20 Lipid Soluble Vitamins

Prof. M. N. Gupta

Objectives

• To understand the role of Vit A in vision
•  To learn about the role of vit D in calcium absorption
•  To learn about how vit K is involved in fibrin clot formation.
•  To learn about status of our information about vit E‟s role in nutrition

Concept Map

3. Description

A strong component of early biochemistry was related to nutrition. Very early, it was realised that human beings (and birds/animals) require some substances other than carbohydrate, fat and protein.

Various diseases led to the discovery of these factors. Broadly, these vitamins were identified as either fat soluble or water soluble. This module will discuss fat soluble vitamins.

It was in 1906 that F. G. Hopkins suggested that animals require some “minimal qualitative factors” similar to the “dielectric” factors which if deficient in the diet can lead to diseases like scurvy and rickets.

Few years later, the involvement of these accessory factors in the utilization of proteins, fats and carbohydrates was established. These small molecular weight compounds were called vitamins by Funk in 1912 (vita: like). Originally spelled as vitamins as Funk believed that anti- beri beri factor in rice polishing was an amine.

Vitamins were divided into four classes: the water soluble (B, C etc) and fat soluble (A,D,E and K). D is strictly a provitamin, as we will see later in this module.

The understanding of the role of water soluble vitamins was gained early. For quite sometime the role of fat soluble vitamins puzzled scientists as unlike water soluble vitamins (especially of B complex), a correlation between their deficiency and a disease was not there. At one time essential fatty acids were called “vitamin F” but that term was soon abandoned.

In 1922, Mc collum‟s group showed that a fat soluble material in butter and cod liver oil contained two vitamins, vitamin A with antixerophthalmic activity and vitamin D with anti racketic activity, The carotenes (α-,β-,γ-) and cryptoxanthins found in higher plants and myxoxanthins in blue green algae were called provitamines. These provitamins when fed to animals were found to get converted into vit A activity.

Xerophthalmia, the syndrome of vitamin A deficiency is typical of rats. However, the role of vit A in visual processes was soon established.

It was also established early that excess of vit A can be toxic to animals if taken in long dosages for a long time. This leads to a serious condition called hypervitaminosis. In fact, this is true of all fat soluble vitamins.

This is because while excess water soluble vitamins can be excreted easily, this is not so with fat soluble vitamins. Milk and food (e.g. breakfast cereals etc) are fortified with A and D but dosage is carefully chosen so that it does not exceed for a person consuming meals which may also contain these vitamins.

Many of the current books refers to vitamin A as vitamin A1 which leaves one wondering whether there is vitamin A2! When people just say vitamin A, it is implied they are talking of vitamin A1.

Feeding of animals β-carotene or A1 produces vitamin A2. Vitamin A2, dehydroform of vitamin A1, arises from vitamin A1. A2 is found in some animal tissues and in large amounts in fresh water fishes. Some of it occurs as ester in the livers.

Neo-vitamin A (13-cis) also occur as fish liver oils. In some crustacea 11-cis isomer (neo-b) is also present abundantly. Of all these forms, vit A, has highest potency.

Carr-Price Reaction: Vitamin A forms blue colour with SbCl3 in chloroform and was used for estimation of vitamin A in early days. Later as spectrophotometers became more freely available, the λmax absorption by vitamin A around 325-328 nm was used to estimate vitamin A.

The extinction coefficient E1%1cm x conversion factor gives the unit of vitamin A per gram of oil. The conversion factor converts the absorption based number to original rat assay. Many workers reported different results from rat assay and hence conversion factors ranging from 1600-2200 have been used. Pure vitamin A (alcohol) has the value of 1750 for this conversion factor, called E-value.

While involvement of vit A in vision is more well known, its deficiency also affects other organs. We referred to epithelial changes in the organs. Actually vit A deficiency also affects growth of bones and teeth. Dry skin and development of skin lesions are known to be associated with vit A deficiency.

Urilithiasis (formation of urinary calculi) also is known to be caused due to deficiency of this vitamin. A range of 40-300 IU per 100ml of plasma has been reported as a safe range. One IU is about 0-3 μg of vit A.

Hypervitaminosis

On the other hand hypervitaminosis often escapes detection. Headache, nausea, vomiting, drowsiness dermatitis, tenderness of bones, alopecia are all known to be associated with hypervitaminosis.

Fish liver oils (oils of black sea bass, swordfish, lingcord and male soupfin shark are among those rich in vit A, cod has reasonable amount) The international standard is vit A acetate of which 0.344 µg corresponds to 1 IU of the vitamin A. This is equal to 0.3 μg of vit A and 0.6 μg of β-carotene. An average person (70 kg weight) requires 5000 IU/day.

Eggs, milk, cheese, green leafy vegetable, corn and carrot are good sources of vitamin A. Synthetic vit A preparation such as vit A palmitate do not have fishy taste and hence are preferred by some people.

The conditions for carrying out rat assay (the biological assay) are given in U S pharmacopoeia (USP). USP reference cod liver oil is given to vit A depleted rats. Another set of such rates are administered the sample. The rate of growth of the two sets is compared.

It is known that animals fail to grow if vitamin A is not given. The changes in the epithelium of many organs of the animal are noticed under vitamin A deficiency. Such organs include salivary glands, the respiratory tract, the genitourinary tract.

Eyes are, of course, the primary organ which is affected. Early deficiency symptoms are enlargement of the eyelids and inflammation of the conjunctiva. Corneal changes finally lead to blindness.

The relationship between vitamin A deficiency and Xerophthalmia was established during world war I. Denmark exported much of its butter and its own children were deprived of it. Xerophthalmia leading to blindness became common. Bloch showed that consumption of cod liver oil (or butter!) cured the symptoms.

Night blindness is the inability to adapt to dim illumination. This is also due to vit a deficiency. If the symptom is detected early before pathological changes occur, night blindness can be corrected by vit A administration. In 1938, G. Wald established the relationship between dark adaption and vit A status.

Retinoic acid is the derivative of vit A which regulates the gene expression in development of epithelial tissues, the drug fretinoin (Retin-A) contains retinoic acid as the active ingredient and is used for treating cases of serves acne and skin conditions.

The visual pigment present in retinal cells of the eye is Retinal or more properly called 11-cis-Retinal. The retinal cells in vertebrates are of two kinds and are distinguished by the nature of the photoreceptor present in them.

Cones are responsible for our vision in bright light and capacity to distinguish colour. Rods help us in seeing things in dim light. The names rods and cones are based upon the shapes of the cells.

A human retina has nearly 3 million cones and a lot more i.e. 100 million rod cells. Light falls onto these photoreceptor cells and that is the primary signal in the visual process. The rod cells contain 11-cis retinal bound to a protein called opsin, the complex is called rhodopsin

The 11-cis retinal is responsible for U.V. spectra of rhodopsin with a λmax of around 500nm. It has exceptionally high molar extinction coefficient of 40,000 cm-1M-1.

Retinal is linked to opsin by a shiff base linkage through an t-NH2 of lysine, the shift base is in the protonated form

                                     R-CH=HN+-(CH2)4-opsin

Figure 5: Structure of 11-cis-retinal, all-trans-retinal and all-trans-retinol

β-Carotene is cleaved to form retinol (vit A1). The visual pigment 11-cis-retinol is its oxidation product. First in the vit A1, all-trans- retinol is oxidised by retinol dehydrogenase which converts –OH group to – CHO group. Next, retinal isomerase converts the specific double bond between C-11 and C-12 to cis configuration.

Rod cells and cone cells of the retina form synapses with bipolar cells which are in turn connected to other nerve cells of the retina. The light signal is transduced to the electrical signal which is transmitted by the fibres of the optic nerve to the brain.

It is the outer segment of the rod cells which has a stack of about 1000 discs of 160 Aº thickness. It is here that rhodopsin is packed. The inner segment, rich in mitochondria and ribosomes is equipped with the capacity of producing ATP rapidly and synthesising protein actively. The discs are renewable with life of one month.

As early as 1938, Selig Hecht showed that a single human rod cell can be excited by even a single photon. That makes rhodopsins exceptionally sensitive photoreceptors.

It was George Wald who in 1958 showed that the primary event as a result of photoexcitation is the conversion of 11-cis-retinal in rhodopsin to all-trans-retinal. This isomerisation results in Schiff base linkage of retinal molecule (with opsin) by about 5 Aº.

The isomerisation of 11-cis-retinal to all-trans-retinal is an extremely fast process taking place within a few picoseconds of photon absorption by rhodopsin. The strained all-trans retinal containing pigment is now called bathorhodopsin.

This, in fact, triggers a series of conformational changes in the visual pigment. Each stage has a different λmax (of absorption spectra) and distinct life times. In metarhodopsin II, the Schiff base is deprotonated and this leads to dissociation of the chromophore all-trans- retinal and opsin.

The trans-retinal does not fit in well with opsin (unlike 11-cis-retinal). However, once photoexcitation ceases, 11-trans-isomer reverts back to 11-cis-retinal which again can bind opsin to regenerate rhodopsin which is then ready for the next photoexciting event.

The plasma membranes of the rod cells has open cation specific channels in the dark. These channels are blocked by light. This does not allow Na+ influx leading to a photoinduced hyperpolarization as the membrane is more +ve from inside.

In the open channel form, the Na+ influx is possible because a Na+-K+ ATPase pump maintains the large electrochemical gradient. The signal due to hyperpolarization is passed from the outer segment to the synapse.

The response of rod cells is proportional to photons absorbed. A single photon absorbed by dark adapted rod closes many hundreds of cation channels and this is equivalent to about 1 mV of hyperpolarization.

So rod cells have high sensitivity. Equally important, if a rod cell is constantly illuminated, it needs more photons to get excited. Conversely, a rod cell which is dark adapted requires very few photons to get excited. This enables us to see in the dark. This adaption also confers on rod cells the capability of perceiving contrast over a 10 thousand fold range of light intensity. (see the X-axis in the above figure).

The signal amplification involved is enormous. One photon absorbed by a dark adapted cone cell stops the flow of a million Na+. As we know, cascade mechanisms are often employed by biological system for amplifying signals.

Rhodopsin R after photooxidation becomes R*. This triggers transducin T-GTP to activate a phosphodiesterase. The activated phosphodiesterase PDE* converts cGMP to 5‟-GMP. The decrease in cGMP concentration is responsible for closing of Na+ specific channel.

Transducin is a peripheral membrane protein with three subunits: α, β and γ with molecular weights of 39, 36 and 8 kD respectively. It is α subunit which has GTP binding site. Rhodopsin after photoxidation actually binds transducin and exchanges its GTP with GDP of the transducin.

The activated phosphodiesterase has the catalytic efficiency of what Knowles called Perfect enzymes. A turnover number of 4200 sec-1 and k2/Km of 6×10 7 M-1sec-1, it is very near to diffusion controlled limit. This is responsible for signal amplification power of the cascade.

11-cis-retinal is the common chromophore in the visual pigment of all the three phyla (molluscs, arthropods and vertebrates) which have image forming eyes. The design of 11-cis-retinal makes this the visual pigment of the choice.

High excitation coefficient of 11-cis retinal in the visible region of electromagnetic spectrum, rapid isomerisation as a result of light stimulus and the adaption in the dark are three valuable design elements. The movement as result of deprotonation of the Schiff base within the molecule is large enough to generate the desired signal.

The β-Carotene, the precursor of visual pigment is widely distributed in biological world. Conjugated double bond systems of carotenoids make these molecules useful even in the microbial world.

The ubiquitous molecule design is confirmed by the fact that octopus rhodopsin can trigger transducin from vertebrates. The design involving rhodopsin and transducin seem to be at least 700 million years old.

Rhodopsin itself is a 40 kDa integral membrane protein with 7 transmembrane helical motif. The –COOH end is towards cytosolic side and is involved in binding of transducin and rhodopsin. The amino terminal is on intradiscal side and glycosylated with N- linked oligosaccharides.

As predicted by the Thomas Young in 1802, photoreception by cone cells involves adsorption of blue, green and red light by the three distinct classes of these cells. The key molecule 11-cis retinal is of course common to all three types of cone cells.

11-cis-retinal forming a Schiff base as a model compound (not with opsin) has λmax of absorption at 440nm. The role of opsin is in shifting this absorption spectra significantly by about 100nm. Proteins are known to significantly tune the properties (and hence function) of these prosthetic group.

The gene sequence of cone cell proteins reveal that the protein sequence in different kinds of cone cells is not vastly different. What is more, their overall structure is very similar to the rod protein rhodopsin.

The genes for green absorbing and red absorbing visual pigment occur next to each other in x-chromosome. The blue absorbing pigment is coded by a gene on an autosome. About 1% men are red blind and 2% are green blind.

Evidences suggests that colour blind males either lack the green gene or contains a hybrid gene of the green and red genes. This is the result of recombination. Some people are known to have array of multiple genes which further confirm this model of colour blindness.

Rickets, previously called Rachitis, is an ancient disease. Glisson in England described it in 1650 as being prevalent in infants. Curiously, the famous London fog was thought to be its cause in early times. The nature of belief turned out to be erroneous but the cause was identified correctly. Fog prevents exposure to sunshine.

Rickets was caused by vit D deficiency. Vit D is actually produced by photochemical reaction of its precursor.

Again, cod liver oil was known very early as useful in rickets. Cod liver oil, apart from vitamin A activity, also contain provitamin D.

Vitamin D

Mellan by in 1918 demonstrate rickets in experimental animals by feeding just milk and porridge to dogs and showed that cod liver oil has an antirachitic factor by curing the experimental rickets.

In 1919, Huldschinsky observed that exposing rachitic children to U.V. rays helps. Soon thereafter, Mc Collum in 1922 showed that cod liver oil factor was involved in Ca deposition.

In 1932, Angus isolated xalline vitamin D by high vacuum distillation of products obtained from U.V. irradiation of ergosterol. The name calciterol was given to what is also called vit D2.

In 1936, Windans isolated 7-dehydrocholestrol, vit D3.

7-dehydrocholestrol is the precursor of vit D3. Exposure of skin (where the precursor is present) to U.V. rays of the sunlight triggers this photochemical conversion to vit-D3 or cholecalciferol.

Enzymes in the liver and kidney converts this inactive vit D3 (which some people prefer to call Previtamin) to 1,25-Dihydroxycholecalciferol. This compound regulates the Ca++ absorption in intestine and controls Ca++ level in kidney and bone. For this reason it is also called a hormone.

Vit D2 (ergocalciferol) is produced by yeast ergosterol irradiation commercially. As both (D2 and D3) have similar effects it is D2 which is a more common supplement and additive.

Practically very little vitamin D is present in milk, grains and vegetables. Butter and fish liver have some.

Albacore tuna, sword fish, lingcord and Halibut have higher potency liver oils.

Just like thyroid and steroid hormones, vit D also operates by its complex with the receptor interacting with hormone response elements on DNA. This regulates the specific gene expressions.

Fortified milk and food thus form the regular source of vit D activity. The recommended daily dose of vit D is 400 IU irrespective of age. 0.025 µg of pure crystalline vit D3 is considered as one IU or one USP unit of vit D.

While rickets is the disease of children, vit D deficiency in adult leads to osteomalacia in which bones soften and weakens. Lack of exposure to sunshine causes osteomalacia as well.

Vitamin K

It was in 1929 that Dam observed that some synthetic diet leads to chicks haemorrhaging. Within few years, in 1935, Dam suggested that the term vitamin K (K for Koagulations) for a factor which must exist in food to protect birds/ animals against bleeding.

Dam found that this factor was present in hog liver and hemp seeds, it was fat soluble. In 1935, Almquist and Stakstad showed vitamin K to be present in fish meal. Alfa alfa also contained this as an unsaponifiable compound in ether extract.

In 1939, vit K was isolated from alfa alfa and fish meal. However two preparations contained different compounds with vit K activity and were called vit K and vit K2 respectively. In the same year, vitamin K was synthesized.

The first pure chemical found to have vit K activity was actually phiocol. Vit K1 and K2 also contain the 1,4 naphthaquinone ring with different substituents. In case of vit K2, the number of isoprene units „n‟ can very as 6, 7 or 9.

The simplest vit K is 2-methyl-1,4- naphthaquinone and is called vit K3. Some water soluble analogs like synkayvite are available. Mostly, these get converted to vit K3 in vivo.

In rats and humans vit K is supplied by intestinal flora which are able to synthesize it and thus vit K is available via intestinal absorption. Infants yet to develop intestinal flora need it and in U.S. newborn are routinely given 1 mg of vit K.

Almquist (1941) described the vit K assay. Chicks depleted with vit K are administered the sample and degree of regeneration of prothrombin is determined. Menadione can also be estimated spectrophotometrically as its 2, 4- dinitrophenylhydrazone derivative.

Blood clots are crosslinked fibrin clot. Fibrin clot is the ultimate product of a cascade pathway. The terminal part of their cascade pathway is:

Vit K converts first 10 glutamate residues on the N-terminal region of prothrombin to γ-carboxyglutamate. These strong chelators of Ca++ helps Ca++ bound prothrombin to attach to phospholipid membranes of damaged blood platelets. Thus triggers the cascade to go towards clot formation.

Dicumarol warfarin are antagonists of vit K and prolong blood clotting time. Dicoumarol was identified as vit K antagonist from the old observation that cattle and hogs which fed on spoiled sweet clover hay bled to death. The disease is called sweet clover disease and factor was identified by Link‟s group in 1941 as Dicoumoral. These vit K antagonists are useful in many surgeries or wherever blood clotting has to be inhibited.

Vitamin E

Vitamin E had been implicated in reproduction since early times. It was in 1927 that vit E was found in nonsaponifiable fraction of lipids from some foods. In 1936, crystalline derivative of vit E were reported: the first vit E to be structurally characterized was α- tocopherol from wheat germ oil.

Fernholz in 1938 determined the structure of α-tocopherol. Same year, Karrer‟s group synthesized it. In due course of time eight naturally occurring tocopherol derivatives were discovered.

The natural tocopherols have d configuration around C-2 asymmetric carbon. Synthetic compound is dl α-tocopherol. Tocopherols are oils but their esters are crystalline.

Oxidation and U.V. light destroys vit E activity. Tocopherols themselves are strong antioxidants. The actual role of vit E has not been clear as deficiency symptoms in humans are not clearly established.

However, vit E is required. In its absence, RBCs are more prone to lysis. Studies showed that high dietary linoleic acid requires vit E. Some correlation with muscular atrophy with vit E deficiency has been reported. Creatinuria also was observed during vit E deficiency.

The picture is somewhat clearer with animals. In female rats, vit E deficiency can disturb the estrons cycle and reproduction. In male rats, degeneration of testes is observed during vit E deficiency. Muscle dystrophy is also clearly seen in rats of all ages and gender.

Wheat germ oil has high content of vit E. Other oils such as from corn, cotton seed and safflower also contain reasonable oil. Unlike vit A and D, fish oils are poor in vit E. Lettuce and alfa alfa are good source of vit E.

Some of the vitamins turned out to be similar to hormones as exemplified with vit D. Of all fat soluble vitamins, molecular level picture is most clear about vitamin A and least clear about vit E. We do know vit E is required but it is difficult to get a clear role as in the case of other vitamins.

What also emerges is that with such simple facilities, some brilliant work was carried out by early biochemists in this area.

Summary

• Sources, assay and physiological importance of vit A.
• Vit D and its role in Ca++ absorption
• Vit K and its involvement in fibrin clot formation
• Vit E as an antioxidants