The Impact of Demographic Characteristics and Lifestyle in the Distribution of Cystatin C Values in a Healthy Greek 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.


Introduction
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 [1][2][3]. 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 [4][5][6]. 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.

Participants.
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 2 , 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 abovementioned criteria and were found eligible to participate in the study.

Other Characteristics.
Participants were classified to the following age categories: 18-44 yrs, 45-64 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 2 ) and to overweight/obese (BMI ≥ 25 kg/m 2 ). 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: 1-10, 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). Current cigarette smoking (yes, no) was also recorded.

Biochemical Characteristics.
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. Blood was immediately transferred to two tubes without anticoagulant (Greiner Vacuette, Cat. no. 455071). 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.

Laboratory Analyses.
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). All measurements were performed on a Roche/Modular Analytics analyzer. Reagents, calibrators, controls, and consumables were purchased from same supplier (Roche Diagnostics GmbH, Sandhofer Straße 116, D-68305 Mannheim, Germany).

Statistical Analysis.
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. Associations between categorical variables were tested using the chi-square test. 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., 18-44, 45-64, and ≥65 years old), obesity status (< or ≥25 kg/m 2 ), 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., Chicago, Il, USA).

Distribution of Cystatin C Levels.
Cystatin C levels varied from 0.50 to 1.21 mg/L among males and from 0.52 to 1.14 mg/L among females. 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. Specifically, cystatin C varied from 0.50 to 1.10 mg/L among males aged 18-44 yrs, from 0.52 to 1.21 mg/L among males aged 45-64 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 2 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/L for current smokers (Table 2). 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. In addition, cystatin C levels varied from 0.52 to 1.10 mg/L among never/rare drinkers and from 0.50 to 1.21 mg/L among drinkers (data not shown here).
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 agerelated differences in serum cystatin C, demonstrating that its levels increase with age [13][14][15][16][17]. Indeed, recent studies define reference values separately for adults and for older individuals. These studies have documented a variation in cystatin C levels as a function of age, probably due to the physiological aging of renal function [18][19][20]. In this study, higher values were noticed among older participants aged over 65 yrs (P < .001).
Gender differences in cystatin C levels have not been consistently observed. Such differences were significant in some studies, especially for adults below 60 years of age [20][21][22], 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 [27]. Cystatin C regulates cathepsin S activity by acting as an endogenous inhibitor. 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 [28].
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."

Conclusions
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. Determination of reference ranges among distinct subpopulations must be taken into consideration.