Clin. Bioehem. 13 (1) 38-40 (1980)

Utilization of a Glycol-Stabilized Liquid NAD for the Measurement of Three Enzymes on the GEMSAEC

PAUL CARTER and MARY E. ROSE Department of Pathology, Wilmington Medical Center, Wilmington, Delaware, 19899, and Department of Chemistry, Veterans Administration Hospital, Wilmington, Delaware, 19805

(Accepted August 22, 1979)

Some analytical parameters have been investigated for a recently described stabilized liquid coenzyme technology in which water-free NAD is dissolved in 1,2 propanediol. Correlation for 108 specimens assayed for AST, ALT and LD with a reference method in which glycol-based N A D was absent was ----- 0.998 with near identical reproducibility over a period of at least 107 days. Mean recovery of exogenous serum enzymes in this linear kinetic assay is 103%. With the option of mixing only the volume of reagent needed for the enzymatic assay, waste can be eliminated as compared to more costly preparations stabilized by lyophilization. Hazards from an impure water supply are avoided since no reconstituting volume is required.

NICOTINAMIDE ADENINE NUCLEOTIDE ( N A D ) is uni- ve r sa l ly accepted as coenzyme of choice for a n u m b e r of a s says p e r f o r m e d in the cl inical l abo ra to ry . Never - theless a number of d i s a d v a n t a g e s m i l i t a t e a g a i n s t i t s use : i t is uns tab le in some aqueous so lu t ions ; i t is uns tab le as a d r i ed p roduc t in a mois t e n v i r o n m e n t ; the ex t inc t ion coef f i c ien t is a f f e c t e d by t r ace heavy meta l s no rma l ly p r e sen t in d is t i l led wa te r . Most labo- r a t o r i e s pu rchase th i s m a t e r i a l lyophi l ized even though f r e e z e - d r y i n g con t r i bu t e s to the h igh cost passed on to the consumer . Recent ly Modrov i t ch c1~ r e p o r t e d t h a t sa l ted NADH~, when solubi l ized in a glycol de r iva t ive , m a i n t a i n e d a one-yea r shel f l i fe p rov ided t h a t r e s idua l w a t e r was removed. In th i s communica t ion we eva lua te such a l iquid polyol co- enzymes p r e p a r a t i o n ava i l ab le commerc ia l ly by do- cumen t ing i ts p e r f o r m a n c e on th ree s u b s t r a t e a s says no rma l ly reques ted in the emergency sect ion of a busy hosp i ta l l abo ra to ry .

MATERIALS AND METHODS

Reagents. Stabilized liquid substrates as well as glycol- stabilized NAD are obtained from Medical Analysis Systems, Camarillo, Co., 93010 and stored a t re f r igera tor temperatures. When combined the working reaction con- centrations are estimated to be: 248 mM L-alanine, 8.5 mM a-ketoglutarate, 745 U / L LDH and 71 ~M NADH for alanine aminotransferase assay (ALT; EC 2.6.1.2); 143 mM L-aspar ta te , 8.5 mM a-ketogiutarate, 745 U / L MDH and 71 .~M NADH for aspar ta te aminotransferase assay (AST; 2.6.1.1.); 79 mM L-lactic acid, 3899 ~M NAD for lactate dehydrogenase assay (LD; EC 1.1.1.27). Correlat ive reference reagents in which the combined substrate-coenzyme mixture is stabilized by lyophilization are obtained from Smith Kline Instruments, Inc., Sunny- vale, CA., 94086. Reaction concentrations are described by the manufac turer to be: 112 mM L-alanine, 9.0 mM

Delivered in pa r t a t Special Meeting on Enzymes, Federat ion of European Biological Societies, Dubrovnik/Cavta t , Apri l , 1979.

a -ke tog lu ta ra te , 333 U / L LDH and 240 ~M NADH for ALT assay; 110 mM aspar ta te , 10 mM a-ketoglutarate , 600 U / L MDH and 200 ~M NADH for AST assay; 93 mM lactate and 3000 ,M NAD for LD assay.

Instrumentation. A GEMSAEC centrifugal analyzer (Electro-Nucleonics, Inc., Fairf ie ld , N.J. 07008) equipped with Rotoloader IV is used for all measurements.

Procedure. All reactions are performed at 30°C at 340 nm in "Auto Rate" reaction mode. The set t ings for ALT and AST are identical: 50 ~l sample volume, 200 ~l f lush volume, 400 ~l reagent volume using 40% of a 1 ml pump. Ini t ial readings occurred at 90 s , with 60 s reading intervals for 4 consecutive readings. The settings for LD are fixed at the following volumes: 20 ~1 sample, 80 ~1 flush (via 40% of 200 ~1 pump) , 600 ~.I reagent (via 60% of 1 ml pump) . Ini t ia l readings occurred at 30 s, with a 30 s reading interval for 5 con- secutive readings. Reference working substrate was re- constituted from ion-free water, while the liquid stabilized NAD was added to its substrate at about 1:150 (VJW) to insure the avai labi l i ty of excess coenzyme.

RESULTS AND DISCUSSION

I t is conceivable t h a t glycol de r iva t i ve s m i g h t in t e r - fe re w i th some enzymat i c procedures . T h e r e f o r e sev- e ra l s e r u m spec imens were ana lyzed by the p roposed method and the c o m p a r a t i v e r e f e rence method in which g lycol -based N A D H was absent . Spec imens were collected f r o m a no rma l and abnorma l hosp i t a l popu la t ion in equal po r t i ons fo r ALT, A S T and LD assay , s to red a t - 2 0 ° C and as sayed as descr ibed . C o m p a r a t i v e s t a t i s t i c a l p a r a m e t e r s by r eg re s s ion ana lys i s as i l l u s t r a t ed in Table 1 reveal good cor- r e l a t i o a and no s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r ences in the technology fo r 108 spec imen a s says over the observed r a n g e e x t e n d i n g f rom no rma l to more t h a n ten-fold the upper l i m i t of normal .

Reproducibility. W i t h i n - r u n prec i s ion was eva lua ted f r o m coef f i c i en t of v a r i a t i o n da t a by 5 dup l i ca te

85

..-/~ ....... o, A //0.~.%,

0 20 40 60 80 100 120 Time (Days)

Fig. i - - Comparative effect of a liquid stabilized ( ) and daily reconstituted lyophilized ( . . . . . ) coenzyme preparation on day-to-day quality control of LD.

UTILIZATION OF A GLYCOL-STABILIZED LIQUID NAD 39

TABLE i

STATISTICAL COMPARATIVE PARAMETERS FOR ENZYME ASSAY WITH (Y) AND WITHOUT (X) A GLYCOL-STABILIZED NAD

ALT AST LD

number of specimens . . . . . . . . . . . 36 36 35 quantitation range (U/L) . . . . . . . 7-139 6-470 49-553 correlation coefficient, r . . . . . . . . . 0.999 0.998 0.999 slope, m, estimating equation . . . . 1.02 0.99 0.96 intercept, b, estimating equation. -0.21 -0.42 5.59 T-probability . . . . . . . . . . . . . . . . . . 0.0001 0.0001 0.0001

TABLE 2

RECOVERY OF EXOGENOUS SERUM ENZYMES (U/L) OBTAINED BY LIQUID STABILIZED COENZYME ASSAY.

assay description . . . . . . . . . . A B C D

AST . . . . . . . . . . . . . . . . . . . . . 83 106.8 109.5 103 ALT . . . . . . . . . . . . . . . . . . . . 53 84.8 88 104 LD . . . . . . . . . . . . . . . . . . . . . . 222 267 273 102

mean 103

Where A = endogenous enzyme present B = predetermined enzyme present (control serum -t-

A) C = enzymatic activity by the proposed method D = recovery in % = C / B X 100

a s s a y s on 2 spec imens each selected f rom the cl ini- cal ly n o r m a l and a b n o r m a l r ange . F o r ALT, th i s da t a y ie lded mean values of 6.5% and 0.3% respec t ive ly ; fo r A S T 3.9% and 0.8% re spec t ive ly ; fo r LD, 1.0% and 0.5% respec t ive ly . Cor re l a t i ve va lues ob ta ined us ing p re - lyophi l i zed coenzyme were 4.4% and 0.6% respec t ive ly fo r A L T ; 2.0% and 1.4% respec t ive ly fo r A S T ; 1.9% and 0.4% respec t ive ly for LD. In no ins tance a r e these values, a v e r a g e d over the ana ly t i ca l

r ange , g r e a t e r t h a n s im i l a r values ob ta ined f r o m a pee r g roup s u m m a r y of a r ecen t enzymology su rvey ci). B e t w e e n - a s s a y p rec i s ion on 2 no rma l and 2 a b n o r m a l spec imens on 5 consecut ive clays y ie lded the fo l lowing mean values fo r glycol s tab i l i zed and non , s t ab i l i zed coenzyme r e spec t ive ly ; fo r A L T 4.3% and 5 . 2 % ; fo r A S T 4.1% and 3 . 4 % ; fo r LD 5.7% and 2.4%. The s i m i l a r i t y in the p a t t e r n of r e p r o d u c i b i l i t y is evidence t h a t the l iqu id - s t ab i l i zed coenzymes p r e p a r a t i o n is nea r to iden t ica l in i t s b e h a v i o u r w i th the pre - lyo- phi l ized produc t . C o m p a r a t i v e long- run v a r i a t i o n over a per iod of 107 days fo r LD is i l l u s t r a t ed in F i g u r e 1. I t is cons i s ten t ly s im i l a r for A L T and A S T as well.

Recovery . Since r e a g e n t concen t r a t ions a r e app re - c iably d i f f e r e n t in the r e f e rence and proposed mix- t u r e s i t was neces sa ry to examine the l i n e a r i t y o f the k ine t i c a s s a y us ing l iquid s t ab i l i zed coenzyme. As r e f e rence m a t e r i a l s a no rma l h u m a n s e r u m base enr iched wi th enzyme a c t i v i t y der ived f rom porc ine and bovine h e a r t ( D u P o n t E leva ted E n z y m e V e r i f i e r , E. I. d u P o n t Co., Wi lming ton , DE 19898) was mixed wi th i nc r e a s ing qua n t i t i e s of no rma l h u m a n based s e r u m ( D u P o n t N o r m a l Enzyme V e r i f i e r ) . The pro- po r t iona l d i lu t ion e f fec t is close to l inea r fo r ALT, A S T and LD as i l l u s t r a t ed in F i g u r e 2. E n z y m e re- covery was de t e rmine d on a pooled se rum specimen collected f rom severa l hosp i t a l p a t i e n t s who were be ing d iagnosed fo r l iver disease . P r e - s t a n d a r d i z e d enr iched control s e r u m ( D u P o n t E leva ted E n z y m e V e r i f i e r ) was added to each spec imen and the re- covery was ca lcu la ted as i l l u s t r a t ed in Table 2. Since es sen t i a l ly all exogenous enzyme was accounted fo r i t is p r e sumed t h a t the glycol based coenzyme has no i n h i b i t o r y e f fec t on the assay .

To date, the m a j o r i t y of commerc ia l ly ava i lab le p re - p a r a t i o n s of N A D a re lyophi l ized which i nhe ren t l y leads to a n u m b e r of p rob lems inc lud ing lo t - to- lo t v a r i a t i o n in the p a c k a g i n g as well as v a r i a t i o n in m e a s u r e m e n t of the r e c o n s t i t u t i n g f lu id and occa- s ional p resence of p a r t i c u l a t e m a t t e r due to incom-

7. THE PROPORTIONAL DILUTION EFFECT FOR 3 ENZYME

ASSAYS IN A GLYCOL-BASED COENZYME MILIEU

o = AST, .= ALD, - = LD R 0.999

300

~ - J

~ 2 O O m

¢o

lOO Q E 1~4 ee

IAI

/ °f

/o.

tOO 20o 30o Enzyme Activity Found U/L

Fig . ~ - - The propor t iona l d i lu t ion effect for 3 enzyme assays in a glycol- based coenzyme mil ieu. ( 0 ) A S T , ( • ) A L T , ( ~ ) , L D r -~ 0.999.

40 CARTER AND ROSE

plete dissolution of components. All these difficulties can be avoided by solvating the coenzyme in a pro- tective glycol to produce a stabilized homogeneous liquid essentially water free and stored at refri- gerator temperature. Frajola and Maurukas (3, have recently documented the stability of human serum enriched with ethylene glycol for quality control. Apparently the effect is somewhat similar when applied to coenzyme preparations as described in this communication in which it is presumed that 6.65 g / L NADH is dissolved in spectroscopically pure 1,2 propanediol to which 10% V/V molecular sieve is added to remove the water'Z~. In our hands analysis of the product available from Medical Analysis Systems revealed only trace water concentration at about 0.13% V/V and glycol concentration of about 99.0% as analyzed by gas chromatography.

With this technology it is to some advantage that workers can avoid waste by proceeding directly to the assay run without having to reconstitute material just prior to use. Thus coenzyme and substrate can be combined as needed. Because of recent restrictions on reagent water duly specified'4~ this technology

should prove especially attractive to smaller labo- ratories which service a remote area where water purity presents a problem. At this writ ing trace quantities of calcium, copper, iron, manganese and some other substances have an appreciable effect on inhibiting enzyme activity (57

REFERENCES

1. Modrovich, I.E., "Stabilized liquid coenzyme pre- paration", Chem. Abstr . 87, 179909, 1977.

2. Grannis, G. E., and Massion, C. C., "The 1977 College of American Pathologist Enzymology Survey", Am. J. CIin. Path. 70, 487, 1978.

3. Frajola, W.J. and Maurukas, J., "A Stable Liquid Human Reference Serum", Health Lab. Sci. 13, 25, 1976.

4. "Reagent Water Specifications", College of American Pathologists, Skokie, Ill. 60077.

5. Winstead, M., "Reagent Grade Water: How, When and Why?" Am. Soc. Med. Techn. 1967, Bellaire, Tex. 77401.

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