Human dimethylarginine dimethylaminohydrolase 1 inhibition by proton pump inhibitors and the cardiovascular risk marker asymmetric dimethylarginine: in vitro and in vivo significance

Abstract

Proton pump inhibitor (PPI)-induced inhibition of dimethylarginine dimethylaminohydrolase 1 (DDAH1), with consequent accumulation of the nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA), might explain the increased cardiovascular risk with PPI use. However, uncertainty exists regarding whether clinical PPI concentrations significantly inhibit DDAH1 under linear initial rate conditions, and whether PPI-induced DDAH1 inhibition significantly increases ADMA in humans. DDAH1 inhibition by esomeprazole, omeprazole, pantoprazole, lansoprazole and rabeprazole was determined by quantifying DDAH1-mediated L-citrulline formation in vitro. Plasma ADMA was measured in PPI users (n = 134) and non-users (n = 489) in the Hunter Community Study (HCS). At clinical PPI concentrations (0.1-10 μmol/L), DDAH1 retained >80% activity vs. baseline. A significant, reversible, time-dependent inhibition was observed with lansoprazole (66% activity at 240 min, P = 0.034) and rabeprazole (25% activity at 240 min, P < 0.001). In regression analysis, PPI use was not associated with ADMA in HCS participants (beta 0.012, 95% CI -0.001 to 0.025, P = 0.077). Furthermore, there were no differences in ADMA between specific PPIs (P = 0.748). At clinical concentrations, PPIs are weak, reversible, DDAH1 inhibitors in vitro. The lack of significant associations between PPIs and ADMA in HCS participants questions the significance of DDAH1 inhibition as a mechanism explaining the increased cardiovascular risk reported with PPI use.

Figure 1

Kinetic plots representing the conversion of ADMA to L-citrulline by DDAH11. Protein linearity (A) and time linearity (B) data were collected in singlicate. For ADMA concentration versus rate (C) each data point is the mean of three singlicate experiments and error bars represent the standard error. The data is represented as an Eadie-Hoffstee transform in (D). The Michaelis-Menten fit is shown as a solid line in panel C and D.

Figure 2

Concentration dependent inhibition of DDAH1 by ZST316 (positive control), esomeprazole (EPZ), lansoprazole (LPZ), omeprazole (OPZ), pantoprazole (PPZ) and rabeprazole (RPZ). Residual DDAH1 activity is reported as percentage of control activity. Each data point represents the mean of at least two single experiments. Error bars represent the standard deviation. *P < 0.01 for trend; ^#P < 0.05 vs. baseline; ^{^}P < 0.01 vs. baseline.

Figure 3

Reversibility of PPI binding to DDAH1. Measurement of DDAH1 activity is expressed as percentage of control activity (incubation with no PPI). Each data point represents the mean of two sets of triplicate experiments (10-fold PPI dilution) or one triplicate experiment (no PPI dilution). Error bars indicate the standard deviation. *P < 0.05; **P < 0.01.

Figure 4

Detection of rabeprazole (RPZ) degradation products. UV chromatograms detected at 280 nm for pantoprazole (PPZ) and RPZ at time 0 (A and B, respectively), after 30 min of incubation (C and D, respectively) and after 4 h of incubation (E and F, respectively). Additional peaks in RPZ chromatograms are attributed to RPZ degradation products and were identified using published mass spectral data (data not shown). The arrow indicates the residual peak for RPZ.