A Short History of Galactose and its Pathophysiological Significance
In nature, galactose occurs in two structurally related forms (anomers), D-galactose and L-galactose. D-Galactose is ubiquitous in glycosidic bonds in the animal kingdom. It is an integral component of glycoproteins, glycolipids and proteoglycans. Glycoproteins and glycolipids (=glycoconjugates) are significant components of the plasma membranes that surround all animal cells. They form a protective barrier around cells; yet they also mediate their contact to the surrounding milieu, the extracellular matrix and neighboring cells. In addition, most serum proteins, with the exception of albumin, are glycoproteins. In plants, glycosidically bonded galactose is an important component of various lectins. Lectins have the ability to bind to sugar-containing structures, but have no enzymatic function.
Systematic studies of galactose began in the middle of the last century with the detection of free galactose in milk (Malyoth et al., 1953), semen (Kubicek and Santavy, 1958), and urine (Montreuil and Boulanger, 1953). Without an exogenic source, blood is galactose-free (Harding and Grant, 1933; Montreuil and Boulanger, 1953). As early as 1910, Lippman detected crystalline galactose on ivy leaves after heavy frost. Two other instances of elevated galactose concentration were found by Henze (1959) in the bark of apple trees in the winter months and by Venkataraman and Reithel (1958) in the ripe fruit of Achras sapota (sapodilla tree).
Bound D-Galactose Saccharides
The simplest saccharide is the disaccharide lactose (glucose-galactose), which has been known for a long time and was described in the first galactose overview by Whittler (1926) and then in 1961 by von Clamp et al. Lactose is present at 0.1 to 8 % in human milk. Not all mammals produce lactose, though cows, some whales, and seals do. In addition to milk, lactose is found in the amniotic fluid (Makepace et al., 1931) and urine of pregnant women, especially in cases of galactostasis (Riffart et al., 1952).
As a structural unit, lactose is also found in glyconjugates (Klenk and Gielen, 1960, Egge, 1960). Lactose was also detected in some fruits and in the pollen of forsythias (Kuhn and Löw, 1949), as well as in shepherd's purse (Nagai, 1960). Oligosaccharides with a lactose-based structure occur in milk as lactotetraose (Kuhn et al., 1955). The lactosamine-based structure (galactose-N-acetylglucosamine) is found in the Lewis blood factors, which play a critical role in the interaction of white blood cells and vascular endothelial cells, and as polylactosamines in tumor cells (Yamashita et al., 1984).
Heterosaccharides, N-and O-Glycans
The N-glycans include serum glycoproteins (all serum proteins except for albumin are glycoproteins) and integral glycoproteins of the plasma membranes found in all cells of the body. The O-glycans include the proteoglycans or mucines, components of all bodily secretions.
Both groups are highly complex compounds. Mucines or mucuses were first designated as such by Hoffbauer (1734) and a short time later by C. Darwin (1778). Lorleberg (1789) and Henle (1790) also recognized the clinical significance of this new class of substance. They distinguished these from albumin and gelatin mainly on the basis of the heat resistance and their formation of precipitates with mercuric chloride. Eichwald (1865) successfully detected carbohydrates in this "mucilaginous material" (Berzelius, 1828) in a sample from Helix pomatia. For further details see A. Gottschalk (1972). Bierry (1929) was the first to detect galactose in an animal organism, which Sørensen and Hauggard confirmed by means of colorimetric methods (1933). Karl Meyer et al. (1937) found galactose in glycoproteins; he was a pioneer in the structural characterization of the O-glycans. Another advancement in methodology occurred with the highly specific enzymatic detection of galactose with galactose dehydrogenase (Wallenfels and Kurz, 1962), which made possible precise determination of galactose in various biological fluids and tissues.
The first structure determination of N-glycans as significant components of serum proteins (except albumin) and integral membrane glycoproteins was carried out by a research team led by Montreuil (Spik et al., 1975). They discovered the pentasaccharide framework of the N-glycans, which represents the core structure of the N-glycans. It consists of a chitobiose bound to a triangular trimannoside structure. The two free mannoses are then each bound to an N-acetylglucosamine. The terminal bound galactose has an important assignment; it is the anchoring sugar for the terminal N-acetylneuraminic acid (or sialic acid), which governs important biological and physical-chemical properties of glycoproteins. Another groundbreaking discovery about the function of monosaccharides in this oligosaccharide structure was made by G. Ashwell and co-workers, who demonstrated that this galactose, when no longer masked or disguised by N-acetylneuraminic acid, determines the biological lifetime of glycoproteins (Morell et al., 1968). This particularly affects serum glycoproteins like immunoglobulins, clotting factors, metal-transport glycoproteins, and hormones such as erythropoietin and others.
These discoveries showed that galactose in the terminal position of glycoproteins acts as a recognition signal for their degradation. The same group also found the receptor responsible for recognizing asialoglycoproteins in the plasma membrane, the asialo or Ashwell receptor (review by Ashwell and Harford, 1982). These surprising discoveries opened a new area of research into the biological function of N-acetylneuraminic acid in glycoproteins. Among its diverse functions, the covering or masking of galactose in glycans is particularly significant. This prevents the galactose-containing molecule or even an entire cell (such as an erythrocyte) from being shunted off for biological degradation. Galactose is thus a necessary molecule for the anchorage of N-acetylneuraminic acid with its various functions and as a signal for biological degradation.
O-Glycans or Mucine-Type Glycans
Whereas N-glycans are always bound with asparagine in the tripeptide (or consensus) sequence asparagine-X-serine/threonine by way of the disaccharide made of two N-acetylglucosamines (chitobiose), the O-glycans are usually bound to serine or threonine in the protein backbone, mostly by means of N-acetylgalactosamine, less frequently via galactose or xylose. No consensus sequence containing the serine/threonine sequence is yet known for this process. The highly complex and high-molecular polysaccharide structure does not seem to have any regularity. Galactose is a consistent component of all O-glycans. In addition, galactose is a structural component in blood types; the B blood group is thus characterized by a terminal galactose (Gibbons et a., 1955).
One peculiarity is the occurrence of galactose in glycogen, which is commonly accepted as a homopolysaccharide made of glucose units. However, prior studies have demonstrated that galactose can be incorporated into glycogen (Schlamowitz, 1951; Nordin and Hanson, 1963). The presence of galactose in glycogen influences its degradation, because glycogen phosphorylase specifically recognizes glucose, not galactose. It is not yet known in what form galactose is eliminated. Its presence in glycogen has the potential to inhibit rapid glycogen degradation.
Biosynthesis of Galactose and Galactosides
In the intermediary metabolism, galactose is made from glucose via the activated monosaccharides UDP-glucose and UDP-galactose by the Leloir pathway (Trucco et al., 1948; Leloir, 1951). In this process, UDP-glucose is converted to 4-epimeric UDP-galactose. E. Fischer postulated reduction equivalents for this pathway (Fischer and Armstrong, 1902), which O. Warburg later identified as DPNH or NADH (Warburg and Negelein, 1929). The metabolism of exogenic, administered galactose occurs by way of its phosphorylation with ATP as the energy donor and the highly specific galactokinase (Kosterlitz, 1937), and the subsequent activation to UDP-galactose by uridylyltransferase and UDP-glucose acting both as coenzyme and energy substrate (Isselbacher et al., 1956). This enzyme is faulty in cases of hereditary galactose intolerance. The attachment of galactose to glycoconjugates occurs by way of galactosyltransferases and UDP-galactose as coenzyme. The first indications of the existence of galacatosyltransferase activity came from the Watkins research group (Watkins and Hassid 1961; 1962). To date, seven different isoenzymes with galactosyltransferase activity have been described. Beta-1,4-galactosyltransferase V moderates the biosynthesis of highly branched complex N-glycans. a characteristic of various tumors (Dennis et al., 1987, Sato et al. 2007). Wallenfels et al. found that the hydrolytically active galactosidase also demonstrates galactosyltransferase activity (Fischer and Armstrong, 1902; Wallenfels et al., 1953), a fact that has found industrial utility, since this reaction requires no energy-rich coenzyme.
Galactose in Lectins
A functionally important animal lectin is galectin-3, a member of the ubiquitous beta-galactoside-binding protein family (Barondes et al., 1994). Galectin-3 regulates cell proliferation in that it is responsible for splicing pre-mRNA. It is strongly linked to tumor progression. It works by means of a shuttle mechanism between thee cytosol and nucleus by way of the importin alpha/beta pathway (Nakahara et al., 2006).
Terminal galactose also plays a functionally important role in a plant lectin, arabinogalactan. This is a proteoglycan and is widely distributed in plants. It is a glucose polymer in a beta 1,3-D-glucosidic bond. In plants it is involved in an essential way in the growth and differentiation processes (Willats and Knox, 1996). In human diseases, arabinogalactan is deployed in viral infections. The receptor of the complementary component C3 is activated and interleukin-3 expression is increased. More recently, there have been indications that it inhibits metastasis of prostate carcinomas (Glinski et al., 2005).
To date, three galactose analogs with biological and diagnostic significance in human or animal systems have been described: 2-desoxy-D-galactose, 2-desoxy-2-fluoro-D-galactose and 2-desoxy-2-amino-D-galactose.
a. 2-Desoxy-D-galactose This synthetic galactose derivative is metabolized by the Leloir pathway, like galactose (Smith and Keppler, 1977), and is incorporated into membrane glycoproteins in the liver and Morris hepatomas of rats. This incorporation surprisingly inhibits the L-fucosylation of glycoproteins (Büchsel et al., 1980). In contrast to galactose, 2-desoxy-D-galactose is toxic. In yeasts, it inhibits respiration, fermentation, and the growth of malignant cells (Landau et al., 1958). Particularly noteworthy are the toxic effects of intrathecally-administered 2-desoxygalactose on the brain; it induces increased morphine tolerance in rats (Richter et al., 1991) and disruption of short-term memory in chickens (Barber and Rose, 1989).
b. 2-Desoxy-2-fluoro-D-galactose Replacement of the hydroxyl group at the C2 position of galactose with fluoride leads to this derivative (Adamson and Marcus, 1972). It has proven itself useful for 18F positron emission tomography (Fukuda et al., 1986). The research team headed by D. Keppler also demonstrated the metabolism of this galactose analog by way of the Leloir pathway (Grün et al. 1990) This fluoride derivative almost completely inhibits N-glycosylation of serum glycoproteins as well as membrane glycoproteins, in contrast to 2-desoxygalactose or 2-desoxy-2-fluoroglucose (Loch et al. 1991, Gross et al., 1992).
c. 2-Desoxy-2-amino-D-galactose (galactosamine) The aminohexose galactosamine does not occur in nature in its free form. In glycosidic linkage, galactosamine is exclusively found in N-acetyl-D-galactosamine, except in some bacteria. Whereas N-acetylgalactosamine is not toxic, the administration of galactosamine leads to severe liver damage. This is the only animal experimental model that closely resembles human viral hepatitis when viewed with an optical microscope (Reutter et al., 1968, Decker and Keppler, 1972). This novel procedure made it possible to increase the endotoxin toxicity in mice by a factor of 104 to 105, and has become the most sensitive endotoxin detection procedure (Galanos et al., 1979).
L-Galactose Among animals, L-galactose is only found in nonvertebrates, in the galactogen of the Weinberg snail (May, 1932) and the egg jellies of sea urchins (HIrohashi et al., 2002). L-Galactose is much more common in the plant realm, particularly in arabidopsis (Rayon et al., 1999; Reuhs et al., 2004) and in special types of rubber (Winterstein, 1898). It has been found to be plentiful in red algae (Oshima and Tollens, 1901). These findings about L-galactose are over 100 years old. More recently L-galactose has been gaining significance as a substrate for the biosynthesis of ascorbate in leaves (Keates et al., 2000; Smirnoff, 2001; Laing et al., 2007).
Galactose and Tumors
As discussed above, the presence of polylactosamine structures is typical of some tumors. It remains to be determined how important this discovery will be to tumor diagnosis. With regard to the possible therapeutic use of galactose in fighting tumor growth, Warburg et al. (1967) have determined that the growth of Ehrlich ascitic tumor cells is inhibited by D-galactose but not by D-glucose. A clinically impressive effect of galactose has been demonstrated by its pre-, intra-, and postoperative infusion in the operative removal of colon carcinomas. Patients who receive this treatment experience far fewer liver metastases than do those who receive no galactose (Kosik et al., 1995).
Galactose and Insulin Resistance
The glucose transporter GluT-4 is the molecule responsible for the regulation of blood-sugar concentration. It is under the control of the insulin receptor, which is, in turn, activated by insulin. When the receptor is activated by insulin (endogenic or, in the case of diabetics, exogenic), GluT-4 is transported from the vesicles within the cells into the plasma membrane, where it accelerates the transport of glucose into the cell (with the exception of hepatocytes). Many diabetics suffer impairment of their insulin receptors and thus underperformance of the glucose transporter GluT-4, because the insulin-mediated signal cascade is continuously disrupted (Hunter and Garvey, 1998). These diabetics can no longer be regulated by means of insulin administration; their blood-sugar concentrations cannot be normalized by giving insulin, and insulin resistance develops.
Insulin resistance is a dangerous state for the patient. It is exacerbated by weight-gain, physical inactivity, mental stress and certain medications. In 1970, Reaven recognnized therapy-resistant increases in blood-sugar concentration as a compensation mechanism for insulin resistance on the one hand, and for hyperinsulinism on the other. The molecule most responsible for insulin resistance is the insulin receptor, whose antenna function is curbed such that it can no longer ensure the delivery of glucose to needy target cells. As a result, the metabolism of the glucose-deficient cells is impaired, which leads to grave deterioration of their functionality. The administration of galactose can improve this reduced functionality, if not completely remove it. The reason for this surprising effect of glucoseâs sister sugar is the insulin-independent uptake of galactose through the glucose transporter GluT-3. After its uptake, galactose can the converted to glucose by means of ubiquitous enzymatic pathways. The blockage of the insulin-dependent GluT-4 is thus avoided, and the cell metabolism can normalize by means of the glucose provided by the conversion of galactose.
Galactose and Alzheimer's Disease
In recent years, many important molecular-biological discoveries have been described with regard to changes in the brain associated with Alzheimer's disease: Descriptions of beta-amyloid and its precursors, the alpha, beta, and gamma secretases, plaques, fibrils, and tangles, to name a few. These important discoveries pushed a previously known, pathophysiologically important finding into the background: the reduced glucose metabolism in the brain cells of Alzheimer's patients. This discovery came from the research team of S. Hoyer (1982), and was further explored in subsequent publications (Hoyer and Lannert, 1999; Frölich et al., 1999; Hoyer 2004). It laid the foundation for the new designation of Alzheimer's disease as type III diabetes mellitus (Gerozissis, 2003; de la Monte, 2005; Salkovic-Petrisic, 2006). These researchers recognized the disrupted function of the insulin receptor and pushed it into the foreground of the pathogenesis of Alzheimer's disease. The disrupted insulin function also induces a state of oxidative stress (de la Monte and Wands, 2006). A striking indicator of the signficance of an intact insulin receptor in Alzheimer's disease was demonstrated by Salkovic-Petrisic et al. (2006) in animal studies in which they shut off the insulin receptors of brain cells by the intrathecal administration of streptozotocin (systemic administration of this substance is also used to induce "ordinary" type II diabetes mellitus). With this procedure, they shut off the memory function in rat brains. However, these memory functions were not lost after administration of streptozotocin if the animals were given galactose in their drinking water during the experiment. These discoveries offer substantial support to the type III diabetes mellitus model of Alzheimer's disease and, moreover, suggest a new treatment for this disease, which involves a strategy similar to that used for type II diabetes mellitus (see above): dietary supplementation with galactose, which is brought into brain cells via the insulin-independent GluT-3 pathway and is then metabolized into glucose.
Galactose and Hepatic Encephalopathy
The liver is the most important organ in the body for the removal of metabolically generated or exogenically introduced toxins (biotransformation system). One of these toxins is ammonia, which is produced in the breakdown of amino acids in gluconeogenesis (the formation of glucose from non-carbohydrates). This liver-specific function serves to ensure an adequate supply of the energy substrate glucose to the brain and the erythrocytes. In this process ammonia equivalents are released as glutamine is broken down. The ammonia is removed by way of the urea cycle (another liver-specific metabolic pathway). When liver function is disrupted (chronic alcoholism, advanced cirrhosis, hepatoma) the highly toxic ammonia can no longer be removed by the damaged liver and penetrates all membranes, including the blood-brain barrier, and into the cells. The toxic effect of ammonia on brain cells leads to restricted brain function, known as hepatic encephalopathy. This condition is expressed with varying degrees of severity. It is certainly within reason that ammonia also affects the insulin receptor function. It has been demonstrated that advanced cases of hepatic encephalopathy can be improved or cured within a surprisingly short time through the administration of galactose. The effect is much faster than that of the more commonly used glucose infusion (Büchsel et al., unpublished). Animal experiments have shown that in rat brains especially, galactose is converted to amino acids. This consumes ammonia equivalents, resulting in an endogenic detoxification of the brain after administration of galactose (Roser, 1991).
Galactose and Glucose-Intolerant Preemies
In glucose-intolerant premature infants, the blood-glucose concentration normalized rapidly after treatment with galactose-containing glucose infusions (Sparks et al., 1982). It is highly likely that the carbohydrate and energy metabolisms in the responsible regulatory centers are normalized so that they can resume their normal function. It is also highly probable that the curative effect occurs by way of the insulin-independent uptake of galactose in the areas of the brain responsible for this regulation.
Galactose and Parkinson's Disease
The morphological substrate in Parkinson's disease is the substantia nigra. Of primary biochemical interest is the reduced synthesis of 3,4-dihydroxyphenylalanine (L-DOPA) from L-tyrosine. DOPA is the precursor for dopamine, which carries out its function in postganglionic neurons. It has recently been possible to uncover a defect in Parkin, an enzyme in the ubiquitin-dependent protein degradation pathway. It has been shown that the key enzyme of L-DOPA synthesis, the activity of mitochondrial tyrosine-3-monooxygenase, is inhibited by the O-glycosidic attachment of N-acetylglucosamine (O-GlcNAc formation) (Bork et al., 2007). Current therapy involves the substitution of the missing L-DOPA. In a number of Parkinson's patients, the administration of galactose over several weeks elicited a surprising effect on the course of the disease (K. Mosetter, unpublished). The mechanism behind this could be a reduction in the formation of O-GlcNAc or induction of the biosynthesis of enzymes that synthesize dopamine.
Galactose and Post-Aggression Syndrome
Post-aggression syndrome is related to burnout syndrome. The cells of the central nervous system are in a state of metabolic stress. It is conceivable that this metabolic situation involves reduced functionality of the insulin receptors with the result that less glucose is transported into the cells of the CNS. In this case as well, galactose is an effective alternative energy substrate to glucose.