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Articles |
Department of Environmental Medicine, Odense University, Winsløwparken 17, DK-5000 Odense C, Denmark.
a Author for correspondence. Fax +45 65 91 14 58; e-mail HR.ANDERSEN{at}Winsloew.OU.DK
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Key Words: indexing terms: superoxide dismutase • glutathione peroxidase • glutahione reductase • catalase • biological variability
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Wide interindividual variations may exist regarding antioxidative capacity, thus affecting individual susceptibility against deleterious oxidative reactions. However, very limited information exists concerning the biological variation of antioxidative enzymes in representative population samples. This study presents intervals for antioxidative enzyme activities from a randomly selected population. All enzyme activities except CAT were measured by automated methods.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To estimate within-subject variations and the effect of fasting and food intake, six healthy volunteers twice delivered blood samples three successive days and thereafter three times once a week. One week later, the six volunteers delivered a fasting blood sample. After this blood sampling a substantial breakfast rich in fat and protein was consumed by all subjects. One hour after this meal a new blood sample was drawn. To investigate the effect of different anticoagulants, venous blood samples from two volunteers were collected into Venoject tubes (VT-100 TK; Terumo, Leuven, Belgium) containing either Na-heparin or K3-EDTA. To minimize the influence of analytical variation, all samples from each individual were analyzed in the same analytical run.
The study followed the guidelines of the Second Helsinki declaration and was approved by the Regional ethical review committee.
blood samples
Venous blood samples were collected from all study subjects into
Venoject tubes with EDTA (0.47 mol/L K3-EDTA) between
1000 and 1800. All individuals were placed in a reclining position for
a minimum of 10 min before blood sampling by the same phlebotomist.
Within 4 h after sampling, the blood was centrifuged at
1000g for 10 min to separate the plasma. The buffy coat was
removed and the remaining erythrocytes were drawn from the bottom,
washed three times in cold saline (9.0 g/L NaCl), and hemolyzed by
adding the same weight of ice-cold demineralized ultrapure (MilliQ plus
reagent grade; Millipore, Bedford, MA) water to yield a 50%
hemolysate. The hemolysates were frozen in 500-µL aliquots at
-80 °C for later analysis.
Erythrocytes isolated from blood samples delivered from 20 healthy blood donors were pooled and hemolyzed after the washing procedure to obtain identical aliquots to be used as control material.
analytical methods
All reagents used were analytical grade. Water used was
demineralized ultrapure of MilliQ plus reagent grade. All reagents
except the phosphate buffers were prepared each day and stored in a
refrigerator at -4 °C. The reagents were equilibrated at room
temperature for 0.5 h before use when the analysis was initiated
or reagent containers were refilled. Phosphate buffers were stable at
-4 °C for 1 month. At the day of analysis the hemolysates were
thawed and diluted 5:1 (by vol) with distilled water and further 2:1
(by vol) with a phosphate buffer containing dithiothreitol (DTT) (100
mmol/L KH2PO4, 1 mmol/L EDTA, 2 mmol/L
DTT, pH 7.4) to a final dilution of 1:20. These 1:20 hemolysates were
used for all analyses. Diluted samples not analyzed immediately were
placed in a refrigerator until analysis later in the day. Protein (Pr)
and hemoglobin (Hb) concentrations and all enzyme activities except CAT
were determined with a Cobas Mira autoanalyzer (F. Hoffmann-La Roche,
Diagnostic Systems, Basle, Switzerland). The methods were modified as
stated below for the autoanalyzer procedure. To obtain optimal accuracy
in pipetting, small volumes of H2O or assay buffer were
pipetted into the cuvettes together with samples and reagents to rinse
the needle. These volumes are included in the final reaction volumes.
All measurements were performed in triplicate.
Assay of CuZn-SOD activity
. Determination of CuZn-SOD
activity was performed by using a kit (Ransod; Randox Labs. cat. no. SD
125, Crumlin, UK) based on the method developed by McCord and Fridovich
(6) coupling an O2·-
generator with an O2·- detector. In the
kit, xanthine and xanthine oxidase are used to generate
O2·- and
2-(4-iodophenyl)-3-(4-nitrophenol)-5-phenyltetrazolium chloride (INT),
which reacts with O2·- to form a red
formazan dye used as detector. CuZn-SOD inhibits the formation of the
formazan dye, and the activity is measured as percent inhibition
compared with a calibration curve with purified SOD. The final
concentrations of the reagents used in the assay were as recommended by
the manufacturer (0.05 mmol/L xanthine and 0.025 mmol/L INT in the main
reagent and 80 U/L xanthine oxidase in the start reagent). The sample
(1:20 hemolysate) was first diluted 10-fold in
KH2PO4 buffer (10 mmol/L; pH 7.0) by the
autoanalyzer and 5 µL (plus 20 µL of H2O) of diluted
hemolysate was added concomitantly with the main reagent (170 µL) to
the cuvette. Absorbance was monitored at 500 nm for 150 s after
addition of xanthine oxidase (25 µL plus 10 µL of H2O)
as start reagent. The final reaction volume was 230 µL. The unit of
activity is defined as the amount of enzyme that inhibits the rate of
the formazan dye formation by 50%.
Assay of GSH-Px activity
. Total activity of GSH-Px was
determined by the coupled enzyme procedure (7) with
tert-butyl hydroperoxide as substrate. In erythrocytes from
adult humans all GSH-Px activity seems to be Se dependent
(8) and therefore the measurements represent Se-dependent
GSH-Px activity. Before analysis the autoanalyzer was programmed to
dilute the sample 10-fold to a 1:200 hemolysate by adding Drabkin's
reagent (double strength) to inhibit the peroxidase activity of Hb. The
main reagent was made by mixing 8.00 mL of
KH2PO4 buffer (100 mmol/L; 1 mmol EDTA/L; pH
7.4), 4.00 mL of GR (5000 U/L, cat. no. G-4751; Sigma Chemical Co., St.
Louis, MO), 2.00 mL of GSH (2.5 mmol/L; Sigma G-4251), and 2.00 mL of
NADPH (2.5 mmol/L; Sigma N-1630). The main reagent (200 µL) and the
sample (25 µL of 1:200 hemolysate plus 10 µL of H2O)
were added to the cuvette and the absorbance at 340 nm was monitored
for 200 s (step A). Then 10 µL of tert-butyl
hydroperoxide (25 mmol/L; Sigma B-2633) (plus 5 µL of
H2O) were added as start reagent. The absorbance was
monitored for another 225 s (step B). The final reaction volume
was 250 µL. The difference in absorbance per minute between steps B
and A was used to calculate the enzyme activity by using a molar
absorptivity of NADPH at 6.22 x 103 L
mol-1 cm-1. The unit is µmol of NADPH
oxidized/min.
Assay of GR activity
. Activity was determined by
following the oxidation of NADPH to NADP+ during the
reduction of oxidized glutathione (GSSG) (9). The main
reagent was prepared by combining 18.00 mL of
KH2PO4 buffer (139 mmol/L, 0.76 mmol/L EDTA; pH
7.4) and 2.00 mL of NADPH (2.5 mmol/L). The sample (20 µL of 1:20
hemolysate plus 20 µL of KH2PO4 buffer), 220
µL of the main reagent, and 5 µL of FAD (315 µmol/L) plus 10 µL
of KH2PO4 buffer were added to the cuvette, and
the absorbance at 340 nm was monitored for 200 s (step A). Then 30
µL of GSSG (22 mmol/L; Sigma G-4376) plus 10 µL of
KH2PO4 buffer were added to start the reaction
and the absorbance was followed for 175 s. The final reaction
volume was 315 µL. The difference in absorbance per minute between
steps B and A was used to calculate the enzyme activity by using a
molar absorptivity of NADPH at 6.22 x 103 L
mol-1 cm-1. The unit is µmol of NADPH
oxidized/min.
Assay of CAT activity
. CAT activity was determined by
the method described by Aebi (10) by using a UV/visible
spectrophotometer (lambda 11; Perkin-Elmer, Norwalk, CT). The
decomposition rate of the substrate H2O2 was
monitored at 240 nm. A molar absorptivity of 43.6 L mol-1
cm-1 was used to calculate the activity. One unit is equal
to 1 µmol of H2O2 decomposed/min.
Hb and Pr concentrations
. Hb and Pr concentrations in
the 1:20 hemolysates were determined spectrophotometrically on the
Cobas Mira by standard kits. The Hb assay (Sigma cat. no. 525-A) is
based on the colorimetric cyanomethemoglobin method, and the Pr assay
(Sigma cat. no. 541–2) is based on the colorimetric biuret method. The
specific enzyme activities were expressed as U/g Hb or U/g Pr.
Quality assurance
. For all enzymes, identical samples of
control material were analyzed repeatedly during each day to adjust for
analytical day-to-day variations.
statistics
Statistical analyses were performed by using the SPSS 6.0 software
(SPSS, Chicago, IL). Goodness of fit to normal distributions were
investigated by probit plots and the Lilliefors test. GSH-Px and CAT
values showed normal distributions. The logarithmic transformation of
CuZn-SOD and GR results also approached the normal distribution.
Multiple regression analysis with backward elimination was used to
investigate the influence of different variables on the enzyme
activities. The criterion for removing variables from the model was
chosen at P >0.10. Spearman's rank correlation coefficient
was used to investigate associations between enzyme activities.
Differences in means between groups were analyzed by the
independent-samples t-test. The effects of fasting and
eating on enzyme activities were analyzed by paired t-test.
Intervals for the enzyme activities were established by using the
program REFVAL 3.42. This program implements the methods for
statistical treatment of reference values recommended by the IFCC.
Two-tailed P values are given throughout.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Within-run CVs express the precision of the autoanalyzer.
Since CAT activity was measured by a manual assay, no within-run CV for
CAT is available. The within-run CVs obtained for the enzyme analysis
on the autoanalyzer by measuring 10 consecutive aliquots of a sample
were always <3%. The within-day variations ranged between 2.1% and
6.5% for the various enzyme analyses. The highest day-to-day
variations were seen for CuZn-SOD and GSH-Px. To minimize the influence
of analytical variations, the values measured were adjusted by using
the control samples as internal standards. Thus the day-to-day
variations for CuZn-SOD, GSH-Px, and GR were reduced considerably
(Table 1
). Within-day CVs for Hb and Pr determinations were <1% and
<2% and day-to-day CVs <3.5% and <2.5% respectively. No
storage-related changes in mean enzyme activities were observed for up
to 6 months at -80 °C. The enzyme activities in the control
hemolysates were therefore considered stable.
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The activities of the four antioxidative enzymes obtained in this study
are shown in Table 2
. The results of the statistical analysis were independent of
whether the enzyme activities were expressed per gram of Pr or gram of
Hb. All enzyme activities were independent of whether the study person
was fasting or not and whether the blood samples were collected into
tubes containing Na-heparin or K3-EDTA as
anticoagulant.
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The within-subject variations are shown in Table 3
. The between-subject variations for the 220 subjects in the
sample group were 11.1% for CuZn-SOD, 16.6% for GSH-Px, 12.3% for
GR, and 9.4% for CAT. Thus, the mean within-subject variation is
approximately half as large for all of these enzymes. The highest
within-subject variations for CuZn-SOD and GSH-Px were almost as large
as the between-subject variation.
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The covariates included in the regression model were age (years), gender (0 = men, 1 = women), smoking (0 = no, 1 = yes), tobacco consumption (g/day), alcohol (drinks/week), caffeine intake (cups of coffee/day; 1 cup of tea = 1/2 cup of coffee), and dietary supplements (0 = no, 1 = yes).
Use of drugs within the last month was reported by 83 individuals. Of these, 32 had used more than one drug. The drugs were classified according to WHO (11) and the most frequently used types of drugs were sex hormones including oral contraceptives (n = 28), analgesics (n = 26), nonsteroidal antiinflammatory drugs (n = 12), diuretics (n = 11), psycholeptics (n = 9), antiasthmatics (n = 8), antibacterials (n = 6), and calcium channel blockers (n = 5). Other drug types were used by less than five individuals. Drug treatment was not included in the regression model since most drug types were only used by a few individuals. In addition, it is not possible in this study to separate effects from drug treatment from effects of the disease on enzyme activities. When means of enzyme activities were compared between groups using specific drugs types and individuals using no drugs, a tendency towards higher GR activity was observed among users of analgesics (P = 0.06). Only groups consisting of 10 or more individuals were analyzed. No other drug-related effects on enzyme activities were observed.
The activity of CuZn-SOD significantly decreased with age (Table 4
, Fig. 1
). None of the other variables recorded had any significant
effect on the CuZn-SOD activity.
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In contrast, no age relation was found for GSH-Px. Daily intake of
dietary supplements was associated with an increased activity of
GSH-Px, whereas the amount of tobacco consumption was negatively
correlated with GSH-Px activity (Table 4
). GSH-Px activity was slightly
higher in women than in men (Table 4
; Fig. 1
). Women used dietary
supplements more frequently than men (43.6% and 27.5% respectively);
therefore the effects of dietary supplements and gender on GSH-Px
activity are difficult to separate. However, the tolerance was high
(0.97) and variance inflation factor low (1.03) for gender in the final
regression model, indicating separate influence on the variance from
the two variables. The mean activity of GSH-Px was significantly lower
in current smokers than in nonsmokers (P = 0.028).
However, some of this difference may be explained by a tendency toward
fewer users of dietary supplements among smokers and a higher level of
tobacco consumption among men than women (data not shown). Thus,
according to the regression analysis, smoking was not significantly
associated with GSH-Px activity when adjustment for other variables was
included (Table 4
).
The activity of GR decreased slightly with age (Table 4
; Fig. 1
) and
with smoking.
The activity of CAT was not appreciably influenced by any of these
variables. The CAT activity as a function of age for both sexes is
shown in Fig. 1
. Alcohol and caffeine intake were not associated with
any of the enzyme activities.
The activity of CuZn-SOD was positively correlated to CAT (r = 0.26; P <0.0001) and GR (r = 0.16; P = 0.02), and CAT was positively correlated to GR (r = 0.21; P = 0.002). No significant correlations were found between GSH-Px and any of the other enzymes measured.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Antioxidative enzyme activities vary among different tissues, and environmental factors might affect the enzyme activities only in susceptible organs. Therefore, the activities found in erythrocytes do not necessarily reflect the antioxidative defense of the whole organism. In this study, variations between individuals are generally substantially larger than within-subject variations, thus suggesting that the enzyme activity in a blood sample may reflect differences in the antioxidative defense.
Most previous studies were designed to compare activities of antioxidative enzymes in groups of patients with certain diseases with matched controls. Observed differences in enzyme activities in most of these studies could constitute a contributory cause of the disease, a result of the disease, or an effect of drug treatment. In addition, analytical differences between laboratories make it difficult to compare the results obtained in different studies. Although most laboratories use methods based on the same methodological principles, the specific assay conditions regarding concentration of reagents, recording time, and temperature differ.
A small number of other studies have addressed the biological variability in antioxidative enzymes (12)(13)(14). However, these studies are apparently not based on randomly selected population groups. In the present study, an age-related decrease was observed in CuZn-SOD and GR activities in erythrocytes. No obvious age-related changes were observed for CAT or GSH-Px, although GSH-Px tended to increase slightly with age. These results are in accordance with a French study (12) based on 167 individuals between 1 month and 67 years of age; a negative correlation between age and activities of CuZn-SOD and GR and a positive correlation between age and GSH-Px activity in erythrocytes were reported. In another French study (13) involving 1782 individuals between 4 and 65 years of age and 54 subjects between 65 and 97 years, the activities of CuZn-SOD, CAT, and GSH-Px in erythrocytes decreased with age, especially in individuals >65 years of age. GR was not investigated. An age-related decrease in CuZn-SOD activity in erythrocytes was also reported from a Polish population (n = 93) between 4 and 80 years of age, and the activities of CAT and GSH-Px increased with age (14). Hence, an age-related decrease in CuZn-SOD activity in human erythrocytes seems to be a rather consistent finding, whereas some discrepancy exists regarding the age relation of the other antioxidative enzymes. Some differences between the studies may be due to factors other than age, since random population samples were not used, and selection bias may have affected the results.
Of the enzymes investigated, GSH-Px seems to be most clearly influenced by life-style and environmental factors, as indicated in this study by intake of dietary supplements and smoking habits. In other studies, smoking habits did not influence GSH-Px activity in erythrocytes (13)(15), but a decreased activity of GSH-Px in plasma has been observed in smokers (16). The higher GSH-Px activity among users of dietary supplements is probably due to higher Se intake from supplements containing minerals (17). A significantly higher GSH-Px activity in women than in men has been found in some studies (13)(18) but not in others (12). In the present study, women tended to have higher GSH-Px activity, but the small difference could be due to chance or to gender-related differences in life-style such as intake of dietary supplements.
Measurement of antioxidative vitamins and other essential nutrients in human blood samples has been recommended to assess the optimal intake (19). However, the application of measurements of antioxidative factors has yet to be elucidated in greater detail. First, reliable methods to measure antioxidative enzymes as well as other antioxidants in human blood samples have to be established and standardized. In addition, the variation in antioxidant concentrations in the general population and the influence of age, gender, life-style, and dietary and environmental factors on these parameters have to be estimated before the role of antioxidants in disease prevention can be further investigated.
In conclusion, this study describes measurement conditions that are suitable and reliable for analyzing antioxidative enzymes in human erythrocytes with a high degree of reproducibility. The advantages of using automated assays include reduced analytical variation, considerable reductions in time, and smaller sample volumes, when compared with manual assays. Intervals for antioxidative enzyme activities in erythrocytes from a randomly selected population have been established. These results can be used in the process of defining reference intervals for the antioxidative enzymes. Depending on the intended use of these reference intervals, the influence of age, gender, state of health, and life-style factors on the enzyme activities should be considered.
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Acknowledgments |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, (- - - -), men; 
, (... . ), women;
(––), total
population.