Metabolism of protocatechuic acid influences fatty acid oxidation in rat heart: New anti-angina mechanism implication

Abstract

Protocatechuic acid (PA), a structurally typical phenolic acid in danshen, shows anti-angina efficacy. But until now, besides scavenging of oxygen free radicals, the understanding of its anti-angina mechanism has been limited. In our study, based on a novel metabolic route of PA identified in rat heart and its influence on fatty acid oxidation (FAO), we proposed a new mechanism for its anti-angina. In detail, three metabolites, catechol methylated metabolite, acyl-coenzyme (CoA) thioester and glycine conjugation, were identified in rat heart. A novel metabolic pathway was confirmed based on several metabolic systems incubated with heart mitochondria, cytosol, microsomes and homogenate. Results indicated that PA was firstly methylated in microsomes and cytosol, which was regarded as the prerequisite step for further metabolism and could be inhibited by tolcapone, and then the resulting methylated metabolite (vanillic acid) diffused into mitochondria where it was converted into acyl-CoA thioester, in similar with FAO. In addition, part of the acyl-CoA thioester was transformed into glycine conjugation, a step also localized within mitochondria. Furthermore, based on isolated rat heart perfusion, it was found that PA markedly decreased FAO, which was shown by higher residual fatty acid level in perfusate (p < 0.05) and lower acy-CoA/CoA ratio in heart (p < 0.05). The FAO inhibiting effect of PA could be largely reversed by its methylation inhibitor tolcapone, indicating the effect was closely related with the identified metabolic pathway of PA in heart. The decrease of FAO may switch heart energy substrate preference from fatty acid to glucose, which is beneficial for ischemia heart.

Introduction

Although not as abundant as in liver, enzymes in heart mediating exogenous or endogenous substances metabolism have been widely reported [1], [2]. Many researches have implicated the association of cytochrome P450 with heart pathology and pharmacology [3], [4]. For drugs in treatment of heart diseases, including β-blockers, calcium antagonists, and angiotensin-converting enzyme inhibitors, all of them were reported to be subject to cytochrome P450-dependent oxidations. Thus, investigating their metabolism in heart will be very important to understand their efficacy and toxicity, such as verapamil [5] and dexrazoxane [6]. Although substantial numbers of herbal drugs are prescribed for heart diseases, such as salvia miltiorrhiza (danshen) and Radix notoginseng, little was known about their metabolism in heart. In our opinion, the investigation of these herbal drugs metabolism in heart may be helpful to understand their pharmacology or toxicology.

Danshen is a well-known Chinese herbal medicine and is used in clinics in China for angina pectoris and myocardial infarction [7], [8]. Chemical constituents study generally divided the compounds in danshen into two groups, phenolic acids (hydrophilic) and diterpenoid tanshinones (lipophilic) [9]. For many years, the lipophilic compounds were regarded as the main contributing factors to danshen activities. Recently, these phenolic acids gained more attention as a large pool of natural antioxidants [10]. Especially in Danshen Injection (a danshen prescription), phenolic acids account for more than 90% of the constituents and nearly no lipophilic constitutes were detected [11]. Thus, the anti-angina efficacy of Danshen Injection should mainly contribute to these phenolic acids. Protocatechuic acid (PA), an active and typical phenolic acid in danshen, has shown obvious anti-angina effect [12], [13]. Structurally similar with other phenolic acids in danshen, PA contains catechol group in benzene ring and carboxylic acid in side chain. Metabolism study indicated that these phenolic acids mainly underwent phase II reaction including methylation, glucuronidation, and sulfonation [14]. Recently, we accidentally found a glycine conjugative metabolite of PA in rat heart, a metabolite that was evidenced to only form within mitochondria according to previous reports [15], [16]. Considering that coenzyme (CoA) thioesters formation is an obligatory step in the course of glycine conjugation, we supposed that there was some association between PA metabolism and heart fatty acid oxidation (FAO), because they both occur in mitochondria and need assistance of CoA factor. Additionally, a body of evidence has convincingly demonstrated that FAO reduction is beneficial for ischemia heart because it shifts heart energy substrate from fatty acid to glucose and enhances the oxygen utilizing efficiency for adenosine triphosphate (ATP) production [17], [18]. The interesting and significant presumption promoted us to investigate the metabolic route of PA in heart and evaluate its relevance to heart FAO.

The present study was designed to investigate PA metabolism in rat heart in vivo and in vitro. The related metabolites were characterized and a novel metabolic pathway was identified based on several metabolic systems incubated with heart cytosol, microsomes, mitochondria and homogenate. Furthermore, the relevance of the metabolic pathway with FAO was also evaluated by isolated heart perfusion assay and a new mechanism for its anti-angina efficacy was proposed.