Aminoguanidine Inhibits Semicarbazide-Sensitive Amine Oxidase Activity: Implications for Advanced Glycation and Diabetic Complications
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Diabetologia (1997) 40:1243-1250 Diabetologia 9Springer-Verlag 1997 Originals Aminoguanidine inhibits semicarbazide-sensitive amine oxidase activity: implications for advanced glycation and diabetic complications P.H. Yu, D.M. Zuo Neuropsychiatry Research Unit, Department of Psychiatry, University of Saskatchewan, Saskatoon, Saskatchewan,Canada Summary Aminoguanidine, a nucleophilic hydrazi- rats following administration of aminoguanidine. ne, has been shown to be capable of blocking the for- The non-hydrazine SSAO inhibitor (E)-2-(4-fluoro- mation of advanced glycation end products. It reduc- phenethyl)-3-fluoroallylamine hydrochloride (MDL- es the development of atherosclerotic plaques and 72974A) has been shown to reduce urinary excretion prevents experimental diabetic nephropathy. We of lactate dehydrogenase (an indicator of nephropa- have'found that aminoguanidine is also quite potent thy) in STZ-induced diabetic rats. Formaldehyde not at inhibiting semicarbazide-sensitive amine oxidase only induces protein crosslinking, but also enhances (SSAO) both in vitro and in vivo. The inhibition is ir- the advanced glycation of proteins in vitro. The re- reversible. This enzyme catalyses the deamination of sults support the hypothesis that increased SSAO- methylamine and aminoacetone, which leads to the mediated deaminafion may be involved il~ structural production of cytotoxic formaldehyde and methylgly- modification of proteins and contribute to advanced oxal, respectively. Serum SSAO activity was reported glycation in diabetes. The clinical implications for to be increased in diabetic patients and positively cor- the use of aminoguanidine to prevent glycoxida- related with the amount of plasma glycated haemo- tion have been discussed. [Diabetologia (1997) 40: globin. Increased SSAO has also been demonstrated 1243-1250] in diabetic animal models. Urinary excretion of methy- lamine is substantially increased in the rats follow- Keywords Aminoguanidine, methylamine, formalde- ing acute or chronic treatment with aminoguanidine. hyde, semicarbazide-sensitive amine oxidase Urinary methylamine levels were substantially in- (SSAO), SSAO inhibitor, diabetes, advanced glyca- creased in streptozotocin (STZ)-induced diabetic tion, glycoxidation, streptozotocin. Glucose reacts chemically with the amino groups of marked changes in the structure and function of vari- amino acids or nucleic acids to form Amadori prod- ous proteins. The formation of AGEs from the Ama- ucts [1], which then rearrange into advanced glyca- dori product is also known to occur via an oxidative tion end products (AGEs) [2]. This process leads to process [3]. It is accelerated in the presence of transi- tion metals and is reduced by ascorbate [4, 5], sug- gesting that the reaction is mediated by free radicals Received: 8 May 1997 and in revised form: 24 June 1997 [6]. There is accumulating evidence that the forma- tion of AGEs increases with aging [7] and is implicat- Corresponding author: Dr. P. H. Yu, NeuropsychiatryResearch ed in the pathogenesis of diabetic atherosclerosis [2]. Unit, Department of Psychiatry, University of Saskatchewan, AGEs can crosslink with collagen and other struct- Saskatoon, Saskatchewan,S7N 5E4 Canada ural and functional proteins [1]. This increases the Abbreviations: SSAO Semicarbazide-sensitiveamine oxidase; STZ streptozotocin; AGEs advanced glycation end products; rigidity of collagen, decreases arterial compliance, LDH lactate dehydrogenase; LDL low density lipoprotein; and enhances hypertension [8, 9]. Advanced glycated MDL-72974A (E)-2-(4-fluoro-phenethyl)-3-fluoroallylamine collagen is capable of covalently trapping LDL [10]. hydrochloride. LDL, once trapped in the arterial wall, is susceptible 1244 RH. Yu, D.M. Zuo: Aminoguanidine inhibits SSAO activity to attack by free radicals and subject to oxidative (MDL-72974A) were gifts from May and Baker Ltd. (Dagen- modification [11]. AGEs may also affect monocyte ham, UK) and Merrell-Dow Research Institute, Cincinnati, migration and induce an inflammatory response [12, Ohio, USA), respectively. All other chemicals were of analyti- 131. cal grade. Aminoguanidine reacts with Amadori fragmenta- Animal experiments. Male Wistar rats (200 g) were used in the tion products and thus prevents glycation and AGE experiments. The animal treatments were in strict accordance formation [14]. It prevents experimental diabetic with the guidelines established by the Canadian Council on nephropathy [15] and inhibits lipid peroxidation in Animal Care and were approved by the University of Sas- vivo [16]. It also blocks the oxidative modification of katchewan Animal Care Committee. The rats were housed in LDL and its subsequent uptake by macrophages in a hanging wire cages with free access to food and water on a 12 h light/dark cycle (lights on 06.00 hours) at a temperature euglycaemic in vitro system [17]. The drug can reduce of 19-20~ Diabetes was induced in animals by a single ad- the development of atherosclerotic plaque without ministration of STZ (60 mg/kg, i.p.). Serum glucose levels altering serum cholesterol levels in cholesterol-fed were assessed in order to ensure that diabetes was induced. rabbits [18], urinary albumin excretion in diabetic The blood glucose levels of the control and STZ-induced dia- rats [19] and regional albumin clearance [20]. betic rats were 1.61 + 0.04 (mean _+ SEM) and 4.74 + 0.35 mg/ Aminoguanidine is a nucleophilic hydrazine. It is ml (from 3.3 to 7.5 mg/ml) respectively. Aminoguanidine (10 mg/kg) was administered daily via intraperitoneal injection well known that hydrazine compounds are capable for 3 weeks. The aminoguanidine treatment was then main- of interacting with carbonyl groups of different bio- tained by including the drug in the drinking water (1 mg/ml). logical constituents, such as glucose, pyridoxal, 6-hy- The control animals were injected with saline and supplied droxydopa and pyrroloquinolinone quinone enzyme with tap water. Twenty-four-hour urine was collected with met- cofactors} etc. SSAO is an enzyme that possesses 6- abolic cages at 24 h, 1 week, 3 week and 7 weeks after the ini- hydroxydopa as a cofactor, and is quite sensitive to tial aminoguanidine treatment. hydrazines. We therefore investigated whether am- Preparation of SSA 0 from rat aorta and human umbilical ar- inoguanidine inhibits SSAO activity. This enzyme ca- tery. SSAO from human umbilical artery [29] and rat aorta talyses the deamination of methylamine, for example, [30] was prepared as previously described. Tissues were thor- and produces toxic formaldehyde and hydrogen per- oughly rinsed with saline, sliced into small pieces and homoge- oxide [21, 22]. Serum SSAO activity has been found nized with a Polytron homogenizer (PT-10-35, at setting 5 for to be substantially increased in diabetes and patients four periods of 5 s on ice) in chilled 0.01 mol/l phosphate buffer with liver diseases [23-26]. A significant positive cor- (pH 6.8). The crude homogenates were centrifuged at 800 g for 10 rain and the supernatants further centrifuged at 32000 g for relation between SSAO activities and plasma gly- 30 min. These final supernatant enzyme preparations were ei- cated haemoglobin levels was observed [26]. SSAO ther used immediately or stored at -70 ~ The enzymes were activity has also been found to be increased in the quite stable under these conditions for at least I month. blood and kidney of diabetic rats (streptozotocin [STZ]-treated) [27] and diabetic sheep (alloxan- Determination of SSAO activity. SSAO activity was deter- treated) [28]. Deamination of methylamine by serum mined by a radio-enzymatic procedure using 14C-labelled ben- zylamine as substrate following our previously described pro- SSAO is cytotoxic to endothelial cells in vitro and cedure [29]. The SSAO enzyme preparations were pre-incu- SSAO inhibitors block this toxicity [29]; It has been bated with clorgyline (1 x 10-4 mol/l) at room temperature for proposed that excessive deamination of methylamine 20 rain to ensure that any monoamine oxidase activity, if pre- may be involved in the initiation of endothelial injury sent, was completely inactivated. The enzyme was then incu- and be related to diabetic complications [29]. In the bated in the presence of benzylamine (5 x 10-5 mol/1, 0.1 ~tCi) present study we describe the inhibition of SSAO by in a final volume of 200/xl at 37 ~ for 30 min. The enzyme re- aminoguandine and the possible involvement of action was terminated by adding 200 ~tl 2 mol/1 citric acid. The oxidized products were extracted into 1 ml toluene:ethyl ace- SSAO in advanced glycation. Alternative actions of tate (1:1, v/v), of which 600 Ixl was then transferred to a count- aminoguanidine are discussed. ing vial containing 10 ml Omnifluor cocktail (New England Nuclear, Boston, Mass., USA). Radioactivity was assessed by liquid scintillation counting (Beckman LS-7500, Fullerton, Ca- lif., USA). One unit of enzyme activity is defined as one nano- Materials and methods mole of product formed per min per mg of protein. Materials. Human umbilical cords were kindly provided by the Determination ofmethylamine. Urinary methylamine was de- Department of Obstetrics and Gynecology, Royal University termined using an HPLC-fluorometric procedure, lsopropyl- Hospital, Saskatoon. Benzylamine, aminoguanidine bicarbon- amine was added as an internal standard to the urine immedi- ate, methylamine hydrochloride, horseradish peroxidase, 3- ately after its collection and the samples were stored at -70 ~ (4,5-dimethylthiazol-2-yl)-2,5-diphenyl