Probing the Synergistic Effect of Pre-Biotics & Iron Fortificants in Anemic
Phase: PHASE1
Conditions: Iron-deficiency
Sponsor: University of Veterinary and Animal Sciences, Lahore - Pakistan

Project Title: Probing the Synergistic Effect of Pre-biotics & Iron Fortificants in Anemic Subjects 
Principal Investigator: Dr. Waqas Ahmed 
NCT No.: NCT03894449 
Date: January 11, 2019 
IRC Ref No. : 
Organization: UVAS, Lahore 
Address: UVAS, Outfall Road, Lahore 
Phone: 0333-5950108 
Other Investigators: Abdul Momin 
Organization: UVAS, Lahore 
Address: UVAS, Outfall Road, Lahore 
Phone: 0300-5332253 
 
MATERIALS AND METHODS 
 
The study will consist of determining the synergistic effect of pre-biotics and iron fortificants in 
anemic human subjects. 
Study Design 
A double blind, randomized controlled trial will be used for the purpose of this study. 
Study Site 
The study will be conducted in Islamabad and/or Lahore, depending on the availability of the 
required sample size with the desired profiles. 
tudy Duration 
The trial will last for 3 months. 
Study Population  
The study population will comprise of university going iron deficient female adults (age 18-25 
years).  
3.3.4.1 Inclusion and Exclusion Criteria 
3.3.4.1.1 Inclusion Criteria 
All willing anemic female adults without any chronic diseases such as diabetes or hypertension will 
be included in the study.  
3.3.4.1.2 Exclusion Criteria 
Married females or those with chronic diseases or those already taking iron and/or prebiotic 
supplements will be excluded from the study. 
3.3.5. Sampling  
3.3.5.1 Sampling Technique  
The technique of convenience sampling will be used for the current study. 
3.3.5.2 Sample Size  
Each group will consist of 15 study subjects for the purpose of convenience and the total sample size 
will be 75. 
 

3.3.6 Treatment Plan 
For this phase of the study, 4 best treatment combinations of pre-biotics and iron fortificants will be 
chosen based on the results of animal trials while one group will be taken as control (Table 1). 
 
Table 1:  Treatment Plan for Anemic Women 
GROUP 
DIET PLAN 
D0 
Control Group (Placebo)  
D1 
Best Treatment Combination 1 
D2 
Best Treatment Combination 2 
D3 
Best Treatment Combination 3 
D4 
Best Treatment Combination 4 
 
Study participants will be randomly chosen to receive either the best treatment combination 1, 2, 3, 4 
or the placebo for 12 weeks on daily basis.  
3.3.7 Efficacy Trials 
Up to three months, blood samples will be collected from overnight fasted women on monthly basis. 
3.3.7.1 Hematological Analysis 
The collected blood samples from all the groups will be subjected to hematological analysis. Blood 
complete picture including RBC indices such as Haemoglobin (Hb) levels and Hematocrit (Hr) levels 
& Leukocytes inlcuding Lymphocytes, Monocytes, Neutrophils and Basophils etc will be evaluated 
as per their respective protocols (Al Haj et al. 2011). 
3.3.7.2 Iron Biomarkers 
Blood serum biomarkers will be determined on the basis of analysis of obtained sera.  
3.3.7.2.1 Serum Folate 
Serum folate of the samples will be measured using the method mentioned by Shuaibi et al (Shuaibi 
et al. 2008). 
3.3.7.2.2 Serum Iron 
Concentration of serum iron concentration will be measured using Atomic Absorption 
Spectrophotometer (Wojciak et al. 2013). 
3.3.7.2.3 Serum Ferritin 
Ciba-Cornings Automated Chemiluminescence System's (ACS 180) commercially available kit will 
be employed to determine the levels of serum ferritin (Kazuaki et al. 2011). 
3.3.7.2.4 Serum Transferrin 
It will be measured as per the procedure menioned by Al-Buhairan and Oluboyede (Al-Buhairan and 
Oluboyede 2000). 

3.3.7.2.5 Total Iron Binding Capacity (TIBC) 
The standard formula for the determination of TIBC will be used; 
TIBC = Transferrin x 24 
3.3.7.2.6 Transferrin Saturation Fraction (TRSF) 
TrSF will be calculated as per the following expression; 
TrSF = Serum Iron Concentration x 100 
 
Total Iron Binding Capacity 
 
3.3.7.3 Liver Function Tests (LFTs) 
LFTs including AST (Aspartate Aminotransferase), ALT (Alanine Aminotransferase), ALP 
(Alkaline Phosphatase) and Total Bilirubin will be conducted (Basuny et al. 2009). 
3.3.7.4 Renal Function Tests (RFTs) 
RFTs including blood urea by GLDH-method and creatinine by Jaffe-method will be measured using  
their respective commercial kits (Jacobs 1996; Thomas 1998). 
 
3.3.7.5 Immunoglobulins 
Total Immunoglobulins will be determined using the technique of ELISA (Enzyme linked 
Immunosorbent Assay). 
3.3.7.6 Statistical Analysis 
In order to determine the level of significance, the statistical analysis of the obtained data will be 
done for the purpose of which, SPSS version 23.0 will be used. CRD will be used during the 
experiment and differences will be considered significant at P - value < 0.05 (Steel and Torrie 1980). 
3.3.7.7 Ethical Considerations 
The approval of the study will be taken from the Institutional Review Committee for Biomedical 
Research of the University of Veterinary & Animal Sciences, Lahore. Moreover, informed written 
consent will be obtained from all the study subjects. In addition to this, privacy and confidentiality of 
the data of all the study subjects will be ensured.