Note: Descriptions are shown in the official language in which they were submitted.
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Method to use a Reactlon By-Product as a
Callbrator for Enzymatlc Assays
S ~I~LI~
l. Fleld of the Inventlon
Method to use pyruvate as a callbrator for a d~agnostlc
lnstrument measurlng alanlne amlnotransferase actlvlty ~herelnafter
ALT). Based on thls method, slmllar callbratlon methods could be
devlsed ~or oth~r enzymes of cllnlcal lmportance.
2. Descrlpt10n ot the Prlor Art
Th~ most com~on m~ns o~ Ass~ylng ALT 0mploys
sp~ctrophot~metr1c ~ssay whlch coupl~s pyruv~te productlon ta the
roactlon cat~ly~d by lactlc dQhydro~nase ~h~rclnafter L~H) ~s
shown ln Flgure l. L~ctle d~hydro~on~se c~t~lyzas the reductlon of
pyruvate ~lth the concurr~nt oxldatlon of HADH. When NADH ls
oxldlz2d, 1t 10SQS adsorptlon at 340 nm and lt ls by loss of
adsorptlon that one tan monltor the orlglnal transamln~se aetlvlty.
~hls coupled assay holds true as a quanttt~tlve measure of alanlne
amlnotransferase act1v1ty as long ~s the coupllng enzyme 1s 1n large
exc~ss relatlve to ALT. Thls assures that the rate llm1tlng fa~t~r
for the oxldatlon of NADH ls the rate that ALT produces pyruvate.
To calculate the actlvlty of ALT 1n terms of ~moles pyruvate
produced per mlnute per l~ter o~ sa~ple, one mathematlcally converts
the chan~e 1n absorpt10n at 340 nm per un1t tlme to actual ~ mles
o~ r~duced XADH per ~lnute~ Th1s calculatlon ls accompl1shed us1ng
~; ~ molar ~bsorptlv1ty const~nt for NADH at 340 nm that relates
absorbance unlts to concentr~tlon. Slnce the relatlonshlp between
NADH and pyruv~te as shown ln Flgure 1 ls l:l, one ~mole of NADH
oxldlzed 1s equlv~lent to one ~ le pyruvate ~the pyruv~te belng
produc~d by the actlon o~ ALr) reduced~ Uslng thls relatlonshlp and
the sample dllutlon ~actor, one can obtaln the ALT actlvlty 1n the
approprlate unlts .
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An automated dlagnostlc 1nstrument deslgned to measure alanlne
amlnotransferase actlvtty 1ncorporates some type of plpettlng scheme
to add reagents and sample lnto a reactlon ~ell. Thls reactlon ~ell
could be an optlcal flow cell. Hhen calculatlng transamlnase
actlv1ty, the volumes del1ver0d by the plpettors and resultlng
sample dllutlon factor are lncorporated lnto the equatlon used for
thls calculatlon. Plpettlng errors, therefore, lmpact the accuracy
of the transamlnase actlvlty calculatlon. From thls 1t ls apparent
that a callbratlon procedure that takes 1nto account plpettlng
lnaccuracy would be deslrable.
Prevlous methods ~or lnstrument callbratlon rely on uslng
standardlzed optlcal ~llters that have constant absorbance value5 at
the wavelength o~ lnter~st. Thls ~ssurcs that th~ lnstrum~nt ls
optlcally correet and th~t u~lng th~ mol~r ~bsorptlvlty constant to
convert absorbance unlts to ~mol~s NADH wlll be valld and
accurate. Thls only addresses the optlcal characterlstlcs of the
1nstrument, however, and does not correct for plpettlng errors.
In addltlon to lnstrumentatlon based on absorptlon, detectlon of
actlvlty can also be done uslng a ~luorlmeter, slnce NADH ls
fluorescent. Brooks & Olken, An Automated Fluorometrlc Method for
rmlnatlon of Lactlc Dehvdrogenase in Serum, ll Clln. Chem. 748
~1965~. ~hlle fluorescence adds 1ncreased sens1tlv1ty, lt does not
have ~ unlversal molar ~ibsorpt1vlty constant to relate fluorescence
unlts to concentratlon of NADH. Because of thls, one needs to
~enerate a standard curve of fluorescence unlts versus NADH
concentrat1On whe~ever the assay 1s run. Unfortunately, NADH
standards ~re not su1tably stable ~nd would requlre frequent
restandardlzatlon. Lowry & Passonneau, "A Flexlble System of
Enzymatlc Analys1s", Chap 1, p 3-20, Aeademlc Press, Ne~ York
~1972). Thls makes NADH based standards lmpractlcal for cllnlcal
dlaynosttcs wlth an ~utomate~ lnstrum~nt.
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3. Summary of the Inventlon
A known ~nalytlcal concentrat10n of pyruvate ls used as a
substltute for an optlcal fllter or NADH as the caltbrator for an
lnstrument ln a dlagnostlc assay. The dlagnostlc assay o~ 1nterest,
S ln thls case, ls the measurament of alanlne amlnotransferase
actlvlty. Pyruvate ls lntroduced to the ALT reagents ln the same
manner as a sample and ls rapldly converted to lactate ~lth
concurrent oxldatlon of NADH (Flgure l). Slnce the stolcheometry ~f
pyruvate turnover to NADH oxldatlon ls l:l, the dlfference ln
absorbance or fluorescent unlts between thls solutlon and a sample
that does not contaln pyruvate ls an exact analytlcal measure
relatlng absorbance or fluoresaanca to NAD~ concantratlon. Thls
ralatlonshlp thQn ls used to calculato tha orlglnAl anzymatlc
actlvlty o~ ALT. The ~dv~nta~es to thls lnventlon lnclu~ ~h~
ellmlnatlon of plpettln~ lnaccuracy ~s d neg~tlva effactor ln
actlvlty determlnatlon, the Qllmlna~lon of preclse optlcal
callbratlon, and tn the case of fluorescence detectlon, replacement
of unstable NADH standards ~lth a stable pyruvate standard.
~I~ILE~ DESC~leIION OF ~E DRAWI~
For a better understandlng o~ the lnventlon as ~ell as other
obJects and ~urther features thereof, reference ls made t~ the
follo~ng detalled dlsclosure of thls 1nventlon taken ln con~unctlon
~1th the accompanylng dra~lngs ~hereln: -
F1gure l shows the react10n scheme 1nvolved 1n the measurement
of alanlne a~1notransferase.
F1gure 2 sho~s the stab111ty data for pyruvate callbrators.
~lgure 3 shows a ccmparlson of standard curves relatlng
~luorescence un1ts to NADH concentrat10n elther d1rectly or wlth the
pyruvate c~llbrator.
F1gure 4 demonstrates the equlvAlency of uslng elther NADH stan-
~ards or the pyruvate c~11brator ln thQ ~luor~sc~nce mode by ~ay of
slopa valu~s ~luoroscence unlts par ~M) over ~ per10d of t~o ~ee~s.
Flgure 5 sho~s the reactlon scheme lnvolvement ln the
measurement of aspartate ~mlnotransferase.
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Flgure 6 shows reactlon schemes ~or pyruvate klnase, creatlne
klnase and glycerol klnase.
~IQILED DEsçB~ oNL~ GE~
If one lntroduces a known analytlcal amount of pyruvate 1n place
S of the sa~ple uslng the normal assay scheme tl.e. same reagents ln
reagent plpettors, same volumes, etc.), the hlgh level of LDH
present ~lll convert all of the pyruvate and therefore an equlvalent
amount of NADH wlthln a very short t1me. The d~fference ln
absorbance from a blank that contalns no pyruvate to a pyruvate
standard wlll be an exact analytlcal measure that relates absorbance
or fluorescent unlts to concentratlon of NADH. Slnce the pyruvate
standard ls dellvered to the reagents ln tha sama manner ~s the
samples, plpettlng lnaccuracy ~lll no longer negatlv~ly lmpa~t th~
accuracy of the ALT actlvlty calculatlon. In addltlon, ~lnce thls
type of callbratl~n ~llmlnatas th~ nq~d to usa th~ molar
absorptlvlty constant to calcutate ~mbles o~ NADH ~thls ls so
because lt ls the pyruvate standard that ts now relatlng absorbance
unlts to ~moles of NADH), preclse optlcal caltbratlon of the
lnstrument ls not requlrad. In the fluorescence mode, callbr~tlon
wlth a pyruvate standard also ellmlnates havlng to use NADH
standards for ca11bration.
T~o crlterla must be met, however, tn order for a pyruvate
c~llbrat10n standard to ~ork. F~rst, the react10n catalyzed by LDH
~n the presence of pyruvate needs to occur relat1vely f~st under the
2~ cond1t~ons of the assay and second, the pyruvate standard solut10n
must be stable so that the analyt1cal concentratlon does not change
over ~n extended per10d of of t1me. ~he second crlter1a 1s of
part1cular s~gn1f1cance, slnce stab111ty of pyruvate and
a-ketoac1ds ln general can be ~ source of error. R. Von Korff,
Pur1ty _on~ LLL~y Qf P~ruvate ~nd ~ ~~sglLl3rI~Q~ 13 Meth.
En2ymol. 519 ~1969~.
~ . ~... . . .. ` ' . .. .. ` ., . ` ` .. ,' .. . .... . ..
`. . . . .. ' . .` . . . . . ' `. . . . . ; . ` . .. . . . . ` . , .... .,- . .' . . . -
.. : . ` . ' . ... . . .. . ` . . . . . . ' , . ' . .: ., . ' ` .; . . . .. .
. ., ,`, .~ ... ,. ` , . ` `, . .' ,. .- .. ` . ` .
. ~ . .. ,.......................... .. ,.... . .... j . ,. .. , . . ;
... . .` ........ .. . ~ ... . . ..
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In general, the pyruvate callbratlon standard ts made by the
process ~ompr1slng admlxlng sodlum acetate, trlhydate, sodlum azlde,
and sodlum pyruvate and ~ater untll al1 soltds dlsso~e and adJustlng
the p~ o~ the admlxture from between ~bout 4.0 to 6.0, ~lth the
S optlmum pH of the flnal mlxture beln~ about 5.5. Thls callbratlon
standard ls stabl~ over an extended p~rlod o~ tlme.
It should be noted that the flnal dllutlon of pyruvate standard
ln the assay solutlon at 1:20, as descrlbed ls not crltlcal, but 15
1n llne wlth the ratlos used ln the present ALT assays. Other
manufacturers flnal dllutlon ls from 1:10 to 1:15 and could easlly
be substltuted. Wlth the 2 mM pyruvate standard, the amount of NADH
oxldlzed would account for approxlmately 80X of the orlgln~l 0.12S
mM NADH us0d ln tha r~actlon mlxtur~ d~scrlb~d heroln. Of
lmportance wlth thl~ 15 that the ~lnal conc~ntratlon of pyruv~t~ not
~xceed the NADH concentratlon. X~ thls ~er~ to occur, all 3~0 nm
absorptlon due to NADH ~ould dlsappQar dS ~ell as the relatlonshlp
of absorbance dtffernece to NADH concentratlon,
E~Ql
In thls example, the materl~ls and lnstrumsntatlon us~d for all
ZO studles are descrlbed along ~lth the procedure for manufacturlng the
pyruvate callbratlon standard and the ~ethod to calculate lts
analytlcal concentratlon. In ~ddltlon, the data and results
addresslng stab~llty and reactlon rate 1ssues are presented.
A. Materl~ls and Instrumentat!on
2~ Sodlum acetate trlhydrate and sodlum azlde were purchased from
Aldrlch Chemlcal Company. Sodlum pyruvate ~as purchased from
Boehrlnger Mannhelm Blochemlcals. Sterile flltratlon ~as done uslng
a 1 L, 0.2 um pore slze Nalgene Dlsposable Fllter (Nalge Company).
Reagents for ALT actlvlty measurements and pyruvate cal1bratlon on a
cl1nlcal chemlstry analyzer were purchased from C1ba-Cornlng.
In-house manu~ctured ALT rQagants ~Pandex~ ALT r~agents A ~nd B
.
together ~lxed wlth dlluted sample ln the ratlo of 1:1:2 ~s composed
of 12.5 mM a-ketoglutarate, 0,125 mM HADH, 0,30 M L-alanlne, l.~
'~ ~ ', ' '`, ,'. ,'' . '.,~ .;'' ,' .'' '. ' ,", ' `' `" ' '"''' " '` ' '' "' ` ' " "
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unlts/mL LDH, and 30 ~M trls, pH 7.8) ~ere used both for the
concentratlon determlnatlon of the pyruvate callbrator and
equlvalency testlng of NADH fluorescence as a functlon of
concentratlon. Hater used ln all experlments and ~or reagent
preparatlon was obtalned through ~n ln-house dQlonl~ed system that
was turther purl~led wlth a Mllllpor~ Q Water System purlfler
(ml~ q H20).
Spectral measurements for stablllty studles and to determl~e
pyruvate callbrator concentratlon ~ere done uslng a Hewlett Packard
dlode array spectrophotometer ~model 84S2A)/HP 9000 serles 300
computer along wlth the manufacturer's Chemstatlon software.
Alanlne trans~m1nase actlvlty measuremQnts were done usln~ a Gllford
SBA 300 autom~te~ cllnlcal chemlstry ~naly~cr. A P~ndex~
fluorescence mlcrotlter plate read~r wlth ~llt~rs approprl~t~ for
monltorlng NADH fluora~cence WAS US~ to test equlval~ncy batween
NADH and the pyruvate callbrator.
B. PreparatlQn of P~ru~ate ~allbratlon Standard ~1 ~ 2 mM)
For ~ 2 mM toncentratlon and 1 L slze volume; 0.680 g ~0.005
moles) sodlum acetate trlhydrate, O,Z00 g sodlum azlde, and 0.2Z~ g
(0.002 molés) of sodlum pyruvate were ~e1ghed together ln a 1 L
flask. To thls ~as added 950 ~L of mllll-Q H2O and a sttr bar. ~he
solutton ~as then st1rred on a magnetlc st1rrer unt11 all sollds
were d~ssol~ed. The pH ~as adJusted to 5.5 1 O.l ~lth 1 N HCL and
brought to 1.~ L ~lnal ~olume. The flnal solut1On ~as ster~le
f11tered, dlvtded 1nto approprlate volumes, ~nd stored at 2-8 ~C.
A 1 ~M concentratlon st~ndard ~as prepared 1n an 1dentlal manner
except for half of the sodlum pyruvate belng added to the flask.
C. ~nalvtlcal ~oncentratlon Determlnatlon of Pvruvate EallbratQr
The actual ~nalytlcal concentratton of pyruvate ~as determlned
by lactlc dehydrogenase dependent turnover of pyruvate to lactate
wlth the concurrant oxldatton of NADH ~1:1 stolch~ometry), Thts wasc~lcul~t~d uslng the A340nm dl~farQnce betwe0n a bu~r b1ank and
the pyruvate callbrator, and the ~olar ~xtlnctlon coefflclent of
NADH (6.22 ~Mrl cm~l).
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To 360 uL of blank bu~fer (30 mM trls, 0.02X sod~um azlde, pH
7.B) was added 40 uL of the pyruvate callbrator follo~ed by 200 uL
each of Pandex~ AL~ A and B reagents ln a l.0 mL seml-mlcro cuvette
~l tm path length). After 5 mlnutes o~ reactlon tlme at 25 C, the
absorpt10n at 340 nm ~AS recorded ~the spectrophotometer was zeroed
~lth blank bv~er). The ~verage o~ three repllcates was accepted as
the absorbance value. Thls procedure was repeated uslng 40 uL of
blank buffer to get an average blank value. Concentratlon of the
pyruvate callbrator ~as calculated uslng the formula below.
Pyruvate callbrator concentratlon ~mM) ~
~ Average A340nm blank - Average A340nm callbrator) X Z0 / 6.22
Illustrated below ls an example caltulatlon:
Wlth an avera~Q A340nm bl~nk ~ 0,790 and an avor~gQ
A340nm callbrator ~ 0.159, pyruvata conc0ntr~t~0n ~mM)
~0.7gO - ~.159) X 20 / 6.~2 ~ 2,03 mM
D. ~
In a l mL cuvette ~tS added ln order 360 uL blank buffer, 40 uL
or pyruvate callbrator ~l or 2 ~M), and Z00 uL Qach of Pandex~ ALT
A and B. For blank measuremants, 40 uL o~ blank buffer ~as
20 substltuted ~or the pyruvate callbrator. After addltlon of all
reagents, the reactlon was allo~ed to progress at 25 C. The
absorbance at 340 nm was measured at one, three, and n1ne mlnutes.
For each tlme polnt the ~verage of four repllcates ~as accepted as
the A340 Yalue-
Table 1 91ves the A340 dlfference from blank ~nd pyruvate
callbrators o~ 1 and 2 mM as A functlon ~f tl~e. The data lndlcates
that for b~th pyruvate callbrator concentratlons the reactlon ls
~reater than ~8X complete ~lthln the f1rst mlnute and lOOX by three
mlnutes. Thls satlfles the crlterla for a rapld reactlon rate.
.. , .. . " i , ,
: ~ , ... .. ..
.. . .
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Table l
s~mLl~ Tlme ~Mlnutes) --~340nm
1 mM Pyruvate 1 0.318
1 mM Pyruvate 3 0.320
S 1 mM Pyr~vate 9 0.320
2 mM Pyruvate l 0.622
2 mM Pyruate 3 0.629
2 mM Pyruvate 9 0.628
10 ~ A340nm ls the dlfference between blank and sample absorbance
values at 340nm. Reactlons ~ere run atcordlng to the procedure
outlln~d ln th~ Example s~ctlon.
E. ~
Pyruvate c~ltbr~tors tl ~nd 2 mM) w~re pr~par~d and thelr
lS ~nalytlcal eoncQntratlon detarmln~d ~s d~scrlbed ~bov~. These
solutlons were then stored at 2-8 C, room temperature, and 37 C.
Per1Odltally, the solutlons ~ere reanalyzed ~or thelr pyruvate
content. Data ~ere collected for 76 days at 2~8C, room
temperature, and S6 days at 37C.
F1gure 2 shows the stablllty data for pyruvate callbrators of
approxlmat~ly 1 and 2 mM concentratlon. As can be seen, there ~s no
105s ln the level of pyruvate ~t any of the temperatures ~or the
length of the study per1Od. In addlt~on, in a separate study, a 2
~M cal~brator has shown no loss ln pyruvate ~ontent after 200 days
~hen stored at 2 - 8 C. These results then ~eet the second
erlter~a of su1table stablllty of the pyruvate callbrator.
Example 2
Example 2 detalls a study atmed at testlng the hypothesls that
the procedure for uslng the pyruvate callbr~tlon standard wlll
el1mlnate ~uch of the error due to plpettlng lnaccuracy.
A. ~
The Gll~ord SBA 300 automated cllnlcal chemlstry analyzer ~as
programed to calculate a NADH converslon factor from the delta
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absorbance change that occurs vla ~he reactlon deserlbed above.
Thls was done us1ng 5, lO, 20, and 30 uL of the pyruvate callbrator,
as dellvered by the sample plpettor, ~lth 0.5 mL of Gllford AL~
reagent dellvered by the reagent plpettor. After 5 m~nutes of
reactlon t~me at 25 C, the absorptlon at 340 nm was recorded. The
average of four repllcates was accepted as the absorbance value.
The formula for calculatlng the converslon factor ls shown belo~.
Pyruvate callbrator converslon factor .
~ Abs3qOnm X total volume ~mL) I umol pyruvate n mM~
Illustrated below ls an example calculatlon
Wlth ~n average Q Abs34onm ~ 0.1162 from 5 uL of 2.05 mM
pyruvate callbrator ~0.01025 umol) plus O.S m~ ALT reayent,
converslon PaGtor ~ 0.1162 X 0.505 / 0.010~5 ~ S.73 mM-l
; Normally, as dlrect~d by the lnstrument manu~acturQr, the molar
extlnctlon co0~1clent ls used al~ne as tha NADH converslon factor
on the assumptlon that the plpette 1s dellverlng accurately. The
value used by the Gllford SBA 300 ls 6.30 mM-l
To determlne the pyruvate callbrator performance ~s compared to
the callbratlon procedure normally used, an ALT standard ~lth an
approxlmate actlv1ty of 80 unlts/mL at 37 C was assayed uslng the
same plpettlng schemes and Gllford ALT reagent as for callbratlon.
The background rate produced from blanks ~ere subtracted from sample
rates ~1th each p1pett1ng scheme so that actlv1ty talculated from
Abs340nm ~as due only from the ALT present 1n the sample 1tself.
~5 7he actual activ1ty calculat~on ~as then done elther ~y the normal
use of the ext1nct1On coeff1c1ent, or wlth the pyruvate callbrator
: determ1ned NADH converslon factor. The ALT standard and blank were
run 1n repl1cates of 4 at 37 C. After the add1tlon of sample and
reagent to the reactlon cup, a lag tlme of three mlnut~s was used to
assure steady-state klnetlcs 1n the ensulhg 45 secQnd r~ad tlme.
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.
The left portlon of Table 2 shows how the converslon factor
varles as a functlon of sample plpettlng volume and 1s 1n reallty an
1ndlcator of the plpettlng accuracy of the SBA 300 Cllnlcal
Chemlstry analyzer. If plpettlng volumes ~ere exact, then the
calculated converslon factor would be the same for eAch and should
be close to the reported coefflclent of 6.22 mM-l ~the value used by
Gllford, as ts shown ln the table, ls 6.30). Hh~le th~s baslcally
ho1ds true for volumes of 10, 20, and 30 uL, the 5 uL settlng ls
slgnlflcantly off from the reported value. It ls 1n thls sltuatlon
that a pyruvate callbrator theoretlcally should glve superlor
results to normal opt1cal callbratlon. The rlght portlon of Table
2, showlng the calculated ALT values for a 80 unlt/L standard,
supports thls con~luslon. For the pyruvate c~llbrator converslon,
all plpettlng volumes glve almost ex~ctly the s2m~ unlts/L v~lue.
Thls ls evldent ln the standard davlatlon o~ the m~ans ~or the
pyruvate and Gllford unlt/L c~lculatlons. It ls partlcularly
strlklng to note the S uL volume sample ~here the Gllford
c~ltulatlon ls excesslvely low, whereas wlth the pyruvate
calculatlon ls ln excellent agreement ~lth the other values.
Table 2
Pyruvate
G1lfordC~llbrator Pyruvate
Volume ConverslonConverslon GllfordCallbrator
(uLl_ ~mM~l? (mM~l ~ Unlts/L Unlts/L
2~ ~ 6.30 5.72 72.3 79.6
6.30 6.16 77.5 7g.2
6.30 6.12 78.4 B0.7
6.30 6.26 .8~.6
Mean 77.2 B0.2
Std Dev 3.5 0.9
Reactlons and calculatlons were done ~ccordlng to proc~dures ln
the Example sectlon
:; . , , , . . . . , . ; ~ ~
::. . - ,, . .: ... . i : . . ... .
:. . . . ; . i. . I
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Example 3 1s a study to test the equ~valency between the
pyruvate callbratlon procedure and a NADH standard curve ~hen
callbratlng a fluorescencè spectrophotometer.
A. ~
In the fluorescence mode, slnce there ls not the luxury of a
molar extln~tlon coefflclent, the use of a NADH ~ased callbrator ~s
essentlal to convert fluorescence unlts (AFU~ to NADH concentra-
tlon. Uslng a mlcrotlter based assay, 4 UL O~ pyruvate callbrator ~
36 uL blank buf~er or 40 uL blank bu~fer were added to 20 uL each of
Pandex~ ALT rea~ents to make 80 uL total volume. The re~ctlon was
allow~d to proceed for S mlnutes at room t~mperatur0 after whlch th~a
AFU was recorded, Thls procedure ls ~ssentl~lly the same ~s above
~or callbrator eoncentratlon dat~rmln~tlon, exc~pt ~or tot~l volume
ln the mlcrotlter fluorescence assay belng l/lO of an absorbance
mode determlnatlon. For performance comparlson, an AFU vs. NADH
standard curve was produced ustng known amounts of NADH 1n t~e range
of 0 to 135 uM ~lth the same buffer components and volumes as above.
The slope values for AFU vs. NADH and AFU vs. pyruvate callbrator
were then compared for equ1valency. Th1s was done over a per1Od of
2 ~eeks. The formula for calculatlng slope 1s shown belo~.
Pyruvate callbrator slope (AFUblank ~ AFUcallbrator)
X 20 / uM pyruvate cat1brator
Illustrated below ls an example calculat~on: -
~lth AFUbl~nk - 13500, - AFUcallbrator ~ 500
and pyruvate callbrator . 2050 uM, slope
(13500 - ~000) X 20 / 2050 Y B2.9
Flgure 3 shows that slope values generated elther by an NADH
st~ndard curve or by the 2 mM pyruvate callbrator 1n the fluore-
scence mode ~re essentlally equlval~nt. In thls partlcular case the
slope value for the NADH standard curve ls ~4.5 wher0~s lt ls ~l.l
for the pyruvate cal1brator. ~he equlvalency of these t~o proce-
dures was tested flve dlfferent tlmes over ~ perlod of t~o ~eeks.
,.: ~ . . . ,. .. -:.. ~. . . , ., . .; . . . . . :. . .. ... .
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As can be seen ln F1gure 4, excludlng day lO, both slope values
are wlthln error o~ each other for each ttme tested. The
dlscrepancy 1n day lO ls ltkely a result from an error 1n plpettlng
or ln manufacturlng of the standard NADH solutlon. The equlvalency
demonstrated here sho~s that a pyruvate standard can replace NADH
standards ln a fluorescence based assay.
Ex~mDle ~
The aspartate amlnotransferase ~herelnafter AST~, ~hlch ls
cllnlcally lmportant 1n monttorlng both heart and llver dysfunctlon,
1s assayed ln a very slmllar manner as the prevlously dlsclosed ALT
assay. ~lth the AST assay, malate dehydrogenase replaces LD~,
aspartate replaces al~nlne, and oxaloacetate replaces pyruvate, as
shown ln Flgure 5. A slmllar callbratlon mQthod ls devlsQd ~here
oxaloacetatQ repl~ces pyruvate as an lnstrumant c~llbrator.
The slmll~rltles between the assays ~or ALT ~nd AST ~re such
that preparatlon o~ the ox~loacetate callbratlon standard ~t l or 2
mM concentratlon would be ld~ntlal ~excludlng dlfferences ln grams
oxaloacetate necessary to glve 0.002 or 0.001 ~oles) to that
descrlbed 1n ExAmple l ~or pyruvate callbrator. In addltlon, the
analytlcal concentratlon determlnatlon of a oxaloacetate callbrator
~ould be carrled out ln a slmllar manner ~s descrlbed for the
pyruvate callbrator ln Example l. The mathematlcal formula used for
the calculatlon would be the same. The only exceptlons to the
procedure would be to substltute AST reagents for ALT reagents.
Exa~ple 5
ALT ls only one example of an enzyme ~hose actlvlty ls measured
by NAD~ reductlon v~a the coupllng to LDH ~ctlvlty. Other enzymes
that are o~ clln1cal lmportance that also utll1ze the NADH/LDH
couple are pyruvate klnase, creatlne klnase, ~nd glycerol klnase.
In the latter two cases, the NADHILDH couple ls e~tended by
1ncludlng pyruvate klnase. The assay sehemes for ~hese three
~nzymes ar~ shown ln Fl~ure 6.
.. , . ,. . . .. , ... ~ .
.... ~ .. . .. , ... i ; . ,, , - ,
:.. . . .. . ,, ; : . , . . - . ,; . ,
- . . ,.. . " , . ,. , ; . . . ~...... . .
: , ;. . . ... . . . ; ,. ., , . i . . , , , , , , , ,: "
w o ~1/13169 z o~j3 8 6~3 PCT/US91/01367
~ ' ; .
- 13 -
The pyruvate catlbrator that ~as manufactured for ALT as
descrlbed ln ~xample 1 wlll work equally well as a callbrator when
assaylng for these other enzymes wlth thelr approprlate reagents.
The 1nventlon has been descrlbed ln detatl w1th partlcular
5 refQrence to the above embodlments. It wlll be understood, ho~ever,
that varlatlons and modlflcatlons can be effected ~lthln the splrlt
and scope of the lnventlon.
~5
