Serum Cys C Assessment in Healthy Indian Population and Chronic Kidney Failure Individuals – A Case Control Study

Introduction

Cys C is from the cysteine protease family. The biochemical characteristics of Cys C enables the molecule to be liberally sieved by the glomeruli. Afterwards, Cys C is also reabsorbed and completely catabolized in the proximal tubule of the nephron. Therefore, no Cys C is returned to the blood, and it is evident that the obtainability of Cys C in human blood is closely related to the biofunctionality of kidneys and thus plays a major part in the identification of renal diseases. Increased Cys C concentration in human blood may indicate a reduced GFR, most likely due to renal diseases.[1, 2, 3] Increase in protein catabolism and dietary factors do not affect the production rate of Cys C. Furthermore, studies have shown that Cys C does not change with increasing age or muscle mass, unlike plasma creatinine. Cys C is therefore identified as an ideal biomarker in individuals with enduring renal impairment.[4, 5, 6] Serum Cys C is a promising alternative for serum creatinine (SCr) in the evaluation of renal function, as part of renal function test (RFT).[10,11] In disorders affecting the filtration rate of kidney, Cys C levels begin to rise proportionately. Creatinine formation depends on and changes significantly in accordance with muscle mass of the body and the diet. Creatinine is filtered in the glomeruli and is also secreted by the renal tubules, contributing to total creatinine excretion. These factors illuminate why creatinine levels may not be a dependable parameter for prompt determination of renal disorders.[2, 3] For these reasons, serum Cys C is recommended as a supreme marker for renal impairment. [12] Cys C has been widely used as a research tool for understanding how kidney function affects health outcomes. In the past several years, researchers have shown a broader interest in Cys C as a clinical test for kidney function. These studies have had direct influence upon the 2012 KDIGO (Kidney Disease: Improving Global Outcomes) clinical practice guidelines relating to the evaluation and management of chronic kidney disease (CKD).[13,14]

The present study focused on assessing and comparing serum Cys C between healthy subjects and individuals with chronic kidney disease (CKD).

Materials and Methods

Sample collection

Healthy subjects (n=30) visiting Padmashree Diagnostic Center, Vijayanagar, Bengaluru for a general checkup were enrolled in the study after explaining all details and obtaining a written informed consent and confirming normal RFT values. Thirty subjects (n=30) with elevated serum creatinine values constituted the CKD disease population. Demographic data including age, sex, weight, etc., of study participants were recorded. The study proposal was approved by Padmashree Institutional Ethics and Review Board.

Subject Selection 

Specimen handling Fresh plain blood specimen was collected and subjected to centrifugation at 2000 rpm for 15 min and clear serum supernatant was used for assay of Cys C. If there was a delay in testing, the specimen was stored at 2-80 C until further analysis. Hemolyzed, icteric, or highly turbid serum specimens were rejected from sample processing. Aliquot of specimen, once thawed, was used for analysis on the same day, and was not subjected to repeated freezing and thawing, to avoid pre-analytical errors. Analytical Method - Latex Enhanced Immunoturbidimetry Assay principle: Serum sample was made to react with a buffer and anti-Cys C coated latex. The rise in turbidity due to formation of antigen antibody complexes during the reaction was measured at 570 nm. By constructing a typical curve from the absorbance of the standards, Cys C concentration of sample was decided. Statistical Analysis Data were analyzed using SPSS Version 19. All values were expressed in mean ± SD. Statistical comparison was performed using student t test, and p<0.05 was considered significant. Results The study population contained of 30 normal subjects (n=30), (Figure 1). Biochemical investigations such as Cys C and creatinine were performed. Serum Cys C levels of healthy individuals were compared with the available reference interval. [BSBECYS C ASSAY KIT MADE IN CHINA] Because establishing the normative data for subjects suffering from renal failure is constrained by ethical clearance, the present study adopted the available reference interval from literature. In this research study, Cyst C levels were assessed in normal subjects. It was observed that levels of sCys C was in the range of 1.03 ± 0.11mg/L in the normal population, comparable to the normal range in the available reference interval.

The study population also consisted of a case group (n=30) of thirty subjects with CKD. The mean levels of serum Cys C was 1.03 ±0.3 mg/L and 4.3±0.34 mg/L in controls and CKD patients, respectively. On comparing serum Cys C levels obtained from controls and patients with CKD, the results underscore that there is a significant (p< 0.001) increase in the value of serum Cys C in CKD patients. Discussion Biochemical analysis of body fluids such as blood/ serum/plasma and urine provide a diagnostic window to evaluate the functional status of the body and study the quality of life of an individual. According to different studies, serum level of CysC is a stronger predictor of renal outcome than serum creatinine level.[1, 2, 3] In a study by Hoek et al, [16] serum Cys C provided better diagnostic accuracy than serum creatinine. KyhseAndersen et al [10] included 27 healthy controls and 24 patients with reduced GFR (<80 ml/min/1.73m2 ) and found a significantly better correlation of serum cys C with GFR and concluded that the diagnostic accuracy of serum Cys C was greater than that of serum creatinine. [10] Radovan et al [17] indicate that serum Cys C is a reliable marker of GFR in patients with mild to moderate impairment of kidney function. The current guidelines emphasize the need to assess kidney function using predictive equations rather than just serum creatinine.[15]

Unfortunately, the commonly assessed parameter for kidney wellbeing is always creatinine clearance and serum creatinine, along with GFR. One of the main reasons include lack of standardization of available kits for serum Cys C, lack of familiarity with the test and costs of Cys C measurement as compared to creatinine measurements.[21] There have been very few Indian studies evaluating the utility of Cys C as a marker for renal function.[18, 19, 20] Therefore, the present study was an attempt to evaluate the utility of Cys C in an Indian sub-population. In this study, nearly 300 percent increase in serum Cys C values was seen in CKD subjects compared to the normal population. Serum Cys C detection and its diagnostic sensitivity suggests the test may soon be a substitute for creatinine estimation as a biochemical marker of choice for monitoring CKD in daily clinical practice. This study also attempted to evolve a reference range in the Indian sub-population, however, this aim requires a higher sample size. However, it is evident from the present investigations that the use of the Cys C diagnostic kit should be according to baseline Cys C levels in a particular area. It also suggests that we need to follow Indian Standard Operating Procedures for manufacturing a diagnostic kit. The findings from this study signify that serum Cys C parameter serves as a better diagnostic index regarding the underlying pathophysiology of kidney diseases. Conclusion To conclude, serum Cys C levels are significantly higher in CKD patients. There are many imperative research questions to be addressed, as work with Cys C moves forward with a focus on clinical decision making. This study proposes that Cys C assay is not a tiresome, time consuming test, nor requires a tertiary care laboratory set up. It is a simple spectrophotometric assay and can be included in a routine health check-up. This work is hoped to result in enhanced plans for observing and handling kidney disease, to reduce its impact on the fitness of those living with the disease. However, the study must be done in a larger population to establish more impactful study results. Conflict of Interest All the authors have contributed equally. The authors of this study have no financial interest nor received any financial support for this research work.

Acknowledgement This research was supported by Padmashree Institute of Medical Laboratory Technology, Padmashree Institute of Clinical Research and Padmashree Diagnostics. We thank all the authors who provided us insights and their expertise, which greatly assisted the research.