Abstract
BACKGROUND: To establish reference intervals (RIs) for red blood cell (RBC) folate, vitamin B12, homocysteine, and 5-methyltetrahydrofolate (5-MTHF) during the first trimester of pregnancy among women in the Qingdao region, stratified by age, body mass index (BMI), and total folate intake categories. METHODS: A cross-sectional study was conducted involving 2,911 pregnant women (9 to 13(+ 6) weeks of gestation) recruited during their first prenatal visit in Qingdao from February 2023 to April 2024. Fasting venous blood samples were collected, and biomarker concentrations were measured using ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) and chemiluminescence immunoassay techniques. Data analysis followed the CLSI C28-A3 guidelines, with subgroup stratification by age, BMI, and total folate intake. RESULTS: The biomarker concentrations are reported as median (interquartile range): Vitamin B12: 525.00 (422.50,663.50) pg/mL; RBC folate: 421.41 (327.14,558.14) ng/mL; homocysteine:6.3 (5.5,7.2) µmol/L, and 5-MTHF: 27.78 (19.28,39.24) ng/mL. Significant age-related and BMI-associated differences in biomarker levels were identified (P < 0.05). Age and BMI related differences in biomarker concentrations were mainly reflected in median values. Women aged ≥ 35 years had higher median levels of vitamin B12 (536.00 vs. 520.00 pg/mL), RBC folate (444.19 vs. 411.16 ng/mL), and 5-MTHF (28.38 vs.27.46 ng/mL), but lower median homocysteine (6.2 vs. 6.3 µmol/L) compared with women < 35 years (P < 0.05). Across BMI categories (underweight, normal weight, overweight, obese), median vitamin B12 varied (584.88, 578.54, 537.52, 532.26 pg/mL, P < 0.001), while RBC folate showed a progressively increasing trend (441.03, 457.18, 477.69, 483.66 ng/mL, P = 0.003). Median homocysteine (6.4, 6.2, 6.3, 6.4 µmol/L) and 5-MTHF (30.61, 30.80, 29.67, 27.42 ng/mL) also differed significantly across BMI groups (P < 0.001). Furthermore, biomarker concentrations demonstrated clear trends across quartiles of total folate intake: RBC folate, vitamin B12, and 5-MTHF increased progressively from Q1 to Q4, while homocysteine decreased (all P < 0.001). CONCLUSION: This study characterized the distribution of RBC folate, vitamin B12, homocysteine, and 5-MTHF concentrations in early pregnancy (9-13(+ 6) weeks) and established RIs across different age, BMI, and total folate intake subgroups in Qingdao population. These findings provide valuable reference data for clinicians and laboratory researchers.