The body fluids
This chapter focuses on body fluids. The human body is regarded as a colloidal solution of great complexity as there is a continuous aqueous phase throughout. The obvious differences in composition that occur from one region to another are maintained by physiological barriers that take the form of membranes and intercellular macromolecules. Except in the case of oxygen, entry of material into the body occurs by absorption across the intestinal epithelium, which is subjected to a constantly changing chemical environment. Apart from the great variety of natural food constituents, it may also be exposed to a tremendous range of synthetic compounds devised by food scientists and pharmaceutical chemists. The life of an intestinal epithelial cell is extremely short, and the whole of the intestinal lining is replaced in rather less than 2 days. Although energy-requiring transport mechanisms ensure that valuable materials such as sugars and amino acids are actively absorbed into the bloodstream, useless and even harmful substances may pass across the intestinal barrier if their physicochemical characteristics are such to allow them to traverse it by passive means. Once water and other materials have been absorbed, they are distributed among the various fluid compartments.
The human body may in a sense be regarded as a colloidal solution of great complexity since there is a continuous aqueous phase throughout. The obvious differences in composition which occur from one region to another are maintained by physiological barriers which take the form of membranes and intercellular macromolecules.
Except in the case of oxygen, entry of material into the body occurs by absorption across the intestinal epithelium which is subjected to a constantly changing chemical environment. Apart from the great variety of natural food constituents it may also be exposed to a tremendous range of synthetic compounds devised by food scientists and pharmaceutical chemists. It is not surprising, therefore, to find that the life of an intestinal epithelial cell is extremely short and that the whole of the intestinal lining is replaced in rather less than 2 days.
Although energy-requiring transport mechanisms ensure that valuable materials such as sugars and amino acids are actively absorbed into the bloodstream, useless and even harmful substances may pass across the intestinal barrier if their physicochemical characteristics are such as to allow them to traverse it by passive means. Once water and other materials have been absorbed they are distributed among the various fluid compartments.
|Solid and semi-solid food||1200||Expired air||350|
|Metabolic water||300||Through the skin||500|
It is very difficult to obtain information about the composition of intracellular fluid under physiological conditions. Not only does it vary from one type of cell to another but also from cell compartment to cell compartment. Nevertheless certain generalizations may be made, such as that the concentration of Na+ and Ca2+ in intracellular fluids is low. This is also true for Cl−, except in the case of red blood corpuscles and the HCl-secreting gastric glands. The chief cellular cations are K+ and Mg2+ and the anions phosphate, sulphate and protein, the bulk of the phosphate being covalently bound to organic material. Data about intracellular pH values are scant but it appears to vary from 4·5 in the cells of the prostate gland to about 8·5 in osteoblasts. Most cells are believed to be slightly more acid than the plasma.
In order to integrate the needs of the various tissues of the body and to maintain the constant conditions required for the survival of the delicate organization of their cells, higher organisms have evolved circulatory systems. In Man the relatively small volume of rapidly circulating blood (about 5 litres) is backed up by 12–15 litres of interstitial or tissue fluid. This continuously exchanges material with the cellular elements both of the blood and tissues. The extracellular fluids are the chief agents through which the homoeostatic mechanisms of the body operate to maintain the necessary balance of water, ions, respiratory gases and metabolites. The precise steady state conditions within which an organism is maintained are characteristic for the species and, whereas the body temperature of Man is held at 37–38°C, that of birds is about 41°C. Similarly while the fasting blood glucose concentration for Man is 4–5 mM that for sheep is 1·7–2·8 mM. It is not known how the various control mechanisms are set to the appropriate value.
The extracellular fluids are essentially solutions of sodium chloride and sodium bicarbonate but they also contain small amounts of calcium, magnesium and potassium, and of phosphate, sulphate and organic ions. Their pH is maintained in the range 7·35–7·45. Organic constituents include glucose, amino acids, urea and a multitude of other compounds in very small amounts. The main difference in composition of the various extracellular fluids lies in their protein contents which range from 7% in plasma down to 0·1% in subcutaneous interstitial fluid.
The composition of the interstitial fluid also differs from that of plasma with respect to other substances which are either inherently indiffusible or rendered indiffusible by combination with the plasma proteins, e.g. lipid, bilirubin and Ca2+ ions.
The main function of the blood is to act as a rapid transport system for oxygen, carbon dioxide, absorbed nutrients, intermediary metabolites, waste products and hormones. It also plays a role in acid-base regulation, water balance and the distribution of body heat and provides defence against infection and intrusion by foreign substances.
Blood consists of about 55% plasma and 45% of formed elements, chiefly red corpuscles but also white corpuscles and platelets. The red corpuscles are mainly concerned with O2 and CO2 transport, the white corpuscles with body defence mechanisms and the platelets with the clotting process.
Although many of the constituents of blood are present in simple solution, others exist in combination with one or other of the blood proteins. For example, almost all the oxygen is combined with the haemoglobin present in the red corpuscles at a concentration of 14–18 g dl−1 in men and 12–16 g dl−1 in women. Carriage of oxygen is the most important commitment of blood and the evolution of blood pigments of one sort or another was essential for the development of large specialized organisms.
Three closely related forms of haemoglobin are found in Man. They are present in different proportions according to the stage of development. The α chain is common to all three forms and their subunit constitution is as follows:
Fetal haemoglobin, the properties of which enable it to take up oxygen from the maternal blood while the fetus is in utero, is gradually replaced by the adult forms. This shows that bone marrow cells possess some form of temporal specificity with respect to the proteins that they synthesize. The α, β, γ and δ polypeptide chains, of which the various forms of haemoglobin are composed, are coded for by different genes, which are believed to have arisen from a common ancestral gene shared with myoglobin.
Haemoglobin is interesting from many points of view and it has probably been studied more extensively than any other protein. Its shape and general characteristics and its relationship to myoglobin have already been described (page 65) but little has been said about the haem group. Myoglobin, which consists of a single polypeptide chain, has one haem group while haemoglobin, which has four globin subunits, correspondingly has four haem groups, each of which can combine with one molecule of oxygen. Globin itself is colourless and contains a greater proportion of basic amino acids than any of the other blood proteins. Although the haemoglobins of all mammals are similar in shape and structure, they show appreciable differences in their amino acid composition, affinity for O2 and other gases, and in their crystalline form. All these differences are attributable to the globin since the haem group is the same in all species. Haem is an iron–porphyrin compound.
The porphyrin ring is a planar structure formed from four pyrrole rings which are joined into a larger ring by me thine (–CH=) bridges. Four methyl (M), two vinyl (V) and two propionate (P) groups are attached to the ring. The highly polar propionate side chains of the haem are situated on the surface of the molecule and are ionized at physiological pH values. The centrally
placed iron atom is bound to the four N atoms of the pyrrole rings leaving two of the six coordination valencies on the iron atom free. In both myoglobin and haemoglobin the iron atom of the haem is directly bonded to a histidine residue. This is known as the proximal histidine. Oxygen binding occurs at the sixth coordination position near which the second important or distal histidine is situated. In deoxyhaemoglobin this position is empty.
Synthesis of haem is readily accomplished from simple precursors. In the first step of porphyrin synthesis succinyl-CoA, which is produced in the citrate cycle and during the metabolism of various amino acids, is condensed with glycine to give δ-aminolaevulinic acid, and it is this reaction which is rate-controlling. Subsequently two molecules of δ-aminolaevulinic acid condense to form porphobilinogen and four molecules of porphobilinogen undergo a series of reactions to produce protoporphyrin which combines with ferrous ions to form haem.
When, at the end of their life span, for unknown reasons the red cells rupture, the haemoglobin is released. It first combines with haptoglobins (page 379) present in the plasma which have a special affinity for Hb and, while combined in this way, the iron is oxidized to the ferric form. Either before or after combination with haptoglobins the haemoglobin is taken up by cells of the reticuloendothelial system, mainly in the liver, spleen and bone marrow. Here it is split into its constituent parts which are independently processed. The globin is broken down to amino acids which pass into the metabolic pool while one of the methine bridges of the porphyrin ring is oxidized, the iron is released and a chain-like tetrapyrrole compound is produced. The first bile pigment to be formed is biliverdin which is green. This is readily reduced to bilirubin which is golden brown and the chief pigment of human bile. The bilirubin is transported from the reticuloendothelial cells to the liver in combination with serum albumin. In the liver it is conjugated with glucuronic acid and the conjugated bilirubin, which is water-soluble, is excreted in bile into the intestine.
which is colourless, but this is subsequently oxidized to orange–yellow stercobilin (urobilin) which contributes to the normal colour of the faeces.
In the haemoglobin molecule, although the four haem groups are separated by appreciable distances, the state of one haem is influenced by that of the other three and these haem–haem interactions are of considerable physiological importance. This can be judged by comparing the properties of haemoglobin and myoglobin. As can be seen from Figure 25.1, the O2 dissociation curve of myoglobin is a simple rectangular hyperbola while that of haemoglobin is sigmoid. The two pigments are well suited for their respective physiological roles. Thus myoglobin acts as a store of O2 in muscles that may be deprived of O2 for relatively long periods. For this purpose it is important that it should take up O2 when it is readily available and release it only when it cannot be obtained from the atmosphere. From the curve it can be seen that at an O2 pressure of 20 mmHg myoglobin is 80% saturated with O2 and only releases significant amounts at O2 pressures below this. Haemoglobin on the other hand, although it also has storage capacity for O2, is essentially a carrier and is required alternately to pick up and release O2 with changes of O2 pressure operating at a higher range.
The difference in properties between the two proteins, whose tertiary structures are very similar (page 67), is due to the superimposed quaternary structure of haemoglobin and the fact that the ease with which any haem group binds O2 is determined by the state of the other three. Starting with deoxyhaemoglobin the first O2 molecule is taken up very slowly, the second and third are taken up more and more readily and the fourth is taken up several hundred times more rapidly than the first; hence the sigmoid shape of the curve.
In deoxyhaemoglobin the iron atom lies about 0·6 Å (0·06 nm) out of the plane of the ring but, on oxygenation, it moves into the plane of the ring and is able to form a strong bond with oxygen (Figure 25.2). This tiny structural change within the subunits is translated into a shift in the relationship between them. One pair of αβ subunits rotates relatively to the other pair causing the β chains to move closer together and the α chains to move slightly away from one another. This change in the quaternary structure on oxygenation and deoxygenation may be regarded as a ‘breathing movement’ at the molecular level.
Not only does haemoglobin have multiple binding sites for oxygen which, as described above, show positive cooperativity, it also combines at different sites with CO2 and H+ ions and also with the substance 2,3-diphosphoglycerate, and a complex relationship exists between the binding of these various ligands. This is physiologically highly advantageous and has the result that when oxygenated blood from the lungs reaches the tissues where H+ and CO2 are being produced, the combined effects of the lower O2 tension and higher H+ and CO2 concentrations ensure that O2 is released in appropriate amounts while, at the same time, CO2 is taken up. The promotion of O2 dissociation by an increase in CO2 tension and therefore of acidity is known as the Bohr Effect and is illustrated in Figure 25.1.
The concentration of 2,3-diphosphoglycerate which is derived from the glycolytic intermediate 1,3-diphosphoglycerate is higher in red corpuscles than in other cells. 2,3-Diphosphoglycerate binds specifically to deoxyhaemoglobin and reduces its O2 affinity so that binding of O2 and 2,3-diphosphoglycerate are mutually exclusive. The 2,3-diphosphoglycerate binds to the central cavity of the haemoglobin molecule which is lined by numerous positively charged groups and crosslinks the two β subunits. But, when the haemoglobin is oxygenated, the central cavity is reduced in size and the 2,3-diphosphoglycerate is extruded.
The effect of the 2,3-diphosphoglycerate is to regulate the O2 binding affinity of haemoglobin in relation to the partial pressure in the lungs. This is of special importance in circumstances when the O2 tension is low, as for example at high altitudes or in patients suffering from hypoxia, i.e. when either the lungs or the circulatory system are not operating efficiently. In such cases the 2,3-diphosphoglycerate concentration in the blood increases and this allows the haemoglobin to release its O2 more readily.
The uptake of O2 by the fetus from the maternal blood is made possible by the fact that fetal haemoglobin (HbF page 371) binds 2,3-diphosphoglycerate less strongly than the maternal haemoglobin (HbA) and consequently has a higher affinity for oxygen.
The modifying effects of O2, CO2, H+ and 2,3-diphosphoglycerate on the properties of haemoglobin provide a good illustration of the extra functional dimension conferred by the evolution of the quaternary level of protein structure.
As already stated when oxygenated blood from the lungs reaches the tissues where acid metabolites are being produced, oxyhaemoglobin tends to lose O2 and the combined effects of the lower O2 and higher CO2 tensions ensure that O2 is released in appropriate amounts.
This reaction is not peculiar to haemoglobin and all proteins with free amino groups will react in this way. The important point about haemoglobin is that the degree to which the reaction takes place depends almost entirely on its state of oxygenation and hardly at all on the CO2 tension. Thus deoxyhaemoglobin readily forms carbamino compounds but when it is converted to oxyhaemoglobin most of the CO2 is released. Although only about 5% of the total CO2 in venous blood is present in the form of carbaminohaemoglobin it represents a very labile form of CO2 and may account for as much as 30% of the CO2 that is taken up in the tissues and released in the lungs.
2. It has a buffering effect. When haemoglobin, which is notably rich in histidine, is oxygenated the resulting conformational changes increase the tendency for specific protonated histidine residues to lose H+, i.e. their pK value in oxyhaemoglobin is 7·16 compared with 7·3 in deoxyhaemoglobin. Thus, in the lungs, the proportion of HbO2 is increased and that of HHb is reduced. Conversely, in the tissues, deoxyhaemoglobin binds H+; at the same time the plasma bicarbonate is increased (see the next paragraph). In this way a large proportion of the acid produced in the course of the tissue metabolism is carried as H+ by haemoglobin in the corpuscles and as bicarbonate in the plasma.
Formation of carbonic acid from H2O and CO2 is a relatively slow process, but within the corpuscles it is speeded up by the presence of the enzyme carbonic anhydrase. As a result the concentration of bicarbonate ions rises more rapidly in the corpuscles than in the plasma and some of them leave the corpuscles in exchange for chloride ions. This chloride-bicarbonate exchange ensures that a large fraction of the acidic CO2 produced in the tissues is carried as plasma bicarbonate. Consequently there is very little increase in the acidity of venous blood.
It can be calculated that, if the respiratory quotient is 0·7 (i.e. 1 volume of O2 is utilized for every 0·7 volume of CO2 produced), all the H+ resulting from the conversion of can be buffered by haemoglobin without any alteration in pH. However, since the respiratory quotient is usually greater than 0·7, other blood buffer systems, notably the Na2HPO4/NaH2PO4 system, also operate to keep the pH change within narrow limits.
In addition to the 13 mol or so of CO2 produced each day most of which is buffered by haemoglobin, small amounts of certain non-volatile acids are produced. These usually account for rather less than 0·1 mol of H+ per day and, since they cannot be eliminated through the lungs, they must be excreted via the kidneys. The non-volatile acids include sulphuric acid, formed by oxidation of the sulphur present in cysteine and methionine, phosphoric acid from phospholipids and phosphoproteins, and lactic acid produced during severe exercise. Appreciable amounts of acetoacetic acid and β-hydroxybutyric acid are released into the blood in ketosis. All these acids are buffered by the plasma bicarbonate according to the following reaction:
The carbonic acid which is produced causes a fall in the NaHCO3/H2CO3 ratio and a slight drop in the pH of the blood. The overall effect is to stimulate the respiratory centre of the brain causing an increase in pulmonary ventilation so that the extra CO2 is lost from the lungs and the normal NaHCO3/H2CO3 ratio is restored. However, in the course of the reaction some of the plasma bicarbonate has been used up so that, although the NaHCO3/H2CO3 ratio and pH are normal, the plasma bicarbonate is reduced. The responsibility for regulating the absolute amount of bicarbonate in the plasma belongs to the kidney (page 395).
In spite of the variety and efficiency of the body’s mechanisms for maintaining a constant pH, disturbances of acid-base balance can and do occur. They may result from gross dietary imbalance, and also from respiratory, metabolic or renal disorders in which there is either too great a production or a failure of elimination of acid or base.
Acidosis is a fairly common condition in which the total concentration of buffer base (chiefly ) is less than normal; alkalosis in which the total concentration of buffer base is greater than normal (i.e. is increased) is far less common. It may occur when diets containing large amounts of vegetables are consumed, as a result of hyperventilation or of continuous vomiting, and after taking alkalizing salts such as potassium citrate and sodium lactate. In these circumstances the increased alkalinity of the blood reaching the brain causes respiration to be depressed so that less CO2 is lost from the lungs. This causes a build-up in PCO2, and the plasma H2CO3 rises until the normal ratio is restored but there is still an increase in total concentration of buffer base. This is reduced to normal by excretion of NaHCO3 by the kidney. Owing to the interplay of the various regulatory mechanisms such conditions are complicated and may be confused by ambiguous terminology.
In prolonged acidosis in which there is a reduction in the pH of the blood, calcium may be drawn from the bones and some of the Na+ of the urine replaced by Ca2+. Although this use of calcium phosphate to neutralize acid is extremely efficient in preventing excessive depletion of the plasma bicarbonate, demineralization of the hard tissues does not do them any good!
In order that haemoglobin may combine reversibly with O2, its iron must be present in the ferrous (Fe2+) state. If the haem is oxidized to the corresponding ferric derivative haematin, a new pigment known as methaemoglobin is produced. Methaemoglobin is brown in colour and, although it contains O2, it is unable to release it in the tissues. Methaemoglobin is not normally found in the blood in appreciable amounts since blood contains the enzyme methaemoglobin reductase which reduces it to haemoglobin. Methaemoglobinaemia may result from exposure to agents which oxidize Fe2+ to Fe3+ including amyl nitrite, nitrates, nitrobenzene and drugs such as salicylates, phenacetin and the sulphonamides.
Haemoglobin has an affinity for carbon monoxide which is 200 or more times that for oxygen. This means that if blood is exposed to a mixture of 1 part of CO and 200 parts of O2 approximately equal amounts of carboxyhaemoglobin and oxyhaemoglobin will be formed. Because of the great stability of carboxyhaemoglobin, the blood is deprived of its O2-carrying power and, when 60–80% of the haemoglobin has been converted into this form, death occurs due to O2 lack. Carboxyhaemoglobin has a bright cherry-red colour which is quite different from the orange-red of oxyhaemoglobin and the purple-red of deoxyhaemoglobin. The pink and healthy-looking colour of victims of carbon monoxide poisoning is very distinctive and is a useful diagnostic feature.
The toxic effects of cyanide and sulphide are chiefly due to combination of these substances with the Fe3+ of cytochrome oxidase so that the final reaction of the respiratory chain is blocked. Cyanide and sulphide, however, also have the effect of converting haemoglobin into cyan-methaemoglobin and sulph-methaemoglobin respectively. Both are stable compounds and once formed can only be removed by complete degradation. Cyan-methaemoglobin is used as a stable and reproducible standard in haemoglobin estimations.
Normal human plasma contains a very complex mixture of proteins amounting to about 7·0–7·5 g/100 ml. Structural and physicochemical data are available on 22 distinct human plasma proteins. A host of others are known to be present in low concentrations and to have important biological functions, but they have not yet been isolated.
For clinical investigations of the plasma proteins serum is commonly used instead of plasma since fibrinogen and fibrin tend to interfere with some of the tests. The serum proteins are usually separated on a variety of materials when five distinct fractions are produced. These are albumin and the α1, α2, β and γ globulin fractions. Typical electrophoretic patterns for normal serum are shown in Figure 25.3 and the percentage composition of the total plasma proteins in Table 25.1.