1 Department of Nephrology and Transplantation, 2 Department of Urology and 3 Department of Laboratory Medicine, University Hospital, Malmö and 4 Department of Urology, University Hospital, Huddinge, Sweden
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Abstract |
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Methods. The study included 20 men on intermittent haemodialysis with low-flux membranes and 25 men on CAPD, without known history of prostate cancer. The control group included 3129 men without known prostate cancer. We analysed fPSA and tPSA in serum by dual-label immunofluorometric assays, from which we calculated %fPSA and cPSA. Serum levels of different PSA forms were adjusted for age and presented as geometric means.
Results. Percent fPSA was significantly higher in patients on either haemodialysis (39.5%) or CAPD (39.6%) compared with controls (28.1%). Haemodialysis patients, but not CAPD patients, had significantly higher mean levels of fPSA. Levels of tPSA and cPSA for haemodialysis or CAPD patients did not differ significantly compared with controls.
Conclusions. Recommended reference ranges for %fPSA, based on men with normal renal function, do not apply to uraemic men on dialysis. In these men, a high %fPSA should not be considered as a sign of benign disease. This is clinically important in the evaluation of dialysis patients for transplantation, as %fPSA is often used as a tool for detection of prostate cancer.
Keywords: haemodialysis; peritoneal dialysis; prostate cancer; PSA; renal failure; renal transplantation
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Introduction |
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The transport of proteins across the glomerular barrier depends on the charge, shape and size of the molecule, as well as the charge and pore-size of the glomerular capillary wall [9]. Low molecular weight proteins (with molecular mass <4050 kDa) are readily eliminated from plasma by glomerular filtration followed by reabsorption and catabolism in proximal tubule cells. Patients with renal failure have a decreased clearance of low molecular weight proteins and their plasma level becomes elevated as renal function decreases. There are several factors that determine levels of plasma proteins in haemodialysis patients. Residual renal function and permeability of dialysis membranes are the main factors involved in the elimination of low molecular weight proteins [10,11]. In patients on continuous ambulatory peritoneal dialysis (CAPD), the efficiency of the dialysis per se, but also residual renal function, is of great importance for clearance of low molecular weight proteins [12]. Clearance of low molecular weight proteins over the peritoneum takes place through large pores. The shape, size and electrical charge of the proteins determine clearance rates.
PSA is a glycoprotein consisting of a single polypeptide chain with an isoelectric point 7. The low molecular mass of fPSA, its predicted globular conformation, and the neutral charge at physiological pH suggest elimination by glomerular filtration. Data from several different studies of fPSA elimination have reported half-lives of 1218 h, which also supports the hypothesis of renal elimination [13]. The route of elimination for cPSA is still uncertain, but reported data suggest mechanisms other than renal clearance due to the high molecular mass and remarkably slow elimination rates [13].
Some previous reports have suggested that patients with severe renal failure treated with haemodialysis may have elevated serum fPSA and higher %fPSA [14,15], but contradictory findings have also been reported [16,17]. Furthermore, it has been shown that haemodialysis may decrease the concentration of fPSA in serum following dialysis sessions performed with high-flux membranes, but not with low-flux membranes [18]. However, there are no previously reported data on CAPD patients and their levels of fPSA and cPSA forms.
Percent fPSA is often used in combination with tPSA to detect prostate cancer in patients with chronic renal failure before they are considered for transplantation and subsequent immunosuppression. We hypothesized that these patients may have a falsely elevated %fPSA, which could diminish the diagnostic usefulness of this parameter in the detection of prostate cancer. The purpose of the present study was to evaluate whether the reference ranges for %fPSA are applicable in patients on haemodialysis with low-flux membranes or CAPD.
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Subjects and methods |
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The haemodialysis group consisted of 20 men (median age 66 years; range 4284) on treatment for at least 1 month with low-flux membranes (Sureflux®, Nipro, Japan; Dicea®, Baxter, USA; Pro® and GFS®, Gambro, Sweden). Dialysis was performed three times weekly. All patients had a Kt/V>1.2. The duration of each dialysis session was from 4 to 5 h and dialysate flow was 500 ml/min, which gives a total dialysate amount of 120150 l during each treatment. The median accumulated treated blood volumes were 81 l (range 56120). Residual renal function was measured in 13 of 20 patients by iohexol clearance, showing a median residual renal function of 0 ml/min/1.73 m2 (range 01.4). Seven men with low urinary production were not evaluated by iohexol clearance. The CAPD group included 25 men (median age 65 years; range 4179) on dialysis for at least 1 month. Median residual renal function was 2.8 ml/min/1.73 m2 (range 08.9). The daily treatment regimen included four to five exchanges of 22.5 l of dialysis fluids, resulting in 812.5 l of dialysate per 24 h.
Informed consent was obtained from all patients.
Controls
In order to obtain a sufficiently large number of controls, we used a total of 3129 healthy men (median age 57 years; range 3380). We used male controls from three different studies to cover the age range of our dialysis patients in this study.
One control group consisted of 1680 healthy men (median age 63 years; range 5570) who had been randomly selected and volunteered to participate in 19881989 in a prostate cancer screening in Stockholm, Sweden [19]. Biopsy criteria were abnormal findings on either digital rectal examination or transrectal ultrasound, or tPSA 10 µg/l. In this group, 307 men underwent prostate biopsy without carcinoma being detected.
The second control group included 63 randomly selected elderly men (median age 73 years; range 7080) from a population study of men in Olmsted County, Minnesota, at the University of Michigan, USA. These men with no history of voiding problems underwent digital rectal examination, serum PSA testing and transrectal ultrasound without prostate cancer being detected [6].
Finally, a third group of controls came from a population study in Malmö, Sweden (the Preventive Medicine Project) enrolling 1386 younger men (median age 47 years; range 3361) who were without any diagnosis of prostate cancer, registered in the Swedish Cancer Registry, up to 23 years after base-line blood sampling [20].
Blood collection and pre-analytical work-up of samples
One blood sample was collected from each CAPD patient. Blood samples were taken both immediately before and after the haemodialysis treatment. The blood samples were allowed to clot for 30 min at room temperature and subsequently centrifuged at 2000 g for 15 min. The serum was then immediately frozen and stored at -20°C, pending analysis. Dialysate was collected 5 min after the start and 5 min before the end of a haemodialysis session. Dialysate was obtained from each bag during a 24 h CAPD session. The collected volumes of dialysate were concentrated 1030-fold before analysis by ultrafiltration on Diaflo membranes with cut-off ranges of 10 kDa (Amicon Corp., Danvers, MA, USA). All dialysates were stored at -20°C.
Analytical methods
A time-resolved fluoroimmunoassay (ProStatusTM PSA Free/Total assay) from Perkin-Elmer Life Sciences (Turku, Finland) was used to measure tPSA and fPSA from which we calculated %fPSA and cPSA (tPSAfPSA=cPSA). This commercial assay is based on a dual-label detection technique, which uses three distinctly uniquely binding and carefully characterized monoclonal anti-PSA antibodies. The lower limit of detection is 0.04 µg/l for fPSA and 0.05 µg/l for tPSA.
The patients in all control groups were analysed with similar assay methods using the same antibody combinations as those used in this study. Correlation coefficients for tPSA vs Tandem-R PSA in the control groups and in our study ranged from 0.97 [6] to 0.99 [19,21].
There were no available measurements of cPSA in all samples from the control groups. However, in our study patients, cPSA was analysed with the commercially available Automated Chemiluminescence System (ACS:180® cPSA assay) from Bayer Diagnostics (NY, USA). We performed simple linear regression analysis in the study group to evaluate whether calculated cPSA (tPSA minus fPSA) correlated with measured cPSA, in order to use calculated cPSA throughout the study.
Residual renal function
Glomerular filtration rates in haemodialysis patients were determined by measuring the plasma clearance of iohexol according to a one-compartment model [22]. Iohexol was analysed by a HPLC technique [23]. The coefficient of variation was 4.03.5% from the lowest standard (31 mg/l) to the highest standard (133 mg/l), respectively.
Patients on CAPD were evaluated regularly by individual peritoneal dialysis capacity (PDCTM). Residual renal function was obtained thereby from creatinine clearance.
Statistical methods
Logarithmic transformation of all variables was performed to fulfil the assumption for normality. For the same reason, our results are presented as geometric means.
As PSA levels are age dependent [6], the values of the different PSA forms were adjusted for age and thereafter expressed as percentage of controls. Statistical analysis comparing the differences between dialysis patients and controls was performed using an analysis of covariance (ANCOVA). A paired t-test was used in the comparison of PSA forms before and after a haemodialysis session.
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Results |
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Simple linear regression analysis between measured cPSA and calculated cPSA showed an excellent correlation (r=0.98) with the equation y=0.063+0.981x, supporting the use of calculated cPSA throughout this study to substitute for the lack of measured cPSA levels. The levels of cPSA in either haemodialysis patients (P=0.69) or CAPD patients (P=0.086) were not significantly different as compared with the controls, but there was a tendency toward lower levels of cPSA in CAPD patients.
Haemodialysis patients were found to have significantly higher serum concentrations of both fPSA and cPSA when serum was collected immediately after termination of the dialysis session, but %fPSA did not differ in samples collected before treatment as compared with post-treatment (data not shown). Finally, we found no detectable levels of the different molecular fractions of PSA in dialysates, either from patients on haemodialysis or CAPD.
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Discussion |
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Haemodialysis patients have been studied by other groups who also noted higher %fPSA [14,15]. However, other researchers have reported conflicting data [16,17] showing unchanged levels of %fPSA in the same category of patients. Yet another study recently presented findings that tPSA, fPSA and %fPSA were unchanged during haemodialysis and that the reference ranges for these parameters were applicable to patients on chronic haemodialysis [24]. It is noteworthy that these authors did not present any comparisons with controls.
As %fPSA involves both fPSA and cPSA, the ratio is dependent on the mechanisms and rates of elimination of these two molecular forms. Increased levels of fPSA as well as decreased cPSA levels result in increased %fPSA. Previously reported elimination kinetics data and the low molecular mass (28 kDa) of fPSA suggests clearance from plasma by glomerular filtration and that decreased renal function may lead to an increased serum level of fPSA, resulting in higher %fPSA. In this study, we show that patients on haemodialysis with low-flux membranes have significantly higher levels of fPSA in serum, indicating decreased elimination of fPSA in patients with severe renal insufficiency and no elimination over low-flux membranes. This is in accordance with the permeability of low-flux membranes considering their low cut-off point (5 kDa). We also found higher levels of fPSA and of cPSA in samples collected immediately post- vs pre-dialysis, which is most likely due to haemoconcentration during haemodialysis. Elimination by the kidneys was excluded due to the findings of very low residual renal function in our haemodialysis patients.
Sasagawa et al. [14] suggested that decreased levels of binding proteins might be the cause of the increased levels of fPSA and %fPSA reported by their group. Douville and Tiberi [15] found both increased levels of fPSA and decreased levels of cPSA as an explanation for the higher %fPSA. We could not demonstrate any significant decrease in cPSA compared with controls in this study as an explanation for the higher %fPSA. In addition, occurrences of up to 10-fold molar excess of -1-antichymotrypsin relative to PSA also makes it less likely that minor differences in blood levels of these binding proteins could significantly increase the fPSA levels in blood.
Our findings are the first to describe levels of fPSA and cPSA in patients on CAPD. The increased %fPSA in CAPD patients may be caused by several factors. The peritoneal membrane has a great variability in pore size. Molecular transport over the peritoneal membrane takes place through different pores; ultra small, small and large pores [12]. Low molecular weight proteins are eliminated through large pores. Our results show that fPSA in serum is not significantly increased in patients on CAPD. This may be explained by either elimination of fPSA over the peritoneal membrane or by renal elimination, or both. It is well known that CAPD patients usually preserve a minor residual renal function for a long time on dialysis compared with haemodialysis patients. This was also noted in our patients, although the residual renal function was very low. The significantly increased %fPSA in CAPD is most likely attributable to a combination of some decrease in levels of cPSA and increased levels of fPSA, even although the differences in either fPSA or cPSA levels were not found to be significant.
Our control material (n=3129) was obtained from three different cohorts. The reason for this is that we wanted to cover the entire age spectrum of our dialysis patients and be able to make age-adjusted comparisons of the serum levels of PSA forms between the dialysis patients and the controls. All analyses of the different PSA isoforms in our study patients and controls were made with the same assay technique and men with diagnosis of prostate cancer were excluded from the control groups. Based on the 5th percentile, the %fPSA considered abnormal is <15% according to Oesterling et al. [6]. This is very close to the %fPSA we found to be considered abnormal (<14%) using our control material of 3129 subjects. Based on our estimates of the 95th percentile for fPSA levels in our control material, five of 25 CAPD patients and seven of 20 haemodialysis patients presented with abnormal fPSA levels.
Our findings are important for patients with chronic renal failure, for several reasons. Patients on haemodialysis have been reported to be at increased risk for prostate cancer [2] and are often considered for renal transplantation. Immunosuppression of transplanted patients is associated with an increased risk of malignancies. Before transplantation, an extensive evaluation of the potential recipient is performed to detect the presence of cancer and in most cases tPSA and %fPSA are used in screening for prostate cancer. However, our findings show that in patients with chronic renal failure on dialysis, a high %fPSA should not be considered as a sign of benign disease. The recommended reference range for %fPSA is based on men with normal renal function and thus not applicable to dialysis patients. In contrast, tPSA is not affected by either haemodialysis or peritoneal dialysis. In these patients, the detection of prostate cancer must be based on tPSA, the image at transrectal ultrasound and digital rectal examination.
There are no reports on the effect of different degrees of renal failure on PSA levels. Further studies are required to determine the level of renal function that is significant for reduction of fPSA elimination, and to obtain reference ranges for fPSA and %fPSA for these patient categories.
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Acknowledgments |
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Notes |
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References |
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