The paraoxonase 1, 2 and 3 in humans

The paraoxonase gene family in humans includes three members: PON1, PON2 and PON3. The products of those three genes are the following enzymes: paraoxonase 1 (PON1), paraoxonase 2 (PON2) and paraoxonase 3 (PON3). PON1 is mainly associated with a high density lipoprotein (HDL). A small amount of this enzyme is also bound to very low-density lipoprotein (VLDL) and postprandial chylomicrons. PON1 possess organophosphatase, arylesterase and lactonase activity and it hydrolyzes many different substrates. It is also known that PON1 may have antiatherogenic function. Compared to the PON1, PON2 and PON3 are much less studied and described. PON2 is ubiquitously expressed intracellular protein, while PON3 is bound to HDL, like PON1. The both enzymes possess antioxidant properties.


In tro duc tion
The ge nes PO N1, PO N2 and PO N3 are the mem be rs of pa raoxo na se ge ne fa mi ly in hu ma ns. The se genes are lo ca ted on the lo ng arm of chro mo so me 7 and they are struc tu ral ly si mi lar. The re is about 70% of iden ti ty in nuc leo ti de sequen ces and about 60% of iden ti ty in ami no acid sequen ces be tween the se three ge nes. PO N1, PO N2 and PO N3 ha ve nine exo ns, howe ver, PO N1 has an extra co don at the po si tion 106 (lysi ne) in exon 4 whi ch is not presen ted in PO N2 and PO N3. From an evo lu tio na ry poi nt of view and ba sed on a struc tu ral ho mo lo gy, PO N2 is the ol de st mem ber of this ge ne fa mi ly, followed by PO N3 and then by PO N1 (1)(2)(3). PO N1 mR-NA is expres sed in li ver whi le PO N3 mR NA is expres sed pri ma ri ly in the li ver but al so in the kidneys. Un li ke PO N1 and PO N3, PO N2's mR NA is ubiqui tous ly expres sed in diff e re nt kin ds of tis sues li ke kid neys, li ver, lun gs, sma ll in tes ti ne, pla cen ta, spleen, sto ma ch and tes tic les. PO N2 mR NA is al so fou nd in the cel ls of the ar te ry wa ll, in clu di ng endot he lial ce ll, smoo th mus cle ce ll and mac rop hages (3).
The struc ture and fun ction of pa raoxo na se 1 PO N1 is a glyco syla ted pro tein con sis ted of 354 ami no acid re si dues wi th an ap pa re nt ma ss of 43-47 kDa. Ma tu re pro tein re tai ns hydrop ho bic sig nal sequen ce on the N-ter mi nal re gion, from whi ch on ly the ini tia tor met hio ni ne re si due is re mo ved (2,4,5). PO N1 is synthe si zed in the li ver, and then sec re ted in to plas ma whe re it is main ly bou nd to hi gh den si ty li pop ro tei ns (HDL). The re tai ned N-ter mi nal sig nal pep ti de is es sen tial for the as socia tion of PO N1 wi th HDL. A sma ll amou nt of PO N1 was al so de tec ted in ve ry low-den si ty li pop ro tein (VLDL) and pos tpran dial chylo mic ro ns (2,6). PON1 pos se ss or ga nop hos pha ta se, aryles te ra se and lac to na se ac ti vi ties and hydro lyzes diff e re nt kin ds of sub stra tes. PO N1 hydro lyzes oxo ns li ke pa raoxon, chlor pyri fos oxon and dia zoxon whi ch are toxic me ta bo li tes of or ga nop hos pha te in sec tici des pa rat hion, dia zi non and chlor pyrip hos. PO N1 al so hydro lyzes ner ve agen ts li ke sa rin and so man. In ad di tion, PO N1 hydro lyzes aro ma tic es te rs li ke Gr dic Raj ko vic M. et al.
The pa raoxo na se 1, 2 and 3 in hu ma ns phe nyla ce ta te, thiop he nyla ce ta te and 2-naphthylacetate and diff e re nt aro ma tic and alip ha tic lac tones as we ll as cyclic car bo na tes li ke ho mo gen ti sic acid lac to ne, di hydro cou ma rin, γ-bu tyro lac to ne and ho mo cystei ne thio lac to ne. PO N1 al so ca talyzes the re ver se reac tion, lac to ni za tion, of γ-and δ-hydroxy-car boxylic aci ds. Fur ther mo re, PO N1 par ti ci pa tes in me ta bo li sm of so me dru gs whi ch con tain lac to ne and cyclic car bo na tes. For example, PO N1 hydro lyzes the un sa tu ra ted cyclic car bona te pru lifl oxa cin to the ac ti ve qui no lo ne an ti biotic, diu re tic spi ro no lac to ne, and hydroxymethylglu ta ryl-CoA re duc ta se in hi bi to rs (me vas ta tin, lo vas ta tin and sim vas ta tin). In ad di tion, it has been re por ted that PO N1 has low le ve ls of pe roxi da se and phos pho li pa se A2-li ke ac ti vi ties (2,3,(7)(8)(9)(10)(11).
Fur ther mo re, it is we ll known that PO N1 pos se ss an tiat he ro ge nic ac ti vi ty, it pro tec ts HDL and lowden si ty li pop ro tein (LDL) from oxi da tion and destroys bio lo gi cal ly ac ti ve oxi di zed li pi ds on li pop rotei ns and in ar te rial cel ls (2,12,13). The en do ge nous sub stra te and the mec ha nis ms of the an tiat he roge nic ac ti vi ty of this en zyme are sti ll lar ge ly unknown. PO N1 is tra di tio nal ly na med pa raoxo na se/ arylesterase. Ne ver the le ss, it was shown ear lier in the text that PO N1 ca ta lyzes the for ma tion and hydro lysis of diff e re nt kin ds of lac to nes. So it is assu med that the na tive ac ti vi ty of PO N1 may be lacto na se ac ti vi ty and that physio lo gi cal sub stra te may be so me lac to nes whi ch are con su med as food ingre dien ts, drug me ta bo li tes (sta ti ns, spi ro no lac tone and glu co cor ti coid γ-lac to nes) and de ri va ti ves of fat ty acid oxi da tion proce ss su ch as 5-hydro-xy6E,8Z,11Z,14Z-eicosatetraenoic acid (5-HE TE) la cto ne that re si des in HDL (13).
PO N1 has two cal cium bin di ng si tes, the one is impor ta nt for the sta bi li ty of en zyme whi le the ot her one is im por ta nt for the ca ta lytic ac ti vi ty. The presen ce of cal cium is requi red for the en zyma tic ac tivi ty and the re mo val of cal cium wi th the che la ti ng agen ts (li ke EDTA or EGTA) ir re ver sib ly des troys PO N1's ac ti vi ty and sta bi li ty (2,14). It was shown that ma ny ami no aci ds re si dues, li ke glu ta mi ne (E53, E195), as par ta te (D54, D169, D183, D269, D279) his ti di ne (H115, H134, H155; H243, H285) and tryptop han (W281) are im por ta nt for or ga nop hospha ta se and aryles te ra se ac ti vi ties. Fur ther mo re, im por ta nt ami no acid re si dues of PO N1 are the  three cystei ne re si dues at po si tion 42, 284 and 353  (C42, C284, C353). C284 is free, whi le C42 and C353 fo rm di sul fi de bo nd (2,15,16). C42 and C353 are impor ta nt for sec re tion and ca ta lytic ac ti vi ty of PO-N1. It was shown that the exchan ge of C42 or C353 wi th ala ni ne re sul ti ng in inac ti va tion and in decrea sed sec re tion of the en zyme (2,16). C284 was con si de red to be the ac ti ve cen ter nuc leop hi le, howe ver re sear ches we re showed that this ami no acid re si due is not spe ci fi cal ly requi red for paraoxo na se/arylesterase ac ti vi ty. The exchan ge of C284 wi th ala ni ne or se ri ne re du ces pa raoxo na se/ arylesterase ac ti vi ty but does not abo li sh the se acti vi ties (2,17). It is as su med that C284 may be lo cated clo se to the ac ti ve cen ter of the en zyme and it may par ti ci pa te in orien ta tion or bin di ng of the sub stra te (17). Fur ther mo re, for the pre ven tion of cop pe r-in du ced LDL oxi da tion, cal cium is not essen tial but C284 is requi red. On the ba sis of expe rimen tal da ta it may be con clu ded that PO N1 has two ca ta lytic si tes, one res pon sib le for hydro lytic ac ti vi ties, and the ot her one res pon sib le for antioxi da nt ac ti vi ty. Howe ver, the exis ten ce of two ca ta lytic si ts has not yet been pro ved so Avi ram and col lea gues as su med that the two ac ti ve si tes of PO N1 are over lap ped (2,17).

Im por tan ce of HDL in sec re tion, sta bi li ty and ac ti vi ty of PO N1
The as so cia tion of PO N1 wi th HDL is ne ces sa ry for main tai ni ng the nor mal se rum ac ti vi ty. The N-termi nal hydrop ho bic sig nal pep ti de is the struc tu ral requi re me nt for boun di ng of PO N1 to this li pop rotein. HDL pro vi des the op ti mal physio lo gi cal accep tor com plex whi ch sti mu la tes sec re tion and sta bi li zes the sec re ted en zyme. HDL al so en su res am phypat hic en vi ron me nt whi ch pro tec ts the N-ter mi nal hydrop ho bic re gion of en zyme and this en vi ron me nt may al so be ne ces sa ry for the inte rac tion of PO N1 wi th sub stra tes (18,19).
The N-ter mi nal sequen ce of PO N1 is si mi lar wi th sig nal sequen ce of sec re tor pro tei ns. Clea va ge of sig nal sequen ces in sec re tor pro tei ns usual ly oc curs in po lar C-ter mi nal fl an ki ng re gion. The ami no aci ds at the po si tio ns -3 and -1 (usual ly sma ll and un char ged re si dues) are cri ti cal for the clea va ge of this sequen ce. In PO N1 the se po si tio ns are oc cu pied wi th lar ge and po lar re si dues, his ti di ne and glu ta mine, whi ch pre ve nt from the clea va ge of this sig nal pep ti de (19). Af ter a re lea se from the ce ll, PO N1 is bou nd at the HDL phos pho li pi ds throu gh its N-termi nal hydrop ho bic sig nal pep ti de. In that way, HDL as su res hydrop ho bic shel ter for the re tai ned sig nal pep ti de in plas ma's aqueous en vi ron me nt (18,19). Ja mes and col lea gues pre sen ted a hypot he ti cal sche me for HDL's me dia ted re lea se of PO N1 from cel ls in their ar tic le. HDL is bou nd on the ce ll membra ne via sca ven ger re cep tor B1 (SR-B1). PO N1, which was in ser ted in to the exter nal fa ce of the ce ll mem bra nes, is then tran sfer red on HDL du ri ng transie nt as so cia tion of the li pop ro tein wi th ce ll (19).
PO N1 is main ly as so cia ted wi th HDL, but this enzyme is al so fou nd on VLDL and pos tpran dial chylomic ro ns, al thou gh to a les ser exte nt. Howe ver, PO-N1 is not as so cia ted wi th LDL (6). The ma jo ri ty of PO N1 is as so cia ted wi th HDL whi ch con tai ns apo lipop ro tein AI (apo AI), whi le HDL whi ch con tai ns clus te rin (apo li pop ro tein J) bou nd to ap proxi ma te ly 30% of to tal PO N1. Apo AI is not ne ces sa ry for bin ding of PO N1 on HDL, howe ver it is im por ta nt for the sta bi li ty and ac ti vi ty of en zymes (20,21).
To tal HDL in plas ma pre sen ts a he te ro ge neous class of li pop ro tei ns whi ch has, a hi gh den si ty (>1.063 g/mL) and a sma ll si ze (Sto ke's dia me ter 5-17 nm) in com mon. By ul tra cen tri fu gal tec hniques HDL can be se pa ra ted in two ma jor sub frac tio ns; HDL 2 and HDL 3 . HDL 2 is lar ge, li pid ri ch and has a den sity ran ge of 1.063-1.125 g/mL whi le HDL 3 is sma ll, li pid poor and has a den si ty ran ge of 1.125-1.210 g/ mL (22)(23)(24). Le ss than 10% of to tal HDL reac ts wi th an ti-PO N1-an ti bo dies whi ch in di ca te that PO N1 is not dis tri bu ted ac ro ss the en ti re HDL spec trum (19). The re sear ches do not ha ve a unique con clusion on the is sue of as so cia tion of PO N1 wi th HDL 2 or HDL 3 . It was as su med that PO N1 fol lows the nor mal me ta bo lic pat hway of HDL. PO N1 is bou nd to HDL 3 , whi ch en lar ges and sub sequen tly transfor ms in to lar ge HDL 2 du ri ng the ac cu mu la tion of li pid com po nen ts. Frac tio na tion of HDL by gel fi ltra tion sup por ts this as sum ption be cau se PO N1 was de tec ted in lar ger size par tic les. Fur ther mo re, when an ti-PO N1 im mu noab sor bed co lu mn was used for the iso la tion of PO N1-con tai ni ng HDL partic les, PO N1 was de tec ted on HDL 2 (19). Un li ke these re sul ts, when ul tra cen tri fu ga tion was used for the se pa ra tion of HDL frac tion PO N1 was de tec ted on smal ler si zed HDL. A pos sib le rea son for the obser ved re su lt is that the ul tra cen tri fu ga tion dis rupts li pop ro tein struc tu re. Du ri ng this pro ce du re the pep ti des whi ch are not tig htly bou nd to HDL, can be strip ped off and ac cu mu la ted in ve ry hi gh densi ty par tic les or even in the li pop ro tein free fraction of plas ma. So it is pos sib le that PO N1 is re distri bu ted du ri ng the ul tra cen tri fu ga tion. (19,(24)(25)(26).
If the met hod of se lec ti ve pre ci pi ta tion is used, the ma jo ri ty of PO N1 is lo ca ted in HDL 3 frac tion (27).

The no n-ge ne tic fac to rs whi ch aff e ct PO N1's ac ti vi ty
PO N1's ac ti vi ty and its con cen tra tion in se rum show lar ge in te r-in di vi dual va ria bi li ty. PO N1's concen tra tion va ries up to 13 ti mes, whi le PO N1's ac tivi ty can va ry up to 40 ti mes. Diff e re nt ge ne tic facto rs, po lymor phis ms in pro mo ter and co di ng region of the PO N1 ge ne, to get her wi th diff e re nt non-ge ne tic fac to rs, bo th aff e ct PO N1's ac ti vi ty and con cen tra tion (2,20,(28)(29)(30).
Die ts ri ch wi th tra ns-un sa tu ra ted fat and mea ls rich in used coo ki ng fat, whi ch con tai ns a hi gh conte nt of oxi di zed li pi ds re du ce PO N1's ac ti vi ty. On the ot her ha nd, the oleic acid from oli ve oil in creases PO N1's ac ti vi ty. The con sum ption of po meg rana te jui ce ri ch wi th po lyphe no ls and ot her an tioxidan ts, re su lts in hig her PO N1's ac ti vi ty. Howe ver, the eff e ct of an tioxi da nt on PO N1's ac ti vi ty requires fur ther re sear ch be cau se of dis cre pan cy in the ob tai ned re sul ts. The con duc ted re sear ches ha ve showed po si ti ve and ne ga ti ve cor re la tion between, for exam ple the con sum ption of vita min C and E or even no as so cia tion be tween the in ta ke of vi ta min C, E and β-ca ro te ne wi th PO N1's ac ti vi ty (20,29,30). Mo de ra te al co hol con sum ption (bear, red wi ne and spi ri ts) re sul ts in in crea sed PO N1's ac ti vi ty and con cen tra tion. The re we re no diff e ren ces between red wi ne, bear and spi ri ts so it was sug gested that the red wi ne po lyphe no ls alo ne are not res pon sib le for this eff e ct. Mo de ra te con sum ption of al co hol drin ks in crea ses the con cen tra tion of HDL and apo AI whi ch may re su lt in ob ser ved ri se of the en zyme's con cen tra tion. Howe ver, so me re sul ts did not show as so cia tion be tween al co hol con sumption and PO N1's ac ti vi ty (20,29,30).
The ac ti vi ty and con cen tra tion of PO N1's in se rum is lower in smo ke rs than in no n-smo ke rs. Ex-smoke rs ha ve PO N1's ac ti vi ty and con cen tra tion si mi lar to tho se fou nd in no n-smo ke rs, whi ch in di ca tes a re ver sib le eff e ct of smo ki ng on PO N1. It was al so shown that mo de ra te al co hol con sum ption or regu lar ly exer ci se can at te nua ted the eff e ct of smoki ng on PO N1 so that the ob ser ved le ve ls of PO N1 in this po pu la tion are si mi lar to tho se by no n-smoke rs (20,29,30).
Fur ther mo re, expo su re to the en vi ron men tal toxins aff ec ts PO N1's ac ti vi ty. For exam ple, the exposu re to or ga nop hos pha te or the expo su re io ni zi ng ra dia tion re sul ts in dec rea se of PO N1's ac ti vi ty (20,29,31,32).
Agi ng al so aff ec ts PO N1's ac ti vi ty. The se rum ac tivi ty af ter bir th is ve ry low, and then it ri ses and achie ves the va lues as in adul ts be tween 6 and 15 mon th of age. On ce it reac hes the adul ts' va lues, PO N1's ac ti vi ty is re la ti ve ly con sta nt du ri ng li fe time. Howe ver, a prog res si ve dec rea se is de tec ted by el der ly sub jec ts (20,29).
Diff e re nt physio lo gi cal con di tio ns aff e ct on PO N1's ac ti vi ty, for exam ple preg na nt wo men ha ve a redu ced ac ti vi ty. Fur ther mo re, PO N1's ac ti vi ty can va ry de pen di ng on diff e re nt pat ho lo gi cal con dition. Lower PO N1's ac ti vi ty was ob ser ved in patien ts wi th in su li n-de pen de nt dia be tes and no nin su li n-de pen de nt dia be tes, in pa tien ts wi th chronic re nal fai lu re un der goi ng hae mo dia lysis, in patien ts wi th rheu ma toid ar thri tis, hyper thyroi di sm, Al zhei mer's di sea se and chro nic li ver di sea se. Redu ced PO N1's ac ti vi ty is al so re la ted to in su lin resis tan ce, hi gh se rum cho les te rol and in fl am ma tion (20,29,31,32).

Po lymor phis ms in co di ng re gion of PO N1 ge ne
Mo re than 160 po lymor phis ms we re iden ti fi ed in re gu la to ry, in tron and co di ng re gio ns of PO N1 gene. It is known that so me of this po lymor phis ms affe ct the PO N1's con cen tra tion and ac ti vi ty (20,29).
Two po lymor phis ms in the co di ng re gion of PO N1 ge ne ha ve been tho roug hly stu died. In Q192R polymor phi sm, the exchan ge of co don CAA to CGA in exon 6 of PO N1 ge ne re sul ts in sub sti tu tion of amino acid glu ta mi ne wi th ar gi ni ne at the po si tion 192. In L55M po lymor phi sm, the exchan ge of co don TTG to ATG in exon 3 of PO N1 ge ne re sul ts in the sub stitu tion of ami no acid leu ci ne to met hio ni ne at the po si tion 55 (33). Q192R ha ve been mo re wi de ly studied out of the se two po lymor phis ms, be cau se the Q192 and R192 al loen zymes ha ve a diff e re nt affi ni ty and ca ta lytic ac ti vi ty towar ds nu me rous sub stra tes (20). The R192 al loen zyme hydro lyzes pa raoxon six times fas ter than Q192 al loen zyme. On the ot her ha nd, the Q192 al loen zyme hydro lyzes sa rin, so man and dia zoxon fas ter than R192 al loen zyme (7,20). The se two al loen zymes are al so diff e re nt in hydrolysis of some lac to nes and car bo na te es te rs. For exam ple, hydro lyze of an ge li co lac to ne and δ-va le ro lac to ne is fas ter wi th Q192 al loen zyme, whi le R192 al loen zyme hydro lyze fas ter, for exam ple, thio lac to nes and γ-butyro lac to ne. Fur ther mo re, the re are no diff e ren ces in hydro lysis ra te for the sa me sub stra tes, for exam ple for phe nyla ce ta te (7,8,20). Q192R po lymor phi sm al so aff ec ts on en zyme's abi li ty to pro te ct LDL from oxida tion in vit ro, whi le Q192 al loen zyme is mo re effi cient than R192 aloen zyme (17,20,34,35).
In vit ro expe ri me nt showed that R192 al loen zyme is mu ch mo re pro tec ti ve again st the toxic eff e ct of pa raoxon, whi le Q192 al loen zyme pro vi des mo re pro tec tion again st the toxic eff e ct of dia zoxon. But, in vi vo expe ri me nt on PO N1-knoc kout mi ce whi ch re cei ved the sa me amou nt of eit her Q192 or R192 al loen zyme, showed that bo th al loen zyme pro vi de the sa me pro tec ti ve eff e ct again st diazoxon. Fur ther mo re, R192 al loen zyme pro vi des mo re pro tec tion again st chlor pyri fos oxon in vi vo, and the sa me eff e ct was ob ser ved in vit ro. Neit her of the se two al loen zymes showed pro tec ti ve eff ect again st pa raoxon in vi vo. It can be con clu ded that PO N1 plays a ma jor ro le in the de toxi fi ca tion of dia zoxon and chlor pyri fos oxon, but not paraoxon in vi vo (36).
It was shown ear lier that ar gi ni ne at the po si tion 192 is an im por ta nt ami no acid re si due of en zyme's ac ti ve cen tre, whi ch can explain the diff e ren ces in en zyme ac ti vi ty of the se two al loen zymes towar ds diff e re nt sub stra tes (20,37). L55M po lymor phi sm does not aff e ct in te rac tion of the en zyme wi th sub stra tes, but aff e cts PO N1's mR NA le ve ls and the con cen tra tion and ac ti vi ty of PO N1. M55 al loen zyme is re la ted to the lower enzyme's ac ti vi ty and con cen tra tion and to the lower le vel of PO N1's mR NA (20,38,39,40). The se two alloen zymes are al so diff e re nt in pro tec tio ns of LDL again st oxi da tion, whe re M55 al loen zyme shows to be mo re pro tec ti ve (34,35). It was shown that L55M po lymor phi sm is in stro ng lin ka ge di sequilib rium wi th po lymor phis ms in the pro mo ter region of PO N1 ge ne, whi ch in fl uen ces PO N1's expression and ac ti vi ty. It was con si de red that the con nection of L55M po lymor phi sm to va ria tion in PO N1's con cen tra tion is a con sequen ce of the ob ser ved linka ge di sequi lib rium. Howe ver, so me la ter re searches showed that the lin ka ge di sequi lib rium does not pro vi de the com ple te expla na tion of L55M polymor phi sm's eff e ct on en zyme's con cen tra tion. Inde pen den tly from -108C>T po lymor phi sm in the pro mo ter re gion of PO N1 ge ne, sub jec ts ca ri ng LL ge no type we re had a hig her PO N1's con cen tra tion than the in di vi dua ls wi th MM ge no type (20,41,42). L55 al loen zyme is mo re sta bi le and re sis ta nt to proteo lysis, whi ch can par tly explain the as so cia tion of this al loen zyme wi th hig her con cen tra tion of PO N1 in se rum. The ana lysis of crystal struc tu re showed that L55 al loen zyme has a key ro le in the cor re ct pac ki ng of the pro tein (20,37,42).
So me ot her po lymor phis ms in the co di ng re gion of PO N1 ge ne we re fou nd, howe ver the se po lymorphis ms are not yet tho roug hly stu died. For example, the exchan ge of iso leu ci ne wi th vali ne at the po si tion 102 in Fin ni sh and the exchan ge of ar gi ni ne wi th glyci ne at the po si tion 160 in Chi ne se (43,44).

The po lymor phis ms in the pro mo ter re gion of PO N1 ge ne
At lea st fi ve po lymor phis ms whi ch are lo ca ted on po si tion -909 (G or C), -832 (A or G), -162 (A or G), -126 (C or G) and -108 (C or T) when the ba se imme dia te ly pre ce di ng the sta rt co don is num be red as "-1", ha ve been iden ti fi ed in the pro mo ter re gion of PO N1 ge ne. The no men cla tu re diff e ren ces for the se po lymor phi sm whi ch oc cur in li te ra tu re (-107/-108, -160/-162, -824/832 and -907/-909) are li-ke ly due to the sma ll va ria tio ns in the sequen ces exa mi ned by the diff e re nt re sear ches' grou ps. Leviev and co-wor ke rs affi r med that -108 and -832 po lymor phis ms ha ve an in fl uen ce on PO N1 expression, whi le -909 po lymor phi sm does not aff e ct the ge ne expres sion (41). Fur ther mo re, Brop hy and collea gues fou nd that the -108, -162 and -909 polymor phis ms aff e ct, whi le -832 and -126 po lymorphis ms do not aff e ct PO N1 expres sion. The diff eren ces in the ob ser ved re sul ts cou ld be a consequen ce of the in te rac tio ns amo ng the po lymorphis ms re sul ti ng in the con text eff ec ts (47). -108C, -832A, -162A and -909G ha ve hig her le ve ls of expres sion than -108T, -832G, -162G and -909C. The va ria tion in pro mo ter ac ti vi ty are physio lo gi cal ly re le va nt sin ce they are cor re la ted to sig ni fi ca nt diffe ren ces in se rum con cen tra tion and ac ti vi ty of PO N1 (20,41,(45)(46)(47). The ana lysis of ea ch po lymorphi sm's in di vi dual con tri bu tion in the pro mo ter region of PO N1 ge ne on PO N1's con cen tra tion and ac ti vi ty, is com pli ca ted be cau se of lin ka ge di sequilib rium be tween them and be tween Q192R and L55M and the pro mo ter re gion po lymor phis ms (2,20,41,46). It is be lie ved that -108C>T po lymorphi sm is the main con tri bu tor to va ria tion of PO N1 in se rum whi ch explain ap proxi ma te ly 23-24% of the to tal va ria tion, whi le -162A>G, -909G>C and -832A>G ha ve a sma ll or no eff e ct on PO N1's le vel (20,46). The pa rt of pro mo ter re gion of ap proxi mate ly 200 bp whi ch con tai ns the po lymor phic po sitio ns -108 and -162 is suffi cie nt for tran scrip tion of PO N1 ge ne (20). Be cau se -108C>T po lymor phis ms see ms to ha ve the hig he st eff e ct on PO N1's va riation in se rum, this po lymor phis ms has been thoroug hly stu died. This po lymor phi sm is lo ca ted in the cen tre of a con sen sus' bin di ng si te for the ubiqui tous tran scrip tion fac tor Sp1 (spe ci fi c protein Sp1). Pre sen ce of T at the po si tion -108 dis rupts the sequen ce whi ch re cog ni zes Sp1 and the bindi ng of this fac tor is wea ker in the pre sen ce of T than C. The pro mo ter ac ti vi ty in the ca se of -108T is sig ni fi can tly lower than in the ca se of -108C but it is pre se nt, in di ca ti ng that this re gion is on ly partly re gu la ti ng the PO N1 tran scrip tion. Out of all the pro mo ter po lymor phis ms, be si de -108C>T, on ly -162A>G po lymor phi sm is lo ca ted wit hin sequences for tran scrip tion fac tor. The po lymor phic po sition -162 is lo ca ted in a con sen sus bin di ng si te for the NF-1 (nuc lear fac to r-1). The pre sen ce of G at the po si tion -162 dis rup ts the sequen ce of the bin di ng si te re sul ti ng wi th lower ge ne expres sion (2,20,41,(45)(46)(47)(48). The po lymor phis ms ha ve been iden ti fi ed in 3'-un tran sla ted re gion of PO N1 ge ne, howe ver, the sig ni fi can ce of the se po lymor phis ms is not yet stu died (2,46).

The dis tri bu tion of PO N1 gene po lymor phis ms in po pu la tion
The frequency of Q192R and L55M alleles are different among populations. The Caucasian population has a higher frequency of Q192 and L55 alleles while the Asian population has a higher frequency of R192 allele and a very low frequency of

PO N1 phe no type and PO N1 sta tus
PO N1's phe no type is de ter mi ned wi th ge ne tic facto rs, po lymor phis ms of PO N1 ge ne, and diff e re nt no n-ge ne tic fac to rs whi ch aff e ct PO N1's ac ti vi ty. Pa raoxo na se PO N1's ac ti vi ty show bi mo dal dis tribu tion and al lows the se pa ra tion of phe no type AA (ho mo zygo te low ac ti vi ty) from phe no types AB (he te ro zygo te) and BB (ho mo zygo te hi gh ac ti vi ty). The met hod usi ng two sub stra tes is mo st wi de ly used to day, and al lows se pa ra tion of all the three phe no type. Ec ker son and co-wor ke rs des cri bed a met hod in whi ch sub stra tes pa raoxon and phenyla ce ta te we re used for de ter mi na tion of PO N1's phe no type. Un li ke the bi mo dal dis tri bu tion of paraoxo na se ac ti vi ty, aryles te ra se ac ti vi ties show unimo dal dis tri bu tion and ra tio of pa raoxo na se/ary-lesterase ac ti vi ties is tri mo dal. The ra tio of the se two ac ti vi ties al lows se pa ra tion of all the three phe no types: AA, AB and BB. Two al loen zymes of PO N1 ha ve a diff e re nt tur no ver num ber for paraoxon and a si mi lar tur no ver num ber for phe nylace ta te (2,50,51). The mo le cu lar bac kgrou nd of polymor phic dis tri bu tion of pa raoxo na se PO N1's ac tivi ty is the Q192R po lymor phi sm. Hum be rt and cowor ke rs iden ti fi ed that the in di vi dua ls wi th B alloen zyme ha ve ar gi ni ne at the po si tion 192, whi le the in di vi dua ls wi th A al loen zyme ha ve glu ta mi ne at this po si tion. The phe no type AA mat ches wi th the ge no type QQ, the phe no type AB wi th the geno type QR and the phe no type BB wi th the ge notype RR (52). Sin ce aryles te ra se ac ti vi ty does not ha ve po lymor phic dis tri bu tion, this ac ti vi ty ser ved for the es ti ma tion of PO N1's con cen tra tion in serum. The aryles te ra se ac ti vi ty cor re la tes wi th PO-N1's con cen tra tion in de pen den tly from ge no types of Q192R po lymor phi sm, whi le pa raoxo na se ac ti vity cor re la tes wi th PO N1's con cen tra tion on ly within the cer tain ge no type of this po lymor phi sm (53)(54)(55)(56). For the de ter mi na tion of PO N1 phe no type ot her sub stra tes can be used in stead of sub stra te phe nyla ce ta te, li ke chlor pyri fos oxon or dia zoxon to get her wi th sub stra te pa raoxon. The be st dis tinction of PO N1's phe no type was ac hie ved wi th substra tes pa raoxon and dia zoxon (2,56).
The re is a wi de va ria tion in PO N1's con cen tra tion and ac ti vi ty be tween in di vi dua ls even wit hin geno type grou ps. The epi de mio lo gi cal stu dies which ana lyze the cor re la tion of PO N1 wi th diff e re nt pat ho lo gi cal con di tion shou ld in clu de the de termi na tion of po lymor phis ms to get her wi th PO N1's sta tus (20). PO N1's sta tus pro vi des a fun ctio nal asses sme nt of the PO N1's 192 al lo for ms and al so provi des the plas ma le vel of PO N1 for the ea ch in di vidual. Al thou gh po lymor phis ms in PO N1 ge ne ha ve the grea te st eff e ct on PO N1 sta tus, diff e re nt non ge ne tic fac to rs al so eff e ct PO N1's con cen tra tion and ac ti vi ty and con tri bu te to the enor mous in terin di vi dual va ria tion. PO N1's sta tus can be de termi ned by the mea su ri ng con cen tra tion and enzyme's ac ti vi ty or by usi ng the met hod wi th two sub stra tes. The two-di men sio nal en zyme ana lysis uti li zi ng pa raoxon and dia zoxon pro vi des the be st de ter mi na tion of PO N1 sta tus (20,29,(56)(57)(58)(59). Pa raoxo na se 2 and pa raoxo na se 3 In con tra st to PO N1, PO N2 and PO N3 en zyme are mu ch le ss stu died. PO N2 is an in tra cel lu lar pro tein wi th re la ti ve mo le cu lar ma ss of ap proxi ma te ly 44 kDa (3). PO N2 mR NA is expres sed in al mo st all human tis sues, wi th the hig he st expres sion in li ver, lun gs, pla cen ta, tes tic les and hea rt. PO N2 mR NA is al so fou nd in the cel ls of the ar te ry wa ll, in clu di ng en dot he lial ce ll, smoo th mus cle ce ll and mac ropha ge. PO N2 is not as so cia ted wi th HDL or LDL (3,49). Al thou gh, PO N2 has the N-ter mi nal sig nal sequen ce li ke PO N1 and PO N3 it ap pea rs that it is lo ca ted in the cel ls as so cia ted wi th the plas ma mem bra ne. It is con si de red that on ly a sma ll amount of PO N2 is sec re ted from the ce ll or that the enzyme may be ra pid ly deg ra ded fol lowi ng sec retion (3,60). PO N2 has got an tioxi da nt pro per ties, lowe rs the in tra cel lu lar oxi da ti ve stre ss and pre vent the ce ll-me dia ted oxi da tion of LDL. Ng and cowor ke rs was de mon stra ted that the ce ll whi ch ove rexpre ss PO N2 oxi da ti ve ly mo di fy LDL to a lesser exte nt. PO N2 not on ly pre ven ts from the oxida tion mo di fi ca tion of LDL, but is al so ab le to rever se the oxi da tion of mi ni mal ly mo di fi ed LDL (mmLDL). LDL and mmLDL whi ch was in cu ba ted wi th cel ls that ove rexpre ss PO N2 ha ve sig ni fi can tly lower le ve ls of li pid hydro pe roxi des and are le ss ab le to in du ce mo no cyte che mo tac tic ac ti vi ty than LDL and mmLDL in cu ba ted wi th con trol cel ls. The ove rexpres sion of PO N2 al so dec rea ses the oxyda ti ve stre ss in the cel ls whi ch we re trea ted with hydro gen pe roxi de or oxi di zed phos pho li pi ds. Sin ce PO N2 is a ubiqui tous ly expres sed in tra cel lular pro tein, it is mo st li ke ly that PO N2 plays a ro le in the re duc tion of in tra cel lu lar or lo cal oxi da ti ve stre ss (3,31,49,60). Two com mon po lymor phis ms we re iden ti fi ed at the po si tio ns 148 and 311 in the PO N2 gene and bo th po lymor phis ms lead to amino acid sub sti tu tion. Ala ni ne or glyci ne cou ld be at the po si tion 148 (A148G) whi le se ri ne or cystei ne cou ld be at po si tion 311 (S311C). A148G po lymorphi sm is re la ted for exam ple wi th va ria tion of to tal and LDL cho les te rol, wi th fas ti ng plas ma glu co se le ve ls and wi th bir th weig ht. S311C po lymor phi sm has been re la ted for exam ple wi th co ro na ry ar te ry di sea se, is che mic stro ke in pa tien ts wi th type 2 dia-be tes mel li tus, Al zhei mer's di sea se and re du ced bone ma ss in pos tme no pau sal wo men (3,31,49).
Out of all the three PON en zymes, PO N3 was disco ve red the la st. PO N3 is pri ma ri ly synthe si zed in the li ver and is as so cia ted wi th HDL in se rum but in mu ch lower le ve ls than PO N1. Be si de in the li ver PO N3 mR NA expres sion was al so de tec ted in the kid ney. PO N3 has mo le cu lar ma ss of ap proxi ma tely 40 kDa and has al so got the an tioxi da nt pro perties. It was shown that PO N3 pre ve nt the for mation of mmLDL and in hi bi ts mmLDL in du ced mono cyte che mo tac tic ac ti vi ty (3,31,49,61). The two po lymor phis ms, at the po si tion 311 and 324, we re iden ti fi ed in PO N3 gene. At the po si tion 311 is se rine or threo ni ne and at the po si tion 324 is glyci ne or as par tic acid. The se po lymor phis ms we re detec ted wit hin the po pu la tion of sout he rn Ita ly but the fun ctio nal con sequen ces of the se po lymorphis ms ha ve not been re por ted (3).
In con tra st wi th PO N1, PO N2 and PO N3 la ck, or have ve ry li mi ted pa raoxo na se and aryles te ra se ac tivi ties, but the bo th en zymes hydro lyze aro ma tic and lo ng-chain alip ha tic lac to nes li ke di hydro couma rin. PO N3 hydro lyzes so me dru gs li ke sta tin lacto nes (lo vas ta tin and sim vas ta tin) and a diu re tic spi ro no lac to ne (2).

Co nclus sion
It is we ll known that PO N1, PO N2 and PO N3 ha ve an tioxi da nt fun ction, but diff e ren ces in en zyme's ac ti vi ty and lo ca li za tion in di ca te that the se tree en zymes ha ve got diff e re nt fun ctio ns in the human bo dy. Howe ver, all the three en zymes sha re an abi li ty to hydro lyze diff e re nt kin ds of lac to nes. PO N1's ac ti vi ty and its con cen tra tion in se rum show lar ge in te r-in di vi dual va ria bi li ty (62) and its physio lo gi cal fun ction and physio lo gi cal sub stra te sti ll re mai ns un known. Fur ther we ll de sig ned studies mu st be con duc ted to iden ti fy its na tu ral substra tes and the mec ha nis ms of ca ta lytic and an tiathe ro ge nic ac ti vi ty.
Po ten tial Con fl ic ts of In te re st: No ne dec la red.