associations have been reported between sleep apnea and systemic hypertension , pulmonary hypertension , ischemic heart disease and stroke .
osa is characterized by recurrent obstructions of the upper airway during sleep , which are followed by lowering of blood hemoglobin oxygen saturation .
this repeated hypoxia and reoxygenation cycle is similar to hypoxia  reperfusion injury , which initiates oxidative stress .
oxidative stress is characterized by an imbalance between oxidant - producing systems and anti - oxidant defence mechanisms , which results in excessive formation of reactive oxygen species ( ros ) .
ros can damage various biomolecules and cellular components , but they also act as important signalling molecules which play roles in induction and activation of multiple genes .
ros can oxidize lipids , proteins , carbohydrates and dna , and thus can alter their functions .
several studies have suggested that osa is associated with increased levels of oxidative stress markers [ 25 ] or decreased antioxidant defence .
mainly the phase of reoxygenation is thought to be the period during which increased production of ros occurs .
it has been established that treating osa patients by continuous positive airway pressure ( cpap ) decreases levels of oxidative stress - related markers .
previous studies found correlation of levels of some molecules ( which are possible markers related to oxidative stress ) and various diseases including chronic renal failure and hemodialysis patients , diabetes mellitus , diabetic kidney disease , arterial hypertension , myocardial ischemic syndromes and atherosclerosis [ 819 ] .
this study was conducted to determine whether these molecules are also altered in osa patients .
we measured levels of soluble receptor for advanced glycation end - products ( srage ) , pregnancy - associated plasma protein - a ( papp - a ) , matrix metalloproteinases 2 and 9 ( mmp-2 and mmp-9 ) and high sensitive c - reactive protein ( hscrp ) .
high crp levels are linked to risk of atherosclerosis and it appears to be a marker for cardiovascular risk .
it was also used as an oxidative stress marker   and a marker of risk of atherosclerosis .
deleterious effects mediated by ages are prevented in part by this receptor lacking the transmembrane domain- soluble rage ( srage ) , which occurs in vivo .
low srage level was described in patients with arterial hypertension   and diabetes mellitus complications .
mmp-2 ( or gelatinase a ) and mmp-9 ( or gelatinase b ) are zn and cu dependent endopeptidases .
mmp-2 and mmp-9 are involved in degradation of collagen type iv , elastin and fibronectin .
ros have been shown to regulate the levels of mmps in various cellular and in vivo systems .
mmps , among others , play a role in many pathological processes in cancerogenesis and cardiovascular diseases .
increase in mmps activity was shown to be associated with the progression of atherosclerotic plaques to their rupture   and recently published elevation of mmp-9 in osa .
copper ( cu ) and zinc ( zn ) are necessary trace element for the body .
copper is a part of an antioxidant enzymes , as are superoxide dismutase ( sod ) , ceruloplasmin , and cytochrome c oxidase .
copper , together with zinc , plays an important role in antioxidant defense mainly as a part of cu / zn sod .
it was shown that cu - deficient rats had higher level of isoprostanes as marker of oxidative stress compared to cu normal rats .
however , it was also found that high levels of cu and other metal elements impair oxidative stress .
low zinc levels are also thought to be associated with increased oxidative stress and reduced antioxidant defense .
adult men with suspicion of osa indicated for nocturnal polygraphy in the sleep laboratory were enrolled in the study .
all subjects signed an informed consent , and the procedures were approved by the local ethics committee .
excluded from the study were patients with the following diseases : stroke or cerebral injury less than 6 month before the study , major cardiological diseases including cardiac insufficiency and manifest coronary artery disease , pulmonary hypertension , and patients with diabetes mellitus treated with insulin . during the night ( from 11 p.m. through 6 a.m. ) patients were examined using the method of nocturnal polygraphy ( monitored parameters were : nasal airflow , thoracoabdominal wall motion , heart rate , blood hemoglobin oxygen saturation , respiratory noises and body position ) .
the nocturnal polygraphy was scored visually according to recommendations of the american academy of sleep medicine ( aasm ) 2007 using 3% blood hemoglobin oxygen saturation decrease as the level of significance .
the following parameters were scored : the apnea / hypopnea index ( ahi  mean number of apneas and hypopneas per hour of subjective sleep duration ) , the oxygen desaturation index ( odi  mean number of oxygen hemoglobin saturation drops of 3% or more per hour of subjective sleep duration ) , mean blood hemoglobin oxygen saturation ( spo2 ) and duration of oxygen blood saturation dip below 90% ( spo2<90% ) . in the morning after polygraphy , fasting venous blood samples were taken .
selected markers were measured : hscrp , papp - a , mmp-2 , mmp-9 , srage , cu , zn .
blood samples were centrifuged for 10 minutes at 1450 g and then serum was frozen and stored until analysis .
cu concentration in the diluted ( 1 + 9 ) serum samples were determined by inductively - coupled plasma mass spectrometry on a pe elan 6000 instrument ( perkin elmer , norwalk , ct , usa ) .
calibration solutions were spiked by the important serum matrix compounds ( nacl , kcl , nano3 , kbr ) .
papp - a and hs - crp were measured by trace ( time resolved amplified cryptate emission ) on the kryptor analyser using standard kits ( brahms , henningsdorf , germany ) .
srage , mmp-2 , mmp-9 ( all kits rd systems , usa ) were assessed with standard elisa ( enzyme - linked immunosorbent assay ) .
we further used standard laboratory methods to measure blood count , minerals , renal function , liver enzymes , lipids , glycemia .
the group comparisons were performed with one - way parametric anova , and the correction for bmi was performed using analysis of covariance ( ancova ) .
multiple regression was used to obtain and compare the effects of individual predictors of oxidative stress - related markers levels .
mean age was 51.1 years ( sd  11.1 ) , mean bmi was 30.8 ( sd  5.3 ) , mean ahi was 31.2 ( sd  22.9 ) , mean odi was 32.2 ( sd  26 ) , mean spo2 was 92.8% ( sd  4 ) , and mean spo2 < 90% was 15.1% ( sd  25 ) of the night .
, we present the number of patients with selected diseases in the whole group : 26 patients arterial hypertension , 2 hyperuricemia , 14 hyperlipidemia , 3 atrial fibrillation , 2 diabetes mellitus ( excluded patients with insulin therapy ) , 4 chronic obstructive pulmonary disease , 4 ischemic cardiac disease and 1 myocardial infarction , 1 stroke .
adult men with suspicion of osa indicated for nocturnal polygraphy in the sleep laboratory were enrolled in the study .
all subjects signed an informed consent , and the procedures were approved by the local ethics committee .
excluded from the study were patients with the following diseases : stroke or cerebral injury less than 6 month before the study , major cardiological diseases including cardiac insufficiency and manifest coronary artery disease , pulmonary hypertension , and patients with diabetes mellitus treated with insulin .
during the night ( from 11 p.m. through 6 a.m. ) patients were examined using the method of nocturnal polygraphy ( monitored parameters were : nasal airflow , thoracoabdominal wall motion , heart rate , blood hemoglobin oxygen saturation , respiratory noises and body position ) .
the nocturnal polygraphy was scored visually according to recommendations of the american academy of sleep medicine ( aasm ) 2007 using 3% blood hemoglobin oxygen saturation decrease as the level of significance .
the following parameters were scored : the apnea / hypopnea index ( ahi  mean number of apneas and hypopneas per hour of subjective sleep duration ) , the oxygen desaturation index ( odi  mean number of oxygen hemoglobin saturation drops of 3% or more per hour of subjective sleep duration ) , mean blood hemoglobin oxygen saturation ( spo2 ) and duration of oxygen blood saturation dip below 90% ( spo2<90% ) .
selected markers were measured : hscrp , papp - a , mmp-2 , mmp-9 , srage , cu , zn .
blood samples were centrifuged for 10 minutes at 1450 g and then serum was frozen and stored until analysis .
cu concentration in the diluted ( 1 + 9 ) serum samples were determined by inductively - coupled plasma mass spectrometry on a pe elan 6000 instrument ( perkin elmer , norwalk , ct , usa ) .
calibration solutions were spiked by the important serum matrix compounds ( nacl , kcl , nano3 , kbr ) .
papp - a and hs - crp were measured by trace ( time resolved amplified cryptate emission ) on the kryptor analyser using standard kits ( brahms , henningsdorf , germany ) .
srage , mmp-2 , mmp-9 ( all kits rd systems , usa ) were assessed with standard elisa ( enzyme - linked immunosorbent assay ) .
we further used standard laboratory methods to measure blood count , minerals , renal function , liver enzymes , lipids , glycemia .
the group comparisons were performed with one - way parametric anova , and the correction for bmi was performed using analysis of covariance ( ancova ) .
multiple regression was used to obtain and compare the effects of individual predictors of oxidative stress - related markers levels .
mean age was 51.1 years ( sd  11.1 ) , mean bmi was 30.8 ( sd  5.3 ) , mean ahi was 31.2 ( sd  22.9 ) , mean odi was 32.2 ( sd  26 ) , mean spo2 was 92.8% ( sd  4 ) , and mean spo2 < 90% was 15.1% ( sd  25 ) of the night .
, we present the number of patients with selected diseases in the whole group : 26 patients arterial hypertension , 2 hyperuricemia , 14 hyperlipidemia , 3 atrial fibrillation , 2 diabetes mellitus ( excluded patients with insulin therapy ) , 4 chronic obstructive pulmonary disease , 4 ischemic cardiac disease and 1 myocardial infarction , 1 stroke .
body mass index ( bmi ) and ventilation parameters were correlated to chosen oxidative stress - related markers for the whole group of patients independent of osa intensity .
we found strong correlations between bmi and levels of mmp-9 ( p0.001 ) , cu ( p=0.035 ) ( figure 1 ) and hscr ( p0.001 ) ( figure 2 ) .
bmi was also positively correlated with fibrinogen ( p=0.019 ) and negatively correlated with srage ( p=0.006 ) ( table 2 ) . concerning ventilation parameters , we found significant positive correlations for hscrp and odi ( r=0.450 , p=0.001 ) ( figure 1 ) , ahi ( r=0.479 , p=0.001 ) and spo2<90 ( r=0.480 , p=0.001 ) .
mmp-9 also correlated positively with odi ( r=0.380 , p=0.008 ) and spo2<90 ( r=0.469 , p=0.001 ) .
positive correlation for cu was found only with odi ( r=0,330 , p=0.020 ) ( figure 2 ) .
significant negative correlations were found for spo2 and markers cu ( r=0.320 , p=0.026 ) , mmp-9 ( r=0.490 , p<0.001 ) , hscrp ( r=0,440 , p=0.002 ) and fibrinogen ( r=0.420 , p=0.020 ) .
srage correlated negatively with ahi ( r=0.290 , p=0.044 ) and odi ( r=0.320 , p=0.027 ) .
there were no correlations between oxidative stress markers and lipid spectrum parameters and other biochemical parameters .
multiple regression showed that strong correlation between oxidative stress - related parameters and osa is explained by connection of these parameters to bmi ( r=0.322 , beta 0.397 , p=0.011 ) .
further patients results were divided into 4 groups according to osa severity , because we wanted to ascertain if markers levels are dependent on ventilation parameters .
group 1 was patients with ahi 5 ( considered as controls without osa ) , group 2 was ahi between 5 and 15 , group 3 was ahi between 15 and 30 , and group 4 was ahi 30 .
values of oxidative stress - related markers according to osa severity are shown in table 3 . by anova test we found significant difference in serum level of cu and hscrp between groups 1 and 4 ( figure 3 and figure 4 ) .
post - hoc tukey hsd testing verified the difference in level of cu between group 1 ( 803.2 ng / ml  113.4 ) and group 4 ( 1045.8 ng / ml  170.1 ) , p=0.023 .
there was also significant difference between serum levels of hscrp between groups 1 and 4 ( 0.9 g / ml  0.8 vs. 4.8 g / ml  3.6 ) , p=0.039 .
oxidative stress is a condition resulting from an imbalance between the prooxidative state and the antioxidant defence .
it is difficult to measure levels of ros , mainly because of their extreme instability and biochemical reactivity , so it is easier to measure oxidative stress indirectly by ros effects on other molecules ( changes in structure , function and quantity of these molecules ) .
this study was designed to assess the impact of osa on novel parameters related to oxidative stress . in our study
higher bmi is associated with increased oxidative stress , independently of other parameters such as osa .
we found strong positive correlation for hscrp , mmp-9 and odi and spo2 < 90% .
we suggest that odi is a stronger predictor of oxidative stress level than is ahi .
the strongest correlation was demonstrated for mmp-9 , hscrp and cu and parameters of osa ( especially odi and spo2 ) .
mmp-9 , hscrp and cu levels are higher in severe osa patients ( according to odi ) and this correlation is further explained by association of ventilation parameters to bmi .
these markers could be used as general biochemical molecules associated with oxidative stress in obese patients .
obesity seems to be primary risk factor of the increased oxidative stress state . according to our results ,
the strongest biochemical markers of oxidative stress in osa patients are cu and hscrp , independently of bmi as verified by analysis of osa subgroups and using bmi as a covariate .
c - reactive protein ( crp ) is an inflammatory marker ; it is thought to be an acute phase reactant .
studies suggest that oxidative stress may have pro - inflammatory effects , but data on the relationship between oxidative stress and crp in healthy persons is sparse .
another study confirmed a positive correlation between crp and ahi and a negative correlation between crp and mean nocturnal saturation .
difference in serum level of hscrp and cu in patients without osa ( ahi 5 ) and patients with severe osa ( ahi 30 ) remains significant even after correction for bmi .
therefore these 2 markers could be used as parameters of oxidative stress in osa , independently of bmi .
increased cu level in severe osa patients and obese patients seems to be a strongly associated factor , but its significance is not well explained .
cu and zn are trace elements important for functioning of the antioxidase system , especially as part of sod .
mmps have a primary function in degradation of the extracellular matrix proteins , and play a role in normal tissue remodeling .
they also perform essential functions at the cell surface involved in signaling , cell survival , and cell death .
mmp-9 was found higher not only in the brain after reperfusion injury but also after liver hypoxia . in our study mmp-9 level
was associated with severity of osa according to odi and mean spo2 and spo2 < 90% .
the interaction between rage and its ligands can cause activation of pro - inflammatory genes by activation of nuclear factor nf-b .
isoforms of the rage protein , which lack the transmembrane and the signaling domain , are commonly referred to as soluble rage ( srage ) , a naturally occurring inhibitor of age - rage action .
there was shown to be a higher level of ages in osa patients , dependently on osa severity .
we found that srage level is associated with ahi , odi and also negatively associated with bmi .
thus we can propose the hypothesis that srage protects against oxidative stress and its level in osa and obese patients is decreased .
osa is an independent risk factor in obesity for increased oxidative stress state as shown by diminishing of some oxidative stress - related markers after cpap therapy .
it is apparent that our group of patients is not homogenous as concerned the comorbidities .
oxidative stress is thought to be associated with many factors including obesity , smoking , age , hypertension , diabetes , and dyslipidemia .
therefore assessment of the relationship between osa and oxidative stress intensity may be influenced by such confounding variables .
osa , unlike obesity , is a treatable risk factor of systemic inflammation and oxidative stress .
our study provides evidence of increased oxidative stress in osa patients , predominantly in obese ones . in patients with severe osa ( ahi over 30 ) , this effect is additive to the influence of obesity .
 summarybackgroundobstructive sleep apnea ( osa ) is a condition contributing to oxidative stress . 
 the aim of this study was to ascertain if there is any connection between osa and novel oxidative stress - related markers . 
 matrix metalloproteinases 2 and 9 ( mmp-2 , mmp-9 ) , high sensitive c - reactive protein ( hscrp ) , pregnancy - associated plasma protein - a ( papp - a ) , soluble receptors for advanced glycation end - products ( srage ) , zinc ( zn ) and copper ( cu ) were measured . 
 further biochemical markers were evaluated.material/methodsfifty-one men suspected for osa indicated for night polygraphy were included . 
 apnea / hypopnea index ( ahi ) , oxygen desaturation index ( odi ) , mean blood hemoglobin oxygen saturation ( spo2 ) and time of blood hemoglobin oxygen saturation below 90% ( spo2 
 < 90% ) were measured . 
 morning venous blood samples were taken.resultsfor body mass index ( bmi ) we found strong positive correlation with levels of cu , mmp-9 , hscrp and fibrinogen , and negative correlation with srage . concerning ventilation parameters , we found positive correlation of odi and spo2 < 
 90% with markers mmp-9 and hscrp . srage level correlated with ahi and odi negatively . 
 spo2 correlated negatively with cu , mmp-9 , hscrp and fibrinogen . 
 there was no correlation between ventilation parameters and markers mmp-2 , papp - a and zn . compared to severity of osa , there was significant difference in levels of hscrp and cu between patients with ahi 5 and ahi 30 independent of bmi.conclusionsmmp-9 , hscrp , srage and cu seem to be strong predictors of oxidative stress in osa patients . 
 the strong correlation between oxidative stress - related markers and osa is elucidated by connection of these to bmi , which is probably a primary condition of oxidative stress , but osa is an additive condition . 
Background
Material and Methods
Study protocol
Sleep study
Biochemical examination
Statistical analyses
Group characteristics
Results
Discussion
Conclusions