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C L IN IC A L C H E MIS TR Y , V o l. 3 6, N o . 2 , 1 99 0 32 5

H ow ev er, th e la ck o f c orre la tio n o f th e p las ma sod ium onday 3 w ith any of the potassium indices tends to inva lida teth e s ic k- ce ll c on ce p t.A lthough w e found no increased osm ola l gaps in p lasm a(F igure 1E ), G roups 2 and 3 on day 1 showed increasedo sm ola l g ap s in u rin e (F ig ure 1G ), which cou ld be taken asevid en ce fo r s ick-cell co nce pt. H ow eve r, ag ain , th e co nce ptwas not supported by the lack of corre la tion of the va lue ofthe plasm a sodium on day 3 w ith the osm ola l gap in urine.A lso, there was no corre lation of the decrease in plasmasod ium w ith the osm olal gap in urine o r p la sm a , a lth ou ghthe p lasm a sodium on day 3 d id corre la te w ith th e o smo la lgap in plasm a on day 2 (Tab le 1).In theory 1 9 , the osmolal gap and potass ium datashou ld produce the strongest evidence for the sick-ce llm echanism . O n th is bas is, the data in th is study have notproduced convinc ing ev idence for th is m echanism operat-ing in the deve lopm ent of hyponatrem ia after AM . if th esick-ce ll m ech anism is op era ting at all, th is s tu dy su gg eststhe m ost like ly tim e is during th e first d ay o f t he illness.The other m echanis tic poss ib ility is that there is asusta ined re lease of AV P over the three days after A M ;so me re ce nt stu die s 4-6,20 su pp ort th is vie w.

W e th an k D r. J. A . Tulloch fo r p e rm is sio n t o s tu d y hi s patients,a nd C h ris tin e B ell, Lindsay Crowe, J oe G ib b, G ra ha m M a tth ew ,a n d A v ri l Ness for car ry ing out t he m a j or it y o f t he as say s.We t hankth e medical an d nu rs ing s ta ff ofthe Co ro na ry C ar e Unit , StracathroHospi tal , fo r co lle ctin g b lo od a nd u rin e sa m ple s fo r t hi s s tu d y.References1. O liver M F. Metabolic responsesduring myocardial infarct ion.II . C lin ic al im p lic a tio n s. C ir cu la tio n 1 9 72 ;4 5 :4 9 1- 50 0 .2 . Vetter NJ , Strange RC, A da m s W , O liv er M F . In itia l m e ta bo licand horm onal response to m yocardia l in farction . L ance t1974;i:284-8.3. Ceremuzynski L. H o rm o n al a n d m e ta b olic r ea ct io n s e vo ke d byacute myoca rd ia l i nf ar ct io n . C irc R es 1981;48:767-76.4. S ch alle r M D , Nicod P, Nussberger, J, e t al. Vasopressine lors

C L IN . C H E M . 3 6 /2 , 3 25 -3 29 (1 99 0)

dinfarctus myocardique a ign : i m p li ca ti ons c li ni ques . S c hwe iz M edWochenschr 1986;116:1727-9.5 . S ch alle r M D , N u ss be rg er J, F ie hl F , e t a l. C lin ica l a nd h em o-dynamic cor re la tes of e le va te d p la sm a a rgi ni ne v asop res si n a ft eracute myocard ia l infarction. Am J C a rd io l 1 9 87 ;6 0 :1 1 78 -8 0 .6 . M cA lpine H M, M orton JJ Leckie B , Rumley A, G illen G ,Dargie H J. N eu ro en do crin e a ctiva tio n a fte r a cu te m yo ca rd ia lin fa rc tio n . B r Heart J 1988;60:117-24.7. Flear C T G , H il to n P . H y po n atr ae m ia a n d s ev e rit y and outcomeof myocardial in fa rc tio n. B r M e d J 1979; i :1242-6.8 . E va ns JR . O sm olal ga ps in urine [Tech Brie f]. C lin C hem1986;32:1415.9. N eary R H . More o n o sm o la l g a ps in urine [Letter]. C lin C hem1986;32:2225-6.10. Evans JR . Y et m ore on o sm ola l ga ps in u rin e [L etter]. C linChem 1987 ;33 :746 .1 1. T ech nico n In stru me nt C orp ., Tarrytown, N Y. Tota l CO2method, N-8b IfH , 1 9 70 .12. M arsh W I , F ingerhut B , M ille r H . Automated a nd m a nu ald ire ct m eth od s fo r th e d eterm in atio n o f b lo od u re a. C lin C he m1965;11:624-7.13. V arley H , Gowanlock All, Bell M , eds. The estim ation ofplasma and urine c re atin in e b y a c on tin uo us-flo w m eth od . In :P ra ctic a l c lin ic al c he m is tr y, 5 th e d ., V o l. 1 . L o nd o n: H e in e m an n ,1980:481-2.14. Gommorri S J. T he m e as ur em e nt o f p la sm a p ho sp ha te . J L abClin M ed 1942;29:955.15 . Varley H , Gowanlock Al , Bell M , eds. T he e stim atio n o fp la sm a a n d u rin e am mo nia u sin g th e B e rt he lo t r ea ct io n . Op.cit.(ref. 13 :835 6.16. C h a n 5 , R a d cli ffe A , J oh n so n A . T h e s er um s od iu m c on ce n tr a-tio n a ft er surgical o pe ra tio n: p re cis io n p erm its p re dic tio ns . B r JSurg 1980;67:711-4.17 . Snedecor GW , Coc h ran WS . Com pari son of means o f i ndepen-den t s am p les w i th different popu la ti on d if fe renc es. I n: S t at is ti ca lm e th od s, 7 th e d., A mes, IA : The Iowa State U n iv er sity P re ss :1980:96-8.18. Ibid. 158-62.19. G ill GV, F le ar C TG . Hyponatraemia. R ecen t A dv C lin B io -c hem 1984 ;3 : 149 -76 .20 . C o l J , Petain M , V an E yll C , C he ro n P , C h arlie r A A, P an le urH Earlyc ha ng es in s od iu m a nd w a te r b ala nc es in p atie nts w itha c ut e m y oc ar dia l in fa rc tio n : r ela tio n sh ip t o haemodynamics an dc re a tin e k in a se . E u r J Cl in I nv est 1984 ;14 :247 -54 .

F alsely Low E stim ation of Trig lycerides in Lipem ic P lasm a by the E nzym atic Trig lycerideM eth od w ith M od ifie d T rin de rs C hro mo ge nMarkD .S . Shephardand Malco lmJ.W hi tingThe enzym atic assay of trig lyceride, based on the use ofL -gl ycerol -3 -phosphate ox idase EC 1 .1 .3 .21 ) a nd a m o difie dTnnde r s ch romogen i nvo lv ing 4 -ch lo rophenol, is s ub je ct tos tro ng n eg ativ e in te rfe re nc e a t c on ce ntr atio ns o f trig ly ce rid e>20 mmol/L , s uc h a s occur in g ro ssly lip em ic p la sm a. Thisinterference is caused by the rap id utilization o f o xy ge n,re su ltin g in th e re actio n b ec om in g tra ns ie ntly a na ero bic. T hedye product already form ed may then be reduced(b le ach ed ) b y a ctin g a s a n a lte rn ative e le ctro n a cce pto r fo r

Depar tment of Biochemistry and Chemical Patho logy , F l indersMedical Centre, Bedford P a rk , S o ut h A u str alia , 5 0 42 , A u s tr alia .Received A u gu st 2 8 th , 1 9 89 ; a c ce p te d O c to b er 1 9 , 1 9 8 9 .

g lyce rol-3 -p ho sp ha te ox id ase . R ed uctio n o f th e d ye le ads toa m arked decrease in final absorbance at 505 nm . G rosslyunderes timated va lues fo r t ri gl ycer ide concen t ra t ions , appar-e ntly w ith in th e lin ear ra ng e o f th e a ss ay , m ay th erefo re b einadvertentlybtainedwithequilibriumethods We suggestth at sa mp le s g ivin g u nex pe cte dly lo w re su lts fo r l ipemicplasm a should be re-assayed after d ilu tion or w ith use of as ma lle r v olu me o f s am ple .A d di tI on a l K e yp h ra se s: g ly cer ol -3 -p hospha te o x id a se electronacceptor indica tor dy e

Colorimetry of h ydro ge n p ero xide by us e o f perox idase -l inked chromogenic system s is an ind ica tor reaction used

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326 CL IN ICALCHEMISTRY ,Vol. 36, N o. 2, 1990

for m any analy tes. These inc lude g lucose 1 , u ric a cid 2 ,and cholesterol 3 , w hich a re ox id ize d b y sp ecific o xid ase sto form hydrogen perox ide. In add ition , the enzym aticassay oft riglyceridesas been recently linked to this typeo f in dic ator re ac tio n, in p refe ren ce to m on ito rin g N AD Hproduction (4 ). T his d eve lop me nt b eca me po ssib le w ith th epu rif ic a tio n o f L -c e -g ly cer ol phospha te ox id ase 5 , and theuse of lipase and g ly ce ro l k in as e to c on ve rt trig ly ce rid e tog lyce ro l 3 -ph osp hate , a s sh ow n be lo w.

Triglyceride + 3 H 20 l ipase glycerol + 3 R CO OH(E C 3.1.3.1)g ly ce ro l k in as eGlycerol + ATP glycero l 3-phosphate + AD P(EC 2.7.1.30)

g ly ce ro l p ho sp ha te o xi da seG lycerol 3 ph osph ate + 02 (E C 1 .1.3.21 )

H202 + dihydroxyacetone phosphateperoxidaseH2O2 + 4-aminophenazone + 4-chlorophenol (E C 1 .11.1.7)

4(benzoquinone-monoamino)-phenazone + 2 H 20 + HC1In a recent batch of pati entsspec imens presented forro utin e e stim ation o f trig lyce rid e, on e p la sm a sample was

observed to be grossly lipem ic. U pon enzymat ic analysis, asu rp risin gly lo w va lu e (fo r trig lyce rid e) o f 5.4 m .m ol/L wa sobta ined for the und ilu ted sam ple. Th is resu lt, althoughwell w ith in the linear range of the assay (upper lim it 12m m oIfL), was c learly inappropriate for this sam ple. W ed es crib e in ve stig atio ns in to th e re aso n fo r g ro ssly u nd ere s-tim ated trig lyceride resu lts obta ined w ith the enzym aticassay of lipem ic p lasm a w hen a m odified Trinders chro-m ogen (6) invo lv ing 4-ch lorophenol is used in an indica torreaction.Ma te ria ls and Me thodsReagents

The reagent k its for enzym atic determ ination of trig lyc-eride were from Boehringer M annheim G m bH Diagnos-tica, M annheim , F .R .G ., and bioM erieux, France. The en-zym atic cholestero l kit also was from Boehringer M ann-heim.Ind ividua l com ponents of the trig lyceride assay-g lyc-e ro l 3 -p ho sp ha te , g ly ce ro l-3 -p ho sp ha te o xid ase , 4 -c hlo ro -phenol, 4-am inophenazone, and peroxidase-were fromS ig ma C he mica l C o., S t. Louis, M O 63178, as were the dyesp-io don itro te tra zo liu m vio le t a nd 2,6-d ich lo rop he no lin -dophenol. G lycero l was obta ined from M ay & Baker Aus-tra lia P ty . L td ., M elb ou rn e, V ic to ria, A ustra lia , an d h yd ro -gen perox ide w as supplied by BD H C hem icals , M elbourne.

R eagent kits w ere prepared according to the m anufac-turers instructions. For the Boehringer M annheim Perio-dochrom Trig lyceride G PO -PAP kit (cat. no. 701904), con-centra tions of com ponents of the reagents used in the testwere as fo llows: (Reagent 1) Tris buffer, 150 m m olJL, pH7.6 ; m agn esiu m su lfa te , 17 .5 mmol/L; E DT A d isod iu m sa lt,1 0 m m oL IL ; 4 -ch loro ph en ol, 3 .5 m mo llL ; so dium ch olate, 1.5g/L; potass ium hexacyanoferrate, 6 m ol/L; a nd (R ea ge nt2) ATP, 0.5 m m ollL ; 4-am inophenazone, 0.35 mmol/L;lipase, 3 kU IL; g lycerophosphate oxidase, 2 .5 kU /L;glycerol kinase, 2 kU /L; and peroxidase, 150 U IL.For the b ioM erieux Trig lyceride PAP 150 kit (ca t. no.

61 23 6 , final co nce ntra tio ns o f com po nen ts o f th e re ag en tsw ere as fo llow s: (R eagent 2) Tris buffer, 1 00 m m o l/L , pH7 .6 ; m ag nesiu m sa lt, 4 m mo lJL ; 4 -ch lo ro phe no l, 5 .4 mmol lL; and (R eagent 3) A TP, 0 .8 m m ol/L ; 4 -a min op he na zo ne ,0 .4 m m o L IL ; lip a se , 1 00 kU IL ; g lyc ero l p ho sp ha te o xid ase ,2 .0 kU IL; g lycero l k inase, 200 U /L; and peroxidase,200 U JL.To study the separate steps in the trig lyceride assay, w eused ind ividua l assay com ponents at the sam e concentra-tions as in the Boehringer-M annheim reagent.

InstrumentationAutom ated assays w ere perform ed in a C obas-B io cen-

trifugal analyzer (R oche D iagnostics S ystem s, N utley, NJ)with Boehringer M annheim reagents and in a M odel 717a na ly ze r (H ita ch i Ltd ., Tokyo, Japan) w ith reagents frombioMerieux. S om e m anu al assays were m onitored in aModel UV-160 reco rd in g sp ectro ph oto meter (ShimadzuCorp. , Kyoto, Japan).Plasma

The plasm a sam ple under study was a gross ly lipem icspecim en co llected from a 52-year-o ld w om an. Its actua ltrig lyceride concentra tion (see below ) was 40 m mol/L. Tostudy ind ividua l steps in the reaction sequence, we pre-pared 40 nunol/L solu tions of g lycero l, g lycero l-3-phos-phate , and hydrogen perox ide in d istilled w ater as substi-tu tes for plasma.Resul ts and D iscuss ionR ea ctio n K in etics w ith L ip em ic P la sm a

A bs or ba nc e d ata fo r the enzym atic trig lyceride assay ofthe lipem ic plasm a were obta ined w ith both the Cobas B ioand the H itachi 717. The reaction kinetics are show n inF igure 1. W ith the instrum ent settings lis ted in Table 1,the C obas B io m onitored the reaction for 5 m m and ca lcu-lated the trig lyceride resu lt on a fina l absorbance read ingtaken at 300 s after the p lasm a was added. The H itachi 717m onitored the reaction for 10 m m , but a lso derived thetrig lycerid e con cen tra tio n fro m a sin gle a bso rba nce valu etaken at 600 s. An obliga tory stirring of the reactionmixture took place in the H itachi 717 at 300 s, even thougha start reagent w as not used.The tim e course of absorbance changes showed that aninitia l rap id increase in the form ation of the Trinder-type

a0

Fig . Abs orb anc erea din gsbtain edithCob as Bio A and H i tac h i71 7 analyzers dur ing assayoftriglyceridenlipemiclasmaR e a ct io n s w er e m o n ito re d a t 3 0 -s i nt er va ls f o r 3 0 0 s o n t he C o b as B io or a t1 2- s i nte rv als f o r 6 00 s o n th e H ita ch i 7 17 . In th e H ita ch i 7 17 th e r ea ctio nm i xt ur e w a s s ti rr ed a t 3 0 0 s arrow

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Cob. . Blo HitachI7172.3 .4.5.6.7 .8 .9 .

10 .11 .12 .13 .14 .15 .16 .17 .18 .19 .

8 . H IT A CH I 717 Tim.(sec)

0 200 400 600 200 400 600 200 400 600 200 400 600

T ab le 1 . In strumen t S ettin gs fo r th e C ob as B io a nd H Ita ch i 7 17 fo r E nz yma tic A ss ay o f T rig ly ce rId es In P la sma

CL IN ICAL CHEM ISTRY, Vo l. 3 6 , No . 2 , 1990 327

UnitsCalcu la t ion fac torStandard 1 concnStandard 2 concnS ta n da rd 3 c on c nLimitTemperature (#{176}C)Type ofanalysisWav e leng th (nm )S am p le v ol ( &L )D ilu e nt v ol ( M L)R e a ge n t v ol ( pL )I nc ubat ion t im e (s)Start r ea g en t v o l ( LL )T im e o f fir st r ea d in g ( s)T im e i nt e rv a l ( s)N u m b er o f r ea d in g sB l ank ing m odeP r in tou t m ode

mmol/L01.861.861.86

1237.0

5050540

30 0600

0.53011

A ss ay c od eS am p le v o lR i vo lu meR 2 v olu m eWavelengthCalib ethodSTD ( 1 ) c oncn -posS T D ( 2) c o nc n- po sS T D ( 3) c o nc n- po sS T D ( 4) c o nc n- po sS T D ( 5) c o nc n- po sS T D ( 6) c o nc n- po sS D li m itDup l ica te l im i tSensi ti vi ty l im i tA bs l im i t ( inc /dec )Prozonel imitExpec ted va lueI nst rumen t f act o r

1 p o in t : 5 0 -05:2300:100: N Oo : 2 0: N O7 00 : 5 05

L inear : 0 : 00 .0 0 :11.86:2

0: 00: 00: 00:00. 1

20 00

0 : In cr ea seo : Lower0 .3 :1 .7 0 m m ol/L

1. 0

quinonemonoimine dye during the firs t 100 s w as follow edby a large decline in absorbance unti l 300 s o f reaction t imeFigure 1 ). In th e H ita ch i 717, a sudden increase in absor-bance was recorded after the 300-s p oin t, c oi nc id en t withstirring of the reaction m ixture . In both instrum ents thetriglyceride concentrationw as ca lcu la ted from the fina labsorbance reading, so it b eca me evident w hy a low resultof 5 .4 m mol/L w as obta ined for this sam ple . However, thereason fo r the steep decline in absorbance was not im me-d ia te ly a pp are nt.T he a ty pica l re actio n kin etics seen w ith the lipem icplasma w ere show n to be concentra tion dependent. S eria ld ilu tions of the plasm a specim en, corresponding to 20% ,40% , 50% , 60% , 80% and 100% of the origina l plasm a, w ereprepared in iso ton ic sa line an d re-ana lyzed in the CobasBio an d the H ita chi 7 17 . A bsorb ance da ta o bta in ed fo r e achd ilu tio n a re plotted fo r the range 50% to 100% in F igure 2.T he re su lts sho w tha t the tru e trig lyce rid e co ncen tra tio n

aS

k C OS AS 8 10

T im e sFig .2 . Absorbance read ings obta ined wi th Cobas -B lo A an d Hitachi717 ana ly z ers dunng assay of t ri g lyceride in ser ia l d i lu t ions ofl ip e m ic p la sm aThe di lu t ions were madeI ns a l ine and correspo ndedo 50% , 60% , 8 0% , and100% of t he o r ig ina l p las ma(a , b , c , an d d, respectively

in th is specim en was -40 m nm ol/L . M ore im portantly , theabnorm al reaction kinetics w ere observed on ly at triglyc-eride concentra tions >20 m mol/L. In a dd itio n, w e fo un dthat th e lin ea r range of the b ioM erieux reagent (upperl imit 15 mmol/L exceeded that o f the Boehringer-Mann-heim reagent (upper lim it 12 mmol/L).P os sib le R ea so ns fo r th e O bs erve d In te rfe re nce

The fo llow ing investiga tions w ere carried out to explainthe negative in terference observed in the enzym atic tri-glyceride assay.

First, spectral analysis of th e reaction products at 1-mmintervals in a r ec ord in g s pe etr op ho to m ete r r ev ea le d t ha tth e decrease in absorbance at 505 rim w as not ascribable toa w avelength sh ift in the absorbance m axim um of thequinonem onoim ine dye product, but instead to a loss of dyebleaching .Second, e ac h s te p in th e r ea ct io n s eq ue nc e wa s e xam in eds ep ara te ly. A 40 mmol/L glyce rol so lu tio n w as su bstitu tedfo r t he lip em ic p lasm a and analyzed as a patients sam ple ;th at is , t he firs t s te p in th e re ac tio n s eq ue nc ew a s bypassed.Absorbance data d isp layed in F igure 3A illustra te thatin te rfe re nc e id en tic al to that o bs erve d w ith th e lipemicplasma w as still observed. G lycero l so lu tions-2, 5, 10, 20,an d 30 m inol/L-w ere a lso ana lyzed and in terference w assh ow n to b e co nce ntra tion d ep en de nt, com me ncin g a t g lyc-ero l concentrations >20 m moIIL . G lycero l phosphate so lu-tions of the same concentrations w ere th en p re pa red an danalyzed to bypass the second step in the reaction se-quence . I nt er fe renc e a t c on ce ntra tio ns > 20 mmol/L wasagain observed. A t 40 mmolJL (Figure 3B , th e effect wasseen within th e f irst 30s of the reaction . F ina lly , a series ofso lutions contain ing 2, 5 , 10, 30, and 40 m mol of hydrogenpe rox id e H2O2 ) per lit er w e re prepared a n d a n al yz ed ; thatis, the reaction was commenced at step 4. At all H2O2concentrations tested (including 40 m mol/L , see F igure3C ), a constant absorbance p la teau w as atta ined, therebyiso lating the interference to the th ird step of the reactionsequence.It has been suggested that th e b le ac hin g o f Trinders dye

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A.G lycero l B .G lycero lPhosphate C .Hydrogen P eroxide5.04.0

0 100 200 300

I I Ia

o too 20 0 30 0 0 10 0 200 300Tim e s

F ig . 3 . Absorbance readings obtained w ith a C o ba s- Bio analyzerd u rin g in cu b atio n o f t he tr ig ly ce rid e a ss a y r ea g en t w ith a 4 0 m m o V Lsa mp le o f g lyc ero l A , g ly ce ro l 3 -p h os ph a te , or hydrogenperox ide CReact ionswere monitored at 30-s in te rva ls f o r300 S

10 15 20 25

1. 8

1.5C . 95% 02

ECU,0IL,00,UC0.00I,.0

1.0

0.58.20% 02

(Air)

328 CL IN ICALCHEM ISTRY , Vo l . 3 6 , No . 2 , 1990

is p ro d uc ed b y re du cin g a ge nts s uc h a s ascorbate 7,8 . Wepredic ted th at e xc es s g ly ce ro l 3 -p ho sp ha te m ig ht b e respon-sib le fo r red uc in g th e q uin on em on oim in e d ye in ou r reac-t ions if oxygen became limiting in th e re ac tio n s eq ue nc e;th at is , t he d ye c ou ld a ct a s a n a lte rna tive ele ctro n a cce pto rto oxygen from th e glycerol phosphate oxidase-glycerolphospha te complex if reac tio ns becam e anae rob ic , a s shownin t he f ollow in g e qu at io n:Glycero l 3-phosphate + quinonem onoim ine dye -*

d ih yd ro xy ac eto ne p ho sp ha te + reduced dyeT he fo llo w in g experiments gave re su lts co nsis te nt w iththis explanat ion.1. A ll oxygen w as remo ve d from an aliquot of assay

reagent in a s ea le d v ia l b y d eg ass ing th e s olu tio n u nd erreduced p re ssu re a nd filling th e v ia l w ith ni t rogen. W hen asample o f 4 0 m m ol/L g lyc ero l 3 -p ho sp ha te w as in je cte d intoth e s olu tio n w hile a na er ob ic c on ditio ns were maintained,n o colo r developed. However, injection of a sample of a 40minol/L solution of hydrogen peroxide resulted in ra pidfo rm ation of a red co lor, follow ed by slow er, com pletebleaching o f th e co lo r, a process th at w as d ep en de nt o n th eprior addition of glycero l 3-phosphate. C ontrol assayss howe d th at d eg as sin g s olu tio ns d id n ot p er s e in ac tiv atethe enzym es in the assay reagent.2. A 15-oL sam ple of a 40 m m ol/L so lu ti on o f g ly cer ol3-p ho sph ate w as a dde d to 9 00 pL of triglyceride rea ge nt ina re co rd in g- sp ec tro ph oto m ete r c uv ette . The a bso rb an ce o f505 nm was read at regu lar in terva ls. C hrom ogen w asformed, but w as then bleached (F igure 4A). After 5 mm ,th e cuvette w as removed from th e spectrophotometer andv ig or ou sly s ha ke n to reoxygenate th e re ag en t. T he cuvettewas r ep la ced and th e absorbance w a s o bs er ve d to increaserapidly and re ac h a constant value, w hich w as s ta ble fo r 1 0m m (Figure 48). Further b leach ing d id not occur becauseth e g ly ce ro l 3 -p ho sp ha te w as d ep le te d. H ow ev er, a dd itio nof a further 15-L a liquot o f g lycero l 3-phosphate againresu lted in a near com ple te loss of dye absorbance at 505nm (F igure 4C).3. The oxygen content o f the assay reagent w as in-creased by bubbling a ir 20 oxygen) or 100% oxygenthrough it for 30 s. On the addition of a sam ple of 40m mol/L g lycero l 3-phosphate , the absorbance increasedafter 30s to a maximum va lue of 1 .7 for a ll assay mixtures

T Im e ( mm )Fig. 4. Form ation and bleaching of quinonem onoim ine dye byv ar io us re la tiv e c on ce ntr atio ns o f glycerol 3-phosphateand oxygenThe reaction w as init iated by adding glycerol3-phosphate a t tim e z er o A .Af te r5 mm, t he reac ti onmix tu rew as oxygenatedby shaking the cuvet te(.At 16 mm ,mo reglycerol3-pho spha tewas added C(Figure 5 ). H ow ev er, n o subsequent b le ac hin g o f d ye product o ccurred w ith reagent bubbled w ith 100 o xygen,whereas a 60% and 9 0 re du ctio n in a bso rb ance w assubsequently observed w ith reagent bubb led w ith a ir anduntreated re ag en t, r es pe ctiv ely (Figure 5 ). Oxygenat ionu po n m ixin g w ou ld explain th e increase in absorbance seenat 300 s for reagent m onitored on the H itachi 717 Figure1B .4. To demonstrate a requirement fo r g lycero l -3 -phos -phate oxidase to m ediate t he b le ach ing effect o f g ly cer ol3-p ho sph ate , w e g en era te d th e sa me q uino ne mon oim in edye w ith use of an enzymat ic cholesterol assayit (Boeh-ringer M annheim C holestero l CHOD PAP kit, cat. no.23669) by incubating a normal pa tien t s sampl e with th er eagen t f or chole s te ro l. T he s peci fi c ox idas e in this reagentsystem was cho lesterol ox idase. A 15-FL aliquot of 40

A . N orm alReact ionCondi t ionsT im e ( mm )

Fig. 5. Format ion and bleach ing of qu inonemonoimine dy e undertheu su al r ea ctio n c on ditio ns A or after oxygenation of the assayr eagen tw i th a i r or oxygen C

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C L IN IC A L C H EM IS TR Y , V ol. 3 6, N o . 2 , 1 99 0 32 9

m m olJL g lycero l 3-phosphate w as added to 900 L of dyeproduced from the cho lestero l kit. In the absence of glycer-o l-3-phosphateoxidase, the g lycero l 3-phosphate d id notb leach th is dye; ev idently, the enzym e is indeed requ ired asa c a ta ly s t.

Further studies revea led that not only the presence buta lso the activity o f glycerol-3-phosphate oxidase w as im -p ortan t, b eca use th e q uin on em ono im in e d ye w as b lea che donly w hen the enzym e activ ity in the assay w as 2.5 kU IL.G lyce ro l-3 -p ho sp ha te o xid as e fro m th re e d iffe re nt b acte ria lsources S trep toc occu s th erm oph ilu s, P edioco ccus sp p. a ndA ero co ccu s uirida ns behaved similarly.5. The effect of substituting other redox dyes for theTrinder-type chrom ogen w as investiga ted. The colorless,o xid iz ed fo rm of io do nitro te tra zo liu m d ye a t a c on ce ntr atio nof 160 m oI/L was reduced by