hypothyroidism is known to be associated with premature atherosclerosis and increased frequency of cardiovascular events [ 13 ] . the increased cardiovascular risk in hypothyroid patients is mainly associated with hypercholesterolemia and an increased low - density lipoprotein cholesterol ( ldl - c ) level , caused by a decrease in the number of ldl - c receptors in the liver [ 46 ] .
however , in hypothyroid patients increased high - density lipoprotein cholesterol ( hdl - c ) also has been observed , as well as changes in size and composition of plasma hdl - c [ 79 ] . lately , it has been suggested that subclinical hyperthyroidism may be not associated with cardiovascular mortality or disorders , except for development of atrial fibrillation .
regardless of possible impact on atherosclerosis progression , hypothyroidism is associated with changes in lipoprotein metabolism , including not only lipid disturbances , but also abnormal plasma protein levels .
an increase in apolipoprotein b and apolipoprotein a   and changes in lipid transfer protein activity   in hypothyreotic patients were observed .
lipid transfer proteins such as cholesteryl ester transfer protein ( cetp ) and phospholipid transfer protein ( pltp ) are responsible for the formation of mature forms of hdl - c and regulate the concentration , size , and composition of the circulating hdl particles . moreover , in some studies cetp activity was positively correlated with total cholesterol ( tc ) , ldl - c , and non - hdl - c levels , whereas pltp activity was related to body mass index ( bmi ) and serum glucose concentration .
increased cetp or pltp mass / activity is considered to be a pro - atherosclerotic factor , mainly related to the decrease in hdl - c level , but also to increased pro - oxidant and pro - inflammatory potential [ 1618 ] .
a decrease in cetp or pltp activity is intended to have anti - atherosclerotic properties , mainly by causing an increase in circulating , small , dense hdl3 particles with increased anti - oxidative activity and increased reverse cholesterol transport efficiency . in some observations cetp activity
less is known about the pltp activity in patients with dysfunction of the thyroid gland .
the aim of this study was to evaluate simultaneously serum cetp and pltp activity in patients diagnosed with hypothyroidism ( studied group ) .
the selection criteria for control group members ( without thyroid dysfunction ) in this case to case study were levels of tc , ldl - c , hdl - c , and triglycerides ( tgs ) similar to those in study group patients .
the next criteria were the following : the same gender , similar bmis , smoking habits , and alcohol consumption .
the differences in lipid transfer proteins between the studied and control groups were related to the level of thyroid - stimulating hormone ( tsh ) .
the study was performed in the group of 202 residents of lower silesian region in poland : 166 women aged 59.610.4 years and 36 men aged 57.910.1 years .
there were 101 patients ( 83 women and 18 men ) diagnosed with hypothyroidism ( ht group ) and 101 healthy volunteers ( 83 women and 18 men ) constituting the control group ( euthyreotic [ et ] group ) . in all subjects , anthropometric data ( body weight , height )
bmi was estimated as the ratio of weight to height squared ( kg / m ) . in the overall population ,
about 23% of women and 33% of men were tobacco smokers , and about 58% of women and 77% men reported moderate alcohol consumption ( women : 1 alcohol unit per day ; men : 14 alcohol units per day ) . because the selection criteria for control group members matched to study patients included the same sex and similar age , bmi , tc , ldl - c , hdl - c , and tgs , these parameters did not differ significantly between ht and et groups .
also , the percentages of smokers or alcohol drinkers among females and males were similar in the studied and control groups .
the tsh level was higher in the study group in comparison with controls ( table 1 ) .
only 10 hypothyreotic women were taking l - thyroxine substitution therapy ; the remaining patients began treatment after this study .
venous blood was taken from subjects after 12 hours of fasting and centrifuged at 1000 g for 20 minutes at 4c .
the thyrotropin in serum was determined using the roche test by electrochemiluminescence method ( normal value : 0.54.0 iu / ml ) .
serum tc , tgs , and hdl - c were measured using the spinreact ( santesteve de bas , girona , spain ) enzymatic assay .
mmol / l ( 400 mg / dl ) by means of the friedewald formula .
the quantolip hdl ( technoclone gmbh , vienna , austria ) precipitation test was used to measure hdl2 and hdl3 cholesterol ( hdl2-c and hdl3-c ) .
the non - hdl - c was calculated as a difference between tc and hdl - c concentrations .
serum cetp and pltp activities were determined using the cetp activity assay kit and the pltp activity assay kit ( biovision research products , 2455-d old middlefield way , mountain view , california , usa ) with the fluorescence spectrophotometer hitachi f-2500 .
the cetp assay uses a synthetic fluorescent ce donor particle and apo - b - containing lipoprotein acceptor particles .
cetp - mediated transfer was determined by an increase in fluorescent intensity in the acceptor .
the serum pltp assay uses a fluorescent phospholipid donor and a synthetic acceptor , and again , pltp - mediated transfer was measured by an increase in fluorescent intensity . for both assays ,
the intra - assay and inter - assay coefficients of variation ranged from 11% to 15% , similar to fluorometric assay procedures that are described by others .
results were presented as mean  standard deviation or median and interquartile range . in the case of normal distribution , t - tests were applied , and a statistical significance between means was calculated using analysis of variance and post hoc tukey s ( checked by least significant difference ) tests . in case of qualitative variables , nonparametric tests were used .
the association between presence of hypothyroidism and lipid metabolism was analyzed in the multivariable linear regression model .
the independent variable was defined as diagnosis of hypothyroidism , and was individually analyzed in relation to lipid transfer protein and lipid parameters .
the following variables were included as potential confounders : age , bmi ( bmi 25 kg / m was treated as normal , bmi > 25 kg / m as overweight , bmi > 30 kg / m as obesity ) , tobacco use ( smokers or nonsmokers ) , alcohol consumption ( moderate drinkers or nondrinkers ) , and coexistence of coronary heart disease or diabetes mellitus ( present or absent ) . three - way analysis of variance ( using presence of hypothyroidism , smoking habits , and alcohol consumption as independent factors ) was also applied with one - dimensional or multidimensional significance tests .
all analyses were conducted using the stat statistical package , version 12.0 ( statistica 12 pl .
the study was performed in the group of 202 residents of lower silesian region in poland : 166 women aged 59.610.4 years and 36 men aged 57.910.1 years .
there were 101 patients ( 83 women and 18 men ) diagnosed with hypothyroidism ( ht group ) and 101 healthy volunteers ( 83 women and 18 men ) constituting the control group ( euthyreotic [ et ] group ) . in all subjects , anthropometric data ( body weight , height )
bmi was estimated as the ratio of weight to height squared ( kg / m ) . in the overall population ,
about 23% of women and 33% of men were tobacco smokers , and about 58% of women and 77% men reported moderate alcohol consumption ( women : 1 alcohol unit per day ; men : 14 alcohol units per day ) . because the selection criteria for control group members matched to study patients included the same sex and similar age , bmi , tc , ldl - c , hdl - c , and tgs , these parameters did not differ significantly between ht and et groups .
also , the percentages of smokers or alcohol drinkers among females and males were similar in the studied and control groups .
the tsh level was higher in the study group in comparison with controls ( table 1 ) .
only 10 hypothyreotic women were taking l - thyroxine substitution therapy ; the remaining patients began treatment after this study .
venous blood was taken from subjects after 12 hours of fasting and centrifuged at 1000 g for 20 minutes at 4c .
the thyrotropin in serum was determined using the roche test by electrochemiluminescence method ( normal value : 0.54.0 iu / ml ) .
serum tc , tgs , and hdl - c were measured using the spinreact ( santesteve de bas , girona , spain ) enzymatic assay .
ldl - c was estimated among patients with a tg concentration lower than 4.52 mmol / l ( 400 mg / dl ) by means of the friedewald formula .
the quantolip hdl ( technoclone gmbh , vienna , austria ) precipitation test was used to measure hdl2 and hdl3 cholesterol ( hdl2-c and hdl3-c ) .
the non - hdl - c was calculated as a difference between tc and hdl - c concentrations .
serum cetp and pltp activities were determined using the cetp activity assay kit and the pltp activity assay kit ( biovision research products , 2455-d old middlefield way , mountain view , california , usa ) with the fluorescence spectrophotometer hitachi f-2500 .
the cetp assay uses a synthetic fluorescent ce donor particle and apo - b - containing lipoprotein acceptor particles .
cetp - mediated transfer was determined by an increase in fluorescent intensity in the acceptor .
the serum pltp assay uses a fluorescent phospholipid donor and a synthetic acceptor , and again , pltp - mediated transfer was measured by an increase in fluorescent intensity . for both assays ,
the intra - assay and inter - assay coefficients of variation ranged from 11% to 15% , similar to fluorometric assay procedures that are described by others .
results were presented as mean  standard deviation or median and interquartile range . in the case of normal distribution , t - tests were applied , and a statistical significance between means was calculated using analysis of variance and post hoc tukey s ( checked by least significant difference ) tests . in case of qualitative variables , nonparametric tests were used .
the association between presence of hypothyroidism and lipid metabolism was analyzed in the multivariable linear regression model .
the independent variable was defined as diagnosis of hypothyroidism , and was individually analyzed in relation to lipid transfer protein and lipid parameters .
the following variables were included as potential confounders : age , bmi ( bmi 25 kg / m was treated as normal , bmi > 25 kg / m as overweight , bmi > 30 kg / m as obesity ) , tobacco use ( smokers or nonsmokers ) , alcohol consumption ( moderate drinkers or nondrinkers ) , and coexistence of coronary heart disease or diabetes mellitus ( present or absent ) .
three - way analysis of variance ( using presence of hypothyroidism , smoking habits , and alcohol consumption as independent factors ) was also applied with one - dimensional or multidimensional significance tests .
all analyses were conducted using the stat statistical package , version 12.0 ( statistica 12 pl .
in the total population of 202 people , the linear , positive correlations between tsh and tc ( r=0.21 ; p<0.01 ) , ldl - c ( r=0.16 ; p<0.05 ) , non - hdl - c ( r=0.21 ; p<0.01 ) , and tgs ( r=0.18 ; p<0.05 ) were shown .
there were no linear correlations between tsh and hdl - c ( p=0.9796 ) , hdl2-c ( p=0.4574 ) , or hdl3-c
( p = 0.3391 ) . on the other hand , at similar concentrations of serum lipids ( tg , tc , ldl - c , non - hdl - c , and hdl - c ) , the differences between hypothyreotic and control groups ( among females and males ) occurred in hdl subclasses ( table 1 ) . in hypothyreotic patients , the hdl2-c concentrations were lower and the hdl3-c concentrations were higher compared with those observed in healthy subjects ( table 2 ) .
serum cetp and pltp activities in hypothyreotic patients were lower ( p<0.001 ) in comparison with those in healthy subjects .
this lowering in the proteins activities was associated with a significant decrease in the hdl2-c level and an increase in the hdl3-c level ; however , in men the increase in hdl3-c was not statistically significant ( table 2 ) . in linear regression analysis a positive correlation between cetp and hdl2-c ( r=0.27 ; p<0.001 ) and between pltp and hdl2-c ( r=0.37 ; p<0.001 ) as well as a negative correlation between cetp and hdl3-c ( r=0.22 ; p<0.01 ) and between pltp and hdl3-c ( r=0.24 ; p<0.001 ) has been demonstrated ( figures 14 ) .
multiple linear regression analyses adjusted for age , sex , bmi , smoking habits , and alcohol drinking showed an association between hypothyroidism and activity of lipid transfer proteins . the significant beta coefficients ( 95% confidence intervals ) for cetp ( p<0.0003 ) and pltp ( p<0.0000001 ) were estimated .
simultaneously , no statistically significant association between thyroid function and lipid classes was observed ( table 3 ) . at the same time
the linear inverse relationship between tsh and cetp ( r = 0.21 ; p<0.01 ) and between tsh and pltp ( r=0.24 ; p<0.001 ) was shown ( figures 5 , 6 ) .
the high values of beta coefficients ( until 0.82 ) estimated for impact of smoking on lipids and the statistically significant impact of alcohol consumption on tgs and hdl3-c ( beta coefficient=0.17 ; p<0.05 ) were the reason for exclusion of smoking and alcohol drinking as confounders and treating them , together with a thyroid dysfunction , as independent variables .
three - way analysis of variance showed that activities of cetp and pltp , being dependent on thyroid function , were not influenced by single confounders such as smoking or alcohol drinking .
however , cetp activity was influenced by the interaction between smoking and moderate alcohol drinking ( table 4 ) .
hypothyroidism is considered to be a condition associated with an increased cardiovascular risk because of changes in lipid metabolism , plasma viscosity , and pro - oxidative and inflammatory potential .
the linear positive correlations observed in this study between tsh and tc , ldl - c , non - hdl - c , and tgs are consistent with the proatherogenic potential of hypothyroidism .
in fact , even within the normal range of tsh values , a linear increase in tc , ldl - c , and tgs has been observed with increasing tsh . in this study ,
the tsh concentration in the studied group was moderately elevated , reaching a maximal value of 7 iu / ml . because of absence of clinical symptoms , subhypothyroidism rather than hypothyroidism could be diagnosed .
the main novelty of this study is an observation that moderate hypothyroidism is associated with simultaneous decrease in cetp and pltp activities .
this association does not depend on lipid pattern , because the ht patients displayed lower lipid transfer proteins compared with healthy subjects , whereas tc , ldl - c , hdl - c , and tgs were similar in both groups .
it can be hypothesized that the reduction of pltp and cetp activities is related directly to the thyroid function .
it was confirmed in our study by linear negative correlations between tsh and cetp , and also between tsh and pltp , but first of all , by a multiple linear regression analysis adjusted for age , sex , bmi , smoking habits , and alcohol drinking .
the association between increased tsh and lipid transfer proteins was strong for cetp ( beta coefficient was significant at the level of 0.0003 ) and very strong for pltp ( p=0.0000001 ; 95% ci ) . decreased cetp activity in clinically manifesting hypothyroidism in adults was observed in some studies [ 68,21 ] . on the contrary ,
however , there are no published data about concomitant changes in pltp mass or activity in patients with hypothyroidism , neither in clinical nor in experimental studies . in the present study ,
more important than the demonstration of a reduction in cetp activity alone was the demonstration of simultaneous reduction in pltp and cetp activities in patients with subclinical hypothyroidism .
the extent of the reduction of pltp activity in patients with hypothyroidism was found to be similar to the reduction of cetp activity .
the role of pltp in lipid metabolism is still under investigation , and most observations have been made on pltp changes in patients diagnosed with diabetes or metabolic syndrome .
it seems that reduced pltp activity , as well as reduced cetp activity , has a protective effect on the cardiovascular system .
the main common mechanism for these effects could be the increase of total hdl - c fraction .
in other studies in ht patients , reduced cetp activity was associated with a decrease in hepatic lipase activity .
both proteins take part in hdl - c metabolism , increasing this fraction through reduction of cholesteryl ester transport from hdl - c to vldl - c and ldl - c .
however , the increase in hdl - c levels does not necessarily indicate an increased antiatherogenic potential , because hypothyroidism can induce dysfunctional hdl - c with increased pro - oxidative and pro - inflammatory properties .
thyroid hormones can influence hdl - c metabolism by increasing the activity of cetp , which transfers cholesteryl esters from hdl2-c to vldl particles , and tgs from vldl particles to hdl2-c .
thyroid hormones also stimulate the lipoprotein lipase , which catabolizes the tg - rich lipoproteins , and the hepatic lipase , which hydrolyzes hdl2-c to hdl3-c and contributes to the conversion of intermediate - density lipoproteins to ldl - c , and the next ldl - c to small , dense ldl particles . in our study , the total hdl - c concentration was similar in subjects with hypothyroidism and euthyreosis .
however , significant changes in hdl - c subfractions in patients with hypothyroidism was observed .
levels were lower and hdl3-c concentrations were higher in the group with hypothyroidism compared with the group with euthyreosis , both in women and , to a lesser extent , in men . in some other studies , clinically manifesting or clinically silent hypothyroidism was connected with an increase in hdl2-c subfractions , which was explained by a reduction in cetp activity and in consequence , attenuated transfer of cholesteryl esters from hdl2-c to hdl3-c . in our study , the hdl2-c subfraction decreased , whereas the hdl3-c subfraction increased ( the ratio of hdl2/hdl3 significantly decreased ) .
it is possible that the cause of this discrepancy in relation to observations from other authors is another degree of thyroid gland dysfunction in studied patients , i.e. , subclinical hypothyroidism in our study versus clinical hypothyroidism in other studies .
if so , changes in cetp , pltp , and hdl - c subfractions constitute very sensitive indicators of thyroid function disorders .
moreover , changes in hdl - c subfractions in patients with subclinical hypothyroidism were directly caused by reduction in cetp and pltp activity , which was demonstrated in a linear regression analysis by a positive correlation between cetp ( pltp ) and hdl2-c , as well as a negative correlation between cetp ( pltp ) and hdl3-c .
simultaneously , there was no effect of subclinical hypothyroidism on hdl - c subfractions ( table 3 ) , as was observed in other studies .
there were no significant differences between smokers and nonsmokers or between alcohol drinkers and nondrinkers in cetp or pltp activity .
on the other hand , the impact of the interaction between smoking and drinking on cetp activity was observed . in the study , neither coexistence of chronic diseases ( such as diabetes type 2 or coronary heart disease ) nor chronic treatment with pharmacological drugs had any significant effect on lipid transfer proteins .
this may be attributed to a relatively small number of patients diagnosed with these diseases ( fewer than 10% ) and a small number of patients treated with pharmacological drugs ( only 10 women were treated with l - thyroxine ) .
reduction in cetp activity with the aim of reducing cardiovascular risk is the subject of continued research using cetp inhibitors [ 3032 ] .
the increase in hdl - c levels reduces the risk of complications of atherosclerosis through various mechanisms , among which the most important one is the increased reverse cholesterol transport . increased concentration of hdl3-c increases the antioxidant and anti - inflammatory potential .
therefore , patients with a moderate increase in tsh reveal simultaneously pro - atherosclerotic changes ( increase in ldl - c , tgs , apolipoprotein b ) as well as anti - atherosclerotic changes ( reduced cetp and pltp activities , increased tc and hdl3-c levels ) in lipid metabolism .
hypothyroidism is associated with decreased activity of cetp and pltp , as well as changes in serum hdl - c subclasses . decreased hdl2-c and increased hdl3-c levels in subjects with hypothyroidism are consequences of decreased activity of lipid transfer proteins .
these changes are early symptoms of lipid disturbances in hypothyroidism because they appear in hypothyreotic patients with normal levels of tc , ldl - c , hdl - c , and tgs .
 backgroundplasma cholesteryl ester transfer protein ( cetp ) activity is often decreased in patients with hypothyroidism , whereas less is known about the phospholipid transfer protein ( pltp ) . 
 we aimed to evaluate simultaneously serum cetp and pltp activity in patients diagnosed with hypothyroidism.material/methodsthe selection criteria for control group members ( without thyroid dysfunction ) in this case to case study were levels of total cholesterol , low - density lipoprotein cholesterol , high - density lipoprotein cholesterol ( hdl - c ) , and triglycerides similar to those in study group patients ( 101 patients diagnosed with hypothyroidism ) . 
 serum cetp and pltp activities were measured by homogenous fluorometric assays using synthetic donor particle substrates.resultsserum cetp and pltp activities in hypothyreotic patients were lower ( p<0.001 ) compared with those in healthy subjects . 
 this lowering was associated with significant changes in hdl - c subclasses : decrease in hdl2- and increase in hdl3 cholesterol levels . 
 multiple linear regression analyses adjusted for age , sex , body mass index , smoking habits , and alcohol drinking showed a strong association between hypothyroidism and activity of lipid transfer proteins . a linear inverse relationship between thyroid - stimulating hormone ( tsh ) and cetp ( r=0.21 ; p<0.01 ) and between tsh and pltp ( r=0.24 ; p<0.001 ) 
 was shown . 
 there also was a positive correlation ( p<0.001 ) between cetp and hdl2 cholesterol ( r=0.27 ) and between pltp and hdl2 cholesterol ( r=0.37 ) . 
 a negative correlation between cetp and hdl3 cholesterol ( r=0.22 : p<0.01 ) and between pltp and hdl3 cholesterol ( r=0.24 ; p<0.001 ) has been demonstrated as well.conclusionsthe decreased hdl2 and increased hdl3 cholesterol levels in subjects with hypothyroidism are consequences of decreased activity of lipid transfer proteins . 
 these changes are early symptoms of lipid disturbances in hypothyroidism . 
Background
Material and Methods
Subjects
Biochemical measurements
Statistical analysis
Results
Discussion
Conclusions