subclinical hypothyroidism ( sh ) , defined as an elevated serum thyroid stimulating hormone ( tsh ) level associated with serum thyroid hormone concentrations within the reference range , is found in 410% of individuals from western populations [ 1 , 2 ] . patients with hypothyroidism have an increased risk of developing atherosclerosis , and the subclinical stage is also considered a risk factor for this disease [ 3 , 4 ] . some investigators have found this connection to be attributed to increased levels of total cholesterol ( tc ) , low - density lipoprotein cholesterol ( ldl - c ) , and apolipoprotein ( apo ) b [ 5 , 6 ] , whereas others did not observe any significant differences [ 7 , 8 ] . depression of metabolism due to hypothyroidism has been reported to decrease oxidant production and thus protects tissues against oxidant damage [ 9 , 10 ] . however , data on the oxidative status of hypothyroidism are limited and controversial [ 1113 ] . lipid peroxidation ( lpo ) is a free radical chain reaction , which is triggered by hydroxyl radical and leads to membrane break . it facilitates the alteration in the protein structure and function and promotes generation of free radicals ( frs ) . lpo is reported to be high in hyperlipidaemia , which is a consistent biochemical feature in hypothyroidism . a study has shown that lpo in subclinical hypothyroid patients was similar to that in normal controls , while another study found increased lpo in hypothyroid patients . the biological oxidative effects of free radicals on lipids , proteins , and dna are controlled by a spectrum of antioxidants . enzymatic protection against reactive oxygen species ( ros ) and the breakdown products of peroxidized lipids and oxidized protein and dna are provided by several enzyme systems such as superoxide dismutase ( sod ) and catalase ( cat ) . sod catalyzes the dismutation of the superoxide anion into hydrogen peroxide ( h2o2 ) , which is then deactivated to water ( h2o ) by catalase or glutathione peroxidase ( gpx ) [ 18 , 19 ] . high - density lipoproteins ( hdls ) inhibit atherosclerosis development mainly by inducing reverse cholesterol transport . however , other antiatherogenic effects of hdl have been reported , because of their apolipoprotein a - i ( apo - ai ) and paraoxonase 1 ( pon1 ) content . arylesterase ( ae ) , one of the enzymatic activities of paraoxonase-1 , is known to play a protective role against peroxidation of ldl and other lipoproteins . given the high prevalence of sh in the general population , it is important to establish whether these alterations of thyroid function entail an oxidative stress and cardiovascular risk . thus , the aim of this study was to assess the oxidative stress biomarkers and investigated their relation with lipid parameters in subjects with hypothyroidism . forty adult subjects from clinical laboratory labimed , santa maria , rs , brazil were recruited for the present study . they were then classified into two groups - control group : 20 healthy subjects ( 47.20  11.73 years ) and the subclinical hypothyroidism ( sh ) group : 20 subjects newly diagnosed ( 49.12  10.85 years ) . sh was defined as an elevated thyrotropin ( tsh ) ( > 4.5  miu / l ) and normal free thyroxine ( ft4 ) level ( 8.722.6  nmol / l ) . exclusion criteria were ( 1 ) lipid - lowering drugs , ( 2 ) antioxidant vitamin supplements , ( 3 ) acetylsalicylic acid , ( 4 ) antihistamines , ( 5 ) antihypertensive , ( 6 ) exposure to high - iodine condition , ( 7 ) smokers , ( 8) alcoholics , ( 9 ) pregnant , ( 10 ) hormone replacement therapy , ( 11 ) diabetes mellitus , and ( 12 ) acute , chronic , or malignant diseases . the protocol was approved by the human ethics committee of the federal university of santa maria ( no . blood samples were collected after 12  h overnight fasting by venous puncture into gray and red top vacutainers ( bd diagnostics , plymouth , uk ) tubes . the samples were centrifuged for 15  min at 2500  g , and aliquots of serum were kept at 20c for maximum of 4 weeks . an aliquot of whole blood was collected into sodium citrate ( 3.2% ) and diluted 1  :  10 in saline solution for measurement of cat and sod activities . thyroid profile was assessed by estimation of serums tsh , t3 , and ft4 that were measured by chemiluminescent immunometric assay on immulite 2000 ( siemens healthcare diagnostics , los angeles , usa ) . miu / l , ft4 were 3.977.2  pmol / l , and t3 was 0.29 serum total cholesterol ( tc ) and triglycerides ( tg ) concentrations were measured using standard enzymatic methods by use of ortho - clinical diagnostics reagents on the fully automated analyzer ( vitros 950 dry chemistry system ; johnson & johnson , rochester , ny , usa ) . high - density lipoprotein cholesterol was measured in the supernatant plasma after the precipitation of apolipoprotein b - containing lipoproteins with dextran sulfate and magnesium chloride as previously described . low - density lipoprotein cholesterol ( ldl - c ) was estimated with the friedewald equation . serum thiobarbituric acid reactive substances ( tbarss ) were measured according to the modified method of jentzsch et al . . serum was added to a reaction mixture containing 1% orthophosphoric acid , an alkaline solution of thiobarbituricacid - tba , followed by heating for 45  min at 95c . after cooling , samples and standards of malondialdehyde ( 0.03  mm ) were read at 532  mda / ml . whole blood catalase ( cat ) activity was determined by the method of aebi   by measuring the rate of decomposition of h2o2 at 240  nm . the spectrophotometric determination was initiated by the addition of sample into an aqueous solution of hydrogen peroxide 0.3  mol / l . cat activity was calculated using the molar extinction coefficient ( 0.0436  cm/mol ) , and results were expressed as u / g hb . whole blood superoxide dismutase activity was measured as described by mccord and fridovich . in this method , a unit of sod is defined as the amount of enzyme that inhibits the rate of adrenalin oxidation by 50% . adrenalin oxidation leads to the formation of the colored product , adrenochrome , which is detected spectrophotometrically . sod activity is determined by measuring the rate of adrenochrome formation , observed at 480  nm , in a reaction medium containing glycine - naoh ( 50  mm , ph 10.0 ) and adrenalin ( 1  mm ) . basal measurements to calibrate the assay were performed in a reaction medium containing 1  ml of glycine - naoh ( 50  mm , ph 10.0 ) and 17  l of adrenalin ( 1  mm ) . serum arylesterase activity was measured using phenylacetate ( sigma co , london , uk ) as the substrate . the phenol formed after the addition of a 40-fold diluted serum sample enzymatic activity was calculated from the molar absorptivity coefficient of the produced phenol , 1310  mcm . one unit of arylesterase activity was defined as 1  mol phenol generated / min under the above conditions and expressed as u / l serum ( , view record in scopus ) . hemoglobin concentrations were measured in whole blood with a pentra 120 analyzer ( abx , montpellier , france ) . the nonparametric mann - whitney u - test was used to compare differences between groups . partial correlations were performed to control the associations between variables for total cholesterol levels . statistical significance was assumed at p < 0.05 . data were analyzed using spss version 11.0 software ( spss inc . , chicago , il , usa ) . there were no significant differences in age and body mass index ( bmi ) between groups . sh patients had significantly higher tsh , tc , ldl - c , and tc / hdl ratio with ft4 normal range than the control group ( table 1 ) . in sh group tbars and cat were significantly higher than controls , while sod did not change , as shown in figure 1 . arylesterase activity was significantly lower in the group with sh , compared with the control group ( figure 1 ) . are did not show any correlation with thyroid hormones , lipids and oxidative stress biomarkers . we observed a positive correlation between tc and tbars ( r = 0.757 , p < 0.0001 ) , tc and cat ( r = 0.650 , p < 0.0001 ) , ldl and tbars ( r = 0.812 , p < 0.0001 ) , ldl and cat ( r = 0.644 , p < 0.0001 ) , and ldl and sod ( r = 0.540 , p the correlations between tsh , t3 , and ft4 with oxidative stress biomarkers are shown in table 3 . tsh was significantly associated with tbars and cat ( r = 0.734 , p = 0.000 ; r = 0.499 , p = 0.004 , resp . ) . in the present study we have demonstrated that patients with subclinical hypothyroidism had altered lipid profiles , reduced are activity , increased lipid peroxidation , and induction of enzymatic defense when compared with control subjects . hypercholesterolemia is a common feature in hypothyroidism since thyroid hormones upregulate ldl - receptor expression . in a substantial number of studies , tc and/or ldl - c seem to be elevated in sh compared with controls [ 3133 ] . in this respect , our results showed that subjects with sh had significantly higher levels of tc , ldl - c , tg , and tc / hdl - c ratio thus displaying a more atherogenic lipid profile when compared with healthy individuals . thyroid hormones may stimulate hydroxymethylglutaryl coenzyme a ( hmg coa ) , the key enzyme of cholesterol biosynthesis , and induce an increased synthesis of cholesterol . additionally , the ldl - c receptor gene contains a thyroid hormone responsive element ( tre ) that could allow triiodothyronine ( t3 ) to modulate the gene expression of the ldl - c receptor resulting in an increase of ldl - c receptor synthesis . thyroid hormones and their function are low in target tissue in sh , and researchers conjectured that sh influences lipid profiles by the above - mentioned mechanism [ 34 , 35 ] . we report here a positive correlation between tsh and total cholesterol and ldl fraction as well as thyroid hormones ( t3 and ft4 ) showing correlation with triglyceride levels . tsh was also associated with deleterious changes in serum lipids , particularly hdl - c , ldl - c , and the ratio of ldl - c to hdl - c as suggested by recent investigations [ 3638 ] . thyroid dysfunctions increase lpo reactions and ros as documented by recent studies [ 39 , 40 ] . such destruction can lead to cell death and to the production of toxic and reactive aldehyde metabolites called free radicals , where malondialdehyde ( mda ) is the most important . it is known that ros would lead to oxidative damage of biological macro10molecules , including lipids , proteins , and dna [ 10 , 42 ] . moreover , tbars was correlated with tsh , tc , and ldl cholesterol . however , after controlling tc levels , the association between tsh and tbars was not significant suggesting that increased lpo could be attributed to lipid levels in hypothyroid status . ( 2008 ) , where a significant correlation between tsh and mda was lost after nullifying the effects of each of the coronary lipid risk factors among hypothyroid subjects . free radical - scavenging enzymes such as sod and cat are the first line of cellular defense against oxidative injury , decomposing o2 and h2o2 before interacting to form a more reactive hydroxyl radical ( oh ) . in addition , our study shows an association between lipid parameters ( ct and ldl ) and cat or sod activities . ( 2010 ) demonstrated that cat was significantly higher in subjects with hypercholesterolemia . on the other hand , some studies have reported no changes in cat activity in hypothyroid patients [ 15 , 44 ] . we also observed that the associations between tsh and tbars , cat , and sod were lost when lipids ( cholesterol ) were annulled . therefore , oxidative stress is likely to be potentially related to the secondary hypercholesterolemia to thyroid dysfunction and not directly to thyroid hormone levels in subclinical hypothyroidism . this result is consistent with our previous study showing that hypercholesterolemia has a stronger influence on the development of oxidative stress in overt hypothyroid ( oh ) patients . it is supposed that ldl particles can be protected from free radical - induced oxidation by an hdl - linked enzyme , paraoxonase 1 ( pon1 ) . pon1 is found in tissues such as liver , kidney , intestine , and also serum . it may possess antiatherogenic and anti - inflammatory properties , resulting from its ability to destroy modified phospholipids and to prevent cumulation of oxidized lipids in lipoproteins . we found lower pon1 arylesterase activity in sh patients than controls , suggesting oxidative stress . in addition , epidemiological evidence demonstrates that low pon1 activity is associated with increased risk of cardiovascular events   and is an independent risk factor for cardiovascular disease . in conclusion , our study shows an increase in the oxidative stress biomarkers in the circulation of patients with subclinical hypothyroidism . oxidative stress biomarkers seem to be associated with secondary hypercholesterolemia to hypothyroidism , whereas hypothyroidism per se does not cause oxidative stress in sh patients . on the other hand , high - plasma lipids can be considered as an oxidation substrate for the oxidative stress . thus , we suggest monitoring of oxidant / antioxidant status and lipid levels in sh patients , because we have found associations between serum tsh levels and serum lipids levels , showing thyroid dysfunction influence on lipid metabolism and consequently on oxidant / antioxidant status in these patients . however , further studies are necessary to evaluate a larger series of patients , with a longer duration of subclinical hypothyroidism .
objective . 
 the aim of the present study was to evaluate the oxidative stress biomarkers in patients with subclinical hypothyroidism ( n = 20 ) and health controls ( n = 20 ) . 
 subjects and methods . total cholesterol ( tc ) , 
 triglycerides ( tgs ) , low - density lipoprotein - cholesterol ( ldl - c ) , high - density lipoprotein cholesterol ( hdl - c ) , thiobarbituric acid reactive substances ( tbarss ) , catalase ( cat ) , superoxide dismutase ( sod ) , and arylesterase ( are ) were analyzed . 
 results . tc , ldl - c , tbars , and cat were higher in subclinical hypothyroidism patients , whereas sod did not change . 
 arylesterase activity was significantly lower in the sh group , compared with the control group . 
 correlation analyses revealed the association of lipids ( tc and ldl - c ) with both oxidative stress biomarkers and thyrotropin ( tsh ) . 
 thyroid hormones were correlated only with triglyceride levels . 
 in addition , tsh was significantly correlated with tbars , cat , and sod . however , no significant correlations were observed after controlling tc levels . conclusions . 
 we found that sh patients are under increased oxidative stress manifested by reduced are activity and elevated lipoperoxidation and cat activity . 
 secondary hypercholesterolemia to thyroid dysfunction and not hypothyroidism per se appears to be associated with oxidative stress in subclinical hypothyroidism .