The overall objective of this study is to evaluate the iron absorption from iron chlorophyllin. Iron deficiency is a public health problem in both developing and industrialized countries. There are several approaches to combat iron deficiency. Most supplements in the present day, to address the problem of iron deficiency, are in the form of iron salts, known as ferrous salts, especially ferrous sulfate. However, we can only usually absorb about 20% of the total iron content in ferrous sulfate. The common strategy of food supplement companies is to increase the amount of iron in the supplements to compensate for the low absorption rate. However, this often causes gastrointestinal side effects. In the present study, we would like to measure the iron bioavailability from sodium iron chlorophyllin, which made up from ferrous salts and chlorophyllin and where we hypothesize that it is absorbed via a different pathway than ferrous sulfate. Via this mechanism, we further hypothesize that sodium iron chlorophyllin will therefore have an enhanced bioavailability and more favorable side effect profile than ferrous sulfate and other iron salts.

New approaches to treat iron deficiency include developing novel iron compounds with possible iron absorption routes that increase iron bioavailability and reduce gastrointestinal side effects. Heme iron is considered to be highly bioavailable (10-20%) and less affected by meal composition than non-heme iron. Heme iron is iron (Fe2+) that is bound to the iron protoporphyrin IX prosthetic groups of proteins, mainly hemoglobin and myoglobin, which are present in animal tissue. Heme is released from hemoglobin during digestion so that it can be taken up by the duodenal enterocytes. The intact iron porphyrin is transported across the brush border membrane by the Heme Carrier Protein 1 (HCP1). Once inside the cell, the iron is released, and it is then likely to enter the low molecular weight pool of iron. The use of heme analogues from vegetable origin could provide an alternative iron source of potentially high bioavailability.

Sodium iron chlorophyllin (SIC) is a water-soluble semisynthetic chlorophyll derivative where the magnesium in the porphyrin ring has been substituted by iron. It is a greenish brown pigment that dissolves in water, alcohols, and chloroform, but not ethers. SIC is known to have a superior processing stability to chlorophyll and is commonly used as food colorings in Asian countries including in Korea and Japan, for foods that do not contain meat or fish. Magnesium Chlorophyllin has an E number of E140 and is approved as a food additive in Europe. Currently sodium iron chlorophyllin is present in the Designated Additives List in Japan with a number 261. Designated additives are those designated by the Minister of Health, Labour and Welfare as substances that are unlikely to harm human health based on Article 10 of the Food Sanitation Act.

Given the porphyrin ring heme-like structure of SIC, it may be an alternative delivery route for iron, suitable also for vegetarians and vegans, yet there are few studies investigating the use of SIC for this purpose.

A recent study by Ding et al. 2019 suggests a positive effect of SIC on hemoglobin (Hb) values in iron-deficiency anemia (IDA) in children and adults after 1 month of treatment. They assume that SIC is taken up by the heme carrier protein. The researchers used shengxuening tablets, also known as SXN, which are based on chlorophyll/porphyrin structures extracted from silkworms in which the Mg is replaced by the Fe and then coadjutants are added. This study provided no data on the absorption pathway of the SXN compound. Thus, it remains unclear if it was taken up by the heme transporter or whether it was digested and the Fe released in the gut lumen to join the non heme Fe pool, and thereby improved Hb in the IDA subjects. The authors also describe Xray diffraction assays to assess if the chlorophyllin porphyrin ring contained Fe and not Mg, however these data are not reported.

Miret et al. (2010) studied the stability of heme-analogous SIC absorption using the Caco-2 cell model. The SIC remained stable and only 5% of the Fe from the compound was released at pH 2 to 4. In-house dissolution studies conducted at the Human Nutrition Laboratory (HNL) show only a 2% release of Fe from the compound at similar pH levels. Using solid phase extraction at a higher pH of 7, similar to that in the duodenum, we observed a 5% Fe loss, which may correspond to free iron or iron liberated from any other binding sites on the chlorophyll structure.

Toyoda et al. (2014) performed a toxicity study of SIC performed in male and female rats with oral administration of SIC in their diet at concentrations of 0%, 0.2%, 1.0%, and 5.0% for 13 weeks. No abnormal clinical signs, no mortality and no abnormal hematological changes were observed in any of the groups during the experiment. Based on the histopathology of the parotid glands, the no-observed-adverse-effect level (NOAEL) of SIC in this study was estimated to be 1.0% (609 mg SIC/kg bodyweight (bw)/day for males and 678 mg/kg bw/day for females). In the present study, we will use 6 mg elemental iron, in 100 mg SIC, 0.002% of the equivalent dose administered by Toyoda et al.

SIC has been produced at the HNL with a 75% incorporation of iron into the chlorophyll. Our protocol is based on a Unilever patent that is no longer active, with in-house adaptation. The SIC has a neutral taste and will be presented as a dark green liquid, being the solution of SIC in water.

The goal of this project is to develop a novel iron compound that can be used as a food supplement. The iron content in our SIC will be limited to 6 mg per dose, which therefore does not exceed the 14 mg daily iron dose stipulated as the legal maximum in Switzerland for food supplements. SIC is already used widely as a food colorant and is safe for human consumption.

We propose to test our SIC against a known comparator, ferrous sulfate, to ascertain whether SIC is an effective iron delivery compound. If SIC, as hypothesized, behaves in a similar way to heme, then commonly used enhancers of iron absorption effective on ferrous sulfate, such as ascorbic acid, will not have an effect on the absorption of iron from SIC

Inclusion Criteria:

• female aged between 18-45 y old;
• SF <80 µg/L;
• BMI 18.5-24.9 kg/m2;
• weight <70 kg;
• signed informed consent;
• able to communicate and comprehend English language

Exclusion Criteria:

• anemia (Hb <12 g/dL);
• inflammation (CRP > 5 mg/L);
• chronic digestive, renal and/or metabolic disease;
• chronic medications (except for oral contraceptives);
• use of vitamin, mineral and pre- and/or probiotic supplements in the previous 2 weeks before study initiation and during the course of the study;
• blood transfusion, blood donation or significant blood loss over the past 4 months;
• pregnancy (tested in serum at screening) or intention to become pregnant;
• lactation up to 6 weeks before study initiation;
• earlier participation in a study using stable isotopes or participation in any clinical study within the last 30 days;
• smoking;
• unwilling to use an effective method of contraception.

Female