Pathophysiology/Complications

O R I G I N A L

A R T I C L E

Plasma F2 Isoprostanes
Direct evidence of increased free radical damage during acute
hyperglycemia in type 2 diabetes
MICHAEL J. SAMPSON, MD1
NITIN GOPAUL, PHD2
ISABEL R. DAVIES, PHD3

DAVID A. HUGHES, PHD3

MARTIN J. CARRIER, PHD2

OBJECTIVE Acute hyperglycemia in type 2 diabetes increases the generation of plasma

8-epi-prostaglandin F2 (8-epi-PGF2) isoprostane, a sensitive direct marker of in vivo free radical
oxidative damage to membrane phospholipids.
RESEARCH DESIGN AND METHODS A total of 21 patients with type 2 diabetes
underwent an oral 75-g glucose tolerance test. Plasma 8-epi-PGF2 isoprostane concentrations
(by gas chromatography [GC]/mass spectrometry [MS]), intralymphocyte reduced-to-oxidized
glutathione ratios, and plasma total antioxidant capacity were measured at baseline and 90 min
after glucose loading.
RESULTS Plasma 8-epi-PGF2 isoprostane concentrations rose significantly (P 0. 010)
from 0.241 0.1 to 0.326 0.17 ng/l after 90 min. Intracellular oxidative balance and plasma
antioxidant capacity did not change in either group.
CONCLUSIONS Plasma concentrations of 8-epi-PGF2 isoprostane increase during
acute hyperglycemia in type 2 diabetes, providing direct evidence of free radicalmediated
oxidative damage and demonstrating a pathway for an association between acute rather than
fasting hyperglycemia and macrovascular risk in type 2 diabetes.
Diabetes Care 25:537541, 2002

cute hyperglycemia after a meal or

glucose load may be an independent predictor of vascular event
rates in type 2 diabetes or impaired glucose tolerance (1 4). One possible mechanism for such a relationship is increased
generation of reactive oxygen species during acute hyperglycemia, leading to acute
oxidative damage to the vascular endothelium, cell membranes, and lipoprotein
components (5).
It has proven difficult to detect increased plasma free radical generation in

human diabetes using, for example, electron spin resonance methods (6), and the
usefulness of other surrogate markers of
oxidative stress has been questioned (7).
The F2 isoprostanes appear to be the best
available marker of lipid peroxidation in
vivo (8). These stable eicosanoids are produced by the enzyme-independent free
radical oxidation of arachidonic acid in
membrane phospholipids and lipoproteins and are generated in conditions of
increased oxidative stress in animal and
human models (8 10). Plasma and urine

From the 1Bertram Diabetes Research Unit, Norfolk and Norwich University Hospital National Health
Service Trust, Norwich, U.K.; 2William Harvey Research Institute, St. Bartholomews Hospital, London, U.K.;
3
Institute of Food Research, Norwich Research Park, Colney, Norwich, U.K.
Address correspondence and reprint requests to Dr. M.J. Sampson, Department of Endocrinology and
Diabetes, Norfolk and Norwich University Hospital NHS Trust, Brunswick Rd., Norwich NR1 3SR, U.K.
E-mail: mike.sampson@norfolk-norwich.thenhs.com.
Received for publication 25 July 2001 and accepted in revised form 30 November 2001.
Abbreviations: ABTS, 2,2-azino-bis-3-ethylbensthiazoline-6-sulfonic acid; BHT, butylated hydroxytoluene; 8-epi-PGF2, 8-epi-prostaglandin F2; GC, gas chromatography; GSH, glutathione; MS, mass spectrometry; TAOS, total antioxidant status.
A table elsewhere in this issue shows conventional and Syste`me International (SI) units and conversion
factors for many substances.

DIABETES CARE, VOLUME 25, NUMBER 3, MARCH 2002

concentrations of F2 isoprostanes are increased in type 2 diabetes (11,12), in direct relationship to measures of chronic
hyperglycemia (12). It is unknown
whether acute hyperglycemia influences
F2 isoprostane generation in type 2 diabetes.
We hypothesized that acute hyperglycemia after a glucose load in people
with type 2 diabetes would lead to acutely
increased generation of plasma F2 isoprostanes, and that this would suggest
one possible pathway between acute hyperglycemia, associated free radical damage, and macrovascular disease in type 2
diabetes.
RESEARCH DESIGN AND
METHODS
Patient selection
After local ethical committee approval
and after obtaining written informed consent, we studied 21 patients with type 2
diabetes. Patients were recruited from the
Bertram Diabetes Center, Norwich, U.K.
(a secondary care diabetes facility), or
from local primary care services if they
were nonsmokers aged between 40 and
70 years and treated with diet or oral hypoglycemic agents alone. Patients were
considered to have type 2 diabetes if they
had been diagnosed after the age of 40
years, had no history of ketosis, and had
stable glycemic control on diet or oral hypoglycemic agents for at least 6 months.
Patients with clinical evidence of coronary artery disease (history of previous
myocardial infarction or angina) and
those receiving insulin were excluded. Patients with microalbuminuria (defined as
an elevated urine albumin-to-creatinine
ratio 2.5 for men and 3.5 for women)
or macroproteinuria (defined as albustixpositive proteinuria) were excluded. Patients omitted all oral hypoglycemic
agents or other medication on the morning of testing and fasted for 15 h before
glucose tolerance tests were undertaken.
All type 2 diabetic patients managed with
diet alone had a fasting venous plasma
537

F2 isoprostanes and acute hyperglycemia in type 2 diabetes

Table 1Baseline data
n
Age (years)
M/F
BMI (kg/m2)
Blood pressure
Diabetes treatment
Diet alone
Sulphonylurea
alone
Metformin
alone
Combination
Other treatment
ACE inhibitor
-blocker
Calcium
channel blocker
HbA1c (%)
Total cholesterol
(mmol/l)
LDL cholesterol
(mmol/l)
Triglycerides
(mmol/l)
HDL cholesterol
(mmol/l)

21
60.6 7.6
9/12
32.3 4.3
148 31/82 11
2
3
7
9
4
1
2
8.5 1.4
5.1 0.9
2.9 1.0
2.3 1.1
1.2 0.3

Data are means SD or n.

glucose 7.0 mmol/l. Clinical details are

shown in Table 1.
Glucose tolerance tests and glycemic
control
All patients underwent a single glucose
tolerance test (75 g oral anhydrous glucose) at 0800, with peripheral blood samples taken at 0, 60, 90, and 120 min from
an indwelling venous cannula. Patients
remained seated throughout the glucose
tolerance test. HbA1c was assessed using a
commercially available kit (Roche, Welwyn, U.K.) on an automated biochemistry
analyzer (Cobas Mira; Roche), with the
normal range quoted as 4.55.7%.
Intracellular oxidative balance
The intracellular glutathione (GSH) redox
cycle between reduced GSH and oxidized
GSH is an effective mechanism in protecting against intracellular oxidative damage
because GSH acts as substrate for antioxidant enzymes and as a free radical scavenger, and the ratio of reduced-tooxidized GSH by mass can be used as an
index of intracellular oxidative stress
(13). A 15-ml sample of whole blood was
collected into EDTA, diluted to twice the
538

original volume with PBS, and layered

onto a 15 ml lymphocyte separation medium (ICN Biomedicals, Basingstoke,
UK). It was then centrifuged at 390g for
30 min at 20C. The lymphocyte layer
was removed and washed twice with PBS,
and the cell pellet was resuspended and
an aliquot removed for cell counting.
GSH was extracted from the lymphocyte
pellet and measured using an enzymatic
recycling method (14) modified for an automated biochemistry analyzer (Cobas
Mira). This ratio was measured at 0 and
90 min during each glucose tolerance test
and expressed as a ratio of reduced to oxidized GSH. The 90-min time point was
chosen as being peak hyperglycemia and,
by extension, the time point most likely to
demonstrate changes in hyperglycemiaassociated oxidative stress
Plasma F2 isoprostane
concentrations
The determination of 8-epi-prostaglandin
F2 (8-epi-PGF 2) was based on previously described methodology (11,15).
Peripheral venous blood (10 ml) was collected into polyethylene tubes containing
a 3.8% (wt/vol) trisodium citrate solution
(blood-to-anticoagulant ratio of 9:1) with
indomethacin (as a cyclooxygenase inhibitor) and butylated hydroxytoluene (BHT;
as a free radical scavenger) at final concentrations of 14 and 20 mol/l, respectively. The sample was allowed to stand
for 30 min at 4C to enable complete inhibition of cyclooxygenase enzymes.
Platelet-poor plasma was obtained by
centrifugation at 1,120g for 15 min at
4C. The plasma was transferred to a
polypropylene screw-cap tube, and BHT
was added at a final concentration of 20
mol/l. The sample was then stored at
80C until analysis. For determination
of plasma total antioxidant status (TAOS),
blood (5 ml) was collected in EDTA tubes
and centrifuged at 1,120g for 15 min at
4C. The plasma was transferred to screwcap polypropylene tubes and stored at
80C until analysis. Plasma samples
were subjected to alkaline hydrolysis for
the measurement of total (sum of free plus
esterified) 8-epi-PGF 2 by GC/MS.
Plasma (0.5 ml) was transferred to a glass
tube, followed by the addition of 8-epiPGF2-d4 as an internal standard (2 ng in
20 ml ethanol). The sample was hydrolyzed with 1.0 mol/l aqueous potassium
hydroxide (0.5 ml) for 30 min at 40C.
Hydrolysis was terminated by the addi-

tion of 0.1 mol/l hydrochloric acid (4.25

ml), and the pH of the sample was adjusted to 7.4 using 0.05 mol/l sodium
phosphate buffer (4.5 ml). Isolation of
8-epi-PGF was carried out by immunoaffinity extraction of the hydrolyzed
plasma. Samples were loaded on an immunoaffinity cartridge (prepared with an
anti 8-epi-PGF2 antiserum) preconditioned with 16 ml sodium phosphate
buffer (0.05 mol/l, pH 7.4). The cartridge
was washed with water (20 ml) to remove
nonretained components, and 8-epiPGF2 was eluted using an acetone and water mixture (95:5 dilution, 5.5 ml). The
immunoaffinity extraction steps were
programmed into an Aspec XL sample
processor (Gilson Medical Electronics,
Villiers-le-Bel, France) and run automatically. The final eluate from the immunoaffinity extraction was dried under
nitrogen and the sample converted to a
perfluorobutyl/trimethylsilyl (PFB/TMS)
derivative. Samples were analyzed by GC/
negative ion chemical ionization/MS using an Autosystem XL GC coupled to a
TurboMass MS (Perkin-Elmer, Beaconsfield, U.K.), with ammonia as reagent
gas. The GC/MS assay has a limit of detection of 10 pg/ml (28 pmol/l) in plasma,
with an intra- and interassay coefficient of
variation of 4.4 and 7.6%, respectively.
Analysis was performed using selected
ion recording of the carboxylate anion
[M-181] at m/z 569 for 8-epi-PGF2 and
m/z 573 for 8-epi-PGF-d4. Quantitative
determination was based on the peak
height ratio of 8-epi-PGF2 against the internal standard. The isolation of 8-epiPGF 2 from plasma is based on the
specific interaction between 8-epi-PGF2
and polyclonal anti 8-epi-PGF2 antibodies, prepared by raising antisera
against 8-epi-PGF2. After derivatization
of the immunoextracted material, quantitation of 8-epi-PGF2 was carried out by
stable-isotope dilution GC/MS with selected ion recording. The measurement of
8-epi-PGF2 provides a direct index of
lipid peroxidation on phospholipid membranes in vivo and reflects oxidative processes within this environment. Oxidative
stress due to hyperglycemia is associated
with the increased generation of oxygenderived radicals, and it is this change in
free radicalmediated oxidation that is
measured through the analysis of F2isoprostanes. The nature of this assay
makes it exceptionally unlikely (if not impossible) that direct interference by
DIABETES CARE, VOLUME 25, NUMBER 3, MARCH 2002

Sampson and Associates

Table 2Changes in oxidative balance and plasma 8-epi-F2 isoprostane during a glucose
tolerance test in 21 subjects with type 2 diabetes
Time (min)

Plasma glucose (mmol/l)

Reduced-to-oxidized GSH ratio
TAOS (%)
Plasma F2 isoprostane (ng/l)
Total cholesterol (mmol/l)
LDL cholesterol (mmol/l)
Triglycerides (mmol/l)
HDL cholesterol (mmol/l)

0
10.7 2.8
12.2 9.1
52.9 15.5
0.241 0.1
5.1 0.9
2.9 1.1
2.3 1.1
1.2 0.3

60
19.9 3.8

90
21.1 3.6
14.1 8.3
50.1 14.9
0.326 0.17*
5.6 1.1
3.1 1.1
1.7 0.6
1.3 0.3

120
19.6 3.8

Data are means 1 SD. *P 0.0102.

plasma glucose or insulin concentrations

in vivo can occur; indeed, these types of
assays are particularly valuable simply because they are not subject to interference
from other constituents present in the assay mix. Plasma concentrations of 8-epiPGF 2 can be expressed in absolute
amounts or normalized to plasma total arachidonic acid. Normalized concentrations
of 8-epi-PGF2 are useful in situations
where significant changes in the lipid profile or plasma fatty acids are expected, and
they are unlikely to provide further information where intervention is limited to an
isolated oral glucose load because even a
high-calorie/highsaturated fat meal does
not influence postprandial plasma total
arachidonic levels (16).
TAOS plasma assay
The total antioxidant status of plasma was
determined by its capacity to inhibit the
peroxidase-mediated formation of the
2,2-azino-bis-3-ethylbensthiazoline-6sulfonic acid (ABTS) radical. In this assay, the relative inhibition of ABTS
formation in the presence of plasma is
proportional to the antioxidant capacity
of the sample. Briefly, plasma (2.5 l) was
incubated for 3 min at 37C in a 96-well
plate with a reaction mixture made up of
(final concentrations) 20 l ABTS (20
mmol/l), 20 l horseradish peroxidase
(30 mU/ml), and 37.5 l PBS (pH 7.4).
The reaction was started by the addition
of 20 ml hydrogen peroxide (final concentration 0.1 mmol/l), and the increase
in absorbance over 6 min was monitored
at 405 nm, using a microplate reader
(model 12605; Anthos Labtech, Salzburg,
Austria). At the end of 6 min, the absorbance due to the accumulation of ABTS
in the test sample was read along with a
DIABETES CARE, VOLUME 25, NUMBER 3, MARCH 2002

control (containing 2.5 l PBS instead of

plasma). The difference in absorbance
(control absorbance minus test absorbance), divided by the control absorbance
(expressed as a percentage) was used to
represent the percentage inhibition of the
reaction.
Measurement of lipid fractions
Plasma lipid profiles were measured at
time 0 and 90 min during each glucose
tolerance test. Lipid profiles were assessed
using commercially available kits (Roche)
on an automated biochemistry analyzer
(Cobas Mira; Roche), with estimation of
LDL cholesterol (17).
Statistical analysis
Data are shown as the means 1 SD, and
all variables were normally distributed.
Differences in individual variables measured more than twice during glucose tolerance test were analyzed by repeatedmeasure one-way ANOVA, with paired t
tests where a significant difference (P
0.05) was found. Otherwise, paired t tests
were used for paired measurements. Relationships between variables were analyzed by simple linear regression or
stepped multiple regression analysis with
entry at P 0.1. Data were analyzed using Apple Macintosh Statview software
(1996).
RESULTS
Clinical details
Clinical details are shown in Table 1. Of
the diabetes group, 17 (81%) were taking
metformin or a metformin-sulfonylurea
combination. Seven patients (33%) were
taking gliclazide, either alone or in combination with metformin, and seven

(33%) were taking antihypertensive medication (Tables 1 and 2).

Oxidative balance during glucose
tolerance test
There were no significant changes in the
intracellular oxidative balance measured
by reduced-to-oxidized GSH ratio or in
the plasma total antioxidant status (P
0.1) (Table 2).
Plasma 8-epi-F2 isoprostane
concentrations in type 2 diabetes
There was a highly significant (P
0.0102) rise in plasma 8-epi-F2 isoprostane concentrations between baseline and
90 min. There were no significant relationships between baseline plasma 8-epiF 2 isoprostane concentrations and
HbA1c (r 0.32, P 0.15), fasting
plasma glucose (r 0.33, P 0.13), or
measures of intracellular oxidative balance and total antioxidant capacity (both
P 0.2). Peak plasma 8-epi-F2 isoprostane concentrations were directly related
only to TAOS at 90 min (r 0.495, P
0.025). Stepwise multiple regression
demonstrated that only TAOS at 90 min
was independently and inversely related
to peak plasma 8-epi-F2 isoprostane (R2
0.248, P 0.025). Finally, the only
variable independently inversely related
to the absolute change in plasma 8-epiF2 isoprostane concentrations during
the glucose tolerance test was TAOS at 90
min (r 0.45, P 0.041), and when
the upper tertile of the 8-epi-F2 isoprostane increment was compared with the
lower tertile, only TAOS at 90 min differed between tertiles (47.8 10.4 vs.
62.9 15.7%, respectively; P 0.023)
(Table 2).
CONCLUSIONS The main finding of this study is that acute hyperglycemia after a glucose load in type 2 diabetes
is associated with an acute increase in
plasma concentrations of 8-epi-F2 isoprostane. This must indicate increased
free radicalmediated generation of these
compounds from arachidonic acid in
membrane and lipoprotein phospholipids (8 12). This provides sensitive and
direct evidence for a link between acute
rather than chronic hyperglycemia and
free radical damage in type 2 diabetes.
Changes in oxidative balance, or in
antioxidant defenses, during acute hyperglycemia have been demonstrated before
in subjects with and without type 2 dia539

F2 isoprostanes and acute hyperglycemia in type 2 diabetes

betes (18 21). Increased LDL oxidizability induced by copper ions (22,23) has
also been reported postprandially in type
2 diabetes, although the difficulties with
this methodology have been reviewed (7),
as has the confounding effect of dietary
oxidized lipids (24). However, these measurements are surrogates, and the present
study demonstrates directly increased
plasma levels of a free radical generated
oxidation product of membrane arachidonic acid during acute hyperglycemia,
independent of dietary intake of oxidized
lipids or dietary isoprostane intake (25).
Increased generation of reactive oxygen
species is a feature of hyperglycemia in
type 2 diabetes and impaired glucose tolerance (5,26), and the increased free radical damage during acute hyperglycemia
demonstrated in this study occurred
without significant changes in intracellular oxidative balance or plasma antioxidant capacity. That plasma total
antioxidant capacity was the sole independent determinant of the increase in
8-epi-F2 isoprostane may suggest that
acute increases in free radical generation
during hyperglycemia do not influence
TAOS, but that it is limited by the variety
of antioxidant defenses contributing to
the TAOS measurements. It is also possible that the TAOS and GSH assays are less
sensitive for detecting changes in oxidative balance or stress compared with the
8-epi-F2 isoprostane assay used here, or
that changes in TAOS and intracellular
oxidative balance occurred before the 90min sampling point used in this study.
Plasma F2 isoprostane concentrations
increased by 34% during acute hyperglycemia, and this is similar to observations
in other models of increased oxidative
damage. For example, Morrow et al. (9)
demonstrated increased plasma esterified
8-epi-F2 isoprostane in heavy smokers,
and smoking cessation led to a 24% reduction in mean 8-epi-F2 isoprostane
concentration within a few weeks. 8-EpiF2 isoprostane may also have biologically important proatherogenic actions, as
well as being a marker for free radical
damage. In vitro, 8-epi-F2 isoprostane at
physiological concentrations promotes
increased message and protein for endothelin-1 (27), promotes platelet adhesion
to collagen in a dose-dependent manner
(28), and antagonizes some actions of nitric oxide (29). Also, increased levels of
8-epi-F2 isoprostane are detectable in
human coronary atherosclerotic plaque
540

(29), particularly in smokers with increased oxidative damage rather than patients with treated hypertension or
dyslipidemia (30), suggesting that 8-epiF2 isoprostane generation may occur
within coronary plaque.
Metanalysis of available epidemiological and prospective studies has shown a
consistent direct relationship between
blood glucose levels and predominantly
cardiovascular mortality (31) in type 2 diabetes, but some data suggest postprandial hyperglycemia may be an
independent predictor of cardiovascular
mortality in type 2 diabetes (13), and
surrogates for macrovascular disease,
such as carotid intimal-medial thickness,
are more closely related to acute rather
chronic hyperglycemia (32), and much of
the controversy over the diagnostic classification of diabetes was based on the
predictive power of post glucose load
hyperglycemia as a marker for increased
vascular risk (33). The present data suggest one possible pathway for this association, by demonstrating increased free
radical damage during acute hyperglycemia. This could promote an increase in
vascular event rates through some of the
mechanisms outlined above, or through
proatherogenic processes sensitive to reactive oxygen species, such as increased
adhesion molecule expression or coronary plaque metalloproteinase expression
(34,35).
The present study demonstrates that
acute hyperglycemia in type 2 diabetes is
associated with a significant increase in
free radicalmediated damage to membrane components, measured by plasma
8-epi-F2 isoprostane concentrations,
and this may be a link between acute hyperglycemia, increased free radical damage, and macrovascular risk in type 2
diabetes.
Acknowledgments We are grateful to Drs.
Greenwood, Heyburn, and Temple for allowing access to their patients, and to the Norwich
and Norfolk Diabetes Trust for funding salary
costs associated with this project.

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