vitamins

Introduction to Vitamins

Vitamins are organic molecules that function in a wide variety of capacities within the body. The most prominent function of the vitamins is to serve as cofactors for enzymatic reactions. The distinguishing feature of the vitamins is that they generally cannot be synthesized by mammalian cells and, therefore, must be supplied in the diet. The vitamins are of two distinct types:
Water Soluble Vitamins

Fat Soluble Vitamins
Thiamin (B1)
B1 Deficiency and Disease

Riboflavin (B2)
B2 Deficiency and Disease

Niacin (B3)
B3 Deficiency and Disease
Pantothenic Acid (B5)
Pyridoxal, Pyridoxamine, Pyridoxine (B6)
Biotin
Cobalamin (B12)
B12 Deficiency and Disease

Folic Acid
Folate Deficiency and Disease

Ascorbic Acid Vitamin A
Gene Control by Vitamin A
Role of Vitamin A in Vision
Additional Roles of Vitamin A
Clinical Significances of Vitamin A

Vitamin D
Clinical Significances of Vitamin D

Vitamin E
Clinical Significances of Vitamin E

Vitamin K
Clinical Significance of Vitamin K
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Thiamin
Thiamin structure

Thiamin is also known as vitamin B1 . Thiamin is derived from a substituted pyrimidine and a thiazole which are coupled by a methylene bridge. Thiamin is rapidly converted to its active form, thiamin pyrophosphate, TPP, in the brain and liver by a specific enzyme, thiamin diphosphotransferase.
Thiamin pyrophosphate

TPP is necessary as a cofactor for the pyruvate dehydrogenase and α-ketoglutarate dehydrogenase catalyzed reactions as well as the transketolase catalyzed reactions of the pentose phosphate pathway. A deficiency in thiamin intake leads to a severely reduced capacity of cells to generate energy as a result of its role in these reactions.

The dietary requirement for thiamin is proportional to the caloric intake of the diet and ranges from 1.0 - 1.5 mg/day for normal adults. If the carbohydrate content of the diet is excessive then an increase in thiamin intake will be required.
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Clinical Significances of Thiamin Deficiency

The earliest symptoms of thiamin deficiency include constipation, appetite suppression, nausea as well as mental depression, peripheral neuropathy and fatigue. Chronic thiamin deficiency leads to more severe neurological symptoms including ataxia, mental confusion and loss of eye coordination. Other clinical symptoms of prolonged thiamin deficiency are related to cardiovascular and musculature defects.

The severe thiamin deficiency disease known as Beriberi, is the result of a diet that is carbohydrate rich and thiamin deficient. An additional thiamin deficiency related disease is known as Wernicke-Korsakoff syndrome. This disease is most commonly found in chronic alcoholics due to their poor dietetic lifestyles.
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Riboflavin
Riboflavin structure

Riboflavin is also known as vitamin B2. Riboflavin is the precursor for the coenzymes, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). The enzymes that require FMN or FAD as cofactors are termed flavoproteins. Several flavoproteins also contain metal ions and are termed metalloflavoproteins. Both classes of enzymes are involved in a wide range of redox reactions, e.g. succinate dehydrogenase and xanthine oxidase. During the course of the enzymatic reactions involving the flavoproteins the reduced forms of FMN and FAD are formed, FMNH2 and FADH2, respectively. The hydrogens of FADH2 are on nitrogens 1 and 5 as indicated in the figure.
Structure of FAD

The normal daily requirement for riboflavin is 1.2 - 1.7 mg/day for normal adults.
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Clinical Significances of Flavin Deficiency

Riboflavin deficiencies are rare in the United States due to the presence of adequate amounts of the vitamin in eggs, milk, meat and cereals. Riboflavin deficiency is often seen in chronic alcoholics due to their poor dietetic habits.

Symptoms associated with riboflavin deficiency include, glossitis, seborrhea, angular stomatitis, cheilosis and photophobia. Riboflavin decomposes when exposed to visible light. This characteristic can lead to riboflavin deficiencies in newborns treated for hyperbilirubinemia by phototherapy.
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Niacin

Nicotinamide

Nicotinic Acid

Niacin (nicotinic acid and nicotinamide) is also known as vitamin B3. Both nicotinic acid and nicotinamide can serve as the dietary source of vitamin B3. Niacin is required for the synthesis of the active forms of vitamin B3, nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+). Both NAD+ and NADP+ function as cofactors for numerous dehydrogenases, e.g., lactate dehydrogenase and malate dehydrogenase.
Structure of NAD+
NADH is shown in the box insert. The -OH phosphorylated in NADP+ is indicated by the red arrow.

Niacin is not a true vitamin in the strictest definition since it can be derived from the amino acid tryptophan. However, the ability to utilize tryptophan for niacin synthesis is inefficient (60 mg of tryptophan are required to synthesize 1 mg of niacin). Also, synthesis of niacin from tryptophan requires vitamins B1, B2 and B6 which would be limiting in themselves on a marginal diet.

The recommended daily requirement for niacin is 13 - 19 niacin equivalents (NE) per day for a normal adult. One NE is equivalent to 1 mg of free niacin).
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Clinical Significances of Niacin and Nicotinic Acid

A diet deficient in niacin (as well as tryptophan) leads to glossitis of the tongue, dermatitis, weight loss, diarrhea, depression and dementia. The severe symptoms, depression, dermatitis and diarrhea, are associated with the condition known as pellagra. Several physiological conditions (e.g. Hartnup disease and malignant carcinoid syndrome) as well as certain drug therapies (e.g. isoniazid) can lead to niacin deficiency. In Hartnup disease tryptophan absorption is impaired and in malignant carcinoid syndrome tryptophan metabolism is altered resulting in excess serotonin synthesis. Isoniazid (the hydrazide derivative of isonicotinic acid) is the primary drug for chemotherapy of tuberculosis.

Nicotinic acid (but not nicotinamide) when administered in pharmacological doses of 2 - 4 g/day lowers plasma cholesterol levels and has been shown to be a useful therapeutic for hypercholesterolemia. The major action of nicotinic acid in this capacity is a reduction in fatty acid mobilization from adipose tissue. Although nicotinic acid therapy lowers blood cholesterol it also causes a depletion of glycogen stores and fat reserves in skeletal and cardiac muscle. Additionally, there is an elevation in blood glucose and uric acid production. For these reasons nicotinic acid therapy is not recommended for diabetics or persons who suffer from gout.
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Pantothenic Acid
Pantothenic Acid

Pantothenic acid is also known as vitamin B5. Pantothenic acid is formed from β-alanine and pantoic acid. Pantothenate is required for synthesis of coenzyme A, CoA and is a component of the acyl carrier protein (ACP) domain of fatty acid synthase. Pantothenate is, therefore, required for the metabolism of carbohydrate via the TCA cycle and all fats and proteins. At least 70 enzymes have been identified as requiring CoA or ACP derivatives for their function.

Deficiency of pantothenic acid is extremely rare due to its widespread distribution in whole grain cereals, legumes and meat. Symptoms of pantothenate deficiency are difficult to assess since they are subtle and resemble those of other B vitamin deficiencies.
Coenzyme A
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Vitamin B6

Pyridoxine

Pyridoxal

Pyridoxamine

Pyridoxal, pyridoxamine and pyridoxine are collectively known as vitamin B6. All three compounds are efficiently converted to the biologically active form of vitamin B6, pyridoxal phosphate (PLP). This conversion is catalyzed by the ATP requiring enzyme, pyridoxal kinase. Pyridoxal kinase requires zinc for full activity thus making it a metaloenzyme.
Pyridoxal Phosphate

Pyridoxal phosphate functions as a cofactor in enzymes involved in transamination reactions required for the synthesis and catabolism of the amino acids as well as in glycogenolysis as a cofactor for glycogen phosphorylase and as a co-factor for the synthesis of the inhibitory neurotransmitter γ-aminobutyric acid (GABA). The requirement for vitamin B6 in the diet is proportional to the level of protein consumption ranging from 1.4 - 2.0 mg/day for a normal adult. During pregnancy and lactation the requirement for vitamin B6 increases approximately 0.6 mg/day.

Deficiencies of vitamin B6 are rare and usually are related to an overall deficiency of all the B-complex vitamins. Isoniazid (see niacin deficiencies above) and penicillamine (used to treat rheumatoid arthritis and cystinurias) are two drugs that complex with pyridoxal and PLP resulting in a deficiency in this vitamin. Deficiencies in pyridoxal kinase result in reduced synthesis of PLP and are associated with seizure disorders related to a reduction in the synthesis of GABA.
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Biotin
Biotin

Biotin is the cofactor required of enzymes that are involved in carboxylation reactions, e.g. acetyl-CoA carboxylase and pyruvate carboxylase. Biotin is found in numerous foods and also is synthesized by intestinal bacteria and as such deficiencies of the vitamin are rare. Deficiencies are generally seen only after long antibiotic therapies which deplete the intestinal fauna or following excessive consumption of raw eggs. The latter is due to the affinity of the egg white protein, avidin, for biotin preventing intestinal absorption of the biotin.
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Cobalamin

Cobalamin is more commonly known as vitamin B12. Vitamin B12 is composed of a complex tetrapyrrol ring structure (corrin ring) and a cobalt ion in the center. Vitamin B12 is synthesized exclusively by microorganisms and is found in the liver of animals bound to protein as methycobalamin or 5'-deoxyadenosylcobalamin. The vitamin must be hydrolyzed from protein in order to be active. Hydrolysis occurs in the stomach by gastric acids or the intestines by trypsin digestion following consumption of animal meat. The vitamin is then bound by intrinsic factor, a protein secreted by parietal cells of the stomach, and carried to the ileum where it is absorbed. Following absorption the vitamin is transported to the liver in the blood bound to transcobalamin II.

There are only two clinically significant reactions in the body that require vitamin B12 as a cofactor. During the catabolism of fatty acids with an odd number of carbon atoms and the amino acids valine, isoleucine and threonine the resultant propionyl-CoA is converted to succinyl-CoA for oxidation in the TCA cycle. One of the enzymes in this pathway, methylmalonyl-CoA mutase, requires vitamin B12 as a cofactor in the conversion of methylmalonyl-CoA to succinyl-CoA. The 5'-deoxyadenosine derivative of cobalamin is required for this reaction.

The second reaction requiring vitamin B12 catalyzes the conversion of homocysteine to methionine and is catalyzed by methionine synthase. This reaction results in the transfer of the methyl group from N5-methyltetrahydrofolate to hydroxycobalamin generating tetrahydrofolate (THF) and methylcobalamin during the process of the conversion.
Vitamin B12
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Clinical Significances of B12 Deficiency

The liver can store up to six years worth of vitamin B12, hence deficiencies in this vitamin are rare. Pernicious anemia is a megaloblastic anemia resulting from vitamin B12 deficiency that develops as a result a lack of intrinsic factor in the stomach leading to malabsorption of the vitamin. The anemia results from impaired DNA synthesis due to a block in purine and thymidine biosynthesis. The block in nucleotide biosynthesis is a consequence of the effect of vitamin B12 on folate metabolism. When vitamin B12 is deficient essentially all of the folate becomes trapped as the N5-methylTHF derivative as a result of the loss of functional methionine synthase. This trapping prevents the synthesis of other THF derivatives required for the purine and thymidine nucleotide biosynthesis pathways.

Neurological complications also are associated with vitamin B12 deficiency and result from a progressive demyelination of nerve cells. The demyelination is thought to result from the increase in methylmalonyl-CoA that result from vitamin B12 deficiency. Methylmalonyl-CoA is a competitive inhibitor of malonyl-CoA in fatty acid biosynthesis as well as being able to substitute for malonyl-CoA in any fatty acid biosynthesis that may occur. Since the myelin sheath is in continual flux the methylmalonyl-CoA-induced inhibition of fatty acid synthesis results in the eventual destruction of the sheath. The incorporation methylmalonyl-CoA into fatty acid biosynthesis results in branched-chain fatty acids being produced that may severely alter the architecture of the normal membrane structure of nerve cells.

Deficiencies in B12 can also lead to elevations in the level of circulating homocysteine. Elevated levels of homocysteine are known to lead to cardiovascular dysfunction. Due to its high reactivity to proteins, homocysteine is almost always bound to proteins, thus thiolating them leading to their degradation. Homocysteine also binds to albumin and hemoglobin in the blood. The detrimental effects of homocysteine are thought to be due to its' binding to lysyl oxidase, an enzyme responsible for proper maturation of the extracellular matrix proteins collagen and elastin. Production of defective collagen and elastin has a negative impact on arteries, bone and skin and the effects on arteries are believed to be the underlying cause for cardiac dysfunction associated with elevated serum homocysteine. In individuals with homocysteine levels above ~12μM there is an increased risk of thrombosis and cardiovascular disease.

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Folic Acid
Folic Acid
positions 7 and 8 carry hydrogens in dihydrofolate (DHF) positions 5-8 carry hydrogens in tetrahydrofolate (THF)

Folic acid is a conjugated molecule consisting of a pteridine ring structure linked to para-aminobenzoic acid (PABA) that forms pteroic acid. Folic acid itself is then generated through the conjugation of glutamic acid residues to pteroic acid. Folic acid is obtained primarily from yeasts and leafy vegetables as well as animal liver. Animal cannot synthesize PABA nor attach glutamate residues to pteroic acid, thus, requiring folate intake in the diet.

When stored in the liver or ingested folic acid exists in a polyglutamate form. Intestinal mucosal cells remove some of the glutamate residues through the action of the lysosomal enzyme, conjugase. The removal of glutamate residues makes folate less negatively charged (from the polyglutamic acids) and therefore more capable of passing through the basal lamenal membrane of the epithelial cells of the intestine and into the bloodstream. Folic acid is reduced within cells (principally the liver where it is stored) to tetrahydrofolate (THF also H4folate) through the action of dihydrofolate reductase (DHFR), an NADPH-requiring enzyme.

The function of THF derivatives is to carry and transfer various forms of one carbon units during biosynthetic reactions. The one carbon units are either methyl, methylene, methenyl, formyl or formimino groups.
Active center of tetrahydrofolate (THF).
Note that the N5 position is the site of attachment of methyl groups, the N10 the site for attachment of formyl and formimino groups and that both N5 and N10 bridge the methylene and methenyl groups.

These one carbon transfer reactions are required in the biosynthesis of serine, methionine, glycine, choline and the purine nucleotides and dTMP.

The ability to acquire choline and amino acids from the diet and to salvage the purine nucleotides makes the role of N5,N10-methylene-THF in dTMP synthesis the most metabolically significant function for this vitamin. The role of vitamin B12 and N5-methyl-THF in the conversion of homocysteine to methionine also can have a significant impact on the ability of cells to regenerate needed THF.
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Clinical Significances of Folate Deficiency

Folate deficiency results in complications nearly identical to those described for vitamin B12 deficiency. The most pronounced effect of folate deficiency on cellular processes is upon DNA synthesis. This is due to an impairment in dTMP synthesis which leads to cell cycle arrest in S-phase of rapidly proliferating cells, in particular hematopoietic cells. The result is megaloblastic anemia as for vitamin B12 deficiency. The inability to synthesize DNA during erythrocyte maturation leads to abnormally large erythrocytes termed macrocytic anemia.

Folate deficiencies are rare due to the adequate presence of folate in food. Poor dietary habits as those of chronic alcoholics can lead to folate deficiency. The predominant causes of folate deficiency in non-alcoholics are impaired absorption or metabolism or an increased demand for the vitamin. The predominant condition requiring an increase in the daily intake of folate is pregnancy. This is due to an increased number of rapidly proliferating cells present in the blood. The need for folate will nearly double by the third trimester of pregnancy. Certain drugs such as anticonvulsants and oral contraceptives can impair the absorption of folate. Anticonvulsants also increase the rate of folate metabolism.
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Ascorbic Acid
Ascorbic Acid

Ascorbic acid is more commonly known as vitamin C. Ascorbic acid is derived from glucose via the uronic acid pathway. The enzyme L-gulonolactone oxidase responsible for the conversion of gulonolactone to ascorbic acid is absent in primates making ascorbic acid required in the diet.

The active form of vitamin C is ascorbate acid itself. The main function of ascorbate is as a reducing agent in a number of different reactions. Vitamin C has the potential to reduce cytochromes a and c of the respiratory chain as well as molecular oxygen. The most important reaction requiring ascorbate as a cofactor is the hydroxylation of proline residues in collagen. Vitamin C is, therefore, required for the maintenance of normal connective tissue as well as for wound healing since synthesis of connective tissue is the first event in wound tissue remodeling. Vitamin C also is necessary for bone remodeling due to the presence of collagen in the organic matrix of bones.

Several other metabolic reactions require vitamin C as a cofactor. These include the catabolism of tyrosine and the synthesis of epinephrine from tyrosine and the synthesis of the bile acids. It is also believed that vitamin C is involved in the process of steroidogenesis since the adrenal cortex contains high levels of vitamin C which are depleted upon adrenocorticotropic hormone (ACTH) stimulation of the gland.

Deficiency in vitamin C leads to the disease scurvy due to the role of the vitamin in the post-translational modification of collagens. Scurvy is characterized by easily bruised skin, muscle fatigue, soft swollen gums, decreased wound healing and hemorrhaging, osteoporosis, and anemia. Vitamin C is readily absorbed and so the primary cause of vitamin C deficiency is poor diet and/or an increased requirement. The primary physiological state leading to an increased requirement for vitamin C is severe stress (or trauma). This is due to a rapid depletion in the adrenal stores of the vitamin. The reason for the decrease in adrenal vitamin C levels is unclear but may be due either to redistribution of the vitamin to areas that need it or an overall increased utilization.
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Vitamin A

Vitamin A consists of three biologically active molecules, retinol, retinal (retinaldehyde) and retinoic acid.

All-trans-retinal

11-cis-retinal


Retinol

Retinoic Acid

Each of these compounds are derived from the plant precursor molecule, β-carotene (a member of a family of molecules known as carotenoids). Beta-carotene, which consists of two molecules of retinal linked at their aldehyde ends, is also referred to as the provitamin form of vitamin A.

Ingested β-carotene is cleaved in the lumen of the intestine by β-carotene dioxygenase to yield retinal. Retinal is reduced to retinol by retinaldehyde reductase, an NADPH requiring enzyme within the intestines. Retinol is esterified to palmitic acid and delivered to the blood via chylomicrons. The uptake of chylomicron remnants by the liver results in delivery of retinol to this organ for storage as a lipid ester within lipocytes. Transport of retinol from the liver to extrahepatic tissues occurs by binding of hydrolyzed retinol to aporetinol binding protein (RBP). the retinol-RBP complex is then transported to the cell surface within the Golgi and secreted. Within extrahepatic tissues retinol is bound to cellular retinol binding protein (CRBP). Plasma transport of retinoic acid is accomplished by binding to albumin.
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Gene Control Exerted by Retinol and Retinoic Acid

Within cells both retinol and retinoic acid bind to specific receptor proteins. Following binding, the receptor-vitamin complex interacts with specific sequences in several genes involved in growth and differentiation and affects expression of these genes. In this capacity retinol and retinoic acid are considered hormones of the steroid/thyroid hormone superfamily of proteins. Vitamin D also acts in a similar capacity. Several genes whose patterns of expression are altered by retinoic acid are involved in the earliest processes of embryogenesis including the differentiation of the three germ layers, organogenesis and limb development.
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Vision and the Role of Vitamin A

Photoreception in the eye is the function of two specialized cell types located in the retina; the rod and cone cells. Both rod and cone cells contain a photoreceptor pigment in their membranes. The photosensitive compound of most mammalian eyes is a protein called opsin to which is covalently coupled an aldehyde of vitamin A. The opsin of rod cells is called scotopsin. The photoreceptor of rod cells is specifically called rhodopsin or visual purple. This compound is a complex between scotopsin and the 11-cis-retinal (also called 11-cis-retinene) form of vitamin A. Rhodopsin is a serpentine receptor imbedded in the membrane of the rod cell. Coupling of 11-cis-retinal occurs at three of the transmembrane domains of rhodopsin. Intracellularly, rhodopsin is coupled to a specific G-protein called transducin.

When the rhodopsin is exposed to light it is bleached releasing the 11-cis-retinal from opsin. Absorption of photons by 11-cis-retinal triggers a series of conformational changes on the way to conversion all-trans-retinal. One important conformational intermediate is metarhodopsin II. The release of opsin results in a conformational change in the photoreceptor. This conformational change activates transducin, leading to an increased GTP-binding by the a-subunit of transducin. Binding of GTP releases the α-subunit from the inhibitory β- and γ-subunits. The GTP-activated α-subunit in turn activates an associated phosphodiesterase; an enzyme that hydrolyzes cyclic-GMP (cGMP) to GMP. Cyclic GMP is required to maintain the Na+ channels of the rod cell in the open conformation. The drop in cGMP concentration results in complete closure of the Na+ channels. Metarhodopsin II appears to be responsible for initiating the closure of the channels. The closing of the channels leads to hyperpolarization of the rod cell with concomitant propagation of nerve impulses to the brain.
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Additional Role of Retinol

Retinol also functions in the synthesis of certain glycoproteins and mucopolysaccharides necessary for mucous production and normal growth regulation. This is accomplished by phosphorylation of retinol to retinyl phosphate which then functions similarly to dolichol phosphate.
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Clinical Significances of Vitamin A Deficiency

Vitamin A is stored in the liver and deficiency of the vitamin occurs only after prolonged lack of dietary intake. The earliest symptoms of vitamin A deficiency are night blindness. Additional early symptoms include follicular hyperkeratinosis, increased susceptibility to infection and cancer and anemia equivalent to iron deficient anemia. Prolonged lack of vitamin A leads to deterioration of the eye tissue through progressive keratinization of the cornea, a condition known as xerophthalmia.

The increased risk of cancer in vitamin deficiency is thought to be the result of a depletion in β-carotene. Beta-carotene is a very effective antioxidant and is suspected to reduce the risk of cancers known to be initiated by the production of free radicals. Of particular interest is the potential benefit of increased β-carotene intake to reduce the risk of lung cancer in smokers. However, caution needs to be taken when increasing the intake of any of the lipid soluble vitamins. Excess accumulation of vitamin A in the liver can lead to toxicity which manifests as bone pain, hepatosplenomegaly, nausea and diarrhea.
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Vitamin D

Vitamin D is a steroid hormone that functions to regulate specific gene expression following interaction with its intracellular receptor. The biologically active form of the hormone is 1,25-dihydroxy vitamin D3 (1,25-(OH)2D3, also termed calcitriol). Calcitriol functions primarily to regulate calcium and phosphorous homeostasis.

Ergosterol

Vitamin D2


7-Dehydrocholesterol

Vitamin D3

Active calcitriol is derived from ergosterol (produced in plants) and from 7-dehydrocholesterol (produced in the skin). Ergocalciferol (vitamin D2) is formed by uv irradiation of ergosterol. In the skin 7-dehydrocholesterol is converted to cholecalciferol (vitamin D3) following uv irradiation.

Vitamin D2 and D3 are processed to D2-calcitriol and D3-calcitriol, respectively, by the same enzymatic pathways in the body. Cholecalciferol (or ergocalciferol) are absorbed from the intestine and transported to the liver bound to a specific vitamin D-binding protein. In the liver cholecalciferol is hydroxylated at the 25 position by a specific D3-25-hydroxylase generating 25-hydroxy-D3 [25-(OH)D3] which is the major circulating form of vitamin D. Conversion of 25-(OH)D3 to its biologically active form, calcitriol, occurs through the activity of a specific D3-1-hydroxylase present in the proximal convoluted tubules of the kidneys, and in bone and placenta. 25-(OH)D3 can also be hydroxylated at the 24 position by a specific D3-24-hydroxylase in the kidneys, intestine, placenta and cartilage.

25-hydroxyvitamin D3

1,25-dihydroxyvitamin D3

Calcitriol functions in concert with parathyroid hormone (PTH) and calcitonin to regulate serum calcium and phosphorous levels. PTH is released in response to low serum calcium and induces the production of calcitriol. In contrast, reduced levels of PTH stimulate synthesis of the inactive 24,25-(OH)2D3. In the intestinal epithelium, calcitriol functions as a steroid hormone in inducing the expression of calbindinD28K, a protein involved in intestinal calcium absorption. The increased absorption of calcium ions requires concomitant absorption of a negatively charged counter ion to maintain electrical neutrality. The predominant counter ion is Pi. When plasma calcium levels fall the major sites of action of calcitriol and PTH are bone where they stimulate bone resorption and the kidneys where they inhibit calcium excretion by stimulating reabsorption by the distal tubules. The role of calcitonin in calcium homeostasis is to decrease elevated serum calcium levels by inhibiting bone resorption.
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Clinical Significances of Vitamin D Deficiency

As a result of the addition of vitamin D to milk, deficiencies in this vitamin are rare in this country. The main symptom of vitamin D deficiency in children is rickets and in adults is osteomalacia. Rickets is characterized improper mineralization during the development of the bones resulting in soft bones. Osteomalacia is characterized by demineralization of previously formed bone leading to increased softness and susceptibility to fracture.
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Vitamin E
α-Tocopherol

Vitamin E is a mixture of several related compounds known as tocopherols. The α-tocopherol molecule is the most potent of the tocopherols. Vitamin E is absorbed from the intestines packaged in chylomicrons. It is delivered to the tissues via chylomicron transport and then to the liver through chylomicron remnant uptake. The liver can export vitamin E in VLDLs. Due to its lipophilic nature, vitamin E accumulates in cellular membranes, fat deposits and other circulating lipoproteins. The major site of vitamin E storage is in adipose tissue.

The major function of vitamin E is to act as a natural antioxidant by scavenging free radicals and molecular oxygen. In particular vitamin E is important for preventing peroxidation of polyunsaturated membrane fatty acids. The vitamins E and C are interrelated in their antioxidant capabilities. Active α-tocopherol can be regenerated by interaction with vitamin C following scavenge of a peroxy free radical. Alternatively, α-tocopherol can scavenge two peroxy free radicals and then be conjugated to glucuronate for excretion in the bile.
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Clinical Significances of Vitamin E Deficiency

No major disease states have been found to be associated with vitamin E deficiency due to adequate levels in the average American diet. The major symptom of vitamin E deficiency in humans is an increase in red blood cell fragility. Since vitamin E is absorbed from the intestines in chylomicrons, any fat malabsorption diseases can lead to deficiencies in vitamin E intake. Neurological disorders have been associated with vitamin E deficiencies associated with fat malabsorptive disorders. Increased intake of vitamin E is recommended in premature infants fed formulas that are low in the vitamin as well as in persons consuming a diet high in polyunsaturated fatty acids. Polyunsaturated fatty acids tend to form free radicals upon exposure to oxygen and this may lead to an increased risk of certain cancers.
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Vitamin K

The K vitamins exist naturally as K1 (phylloquinone) in green vegetables and K2 (menaquinone) produced by intestinal bacteria and K3 is synthetic menadione. When administered, vitamin K3 is alkylated to one of the vitamin K2 forms of menaquinone.
Vitamin K1


Vitamin K2
"n" can be 6, 7 or 9 isoprenoid groups
Vitamin K3

The major function of the K vitamins is in the maintenance of normal levels of the blood clotting proteins, factors II, VII, IX, X and protein C and protein S, which are synthesized in the liver as inactive precursor proteins. Conversion from inactive to active clotting factor requires a posttranslational modification of specific glutamate (E) residues. This modification is a carboxylation and the enzyme responsible requires vitamin K as a cofactor. The resultant modified E residues are γ-carboxyglutamate (gla). This process is most clearly understood for factor II, also called preprothrombin. Prothrombin is modified preprothrombin. The gla residues are effective calcium ion chelators. Upon chelation of calcium, prothrombin interacts with phospholipids in membranes and is proteolysed to thrombin through the action of activated factor X (Xa).

During the carboxylation reaction reduced hydroquinone form of vitamin K is converted to a 2,3-epoxide form. The regeneration of the hydroquinone form requires an uncharacterized reductase. This latter reaction is the site of action of the coumarin based anticoagulants such as warfarin (trade name = Coumadin®).
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Clinical Significances of Vitamin K Deficiency

Naturally occurring vitamin K is absorbed from the intestines only in the presence of bile salts and other lipids through interaction with chylomicrons. Therefore, fat malabsorptive diseases can result in vitamin K deficiency. The synthetic vitamin K3 is water soluble and absorbed irrespective of the presence of intestinal lipids and bile. Since the vitamin K2 form is synthesized by intestinal bacteria, deficiency of the vitamin in adults is rare. However, long term antibiotic treatment can lead to deficiency in adults. The intestine of newborn infants is sterile, therefore, vitamin K deficiency in infants is possible if lacking from the early diet. The primary symptom of a deficiency in infants is a hemorrhagic syndrome.

Molecular medicine

Introduction

Modern molecular medicine encompasses the utilization of many molecular biological techniques in the analysis of disease, disease genes and disease gene function. The study of disease genes and their function in an unaffected individual has been possible by the development of recombinant DNA and cloning techniques. The basis of the term recombinant DNA refers to the recombining of different segments of DNA. Cloning refers to the process of preparing multiple copies of an individual type of recombinant DNA molecule. The classical mechanisms for producing recombinant molecules involves the insertion of exogenous fragments of DNA into either bacterially derived plasmid (circular double stranded autonomously replicating DNAs found in bacteria) vectors or bacteriophage (viruses that infect bacteria) based vectors. The term vector refers to the DNA molecule used to carry or transport DNA of interest into cells.
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Common Enzymes used in Molecular Biology
Enzyme(s)

Activity

Comments
Restriction endonucleases recognize specific nucleotide sequences and cleaves the DNA within or near to the recognition sequences see below
Reverse transcriptase (RT) retrovirally encoded RNA-dependent DNA polymerase used to convert mRNA into a complimentary DNA (cDNA) copy for the purpose of cloning cDNAs
RNase H recognizes RNA-DNA duplexes and randomly cleaves the phosphodiester backbone of the RNA used primarily to cleave the mRNA strand that is annealed to the first strand of cDNA generated by reverse transcription
DNA polymerase synthesis of DNA used during most procedures where DNA synthesis is required, also used in in vitro mutagenesis
Klenow DNA polymerase proteolytic fragment of DNA polymerase that lacks
the 5'——> 3' exonuclease activity used to incorporate radioactive nucleotides into restriction enzyme generated ends of DNA, also can be used in place of DNA polymerase
DNA ligase covalently attaches a free 5' phosphate to a 3' hydroxyl used in all procedures where to molecules of DNA need to be covalently attached
Alkaline phosphatase removes phosphates from 5' ends of DNA molecules used to allow 5' ends to be subsequently radiolabeled with the γ-phosphate of ATP in the presence of polynucleotide kinase, also used to prevent self-ligation of restriction enzyme digested plasmids and lambda vectors
Polynucleotide kinase introduces γ-phosphate of ATP to 5' ends of DNA see above for alkaline phosphatase
DNase I randomly hydrolyzes the phosphodiester bonds of double-stranded DNA is used in the identification of regions of DNA that are bound by protein and thereby protected from DNase I digestion, also used to identify transcriptionally active regions of chromatin since they are more susceptible to DNase I digestion
S1 Nuclease exonuclease that recognizes single-stranded regions of DNA used to remove regions of single strandedness in DNA or RNA-DNA duplexes
Exonuclease III exonuclease that removes nucleotides from the 3' end of DNAs used to generate deletions in DNA for sequencing, or to map functional domains of DNA duplexes
Terminal transferase DNA polymerase that requires only a 3'-OH, lengthens 3' ends with any dNTP used to introduce homopolymeric (same dNTP) tails onto the 3' ends of DNA duplexes, also used to introduce radiolabeled nucleotides on the 3' ends of DNA
T3, T7, and SP6 RNA polymerases bacterial virus encoded RNA polymerase, recognize specific nucleotide sequences for initiation of transcription used to synthesize RNA in vitro
Taq and Vent DNA polymerase thermostable DNA polymerases used in PCR
Taq and Vent DNA ligases thermostable DNA ligases used in LCR

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Restriction Endonucleases

Restriction endonuclease are enzymes that will recognize, bind to and hydrolyze specific nucleic acid sequences in double-stranded DNA. The term restriction endonuclease was given to this class of bacterially derived enzymes since they were identified as being involved in restricting the growth of certain bacteriophages. Bacteria are capable of modifying specific sequences within their genomes by methylation which prevents their own DNA from being recognized by the restriction enzymes encoded by their genomes. This process is termed modification and restriction. Infecting bacteriophage DNA is not modified and, hence, will be digested by the restriction endonucleases present in the bacterium.

The key to the in vitro utilization of restriction endonucleases is their strict nucleotide sequence specificity. The different enzymes are identified by being given a name indicating the bacteria from which they were isolated, e.g. the enzymes EcoRI which recognizes the sequences, 5'–GAATTC–3', was isolated from Escherichia coli. One unique feature of restriction enzymes is that the nucleotide sequences they recognize are palindromic, i.e. they are the same sequences in the 5'——>3' direction of both strands. Some restriction endonucleases make staggered symmetrical cuts away from the center of their recognition site within the DNA duplex, some make symmetrical cuts in the middle of their recognition site while still others cleave the DNA at a distance from the recognition sequence. Enzymes that make staggered cuts leave the resultant DNA with cohesive or sticky ends. Enzymes that cleave the DNA at the center of the recognition sequence leave blunt-ended fragments of DNA.

Any two pieces of DNA containing the same sequences within their sticky ends can anneal together and be covalently ligated together in the presence of DNA ligase. Any two blunt-ended fragments of DNA can be ligated together irrespective of the sequences at the ends of the duplexes.
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DNA Sequencing

Sequencing of DNA can be accomplished by either chemical or enzymatic means. The original technique for sequencing, Maxam and Gilbert sequencing, relies on the nucleotide-specific chemical cleavage of DNA and is not routinely used any more. The enzymatic technique, Sanger sequencing, involves the use of dideoxynucleotides (2',3'-dideoxy) that terminate DNA synthesis and is, therefore, also called dideoxy chain termination sequencing.

The Sanger DNA sequencing protocol utilizes dideoxynucleotides (ddNTPs) to terminate chain elongation during the in vitro synthesis of DNA from a cloned template. Synthesis is initiated using a specific oligonucleotide primer. During the synthesis reaction a radioactive nucleotide (usually dATP) is incorporated into the elongating strands. Four separate reactions are carried out simultaneously, each of which contains all 4 dNTPs and a single ddNTP. The higher the concentration of ddNTP the more frequently chain elongation will terminate. Therefore, one can regulate the extent of sequence information obtainable by varying the dNTP/ddNTP ratio. Following the extension reactions the products are resolved by electrophoresis in a denaturing (urea) polyacrylamide gel. The results are obtained when the gel is dried and exposed to x-ray film. Bands near the bottom of the gel represent short reaction products (ie closest to the 3'-end of the primer) and those near the top the longest products.

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Background to Cloning

Any fragment of DNA can be cloned once it is introduced into a suitable vector for transforming a bacterial host. Cloning refers to the production of large quantities of identical DNA molecules and usually involves the use of a bacterial cell as a host for the DNA, although cloning can be done in eukaryotic cells as well. cDNA cloning refers to the production of a library of cloned DNAs that represent all mRNAs present in a particular cell or tissue. Genomic cloning refers to the production of a library of cloned DNAs representing the entire genome of a particular organism. From either of these types of libraries one can isolate (by a variety of screening protocols) a single cDNA or gene clone.

In order to clone either cDNAs or copies of genes a vector is required to carry the cloned DNA. Vectors used in molecular biology are of two basic classes. One class of vectors is derived from bacterial plasmids. Plasmids are circular DNAs found in bacteria that replicate autonomously from the host genome. These DNAs were first identified because they harbored genes that conferred antibiotic resistance to the bacteria. The antibiotic resistance genes found on the original plasmids are used in modern in vitro engineered plasmids to allow selection of bacteria that have taken up the plasmids containing the DNAs of interest. Plasmids are limited in that in general fragments of DNA less than 10,000 base pairs (bp) can be cloned. In practice fragments of around 5,000 bp are the limit.

The second class of vectors are derived from the bacteriophage (bacterial virus) lambda. This virus is capable of both lysogeny (integration into the host genome) and lysis (infection followed by lysis of the infected host). The genes required for lysogeny have been removed from the lambda based vectors in order to allow only the lytic life cycle to take place. The advantage to lambda-based vectors is that they can carry fragments of DNA up to 25,000 bp. In the analysis of the human genome even lambda-based vectors are limiting and a yeast artificial chromosome (YAC) vector system has been developed for the cloning of DNA fragments up to 500,000 bp (see below).
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cDNA Cloning

cDNAs are made from the mRNAs of a cell by any number of related techniques. Each technique consists of first reverse transcription of the mRNA followed by synthesis of the second strand of DNA and insertion of the double-stranded cDNA into either a plasmid or lambda vector for cloning. This process creates a library of cloned cDNA representing each mRNA species. Screening of the library for a particular cDNA clone is accomplished using nucleic acid or protein-based (proteins or antibodies) probes. cDNA libraries can also be screened by biological assay of the products produced by the cloned cDNAs. Screening with proteins, antibodies or by biological assay are mechanisms for analysis of the expression of proteins from cloned cDNAs and is given the term expression cloning. Nucleic acids probes can be generated from DNA (including synthetic oligonucleotides, oligos) or RNA. Nucleic acid probes can be radioactively labeled or labeled with modified nucleotides that are recognizable by specific antibodies and detected by colorimetric or chemiluminescent assays.

Typical process for production and cloning of cDNA. This example shows the use of a specific primer-adapter containing the sequences for the restriction enzyme NotI in addition to the poly(T) for annealing to the poly(A) tail of the RNA. It is possible to use only poly(T), or poly(T) with other restriction sites or random primers (a mixture of oligos that contain random sequences) to initiate the first strand cDNA reaction. In some cases poly(T) priming does not allow for extension of the cDNA to the 5'-end of the RNA, the use of random primers can overcome this problem since they will prime first strand synthesis all along the mRNA. This technique shows the ligation of EcoRI adapters followed by EcoRI and NotI digestion. This process allows the cDNAs to all be cloned in one direction, termed directional cloning.

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Genomic Cloning

The majority of genomic cloning utilizes lambda-based vector systems. These vector systems are capable of carrying 15-25,000 bp of DNA. Cloning slightly larger fragments of genomic DNA can be accomplished using a chimeric plasmid-lambda vector system termed a cosmid. Cosmid vectors contain only the cos (cohesive) ends of the lambda genome (required for packaging the DNA into infectious virus particles) along with a plasmid antibiotic resistance gene and origin of DNA replication. Since approximately 30,000 bp of lambda DNA have been removed from cosmid vectors, larger genomic DNA fragments can be cloned. Still larger genomic DNA fragments can be cloned into YAC vectors (see below).

Genomic DNA can be isolated from any cell or tissue for cloning. The genomic DNA is first digested with restriction enzymes to generate fragments in the size range that are optimal for the vector being utilized for cloning. Given that some genes encompass many more base pairs than can be inserted into conventional lambda or cosmid vectors, the clones that are present in a genomic library must be overlapping. In order to generate overlapping clones, the DNA is only partially digested with restriction enzymes. This means that not every restriction site, present in all the copies of a given gene in the preparation of DNA, is cleaved. The partially digested DNA is then size-selected by a variety of techniques (e.g. gel electrophoresis or gradient centrifugation) prior to cloning. Screening of genomic libraries is accomplished primarily with nucleic acid-based probes. However, they can be screened with protethat are known to bindpecific sequences of DNA (e.g. transcription factors). A typical genomic DNA cloning protocol is diagrammed below.

Diagrammatic representation of a hypothetical gene present in a preparation of genomic DNA. The boxes indicate exons and the lines separating the boxes represent introns. The bold arrows indicate the positions of restriction enzyme sites, e.g. Sau3AI. Following partial enzyme digestion a wide range of different fragments of the gene will be generated, 4 possible fragments are indicated. Fragments in the size range of 15 - 25 kilobase pairs (kbp) are purified by gel electrophoresis or gradient centrifugation and ligated into a lambda vector. The DNA is packaged into phage particles in vitro and used to infect E. coli.

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Cloning Genomic DNA in YAC Vectors

YAC vectors allow the cloning, within yeast cells, fragments of genomic DNA that approach 500,000 bp. These vectors contain several elements of typical yeast chromosomes, hence the term YAC. The YAC vectors contain a yeast centromere (CEN), yeast telomeres (TEL), telomeres are the specific sequences that are present at the ends of chromosomes and are necessary for replication) and a yeast autonomously replicating sequence (ARS). Yeast ARSes are essentially origins of replication that function in yeast cells autonomously from the replication of yeast chromosomal replication origins. YAC vectors also contain genes, (e.g. URA3, a gene involved in uracil synthesis) that allow selection of yeast cells that have taken up the vector. In order to propagate the vector in bacterial cells, prior to insertion of genomic DNA, YAC vectors contain a bacterial replication origin and a bacterial selectable marker such as the gene fro ampicillin resistance.

In the cloning of genomic DNA in a typical YAC vector, the genomic DNA is partially digested with EcoRI and fragments in the range of 400 - 500 kilobase pairs (kbp) are purified by pulsed field gel electrophoresis, PFGE. The YAC vector is digested with EcoRI and BamHI which places the telomere sequences at the ends of the linearized vector. The small BamHI fragment is separated from the rest of the YAC vector by standard gel electrophoresis. The genomic DNA is then ligated into the vector and then used to transform yeast cells.

Diagrammatic representation of a typical YAC vector used to clone genomic DNA. The vector contains yeast telomeres (TEL), a centromere (CEN), a selectable marker (URA3), and autonomously replicating sequences (ARS) as well as bacterial plasmid sequences for antibiotic selection and replication in E. coli.

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Analysis of Cloned Products

The analysis of cloned cDNAs and genes involves a number of techniques. The initial characterization usually involves mapping of the number and location of different restriction enzyme sites. This information is useful for DNA sequencing since it provides a means to digest the clone into specific fragments for sub-cloning, a process involving the cloning of fragments of a particular cloned DNA. Once the DNA is fully characterized cDNA clones can be used to produce RNA in vitro and the RNA translated in vitro to characterize the protein. Clones of cDNAs also can be used as probes to analyze the structure of a gene by Southern blotting or to analyze the size of the RNA and pattern of its expression by Northern blotting. Northern blotting is also a useful tool in the analysis of the exon-intron organization of gene clones since only fragments of a gene that contain exons will hybridize to the RNA on the blot.
Southern Blotting:

Southern blotting is the analysis of DNA structure following its attachment to a solid support. The DNA to be analyzed is first digested with a given restriction enzyme then the resultant DNA fragments are separated in an agarose gel. The gel is treated with NaOH to denature the DNA, then the NaOH is neutralized. The DNA is transferred from the gel to nitrocellulose or nylon filter paper by either capillary diffusion or under electric current. The DNA is fixed to the filter by baking or ultraviolet light treatment. The filter can then be probed for the presence of a given fragment of DNA by various radioactive or non-radioactive means.
Northern Blotting:

Northern blotting involves the analysis of RNA following its attachment to a solid support. The RNA is sized by gel electrophoresis then transferred to nitrocellulose or nylon filter paper as for Southern blotting. Probing the filter for a particular RNA is done similarly to probing of Southern blots.
Western Blotting:

Western blotting involves the analysis of proteins following attachment to a solid support. The proteins are separated by size SDS-PAGE and electrophoretically transferred to nitrocellulose or nylon filters. The filter is then probed with antibodies raised against a particular protein.
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Restriction Fragment Length Polymorphism (RFLP) Analysis

The genetic variability at a particular locus (gene) due to even minor base changes can alter the pattern of restriction enzyme digestion fragments that can be generated. Pathogenic alterations to the genotypic can be due to deletions or insertions within the gene being analyzed or even single nucleotide substitutions that can create or delete a restriction enzyme recognition site. RFLP analysis takes advantage of this and utilizes Southern blotting of restriction enzyme digested genomic DNA to detect familial patterns of the fragments of a given gene, detectable by screening the Southern blot with a probe corresponding to the gene of interest. A classic example of a disease detectable by RFLP is sickle cell anemia.

Sickle cell anemia results (at the level of the gene) from a single nucleotide change (A to T) at codon 6 within the β-globin gene. This alteration leads to a glu (G) to val (V) amino acid substitution, while at the same time abolishing a MstII restriction site. As a result a β-globin gene probe can be used to detect the different MstII restriction fragments. It should be recalled that there are two copies of each gene in all human cells, therefore, RFLP analysis detects both copies: the affected alelle and the unaffected allele.

Size variability in detectable fragments within a family pedigree indicate differences in the pattern of restriction sites within and around the gene being analyzed. RFLP patterns are inherited and segregate in Mendelian fashion thus, allowing their use in genotyping such as in cases of paternity dispute or in criminal investigations.

Another form of DNA polymorphism detectable by classical RFLP mapping results from the inherited variations in the number of tandemly repeated DNA sequence elements that are from 2 to 60 bp in length. The number of repeats is also variable from 2 to 40 copies. These elements are termed variable number tandem repeats (VNTR). When restriction enzyme digestion cuts DNA flanking the VNTRs, the lengths of the resultant fragments will be variable depending upon the number of repeats at a given locus. Many different VNTR loci have been identified and are extremely useful for DNA fingerprint analysis such as in forensic and paternity identity cases.

Diagrammatic respresentation of an RFLP analysis for the presence of the sickle-cell locus. Genomic DNA is isolated and digested with the restriction enzyme MstII. One MstII site is lost at the sickle-cell locus. The DNA is then Southern blotted and analyzed with a β-globin-specific probe corresponding to sequences at the 5'-end of the gene. Individuals homozygous for the normal globin genes will exhibit a single hybridization band since both maternal and paternal genes are unaffected. Heterozygotes will exhibit the normal band and the larger sickle-cell gene band. Homozygous sickle-cell individuals will exhibit a single larger hybridization band.

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The Polymerase Chain Reaction (PCR)

The PCR is a powerful technique used to amplify DNA millions of fold, by repeated replication of a template, in a short period of time. The process utilizes sets of specific in vitro synthesized oligonucleotides to prime DNA synthesis. The design of the primers is dependent upon the sequences of the DNA that is desired to be analyzed. The technique is carried out through many cycles (usually 20–50) of melting the template at high temperature, allowing the primers to anneal to complimentary sequences within the template and then replicating the template with DNA polymerase. The process has been automated with the use of thermostable DNA polymerases isolated from bacteria that grow in thermal vents in the ocean or hot springs. During the first round of replication a single copy of DNA is converted to two copies and so on resulting in an exponential increase in the number of copies of the sequences targeted by the primers. After just 20 cycles a single copy of DNA is amplified over 2,000,000 fold.

The polymerase chain reaction can be used to amplify both double and single stranded (eg the products of a reverse transcription reaction, RT-PCR) DNA. The template is mixed with specific or degenerate primers, dNTPs, polymerase buffer including MgCl2 and thermostable DNA polymerase. The template is denatured at high temperature (eg 95°C) and then cooled to a temperature that will allow optimal primer binding. The reaction temperature is then raised to that optimal for the DNA polymerase (eg 72°C) whereby the primers are extended along the template. This series of steps is carried out 20–30 times leading to exponential amplification of the target template. The amplification is so great that the reaction products can be visualized following gel electrophoresis.

The products of PCR reactions are analyzed by separation in agarose gels followed by ethidium bromide staining and visualization with uv transillumination. Alternatively, radioactive dNTPs can be added to the PCR in order to incorporate label into the products. In this case the products of the PCR are visualized by exposure of the gel to x-ray film. The added advantage of radiolabeling PCR products is that the levels of individual amplification products can be quantitated.

PCR can be used in the analysis of disease genes by being able to amplify detectable amounts of specific fragments of DNA. The amplified fragments from disease genes may be larger, due to insertions, or smaller, due to deletions. The dramatic amplification of DNA by PCR allows the analysis of disease genes in extremely small samples of DNA. For example, only a small number of fetal cells need be extracted from amniotic fluid in order to analyze for the presence of specific disease genes. Additionally, single point mutations can be detected by modified PCR techniques such as the ligase chain reaction (LCR) and PCR-single-strand conformational polymorphisms (PCR-SSCP) analysis. The PCR technique also can be used to identify the level of expression of genes in extremely small samples of material, e.g. tissues or cells from the body. This technique is termed reverse transcription-PCR (RT-PCR)
Examples of Inherited Disorders Detectable by PCR
Disease

Affected Gene
Severe-combined immunodeficiency, SCID adenosine deaminase (ADA)
Lesch-Nyhan syndrome hypoxanthine-guanine phosphoribosyltransferase (HGPRT)
α1-Antitrypsin deficiency α1-Antitrypsin
Cystic fibrosis cystic fibrosis transmembrane conductance (CFTR) protein
Fabry disease α-galactosidase
Gaucher disease acid β-glucosidase (glucocerebrosidase)
Sandhoff disease hexosaminidase A and B
Tay-Sachs disease hexosaminidase A

Familial hypercholesterolemia (FH) LDL receptor
Glucose-6-phosphate dehydrogenase deficiency glucose-6-phosphate dehydrogenase
Maple syrup urine disease branched-chain α-keto acid dehydrogenase
Phenylketonuria (PKU) phenylalanine hydroxylase
Ornithine transcarbamylase deficiency ornithine transcarbamylase
Retinoblastoma (Rb) RB gene product, pRB
Sickle-cell anemia point mutation in β-globin
β-Thalassemia mutations in β-globin gene that result in loss of synthesis of protein
Hemophilia A Factor VIII
Hemophilia B Factor IX
von Willebrand disease von Willebrand factor (vWF)

Reverse Transcription-PCR (RT-PCR)

RT-PCR is a rapid and quantitative procedure for the analysis of the level of expression of genes. This technique utilizes the ability of reverse transcriptase (RT) to convert RNA into single-stranded cDNA and couples it with the PCR-mediated amplification of specific types of cDNAs present in the RT reaction. The cDNAs that are produced during the RT reaction represent a window into the pattern of genes that are being expressed at the time the RNA was extracted.

Total cellular RNA can be extracted from tissues or cells by any of several techniques and used as a template for RT. In most cases the RNA is primed using random primers. A small aliquot of the RT reaction is then added to a PCR reaction containing primers specific to the sequences one wishes to amplify. The products of the RT-PCR can be then be visualized as described above for standard PCR.
PCR-Single-Strand Conformation Polymorphism (PCR-SSCP)

Many inherited disorders are due to single nucleotide changes within critical regions of the affected gene (eg sickle cell anemia). The PCR-SSCP technique can detect single mutations in genes due to the altered conformation mobility of the single strands of DNA (within an electrophoresis gel) harboring the mutation relative to the wild-type strands that do not. Specific PCR primers are made that span the sequences of a given disease gene where a mutation is known to exist and the region amplified by PCR. The same region of the wild-type gene is PCR amplified. The two strands of wild-type PCR product will migrate differently than the two strands of mutant PCR product. Even single point mutations lead to the strands of amplified DNA existing in different conformations which alter their mobility when subjected to electrophoresis in non-denaturing gels.

In order to accurately visualize the PCR products following gel electrophoresis either the primers are radioactively labeled or radioactive nucleotides are incorporated into the PCR products. The PCR products are separated in a polyacrylamide gel and visualized by exposure of the gel to x-ray film. Individuals that are homozygous wild-type at the locus being analyzed will exhibit two distinct bands in the gel as will those individuals that are homozygous mutant. However, due to the nucleotide change the mutant PCR products will migrate with different mobilities in the gel. Individuals that are heterozygous will exhibit a pattern consisting of all four bands.

PCR-SSCP analysis of normal and sickle cell β-globin genes. The A ---> T mutation is indicated in blue. The region surrounding the mutation is PCR amplified and the products separated on a non-denaturing polyacrylamide gel. The PCR products from the wild-type locus and the sickle cell locus will migrate differently due to sequence-specific conformations. Persons homozygous normal will display two bands as will homozygous sickle cell persons (although of different sizes than normal). Persons heterozygous at the sickle cell locu will display four bands.

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The Ligase Chain Reaction (LCR)

The LCR is another technique that allows detection of single point mutations in disease genes. The technique utilizes a thermostable DNA ligase to ligate together perfectly adjacent oligos. Two sets of oligos are designed to anneal to one strand of the gene at the site of the mutation, a second set of two oligos anneals to the other strand. The oligos are designed such that they will only completely anneal to the wild-type sequences. In the example shown below for the sickle-cell mutation, the 3' nucleotide of one oligo in each pair is mismatched. This mismatch prevent the annealing of the oligos directly adjacent to each other. Therefore, DNA ligase will not ligate the two oligos of each pair together. With the wild-type sequence the oligo pairs that are ligated together become targets for annealing the oligos and, therefore, result in an exponential amplification of the wild-type target. Given that prior sequence knowledge is required in order to detect point mutations in disease genes, the LCR technique is utilized for the diagnosis of the presence of a mutant allele in high risk patients.
The LCR technique used to analyze the sickle-cell locus.
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Microarray Analysis

Microarray analysis involves the use of what are commonly called "gene chips" to determine the expression of a large set of genes at the same time in a single experiment. Gene chips can be purchased from several different companies, eg Affymetrix, or they can be custom prepared in laboratories with the proper equipment. Affymetrix gene chips are created through the covalent attachment of synthetic oligonucleotides (oligos) to a small surface. In general, there are 20 or more different oligos on the chip corresponding to different regions of each gene to be analyzed. In addition, a set of oligos that each contain a nucleotide mismatch are included as negative controls for each gene. The technology of creating gene chips is such that there can be 10's of thousands of different genes represented on a single chip approximately 2cm square.
Affymetrix Gene Chips

Although there are numerous uses for gene chips, the most common experiment involves a comparison of the expression of the genes on the chip between two samples, e.g. cancer cells and normal cells. The assay is carried out by preparing RNA from each sample and converting the RNA to cDNA in the presence of fluorescent nucleotides. For example, one RNA sample is converted to cDNA with a green fluorescent nucleotide and the other RNA sample is converted to cDNA in the presence of a red fluorescent nucleotide. These "tagged" cDNA preparations are called "targets" and equal amounts of each target preparation are mixed together and then hybridized to the gene chip. After washing off the unhybridized targets and image processing of the chip one will see spots that are only green, only red, or a color in between that represents a mix of some red and some green. Thus, some spots will be yellow, some will be orange or degrees of these intermediate combination colors. Spots that are only red indicate that the gene was expressed only in the source of the red labeled targets and visa versa for green spots. Intermediate colors indicate different levels of expression of a gene in both samples. Using a computer to determine hybridization intensity one will get a complete picture of the level of expression of each gene on the chip in each RNA preparation.
Example of a Custom Spotted DNA Array Result

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Transgenesis

Transgenesis refers to the process of introducing exogenous genes into the germ line of an organism. The first successful transgenesis experiments were carried out in mice. One relatively well known experiment involved the introduction of the rat growth hormone gene into the germ line of mice. These transgenic mice grew to twice their normal size.

To create a transgenic animal the gene of interest must be passed from generation to generation, i.e. it must be inherited in the germ line. To accomplish this with mice or livestock animals, vectors containing the gene of interest with appropriate regulatory elements (e.g. the β-lactoglobulin promoter if expression of the transgene in the milk is desired) are injected into the nucleus of fertilized eggs. The eggs are then transplanted into the uterus of receptive females for development of the potential transgenic offspring. In order to test the resultant animal for germ line transmission of the transgene the chromosomal DNA of their offspring is tested for the presence of the transgene. If the transgene exhibits Mendelian inheritance then it is being transmitted in the germ line.

Currently the process of transgenesis is being utilized in both the plant and livestock industries. The aim of the majority of these experiments is to generate plants and animals that are more resistant to diseases and infections. However, some transgenic farm animal such as sheep and cows are being developed in order to obtain high levels of expression of therapeutically important proteins during milk synthesis. This allows large amounts of the protein of interest to be purified from the milk of the transgenic animals.
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Gene Therapy

Transgenesis with humans would allow for the elimination of disease genes in a population of offspring, however, technical as well as ethical issues likely will prevent any transgenic experiments to be carried out with human eggs. Therefore, the ability to replace known disease genes with normal copies in afflicted humans is the ultimate goal of gene therapy. Human gene therapy protocols aim to introduce correcting copies of disease genes into somatic cells of the affected individual. Expression of a correct copy of an affected gene in somatic cells prevents transmission through the germ line, thereby, avoiding many of the ethical issues of transgenesis. This is analogous to treatment of individuals by organ or tissue transplantation.

The most common techniques utilized in gene therapy studies is the introduction of the corrected gene into bone marrow cells, skin fibroblasts or hepatocytes. The vectors most commonly utilized are derived from retroviruses and utilize only the transcriptional promoter regions of these viruses (the LTRs) to drive expression of the gene of interest. The advantage of retroviral-based vector systems is that expression occurs in most cell types.

A number of human inherited disorders have been corrected in cultured cells and several diseases (e.g. malignant melanoma and severe combined immunodeficiency disease, SCID) are currently being treated by gene therapy techniques indicating that gene therapy is likely to be a powerful therapeutic technique against a host of diseases in coming years.
Human Disorders Treated in Cultured Cells by Gene Therapy
Disorder

Affected Gene
SCID adenosine deaminase (ADA)
SCID purine nucleoside phosphorylase (PNP)
Lesch-Nyhan syndrome hypoxanthine-guanine phosphoribosyltransferase (HGPRT)
Gaucher disease acid β-glucosidase (glucocerebrosidase)
Familial hypercholesterolemia (FH) LDL receptor
Phenylketonuria (PKU) phenylalanine hydroxylase
β-Thalassemia β-Globin
Hemophilia B Factor IX

back to the top Return to The Medical Biochemistry Page Michael W. King, Ph.D / IU School of Medicine / miking at iupui.edu
Last modified: February 12, 2009

Laplas Law Application in Physiology

LaPlace's Law The larger the vessel radius, the larger the wall tension required to withstand a given internal fluid pressure. For a given vessel radius and internal pressure, a spherical vessel will have half the wall tension of a cylindrical vessel.Why does the wall tension increase with radius?Alveoli of the Lungs The oxygen exchange in the lungs takes place across the membranes of small balloon-like structures called alveoli attached to the branches of the bronchial passages. These alveoli inflate and deflate with inhalation and exhalation. The behavior of the alveoli is largely dictated by LaPlace's law and surface tension. It takes some effort to breathe in because these tiny balloons must be inflated, but the elastic recoil of the tiny balloons assists us in the process of exhalation. If the elastic recoil of the alveoli is compromised, as in the case of emphysema, then it is difficult to exhale forcibly.
The difficulty of inspiration during the baby's first breath is great because all the balloons must be inflated from a collapsed state. Inflation of alveoli
Respiratory System





Inflating the Alveoli Inflating the alveoli in the process of respiration requires an excess pressure inside the alveoli relative to their surroundings. This is actually accomplished by making the pressure in the thoracic cavity negative with respect to atmospheric pressure. The amount of net pressure required for inflation is dictated by the surface tension and radii of the tiny balloon-like alveoli. During inhalation the radii of the alveoli increase from about 0.05 mm to 0.1 mm . The normal mucous tissue fluid surrounding the alveoli has a nominal surface tension of about 50 dynes/cm so the required net outward pressure is: The remarkable property of the surfactant which coats the alveoli is that it reduces the surface tension by a factor of about 15 so that the 1 mmHg pressure differential is sufficient to inflate the alveoli. Other factors affecting the remarkable efficiency of oxygen transport across the lung membranes is characterized in Fick's Law.
IndexLaPlace's law conceptsReferenceShier, et al.Ch 19
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Surfactant Role in Respiration One of the remarkable phenomena in the process of respiration is the role of the fluid coating the walls of the alveoli of the lungs. This fluid, called a surfactant, lowers the surface tension of the balloon-like alveoli by about a factor of 15 compared to the normal mucous tissue fluid in which they are immersed. There appears to be a nearly constant amount of this surfactant per alveolus, so that when the alveoli are deflated it is more concentrated on the surface. Since the surface-tension-lowering effect of the surfactant depends on this concentration, it diminishes the required pressure for inflation of the alveoli at their most critical phase. For a given surface tension, the pressure to inflate a smaller bubble is greater. It is the surfactant which makes possible the inflation of the alveoli with only about 1 mmHg of pressure excess over their surroundings. The baby's first breath depends upon this surfactant and is made more difficult in premature infants by the incomplete formation of the surfactant.
IndexLaPlace's law conceptsReferenceShier, et al.Ch 19
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Alveoli and Exhalation The alveoli of the lungs act much like balloons in that there is some effort involved to inflate them, but when the inflating pressure is released, the recoil of the elastic walls provides the pressure necessary to deflate them. The lungs are suspended in the thoracic cavity which is normally at a slight negative pressure. When the diaphragm is lowered, that pressure becomes more negative and the lungs expand into the cavity. Air from the atmosphere moves into the resulting partial vacuum and inflates the alveoli. One is aware of the effort, but it is not extreme as in the case of the baby's first breath . Once the alveoli are fully inflated, exhalation can be accomplished by merely relaxing the diaphragm, since the wall tension in all the tiny alveoli will act to force the air out of them. By forcing the diaphragm upward, we can exhale forcefully by adding the diaphragm effort to the recoil of the elastic alveoli. In diseases like emphysema, the elasticity of the alveoli is lost and exhalation becomes a laborious process.
IndexLaPlace's law conceptsReferenceShier, et al.Ch 19
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The Baby's First Breath Everyone knows that it is much more difficult to blow up a balloon for the first time. Why is that? For one thing, the applied pressure does not create much tension in the walls of a small balloon to start the stretching process necessary for inflation. According to LaPlace's law, the wall tension will be twice as large for a balloon of twice the radius. If it takes a certain applied pressure to overcome the elasticity of the large balloon and cause it to expand further, it will take twice as much pressure to start to expand the smaller balloon. All this makes it difficult for the baby to take its first breath -- all the balloons are small! The alveoli of the lungs are collapsed in the fetus and must be inflated in the process of inhalation. Thus the traditional spank on the bottom of the newborn to make him/her mad enough to make the effort for the first breath. Further difficulties are encountered by premature infants because the surfactant fluid which coats the alveoli to give them the appropriate wall tensions is formed in the later stages of pregnacy. Until that point, the alveoli are coated with fluid which has essentially the surface tension of water, much higher than that of the normal surfactant.




Emphysema The disease of the lungs called emphysema or chronic obstructive pulmonary disease (COPD) results in the enlargement of the alveoli of the lungs as some are destroyed and others either enlarge or combine. The disease is one of the destructive effects of long-term smoking, but sometimes occurs in non-smokers. If the normal inhalation process inflates the alveoli to a larger radius, the implications of LaPlace's law are that the wall must have lost much of its elasticity. Normally it would take twice the pressure to inflate a constant tension membrane to twice its radius. Typically, the wall tension of the healthy alveoli is determined by the surface tension of the liquid which coats them, and with a uniform coating (called a surfactant), they will all inflate to a similar radius. The enlarged alveoli in the emphysema patient imply less elastic recoil during the process of exhalation. Exhalation requires effort from the diaphragm and in advanced stages of the disease, a patient will not be able to blow out a match. Besides the loss of elasticity of the alveolar walls, the larger size of the compartments implies a smaller surface area for a given volume. Because the oxygen exchange from the air to the blood is proportional to the area of the exchange membrane, this diminishes the rate of oxygen transfer.




Tension in Arterial Walls The tension in the walls of arteries and veins in the human body is a classic example of LaPlace's law. This geometrical law applied to a tube or pipe says that for a given internal fluid pressure, the wall tension will be proportional to the radius of the vessel. The implication of this law for the large arteries, which have comparable blood pressures, is that the larger arteries must have stronger walls since an artery of twice the radius must be able to withstand twice the wall tension. Arteries are reinforced by fibrous bands to strengthen them against the risks of an aneurysm. The tiny capillaries rely on their small size.
Demonstration with balloon





Capillary Walls The walls of the capillaries of the human circulatory system are so thin as to appear transparent under a microscope, yet they withstand a pressure up to about half of the full blood pressure. LaPlace's law gives insight into how they are able to withstand such pressures: their small size implies that the wall tension for a given internal pressure is much smaller than that of the larger arteries. Given a peak blood pressure of about 120 mmHg at the left ventricle, the pressure at the beginning of the capillary system may be on the order of 50 mmHg. The large radii of the large arteries imply that for pressures in that range they must have strong walls to withstand the large resulting wall tension. The larger arteries provide much less resistance to flow than the smaller vessels according to Poiseuille's law, and thus the drop in pressure across them is only about half the total drop. The capillaries offer large resistances to flow, but don't require much strength in their walls




Danger of Aneurysms The larger arteries of the body are subject to higher wall tensions than the smaller arteries and capillaries. This wall tension follows the dictates of LaPlace's law, a geometrical relationship which shows that the wall tension is proportional to the radius for a given blood pressure. If an artery wall develops a weak spot and expands as a result, it might seem that the expansion would provide some relief, but in fact the opposite is true. In a classic "vicious cycle", the expansion subjects the weakened wall to even more tension. The weakened vessel may continue to expand in what is called an aneurysm. Unchecked, this condition will lead to rupture of the vessel, so aneurysms require prompt medical attention.A localized weak spot in an artery might gain some temporary tension relief by expanding toward a spherical shape, since a spherical membrane has half the wall tension for a given radius. Minimizing membrane tension is why soap bubbles tend to form a spherical shape. But for an expanding artery, forming a near-spherical shape cannot be depended upon to give sufficient tension relief. Demonstration with balloo

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Respiration

BIOL 238

Class Notes

The Respiratory System
Respiration involves several components:

Ventilation - the exchange of respiratory gases (O2 and CO2) between the atmosphere and the lungs. This involves gas pressures and muscle contractions.

External respiration - the exchange of gases between the lungs and the blood. This involves partial pressures of gases, diffusion, and the chemical reactions involved in transport of O2and CO2.

Internal respiration - the exchange of gases between the blood and the systemic tissues. This involves the same processes as external respiration.

Cellular respiration - the includes the metabolic pathways which utilize oxygen and produce carbon dioxide, which will not be included in this unit.
Structure of the respiratory system:

The upper respiratory division (See Figure 22.3) includes the nasal cavity [lateral view] [medial view] and pharynx. Air enters the nasal cavity through the external nares and passes through the narrow channels (meatus) created between the nasal conchae. See [coronal section of ethmoid] This exposes the air to the mucosa which warms and moistens the air as it passes through the entire system. The mucosa also removes particulates, dust, pollen, etc. and moves it toward the esophagus. In the nasal cavity the respiratory mucosa is partly ciliated and partly non ciliated and the cilia beat downward. [See olfactory epithelium] The upper portion of the nasal cavity has olfactory receptors whose nerves pass through the foramina in the cribriform plate to the olfactory bulb of the brain. The nasal cavity narrows at the internal nares before joining with the pharynx. The pharynx is divided into three portions: the nasopharynx, the oropharynx and the layngopharynx which connect to the nasal cavity, oral cavity and larynx respectively.

The nasopharynx has the pharyngeal tonsils (lymph nodes) and the opening into the eustachian canal (a.k.a. pharyngotympanic tube or internal auditory canal). This canal allows equalization of pressure between the atmosphere and the middle ear. This is important when increasing or decreasing in altitude and when atmospheric pressure changes for any reason. Inability to equalize pressure can impair hearing as occurs when the mucosa is inflamed due to a respiratory infection.

The lining continues to be ciliated into the nasopharynx and then changes to non-keratinized stratified squamous in the oropharynx and laryngopharynx where protection from ingested food is important.
The larynx (Figure 22.4) lies atop the respiratory tree and contains the voice box. Supporting the larynx is the hyoid bone which connects to the tongue above and to the thyroid cartilage below. When swallowing occurs, the hyoid bone, which does not articulate with any other bones, hinges upward and the larynx tilts backward. [See laryngeal histology] This causes the epiglottis to more effectively shield the glottis and prevent aspiration of food or liquid. The thyroid cartilage is the largest laryngeal cartilage and projects anteriorly as the "Adam's Apple". The thyroid cartilage is open posteriorly and the vocal folds (true vocal cords) run along its inside antero-posteriorly. Men tend to have deeper voices and more pronounced "Adam's Apples" due to the increased length of the thyroid cartilage and vocal fold. Tension on the vocal cords determines the pitch of the voice as air is pushed up between them. This tension in turn is controlled by cartilages attached to the back of the cords called the arytenoid cartilages. These cartilages swivel to change vocal cord tension. Vocal cord tension is controlled by arytenoid and other muscles [See laryngeal muscles] which contract or relax to increase (for high pitches) or decrease (for low pitches) tightness. Lying above the vocal folds are the vestibular folds (false vocal cords) which are narrow ridges protecting the vocal cords from aspirated materials. The glottis is the opening between the vocal cords and the passageway for air into the trachea.
The Conducting Zone - area which conducts air into the lungs. No gas transport occurs here. Also called the anatomical dead space. See [Conducting Zone Hierarchy]

The trachea, See (Figure 22.5) or windpipe, is the beginning of the respiratory tree. It has cartilages shaped like Cs (or Us) with the open portion posterior. The trachea is lined (See tracheal lining) with pseudostratified ciliated columnar epithelium (p.c.c.e.) which continues into the bronchi. The cilia of this mucosa beat upward to carry particulates up and out into the esophagus. The submucosa of the trachea contains seromucous glands to produce an abundance of mucus for the mucociliary escalator, the mechanism by which the mucus is moved up and out of the respiratory tract. The membraneous portion of the trachea on the posterior side contains the majority of the trachealis muscle and is flexible to accommodate the esophagus nestled immediately behind the trachea.
The trachea, See [Respiratory Tree], branches into the primary bronchi (See Figure 22.7) which lead to each lung, then branching to the secondary bronchi leading to the lobes (2 on the left, three on the right), then the tertiary bronchi to the 18 broncho-pulmonary segments (8 on the left, 10 on the right). Each broncho-pulmonary segment includes a branch of the pulmonary artery, pulmonary vein, and a tertiary bronchus. These segments act as structural and functional units in the lung to maintain blood flow and gas transport. The cartilages change from C-rings to plate cartilages in the bronchi while the walls get thinner. PCCE continues. See [Zones of the Respiratory Tree]

Tertiary bronchi lead to the large bronchioles. Large bronchioles have little or no cartilage and the mucosa becomes simple ciliated columnar epithelium. Large bronchioles lead to the terminal bronchioles which have simple cuboidal epithelium. Goblet cells continue through the mucosa providing continued mucus secretion.
The Respiratory Zone - this area has thin simple squamous epithelial walls (the respiratory bronchioles start out as simple cuboidal and soon become simple squamous), no mucosa, and permits gas transport into and out of the blood.

Respiratory bronchioles begin this zone, leading to alveolar ducts and then alveolar sacs. These sacs are a connected system of thin-walled chambers called alveoli. The alveoli increase the surface area for contact with blood vessels. (See Figure 22.8, modified) The structure of the lungs is composed of these spongy-appearing alveolar sacs, together with bronchial passageways and blood vessels. See the following for highly magnified views of alveoli and associated cells and tissues: [alveoli 1][alveoli 2] Look for the numerous capillaries in these closeups. They are indicated by the red blood cells inside them.
Alveolar capillaries are more numerous and densely arranged than in most any other organ. They form an area of contact with the alveolar walls of (depending on which source you read) from 50 to 400 sq. meters. (See Figure from class) Upon this area, at rest, approximately a liter and a half of blood is distributed. (This amount increases with exercise). A dense network of capillaries surrounds each alveolus to produce the respiratory membrane (See Figure 22.9) consisting of the capillary wall (endothelium), the alveolar wall (simple squamous epithelium), and the basement membranes of each. These basement membranes are composed of a basal lamina with collagen fibers and a small amount of elastic tissue. The elastic tissue gives the lungs their elasticity or recoil. The structure of the respiratory membrane permits gas transport while preventing much water from entering the alveoli from the capillaries. Since interstitial fluid in the lungs would be disastrous to respiratory function, any fluid which does enter is removed by the lymph system. All the moisture in the alveolar sacs is the result of evaporation from the mucosal lining of the respiratory passages above. In addition to the Type I alveolar cells (the simple squamous cells of the alveolar wall) there are Type II alveolar cells present which secrete surfactant. Surfactant acts to break the surface tension (cohesiveness) of water which would cause collapse of the alveoli during expiration. This happens in premature infants whose respiratory systems have not matured, a condition called Infant Respiratory Distress Syndrome (IRDS) (DEF) or Hyaline Membrane Disease . Macrophages are also present which phagocytize bacteria that make it into the alveoli.
(See Figure 22.10 and 22.12) The lungs are surrounded by a double layered pleural membrane or sack. The inner layer is called the visceral pleura and is attached to the surface of each lung. The outer layer is the parietal pleura and is attached to the lining of the thorax and the mediastinum. It adheres to the inner wall of the ribcage and to the upper surface of the diaphragm. The space between these layers, sometimes called the pleural space or cavity, is really only a potential space, normally just the layer of serous fluid secreted by the membranes. The serous fluid binds the two layers together so that if the parietal layer is expanded by movement of the ribcage or diaphragm, so is the visceral pleura and so are the lungs.
Ventilation is composed of two parts: inspiration and expiration. Each of these can be described as being either quiet, the process at rest, or forced, the process when active such as when exercising. Boyle's Law states that the volume and pressure of a gas are inversely proportional. If the volume of the gas increases, its pressure will decrease. If the volume decreases, its pressure will increase. The movement of air in ventilation occurs as a result of the pressure gradient produced when the volume of the lungs increases or decreases. The following table describes the events which produce this pressure gradient:
Quiet inspiration:

The diaphragm contracts, this causes an increase in volume of the thorax and the lungs, which causes a decrease in pressure of the thorax and lungs, which causes air to enter the lungs, moving down its pressure gradient. Air moves into the lungs to fill the partial vacuum created by the increase in volume.
Forced inspiration:

Other muscles aid in the increase in thoracic and lung volumes.

The scalenes - pull up on the first and second ribs.

The sternocleidomastoid muscles pull up on the clavicle and sternum.

The pectoralis minor pulls forward on the ribs.

The external intercostals are especially important because they spread the ribs apart, thus increasing thoracic volume. (See Figure 22.12) It's these muscles whose contraction produces the "costal breathing" during rapid respirations.
Quiet expiration:

The diaphragm relaxes. The elasticity of the muscle tissue and of the lung stroma causes recoil which returns the lungs to their volume before inspiration. The reduced volume causes the pressure in the lungs to increase thus causing air to leave the lungs due to the pressure gradient.
Forced Expiration:

The following muscles aid in reducing the volume of the thorax and lungs:

The internal intercostals - these compress the ribs together (See Figure 22.12).

The abdominus rectus and abdominal obliques: internal obliques, external obliques- these muscles push the diaphragm up by compressing the abdomen.
Respiratory output is determined by the minute volume, calculated by multiplying the respiratory rate time the tidal volume.

Minute Volume = Rate (breaths per minute) X Tidal Volume (ml/breath)

Rate of respiration at rest varies from about 12 to 15 bpm. Tidal volume averages 500 ml (See Figure 22.16 or Lung Volumes Table below)

Assuming a rate of 12 breaths per minute and a tidal volume of 500, the restful minute volume is 6000 ml. Rates can, with strenuous exercise, increase to 30 to 40 bpm and volumes can increase to around half the vital capacity.

Not all of this air ventilates the alveoli, even under maximal conditions. The conducting zone volume is about 150 ml and of each breath this amount does not extend into the respiratory zone. The Alveolar Ventilation Rate, AVR, is the volume per minute ventilating the alveoli and is calculated by multiplying the rate times the (tidal volume-less the conducting zone volume).

AVR = Rate X (Tidal Volume - 150 ml)

For a calculation using the same restful rate and volume as above this yields 4200 ml.

Since each breath sacrifices 150 ml to the conducting zone, more alveolar ventilation occurs when the volume is increased rather than the rate.
During inspiration the pressure inside the lungs (the intrapulmonary pressure) decreases to -1 to -3 mmHg compared to the atmosphere. The variation is related to the forcefulness and depth of inspiration. During expiration the intrapulmonary pressure increases to +1 to +3 mmHg compared to the atmosphere. The pressure oscillates around zero or atmospheric pressure. See (Figure 22.14)

The intrapleural pressure is always negative compared to the atmosphere. This is necessary in order to exert a pulling action on the lungs. The pressure varies from about -4 mmHg at the end of expiration, to -8 mmHg and the end of inspiration.
The tendency of the lungs to expand, called compliance or distensibility, is due to the pulling action exerted by the pleural membranes. Expansion is also facilitated by the action of surfactant in preventing the collapse of the alveoli.

The opposite tendency is called elasticity or recoil, and is the process by which the lungs return to their original or resting volume. Recoil is due to the elastic stroma of the lungs and the series elastic elements of the respiratory muscles, particularly the diaphragm.

NOTE: Click on names for comprehensive review of respiratory conditions or on (def) for expanded definition. Also see the Respiratory Pathology Images

Conditions which interfere with compliance or elasticity are called restrictive disorders. Examples are emphysema (def), which increases compliance and decreases elasticity, and fibrosis (def), which reduces both.

In emphysema the buildup of toxins from cigarette smoke and resulting mucus production leads to destruction of the alveolar and capillary walls and fibrosis of the tissue. This produces large thick-walled chambers replacing the normal small thin-walled alveoli. It results in a larger volume in the lungs but impaired gas transport and reduced ability to expire the trapped air. Many emphysema sufferers have the characteristic "barrel chest" as a result. Carbon dioxide tends to increase in alveolar air and in the blood, in some individuals interfering with normal respiratory stimuli and responses.

Pulmonary or cystic fibrosis (def) produces thickened mucus secretions which block the airways and scar tissue often develops in place of the normal elastic stroma. This is both restrictive and obstructive in its effects.

Restrictive disorders reduce the volume which can be ventilated as seen in a reduced vital capacity. (See below and Figure 22.16)

In contrast, obstructive disorders such as bronchitis (def) and asthma (def) reduce the size of the bronchial passages thus interfering with the airflow. [See COPD below]

In bronchitis an inflammation results from respiratory infection or irritation from smoke or pollution.

Asthma is like an allergic reaction in which inflammation also occurs together with a constriction of the bronchial passages.

Purely obstructive disorders do not technically reduce the volume which can be ventilated but they reduce the rate of ventilation by increasing the resistance to airflow. They will show a normal vital capacity but a reduced FEV1, (Forced Expiratory Volume) the percentage of the vital capacity which can be expelled in the first second. Normal FEV1 is 75% or greater.

Many times disorders are both obstructive and restrictive. For example:

obstructive emphysema - in addition to the enlarging and thickening of alveolar walls many of the passageways collapse producing obstructive effects as well.

COPD Chronic Obstructive Pulmonary Disease - a combination of emphysema, bronchitis and asthma, has components of each.

Other respiratory conditions:

Pneumonia

SIDS - Sudden Infant Death Syndrome
Respiratory volumes See (Figure 22.16)

Tidal Volume TV: Volume of a single breath, usually at rest.

Inspiratory Reserve Volume IRV: Volume which can be inspired beyond a restful inspiration.

Expiratory Reserve Volume ERV: Volume which can be expired beyond a restful expiration.

Residual Volume RV: Volume remaining in the lungs after a maximum expiration. This volume keeps the alveoli inflated.

Vital Capacity: The vital capacity (VC) is the maximum volume which can be ventilated in a single breath. VC= IRV+TV+ERV. VC varies with gender, age, and body build. Measuring VC gives a device for diagnosis of respiratory disorder, and a benchmark for judging the effectiveness of treatment.

Vital Capacity is reduced in restrictive disorders, but not in disorders which are purely obstructive.

The FEV1 is the % of the vital capacity which is expelled in the first second. It should be at least 75%. The FEV1 is reduced in obstructive disorders.

Both VC and the FEV1 are reduced in disorders which are both restrictive and obstructive. (See above)
Movement of the respiratory gases is due to diffusion. Diffusion results from a concentration gradient which is expressed for gases as the difference in partial pressures.

Dalton's Law of Partial Pressures states that The partial pressure of a gas in a mixture is calculated by multiplying the fraction occupied by the gas times the total pressure of the mixture.

The mixture of gases under consideration is the air and Table 22.2 shows the partial pressures of its gases at STP (standard temperature and pressure at sea level).

Oxygen is present at nearly 21% of ambient air. Multiplying .21 times 760 mmHg (standard pressure at sea level) yields a pO2 of about 160. Carbon dioxide is .04% of air and its partial pressure, pCO2, is .3.

As Figure 22.17 shows there is a partial pressure gradient between inspired air and the alveolus with alveolar air having a pO2 of 104 and a pCO2 of 40. So oxygen diffuses into the alveoli from inspired air and carbon dioxide diffuses from the alveoli into air which will be expired. This causes the levels of oxygen and carbon dioxide to be intermediate in expired air when compared to inspired air and alveolar air. Some oxygen has been lost to the alveolus, lowering its level to 120, carbon dioxide has been gained from the alveolus raising its level to 27.

Likewise a concentration gradient causes oxygen to diffuse into the blood from the alveoli and carbon dioxide to leave the blood. This produces the levels seen in oxygenated blood in the body. When this blood reaches the systemic tissues the reverse process occurs restoring levels seen in deoxygenated blood.
The effect of pO2 on hemoglobin saturation - the Oxyhemoglobin Dissociation Curve. Figure 22.20

This curve illustrates the % saturation of hemoglobin at different levels of pO2 . Because the curve is flat on top, changes in pO2 in this region do not result in significant differences in % saturation of hemoglobin. At sea level with a pO2 in oxygenated blood of 100+ (as seen earlier) the % saturation of hemoglobin is 98%. At 5000' the pO2 in oxygenated blood has decreased to about 83, but the saturation of hemoglobin has gone down only a few % to 95-97.

Deoxygenated blood is in the steeper portion of the curve. At sea level the pO2 in deoxygenated blood is 40. This results in saturation of hemoglobin of about 75%. This means that about 1/4 of the oxygen carried by hemoglobin has been released to the tissues. The remaining oxygen on hemoglobin represents a significant reservoir which can be delivered to the tissues during hypoxic stress. At 5000' the pO2 is about 33. This results in a saturation of hemoglobin of 65%. More oxygen has been unloaded (dissociated) from hemoglobin at altitude. The result of lowered pO2, whether as a result of altitude or hypoxic stress is that more oxygen is unloaded to the tissues from the oxygen reservoir.
The effect of pH on the Oxyhemoglobin Dissociation Curve. (Figure 22.21).

The effect of lowered pH steepen the curve resulting in greater dissociation or unloading of oxygen from hemoglobin. Higher pH flattens the curve resulting is reduced dissociation or unloading of oxygen from hemoglobin. This is due to a coupling of chemical reactions in which the reduction in pH which occurs as the blood picks up CO2 in the systemic tissues enhances the unloading of oxygen from hemoglobin. Likewise the increase in pH accompanying release of CO2 from the blood to alveolar air causes hemoglobin to load up with oxygen.
The Bohr Effect describes the result of increasing CO2 in causing more oxygen unloading from hemoglobin. [See Bohr Effect, Figure 22.22] It results from two circumstances: 1) the effect of lowering pH as described above, and 2) the effect of carbaminohemoglobin in stimulating oxygen unloading. The corollary to the Bohr Effect is called the Haldane Effect. See (Figure 22.23) This effect states that a reduction of oxygen on the hemoglobin molecule allows for more CO2 transport as carbaminohemoglobin.
Ventilation-Perfusion Coupling

These mechanisms optimize the perfusion and airflow in a portion of the lungs to maximize efficiency of gas transport.

When oxygen increases in the blood that is perfusing a section of lung (meaning that area is well ventilated), the vessels leading to that section dilate, and vessels leading to areas which are not as well ventilated (meaning blood in those areas will contain less oxygen) are constricted. This is the autoregulation we discussed in the circulation section

Carbon dioxide regulates the airflow. When carbon dioxide increases in the blood of a section of lung it triggers a reflex dilation of the bronchial passages serving that area.

So not only will blood flow be directed to the best ventilated areas, but airflow will be maximized as well.
Control of respiration: (See Figures 22.24, 22.25)

The respiratory center is located in the medulla of the brainstem, with contributions from the pons. Within the medulla are the DRG (dorsal respiratory group) and the VRG (ventral respiratory group). The DRG sends stimuli to the muscles of inspiration: the diaphragm, external intercostals, and other. The VRG sends stimuli to the muscles of expiration: the internal intercostals and abdominal muscles. Since these muscles act only in forced expiration the VRG is only active then, while the DRG acts in both quiet and forced respiration. These muscles are all skeletal muscles and the motor control of respiration, whether quiet or forced, is a voluntary function. The autonomic nervous system does not control the muscles of respiration. The medulla controls the rhythmicity of respiration.

The pons sends stimuli to the medulla to regulate the rate and depth. The pneumotaxic center increases the rate by shortening inspirations. The apneustic center (its function is established but its location is not) increases the depth and reduces the rate by prolonging inspirations.

Inputs to these centers come from several locations:

1) peripheral chemoreceptors - located in the aortic sinus and carotid sinus, respond to increased carbon dioxide and decreased pH (which are related as discussed earlier).

Although the peripheral chemoreceptors include receptors to oxygen, oxygen levels are secondary stimuli at best, normally not important in triggering increased respiration. For healthy people the levels of oxygen would never decrease to the point of stimulating respiration. However, this can apparently happen in certain disorders such as emphysema, in a condition known as hypoxic drive. This happens because CO2 levels become permanently elevated and the CO2 receptors cease responding. This is somewhat controversial, but some believe that administering oxygen can actually suppress respiration in someone with hypoxic drive.

2) central chemoreceptors - located in the medulla. Their primary stimuli are decreased pH and increased carbon dioxide. pH is especially important since carbon dioxide does not readily diffuse into brain tissue but affects it through its impact on hydrogen ions.

3) muscle contraction - when you exercise there is a direct stimulus to the respiratory center from active muscles and joint receptors. This causes increased respiration before blood chemistry actually changes enough to demand it.

4) higher brain centers - from the voluntary motor center for voluntary control over respiration, and from the hypothalamus for control in response to emotional stimuli and body temperature.

5) vagal afferents - the only autonomic part of respiration, these send stimuli from stretch receptors in the lungs and from irritant receptors. The stretch receptors allow a reflex which prevents overinflation called the Hering-Breuer Reflex, much as a skeletal muscle reflexively changes from one action to another. The irritant receptors act to produce coughing, hiccups, etc.

The following table summarizes the responses to various stimuli on the respiratory system:
Stimulus Response
primary: increased blood CO2, decreased pH,

secondary: decreased O2 (only in certain abnormal conditions*)

These stimuli can come from respiratory or non-respiratory causes
hyperventilation

(increase in rate and/or volume)
decreasing CO2, increasing pH,

increased O2 (only in certain conditions*)
hypoventilation
voluntary (whether intentional or not)

hyperventilation
reduced CO2 in blood, increased pH

reduced stimulus for respiration
voluntary hypoventilation increased CO2, decreased pH

increased stimulus for respiration.
* This is believed to occur only in certain conditions in emphysema in which an oxygen drive is present.

Revised: October 01, 2006

Structure and Properties of the Filtration Barrier: Overview

As described in Chapter 3 of this section, the filtration barrier of the glomerular capillaries consists of three major elements. The first of these is the endothelial cells that line the inside of the glomerular capillary. The second is the basement membrane of the capillary itself, and the third is the epithelial cells containing podocytes or foot process projections that lie on the outside of the capillary in the urinary space of Bowman’s capsule. The glomerular filtration barrier is about 1000 times more porous than other capillaries. It excludes cells and behaves as a molecular sieve restricting solute filtration based on molecular size, shape and charge.

Endothelium:

The nuclei of the endothelial cells are usually found in an area of the basement membrane that is attached to the messangium. The remainder of each cell is distributed around the inner wall of the glomerular capillary. The endothelial cell cytoplasm becomes quite thin and contains 70 nm pores called fenestrae. Thin single membranes, possibly of a protein-polysaccharide film, cover these fenestrae. These are highly permeable, and do not pose a significant barrier to the movement of even large molecules but do exclude passage of cellular elements of the blood

Basement Membrane: The basement membrane of the glomerular capillary consists of three layers, but these layers do not contain pores. In the middle of the basement membrane is a dense inner layer called the lamina densa. The lamina densa separates two thinner layers, the lamina rara interna, nearest the capillary lumen, and the lamina rara externa nearest the urinary space. The lamina densa is made of type IV collagen which selectively filters molecules between the fibers based on size. The lamina rara layers contain heparin sulfate, a polyanionic molecule that may act as a charge barrier to large negatively charged molecules such as protein Epithelium; Cell Types: Two types of epithelial cells are found within the urinary space of Bowman’s capsule. The first of these are the parietal epithelial cells that line the inside of Bowman’s capsule. These cells are not part of the filtration barrier. The second type of epithelial cells are the visceral epithelial cells or podocytes, which rest on the basement membrane of the glomerular capillary and which are the largest of the cells in the glomerulus. Extending from the main cell body of the podocytes are primary processes from which pedicels or foot processes extend and actually contact the lamina rara externa of the basement membrane. Additional pedicels also arise from secondary and tertiary processes. Glomerular Filtration: Composition of Glomerular Filtrate The glomerular filtration barrier allows fluid to be filtered at a high rate while remaining nearly impermeable to cells and larger molecules. Up to molecular weights of about 7000 daltons molecules are freely filtered across the barrier. As molecular weight increases above 7000 daltons filterability decreases progressively and essentially ceases at molecular weights of about 70000. Plasma albumin, with a molecular weight of 66000 daltons is very poorly filtered and appears in the filtrate at about 0.02% of its concentration in plasma. The resultant glomerular filtrate is, as expected, very close to plasma in composition of small solutes, while being nearly devoid of protein. Because the contribution of protein to total plasma osmolarity is quite small and that of the filtrate even less the filtrate is essentially isosmotic with the plasma from which it is derived. In animals with easily accessible surface glomeruli, the micropuncture technique has allowed the direct sampling of the glomerular filtrate. These studies have shown that small molecules such as inulin ( molecular weight 5500) are filtered freely while larger molecules such as albumin are cleared at a rate thGlomerular Filtration Rate: The glomerular filtration rate (GFR) is defined as the volume of plasma filtered by all the glomeruli in a given period of time. In the discussion of renal function the GFR is often referred to as the "First Factor." This is because the GFR determines the volume of fluid, both water and solutes, available to the nephron to act on in performing its major function of regulation of water and electrolyte balance. In the normal adult male, the GFR is equal to about 125 ml/minute. In the normal adult female the GFR is 10% less. At this GFR, about 180 L of fluid are filtered in 24 hours. Urine output, however, is only about 1 to 2 L per day. From these values it may be assumed that about 99% of the glomerular filtrate is reabsorbed by the renal tubules. at is less than 1% of inulin. Forces Driving the Glomerular Filtration Rate The formula on the right characterizes the GFR in terms of the forces that contribute to filtration across the glomerular capillary wall. These forces are essentially the same as those affecting movement across the other capillaries in the body and are as follows: KF = filtration coefficient which is a product of the glomerular capillary permeability and the glomerular capillary surface area; PGC = mean capillary hydraulic pressure; PT = mean tubule hydraulic pressure; PGC = oncotic (protein osmotic) pressure in the plasma in the glomerular capillaries; and P T = oncotic pressure of the glomerular filtrate in the renal tubules. Because filtrate protein concentration is very low P T is low and plays a minimal role in determining the force driving filtration. Measurement of Glomerular Filtration Rate