AN MSC PROJECT PROPOSAL PRESENTATION

On

MOLECULAR ACCOUNT OF THE THERAPEUTIC EFFECTS OF

CALCIUM AND CALCIUM-D-GLUCARATE ON MONOSODIUM
GLUTAMATE INDUCED FIBROID IN FEMALE WISTAR RAT

By
ESSIEN ANIEBIET UDUAK (19PCP02022)
Supervisor: Prof. I. S. AFOLABI

FEBRUARY 2021
 OUTLINE

INTRODUCTION
STATEMENT OF PROBLEM
JUSTIFICATION
AIMS
 OBJECTIVES
MATERIALS AND METHOD
CONTRIBUTIONS TO KNOWLEDGE
REFERENCE
 INTRODUCTION
Uterine fibroids are the most common benign uterine tumors, with an
estimated incidence of 20–40% in women during their reproductive
years (Eltoukhi et al., 2014).
This benign tumor originates from the uterine muscle cells and
consist of large amounts of extracellular matrix (Sankaran, 2008).
 STATEMENT OF PROBLEM

 Fibroid is present in 5-10% of infertile patients and may be the sole

cause of infertility in 1-2.4% (Guo, 2012).
Dyspareunia and non-cyclic pelvic pain (Anne, 2012).
Treatment of uterine fibroids includes myomectomy, hysterectomy,
hormone replacement therapy and uterine artery embolization;
 JUSTIFICATION

Uterine fibroids surgical treatments are expensive and also have side effects.
They affect reproductive health, fertility, and birth rates and may even lead to
death, which indicates a need for more understanding on how/why these
tumors develop and grow as well as a need for the non-surgical and less
expensive prevention/treatment.
 AIMS

This work aims to ascertain the effectiveness of calcium-d-glucarate as either

a preventive or curative treatment measure for fibroid.

Following this the Biochemical role of selected minerals as well as the likely

mineral deficiencies contributing to the development of uterine fibroid may

be elucidated.
 OBJECTIVES

To ascertain the biochemical role of selected minerals during/in the

development of fibroid among women.

To identify the likely mineral deficiencies contributing to the

development of uterine fibroid in women.

To identify possible relationship/contribution of Atpase gene and

lactate dehydrogenase(LDH) gene to the development of uterine
fibroid in women.
 MATERIALS AND METHOD
Monosodium glutamate, mineral formulated diet and calcium d-
glucarate supplement (to be sourced), healthy animals (FUNAB
Abeokuta), animal feed (commercial feeds), ceramic plates, bottle
water, beddings, EDTA and heparin bottles, Eppendorf tubes.
Formalin, formaldehyde.

Total RNA isolation kit , Buffer of the above kit.

 EXPERIMENTAL DESIGN AND ADMINISTRATION OF DIETS TO THE EXPERIMENTAL ANIMALS

Pre-Fibroid induced treatments phase

 Group 1 shall be administered with distilled water to serve as normal control.

 Group 2 shall be administered with 750 mg/kg bwt monosodium glutamate to serve as negative control group.
 Group 3 - shall be administered with the calcium-d-glucarate (35.0 mg/kg bwt.) formulated diet and
subsequently the daily administration of the 750 mg/kg bwt monosodium glutamate.

 Group 4 - shall be administered with the calcium (9.2 µL/ g bwt.) formulated diet and subsequently the 750
mg/kg bwt. monosodium glutamate.

 Group 5 shall be administered with a combination of the calcium formulated diet (9.2 µL/ g bwt.) and the
calcium-d-glucarate supplement (35.0 mg/ kg bwt) and subsequently the 750 mg/kg bwt. monosodium glutamate.
 EXPERIMENTAL DESIGN AND ADMINISTRATION OF DIETS TO THE
EXPERIMENTAL ANIMALS (CONT’D)

Post-Fibroid induced treatments phase

 Group 1 shall be administered with distilled water to serve as normal control.
 Group 2 shall be administered with 750 mg/kg bwt monosodium glutamate to serve as negative control
group.
 Group 3 shall be administered with 750 mg/kg bwt monosodium glutamate and subsequently the calcium-
d-glucarate (35.0 mg/ kg bwt) based diet.
 Group 4 shall be administered with 750 mg/kg bwt monosodium glutamate and subsequently the calcium
(9.2 µL/ g bwt.) based diet.
 Group 5 shall be administered with a combination of the calcium formulated diet (9.2 µL/ g bwt.) and the
calcium d glucarate supplement (35.0 mg/ kg bwt).
 INDUCTION
Induction of fibroid using monosodium glutamate.
Leiomyomas will be induced by the daily administration of a
single dose of 750mg/kg monosodium glutamate solution via
the intraperitoneal route following the method of Eze Steven,
(2019).
They will thereafter be sacrificed on the 14th, 28th and 42nd
day of induction.
Acclimatization of rat will be for a period of 2-4 weeks.
 ANALYSIS
Determination of Mineral concentration

The mineral concentration will be determined by serum digestion

using the AOAC wet-ash method described by Boyer 1984, and the
mineral content will be determined using the Atomic Absorption
Spectrophotometer (Shimadzu, model AA-7000) following the
method described by de Ruig 1986.
 ANALYSIS(CONT’D)
Molecular Analysis for ATPase and LDH gene

Total RNAs will be individually extracted using the Ambion Magmax™-96 total
RNA isolation kit (Life Sciences) according to the manufacturer’s instructions.

Briefly, 100 mg of tissue will be placed in the lysis solution and homogenized in
Qiagen Tissuelyzer™ for a period of 30s. Nucleic acids will be captured onto
magnetic beads, washed and treated with DNase.

Total RNA will then be eluted in 50 μl elution buffer. RNA quality and
concentration will be measured using an RNA Pico chip on an Agilent
Bioanalyzer (Söllner et al., 2017).
 ANALYSIS(CONT’D)
Histopathological Examination

Histological assessment of tissues will carried out. Tissues will be excised and
stored in 10% formalin. They will be embedded in paraffin section and stained

with hematoxylin and eosin (H&E). Histopathological changes will be

evaluated by a pathologist.
Statistical Analysis

The experiments will be done in three replicates and result will be represented
as mean ± standard deviation (SD). The data will be analyzed using analysis of

variance. The results having P <0.05 will be considered statistically significant.

 CONTRIBUTIONS TO KNOWLEDGE
At the end of this research,

This work will help to improvise nutritional strategies for the

management of fibroid.

This work will help to identify possible relationship between the

Atpase gene and lactate dehydrogenase(LDH) gene and it contribution
to the development of uterine fibroid in women.
 REFERENCE
 Afolabi, I. S., Nwachukwu, I. C., Ezeoke, C. S., Woke, R. C., Adegbite, O. A., Olawole, T. D., and Martins, O. C
(2018). Production of a new plant-based milk from Adenanthera pavonina seed and evaluation of its nutritional

and health benefits. Frontiers in Nutrition, 5:9.

 Anne, Z., David, B., Christoph, G., Matthias, S and Katharina, G (2012). Prevalence, symptoms and management
of uterine fibroids: an international internet-based survey of 21,746 women, BMC Women's Health. 12:6.

 Ciavattini, A., Di-Giuseppe, J., Stortoni, P., Montik, N., Giannubilo, R., Litta, P and Ciarmela, P (2013). Uterine
Fibroids: Pathogenesis and interactions with endometrium and endomyometrial junction. Obstetrics and

Gynecology International, 1–11.

 De Ruig, W (1986). Atomic absorption spectrometric determination of calcium, copper, iron, magnesium,
manganese, potassium, sodium, and zinc in animal feeding stuffs: Interlaboratory collaborative studies. Journal

of AOAC International, 69(6):1009–13.

 REFERENCE(CONT’D)
Douglas, C. and Hall, M (2001). Nutritional Influences on Estrogen Metabolism. Advanced Nutrition
Publications, Incorporated.

Eltoukhi, H., Modi, M., Weston, M., Armstrong, A and Stewart, E (2014). The health disparities of
uterine fibroids for African American women: A public health issue. The American Journal of Obstetrics

and Gynaecology; 210 (3):194e9.

Eze-Steven, P (2019). Effects of Spondias mombin and Aspilia africana Aqueous extracts on rats with
monosodium Glutamate Induced leiomyoma. Cross Currents. 1: 26-32.

Guo, C and Segars, J (2012). The impact and management of fibroids for fertility. Obstetrics and
Gynecology Clinics of North America, 39(4), 521–533.
 REFERENCE(CONT’D)
Harrington, J., Young, D., Essader, S., Sumner, S and Levine, K (2014). Analysis of human serum and whole
blood for mineral content by ICP-MS and ICP-OES: Development of a mineral omics method. Biological

Trace Element Research, 160(1)132–142.

Ibegbulem, C., Agha, N and Emeka-Nwabunnia, I. (2012). Unutilized energy reserves and mineral contents
of fibroid tissues suggesting perturbed membrane transport processes. African Journal of Biochemistry

Research Vol. 6(4)50-54.

Khan, A., Shehmar, M and Gupta, J (2014). Uterine fibroids: current perspectives. International Journal of
Women’s Health, 95.

Olubukola, T., Obiekwe, M and Olorunsogo, O (2019). Protective effects of alpha stone on monosodium
glutamate-induced uterine hyperplasia in female wistar rats. Journal of Ayurveda and Integrative Medicine,

11(3)217-223.
 REFERENCE(CONT’D)
Song, K., Yu, X., Lv, T., Chen, Y., Diao, Y., Liu, S and Yao, Q (2018). Expression and
prognostic value of lactate dehydrogenase-A and -D subunits in human uterine myoma and

uterine sarcoma. Medicine, 97:14.

Stewart, E., Cookson, C., Gandolfo, R and Schulze-Rath, R (2017). Epidemiology of

uterine fibroids: a systematic review. BJOG: An International Journal of Obstetrics &

Gynaecology, 124(10)1501–1512.

Styer, A and Rueda, B (2016). The epidemiology and genetics of uterine leiomyoma. Best
Practice and Research Clinical Obstetrics and Gynaecology, 34:3–12.
THANK
YOU