The Journal of Clinical Endocrinology & Metabolism Vol. 83, No. 1 140-143
Copyright © 1998 by The Endocrine Society
Plasma Cholesteryl Ester Transfer Protein Activity in Hyper- and Hypothyroidism1
K. C. B. Tan,
S. W. M. Shiu and
A. W. C. Kung
Department of Medicine, University of Hong Kong, Hong Kong
Address all correspondence and requests for reprints to: Dr. Kathryn C. B. Tan, Department of Medicine, Queen Mary Hospital, Pokfulam Road, Hong Kong.
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Abstract
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Thyroid dysfunction is associated with multiple changes in lipoprotein
metabolism, and we have determined the effects of thyroid dysfunction
on plasma cholesteryl ester transfer protein (CETP) activity. CETP is a
plasma protein that mediates the exchange of cholesteryl ester and
triglyceride between plasma lipoproteins and plays an important role in
high-density lipoprotein metabolism and in the reverse cholesterol
transport pathway. Plasma CETP activity was assayed in 18 hyperthyroid
and in 17 hypothyroid patients, before and after treatment, by
measuring the transfer of cholesteryl esters from exogenous
radiolabeled high-density lipoprotein to apolipoprotein B-containing
lipoproteins. Plasma CETP activity was increased in hyperthyroid
patients, compared with their matched controls (22.11 ± 8.92%
transferred/5 µL·4 h vs. 16.75 ± 6.48,
P < 0.05), whereas in hypothyroid patients, plasma
CETP activity was decreased (11.14 ± 4.84% transferred/5
µL·4 h vs. 17.26 ± 7.13, P
< 0.01). Plasma CETP activity decreased after treatment of
thyrotoxicosis, although a significant change was observed, mainly in
the severely thyrotoxic patients with free T4 > 100 pmol/L
(n = 11, 25.61 ± 8.12% transferred/5 µL·4 h
vs. 21.71 ± 7.84, P < 0.05).
In the hypothyroid patients, there was a significant increase in plasma
CETP activity after thyroxine replacement (11.14 ± 4.84%
transferred/5 µL·4 h vs. 15.46 ± 6.71,
P < 0.01). There was a strong positive correlation
between log(free T4) and plasma CETP activity (r =
0.51, P < 0.001). In summary, both hyper- and
hypothyroidism are associated with significant changes in plasma CETP
activity, and these changes are corrected when the patients have been
rendered euthyroid.
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Introduction
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IT IS WELL recognized that thyroid
dysfunction has a major impact on lipoprotein metabolism. The main
effect of hyperthyroidism seems to be an enhanced elimination of
very-low-density lipoprotein (VLDL), low-density lipoprotein (LDL), and
probably also high-density lipoprotein (HDL); and patients tend to have
low levels of these lipoproteins (1, 2, 3). In contrast, hypothyroid
patients have high levels of intermediate-density lipoprotein, LDL, and
HDL (2, 4, 5). Thyroid hormone seems to modulate LDL receptor activity
both in vitro and in vivo, and this accounts for
the changes in LDL levels seen in hyper- and hypothyroidism (6, 7, 8). The
activities of the lipolytic enzymes, lipoprotein lipase (LPL) and
hepatic lipase (HL), also are influenced by thyroid hormones. In
hyperthyroidism, LPL has been reported to be normal and HL is increased
(9, 10), whereas in hypothyroidism, both LPL and HL are decreased
(10, 11, 12). Two recent studies suggest that changes in plasma cholesteryl
ester transfer protein (CETP) activity are seen also in hypothyroidism
(13, 14), but there is currently no data in the literature on the
effect of hyperthyroidism on plasma CETP activity. CETP is a
hydrophobic glycoprotein that mediates the net transfer of neutral
lipids between lipoproteins by stimulating the hetero-exchange of
cholesteryl esters and triglycerides. It redistributes cholesteryl
esters formed by lecithin:cholesterol acyltransferase (LCAT) in HDL to
the less dense apolipoprotein B (apoB)-containing lipoproteins.
Therefore, it plays an important role in the metabolism of HDL and
apolipoprotein A-I(apoA-I) and in the reverse cholesterol transport
pathway (15). The objective of the present study was to investigate the
effects of thyroid dysfunction on CETP by studying plasma CETP activity
in both hyperthyroid and hypothyroid patients before and after they had
been rendered euthyroid with medical treatment.
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Subjects and Methods
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Patients with thyroid dysfunction were recruited from the
Thyroid Clinic of the University of Hong Kong. Eighteen female patients
with active Graves’ disease and 17 patients with newly diagnosed
hypothyroidism were recruited. The etiology of hypothyroidism is as
follow: 8 patients with autoimmune thyroid disease, 4 patients had
previous partial thyroidectomy, 4 patients had a past history of
radioiodine therapy, and 1 patient was a cretin. Each patient was
matched with a control subject [matched for age, sex, and body mass
index (BMI)] recruited amongst the hospital staff and their relatives.
All subjects had fasting blood samples taken for the measurement of
lipids, apolipoproteins, CETP, free T4 (FT4),
and TSH. The hyperthyroid patients were started on antithyroid drug,
and T4 replacement was started in the hypothyroid patients.
All parameters were measured again in the patient groups after 3–4
months of treatment, when they had been rendered euthyroid. All
subjects gave their informed consent, and the protocol was approved by
the Ethics Committee of the University of Hong Kong.
Plasma total cholesterol and triglycerides were determined
enzymatically (Boehringer Mannheim, Mannheim, Germany) on a Hitachi 717
analyzer (Boehringer Mannheim). HDL-cholesterol was measured by the
same method after precipitation of apo B-containing lipoproteins with
polyethylene glycol (PEG) 6000. LDL-cholesterol was calculated by the
Friedewald equation. Serum apo A-I and apo B were measured by rate
nephelometry using the Beckman Array System (Beckman Instruments, Brea,
CA). FT4 was measured by competitive immunoassay on the ACS
180 (Chiron Diagnostics Corp., East Walpole, MA). TSH was measured by a
two-site chemiluminometric immunoassay on the ACS 180.
Plasma CETP activity was measured by an isotopic method, as described
by Freeman et al., with minor modifications (16). HDL
isolated from pooled normal plasma labeled with
[3H]cholesteryl oleate was used as donor and a
combined VLDL/LDL fraction was used as acceptor. [3H]HDL
was prepared by incubating 20 mL of dialyzed-density (>1.125 g/mL)
plasma infranatant (isolated from pooled normal fasting plasma by
ultracentrifugation) with [3H]cholesteryl oleate (200
µCi; 45 Ci/mmol) for 18 h at 37 C under nitrogen.
[3H]HDL was then isolated by density gradient
ultracentrifugation in a Beckman TLA-100.4 rotor at 100,000 rpm for
4 h and further purified by a second ultracentrifugation. The
harvested [3H]HDL was loaded onto an 80-mL (1.5 cm
x 45 cm) CL-4B gel filtration column and eluted overnight with
Tris-saline buffer. Samples of the fractions were counted by
scintillation counting and the radioactive peaks pooled and stored.
A combined VLDL/LDL fraction (density < 1.063 g/mL) was isolated
from pooled normal fasting plasma by ultracentrifugation at 1.063 g/mL
in a Beckman SW40 rotor at 40,000 rpm for 18 h and dialyzed
against Tris-saline buffer before being used as acceptor
lipoproteins.
Five microliters of plasma was incubated with [3H]HDL (25
µg protein) and VLDL/LDL (40 µg protein) for 4 h at 42 C. The
assay was stopped by placing on ice, and HDL was separated by
precipitation with heparin/MnCl2 (17). The precipitate was
separated by centrifugation and radioactivity of the supernatant
measured. Cholesteryl ester transfer activity was expressed as percent
transferred from supernatant to pellet. Plasma CETP activity of each
sample was assayed in duplicate, and pre- and posttreatment samples
were assayed in the same run. The intra- and interassay coefficients of
variation were 2.9% and 6.6%, respectively.
Results in this study were expressed as the means and SD
when the data were normally distributed. FT4 and TSH were
expressed as median and range because of their skewed distribution.
Comparisons between patients and their matched controls were done by
using two-tailed unpaired t test. The longitudinal analysis
of each variable, pre treatment and post treatment, in the patient
groups was evaluated by paired t test. Associations between
different parameters were determined by Pearson correlation
coefficients. The statistical package RS/1 (Bolt Beranek and Newman,
Cambridge, MA) was used for data analysis.
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Results
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The hyperthyroid patients and their control group were well
matched and their fasting lipid and apolipoproteins are shown in Table 1
. TSH was suppressed to less than 0.03
mIU/L in all the hyperthyroid subjects at diagnosis, and plasma CETP
activity was significantly higher than the controls (Fig. 1). After treatment, total cholesterol,
LDL cholesterol, and apo B increased significantly, compared with
baseline (Table 1). The effect of treatment on plasma CETP activity is
shown in Fig. 2. A significant reduction
in plasma CETP activity was observed mainly in the severely thyrotoxic
patients with FT4 > 100 pmol/L (n = 11,
25.61 ± 8.12% transferred/5 µL·4 h vs. 21.71
± 7.84, P < 0.05).
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Figure 1. Plasma CETP activity in hyper- and
hypothyroid patients vs. their matched controls. Values
are means ± SD. *, P < 0.05
vs. matched controls; **, P < 0.01
vs.. matched controls.
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The clinical characteristics and fasting lipid, apolipoproteins of
the hypothyroid patients, and their matched controls are shown in Table 2. Plasma CETP activity was decreased,
compared with the controls (Fig. 1
). After T4 replacement,
total cholesterol, LDL cholesterol, HDL cholesterol, apo A-I and B
all decreased significantly (Table 2), whereas a significant rise was
seen in plasma CETP activity (11.14 ± 4.84% transferred/5
µL·4 h vs. 15.46 ± 6.71, P <
0.01) (Fig. 3). The increase in plasma
CETP activity after T4 replacement remained statistically
significant, even when the two patients whose TSH level had not
normalized were excluded from the analysis.
There were significant alterations in BMI after treatment in both the
hyper- and hypothyroid patients, but the changes in BMI did not
correlate with changes in lipid profiles or CETP activity in either
group of patients. None of the changes observed in fasting lipids after
treatment correlate with the changes in plasma CETP activity. To
determine whether there is any relationship between plasma CETP
activity and thyroid hormone, correlation analysis was performed using
combined data from the controls and the baseline data of the hyper- and
hypothyroid patients. FT4 concentration was log-transformed
prior analysis because of the skewed distribution. There was a strong
positive correlation between log(FT4) and plasma CETP
activity (r = 0.51, P < 0.001).
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Discussion
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Lipoprotein abnormalities are commonly seen in patients with
thyroid dysfunction, because thyroid hormone affects the activities of
receptors and enzymes involved in lipoprotein metabolism. Thyroid
hormone is known to stimulate LDL receptor activity, and it also
affects the activities of the lipolytic enzymes and of LCAT (8, 9, 10). We
have shown in our study that thyroid hormone also has an effect on
plasma CETP activity. There was a strong correlation between thyroid
hormone and plasma CETP activity, which was increased in
hyperthyroidism and decreased in hypothyroidism. Plasma CETP is known
to play a role in HDL metabolism, and abnormalities in CETP activity
can result in changes in HDL. For instance, CETP deficiency, caused by
mutations in the CETP gene, is associated with high plasma levels of
HDL-C (15). HDL tends to be high in patients with hypothyroidism (2, 4), and we demonstrated a significant drop in HDL-C in our hypothyroid
patients after treatment. However, we did not find any correlation
between changes in plasma CETP activity and changes in HDL-C or other
lipid levels after treatment. This may be because the changes in HDL
seen in thyroid dysfunction are also partly caused by alterations in
LPL and HL (9, 10, 11).
The increase in plasma CETP activity in hyperthyroidism shown by our
study has not been previously reported. This study also confirms that
plasma CETP activity is reduced in hypothyroidism. Using a similar
isotopic method, Dullaart et al. (13) demonstrated that
plasma CETP activity was 15% lower in the hypothyroid state, compared
with euthyroid state, after T3 supplementation. However,
they were unable to find any differences between plasma CETP activity
of their 13 hypothyroid patients and that of the 26 euthyroid controls.
The authors attributed this lack of difference to the fact that plasma
CETP activities of the patients and the controls were not assayed in
the same run. Another study by Ritter et al. (14) reported
that the rate of cholesteryl ester transfer in hypothyroid patients was
decreased, and the reduction in cholesteryl ester transfer was
secondary to acceptor lipoprotein changes in the hypothyroid
state and not to changes in the concentration of CETP itself. Because
Ritter et al. employed a different method to measure CETP,
their results cannot be directly compared with ours or that of Dullaart
et al. The mass transfer assay used by Ritter et
al. measures the rate of transfer of cholesteryl esters between
HDL and apo B-containing lipoproteins, using each subject’s own
endogenous lipoproteins as substrates. Hence, this assay is not only
affected by the concentration of CETP but also by the concentration and
composition of endogenous plasma lipoproteins. The isotopic assay,
using standardized amounts of either radiolabeled exogenous
lipoproteins or chemically-labeled cholesteryl ester analogs, minimizes
the effect of endogenous lipoproteins on plasma CETP activity; and the
result has been shown to correlate with plasma concentration of CETP
measured by RIA (18). Another possible explanation for the lack of
change in plasma CETP concentration after treatment of hypothyroidism
in Ritter’s report might be the relatively short duration of
hypothyroidism of their studied subjects. Eight of their 10 patients
were hyperthyroid and received ablative doses of radioactive iodine
about 1 month before the study.
Thyroid hormone is known to regulate the transcription of several genes
and, consequently, influence the synthesis of these proteins. In
vitro study has shown that LDL receptor is regulated at the
messenger RNA (mRNA) level by thyroid hormone, and LDL receptor mRNA
increases by more than 50% in hyperthyroid rats (8, 19). Thyroid
hormone also stimulates the transcription of apo A-I (20), whereas HL
gene expression is relatively resistant to alterations in thyroid
status (8). Whether thyroid hormone causes an increase in CETP
activity, by stimulating CETP gene expression, remains to be
determined.
In conclusion, thyroid hormone has multiple effects on lipoprotein
metabolism. In addition to the previously described effects on LDL
receptor and lipolytic enzymes, we have shown that thyroid hormone also
affects CETP. Plasma CETP activity is increased in patients with
hyperthyroidism and reduced in patients with hypothyroidism, and these
changes are corrected when the patients have been rendered
euthyroid.
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Acknowledgments
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The authors are grateful to Ms. Betty Chu, Dr. Richard Pang, and
staff of the Clinical Biochemistry Unit for their technical
assistance.
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Footnotes
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1 This study was supported by Grant CRCG 337/041/0052 from the
Committee on Research and Conference Grants of the University of Hong
Kong. 
Received June 17, 1997.
Revised September 23, 1997.
Accepted October 1, 1997.
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Abstract
Introduction
