Inhibition of heme peroxidases by melamine

Abstract

In 2008 melamine-contaminated infant formula and dairy products in China led to over 50,000 hospitalizations of children due to renal injuries. In North America during 2007 and in Asia during 2004, melamine-contaminated pet food products resulted in numerous pet deaths due to renal failure. Animal studies have confirmed the potent renal toxicity of melamine combined with cyanuric acid. We showed previously that the solubility of melamine cyanurate is low at physiologic pH and ionic strength, provoking us to speculate how toxic levels of these compounds could be transported through the circulation without crystallizing until passing into the renal filtrate. We hypothesized that melamine might be sequestered by heme proteins, which could interfere with heme enzyme activity. Four heme peroxidase enzymes were selected for study: horseradish peroxidase (HRP), lactoperoxidase (LPO), and cyclooxygenase-1 and -2 (COX-1 and -2). Melamine exhibited noncompetitive inhibition of HRP (K(i) 9.5 ± 0.7 mM), and LPO showed a mixed model of inhibition (K(i) 14.5 ± 4.7 mM). The inhibition of HRP and LPO was confirmed using a chemiluminescent peroxidase assay. Melamine also exhibited COX-1 inhibition, but inhibition of COX-2 was not detected. Thus, our results demonstrate that melamine inhibits the activity of three heme peroxidases.

Figure 1

Structure of melamine (2,4,6-triamino-1,3,5-triazine [108-78-1)].

Figure 2

The enzymatic reactions of (a) horseradish peroxidase, (b) lactoperoxidase, and (c) cylooxygenases.

Figure 3

(a) Plots of velocity (V) of oxidized ABTS formation versus ABTS concentration (ABTS) with melamine concentration ranged from 0.5–2 mM. (b) Lineweaver-Burk plots of velocity (V) of oxidized ABTS formation versus ABTS concentration (ABTS) with melamine concentration ranged from 0.5–2 mM showing noncompetitive inhibition.

Figure 4

(a) Michaelis-Menton plots of the velocity of the lactoperoxidase-catalyzed reaction versus KI concentration with 6–15 mM melamine. (b) Lineweaver-Burk plots showing mixed model inhibition (primarily competitive) of lactoperoxidase by melamine.

Figure 5

Plots of chemiluminescent intensity for reactions catalyzed by (a) horseradish peroxidase or (b) lactoperoxidase showing inhibition by melamine. Dotted lines depict 95% confidential bands for linear trends.

Figure 6

Effects of melamine on peroxidase activity of (a) COX-1 and (b) COX-2.