• ELSEVIER SCIENCE
`
`IRELAND
`
`Chemico-Biological interactions 90 ( 1994) 1-12
`
`Chemleo-8lologlcol
`lntllroction.f
`
`Influence of various compounds on the
`degradation of hyaluronic acid by a
`myeloperoxidase system
`
`Sven Lindvall*, Gunilla Rydell
`Infection and Immunology, Preclinical R & D, Astra Arcus AB, S-151 85 Soderiilje, Sweden
`
`(RCQCived 6 November 1992; revision received 10 May 1993; accepted 11 May 1993)
`
`Myeloperoxid.a$e in the presence of 0. 7 mM hydrogen peroxide degrades hyaluronic by a
`mecbar.usm which involves iron. Degradation is enhanced in the presence .of chloride ion,
`which is attributed to the formation of hypochJorous acid. Myeloperoxida!H'-dcpendcnt de(cid:173)
`gradation of hyaluronic acid . is inhibited by superoxide · dismutase, desferrioxamine, iodide
`ion, bromide ion, mannitol, histidine and various antiinflammatory agents. The destructing
`agent is presumably the hydroxyl radical.
`
`Key · words: Myelopcroxidase; Free radicids; Ir.on; Hypochlorous acid; Anti•inflammatory
`agents
`
`1. Introduction
`
`Many factors have been recognized which activate the polymorphonuclear
`leucocytes (PMN) to produce tissue damage (1,2). Perturbation of the· PMN mem(cid:173)
`brane which starts the oxidative metabolism of tbe cells, i.e., tbe respiratory burst,
`is essential for this activation (3). Tbe respiratory burst is associated with the re.lease
`and furd1er generation of the highly tox:ic and reactive ox:ygen-ccmtaining species
`H 20 2, 0 2 -;-, 10 2, OH• and HOCl into the phagosome with the ingested foreign
`material as· well as into the extracellular. space 14-9). During phagocytosis some
`
`• Corresponding 11-uthor, Fribovagen 2C, 15144 Soderiilje, Sweden.
`
`0009~2797/94/S07.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved.
`SSDJ 0009-2797(93)03202-6
`
`Exhibit 1114
`Prollenium v. Allergan
`IPR2019-01505 et al.
`
`

`

`2
`
`S. Lindva/1 and G. Rydell I Chem.-Biol. lnreract. 90 (1994) J-12
`
`myeloperoxidase (MPO) is also released from the PMN into the extracellular space
`where the enzyme mediates the iodination of plasma proteins [IOJ.
`Myeloperoxidase, H 2O 2 and a halide form a potent antimicrobial and cytotoxic
`system. It · is suggested that release of MPO and H 2O2 from polymorphonuclear
`leucocytes results in a peroxidase-mediated leucocytic injury which may be an
`important feature of the inflammatory response (11). The release of radicals and en(cid:173)
`zymes into the s-urrounding environment by phagocytic cells is a potential cause of
`damage to the tissues and chronic inflammatory disease results when this damage
`gets out of hand. The destructive effects depend on the formation of hydroxyl
`radicals (12) .
`A complex of hydrogen peroxide with MPO has been described ( 13) which has
`been classified as peroxidase Compound II (l4]. On addition of increasing concen(cid:173)
`trations of H202 to MPO it has been shown that three compounds are formed:
`compounds I, II and Ill. When using H 2O 2 concentrations below 0.5 mM, Com(cid:173)
`pound H is formed, which up~>n further addition of ff 2O2 is convened to c;om(cid:173)
`pound JU [15). Compound III is also formed on addition of 0 2 7
`to MPO [16].
`Evidence has be.en presented that superoxide anion radicals a.re generated from
`is
`hydrogen peroxide .in the presence of MPO and that it is conceivable that 0 2 7
`generated in .the reduction ofCompoundI to Compound n as well as in the reduc(cid:173)
`tion of Compound . n . to the . native enzyme ll 7].
`Studies on tbef onnation of hydroxyl radicals have been performed using the
`superoxide-fonning enzyme xanthine oxidase. The substrates used were methional
`U 8l as well asdeoxyribose, benzoate ora-keto-'Y-methiolbutyric acid (19]. In the lat(cid:173)
`ter cases iron was used as a catalyst. Hyaluronic acid has also been used in studies
`on the formation of OH· by xanth.ine oxidase and the decrease in the viscosity of
`a solutionofthe acid was related to the extent of formationof hydroxyl radicals (20) ,
`In the present study, hyaluronic acid has been used as substrate. The production
`of. OH . •. by Mf>O . in a gystem containing high concentrations of H202. and NaCl
`and with iron as a catalyst has been investigated. lnaddition, the influence ofvarjous
`compounds on the formation of the . hydroxyl radicals as . well . as . their effect on the
`radicals themselves · has been • studied.
`
`2. Experimental
`
`2. I. Materials
`Myeloperoxidase (EC l.1 LI. 7) was isolated from granulocytes from healthy
`donors [21) and had a UV-absorption ratio A,t~of A 280 9f 0.82. Superoxide dismutase
`was obtained from Sigma Chemical Co., St. Louis, USA.
`The potassium salt ofhyaluronic acid, which had been isolated from human um(cid:173)
`bilical cord, was purchased from Sigma ,Chemical Co. Analysis of Cu and Fe in a
`solution of LS rng hyaluronic acid per rnl in 0.1 M phosphate buffer by means of
`atomic absorption spectroscopy showed a content of 0.07 µg Cu/ml and 0.35 .µg
`Fe/ml. The buffer contains 0.08 and 0.09 µglrnl of Cu and Fe, respectively. A solu(cid:173)
`tion of l.6 M NaCl in buffer had a content of 0.35 µ,g Fe/ml analysed by use of
`polarography with a hanging mercury drop electrode. Analysis of c1- by poten(cid:173)
`tiometric titration with si~ver nitrate showed that the hyaluronic acid solution con(cid:173)
`tains 80 µM and the buffer < 3 µ,M c1-.
`
`

`

`S. Lirulvall and G. Rydell/Chem.-Blo/. Interact. 90 (1991) 1-12
`
`3
`
`Pesf errioxamine was obtained from Ciba-Geigy, Switzerland, mannitol, rein st,
`from Merck Schuchard, Germany, 6-chloro-3-hydrazinopyridazine (ABC 907) from
`Maybridge Chemical Co. Ltd., UK, a-histidine, p-hydroxybenzoic acid and p(cid:173)
`hydroxyacetanilide (Paracetamol) from Fluka, Buchs, Switzerland. Salicylic acid,
`acetylsalicylic acid and diethylaminoaceto-2,6-xylidide (Lidocaine) were provided by
`AB Astra, Sweden. 2-(4-lsobutylphenyl) ptopionic acid (Ibuprofen) and 4-butyl-l,
`2'-diphenyl-3,5-pyrazolidinedione (Phenylbutazone) were purchased from Sigma
`Chemical Co., USA.
`
`2.2. Methods
`A mixture of 1375 µl 0.1 M phosphate buffer, pH 6.9, (compounds studied were
`dissolved in the buffer), 2000 µl of a freshly prepared solution of hyaluronic acid in
`buffer(l.5 mg/ml), 500 µI of a l.6-M solution of NaCl in buffer and 25 µl of a 6.9
`µM solution of MPO is thoroughly shaken. The reaction is started by adding 100
`µ1 ofa 0.028.M solution of 1-120 2 in buffer. 3,0 ml ofthis mixture are immediately
`transferred to an Ostwald viscosimeter with a outflow time of 28.4 s for the phos(cid:173)
`phate buffer. The outflow time of the mixture was measured 1,3,5,7,9 and 15 min
`aftet the addition of the ff20 2 solution. AU samples were maintained at 25° in a
`thermostat. The specific viscosity was calculated according to the formula
`
`;:;: outflo~ time reaction .solution _ I (22).
`"1s
`outflow time solvent ·
`P
`The values given in figures and tables are l/"1sp at v~rious times after ttie addition
`·
`of H 20 2 from which the l/178" value for the substrate is subtracted. · -
`
`3. Results
`
`Addition of MPO, NaCl and H 20 2 to a solution of hyaluronic acid reduces the
`viscosity of this solution to a high degree. Omission of the enzyme as well as using
`a .boiled solution of the enzyme results in a considerable decrease in the degradative
`effect. When NaCl is omitted, the degradative effect is lowered to a certain degree
`(Fig. 1). On omission of H20 2 from the system, no change in the viscosity could be
`observed.
`Replacement of NaCl (200 mM) with KI or KBr at a concentration above 2.0 mM
`inhibits the degradative effect almost completely. Even in the . presence of NaCl,
`these two halides in}:tibited the degradative effect. As can be seen in Table I, iodide
`has a strong inhibitory effect at a concentration of 5.0 µM. Almost complete inhibi(cid:173)
`tion of the degradative effect was observed at concentration of 50.n µM. Bromide
`ion does not show such a pronounced effect. At a concentration of 200.0 µM, the
`extent of degradation is still 70%. Almost complete inhibition occurred at a concen(cid:173)
`tration of 2.0 mM when200 mM NaCl was present It was observed that the reaction
`mixture became coloured when iodide was added indicating liberation of I2 during
`the process.
`As degradation of hyaluronic acid by : the superoxide anion-generating enzyme,
`xanthine oxidase,
`is
`inhibited by
`the OH· -scavenger mannitol, superoxide
`dismutase, or the iron-complexing compound desferrioxamine 120,23-251, the influ-
`
`

`

`4
`
`S. Lindva/1 and G. Ryde/l lChem.-Biol. Interact. 9() (1994) 1-12
`
`1.00
`
`It
`E
`
`-
`
`0.50
`
`com~te
`MPO•System
`
`NaCl
`omitted
`
`MPO
`omitted or
`boiled
`MPO
`
`----4--1---t-f-----I
`
`O..Jooie::.:,'-------.--------------.----..._..._ __ -,-
`15
`10
`5
`0
`Minutes
`Fig. l . The degradative effect on hyaluronic acid by the MP().system and the influence of MPO and NaCl
`on this system, The mean values and ranges of six separate determinations are given.
`
`/
`
`ence of these compounds on the MPO-system was studied. As can be seen in Fig.
`2. a concentration of 2.0 rnM mannitol inhibits the degradative effect of the MPO(cid:173)
`system by about 50%. At a concentration of 200.0 mM the inhibition is complete.
`ln Fig. 3. the effect of superoxide dism-utase and desferrioxamine is clemonstratecl.
`When 10.0 µg superoxide dismutase wasadded, there was almost complete inhibition
`of the degradative effect by t.he complete MPO-system. Similar results were obtained
`when. >0,2 mM desferrioxami11e was add~d. This chelator .• eliminates . the con(cid:173)
`tamjnating iron which is present· at a concentration of 4S 14M in. the reaction mixture
`126). The low degradative activity seen when the enzyme was omitted from the reac-
`
`Table I
`The viscosity (llti,p) of a solution of hyaluronic acid after degradation by a MPO•system containing
`various concentrations of Kl and KBr
`
`C<>nc.
`µM
`
`0.0
`5.0
`10.0
`50.0
`200.0
`
`Kl
`
`1 '1sp
`
`0.62 (0.62,0.62)
`0.17 . (0.14,9,.19)
`0.09 (0.08,0. 10)
`0.05 (0.04,0.06)
`0.02 (0.01,0.02)
`
`%,
`
`100
`27
`15
`8
`3
`
`KBr
`
`l/>1sp
`
`0.71 (0.09,0.72)
`0.65 .(0;59,0.71)
`0.61 (0.58,0.63)
`0.56 (0.54,0.,57)
`0 .50 (0.48,0.51)
`
`%
`
`100
`92
`86
`79
`71
`
`Reaction times in minutes. Mean values and range of duplicate experiments. The (1/'lsp) seen after addi-
`tion of the compound in pen;:cnt of the control is al$O given.
`.
`
`

`

`S. 1,,,lnthall and G. Rydell/ Chem.~Biol. Interact. 90 ( 1994) 1-12
`
`s
`
`1.00
`
`0.60
`
`/
`
`Complete
`-----------lC MPO-ayst.-n (A)
`
`x
`
`A+O.SmM
`. . • - -•--_.;,_..,... ___ _... mannttol
`
`_
`-r.:>
`
`________ -., A+ 2.0 mM
`mannltol
`
`-----x ----·
`.------
`o-----c,-
`0_,,,/"
`o D 01------------0 A .. + 20.0 mM
`
`mannttol
`
`5
`
`· 1D
`
`Minutes
`
`15
`
`Fig. 2. Influence of mannitol on the desradation (tf hyaluronic acid by the MJ>O.system. The mean values
`. of two separate de.terminations arc given.
`
`tion mixture, was almost complete inhibited on addition of either superoxide
`dismutase or. desferrioxallline (Fig. 3).
`The results from. the studies on the influence of various compounds on fhede(cid:173)
`gradation of hyaluronic acid by the µse. of the MPO~system shQwthat one. group of
`these agents .for . the •. most part acts in the same manner as mannitol .. In Table 2 it
`is shown that these compounds inhibit the viscosity..(lecr~asi-,g effect of the MPO(cid:173)
`system in the order paracetamol > lidocaine . > salicylic acid > p,-hydroxybenzoic
`acid > . ibuprofen > acetyls.alicylic acid. A difference was seen in the inhibitory pat•
`tern . at low concentrations of paracetamol and p-hydroxybenzoic acid compared to
`the other compounds. These two compounds · wit.h a hydroxyl group in th.e para ..
`position have initially a low jnhibitory. effect onJhe degradation of byflluronic acid,
`but an increase ofthe inhibition could beseen. during the following minutes. How(cid:173)
`ever. at higher .concentrations o.f these two compounds, the degree of inhibition re(cid:173)
`mained constant during the experimental period as for the other eompoumJs in t.his
`group.
`An.,tber group o.f compounds "'--bistidine, phenylbutazone and ABC907-· inhi(cid:173)
`bit the degradation ofhyaluronic acid by the MPQ .. system in quite a diff eren.t man(cid:173)
`ner. When .these compounds were added to the system, a high degree of inbjbition
`was. initially observed, but was . followed by a decrease . in inhibition during the
`following minutes. As can be seen in Table 3, the increase in the degradation at a
`certain concentration of the compounds is the same as that seen when the enzyme
`is omitted from the reaction mixture. The results show also that ABC 907 initially
`
`

`

`6
`
`S. Li11dvall and G. Rydell/Chem.-Biol. /111eract. 90 (/994) 1-12
`
`1.00
`
`A.
`
`G.
`~ 0.50
`,,_
`
`1--I____.--i
`~1-:::-J:=-.... ----I--------------~
`
`MPO
`omitted (B)
`
`A and B +
`200µ11
`DNferrloxamme
`
`0-fll-----·:·;.;.-.... -_;;. ____ --r ______ _,__...,,_ ________ ..,..
`
`0
`
`1.00
`
`B.
`
`5
`
`Mlnuhta
`
`10
`
`15
`
`MPO
`x~x omltted(B)
`
`..
`I' E" 0.50
`
`0
`
`0
`
`. -0--------------0 B + 10.0 µg
`::i----·• . . ·. · ..
`==~ ...,.,_ ...... o~ .... ~ .... 0 - - - -
`s
`
`A+ 10.0 µg
`.
`. ·.
`---IC - - -
`~ • ____ •----•·-----------------• SOD/ml
`SOD/1111
`-
`ro
`
`u
`
`Minutes
`
`Fig. 3 (A), influence or desrerrioxamine on the degradation ofhyaluronic acid by the MPO-syiitem and
`the system without MPO. Mean values and ranges or three separate determinations. (B), influence of
`superoxide disrnutase (SOD) on the degradation of hyaluronic acid by the MPO-system and the system
`without MPO. Mean values of two separate determinations.
`
`has a pronounced inhibitory effect on the complete MPO-system at a concentration
`as low as t .S, µM.
`
`4. Discussion
`
`It is obvious from the present study that the MPO-system used degrades
`hyaluronic acid. A high concentration of hydrogen peroxide, superoxide anion radi-
`
`

`

`S. Undvall and G. Rydell/ Chem.-Biol. Interact. 9() ( 1994) 1~12
`
`7
`
`Table 2
`The viscosity (11'1,p) of a solution of hyaluronic acid in percent of the control after degradation by a
`MPO-system containing various compounds at a reaction time of 5 min
`
`Cone.
`pM
`
`Salicylic
`acid
`
`Acctylsalcylic Ibuprofen
`acid
`
`Lidpcaine
`
`Paracetamol
`
`p-Hydroxy-
`bcnzoic acid
`
`0
`15.0
`50.0
`100
`150
`200
`300
`500
`1000
`
`100
`
`100
`
`100
`
`100
`
`48 (47,48)
`
`too (100,100)
`
`76 (73,78)
`
`57 (56,57)
`32 (30,34)
`
`36 (34,38)
`
`17 (16,18)
`
`60 (55,65)
`
`65 (65,65)
`14 (12,15)
`
`34 (29,37)
`34 (31,37)
`
`14 (12,15)
`
`100
`51 (49,52)
`
`100
`100 (97,100)
`
`18 (17,19)
`
`65 (65,65)
`
`17 (17)
`
`47 (~.48)
`
`cal and iron seems to be e.ssential for this effect. Evidence for hydroxyl radical as
`the degradative species is supported by the fact that a concentration of 20.0 mM of
`the OH· -scavenger mannitol nearly · completely inhibits the degradation of
`hya)uronic acid,
`It · has • been shown that 0 2 • is formed when H20 2 is added to the native enzyme
`(17). Taking this result into consideration. it may be assumed that in the .present
`study, addition of H20 2 at a concentration of 0. 7 mM to the c1- free MPO-system,
`which contains some contaminatjns iron, .results in .formation of OH· according to
`the Haber-Weiss-reaction. The small hon-enzymatic degradative effect seen on omis(cid:173)
`sion of the enzyme is also dependent on iron and 0 2 7 and is started by the slow
`reaction Fe 3++H202 - Fe2+ + 02· + u+.
`
`Table 3
`The viscosity ( ll'lsp) of a solution of hyaluronic acid in percent of the control. after depolymeri:zation by
`a MPO-system containing various compounds
`
`Compounds
`
`Cone.
`pM
`
`Minutes after addition of H202
`
`5
`
`9
`
`15
`
`Histidine
`
`Phenylbutazone
`
`ABC 907
`
`MPO-omitted
`
`0
`200
`2000
`20000
`100
`200
`500
`I.S
`15.0
`150
`
`100
`27 (27,29)
`3 (2,3)
`0
`64 (63,64)
`44 (41,47)
`24 (24,24)
`30 (26,34)
`12 (10,13)
`8 (8,8)
`6
`
`100
`41 (40,41)
`12(11,13)
`0
`75 (73,78)
`54 (52,57)
`28 ·(27,29)
`54 (S3,54)
`35 (35,35)
`17 (15, 19)
`14
`
`100
`54 (53,55)
`19 (18,20)
`0
`86(85,87)
`64 (62,66)
`32 (30,33)
`68 (67,68)
`47 (4(),48)
`26 (23,28)
`22
`
`100
`58 (58,58)
`25 (23,26)
`0
`91 (91,91)
`72 (7l,73)
`34 (34,34)
`82 (81,82)
`63 (62,63)
`37 (35,39)
`34
`
`

`

`8
`
`S. Lindvall and G. Rydell/ Chem.-Biol. Interact. 90 ( 1994) J-12
`
`Native MPO reacts with H 20 2 to form Compound l, which oxidizes chloride to
`hypochlorous acid with concomitant regeneration of the native enzyme. At neutral
`pH it has been found that as the [H202)/[CI-J ratio was increased, HOCI formation
`decreases and Compound ll accumulates . (29) . . Compound U is a inactive form of
`MPO with regard to the formation of HOCI. The MPO-H20rCl--system used in
`the present study contains 200 mM NaCl which meansthat the ratio [H20 2/[CI-J is
`more favourable for the formation of HOCl at the high H20rconcentration that
`has been used. It has been shown that using 200 mM KCI and various concentrations
`of H20 2 at neutral pH, the inactivation of MPO was dependent upon both time and
`concentration of H 20 2 and mainly a result of the reaction of Compound I with
`H 20 2 producing Compound II [30]. This event may be the explanation for the
`levelling off of degradation with time seen when the complete MPO-system is used.
`Our findings implicate the involvement of HOCl in the formation of OH· . Fµr(cid:173)
`ther evidence for this sµggestion is the inhibiting influence of histidine on the
`degradative effect of the MPO-H20rCI --system. The reason may be that this
`amino acid acts as a scavenger of HOCl and th¢reby inhibits the fonnation of OH ..
`Furthermore. it has been found that histidine inhibits the chlorination of the primary
`amine (Z)-3-(4-bromphenyl)-3-(3-pyridyl~alJylamine (31 ].
`Desferrioxamin~, the iron chelator which remo.ves the iron ca.talyst for OH· pro(cid:173)
`duction, also reacts readily with HOCI/OCJ- [32]. In the MPO-H20rCJ- system,
`desferriox.amine may 3ct as a scavenger ofthe HOCl .formed thereby inhibiting the
`degradation of hyaluronic acid. In addition to iron, 0 2 -=- has been shown to be es(cid:173)
`sential for ,OH • formation. The only chlorine-containing species which reacts with
`0 2-;- at an appreciable rate is HOCl according to the mechanism 0 2 -=- + HOCl -
`OH· + 10 2 + c1- [33}. The inhibiting effect of · desferrioxamine in the present
`study may therefore be dependent on two separate mechanisms. Jo the MPO-systeJJl
`when 01- is omiUed, the compound may act as a scavenger • for iron, and m the
`Neit.her Br- nor I - could replace c1- in the MPO-system <Used. Instead, these
`MPO H202-.c1--system it may also act as a scavenger for HOCl . formed •.
`ions inhibit the degradative effect. The explanation may be that they react with OH·
`and thereby prewmt its .deteriouseffects. This suggestion i~ supported by the factthat
`the specific rate constants at pH 7 for th.is interaction of OH · with Br'"' and 1- are
`in the region of 109 and 10 10, respectively. For c1- this value is 103 (34). The reac(cid:173)
`tion between 1- and ILOH' provides 1 · which is capable of forming a covalent
`linkage to tyrosine residues [35). The reason for the inhibitory effect seen in the pre(cid:173)
`sent study when c1- is replaced l,Y .I"""' may also be that . the r formed reacts with
`MPO in such a way as to inhibit the generation of OH· . lt is shown that the catalysis
`of iodination by peroxidase and }{20 2 can be enhanced by chloride and that the
`iodination system required supplementation by Fe 3+ [35). These results may sup(cid:173)
`port the suggestion that Cl"" and J'· together in the MPO-system used increase the
`inhibition of the degradative effect because of .increased formation of F . . The
`possibility that OH· plays a role in biological iodination reactions has been discuss(cid:173)
`ed (35] and the results of the present study are in agreement with sµch a suggestion.
`Radicals and enzymes released from phagocytic cells caµse inflammatory process(cid:173)
`es [ 12) and inflammation is associated with accumulation and activation of neutro(cid:173)
`phils with the subsequent release of oxygen species including OH · . The release of
`
`

`

`S. L/ndyal/ and G. R:,,t:kll I Chem.-Blol. /nJeract. 90 (/!>94) 1-12
`
`9
`
`inflammatory mediators from neut,rophils may include the MPQ.,system with the for(cid:173)
`mation of OH· in the ways that have ~n discussed above. Therefore, some anti(cid:173)
`inflammatory agents have been included in the studies on. the · influence of various
`compounds on the MPO-system used.
`Of the compounds with effects like mannitol, i.e., OH· scavengers, acetylsalicylic
`acid has a low inhibitory effect on the degradation of hyaluronic acid by tbe >MPO(cid:173)
`system. These results agree with those from studies on the influence of various com(cid:173)
`poun(is on the degradation of synovial fluid induced by the reaction between hypc:>x(cid:173)
`anthine and xanthine oxidase (37). However, the non-"acetyhited compound salicylic
`. acid has a higher inhibitory effect, This correspc:>nds well to the results reported
`which showed that salicylic•acid •inhibits the •degradation ofhyaluronic·acid caused
`by aut®xidation of ascorbic acid at a concentration where acetylsalicylic acid bad
`no effect [38]. In the reaction between saUcylic acid and hydroxyl radicals the hy(cid:173)
`droxyl group acti.vates the aromatic ring toward.s .hydroxylation by OH • •.
`The results obtained at a low concentration of paracetamol and p-hydtQxybenzoic
`acid>are noteworthy. Oxidation of paracetamol to the correspc:>nding phenoxyl free
`radical and N-acetyl-p-benzoquinoneimine by rnafilrllalian peroxidases has been
`discussed previously (39). The reason for the increase in inhibitory activity with
`reacts with these com(cid:173)
`experimental time . may be that the initially formed 0 2 7
`pounds by withdrawal of an electron > giving the phenoxyl free ra'1ical and H 20 2
`followed by <a fµrther similar withdrawal . of a electron bY ()2 7
`. f tQm • the radical
`formed in th~ case of paracetamol. The ra.dical formed from p-by4roxybenzoic acid
`may instead tea.ct with OH· leading to the formation of3,4-<l.ibydtoxybenzoic acid.
`These continuous rea.ctions may be the tea.son fof the observed increased inhibition
`of degradation·of hyaluronic acid at low concentrations by removing 0 2 -=- · from its
`function as a reducing agenffor Fe3•. In this way, the formation of OH" will be
`prevented. At higher concentrations these two · comPQunds may also act as
`scavengers of hydroxyl fadicals.
`Lidocaine has a inhibitory effect on the depolymerization of hyaluronfo acid bY
`the MPO-system similar to that of mannitol. This property of lidocaine may explain
`the • protective effect of a eutectic lid®aine/prilocaine composition· on the dermal •re(cid:173)
`sponse to higlt-¢nergy irradiation (40). >Lidocaine may inhibit an initially formed
`OH· from initiating Jipid peroxidation in the cell membrane.
`Regarding phenylbutazone, it bas been found that this compound donates a single
`electron to prostaglandin H synthetase during hydrogen peroxide reduction. The
`resulting phenylbutazone radical, however, traps molecular oxygen to yield a perox•
`yl . radical, a phenylbutazone hydroperoxide, en route to the stable end product 4-
`hydroxyphenylbutazone [4l,42]. It may be postulated that by donating an electron
`to the MPO-system, phenylbutaz:one primarily acts as a inhibitor of the. enz}'Jllatic
`generation of 0 2 7 and thereby inhibits the meehanisms for formatior1 of OH· .
`This compound has also been reported to be a potential scavenger of HOC1 [43),
`which supports the • assumption that HOCl is involved in the formf:ltion of OH · .
`In studies 011 the jnfluence of various compc:>unds on the chlorinating activity of
`MPO it was found that ABC 907 i11hil:>its this reaction in a non-competitive way [3 l].
`lt has beeri shown that catalase oxidizes phenylhydrazine resulting in a phenyl radi(cid:173)
`cal that inactivates the enzyme by reacting with the prostetic heme group in the
`
`

`

`10
`
`S , Llndvall and G. Rydell/Chem.-Biol. Interact, 90 (1994) 1-12
`
`enzyme(44J. The increase in the degenerative effect with tirne at the highest concen(cid:173)
`tration of ABC 907 shows that this compound does not scavenge OH· formed by
`the non-enzymatic mechanism. Since ABC 907 neither scavenges OH· nor interferes
`with HOCl, the inhibition by this compound may be dependent on· the mechanisms
`seen in the studies on catalase.
`It is apparent that various mechanisms are participating in formation of OH· by
`the MPO-system. The superoxide anion radical js involved in the two mechanisms
`discussed in this paper. With regard to iron it is found that the reaction mixture con(cid:173)
`tains 4.5 µ.m whichis in the same range given for normal synovial fluid [26) and 'free
`iron' in rheumatoid synovial fluid [45].
`It has been shown that various antiinflammatory agents inhibit the degradation
`of hyaluronic acid by the MPO-H20rCl--system indicating an influence on the
`fonnation of OH · or on the radical itself. It may therefore be postulated that MPO
`released from polymorphonuclear leucocytes into extra~Uular space, in addition to
`other mechanisms, participates in the inflammatory process by forming OH· at
`critical sites.
`
`5. A,:knowleclgements
`
`We thank Dr. Brian Pring for advice and discussions as wen as for linguistic
`correction of this paper.
`
`6. References
`
`C.G. C:::ochr11ne, Immunologic tissue injury mediated by neutrophilic leukocytes, Adv, lmmun<>I,. 9
`(1968) 97-162.
`2 J.M. Goldstein, Polymorphonuclear1e1.1kocyte lysosome and immune tissue injury, Prog. Allergy, 20
`(1976) 310--340.
`3 A.J .• $.barra a.nd •M·L· · KarnovskY•. Tne.bjoc;:ltemical .basis.of pha~ocytosis.•·1:•.Metabolic changes dur(cid:173)
`ing the jngestion of particles by polymorphonuclear> leukocytes, • J. Biol, • Chem;;> 234 • ( 1959)
`1355-1362,
`4 O.Y.N. Iyer, D.M.F. Islam and J.H. Quastel, Biochemical aspects of phagocyfosis, Nature. 192
`( l~I) 535-541,
`5 B.M, 8abior, R.S. Kipnes and J.T. Cumutte, Biological defense mechanisms. The production by leu(cid:173)
`kocytes ofsupetoxide. A potential bactericidal 11gent, J. Clin. Invest, 52 (1972) 741-744.
`6 R.C. Allen, R.L. Stjemhomand R.H. Steele. Evidence for the generation of an electronic excitation
`state(s) iR h1.1man polymorphonuclear leukocytes and its participation in bactericidal activity,
`Biochem. • Biophys. Res. Commun .• 47 ( 1972) . 672-684.
`7 A.J. Tauber and B.M. Babior, Evidence for hydroxyl radical production by human neutrophils, J.
`Clin. Invest, 60 (1977) 374-379.
`8 K. Agner, Piological eff~ts of hypoc;:hlorui. acid formed by 'MPO'-pero/tidation in the presence of
`chloride ions, in: A. A keson • and A .• Ehrenberg (Eds.), • Stnu,ture and function of O/tidation-
`reductiort enzymes. PergamonPress. oxford, 1972, pp. 329=335.
`.
`.
`.·
`·.·
`... . . . .
`.
`9 J.M. Zglic:eynski, ·T. Stelmaseynska, J . . Qomansk.iand W. Qstrowski, Chloramines.as inte.r:mediates
`of oxidation re11ction of amino .. acids by myeloperoxidase, Biochem. Biophys. Acta, 235 (1971)
`419 .... 424.
`10 H. Odeberg, T. Olofsson and I. Olsson, Myelo~roxidase-mediated extracellular iodination during
`phagocytosis in granulocytes, Scand. J. Haematol., 12 ( 1974) I 55-160.
`.
`11 R.A. Clark and S.J. Klebanoff, Myelc;,pero,r,idase-H2Orhalide system: cytotoxic effect on human
`blood leukocytes, Blood, 50 (1977) 65-70.
`
`

`

`S. Llndva/1 and G. ·Rydell I Chem.-,Biol. Interact. 90 ( 1994) 1-12
`
`11
`
`12 B. Halliwell, Hypothesis: production of supcroxide, hydrogen pcrox.ide and hydroxyl radicals by
`phagocytic cells: a cause of chronic inOammatory disease? Cell. Biol. Int. Rep;; 6 (1982) 529-542.
`13 K.. Agner, Verdoperoxidase. A fermentisolated from leukocytes, Acta Physiol. Scand., 2 (Suppl. 8):
`(1941) 1-62.
`14 B. Chance, The iron-containing enzyme. C. The enzyme-substrate compounds and mechanisms of
`action of the hydropcroxidase in the enzymes. in: J.B. Sumner and K. Myrbii.ck (Eds.), Vol. 11, Part
`I, Academic Press, New York 1951, pp. 428-453.
`15 T. Odajima and I. Yamazaki, Myeloperoxidase of the leukocyte of normal blood. I. · Reaction of
`myeloperoxidase with hydrogen peroxide, Biochem. Biophys. Acta, 206 (1970) 71-75.
`16 T. Odajima and I. Yamazaki, Myelopero;,i.idase of the leukocyte of normal blood; 111. The reaction
`of ferric myeloperoiidase with supero;,i.ide anion, Biochem. Biophys. Acta, 284 (1972) 355-359.
`17 H. tt®gland, i-U: •. Dekker, C. van Reil, A. van K.iulenburg, A.O. Muissers and R. Wever, A steady(cid:173)
`state study on the formation of Compounds n and HI of myeloperoxidase, Bfochem. Biophys. Acta,
`955 (19118) 337 ... 345_
`18 c. Beauchamp and I. Fridovich, A mechanismfonhe production ofttbylene from mcthional. The
`generation of the hydroxyl radical by xanthine oxidlUIC, J. Biol. Chem., 245 (1970) 4641-4646.
`19 C.C. Winterboutn, · Myefoperoxidase as an effective inhibitor of hydroxyl radical production. · Im(cid:173)
`plications for the oxidative reactions of neutrophils, J . Clin, Invest., 78 (1986) 545-550.
`20 J.M. McCord, Free radicals and inflammation: Protection of synovial fluid by superoxide dismutase,
`Science, 185 ( 1974) 529--531.
`21 B .. E. SvenssQn, K. Dosrieij, S. Lindvall and O. Rydell, Peroxidase and pcroxidase-oxidase activities
`of isolated human .myefopeioxidase, Biochem. J., 242 ·(1987) ·673-680.
`22 E. Hultin, A viseosirnetric method for determination of enzymatic activity, Svens.k Kem. Tidskr., 58
`(l 946)28l--235.
`23 B. HalliweU, $uperQxide-dependent fonnalion of hydroxyl radicals in the p~11¢n¢e of iron salts. lts
`fole in degradation ofhyaluronic acid by a superoxide genera.ting system, FEBS Lett., 96 (1978)
`238-242.
`. . . .
`. . .
`.
`. .
`.
`.
`24 R.R.A.Oreenwald and W.W. Moy, Effect ofoitygen-derived free radicals on hyaluronic acid, Arth•
`ritis Rheum., 23 ( 1980) 455-463.
`J .M.C. Gutteridge, R. Richmond and B. Halliwell, Inhibition of the iron catalysed formation of hy(cid:173)
`droxyl radicals frorri supero:x.ide and of lipid peroxid11tiOn by< desferrioxamine, Bioc.hem. J., 184
`,
`(1979) 469-472.
`26 S,F. Wong, 8. Halliwell, R· Richmond and W.R. Skowroneck, The role of superoxide and hydroxyl
`radicals in the degradation of hyaluronic acid induced by<rrietal ions and ascorbic acid. J. lnorg.
`Biochem., 14 (1981) 121-134.
`27 F •• Ha.ber and J. •Wei:ss, The catalytic decomposition of hydrogen .peroxide by.iron salts, Proc. Soc.
`Ser. A, 141(19)4> 332-351.
`28 B, Iialtiwell an4 M.C. Outteridge, C>x.ygen toxicity, oxygen radicals, transition metals and disease,
`BiQChem. J., 219 (1984) 1-14. .
`.
`29 A.J. • Kettle · and • Ch. Winterbourn, Jnfiuence • Qf superoxide on myelopefoxidase · kinetics measured
`with a hydrogen peroxide electrode, Biochem. J., 263 ( l989) 823-828.
`30 K,M,N. Zuurbier, A.R,J. Bllkkenis.t, R. Wever and A.O. Muijsers. The chlorinating activity of
`human myeloperozidase: high initial activity at neutral pH value on activation by elecfrori donors,
`Biochcm. Biophys. Acta, 1037 (1990) No-146.
`31 S. Lindvall and O. Rydell, Influences of tbiols on chlorinating effect by myeloperoxidase, Chem.-
`Biol. Interact., (1993) in press.
`.
`32 M.C. M. Visser and J.C. Fantone, Inhibition of h;ypochl<>rous acid•mediated reactions by desferriox(cid:173)
`amine, JmplicatlQnS for the mechanism of cellular injury by m:utrophils Free Rad. Biol. Med .. 8
`(199()) 331-337.
`33 C. Long and B.H ,J. Bielski, Rate Qf reaction ohuperoxide radical with chloride-containing species,
`J. Phys. <;:hem., 84 (1980) 555-557.
`34 M . . Anbar and p, Neta, A compilati<>n of specific bimolecular rate constant!. for the reaction or
`hydrated electrons, hydrogen atoms and hydroJ1.:yl radicals with • inorganic and organic compounds
`in aqueous solution, Int. J. Appl. Radial. l.sotopes, 18 (1957) 493:-523.
`35 S.J. K.lcbanofT, Iodination catalyzed by xanthine oxidase system: role of hydroxyl radicals.
`Biochemistry, 21 (1982) 4110-4116.
`
`25
`
`

`

`12
`
`S. Lindval/ and G. Rydell /Chem.-Blol. Interact. 90 (1994) 1-12
`
`36 R.F. Del Maestro, Role ofsuperoxide anion in microvascular permeability and leukocyte behaviour,
`Can. J. Physiol. Pitarmacol., 00 (1982) 1406-1414.
`37 P. Puig-Parcllada and J.M. Planas, Synovial nuid degradation indu~d by frf.'ll radicals. In vitro ac(cid:173)
`tion of several free radical scavengers and anti-inflammatory drugs, Biochem. Pharmacol., 27 (1978)
`.535-537.
`.;
`38 G. Mat$umura, A. Herp and W. Pig.i,an, Oepolymeri:uuion of hyaluronic acid by antioxidant and
`radiations, Radiat. Res., 28 (1966) 735-752,
`39 R.P. Mason and V, Fisclter, Free>radicals of acetaminophen: their subsequent reactions and toxico•
`logical signifi<:ance, Fed. Proc, 28 (1986) 2493-2499.
`40 L. Ohlsen, H. Evers, K. Segerstrom and S. Graffman, Local anaesthetic