What is lactoferrin?

Lactoferrin is a monomeric glycoprotein of about 80 kilodaltons, built from a single polypeptide chain of roughly 690 amino acid residues that folds into two lobes of similar architecture. Amino acid sequence places it in the transferrin family, a group of proteins defined by their capacity to bind iron.

The molecule, as the crystallographers found it

Anderson and colleagues refined the structure of human lactoferrin at 2.8 ångström resolution in 1989. Their model accounts for 681 of the 691 residues, two ferric iron ions and two carbonate ions. The two lobes, representing the N-terminal and C-terminal halves of the chain, fold almost identically: superposing one on the other leaves a root-mean-square deviation of 1.32 ångströms across 285 of 330 alpha-carbon positions, and the differences that remain sit in surface loops. Each lobe divides into two dissimilar domains, one built on a six-stranded beta-sheet and one on a five-stranded sheet, with the iron site in the cleft between them.

That architecture is not peculiar to humans. Crystal structures have since been solved for lactoferrin from cow, horse, buffalo and camel, and the three-dimensional fold is highly conserved across all of them, with differences only in detail.

The iron site, and why the grip survives acid

Each iron atom is coordinated by four protein ligands, two tyrosine residues, one aspartate and one histidine, together with the carbonate ion, which binds the metal in a bidentate mode. The anion sits in a pocket between the iron and two positively charged groups on the protein, and may neutralize that charge before iron arrives.

The behavior that separates lactoferrin from its close relative serum transferrin is what happens as acidity rises. Transferrin releases its iron, which is how cells collect it. Lactoferrin keeps hold much further down the pH range. Anderson and colleagues traced that greater acid stability to residues on the far side of the iron site, near the strands crossing between the domains, which could provide secondary anion binding sites. Binding and release are also accompanied by domain movements that close or open the site, and the two lobes influence each other as they do it.

The protein exists in an iron-free form, called apo-lactoferrin, and an iron-loaded form, holo-lactoferrin. Which form a supplement or a milk fraction contains is often unstated, and the distinction matters for any argument that rests on iron withholding.

How much is in milk, and in which milk

Figures for lactoferrin concentration circulate widely without saying which milk they describe. The gap between human colostrum and bovine milk runs to more than an order of magnitude, so a number without its source is close to useless.

Yang and colleagues drew their 824 samples from a cross-sectional study of 6,481 lactating women across eleven Chinese provinces and regions between 2011 and 2013. The design compares groups of women at different stages rather than following the same women down the curve, so it describes the fall across lactation without tracking it in any individual. Maternal body mass index, age, mode of delivery, parity, protein intake and serum albumin showed no correlation with milk lactoferrin. Geography and ethnicity did: milk from Dai women averaged 1.80 g/L against 0.99 g/L from Tibetan women.

The bovine figures come from 198 Chinese Holstein cows with no sign of mastitis for two months. Concentration tracked stage of lactation and fell as daily milk yield rose, which is part of why single published values for cow's milk disagree with each other so often. Comparisons across lactoferrin sources inherit that problem. The human rows above rest on UPLC/MS and the bovine row on ELISA, so part of the gap between them belongs to the assay. The table omits bovine colostrum, which a 1988 lactation series puts at 0.83 g/L falling to 0.09 g/L by the third week; the full set of figures with every method stated sits in the reference table on lactoferrin concentration. The assay is only half of what separates those rows. The two proteins are 69 percent identical and carry different sugar chains, and human lactoferrin sets out the sequence comparison and the three trials that have given the human form to people.

The second origin

Masson, Heremans and Schonne identified lactoferrin as one of the major proteins of human and guinea pig neutrophils in 1969, matching its electrophoretic, antigenic and iron-combining properties against the protein isolated from milk. Immunochemical measurement put the content at 3 micrograms per million human neutrophils. Cultured guinea pig bone marrow and spleen made the protein rather than merely storing it, and immunohistochemistry located its first appearance at the promyelocyte stage.

Because neutrophils carry it and release it where they gather, lactoferrin in a sample of stool reports on neutrophil traffic into the gut. That is the basis of the fecal lactoferrin assay, covered separately. The assay-dependence that makes the two rows above awkward to compare runs through that literature as well: the published thresholds for the lactoferrin test are quoted in micrograms per gram of stool by some studies and micrograms per milliliter by others, with no conversion given.

Where the antibacterial reputation comes from

The older explanation follows from the iron binding. Holding iron in a form bacteria cannot reach restricts the growth of organisms that depend on it, and that is where the antimicrobial reputation began.

A second mechanism was found in 1992 and works without iron. Bellamy and colleagues cut human and bovine lactoferrin with gastric pepsin and isolated an antimicrobial peptide from a region near the N-terminus, away from the iron sites. The active sequence is a loop of 18 residues closed by a disulfide bond between cysteines 20 and 37 in human lactoferrin, or 19 and 36 in bovine. Synthetic copies killed Gram-negative and Gram-positive bacteria at concentrations between 0.3 and 3.0 micromolar, with the bovine peptide the more potent of the two, and the effect was lethal rather than merely inhibitory.

Those experiments used isolated bacteria in a tube. The distance between that result and a measurable effect in a person who swallows a capsule is where most claims about lactoferrin come apart. This site grades each claimed effect on its own evidence, and the record so far is mixed: the graded review of lactoferrin benefits sets out which claims human trials have supported and which they have tested and failed to confirm.

The mechanisms themselves divide the same way. A summary of what lactoferrin does places each function at the level where it was demonstrated, and the division is sharper than the literature usually admits: the iron binding comes from crystallography of the purified protein, the release from neutrophil granules from microscopy of human cells, and the receptor that supposedly carries milk iron into an infant gut from suckling piglets, mouse intestine and a cell line.