REVIEW the Role and Potential of Sialic Acid in Human Nutrition

REVIEW The role and potential of sialic acid in human nutrition

B Wang1* and J Brand-Miller1

1Human Nutrition Unit, School of Molecular and Microbial Biosciences, University of Sydney, NSW, Australia

Sialic acids are a family of nine-carbon acidic monosaccharides that occur naturally at the end of sugar chains attached to the surfaces of cells and soluble proteins. In the human body, the highest concentration of sialic acid (as N-acetylneuraminic acid) occurs in the brain where it participates as an integral part of ganglioside structure in synaptogenesis and neural transmission. Human milk also contains a high concentration of sialic acid attached to the terminal end of free oligosaccharides, but its metabolic fate and biological role are currently unknown. An important question is whether the sialic acid in human milk is a conditional nutrient and confers developmental advantages on breast-fed infants compared to those fed infant formula. In this review, we critically discuss the current state of knowledge of the biology and role of sialic acid in human milk and nervous tissue, and the link between sialic acid, breastfeeding and learning behaviour. European Journal of Clinical Nutrition (2003) 57, 1351–1369. doi:10.1038/sj.ejcn.1601704

Keywords: sialic acid; ganglioside; sialyl-oligosaccharides; human milk; infant formula; breastfeeding

Introduction promising new candidate is sialic acid (also known as The rapid growth and development of the newborn infant N-acetylneuraminic acid), a nine-carbon sugar that is a puts exceptional demands on the supply of nutrients. Any structural and functional component of brain gangliosides deficit has profound effects on somatic growth and organ and correlates with the amounts of DHA and total long- structural and functional development, especially the brain. chain polyunsaturated fatty acids in the ceramide tail of Brain growth, including cell number, organ structural and brain gangliosides. Sialic acid may be a conditionally synaptic connectivity, etc, reaches its peak at 26 weeks essential nutrient in infancy, if demand outstrips the rate gestation and then continues at a high rate throughout the of endogenous synthesis. first year of life (Uauy and Peirano, 1999; Uauy et al, 2001). The rapid initial growth of the brain exceeds that of other body tissues. At 6th month gestation, it comprises 21% of Breastfeeding and cognition total body weight and 15% at term (Friede, 1989). The brain Several studies show that children who were breast-fed as weight more than doubles during the first 9 postnatal babies attain higher scores on intelligence tests than those months to reach over 90% of the adult weight by the 6th who were bottle-fed (Rodgers, 1978; Fergusson et al, 1982; year. Once the time for the critical period of brain growth has Lucas et al, 1992, 1998). On average, scores are 2–9 points passed, it cannot be restarted. The challenge is accentuated higher, a difference that is considered biologically signifi- in the premature infants, particularly in relation to nutri- cant. The difference becomes more pronounced as the tional support for brain growth. duration of breastfeeding increases (Dewey et al, 1995). Recently, neurodevelopmental research has focused atten- Lucas et al (1992) reported that preterm infants fed human tion on the role of long-chain polyunsaturated fatty acids milk in the first month of life have an 8-point advantage in (LCPUFAs), and particularly docosahexaenoic acid (DHA) in verbal IQ at 7–8 y of age compared with infants fed standard improving visual acuity and cognitive ability in preterm infant formulas. In a large cohort study of several thousand infants (Carlson et al, 1996; Gibson, 1999). There are many adults, sentence completion, reading ability, and vocabulary factors, however, that may support brain growth. One were all related to patterns of infant feeding (Richards et al, 1998). In a retrospective study, Menkes (1977) found a significantly greater incidence of bottle feeding among *Correspondence: B Wang, Human Nutrition Unit, School of Molecular learning-disabled children than among controls being and Microbial Biosciences, G08 University of Sydney, NSW 2006, Australia. followed for other neurological symptoms. Rodgers (1978) E-mail: [email protected] described a large, stratified sample of British children. The role and potential of sialic acid B Wang and J Brand-Miller 1352 Covariates included social class, parental interest in educa- but also in physiological, biochemical, and psychological tion, material home conditions, parental education, family parameters, early nutrition may modulate nervous system size and birth rank, and age at weaning. After control of development. Infant formulas are the sole source of nutri- confounding variables, there was a significant advantage to tion for infants who are not breast-fed and differences breast-fed children on a picture vocabulary test at 8 y of age between them may be important. In a randomised multi- and on nonverbal ability, mathematics, and sentence center study (Lucas et al, 1990), preterm infants fed a completion at 15 y. A few studies have examined reading standard term formula for 1 month performed more poorly ability or school attainment, and breast-fed children tended at 18 months than those given a nutrient-enriched preterm to do better (Ounsted et al, 1984; Rogan & Gladen, 1993; formula. Those infants given the enriched formula had 15 Horwood & Fergusson, 1998). The most recent follow-up points higher score than those infants given standard term study in a New Zealand cohort of 1000 children reported formula. Recently, neurodevelopmental research has focused that breastfeeding is associated with small but detectable on the role of LCPUFAs, and particularly, docosahexaenoic increases in cognitive ability and academic achievement, acid (DHA) (Makrides et al, 1994, 1995; Lanting & Boersma, extending from 8 to 18 y (Horwood & Fergusson, 1998). The 1996). Clinical studies in which infant formula was supple- difference was significant after adjustment for social and mented with DHA suggested possible improvements in family history, including maternal age, education, social visual acuity and cognitive ability in preterm infants economic status, marital status, smoking during pregnancy, (Carlson et al, 1994, 1996). Infants fed with formula family living conditions, and family income, and perinatal containing DHA had significantly better visual-evoked factors, including gender, birth weight, child’s estimated potential scores than infants fed with control formula gestational age, and birth order in the family. Not all studies without DHA at both 16 and 30 weeks (Makrides et al, yield significant differences after adjustment (Sandra et al, 1995). DHA may be the limiting factor in milk formula 1999). A meta-analysis of 20 controlled studies showed that because the brain, in particular the visual cortex, lays down breastfeeding was associated with a 3.2 point higher large amounts in the first year of life. However, if diet is a key cognitive development score than formula feeding after to mental development, then there are many factors besides adjusting for significant covariates. The IQ advantage DHA that may be important, such as enzymes, hormones, increased with duration of breastfeeding, reaching a plateau growth factors, and sialic acid, which are found in human at 4–6 months of age. Low birth weight infants received the milk but poorly represented in the milk of other species and greatest benefits (Anderson et al, 1999). More recently, in infant formulas (Goldman & Garza, 1987). Of these, sialic Mortensen et al (2002) also reported that duration of acid is most intriguing because of its simultaneous presence breastfeeding was associated with significantly higher scores in large amounts in both human milk and human grey on all components of the Wechsler adult intelligence scale. matter. There is little direct evidence for a causative mechanism whereby breastfeeding might enhance or, conversely, bottle feeding might impair cognitive growth. Rodgers (1978) Structure and state of sialic acid in nature suggested possible mediating factors might be differences It is now known that sialic acids comprise a family of 43 between breast and bottle milk osmotic load or protein and naturally occurring derivatives of the nine-carbon sugar lipid concentrations or differences in the feeding situation neuraminic acid (5-amino-3,5-dideoxy-D-glycero-D-galacto- such as infection risk and psychological effects. Menkes nonulsonic acid) (Schauer et al, 1995; Schauer & Kamerling, (1977) proposed that tyrosinemia due to increased protein 1997). One branch of the sialic acid family is N-acetylated to levels in formula milk might produce an increased incidence form N-acetylneuraminic acids (Neu5Ac, NANA, Sia), which of learning disabilities in bottle-fed children. Improved are the most widespread form of sialic acid and almost the maternal–child interaction and reduced morbidity could only form found in humans (Figure 1). The other branch is account for the effect or contribute to it (Grantham- based on N-glycolylneuraminic acids (Neu5Gc) which are McGregor et al, 2000). Interpretation is further complicated common in many animal species (best investigated in by associations between breastfeeding, social status and porcine tissues), but not found in humans except in the education of parents. Women who make a decision to case of a particular cancer (Schauer et al, 1995) (Figure 1). breastfeed are often better educated with positive health Sialic acid molecules can be substituted in more than one attitudes concerning immunisation and smoking, and may position. O-substitution at C4, -7, -8 and -9 (O-acetyl, O- provide a more desirable environment for their young to methyl, O-sulphate, and phosphate groups) or the introduc- develop intellectually (Lucas et al, 1990, 1992). Statistical tion of a double bond between C-2 and C-3 can give rise to a adjustment for these associations may not remove the full wide variety of possible isomers (Varki & Diaz, 1984). An confounding effect of all these factors. unusual modification is an additional hydroxyl group It is highly possible that any benefit from breastfeeding is present instead of the amino function at position 5 of the due to the unique nutritional content of human milk. Since sugar, leading to 2-keto-3-deoxy-nonulosonic acid (Keto- the brain is undergoing rapid development during the first deoxynonulosonic acid, Kdn). This component has been few weeks or months after birth, not only in anatomic terms found in fish eggs (Caviar) (Varki, 1992).

European Journal of Clinical Nutrition The role and potential of sialic acid B Wang and J Brand-Miller 1353 Sialic acids rarely occur free in nature. They are more the liver is initially low, rising to a maximum 14–21 days commonly present as components of oligosaccharide chains after birth (Dickson & Messer, 1978). The evidence from of mucins, glycoproteins, and glycolipids. They usually other animal models, such as mice and guinea pigs, also occupy terminal, nonreducing positions of oligosaccharide indicates that enzyme activity is correlated with age and chains of complex carbohydrates on outer and inner developmental stage (Gal et al, 1997). Thus, we postulate membrane surfaces in various linkages, mainly to galactose, that the human infant liver may also have a limited capacity N-acetylgalactosamine, and other sialic acid moieties, where for synthesising sialic acid during early postnatal life (Wang they are highly exposed and functionally important. et al, 2001a, b). Cells from higher animals and various microorganisms produce sialic acid in a long pathway starting from glucose. An outline of the many reactions involving the sialic acids is Distribution of sialic acid in nature and the shown in Figure 2. All mammals, including human beings, human body have the capacity to synthesise sialic acid in all tissues from Sialic acids are found in all vertebrates (Mammalia, Aves, simple sugar precursors. However, in young rats, the activity Reptilia, Amphibia, Pisces) and within each organism they of the key enzyme UDP-N-acetylglucosamine-2-epimerase in are present in essentially all tissues (Corfield et al, 1982;

2 Figure 1 C5-conformation of N-acetylneuraminic acid (Neu5Ac, NANA, right) and N-glycoloylneuraminic acid (Neu5Gc, left).

Figure 2 Schema showing the metabolism of sialic acids modified from Varki et al (1999): (1) Glycosamine-6-phoshate synthase; (2) Glucosamine-6-phosphate N-acetyltransferase; (3) N-acetylglucosamine-6-phosphate mutase; (4) UDP-N-acetylglucosamine pyrophos- phorylase; (5) *key enzyme: UDP-N-acetylglucosamine-2-epimerase; activity is initially low in young rats; (6) N-acetylmannosamine kinase; (7) N-acetylneuraminate-9-phosphate synthase; (8) N-acetylneuraminate-9-phosphatephatase; (9) CMP-N-acetylneuraminate synthetase; (10) monooxygenase.

European Journal of Clinical Nutrition The role and potential of sialic acid B Wang and J Brand-Miller 1354 Warren, 1994), but not generally found in plants, prokar- particularly in patients with the disease sialuria, where up to yotes, or invertebrates (Varki et al, 1999). Certain strains of 7 g of sialic acid can be eliminated in 1 day (Montreuil et al, bacteria contain large amounts of sialic acid in their capsular 1968). Sialic acid is a significant component in all salivary polysaccharides. The erratic distribution of sialic acids mucins. Indeed, the name sialic acid is derived from the among bacteria and possibly certain protozoa, suggests that Greek ‘sialos’ meaning saliva. The structure of the mucin the enzymes responsible for their synthesis and metabolism consists of a protein core with varying quantities of attached were fortuitously acquired during association with animal oligosaccharide side chains. The oligosaccharide component cells (Warren, 1994). may constitute up to 75% of the total mucin molecule The mammalian central nervous system has the highest although not every carbohydrate chain carries a terminal concentration of sialic acid. The majority (65%) is present in sialic acid residue (Corfield et al, 1982). Recently, we gangliosides and glycoproteins (32%) with the remaining 3% demonstrated that breast-fed preterm infants in the first 3 as free sialic acid (Brunngraber et al, 1972). However, months of life had almost twice the levels of bound sialic gangliosides are not uniformly distributed within the human acid in their saliva as formula milk-fed preterm infants. body (Rosenberg, 1995; Schauer & Kamerling, 1997). Gang- Feeding per se did not alter the concentration of sialic acid in lioside concentration in brain grey matter is 15 times that of saliva, ruling out contamination of saliva by milk. Further- large visceral organs such as liver, lung, and spleen and 500 more, we found no differences in sodium or potassium times greater than intestinal mucosa (Table 1). Human adult concentration in the saliva of the two groups, which might cerebral cortex (particularly frontal cortex) and cerebellar grey matter have approximately 3 times more than the corresponding white matter (Svennerholm, 1980). Our Table 1 The concentration of ganglioside-bound sialic acid in the brain and large visceral organs of human adults previous study demonstrated that total sialic acid concentration in the human brain was almost two to four times that of Ganglioside-bound Reference eight other animal species. The rank order of decreasing sialic acid (mg/g wet wt) brain sialic acid after humans was rat, mouse, rabbit, sheep, cow, and pig. In a 2-y-old chimpanzee, the brain cortex sialic Brain acid concentration (Wang et al, 1998) was about one-third of Cerebral grey matter 890 Wang et al (1998) Cerebral white matter 275 Ueno et al (1978) the level of a human infant of the same age (Svennerholm Cerebellar grey matter 660 Svennerholm (1980) et al, 1989). Also, the sialic acid concentration in left lobe of Spinal cord 87 Ueno et al (1978) the brain cortex was 22% higher than that of the right lobe Cerebral myelin 63a Ueno et al (1978) (Wang et al, 1998), possibly because the different brain Retina 31–46 Svennerholm (1980) Peripheral nerve 11–25 Svennerholm (1980) regions perform different neurological functions (Suzuki, 1965). Brain ganglioside sialic acid has implications for Organ evolutionary development and intellectual capacity. The Spleen 62–93 Svennerholm (1980) sialic acid moieties of gangliosides and glycoproteins in the Placenta 31–62 Svennerholm (1980) Thyroid 19–31 Svennerholm (1980) frontal cortex play both a structural and functional role and Liver 15–31 Svennerholm (1980) probably participate in a variety of cellular events, such as Small intestinal 15–25 Svennerholm (1980) cell recognition, cell-to-cell contact formation, receptor muscular layer binding and modulation, immunological properties, and Kidney 9–19 Svennerholm (1980) Skin 9–11 Svennerholm (1980) biosignal transduction. Fat tissue 3–5 Svennerholm (1980) Sialic acids also exist in many human body fluids Small intestinal mucosa 1.5–2 Svennerholm (1980) including saliva, gastric juice, serum, urine, tears, and human milk (Table 2). Free sialic acid is found in urine, amg Sialic acid per 100 mg dry myelin.

Table 2 Sialic acid concentration in the body fluids of human adults

Fluid Total sialic acid (mmol/l) Reference

Human milk (full term) 3.72 (colostrum) 1.48 (one mo milk) Wang et al (2001b) Serum 2.3 Waters et al (1992), Hayakawa et al (1993) Tears 0.8–1.8 Kuizenga et al (1990) (enzyme hydrolysed) Gastric aspirates 0.3 Corfield et al (1993) Saliva (full term) 0.2 (4–5 months) Tram et al (1997) Saliva (preterm term) 0.24 (4–5 months) Wang et al (2001a) Urine 0.2 Hayakawa et al (1993) Amniotic fluid 0.2 Hayakawa et al (1993) Cerebral fluid 0.05 Hayakawa et al (1993)

European Journal of Clinical Nutrition The role and potential of sialic acid B Wang and J Brand-Miller 1355 otherwise have suggested an inherent difference between the synchronously altering neuronal activity (Schmidt, 1989). saliva of the two groups (Wang et al, 200la). Similar results The carbohydrates in brain protein have been correlated were found in 4–5 months old full-term infants (Tram et al, with operant conditioning training of pigeons, higher 1997). Sialo-compounds in the body fluids may play a role in glycoprotein levels being associated with higher learning the structural and functional protection of the mucosal ability (Bogoch, 1970). surfaces. Sialic acid concentrations in body fluids may also Sialic acid may be a negative modulator of the interactions reflect metabolic status and body tissue levels. The saliva of mediated by the protein part of neural cell adhesion pregnant women increases in sialic acid concentration molecule (N-CAM) (Sadoul et al, 1983). Thus, sialic acid has during the course of gestation (Salvolini et al, 1998). a possible role in cell-to-cell interaction (Rosenberg, 1995) and has even been postulated to be the actual receptor for neurotransmitters in the central nervous system (von Itzstein & Thomson, 1997). Sialic acid and cognition Neural cell membranes contain 20 times more sialic acid than other types of membranes, indicating that sialic acid has a clear role in neural structure (Schauer, 1982). Ganglio- Sialic acid and infection sides in nervous tissue contain sialic acid in the form of Human milk is a rich source of sialic acid containing glycosphingolipids, which are found in highest concentra- oligosaccharides. Human milk oligosaccharides (HMOs) tions in the cerebral cortex of the human brain (Rosenberg, have been shown to inhibit microbial pathogens in vitro 1995). It has been proposed that sialo-compounds play a role and in vivo (Newburg, 1999). Harmful bacteria, viruses, and in the structural and functional establishment of synaptic other pathogens use cell surface carbohydrates as sites for pathways (Schauer, 1982). Cell membranes containing sialic recognition and binding to their target host cell, the first acid contribute to the negative charge of the membrane step in infection. Oligosaccharide sequences on soluble (Rosenberg, 1995). Cell aggregation may be prevented by the glycoconjugates such as the mucins can act as ‘decoys’ for repulsive effects of the negatively charged sialic acid, as was microorganisms and parasites (Rosenberg, 1995; Newburg, observed when studying the adhesion of culture cells to their 1999). Thus, pathogenic organisms attempting to gain access substratum (Schauer & Kamerling, 1997). Cell adhesion may to mucosal membranes might first encounter their cognate be facilitated via positively charged substances or Ca2 þ oligosaccharide ligands attached to soluble mucins. Upon bridges which break the repulsive effect of sialic acids. binding to these sequences, they are swept away by ciliary Furthermore, binding of Ca2 þ to ganglioside sialic acid is of action, leaving the mucosal cell untouched. In these cases, great importance in the function of nervous tissues (Schauer, the host may successfully turn the specificity of the 1982; Rosenberg, 1995). Gangliosides are localised in clusters pathogen receptor to its own advantage (von Itzstein & on neuronal and especially synaptic membranes in the Thomson, 1997; Newburg, 1999). The potential of sialic vicinity of a membrane-bound calcium pump, thus facilitat- acids as antimicrobials is clearly enormous. Sialylated ing the supply of these ions for neuronal cells. Calcium– oligosaccharides in human milk can act as highly specific ganglioside interactions may modulate neuronal functions, receptors for a variety of viruses, bacteria, and parasites as not only for the short-term process of synaptic transmission reviewed by Varki (1993). Both free and bound sialylated of information, but also for long-term events of neuronal oligosaccharides in human milk prevent the binding of adaptations including storage of information. rotavirus (Varki, 1993) and cholera toxin (Idota et al, 1995) Brain glycoproteins are also important. The expression of associated with infant diarrhoea, as well as Escherichia coli brain function in learning and memory is related to the strains associated with neonatal meningitis and sepsis proposed higher recognition functions of the brain glyco- (Parkkinen et al, 1983). An understanding of the role that proteins (Bogoch, 1977). The sialoglycopeptides are highly these highly functionalised carbohydrates play in the concentrated in the synaptosomal fraction (Garcia-Segura pathogenicity of infectious microorganisms is crucial to the et al, 1978) and there is evidence for involvement of sialic development of infant nutrition. acid containing brain glycoproteins (ependymins) in mem- Breastfeeding has been associated with a lower incidence ory formation (Bogoch, 1977; Schmidt, 1989). In goldfish, of morbidity and mortality, especially of enteric disease, ependymin synthesis is preferentially upregulated during otitis media, and respiratory infection (Newburg, 2000). One learning tasks and treatment with antiependymin antiserum mechanism is believed to be the HMOs. In the absence of up to 24 h following training inhibits memory formation in HMOs, as in formula-feeding, pathogens are able to bind to these tasks (Schmidt, 1989). The mechanism by which these the membrane-bound receptors of their host target cell, proteins mediate memory formation is not clear, but the adhere to the cell surface and consequently gain entry into primary structure of ependymin comprises N-glycosylation the cell to cause infection. In breast-fed infants, pathogens sites, giving rise to glycoproteins of variable carbohydrate bind to human milk receptor analogs or ‘decoys’ (HMOs) content. Ependymin contains clusters of negatively charged and not to the host cell, thus preventing infection. HMOs amino acids suitable for binding of calcium ions and may protect the infant from harmful pathogens during the

European Journal of Clinical Nutrition The role and potential of sialic acid B Wang and J Brand-Miller 1356 first few months of life, before the immune system is fully gangliosides were localised in the nerve ending, but more mature and before a stable colonic microflora has been recent work in different laboratories (Ledeen, 1978) suggests established, while providing an environment that stimulates that gangliosides are distributed over a large part of the the growth of commensal bacteria. It is not so surprising, neuronal surface. Human brain ganglioside concentration perhaps, that human milk contains many agents, including and distribution change significantly during development. these carbohydrate ‘decoys’ that protect against disease. The concentration increases approximately three times from the 10th gestational week to the age of about 5 y. Ganglioside GM1 and GD1a increase 12–15 times during the same period. GT1b is the major ganglioside during the third to Sialic acid in brain gangliosides fifth gestational month, thereafter its concentration drops Gangliosides are complex glycosphingolipids, which make rapidly to term, and then increases again up to 50 y of age up 10% of the total lipid mass in the brain and contain (Svennerholm et al, 1989). There are also significant regional different numbers of negatively charged sialic acid moieties. differences in the gangliosides patterns (Suzuki, 1965; Brain tissue is unique in that the quantity of lipid-bound Kracun et al, 1984). Different neuronal and glial cell types sialic acid exceeds that of the protein-bound fraction. contain specific and characteristic sets of gangliosides (Byrne Gangliosides are hybrid molecules composed of a hydro- et al, 1988). philic sialyl oligosaccharide and a hydrophobic ceramide portion that consists of sphingosine and fatty acids (Figure 3) (Svennerholm et al, 1994; Rosenberg, 1995). The ceramide is Gangliosides and memory formation an N-acetyl-sphingosine in which the acetyl residue is The precise functional role of gangliosides is still poorly usually a saturated fatty acid with a chain length greater understood. However, gangliosides have been hypothesised than 14 carbons. C14–C18 predominate in certain sources to be involved in the formation of memory. It is generally and C20–C26 in others (Cabezas & Calvo, 1984). Among 100 accepted that the neuronal transmembraneous calcium carbohydrates in nature, only six, that is glucose (Glc), exchange can be modulated in a complex way by external galactose (Gal), N-acetylglucosamine (GlcNAc), N-acetylga- factors, such as physical parameters (lateral surface pressure lactosamine (GalNAc), fucose (Fuc), and sialic acid (NeuAc or and temperature) or chemical parameters such as ion milieu, NeuGc), contribute to construction of gangliosides (the same enzyme activities, hormones, and drugs (Rahmann, 1989). six are used in the construction of HMOs). In addition, most Gangliosides are highly accumulated in a complex composi- biological properties of glycosphingolipids appear to be more tion in the outer leaflet of synaptic membranes. They possess influenced by particular structures of their oligosaccharides special physicochemical properties particularly in their than the ceramide composition (Wiegandt, 1994). interaction with calcium. Gangliosides bind calcium ions At least 100 different ganglioside structures have been via electrostatic interactions between the ions and sialic acid established during the last 20 y, but in higher vertebrates 80– residues (Leskawa & Rosenberg, 1981) so they may act as an 90% of the total gangliosides in the brain are GM1, GD1a, extracellular storage mechanism for calcium (Romer & GD1b, and GT1b (Rosenberg, 1995). Variation of sialic acid Rahmann, 1979). Calcium is required for synaptic transmis- in gangliosides is well documented. All human brain gang- sion and probably activates second messenger pathways to liosides contain the Neu5Ac form of sialic acid (Haverkamp induce synaptic potentiation (Castillo et al, 1994). This et al, 1977). Gangliosides are widely distributed in most storage hypothesis is supported by experimental treatment vertebrate tissues. For a long time, it was believed that of frog spinal cord neurons with neuraminidase enzyme.

GM3 GM2 GM1 GD1a GD1b GT1b

Polar Oligosaccharide Head groups

Hydrophobic Core of lipid bilayer

Figure 3 The chemical structures of some gangliosides showing the position of sialic acid: - , sialic acid (NANA, Sia); , glucose; , galactose; , N-acetylgalactosamine.

European Journal of Clinical Nutrition The role and potential of sialic acid B Wang and J Brand-Miller 1357 Neuraminidase cleaves sialic acid residues from gangliosides to the extracellular space influences the excitability of retinal and results in increased postsynaptic activity in response to neurons in culture (Fernandes de Lima et al, 1997). synapse activation not unlike that observed in long-term Gangliosides are associated with neuronal growth and potentiation (LTP), presumably because liberated sialic acid development. The cell produces ‘plasma membrane residues release bound calcium, which activates transmitter elements’ which contain a high proportion of gangliosides release (Romer & Rahmann, 1979). The addition of calcium and allow axonal growth (Rosenberg, 1995). Gangliosides are to ganglioside monolayers induces changes in surface required in areas of membrane extension for axonal and potential, condensation, surface pressure, and other physical dendritic growth, and applied exogenous gangliosides are properties, possibly as a result of ganglioside oligosaccharide inserted into the membrane, increasing both neuritogenesis chains forming cross-bridges with calcium ions (Leskawa & and synapse formation (Rosenberg, 1995). Lack of sufficient Rosenberg, 1981; Beitinger et al, 1989). Gangliosides can gangliosides, such as observed in the presence of ethanol, is modify the properties of the plasma membrane, for example associated with striking loss of neurites (Miller, 1993). to increase membrane fluidity (Esmann et al, 1988; Beitinger Devascularising neural lesions involve a loss of functional et al, 1989). Gangliosides also activate growth-stimulating synaptic connections between neurons, a process which molecules like nerve growth factor, which may mediate this probably also occurs during ageing. Ganglioside-mediated effect (Rosenberg & Noble, 1994). protection against memory loss in these situations seems Neuronal gangliosides also interact with membrane-bound likely to be the result of formation of replacement synaptic proteins (Esmann et al, 1988). Electron microscopic studies connections (Rahmann 1995). Figure 4 summarises the indicate that in the presence of calcium, gangliosides form mechanisms by which gangliosides could be involved in aggregates around membrane-associated peptides. This find- memory formation. ing supports the hypothesis that gangliosides form annuli Exogenously applied gangliosides are taken up by neurons around synaptic membrane ion channels to regulate move- and integrated into the plasma membrane in the same way ment of ions through the membrane (Rahmann & Rah- as endogenous gangliosides (Morgan & Winick, 1981; mann, 1992). The presence of gangliosides increases the Dreyfus et al, 1984) and have been shown to influence the conductance of membrane-bound ion carriers, where greater activity of a number of types of neural circuits (Agnati et al, conductance is associated with increased ganglioside com- 1983; Silva et al, 1999). Recent studies in rodents demon- plexity and polarity. Application of exogenous gangliosides strate that gangliosides given intraperitoneally can protect

Neuronal gangliosides

Interact with Neuronal growth membrane bound protein

Influence Extension of Bind calcium neuronal axons and Influence excitability dendrites Enhance membrane growth flexibility and factor fluidity activity Formation of Change functional membrane synaptic Control permeability to connections extracellular calcium calcium concentration

Formation of neural cricuits during Changes in development and following age or injury- synaptic strength related deficits

MEMORY FORMATION

Figure 4 Mechanisms for possible involvement of neuronal gangliosides in memory formation. Unbroken lines indicate known or well supported associations. Broken lines indicate proposed functions.

European Journal of Clinical Nutrition The role and potential of sialic acid B Wang and J Brand-Miller 1358 against or reverse age- or lesion-induced memory deficits. colostrum have been identified so far, with almost 55% of This effect is observed in animals with various impairments, the free oligosaccharides being sialylated (Table 5). Eight including genetic, lesion-induced, and age-related cognitive structures are the same as human milk sialyl-oligosacchar- deficits, and is observed in tasks requiring either simple recall ides (based on Neu5Ac), while three are different, being or spatial memory from a few hours to several weeks after based on Neu5Gc. initial training. The findings of these studies are summarised in Table 3. Gangliosides have been used to treat specific central nervous system lesions. Serum albumin has functions Human milk and cow’s milk: quantitative and qualitative in the transport of gangliosides across the blood-brain barrier aspects (BBB), since measurements of the ability of labelled albumin Human milk contains significantly higher levels of sialic acid (albumin adheres to gangliosides) to cross the BBB indicate than bovine milk or any type of infant formulas. However, that it may cross by fluid-phase endocytosis (Poduslo & knowledge of the amino sugar content and distribution in Curran, 1994; Schengrund & Mummert, 1998). Normal rat human milk is still limited, partly due to the absence of pups treated with gangliosides displayed improved learning standardised methods of analysis (Atkinson & Lo¨nnerdal, performances and increased cortex levels of acetylcholine 1995). We investigated the milk of 20 full-term mothers and esterase as compared to untreated animals (Karpiak & 14 preterm mothers (mean gestational age 3173 weeks) at Mahadik, 1990). On the other hand, some studies showed four stages of lactation (colostrum, transition, 1 and 3 gangliosides improve memory only in the presence of a months) and compared with 21 different formulas. A Bio-Gel specific impairment (Agnati et al, 1983; Fong et al, 1997). P-2 column was used to separate sialic acid from milk and a colorimetric method was used to quantitate the amount (Wang et al, 2001b). We found total sialic acid levels were Sialic acid in the diet highest in colostrum (3.7270.15 mmol/l in full term and Human milk oligosaccharides (HMOs) 4.2770.15 mmol/l in preterm) and decreased over time. The The complex free oligosaccharides of human milk consist of downward time trend was evident in the full data set and in glucose, galactose, fucose, N-acetylglucosamine, and sialic the 15 full-term mothers and five preterm mothers who had acid residues. HMOs are the third largest solid component complete data for all timepoints. By 3 months only about after lactose and fat (Jensen, 1995). The highest concentra- 20% of the initial content of sialic acid was present in both tion can be found in colostrum (22–24 g/l), but even mature groups. Preterm milk contained about 13–23% more total milk contains oligosaccharides at levels of 13 g/l (Coppa et al, sialic acid than full-term milk during first 3 months 1993). Preterm human milk may contain more than in full- lactation. Carlson (1985) determined the sialic acid concen- term milk: 26 g/l on day 4 and 17 g/l on day 30 after giving tration in human milk at various lactational intervals using a birth (Coppa et al, 1997). colorimetric method and noted an exponential decay with Approximately 88 different HMOs (Newburg & Neubauer, time. The concentration was highest at 0–2 weeks (approxi- 1995; Chaturvedi et al, 1997; Shen et al, 2000) have been mately 3.56 mmol/l) and dropped to 0.44 mmol/l at 10–28 isolated and characterised, with about 50% 38 of 88 being weeks lactation where it plateaued. Kawakami (1997) assayed sialylated (Table 4). These have a core structure consisting of 2700 milk samples from over 2000 Japanese mothers at 3 a lactose unit at the reducing end and at least one N- days–16 months postpartum using an HPLC method. Sialic acetylneuraminic acid unit at the nonreducing end. Sialyl- acid content decreased from about 4.85 mmol/l on day 3–5 lacto-N-tetraose c (LSTc), 60-sialyllactoses (60-SL) and disia- to 1.03 mmol/l on day 120–240. Using the colorimetric lyllacto-N-tetraose (DSLNT) are the most representative method, Brand Miller et al (1994) found similarly high constituents among sialyl-oligosaccharides, their concentra- concentrations of oligosaccharide-bound sialic acid in tion in human milk being 1050, 590, and 800 mg/l in human milk in the first month of lactation with levels colostrum and 120, 240, and 630 mg/l at 90 days lactation, declining by 70% over 3 months. At 1 month lactation, we respectively (Coppa et al, 1999). They constitute 60–70% of found a two-fold variation in the milk of full-term mothers the weight of all sialyl-oligosaccharides (w/w) in human milk (range 1.29–2.62 mmol/l) and a three-fold variation among (Coppa et al, 1999). All sialyl-oligosaccharides show a gradual preterm mothers (range 1.37–4.04 mmol/l). Our previous decrement as lactation progresses, the concentration at day studies also demonstrated a three-fold difference in levels 90 being about 30–50% of that present in colostrum. among 10 mothers (Brand Miller et al, 1994). Sialic acid However, the content of 30-sialyllactoses (30-SL) (Figure 5) therefore appears to be one of the most variable fractions of in human milk is relatively stable (100–170 mg/l) through- human milk. The reason for the wide range remains out lactation. unknown, but may represent genetic differences in synthe- In contrast to human milk, cow’s milk contains few free tic capacity or in environmental exposure to infective oligosaccharides, with only 0.025 g/l in colostrum and microorganisms. 0.01 g/l in mature milk (Urashima, unpublished data). In human milk, most sialic acid (ie about 73%) is bound to Sialyl-lactose is the major component (Kunz & Rudloff, free oligosaccharides and this proportion remains fairly 1993). In all, 20 different oligosaccharides in bovine constant throughout lactation despite an overall decline in

European Journal of Clinical Nutrition Table 3 Exogenous ganglioside administration alleviates specific memory deficits in rodents

Reference Deficit Ganglioside administered Type of memory Memory test Controls Notes

Fagioli et al (1990) Genetic deficit in GM1 Medium-term (72 h) Retention of passive No difference in general Suggests GM1 increases cholinergic neural avoidance of shock: activity levels or shock cholinergic neural systems (C57BL/6 mouse Animals must remember sensitivity between parameters. strain). to remain stationary to experimental groups and avoid a mild electrical saline-injected animals. shock. Silva et al (1998) Amnesia induced by GM1 Medium-term and spatial Passive avoidance, plus Saline-injected rats. GM1 improved the scopolamine (cholinergic maze tests. memory of aged but not antagonist) in rats and adult rats. mice. Inokuchi et al (1997) Ischemia-induced L-PDMP (synthetic Spatial 8-Arm radial maze test: Nonischemic rats. No Treatment also increased cognitive deficits in rats. ceramide analog: Animals must remember controls for ischemia- p42 MAPK, which is Stimulates ganglioside which arms they have induced motor deficits. involved in synaptic bio-synthesis) not yet visited. transmission. Glasier et al (1999) Unilateral entorhinal GM1 Short-term Hebb-Williams maze Sham-operated rats and Suggests GM1 may be

cortex (EC) lesions in tasks. saline-injected, lesioned involved in the neuronal Brand-Miller J acid and sialic Wang of B potential and role The rats. rats. No controls for sprouting observed after associated motor deficits. EC lesions. Elliott et al (1989) Neocortical lesions in GM1 Spatial Morris water maze Sham-operated animals. GM1 improved task rats. GM1 had no effect on acquisition and motor activity or retention. coordination. Fong et al (1997) Age-related cognitive GM1 Spatial Morris water maze Saline-treated senescent Effects of GM1 on deficits in senescent rats. rats, and saline-treated memory observed in young rats. No difference aged rats only, not in was observed in swim younger controls. speed or body weight. Silva et al (1996) Age-related cognitive GM1 Long-term (several Saline-injected old rats, Other evidence suggests deficits in senescent rats. weeks) saline-injected young age-related memory rats. deficits are due to cholinergic impairment.

Gangliosides are typically administered intraperitoneally at 10–50 mg/kg, daily for 7–21 days, with memory training and testing during or following this period. uoenJunlo lnclNutrition Clinical of Journal European 1359 The role and potential of sialic acid B Wang and J Brand-Miller 1360 Table 4 Structure of sialyl-oligosaccharides in human milk

I. Lactose 30-Sialyllactose Neu5Ac (a2-3) Gal (b1-4) Glc Kuhn and Brossmer (1959) 60-Sialyllactose Neu5Ac (a2-6) Gal (b1-4) Glc Kuhn (1959) 30-Sialyllactosamine Neu5Ac (a2-3) Gal (b1-4) GlcNAc Shen et al (2000) 60-Sialyllactosamine Neu5Ac (a2-6) Gal (b1-4) GlcNAc Shen et al (2000) N-Acetylneuramin lactose sulfate Neu5Ac (a2-3) Gal-6-SO4 (b1-4) Glc Sturman et al (1985) Monofucosylmonosialyllactose Neu5Ac (a2-3) Gal ((b1-4) Glc’Fuc (al-3) Gronberg et al (1989) II. Lacto-N-tetraose Sialyllacto-N-tetraose (a) Neu5Ac (a2-3) Gal (bl-3) GlcNAc (bl-3) Gal (b1-4) Glc Kuhn and Gauhe (1965) Sialyllacto-N-tetraose (b) Neu5Ac (a2-6) GlcNAc (bl-3) Gal (b1-4) Glc Kuhn and Gauhe (1965) Gal (b1-3)s Sialyllacto-N-tetraose (c) Neu5Ac (a2-6) Gal (b1-4) GlcNAc (bl-3) Gal (b1-4) Glc Shen et al (2000) Disialyllacto-N-tetraose Neu5Ac (a2-6) Glc (bl-3) Gal (bl-4) Glc Grimmonprez and Montreuil (1968) Neu5Ac (a2-3) Gal (bl-3)s Disialyltetraose Neu5Ac (a2-3) Gal b(l-3) GalNAc Shen et al (2000) Neu5Ac (a2-6)s Monosialylmonofucosyllacto-N-tetraose Neu5Ac (a2-6) Glc (bl-3)-GlcNAc (bl-3) Gal (b1-4) Glc Wieruszeski et al (1985) Fuc (al-4)s Monofucosylmonosialyllacto-N-tetraose Neu5Ac (a2-6)-GlcNAc (bl-3) Gal (b1-4) Glc Wieruszeski et al (1985) Fuc (al-2) Gal (bl-3)s Neu5Ac (a2-6)r Monofucosyldisialyllacto-N-tetraose Neu5Ac (a2-3) Gal (bl-3) GlcNAc (bl-3) Gal (b1-4) Glc Kitagawa et al (1991) Fuc (al-4)s III. Lacto-N-neotetraose Sialyllacto-N-neotetraose Neu5Ac (a2-6) Gal (b1-4) GlcNAc (bl-3) Gal (b-4) Glc Kuhn and Gauhe (1965) Monofucosylmonosialyllacto-N-neotetraose Neu5Ac (a2-6) Gal (bl-4) GlcNAc (bl-3) Gal (b1-4) Glc Smith et al (1987) Fuc (al-3)s IV. Lacto-N-Hexaose Monosialyllacto-N-hexaose Neu5Ac (a2-6) Gal (bl-4) GlcNAc (bl-6)-Gal (bl-4) Glc Kobata and Ginsburg (1972a) Gal (bl-3) GlcNAc (bl-3)s Disialyllacto-N-hexaose I Neu5Ac (a2-3) Gal (bl-3) GlcNAc (b1-3) Gal (bl-4) Glc Kitagawa et al (1991) Neu5Ac (a2-6)Gal (bl-4) GlcNAc (bl-6)s Disialyllacto-N-hexaose II Neu5Ac a(2-6)r Kitagawa et al (1991) Neu5Ac (a2-3)Gal (bl-3) GlcNAc (bl-3) Gal (bl-4) Glc Gal (bl-4) GlcNAc (bl-6)s Monofucosylmonosialyllacto-N-hexaose Neu5Ac (a2-6)Gal (bl-4) GlcNAc (bl-6) Gal (bl-4) Glc Yamashita et al (1977) Fuc (al-2)Gal (bl-3)GlcNAc (bl-3)s Fuc (al-3) Gal (bl-4) GlcNAc (bl-6) Gal (bl-4) Glc Monofucosyldisialyllacto-N-hexaose I Neu5Ac (a2-3) Gal (bl-3) GlcNAc (bl-3)s Yamashita et al (1976) Neu5Ac (a2-6)s Neu5Ac (a2-6)r Monofucosyldisialyllacto-N-hexaose II Neu5Ac (a2-3) Gal (bl-3) GlcNAc (bl-3) Gal (bl-4) Glc Kitagawa et al (1991) Gal (bl-4) GlcNAc (bl-6)s Fuc (a l-3)s Fuc (al-3)r Gal (bl-4) GlcNAc (bl-6)r Monofucosyldisialyllacto-N-hexaose III Neu5Ac (a2-3) Gal (bl-3) GlcNAc (bl-3) Gal (bl-4) Glc Yamashita et al (1976) Neu5Ac (a2-6)s Neu5Ac (a2-6)r Gal (bl-3) GlcNAc (bl-3) Gal (bl-4) Glc Monofucosylmonosialyllacto-N-hexaose I Gal (bl-4) GlcNAc (bl-6)s Gronberg et al (1992) Fuc (a l-3)s Neu5Ac (a2-3) Gal (bl-3) GlcNAc (bl-3) Gal (bl-4) Glc Monofucosylmonosialyllacto-N-hexaose II Gal (bl-4) GlcNAc (bl-6)s Gronberg et al (1992) Fuc (al-3)s Fuc (a1-4)r Monofucosylmonosialyllacto-N-hexaose III Gal (bl-3) GlcNAc (b1-3)r Gronberg et al (1992) Neu5Ac (a2-6) Gal (bl-4) GlcNAc (bl-6) Gal (bl-4) Glc Fuc (al-4)r Difucosylmonosialyllacto-N-hexaose Fuc (a1-2) Gal (bl-3) GlcNAc (bl-3)r Gronberg et al (1992) Neu5Ac (a2-6) Gal (bl-4) GlcNAc (bl-6) Gal (bl-4) Glc V. Lacto-N-neohexaose Neu5Ac (a2-6) Gal (bl-4) GlcNAc (bl-6)-Gal (bl-4) Glc Kobata and Ginsburg (1972b) Monosialyllacto-N-neohexaose I Gal (bl-4) GlcNAc (bl-3)s Monosialyllacto-N-neohexaose II Neu5Ac (a2-6) Gal (bl-4) GlcNAc ((bl-3) Gal ((bl-4) Glc Gronberg et al (1989) Gal (bl-4) GlcNAc (bl-6)s

European Journal of Clinical Nutrition The role and potential of sialic acid B Wang and J Brand-Miller 1361 Table 4 (Continued )

Disialyllacto-N-neohexaose Neu5Ac (a2-6) Gal (bl-4) GlcNAc (bl-6)-Gal (bl-4) Glc Gronberg et al (1992) Neu5Ac (a2-6) Gal (b1-4) GlcNAc (bl-3)s Fuc (al-3)r Monofucosylmonosialyllacto-N-neohexaose Gal (bl-4) GlcNAc (bl-6) Gal (bl-4) Glc Gronberg et al (1989) Neu5Ach (a2-6) Gal (bl-4) GlcNAc (bl-3)s Neu5Ac (a2-6) Gal (bl-4) GlcNAc (bl-6)r Kobata and Ginsburg (1972b) Monofucosylmonosialyllacto-N-neohexaose Fuc (al Gal (bl-4) GlcNAc (bl-3) Gal (bl-4)Glc Fuc (a1-2) Gal (bl-4)r Difucosylmonosialyllacto-N-neohexaose Fuc (a1-3) GlcNAc (bl-6) Gal (bl-4)Glc Gronberg et al (1989) Neu5Ac (a2-6) Gal (bl-4) GlcNAc (bl-3)s Neu5Ac (a2-6/3) Gal (bl-4) GlcNAc (bl-6) Gal (bl-4)Glc Monofucosyldisialyllacto-N-neohexaose Fuc (al-3) GlcNAc (bl-3)s Yamashita et al (1976) Neu5Ac (a2-3/6) Gal (bl-4)s VI. Lacto-N-octaose Monofucosylmonosialyllacto-N-octaose Gal (bl-4) GlcNAc (bl-3) Gal (bl-4) GlcNAc (bl-6)r Kitagawa et al (1993) (sialyl Lea) Neu5Ac (a2-3) Gal (bl-3) GlcNAc (bl-3) Gal (bl-4) Glc Fuc (a1-4)s VII. iso-N-octaose Trifucosylmonosialyl-iso-lacto-N-octaose Fuc (al-3)r Kitagawa et al (1993) (sialyl Lea) Fuc (a1-2) Gal (bl-3) GlcNAc (bl-3) Gal (bl-4) GlcNAc (bl-6) Neu5Ac (a2-3) Gal (bl-3) GlcNAc (bl-3) Gal (bl-4) Glcs Fuc (a1-4)s VIII. Deviant structures Neu5Ac (a2-3) Gal (bl-3)r Lactose-containing Gal (bl-4) GlcNAc (bl-6) Gal (bl-4) Glc Gronberg et al (1992) Fuc (a1-3)s Fuc (a1-4)r Nonlactose structures Neu5Ac (a2-3) Gal (bl-3) GlcNAc Kitagawa et al (1990) Fuc (a1-4) GlcNAc (bl-3) Gal Neu5Ac (a2-3) Gal (bl-3)s

Table 5 Structure of sialyl-oligosaccharides in bovine milk

Neu5Ac (a2-3) Gal Kuhn and Gauhe (1965) Neu5Ac (a2-3) Gal (b1-4) Glc Veh et al (1981) Neu5Ac (a2-6) Gal (b1-4) Glc Kuhn and Gauhe (1965) Neu5Gc (a2-3) Gal (b1-4) Glc Kuhn and Gauhe (1965) Neu5Gc (a2-6) Gal (b1-4) Glc Veh et al (1981) Neu5Ac (a2-6) Gal (b1-4) GlcNAc Kuhn and Gauhe (1965) Figure 5 Chemical structure of 30-sialyllactose. Neu5Gc (a2-6) Gal (b1-4) GlcNAc Veh et al (1981) Neu5Ac (a2-3) Gall-3Gall-4Glc Parkkinen and Finne (1987) Neu5Ac (a2-8) Neu5Ac (a2-3) Kuhn and Gauhe (1965) Gal (b1-4) Glc absolute amounts. In contrast, infant formulas contain most Neu5Ac (a2-6) Gal (b1-4) GlcNAc-1-PO4 Parkkinen and Finne (1987) Neu5Ac (a2-6) Gal (b1-4) GlcNAc-6-PO Parkkinen and Finne (1987) sialic acid bound to glycoproteins (70%) (Wang et al, 2001b). 4 Carlson (1985) made similar observations. Human milk also contains glycolipid sialic acid at low levels (0.016 mmol/l in colostrum and 0.006 mmol/l in mature milk) (Rueda et al, 2000). Both glycoprotein and glycolipid fractions of human 1996b; Sanchez-Diaz et al, 1997). This is two times higher milk contain lower levels of sialic acid than that of the free than that in cow’s milk and infant formulas (Pan & Izumi, oligosaccharide fraction. 2000). Furthermore, the content of GD3 in human milk was Determination of sialic acid levels in infant formulas higher in colostrum than in mature milk, and tended to be established that most sialic acid occurs in the glycoprotein higher in preterm than in term colostrum. In contrast, the fraction, and that the content of sialic acid in the GM3 content was higher in mature milk than in colostrum, oligosaccharide fraction is dependent on the whey: casein and was also higher in term than in preterm milk (Rueda et al, ratio of the formula (Neeser et al, 1991; Wang et al, 2001b). 1996a, b). GD3 is usually detected in developing tissues, Bovine mature milk, which is currently used to produce whereas GM3 is more abundant in mature tissues (Yu et al, infant formulas has a low sialic acid content of 0.05– 1989). In cow’s milk, GD3 was the major ganglioside 0.62 mmol/l (Carlson, 1985; Neeser et al, 1991; Sanchez-Diaz followed by GM3. Other gangliosides amounted to no more et al, 1997; Wang et al, 2001b), equivalent to less than than 20% of the total ganglioside content (Pan & Izumi, one-quarter of the level in mature human milk (1 month

European Journal of Clinical Nutrition The role and potential of sialic acid B Wang and J Brand-Miller 1362 lactation) (Wang et al, 2001b). Preterm formulas contained The source of the protein-bound sialic acid therefore changes the most sialic acid (0.6370.12 mmol/l) followed by the throughout lactation from predominantly whey proteins in ‘follow-on’ formulas (0.4370.03 mmol/l). Soy formulas do the beginning to the caseins as lactation proceeds. In not contain any detectable residue of sialic acid (Carlson, contrast, there is only 10% carbohydrates in cow’s milk k- 1985). We found soy formulas appeared to contain some casein, but the absolute amount is higher (3.3 g/l) than in sialic acid (0.0570.003 mmol/l) although this likely repre- mature human milk (1–3 g/l) (Rudloff & Kunz, 1997). sents interfering substances such as quinic acid. Cow’s milk- The structure of sialic acid in human milk consists only of based formulas containing 60% whey (ie whey:casein ratio in Neu5Ac and the dominant N-acetylneuraminic acid is linked protein component ¼ 60:40) contained more sialic acid to the galactose via an alpha 2-6 bond. Cow’s milk contains (0.3770.01 mmol/l) than those containing 20% whey both Neu5Ac and Neu5Gc structures and the dominant (0.2170.02 mmol/l). The majority of sialic acid in infant galactose is linked by a 2-3 bond (Ebner & Schanbacher, formulas is bound to protein (70%) followed by free 1974). Puente and Hueso (1993) showed that the Neu5Gc oligosaccharides (28%), with only 1% in the free form (Wang content in bovine milk is high in colostrum (32% of the total et al, 2001b). Thus infants fed bovine milk, bovine-based sialic acid content) and decreases until the end of the first formula or soybean-derived formulas receive little or no month. The Neu5Gc content in bovine milk gangliosides sialic acid, while breast-fed babies receive an early diet rich in shows a similar profile. Neu5Gc has never been detected in sialic acid. normal human tissue (Shaw & Schauer, 1988), although it The large amount of sialic acid in human milk compared has recently been reported to occur in malignant samples with formulas based on cow’s milk results from major (Rosenberg, 1995). Differences in the chemical structure of differences in both the amount and types of glycoproteins sialic acid in human vs cow’s milk are likely to influence its and oligosaccharides in the milk of the two species. Mature bioavailability. The amounts and structures of sialic acid in human milk has a whey:casein ratio of 60:40, compared with human milk and cow’s milk are summarised in Table 6. a ratio of approximately 20:80 in cow’s milk. Because casein Two types of formula are currently in wide use for regular synthesis is not initiated in the first few days of lactation infant feeding: those based on cow’s milk and those (Kunz & Lo¨nnerdal, 1990), whey proteins make the largest formulated from protein isolates and hydrolysates. Most of contribution to total protein at the beginning of lactation, the protective glycoconjugates and oligosaccharides in hu- particularly secretory IgA (which contains 12% carbohy- man milk are not found in cow’s milk or protein isolates. drate) and lactoferrin (6% carbohydrate) (Kunz & Lo¨nnerdal, Furthermore, processing excludes the milk fat globule 1990; Rudloff & Kunz, 1997). The carbohydrate side chains membrane from the final product. Therefore, common of human lactoferrin consist of an N-acetyl-lactosamine infant formulas would not be expected to contain the high glycan with sialic acid in the terminal position (Spik et al, levels of glycolipids, glycosaminoglycans, mucins, and other 1982). Cow’s milk, however, is low in lactoferrin and the membrane-associated materials found in human milk. glycan side chains show pronounced differences to human Recently, we analysed 25 brain frontal cortex samples from lactoferrin. The glycosylated caseins (eg k-casein contains infants who died of sudden infant death syndrome (Wang 40–60% carbohydrate) are a major source of protein-bound et al, 2001c). A total of 12 infants were breast-fed, nine sialic acid in mature human milk (Rudloff & Kunz, 1997). formula-fed, and the rest unknown. We found there was a

Table 6 The amounts and structures of oligosaccharides in human milk vs cow’s milk

Colostrum Mature milk Reference

Human milk Oligosaccharides (g/l) 24 12–14 Newburg and Neubauer (1995) Number of structures identified 88 88 Newburg and Neubauer (1995), Chaturvedi et al (1997), Shen et al (2000) Number of sialylated types (as %) 38 (43%) 38 (43%) Newburg and Neubauer (1995) Total sialic acid content (mmol/l) 3.7270.15 1.4870.07 Wang et al (2001b) Structure of sialic acid 100% N-acetylneuraminic acid (Neu5Ac) Spik et al (1982) Newburg and Neubauer (1995) Linkage of Neu5Ac to the galactose 2-6 bond (28 of 38) or 2-3 (19 of 38) Newburg and Neubauer (1995)

Cow’s milk Free oligosaccharides (g/l) 0.025 0.01 Tadaus unpublished data Number of structures identified 20 4 Urashima et al (2001) Number of sialylated types (as %) 11 (55%) 0 Urashima et al (2001) Total sialic acid content (mmol/l) 0.48 (cow’s milk based infant formulas) Wang et al (2001b) Structure of sialic acid 73% Neu 5Ac 0 Urashima et al (2001) 27% Neu5Gc 0 Linkage of Neu5Ac or Neu5Gc to the galactose 2-3 bond (5 of 11), 2-6 bond (6 of 11) Urashima et al (2001)

European Journal of Clinical Nutrition The role and potential of sialic acid B Wang and J Brand-Miller 1363 significant positive correlation between protein-bound sialic (about 9:1 in urine and 62:1 in feces) (Newburg, 2000). acid and age at death in breast-fed infants, but not formula- Furthermore, oligosaccharides in urine and feces of artifi- fed infants. After adjusting for age at death and gender as a cially fed infants have a different pattern from that of breast- covariate, the average concentration of ganglioside-bound fed infants. It is possible that sialidases of bacterial origin and protein-bound sialic acid in breast-fed infants was 32% cleave sialic acid residues from milk oligosaccharides in the and 22% higher than that in formula-fed infants (P ¼ 0.013 colon. However, it is not known if sialic acid can be absorbed and 0.01, respectively). Our study therefore implies that across the colonic mucosa. nutrition might influence the availability and incorporation The high concentration of sialyloligosaccharides in hu- of sialic acid into sialic acid containing compounds. man milk is intriguing, considering the multitude of Interestingly, we also found that ganglioside-bound sialic developmental changes occurring in the brain and other acid in all 25 brain samples was significantly correlated with organs during early infancy. Human milk sialic acid is the proportion of DHA, total n-3 PUFA and total LCPUFA in highest during early lactation, a time when the brain is the ceramide tail. Examining the dietary groups separately, taking up sialic acid most rapidly (Svennerholm et al, 1989). the correlations remained significant only in those who were In addition, deficits in protein or energy in rats during breast-fed, particularly with regard to DHA and AA. The gestation are associated with decreased ganglioside sialic acid correlations therefore imply that sialic acid and LCPUFA are in the offspring and poor learning behaviour (Morgan & interdependent building blocks for neural tissues involved in Naismith, 1982). higher cognitive function. Conceivably, both work together to increase the fluidity and functionality of neuronal membranes. Exogenous administration of sialic acid The effect of repeated administration of sialic acid during periods of maximal brain ganglioside accumulation was the Digestion and absorption of sialic acid subject of extensive research as early as 20 y ago. Morgan and Once digested, the constituents of the glycoproteins and Winick (1980a) examined undernourished and well-fed rat glycolipids provide a source of amino acids, fatty acids, and pups injected intraperitoneally with 1 mg N-acetylneurami- sialic acid for absorption. At present however, we know little nic acid per 50 g body weight daily from 14 to 20 days about the mechanisms and extent of digestion of sialic acid postnatally. The results showed the administration of sialic containing compounds. acid was associated with an increase in cerebral and Hydrolysis of sialic acid containing carbohydrates in the cerebellar ganglioside and glycoprotein sialic acid concen- intestine is possible because sialidase activity in mucosal tration and higher scores in the maze test compared with homogenates has been shown to be high during the suckling controls. These changes occurred without affecting brain period in several species and positively correlated to the weight, cell size, cell number and DNA, RNA or protein sialic acid content of the respective milks (Dickson & Messer, content. Furthermore, these effects were shown to persist 1978). Sialic acid appears to be cleaved after uptake into the into adulthood (Morgan & Winick, 1980a). Interestingly, tissues, probably by Neu5Ac lyase to N-acetylmannosami- early environmental stimulation produced the same effects ne þ pyruvic acid. N-acetylmannosamine is then used for the (Morgan & Winick, 1980b). The authors concluded that resynthesis of sialic acid containing compounds (Schauer, since environmental stimulation and administration of sialic 1982). Sialic acid always occupies the terminal position of acid brought about the same changes in brain sialic acid milk oligosaccharides, and the bond may be cleaved even if content, it was possible that brain sialic acid played the remainder of the chain resists digestion. Autohydrolysis an important role in learning ability (Morgan & Winick, has also been claimed because sialic acids are relatively 1980a, b). strong acids (pKa 2.2–3.0) (Schauer & Kamerling, 1997). Rat The effect of administration of sialic acid on brain intestinal cell walls are highly permeable to free sialic acid. subcellular localisation was also studied by Morgan and Radioactively labelled forms of sialic acid and sialyl lactose Winick (1981). The pregnant rats were either well-fed (200 g were found to be well absorbed (B90%) by rat pups, 30% casein/kg) or protein restricted (100 g casein/kg) throughout being retained in the body, and 3–4% in the brain after 6 h gestation. Each of the low and high protein groups was given (No¨hle & Schauer, 1981). However, Brand-Miller et al (1998) their respective diet for the first 11 days of lactation. On day demonstrated with breath hydrogen methodology that most 12 of lactation, 2.5 mCi14 Neu5Ac/kg body weight was HMOs resist digestion in the small intestine of breast-fed injected intraperitoneally into the pups. After 1 h, the brains infants and undergo fermentation in the colon. Studies were analysed for sialic acid in nuclear, myelin-rich, in vitro have also confirmed that sialic acid is not released synaptosomal, mitochondrial, and microsomal fractions. from the incubation of HMOs with pancreatic and mucosal The results showed that 80% of the labelled Neu5Ac enzyme mixtures (Engfer et al, 2000). The quantities of incorporated into the brains was found in the synaptosomal oligosaccharides in the urine and feces of breast-fed infants fraction in both the well-fed and protein restricted animals. are also much higher than those in formula-fed infants The remainder was distributed among the other subcellular

European Journal of Clinical Nutrition The role and potential of sialic acid B Wang and J Brand-Miller 1364 fractions in proportion to their total Neu5Ac content. Well- 42 weeks of gestation. They showed significant correlations fed animals had levels 50–60% higher incorporation than (Po0.01) between maternal and retroplacental blood on the the protein-restricted group in all fractions. The results one side and between maternal and the cord blood on the suggested that sialic acid exerts its effects on learning other side. This suggests that the mother synthesises much behaviour via the synaptic membrane. of the sialic acid, which crosses the placenta to contribute Carlson and House (1986) investigated both oral and the fetal growth in the third trimester. The high concentra- intraperitoneal administration of sialic acid. Beginning on tion in early human milk (but not infant formula) may day 14 of life, the rat pups were administrated sialic acid by provide for continuing need. intraperitoneal (i.p.) injection or via a feeding catheter with 1.0 mg on days 1 and 2, 1.2 mg on the remaining days, or approximately 20 mg/kg body weight per day for 8 con- Medical significance of disturbances in sialic acid secutive days. The results showed administration of sialic metabolism acid by both routes resulted in significantly more cerebral Disturbances in the metabolism of sialic acid, either due to and cerebellar ganglioside and glycoprotein sialic acid than genetic error or at the post-translational level, may impair the control group. Both routes were similarly effective. physiological function and lead to disease. A number of rare Earlier studies have shown that a diet lower in essential disorders that involve sialic acid accumulation and defi- fatty acids (EFA) (100 g hydrogenated coconut oil/kg) or ciency in humans have been described. Deficiencies of sialic deficient in protein decreased the amount of brain ganglio- acid in brain glycoprotein and ganglioside are related to side and glycoprotein as measured by sialic acid concentra- mental retardation, but severe deficiency has not been tion (Merat & Dickerson, 1973; Karlsson & Svennerholm, described, perhaps because inability to use sialic acid may 1978; Morgan et al, 1981). These effects were associated with be lethal (McVeagh & Brand Miller, 1997). depressed activities of sialidase and CMP-Neu5Ac synthetase In studies on impairment of human brain and glycocon- in the brain (Morgan et al, 1981). In addition, pregnant rats jugates in congenital athyroidism, protein-bound and gang- fed a diet with low protein or low EFA content produced lioside-bound sialic acid in grey matter was 60 and 11 % offspring with altered brain ganglioside distribution and lower respectively than that of control group (death from decreased sialic acid content (Berra et al, 1981). non-neurological disease) (Annunziata et al, 1983). There Oral and intravenous administration of radioactively was a decrease in GD1b, GT1, and GQ1, however GM2, GD3, labelled forms of both sialic acid and sialyl-lactose (a and GM1 increased compared to controls. These results infer trisaccharide) were found to be well absorbed (B90%) by that some types of brain ganglioside (maybe GD1b or GQ1) 20-day-old rat pups, 30% being retained in the body, and 3– play a more important role in intelligence. The decrease of 4% in the brain after 6 h (No¨hle & Schauer, 1981). Our group glycoprotein sialic acid suggested an impairment of cell demonstrated that 0.23% of a labelled dose of sialic acid was maturation (Annunziata et al, 1983). taken up into the brain of 3-day-old piglets by 2 h after In schizophrenia, Campbell et al (1967) found the more intravenous administration (Downing et al, 2001). However, serious the psychosis, the lower the sialic acid content in the older animals did not show significant incorporation of glycoproteins of the cerebrospinal fluid. When the schizo- labelled sialic acid after acute dosing (No¨hle & Schauer, phrenic state was treated successfully, the sialic acid content 1981). Morgon and Winick (unpublished results) also found rose to normal values. Edelfors (1981) showed that chronic that when sialic acid was injected after 30 days of age (by lithium treatment in animal models of schizophrenia caused which time the rat brain had completed its growth), neither changes in the sialic acid content of rat synaptosomes. behaviour nor brain biochemistry was affected. Because Edelfors (1981) concluded that the increased content of sialic brain growth is time dependent, the effect of administrated acid in both glycoprotein and gangliosides in the synaptic sialic acid is likely to be time dependent (Morgan, 1990). membrane may alter electric properties, leading to improvements in schizophrenic patients. Interference with the synthesis and function of gangliosides and sialoglycoproteins during development appears to Pregnancy contribute to brain dysfunction in phenylketonuria (PKU) Pregnancy is associated with an increase in concentration of (Loo et al, 1985). In the offspring of untreated PKU mothers, sialic acid in maternal saliva and plasma. Sialic acid increases there is a noticeable disruption of the normal ganglioside from about 50 mg/l at 10 weeks gestation to over 150 mg/l at pattern (delayed drop in GQlb and GD3 and slower rise in 21–40 weeks gestation in saliva corresponding to the period GM1 and GD1 a) and a significant reduction of sialoglyco- of rapid sialic acid accumulation in the fetal brain (Salvolini proteins. et al, 1998). Plasma increases from 507793 mg/l at the onset Alzheimer’s disease and older Down’s syndrome typically of pregnancy to 6377152 mg/l at term or early postpartum occurs after 65 y of age, but can also affect people in their 40s in plasma (Alvi et al, 1988). Briese et al (1999) measured the and 50s (Warren, 1994). Degenerative and progressive, this sialic acid concentration in maternal, retroplacental, and disease involves the brain, resulting in loss of memory and cord blood samples of 126 pregnant women between 28 and cognitive function. Alzheimer’s patients have been found to

European Journal of Clinical Nutrition The role and potential of sialic acid B Wang and J Brand-Miller 1365 have decreased sialyltransferase activity in serum that affects acid in human milk may contribute significantly to greater the a-2,3-linked sialic acid in serum glycoproteins. Ganglio- sialydation of gangliosides and glycoproteins in body fluids, side sialic acid content in cerebral cortex was also decreased tissues and brain glycoconjugates in breast-fed infants and (Sorbi et al, 1987). It can only be assumed that the defects contribute to the observed neurological and intellectual also occur in neuronal tissue and contribute to the disease advantages of breastfeeding over formula feeding. These also process. Serum sialyltransferase may thus be an early have important implications for the formulation of artificial biochemical marker of neurodegeneration (Schauer & milk for the feeding of newborn infants, particularly preterm Kamerling, 1997). infants. In the rare genetic disease sialuria, there is an over- production of free sialic acid with massive excretion in the urine (5–7 g/day) (Fontaine et al, 1968; Seppala et al, 1991). In Salla disease, free sialic acid, formed by cleavage of References glycoproteins and glycolipids by lysosomal neuraminidase, Agnati LF, Fuxe K, Benfenati F, Battistini N, Zini I & Toffano G (1983): cannot be transported out of the lysosome where it Chronic ganglioside treatment counteracts the biochemical signs of dopamine receptor supersensitivity induced by chronic halo- accumulates (Renlund et al, 1986). Infantile free sialic acid peridol treatment. Neurosci. Lett. 40, 293–297. storage disease produces the same defects as in Salla’s disease, Alvi MH, Amer NA & Sumerin I (1988): Serum 5-nucleotidase and but is more severe and leads to early death (Mancini et al, serum sialic acid in pregnancy. Obstet. Gynecol. 72, 171–174. Anderson JW, Johnstone BM & Remley DT (1999): Breast-feeding and 1992). cognitive development: a meta-analysis. Am. J. Clin. Nutr. 70(4), Adult humans do not express significant levels of Neu5Gc 525–535. in normal cells, but it has been found in certain human Annunziata P, Federico A, D’Amore I, Corona RM & Guazzi GC tumors and tumor cell lines (Schauer et al, 1995; Reuter & (1983): Impairment of human brain development: glycoconjugate and lipid changes in congenital athyroidism. Early Hum. Dev. 8, Gabius, 1996). Spontaneously occurring antibodies to 269–278. Neu5Gc occur in patients with cancer and with certain Atkinson SA & Lonnerdal B (1995): Nonprotein nitrogen fractions of infectious diseases, as well as in chickens with Marek’s human milk. In Handbook of Milk Composition, ed. RG Jensen, pp disease, a malignant herpes virus infection (Varki et al, 1999). 369–385. San Diego: Academic Press. Beitinger H, Vogel V, Mobius D & Rahmann H (1989): Surface In tumour metastasis, an increase in the level of cell surface potentials and electric dipole moments of ganglioside and sialylation in certain cell lines has been found to be linked to phospholipid bilayers: contribution of the polar headgroup at their metastatic potential (Troy, 1995). The increased the water/lipid interface. Biochim. Biophys. Acta 984, 293–300. sialylation may contribute to the survival of the metastasis- Berra B, Lindi C, Omodeo-Sale F, Beltrame D & Cantone A (1981): Effect of maternal diet on ganglioside distribution in fetal rat ing cells in the circulatory system, and to adhesion of the brain. J. Nutr. 111, 1980–1984. cells to the endothelium at a secondary site (von Itzstein & Bogoch S (1970): Glycoproteins of the brain of the training pigeon. Thomson, 1997). In Protein Metabolism of the Nervous System, ed. A Lajtha, pp 535– Sialic acid levels in blood are also used as a marker of the 569. New York: Plenum Press. Bogoch S (1977): Recognins and their chemoreciprocals. In Behavior- acute-phase response proteins. Some acidic glycoproteins, al Neurochemistry. eds FV DeFeudis & JMR Delgado, p 270. New for example, fibrinogen and haptoglobin, are glycoproteins York:Spectrum Pubs. with sialic acid as the terminal sugar which increase Brand-Miller JC, McVeagh P, McNeil Y & Messer M (1998): Digestion markedly in response to cell injury (Taniuchi et al, 1981). of human milk oligosaccharides by healthy infants evaluated by the lactulose hydrogen breath test. J. Pediatr. 133, 95–98. Elevated plasma sialic acid concentration is strongly related Brand Miller JC, Miller JJ, McVeagh P & Bull S (1994): The to the presence of microvascular complications in type I oligosaccharide composition of human milk: temporal and diabetes (Crook et al, 2001) and cardiovascular morbidity in individual variations in monosaccharides components. J. Pediatr. the general population (Lindberg et al, 1991). 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(1988): Ganglioside content of astroglia and neurons isolated from The nutritional and biological roles of sialic acids in maturing rat brain: consideration of the source of astroglial human milk and other dietary sources (eg fish eggs) are still gangliosides. Brain Res. 461, 87–97. Cabezas JA & Calvo P (1984): Gangliosidos. Sci. Am.(Spanish ed) 6, not fully understood. The higher levels of sialic acid in 86–95. human milk, together with the higher content in saliva of Campbell RJ, Bogoch S, Scolaro MJ & Belval PC (1967): Cerebrospinal breast-fed compared with formula-fed infants, suggests that fluid glycoproteins in schizophrenia. Am. J. Psychiatry 123, dietary sources are absorbed. The higher level of ganglioside 952–962. Chaturvedi P, Warren CD, Ruiz-Palacios GM, Pickering LK & New- and protein-bound sialic acid in brain frontal cortex of burg DS (1997): Milk oligosaccharide profiles by reversed-phase breast-fed infants has important implications. 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