(M1030-05-32) Synthesis, Characterization, and Stability of Iron-Chlorophyll Derivatives (ICDs)

Purpose: Iron deficiency anemia impacts 2 billion people worldwide. Current prevention and treatment strategies heavily rely on inorganic iron-fortified foods and supplements. However, inorganic iron has low solubility which causes poor bioavailability, and can cause gastrointestinal issues. In contrast, heme iron (rich in red meat) has 2-10x better bioavailability without associated side-effects. Multiple supplements are marketed in China and Japan as iron chlorophyll derivatives (ICDs), structurally analogous to heme with the protoporphyrin chelating iron instead of Mg. However, a random sampling of these products reveals 0-50% of the iron to be ICD-bound. To evaluate the effectiveness of ICDs to deliver iron in pig and human models, we sought to synthesize and chemically characterize ICDs with >95% of the iron ICD-bound. We also evaluated shelf stability using an accelerated model.

Methods: Chlorophylls were isolated from spinach using acetone and ethyl acetate, and converted into pheophytins using acetic acid. Afterward, they were refluxed with ferric chloride to produce ICDs. The mixture containing ICDs was further saponified to enhance stability and purity. The ICDs in the product were characterized using HPLC-DAD MS, and MS/MS. Total iron measurements of the ICD-containing product were conducted using AAS, and percent of remaining free iron was assessed via a ferrozine assay. Accelerated shelf-stability studies were also conducted under atmospheric O₂ and argon. ¹H, ¹³C, and 2D NMR analyses are ongoing.

Results: ICDs were successfully synthesized in the extract. Based on HPLC retention times relative to authentic standards of chorophyll derivatives, UV-Vis, MS, and MS/MS spectra, we putatively identified two primary derivatives, iron pheophorbide a and b, in the mixture. Based upon the difference in AAS and ferrozine measures, we established that >98% of the iron in the final product was ICD-bound. ICD purity in the saponified final product was ~15%. Shelf stability testing at 75% humidity at 40°C for 4 weeks (equivalent to 4 months of shelf life) revealed 85% of ICD loss under atmospheric oxygen, and 25% ICD loss under argon. In contrast, when backfilled with argon and stored at -80 °C over 6 months, ICDs in the product were 75% stable.

Conclusion: We successfully synthesized and putatively characterized both labelled and unlabeled ICD product and identified current limitations of storage. This method has been leveraged to synthesize ⁵⁴Fe-ICD product for a pilot clinical trial in iron-deplete women, to compare iron delivery from heme. Results of the human study will determine whether ICDs should be considered as a supplemental source of iron (and thus whether additional purification and storage strategies should be explored).

References: Kang et.al., Synthesis, characterization, and functional properties of chlorophylls, pheophytins, and Zn-pheophytins. Food Chem.15;245:943-950. doi: 10.1016/j.foodchem.2017.11.079.
Kopec et.al., LC-MS metabolomics reveals how food proteins affect iron absorption using in vitro digestion coupled with a Caco-2 cell intestinal model. Ohio Mass Spectrometry Symposium. October 15, 2024, Columbus, OH.

Acknowledgements: We thank the Bill & Melinda Gates Foundation and USDA Multistate project #W5122 for providing funding to support the work. We thank 3i Innovations, Inc. for their assistance with the accelerated shelf-stability studies.