Brief Discussion Report - Calcium and its use in biology

INTRODUCTION

Calcium, like many other "inorganic elements" in biological systems, has during the last decade become the subject of much attention both by scientists and by the general public. The presence and central role of calcium in mammalian bones and other mineralized tissues were recognized soon after its discovery as an element by Davy in 1808. Much later, the insight arrived that Ca ²⁺ ions could play an important role in other tissues as well. Experiments of great historical influence were performed by the British physiologist Sidney Ringer a little over a century ago. He was interested in the effects of various cations on frog-heart muscle and somewhat serendipitously discovered that Ca²⁺ ions, everpresent in the tap water distributed in central London, in millimolar concentrations were necessary for muscle contraction and tissue survival.

 History

Lime as building material was used since prehistoric times going as far back as 7000 to 14000 BC. Significant statues made from lime plaster date back into the 7 millennia BC were found in 'Ain Ghazal. The first dated lime kiln dates back to 2500 BC and was found in Khafajah mesopotamia. Calcium (from Latin calx, genitive calcis, meaning "lime") was known as early as the first century when the Ancient Romans prepared lime as calcium oxide. Literature dating back to 975 AD notes that plaster of paris (calcium sulfate), is useful for setting broken bones. It was not isolated until 1808 in England when Sir Humphry Davy electrolyzed a mixture of lime and mercuric oxide.Calcium metal was not available in large scale until the beginning of the 20th century.

Facts about calcium

Calcium is a chemical element with symbol Ca and atomic number 20. Calcium is a soft gray alkaline earth metal, fifth most abundant element by mass in the Earth's crust. The ion Ca²⁺ is also the fifth most abundant dissolved ion in seawater by both molarity and mass, after sodium, chloride, magnesium, and sulfate. Free calcium metal is too reactive to occur in nature. Calcium is produced in the explosions at the end of the life of massive stars Calcium is essential for living organisms, in particular in cell physiology, where movement of the calcium ion into and out of the cytoplasm functions as a signal for many cellular processes. As a major material used in mineralization of bone, teeth and shells, calcium is the most abundant metal by mass in many animals.

 Characteristics

In chemical terms, calcium is reactive and soft for a metal, though harder than lead, it can be cut with a knife with difficulty. It is a silvery metallic element that must be extracted by electrolysis from a fused salt like calcium chloride. Once produced, it rapidly forms a gray-white oxide and nitride coating when exposed to air. In bulk form (typically as chips or "turnings"), the metal is somewhat difficult to ignite, more so even than magnesium chips; but, when lit, the metal burns in air with a brilliant high intensity orange-red light. Calcium metal reacts with water, generating hydrogen gas at a rate rapid enough to be noticeable, but not fast enough at room temperature to generate much heat, making it useful for generating hydrogen. In powdered form, however, the reaction with water is extremely rapid, as the increased surface area of the powder accelerates the reaction with the water. Part of the reason for the slowness of the calcium–water reaction is a result of the metal being partly protected by insoluble white calcium hydroxide; water solutions of acids, where this salt is soluble, calcium reacts vigorously. With a density of 1.54 g/cm3, calcium is the lightest of the alkaline earth metals; magnesium (specific gravity 1.74) and beryllium (1.84) are denser though lighter in atomic mass. From strontium onward, the alkali earth metals become denser with increasing atomic mass.

 Occurrence

Calcium is not naturally found in its elemental state. Calcium occurs most commonly in sedimentary rocks in the minerals calcite, dolomite and gypsum. It also occurs in igneous and metamorphic rocks chiefly in the silicate minerals: plagioclases, amphiboles, pyroxenes and garnets.

Food sources

Calcium amount in foods, 100 g:

1.  parmesan (cheese) = 1140 mg
2.    milk powder = 909 mg
3.  Cheddar cheese = 720 mg
4.   tahini paste = 427 mg
5.   molasses = 273 mg
6.   hazelnuts = 114 mg
7. almonds = 234 mg
8. sesame seeds (unhulled) = 125 mg
9.    nonfat cow milk = 122 mg
10.  plain wholemilk
11.   yogurt = 121 mg
12.   skimmed milk cheese = 90 mg
13.   brown sugar = 85 mg
14.   lentils = 79 mg
15.   wheat germs = 72 mg
16.   pigeon peas = 62.7 mg
17.   eggs, boiled = 50 mg
18.   chickpeas = 53.1
19.   flour = 41 mg
20.   orange = 40 mg

by a hormone acting directly on the channel, or by a small molecule released intracellularly when a hormone is attached membrane bound receptor. Some channels may be switched on by voltage gradients, and both these mechanisms may operate concurrently. Increased intracellular Ca⁺ levels must eventually be brought back to the base levels, in some cells very quickly. The ions could be transported out of the cell or back into the Ca²⁺ - rich organelles. This transport will be against an electrochemical potential gradient, and thus requires energy. There are many possibilities for different forms of Ca²⁺ ions transport and regulation in living systems.

Absorption and excretion

·         Usual intakes is 1000 mg/day.

·         About 35 % is absorbed (350 mg/day) by the intestines.

·         Calcium remaining in the intestine is excreted in the feces.

·         250 mg/day enters intestine via secreted gastrointestinal juices and sloughed mucosal cells

·         90 % (900 mg/day) of the daily intake is excreted in the feces.

·         10 % (100 mg/day) of the ingested calcium is excreted in the urine.

Transport and regulation -

Ø  Calcium levels in mammals are tightly regulated, with bone acting as the major mineral storage site. Calcium ions, Ca²⁺, are released from bone into the bloodstream under controlled conditions.

Ø  Calcium is transported through the bloodstream as dissolved ions or bound to proteins such as serum albumin.

Ø  Parathyroid hormone secreted by the parathyroid gland regulates the resorption of Ca²⁺ from bone, reabsorption in the kidney back into circulation, and increases in the activation of vitamin D₃ to Calcitriol.

Ø  Calcitriol, the active form of vitamin D₃, promotes absorption of calcium from the intestines and the mobilization of calcium ions from bone matrix.

Ø  Calcitonin secreted from the parafollicular cells of the thyroid gland also affects calcium levels by opposing parathyroid hormone; however, its physiological significance in humans is dubious.

Ø  Calcium storages are intracellular organelles, that constantly accumulate Ca²⁺ ions and release them during certain cellular events.

Ø  Intracellular Ca²⁺ storages include mitochondria and the endoplasmic reticulum.

Plantae

Stomata closing

When ABA signals the guard cells, free Ca2+ ions enter the cytosol from both outside the cell and internal stores, reversing the concentration gradient so the K+ ions begin exiting the cell. The loss of solutes makes the cell flaccid and closes the stomatal pores.

Cellular division

Calcium is a necessary ion in the formation of the mitotic spindle. Without the mitotic spindle, cellular division cannot occur. Although young leaves have a higher need for calcium, older leaves contain higher amounts of calcium because calcium is relatively immobile through the plant. It is not transported through the phloem because it can bind with other nutrient ions and precipitate out of liquid solutions.

Structural roles

Ca2+ ions are an essential component of plant cell walls and cell membranes, and are used as cations to balance organic anions in the plant vacuole. The Ca2+ concentration of the vacuole may reach millimolar levels. The most striking use of Ca2+ ions as a structural element in plants occurs in the marine coccolithophores, which use Ca2+ to form the calcium carbonate plates, with which they are covered.

Calcium is needed to form the pectin in the middle lamella of newly formed cells.

Calcium is needed to stabilize the permeability of cell membranes. Without calcium, the cell walls are unable to stabilize and hold their contents. This is particularly important in developing fruits. Without calcium, the cell walls are weak and unable to hold the contents of the fruit. Some plants accumulate Ca in their tissues, thus making them more firm. Calcium is stored as Caoxalate crystals in plastids. Calcium coordination plays an important role in defining the structure and function of proteins.

 An example a protein with calcium coordination is von Willebrand factor (vWF) which has an essential role in blood clot formation process. It is discovered using single molecule optical tweezers measurement that calciumbound vWF acts as a shear force sensor in the blood. Shear force leads to unfolding of the A2 domain of vWF whose refolding rate is dramatically enhanced in the presence of calcium.

Intracellular Ca21 and Cellular Pathology

There are numerous examples of Ca21 being involved in cellular pathology and even cell death. By using Ca21 as an intracellular messenger, cells walk a delicate tightrope

between life and death. When the homeostatic mechanisms responsible for regulating cellular Ca21 are compromised, cells will die. This can be either in a disordered manner by necrosis, or by the more deliberate apoptotic mechanism.  It is possible that only subtle changes in Ca21 signalling are necessary to have a dramatic pathological effect on cells.

For example, the effect of many InsP3-generating hormones on cardiac myocytes appears to be to activate elementary Ca21 signals that do not coincide with the electrical pacing of the heart. Individually, such ‘spontaspontaneous’ elementary Ca21 signals do not pose a problem. However, over time they have an integrated effect and can begin to interfere with gene transcription, possibly leading to hypertrophy of the heart. In addition, at sufficient frequencies, the elementary Ca21 signals may be able to

generate electrical signals by altering the activity of electrogenic processes.

For example Na1/Ca21 exchange, at the PM. Such spontaneous electrical activity can lead to cardiac arrhythmias and sudden heart death. Another well-known example of Ca21 signalling exceeding physiological requirements and becoming pathological is glutamate-induced excitotoxicty in the brain. This arises owing to excessive activation of NMDA receptors, and is a major cause of neuronal death associated with stroke and is chaemia.

Dietary supplements

Calcium supplements are used to prevent and to treat calcium deficiencies. Office of Dietary Supplements (National Institutes of Health) recommends that no more than 600 mg of supplement should be taken at a time because the percent of calcium absorbed decreases as the amount of calcium in the supplement increases. It is therefore recommended to spread doses throughout the day. Recommended daily calcium intake for adults ranges from 1000 to 1300 mg. Calcium supplements may have side effects such as bloating and constipation in some people. It is suggested that taking the supplements with food may aid in nullifying these side effects. Vitamin D is added to some calcium supplements. Proper vitamin D status is important because vitamin D is converted to a hormone in the body, which then induces the synthesis of intestinal proteins responsible for calcium absorption.

The absorption of calcium from most food and commonly used dietary supplements is very similar.This is contrary to what many calcium supplement manufacturers claim in their promotional materials.

Milk is an excellent source of dietary calcium for those whose bodies tolerate it because it has a high concentration of calcium and the calcium in milk is excellently absorbed.

Soymilk and other vegetable milks are usually sold with calcium added so that their calcium concentration is as high as in milk.

Also different kind of juices boosted with calcium are widely available.

Calcium carbonate is the most common and least expensive calcium supplement. It should be taken with food, and depends on low pH levels (acidic) for proper absorption in the intestine. Some studies suggests that the absorption of calcium from calcium carbonate is similar to the absorption of calcium from milk.

Antacids frequently contain calcium carbonate, and are a commonly used, inexpensive calcium supplement.

Coral calcium is a salt of calcium derived from fossilized coral reefs. Coral calcium is composed of calcium carbonate and trace minerals.

Calcium citrate can be taken without food and is the supplement of choice for individuals with achlorhydria or who are taking histamine2 blockers or proton pump inhibitors. Calcium citrate is about 21% elemental calcium. 1000 mg will provide 210 mg of calcium. It is more expensive than calcium carbonate and more of it must be taken to get the same amount of calcium.

 Intracellular Ca²⁺ Homeostatis

The calcium ion (Ca²⁺) is an almost universal intracellular messenger, controlling a diverse range of cellular processes, such as gene transcription, muscle contraction and cell proliferation (for reviews, see Berridge, 1993; Petersen et al., 1994; Clapham, 1995; Berridge et al., 1998).  In most cells, Ca²⁺  has its major signalling function when it is elevated in the cytosolic compartment. From there it can also diffuse into organelles such as mitochondria and the nucleus. The Ca²⁺  concentration inside cells is regulated by the simultaneous interplay of several counteracting processes, which can be divided into Ca²⁺  ‘on’ and ‘off’ mechanisms depending on whether they serve to increase or decrease cytosolic Ca²⁺.

The Ca²⁺  ‘on’ mechanisms include channels located at the plasma membrane (PM) which regulate the inexhaustible supply of Ca²⁺  from the extracellular space, and channels on the endoplasmic reticulum and sarcoplasmic reticulum (ER and SR, respectively) which release the finite intracellular Ca²⁺  stores. A more diverse set of ‘off’ mechanisms is employed by cells to remove Ca²⁺ from the cytoplasm. These include Ca²⁺  ATPases on the PM and ER/SR, in addition to exchangers that utilize gradients of other ions to provide the energy to transport Ca²⁺ out of the cell, e.g. Na⁺/ Ca²⁺  exchange. Occasionally, some of the ‘off’ mechanisms contribute to cytosolic Ca²⁺ increases, for example ‘slippage’ of Ca²⁺ through Ca²⁺ ATPases and reverse-mode Na⁺/ Ca²⁺ exchange. Organelles other than the ER and SR may also play important roles in Ca²⁺ homeostasis by sequestering or releasing Ca²⁺. For example, mitochondria have been shown to limit the amplitude of cytosolic Ca²⁺ increases by rapidly sequestering Ca²⁺   and then more slowly return it to the cytoplasm.

When cells are at rest, the balance lies in favour of the ‘off’ mechanisms, thus yielding an intracellular Ca²⁺ concentration of 100 nmolL^-1. However, when cells are stimulated by various means (e.g. depolarization, mechanical deformation or hormones) the ‘on’ mechanisms are activated and the cytosolic Ca²⁺   concentration increases to levels of micro-molL^-1 or more. It is important to point out that not all cells employ each of the ‘on’ and ‘off’ mechanisms described in. Instead, different cell types express various combinations of these channels, ATPases and exchangers to suit their physiology (Berridge, 1993). The diversity of Ca²⁺   ‘on’ and ‘off’ mechanisms underlies the huge variability in the characteristics of Ca²⁺ signals recorded in different cell types.

Recommended adequate intake by the IOH for calcium

Role of Ca²⁺ in muscle contraction

Functions Of Ca²⁺ in Muscle Contraction – 

Some of the important function of calcium are –

In smooth muscle contraction – The smooth muscle cell membrane has for more voltage gated Ca²⁺ ions that causes contraction enter the muscle cell from the extra cellular fluid at the time of action potential.

In Cardiac Muscle contraction – The strength of contraction of Cardiac muscle depends to a great extent on the contraction of Ca²⁺ ion in the extracellular fluids. The reason for this is that the end of transverse tubules open directly to the outside of the cardiac muscle fibers and thus allowing the some extracellular fluid to percolate through the tranverse tubules as well.

In Skeletal muscle contraction – Ca²⁺ ions initiate attractive forces between actin and myosin filaments causing them to slide along side each other, which is contractile process. The actin potential travels along the muscle fiber membrane in the same way that action potentials travels along nerve membrane.

Role of Ca²⁺ in blood coagulation

Ca²⁺ ions are required for promotion and acceleration of all the blood clotting reactions. Therefore in the absence of Ca²⁺ ions blood clotting by either process does not occur. The clotting takes place in following steps –

1)            In response to the rupture of the vessel complex cascade of chemical reaction occurs in the blood which results the formation of a complex of substances collectively called prothrombin activater.

2)            Prothrombin activator catalyses the conversion of prothrombin into thrombin.

3)            The thrombin acts as an enzyme to convert fibrinogen into fibrin fibers that enmesh platelets blood cells and plasma to form the clot.

Formation of bones

Bone is composed of a tough organic matrix that is gently strenghthend  by deposits of Ca²⁺ salts. An average compact bone consists of ~70 % salts & 30% matrix. The formula for major crystalline salts in Ca₁₀(PO₄)₆(OH)₂ which is known as hydroxyapatite.

Formation of teeth

The salts of teeth are composed of hydroxyapatite with observed carbonate & various cations together in a hard crystalline substances.

Deposition and reabsorption occur mainly in the Dentine and cementum & to a very limited extent in the enamel. In enamel of the teeth Ca²⁺ is present as fluoroapatite         [ 3Ca₃(Po₄).CaF₂].

Molecular Aspects of Ca²⁺ regulated intracellular processes

In order to influence the cellular machinery the Ca²⁺ions must interact with different proteins i.e. intracellular ca receptors proteins in order to function. These are –

1.    Their Ca²⁺ affinity must be such that their Ca²⁺ binding sites are essentially unoccupied at resting levels of free Ca²⁺ & occupied at levels reached upon stimulus.

2.    Ca²⁺ must exert its function in the presence of a number of other ions like K⁺, Mg⁺ etc.

3.    A Ca²⁺receptors must undergo some kind of conformation change that either alters its interaction with other molecules or changes its activity if it is an enzyme.

4.    There are kinetic consideration also i.e. the receptor must be able to interact swiftly  ( with in few milliseconds).

The best known intracellular Ca²⁺ receptor protein are –

1.    Calmodulin (CaM)

2.    Protein kinase C (PKC)

3.    Troponin (TnC)

Calmodulin (CAM) –

CaM is a small acidic protein. It is present in all eukaryotic cells. The three dimensional x-ray structure of brain CaM has been extensively studied. Spectroscopic evidences have shown that the first two Ca ions are bound in the C-terminal domain while other ions like Mg in the N-terminal domain. The rate of dissociation of Ca²⁺ form the (Ca)₄ CaM complex have been stidied by NMR as well as by stopped flow technique.

Fast and slow processes are observed both corresponding to the release of two Ca²⁺ ions.

The two important features of this protein are-

1)            The binding of Ca²⁺ to CaM is quite likely co operative.

2)            The effective Ca²⁺ affinity will be different in presence of the target proteins.

Protein kinase C (PKC)-

The activity of PKC is regulated by three factors-

1)            Phospholipids

2)            Diacyl-glycelol

3)            Ca²⁺ ions

The high activity form of PKC is presumably membrane bound, where as low activity form may be partly cytosolic.

Troponin (TnC) –

It occurs in muscle cells in the Ca²⁺ binding subunit of tropnin.

Skeletal muscle TnC can bind form Ca ions while cardiac-TnC can bind only three Ca²⁺ ions. Both sTnC & cTnC have two high affinity Ca binding sites called the Ca-Mg sites.

NMR studies shows that the binding of both sTnC & cTnC undergo significant conformation changes.

Recently few intracellular binding protein have been discovered these are *-

1)            Paralbumin – function as buffering Ca²⁺ in muscle cells.

2)            Calbindin D_9k- intracellular low molecular weight Ca²⁺ ion binding protein.

3)            Calbindin D_28k- intracellular low molecular weight Ca²⁺ ion binding protein.

Extracellular Ca²⁺ binding protein

Ca²⁺ions in extracellular fluids plays a very different role from that inside the cell because the Ca²⁺ concentration in extracellular fluid is usually higher then intracellular concentration. One important aspect of Ca²⁺ in mammals is its role in the blood coagulation system.

For new type of amino acids γ- carboxyglutamic acid (Gla) & β-hydroxy aspartic acid (Hya) are desired by nature as a Ca²⁺ ligand.

Several extracellular enzymes have one or more Ca²⁺ ions as integral parts of their structure. In some the Ca²⁺ is bound at a near the active cleft & appears necessary for maintaining the catalytic activity whereas others enzymes shows catalytic activity even in the absence of Ca²⁺ (like Trypsin) Trypsin has one Ca²⁺ binding site with four ligands donated by the protein & two water molecules making the site octahedral.

Few other enzymes like pancreatic phospholipase A₂ also have Ca²⁺ and are essential for activity.  

Recent advances

Recent advances in elucidating the structural features of calcium channels, pumps, and translocases have provided a greater understanding of the molecular mechanisms through which the movement of Ca²⁺ is mediated, as well as the basis for understanding how mutations in them are linked to Ca²⁺ signaling disorders. The minireviews in the series entitled “Ins and Outs of Calcium Transport” will explore recent advances in understanding these structural details. A second related series entitled “Calcium Function and Disease” will explore recent advances in understanding the linkages between known calcium signaling systems and disease processes in animals.

 Hazards and toxicity

Compared with other metals, the calcium ion and most calcium compounds have low toxicity. This is not surprising given the very high natural abundance of calcium compounds in the environment and in organisms. Calcium poses few serious environmental problems, with kidney stones the most common side effect in clinical studies. Acute calcium poisoning is rare, and difficult to achieve unless calcium compounds are administered intravenously. For example, the oral median lethal dose (LD50) for rats for calcium carbonate and calcium chloride are 6.45 and 1.4 g/kg, respectively.

Calcium metal is hazardous because of its sometimes violent reactions with water and acids. Calcium metal is found in some drain cleaners, where it functions to generate heat and calcium hydroxide that saponifies the fats and liquefies the proteins (e.g., hair) that block drains. When swallowed calcium metal has the same effect on the mouth, esophagus and stomach, and can be fatal.

 Summary of the topic 

The universality and versatility of Ca²⁺ as an intracellular messenger is illustrated. It is striking that a simple ion such as Ca²⁺ can regulate so many different cellular processes. The central role of Ca²⁺ in cell biology essentially arises owing to the utilization of a Ca²⁺   signalling toolkit, whereby cells employ specific Ca²⁺   ‘on’ and ‘off’ mechanisms selected from a diverse array of channels, pumps and exchangers. Subtle modulation of the amplitude or the temporal/spatial presentation of Ca²⁺   signals can differentially regulate Ca²⁺-sensitive processes within the same cell. However, cells must utilize Ca²⁺ with care, since it can also trigger deleterious processes that culminate in cell death.

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