New! The Zinc Link:
Zinc's Separate, Critical Role in Copper-Iron Metabolism, Linking Ceruloplasmin with Transferrin

by Jon Sasmor RCPC (Mineral Guide, MinBalance LLC)
Updated February 1, 2022

Background: Why Ceruloplasmin?

As many of you know, ceruloplasmin is the enzyme at the heart of the Root Cause Protocol (RCP) nutrition program. RCP aims to increase the function of ceruloplasmin as a ferroxidase: oxidizing iron, mobilizing iron, and protecting from excess iron causing oxidative stress and inflammation.

My notes reflect that RCP arose when Morley Robbins, founder of the Magnesium Advocacy Group, read Dr. Ray Peat's writing about ceruloplasmin. Dr. Peat said he had never had seen a diet solely designed to boost ceruloplasmin function. Morley Robbins decided to try making that diet. At first, the new diet was called "The Steps to Increase Ceruloplasmin".

In late 2015, Morley Robbins published a post entitled, "If the Sun is the ‘center’ of our Universe, I’m coming to regard Ceruloplasmin as the ‘Sun’ of our universe of metabolic activity" (Robbins, Dec. 21, 2015).

By 2016, Morley Robbins had developed "the Root Cause Protocol" (RCP) to address the root cause of oxidative stress and inflammation: mineral imbalance and lack of ceruloplasmin.

Morley Robbins' relentless research led him to conclude that magnesium alone wasn't the solution. Instead,

"the lack of ceruloplasmin prevents our bodies from being able to metabolize iron. And this iron-ic dynamic, in turn, creates oxidative stress, which is at the root of both persistent fatigue and chronic disease. This relentless process of oxidative stress is also the metabolic cause of the ubiquitous loss of magnesium."

(Robbins, 2021, p. 10).

"the root cause of oxidative stress is 'cellular dysfunction' caused by an imbalance of three key minerals: copper, iron, and thus magnesium, compounded by a lack of ceruloplasmin, which prevents our ability to metabolize energy, recycle iron in the mitochondria, and, therefore, prevent harm caused by iron's inevitable interactions with oxygen in our tissues and blood."

(Robbins, 2021, p. 12).

RCP continues to grow and develop. RCP continues to focus on boosting the ferroxidase activity of ceruloplasmin. For more details, please download the official RCP Handbook (RCP Institute, LLC, 2021).

RCP has delivered outstanding results, and has reached hundreds of thousands of people, on social media, in trainings, webinars, video series, forums, and other RCP programs.

The importance of ceruloplasmin is catching on in the mineral balancing consultation field, even beyond RCP. It was notable that, at the 2021 HTMA Summit, many of the speakers mentioned ceruloplasmin and/or bioavailable copper. This was true even though many of the speakers were presenting an overall unfavorable view of copper.

Ceruloplasmin plays a crucial role in the body's mineral balance. It's one of the linchpins of copper-iron regulation. That's why you may be intrigued to learn about zinc's newly proposed vital involvement in ceruloplasmin's function.

As background, let's first review the minerals with previously known functions in ceruloplasmin.

Previously Known Roles of Minerals in Ceruloplasmin Function

Bento et al. (2007) identified structural roles of minerals in ceruloplasmin, as follows:

Copper (Cu) occupies at least six binding sites in ceruloplasmin. Copper-loaded ceruloplasmin (holo-CP) acts as a ferroxidase to oxidize iron, as well as many additional functions.

Iron (Fe) binds to two binding sites near the surface of ceruloplasmin. Ceruloplasmin then oxidizes iron from +2 to +3 oxidation state, to allow iron to be transported through the blood by transferrin.

Cobalt (Co) may at times displace copper from certain of its binding sites in ceruloplasmin.

Calcium (Ca) has a place in the structure of ceruloplasmin. Calcium may assist binding of holo-ceruloplasmin to red blood cells.

Sodium (Na) likely occupies three structural sites in ceruloplasmin. These sodium ions support the rigidity of three protuberances which limit access at ceruloplasmin's oxidation sites. Access is restricted to metal cations such as iron, as well as to biogenic amines such as norepinephrine, epinephrine, dopamine and serotonin. Larger molecules are excluded.

New! The Zinc Link

How Zinc Helps Copper-Loaded Ceruloplasmin Transfer Iron(III) to Transferrin

According to the recently published, groundbreaking research of Sakajiri et al. (2021):

Zinc (Zn) forms a link between ceruloplasmin and transferrin. The zinc link permits the safe, efficient transfer of iron(III) from ceruloplasmin to transferrin.

Three zinc ions bind each ceruloplasmin molecule, and also bind to apo-transferrin (transferrin without iron(III)). Two ceruloplasmin molecules bind to each transferrin molecule, one on each side, via tri-zinc links. Each ceruloplasmin transfers an iron(III) to transferrin.

Once the transfer of both iron(III)'s has completed, transferrin changes shape. As a result of the change in shape, iron-loaded transferrin dissociates from ceruloplasmin.

The zinc links between ceruloplasmin and transferrin ensure that iron(III) is escorted safely at all times. This mechanism prevents the release of reactive unbound iron (III) into the blood, during the transfer.

What Happens to Ceruloplasmin Function in the Absence of Zinc

Sakajiri et al. (2021) found that the ferroxidase function of ceruloplasmin was unaffected by absence of zinc. If there's no zinc around, iron still binds to the ceruloplasmin and is oxidized.

Copper, rather than zinc, is involved with the ferroxidase function. Copper is needed in ceruloplasmin to oxidize iron(II) to iron(III).

In contrast, zinc is then needed to safely transfer iron(III) from ceruloplasmin to transferrin.

Without zinc, the transfer of iron(III) to transferrin cannot complete with full effectiveness.

Also, without zinc, the release of dangerous unbound iron(III) increases.

Thus, both copper and zinc are critical to ceruloplasmin's safe processing of iron.

Further Details from the Authors: Zinc and Copper Play Clearly Different Roles in Ceruloplasmin's Function

At first, I wondered if zinc might be partly substituting for copper in copper deficiency, rather than playing its own necessary role.

However, in kind response to questions, corresponding author Dr. Takaki Yamamura made clear that zinc is playing its own separate, vital role in ceruloplasmin's function, distinct from the role of copper.

With Dr. Yamamura's permission to reprint, here are Dr. Yamamura's complete answers:

Did the experiments and computer models use apo-CP, or was it holo-CP (with or without copper)?
- We used holo-CP.
Do zinc and copper bind the same sites on CP, or different sites?
- Zinc binds to sites different from copper.
Do zinc and copper compete for binding, or do zinc and copper bind simultaneously?
- Zinc is not considered to compete with copper, because zinc and copper bind to different sites. Copper is stronger than that of zinc, because when using a chelate (Chelex 100), zinc was removed from CP, but copper was not. Cp has different sites for zinc and copper binding, so it doesn't know whether both bind simultaneously to CP.
Can copper perform the same function proposed here for zinc? Might zinc be substituting for copper in this role, due to copper deficiency? Or, do both copper and zinc have separate, essential roles for CP's functions oxidizing Fe(II) and then transferring Fe(III) to transferrin?
- Copper did not perform as well as zinc for the interaction of CP with apo-transferrin. We examined various metal ions, and zinc ion showed highest activity. I don't think that zinc can replace copper, because copper and zinc have different functions. Copper has the role of oxidizing Fe(II). The addition of zinc to the CP solution did not enhance its oxidative capacity. Our research results show that zinc plays a role as a mediator of the interaction between CP and apo-transferrin to transfer safely and efficiently Fe(III) from CP to apo-transferrin.

Possible Further Research

As Sakajiri et al. (2021) note, they performed the experiments at 25˚C instead of body temperature 37˚C because the reactions proceeded too fast to measure at 37°C. The reactions theoretically could proceed differently at 25˚C than at 37˚C, but there's no known reason why they would.

The authors note that ferroportin (FPN1) exports iron(II) from the cell, prior to ceruloplasmin's oxidation of the iron(II) to iron(III) and the transfer of iron(III) to transferrin. The iron(II) exported by ferroportin also is highly toxic and reactive in body fluids, and must be kept under guard. Sakajiri et al. (2021) propose that additional research could investigate whether a ternary complex may be formed between ferroportin, zinc-bound ceruloplasmin, and apo-transferrin. They suggest that zinc(II) binding of ceruloplasmin also may allow a bond to form with ferroportin, to create such a ternary complex.

Sakajiri et al. (2021) also suggest further research into clininal data investigating relationships between zinc and non-transferrin-bound iron levels.

Copper, Zinc, Iron, Ceruloplasmin, and Transferrin: A Symphony of Nature's Resourcefulness in Using the Minerals

Copper, zinc, iron, ceruloplasmin, and transferrin work together in an intricate dance. Together, they illustrate Nature's magnificent orchestration of the minerals:

Copper assists ceruloplasmin to oxidize iron from +2 to +3 oxidation state.
Zinc assists the transfer of the +3 form of iron from ceruloplasmin to transferrin.

Let's turn to the practical side. How do we use diet and supplementation to support Nature's symphony of minerals?

Practical Applications: How to Support Nature's Symphony of Minerals

As discussed above, Sakajiri et al. (2021) have unveiled an important, previously unknown role for zinc!

In ceruloplasmin, we can see an important example of the beautifully balanced symphony of the minerals.

This is why we obtain copper, zinc, iron, and many other transition metals, in natural amounts and proportions, from the organic ancestral whole foods diet which is part of the Root Cause Protocol!

Zinc Sources in the Organic Ancestral Whole Foods Diet

Grass-fed beef liver provides an excellent source of zinc, in natural proportions with copper, iron, retinol-A, B vitamins, and many other nutrients. Together, all these nutrients help make ceruloplasmin work to oxidize iron and move iron safely.
A small serving (4-5 oz) of grass-fed beef or lamb meat, 3-4 times weekly, also provides an outstanding source of zinc. White meat usually contains less zinc than red meat.
Pastured egg yolks provide a good source of zinc. Like many other nutrients, almost all the zinc is concentrated in the egg yolk; there is little zinc in the egg white.
Shellfish, especially oysters, are among the richest natural sources of zinc and copper. Be careful about sourcing due to potential presence of toxic metals from polluted environments.
Many plant foods (such as nuts, seeds, and legumes, properly prepared) contain zinc. Phytates in plant foods may limit absorption of zinc and other nutrients. Therefore, prepare plant foods with traditional methods of soaking, sprouting, and cooking; and also include in your diet animal zinc sources from the list above.

A Symphony Doesn't Like Certain Members Playing Way Too Loud

When megadoses are added as fortification or supplements, imbalances of other minerals occur. Megadoses are extremely common for iron and zinc!

Megadoses muck up the orchestra, so Nature's balanced symphony of minerals doesn't sound so good anymore. With megadoses, ceruloplasmin and other enzymes may have extra of some things they need, but they have lost the balance with other needed minerals.

In the long term, things don't work as well when we put in megadoses of certain ingredients that we think are important. Later, it turns out we find other ingredients equally important; and we need balance among all the minerals.

Therefore, RCP recommends stopping synthetic megadoses of zinc, iron, and other transition metals. Instead, eat natural food-based mineral sources, as part of the Root Cause Protocol. Eat minerals in Nature's balance.

Conclusion

Many of us have experienced bad results from megadoses of supplements like zinc, vitamin D, and iron. However, zinc is an important nutrient, and Sakajiri et al. (2021) have uncovered a specific, vital role for zinc in ceruloplasmin's function.

Copper helps ceruloplasmin oxidize iron; whereas zinc helps transfer iron to transferrin.

Zinc, copper, and iron all are needed together, interconnected in ceruloplasmin's work. And ceruloplasmin's function may be the root cause of many of our concerns!

We can give thanks for Nature's symphony of minerals by obtaining zinc, copper, iron, and other transition metals in Nature's proportions from Nature's food sources.

This article is here to celebrate the Zinc Link discovery of Sakajiri et al. (2021), and to help share the Zinc Link with those learning about ceruloplasmin for nutrition programs.

Bonus: In Greater Detail: The Zinc Link Experiments of Sakajiri et al. (2021)

Sakajiri et al. (2021) researched the zinc link with a series of experiments. Their published article includes experimental details and figures with data. A brief summary appears below. Their series of experiments shows resourcefulness, thoroughness, and ingenuity:

Formation of iron-loaded transferrin, with and without ceruloplasmin and zinc(II). Rates and completion of reactions were compared, for production of diferric holo-transferrin (Fe(III)₂TF, or transferrin loaded with two iron(III) ions) from iron(II) and apo-transferrin (transferrin without iron). Ceruloplasmin and zinc(II) each were included or excluded, and the ingredients were combined in different orders. Ceruloplasmin catalyzed the formation of iron-loaded transferrin. However, when ceruloplasmin was present, the reaction ran only to partial saturation unless zinc(II) also was present to permit full saturation. The coexistence of ceruloplasmin and zinc(II) enhanced the formation of iron-loaded transferrin.
Effect of zinc(II) on oxidative capacity of ceruloplasmin. The addition of zinc(II) scarcely affected the rate at which p-phenylenediamine (PPD) was oxidized by holo-ceruloplasmin. Thus zinc(II) appears to leave the ferroxidase function of ceruloplasmin unaffected. (Note: PPD oxidation by ceruloplasmin is a common spectrophotometric method for assessing ceruloplasmin's ferroxidase function. PPD oxidation by ceruloplasmin closely correlates with iron(II) oxidation by ceruloplasmin. See Johnson et al., 1967).
Human plasma with and without zinc(II). A chelator was used to remove zinc from human plasma. Almost all the copper remained after chelation. When iron(II) was added, iron-loaded transferrin formation was measured by absorbance at 465 nm. At 240 seconds, absorbance was 62% without zinc(II), compared with the same experiment including zinc(II) added. More iron-loaded transferrin was formed in human plasma when zinc was present.
Effect of zinc(II) on dangerous non-transferrin-bound iron in plasma. Iron(II) was mixed with human plasma and apo-transferrin at variable zinc(II) concentrations. Higher zinc(II) sharply reduced the non-transferrin-bound iron in plasma. Thus, the authors suggest that zinc deficiency promotes accumulation of the unbound, toxic iron in blood due to poor formation of the iron-loaded transferrin.
Comparative zinc(II) binding to ceruloplasmin and apo-transferrin. Zinc(II) was combined with ceruloplasmin and apo-transferrin, together or in either order. Kinetics favored momentary binding of zinc(II) with transferrin. However, thermodynamicaly, the zinc(II)-ceruloplasmin complex was more stable, and quickly predominated.
Binding affinity measurements. Using isothermal titration calorimetry, equilibrium dissociation constants were measured. Zinc(II) bound to ceruloplasmin with ~16-fold higher affinity than to transferrin. Stoichiometry showed as three zinc(II) per ceruloplasmin; and two zinc(II) per transferrin. The curves showed ceruloplasmin having one set of independent binding sites, whereas transferrin showed sequential binding.
Measurements of ceruloplasmin-transferrin binding, with and without zinc(II). Using isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR), ceruloplasmin was mixed with transferrin (both apo- and iron-loaded). In the absence of zinc(II), no direct interaction occurred between ceruloplasmin and transferrin. When zinc(II)-bound transferrin was introduced with ceruloplasmin, SPR showed binding occurred. The zinc would have quickly bound to ceruloplasmin; then the zinc-bound-ceruloplasmin attached to apo-transferrin. ITC analysis confirmed that a mixture of ceruloplasmin, zinc(II), and apo-transferrin produced a ceruloplasmin-transferrin bond. The equilibrium dissociation constant was measured, and showed a strong enough interaction to occur at physiological concentrations of transferrin. The stoichiometry was 0.64 apo-transferrin per ceruloplasmin. However, when zinc(II)-bound ceruloplasmin was mixed with iron(III)-loaded transferrin, little interaction occurred. Thus, ceruloplasmin bound to transferrin only in the presence of zinc(II), and only when transferrin wasn't yet loaded with two iron(III) ions. Ceruloplasmin must bind zinc(II) to link with apo-transferrin.
Computational modeling of the formation of ceruloplasmin-zinc(II)-apo-transferrin complex. Using the Metal Ion-Binding Site Prediction method, 44 candidate zinc-binding sites in ceruloplasmin were identified. Since the ITC analysis above showed ceruloplasmin binding three zinc(II) ions, three binding sites were selected with high validity and location near the surface of ceruloplasmin. Software was used to prepare a 3D model of the docking of Zn(II)-bound ceruloplasmin with apo-transferrin. Since ITC analysis above showed 0.64, or approximately 0.5, apo-transferrin per ceruloplasmin, the model showed 2 zinc(II)-bound ceruloplasmins binding each apo-transferrin. The details of the model, including which ions interact with which amino acids on each molecule, are presented in Sakajiri et al. (2021), along with neat graphical representations. Iron(III) traverses a specified escorted path from ceruloplasmin into transferrin, which avoids contact with body fluids. The model also shows how transferrin changes geometry once loaded with iron(III), in a way that makes binding with zinc(II)-bound ceruloplasmin become difficult. Thus, the successful loading of iron(III) onto transferrin breaks apart the linked complex that had formed for the purpose of the transfer.

References

Bento, I., Peixoto, C., Zaitsev, V. N., & Lindley, P. F. (2007). Ceruloplasmin revisited: structural and functional roles of various metal cation-binding sites. Acta Crystallographica Section D: Biological Crystallography, 63(2), 240-248. https://doi.org/10.1107/S090744490604947X

Johnson, D. A., Osaki, S., & Frieden, E. (1967). A micromethod for the determination of ferroxidase (ceruloplasmin) in human serums. Clinical Chemistry, 13(2), 142-150. https://doi.org/10.1093/clinchem/13.2.142

RCP Institute, LLC. (2021, October 4). RCP handbook, Version 10.0. https://therootcauseprotocol.com/handbook-download/

Robbins, M. M. (2015, December 21). Iron toxicity post #11: If the Sun is the ‘center’ of our Universe, I’m coming to regard Ceruloplasmin as the ‘Sun’ of our universe of metabolic activity. The Root Cause Protocol. https://therootcauseprotocol.com/mag-pie-alert-13-toxicity-of-iron/

Robbins, M. M. (2021). Cure: Your fatigue: How balancing 3 minerals and 1 protein is the solution that you're looking for. Gatekeeper Press. https://www.amazon.com/gp/product/1662910282

Sakajiri, T., Nakatsuji, M., Teraoka, Y., Furuta, K., Ikuta, K., Shibusa, K., Sugano, E., Tomita, H., Inui, T., & Yamamura, T. (2021). Zinc mediates the interaction between ceruloplasmin and apo-transferrin for the efficient transfer of Fe (III) ions. Metallomics, 13(12), mfab065. https://doi.org/10.1093/mtomcs/mfab065

New! The Zinc Link: Zinc's Separate, Critical Role in Copper-Iron Metabolism, Linking Ceruloplasmin with Transferrin