Serum Prostate-specific antigen and Alkaline phosphatase
concentration in Prostate cancer and Benign Prostatic
Hyperplasia correlated with their salivary concentration
Abstract
Objectives: In recent years, the use of saliva as a specimen, due to its non-invasive and easy access
increased. However, their roles in the diagnosis have not been confirmed yet. Therefore, we conducted to
study the relationship between serum, saliva PSA and ALP.
Methods: Our case and control study included 20 prostate cancer patients (PCa) and 20 benign prostatic
hyperplasia (BPH) subjects. The levels of PSA and ALP in samples were measured with enzyme-linked
immunosorbent assay and Biochemical kits, respectively. In this study, statistical tests of Mann-Whitney
test and Spearman correlation coefficients were used.
Results: Significant difference between PCa and the BPH in terms of PSA concentration in serum and
saliva were observed. As well as, the serum PSA concentration in PCa and BPH groups was positive and
statistically significant correlated with salivary PSA concentration (r=0.431, P<0.05), (r=0.510, P<0.05)
respectively. The ALP activity in serum was no statistically significant between PCa and the BPH groups.
While, in saliva were statistically significant between two groups. The correlation between salivary and
serum ALP concentration in PCa group and BPH group was positive and no significant (r=0.037,
P<0.05), (r=0.213, P<0.05) respectively.
Conclusion: According to the results of the present study, the sample of saliva can substitute for serum
in the diagnosis and monitoring of prostate cancer. However, further studies are recommended.
Keywords: PSA; ALP; Saliva; Prostate cancer; Benign Prostatic Hyperplasia.
Introduction
Prostate cancer (PCa) is the most common form of malignancy in elderly males and a major
cause of cancer deaths after lung cancer (1). Benign prostatic hyperplasia (BPH) is a noncancerous condition prostate, as a result of hyperplasia prostate stromal and epithelial cells which
often occurs naturally in older men(2). Prostate specific antigen (PSA) is a glycoprotein that is
the serine protease family also known as γ-seminoprotein and kallikrein III. This compound
mainly produced by the epithelial cells of the prostate gland. Blood contains two different forms
of PSA: free PSA and complex PSA (complexed with α1-antichymotrypsin). Normally, most of
them are complexes (80-90%) and the remainder as uncomplex (3). According to previous
studies measuring the total PSA can be useful aids in the diagnosis, screening, prognosis,
monitoring of PCa as well as differentiating it from BPH (4).
Alkaline phosphatase (ALP) is phosphor‑hydrolytic enzyme that hydrolysis phosphate groups
from molecules including, proteins, nucleotides and alkaloids. Although, this enzyme is present
throughout the body tissues but there are mainly in bone and liver tissue (5). According to a
study by Robinson et al (6), serum ALP levels are often elevated in patients with metastatic PCa,
as well as this enzyme with PSA can be useful for monitoring and prognosis the PCa (5).
The saliva produced by the salivary gland is including the submandibular, sublingual and parotid
glands. Saliva contains a large number of hormones, antibodies, enzymes, growth factors and
antimicrobial constituents(7, 8). Many of these compounds in saliva are obtained from the blood,
because salivary gland produce and secrete saliva utilizing blood materials (9-11). So, saliva can
show the body's physiological function such as serum(12). For example, Laidi et al (13), showed
that there was positive correlation between serum and saliva CA 15-3 concentrations.While, CA
15-3 was not produced by the salivary gland. Based on studies, saliva can be used to measure
Protein markers such as: CA 125 in ovarian cancer (14), EGF in breast cancer (15), M2BP,
MRP14, and catalase in oral cancer as well as nucleic acid molecules markers: such as hsa-miR21, hsa-miR-23a, hsa-miR-23b and miR-29c in pancreatic cancer (16), miR-203 in Colorectal
cancer (17) etc. Also, saliva have many advantages comparing to other bodily fluids: Collecting
saliva is inexpensive, non-invasive approach, no painful, safe and does not require the skill and
special equipment, also without the patient suffer can be several times the sampling (21-18).
Thus, the saliva can be used as a laboratory sample better than other bodily fluids such as urine
and serum. Therefore, the aim of this study was to determine whether there is a relationship
between serum and salivary concentration of the protein PSA and ALP in PCa with BPH.
Methods
Study design
Our case-control study that performed from May 2015 to September 2015, the study groups
included 20 cases (Pca) and 20Control (BPH) group, which were selected from hospital
Ayatollah Khansari in Arak, IR Iran. PCa consider as a group that has a high-serum PSA
concentration and BPH group were considered as a low-serum PSA concentration. According to
the protocol ethics committee on medical research, Arak University of Medical Sciences, which
was consistent with the Helsinki Declaration, all of the participants in the study were asked to,
completed a consent form of demographic and information questionnaire before sample
collection. The age of PCa and BPH subjects were matched; those with age ranging from 60 to
80 were selected. Sample size was calculated based on a pilot study and according to two mean
comparison formula.
The Inclusions and Exclusion criteria for the PCa group and BPH group were
as follows
Inclusions criteria
PCa group: with prostate cancer and without metastasis, chemotherapy and Surgery, capable of
giving informed consent, Lack of specific diseases (HIV , …) and no oral and dental disease.
BPH group: Without a history of prostate cancer that has been diagnosed by a specialist, capable
of giving informed consent, Lack of specific diseases (HIV,…) and no oral and dental disease.
Exclusion criteria
PCa group: with benign tumors or already treated, having metabolic, liver, oral and dental
disease
BPH group: with prostate cancer that has been diagnosed by a specialist, having metabolic, liver,
oral and dental disease
NOTE: The Inclusions and Exclusion criteria for this study was achieved by accurate laboratory
analysis and clinical history and under the supervision of consultant physician in urology
medicine.
Blood and Saliva sample collection
Blood was collected from the peripheral vein (5 ml) of each participant, then blood was put into
van cover tube and it was allowed to clot, after clotting the blood sample were centrifuged for 5
min at 3000 rpm to obtain clear serum which was stored at -70°C until estimation of serum PSA
and ALP. In this study, unstimulated saliva was collected as follows: Firstly, each participant
were asked to clean the lip area and refrain from drinking, eating, smoking or oral hygiene
procedures for 2 hr before their saliva collection, as well as rinse her mouth with plain water
several times and seat before collecting for ~15 min before to collection. In general, that the time
of sample collection was 5 minutes and each participant donated ~5–10 ml of saliva. All saliva
samples were centrifuged for 10 min at 3000 rpm to obtain clear supernatant which was stored at
-70°C for later determination of saliva PSA and ALP. All serum and saliva samples were aliquot
in tubes then coded for each test.
Measurement of PSA and ALP
The determination of total PSA was carried out using sandwich enzyme-linked immunosorbent
assay (ELISA) in accordance with the manufacturer’s instructions. The kit was purchased from
Bioassay technology laboratory (Shanghai, China), and is designed to quantify the PSA
concentration in biological fluids (serum, saliva, cell lysate and…). The minimum PSA detection
level reported by the manufacturer was 0.03ng/ml. Intra-and inter-assay coefficients of variation
were <8 and <10%, respectively. Sample absorbance at 450 nm was measured with an ELX 800
ELISA reader. Total ALP activities were measured by using PARS kit procedure (Tehran,
IR.Iran). Kinetic determination level of ALP were as follow reaction: colorless 4-nitrophenyl
phosphate converts into a yellow 4-nitrophenol and inorganic phosphate by ALP in samples,
finally the yellow solution absorbance at 405 nm wavelength was assessed by a
spectrophotometer. The minimum ALP detection level reported by the manufacturer was 3U/L.
Statistical analysis
The normal distribution of data was assessed using D’Agostino test. Mann–Whitney test was
employed to calculate and compared the means of non-normally distributed and standard error
deviation (SEM) of factors separately for each group of participant and between two groups.
Spearman correlation coefficients (non-normally distributed data) were used to determine the
relationship between serum, saliva PSA and ALP concentration for each person participating in
the study. All data are expressed as means ± SEM and a value of p < 0.05 was considered
statistically significant. All Statistical analysis was performed by the software Graph Pad Prism
software (Version 5.00).
Result
Demographic characteristics of the study groups are summarized in Table 1. The mean ± SEM
age of PCa and BPH participants were 63.95 ± 2.76 and 63.45 ± 2.61 years respectively. Which
was not significantly different between PCa and BPH groups (p<0.05). All the 40 participants
were married. The two groups in terms of smoking, alcohol consumption, and education level
were compared: 35% PCa and 20% BPH group were smoker but none of the two groups did not
consume alcohol. Based on the information obtained, 45% participating have a higher education
level (all were in control group). 35% of cases, 20% of control group were bachelor and 65% of
cases, 35% of control group were primary education.
Table 1: Demographic data based on participants questionnaire
Case(PCa)
Control(BPH)
n=20
n=20
63.95 ± 2.76
63.45 ± 2.61
0.8
0
45%
-
Bachelor
35%
20%
-
Primary
65%
35%
-
Married
100%
100%
-
Tobacco usage
35%
20%
-
Alcohol usage
0%
0%
-
Age( Year)
High
Education
p value
Data are expressed as mean ± SEM and P < 0.05 is statistically significant
PCa: Prostate cancer; BPH: Benign prostatic hyperplasia
The results showed that there was a significant difference between PCa and the BPH in terms of
PSA concentration in serum and saliva (Table 2). The PSA concentration in serum PCa (5.32 ±
1.23) were higher than serum BPH (1.52 ± 0.14), which was statistically significant (P<0.0002).
As well as, The PSA concentration in saliva PCa (1.77 ± 0.3) were higher than saliva BPH (0.59
± 0.36), that was statistically significant (P<0.0004). But in general, serum PSA concentration in
PCa and BPH groups, it was more of the salivary concentration.
Table 2: Mann–Whitney test comparing serum and salivary PSA concentration between PCa and
BPH groups.
Case(PCa)
Control(BPH)
p value*
n=20
n=20
PSA (Serum)
5.32 ± 1.23
1.52 ± 0.14
0.0002
PSA (Salivary)
1.77 ± 0.3
0.59 ± 0.36
0.0004
Data are expressed as mean (ng/ml) ± SEM and *P < 0.05 is statistically significant
PCa: Prostate cancer; BPH: Benign prostatic hyperplasia; PSA: Prostate specific antigen
The ALP activity in serum was no statistically significant between PCa and the BPH groups,
while, the ALP activity in saliva was statistically significant between two groups (Table 3).
However, The ALP activity in serum PCa (245.5 ± 60.29) were higher than serum BPH (173.0 ±
7.94), which was no statistically significant (P<0.05), as well as, the ALP activity in saliva PCa
(118.4 ± 17.23) were higher than saliva BPH (35.35 ± 16.03), but that was statistically
significant (P<0.05). Finally, according to study's findings ALP activity in serum and salivary
was higher in PCa compared to that of BPH.
Table 3: Mann–Whitney test comparing serum and salivary ALP activity between PCa and BPH
groups.
Case(PCa)
Control(BPH)
p value*
n=20
n=20
ALP(serum)
245.5 ± 60.29
173.0 ± 7.94
0.8
ALP(salivary)
118.4 ± 17.23
35.35 ± 16.03
0.001
Data are expressed as mean (ng/ml) ± SEM and *P < 0.05 is statistically significant
PCa: Prostate cancer; BPH: Benign prostatic hyperplasia; ALP: Alkaline phosphatase
A)
B)
6
8
PSA in saliva (ng/ml)
PSA in saliva (ng/ml)
The correlation between serum PSA and saliva PSA concentration of participants in the study are
shown separately in Figure 1. In PCa group (Figure1a), serum PSA level was correlated
significantly positively with saliva PSA (r=0.431, P<0.05).Moreover, in BPH group, serum PSA
level was correlated significantly positively with saliva PSA (r=0.510, P<0.05; Figure1b).
r= 0.431
P<0.05
4
2
r= 0.510
P<0.05
6
4
2
0
0
0
5
10
15
PSA in Serum (ng/ml)
20
0
1
2
3
PSA in Serum (ng/ml)
Figure 1: Correlation Curves of serum and salivary PSA in PCa and BPH groups . The serum PSA concentration
showed significantly correlated with the salivary PSA concentration in two groups. Whereas, this correlation is
positive for PCa, Figure 1a: (r= 0.431, P<0.05) and BPH, Figure 1b: (r=0.510, P<0.05) groups respectively.
Spearman's correlation coefficient (r) was used to assess the correlation between serum and saliva PSA.*P-value is
for spearman correlation, P < 0.05 is statistically significant.
According to observations in this study serum ALP concentration in PCa and BPH groups was
no significantly correlated with salivary ALP concentration (Figure 2).However, as shown in
Figure 2a, in PCa group between ALP concentrations in serum and salivary positive correlation
was found (r= 0.037, P<0.05). As well as, in BPH group (Figure 2b) there was a positive
correlation in serum and salivary ALP concentrations (r=0.213, P<0.05).
B)
A)
300
200
150
r= 0.037
P<0.05
100
50
0
ALP in Saliva (U/L)
ALP in Saliva (U/L)
250
200
r= 0.213
P<0.05
100
0
0
500
1000
ALP in serum(U/L)
1500
0
100
200
300
ALP in serum(U/L)
Figure 2: Correlation Curves of serum and salivary ALP in PCa and BPH groups. There was no significant
correlation between serum and salivary ALP concentration in two groups. But, this correlation is positive for PCa,
Figure 2a: (r= 0.037, P<0.05) and BPH, Figure 2b: (r=0.213, P<0.05) groups respectively. Spearman's correlation
coefficient (r) was used to assess the correlation between serum and saliva PSA.*P-value is for spearman
correlation, P < 0.05 is statistically significant.
Discussion
There are many biological samples, but some of them are very important and acceptable for
patients and physician (21). Since prostate cancer occurs in adult males, the use of saliva for
diagnostic and monitoring tests (Such as measuring PSA ) as well as metastatic diagnostic tests
(Such as measuring ALP ) to be useful in these patients because it is inexpensive, non-invasive,
simple and requires no special skills to collect (22, 23). Recently, interest in saliva as a biological
sample for measure tumor markers has increased exponentially (21). In this study total PSA and
total ALP were measured in serum and saliva samples to investigate the relationship between
changes in serum and saliva.
The results of our study showed that there was a significant difference between PCa and BPH in
terms of PSA concentration in serum and saliva, as well as in two groups, serum PSA level was
correlated significantly positively with saliva PSA level. Based on the results from the
measurement of ALP in the saliva and serum of two groups, the ALP activity in serum was no
statistically significant between PCa and BPH groups. While, the ALP activity in saliva was
statistically significant between two groups. However, there was positive correlation between
serum and saliva for ALP in the two groups.
Study conducted on healthy men without prostate cancer, finding that there was no correlation
between the total PSA level in saliva and serum(24). As well as, Turan et al (25), reported that
there wasn’t correlation between serum and salivary PSA levels in normal subjects or in patients
with BPH and prostate cancer. In contrast, Shiiki et al (26), Showed a positive and significant
correlation between the total PSA in the saliva and serum in the high-serum PSA concentration
group but no correlation in the low-serum PSA concentration group. This report demonstrated
that salivary level of PSA is linked to the amount of blood. Furthermore, the results of our study
showed a significant positive correlation between salivary and serum concentrations of PSA, in
two groups of PCa (high-serum concentrations of PSA) and BPH (low-serum PSA
concentration). In other hand, in another study that was conducted on healthy women during the
menstrual cycle showed that serum PSA concentrations were positively correlated with salivary
PSA concentrations(27).
ALP is an intracellular enzyme that there is in most tissues of the body and clinically increases in
serum at the time of severe cellular damage such as malignancies. However, measuring this
enzyme in serum usually used to detect metastasis. For example, a study reported by Wang et al
(28), that the ALP concentrations in patients with metastatic prostate cancer and those who have
hormone therapy can be greatly increased. Some studies have measured ALP activity in the
saliva, such as report of Jazaeri et al (5), in healthy subjects to evaluate the association of ALP
with calcium and phosphate concentrations. As well as, salivary ALP level in smokers, diabetics
and cancer patients were measured by the Prakash (23). Now, according to the results of the
above studies, Due to the measurement of this enzyme in saliva, also the changes in prostate
patients, we examined the relationship between serum and saliva. The results of our study
showed that the greater the level of ALP in the saliva of PCa group compared to BPH group.
More importantly, there was a positive correlation between salivary and serum concentrations of
ALP in the two groups, but this correlation was not significant.
In conclusion, we demonstrated that salivary and serum concentration of PSA is higher levels in
patients group than the control group. In addition, serum concentrations of PSA in the two
groups were higher than concentrations in saliva. On the other hand, there is a significant and
positive correlation between serum and salivary of PSA in the two groups. In this study, ALP
activity in serum and saliva were higher in PCa compared with BPH, although, serum
concentrations of ALP in the two groups were higher than concentrations in saliva but not
significant. Results of correlation showed that there were no significant and positive correlation
between serum levels of ALP and the saliva. Based on the results mentioned above, saliva can
reflect serum levels of PSA and ALP in prostate patients. However, further studies in other
diseases, differnt parameters and a larger sample size are needed to confirm these dicission.
Acknowledgments
This research was supported by a grant of deputy in research and technology, Arak Medical
Science University, with ethic permission number IR.ARAKMU.REC.1394.2. The authors are
grateful all the participants took part in this study.
References
1.
Al Nemer AM, Aldamanhori RB. Prostatic diseases under focus in a university hospital in Eastern
Saudi Arabia: A 15-year experience. Saudi Med J. 2015;36(11): 1319–1323
2.
Abdel-Meguid TA, Mosli HA, Al-Maghrabi JA. Prostate inflammation. Association with benign
prostatic hyperplasia and prostate cancer. Saudi Med J. 2009;30(12):1563-1567.
3.
Mikolajczyk SD, Marks LS, Partin AW, Rittenhouse HG. Free prostate-specific antigen in serum is
becoming more complex. Urology. 2002;59(6):797-802.
4.
Schröder FH. Review of diagnostic markers for prostate cancer. Recent Results Cancer Res.
2009;181:173-82.
5.
Jazaeri M, Malekzadeh H, Abdolsamadi H, Rezaei-Soufi L, Samami M. Relationship between
Salivary Alkaline Phosphatase Enzyme Activity and The Concentrations of Salivary Calcium and
Phosphate Ions. Cell J. 2015;17(1):159.
6.
Robinson D, Sandblom G, Johansson R, Garmo H, Stattin P, Mommsen S, et al. Prediction of
survival of metastatic prostate cancer based on early serial measurements of prostate specific antigen
and alkaline phosphatase. J Urol. 2008;179(1):117-23.
7.
Zelles T, Purushotham K, Macauley S, Oxford G, Humphreys-Beher M. Concise review: saliva and
growth factors: the fountain of youth resides in us all. J Dent Res. 1995;74(12):1826-32.
8.
Rehak NN, Cecco SA, Csako G. Biochemical composition and electrolyte balance of"
unstimulated" whole human saliva. Clin Chem Lab Med. 2000;38(4):335-43.
9.
Drobitch RK, Svensson CK. Therapeutic drug monitoring in saliva. An update. Clin
Pharmacokinet. 1992;23(5):365-79.
10.
Haeckel R, Hänecke P, editors. The application of saliva, sweat and tear fluid for diagnostic
purposes. Annales de biologie clinique. 1993; 51(10-11):903-910.
11.
Jusko WJ, Milsap RL. Pharmacokinetic Principles of Drug Distribution in Salivaa. Annals of the
New York Academy of Sci. 1993;694(1):36-47.
12.
Miller S. Saliva testinga nontraditional diagnostic tool. Clin Lab Sci. 1993;7(1):39-44.
13.
Laidi F, Bouziane A, Lakhdar A, Khabouze S, Amrani M, Rhrab B, et al. Significant correlation
between salivary and serum Ca 15-3 in healthy women and breast cancer patients. Asian Pac J Cancer
Prev. 2014;15(11):4659-62.
14.
Di-Xia C, Schwartz PE, FAN-QIN L. Saliva and serum CA 125 assays for detecting malignant
ovarian tumors. Obstet Gynecol. 1990;75(4):701-4.
15.
Navarro MA, Mesía R, Díez-Gibert O, Rueda A, Ojeda B, Alonso MC. Epidermal growth factor in
plasma and saliva of patients with active breast cancer and breast cancer patients in follow-up
compared with healthy women. Breast Cancer Res Treat. 1997;42(1):83-6.
16.
Humeau M, Vignolle-Vidoni A, Sicard F, Martins F, Bournet B, Buscail L, et al. Salivary microRNA
in pancreatic cancer patients. PloS one. 2015;10(6):1-13.
17.
Gallo A, Tandon M, Alevizos I, Illei GG. The majority of microRNAs detectable in serum and saliva
is concentrated in exosomes. PloS one. 2012;7(3):1-7.
18.
Malon RS, Sadir S, Balakrishnan M, Córcoles EP. Saliva-based biosensors: noninvasive monitoring
tool for clinical diagnostics. Biomed Res Int. 2014;2014:1-20
19.
Chiappin S, Antonelli G, Gatti R, Elio F. Saliva specimen: a new laboratory tool for diagnostic and
basic investigation. Clin Chim Acta. 2007;383(1):30-40.
20.
Malamud D, Tabak L. Saliva as a diagnostic fluid. BMJ. 1992; 305(6847): 207–208.
21.
Liu J, Duan Y. Saliva: A potential media for disease diagnostics and monitoring. Oral Oncol.
2012;48(7):569-77.
22.
Dabra S, Singh P. Evaluating the levels of salivary alkaline and acid phosphatase activities as
biochemical markers for periodontal disease: A case series. Dent Res J (Isfahan). 2012;9(1): 41–45.
23.
Prakash AR, Indupuru K, Sreenath G, Kanth MR, Reddy AVS, Indira Y. Salivary alkaline
phosphatase levels speak about association of smoking, diabetes and potentially malignant diseases???.
J Oral Maxillofac Pathol. 2016;20(1):66-70.
24.
Ayatollahi H, Darabi Mahboub MR, Mohammadian N, Parizadeh MR, Kianoosh T, Khabbaz Khoob
M, et al. Ratios of free to total prostate-specific antigen and total prostate specific antigen to protein
concentrations in saliva and serum of healthy men. Urol J. 2009;4(4):238-41.
25.
Turan T, Demir S, Aybek H, Atahan O, Tuncay OL, Aybek Z. Free and Total Prostate–Specific
Antigen Levels in Saliva and the Comparison with Serum Levels in Men. Eur Urol. 2000;38(5):550-554.
26.
Shiiki N, Tokuyama S, Sato C, Kondo Y, Saruta J, Mori Y, et al. Association between saliva PSA and
serum PSA in conditions with prostate adenocarcinoma. Biomarkers. 2011;16(6):498-503.
27.
Aksoy H, Akçay F, Umudum Z, Yildirim AK, Memisogullari R. Changes of PSA concentrations in
serum and saliva of healthy women during the menstrual cycle. Ann Clin Lab Sci. 2002;32(1):31-6.
28.
Wang Z, Wang X. [Relationship of serum prostate-specific antigen and alkaline phosphatase
levels with bone metastases in patients with prostate cancer]. Zhonghua Nan Ke Xue. 2005;11(11):8257.