Carbohydrates

Although a major part of histotechnology is based on the staining of proteins in one way or another, proteins are not the only biological material with which we are concerned. Both carbohydrates and lipids are significant in both healthy and disease states, and it is important that their demonstration be reliable. Before that is possible an understanding of the nature of the materials is necessary. This page discusses the structure of carbohydrates as they relate to human histology. It is not an exhaustive explanation but will provide the background for demonstrating these materials.

Carbohydrates are carbon compounds with hydrogen and oxygen in a 2:1 ratio, from which the name is derived, i.e. watery carbon or hydrated carbon. The 2:1 ratio of hydrogen to oxygen is, of course, H2O, the formula for water. The term carbohydrate is also used to refer to compounds derived from these by oxidation or reduction, by the addition of amino, carboxyl and phosphate groups, or by their being linked with proteins and lipids.

Monosaccharides
The basic unit for carbohydrates is a sugar. There are numerous examples of sugars, which are more properly called monosaccharides and have the general formula of Cn(H2O)n. The prefix mono means "one" or "single", while the word saccharide means it is a sugar, i.e. it is a compound composed of a single sugar. The ending ide in this context means "in the class of", so a monosaccharide is a compound composed of a single molecule of sugar and classed as a carbohydrate.

In addition to "mono", other prefixes which denote numbers may be encountered. The prefix di means two, so a disaccharide is a carbohydrate composed of two sugar molecules, and tri denotes carbohydrates with three sugar molecules i.e. a trisaccharide. The word Oligosaccharide means "a few sugars" and refers to carbohydrates with several sugar molecules. Polysaccharide means "many sugars" and refers to carbohydrates composed of large numbers of sugar molecules, such as glycogen, cellulose or starch.

The names of carbohydrates usually end with ose, e.g. sucrose, which is more commonly known as table sugar or cane sugar, and is a disaccharide composed of one molecule of glucose and one molecule of fructose. Due to the fact that they contain hydroxyl groups, sugars are sometimes referred to as alcohols or ols.

If sugars contain an aldehyde (-CHO) they may be referred to as aldoses or aldehyde sugars. If they contain a ketone (-C=O) they may be referred to as ketoses, keto or ketone sugars. Of the two formulas shown, the leftmost is a general formula for an aldose and the second is for a ketose.

LengthName
3 carbon chaintriose
4 carbon chaintetrose
5 carbon chainpentose
6 carbon chainhexose
7 carbon chainseptulose

Monosaccharides may also be grouped according to the number of carbon atoms linked together in a chain making up their molecule. The pentoses are an important group as ribose and deoxyribose, both pentoses, are incorporated into RNA and DNA respectively. Similarly, hexoses are important as the group which contains glucose, galactose and fructose, all sugars important to human metabolism. Glucose and galactose are aldoses, while fructose is a ketose.


Glucose

Galactose

Fructose

The carbon atoms in sugars are often numbered so that it may be specified where in the molecule a reaction has occured. Carbon number 1 is the topmost carbon in each case in the formulas above, i.e. the CHO for glucose and galactose, and the CH2OH for fructose. The other carbon atoms are then sequentially numbered downwards. The way the formulas have been illustrated above is called the Fischer projection.

A comparison of the formulas for glucose and galactose shows the difference between them to be that one of the carbons (number 4) has "HCOH" in glucose and "HOCH" in galactose. However, this difference could be at each of the four carbons (numbers 2 to 5) giving 16 possible monosaccharides, known as stereoisomers.


Allose

Altrose

Glucose

Mannose

Gulose

Idose

Galactose

Talose

Of these, eight will be mirror images of the other eight and are known as enantiomers of each other. They are grouped according to carbon 5. Eight of them will have "HCOH" there, and the remaining eight will have "HOCH". Those with "HCOH" are the D form of each monosaccharide, and those with "HOCH" are the L form. Note that the "D" and "L" are in capital or uppercase letters. The eight formulas immediately above are all of the D-enantiomers, and "D-" could be put as a prefix to the names given, i.e. D-glucose, D-galactose, etc. The two formulas below show the difference between the "D" and "L" enantiomers, using glucose as an example. D-glucose is probably the most widely distributed hexose in biological systems, certainly it is the one we are most concerned with in anatomic pathology.


D-glucose

L-glucose

Designations as the "D" or "L" enantiomer is based on a similarity to the positions of the groups relative to each other in the structure of glyceraldehyde. In that compound the "D" and "L" refer to the optical rotation of light in a solution, but this is not carried over to the monosaccharides and the lowercase d and l have been used to designate the optical refraction of the specific molecules. These optically active compounds may exist as mixtures of both the "D" and "L" forms, in which case they are designated "DL". If present in equal amounts they become optically inactive as the right and left rotations of light cancel each other out, in which case they are called racemic mixtures.

If light is passed through a solution of a monosaccharide, it will be rotated to either the right or the left. If to the right it is designated with the plus sign + or the lowercase d. If to the left, the minus sign or lowercase l is used. The lowercase "d" is short for "dextro", meaning "right" and the "l" is short for "levulo" or "laevulo", meaning "left". The prefix "dextro" gave rise to the term "dextrose" for the most commonly encountered form of glucose, dextro-rotatory D glucose, illustrated just above. Similarly, the prefix "levulo" gave rise to the name "levulose" for fructose, or fruit sugar. Although sometimes seen, both "dextrose" and "levulose" as identifying names for glucose and fructose are deprecated and should be avoided in scientific use.

Ring forms
Besides the Fischer projection to illustrate the structure of carbohydrates, illustrations using rings are frequently seen. Known as the Haworth projection, they are commonly encountered. There are two types of ring formula, those based on pyranose, which has a six member ring (5 carbons and an oxygen), and those based on furanose, which has a 5 member ring (4 carbons and an oxygen).


D-glucose

D-glucopyranose

D-glucofuranose

As can be seen, a pyranose ring is formed from the hydroxyl of carbon 5 reacting with the aldehyde of carbon 1 forming a hemiacetal. In the case of the furanose, the reaction is between the aldehyde of carbon 1 and the hydroxyl of carbon 4. The hydroxyl group on carbon 1 of glucopyranose, called the glycosidic hydroxyl, is reactive and can form compounds with other monosaccharides and alcohols. These are called disaccharides if with another monosaccharide or a glycoside if with a non-carbohydrate ol, methanol for instance.


α-D-glucopyranose

β-D-glucopyranose

Two different pyranose molecules can be formed from glucose, depending on whether the hydroxyl next to the oxygen is on the opposite side of the molecule as the CH2OH, in which case it is known as α-D-glucopyranose (the formula on the left), or on the same side, when it is known as β-D-glucopyranose (the formula on the right).

Similar compounds for keto sugars are shown below, using fructose as an example.


α-D-fructopyranose

α-D-fructofuranose

There is an alternate way of drawing monosaccharide rings, shown in the diagram on the left, below. This is sometimes called the "chair" structure, and it is designed to show the relationships between bond angles more clearly. It is of the same α-D-glucopyranose shown in the diagram next to it. The third diagram, on the right, gives the usual numbering sequence for monosaccharide rings.


α-D-glucopyranose chair

α-D-glucopyranose ring

Numbering

Keep in mind that when the term "glucose" is used in anatomic pathology it usually refers to α-D-glucopyranose, as that is the most common form encountered.

Disaccharides
Monosaccharide units can combine with each other to form disaccharides, "di" meaning "two". Probably the most familiar will be sucrose, or cane sugar, and lactose, or milk sugar. Sucrose is illustrated as a product of α-D-glucose and β-D-fructose, and lactose is illustrated as a product of β-D-galactose and β-D-glucose.


Sucrose

Lactose

Polysaccharides
In human tissue the polysaccharide encountered is glycogen. In vegetable tissues it is starch and cellulose. Some animals and fungi contain chitin. All are different kinds of polysaccharides. Glycogen, starch and cellulose are polymers of glucose, while chitin is a polymer of N-acetylglucosamine, a modified glucose.

The polysaccharide that is most important in anatomic pathology is glycogen, a polysaccharide very similar to starch. It is made up of many glucose units, perhaps a quarter to half a million or more. These are joined in long chains by 1-4 glycosidic links, i.e. they are linked together at carbon atoms 1 and 4 by the glycosidic hydroxyl. Every ten or so glucose units there is a 1-6 glycosidic link which causes the glycogen chain to branch.


Glycogen

One of the the major polysaccharides found in plants is starch, which includes two slightly different compounds. One is amylose, a non-branching polysaccharide composed of large numbers of 1-4 linked glucose units. The other is amylopectin, a branching polysaccharide similar in structure to glycogen but with fewer 1-6 linked branches and two to three times as many glucose units between branches. Amylopectin is sometimes used as a section adhesive.

Cellulose is another glucose polysaccharide found in plants. This is a very widely distributed material as it forms a major part of the structure of plants. It is also a non-branching polymer of glucose units linked via 1-4 bonds, but in this case they are β-glycosidic bonds rather than the α-glycosidic bonds of glycogen and starch (see the diagrams above of α-D-glucopyranose and β-D-glucopyranose). Cellulose polymers contain several hundred to several thousand glucose units with hydrogen bonds between units which stabilise the structure and make it more rigid.

Chitin is a polysaccharide found in fungi, the shells of crustaceans and some other animals. It is a polymer composed of N-acetylglucosamine rather than glucose. The N-acetylglucosamine units are linked via 1-4 β-glycosidic bonds similar to cellulose.

All of these polysaccharides can be encountered in anatomic pathology, either as a natural component of the tissue, as a consequence of disease (chitin in fungal cell walls), contamination from latex gloves (starch granules) or by chance (wood splinters, food residue). Fortunately, they all look different microscopically so even though all of them may be PAS positive, their visual appearance makes them easy to identify.

The presence of N-acetylglucosamine in chitin highlights the fact that groups other than another monosaccharides can attach to a monosaccharide. A commonly encountered material composed of a glucose molecule with an amino group attached is glucosamine, a dietary supplement often taken by those with arthritis and a common component of the mucins, glycolipids and glycoproteins. If the amino group itself has an attached carboxyl group (N-acetyl-) the compound formed is N-acetylglucosamine. As noted, the polymer of this compound is the polysaccharide chitin.


Glucosamine

N-acetylglucosamine

A common group of compounds are the sialo-mucins, widely distributed as components of epithelial mucins. Sialomucins contain sialic acids (neuraminic acids), which are nine carbon amino sugars containing carboxyl groups. Two of these, N-acetyl- and N-glycoloyl- neuraminic acids, are illustrated below and the numbering of the nine carbons is shown on the left.


Numbering

N-acetyl-neuraminic acid

N-glycoloyl-neuraminic acid

Carboxyl groups are not the only acid groups which can form compounds with monosaccharides. Compounds with phosphate groups are quite common. Glucose-1-phosphate and glucose-6-phosphate are examples. The difference between these two compounds is which carbon has the phosphate group attached. Both are important in cellular metabolism, energy storage, etc.

It is also not a case of either one group or another group attached to a monosaccharide. It can easily be a case of one group and another. A case in point is N-acetylglucosamine-6-phosphate, i.e. glucose with an amino and carboxyl group attached at carbon 2, and a phosphate group at carbon 6. Compare the formula for N-acetylglucosamine and the formula for glucose-6-phosphate below.


Glucose-1-phosphate

Glucose-6-phosphate

N-acetylglucosamine-6-phosphate

In addition to adding carboxyl groups to the molecule, both the terminal aldehyde of aldose sugars and the terminal hydroxyl can be oxidised to a carboxyl group. This gives three possibilities. If the terminal aldehyde alone is changed it forms an aldonic acid. If the terminal hydroxyl is changed it forms a uronic acid. If both are changed it is called an aric or saccharic acid. These are illustrated below with glucose.


Gluconic acid

Glucuronic acid

Glucaric acid

Glucuronic acid is a common constituent of mucins. So is N-acetylglucosamine. Together they form the disaccharide hyaluranon, also known as hyaluronic acid with the two monosaccharides being linked by bonds alternating between the 1-3 and 1-4 positions. The polymer of this compound, in conjunction with protein, forms the basis of hyaluronic acid containing acid mucopolysaccharides. Another class of acid mucopolysaccharides are the sialomucins, already mentioned.


Hyaluranon

As well as carboxylated acid mucopolysaccharides there are sulphated mucopolysaccharides. These are formed from monosaccharides containing sulphate groups. Chondroitin sulphates are examples, and contain sulphated galactosamines in conjunction with glucuronic acid. Chondroitin-4-sulphate and chondroitin-6-sulphate are examples differing in which carbon has the sulphate group. Chondroitin-4-sulphate is called chondroitin sulphate type A and chondroitin-6-sulphate is type C. Chondoitin sulphate is found in cartilage, and is used as a dietary supplement for people with arthritis. Dermatan sulphate is similar but contains iduronic acid instead of glucuronic acid, and is found in the skin. Heparin is also a related compound which contains iduronic acid and is more strongly sulphated. It is an anticoagulant found in mast cells and often used for their demonstration. Like chondroitin sulphates and dermatan sulphate, heparin is strongly metachromatic with the azures and related dyes.


Chondroitin-4-sulphate

Chondroitin-6-sulphate

Dermatan sulphate

Heparin

Pentose sugars
So far the discussion has focused on hexoses, particularly glucose and how it is modified for different purposes. There is another group of compounds which are very important but are based on the pentose sugars, ribose and deoxyribose. Deoxyribose has one less hydroxyl group than ribose, with hydrogen replacing it. This is shown in the diagrams below. Ribose and deoxyribose are both shown as they are usually represented and next to that the same molecules is shown with the hydrogen atoms explicitly included to emphasise the oxygen which has been removed in deoxyribose at carbon 2. The numbering scheme is shown to the left.


Ribose

Ribose-explicit
 
Deoxyribose

Deoxyribose-explicit

Both ribose and deoxyribose are important as they are the basis of RNA (ribosenucleic acid) and DNA (deoxyribosenucleic acid) respectively. However, that is not their only function. They are also important in energy transfer and cellular respiration.



 

Reference
Almost any modern organic chemistry reference text would contain much the same information.

Diem, K. and Lentner, C., (1970)
Scientific tables
Ciba-Giegy, Basle, Switzerland.

Rensselaer Polytechnic
Carbohydrates and polysaccharides

Scientific Psychic
Carbohydrates

Wikipedia articles
Glyceraldehyde Hexose Glucose Pyranose Sucrose
Lactose Glycosidic bond Glycogen Starch Amylose
Amylopectin Cellulose Chitin Sialic acid Gluconic acid
Glucuronic acid Hyaluranon Chondroitin sulphate Dermatan sulphate Heparin
Ribose Deoxyribose

 


 

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