Transcript Carbohydrates

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.