Serum and Saliva Concentrations of Biochemical Parameters in Men With Prostate Cancer and Benign Prostate Hyperplasia

Hyder Farahani, PhD; Mona Alaee, MSc; Jamal Amri, MSc; Mahmoud-Reza Baghinia, MD; Mohammad Rafiee, PhD


Lab Med. 2020;51(3):243-251. 

In This Article

Materials and Methods

Study Population

This case-control study was performed on 40 male Iranian patients aged 50 to 55 years. The study subjects were divided into 2 groups: case individuals (20 subjects with PC) and control individuals (20 subjects with BPH) who were admitted to Khansari Hospital, a referral center in Arak, Iran. All participants provided written informed consent for participation in the study. The study protocol was approved by the ethics committee of the Arak University of Medical Sciences (ethics permission number IR.ARAKMU.REC.1394.2).

Inclusion and Exclusion Criteria

The inclusion criteria for the PC group included age between 50 and 55 years; histologically confirmed PC; and had not received any chemotherapy, radiotherapy, or surgical procedures; absence of specific diseases, such as other malignant diseases and infections; and had not had any oral or dental diseases. Inclusion criteria for the BPH group included age between 50 and 55 years; histologically confirmed BPH; absence of previous malignant neoplasms; lack of specific diseases, such as infections; and no history of oral or dental diseases. The exxclusion criteria for the PC group included having metastatic disease or already-treated tumors, previous chemotherapy or radiotherapy, having had a history of systemic diseases, and existence of oral or salivary-gland diseases. Exclusion criteria for the BPH group included diagnosed PC and existence of oral or salivary-gland disorders or systemic diseases.

Blood and Saliva Specimen Collection

For ethical reasons, we collected only general characteristics of participants. Also, we collected 5 mL of whole blood from a peripheral vein in each participant. Then, the blood was allowed to clot in a clean glass tube for 30 minutes at room temperature. After clotting, the blood specimens were centrifuged at 3000 rpm for 5 minutes to obtain a clear serum, which was stored at −70°C.

In this study, unstimulated saliva was collected as follows: each participant was asked to clean their own lip area and refrain from drinking, eating, smoking, and oral-hygiene procedures for 2 hours before saliva collection. Then, participants were asked to rinse their mouths with plain water several times and to sit for approximately 5 minutes before saliva collection. Generally, the time of specimen collection was 5 minutes; each participant donated 5 to 10 mL of saliva. The saliva specimens were centrifuged at 3000 rpm for 10 minutes to obtain a clear supernatant, which was stored at −70°C until the time of assay.

Analysis of Salivary and Serum Parameters

Serum and salivary concentrations of PSA, B2M, CK-BB, and MT were determined using the sandwich enzyme-linked immunosorbent assay (ELISA) kit in accordance with the manufacturer-provided instructions. These kits were designed to quantify PSA, B2M, CK-BB, and MT concentrations in biological fluids (serum, saliva, cell lysate, and urine). Specimen absorbance at 450 nm was measured with an ELISA reader (ELX 800 TM ELISA reader; BioTek Instruments, Inc). The serum and salivary creatinine (Jaffe method) and urea (ultraviolet [UV] kinetic method) were measured spectrophotometrically using commercial kits. Zinc concentrations in serum and salivary specimens were determined using a commercial chemical colorimetrical assay kit. Finally, the specimen absorbances were assessed at 500, 578, and 425 nm using a spectrophotometer (Jenway 6505, European Union model; Cole-Parmer) for creatinine, urea, and zinc, respectively.

Statistical Analysis

The Fisher exact and Pearson χ2 tests were used to analyze the descriptive information of the PC group versus the BPH group. The normal distribution of data was assessed using the D'Agostino test. Because the data had been non-normally distributed, we used the Mann-Whitney U test to calculate and compare the means (SEMs) of saliva and serum parameters separately for each group. Spearman correlation coefficients (non-normally distributed data) were used to determine the relationship between serum and salivary parameters. We applied receiver operating characteristic (ROC) analysis to appraise the diagnostic potential of salivary biochemical markers, compared with serum, and to correctly separate the participants into cases and controls (ie, to find out whether salivary PSA, B2M, CK-BB, MT, zinc, creatinine, and urea concentrations can distinguish patients with PC from those with BPH).

The cutoff-values curve was determined based on the best trade-off between the sensitivity and specificity, and the overall performance of the test was evaluated by the total area under the curve (AUC). A P value of less than .05 was considered statistically significant. All statistical analysis was performed using GraphPad Prism software (Version 6.00; GraphPad Software) and MedCalc software (Version 6.00; MedCalc Software bvba).