Ingestion of the Non-Nutritive Sweetener Erythritol, but Not Glucose, Enhances Platelet Reactivity and Thrombosis Potential in Healthy Volunteers—Brief Report

The human study abided by the Declaration of Helsinki, and all subjects provided written informed consent. The Institutional Review Board of the Cleveland Clinic approved the erythritol intervention study (IRB 21-005). For this study, subjects were recruited from the Cleveland, OH, area and received renumeration for participation. Subjects were recruited by several methods, including (but not limited to) posted advertisements and personal interactions with employees at the Cleveland Clinic (by ad or direct solicitation, not clinic setting). All advertising materials used for this study were approved by the Cleveland Clinic IRB. Plasma levels of erythritol were assessed using stable isotope dilution liquid chromatography tandem mass spectrometry using methods specifically designed to distinguish erythritol from its structural isomers, as described previously.³ Aggregometry studies in platelet-rich plasma were performed as described previously.⁵ Platelet-rich plasma was isolated before and immediately (30 minutes) after ingestion of erythritol or glucose from blood anticoagulated with sodium citrate (0.109 mol/L). Platelet aggregation was induced by adding the agonists ADP (up to 5 µmol/L, catalog No. 384; Chronolog, Havertown, PA) and TRAP6 (thrombin activator peptide 6; TFLLR-NH2 [Thr-Phe-Leu-Leu-Arg-NH2], up to 10 µmol/L, catalog No. 464; Tocris, Bristol, United Kingdom) as indicated. For all platelet aggregometry experiments, blood was always processed within ≈30 minutes of collection, and isolated platelets were used within 120 minutes of isolation. Serotonin levels were quantified by liquid chromatography tandem mass spectrometry, and CXCL4 (C-X-C motif ligand-4) concentrations were analyzed via ELISA (R&D Systems, MN).³

This single-center trial was approved by the Cleveland Clinic Institutional Review Board. Following subject consent (nonsmokers without cardiovascular disease, hypertension, or diabetes, with normal renal function, no history of recent [1 month] antiplatelet medication, and no clinical history of bleeding, bruising, or documented bleeding disorder; Table S1), blood was drawn after overnight fast and 30 minutes following consumption of water mixed with 30 g of either glucose (n=10 subjects, 30.1±11 years of age, 40% male) or erythritol (n=10 subjects, 30.5±8 years of age, 50% male), a quantity commonly found in erythritol-sweetened foods and the daily intake of some subjects based on 2013 to 2014 National Health and Nutrition Examination Survey data and Food and Drug Administration filings.^4,6

Following erythritol consumption, postprandial circulating erythritol levels were increased >1000-fold compared with baseline levels (median, 6480 [interquartile range, 5930–7300] versus 3.8 [interquartile range, 3.4–3.9] μmol/L; P<0.0001). In contrast, circulating erythritol levels remained similar before versus after glucose consumption (3.0 [2.6–4.0] versus 2.9 [2.7–3.8] μmol/L; P=0.87), while glucose levels were modestly increased (87 [82–93] versus 127 [122–132] mg/dL; P=0.002). A striking increase in platelet aggregation responses to multiple submaximal levels of both ADP and TRAP6 was observed following erythritol ingestion (Figure 1). Every subject showed the same effect (increased responsiveness with erythritol consumption; Figure S1). In contrast, glucose consumption had no effect on platelet aggregation (Figure 1; Figure S2), in line with previous in vitro studies showing that much higher glucose concentrations (>400 mg/dL) are needed to enhance agonist-induced platelet activation.⁷ The erythritol-dependent increase in platelet responsiveness showed significant correlation among all subjects (erythritol levels versus agonist-induced aggregation; Spearman ρ, 0.65 and 0.68; P<0.0001 each, for ADP and TRAP6, respectively). Erythritol ingestion also markedly enhanced stimulus-dependent release of both the dense granule marker serotonin (P<0.0001 for TRAP6 and P=0.004 for ADP) and the α-granule marker CXCL4 (P<0.0001 for TRAP6 and P=0.064 for ADP; Figure 2). In contrast, no significant increases were observed in stimulus (ADP and TRAP6)-dependent release of either serotonin or CXCL4 following glucose ingestion (Figure 2).

Upon reevaluation of erythritol (E968) as a food additive, the European Food Safety Authority recently noted that further studies are needed to test the direct effects of erythritol exposure in humans.⁸ The present studies show that a standard serving of erythritol enhances platelet responsiveness in healthy subjects with normal renal function. Moreover, erythritol consumption led to statistically significant enhancement in multiple indices of stimulus-dependent platelet responsiveness, including ADP- and TRAP6-induced aggregation (at multiple submaximal doses examined), as well as α-granule and dense granule release, in every subject examined. When coupled with recently reported large-scale clinical studies and mechanistic animal model studies linking erythritol to cardiovascular disease risks,³ the present findings raise concerns that erythritol consumption in humans may provoke a direct prothrombotic effect. They also suggest reevaluation of the safety of erythritol as a food additive with Generally Recognized as Safe designation should be considered.

Our present studies have several limitations. First, while the effects of erythritol ingestion were uniformly observed in every subject enrolled and were statistically significant, the study size was small and replication of results with different cohorts is needed. Further, we did not test long-term changes in platelet function following erythritol consumption. This was because in previous pharmacokinetics data, it was observed that erythritol is rapidly absorbed, with circulating erythritol levels being increased within 15 minutes of ingestion. Further, both in vitro and ex vivo (whole blood) studies showed enhanced platelet responsiveness elicited with erythritol within 15 minutes of exposure.³ Given that the lifespan of a platelet is only 5 to 7 days, we focused our studies on examining the impact of acute dietary exposure. Our protocol had 2 advantages: it both minimized inconvenience to volunteers by enabling collection of both (baseline and postprandial) samples at the same visit/day, and it reduced variation in intake for each participant to be able to compare changes in platelet responsiveness within a given subject before versus after exposure and also across comparable exposures between subjects. The chronic effects of erythritol intake warrant further investigation. As with any study, another limitation is the possibility for residual confounding by unknown factors. Use of a glucose control arm with comparable study design (baseline versus 30-minute postprandial blood collection) that showed no enhancement of platelet responsiveness in subjects with any functional measure indicated that the findings are specific to erythritol.

While intervention studies with sweeteners monitoring thrombosis-relevant phenotypes have not yet been reported, several studies have tested whether alternative dietary interventions modulate platelet function (ie, adhesion, activation, and aggregation), mainly with the goal of improving primary and secondary preventive efforts. Among a large number of studies, most evidence for platelet-inhibitory effects comes from studies involving supplementation with polyunsaturated fatty acids⁹ and plant polyphenols.¹⁰ Other studies tested food constituents, such as olive oil,^11,12 garlic,¹³ and tomato extract,¹⁴ with the latter found to induce ≈30% platelet inhibition compared with aspirin. Despite their ever-growing use, the limited studies on sugar substitutes have primarily focused on glucose control and weight reduction. The present studies showing heightened platelet responsiveness following erythritol ingestion are consistent with recent clinical associations observed between erythritol levels and incident major adverse cardiac event risks in observational cohort studies and in mechanistic cell and animal model studies.³ They also are consistent with similar clinical and mechanistic findings recently reported with the sugar alcohol xylitol.¹⁵ Further studies exploring the cardiovascular safety of sugar alcohols like erythritol as a sugar substitute, particularly among subjects at heightened thrombosis risk, seem prudent.