Several methods for measuring the enzymic reduct’ion of ferrihemoglohin in red blood cells have been described (2,3,5,6). Scott (5) measured erythrocyte diaphorase, a NADH-dependent reductase, utilizing 2,6-dichlorobenzcnoneindophenol as a final electron acceptor. A more direct method for determining NADH-methemoglobin reductase was presented by Hegesh et nl. (2,4). This method utilizeP ferrihemoglobin itself as the electron acceptor. A comparison of these two methods was presented by Scott (1) , who found that the methods gave equivalent results. In the development of an automated procedure for NADH-methemoglobin rcductase (MetHb rcduct,ase), the manual method of Hcgesh et al. (2) was chosen for aclaptation since it appears to be more specific and preparation of the red blood cells is less time consuming (5). The automated procedure described here allows rapid and precise measurement of studies more MetHb reductase act,ivity, and make s large population feasible.
All chemicals were reagent grade, and distilled, dcionizetl water was used in all solutions. (A) A solution of 3.33 m&1 EDTA, 0.33 mM sodium citrate, and 0.5 mM K,Fe(CN)a, was adjusted to pH 5.2 and stored at 4”. Brij 35 (0.5 ml/liter) was added before the reagent was used. (B) The hemoglobin substrate was prepared from recently outdated banked blood according to Hegcah et ~11.(21, except that 0.8 gm diethyiaminoethylcellulose (DEAE, Wha-tman D 11) was added to each 10 ml of washed, packed cells along with 30 ml of H,O. The hemoglobin sub&rate, freed of MetHb rcductase, was then filtt~retl through glass n-001 to 72
remove any remaining DEAE. The hemoglobin concent,ration was measured according to Hegesh et nl. (2)) and adjusted to a final concentration of 0.50 gm/100 ml. The substrate was analyzed for residual MctHb reductase act#ivity by the automated method dcscrihed hew. It was empirically determined that more consietcnt results arc ohtnincd 1)~ aging tllc substrate at, 4” for 4-7 (lays before uw. (C) 2.0 ml11 NXDH (A grade, (‘alhiocherq, Los Angeles, California) in HZ0 was made fresh daily. (D) Samples of whole l)loo~l wore taken for cnzymr analysis using either EDTA (1 mg,/ml I or soclium lieparin (IO USP units/ml) as anticoagulant,. The sample 1s wcw centrifuged, t,lic plasma was rt~movcd, and, unless otherwise spwifkcl, 100 pl of p~cketl ret1 blood ~11s was mixed wit’h 2.9 ml of H,C). A portion of this sample was used for t’he analysis. A 100 pl syringe (Drummontl Microtrol. Helena Lahoratorics, A4llen Park, AIichigan)l provc(1 to 1~ most wcful in dclircring pa&cd cells.
A schematic design for the automat& measurement of MetHb recluctane activity is shown in Fig. 1. Technicon AutoAnalyzer (Technicon Inkument Corporation, Tarrytown, New York) equipment was used to construct’ t’he analytical system. The best sampling speed was 100 per hour (1 : 1 sample to mash 1 with tlic sampling tray arranged such that. sample cups containing w?-:ltci altcwiated with cups containing diluted red blood cells. In this way, one may analyze 50 samples per hour and yet maintain adequate resolution hetwccn samples. The determination of MctHh rctluctase activity according to the design in Fig. 1 proceeds as follows: Hemoglobin substrate (B), 0.6 ml/min, and reagent (A), 0.8 ml/min, are mixed in mixing coils for 15 min at room temperature ant1 t’lien iiicul)atccl at 37” for 2 min. One-third of tlic mixture is t’hen puml~~d through the flow cell of the first calorimeter, where the optical density of the l~lctllemoglobin-fcrricyanide complex is monitored at 580 mp; the remaining two-thirds of the mixture (with air hul~hle) is incubatc~cl again at 37” for 5 min. Anot.her one-third of the mixture is then pumped through the sccontl calorimeter flow roll (also monitored at 580 mp I with the remaining one-third of thr mixture (with air hubblc) going to n-&c. Both coloriiwtcw :\r(’ atljustctl to give a rccorder baseline of 9OGlOW~ 7’.
FIG. 1. Schematic tluctasc activity.
of system for measuring
This adjustment was necessary since usually the colorimct,ers would not give equivalent peak responses when adjusted to equivalent baseline settings. These adjustments arc indicated by part B (the six initial peaks) of Fig. 2. Determination of enzyme activity is initiated by adding NADH to the system (see arrow, Fig. 2). Figure 2 shows six peaks labeled “N.” The lower three represent8 the initial calorimeter’s response to a sample of normal k)lood in kiplicatc. The higher three “K” peaks represent the second colorimctcr’s rcsponsc to the same Moot1 sample (in triplicate) aftw incwb:~tioll at 37”. \Vhw the, leak Iwight, v:~luc~sfor the corrcsponding ~)c~~ksarc’ sul)tractecl ow ol)t:iilw a (‘h:nlg(l in absorbanc~~that is wlatrd to ;IlctHb rcrluctase activity during a sl)wifieci t)imc>of incubatJion ~1.37”. This (*11:t11~(~ill :~hso14~:ttiw is vx~)r(w(~~l ~II this rqwrt 3s 111 X 10:: pt’r iiiillllt(~.
FIG. 2. Typic,nl c*lrnrL rrcouling : 13. ~~lnnl;-r~o KADH :trl(lcd: KAADH trddd tinlr intlic~xtcvl 13y xrmv; Xi. Irtrrnd I~lootl in trildicxtcy : (‘. cmd hloml.
Also sl~own in Fig. 2 MC six lady!: lal~~lctl “C’.” Thwc rcprcwnt triplicate initial and final nwasureu~cnt~of RletHh reductaw act,ivitg in one sample of cord blood. *ill san~pl~~swere uw;wurecl in tril)lic:ltc unless otherwise slwcifictl. REWLTS
The automated sy&w tlescribd in Fig. 1 allows for only two incasuremerits to determiuc the rate of enzynle activity. It was therefore necessary to determine during what, time period the reduction of substrate proceeds linearly, ad then to adjust the time of incubation accordingly. From the data illustrated in Fig. 3, a linear increase in reduced substrate monitored at, 580 mp, is olwervccl beginning at 2 min and extending to
3CoR 0x 4
FIG. 3. Time course of substrate redwtion during incubation blood cells were diluted 1:30 to provide enzyme, nnd substrate 0.50 gm/lOO ml.
at 37”. Packed concenttxtion
8 min of reaction at 37”. In subsequent experiments, the initial colorimetric measurement was made at’ 2 min of incubation and t.he final measurement was made at 7 min to give an incubation time of 5 min. NASH is mixed with substrate, enzyme, and reagent for 2 min at room temperature prior to incubation at 37”. To determine the dilution of red blood cells to be used in measuring enzyme activity, the MetHb reductase activity of several dilutions was measured. The data in Fig. 4 indicate the enzyme act,ivity is related linearly to microliters of packed red blood cells per milliliter of react’ion mixture up to approximately 1 ,JJ.~of packed cells. The volume of packed red cells was obtained by considering the appropriate dilution factor (usually 1:30), the rate at which the reagents are pumped (1.6 ml/min) , the rate at which the diluted cells are pumped (0.16 ml/min), and the sampling time (15 set) . Using these conditions, a 1: 30 dilution of packed red blood cells as the sample is equivalent to mixing 0.8 ~1 of packed cells with 1 ml of reaction mixture. Therefore, a 1: 30 (or greater) dilution of packed cells gives an enzyme concentration wit’hin the linear portion of the curve shown in Fig. 4. The enzyme activity corresponding to this dilution, AA/min = 0.045, compares well with that of 0.050 found by the manual method (2). Since only 40 ~1 of diluted cells is pumped, adequate enzyme activity could 1)~ obtained with a 1 :30 tlilution of F, 1~1of l~c~kcd red blood cells
I 0.75 OF PACKED REACTION
4. MetHh reductasc activity of pa&cd cells corrwpond;; concentration 0.50 gm/lOO
I 1.0 CELLS
I 1.25 /ml
xs related to conwntration to 0.9 ~1 packed cells/ml ml.
of enzyme. reaction
A 1:30 mixture.
FIG. 5. MetHh rcductase a 1:30 dilution of packed
activity red blood
as related to substrate cells served as source
concentration. of mzymr.
‘l’llr srlhstl~atr (~011(~~~1111~:11iotl :t1 \vliic*l~ litl(l:(r txt(as :trc ot)t:titi(vl \v;i,s tletcrminetl by measuring 121vtH I) rc~(lrl&sc~ nctivit\r at, yttrious: su})strate concentrations (Fig. 5). As the hemoglobin concentration was changecl, the concentration of Iwt~assiurn fcrricyanitle was also changed so a:: to maintain a molar ratio of 4: 1 (K,,Fe( C’N),: Hb) as rccommencl~~~l by Hegesh et al. (2). The hemoglobin conccnt,rations given in Fig. 5 xvcrc calculated from the hemoglobin substrate (rcagcnt rJ) conccnt,rations, the dilutions by reagent A ( I : 2.5 j , the NADH solution (1: l.l), and the hemoglobin added along wit’h the enzyme sample. When no ht~moglobin other than that contained in the diluted cells (1:30) was atl~lctl to the system, a substrate conccntrat’ion of 24 pM was obtained. For measuring MetHb reductasc activity, a Fubstratc concentration of 74 pM was selected. To obtain t’his concent,ration, reagent (B) was adjusted t’o 0.50 gm/lOO ml (assuming 68,000 for the molecular weight of hemoglobin). This concentration, with t,llcl a(ltlitioll of the substrate contained in the diluted red blood cells, is within the range where linear also reaction rates are obsrrved (Fig. 4). Thi s substrate concentration allows adjustment of the recorder baseline to 9&100$% 7’. Several dilutions of red cells from one individual were analyzed for MetHb reductase by two nwthods: this scmiautoiiiatetl procedure antl t#he manual method of Hegesh et rll. (2). The tlata arc shown in Fig. 6. The multiple correlation coefficient is 0.998, and is highly significant as tested by the ANOV. This indicates an excellent agreement’ brtwwn the two methods over this range of fiIct,Hb rwluctase activity.
FIG. 6. MetHb rcductase activity measured by two methods: this procedure and that of Hegesh et al. (2). Several dilutions of cells vidual were used as enzyme source. Data points are sveragrs measurements.
semiautomakd from one indiof triplicate
--0 z,0 -3 s ? JC! : x 3;, z
7. MetHh reductnse and that of Scott
. ..* . *. . -. .. 2.. . . ..“. . . ... .; . .. . ..*:I . .. . -
activity measured by two m&hods: this semiautomated Cl). 811 data points are single mcasuremcnts of different
This ~cmiautorrl:ttccl proccdurc wne compared with the manual method of Scott (1) by analyzing the red cells from 76 individuals by both methods. An analysis of t,l~e data in Fig. 7 gives a multsiple correlation coefficient. of 0.446, which is signific*nnt as t)ested by t,he ANOV, indicating good agreement betwecxn this mcthocl and that of Scott (1 1. The magnitude of both “between day” and “within day” variations on tlw measurement of MctI-fb reduct,ase activity by this method is given in Table 1. Individuals .J.p\I. and D.S. were analyzed during two 4-day intervals and the per cent, deviations of the analysts w(‘re 15 and Scjo, rcsprctircly. These deviations compwrc well with the deviation from the mean of repeat,ed analyses of subject, ,J.hl. il2%:1 on day 1 of the experiment (Table 1) . Student f test, indicates no significant difference between the means of “within (lay-” and “bekvccn days” analyses of subject J.M. (0.40 > p > 0.35). It appears t’hen that) the precision of the met,hocl over a two ~ck period rompares well with the precision of the analysis on the first day of tllat J)c>rio(l.Tli(~ variation:: compart~ very favorably with tho.sc~for t11c~nl:i~iu:tI ~llc~tllocl,~ 01’ Ht~gcdi :iricl Gott as rrport~fd hy scwtt ( I )
Redllctase Ilttervals alld
TABLK 1 Activity Jleasured (2) Several Times
(1 ) I)aily drlring in 011~ I):~)
67,' 7:s 7:;
44,L ::X 37 49 64 .iO 42 40 :;S 41 :;s
67 66 .i7 37
n Activity * Substrat,e
equals AA X lo” per minute. was 7 days old OII day 1. All blood
at 4” for 24 hr heforr
ever, for a more detailed look at asp&s of this enzyme deficiency, such as K, values, one must resort, to manual methods in order to maintain more flexibility and control of reaction variables. SUMMARY
A semiautomated method for measuring NASH-methemoglobin reductase is presented. The method is patterned after the manual method of Hegesh et al. (2). Hemoglobin substrate concentration is O.FiO gm/ 100 ml and a 1: 30 dilution of packed red blood cells serves as the enzyme source. Fifty samples per hour may be measured using as little as 5 ~1 of of this method compares packed cells. The precision and accuracy favorably with the manual tnrthods of Hegrsh rf nl. (2) and Scott (1).