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INTRODUCTION
On exposure to hydrogen peroxide, metmyoglobin and methaemoglobin are activated to ferryl
states [1, 2] in which the iron is one oxidizing
equivalent above the original level and one oxidizing equivalent is on the surface of the protein [1] .
With reducing agents (electron or hydrogen donors)
these species are reduced back to metmyoglobin or
methaemoglobin [2-4].
In this study we have exploited the peroxidase
activity of metmyoglobin combined with its interaction with a phenothiazine compound to form a
radical cation intermediate [5], to establish a
method for measuring antioxidant status [6] . The
method derives from the observation that when 2,2'azinobis-(3-etbylbenzothiazoline-6-sulphonie acid)
(ABTS) is incubated with a peroxidase (such as
metmyoglobin) and hydrogen peroxide, the relatively long-lived radical cation, ABTS'* [7], is
Applying this method, the total plasma antioxidant status of pre-term babies has been monitored and compared with that of term babies and
maternal levels . The results show that infants born
at 27 f 2 weeks have a significantly depressed total
Key vror-0e antloxldaM, 7.t'~bnh0{3-ethylhenaathiamlinedaulphenir add), ferryl myotbbin, free radinis, premature neonata, Trobr .
Abhredations: ABTS, 7,1'ealnohis{3ethylbenmohluollnedaulphonic uid) ; TEAC, Trolox equivalent antlaxldant Upacity ; TRAP, totel perevyl redinkrapplnt entlavidant
parameter.
Correrpondence : Dr Gtherlne Rlcefvan,, Free Radical Research Group, Divelon of Biahemlrtry, UMDS-Guy's Campm, St Thoma Strest, Lendon SEI 9RT, U .K .
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Spsetroscopic assay
Patients
Babies were recruited at the time of delivery when
cord venous blood samples were collected . Further
venous blood samples (0.5ml) were obtained on day
5 in term babies at the time of the Guthrie test and
in pre-term babies at the time of routine blood
sampling.
Term babies (3"1 weeks gestation) weighed
2.20-3 .92kg; pre-term babies (25-29 weeks gestation) weighed 0.73-1 .46kg . Three of the premature
babies subsequently died of their complications
(intraventicular haemorrhage, group B streptococcal
sepsis, necrotizing enterocolitis) and three developed
bronchopulmonary dysplasia . The remaining ten
premature infants did well after their initial period
of ventilation. Each premature infant was treated
with surfactant (Exosurf), which showed no activity
when introduced into the antioxidant assay as a
sample, and penicillin and gentamicin, which both
showed moderate antioxidant activity. None of
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1 .0 1 .5
Trolox sonm . (mmol/I)
2.0
2.5
855
900
Table I . TEAC .alues
TEAC
8D
DeAerrioxemine
gllirubin
7.96
1 .50
4
3
0 .09
0I3
Urae
1 .02
0,06
Ascorbate
s70copharal
Glutathione
Albumin
EDTA
Mannitol
Gluaose
0.99
0.97
0 .90
0 .63
0 .05
0.00
0.00
S
3
3
3
3
3
2
0.04
0.01
0.03
0.02
0.01
Edanol
Heparin
Ura
0.00
0.W
0A0
3
5
3
Creatinine
OA0
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Two control pools were prepared from eommercial freeze-dried quality control sera . The results for
intra-assay precision when analysed repeatedly in a
single assay, or inter-assay precision, obtained by reanalysing the pools on 20 sequential days, are
shown in Table 2 The data indicate the ability to
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N. J . Miller et al.
Tahle L Estittuts d anatyaal perfarmaaee
Migh panl
Intrrauay prec is ion
Na
Mean
SD
Coefficient
of variation (%)
Interassay precislon
No.
Mean
SD
CaaMclenr of verlatlan (X)
Low pad
25
25
' 1 .51
0.008
0.83
0.013
0,54
1 .59
30
1 .52
0.055
3 .6
20
0.84
0.050
6.1
DISCUSSION
The derivation of a TEAC value, as shown above,
provides a method for the comparison of antioxidant activity among groups of drugs, provided
they are water-soluble (or can be solubilized), or
body fluids, such as plasma . The method thus has
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mmsuraman
ot
aaiazidan
npaity
411
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N. J. Miller et al .
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