Effect of Obesity on Plasma Alkaline Phosphatase Activity in Breast Cancer

Background: Breast cancer is most common cancer in women. Obesity is one of related-risk factor in breast cancer. In obese normal subjects, alkaline phosphatase (ALP) has been studied. However, there is no previous study investigate the association between ALP and obesity in breast cancer and its correlation with other clinical characteristics. Therefore, the objective of present study is to investigate the association between ALP and clinical characteristics in generally and obesity in particularly. Methods: A cross-study 111 new diagnosed breast cancer patients was included. Plasma ALP was measured in different subgroups: patients age <40 vs >40, premenopausal vs postmenopausal, estrogen receptor-positive (ER+) vs estrogen receptor negative (ER-), metastasis vs non-metastasis and obese vs non-obese patients. Results: Significant increasing on plasma ALP were shown between groups of each age, menopausal status, metastasis, and obesity (p< 0.05, p< 0.05, p< 0.01 and p< 0.05) respectively. Positive correlation was observed between plasma ALP and age, menopausal status, metastasis, and obesity (r: 0.616, p< 0.05; r: 0.667, p< 0.01; r: 0.691, p< 0.005; and r: 0.627, p< 0.01). Multiple regression analysis was indicated that ALP can be determined by menopausal status, metastasis, and obesity (βCoefficient = 0.428, p< 0.01; β-Coefficient = 0.534; p< 0.001; β-coefficient= 0.545; p= 0.005), respectively. Conclusions: Together, the relation between ALP and obesity indicates that ALP could have a role in maturation of preadipocytes of breast cancer patients. Further investigations are needed to confirm that there could be a potential hormonal link between ALP and obesity in breast cancer patients.


Introduction
Breast cancer is one of the most frequent cancers in women. It is well known that breast cancer is second common and second related death in women (1). Globally, the total number of deaths is 535,341. In Iraq, the total number of death due to breast cancer is 2623 in 2016 (2). It was reported that 909 cases were died because of late diagnosis in Iraq (3)(4)(5). Therefore, investigation for a cheap and easily detectable biomarker, which can measure in simple laboratory, is an urgent need.
Obesity is one of the most important risk factor of breast cancer (6,7). It is considered that adipose tissue is an endocrine organ which secretes many factors (8). Alkaline phosphatase (ALP) is an enzyme that hydrolyses phosphate in alkaline medium. ALP isoenzymes are expressed in different organs, such as bone, liver, intestine, bile duct, kidney, germ cell and placenta (9)(10)(11). In last decade, it was shown that ALP was expressed by preadipocytes which were isolated from abdominal subcutaneous adipose tissue in human and murine preadipocytes cell line 3T3 L1 (12,13). It was hypothesized that ALP may have a role in maturation of preadipocytes to adipocytes (14)(15)(16).
Furthermore, the presence of ALP in preadipocytes suggested that adipose tissue is one of the sources of ALP in blood circulation (17). In healthy subjects, it was found that serum ALP level is higher in obese in compared to non-obese (17). In cancer patients, it has been suggested that ALP as a potential tumor marker to predict bone metastasis in bladder (18) and breast cancer (19). However, there is limited clinical evidence about ALP and obesity in breast cancer patients. Therefore, the aim of the present study is to focus on the relation between ALP and clinical characteristics of breast cancer patients generally and ALP with obesity in particularly.

Study design
This study was conducted at Al-Amal National Hospital of Cancer Management in period from November 2018 to June 2019. The protocol of study was approved by ethics committee (27280) in the Ministry of Health in Baghdad, Iraq. The consent was taken from each patient before blood collection. A cross-sample, 111 new diagnosed breast cancer patients was included in the study. Patients, who treated with hormonal therapy, chemotherapy, and radiotherapy, were excluded from study. The clinical characteristics, which included age, menopausal status, breast cancer subtypes; estrogen receptor positive (ER+) and estrogen receptor negative (ER-), and metastasis, were informed form pathological report of each patient. BMI was measured by dividing the body weight in kilogram (kg) by body height in meter square (m 2 ). Patients, who have BMI more than or equal 30 kg/m 2 , were categorized as obese and patients, who have BMI less than 30 kg/m 2 , were categorized as non-obese.

Sample collection
Five ml of blood was collected from peripheral venous blood in heparin-plasma tube. The blood was centrifuged at 3000 g for 10 min. Plasma blood was stored at -20 ºC. The activity of total ALP was measured by Kinetic method. Twenty microliters of the plasma were incubated with 1000 µl solution which contains 10 mmole/l of p-Nitrophenyl phosphate, 0.25 mole/l of diethanolamine and 0.125 mmole/l of MgCl2 at pH 10.35±0.2. The absorbance of solution was measured after 1, 2, and 3 minutes. ALP activity was calculated by dividing the change in absorbance by time and then multiple by 2757 factor according to kit instruction of HUMAN company.

Statistical analyses
Normality of ALP measurement was tested by Kolmogorov-Smirnov test. ALP activity was expressed by mean±SD. The differences in ALP levels were performed by unpaired two-tailed tstudent's test. The Pearson correlation was used for testing the relation between continuous variables and point bi-serial correlation for testing the relation continuous variable (ALP) and between dichotomous variables. The significant difference was considered when p value was less than 0.05. Statistical analyses were performed by IBM SPSS version 23.0 and GraphPad Prism 8.0.2.

Results
In the present study, some clinical features of breast cancer patients were investigated. The percentage of patients in each subclass of age, menopausal status, breast cancer subtypes, metastasis status and BMI are shown in Table 1. The difference in ALP level between each clinical parameters subtype was tested by student t-test, as it was shown in Table 2. The significant difference (p< 0.05) was found in ALP level between breast cancer patients who their ages were less than 40 years old when compared to patients who their ages were more or equal 40 years old. In addition, ALP level was significantly increased in post-menopausal breast cancer when compared to pre-menopausal status (p< 0.05). ALP level was slightly higher in ER+ than in ERbreast cancer. Metastasis breast cancer patients have significantly higher ALP level than nonmetastasis breast cancer patients (p< 0.01). In addition, a significant increase (p< 0.05) in ALP level was found in obese patients compared to non-obese patients. To study the relation between ALP and some clinical characteristics of breast cancer patients, a Pearson correlation was performed. As it was shown in (Table 3, Fig. 1

Discussion
There are substantial evidences suggest that ALP is potential tumour marker in bladder and breast cancer (18,19). However, there are limited studies investigate the relation between ALP and obesity in breast cancer patients. Therefore, the present study aims to investigate the relation between ALP and clinical characteristics of breast cancer patients generally and between ALP and obesity in particularly.
Our results were found significant difference in ALP level between older breast cancer patients when compared to young patients. In addition, ALP level was significantly increased in post-menopausal breast cancer when compared to pre-menopausal status. This result is in consistent with previous studies which were done in normal subjects (17,20,21). The explanation of this could be because bone damage in postmenopausal women lead to increase ALP level (17,21). Interestingly, there is slightly increasing difference between ALP levels in ER+ in comparison with ER-breast cancer patients. This data suggests that there might be a potential hormonal effect on ALP activity. Furthermore, it was shown that metastasis breast cancer patients have significantly higher ALP level than nonmetastasis breast cancer patients. This data agrees with previous studies (19,22) which suggested that measurement of ALP is an accurate and reliable marker.
Previous studies, which investigated ALP level in normal subjects, found that ALP level in obese is higher than in non-obese (17,20). However, there are other studies reported that body weight has no effect on ALP level in normal subject (23,24). This is the first study that investigates the effect of obesity on ALP level in breast cancer patients. The current study shows a significant increase in ALP level in obese patients compared to non-obese patients. This could be supporting the hypothesis that ALP has a potential role in maturation of preadipocytes (17,25).
Furthermore, a positive correlation was found between ALP and age, and menopausal status These results confirmed the previous studies (19,24). Moreover, a positive correlation between ALP and menopausal status was confirmed by multiple linear regression. The explanation of these results could be due to increasing age and after menopausal the bone damage increases leading to elevate ALP level (24).
Our study found a positive significant association between ALP level and metastasis. Furthermore, metastasis is mainly an independent variable that can be determined by ALP. This result could be explained that cancer cells may spread and settle in bone, liver, intestine, and brain in which ALP isoenzyme is expressed and led to elevate total ALP level.
Interestingly, there is a marginal association between ALP level and breast cancer subtypes. Furthermore, a positive correlation was shown between ALP level and obesity. These results suggest that an estrogenic hormonal effect could be link between ALP and both ER+ breast cancer and obesity. In addition, multiple regression analysis showed that ALP can be determined by BMI. The possibly explanation of these findings could be because adipose tissue is considered the main source of estrogen especially after menopausal (26). Furthermore, it was found previously that there is a potential regulation effect of estrogen on ALP expression in bone marrow stromal cell line (27). Further investigations are needed to confirm the relation between ALP and ER+ and between ALP and obesity.
The limitations in this study are firstly; there is high percent of postmenopausal breast cancer patients in compare to premenopausal patients. Secondly, it lacks the measurement of other ALP isoenzymes. Thirdly, it lacks study the estrogen level. Finally, the number of patients is small. Nevertheless, the current study highlights promising results which could help further in establish cheap and easily measurable biomarker for early detection obese breast cancer patients.
The activity of ALP increases with older, in postmenopausal patients and in metastasis breast cancer patients. Interestingly, the observed positive association between ALP and obesity indicates that ALP could have a role in maturation of preadipocytes of breast cancer patients. Further investigations are needed to confirm that there could be a potential hormonal link between ALP and obesity.