Saied M. I. Al-Dalaen*, Abdul-Wahab R. Hamad, Faker Al-Ani and Fawaz Al-Saraireh

College of Pharmacology, Mutah University, Jordan.

Corresponding Author E-mail : aldalaensm@yahoo.com

DOI : https://dx.doi.org/10.13005/bpj/1858

Abstract

Proteins play a central role in cell function and cell structure, serum contains a mixture of proteins differing in origin and function, the amount of protein in the vascular compartment depends on the balance between the rate of synthesis and the rate of catabolism or loss. It is a well established and evidence-based fact that serum (plasma proteins) levels may suffer changes during a neoplastic disease process. A prospective study to explore the serum and cerebrospinal fluid (CSF) proteins levels in the setting of primary brain tumours (PBT) among Iraqi persons suffering the latter condition and to have it compared with hydrocephalic and healthy individuals. This study had been conducted at both University and Neurosurgical Dept. between November 2018 and October 2019.  Out of the 107 patients suffering from primary brain tumors with an age range 2-75 years (mean 35, the standard SD ± 19), 56 were males (52.33%), and 51 were females (47.66%).  Although 89% of patients were under the age of 60 years, however, the most affected age group was 31-40 years (17.75%).Fifty age- and sex-matched patients with hydrocephalus (non-neoplastic disease) were used as control group in CSF measurements.Forty age- and sex-matched normal subjects were used as controls (in serum). A highly significant increase in total protein levels in serum of primary brain tumor patients was noticed when compared to that normal subject.  Elevation in total protein content in malignant tissue was also observed in comparison to benign tissue. Serum proteins levels changed in PBT.

Keywords

Cerebrospinal Fluid (CSF); CSF Proteins; PBT; Plasma Protein(s)

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Copy the following to cite this article: Al-Dalaen S. M. I, Hamad A. W. R, Al-Ani F, Al-Saraireh F. Evaluation of Proteins in Serum and Cerebrospinal Fluid in Primary Brain Tumors. Biomed Pharmacol J 2020;13(1).

Copy the following to cite this URL: Al-Dalaen S. M. I, Hamad A. W. R, Al-Ani F, Al-Saraireh F. Evaluation of Proteins in Serum and Cerebrospinal Fluid in Primary Brain Tumors. Biomed Pharmacol J 2020;13(1). Available from: https://bit.ly/37Dk56N

Introduction

Proteins play a central role in cell function and cell structure,^(1-2) serum contains a mixture of proteins differing in origin and function, the amount of protein in the vascular compartment depends on the balance between the rate of synthesis and the rate of catabolism or loss.   Hepatocytes synthesize many plasma proteins ^(3-4), those of the complement system are synthesized both in these cell, and by macrophages.

Control of growth and differentiation: controlled sequential expression of genetic information is essential for the orderly growth and differentiation of cells. In higher organisms, growth and differentiation are controlled by growth factor proteins. Hormones coordinate the activities of different cells in multicellular organisms, many of them, such as insulin ⁽⁵⁾ and thyroid stimulating hormone ^{(1, 4)} are proteins.

All proteins normally present in cerebrospinal fluid (CSF) are derived from serum.   Only a few proteins, such as beta-trace protein and gamma-trace protein (Cystatin C), are synthesized intrathecally (6).

Protein entry into the CSF is thought to be mainly dependent on pinocytosis by the capillary endothelial cells of the brain and spinal cord that constitute the blood-brain barrier (7).  The normal protein content of CSF is several fold lower compared to serum and depends upon the relative exclusion of macromolecules by the blood-brain barrier (8).

The identification of proteins in the CSF that are secreted by the tumor or its environment may reveal cellular mechanisms relevant to cancer biology.  Also, it may result in the development of new tumor markers and may ultimately target new therapies.  Protein expression profiling has be suitable a valuable tool in obtaining information about the state of protein circuits inside tumor

The spread of cancer into the brain and central nervous system is a serious problem leading to neurological symptoms and rapid mortality.  Malignant cells in the brain and CSF derived from primary neural cancers can be ability to detect and characterize these cells, this is allow us to answer important questions about metastatic through identification and characterization of the cancer cell populations capable of infiltrating the CSF. Cancer may reach the CSF through hematogenous spread, direct extension from the tumor itself, or by migration along perineural or perivascular spaces (10, 11)

Controlled sequential expression of genetic information is essential for the orderly growth and differentiation of cells. In higher organisms, growth and differentiation are controlled by growth factor proteins. Hormones coordinate the activities of different cells in multicellular organisms, many of them, such as insulin ^(18-19) and thyroid stimulating hormone ^(1,4) are proteins.

The aim of the study is to investigate plasma proteins in the context of PBT among a group of patients in their sera and CSF and to compare the CSF figures with that of cohort of hydrocephalic patients and sera of another cohort of healthy volunteers.

Materials and Methods 

This study had been conducted between November 2018 and October 2019 at both The University and Neurosurgical Dept.  Patients were evaluated by full medical history to exclude any existing systemic disease that may affect the parameters to be diagnosed, particularly diabetes, liver disease, renal disease and chronic drug intake, other wise the patient was excluded from the study.  Ages and sex-matched normal subjects were used as controls (in serum).

Out of the 107 patients suffering from primary brain tumors with an age range 2-75 years, (mean 35, the standard SD ± 19), 56 were males and 51 were females.   Although 89% of the patients were under the age of 60 years, however, the most affected age group was 31-40 years (Table 1).

Fifty age- and sex-matched patients with hydrocephalus (non-neoplastic disease) were used as control group in CSF measurements. 

Duration of the Disease

The duration of the disease range from <1 – >9 years.  The majority of the patients were presented within less than 1-year from the onset of symptoms. 

Histological Findings and Grading  

The age and sex distribution in 110 primary brain tumors patients is shown in Table 1. Histological grading of the malignant varieties has shown that the highest percentage was that of grade IV (34%) followed by grade III (27%) giving a total percentage of 61%.

Chemical and Reagents     

All Chemical and standard solutions used in this work, were the highest analytical grade obtained from commercial source, and used without further purification. All volumetric glassware were cleaned in a solution of 5 N HCL for at least 24 hrs, then washed repeatedly in deionized water prior to use.

Total protein kit, and Albumin kit were obtained from Randox Laboratories Ltd. United Kingdom.

Table 1: Distribution of PBT patients according to age and sex.

Age (years)	Male	Female	Total
1-10	5 (41.66%)	7 (58.33%)	12 (11.21%)
11-20	10 (58.82%)	7 (41.17%)	17 (15.88%)
21-30	9 (60%)	6 (40%)	15 (14.01%)
31-40	10 (52.63%)	9 (47.36%)	19 (17.75%)
41-50	8 (44.44%)	10 (55.55%)	18 (16.82%)
51-60	8 (57.14%)	6 (42.85%)	14 (13.08%)
61-70	4 (44.44%)	5 (55.55%)	9 (8.41%)
>70	2 (66.66%)	1 (33.33%)	3 (2.80 %)
Total	56 (52.33%)	51 (47.66%)	107 (100%)

Sample Collection and Preparation

The 107 patient included in this study had fasted for 8 to 12 hrs. before surgery.  The operation is usually performed under a general anesthetic and endotracheal intubation. 

Tumour Tissue

At craniotomy operation, while tumour removal is being done, a sufficient amount of the abnormal growth is kept for processing and tissue protein estimation. 

CSF

A CSF specimen (3 to 4 ml) was collected in a plastic specimen container catheter following opening the dura and arachnoid matters or via a ventricular puncture.

Control group: fifty patients with hydrocephalus were included as a control group; CSF samples were collected via a ventricular catheter that was used in treatment. The CSF specimens were collected in plastic containers, promptly frozen, and stored at (-20 ˚C) until analysis.

Assays were done within one week to one month of collection at the laboratories of The Neurosurgery Hospital, and The research center. 

Serum

About 5 ml venous blood was drawn aseptically into sterile test tube with silicon coated, by utilizing disposable needle and plastic syringes. The blood was allowed to clot (10 minutes), centrifuged at 4000 rpm for 15 min.  Serum sample were immediately transferred into four tube and frozen at (-20˚C) for subsequent analysis, haemolyzed samples were discarded.

One milliliter of venous blood, after clothing, was centrifuged at 600 rpm for 10 min. serum was diluted with 0.3 ml (0.2 M) sodium potassium phosphate buffer (pH8.4) and centrifuged for 20 min. at 1000 rpm thoroughly to remove protein. The filtrate was kept frozen at (-20˚C) until analyzed. (20-100 ml) aliquot of the filtrate was used for HPLC analysis.

Results

Data of proteins in Malignant, Benign, CSF and in serum albumin concentration are shown in tables 2, 3, and 4.

Table 2: Mean protein concentration in malignant and benign Tissue of PBT patients.

Malignant tumour tissue Mean ±SD (g/dl)	Benign tumour tissue Mean ±SD (g/dl)	P value
4.5251±0.9216	1.5472±0.9680	<0.01

 

Table 3: Mean CSF protein concentration in malignant and benign PBT patients.

CSF of malignant tumoursMean ±SD(mg/dl)	CSF of benign tumoursMean ±SD(mg/dl)	P value
156.7741±46.4748	77.9791±14.0174	<0.01

 

Table 4: Mean serum albumin concentration in PBT patients and normal subjects.

Patient serumMean ±SD(g/dl)	Normal serumMean ±SD(g/dl)	P value
2.2186±0.533 2	3.908±0.724	<0.01

Discussion

Total Serum Protein

A highly significant increase in total protein levels in serum of primary brain tumor patients was noticed when compared to that normal subject (Table 4).  Elevation in total protein content in malignant tissue was also observed in comparison to benign tissue (12).

This increase could be explained on the basis that the whole body of cancer patients is engaged in protein synthesis of various forms like: C-reactive, proteins, tumor markers, enzymes, and immunoglobulins and other proteins material.  Our results  are in agreement with Fiandra et al 1993 and Shrotriya et al. 2015.(13,14)  who found that patients with neoplasm had higher values of total protein (P<0.01).

In addition, patients with oral squamous cell carcinoma had also markedly increased total protein concentrations (15).  While Nagashima and Schreiber ⁽1984) grouped several plasma proteins as acute phase reactant (APR), which significantly rise, rise during inflammation and neoplasms.

On the other hand, it was found that the increase in synthesis of acute phase reactant (APR) is accompanied by a decrease in the synthesis of prealbumin, albumin and transferring, which are so called negative APR. inflammatory tissue lesions generally induce changes in the concentrations of various  serum proteins. The acute phase reactants may be increased, albumin may be decreased and the immunoglobulin production may be enhanced (17, 18, 19).

The increase in total protein concentrations, in this study, indicated that synthesis of APR proteins had exceeded the synthesis of negative APR, this imbalance leads to a marked increase in total protein levels.

Total Protein in CSF

In the current study, it was found a significant increase in total protein concentration in CSF of malignant PBT (156.77± 46.47 mg/dl) compared to that of benign nature (77.97±14.01) as shown in table 3, with statistical significance (p<0.01).

The CSF protein concentration depends of the serum protein concentration, permeability of the blood-CSF barrier, and immunoglobulin synthesis (20) Abnormal concentrations of the serum proteins influence the corresponding protein levels in CSF, this is due to increased capillary permeability, with a similar increase in the permeability of the blood- brain barrier, which may be demonstrated by finding relatively high molecular weight proteins not normally present in CSF

The non –specific pattern is associated with a large number of inflammatory conditions, but may sometimes aid diagnosis such a pattern may be due to: cerebral tumors, multiple sclerosis and increased intrathecal immunoglobulin synthesis (acute phase reactants) (21, 22) . 

Albumin

Reduced serum albumin concentration is a common finding in patients with neoplastic diseases. Hypo-albuminamia can appear early in the course of the disease, and indeed it may occur despite normal nutrition and even without effusion, protein loss or evident clinical signs of liver damage.(23, 24).   The presence hypoalbuminaemia has been widely confirmed in the malignant lymphomas (25), prostatic cancer ⁽²⁶⁾, melanoma ⁽²⁷⁾, colorectal cancer⁽²⁸⁾ and leukemia.⁽²⁹⁾

It is well established that the acute phase reaction, usually involved interleukins, tumor necrosis factor (TNF), and C-reactive proteins causing a reduction in the concentration of albumin is associated with the risk and development of cancer (30).

However, it has been shown that TNF may increase the permeability of the microvasculature, thus allowing an increased trans-capillary passage of albumin (31) and hence a lowing of the serum albumin concentrations

The present study demonstrated a significant decrease in albumin concentration in serum of patients with primary brain tumors (2.21±0.53 g/dl) in comparison to normal group (3.90±0.72 g/dl) with statistical significance (p < 0.01) as seen in table (4). 

Conclusions

Both the serum and CSF have shown a significant increase in the total proteins while, on the contrary to that, the albumin value has been less than normal.

Acknowledgements

The authors are indebted to all those who have assisted in the research, namely the staff neurosurgeons at the University and the Neurosurgical Dept, the personnel at the Medical Research Centre at College of Pharmacy, Muta University, Jordan.

 References

1. Suzanne Clanc, 2008. The central dogma of molecular biology suggests that the primary role of RNA is to convert the information stored in DNA into proteins. In reality, there is much more to the RNA story. Nature Education 1(1):102.
2. Adams J. The proteasome: structure, function, and role in the cell. Cancer Treat Rev. 2003 May; 29 Suppl 1:3-9.
   CrossRef
3. Jacek R.Wiśniewski^aAnnaVildhede^bAgnetaNorén^cPerArtursson. In-depth quantitative analysis and comparison of the human hepatocyte and hepatoma cell line HepG2 proteomes. Journal of Proteomics; 136, 16 March 2016, Pages 234-247.
   CrossRef
4. Lodish, A. Berk, S. Zipursky, P. Matsudaira, D. Baltimore, J. Darnell Molecular Cell Biology, (fourth ed.), W.H. Freeman, New York (2000).
   CrossRef
5. R. Wisniewski, D. Rakus Multi-enzyme digestion FASP and the ‘Total Protein Approach’-based absolute quantification of the Escherichia coliproteome. J. Proteome, 109 (2014), pp. 322-331.
   CrossRef
6. Bishoy El-Aarag, Azza M Abdu-Allah, Mohamed Aid Abo-Alfa and Ibrahim El Tantawy El Sayed Serum Beta-Trace Protein and Cystatin C as Biomarkers for Renal Dysfunction in Patients with Chronic Kidney Disease.  J Mol Biomark Diagn 2018, Vol 9(4): 399
   CrossRef
7. Fishman RA. Cerebrospinal fluid in diseases of the nervous system. Philadephia:  B. Saunders Company, 1992.
8. Ping-Pin Zheng, Cees J.J. Avezaat and Peter A.E. Sillevis Smitt. Identification of Tumor-Related Proteins by Proteomic Analysis of Cerebrospinal Fluid from Patients with Primary Brain Tumors. Journal of Neuropathology & Experimental Neurology, Volume 62, Issue 8, 1 August 2003.
   CrossRef
9. Liotta LA, Kohn EC. The microenvironment of the tumour-host interface. Nature 2001; 411:375–
   CrossRef
10. Chamberlain MC. Neoplastic Meningitis. The Oncologist. 2008; 13:967–977.
    CrossRef
11. Mammoser AG, Groves MD. Biology and therapy of neoplastic meningitis. Curr Oncol Rep. 2010; 12:41– 49.
    CrossRef
12. Rasouli M¹, Okhovatian A, Enderami A. Serum proteins profile as an indicator of malignancy: multivariate logistic regression and ROC analyses. Clin Chem Lab Med. 2005; 43(9):913-8.
    CrossRef
13. Fiandra U., Bo M., Fonte G., Poli L., and Fabris F.; Cancer Detection and Prevention ; 1993 ; 17 (1) : 1.
14. Shiva Shrotriya, Declan Walsh, Nabila Bennani-Baiti,Shirley Thomas, Cliona Lorton.  C – reactive protein is an Important Biomarker for Prognosis Tumor Recurrence and Treatment Response in Adult Solid Tumors: A Systematic Review. 2015.
    CrossRef
15. Zhi Wang, Lu Jiang, Canhua Huang, Zhengyu Li, Lijuan Chen, Lantu Gou, Ping Chen, Aiping Tong, Minghai Tang, Feng Gao, Jun Shen, Yuanyuan Zhang, Jingping Bai, Min Zhou, Di Miao, and Qianming Chen. Comparative Proteomics Approach to Screening of Potential Diagnostic and Therapeutic Targets for Oral Squamous Cell Carcinoma. Mol Cell Proteomics. 2008 Sep; 7(9): 1639–1650.
    CrossRef
16. Nagashima M. , and Schreiber G. ; Am. Assoc. Clin. Chem.; 1984; 1: 1.
17. Whicher J. ; Abnormalities of plasma protiens In : Biochemistry in Clinical Practice . Williams D. , and Marks V. Eds. London , William Heinemann ; 1983
18. Anurag Markanday. Acute Phase Reactants in Infections: Evidence-Based Review and a Guide for Clinicians. Open Forum Infectious Diseases, Volume 2, Issue 3, 1 September 2015.
    CrossRef
19. Sachin Jain, Vidhi Gautam, and Sania Naseem. Acute-phase proteins: As diagnostic tool. J Pharm Bioallied Sci. 2011; 3(1): 118–127.
    CrossRef
20. Daniel B., Jean- Francois B. , Claire N. , Philippe M. , and Martine M. ; Clin. ; 2000; 46 (3): 399.
21. Hansotto Reiber. Proteins in cerebrospinal fluid and blood: Barriers, CSF flow rate and source-related dynamics. Restorative Neurology and Neuroscience 21 (2003) 79–96 79.
22. Hansotto Reiber. Flow rate of cerebrospinal fluid (CSF) – a concept common to normal blood-CSF barrier function and to dysfunction in neurological diseases. Journal of the Neurological Sciences,1994; 122 (1094) 189 203.
    CrossRef
23. Mazzaferro EM, Rudloff E, Kirby R: the Role of Albumin Replacement in the Critically Ill Veterinary Patient. J Vet Emerg Crit Care12(2):113-124, 2002.
    CrossRef
24. Daniel B., Jean- Francois B., Claire N, Philippe M., and Martine M.; Clin. ; 2000; 46 (3): 399.
25. Samuel W D Merriel Robert Carroll Fergus Hamilton William Hamilton. Association between unexplained hypoalbuminaemia and new cancer diagnoses in UK primary care patients. Family Practice, Volume 33, Issue 5, 1 October 2016, Pages 449–452,
    CrossRef
26. Berry W. , Laszlo J. , Cox E. , Walker A. , and Paulson D. ; Prognostic factors in metastatic and hormonally unresponsive carcinoma of the prostate. Cancer ; 1979 ; 44 763.
    CrossRef
27. Matthew N. Sirott, Prognostic Factors in Patients with Metastatic Malignant Melanoma A Multivariate Analysis. CANCER,1993, 72 (10): 3091- 3098.
    CrossRef
28. Heys S. , Walker L. , Deehan D. , and Ermin O. ; Risk Factors and Postoperative Morbidities in Colon Cancer Patients with Preoperative Hypoalbuminemia. J R Coll Surg Edinb 1998; 43:163-8.
29. Keating K. , Smith T. , Gehan E. , Merdie K. , Bodey G. , and Freireich E. ; A four-year experience with anthracycline, cytosine arabinoside, vincristine and prednisone corn hina- tion chemotherapy in 325 adults with acute leukemia. Cancer 1982; 47: 457.
    CrossRef
30. Daniel B., Jean- Francois B. , Claire N. , Philippe M. , and Martine M. ; Clin. Chem; 2000; 46 (3): 399.
31. Goldberg DM, Brown D. Biochemical tests in the diagnosis, classification, and management of patients with malignant lymphoma and leukemia. Clin Chim Acta 1987; 169:1-76.
    CrossRef

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