cystatin c is a nonglycosylated , low molecular weight ( 13.250  da ) , basic protein that is a member of the cystatin superfamily of cysteine protease inhibitors [ 13 ] .
it consists of 120 amino acids , it is produced by all nucleated cells at a constant rate , and it is excreted by the kidneys by free glomerular filtration and complete tubular reabsorption and degradation [ 46 ] .
therefore , serum concentration levels of cystatin c are almost totally dependent on the glomerular filtration rate and  unlike serum creatinine levels which increase after glomerular filtration rate has fallen by approximately 50%even a slight reduction in glomerular filtration rate causes a rise in serum cystatin c [ 7 , 8 ] .
besides its usefulness as a marker of renal function , serum cystatin c appears to be a prognostic marker of cardiovascular events and death among elderly persons without chronic kidney disease [ 9 , 10 ] .
therefore , it is important to establish reference values of cystatin c not only for nephrologists , but for cardiologists as well . in this study , serum cystatin c concentrations were measured in a healthy greek adult population ,   and reference intervals were derived after taking under consideration sources of variation for this population .
between april 2009 and january 2010 , a total of 490 consecutive adults ( 85% participation rate ) , who had visited the  polykliniki  general hospital for an annual health check , agreed to participate in the study .
the retrieved data were confidential ,   and the study followed the ethical considerations provided by the world medical association ( 52nd wma general assembly , edinburgh , scotland , october 2000 ) .
moreover , the institutional review board approved the design , procedures , and aims of the study ( ga 23/14.05.2009 ) .
all participants were informed about the procedures of the study and agreed to participate providing written informed consent .
subjects who reported chronic diseases such as renal failure , diabetes mellitus , cardiovascular diseases , cancer , thyroid dysfunctions , or pregnancy were excluded as well as those treated with drugs that may influence renal function or cystatin c concentrations ( i.e. , antihypertensives , diuretics , antiinflammatory agents , hypoglycemic agents , anticonvulsants ,   and antibiotics ) .
other exclusion criteria were : ( 1 ) fasting serum glucose 126  mg / dl , ( 2 ) systolic blood pressure 140  mmhg or diastolic blood pressure 90  mmhg , ( 3 ) body mass index ( bmi ) 18.5 or 30 , to avoid the extremes of body size , and ( 4 ) egfr ( glomerular filtration rate ) < 60/ml / min/1.73  m , a threshold defining chronic kidney disease ( ckd ) by the independent international kidney disease improving global outcomes organization ( kdigo ) using the simplified modification of diet in renal disease study ( mdrd ) equation [ 11 , 12 ]
. therefore , 279 individuals ( 40  13 yrs , 37% males ) fulfilled the above - mentioned criteria and were found eligible to participate in the study .
participants were classified to the following age categories : 1844  yrs , 4564  yrs , and 65  yrs .
waist circumference and height ( without shoes ) were measured to the nearest 0.5  cm , and weight was measured with a lever balance , to the nearest 100  g , without shoes , in light undergarments . body mass index ( bmi ) was then calculated as weight in kilograms divided by the square of standing height in meters .
participants were then classified in those with normal values of bmi ( i.e. , < 25  kg / m ) and to overweight / obese ( bmi  25  kg / m ) .
with respect to lifestyle characteristics , participants were asked to fill in a 10-grade scale range regarding their physical activity status ( grade of scale used : 110 , where 1 denotes sedentary lifestyle and 10 daily hard activity of at least 30 minutes ) .
participants with score 6 were classified as with low / moderate activity , while those with score > 6 were considered as highly active .
alcohol consumption was assessed as the self - reported number of drinks per week and participants were categorized as never / rare ( i.e. , 0 - 1 drink / week )   and current drinkers ( i.e. , > 1 drink / week ) .
systolic and diastolic blood pressures , together with fasting serum glucose , serum creatinine ,   and cystatin c , were measured in all participants .
blood pressure was measured by the same physician using a standard mercury sphygmomanometer on the right arm of the seated subject .
venipuncture was performed for each participant , early in the morning ( between 07:00  am and 11:00  am ) , after a 12-hour fasting period by applying a natural latex rubber strap and using a 20  ml syringe .
samples were left undisturbed for 20 minutes to clot and then centrifuged at 4000 rotations per minute ( relative centrifugal force : rcf 2.7 ) for 10 minutes so as to obtain serum .
glucose was determined via an enzymatic colorimetric test ( glucose oxidase pap , trinder endpoint reaction , god - pap ) .
serum creatinine was determined via a kinetic colorimetric assay based on the reaction of creatinine with picric acid in alkaline solution and cystatin c via a particle - enhanced immunoturbidimetric assay . in the latter case ,
human cystatin c agglutinates with latex particles coated with anticystatin c antibodies , and the aggregate is determined turbidimetrically at 546  nm .
reproducibility in the lab has been determined using human samples and controls in an internal protocol .
for the above mentioned tests , within run and between day , coefficients of variation ( cv ) were less than 8% .
two levels of control sera were used for these assays ( precinorm u plus 12149435122 & precipath u plus 12149443122 for glucose and serum creatinine , cystatin c control set 04975936190 for cystatin c ) .
control recovery for all tests was very close to the recommended target values ( tv  5% ) .
accuracy of results is further supported by participation in suitable external quality assurance program for glucose and serum creatinine ( eseap ) .
reagents , calibrators , controls , and consumables were purchased from same supplier ( roche diagnostics gmbh , sandhofer strae 116 , d-68305 mannheim , germany ) .
continuous variables are presented as mean  standard deviation and categorical variables as absolute ( n ) and relative frequencies ( % ) .
comparisons of continuous variables between groups of study were performed using the independent samples t - test and the analysis of variance , after controlling for the normality of the distribution .
distribution of cystatin c levels was presented for the 2.5th , 5th , 10th , 25th , 50th ( median ) , 75th , 90th , 95th , and 97.5th percentile .
subgroups analyses were performed by gender , age category ( i.e. , 1844 , 4564 , and 65 years old ) , obesity status ( < or 25  kg / m ) , current smoking ( yes versus   no ) , alcohol drinking ( yes versus no ) , and physical activity status ( low / moderate versus   high ) . all statistical analyses were performed using the spss version 14 ( spss inc . ,
thirty - seven percent of participants were male , while the mean age between genders did not differ ( 40  14 for males , 40  13 for females , p = .81 ) .
differences were not observed as well in physical activity status between genders ( i.e. , 5.8  2.1 for males versus   5.9  2.3 for females , p = .91 ) .
percentage of current smokers was similar between males ( 34% ) and females ( 36% ) ( p = .79 ) .
approximately 2/3 of males and 27% of females were considered as overweight / obese ( p < .001 ) , while 85% of males and 73% of females were current drinkers ( p = .02 ) .
cystatin c levels varied from 0.50 to 1.21  mg / l among males and from 0.52 to 1.14  mg /
in addition , cystatin c levels were normally distributed among the 176 healthy women and 103 healthy men that comprised the studied population ( figure 1 ) .
the distribution of cystatin c levels among age groups between the two genders is presented in table 1 . in general , higher levels were observed for males as compared to females in all age groups .
mg / l among males aged 1844  yrs , from 0.52 to 1.21  mg / l among males aged 4564  yrs , and from 0.83 to 1.18 
mg / l among males over 65  yrs . among females , the cystatin c levels varied from 0.52 to 1.05 ,
0.61 to 1.12 , and 0.74 to 1.14  mg / l , respectively , for each age group ( table 1 ) . the distribution of cystatin c levels was furthermore examined according to obesity and current smoking status .
levels of cystatin c varied from 0.52 to 1.12  mg / l for participants with bmi < 25  kg / m and from 0.50 to 1.21  mg / l for overweight / obese participants .
moreover , cystatin c levels varied from 0.50 to 1.21  mg / l for never or ex - smokers and from 0.52 to 1.18  mg /
regarding    the distribution of cystatin c levels according to physical activity status , this varied from 0.50 to 1.18  mg / l among participants with low physical activity and from 0.52 to 1.21  mg / l among highly active participants .
differences in mean values of cystatin c levels were observed between genders , age group , smoking , and obesity status . in particular , higher values were noticed among males ( p = .04 ) , older participants aged over 65  yrs ( p <
.001 ) , current smokers ( p < .001 ) , and overweight / obese participants ( p = .03 ) .
in contrast , cystatin c levels did not seem to differ regarding alcohol drinking status and physical activity status ( p = .61 , p = .95 , resp . ) ( table 3 ) .
various physiological sources of variation for serum cystatin c such as age , gender , bmi , cigarette smoking , and alcohol consumption have been reported based on analysis of healthy adult populations .
most studies have shown age - related differences in serum cystatin c , demonstrating that its levels increase with age [ 1317 ] .
these studies have documented a variation in cystatin c levels as a function of age , probably due to the physiological aging of renal function [ 1820 ] . in this study ,
higher values were noticed among older participants aged over 65 yrs ( p < .001 ) .
such differences were significant in some studies , especially for adults below 60 years of age [ 2022 ] , but not significant in others [ 13 , 23 ] . in this study ,
gender differences in cystatin c were revealed , with higher values of cystatin c among males as compared with females .
the findings of this study , regarding the influence of smoking in the levels of cystatin c , have been also confirmed by other studies [ 13 , 24 ] .
it seems likely that smoking is an independent source of variation for cystatin c , although the mechanism is not known .
previous studies with respect to positive association of cystatin c levels with bmi support the above mentioned results of this study [ 25 , 26 ]
. specifically , higher values of cystatin c levels among overweight / obese individuals in comparison with normal weight individuals were revealed .
laboratory studies have examined the expression of cathepsin s as a new biomarker of adiposity and have shown that human adipose tissue secretes and expresses cathepsin s , which is upregulated in obesity .
it has been found that cystatin c secretion increased and cathepsin s decreased during preadipocyte differentiation , suggesting a possible role of cystatin c in adipogenesis .
alcohol consumption and physical activity did not seem to influence cystatin c levels in the studied population .
this is in accordance with most studies that have been conducted until today [ 13 , 29 , 30 ] .
as it is clearly mentioned on the package insert of the commercial assay for cystatin c  each laboratory should investigate the transferability of the expected values to its own patient population and if necessary determine its own reference ranges . 
this is  to the best of our knowledge  the first study for determination of cystatin c reference values in greek population . in the era of newly   discovered properties and clinical significance of cystatin c ,
factors such as age , gender , bmi ,   and cigarette smoking need to be considered when interpreting serum cystatin c values even in a carefully selected healthy adult population .
 
 background . 
 the aim of the present study was to examine sources of variation for serum cystatin c in a healthy greek population . 
 methods . cystatin c together with basic clinical chemistry tests was measured in a total of 490 adults ( 46  16  yrs , 40% males ) who underwent an annual health check . 
 demographic , anthropometric , and lifestyle characteristics were recorded . 
 results . 
 higher values of cystatin c were observed among males ( p = .04 ) , participants aged over 65 years ( p < .001 ) , current smokers ( p = .001 ) and overweight / obese participants ( p = .03 ) . on the contrary , alcohol consumption and physical activity seemed to have no influence on cystatin c levels ( p = .61 ; p = .95 , resp . ) . 
 conclusions . in interpreting serum cystatin c values in a healthy adult population , age , gender , body mass index , and cigarette smoking need to be considered , and determination of reference ranges among distinct subpopulations seem to be prudent . 
1. Introduction
2. Materials and Methods
3. Results and Discussion
4. Conclusions