Oxidative stress and high-density lipoprotein function in Type I diabetes and end-stage renal disease.

Abstract

In a cross-sectional study, oxidative stress in high vascular disease risk groups, ESRD (end-stage renal disease) and Type I diabetes, was assessed by measuring plasma protein carbonyls and comparing antioxidant capacity of HDL (high-density lipoprotein) as pertaining to PON1 (paraoxonase 1) activity and in vitro removal of LPO (lipid peroxides). ESRD subjects on haemodialysis (n=22), Type I diabetes subjects (n=20) without vascular complications and healthy subjects (n=23) were compared. Plasma protein carbonyls were higher in ESRD patients [0.16 (0.050) nmol/mg of protein; P=0.001; value is mean (SD)] relative to subjects with Type I diabetes [0.099 (0.014) nmol/mg of protein] and healthy subjects [0.093 (0.014) nmol/mg of protein]. Plasma PON1 activity, with and without correction for HDL-cholesterol, was lower in diabetes but did not differ in ESRD compared with healthy subjects. Plasma PON1 activity, without correction for HDL, did not differ between the three groups. In ESRD, plasma PON1 activity and plasma protein carbonyl concentrations were inversely related (r=-0.50, P<0.05). In an in vitro assay, LPO removal by HDL in ESRD subjects was greater than HDL from healthy subjects (P<0.01), whereas HDL from patients with Type I diabetes was less effective (P<0.01). Efficacy of LPO removal was unrelated to plasma PON1 activity, in vitro glycation or mild oxidation, but was impaired by marked oxidation and glycoxidation. Protein carbonyl levels are increased in ESRD but not in complication-free Type I diabetes. HDL antioxidant function is increased in ESRD, perhaps a compensatory response to increased oxidative stress, but is lower in Type I diabetes. HDL dysfunction is related to glycoxidation rather than glycation or PON1 activity.

Full text

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Clinical Science (2005) 108, 497–506 (Printed in Great Britain)
497
Oxidative stress and high-density lipoprotein
function in Type I diabetes and end-stage
renal disease
George KALOGERAKIS∗1, Arthur M. BAKER∗1, Steve CHRISTOV∗,
Kevin G. ROWLEY∗, Karen DWYER∗, Christine WINTERBOURN†,
James D. BEST∗and Alicia J. JENKINS∗
∗Department of Medicine, The University of Melbourne, St. Vincent’s Hospital, Fitzroy, 3065, Melbourne, Australia,
and †Christchurch School of Medicine and Health Sciences, Department of Pathology, Free Radical Research Group,
Christchurch Hospital, Christchurch, New Zealand
ABSTRACT
In a cross-sectional study, oxidative stress in high vascular disease risk groups, ESRD (end-
stage renal disease) and Type I diabetes, was assessed by measuring plasma protein carbonyls
and comparing antioxidant capacity of HDL (high-density lipoprotein) as pertaining to PON1
(paraoxonase 1) activity and in vitro removal of LPO (lipid peroxides). ESRD subjects on
haemodialysis(n=22),TypeIdiabetessubjects(n=20)withoutvascularcomplicationsandhealthy
subjects (n=23) were compared. Plasma protein carbonyls were higher in ESRD patients [0.16
(0.050) nmol/mg of protein; P=0.001; value is mean (SD)] relative to subjects with Type I diabetes
[0.099 (0.014) nmol/mg of protein] and healthy subjects[0.093 (0.014) nmol/mgof protein]. Plasma
PON1 activity, with and without correction for HDL-cholesterol, was lower in diabetes but did
not differ in ESRD compared with healthy subjects. Plasma PON1 activity, without correction
for HDL, did not differ between the three groups. In ESRD, plasma PON1 activity and plasma
protein carbonyl concentrations were inversely related (r=−0.50, P<0.05). In an in vitro assay,
LPO removal by HDL in ESRD subjects was greater than HDL from healthy subjects (P<0.01),
whereas HDL from patients with Type I diabetes was less effective (P<0.01). Efficacy of LPO
removal was unrelated to plasma PON1 activity, in vitro glycation or mild oxidation, but was
impaired by marked oxidation and glycoxidation. Protein carbonyl levels are increased in ESRD but
not in complication-free Type I diabetes. HDL antioxidant function is increased in ESRD, perhaps
a compensatory response to increased oxidative stress, but is lower in Type I diabetes. HDL
dysfunction is related to glycoxidation rather than glycation or PON1 activity.
INTRODUCTION
Cardiovascular morbidity and mortality is increased in
people with Type I diabetes [1,2] and also inpatients with
ESRD (end-stage renal disease) [3,4]. One proposed
mechanism for the increased cardiovascular disease risk
in these two conditions is increased oxidative stress. In
diabetes, this may be mediated through carbonyl stress,
Key words: end-stage renal disease, high-density lipoprotein, lipid peroxide, oxidative stress, paraoxonase 1, protein carbonyl,
Type I diabetes.
Abbreviations: AGE, advanced glycation end-product; BHT, butylated hydroxytoluene; CHD, coronary heart disease;
CV, coefficient of variation; DNP, 2,4-dinitrophenylhydrazine; ESRD, end-stage renal disease; FOX2, ferrous oxidation of
Xylenol Orange (version 2); HbA1C, glycated haemoglobin; HDL, high-density lipoprotein; LDL, low-density lipoprotein;
LPO, lipid peroxide; PAF, platelet-activating factor; PAF-AH, PAF-acetyl hydrolase; PON1, paraoxonase 1; RBC, red blood cell;
TPP, triphenylphosphine.
1These authors contributed equally.
Correspondence: Dr Alicia Jenkins (email jenkinsa@medstv.unimelb.edu.au).
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G. Kalogerakis and others
accelerating glycoxidation and lipid peroxidation, re-
sulting in AGEs (advanced glycation end-products) [5].
Carbonylgroups(aldehydesorketones)areproducedon
proteins as a result of oxidation of amino acid residues or
by covalent attachment of aldehydes, such as 4-hydroxy-
nonenal[5].However,thepresenceofincreasedoxidative
stress in human diabetes is controversial due to the lack
of conclusive evidence from studies using well-validated
markers.InESRD,thereisclearerevidencethatoxidative
stress, mediated by carbonyl formation, is a major con-
tributor to the atherosclerotic process [6,7]. To establish
whether plasma oxidative stress is increased in these
two conditions, an assay of protein carbonyl levels was
utilized.
One of the main defences against atherosclerosis is
HDL (high-density lipoprotein). As well as its role in
reverse cholesterol transport, HDL has an array of anti-
oxidant mechanisms which may prevent the formation
and promote removal of LPOs (lipid peroxides) from
other lipoproteins [e.g. the pro-atherogenic LDL (low-
density lipoprotein)] and from cell membranes [8–10].
The HDL-associated antioxidant enzyme PON1 (para-
oxonase 1) is believed to be important in retarding
atherosclerosisandhasawiderangeofactivitydependent
on genotype and phenotype factors [11–13]. To examine
further the importance of oxidative stress in groups with
a high risk of vascular disease, a bioassay was used in the
present study to investigate whether the LPO-removing
efficacy of HDL was altered in ESRD and Type I dia-
betes, and the relationships between HDL function,
serum HDL-cholesterol levels and PON1 activity were
evaluated.
METHODS
Subjects
For the protein carbonyl study, subjects with Type I
diabetes (n=20) were recruited from the Diabetes Out-
patient Clinics, and subjects with ESRD (n=22) were
from the Haemodialysis Centre of St Vincent’s Hos-
pital, Melbourne, Australia. Healthy control subjects
(n=23) were chosen from a group of volunteers and
hospital staff completing a health screening survey ques-
tionnaire. For the HDL function study, subjects were re-
cruitedbythesamemethodandconsistedofcontrolsub-
jects (n=14), subjects with Type I diabetes (n=15)
and subjects with ESRD (n=14). Three additional
healthy subjects with low HDL levels were also recruited
for further analysis of observations made during the
study.
This study was approved by the Human Research
Ethics Committee of St Vincent’s Hospital, Melbourne,
and each participant gave written informed consent.
Blood (50–75 ml) was taken after an overnight fast
(during which water was permitted) and prior to pre-
scribed medication. Biochemical characterization tests
(performed by the St. Vincent’s Hospital Clinical Chemi-
stry Department) included fasting lipids, renal and liver
function tests, glucose and HbA1C (glycated haemo-
globin). Plasma was prepared by centrifugation (2278 g
at 4◦C for 20 min) and aliquots of plasma and serum
were frozen at −86◦C until analysis of plasma protein
carbonyls and serum PON1 activity. Lipoproteins were
prepared from fresh plasma on the day of collection,
and the LPO removal assay performed the next day (as
described below).
Plasma protein carbonyl measurement
Plasma protein carbonyls were determined by ELISA,
using a method described previously [14,15], involving
derivatization with DNP (2,4-dinitrophenylhydrazine)
and detection with an anti-DNP-biotinylate conjugate,
followed by streptavidin/biotin–horseradish peroxidase.
Intra- and inter-assay CV (coefficients of variation) were
8.0 and 4.5% respectively, at a standard concentration
in healthy control subjects. The limit of detection was
0.05 nmol/mg of protein.
RBC (red blood cell) membrane
preparation
Human RBC ghosts were prepared by a modification of
the method of Dodge and Phillips [16]. Briefly, centri-
fugation (1468 g at 4◦C for 20 min) was used to separate
the RBC pellet from the blood of fasted healthy sub-
jects. The RBC pellet was washed in buffer [10 mmol/l
Na2HPO4 and 300 mmol/l NaCl, pH 8.0] three times
(749 g at 4◦C for 25 min) and a final time in the same
buffer at 15849 g for 10 min. After washing, RBCs were
lysed with the same buffer as above, but without NaCl.
The lysed RBCs were washed three times (15849 g for
25 min) with this buffer. Repeated spins (41015 g
for 25 min) of the lysed RBCs were performed until the
RBC membrane pellet was washed free of haemoglobin
and was visibly white. The RBC membranes were then
washed twice (41015 g for 15 min) in PBS. All centri-
fugations were carried out at 4◦C. The protein concen-
trationoftheRBCmembraneswasassessedbythemodi-
fied method of Lowry et al. [17], and diluted to a final
concentrationof1 mg/ml.Thispreparationwasthenoxi-
dized in a UV-crosslinker (Fisher Biotek, Spectroline,
Westbury, NY, U.S.A.) for 90 min at the unit’s standard
level of intensity. Protein concentrations were assessed
again, and both oxidized and unoxidized samples were
aliquoted (250 µg) and stored at −86◦C. Preliminary
studies (results not shown) demonstrated the stability of
oxidized RBC membranes prepared and stored in this
manner.
Lipoprotein preparation
Whole blood was collected into lithium-heparin tubes
and HDL and LDL were isolated. Plasma density was
adjusted to 1.3 g/ml with KBr and overlayed with
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Oxidative stress and HDL function in Type I diabetes and end-stage renal disease
499
d=1.006 g/ml PBS buffer, followed by vertical spin
ultracentrifugation using a Beckman ultracentrifuge
(401747 g at 4◦C for 90 min; VTi65.1 rotor). To preserve
PON1activity,allbuffersusedinlipoproteinpreparation
and the LPO removal assay were free of EDTA and con-
tained 1 mmol/l CaCl2. For the patient study, HDL and
LDL samples were buffer-exchanged into PBS (1 mmol/l
CaCl2) by size-exclusion chromatography using PD-
10 columns (Amersham Biosciences, Uppsala, Sweden).
For the in vitro-modified lipoprotein studies, HDL and
LDL samples were buffer-exchanged into PBS (1 mmol/l
CaCl2) using 100000 MrCentricons (Millipore, Bedford,
CA, U.S.A.). Samples from both protocols were then
concentrated using Centricons. Protein concentrations
were assessed by the modified method of Lowry et al.
[17]. Lipoproteins were stored overnight under nitrogen
in the dark at 4◦C.
Lipoprotein modification and
characterization
Glycation of HDL was achieved by incubating HDL
(4.35 mg/mlofprotein)ina50mmol/ld-glucosesolution
with BHT (butylated hydroxytoluene; 80 mmol/l) under
nitrogen for 4 days at 37◦C. Glycoxidation of HDL
(glycationandoxidation)wasachievedwiththeomission
of BHT and nitrogen under the same conditions de-
scribed above. Mildly oxidized HDL was produced by
incubating8 mlofHDL(1.0 mgofprotein/ml)inatissue
culture plate (Corning, Acton, MA, U.S.A.) under air
in a 37◦C incubator for 22 h. A UV crosslinker (Fisher
Biotek) was used to generate UV-oxidized HDL by
oxidizing 1.0 ml of 1.0 mg of protein/ml HDL for 45 min
at the unit’s standard intensity level. Loss of coloration
of HDL preparations was noted on UV oxidation, in
keeping with loss of the lipid-soluble antioxidant caro-
tenoids. Inactivation of PON1 on HDL was achieved by
placing HDL under nitrogen for 20 min in a water bath
preheatedto60◦C.NativeHDLwaskeptundernitrogen
in the dark at 4◦C. The extent of HDL oxidation for
each condition was quantified by absorbance at 234 nm,
FOX2 [ferrous oxidation of Xylenol Orange (version 2)]
assay, fluorescence (excitation 360 nm/emission 430 nm)
and RF(ratio of electrophoretic mobility). Lipoprotein
preparations were electrophoresed on native agarose gels
(HelenaLaboratories,Beaumont,TX,U.S.A.)toconfirm
lipoprotein purity and to assess the extent of oxidative
modification, based on electrophoretic mobility relative
to native lipoproteins.
PON1 and arylesterase activities
PON1 activity was determined by the rate of hydrolysis
of paraoxon and of phenylacetate as described previously
[18,19].ConsentforPON1genotypingwasnotobtained.
We demonstrated that PON1 activity does not differ
between serum and plasma prepared from lithium-
heparintubes(resultsnotshown).Themethodusedisnot
a salt-stimulated assay. Before analysis of PON1 activity,
sampleswerefirstincubatedwitheserine(finalconcentra-
tion, 5 µmol/l) for 10 min at room temperature to reduce
butyrylcholinesterase activity, which may be increased
in diabetes. PON1 activity was measured by adding
100 µlofassaybuffer[100 mmol/lTris/HCl(pH 8.0)and
2 mmol/l CaCl2] to wells of a 96-well plate followed by
6 µl of each (serum or lipoprotein) sample. A portion
(100 µl)oftheworkingparaoxonsolution(2 mmol/l)was
added to each well, and the rate of change of absorbance
(units/min), which represents p-nitrophenol production
(molar absorption coefficient=18053 M−1·cm−1), was
determined using a plate reader (Biotek Instruments,
Winooski, VT, U.S.A.) at A412at 19 s intervals for 6 min
at 25◦C. The units (units/l) represent the rate of p-nitro-
phenol production (nmol) per min per litre of plasma.
Intra- and inter-assay CVs were 4.6% and 6.8% respect-
ively. Arylesterase activity was assessed by incubating
2 ml of assay buffer [30 mmol/l Tris/HCl (pH 8.0) and
1.5 mmol/l CaCl2] with 5 µl of sample at 25◦C for
5 min. A portion (1 ml) of phenylacetate (20 mmol/l)
was then added. Reaction tubes were quickly vortexed,
and 300 µl from each tube was transferred to a UV 96-
well plate (Costar, New York, NY, U.S.A.), and the
rate of hydrolysis of phenylacetate production (molar
absorption coefficient=1310 M−1·cm−1) was read on a
plate reader at A270at 19 s intervals for 3 min at 25◦C.
The units (units/ml) represent the rate of conversion of
phenylacetate (µmol) per min per ml of plasma. Intra-
and inter-assay CVs were 2.7% and 3.7% respectively.
For the isolated HDL PON1, the units were calculated
as above for each substrate, but instead of being unit/litre
of plasma were units/µg of HDL protein.
Lipoprotein incubation with oxidized
RBC membranes
Frozensamples(250 µg)ofbothoxidizedandunoxidized
RBC membranes were thawed to room temperature.
Lipoprotein samples (500 µg) were then added to the
tubes containing oxidized RBC membranes. Tubes con-
taining oxidized RBC membranes with no added lipo-
proteins were used to establish a baseline value of the
amount of lipid hydroperoxides present, with that in
the oxidized preparation being taken as 100%. Levels
in unoxidized RBC membrane preparations were at low
or undetectable levels. Samples were mixed by gentle
inversion,andwereplacedinthedarkina37◦Cincubator
for 2 h. Each condition was evaluated in triplicate. Four
subjects were studied in each run, which always included
a control subject. Intra- and inter-assay CVs were 5.7%
and 3.0% respectively.
Assessment of LPO removal by HDL from
oxidized RBC membranes
Samples were removed from the 37◦C incubator after
2 h, and lipid hydroperoxide content was assessed using
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G. Kalogerakis and others
a modified version of the FOX2 assay as described
by Nourooz-Zadeh et al. [20] and Deiana et al. [21].
Briefly,180 µlfromeachsamplewasremovedanddivided
equally between two microfuge tubes. A portion [10 µl
of 100 mmol/l TPP (triphenylphosphine)] was added
to one tube, and 10 µl of methanol added to the other to
create a control for each sample. All tubes were vortexed
and incubated in the dark (37◦C for 2 h). FOX2 reagent
(900 µl) was added to each sample, tubes were vortexed
andincubatedfor30 mininthedarkatroomtemperature.
Samples were then centrifuged at 9940 g for 5 min.
Supernatantsweretransferredtoa96-wellplate(NUNC,
Roskilde, Denmark), and A560was read. The difference
betweenthe‘noTPP’sampleand‘TPP’samplewascalcu-
lated as the amount of authentic hydroperoxides present.
Statistics
All variables are expressed as means (S.D.), unless indi-
cated otherwise. For comparisons between the three
groups, one-way ANOVA was used. When appropriate,
a post-hoc test comparing groups was performed by the
Bonferronimethod.AllanalysesincludedtheRyanJoiner
normality test using standardized residuals. For any
groups of data not normally distributed, a logarithmic
transformation was performed and the normality test re-
applied. Correlations between variables were calculated
by the Pearson correlation coefficient (r). Prevalence of
PON1 phenotype categories across patient groups was
tested by χ2analysis. P<0.05 was taken as statistically
significant.
RESULTS
Clinical and biochemical parameters
Clinical and biochemical parameters of the three study
groups are shown in Table 1. Overall there was a signi-
ficant difference in the age of the Type I diabetic and
ESRD subjects compared with controls. There was no
correlation between age and the key variables of the
study, i.e. protein carbonyls, PON1 activity and HDL
LPO removal. None of the Type I diabetic subjects had
microvascular or macrovascular complications. Three of
theESRDsubjectsweresmokers,sixhadTypeIIdiabetes
andeighthadCHD(coronaryheartdisease).Exclusionof
any or all of these subjects did not affect the comparative
significance of any of the variables measured. All of
the ESRD subjects, but none of the Type I diabetic or
control subjects, were receiving vitamin E and vitamin C
supplementation as part of their routine management.
Asexpected,TypeIdiabeticsubjectshadhigherfasting
glucose and HbA1C levels than controls. The overall
glycaemic control in the Type I diabetic group was good
[7.8 (1.2)%], with the highest HbA1Cbeing 9.9% and
the lowest 5.7%. Glucose and HbA1Cwere also higher
in the ESRD group, which included six subjects with
Table 1
subjects and subjects with Type I diabetes or ESRD in the
plasma protein carbonyl study and the LPO removal by HDL
study
Values are means (S.D.).
‡P <0.05 compared with Type I diabetes; ?P <0.01 compared with ESRD.
Control
Demographic and biochemical features of control
∗P <0.05, †P <0.01 compared with control;
Type I diabetes ESRD
Plasma protein carbonyl study
n (female/male)
Age (years)
BMI (kg/m2)
Total cholesterol (mmol/l)
LDL-cholesterol (mmol/l)
HDL-cholesterol (mmol/l)
Triacylglyerols (mmol/l)
Albumin (g/l)
Total protein (g/l)
Bilirubin (µmol/l)
Creatinine (mmol/l)
Urea (mmol/l)
Fasting glucose (mmol/l)
HbA1C(%)
LPO removal by HDL study
n (female/male)
Age (years)
BMI (kg/m2)
Total cholesterol (mmol/l)
LDL-cholesterol (mmol/l)
HDL-cholesterol (mmol/l)
Triacylglyerols (mmol/l)
Creatinine (mmol/l)
Urea (mmol/l)
Fasting glucose (mmol/l)
HbA1C(%)
11/12
42.5 (11.3)
24.8 (4.4)
5.6 (0.80)
3.6 (0.72)
1.5 (0.40)
1.1 (0.40)
44 (4.0)
76 (5.2)
13 (7.3)
0.09 (0.01)
5.6 (1.3)
5.0 (0.68)
4.9 (0.54)
11/9
32.6 (11.3)∗
24.4 (3.5)
5.5 (1.00)
3.3 (0.90)
1.6 (0.53)
1.3 (0.83)
43 (4.5)
72 (5.5)∗
7.8 (4.3)∗
0.09 (0.02)
5.2 (1.0)
14.6 (5.3)†
7.8 (1.2)∗
8/14
60.8 (17)†‡
25.2 (4.2)
5.6 (1.9)
3.0 (1.6)
1.2 (0.50)‡
1.9 (1.0)†‡
37 (6.8)†‡
71 (10)
6.9 (3.6)†
0.85 (0.3)†‡
24 (6.8)†‡
7.3 (3.7)‡
6.1 (1.8)∗‡
7/7 6/9 7/7
38 (12)
24.3 (5.1)
5.4 (0.78)
3.2 (0.45)
1.7 (0.41)
1.0 (0.41)
0.09 (0.01)
5.6 (1.5)
5.0 (0.68)
4.6 (0.36)
34 (12)
24.7 (3.9)
5.4 (0.9)
3.3 (0.68)
1.5 (0.50)
1.2 (0.55)
0.09 (0.02)
5.2 (1.1)
13 (5.3)†?
7.6 (1.1)†?
57 (15)†‡
25.0 (4.1)
4.9 (1.0)
2.7 (0.87)
1.3 (0.57)
1.9 (0.65)†‡
0.82 (0.22)†‡
21 (6.2)†‡
5.9 (1.1)
5.2 (0.63)
Type II diabetes. The only statistically significant differ-
ences in the lipid profiles of the three groups were lower
HDL-cholesterol concentrations and higher triacylgly-
cerol (triglyceride) concentrations in the ESRD subjects
compared with the controls.
Plasma protein carbonyls
The plasma protein carbonyl concentrations (Table 2)
were significantly higher in the ESRD group compared
with the control and diabetes groups. When the ESRD
subjects who also had diabetes and/or who smoked were
takenoutofthecohort,theremainingESRDsubjectsstill
had significantly higher protein carbonyl concentrations
[0.15(0.028)nmol/mgofprotein]comparedwiththecon-
trol group [0.093 (0.014) nmol/mg of protein; P<0.01].
ESRD subjects with CHD [0.17 (0.03) nmol/mg of pro-
tein] did not have higher protein carbonyls than those
withoutCHD[0.16(0.06)nmol/mgofprotein;P>0.05].
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Oxidative stress and HDL function in Type I diabetes and end-stage renal disease
501
Table 2
diabetes or ESRD
Values are means (S.D.).∗P <0.05, †P <0.01 and ‡P =0.09 compared with control; ¶P <0.05 compared with Type I diabetes;
?P <0.01 compared with ESRD.
Control
Plasma protein carbonyls and PON1 parameters in control subjects and subjects with Type I
Type I diabetesESRD
Protein carbonyl study
Plasma protein carbonyls (nmol/mg of protein)
Plasma PON1 activity
Paraoxon hydrolysis (units/l)
Phenylacetate hydrolysis (units/ml)
PON1/HDL-cholesterol (units/mmol)
Paraoxon hydrolysis
Phenylacetate hydrolysis
LPO removal by HDL study
HDL PON1 (units/µg of HDL protein)
Paraoxon hydrolysis
Phenylacetate hydrolysis
0.093 (0.014) 0.099 (0.014) 0.16 (0.050)†¶
28.6 (14.9)
125.2 (24.0)
18.8 (10.3)∗
129.5 (30.8)
27.3 (15.0)
87.8 (25.7)
21.4 (13.5)
84.6 (35.9)
13.3 (8.4)∗?
88.4 (36.9)∗?
24.9 (18.2)
74.3 (30.1)
5.7 (3.2)
27.7 (7.2)
3.5 (1.7)‡
26.9 (5.9)
5.6 (2.7)
21.8 (8.2)∗
There was no significant correlation between protein car-
bonyl levels and the number of years on haemodialysis.
There was no significant correlation between age and
protein carbonyl levels in the control, Type I diabetic
and ESRD groups, as separate groups or when com-
bined. A subgroup analysis of age-matched controls
(mean, 50 years of age; n=14) and ESRD subjects (mean,
52 years of age; n=14) still showed a highly significant
(P<0.001) elevation of protein carbonyl levels in ESRD
[0.18 (0.06)] compared with controls [0.091 (0.01)].
Plasma protein carbonyl levels did not differ between
subjects with complication-free Type I diabetes and hea-
lthycontrols.Inthediabetesgroup,therewasasignificant
negative correlation between plasma protein carbonyl
concentration and HbA1C(r=−0.49, P<0.05). For the
three groups, there was no statistically significant corre-
lation between plasma protein carbonyls and age, serum
albumin or total serum protein.
Plasma PON1 activity (specifically as assessed by
paraoxon hydrolysis) was significantly lower in the dia-
betes group compared with healthy controls. Only the
ESRDgroup,withhighproteincarbonyllevels,displayed
a statistically significant negative correlation (r=−0.50,
P<0.05) between plasma PON1 activity (by paraoxon
hydrolysis) and plasma protein carbonyl concentra-
tions.
HDL removal of LPO
The efficacy of LPO removal by HDL from subjects
with diabetes was significantly impaired relative to the
control group (Figure 1). There was no significant differ-
enceintheLPOcontentoftheisolatedHDLfromTypeI
diabetic subjects [0.54 (0.22) µmol/ml] compared with
the control group [0.33 (0.29) µmol/ml; P>0.05] nor did
it correlate with removal of LPO or PON1 activity. The
associated PON1 activity (specifically paraoxon hydro-
Figure 1
tein in the control, Type I diabetic and ESRD groups
100% represents removal of all LPOs.∗P <0.01 compared with control group;
†P <0.01 compared with the Type I diabetic group. IDDM, Type I diabetes.
Percentage of LPO removal by 250 µg of HDL pro-
lysis) of the isolated HDL used in this bioassay tended
to be lower in subjects with Type I diabetes compared
with the control group (Table 2), but this did not reach
statistical significance. HDL PON1 phenotype charac-
terization was performed using the ratio of paraoxon
and phenylacetate hydrolysis to categorize phenotype
as ‘low activity’ or ‘intermediate/high activity’, which
was not significantly different between the three groups.
Specifically, further analysis of phenotype distributions
between the three groups, in particular the proportion
of high-activity phenotype (representing PON1−192QR
or RR) tended to be lower in Type I diabetes (20%)
compared with controls (50%) and ESRD (57%), but
this variation was not statistically significant (P=0.097).
Within the group with Type I diabetes, efficacy of HDL
removal of LPO did not correlate with PON1 activity
(paraoxon or phenylacetate hydrolysis) of plasma or of
isolated HDL, nor was it related to fasting blood glucose
or HbA1C(results not shown).
C ?2005 The Biochemical Society