Carbohydrates Resources: • McMurry, Organic Chemistry, Ch. 25 • Nielsen, Food Analysis, Ch. 10 • Dewick, Medicinal Natural Products, Ch. 8 • Belitz, Food Chemistry Carbohydrates Carbohydrates or “sugars” are polyhydroxy aldehydes or ketones Sugars contain several chiral centers, allowing for many possible stereoisomers Carbohydrates include both simple sugars and polymers of sugar subunits which can be hydrolyzed to simple sugars Produced through photosynthetic reactions in plants: 6 CO2 + 6 H2O + energy C6H12O6 + 6 O2 Single sugar unit = monosaccharide ex: glucose = primary animal energy source Two sugar units = disaccharide ex: lactose = milk sugar 5 units or more = oligosaccharide or polysaccharide ex: amylose = starch Monosaccharides Isomerism and classification: • Number of stereoisomers = 2n where n = # of chiral carbons • Enantiomers are assigned D or L notations based on comparison to D and L-glyceraldehyde. Natural sugars are generally the D – enantiomers • (+) or (-) designation must be determined by optical rotation • Structures are classified by number of carbons (pentose, hexose, heptose, etc) or type of functional group at the anomeric carbon (aldose or ketose) D-Diastereomers of the aldoses Cyclization occurs in solution to form a and b anomers • Hemiacetal formation occurs between anomeric C and another C in the chain • The a and b anomers are in equilibrium with open chain form, will interconvert, but the b anomers are more stable • Most common aldoses and ketoses prefer to form 5 or 6-member rings (furanoses and pyranoses) Fructose, pentoses can form pyranose or furanose rings Major carbohydrates in food • D-glucose (dextrose) and D-fructose are the major monosaccharides, others only present in minor amounts • Table 10-1 (Nielsen) • Sucrose (fruits, vegetables), Lactose (dairy) and maltose (malt, corn syrup) are the major disaccharides • Oligosaccharides and polysaccharides are mainly composed of glucose, but may include fructose or galactose (e.g. raffinose & stachyose in beans) • Sugar alcohols, gums, hydrocolloids are carbohydate based substances often added to foods as humectants or thickening agents Natural Sweeteners • Sucrose is obtained from a variety of sources, including sugar cane (Saccharum officinarum; Graminae/Poaceae), sugar beet (Beta vulgaris; Chenopodiaceae), and sugar maple (Acer saccharum; Aceraceae). • Invert sugar (equimolar mixture of glucose and fructose) is obtained from sucrose by hydrolysis with acid or the enzyme invertase, during which the optical activity changes from + to −. Fructose, which is sweeter than glucose or sucrose, can be isolated from this mix. • High fructose corn syrup is a mixture of fructose and glucose containing up to 90% fructose, produced by enzymatic hydrolysis/ isomerization of starch. • The sugar alcohol D-sorbitol is isolated from the berries of the mountain ash (Sorbus aucuparia; Rosaceae) or prepared by reduction of glucose or fructose. It is half as sweet as sucrose, but not readily metabolized in the body and is used mainly in sugar-free gum and candy. Glycoside formation The formation of di- and oligosaccharides (typically two to five monomers) and polysaccharides is dependent on the generation of an activated sugar bound to a nucleoside diphosphate. The nucleoside diphosphate most often employed is UDP, but ADP and GDP are sometimes involved. Mechanism is SN2 reaction of a particular OH group on the incoming sugar and the anomeric C on the UDPsugar. Formation of alinked glycosides involves initial enzyme-catalyzed SN2 displacement of UPP group. Glycoside formation Sugar units are linked together or linked to other entities in natural products called “aglycone” moiety, through formation of glycosidic bonds Glycosides are acetals or ketals formed by reaction of hemiacetal carbon (anomeric C) with an OH group on the other molecule Glycosides of natural compounds are formed to increase water-solubility Glycosidic linkages can be hydrolyzed by acid to release the aglycone OH OH HO O OH CH2 O HO O OH O OH HO Quercetin galactoside, the primary flavonol glycoside in cranberries Glycyrrhizic acid, a saponin Common types of glycosidic linkages in di- and polysaccharides In nature, enzymes catalyze reactions between specific anomers of monosaccharides. Some glycosidic linkages are more easily digested/metabolized by humans, e.g. a-1,4 linkages: The C1 carbon of the a-anomer of one sugar links to C4 of the next unit Ex: maltose b-1,4: The C1 carbon of a b-anomer links to C4 of next unit Ex: cellobiose lactose Polysaccharides • Play 2 major roles in organisms: food reserves and structural elements • Plants store glucose mainly as starch (amylose & amylopectin) • Humans store glucose in the form of glycogen, similar in structure to amylopectin, but with more branching • Branching in amylopectin and glycogen is achieved by enzymatic removal of a portion of the α1→4 linked straight chain containing several glucose residues, then transfer of this short chain to a 6-hydroxyl group. • While humans can digest starch and use the glucose for energy, the plant polysaccharides that make up dietary fiber are indigestible Starch structure Starch is a glucose storage form in plants, composed of amylose & amylopectin Amylose contains 1000-2000 glucose units in a-1,4-linkages forming a long chain with a spiral or helical 3-D structure. Amylose can be digested by humans in enzymatic reactions similar to acidcatalyzed hydrolysis of the glycosidic linkages. Amylopectin is a major component of starch that may contain up to a million glucose units connected by a-1,4-linkages with a-1,6-linkages leading to branches Cellulose • Structural polysaccharide component of plants, found in cell walls • Contains up to 3000 glucose units per molecule, with all b-1,4 linkages, leading to straight chains, no helix • Chains line up side by side to form rigid layers or fibers held together by hydrogen bonding between the many OH groups • A major component of insoluble dietary fiber, cellulose absorbs water very effectively Other constituents of dietary fiber Insoluble fiber contains lignins, which are phenylpropanoid polymers that cross-link polysaccharides (recall dimers are lignans) Other soluble and insoluble constituents are primarily carbs: • hemicelluloses (amorphous oligomers of various sugars) • pentosans – arabinoxylan, etc. are small oligomers of pentoses • b-glucans and glucofructans – oligomers of glucose & fructose • pectins – a-1,4-D-galacturonate chains • gums & mucilage – large oligomers of sugars, uronic acid - absorb water to form sticky (gums) or slippery (mucilage) mass Carbohydrate components of some fiber constituents taken from Basic Medical Biochemistry, Marks, Marks and Smith Dextrans Glucose polysaccharide characterized by a-1,6 linkages in the main chain, with side chains bound by a-1,3, a-1,4 or a-1,6 linkages Produced from sucrose by action of dextran sucrase (D-fructose-2glucosyl transferase) found in some bacteria Sucrose most often produced from sugar cane, corn or beets Used as a thickening and stabilizing agent in baked goods, candy, ice cream Maillard reaction: caramelization and browning of foods • The Maillard reaction is a reaction between any amino acid and reducing sugar found in foods, usually requiring heat. • Known as non-enzymatic browning, Maillard reaction is a key process in the preparation or presentation of many foods (plant and animal-based) • The carbonyl group of the sugar reacts with the nucleophilic amino group of the amino acid, forming a complex mixture of diverse and often not wellcharacterized molecules • These diverse structures result in a range of odors and flavors in browned foods A typical Maillard reaction occurs in three main steps: 1. Initial step: formation of the N glycoside of the amino acid (H2N-R’) 2. An iminium ion forms, then isomerizes forming a ketosamine (Amadori rearrangement) These Amadori compounds can be detected in many foods reductone formation 3. The Amadori products are oxidized to a-dicarbonyls (reductones) found in caramel, or produce short chain hydrolytic fission products such as diacetyl (shown) These may react further with amino acids and undergo the Strecker degradation (left) producing aldehydes. From Belitz, Food Chemistry and http://www.chm.bris.ac.uk/webprojects2002/rakotomalala/maillard.htm Why are cooked onions sweeter than raw? Why don’t they make you cry? • Browning onions (Allium sepa) doesn’t create more sugar – sugars are destroyed by Maillard chemistry! • Frying/sauteing onions creates bispropenyl disulfide, which has a sweet smell and taste. Maillard products provide additional color & taste. • Many of the pungent onion volatiles (thiols, thials and sulphoxides) are destroyed in cooking – for example propanethial oxide, the lachrymator responsible for making cooks cry when chopping! http://oss.mcgill.ca/yasked/onions.pdf Some health conditions related to sugar metabolism • Lactose intolerance: The enzyme required to hydrolyze the b-1,4 linkage of lactose is lactase; this enzyme is either missing at birth or production decreases over the years • Galactosemia: Lack of the enzyme required to convert galactose to glucose for use in the human body causes buildup of galactose in the bloodstream, brain malfunction • Type 2 diabetes/metabolic syndrome: results from insulin resistance – body stops using or producing insulin effectively sugars are not taken up by tissues increased blood sugar results in diabetes, increasing CVD risk (linked to sugar intake, obesity) • Periodontal disease from plaque buildup results from conversion of dietary sucrose into the polysaccharide dextran (a-glucans containing a-1,3 and a-1,6-linkages of glucose) by bacteria Carbohydrate analysis • Both qualitative and quantitative analyses are needed to provide nutritional information for foods • Nutrition Facts labels categorize carbs by – – – – Total Carbohydrate (g) and % RDA Dietary Fiber (g) and % RDA Sugars (g) – glucose, fructose, sucrose & lactose remainder includes mainly starch • RDA for a 2000-calorie-per-day diet: • 300 g carbohydrates, 25 g of which should be fiber • Total carbohydrate content of food is determined by difference: • total weight of food – sum of the weights of proteins, fats, moisture and ash Evolution of analytical methods for carbohydrates • Early methods were primarily colorimetric methods including the tests for reducing sugars employing Cu2+ chemistry (Benedict’s or Fehling’s tests) • These tests were also used to screen for diabetes by measuring sugar in the urine (Clinitest reagent) Newer methods AOAC methods include • Paper chromatography or TLC on cellulose • GC of derivatized sugars • HPLC methods Newer methods in development include • NMR / NIR • immunoassay • capillary electrophoresis • MS Sample preparation • Drying & determination of water content (vacuum oven) • Grinding • Defatting – Soxhlet extraction of lipids using 95:5 CHCl3/MeOH or hexane • Mono, di and oligosaccharides are extracted using hot 80% EtOH with CaCO3, then filtered (excludes polysaccharides and other polymeric materials) • Pigments, phenolics, organic acids & amino acids are removed from 80% EtOH extract by ion-exchange chromatography – most contaminants are adsorbed by the resin and the sugars stay in solution • Extract is rotovapped to dryness. Somogyi-Nelson Method for quantification of reducing sugars • Improvement on Benedict’s/Fehling’s method • Same principle: aldehyde group of reducing sugar reduces Cu2+ Cu+ in basic solution • Ketoses (e.g. fructose) enolize to aldoses in basic solution so they are also reducing sugars • Anomeric C must be labile (e.g. not tied up in an acetal linkage), so oligo or polysaccharides must be hydrolyzed to monosaccharides first • Reagent: arsenomolybdate complex produced from ammonium molybdate [(NH4)6Mo7O24] rxn with sodium arsenate (Na2HAsO7) • Cu+ reduces the arsenomolybdate to a stable blue product • Absorbance at 520 nm is read and samples are quantified against a standard curve of glucose HPLC analysis • Can be used for qualitative and quantitative analysis of soluble mono and oligosaccharides • Polysaccharides can also be analyzed after acid hydrolysis • Since sugars have no chromophore, UV-vis detection is not effective • Detection by refractive index (RI) is most common • Advantage: wide range of concentrations • Disadvantages: gradient elution cannot be used due to change in refraction of solvent – RI is affected by changes in flow, pressure and temperature. • For sub-microgram quantities, RI is not sensitive enough, Pulsed-amperometric electrochemical detection is used – PAD works by oxidizing the sugar functional groups at high pH – gradient separation can be used HPLC analysis Columns • Reversed-phase (C18 or phenyl-bonded) can be used for mono, di & trisaccharides but monosaccharide retention times are short and resolution may be poor due to presence of anomers – More effective for polysaccharides (e.g. maltodextrins), see Fig 10-5 • Normal-phase is more effective due to greater association of carbs with a polar stationary phase – Silica gel is usually amine-bonded – Isocratic elution with 50-85% acetonitrile-water elutes monosaccharides & sugar alcohols followed by disaccharides and larger HPLC analysis Columns, cont’d • Anion-exchange packings – Carbohydrates have pKas in 12-14 range (weakly acidic) and at high pH some OH groups may be ionized, allowing separation – Most often used with electrochemical detection – Size affects retention time; larger carbs elute more slowly • Cation-exchange packings – Dowex-type sulfonated polystyrene resins are used with water/MeOH or water/CH3CN as mobile phase – Metal counter-ion added (Ca2+ or Ag+) – Columns work more effectively at high T (> 80oC) – Larger carbohydrates elute more quickly; monosaccharides adsorb more strongly and are better resolved Enzymatic methods • Used primarily for starch determination in food • Total starch determination is by complete enzymatic hydrolysis of starch to D-glucose which is then quantified • Amylases must be purified to eliminate nonspecific sources of D-glucose (cellulose, sucrose, etc.) • The D-glucose produced is determined with a glucose oxidase/peroxidase (GOPOD) reagent • Starches that are resistant to digestion because they are physically trapped in a food matrix or chemically modified will negatively affect the results • Starch “retrogradation” or formation of polycrystalline structures by re-alignment of starch polymers is associated with “stale” breads/bakery products Analysis of gums & polysaccharides • Complicated due to variation in size, MW, structure and monosaccharide units • Solubility and ionic behavior varies • Methods approved for some but not all non-starch polysaccharides • Extraction of polysaccharides may be performed as shown in Fig. 10-11 • Characterization involves acid-catalyzed hydrolysis and analysis of the monosaccharide constituents – composition can be determined but exact structures may be unknown. • Pectin for example may vary in esterification of galacturonic acid units making up the main chain, composition of chains Analysis of dietary fiber • Although dietary fiber cannot be digested, its consumption is important to maintain good health • Colon health - reduces cancer risk, aids in regular excretion • Cardiovascular health - helps normalize blood lipid levels • Can slow the absorption of glucose, reducing need for insulin production (may reduce risk of insulin resistance) Analysis of total dietary fiber • Flow chart Figure 10-12: AOAC method for insoluble, soluble and total dietary fiber (gravimetric method) • Sample is digested with a series of enzymes (amylase, protease, glucoamylase) • Filtration & washing separates soluble (filtrate) from insoluble fiber (solids) • Soluble fiber is precipitated from filtrate with EtOH, then washed, dried & weighed • Insoluble fiber content determined by drying and weighing the solids • Weighed samples are ashed to determine residual protein (subtracted from initial weight) • Chemical methods can also be used to digest fiber and measure monosaccharide content gravimetrically. Why I eat oatmeal every morning... Oats are a great source of soluble and insoluble fiber! According to the American Cancer Society: 1. Insoluble fiber attacks certain bile acids, reducing their toxicity. 2. Soluble fiber may reduce LDL cholesterol without lowering HDL cholesterol. 3. Soluble fiber slows down the digestion of starch, which helps to avoid the sharp rises in blood sugar that usually occur following a meal. 4. It has been found that those who eat more oats are less likely to develop heart disease. 5. Some phytochemicals in oats (lignans, phenolics) may have cancer-fighting properties. 6. Oats are a good source of many nutrients including protein, vitamin E, zinc, selenium, copper, iron, manganese and magnesium.