Detailed Study Description Sampling, Eligibility Criteria, and Sample Size

Participant recruitment will be initiated through social media campaigns targeting
students from Ariel University and nearby academic institutions. Eligible participants
will be healthy adults aged 20-45 years who have adhered for at least one year to one of
three self-identified dietary patterns:

Plant-based diet (vegetarian or vegan; target 20% vegans),

Omnivorous diet ("regular" mixed diet), or

Low-carbohydrate diet (animal-based dietary patterns such as paleo or ketogenic).

Dietary pattern will be verified using a validated Food Frequency Questionnaire (FFQ)
developed by the Israeli Ministry of Health, Department of Nutrition (adapted from Berman
et al., 2023).

Exclusion criteria include obesity (BMI ≥ 30 kg/m²), underweight (BMI ≤ 18.5 kg/m²), or
any chronic disease, verified via self-report and confirmed by a physician's medical
summary.

Following informed consent, participants will complete the FFQ through the NutRatio®
dietary-analysis software to confirm diet pattern. A brief phone interview and an
instructional video will orient participants to study procedures and eligibility
requirements.

We aim to recruit 250 participants in total, evenly distributed across the three diet
groups. Participants will be frequency-matched by age (± 5 years), BMI (± 3 kg/m²), and
smoking status, given smoking's established association with metal exposure. Sample size
is based on ANOVA (Cohen's f = 0.25, medium effect size).

Measurement Day Protocol

Eligible participants will be scheduled for a standardized assessment session (~2 hours)
at the MetaboLab, Department of Nutritional Sciences, Ariel University.

Short-term dietary intake: Participants will complete a 3-day food record (two weekdays +
one weekend day) using NutRatio software within two weeks before their visit. The tool
integrates USDA (SR-28), Israeli Ministry of Health (2017), and updated food label
databases (2024). Participants will photograph each meal before and after eating, upload
photos to the research team, and record foods immediately after consumption. The team
will review entries daily, ensuring completeness and logical accuracy; missing or unclear
data will be clarified by phone the next day.

NutRatio will quantify energy, macronutrients (including fatty-acid and amino-acid
profiles), dietary fiber, selected vitamins (e.g., folate, B-12), and minerals (e.g.,
calcium, iron, zinc). Dietary metal exposure will be estimated from the reported foods
using both NutRatio and additional reference databases. These values will later be
compared to international toxicological thresholds (e.g., U.S. CDC, German Environment
Agency).

Food Sampling for Toxic Metal Assessment

Based on correlations between reported intake and measured blood or urine metal levels,
food groups most associated with each metal will be identified. Representative items will
then be sampled for laboratory analysis.

For example, if mercury levels correlate strongly with fish intake, approximately six
fish species or brands most commonly consumed by high-exposure participants will be
purchased and analyzed. The type and brand of food will be confirmed through participant
follow-up when necessary.

Each food item will undergo chemical digestion and quantification of metals (cadmium,
lead, chromium, arsenic, mercury). Measured concentrations will be compared with
international datasets including EFSA, Codex Alimentarius, U.S. NCEH, and the Israeli
Ministry of Health (Food Control Services, 2016).

Anthropometric and Demographic Measures

Participants will attend the study visit after an overnight fast (water only).
Measurements will include:

Weight and height (standardized protocols),

Body composition using InBody 570 (Biospace, Korea), a validated bioelectrical impedance
analyzer providing body-fat, muscle-mass, and water-compartment data (Miller et al.,
2016; Sirirat et al., 2020).

Questionnaires will collect demographic information, health history, smoking,
occupational exposures (including military service), and lifestyle factors related to
environmental pollutant exposure. These tools are adapted from Israel's national
heavy-metal survey (Berman et al., 2023) and the European PARC initiative
(https://www.eu-parc.eu

).

Biological Sample Collection and Chemical Analysis

Blood (whole and serum) and urine samples will be collected using BPA-free tubes and
stored at -20 °C until analysis to prevent chemical degradation. Serum will be separated
by controlled thawing (2-8 °C overnight), gentle mixing, clotting (30 min),
centrifugation (15 min, 2000 rpm), and pipette aspiration. Whole-blood samples will be
homogenized by vortex.

Samples (blood, serum, urine, and food) will be digested using analytical-grade nitric
acid and hydrogen peroxide in a microwave digestion system following EPA 3052 protocols
(Bocca et al., 2003). Blank samples will accompany each batch to determine limits of
detection and quantification. Quality control will include certified reference materials
and inter-laboratory validation using split samples.

Primary analyses will be conducted at Ariel University's Trace Metal Laboratory (Co-PI:
Dr. Daniel Palchan). Additional or confirmatory analyses may occur at the Ministry of
Health laboratory (Dr. Tamar Berman), Hebrew University, Weizmann Institute, or
Geological Survey of Israel, depending on instrument availability.

Trace-element quantification will be performed using Inductively Coupled Plasma-Mass
Spectrometry (ICP-MS) according to validated methods (Laur et al., 2020). Analytical
protocols will align with CDC DLS 3040.1-01. Metal concentrations in blood and urine will
be compared with reference values from national (Berman et al., 2023) and international
surveys (CDC 2018; EFSA 2010; German Environment Agency 2023).

Results will be evaluated against Human Biomonitoring (HBM-I, HBM-II) thresholds (German
Environment Agency 2023). Creatinine will be measured for urinary normalization.

Data Integration and Statistical Analysis

All data sources-FFQ, 3-day food record, anthropometric measures, biological samples, and
chemical analyses-will be integrated into a unified database. Statistical analyses will
examine relationships between dietary pattern and biomarker concentrations using ANOVA,
multivariable regression, and correlation analyses. Dietary estimates of metal exposure
will be compared with both measured biological levels and concentrations found in
representative food samples.

Potential confounders (age, BMI, smoking, and occupational exposure) will be adjusted for
in multivariate models. Sensitivity analyses will explore differences by sex and by vegan
versus vegetarian subgroups within the plant-based group.

Pilot Study

A pilot phase involving 10 volunteers will precede full data collection. The pilot will
test recruitment, informed-consent communication, FFQ completion, anthropometric
assessment, biospecimen collection, and data-entry logistics. It will also be used to
optimize sample-preparation protocols in the Trace Metal Laboratory.

If full analytical workflows cannot yet be implemented, mock data for metal levels will
be generated to validate database structure and preliminary statistical pipelines.
Biological samples from the pilot will be stored frozen for later analysis once full
ICP-MS procedures are operational.