Beta-casein variants, beta-casomorphin-7 and infants
Hypotheses concerning biological responses to A1 beta-casein cows’ milk protein pivot about the release of the exogenous opioid peptide, beta-casomorphin-7 (BCM-7). This occurs via the incomplete digestion of A1 beta-casein(1–3) and its subsequent absorption into circulation. BCM-7 production is influenced by the amino acid present at position 67 of the beta-casein protein chain(4). A1 beta-casein has a histidine at this position while the other major beta-casein invariant, the A2 beta-casein type has a proline at position 67(4). Due to this amino acid variation, A1 beta-casein releases BCM-7 following normal enzymatic digestion(1–3,5,6), whereas A2 beta-casein does not or if it does, it happens at a very low rate(4).
Opioid peptides play a role in various biological processes, including respiration, analgesia, constipation and behaviour(7). However, as noted by Maklakova et al. (1993)
“Depending on the type of peptide, the mode of its administration into the organism, the species of experimental animal, and the peptide dose, it is possible to register an entire spectrum of effects, ranging from significant motor excitation to a complete inhibition of locomotor activity and catatonia”(8).
A1 beta-casein is present in cows’ milk and its products, including infant formula, but today some cows are selected for their genes to produce only the A2 type beta-casein type and not the A1 type, and these cows produce milk which is A1 protein free (e.g. a2 Milk® in Australia). Notably, human milk beta-casein and bovine A2 beta-casein share a proline at their aligned residues(9), (Figure 1).
Figure 1: Human beta-casein and A2 beta-casein share a proline at their aligned residues
Human BCM-7 has been identified in human breast milk, which shows that before the digestion of the human beta-casein in an infants’ gut, human BCM – 7 is present. Other human beta-casomorphins have also been found in the blood of pregnant and lactating women(11), but not in men or non-pregnant women, leading researchers to suggest that human BCM-type mammary products have physiological importance during pregnancy or after parturition(10,11). Importantly, human and bovine BCM-7 differ by two amino acids at positions four and five of the peptide (Figure 2). These structural differences affect the opioid activity of BCM-7(9) with bovine milk beta-casomorphins shown to be at least ten times more potent (i.e. greater binding affinity to mu-opioid receptors) than human beta-casomorphins(12–14). This appears to have consequences on the function of each variant.
Figure 2: Human derived BCM-7 structure [ H-Tyr51-Pro52-Phe53-Val54-Glu55-Pro56-Ile57-OH ] versus bovine A1 beta-casein derived BCM-7 structure [ H-Tyr60-Pro61-Phe62-Pro63-Gly64-Pro65-Ile66-OH ].
Recent studies have found bovine BCM-7 in the blood of human babies(15,16), while earlier research detected beta-casomorphin materials in the cerebrospinal fluid and blood of some lactating women and some female controls(17). The full implications of bovine BCM-7 absorption into human infant circulation requires ongoing research. However, elevated circulating human BCM-7 has been correlated with beneficial developmental outcomes in breastfed infants, while the opposite has been observed in their formula fed counterparts with elevated bovine BCM-7 levels(16).In addition, Jarmolowska et al. (2007) have shown that the concentration of human BCM-7 in colostrum (produced in the first days of lactation) is seven times higher than in human milk at two months of age (10), which suggests that a human infant’s requirement for human BCM-7 is significantly lower at two months compared with the first few days of life and that exposure to the more potent bovine BCM-7 at around two months of age may be undesirable.
Binding and rodent studies suggest that human and bovine BCM-7 may have central and/or peripheral effects via their ability to modulate the serotoninergic system, in addition to their known effect on the opioid system(18–20). Earlier rodent studies suggest an effect of shorter bovine BCM-7 derivatives on dopamine mediated processes(21,22). More recent animal research suggests that bovine BCM-7 may affect neurochemically mediated maternal-offspring bonding(8, 23–25) and learning, and environmental adaptation behaviour in infant and young mammals(26–28).
Currently, the role of bovine BCM-7 in the health and development of human infants is the topic of extensive scientific debate. Such debate was stimulated with the study by Kost et al. (2009), which showed that bovine BCM-7 could be measured in the blood of infants fed beta-casein containing infant formula(16). They also reported that higher blood levels of bovine BCM-7 found in some infants was correlated with delays in psychomotor development(16). Furthermore, babies who were unable to metabolise BCM-7 rapidly and thus eliminate it from their systems quickly, were particularly at risk.
Prior to the research by Kost et al. (2009), it had been argued that BCM-7 did not pass into human blood due to rapid proteolysis(29), even though BCM-7 and other related immunoreactive materials had been found in the blood of newborn calves(30) and dogs(31). In adult human males, it has also been shown that following casokefamide ingestion (a synthetic beta-casomorphin analogue), casokefamide-like-immunoreactivity was detected in blood within 60 minutes(32). Taken together, these data suggest that mechanisms exist by which beta-casomorphins are absorbed into the blood of both infant and adult humans. There are a range of physiological mechanisms by which this might occur(33–38). Further, the transport rate of beta-casomorphins has been shown to accelerate with increasing hydrophobicity(37), which suggests a faster rate of transfer for the more hydrophobic bovine relative to human beta-casomorphins. However, the precise mechanism of beta-casomorphin transfer from the gut to circulation has yet to be determined.
The latest research by a Polish research group (15) confirms the presence of BCM-7 in the blood of babies, but also suggests that BCM-7 may be a risk factor for apnoea (expressed as “apparent life threatening events”), compared to a group of healthy infants. In this study, blood BCM-7 levels in “at risk” apnoea infants were on average three times higher compared with normal infants. This work is consistent with earlier animal studies that have shown BCM-7 induces apnoea and irregular breathing in newborn rabbits and adult rats(39), and that the beta-casomorphin analogue morphiceptin has an effect on the brain stem respiratory center in dogs(40). Notably, mu-opioid receptors are expressed heavily in the brainstem(41) and BCM-7 is a known mu-opioid receptor ligand(42–45). This research also identified that “at risk” infants had low blood levels of DPP-4,the enzyme that metabolises BCM-7(46–48).
In the “at risk” infants, DPP-4 levels were 58% of those found in healthy babies. Furthermore, in the normal infants, BCM-7 was positively associated with DPP-4 levels (which leads to faster BCM-7 elimination). This relationship is absent in the “at risk” babies. The statistics associated with these findings are strong (i.e. p<0.001 in each case). Not surprisingly, this study also showed that BCM-7 levels were higher in babies fed formula which was high in casein, compared to those fed formula that was low in casein (BCM-7 can only be derived from casein and not from other milk proteins). However, it could be regarded as surprising that bovine BCM-7 was also found in the blood of 1–4 month old babies who were breastfed. The suggestion by the researchers is that bovine BCM-7 from milk ingested by lactating mothers can be transferred directly to the mothers’ milk.
Other research in human infants conducted by a Czechoslovakian research group supports the idea that bovine BCM-7 may be an early contributor to heart disease later in life. Steinerova et al. (2004) have shown that formula fed infants (beta-casein containing formula) have elevated serum levels of antibodies to oxidized LDL relative to breastfed babies(49). Given that BCM-7 has been shown to oxidise LDL in vitro(50), it is possible that the differences in oxidised LDL between the formula and breastfed babies are mediated via A1 beta-casein derived BCM-7. Although the researchers in this study did not assess serum BCM-7 levels, they did suggest that
“As human milk does not contain beta-casein A1 and infant formulas are based on bovine milk, we can express a hypothesis that beta-casein A1 is the substance, which caused increased production of IgoxLDL”(49).
The same research group has also identified that antibodies to oxidized LDL in formula fed infants have high affinity to A1 beta-casein and BCM-7 relative to A2 beta-casein and maternal milk in vitro(51), but the significance of this remains to be elucidated.
Given the growing body of evidence surrounding the significant health implications of A1 beta-casein derived BCM-7, the call for this issue to be taken seriously and thoroughly examined impartially and without industry interference is compelling.
- De Noni I (2008). Release of b-casomorphins 5 and 7 during simulated gastro-intestinal digestion of bovine b-casein variants and milk-based infant formulas. Food Chemistry. 110(4), 897–903.
- Jinsmaa Y, Yoshikawa M (1999). Enzymatic release of neocasomorphin and beta-casomorphin from bovine beta-casein. Peptides. 20(8), 957–62.
- Ul Haq M.R, Kapila R, Kapila S (2015). Release of beta-casomorphin-7/5 during simulated gastrointestinal digestion of milk beta-casein variants from Indian crossbred cattle (Karan Fries). Food chemistry. 168, 70–9.
- Scientific Report of EFSA prepared by a DATEX Working Group on the potential health impact of ß-casomorphins and related peptides. EFSA Scientific Report (2009). 231, 1–107 [cited April 2016]. Available from: http://edepot.wur.nl/8139
- De Noni I, Cattaneo S (2010). Occurrence of beta-casomorphins 5 and 7 in commercial dairy products and in their digests following in vitro simulated gastro-intestinal digestion. Food Chemistry. 119(2), 560–6.
- Hartwig A, Teschemacher H, Lehmann W et al. (1997). Influence of genetic polymorphisms in bovine milk on the occurence of bioactive peptides. Seminar on milk protein polymorphism. IDF special issue no. 9702. International Dairy Federation, Bruss els. 459–60.
- Bodnar R.J (2008). Endogenous opiates and behavior: Peptides. 30(12), 2432–79.
- Maklakova A.S, Dubynin V.A, Levitskaia N.H, et al. (1993). The behavioral effects of beta-casomorphin-7 and its des-Tyr analogs. Biull Eksp Biol Med. 116(8), 155–8.
- Hamosh M, Hong H, Hamosh P (1989). beta-Casomorphins: milk-beta-casein derived opioid peptides. In: Lebenthal E, ed. Textbook of Gastroenterology and Nutrition in Infancy, ed 2. New York: Raven Press; 143–50.
- Jarmolowska B, Sidor K, Iwan M, et al. (2007). Changes of beta-casomorphin content in human milk during lactation. Peptides. 28(10), 1982–6.
- Koch G, Wiedemann K, Drebes E, et al. (1988). Human beta-casomorphin-8 immunoreactive material in the plasma of women during pregnancy and after delivery. Regul Pept. 20(2), 107–17.
- Brantl V (1984). Novel opioid peptides derived from human beta-casein: human beta-casomorphins. Eur J Pharmacol. 106(1), 213–4.
- Koch G, Wiedemann K, Teschemacher H (1985). Opioid activities of human beta-casomorphins. Naunyn Schmiedebergs Arch Pharmacol. 331(4), 351–4.
- Herrera-Marschitz M, Terenius L, Grehn L, Ungerstedt U (1989). Rotational behaviour produced by intranigral injections of bovine and human betacasomorphins in rats. Psychopharmacology. 99(3), 357–61.
- Wasilewska J, Sienkiewicz-Szlapka E, Kuzbida E, et al. (2011). The exogenous opioid peptides and DPPIV serum activity in infants with apnoea express ed as apparent life threatening events (ALTE). Neuropeptides. 45(3), 189–95.
- Kost N.V, Sokolov O.Y, Kurasova O.B, et al. (2009). Beta-casomorphins-7 in infants on different type of feeding and different levels of psychomotor development. Peptides. 30(10), 1854–60.
- Lindstrom L.H, Nyberg F, Terenius L, et al. (1984). CSF and plasma beta-casomorphin-like opioid peptides in postpartum psychosis. Am J Psychiatry. 141(9), 1059–66.
- Sokolov O.Y, Pryanikova N.A, Kost N.V, et al. (2005). Reactions between beta-casomorphins-7 and 5-HT2-serotonin receptors. Bull Exp Biol Med. 140(5), 582–4.
- Sokolov O.Y, Kost N.V, Zolotarev Y.A, et al. (2006). Influence of human B-casomorphin-7 on specific binding of 3H-spiperone to the 5-HT2- receptors of rat brain frontal cortex. Protein Pept Lett. 13(2), 169–70.
- Zozulia A.A, Meshavkin V.K, Sokolov O, Kost N.V (2009). Naloxone-induced suppression of the behavioral manifestation of serotoninergic system hyperactivation by beta-casomorphins-7 in mice. Eksp Klin Farmakol. 72(2), 3–5.
- Ruthrich H.L, Grecksch G, Matthies H (1992). Influence of modified casomorphins on yawning behavior of rats. Peptides. 13(1), 69–72.
- Ruthrich H.L, Grecksch G, Matthies H (1993). Influence of betacasomorphins on apomorphine-induced hyperlocomotion. Pharmacology, biochemistry, and behavior. 44(1), 227–31.
- Stovolosov I.S, Dubynin V.A, Kamensky A.A (2011). Role of the brain dopaminergic and opioid system in the regulation of “child’s” (maternal bonding) behavior of newborn albino rats. Bull Exp Biol Med. 150(3), 281–5.
- Dubynin V.A, Ivleva Y.A, Stovolosov I.S, et al. (2007). Effect of betacasomorphines on mother-oriented (“child’s”) behavior of white rats. Dokl Biol Sci. 412, 1–4.
- Dobryakova Y.V, Dubynin V.A, Ivleva Y.A, et al. (2005). Effect of opioid antagonist naloxone on maternal motivation in albino rats. Bull Exp Biol Med. 140(1), 10–2.
- Dubynin V.A, Malinovskaia I.V, Beliaeva Iu A, et al. (2008). Delayed effect of exorphins on learning of albino rat pups. Biol Bull. 35(1), 43–49.
- Dubynin V.A, Malinovskaya I.V, Ivleva Y.A, et al. (2000). Delayed behavioral effects of beta-casomorphin-7 depend on age and gender of albino rat pups. Bull Exp Biol Med. 130(11), 1031–4.
- Maklakova A.S, Dubynin V.A, Nazarenko I.V, et al. A (1995). Effects of ß-casomorphin-7 on different types of learning in white rats. Bulletin of Experimental Biology and Medicine. 120(5), 1121–4.
- Read L.C, Lord A.P, Brantl V, Koch G (1990). Absorption of betacasomorphins from autoperfused lamb and piglet small intestine. Am J Physiol. 259(3 Pt 1), G443–52.
- Umbach M, Teschemacher H, Praetorius K, et al. (1985). Demonstration of a beta-casomorphin immunoreactive material in the plasma of newborn calves after milk intake. Regul Pept. 12(3), 223–30.
- Singh M, Rosen C.L, Chang K.J, Haddad G.G (1989). Plasma betacasomorphin- 7 immunoreactive peptide increases after milk intake in newborn but not in adult dogs. Pediatr Res. 26(1), 34–8.
- Schulte-Frohlinde E, Reindl W, Bierling D, et al. (2000). Effects of oral casokefamide on plasma levels, tolerance, and intestinal transit in man. Peptides. 21(3), 439–42.
- Ganapathy V, Miyauchi S (2005). Transport systems for opioid peptides in mammalian tissues. The AAPS Journal. 7(4), E852–6.
- Husby S, Jensenius J.C, Svehag S.E (1985). Passage of undegraded dietary antigen into the blood of healthy adults. Quantification, estimation of size distribution, and relation of uptake to levels of specific antibodies. Scand J Immunol. 22(1), 83–92.
- Heyman M, Desjeux J.F (1992). Significance of intestinal food protein transport. J Pediatr Gastroenterol Nutr. 15(1), 48–57.
- Iwan M, Jarmolowska B, Bielikowicz K, et al. (2008). Transport of micro-opioid receptor agonists and antagonist peptides across Caco-2 monolayer. Peptides. 29(6), 1042–7.
- Shimizu M, Tsunogai M, Arai S (1997). Transepithelial transport of oligopeptides in the human intestinal cell, Caco-2. Peptides. 18(5), 681–7.
- Sienkiewicz-Szlapka E, Jarmolowska B, Krawczuk S, et al. (2009). Transport of bovine milk-derived opioid peptides across a Caco-2 monolayer. Int Dairy Journ. 19, 252–7.
- Hedner J, Hedner T (1987). beta-Casomorphins induce apnea and irregular breathing in adult rats and newborn rabbits. Life Sci. 41(20), 2303–12.
- Haddad G.G, Schaeffer J.I, Chang K.J (1984). Opposite effects of the deltaand mu-opioid receptor agonists on ventilation in conscious adult dogs. Brain Res. 323(1), 73–82.
- Xia Y, Haddad G.G (1991). Ontogeny and distribution of opioid receptors in the rat brainstem. Brain Res. 549(2), 181–93.
- Brantl V, Teschemacher H, Henschen A, Lottspeich F (1979). Novel opioid peptides derived from casein (beta-casomorphins). I. Isolation from bovine casein peptone. Hoppe Seylers Z Physiol Chem. 360(9), 1211–6.
- Taira T, Hilakivi L.A, Aalto J, Hilakivi I (1990). Effect of beta-casomorphin on neonatal sleep in rats. Peptides. 11(1), 1–4.
- Zoghbi S, Trompette A, Claustre J, et al. (2006). beta-Casomorphin-7 regulates the secretion and express ion of gastrointestinal mucins through a mu-opioid pathway. Am J Physiol Gastrointest Liver Physiol. 290(6), G1105–13.
- Teschemacher H, Koch G, Brantl V (1997). Milk protein-derived opioid receptor ligands. Biopolymers. 43(2), 99–117.
- Kreil G, Umbach M, Brantl V, Teschemacher H (1983). Studies on the enzymatic degradation of beta-casomorphins. Life Sci. 33(Suppl 1), 137–40.
- Lambeir A.M, Durinx C, Scharpe S, De Meester I (2003). Dipeptidylpeptidase IV from bench to bedside: an update on structural properties, functions, and clinical aspects of the enzyme DPP IV. Crit Rev Clin Lab Sci. 40(3), 209–94.
- Mentlein R (1999). Dipeptidyl-peptidase IV (CD26)–role in the inactivation of regulatory peptides. Regul Pept. 85(1), 9–24.
- Steinerova A, Korotvicka M, Racek J, et al (2004). Significant increase in antibodies against oxidized LDL particles (IgoxLDL) in three-month old infants who received milk formula. Atherosclerosis. 173(1), 147–8.
- Torreilles J, Guerin M.C (1995). Casein-derived peptides can promote human LDL oxidation by a peroxidase-dependent and metal-independent process. C R Seances Soc Biol Fil. 189(5), 933–42.
- Steinerova A, Racek J, Korotvicka M, Stozicky F (2009). Beta-casein A1 is a Possible Risk Factor for Atherosclerosis. Atherosclerosis Supplements, 10(2).