Diabetologia 9 Springer-Verlag 1997

Transcription

1 Dabetologa (1997) 4: Dabetologa 9 Sprnger-Verlag 1997 rgnals Amnoguandne nhbts semcarbazde-senstve amne oxdase actvty: mplcatons for advanced glycaton and dabetc complcatons P.H. Yu, D.M. Zuo Neuropsychatry Research Unt, Department of Psychatry, Unversty of Saskatchewan, Saskatoon, Saskatchewan, Canada Summary Amnoguandne, a nucleophlc hydrazne, has been shown to be capable of blockng the formaton of advanced glycaton end products. It reduces the development of atherosclerotc plaques and prevents expermental dabetc nephropathy. We have'found that amnoguandne s also qute potent at nhbtng semcarbazde-senstve amne oxdase (SSA) both n vtro and n vvo. The nhbton s rreversble. Ths enzyme catalyses the deamnaton of methylamne and amnoacetone, whch leads to the producton of cytotoxc formaldehyde and methylglyoxal, respectvely. Serum SSA actvty was reported to be ncreased n dabetc patents and postvely correlated wth the amount of plasma glycated haemoglobn. Increased SSA has also been demonstrated n dabetc anmal models. Urnary excreton of methylamne s substantally ncreased n the rats followng acute or chronc treatment wth amnoguandne. Urnary methylamne levels were substantally ncreased n streptozotocn (STZ)-nduced dabetc rats followng admnstraton of amnoguandne. The non-hydrazne SSA nhbtor (E)-2-(4-fluorophenethyl)-3-fluoroallylamne hydrochlorde (MDL A) has been shown to reduce urnary excreton of lactate dehydrogenase (an ndcator of nephropathy) n STZ-nduced dabetc rats. Formaldehyde not only nduces proten crosslnkng, but also enhances the advanced glycaton of protens n vtro. The results support the hypothess that ncreased SSAmedated deamnafon may be nvolved l~ structural modfcaton of protens and contrbute to advanced glycaton n dabetes. The clncal mplcatons for the use of amnoguandne to prevent glycoxdaton have been dscussed. [Dabetologa (1997) 4: ] Keywords Amnoguandne, methylamne, formaldehyde, semcarbazde-senstve amne oxdase (SSA), SSA nhbtor, dabetes, advanced glycaton, glycoxdaton, streptozotocn. Glucose reacts chemcally wth the amno groups of amno acds or nuclec acds to form Amador products [1], whch then rearrange nto advanced glycaton end products (AGEs) [2]. Ths process leads to Receved: 8 May 1997 and n revsed form: 24 June 1997 Correspondng author: Dr. P. H. Yu, Neuropsychatry Research Unt, Department of Psychatry, Unversty of Saskatchewan, Saskatoon, Saskatchewan, S7N 5E4 Canada Abbrevatons: SSA Semcarbazde-senstve amne oxdase; STZ streptozotocn; AGEs advanced glycaton end products; LDH lactate dehydrogenase; LDL low densty lpoproten; MDL-72974A (E)-2-(4-fluoro-phenethyl)-3-fluoroallylamne hydrochlorde. marked changes n the structure and functon of varous protens. The formaton of AGEs from the Amador product s also known to occur va an oxdatve process [3]. It s accelerated n the presence of transton metals and s reduced by ascorbate [4, 5], suggestng that the reacton s medated by free radcals [6]. There s accumulatng evdence that the formaton of AGEs ncreases wth agng [7] and s mplcated n the pathogeness of dabetc atheroscleross [2]. AGEs can crosslnk wth collagen and other structural and functonal protens [1]. Ths ncreases the rgdty of collagen, decreases arteral complance, and enhances hypertenson [8, 9]. Advanced glycated collagen s capable of covalently trappng LDL [1]. LDL, once trapped n the arteral wall, s susceptble

2 1244 RH. Yu, D.M. Zuo: Amnoguandne nhbts SSA actvty to attack by free radcals and subject to oxdatve modfcaton [11]. AGEs may also affect monocyte mgraton and nduce an nflammatory response [12, 131. Amnoguandne reacts wth Amador fragmentaton products and thus prevents glycaton and AGE formaton [14]. It prevents expermental dabetc nephropathy [15] and nhbts lpd peroxdaton n vvo [16]. It also blocks the oxdatve modfcaton of LDL and ts subsequent uptake by macrophages n a euglycaemc n vtro system [17]. The drug can reduce the development of atherosclerotc plaque wthout alterng serum cholesterol levels n cholesterol-fed rabbts [18], urnary albumn excreton n dabetc rats [19] and regonal albumn clearance [2]. Amnoguandne s a nucleophlc hydrazne. It s well known that hydrazne compounds are capable of nteractng wth carbonyl groups of dfferent bologcal consttuents, such as glucose, pyrdoxal, 6-hydroxydopa and pyrroloqunolnone qunone enzyme cofactors} etc. SSA s an enzyme that possesses 6- hydroxydopa as a cofactor, and s qute senstve to hydraznes. We therefore nvestgated whether amnoguandne nhbts SSA actvty. Ths enzyme catalyses the deamnaton of methylamne, for example, and produces toxc formaldehyde and hydrogen peroxde [21, 22]. Serum SSA actvty has been found to be substantally ncreased n dabetes and patents wth lver dseases [23-26]. A sgnfcant postve correlaton between SSA actvtes and plasma glycated haemoglobn levels was observed [26]. SSA actvty has also been found to be ncreased n the blood and kdney of dabetc rats (streptozotocn [STZ]-treated) [27] and dabetc sheep (alloxantreated) [28]. Deamnaton of methylamne by serum SSA s cytotoxc to endothelal cells n vtro and SSA nhbtors block ths toxcty [29]; It has been proposed that excessve deamnaton of methylamne may be nvolved n the ntaton of endothelal njury and be related to dabetc complcatons [29]. In the present study we descrbe the nhbton of SSA by amnoguandne and the possble nvolvement of SSA n advanced glycaton. Alternatve actons of amnoguandne are dscussed. Materals and methods Materals. Human umblcal cords were kndly provded by the Department of bstetrcs and Gynecology, Royal Unversty Hosptal, Saskatoon. Benzylamne, amnoguandne bcarbonate, methylamne hydrochlorde, horseradsh peroxdase, 3- (4,5-dmethylthazol-2-yl)-2,5-dphenyl tetrazolum bromde and semcarbazde hydrochlorde were purchased from Sgma, (St. Lous, Mo., USA). [7-14C]-Benzylamne, [a4c]-methylamne and [methyl-3h]-thymdne were obtaned from Amersham (akvlle, ntaro, Canada). (Clorgylne [N-(2, 4-dchlorophenoxy-n-propyl)-N-methylpropargylamne HC1] and (E)-2-(4-fluorophenethyl)-3-fluoroallylamne hydrochlorde (MDL-72974A) were gfts from May and Baker Ltd. (Dagenham, UK) and Merrell-Dow Research Insttute, Cncnnat, ho, USA), respectvely. All other chemcals were of analytcal grade. Anmal experments. Male Wstar rats (2 g) were used n the experments. The anmal treatments were n strct accordance wth the gudelnes establshed by the Canadan Councl on Anmal Care and were approved by the Unversty of Saskatchewan Anmal Care Commttee. The rats were housed n hangng wre cages wth free access to food and water on a 12 h lght/dark cycle (lghts on 6. hours) at a temperature of 19-2~ Dabetes was nduced n anmals by a sngle admnstraton of STZ (6 mg/kg,.p.). Serum glucose levels were assessed n order to ensure that dabetes was nduced. The blood glucose levels of the control and STZ-nduced dabetc rats were (mean _+ SEM) and mg/ ml (from 3.3 to 7.5 mg/ml) respectvely. Amnoguandne (1 mg/kg) was admnstered daly va ntrapertoneal njecton for 3 weeks. The amnoguandne treatment was then mantaned by ncludng the drug n the drnkng water (1 mg/ml). The control anmals were njected wth salne and suppled wth tap water. Twenty-four-hour urne was collected wth metabolc cages at 24 h, 1 week, 3 week and 7 weeks after the ntal amnoguandne treatment. Preparaton of SSA from rat aorta and human umblcal artery. SSA from human umblcal artery [29] and rat aorta [3] was prepared as prevously descrbed. Tssues were thoroughly rnsed wth salne, slced nto small peces and homogenzed wth a Polytron homogenzer (PT-1-35, at settng 5 for four perods of 5 s on ce) n chlled.1 mol/l phosphate buffer (ph 6.8). The crude homogenates were centrfuged at 8 g for 1 ran and the supernatants further centrfuged at 32 g for 3 mn. These fnal supernatant enzyme preparatons were ether used mmedately or stored at -7 ~ The enzymes were qute stable under these condtons for at least I month. Determnaton of SSA actvty. SSA actvty was determned by a rado-enzymatc procedure usng 14C-labelled benzylamne as substrate followng our prevously descrbed procedure [29]. The SSA enzyme preparatons were pre-ncubated wth clorgylne (1 x 1-4 mol/l) at room temperature for 2 ran to ensure that any monoamne oxdase actvty, f present, was completely nactvated. The enzyme was then ncubated n the presence of benzylamne (5 x 1-5 mol/1,.1 ~tc) n a fnal volume of 2/xl at 37 ~ for 3 mn. The enzyme reacton was termnated by addng 2 ~tl 2 mol/1 ctrc acd. The oxdzed products were extracted nto 1 ml toluene:ethyl acetate (1:1, v/v), of whch 6 Ixl was then transferred to a countng val contanng 1 ml mnfluor cocktal (New England Nuclear, Boston, Mass., USA). Radoactvty was assessed by lqud scntllaton countng (Beckman LS-75, Fullerton, Calf., USA). ne unt of enzyme actvty s defned as one nanomole of product formed per mn per mg of proten. Determnaton ofmethylamne. Urnary methylamne was determned usng an HPLC-fluorometrc procedure, lsopropylamne was added as an nternal standard to the urne mmedately after ts collecton and the samples were stored at -7 ~ untl use. Urne samples (.5 ml) were pre-purfed by applcaton to a small column of CG-5 amberlte (.5 x 2.5 cm), rnsed wth 1 ml water and the amnes then eluted wth 2 ml 1 mol/1 HCI. The samples (1 ~tl) were dervatzed wth o- phthaldaldehyde (PA)(1 ~tl) under alkalne condtons (ph 1.4), and 5 ~tl of the dervatzed sample eluates were separated n a Shmadzu HPLC system (Sl-9A auto njector;

3 P.H. Yu, D.M. Zuo: Amnoguandne nhbts SSA actvty Tokyo, Japan) equpped wth a pre-column dervatzaton program. The fluorescent amne dervatves were separated n an analytcal Ultrasphere I.E column packed wth octadecylbonded sphercal-5 mcron slca partcles (25 x 4.6 mm nternal dameter) (Beckman, Toronto, ntaro, Canada). The column was eluted wth 55 % methanol at a flow rate of 1. ml/ ran usng a SSI 222B solvent delvery system (State College, Pa., USA). For quanttatve assessment, a programmable fluorescence detector (Hewlett Packard, HPt46A, Mssssauga, ntaro, Canada) wth exctaton at 36 nm and emsson at 445 nm was employed. The sgnals of peak area were ntegrated usng a Spectra-Physcs SP-429 ntegrator. The retenton tmes for methylamne and so-propylamne are 11.9 and 29.2 mn, respectvely. The recovery of methylamne from the on exchange chromatography was approxmately 8 %. The peak ratos of methylamne/sopropylamne (nternal standard) were used for the calculaton of urne levels of methylamne. Determnaton of urnary LDH actvty. The assay s based on the producton of NADH from ncotnamde adenne dnucleotde (NAD +) durng the converson of lactate to pyruvate by LDH. NADH was measured spectrophotometrcaliy at 34 nm. All assays were performed n 96-well plates. An alquot of urne (5 txl) was ncubated n the presence of 5 ~tl NAD* (1 mg/ml) and 1 F1 lactate (1%) n.5 tool/1 phosphate buffer, ph 9.. The rates of NADH formaton (mll-optcal densty per ran [rod/ran]) were montored usng a UVmax knetc mcroplate reader (Molecular Devces, Menlo Park, Calf., USA). Proten glycaton n vtro. In vtro glycaton was estmated accordng to Edelsten and Brownlee [14]. The glycaton reactons were conducted at 37 ~ n.8 tool/1 sodum phosphate buffer (ph 7.8) contanng.12 mg/ml penclln-g,.1 mg/ml gentamen,.25 xg/ml fungazone,.2 mg/ml polymyxn B, mmol/l EDTA and 1 mmol/l phenylmethylsulphonyl fluorde. RNase (1 mg/ml) was ncubated for dfferent tme perods wth.5 mol/1 glucose n the presence or absence of formaldehyde (.5 %). AGE formaton was determned fuorospectrometrcally at an exctaton-emsson wavelength of 37/45 nm Rat aorta S S A 121-1{ '~ 8" > t~ -~ n Results Inhbton o f SSA actvty by amnoguandne n vtro. A s can b e s e e n f r o m F g u r e 1, a m n o g u a n d n e s qute p o t e n t at nhbtng the actvtes of S S A obt a n e d f r o m b o t h h u m a n umblcal arteres a n d r a t a o r t a e. T h e IC5 values w e r e e s t m a t e d to b e 1 x 1-5 a n d 2 x 1-5 mol/1 wth r e s p e c t to the r a t a o r t a a n d the h u m a n umblcal e n z y m e s. Effect of amnoguandne on rat kdney and aorta S S A n vvo, The S S A actvty n r a t k d n e y a n d a o r t a was d e t e r m n e d 3 h a f t e r a sngle n j e c t o n of v a r o u s d o s e s of a m n o g u a n d n e. A s c a n b e s e e n f r o m Fgure 2, S S A actvty s sgnfcantly n h b t e d b y t h e d r u g n b o t h tssues. T h e EDs v a l u e s w e r e Human umblcal artery SSA 1 "6 16' Amnoguandne Amnoguandne Fg. 1. Inhbton of the actvtes of SSA obtaned from both human umblcal arteres and rat aortae by amnoguandne. Enzyme actvtes were measured by a radoenzymatc method usng benzylamne (1 x 14 tool/l) as substrate. The specfc actvtes of the untreated control enzymes were and (nmol 9mn -1. mg proten -1) for rat aorta and human umblcal artery SSA respectvely. The enzymes were pre-ncubated n the absence or presence of dfferent concentratons of amnoguandne for 2 mn at room temperature before addton of substrates 5.~ e-" e~ o9 ~'~ ' ~" " Amnoguandne (mg/kg,,p.).2- Statstcal analyss. The results were assessed usng analyss of varance (ANVA) followed by Newman-Keuls multple comparsons. In general, the null hypothess used for all analyses was that the factor has no nfluence on the measured varable and sgnfcance was accepted at the over 95 % confdence level o_ rrv Amnoguandne (mg/kg,.p,) Fg.2. Inhbton of amnoguandne on rat kdney and aorta SSA actvty n vvo. Rat kdney and aorta tssues were collected 3 h after the ntrapertoneal admnstraton of dfferent doses of amnoguandne. Values are mean + SEM of three anmals at each dose. *p <.5, **p <.1 e s t m a t e d to b e 1 a n d 5 m g / k g.p. njecton for the rat k d n e y a n d a o r t a S S A, respectvely. Mode o f nhbton o f human umblcal SSA by amnoguandne. The nhbton o f h u m a n umblcal

4 1246 EH. Yu, D.M. Zuo: Amnoguandne nhbts SSA actvty SSA actvty by amnoguandne was qute fast. Approxmately 9 % of the actvty was nhbted wthout prencubaton of the enzyme wth amnoguandne; complete nhbton could be obtaned wth a 15 mn prencubaton. After ncubaton of the enzyme wth amnoguandne for 3 mn, the unreacted nhbtor was removed by gel fltraton through a small Sephadex G-25 column (PD-1, Pharmaca). SSA actvty was not recovered after the gel fltraton. Untreated SSA actvty was recovered after the same procedure, thus ndcatng that the nhbton of SSA actvty by amnoguandne s rreversble. Effect of amnoguandne on methylamne excreton. A sngle dose of amnoguandne (25 mg/kg) was admnstered to rats and the total urne excreted durng the followng 24 h collected. The level of methylamne, a typcal SSA substrate, n the urne, was sgnfcantly ncreased. The urnary level of methylamne after treatment wth amnoguandne s qute comparable to that from anmals treated wth the potent selectve SSA nhbtor MDL-72974A (2 mg/ kg) [291 (Fg. 3). Chronc effect of amnoguandne on methylamne excreton n control and dabetc rats. As can be seen from Fgure 4 the urnary excreton of methylamne substantally ncreased at 1, 3 and 7 weeks after chronc amnoguandne treatment n both control and dabetc rats. The ncrease ranged from 5- to 1- fold based on ether total methylamne excreton rat -1 (Fg. 4, upper panel) or per mg creatnne (Fg.4, lower panel). The magntude of ncrease of methylamne excreton was much more pronounced followng chronc treatment than a sngle acute treatment (Fg.3), ndcatng that multple treatments wth amnoguandne may produce cumulatve and complete nhbton of SSA actvty. The total methylamne excreton was slghtly ncreased n the STZ-nduced dabetc rats wthout amnoguandne treatment (but only sgnfcant at 7 weeks). When SSA actvty was blocked by amnoguandne, however, the methylamne levels were sgnfcantly hgher n the STZ-nduced dabetc rats at dfferent tme followng amnoguandne treatment. There was a trend toward further ncrease of methylamne excreton n the dabetc anmals wth prolonged llness. Effect of chronc treatment of SSA nhbtor on the urnary excreton of LDH. Urnary LDH has been used as a marker of nephropathy [31]. As can be seen from Table 1 ts actvty was not detected n the control rats, but became detectable by the fourth week and further ncreased by week 12 n STZ-nduced dabetc rats. MDL-72974A was found to reduce the urnary excreton of LDH after 4 weeks of Fg.3. Effect of amnoguandne and the selectve SSA nhbtor MDL-72974A on urnary methylamne levels n rats. Amnoguandne (25 mg/kg), MDL-72974A (2 mg/kg) and salne were admnstered.p. to rats and then 24 h urne was collected. Methylamne was assessed by an HPLC-fluorometrc procedure as descrbed n Methods. Creatnne was determned usng the pcrc acd method. Values are mean + SEM of three anmals at each dose; * p <.1 Fg.4. Subchronc effect of amnoguandne (AG) on urnary methylamne levels n dabetc and control rats. Amnoguandne (1 mg. kg -1. day -1) and salne were admnstered.p. daly to rats for 3 weeks. The anmals were contnuously orally treated wth amnoguandne (. e. the drug was ncluded n the drnkng water [1 mg/ml]). Twenty-four h urne was collected n.5 N HC1 at 1 week and 3 and 7 weeks after amnoguandne treatment. Internal standard sopropylamne was added to each sample and the urne was kept frozen at -7 ~ untl assay. Methylamne was assessed by an HPLC-fluorometre procedure as descrbed n Methods. Creatnne was determned usng the pcrc acd method. Values are mean + SEM of sx anmals n each group; a) sgnfcantly dfferent from the correspondng amnoguandne untreated controls; b) sgnfcantly dfferent from the correspondng non-dabetc controls

5 EH. Yu, D.M. Zuo: Amnoguandne nhbts SSA actvty Table 1. Effect of SSA nhbtor (MDL-72974A) on STZ-nduced nephropathy Treatment Control (n = 9) MDL-72974A (n = 9) STZ (n = 9) MDL STZ (n = 9) mu LDH actvty/mg creatnne EE o 8- Week 1 Week 4 Week 12 L ".15 _+.2 a,b 2.22 _+.3 a,b a a L.U STZ-nduced dabetc rats were ether njected wth MDL72974A (2 mg/kg,.p.) or salne every second day. Smlar salne njectons were also gven to non-dabetc control anmals. A t the end of weeks 1, 4 and 12, a 24 h urne was collected from each anmal. The assay of L D H actvty s descrbed n the Methods. ap <.1, n comparson to controls; b p <.25 n comparson to STZ group. Number of expermental anmals ndcated n parentheses. Statstcs were performed usng twoway A N V A followed by Newman-Keuls multple comparsons treatment n the dabetc rats. MDL-72974A alone was, however, also shown to ncrease the urnary L D H n the rats followng 12 weeks of chronc treatment. MDL-72974A does not affect blood glucose (result not shown). 9 9 [] o RNase + Glucose / RNase + Glucose + Formaldehyde RNase + Formaldehyde J9 RNase E 6. w ~ EffeCt or formaldehyde on proten glycaton n vtro. 9 ~ 4- e-" ffl ~ 2- ~ o ; ; 1'o Tme (days) Fg.& Effect of formaldehyde on advanced glycaton of bovne RNase n vtro. RNase (2 mg/ml) was ncubated wth glucose (.5 moll1) n the presence or absence of formaldehyde (.8 %) at 37 ~ Florescence (Ex 37/Em 45 nm) was measured at dfferent tme perod. Results are of duplcate experments H Bovne RNase was ncubated wth glucose n the absence or presence of a small amount of formaldehyde (.8%). The degree of advanced glycaton was montored on dfferent days. As shown n Fgure 5, formaldehyde nduced a twofold ncrease n advanced glycaton, whle formaldehyde by tself dd not seem to form any fluorescent adducts wth the bovne RNase. 9 I H II I 9.:.... N - C - C... SSA 'C,u (11) CH 2 ""--~ + NH 2 II HzN-NH-C-NH~ - H H --,-,.... N -? - C... SSA C,u (11) CH 2 Dscusson The results clearly ndcate that amnoguandne s a potent, rreversble nhbtor of human and rat SSA both n vtro and n vvo. SSA s a copper-dependent enzyme and probably possesses a 6-hydroxydopa (or pyrdoxal, pyrroloqunolne qunone) moety as cofactor [32]. As ndcated n Fgure 6 amnoguandne, as s the case for other hydraznes [33], can react wth the 3-keto group of the phenyl rng of 6-hydroxydopa (as well as pyrdoxal or pyrroloqunolne qunone groups) and form covalently a hydrazone adduct. The enzyme s therefore rreversbly nactvated by amnoguandne. Amnoguandne blocks advanced proten glycaton n vtro [14]. Whether or not ths drug also nhbts advanced glycaton n vvo remans to be clarfed. Nevertheless amnoguandne sgnfcantly reduces STZ-nduced albumnura [19], regonal albumn clearance [2], lpd peroxdaton n vvo [16], and t retards the development of expermental dabetc nephropathy [15]. These studes not only ndcate NH 2 II N-NH-C-NH~ + H2 - Fg. 6. Scheme showng that amnoguandne, as wth other hydraznes, can react wth the 3-keto group of the phenyl rng of 6-hydroxydopa and form covalently a hydrazone adduct the mportance of the formaton of AGEs n the aetology of complcatons assocated wth dabetes, but also ndcate that the preventon of the formaton of AGEs may be a useful way of ameloratng dabetc complcatons. In these n vvo studes multple daly doses of amnoguandne (. e. from 25 to 5 mg/kg) were used [15, 16, 18-2, 34]. We have shown that a sngle dose of amnoguandne (1mg/kg) almost completely blocks aorta and partally blocks kdney SSA actvty n rats. There s lttle doubt that the multple large doses of amnoguandne, that were employed n those prevous studes, would effectvely nhbt SSA actvty. We have shown that amnoguandne admnstered chroncally caused a substantally

6 1248 RH. Yu, D.M. Zuo: Amnoguandne nhbts SSA actvty greater ncrease of methylamne excreton, a typcal SSA substrate (Fg. 4) than a sngle acute treatment. A complete nhbton of SSA actvty was detected after such chronc treatment (results not shown). Amnoguandne has also been found to nhbt ntrc oxde synthase [35]. Ntrc oxde producton seems to be nvolved n the pathogeness of early dabetc vascular dysfuncton and nhbton of ts actvty by amnoguandne was proposed to be related to the preventon of AGE formaton [36]. The n vvo effects of amnoguandne seem to be more complcated than smply preventng drect chemcal glycaton. Amnoguandne has been wdely used as a damne oxdase nhbtor [37]. Ths s an mportant enzyme regulatng polyamne homeostass and related to several mportant physologcal functons [38]. Amnoguandne, a reactve hydrazne, seems capable of reactng wth pyrdoxal phosphate or the pyrdoxal enzymes and form hydrazone adducts. Hydrazne dervatves also form DNA adducts [39] and have been clamed to be carcnogenc and genotoxc to the lver and other tssues [4]. The potental secondary effects of any hydrazne drugs should not be totally gnored. It s nterestng to note that n dabetes not only s plasma SSA actvty ncreased [24-28], but t also postvely correlates wth plasma glycated haemoglobn and urnary albumn excreton [26]. ur fndngs that MDL-72974A, a selectve non-hydrazne SSA nhbtor, reduces urnary LDH excreton :(a nephropathy ndcator) n STZ-nduced dabetc'rats and that amnoguandne strongly nhbts SSA actvty n vvo, suggest that SSA-catalysed deamnaton may be related to some of the dabetc complcatons, such as nephropathy. Unfortunately ths study was complcated due to the effect of MDL-72974A on the urnary LDH excreton. The apparent ncrease of proten glycaton by formaldehyde at low concentraton n vtro (Fg.5) also suggests the nvolvement of SSA-medated deamnaton n the formaton of AGEs. The mechansm of the formaldehyde effect on proten glycaton remans to be explored. Formaldehyde by tself s a well-known proten fxaton agent, whch s capable of nducng proten crosslnkng due to formaton of methylene brdges of protens and thus nducng modfcaton of mportant structural and functonal protens. The ncreased producton of formaldehyde n the blood from deamnaton of methylamne could be an alternatve mode of ncreasng blood vessel hardenng, whch s consdered to be a major problem assocated wth dabetc complcaton and agng. Interestngly aldehyde dehydrogenase s known to be absent n the plasma [41], therefore, formaldehyde generated n the blood could not be quckly detoxfed. Advanced glycaton s an oxdatve process n whch free radcals are nvolved [3-6]. In fact, dabetes has been consdered to be a state of ncreased oxdatve stress (a dsturbance of balance between oxdatve stress factors and antoxdant factors n favour of the former) [4]. Increases n the levels of crculatng lpd peroxdes [42], conjugated denes [43], and malonaldehyde [16] have been found to be assocated wth dabetc atheroscleross. Ths hypothess has been confrmed n a recent fndng that hydroxyl free radcal s ncreased n blood and tssues n STZnduced dabetc rats [44]. It s, however, not yet known what trggers the ncrease n the oxdatve stress n dabetes. The ncrease n SSA actvty n serum or kdney tssues of dabetc patents and anmals [23-28] may relate to ths ncrease n oxdatve stress due to excessve deamnaton. Deamnaton reactons lead to the producton of hydrogen peroxde, whch s n turn converted to toxc hydroxyl free radcals va the Fenten reacton n the presence of transton metals [45]. It s nterestng to note that SSA s selectvely located n vascular smooth muscles [46, 47], cartlage [48] and the retna [49] and also crculates n the blood [23-28]. These tssues are known to be vulnerable to glycaton and damage n dabetc patents. SSA catalyses the deamnaton of endogenous alkylamnes, such as methylamne [21, 22] and amnoacetone [5]; toxc formaldehyde and methylglyoxal are produced respectvely. SSA-medated deamnaton of methylamne has been demonstrated to be cytotoxc to endothelal cells n vtro [29]. It was also observed that formaldehyde, derved from deamnaton of methylamne, forms macromolecular adducts and nduces proten cross-lnkng n vvo [51]. It s ntrgung that urnary methylamne excreton s enhanced n the dabetc rats (Fg. 4). Ths observaton s n fact consstent wth a classc 1935 report [52]. In ths study only three patents were analysed wth a rather prmtve method. We found approxmately twofold ncrease of urnary methylamne n the dabetc rats, when SSA actvty was blocked by chronc admnstraton of amnoguandne. If SSA actvty was not blocked, methylamne would be quckly metabolzed. The ncrease of methylamne metabolsm n the dabetc rats was less clearly evdent. It s possble that the ncreased SSA actvty n the dabetes may be a result of up-regulaton due to ncrease of SSA substrates,.e. methylamne or amnoacetone. It remans to be establshed how methylamne s ncreased n dabetes. Blood methylglyoxal level was found to be sgnfcantly ncreased n dabetc patents [53]. Ths actve endogenous aldehyde also bnds and modfes protens under physologcal condtons [54] and has been consdered to be cytotoxc and related to dabetc complcatons [55]. Amnoguandne prevents dabetes-nduced arteral wall proten crosslnkng [56] and reduces dabetc nephropathy [15]. Amnoguandne reacts wth methylglyoxal under physologcal

7 P.H. Yu, D.M. Zuo: Amnoguandne nhbts SSA actvty 1249 condtons [57]. It seems to be reasonable to propose that amnoguandne can also chemcally react wth and detoxfy toxc aldehydes. In concluson excessve SSA-medated deamnaton,. e. n dabetes, would ncrease the producton of toxc aldehydes, such as formaldehyde and methylglyoxal, and enhance oxdatve stress, whch are n favour of nducton of advanced proten glycaton. Amnoguandne can effectvely block SSA actvty and/or react wth the toxc aldehyde products. Ths may be, at least partly, related to the reducton of dabetc expermental albumnura and nephropathy. More nvestgaton s requred to substantate ths hypothess. Acknowledgements. We are grateful to the Medcal Research Councl of Canada, the Canadan Dabetes Assocaton and Saskatchewan Health for fnancal support, and Danne Young and Keth Jay for techncal assstance. References 1. Brownlee M, Ceram A, Vlassara H (1988) Advanced glycosylaton end products n tssue and the bochemcal bass of dabetc complcatons. N Engl J Med 318: Bfownlee M (1992) Non-enzymatc glycosylaton of macromolecules: prospects of pharmacologcal modulaton. Dabetes 41[Suppl 2]: Fu MX, Knecht KJ, Thorpe SR, Baynes JW (1992) Role of oxygen n cross lnkng and chemcal modfcaton of collagen by glucose. Dabetes 41[Suppl 2]: Baynes JW (1991) Role of oxdated stress n development of complcatons n dabetes. Dabetes 4: Hunt JV, Bottoms MA, Mtchnson MJ (1993) xdatve alteraton n the expermental glycaton model of dabetes melltus are due to proten-glucose adduct oxdaton. Bochem J 291: Hunt JV, Smth CCT, Wolff SP (199) Autooxdatve glycosylaton and possble nvolvement of peroxdes and free radcals n LDL modfcaton by glucose. Dabetes 39: Monner VM, Kohn RR, Ceram A (1984) Accelerated age related brownng of human collagen n dabetes melltus. Proc Natl Acad Sc USA 81: Lo SK, Janakdev K, La L, Malk AB (1993) Hydrogen peroxde-nduced ncrease n endothelal adhesveness s dependent on ICAM-1 actvaton. Am J Physol 264: L46-L Uustupa MIJ, Nskanen LK, Sttonen, Voutlanen E, Pyorala K (1993) Ten-year cardovascular mortalty n relaton to rsk factors and abnormaltes n lpoproten composton n type 2 (non-nsuln-dependent) dabetc and nondabetc subjects. Dabetologa 36: Brownlee M (1985) Mcrovascular dsease and related abnormaltes: Ther relaton to control of dabetes. In: Marble A et al. (eds) Josln's dabetes melltus. Lea & Febger, Phladelpha pp Mullarkey CJ, Edelsten D, Brownlee M (199) Free radcal generaton by early glycaton products: a mechansm for accelerated atherogeness n dabetes. Bochem Bophys Res Comm 173: Brett J, gawa S, Krsten M, Radoff S, Vlassara H, Stern D (199) Advanced glycosylaton end products selectvely attract monocytes to mgrate across endothelal cell monolayers and nduce actvaton and growth factor elaboraton. Crculaton 82[Suppl 3]: 92 (Abstract) 13. Vlassara H, Makta Z, Rayfeld E, Fredman E, Ceram A, Morgelo S (199) In vtro advanced glycaton as a sgnal for monocyte mgraton n vessel wall: role n dabetes and agng. Crculaton 82[Suppl 3]: 92 (Abstract) 14. Edelsten D, Brownlee M (1992) Mechanstc studes of advanced glycosylaton end product nhbton by amnoguandne. Dabetes 41: Souls-Lparota T, Cooper M, Papazoglou D, Clarke B, Jerums G (1991) Retardaton by amnoguandne of development of albumnura, mesangal expanson and tssue fluorescence n streptozotocn nduced dabetc rats. Dabetes 4: Bucala R, Makta Z, Koschnsky T, Ceram A, Vlassara H (1993) Lpd advanced glycaton: pathway for lpd oxdaton n vvo. Proc Natl Acad Sc USA 9: Pcard S, Parathasarathy S, Fruebs J, Wtzum JL (1992) Amnoguandne nhbts oxdatve modfcaton of low densty lpoproten and the subsequent ncrease n uptake by macrophage scavenger receptors. Proc Natl Acad Sc USA 89: 'Bren RE, Panangotopoulos S, Cooper MS, Jerums G (1992) Ant-atherogenc effect of amnoguandne, an nhbtor of advanced glycaton. Dabetes 4[Suppl 1] 16A (Abstract) 19. Itakura M, Yoshkawa H, Banna C et al. (1992) Amnoguandne decreases urnary albumn and hgh-molecular weght protens n dabetc rats. Lfe Sc 49: Hujberts MSP, Wolffenbuttel BHR, Crjns FRL et al. (1994) Amnoguandne reduces regonal albumn clearance but not urnary albumn excreton n streptozotocndabetc rats. Dabetologa 37: Yu PH (199) xdatve deamnaton of alptaatc amnes by rat aorta semcarbazde-senstve amne oxdase. J Pharm Pharmacol 42: Boor PJ, Trent MB, Lyles GA, Tao M, Ansar GAS (1992) Methylamne metabolsm to formaldehyde by vascular semcarbazde-senstve amne oxdase. Toxcology 73: McEwen CM Jr, Castell D (1967) Abnormaltes of serum monoamne oxdase n chronc lver dsease. J Lab Cln Med 7: Nlsson SE, Trydng N, Tufvesson G (1968) Serum monoamne oxdase n dabetes melltus and some other nternal dseases. Acta Med Scand 184: Trydng N, Nlsson SE, Tufvesson Get al. (1969) Physologcal and pathologcal nfluences on serum monoamne oxdase level. Scan J Cln Lab Invest 23: Boomsma E Derkx FHM, van den Meracker AH, Veld AJM, Schalekamp MADH (1995) Plasma semcarbazdesenstve amne oxdase actvty s elevated n dabetes melltus and correlates wth glycosylated haemoglobn. Cln Sc 88: Hayes BE, Clarke DE (199) Semcarbazde-senstve amne oxdase actvty n streptozotocn dabetc rats. Res Comm Chem Pathol Pharmacol 69: Ellott J, Fowden AL, Callngham BA, Sharman DE Slver M (1991) Physologcal and pathologcal nfluences on sheep blood plasma amne oxdase: effect of pregnancy and expermental alloxan-nduced dabetes melltus. Res Vet Scence 5: Yu PH, Zuo DM (1993) Methylamne, a potental endogenous toxn for vascular tssues: formaton of formaldehyde va enzymatc deamnaton and the cytotoxc effects on endothelal cells. Dabetes 42:594-63

8 125 EH. Yu, D.M. Zuo: Amnoguandne nhbts SSA actvty 3. Yu PH, Zuo DM (1991) (E)-4-Fluoro-beta-fluoroethylene benzene butamne (MDL-72974A) as hghly potent nhbtor for semcarbazde-senstve amne oxdase from vascular tssues and serum of dfferent speces. Bochem Pharmacol 43: Chounard S, Vau C (1992) Reversblty of renal tubular dysfuncton n streptozotocn-nduced dabetes n the rat. Can J Physol Pharmacol 7: Jane SM, Mu D, Wemmer D et al. (199) A new redox cofactor n eukaryotc enzymes: 6-hydroxydopa at the actve ste of bovne serum amne oxdase. Scence 248: Jane SM, Klnman JP (1991) An nvestgaton of bovne serum amne oxdase actve ste stochometry: evdence for an amnotransferase mechansm nvolvng two carbonyl cofactors per enzyme dmer. Am Chem Soc 3: Nchols K, Mandel TE (1989) Advanced glycosylaton end-products n expermental murne dabetc nephropathy: effect of slet sograftng and of amnoguandne. Lab Invest 6: Corbett JA, Tlton RG, Chang K et al. (1992) Amnoguandne, a novel nhbtor of ntrc oxde formaton, prevents dabetc vascular dysfuncton. Dabetes 41: Tlton RG, Chang K, Hason KS et al. (1993) Preventon of dabetc vascular dysfuncton by guandnes; nhbton of ntrc oxde synthase versus advanced glycaton end-product formaton. Dabetes 42: Tamura H, Horke K, Fukura H, Watanabe T (1989) Knetc studes on the nhbton mechansm of damne oxdase from porcne kdney by amnoguandne. J Bochem (Tokyo) 15: Seler N (1995) Polyamne oxdase, propertes and functons. Prog Bran Res 16: Mathson BH, Murphy SE, Shank R (1994) Hydralazne and other hydrazne dervatves and the formaton of DNA adducts. Toxcol Appl Pharmacol 127: Bosan WS, Shank RC, MacEven JD, Gaworske CL, Newberne PM (1987) Methylaton of DNA guanne durng the course of nducton of lver cancer n hamsters by hydrazne or dmethylntrosamne. Carcnogeness 8: Helander A, Tottmar (1987) Metabolsm of bogenc aldehydes n solated human blood cells, platelets and n plasma. Bochem Pharmacol 36: Sato Y, Hotta N, Sakamonto N, Matsuoka S, hshn N, Yag IK (1979) Lpd peroxde levels n plasma of dabetc patents. Bochem Med 21: Jennngs PE, Jones AF, Florkousk CM, Lunc J, Barnett AH (1987) Increased dene conjugates n dabetc subjects wth mcroangopathy. Dabet Med 4: hkuwa T, Sato Y, Nao M (1995) Hydroxyl radcal formaton n dabetc rats nduced by streptozotocn. Lfe Sc 56: Gutterdge JMC (1994) Hydroxyl radcals, ron, oxdatve stress and neurodegeneraton. Ann N Y Acad Sc 738: Lyles GA, Sngh I (1985) Vascular smooth muscle cells: a major source of the semcarbazde-senstve amne oxdase of the rat aorta. J Pharm Pharmacol 37: Wbo M, Duong AT, Godfrand T (198) Subcellular locaton of semcarbazde-senstve amne oxdase n rat aorta. Eur J Bochem 112: Lyles GA, Berte KH (1987) Propertes of a semcarbazdesenstve amne oxdase n rat artcular cartlage. Pharmacol Toxcol [Suppl] 1: Zuo, DM, Yu, PH (1993) Semcarbazde-senstve amne oxdase and monoamne oxdase n rat bran mcrovessels, mennges, retna and eye sclera. Bran Research Bull 33: Lyles GA, Chalmers J (1992) The metabolsm of amnoacetone to methylglyoxal by semcarbazde-senstve amne oxdase n human umblcal artery. Bochem Pharmacol 31: Yu PH, Zuo DM (1995) Formaldehyde produced endogenously va deamnaton of methylamne; a potental rsk factor for ntaton of endothelal njury. Atheroscleross 12: Kapeller-Adler R, Toda K (1932) lslber das Vorkommen von Monomethylamne m Harn. Bochem Zeschrft 248: McLellan AC, Phllps SA, Thornalley PJ (1992) The assay of methylglyoxal n bologcal systems by dervatzaton wth 1,2-damno-4,5-dmethoxybenzene. Anal Bochem 26: Lo TWC, Westwood ME, McLellan AC Selwood T, Thornalley PJ (1994) Bndng and modfcaton of protens by methylglyoxal and physologcal condtons. J Bol Chem 269: Thornalley PJ (199) The glyoxalase system: new developments towards functonal characterzaton of a metabolc pathway fundamental to bologcal lfe. Bochem J 269: Brownlee M, Vlassara H, Kooney P, Ulrch P, Cera A (1986) Amnoguandne prevents dabetes-nduced arteral wall proten cross-lnkng. Scence 232: Lo TWC, Selwood T, Thornalley PJ (1994) The reacton of methylglyoxal wth amnoguandne under physologcal condtons and preventon of methylglyoxal bndng to plasma protens. Bochem Pharmacol 48: