The Role of Prolidase Activity in the Diagnosis of Uremic Bone Disease

Abstract

The derangements in bone metabolism in patients with chronic renal failure (CRF) are summarized as uremic bone disease (UBD). In this study, we planned to determine the serum prolidase to compare it with the other biochemical markers. This study was performed on 44 patients (19 females, 25 males, mean age = 56.8 ± 15.6 years) with end-stage renal disease (ESRD). The patients were divided into three groups according to serum bone alkaline phosphatase (bAP) levels. The patients whose bAP was ≥77 U/L were accepted as having high-turnover UBD (n = 18), the patients whose bAP was ≤50 U/L were accepted as having low-turnover UBD (n = 14), and the patients whose bAP levels were between these two values were accepted as having bone disease with normal turnover (n = 12). The serum prolidase levels did not increase in patients with ESRD. There were no significant differences between the serum prolidase levels of patients according to types of the UBD (p > 0.05). Kidney is the most prolidase-rich tissue of the human body. The serum prolidase activity is low in all patients with ESRD, irrespective of the type of UBD. Therefore, we concluded that prolidase had no value in the diagnosis of UBD.

Keywords:

• prolidase
• bone alkaline phosphatase
• TRAP
• dialysis
• uremic bone disease

INTRODUCTION

Chronic renal failure (CRF) is a syndrome characterized by progressive and irreparable nephron loss. The kidney plays an important role in the regulation of mineral balance. In situations where kidney function is totally impaired, as in end-stage renal disease (ESRD), bone disorders, from mild to increasingly severe in degree, may occur.[1] These disorders in the bone, occurring as a result of renal failure, are generally grouped together under the heading uremic bone disease (UBD). Reliable and non-invasive methods that can be used in the measurement of bone metabolism in uremic patients are being researched. Prolidase (EC 3.4.13.9) is an enzyme that destroys the imidodipeptide proline (Pro) or hydroxyproline (Hyp) in the C terminal, and plays a role in protein digestion in the intestinal mucosa.[2] Some 90% of the bone matrix consists of type I collagen. Twenty-five percent of type I collagen contains Pro and Hyp.[3] In situations in which the collagen cycle accelerates, an increase is expected in serum prolidase activity. Prolidase activity is found in many tissues, including the kidneys, lower intestinal mucosa, uterus, brain, muscle tissue, erythrocytes, and plasma. The highest activity is in the kidney tissue.[4] Normal plasma prolidase level is below 1000 U/L. Levels in excess of 15,000 U/L have been reported in chronic liver patients.[5] Very few studies have been performed on whether or not prolidase activity can be used as an indicator in the determination of the bone cycle. To our knowledge, the prolidase activity in UBD sufferers has not been investigated. Our aim in this study was to compare serum prolidase activity with other UBD determinants.

MATERIALS AND METHODS

This study was performed on 44 end-stage renal disease (ESRD) patients on dialysis for more than 6 months (19 female, 25 male, average age 56.8 ± 15.6 years). The mean duration of renal replacement therapy (RRT) was 41.6 ± 41.8 months. Diabetes mellitus was the leading cause of ESRD (30%) (Table 1). All participating subjects gave informed consent. Serum prolidase, bone alkaline phosphatase (bAP), and tartrate resistant acid phosphatase (TRAP) levels were determined before a standard mid-week hemodialysis session. Venous blood samples were placed into a straight tube, serum was separated by centrifugation at 3000 rpm for 15 min, and specimens were conserved at −70°C. Prolidase activity measurement was carried out on a Beckman Coulter DU 500 device (Philadelphia USA) using a modified Myara technique.[6] The bAP was investigated manually on a Beckman BTS-310 spectrophometry device (Biosystem S.A., Barcelona, Spain) using the heat inhibition technique; and TRAP was investigated manually with a Beckman BTS-310 spectrophotometry device (Biosystem S.A., Barcelona, Spain) using PNPP substrate at acid pH. Patients were divided into three groups based on bAP levels. Patients with bAP levels ≥77 U/L (n = 18, average age 58.3 ± 13.0) were classified as high turn-over bone disease (Group HBD); patients with bAP levels ≤50 U/L (n = 14, average age 59.0 ± 15.9) were classified as low turn-over bone disease (Group LBD), and patients with bAP levels between these values (n = 12, average age 51.9 ± 18.7) were classified as having normal bone turn-over (Group NBD) (85, 87), and the prolidase levels of these groups were compared. Data were analyzed using the SPSS (Statistical Package for Social Sciences) for Windows 10.0. All data were expressed as mean values ± SD. Subgroup compatibility with normal distribution was examined for comparisons between groups. Student's t test was performed for the comparison of bone markers of each group, and data, which were not distributed normally, were analyzed by a Mann-Whitney U test. Pearson's method was used in correlation analyses. The confidence interval was 95% and changes were considered to be statistically significant at the p < 0.05 level.

Table 1 Characteristics of ESRD patients

No. of patients	44
Women	19 (43%)
Men	25 (57%)
Interval of age (year)	21–81
Age (years)	56.8 ± 15.6
Duration of RRT (month)	41.6 ± 41.8
Primary renal disease (%)
Diabetes mellitus	13 (30%)
Chronic glomerulonephritis	7 (16%)
Hypertension	6 (14%)
Chronic pyelonephritis	3 (7%)
Polycystic kidney disease	3 (7%)
Amyloidosis	2 (4%)
Urolithiasis	2 (4%)
Unknown origin	8 (18%)

RESULTS

Forty-four ESRD patients (25 male, 19 female) were included in the study. Characteristics of the ESRD patients are shown in Table 1, and case distribution according to UBD type is shown in Table 2. Group HBD, Group NBD, and Group LBD variables were compared with each other. Compared with those of Group NBD, bAP and TRAP levels were high in Group HBD (P < 0.05). When Group HBD was compared with Group LBD, there was no significant difference between age and prolidase levels (P > 0.05), but bAP and TRAP levels were significantly high in Group HBD (P < 0.05). No statistically significant difference was determined between Group NBD and Group LBD variables (P > 0.05). A positive correlation was determined between bAP levels and RRT duration (r = 0.371, P < 0.05) and TRAP (r = 0.485, P < 0.001) levels.

Table 2 The comparisons of the bone turnover markers between groups

Variable	HBD (bAP ≥77 U/L)	NBD (bAP 51–76 U/L)	LBD (bAP ≤50 U/L)
Age (years)	58.3 ± 13.1	51.9 ± 18.7	59.0 ± 15.9
Number of patients (%)	18 (40.9%)	12 (27.3%)	14 (31.8%)
Duration of RRT (months)	57.8 ± 49.5	33.3 ± 38.7	27.8 ± 26.8
Prolidase (U/L)	936 ± 185 (NS)	952 ± 152 (NS)	941 ± 176 (NS)
bAP (U/L)	180 ± 180 **	60 ± 8	38 ± 12
TRAP (U/L)	20.2 ± 13.6 ***	11.4 ± 6.6	10.7 ± 4.9

*Positive correlation was determined between bAP levels and RRT duration (r = 0.371, P < 0.05) and bAP (r = 0.955, P < 0.001) and TRAP (r = 0.485, P < 0.001) levels.

**bAP levels were significantly high in Group HBD (P < 0.05).

***TRAP levels were significantly high in Group HBD (P < 0.05).

(NS) Not significant.

Ω No statistically significant difference was determined between Group NBD and Group LBD variables (P > 0.05).

DISCUSSION

In this study we intended to investigate the relationship between serum prolidase levels and biochemical indicators in ESRD in order to determine UBD type. It has been reported that in studies on adult dialysis patients, plasma bAP levels greater than 77 U/L (20 ng/mL) were linked to histological or biological diagnosis of secondary hyperparathyroidism (sHPT) or HBD.[7],[8] In one study on 41 hemodialysis patients on whom bone biopsy was performed, a bAP level of less than 51 U/L was reported as showing 100% sensitivity in LBD estimation, 94% as specificity, and 72% as a positive predictive value.[9] Based on this knowledge, three groups were set up according to serum bAP levels. In many studies to date, bAP levels have been found to be more sensitive in the evaluation of bone renewal in CRF patients than total alkaline phosphatase (tAP), osteocalcin, and osteonectin.[7],[8],[10] It has been stated that bAP demonstrates a better correlation than tAP and intact parathormone (iPTH) with histomorphometric parameters as it does with osteoclast and osteoblast surfaces and bone trabecular density, metabolically the most active part of bone.[7],[8],[10],[11] In another study performed using electrophoretic methods, a positive correlation between serum TRAP and bAP levels was reported.[12] In our study, a positive correlation was determined between bAP levels and RRT duration and TRAP levels. In a study on a group of patients administered peritoneal dialysis, Couttenye et al. determined the strongest relationship between bAP and TRAP.[13] It is known that TRAP dephosphorylates various bone matrix proteins, such as osteopontin and sialoprotein.[7],[14] It has been shown in several studies that there is a correlation between serum TRAP activity and osteoclast number and percentage of eroded bone surface.[7],[15],[16] iPTH exhibits a good correlation with bone histomorphometric parameters, but it fails to provide enough information for definitive diagnosis.[17],[18] iPTH was not, therefore, adopted as an analysis criterion in our study.

In the same way that no studies on serum prolidase activity in UBD patients were encountered in the literature, there are very few studies on whether it can be used as an indicator in the determination of the bone cycle. It was determined in a study by Iyidogan et al. that serum prolidase activity increases in postmenopausal women compared to premenopausal women. It was thought that this increase may be linked to bone collapse occurring postmenopausally.[2] Erbagci et al. found lower serum prolidase activity in osteoporotic patients diagnosed Type 2 DM compared to healthy controls.[19] In our study, serum prolidase levels were not found to have a correlation with other study variables (P > 0.05). No statistically significant difference was determined among the groups in comparisons according to UBD types (P > 0.05).

The highest prolidase activity in the body is in the kidney. The functional kidney tissues of ESRD patients, and therefore prolidase activity being at a low level independent of type of UBD, led us to think that prolidase was of no value in UBD diagnosis. However, the number of patients in this study was small and bone biopsy was not performed. Therefore, it was concluded that the clarification of the diagnostic value of prolidase would be more appropriate through wider ranging studies also including bone biopsy.