N-Acetylcysteine inhibits platelet-monocyte conjugation in patients with type 2 diabetes with depleted intraplatelet glutathione: a randomised controlled trial

Abstract

Aims/hypothesis: The aim of this study was to determine whether oral dosing with N-acetylcysteine (NAC) increases intraplatelet levels of the antioxidant, glutathione (GSH), and reduces platelet-monocyte conjugation in blood from patients with type 2 diabetes.

Methods: In this placebo-controlled randomised crossover study, the effect of oral NAC dosing on platelet-monocyte conjugation and intraplatelet GSH was investigated in patients with type 2 diabetes (eligibility criteria: men or post-menopausal women with well-controlled diabetes (HbA(1c) < 10%), not on aspirin or statins). Patients (n = 14; age range 43-79 years, HbA(1c) = 6.9 ± 0.9% [52.3 ± 10.3 mmol/mol]) visited the Highland Clinical Research Facility, Inverness, UK on day 0 and day 7 for each arm of the study. Blood was sampled before and 2 h after oral administration of placebo or NAC (1,200 mg) on day 0 and day 7. Patients received placebo or NAC capsules for once-daily dosing on the intervening days. The order of administration of NAC and placebo was allocated by a central office and all patients and research staff involved in the study were blinded to the allocation until after the study was complete and the data fully analysed. The primary outcome for the study was platelet-monocyte conjugation.

Results: Oral NAC reduced platelet-monocyte conjugation (from 53.1 ± 4.5% to 42.5 ± 3.9%) at 2 h after administration and the effect was maintained after 7 days of dosing. Intraplatelet GSH was raised in individuals with depleted GSH and there was a negative correlation between baseline intraplatelet GSH and platelet-monocyte conjugation. There were no adverse events.

Conclusions/interpretation: The NAC-induced normalisation of intraplatelet GSH, coupled with a reduction in platelet-monocyte conjugation, suggests that NAC might help to reduce atherothrombotic risk in type 2 diabetes.

Funding: Chief Scientist Office (CZB/4/622), Scottish Funding Council, Highlands & Islands Enterprise and European Regional Development Fund.

Trial registration: isrctn.org ISRCTN89304265.

Fig. 1

Diagrammatic representation of the study design

Fig. 2

Effect of NAC (a; p = 0.011; n = 14 by paired Student’s t test) and placebo (b; p > 0.05) on platelet–monocyte conjugation after 2 h and after 7 days of daily oral dosing (c; p = 0.010 for NAC vs placebo; n = 13 by repeated measures two-way ANOVA). NAC, but not placebo, was found to reduce platelet–monocyte conjugation at 2 h after oral dosing and the reduction compared with placebo was significant across the 7 day dosing period (black bars, NAC; white bars, placebo.) Insufficient blood was drawn from one patient at one visit and data from that patient was excluded from (c)

Fig. 3

Effect of NAC (a) and placebo (b) on plasma microparticle count after 2 h. There was a significant increase in microparticle count at 2 h in the placebo arm of the study (p = 0.047 by paired Student’s t test) but a trend towards a reduction (p = 0.09) in the NAC arm (n = 14). Over a 7 day dosing period (c), there was a significant inhibition of microparticle count between NAC and placebo (black bars, NAC; white bars, placebo: p = 0.016 for NAC vs placebo; n = 13 by repeated measures two-way ANOVA) Insufficient blood was drawn from one patient at one visit and data from that patient was excluded from (c)

Fig. 4

Effect of NAC on intraplatelet GSH. Neither NAC (a) nor placebo (b) had a significant impact on platelet GSH (p > 0.05 for both; n = 12). There was, however, a significant negative correlation between the NAC-induced increase in platelet tGSH at 2 h and the baseline tGSH (c: black symbols; p = 0.0007, r = 0.82, r ² = 0.66; n = 12), which was not reproduced in the placebo arm (white symbols; p = 0.97, r = 0.01, r ² = 0.0002; n = 12). The level at which NAC had no effect was estimated to be 120 nmol GSH/mg protein (c: dotted line; this value was used to subdivide patients into tGSH replete (>120 nmol/mg protein) and deficient <120 nmol/mg protein) for further analysis (d, e). Subgroup analysis for individuals with baseline tGSH of <120 nmol/mg protein (black bar; n = 6) and those above this value (white bar; n = 6) clearly showed that GSH is only increased by NAC administration in those with depleted GSH (d: *p < 0.05; **p < 0.01;***p < 0.001 ); placebo failed to show the same trend (e) Insufficient sample was available from two patients for GSH analysis

Fig. 5

Correlation between baseline tGSH (a) and HbA_1c (b) and platelet–monocyte conjugation (%). There was a significant negative correlation between tGSH (p = 0.047, r = 0.58, r ² = 0.34; n = 12) and platelet–monocyte conjugation (a) but no correlation between HbA_1c and platelet–monocyte conjugation (b: p = 0.225, R = 0.044, r ² = 0.05; n = 14). To convert values for HbA_1c in % to mmol/mol, subtract 2.15 and multiply by 10.929. Insufficient sample was available from two patients for GSH analysis