Long-term Iron Absorption and Loss in Women

1. Research Objective To study iron absorption, loss, and long-term iron gain in overweight or obese middle-aged Thai women compared with normal-weight middle-aged Thai women.
2. Research Methodology 2.1 Study design: This study is a longitudinal study comparing long-term iron absorption and loss between overweight or obese middle-aged Thai women and normal-weight middle-aged Thai women.

2.2 Study population: The study population consists of 64 women aged 40-50 years, divided into two groups: 32 overweight or obese women, and 32 normal-weight women.

2.3 Research Procedures 2.3.1 Before data collection: The research protocol is submitted for approval to the Central Institutional Review Board, Mahidol University. All data collectors will be trained prior to fieldwork. Upon receiving ethical approval, recruitment posters will be used to invite participants.

Screening process:

1. Interested individuals meeting preliminary eligibility criteria will be informed about study details, sign informed consent, and schedule the first visit for screening and blood collection.

2. Visit 1 (Screening):

   • Collect demographic and health history, measure weight, height, and body composition.
   • Collect 5 mL blood sample for Hb, SF, CRP, AGP, and hemoglobin typing.
   • Perform urine pregnancy test.
   • Administer one 400 mg albendazole tablet to prevent parasitic infection.

Iron isotope administration and follow-up:

2.3.2 Eligible participants will return within 1-2 weeks for the second visit.

Visit 2 (Month 0; M0):

Participants will consume a 15 mg ⁵⁷Fe isotope solution followed by 100 mg vitamin C drink, then fast for 3 hours.

A snack (plain yogurt and water) will be provided 1.5 hours after ingestion. A 24-hour dietary recall and semi-quantitative food frequency questionnaire (FFQ) will be conducted, with two additional 24-hour recalls by phone (2 weekdays and 1 weekend day).

Participants will then enter a 1-year equilibration period, recording illness and menstrual data monthly. The research team will monitor via LINE or email.

Visit 3 (Month 9; M9):

Collect fasting blood (5 mL) for Hb, glucose, HbA1c, and lipid profile; measure weight, BP, and conduct 24-hour recall (plus two by phone).

Visit 4 (After 1 year; WK0):

Collect fasting blood (10 mL) for Hb, SF, TfR, CRP, AGP, erythrocyte isotopic enrichment.

Measure weight, body composition, pregnancy test, and dietary recalls (3 days) and FFQ.

Visits 5-7 (WK8, WK16, WK24):

For eligible participants, repeat blood tests (7 mL) for Hb, SF, TfR, CRP, AGP, isotopic enrichment, anthropometry, pregnancy test, 3-day dietary recalls, and FFQ.

2.4 Research Instruments

1. General information and medical history questionnaire, 24-hour dietary recall, and FFQ.

2. Illness and menstrual record forms.

3. Anthropometry and clinical measures:

   • Weight: InBody digital scale
   • Height: Harpenden stadiometer
   • Body composition: Bioelectrical Impedance Analyzer (BIA; InBody)
   • Blood pressure: Omron digital sphygmomanometer

4. Laboratory analyses:

   • Hb: Complete Blood Count (CBC) using flow cytometry
   • SF, TfR, CRP, AGP: ELISA (SFa calculated from SF, CRP, AGP)
   • Hemoglobin typing: Capillary electrophoresis
   • Fasting glucose: Enzymatic method
   • HbA1c: Turbidimetric inhibition immunoassay
   • Lipids: Enzymatic colorimetric and homogeneous methods
   • Iron isotopes: Inductively Coupled Plasma Mass Spectrometry (ICP-MS) 2.5 Data Analysis Nutrient intake (energy, carbohydrate, protein, fat, iron, zinc, calcium, vitamin C, vitamin A, and fiber) will be calculated using INMUCAL software.

Dietary patterns will be analyzed using Principal Component Analysis (PCA).

The average iron absorption (Fe_abs) over the study period will be calculated as:

Feabs=kabs×FetotalFe_{abs} = k_{abs} \times Fe_{total}Feabs=kabs×Fetotal where Fe_total is the total body iron at the midpoint estimated from the regression of Fe_total over time, and k_abs is the fractional iron absorption rate derived from the slope of the regression line of ln(⁵⁷Fe) vs. time.

Average iron loss (Fe_loss) is calculated similarly:

Feloss=kloss×FetotalFe_{loss} = k_{loss} \times Fe_{total}Feloss=kloss×Fetotal

Iron gain (Fe_gain) is calculated as:

Fegain=Feabs-FelossFe_{gain} = Fe_{abs} - Fe_{loss}Fegain=Feabs-Feloss Differences between groups in general characteristics, biochemical parameters, and iron absorption/loss slopes will be tested using Independent t-test or Mann-Whitney U-test, as appropriate.

A Linear Mixed-Effects Model will be used to analyze group differences in longitudinal changes of iron absorption, loss, and gain.

Statistical significance will be set at p < 0.05.