Atherosclerosis 174 (2004) 141–149

The effect of high dose atorvastatin therapy on lipids and lipoprotein

subfractions in overweight patients with type 2 diabetes
J.M. Lawrence a,b,∗ , J. Reid a,b , G.J. Taylor b , C. Stirling a , J.P.D. Reckless a,b
a Diabetes and Lipid Research, Wolfson Centre, Royal United Hospital, Wolfson Centre Combe Park, Bath BA1 3NG, UK
b Department of Medical Sciences, University of Bath, Bath, UK

Received 19 September 2003; received in revised form 15 December 2003; accepted 21 January 2004

Abstract

Few data are available on the effects of high dose statin therapy on lipoprotein subfractions in type 2 diabetes. In a double blind randomised
placebo-controlled trial we have studied the effects of 80 mg atorvastatin over 8 weeks on LDL, VLDL and HDL subfractions in 40 overweight
type 2 diabetes patients. VLDL and LDL subfractions were prepared by density gradient ultracentrifugation. Triglycerides, cholesterol, total
protein and phospholipids were measured and mass of subfractions calculated. HDL subfractions were prepared by precipitation. Atorvastatin
80 mg produced significant falls in LDL subfractions (LDL1 66.2 mg/dl:36.6 mg/dl, LDL2 118:56.6 mg/dl, LDL3 36.9:19.9 mg/dl all P < 0.01
relative to placebo) and VLDL subfractions (VLDL1 55:22.1 mg/dl, VLDL2 40.1:19.1 mg/dl, VLDL3 52.6:30 mg/dl all P < 0.01 relative
to placebo). There was no change in the proportion of LDL present as LDL3 . There was a reduction in the proportion of VLDL as VLDL1
and a reciprocal increase in the proportion as VLDL3 . Changes in VLDL subfractions were associated with changes in lipid composition,
particularly a reduction in cholesterol ester and a reduction in the cholesterol ester/triglyceride ratio. Effects on HDL subfractions were largely
neutral. High dose atorvastatin produces favourable effects on lipoprotein subfractions in type 2 diabetes which may enhance antiatherogenic
potential.
© 2004 Elsevier Ireland Ltd. All rights reserved.

Keywords: Diabetes; Lipoprotein subfractions; Atorvastatin; Statin; Small dense LDL; VLDL; HDL

1. Introduction ment as patients with elevated cholesterol, suggesting that

Diabetes is associated with a several fold increased risk of
macrovascular disease [1,2]. Much of the increased risk in
these patients is due to the clustering of well established risk
factors, particularly hypertension and dyslipidaemia. Over
the past decade there has been increasing interest in recog-
nising and treating hyperlipidaemia in an attempt to reduce
the burden of macrovascular disease. Subgroup analyses of
secondary prevention trials using HMG CoA reductase in-
hibitors or statins have shown that patients with diabetes
benefit at least as much as other patient groups in terms of
event reduction [2]. Recent evidence from the Heart Protec-
tion Study has extended this to primary prevention [3]. Many
patients included in HPS had ‘normal’ cholesterol levels at
randomisation but still accrued the same benefit from treat-
∗ Corresponding author. Tel.: +44-122-582-4125;
fax: +44-122-582-4279.
E-mail address: mpsjml@bath.ac.uk (J.M. Lawrence).
there is no level below which cholesterol lowering is not
beneficial in high risk patients. The likelihood is that current
guidelines will be modified to reflect this new evidence, with
more stringent targets for cholesterol lowering. If treatment
targets are reduced, an increasing number of patients will
require and benefit from higher dose lipid lowering therapy
to reach target.
Dyslipidaemia in diabetes is characterised by raised
triglycerides and reduced HDL cholesterol. Although the
concentration of LDL cholesterol is typically not elevated,
there are often qualitative changes in LDL subfractions with
an increase in the proportion of LDL occurring as small
dense LDL or LDL3 [4]. For a given LDL cholesterol con-
centration there will be more LDL particles when patients
have more LDL3 . Increased LDL3 has been associated with
increased rates of myocardial infarction [5]. Part of the in-
creased macrovascular risk in diabetes may be explained by
these qualitative changes in lipoprotein subfractions, and as
well as considering reductions in total and LDL cholesterol,

0021-9150/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.atherosclerosis.2004.01.016
142 J.M. Lawrence et al. / Atherosclerosis 174 (2004) 141–149
much interest in diabetic dyslipidaemia has been focussed
on drug effects on the lipoprotein subfraction distribution.
There have been a number of studies assessing the effects
of statins on lipoprotein subfractions in type 2 diabetes
producing variable results [6]. However, the effects of high
dose statin therapy have not been extensively studied. The
present study was designed to assess the effects of high
dose atorvastatin on lipids and lipoprotein subfractions in
overweight patients with type 2 diabetes.
2. Materials and methods
2.1. Patients
Forty four patients met the inclusion criteria and agreed
to take part in the study. Eligible patients were those aged
45–80 with type 2 diabetes, a body mass index (BMI)
>27 kg/m2 , a total cholesterol >5 mmol/l and not currently
prescribed lipid lowering therapy. Patients had to be on
diet alone or on oral hypoglycaemic agents in monotherapy
or combination therapy with metformin and/or a sulpho-
nylurea with an HbA1c <10%. Patients were excluded if
prescribed insulin or a thiazolidinedione (pioglitazone or
rosiglitazone), if they had previously been intolerant of
statin therapy or if a statin was contraindicated (ALT >
three times the upper limit of normal). Patients were also
excluded if there was a clear indication for instituting lipid
lowering therapy within 4 months, as it was considered un-
ethical to potentially treat higher-risk patients with placebo
for the duration of the study. This included patients with
manifest macrovascular disease and also those with a cal-
culated coronary heart disease (CHD) risk over the next 10
years of greater than 30% in line with current recommenda-
tions of the Joint British Societies Guidelines on Prevention
of Coronary Heart Disease [7]. Women of childbearing
age were not to be excluded if sterilised or using adequate
contraception, but no pre-menopausal women entered the
study. The study had the ethical approval of the institutional
research ethics committee and written informed consent
was obtained from all patients.
2.2. Study design
The study was a double blind, randomized, placebo-
controlled design. At screening, eligible patients were given
dietary advice in line with the AHA step 1 diet and entered a
6-week dietary run-in phase. Patients were then randomised
to receive atorvastatin 80 mg daily or placebo for a total of 8
weeks, with a visit at 4 weeks post-randomisation for safety
monitoring. At all visits patients were asked to attend fasting
from at least midnight the night before. At randomisation
and at the end of the study 20 ml of blood was drawn into
EDTA for measurement of lipoprotein subfractions. Plasma
was separated by centrifugation at 1000 × g for 20 min at
4 ◦ C. A 1 ml sample was stored at 4 ◦ C for no longer than
4 days and used for preparation of HDL subfractions. Five
milliliters were stored at −70 ◦ C for later preparation of
LDL and VLDL subfractions (see Section 2.4). Previous
studies have established that lipoprotein subfractions are
stable in plasma frozen at –70 ◦ C for at least 29 months [8].
2.3. Safety monitoring
Very few adverse events were reported. Four patients
withdrew from the study, two during the dietary run-in. One
in the atorvastatin arm withdrew as a result of a significant
increase in ALT (to >10 times the upper limit of normal)
seen at the 4-week post-randomisation visit. This returned
to normal over 3 months following drug withdrawal. One
in the placebo arm withdrew as a result of developing a
generalised, itchy rash after 2 weeks. A second patient on
atorvastatin had an increase in ALT to twice the upper limit
of normal at 4 weeks but this had returned to normal at 8
weeks. Three patients in the placebo arm and one in the
atorvastatin arm of the study reported generalised muscle
aches but in all cases the CK was normal and no patient
withdrew as a result. One patient in the atorvastatin arm
complained of mild indigestion and one in the placebo arm
complained of generalised arthralgia. Twenty patients in
each group completed the study.
2.4. Laboratory methods
Analyses of HbA1c, renal function, liver function, glu-
cose, full blood count and initial lipid measurements
were undertaken in the central laboratories of the Royal
United Hospital, Bath. The HbA1c was measured using
high-performance liquid chromatography (DCCT aligned
non-diabetic normal 4–6%). All other analyses were carried
out in the laboratory of the Diabetes and Lipid Research
Department, University of Bath. High and low control
sera (Wako chemicals) were run for each patient to ensure
quality control.
2.5. HDL subfractions
Total HDL and HDL3 were separated by a double pre-
cipitation technique first described by Gidez et al. [9]. One
milliliter EDTA-plasma was diluted 1 to 1 with 0.15 mol/l
NaCl. Apolipoprotein B containing lipoproteins were pre-
cipitated in a first step by adding heparin-manganese chlo-
ride and HDL2 was precipitated in a second step by adding
dextran sulphate. Aliquots of the supernatant at each stage
were stored at −20 ◦ C for batch analysis of triglycerides
(GPO-DAOS method; Wako chemicals: interassay coef-
ficient of variation (cv) 4.4%), cholesterol (CO HDAOS
method; Wako chemicals: interassay cv 2.9%), apolipopro-
tein AI (turbidometric immunoassay; Wako chemicals:
interassay cv 2.1%) and apolipoprotein AII (turbidomet-
ric immunoassay; Wako chemicals: interassay cv 3.1%).
 J.M. Lawrence et al. / Atherosclerosis 174 (2004) 141–149 143

Table 1
Preparation of density stock solutions for density gradient ultracentrifu-
gation
Density Milliliters of density Milliliters of density
1.006 g/ml solution 1.182 g/ml solution
1.0998 50 55.77
1.0860 50 41.67
1.0790 75 53.16
1.0722 75 45.22
1.0641 75 36.99
1.0588 100 42.86
Density 1.006 g/ml stock solution: 22.7916 g NaCl and 0.2 g EDTA dis-
solved in distilled water to make up a total volume of 2 l. Density
1.182 g/ml stock solution: 125.23 g NaBr dissolved in density 1.006 g/ml
stock solution to make a total volume of 500 ml.
Results for HDL2 were calculated by subtracting results for
HDL3 from those for total HDL.
2.6. LDL and VLDL subfractions
LDL and VLDL subfractions were separated by density
gradient ultracentrifugation at 23 ◦ C using an SW40 rotor in
a Beckman L8-M ultracentrifuge with maximum accelera-
tion and no deceleration. The method used in our laboratory
is an adaptation of that first described by Lindgren et al. in
1972 [10]. The preparation of density stock solutions is sum-
marised in Table 1. Densities of all solutions were checked
with a densitometer (DMA 35; Paar Scientific Limited).
Centrifuge tubes were coated with polyvinyl alcohol
as previously described to enable the density solutions to
gravity feed down the tubes. EDTA-plasma was allowed to
thaw to room temperature from −70 ◦ C and the density was
then adjusted to 1.118 g/ml by adding 0.341 g NaCl to 2 ml
plasma. 0.5 ml of 1.182 g/ml stock solution was pipetted
into the centrifuge tube. Two milliliters density-adjusted
plasma was layered on top using a peristaltic pump. This
was followed by 1 ml of 1.0988 g/ml solution, 1 ml of
1.086 g/ml solution, 2 ml of 1.079 g/ml solution, 2 ml of
1.0722 g/ml solution, 2 ml of 1.0641 g/ml solution and 1.5
ml of 1.0588 g/ml solution. Table 2 shows the run times
for each subfraction. At the end of each run the top layer
was removed with a pipette (Table 2) and the same volume
was replaced with 1.0588 g/ml stock solution prior to the
next run.
Each fraction was aliquoted and stored at −20 ◦ C for later
measurement of triglycerides, cholesterol, apolipoprotein B
(turbidometric immunoassay; Wako chemicals: interassay
cv 4.6%), total protein (pyrogallol red; Randox laboratories:
interassay cv 4.2%), free cholesterol (enzymatic colorimetric
method [COD-PAP]; Wako Chemicals: interassay cv 5.3%)
and phospholipids (enzymatic colorimetric method; Wako
chemicals: interassay cv 3.3%).
Total mass of each fraction was calculated by adding
triglycerides, cholesterol, total protein and phospholipids.
Total cholesterol, apolipoprotein B and triglycerides were
also measured in serum collected at randomisation and at
the end of the study.
2.7. Statistical methods
Data were analysed using SPSS version 11. All normal
data are expressed as mean ± standard deviation. Triglyc-
erides were log transformed prior to analysis. Between
group comparisons were made using Analysis of Vari-
ance (ANOVA) adjusting for baseline values. Within group
comparisons were made using Student’s t-test.
3. Results
A total of 40 patients completed the study, 20 in each
group. There were no significant demographic differences
between groups at baseline (Table 3). One patient in the
placebo group was found to have attended at the beginning
and end of the study non-fasting and their results have not
been included in the analysis.
Eighty milligrams atorvastatin resulted in significant re-
ductions in cholesterol, triglycerides, apolipoprotein B and
LDL cholesterol relative to placebo (Table 4). The mean
within patient reduction in cholesterol was 39.7% (±9%),
in LDL cholesterol was 51% (±12%), in triglycerides was
32% (±20%) and in apoB was 35.1% (±7.3%) on atorvas-
tatin. This compared with no change in cholesterol, LDL
cholesterol and apoB and a non-significant 7.7% fall in
triglycerides with placebo. The mean within patient change
Table 3
Baseline demographic data
Atorvastatin group Placebo group

Table 2 Age (year) 63 (±9) 61 (±9)

Run times for preparation of lipoprotein subfractions Body mass index (kg/m2 ) 31.1 (±5.6) 34.6 (±6.3)
Sex 10 female 9 female
Fraction Speed (rpm) Time Volume
(hours–minutes) removed (ml) Diabetes treatment
Metformin 6 3
Chylomicrons 15,000 1–45 0.5
Sulphonylurea 4 5
VLDL1 28,000 1–30 0.5
Combination 4 5
VLDL2 28,000 1–14 0.5
VLDL3 17,000 16–29 0.5 HbA1c (%) 6.9 (±0.96) 6.7 (±0.9)
LDL1 39,000 2–17 1 Antihypertensive treatment 10 14
LDL2 40,000 5–13 1 Previous macrovascular disease 1 1
LDL3 30,000 13–23 1 Previous microvascular disease 1 0
144 J.M. Lawrence et al. / Atherosclerosis 174 (2004) 141–149

Table 4
Effects of high dose atorvastatin on basic lipid parameters
Atorvastatin group Placebo group

Start End Mean within Start End Mean within

patient change patient change
Cholesterol (mg/dl) 218 (±22.5) 132.2 (±15.1)a −86.1 (±21.6)c 227.1 (±48.9) 218.9 (±28.1) −7.7 (±34.9)
Triglycerides (mg/dl) 171.2 (±100.7) 107.5 (±46.4)a −63.7 (±70.0)c 181.9 (±65.8) 167.7 (±86.4) −14.6 (±54.1)
HDL cholesterol (mg/dl) 50.3 (±8.0) 47.3 (±8.1)b −3.0 (±6.0) 47.9 (±8.4) 48.6 (±8.7) +0.7 (±4.9)
Apolipoprotein B (mg/dl) 96.3 (±10.4) 62.5 (±8.9)a −33.9 (±8.2)c 99.8 (±15.3) 98.9 (±11.8) −0.9 (±9.9)
Cholesterol/HDL ratio 4.44 (±0.86) 2.84 (±0.44)a −1.59 (±0.76)c 4.77 (±1.36) 4.64 (±1.35) −0.14 (±0.9)
a P < 0.01 within group end vs. start.
b P < 0.05 within group end vs. start.
c P < 0.01 atorvastatin vs. placebo.

in HDL cholesterol on atorvastatin was −5.5% compared
with a 1.7% rise on placebo. Although the within group
change was statistically significant on atorvastatin, the dif-
ference between atorvastatin and placebo was not significant
(P = 0.09). There was also a significant reduction in the
cholesterol/HDL ratio, with a mean within patient reduction
of 34% (±11.2%) with atorvastatin compared with a mean
within patient reduction of 3.7% (±13.1%) on placebo (P <
0.001 atorvastatin versus placebo).
Use of atorvastatin was associated with a significant re-
duction in total LDL and also each of the LDL subfractions
compared with placebo (Table 5). Associated with this there
was no change in the LDL subfraction distribution, with the
same proportion of LDL present as LDL1 and LDL3 before
and after treatment in both patient groups.
There were significant reductions in all VLDL fractions
with atorvastatin compared with small non-significant re-
ductions across the board in the placebo group (Table 6).
The proportion of VLDL as large VLDL1 was significantly
reduced on atorvastatin. This reduction was associated with
a reciprocal non-significant increase in the proportion of
VLDL as VLDL3 . Similar, smaller and non-significant
effects were also seen in the placebo group. The mean
within patient change on atorvastatin in the proportion of
VLDL present as VLDL1 was −7.3% (±8%) compared
with −2.0% (±7.5%) on placebo (P = 0.035 atorvastatin
versus placebo). The mean within patient change in the
proportion of VLDL present as VLDL3 on atorvastatin was
+8.8% (±12%) compared with +3.0% (±8.7%) on placebo
(P = 0.1 atorvastatin versus placebo).
In all VLDL fractions atorvastatin was associated with
significant reductions in cholesterol, triglycerides and
cholesteryl ester (CE) compared with placebo (Tables 6
and 7). There was a reduction in the relative proportion of
cholesteryl ester and a relative increase in the proportion
of triglyceride in all VLDL subfractions on atorvastatin.
Smaller but significant changes were also seen on placebo
reflecting the non-significant fall in total VLDL mass. The
mean within patient change in the CE/total cholesterol ratio
and the CE/triglyceride ratio were significantly greater with
atorvastatin than placebo in VLDL3 .
There was no difference in the two groups in the change
in HDL2 but there was a significant difference in HDL3
(atorvastatin mean within patient change −2% compared
with +7.9% on placebo: between group comparison P =
0.003) (Table 8). A significant fall in total apoA1 was seen
on atorvastatin compared with a non-significant increase on
placebo. This effect was largely due to differences between
apoA1 in HDL3 (P = 0.03 between group comparison ator-
vastatin versus placebo) with no significant difference be-
tween groups in apoA1 in HDL2 . The apoAI/AII ratio was
increased in total HDL, HDL2 and HDL3 on atorvastatin,
but these changes were not significant and there were no
differences between the atorvastatin and placebo groups.

Table 5
Effects of high dose atorvastatin on LDL subfractions
Atorvastatin group Placebo group

Start End Mean within Start End Mean within

patient change patient change
Total LDL mass (mg/dl) 221.8 (±34) 113.1 (±28)a −108.7 (±30.4)b 239.7 (±45.9) 251.6 (±39.5) +11.9 (±30.5)
LDL1 mass (mg/dl) 66.2 (±14.3) 36.6 (±12.6)a −29.7 (±11.5)b 68.8 (±16) 72.3 (±14.9) +3.5 (±13.5)
LDL2 mass (mg/dl) 118 (±26.5) 56.6 (±14.2)a −62.1 (±21.6)b 127.1 (±33.3) 133.7 (±32.1) +7.1 (±27.1)
LDL3 mass (mg/dl) 36.9 (±14.5) 19.9 (±13.4)a −16.9 (±12.3)b 43.8 (±18) 45.6 (±22.7) +1.8 (±23.1)
LDL1 /total LDL (%) 30 (±5) 32 (±7) +2.6 (±7) 29 (±5) 29 (±5) −0.1 (±9)
LDL3 /total LDL (%) 17 (±7) 17 (±8) +0.2 (±6) 18 (±6) 18 (±9) +0.2 (±3)
a P < 0.01 within group end vs. start.
b P < 0.01 atorvastatin vs. placebo.
 J.M. Lawrence et al. / Atherosclerosis 174 (2004) 141–149 145

Table 6
Effect of high dose atorvastatin on VLDL subfractions
Atorvastatin group Placebo group

Start End Mean within Start End Mean within

patient change patient change
Total VLDL (mg/dl) 147.7 (±86.7) 71.1 (±44.3)b −76.5 (±55.5)d 151.8 (±69.6) 139.5 (±85) −12.3 (±58.2)
VLDL1 (mg/dl) 55.0 (±46.5) 22.1 (±19.1)b −32.9 (±31.7)d 51.9 (±31.0) 45.0 (±37.7) −6.9 (±29.6)
VLDL2 (mg/dl) 40.1 (±29.3) 19.1 (±14.4)b −21.0 (±20.2)d 38.9 (±26) 36.1 (±28.7) −2.9 (±17.0)
VLDL3 (mg/dl) 52.6 (±16) 30.0 (±14.0)b −22.6 (±11.1)d 60.9 (±18.6) 58.5 (±27) −2.5 (±17.8)
VLDL1 /total VLDL (%) 33.4 (±9.5) 26.1 (±12.4)b −7.3 (±8.0)c 32.8 (±8.2) 29.8 (±9.0) −2.0 (±7.5)
VLDL3 /total VLDL (%) 41.3 (±13.2) 50.2 (±17.5)b +8.8 (±12.0) 43.5 (±11.0) 46.1 (±12.3) +3.0 (±8.7)
Total VLDL cholesterol (mg/dl) 27.3 (±12.9) 11.0 (±5.9)b −16.3 (±9.0)d 29.4 (±12.0) 25.4 (±13.0)a −4.1 (±8.6)
Total VLDL triglyceride (mg/dl) 74.3 (±49.3) 39.6 (±26.0)b −34.7 (±31.1)d 73.3 (±35.0) 71.6 (±53.4) −1.7 (±36.6)
VLDL1 triglyceride (mg/dl) 33.9 (±27.9) 15 (±12.2)b −19.0 (±18.7)d 31.4 (±17.2) 29.6 (±26.2) −1.8 (±20.8)
VLDL2 triglyceride (mg/dl) 22.1 (±16.3) 12 (±8.8)b −10.2 (±10.9)d 20.9 (±13.3) 20.9 (±17.1) +0.0 (±10.0)
VLDL3 triglyceride (mg/dl) 18.2 (±7.2) 12.6 (±6.5)b −5.6 (±5.0)c 21.1 (±7.3) 21.0 (±11.4) +0.0 (±8.4)
VLDL1 cholesterol (mg/dl) 6.3 (±6.4) 2.1 (±1.8)b −4.3 (±4.8)d 5.9 (±4.0) 4.5 (±3.5) −1.4 (±3.1)
VLDL2 cholesterol (mg/dl) 6.6 (±4.3) 2.5 (±1.8)b −4.2 (±3.1)d 6.8 (±4.4) 5.4 (±4.1) −1.4 (±2.9)
VLDL3 cholesterol (mg/dl) 14.4 (±4.0) 6.6 (±2.8)b −7.8 (±3)d 16.8 (±5.1) 15.5 (±6.9) −1.6 (±3.8)
a P < 0.05 within group end vs. start.
b P < 0.01 within group end vs. start.
c P < 0.05 atorvastatin vs. placebo.
d P < 0.01 atorvastatin vs. placebo.

Table 7
Effect of high dose atorvastatin on core lipid make-up in VLDL subfractions
Atorvastatin Placebo

Start End Mean within Start End Mean within

patient change patient change
VLDL1 CE (mg/dl) 4.1 (±4.0) 1.2 (±1)b −2.9 (±3.2)c 3.2 (±2.2) 2.3 (±1.8)a −0.9 (±1.9)
VLDL2 CE (mg/dl) 4.5 (±2.8) 1.7 (±1.3)b −2.9 (±2)c 4.2 (±2.7) 3.1 (±2.3)a −1.1 (±1.9)
VLDL3 CE (mg/dl) 9.7 (±2.8) 4.3 (±1.8)b −5.4 (±2.2)c 11.1 (±3.7) 10.2 (±4.8) −0.9 (±2.5)
VLDL1 CE/TC (%) 54.7 (±4.7) 43.9 (±10.2)b −10.8 (±9.6) 53.8 (±7.2) 49.4 (±7.5)a −4.5 (±7.7)
VLDL2 CE/TC (%) 61.0 (±4.9) 51.5 (±19.3) −9.5 (±17.3) 61.5 (±4.2) 56.1 (±7)b −5.4 (±6)
VLDL3 CE/TC (%) 65.8 (±1.8) 59.5 (±4.5)b −6.2 (±3.6)c 65.5 (±3.2) 65 (±4.5) −0.5 (±3.6)
VLDL1 CE/Trig 0.09 (±0.03) 0.06 (±0.02)b −0.03 (±0.02) 0.1 (±0.03) 0.08 (±0.02)b −0.02 (±0.02)
VLDL2 CE/Trig 0.18 (±0.04) 0.11 (±0.05)b −0.07 (±0.05) 0.2 (±0.05) 0.15 (±0.05)b −0.05 (±0.04)
VLDL3 CE/Trig 0.53 (±0.14) 0.3 (±0.1)b −0.20 (±0.12)c 0.55 (±0.16) 0.52 (±0.18) −0.03 (±0.15)
a P < 0.05 within group end vs. start.
b P < 0.01 within group end vs. start.
c P < 0.01 atorvastatin vs. placebo.

4. Discussion Increased LDL has been clearly associated with increased

Effects of high dose atorvastatin on lipoprotein subfrac-
tion distribution have been assessed in overweight patients
with type 2 diabetes and the highly significant reductions in
total & LDL cholesterol and triglycerides confirm the DALI
study results using high dose atorvastatin [11]. Additionally,
this study has also shown significant reductions in all LDL
and VLDL subfractions. The reduction in total VLDL mass
with high dose atorvastatin was associated with changes in
the core lipid composition and in VLDL subfraction dis-
tribution, with a reduction in the proportion of VLDL as
VLDL1 . In contrast, no change in subclass distribution was
observed in LDL. This study has not shown a benefit of
high dose atorvastatin on total HDL and HDL subfractions
in type 2 diabetes and the results have suggested a negative
effect on apoAI in total HDL and particularly HDL3 .
risk of atherosclerosis. Small dense LDL is thought to have
the greatest atherogenic potential as it is more prone to
oxidation and glycaemic modification [12] binds less well
to hepatic LDL receptors [13] and is more likely to bind
to the extracellular matrix and be taken up by scavenger
macrophages in atherosclerotic plaques [14]. Indeed, there
are now several epidemiological studies that support the no-
tion that small dense LDL is particularly atherogenic [5].
Nevertheless, increased levels of all subfractions are associ-
ated with increased risk. In this study the large reductions
in LDL across the board, including LDL3 , induced by high
dose atorvastatin would be expected to reduce atherosclero-
sis risk.
The subfraction distribution of LDL is intrinsically linked
with the metabolism of VLDL particles. Kinetic stud-
ies have shown that LDL3 is largely derived from large
146 J.M. Lawrence et al. / Atherosclerosis 174 (2004) 141–149

Table 8
Effect of high dose atorvastatin on HDL subfractions
Atorvastatin group Placebo group

Start End Mean within Start End Mean within

patient change patient change
HDLT cholesterol (mg/dl) 50.3 (±7.9) 47.3 (±8.1)a −3.0 (±6.0) 47.9 (±8.4) 48.6 (±8.7) +0.7 (±5.0)
HDL2 cholesterol (mg/dl) 17.4 (±6) 15.6 (±6.2) −1.8 (±5.5) 14.9 (±6.1) 13.1 (±4.7) −1.9 (±4.9)
HDL3 cholesterol (mg/dl) 32.9 (±6.2) 31.7 (±4.4) −1.2 (±5.8) 33.9 (±4.6) 36.9 (±2.3)a +3.1 (±3.2)
HDL2 /HDL3 0.55 (±0.2) 0.5 (±0.2) −0.05 (±0.2) 0.45 (±0.2) 0.36 (±0.14)a −0.1 (±0.2)
HDLT apo AII (mg/dl) 37.5 (±7.3) 33.9 (±9.4) −3.6 (±4.5)b 39.3 (±9) 41.8 (±8.3) +2.3 (±5.6)
HDL3 apo AII (mg/dl) 26.3 (±5.7) 23.9 (±5.9) −2.4 (±7.2) 27.1 (±5.9) 30.1 (±7.3) +3.0 (±7.2)
HDL2 apo AII (mg/dl) 11.2 (±5.3) 10 (±7.6) −1.2 (±8.1) 12.2 (±6.9) 11.7 (±6.6) −0.6 (±5.1)
HDLT apo AI (mg/dl) 134.3 (±18.2) 124.9 (±21.2)a −9.4 (±7.9)c 135.8 (±16.6) 142.5 (±16.7) +6.7 (±9.6)
HDL3 apo AI (mg/dl) 84.9 (±14.2) 83.3 (±15) −1.6 (±17)c 91.2 (±19) 102.4 (±14.7)a +11.3 (±19.4)
HDL2 apo AI (mg/dl) 49.4 (±16.2) 41.6 (±18.6)a −7.8 (±16.2) 44.6 (±19.1) 40.1 (±12.4) −4.5 (±19.3)
HDLT apoAI/AII 3.7 (±0.5) 3.9 (±0.9) +0.2 (±0.8) 3.6 (±0.6) 3.5 (±0.5) −0.1 (±0.7)
HDL3 apoAI/AII 3.3 (±0.6) 3.5 (±0.53) +0.2 (±0.6) 3.4 (±0.5) 3.5 (±0.6) +0.2 (0.5)
HDL2 apoAI/AII 5.2 (±1.8) 5.9 (±4.5) +0.7 (±4.7) 5.2 (±3.9) 4.5 (±2.4) −0.7 (±4.7)
a P < 0.05 within group end vs. start.
b P < 0.05 atorvastatin vs. placebo.
c P < 0.01 atorvastatin vs. placebo.

triglyceride-rich VLDL1 , whilst other LDL subclasses ap-
pear to result from either direct secretion from the liver
or by delipidation and intravascular remodelling of other
VLDL subclasses [15]. Large VLDL have a longer resi-
dence time because of size and because they are less well
recognised by lipoprotein lipase, and are therefore subject
to more cholesterol ester–triglyceride interchange through
cholesterol ester transfer protein. A proportional decrease
in triglyceride-rich VLDL1 might be expected to be associ-
ated with a shift in LDL from LDL3 to LDL1 . The lack of
effect seen in this study may be explained by the relatively
low levels of LDL3 present initially, as there is evidence
from some studies [16] but not all [6] that treatment with
statins will have the greatest effect on whichever LDL sub-
class predominates. Alternatively, preferential clearance of
larger, less dense LDL fractions may explain the lack of
effect on LDL subfraction distribution. Statins upregulate
expression of LDL receptors in the liver. LDL1 and LDL2
bind to LDL receptors with greater affinity than LDL3 and
are therefore cleared more rapidly from the circulation.
Previous studies assessing the effects of atorvastatin, and
indeed other statins, on LDL subfraction distribution have
produced contrasting results. This may partly reflect the dif-
ferent methods of assessment as well as variations in patient
groups. In a study comparing atorvastatin 10 mg per day
with fenofibrate in non-diabetic patients with mixed dyslip-
idaemia, the mean size of LDL particles on polyacrylamide
gradient gel electrophoresis was shown to increase signifi-
cantly on both drugs [17] Similar results with atorvastatin
have been seen in another study using gradient gel elec-
trophoresis in patients with familial hypercholesterolaemia.
Using 40 and 80 mg of atorvastatin, increases in LDL peak
particle diameter were seen, but this effect was only evident
in men [18]. A study using nuclear magnetic resonance spec-
troscopy to compare the effects of atorvastatin and niacin
on low-density lipoprotein subfractions in non-diabetic pa-
tients with a combined dyslipidaemia suggested that 10 mg
atorvastatin per day, whilst reducing all LDL subclasses, has
a particularly favourable effect on small LDL particles [19].
This contrasts with a similar study in patients with diabetes
and a mixed dyslipidaemia where 10 mg atorvastatin per day
was shown to produce a reduction of LDL subclasses across
the board with no particular beneficial effects on small dense
LDL [20]. In the largest study to date, using NMR to assess
the effects of 10 mg atorvastatin on lipoprotein subclasses in
patients with diabetes who have survived myocardial infarc-
tion, although LDL was reduced significantly overall, there
were no convincing preferential effects of treatment on sub-
fraction distribution [6].
VLDL metabolism may be important in atherogenesis.
In addition to a role in determining the density distribu-
tion of LDL, VLDL remnants, formed as large VLDL is
metabolised, are believed to be particularly atherogenic.
Few data are available assessing the effects of atorvastatin
on VLDL subfractions in diabetes. In the one previous
study in diabetes assessing the effects of 10 mg atorvastatin,
and using NMR to measure subfractions, VLDL particle
numbers were reduced overall, with a particular effect
seen on small and medium VLDL particle concentrations
[6]. Previous studies in combined hyperlipidaemia with
10–40 mg of atorvastatin have shown significant reduc-
tions in ultracentrifugally-determined VLDL subfractions
[16,21,22]. Guerin et al. using 10mg atorvastatin, showed
a greater effect on VLDL1 compared with other subfrac-
tions [16] but the other studies have failed to confirm the
changes in subfraction distribution. Mechanisms by which
triglycerides are reduced by atorvastatin may vary. Whilst
animal studies have shown a reduction in VLDL synthesis
with a limited effect on catabolism of VLDL [23] studies
in patients with visceral obesity, the metabolic syndrome
 J.M. Lawrence et al. / Atherosclerosis 174 (2004) 141–149
and combined hyperlipidaemia have suggested increased
catabolism of VLDL subfractions is more important [21,24].
Part of this effect may be due to direct clearance of VLDL
particles across the density spectrum by binding to the in-
creased number of LDL receptors, whilst part of the effect
may be due to increased conversion of VLDL subfractions
to LDL, which are then cleared from the circulation [21].
The importance of the change in core lipids within
each VLDL fraction is unclear. The fall in the propor-
tion of cholesterol present as CE and the reduction in
the CE/triglyceride ratio particularly seen in VLDL1 and
VLDL3 is consistent with the reduced cholesteryl ester
transfer protein activity which has been seen in previous
studies using 10–80 mg atorvastatin [16]. CETP plays a key
role in remodelling of lipoproteins by promoting the ex-
change of triglycerides and cholesteryl esters between HDL
and VLDL and between VLDL and LDL. The reduced
CETP activity with statins is thought to relate largely to
the reduced availability of acceptors of CE, although there
is also some evidence that CETP gene expression may be
altered.
The total cholesterol/HDL cholesterol ratio is one of the
strongest predictors of macrovascular risk, and high dose
atorvastatin therapy in our study showed the expected sig-
nificant reduction compared to placebo, but no benefit on
HDL cholesterol per se. Although not seen universally [25]
a number of studies have shown a negative dose-response
effect of atorvastatin on HDL cholesterol [26–29] with most
studies showing either a small increase or neutral effect of
high dose atorvastatin. There is also some evidence that, par-
ticularly at higher doses, atorvastatin may be less effective
in reducing HDL than other statins [26,29,30]. Studies of
the effects of atorvastatin on HDL in diabetes have produced
contrasting results. Whilst the effect of 10 mg of atorvastatin
was largely neutral in the study of Soedamah-Muthu et al.
[6] in the DALI study 80 mg atorvastatin over 30 weeks in-
creased HDL by 5.2% from baseline, a result very similar to
that seen with 10 mg. The discrepancy between our results
and those of the DALI study may relate to the higher mean
HDL in our study at baseline.
Increased macrovascular risk has also been associated
in epidemiological studies with reduced HDL2 , a reduced
HDL2 /HDL3 ratio and reduced apolipoprotein AI, although
it remains unclear whether these are independent risk factors
[31] Our results were neutral when comparing the effects
of atorvastatin with placebo on HDL2 and the HDL2 /HDL3
ratio. However, a significant reduction in apoAI relative
to placebo was seen in both total HDL and HDL3 . Ef-
fects of atorvastatin on apoAI levels have varied between
studies. Whilst some have suggested benefit [32,33] oth-
ers have shown no change [16,34] or a reduction [27]. As
with total HDL cholesterol, there is evidence of a negative
dose-response effect of atorvastatin on apoA1 [28,30,35] and
evidence that other statins may be more effective in increas-
ing apoA1, particularly at higher doses [30,35,36]. Little ev-
idence is currently available on the effects in diabetes. In one
147
study comparing atorvastatin 20 mg with fenofibrate over 20
weeks in patients with type 2 diabetes and combined hyper-
lipidaemia, no change was seen in apoA1 in those on ator-
vastatin [34]. In a very recent study comparing the effects
of atorvastatin (up to 80 mg per day) with simvastatin (up
to 80 mg per day) in patients with the metabolic syndrome,
20 mg of atorvastatin was associated with small but signif-
icant increase in apoAI of 3% (95% CI 1–4%). This com-
pared with a neutral effect of 40 mg, and a negative effect
of 80 mg, with a mean reduction in apoAI of 5% (95% CI
−6 to −3%) at the highest dose [27]. It has been suggested
that the reduced apo AI effect of atorvastatin compared with
other statins might reflect a differential effect on lipopro-
tein or hepatic lipases, or reflect the longer plasma half life
of atorvastatin [35] but rosuvastatin with a longer half life
has positive HDL effects. In animal studies it has also been
shown that, although atorvastatin does increase the rate of
apoAI synthesis, it has a much greater effect on clearance at
high doses [37]. In addition, atorvastatin therapy results in
a significant reduction in apoE, with a selective reduction in
the proportion of apoE in HDL. The reason for the differen-
tial change is unclear but it has been speculated that this may
relate to accelerated catabolism of apoE-rich HDL particles
which are cleared via LDL receptors [38]. Whilst it may be
attractive to speculate that the greater upregulation of LDL
receptors by atorvastatin 80 mg daily may result in greater
clearance of apoE containing HDL and potentially explain
the fall in apoAI in our study, comparisons of the effects
of atorvastatin 20 and 80 mg in patients with hypertriglyc-
eridaemia did not suggest a dose related decrease in apoE
in HDL in the study of Le et al. [38]. HDL kinetic studies
using atorvastatin in man and the effects of atorvastatin on
apoE in diabetes have not been studied.
As targets for lipid lowering in high risk patients are re-
duced, increasing numbers will be treated with high dose
therapy. Whilst it is established that increasing the dose of
drug therapy is effective in producing further reductions in
total cholesterol, LDL cholesterol and triglycerides, there
is less evidence relating to possible qualitative changes in
lipoprotein subfractions on high drug doses. Our study is the
first to assess the effects of high dose atorvastatin therapy
on lipoprotein subfractions in patients with type 2 diabetes.
LDL and VLDL subfractions have been reduced across the
board with a relative reduction in VLDL1 compared with
VLDL3 , all of which would be expected to be antiathero-
genic. The fall in apoAI with high dose atorvastatin seen
here, along with the previously observed differential effect
of atorvastatin and other statins on HDL and apoAI, partic-
ularly at high doses, could potentially limit the antiathero-
genic potential of high dose therapy. It would be informative
to further study kinetics of HDL in type 2 diabetes, espe-
cially looking at the effects of high versus low dose atorvas-
tatin therapy to see whether our results are confirmed and
elucidate further possible mechanisms for the changes ob-
served. Limited data suggest that aggressive lipid lowering
with high dose atorvastatin may be more effective than less
148 J.M. Lawrence et al. / Atherosclerosis 174 (2004) 141–149
aggressive therapy with simvastatin in reducing progression
of atherosclerosis [39]. Further studies comparing high and
low dose atorvastatin and high doses of atorvastatin with
high doses of other statins in different at risk groups will
be needed to assess whether reduction in atherosclerosis is
attenuated by the observed changes in HDL with high dose
atorvastatin.
Acknowledgements
The study was supported by an unencumbered grant-in-aid
from Pfizer Pharmaceuticals to the diabetes and lipid re-
search department of the Universityof Bath.
References
[1] Kannel WB, McGee DL. Diabetes and cardiovascular disease. The
Framingham Study. JAMA 1979;241:2035–8.
[2] Pyorala K, Pedersen TR, Kjekshus J, et al. Cholesterol lowering with
simvastatin improves prognosis of diabetic patients with coronary
heart disease. A subgroup analysis of the Scandinavian Simvastatin
Survival Study (4S). Diabetes Care 1997;20:614–20.
[3] MRC/BHF Heart Protection Study of cholesterol lowering with
simvastatin in 20,536 high-risk individuals: a randomised placebo-
controlled trial. Lancet 2002;360:7–22.
[4] Sniderman AD, Scantlebury T, Cianflone K. Hypertriglyceridemic
hyperapoB: the unappreciated atherogenic dyslipoproteinemia in type
2 diabetes mellitus. Ann Intern Med 2001;135:447–59.
[5] Austin M, Breslow JL, Hennekens CH, et al. Low-density lipoprotein
subclass patterns and risk of myocardial infarction. JAMA 1988;
260:1917–21.
[6] Soedamah-Muthu SS, Colhoun HM, Thomason MJ, et al. The effect
of atorvastatin on serum lipids, lipoproteins and NMR spectroscopy
defined lipoprotein subclasses in type 2 diabetic patients with is-
chaemic heart disease. Atherosclerosis 2003.
[7] Wood D, Durrington P, Poulter N, et al. Joint British recommen-
dations on prevention of coronary heart disease in clinical practice.
Heart 1998;80(Suppl 2):S1–S29.
[8] Hokanson JE, Austin MA, Brunzell JD. Measurement and clinical
significance of low-density lipoprotein subclasses. In: Rifai N, War-
nick GR, Dominiczak MH., editors. Handbook of Lipoprotein Test-
ing. Washington: AACC Press; 1997. p. 267–82.
[9] Gidez LI, Miller GJ, Burstein M, Slagle S, Eder HA. Separation and
quantitation of subclasses of human plasma high density lipoproteins
by a simple precipitation procedure. J Lipid Res 1982;23:1206–
23.
[10] Lindgren FT, Jensen LC, Hatch FT. The isolation and quantitative
analysis of serum lipoproteins. In: Nelson GJ, editors. Blood lipids
and lipoproteins. New York: Wiley/Interscience; 1972. p. 181–274.
[11] The Diabetes Atorvastatin Lipid Intervention (DALI) Study Group.
The effect of aggressive versus standard lipid lowering by atorvas-
tatin on diabetic dyslipidaemia. The DALI study: a double-blind,
randomised, placebo-controlled trial in patients with type 2 diabetes
and diabetic dyslipidaemia. Diabetes Care 2001;241:335–41.
[12] Chapman MJ, Guérin M, Bruckert É. Atherogenic, dense low-density
lipoproteins. Pathophysiology and new therapeutic approaches. Eur
Heart J 1998;19:A24–30.
[13] Lund-Katz S, Laplaud PM, Phillips MC, Chapman MJ. Apolipopro-
tein B-100 conformation and particle surface charge in human LDL
subspecies: implication for LDL receptor interaction. Biochemistry
1998;37:12867–74.
[14] Galeano NF, Al-Haideri M, Keyserman F, Rumsey SC, Deckel-
baum RJ. Small dense low density lipoprotein has increased affinity
for LDL receptor-independent cell surface binding sites: a poten-
tial mechanism for increased atherogenicity. J Lipid Res 1998;39:
1263–73.
[15] Packard CJ, Shepherd J. Lipoprotein heterogeneity and apolipoprotein
B metabolism. Arterioscler Thromb Vasc Biol 1997;17:3542–56.
[16] Guérin M, Lassel TS, Le Goff W, Farnier M, Chapman MJ. Action
of atorvastatin in combined hyperlipidemia preferential reduction of
cholesteryl ester transfer from HDL to VLDL1 particles. Arterioscler
Thromb Vasc Biol 2000;20:189–97.
[17] Melenovsky V, Malik J, Wichterle D, et al. Comparison of the effects
of atorvastatin or fenofibrate on nonlipid biochemical risk factors and
the LDL particle size in subjects with combined hyperlipidaemia.
Am Heart J 2002;144:G1–7.
[18] Hoogerbrugge N, Jansen H. Atorvastatin increases low-density
lipoprotein size and enhances high-density lipoprotein cholesterol
concentration in male, but not in female patients with Familial Hy-
percholesterolemia. Atherosclerosis 1999;146:167–74.
[19] McKenny JM, McCormick LS, Schaefer EJ, Black DM, Watkins ML.
Effect of niacin and atorvastatin on lipoprotein subclasses in patients
with atherogenic dyslipidemia. Am J Cardiol 2001;88:270–4.
[20] Frost RJA, Otto C, Geiss HC, Schwandt P, Parhofer KG. Effects of
Atorvastatin versus Fenofibrate on lipoprotein profiles, low-density
lipoprotein subfraction distribution, and hemorheologic parameters
in type 2 diabetes mellitus with mixed hyperlipoproteinemia. Am J
Cardiol 2001;87:44–8.
[21] Forster LF, Stewart G, Bedford D, et al. Influence of atorvastatin
and simvastatin on apolipoprotein B metabolism in moderate com-
bined hyperlipidemic subjects with low VLDL and LDL fractional
clearance rates. Atherosclerosis 2002;164:129–45.
[22] Guerin M, Egger P, Soudant C, et al. Dose-dependent action of
atorvastatin in type IIB hyperlipidemia: preferential and progres-
sive reduction of atherogenic apoB containing lipoprotein subclasses
(VLDL-2, IDL, small dense LDL) and stimulation of cellular choles-
terol efflux. Atherosclerosis 2002;163:287–96.
[23] Burnett JR, Wilcox LJ, Telford DE, et al. Inhibition of HMG-CoA
reductase by atorvastatin decreases both VLDL and LDL apolipopro-
tein B production in miniature pigs. Arterioscler Thromb Vasc Biol
1997;17:2589–600.
[24] Chan DC, Watts GF, Barrett PHR, et al. Mechanism of action of
a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor on
apolipoprotein B-100 kinetics in visceral obesity. JCEM 2003;87:
2283–9.
[25] Lea AP, McTavish D. Atorvastatin. A review of its pharmacology
and therapeutic potential in the management of hyperlipidaemias.
Drugs 1997;53:828–47.
[26] Schneck DW, Knopp RH, Ballantyne CM, et al. Comparative effects
of rosuvastatin and atorvastatin across thier dose ranges in patients
with hypercholesterolemia and without active arterial disease. Am J
Cardiol 2003;91:33–41.
[27] Hunninghake DB, Ballantyne CM, Maccubbin DL, et al. Compara-
tive effects of simvastatin and atorvastatin in hypercholesterolemic
patients with characteristics of metabolic syndrome. Clin Ther
2003;25:1670–86.
[28] Schaefer EJ, McNamara JR, Tayler T, et al. Effects of atorvastatin
on fasting and postprandial lipoprotein subclasses in coronary heart
disease patients versus control subjects. Am J Cardiol 2002;90:689–
96.
[29] Karalis DG, Ross AM, Vacari RM, Zarren H, Scott R. On behalf of
the CHALLENGE study group. Comparison of efficacy and safety
of atorvastatin and simvastatin in patients with dyslipidaemia with
and without coronary heart disease. Am J Cardiol 2002;89:667–71.
[30] Crouse JR, Frohlich J, Ose L, Mercuri M, Tobert JA. Effects of
high doses of simvastatin and atorvastatin on high-density lipoprotein
cholesterol and apolipoprotein A-I. Am J Cardiol 1999;83:1476–7.
 J.M. Lawrence et al. / Atherosclerosis 174 (2004) 141–149
[31] Silverman DI, Ginsburg GS, Pasternak RC. High-density lipoprotein
subfractions. Am J Med 1993;94:636–45.
[32] Alaupovic P, Heinonen T, Shurzinske L, Black DM. Effect of a new
HMG-CoA reductase inhibitor, atorvastatin, on lipids, apolipoproteins
and lipoprotein particles in patients with elevated serum cholesterol
and triglyceride levels. Atherosclerosis 2003;133:123–33.
[33] Guerin M, Egger P, Le Goff W, Soudant C, Dupuis R. Atorvas-
tatin reduces postprandial accumulation and cholesteryl ester trans-
fer protein-mediated remodeling of triglyceride-rich lipoprotein sub-
species in type IIB hyperlipemia. JCEM 2002;87:4991–5000.
[34] Athyros VG, Papageorgiou AA, Athyrou VV, Demitriadis DS, Kon-
topoulos AG. Atorvastatin and micronised fenofibrate alone and in
combination in type 2 diabetes with combined hyperlipidaemia. Di-
abetes Care 2002;25:1198–202.
[35] Kastelein JJP, Isaacsohn JL, Ose L, et al. Comparison of effects of
simvastatin versus atorvastatin on high density lipoprotein cholesterol
and apolipoprotein A-1 levels. Am J Cardiol 2000;86:221–3.
149
[36] Davidson M, Ma P, Stein EA, et al. Comparison of effects
on low-density lipoprotein cholesterol and high-density lipoprotein
cholesterol with rosuvastatin versus atorvastatin in patients with
type IIa or IIb hypercholesterolemia. Am J Cardiol 2002;89:268–
75.
[37] Rashid S, Uffelman KD, Barrett PHR, Lewis GF. Effect of ator-
vastatin on high-density lipoprotein apolipoprotein A-I produc-
tion and clearance in the New Zealand White Rabbit. Circulation
2002;106:2955–60.
[38] Le N-A, Innis-Whitehouse W, Li X, et al. Lipid and apolipoprotein
levels and distribution in patients with hypertriglyceridemia: effect of
triglyceride reductions with atorvastatin. Metabolism 2000;49:167–
77.
[39] Smilde TJ, Van Wissen S, Wollersheim H, et al. Effect of aggressive
versus conventional lipid lowering on atherosclerosis progression in
familial hypercholesterolaemia (ASAP): a prospective, randomised,
double-blind trial. Lancet 2001;357:577–81.