Brucellar Spondylitis causes Alteration in Serum protein and Electrophoretic pattern

Ekhlass M. Taha¹*, Muzahem Mohialdeen Taha², Jinan Hameed Abu-Shana³, Saleh M. Rahim⁴, Mays Mohammed Abdullah⁵

¹University of Baghdad College of Science for Women Department of Chemistry,

Al-Jadriya, Baghdad, Iraq, Postal Codes: 10071.

²Kirkuk University, Kirkuk, Iraq: Postal Codes: 36001. Consultant Orthopedic and Spine Surgeon- Erbil International Hospital.

³Al-Iraqia University- College of Medicine, Baghdad, Iraq, Postal Codes: 10053.

⁴Kirkuk University, Kirkuk, Iraq: Postal Codes: 36001.

⁵University of Baghdad College of Science for women Department of Chemistry, Al-Jadriya, Baghdad, Iraq.

*Corresponding Author E-mail: ekhlassch@yahoo.com, ikhlasmb_chem@csw.uobaghdad.edu.iq

ABSTRACT:

Background and Objectives: The frequency of Brucellosis is expanding in the Middle East particularly in Iraq. Spondylitis is the most incessant difficulty, yet it is hard to analyze in light of the fact that its clinical side effects are vague. The cross-sectional study was conducted to evaluate abnormal serum protein electrophoresis patience living with brucellar spondylitis and it is related to the disease. This study shares out the distribution of protein pattern in Brucellar spondylitis patients. Methods: The current study included a total number of 40 voluntary subjects covers of 25 cases and 15 controls. Total serum protein was determined using the biuret method, serum albumin was determined using bromocresol green method, Serum protein electrophoresis was determined using SAS-MX serum protein gel. Results: Total proteins, Alpha 1 Globulin, and Gamma Globulin were significantly increased in Brucellar spondylitis when compared to controls. Ratio Albumin/Gamma was significantly decreased in Brucellar spondylitis compared to control. Albumin, Alpha 2 Globulin, and Beta Globulin did not appear significant difference. According to the multivariable regression analysis the level of Alpha 1 Globulin was highly related to total protein in Brucellar spondylitis. The cluster analysis showed that the investigated parameters organized into four clusters in the serum of patients with Brucellar spondylitis. Conclusion: we conclude that Total proteins, Alpha 1 Globulin, and Gamma Globulin have altered in patients Brucellar spondylitis when compared to control, Also Alpha 1 Globulin was associated with total protein in the studied cases.

KEYWORDS: Brucellar spondylitis, protein electrophoresis, Alpha 1 Globulin, MRI.

INTRODUCTION:

Brucella species are gram-negative bacilli that are transmitted to people either by the utilization of unpasteurized milk or different items from tainted creatures or by direct contact with contaminated creatures¹.

Spondylitis is the most common and significant clinical type of osteoarticular attacks adults who infected with Brucella species². Spondylitis might be hard to identify. The clinical appearance of brucellosis in humans is complex and unspecific, and thus, diagnostic laboratory corroboration is necessary for the proper care of the patient. Brucellar infections can be diagnosed by history, serology, nucleic acid amplification, Xray and NMR resonance^3,4. NMR method is represented as a professional method for detection of several diseases^5,6,7,8.Recently VirB12 recombinant protein investigated for possibility used it as a diagnostic test⁹. Generally, total serum protein gives insight into general health and, it is involved in most infected¹⁰. The measurement of serum proteins is a valuable instrument for diagnosing and checking different infections that include changes in the levels of these proteins¹¹. Also assessment of serum proteins and their electrophoretic fractions is a good technique in the analysis of several illnesses in people¹². The consequences of serum protein electrophoresis can be one of the most valuable demonstrative guides in a wide range of illness, including inflammatory diseases. Protein electrophoretic fractions are included Albumin, alpha fraction, Beta fraction, and gamma fraction, each fraction has a significant role in the human body. Albumin, is the key blood serum macromolecule, has multiple necessary physiological functions together with maintenance of the colloidal osmotic pressure, binding of a large form of compounds, and provision of the majority of plasma antioxidant activity. Thus, a correlation between albumin concentration and health may well be expected. In fact, there's an associate surprisingly strong correlation between albumen concentration and mortality risk¹³. Concentrations of serum globulin include fractions of alpha, beta, and gamma globulin dependent on electrophoretic fractionation of the protein. In alpha and beta fractions, acute-phase proteins, complements, and fibrinogen migrate, and immunoglobulins (Igs), especially IgG, migrate in the gamma globulin portion¹⁴. Several diseases make an alteration in alpha 1-protein fraction like malignancy and acute inflammation, these diseases increase the alpha 1 protein band¹⁵. Other diseases like renal and epithelial cancer appear changes in the content of β-globulins¹⁶. Alpha and gamma globulins are inflammatory parameters, they are associated with so many diseases, low albumin and high globulin are both independent prognostic factors for various cancers. Many factors can, however, interfere with the albumin and globulin levels. The albumin – globulin ratio conceded as a good prognostic factor for a systemic response to inflammation¹⁷. The objective of this study is to explore the general profiles of serum proteins by serum protein electrophoresis in order to comprehend serum protein profiles as symptomatic markers in Brucellar spondylitis.

MATERIAL AND METHODS:

Study population:

This study was carried out in the Biochemistry lab, after collecting samples from outpatient clinics in Serdem private hospital and Erbil international hospital in the period from June 2018 to August 2019. After taking consent from the Patients and institutional ethical approval. We have taken 15 controls as normal patients and 25 cases as Brucellar spondylitis patients we estimated the levels of serum proteins. The patients were diagnosed by an orthopedic and spine surgeons based on their clinical manifestations, serology tests, X-ray and MRI.

Samples collection and processing:

Around 10ml of blood was collected using standard techniques from each participant in the fasting state. Blood samples were collected in a tube without anticoagulants at room temperature. Then serum was separated.

Determination of total protein:

Total protein was estimated using a biuret method kit that supplied by BIOLABO manufacturer. The method depends on the reaction of protein, peptide bond with Cu within an alkaline solution to form color complex the reagents content 370 mmol/L Sodium hydroxide, A 10 mmo/L of Na-K Tartrate, A 3 mmol/L Potassium iodide, and Copper II sulfate 3 mmol/L.

Determination of Albumin:

Albumin was determined using the Bromocresol green method, using the kit supplied by Elabscience Biotechnology Inc. This method depended on the combination between albumin and Bromocresol green in pH4 to form a complex called albumin-BCG, this complex develops yellowish-green color. The serum albumin concentration can be calculated by measuring the OD value at 628 nm.

For both total protein and Albumin, the standard curve was performed.

Serum protein electrophoresis:

Serum protein electrophoresis were done by Helena Electrophoretic apparatus in cases and controls. Agarose gel is ready for use, Tris/barbital Buffer was used after diluting 1:4 fold.

Rose Bengal test:

The Rose Bengal test was performed for all studied subjects as described previously^18,19.

IgG and IgM ELISA kit:

ALL subject sera were tested for IgG and IgM antibodies for brucella using ELISA.

Statistical analysis:

Statistical analysis program (SPSS 25) has been performed for analysis the results. The general descriptive statistic was used to describe the main findings; Student t-test, chi-square test and, multi regression analysis has been used to identify the significant differences. Dendrogram have been used to identify the similarity between variables

RESULTS AND DISCUSSION:

Demographic elements for the donors:

The study included a total 40 subjects. Out of them 25 are patients and 15 are controlled.

The demographic characteristic percentage associated with the brucellar spondylitis patients and matching healthy donors was summarized in chart 1. To identify the significance level for each variable in testing subject’s chi-square test have been applied with significant level p value <0.005. There was an association between the disease and the diagnostic test Since the p-value was less than what we had chosen in the test, so reject the null hypothesis. Also, the associations had been proved between the disease and gender as well as to the association between the disease and the age. Test findings for human brucellosis diagnosis have been analyzed. The titrated Rose Bengal test in sera of patients with acute brucellosis was positive for 47.5 percent, and in sera of patients with acute brucellosis 15 percent were negative. Immunoglobulin G (IgG) and IgM commercial enzyme immunoassays (ELISA) did not show specific antibodies in 7.5%, and 12.5% in patients respectively. ELISA's, sensitivity doesn't surpass that of standardized tests. Concepción Gómez et al discovered a related finding²⁰. Another study concludes that ELISA offers a critical favorable position over traditional serological strategies in the finding of brucellosis in endemic regions²¹. The presented results showed that 55% of brucellar spondylitis patients’ were diagnosed using MRI, and 7% of patients’ showed negative results. In 2018 magnetic resonance imaging (MRI) was used for distinguishing between pyogenic spondylitis and brucellar spondylitis²². Other researchers concluded that NMR is the best quality level for the conclusion and follow-up of brucellar^23,24.

Table 1 described the total protein and it is fractions, distribution in patient’s and control. Mean of total proteins in Brucellar spondylitis 8.0±0.065g/dl and control 7.045±0.2 respectively. Total proteins significantly increase in Brucellar spondylitis cases compared to controls. The total proteins picture is characteristic, not of any specific disease, but of the host's reaction to an infection like the development of any resistance to a period of time. Albumin in patients 4.35±0.09 and control 4.03±0.12 respectively. Albumin did not alter in patient’s pictures and, did not show any significant p-value 0.4. Mean of Alpha 1 Globulin in Brucellar spondylitis 0.333±0.024 and, control 0.184± 0.013 respectively. Alpha 1 Globulin significantly increased in case of Brucellar spondylitis compare to control p-value 0.000. Whereas Alpha 2 Globulin slightly increase in patients compared to control with no significant p-value 0.19. The Alteration in Alpha 1 Globulin, and Alpha 2 Globulin levels in Brucellar spondylitis may be due to the association of immunoglobulin and cellular immunity in the process. Mean of Beta Globulin in Brucellar spondylitis 0.946± 0.04 and control 1.04±0.034 respectively. Beta Globulin non significantly decreased compared to control p- value 0.07. Mean of Gamma Globulin in Brucellar spondylitis 1.5±0.03 and control 0.916±0.074 respectively. Gamma Globulin highly increased in case of Brucellar spondylitis as compared to control. The increment in Gamma Globulin is highly significant with p- value 0.000. The increased in Glubulin level can be attributed to immunologic response to Brucellar spondylitis that elicits the production of gamma globulins as reported by other authors in patients with a different type of brucellosis². The authors also reported high levels of IgG and IgM in brucellosis. Mean of Ratio Albumin/ Gamma in Brucellar spondylitis 2.8 ±0.11 and, in control 4.6±0.21 respectively. Ratio Albumin / Gamma significantly decreased in Brucellar spondylitis than in that of control p-value 0.000. This change associated with immunization and, a defense mechanism, comes as a result of an increase in the globulins of the blood sometimes a complete inversion of the normal albumin-globulin ratio is observed. This may be caused by ether living and dead organisms or by bacterial endotoxins. A rise in the globulins might occur long before the human develops immune bodies in any appreciable concentration. The progress of an infection is usually associated with marked changes in the serum proteins, especially in gamma globulin and Ratio Albumin/ Gamma. In general, serum proteins were affected by several points like adrenocortical hormones, the protein-catabolic reaction that follows an infection. The stage of infections.

Multivariable Regression analysis:

Multivariate analysis revealed that the significant predictor of total proteins in Brucellar spondylitis was Alpha 1 Globulin, whereas the significant predictors of total proteins in control were Albumin, Alpha 2 Globulin, Gamma Globulin and, Ratio Albumin/Gamma as shown in table 2.

Table 2 Summary of multivariable regression analysis, the dependent variable is total proteins.

Parameter	Brucellar spondylitis	Control
Albumin	Not dependent	↑↑
Alpha 1 Globulin	↑↑↑	Not dependent
Alpha 2 Globulin	Not dependent	↑↑↑
Beta	Not dependent	Not dependent
Gamma Globulin	Not dependent	↑↑↑
Ratio Albumin / Gamma	Not dependent	↑↑↑

using alpha level 0.05:

The cod ↑↑↑ represent t test equal or more than 3 and less than 3.5.

The cod ↑↑ represent t test equal or more than 2.5 and less than 3.

The cod ↑ represent t test equal or more than 2 and less than 2.5.

The cod ↓ represent t test equal or more than -2.5 and less than -2.

The cod ↓↓ represent t test equal or more than -3 and less than -2.5.

The cod ↓↓↓ represent t test equal or more than -3.5 and less than -3.

Multivariate analysis was repeated Using albumin to globulin ratio as dependent variable table 3. The result showed the most significant predictor of Albumin/ Gamma ratio in Brucellar spondylitis was Albumin, Alpha 2 Globulin, and Gamma Globulin whereas the significant predictors of Albumin/Gamma ratio in control were total proteins, and Albumin, whereas Alpha 2 Globulin and Gamma Globulin were conected negatively Albumin / Gamma ratio.

Table 3. Summary of multivariable regression analysis, the dependent variable is the Ratio of Albumin / Gamma.

Parameter	Brucellar spondylitis	Control
Total proteins.	Not dependent	↑↑↑
Albumin	↑↑↑	↑↑
Alpha 1 Globulin	Not dependent	Not dependent
Alpha 2 Globulin	↑	↓↓↓
Beta	Not dependent	Not dependent
Gamma Globulin	↑↑↑	↓↓↓

using alpha level 0.05:

The cod ↑↑↑ represent t test equal or more than 3 and less than 3.5.

The cod ↑↑ represent t test equal or more than 2.5 and less than 3.

The cod ↑ represent t test equal or more than 2 and less than 2.5.

The cod ↓ represent t test equal or more than -2.5 and less than -2.

The cod ↓↓ represent t test equal or more than -3 and less than -2.5.

The cod ↓↓↓ represent t test equal or more than -3.5 and less than -3.

The cluster analysis of Multivariate:

Cluster Analysis have been used to classify the variables as groups depends on their role in the studied patients or model, several methods is used to achieve the analyses, whereas the common one is wards method, to achieve the aimed of the current study, dendrogram shape have been used. Chart 1 A described the multivariable cluster analysis using Patient data. The results clearly identified the variables classified into four clusters. The first cluster included the most well-known diagnostic tests, as well as some fractions of proteins (alpha 2, gamma globulin, Beta and alpha 1). Cluster two included Albumin and albumin to globulin ratio. Cluster three included only total albumin. A fusion of the first cluster with the second one takes place to form a single group. Chart 1 B described the multivariable cluster analysis using control data. The results clearly identified that the variables classified into five clusters. Cluster one included the most well-known diagnostic tests, X-ray, MRI examination, IGM, IgG, Rose Bengal test, Back-pain, Age, and gender. Cluster two included alpha 2, gamma globulin, Beta and alpha 1. Cluster three included Albumin and albumin to globulin ratio. Whereas the four one included only total albumin. A fusion of cluster three and four takes place to form a single group and fusion of cluster two and one takes place to form a single group.

Chart 1: Cluster diagram of study variables in the serum of A: Brucellar spondylitis Patients’ and B: control group.

FUNDING:

This study was not funded, It was fully supported by the authors.

ETHICAL APPROVAL:

The studies have been approved by Committee of the University of Baghdad College of science for women, and have been performed in accordance with the ethical standard as laid down in 1964 declaration of Helsinki statement and its later corrections or comparable ethical standards, also accordance to the ministry of Iraqi health protocols.

Informed consent was obtained from all individual participants included in the study.

DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST:

NO potential conflicts.

ACKNOWLEDGEMENTS:

We owe our deep gratitude to university of Baghdad college of science for women., Serdem private hospital and Erbil international hospital

CONCLUSION:

The results of the present study of estimation of serum proteins in Brucellar spondylitis showed that Total proteins, Alpha 1 Globulin, and Gamma Globulin significantly elevated in patients when compared to control, moreover Alpha 1 Globulin was associated with total protein in the studied cases. All diagnostic tests were organized into 4 clusters in patient group which indicated that all the studied parameters were close to each other in diagnosis the patients with Brucella included Alpha 1 Globulin.

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Received on 25.11.2021 Modified on 27.01.2022

Research J. Pharm. and Tech 2022; 15(10):4733-4738.

DOI: 10.52711/0974-360X.2022.00795

Parameter	Brucellar spondylitis	Control	t- value	p- value
Parameter	Mean ± Std. Error	Mean ± Std. Error	t- value	p- value
Total proteins	8.0 ± 0.065^**	7.045 ± 0.2	4.85	0.000
Albumin	4.358 ± 0.091	4.038 ± 0.12	2.09	0.4
Alpha 1 Globulin	0.333 ± 0.024^**	0.184 ± 0.013	5.4	0.000
Alpha 2 Globulin	0.861 ± 0.034	0.7793 ± 0.051	1.32	0.19
Beta	0.946 ± 0.04	1.04 ± 0.034	-1.82	0.07
Gamma Globulin	1.5 ± 0.03^**	0.916 ± 0.074	8.0	0.000
Ratio Albumin/Gamma	2.8 ±0.11^**	4.6 ± 0.21	-7.4	0.000