry responses of human PRP with varying concentrations of xylitol and fixed submaximal concentration of ADP (2 μM, D) and TRAP6 (5 μM, E) with lines and whiskers representing medians (IQR). P-values were calculated by two-sided Kruskal–Wallis (K.W.) test with Dunn’s post hoc test. *P < .05, **P < .01, ***P < .001. Bar graph data are represented as means (±SD). P-values were calculated by two-tailed Mann–Whitney test

Figure 3

Xylitol increases stimulus-dependent intracellular calcium release and markers for activation in human platelets. (A) Representative fluorescent signal showing thrombin (0.02 U)-induced changes in intracellular calcium release in Fura 2-filled washed human platelets incubated with xylitol. (B) Fold change (relative to vehicle) in peak Fura 2 fluorescence following submaximal (0.02 U) thrombin stimulation at the indicated concentrations of xylitol in washed human platelets. Bars show mean with SEM indicated by whiskers. P-values were calculated by two-sided Kruskal–Wallis test with Dunn’s post hoc test. *P < .05; **P < .01; ***P < .001. (C) ADP-induced changes in P-selectin surface expression in washed human platelets pre-incubated with the indicated concentrations of xylitol. Plotted are interquartile ranges (boxes). The line in the box is the median, and whiskers represent minimum and maximum values. P-values were calculated by two-sided Kruskal–Wallis test with Dunn’s post hoc test. *P < .05; **P < .01; ***P < .001, **** P < .0001. (D) ADP-induced changes in GP IIb/IIIa (PAC-1 antibody staining) in washed human platelets pre-incubated with the indicated concentrations of xylitol. Plotted are interquartile ranges (boxes). The line in the box is the median, and whiskers represent minimum and maximum values. P-values were calculated by two-sided Kruskal–Wallis test with Dunn’s post hoc test. *P < .05; **P < .01; ***P < .001, ****P < .0001. (E, left) Representative fluorescent images of platelet–leucocyte aggregates (BF, bright field, CD45 in green, P-selectin in yellow, CD41 in red, merged image) in human whole blood stimulated with TRAP6 (7.5 μM). (Right) Numbers of platelet–leucocyte aggregates (CD45+, P-selectin+, CD41+) quantified by image stream in human whole blood incubated with indicated concentrations of xylitol at baseline (blue circles) and stimulated with 7.5 μM TRAP6 (red circles) relative to vehicle control with TRAP6. N numbers shown for donors for TRAP-stimulated blood samples, for unstimulated samples n = 6–8. P-values were calculated by two-sided Kruskal–Wallis test with Dunn’s post hoc test. *P < .05; **P < .01; ***P < .001

Figure 4

Xylitol enhances in vivo clot formation. (A) Human platelet adhesion in whole blood to a collagen-coated microfluidic chip surface under physiological shear conditions ± xylitol. Representative images of platelet (green) adhesion at the indicated times (scale bar, 50 μm). P-values were calculated by two-way repeated measures ANOVA with Šídák’s post hoc test. Overall, P-value (xylitol effect) is shown in black, and Šídák’s post hoc test P-values are shown in red over the three follow-up times. Data is represented as means (±SEM). (B) Representative micrographs of carotid artery thrombus formation at the indicated time points following FeCl₃-induced carotid artery injury (scale bar, 200 μm) and time to cessation of blood flow in mice from indicated groups i.p. injected with vehicle or xylitol. Bars represent means, and two-sided P-values were calculated by Mann–Whitney test. Plasma xylitol concentrations in both groups are indicated as means (±SEM)

Figure 5

Effect of routine dietary xylitol challenge on platelet responsiveness in healthy subjects. (A and B) Study participants (n = 10) were given 30 g of xylitol in a drink. Before and 30 min after the xylitol challenge, PRP was rapidly isolated, and platelet aggregometry was performed using different concentrations of ADP (A) and TRAP6 (B). Shown are aggregation responses of paired samples (baseline and post-xylitol) that were analysed together. Boxes represent interquartile ranges (IQR) with the notch indicating the median. Lower whiskers represent the smallest observation (≥25% quantile − 1.5 × IQR) and upper whiskers the largest observation (≤75% quantile + 1.5 × IQR). The total number of replicates (multiple replicates per donor) and the total number of individual donors for each agonist concentration are indicated. P-values were calculated with the Kruskal–Wallis (K.W.) test with a Dunn post hoc analysis

Figure 6

Effect of dietary xylitol exposure on platelet responsiveness in individual subjects. (A and B) Platelet aggregation responses in PRP from each subject in response to submaximal concentration of ADP (2 μM, A) and TRAP6 (7.5 μM, B) before and after xylitol exposure. Shown are aggregation responses of paired samples (baseline and post-xylitol) that were analysed together. Boxes represent interquartile ranges (IQR) with the notch indicating the median. Lower whiskers represent the smallest observation (≥25% quantile − 1.5 × IQR) and upper whiskers the largest observation (≤75% quantile + 1.5 × IQR). The total number of replicates per individual donor is indicated. All subjects showed significant differences in agonist-induced aggregation (P < .05) for pairwise comparison (pre- vs. post-xylitol exposure) except for Subject 1 with only three replicates, and Subject 10 with four replicates showed P = .07 for TRAP6 stimulation. P-values were calculated with the Kruskal–Wallis (K.W.) test with a Dunn post hoc analysis