Sodium and water balance

Sodium and water metabolism are closely interrelated both physiologically and clinically, and play a major role in determining the osmolality of serum.

Water constitutes approximately 60% of body weight in men and 55% in women (women have a greater proportion of fat tissue which contains little water). Approximately two-thirds of body water is found in the intracellular fluid (ICF) and one-third in the extracellular fluid (ECF). Of the ECF 75% is found within interstitial fluid and 25% within serum (Fig. 6.1). Total body water is regulated by the renal action of antidiuretic hormone (ADH), the renin angiotensin–aldosterone system, noradrenaline/norepinephrine and by thirst which is stimulated by rising plasma osmolality.

Fig. 6.1 Approximate distribution of water in a 70 kg man.

In general, water permeates freely between the ICF and ECF. Cell walls function as semipermeable membranes, with water movement from one compartment to the other being controlled by osmotic pressure: water moves into the compartment with the higher osmotic concentration. The osmotic content of the two compartments is generally the same, that is, they are isotonic, which ensures normal cell membrane integrity and cellular processes. However, the kidneys are an exception to the rule.

The osmolality of the ECF is largely determined by sodium and its associated anions, chloride and bicarbonate. Glucose and urea have a lesser, but nevertheless important, role in determining ECF osmolality. Protein, especially albumin, makes only a small (0.5%) contribution to the osmolality of the ECF but is a major factor in determining water distribution between the two compartments. The contribution of proteins to the osmotic pressure of serum is known as the colloid osmotic pressure or oncotic pressure.

The major contributor to the osmolality of the ICF is potassium.

The amount of water taken in and lost by the body depends on intake, diet, activity and the environment. Over time the intake of water is normally equal to that lost (Table 6.2). The minimum daily intake necessary to maintain this balance is approximately 1100 mL. Of this, 500 mL is required for normal excretion of waste products in urine, whilst the remaining volume is lost via the skin in sweat, via the lungs in expired air, and in faeces. The kidneys regulate water balance, water being filtered, then reabsorbed in variable amounts depending primarily on the level of ADH.

Potassium

The total amount of potassium in the body, like sodium, is 3000 mmol. About 10% of the body potassium is bound in red blood cells (RBCs), bone and brain tissue and is not exchangeable. The remaining 90% of total body potassium is free and exchangeable with the vast majority having an intracellular location, being pumped in and out by Na/K-ATPase pumps. This is controlled by mechanisms aimed at ensuring stable intracellular to extracellular ratios, and hence correct muscular and neuronal excitability. Only 2% of the exchangeable total body potassium is in the ECF, the compartment from where the serum concentration is sampled and measured. Consequently, the measurement of serum potassium is not an accurate index of total body potassium, but together with the clinical status of a patient it permits a sound practical assessment of potassium homeostasis.

The serum potassium concentration is controlled mainly by the kidney with the gastro-intestinal tract normally having a minor role. The potassium filtered in the kidney is almost completely reabsorbed in the proximal tubule. Potassium secretion is largely a passive process in response to the need to maintain membrane potential neutrality associated with active reabsorption of sodium in the distal convoluted tubule and collecting duct. The extent of potassium secretion is determined by a number of factors including:

As described above, both potassium and hydrogen can neutralise the membrane potential generated by active sodium reabsorption and consequently there is a close relationship between potassium and hydrogen ion homeostasis. In acidosis, hydrogen ions are normally secreted in preference to potassium and potassium moves out of cells, that is, hyperkalaemia is often associated with acidosis, except in renal tubular acidosis. In alkalosis, fewer hydrogen ions will be present and potassium moves into cells and potassium is excreted, that is, hypokalaemia is often associated with alkalosis.

The normal daily dietary intake of potassium is of the order of 60–200 mmol, which is more than adequate to replace that lost from the body. It is unusual for a deficiency in intake to account for hypokalaemia. A transcellular movement of potassium into cells, loss from the gut or excretion in the urine are the main causes of hypokalaemia.

Calcium

The body of an average man contains about 1 kg of calcium and 99% of this is bound within bone. Calcium is present in serum bound mainly to the albumin component of protein (46%), complexed with citrate and phosphate (7%), and as free ions (47%). Only the free ions of calcium are physiologically active. Calcium metabolism is regulated by 1,25-dihydroxycholecalciferol (vitamin D) which, when serum calcium is low, is secreted to promote gastro-intestinal absorption of calcium, and by parathyroid hormone (PTH) which is inhibited by increased serum concentrations of calcium ions. PTH is secreted in response to low calcium concentrations and increases serum calcium by actions on osteoclasts, kidney and gut.

The serum calcium level is often determined by measuring total calcium, that is, that which is free and bound but the measurement of free or ionised calcium offers advantages in some situations.

In alkalosis, hydrogen ions dissociate from albumin, and calcium binding to albumin increases, together with an increase in complex formation. If the concentration of ionised calcium falls sufficiently, clinical symptoms of hypocalcaemia may occur despite the total serum calcium concentration being unchanged. The reverse effect, that is, increased ionised calcium, occurs in acidosis.

Changes in serum albumin also affect the total serum calcium concentration independently of the ionised concentration. A variety of equations are available to estimate the calcium concentration and many laboratories report total and adjusted calcium routinely. A commonly used formula is shown in Fig. 6.2. Caution must be taken when using such a formula in the presence of disturbed blood hydrogen ion concentrations.

Fig. 6.2 Formula for correction of total serum calcium concentration for changes in albumin concentration: albumin concentration = [alb] (albumin units = g/L); calcium concentration = [Ca] (total calcium units = mmol/L).

Phosphate

About 85% of body phosphate is in bone, 15% in ICF and only 0.1% in ECF. Its major function is in energy metabolism. Serum levels are regulated by absorption from the diet, which is partly under the control of vitamin D, and PTH which controls its excretion by the kidney and resorption from bone. The recent identification of genes encoding for renal phosphate transporters or associated proteins, and the discovery of a new hormone, fibroblast growth factor 23 and its emerging role in the bone–kidney axis which regulates systemic phosphate, has improved knowledge of homeostasis.

Magnesium

Magnesium is an essential cation, found primarily in bone, muscle and soft tissue. About 1% of the total body content is in the ECF. As an important cofactor for numerous enzymes and ATP, it is critical in energy requiring metabolic processes, protein synthesis, membrane integrity, nervous tissue conduction, neuromuscular excitability, muscle contraction, hormone secretion and in intermediary metabolism. Serum magnesium levels are usually maintained within a tight range (0.7–1.0 mmol/L). Although a serum concentration of less than this usually indicates some level of magnesium depletion, serum levels may be normal in spite of low intracellular magnesium due to magnesium depletion. Hypocalcaemia is a prominent manifestation of moderate to severe magnesium deficiency in humans.

Hypomagnesaemia is frequently seen in critically ill patients. Causes include excessive gastro-intestinal losses, renal losses, surgery, trauma, infection, malnutrition and sepsis. The drugs most likely to induce significant hypomagnesaemia are cisplatin, amphotericin B and ciclosporin, but it is also a potential complication of treatment with amikacin, gentamicin, laxatives, pentamidine, tobramycin, tacrolimus and carboplatin. A hypomagnesaemic effect of furosemide and hydrochlorothiazide is questionable and routine monitoring and treatment are not required. Use of digoxin has been associated with hypomagnesaemia, possibly by enhancing magnesium excretion, which may predispose to digoxin toxicity, for example, dysrhythmias.

Where treatment is indicated, oral supplements are available but because of their slow onset of action and gastro-intestinal intolerance the intravenous route is often preferred and especially in critically ill patients with severe symptomatic hypomagnesaemia.

Hypermagnesaemia is most commonly caused by renal insufficiency and excess iatrogenic magnesium administration.

Creatinine

Serum creatinine concentration is largely determined by its rate of production, rate of renal excretion and volume of distribution. It is frequently used to evaluate renal function.

Creatinine is produced at a fairly constant rate from creatine and creatine phosphate in muscle. Daily production is a function of muscle mass and declines with age from 24 mg/kg/day in a healthy 25-year-old to 9 mg/kg/day in a 95-year-old. Creatinine undergoes complete glomerular filtration with little reabsorption by the renal tubules. Its clearance is, therefore, usually a good indicator of the glomerular filtration rate (GFR). As a general rule, and only at the steady state, if the serum creatinine doubles this equates to a 50% reduction in the GFR and consequently renal function. The serum creatinine level can be transiently elevated following meat ingestion, but less so than urea, or strenuous exercise. Individuals with a high muscle bulk produce more creatinine and, therefore, have a higher serum creatinine level compared to an otherwise identical but less muscular individual.

The value for creatinine clearance is higher than the true GFR due to the active tubular secretion of creatinine. In a patient with a normal GFR, this is of little significance. However, in an individual in whom the GFR is low (<10 mL/min), the tubular secretion may make a significant contribution to creatinine elimination and overestimate the GFR. In this type of patient, the breakdown of creatinine in the gut can also become a significant source of elimination. Some drugs including trimethoprim and cimetidine inhibit creatinine secretion, reducing creatinine clearance and elevating serum creatinine without affecting the GFR.

Urea

The catabolism of dietary and endogenous amino acids in the body produces large amounts of ammonia. Ammonia is toxic and its concentration is kept very low by conversion in the liver to urea. Urea is eliminated in urine and represents the major route of nitrogen excretion. The urea is filtered from the blood at the renal glomerulus and undergoes significant tubular reabsorption of 40–50%. This tubular reabsorption is pronounced at low rates of urine flow but is reduced in advanced renal failure. Serum urea is a less reliable marker of GFR than creatinine. Urea levels vary widely with diet, rate of protein metabolism, liver production and the GFR. A high protein intake from the diet, tissue breakdown, major haemorrhage in the gut, and consequent absorption of the protein from the blood, and corticosteroid therapy may produce elevated serum urea levels (up to 10 mmol/L). Urea concentrations of more than 10 mmol/L are usually due to renal disease or decreased renal blood flow following shock or dehydration. As with serum creatinine levels, serum urea levels do not begin to increase until the GFR has fallen by 50% or more.

Production is decreased in situations where there is a low protein intake and in some patients with liver disease. Thus, non-renal as well as renal influences should be considered when evaluating changes in serum urea concentrations.

Arterial blood gases

Arterial blood gas analysis provides a rapid and accurate assessment of oxygenation, alveolar ventilation and acid–base status, the three processes which maintain pH homeostasis. The maintenance of arterial CO₂ tension (PaCO₂) depends on the quantity of CO₂ produced in the body and its removal through alveolar ventilation. High PaCO₂ (>6.1 kPa) indicates alveolar hypoventilation and low PaCO₂ (<4.5 kPa) implies alveolar hyperventilation.

The adequate delivery of oxygen to the tissues depends upon the cardiopulmonary system, arterial oxygen tension (PaO₂), oxygen concentration in inspired air and haemoglobin content and its affinity for oxygen. Oxygen saturation is measured by pulse oximetry or by arterial blood gas analysis. Hypoxaemia is defined as a PaO₂ of less than 12 kPa at sea level in an adult patient breathing room air.

Bicarbonate and acid–base

Bicarbonate acts as part of the carbonic acid–bicarbonate buffer system, which is important to maintain acid–base balance and thus the pH of the blood. pH homeostasis is accomplished through the interaction of lungs, kidneys and blood buffers. This interaction is best represented by the Henderson–Hasselbalch equation, an equation by which the pH of a buffer solution, blood plasma being one, can be determined (Fig. 6.3).

Fig. 6.3 The Henderson–Hasselbalch equation. pH, plasma pH; pK_a, negative log to base 10 of the apparent overall dissociation constant of carbonic acid; [HCO₃^–], plasma bicarbonate concentration; α, solubility of carbon dioxide in blood at 37 °C; pCO₂, partial pressure of carbon dioxide in blood.

Plasma bicarbonate is controlled mainly by kidney and blood buffers. The lungs control the PaCO₂.

In metabolic acidosis such as that which occurs in renal failure, diabetic ketoacidosis or salicylate poisoning, bicarbonate levels fall. In metabolic alkalosis, the plasma bicarbonate concentration is high. This can occur, for instance, when there is a loss of hydrogen ions from the stomach, as in severe vomiting, or loss through the kidneys, as in mineralocorticoid excess or severe potassium depletion. In the latter situation, an increase in sodium reabsorption in the kidney results in bicarbonate retention and a loss of hydrogen ions. The blood buffer system of carbonic acid/bicarbonate base can act immediately to prevent excessive change in pH. The respiratory system takes a few minutes but the kidneys up to several days to readjust H⁺ ions concentration.

Glucose

The serum glucose concentration is largely determined by the balance of glucose moving into, and leaving, the extracellular compartment. In a healthy adult, this movement is capable of maintaining serum levels below 10 mmol/L, regardless of the intake of meals of varying carbohydrate content.

The renal tubules have the capacity to reabsorb glucose from the glomerular filtrate, and little unchanged glucose is normally lost from the body. Glucose in the urine (glycosuria) is normally only present when the concentration in serum exceeds 10 mmol/L, the renal threshold for total reabsorption.

Normal ranges for serum glucose concentrations are often quoted as non-fasting (<11.1 mmol/L) or fasting (3.3–6.0 mmol/L) concentration ranges. Fasting serum glucose levels between 6.1 and 7.0 mmol/L indicate impaired glucose tolerance. When symptoms are typical of diabetes, a fasting level above 7.0 mmol/L or a 2 h post-glucose or random serum glucose level ≥11.1 mmol/L is consistent with a diagnosis of diabetes. Other signs and symptoms, if present, are notably those attributable to an osmotic diuresis, and will suggest clinically the diagnosis of diabetes mellitus.

Glycated haemoglobin

Glucose binds to a part of the haemoglobin molecule to form a small glycated fraction. Normally, about 5% of haemoglobin is glycated, but this amount is dependent on the average blood glucose concentration over the lifespan of the red cells (about 120 days) and where red cell lifespan is reduced this leads to low glycated haemoglobin levels. The major component of the glycated fraction is referred to as HbA_1C.

Measurement of HbA_1C is well established as an indicator of chronic glycaemic control in patients with diabetes. Several methods exist for its determination, but the International Federation of Clinical Chemistry and Laboratory Medicine has recently approved a reference measurement procedure which is analytically specific. As a consequence, laboratories are moving over to measure the substance fraction of the valyl-I-fructosylated haemoglobin β-chains with the unit millimole per mole, and away from the non-specific HbA_1C as measured by various non-standardised laboratory procedures.

Uric acid

The production of uric acid, the end product of purine metabolism, is catalysed by xanthine oxidase, an enzyme linked to oxidative stress, endothelial dysfunction and heart failure. The purines, which are used for nucleic acid synthesis, are produced by the breakdown of nucleic acid from ingested meat or synthesised within the body.

Monosodium urate is the form in which uric acid usually exists at the normal pH of body fluids. The term urate is used to represent any salt of uric acid.

Two main factors contribute to elevated serum uric acid levels: an increased rate of formation and reduced excretion. Uric acid is poorly soluble and an elevation in serum concentration can readily result in deposition, as monosodium urate, in tissues or joints. Deposition usually precipitates an acute attack of gouty arthritis. The aim of treatment is to reduce the concentration of uric acid and prevent further attacks of gout. It has been hypothesised that measurement of urate could serve as a marker of cardiovascular risk because the serum uric acid level is an independent predictor of all causes of mortality in patients at high risk of cardiovascular disease, independent of diuretic use.

Albumin

Albumin is quantitatively the most important protein synthesised in the liver, with 10–15 g/day being produced in a healthy man. About 60% is located in the interstitial compartment of the ECF, the remainder in the smaller, but relatively impermeable, serum compartment where it is present at a higher concentration. The concentration in the serum is important in maintaining its volume since it accounts for approximately 80% of serum colloid osmotic pressure. A reduction in serum albumin concentration often results in oedema.

Albumin has an important role in binding, among others, calcium, bilirubin and many drugs. A reduction in serum albumin will increase free levels of agents which are normally bound and adverse effects can result if the ‘free’ entity is not rapidly cleared from the body.

The serum concentration of albumin depends on its rate of synthesis, volume of distribution and rate of catabolism. Synthesis falls in parallel with increasing severity of liver disease or in malnutrition states where there is an inadequate supply of amino acids to maintain albumin production. Synthesis also decreases in response to inflammatory mediators such as interleukin. A low serum albumin concentration will occur when the volume of distribution of albumin increases, as happens, for example, in cirrhosis with ascites, in fluid retention states such as pregnancy or where a shift of albumin from serum to interstitial fluid causes dilutional hypoalbuminaemia after parenteral infusion of excess protein-free fluid. The movement of albumin from serum into interstitial fluid is often associated with increased capillary permeability in post-operative patients or those with septicaemia.

Other causes of hypoalbuminaemia include catabolic states associated with a variety of illnesses and increased loss of albumin, either in urine from damaged kidneys, as occurs in the nephrotic syndrome, or via the skin following burns or a skin disorder such as psoriasis, or from the intestinal wall in a protein-losing enteropathy. The finding of hypoalbuminaemia and no other alteration in liver tests virtually rules out hepatic origin of this abnormality.

Albumin’s serum half-life of approximately 20 days precludes its use as an indicator of acute change in liver function but levels are of prognostic value in chronic disease.

An increase in serum albumin is rare and can be iatrogenic, for example, inappropriate infusion of albumin, or the result of dehydration or shock.

A shift of protein is known to occur physiologically when moving from lying down to the upright position. This can account for an increase in the serum albumin level of up to 10 g/L and can contribute to the variation in serum concentration of highly bound drugs which are therapeutically monitored.

Bilirubin

At the end of their life, RBCs are broken down by the reticuloendothelial system, mainly in the spleen. The haemoglobin molecules, which are subsequently liberated, are split into globin and haem. The globin enters the general protein pool, the iron in haem is reutilised, and the remaining tetrapyrrole ring of haem is degraded to bilirubin. Unconjugated bilirubin, which is water insoluble and fat soluble, is transported to the liver tightly bound to albumin. Unconjugated hyperbilirubinaemia in adults is most commonly the result of haemolysis, or Gilbert’s syndrome due to genetic defects in UDP-glucronyltransferase. It is actively taken up by hepatocytes, conjugated with glucuronic acid and excreted into bile. The conjugated bilirubin is water soluble and secreted rapidly into the gut where it is broken down by bacteria into urobilinogen, a colourless compound, which is subsequently oxidised in the colon to urobilin, a brown pigment excreted in faeces. Some of the urobilinogen is absorbed and most is subsequently re-excreted in bile (enterohepatic circulation). A small amount is absorbed into the systemic circulation and excreted in urine, where it too may be oxidised to urobilin. The presence of increased conjugated bilirubin is usually a sign of liver disease.

The liver produces 300 mg of bilirubin each day. However, because the mature liver can metabolise and excrete up to 3 g daily, serum bilirubin concentrations are not a sensitive test of liver function. As a screening test they rarely do other than confirm the presence or absence of jaundice. In chronic liver disease, however, changes in bilirubin concentrations over time do convey prognostic information.

An elevation of serum bilirubin concentration above 50 μmol/L (i.e. approximately 2.5 times the normal upper limit) will reveal itself as jaundice, seen best in the skin and sclerae. Elevated bilirubin levels can be caused by increased production of bilirubin (e.g. haemolysis, ineffective erythropoiesis), impaired transport into hepatocytes (e.g. interference with bilirubin uptake by drugs such as rifampicin or due to hepatitis), decreased excretion (e.g. with drugs such as rifampicin and methyltestosterone, intrahepatic obstruction due to cirrhosis, tumours, etc.) or a combination of the above factors.

The bilirubin in serum is normally unconjugated, bound to protein, not filtered by the glomeruli and does not normally appear in the urine. Bilirubin in the urine (bilirubinuria) is usually the result of an increase in serum concentration of conjugated bilirubin and indicates an underlying pathological disorder.

Enzymes

The enzymes measured in routine LFTs are listed in Table 6.1. Enzyme concentrations in the serum of healthy individuals are normally low. When cells are damaged, increased amounts of enzymes are detected as the intracellular contents are released into the blood.

It is important to remember that the assay of ‘serum enzymes’ is a measurement of catalytic activity and not actual enzyme concentration and that activity can vary depending on assay conditions. Consequently, the reference range may vary widely between laboratories.

While the measurement of enzymes may be very specific, the enzymes themselves may not be specific to a particular tissue or cell. Many enzymes arise in more than one tissue and an increase in the serum activity of one enzyme can represent damage to any one of the tissues which contain the enzymes. In practice, this problem may be clarified because some tissues contain two or more enzymes in different proportions which are released on damage. For example, alanine and aspartate transaminase both occur in cardiac muscle and liver cells, but their site of origin can often be differentiated, because there is more alanine transaminase in the liver than in the heart. In those situations where it is not possible to look at the relative ratio of enzymes, it is sometimes possible to differentiate the same enzyme from different tissues. Such enzymes have the same catalytic activity but differ in some other measurable property, and are referred to as isoenzymes.

The measured activity of an enzyme will be dependent upon the time it is sampled relative to its time of release from the cell. If a sample is drawn too early after a particular insult to a tissue there may be no detectable increase in enzyme activity. If it is drawn too late, the enzyme may have been cleared from the blood.

Alkaline phosphatase

Alkaline phosphatase is an enzyme which transports metabolites across cell membranes. Alkaline phosphatases are found in the canalicular plasma membrane of hepatocytes, in bone where they reflect bone building or osteoblastic activity, and in the intestinal wall and placenta, kidneys and leucocytes. Each site of origin produces a specific isoenzyme of alkaline phosphatase, which can be electrophoretically separated if concentrations are sufficiently high. Hepatic alkaline phosphatase is present on the surface of bile duct epithelia.

Disorders of the liver which can elevate alkaline phosphatase include intra- or extra-hepatic cholestasis, space-occupying lesions, for example, tumour or abscess, and hepatitis. Drug-induced liver injury, for example, by ACE inhibitors or oestrogens, may present with a cholestatic pattern, that is, a preferential increase in alkaline phosphatase.

Physiological increases in serum alkaline phosphatase activity also occur in pregnancy due to release of the placental isoenzyme and during periods of growth in children and adolescents when the bone isoenzyme is released.

Pathological increases in serum alkaline phosphatase of bone origin may arise in disorders such as osteomalacia and rickets, Paget’s disease of bone, bone tumours, renal bone disease, osteomyelitis and healing fractures. Alkaline phosphatase is also raised as part of the acute-phase response, for example, intestinal alkaline phosphatase may be raised in active inflammatory bowel disease. If in doubt, the origin of the enzyme can be indicated by assessment of γ-glutamyl transpeptidase (see next section) or electrophoresis to separate alkaline phosphatase isoenzymes.

Transaminases

The two transaminases of diagnostic use are aspartate transaminase (AST; also known as aspartate aminotransferase) and alanine transaminase (ALT; also known as alanine aminotransferase). These enzymes catalyse the transfer of α-amino groups from aspartate and alanine to the α-keto group of ketoglutaricacid to generate oxalacetic and pyruvic acid. They are found in many body tissues, with the highest concentration in hepatocytes and muscle cells. In the liver, ALT is localised solely in cytoplasm whereas AST is cytosolic and mitrochondrial.

Serum AST levels are increased in a variety of disorders including liver disease, crush injuries, severe tissue hypoxia, myocardial infarction, surgery, trauma, muscle disease and pancreatitis. ALT is elevated to a similar extent in the disorders listed which involve the liver, though to a lesser extent, if at all, in the other disorders. In the context of liver disease, increased transaminase activity indicates deranged integrity of hepatocyte plasma membranes and/or hepatocyte necrosis. They may be raised in all forms of viral and non-viral, acute and chronic liver disease, most markedly in acute viral, drug induced (e.g. paracetamol poisoning), alcohol related and ischaemic liver damage. Non-alcoholic fatty liver disease is now the most common cause of mild alteration of aminotransferase levels in the developed world.

γ-Glutamyl transpeptidase

γ-Glutamyl transpeptidase (Gamma GT; also known as γ-glutamyl transferase) is present in high concentrations in the liver, kidney and pancreas, where it is found within the endoplasmic reticulum of cells. It is a sensitive indicator of hepatobiliary disease but does not differentiate a cholestatic disorder from hepatocellular disease. It can also be elevated in alcoholic liver disease, hepatitis, cirrhosis and non-hepatic disease such as pancreatitis, congestive cardiac failure, chronic obstructive pulmonary disease and renal failure.

Serum levels of γ-glutamyl transpeptidase activity can be raised by enzyme induction by certain drugs such as phenytoin, phenobarbital, rifampicin and oral contraceptives.

Serum γ-glutamyl transpeptidase activity is usually raised in an individual with alcoholic liver disease. However, it can also be raised in heavy drinkers of alcohol who do not have liver damage, due to enzyme induction. Its activity can remain elevated for up to 4 weeks after stopping alcohol intake.

Although it lacks specificity, it has a high sensitivity for liver disease, and is thus useful for identifying the cause of a raised alkaline phosphatase level.

Ammonia

The concentration of free ammonia in the blood is very tightly regulated and is exceeded by two orders of magnitude by its derivative, urea. The normal capacity for urea production far exceeds the rate of free ammonia production by protein catabolism under normal circumstances, such that any increase in free blood ammonia concentration is a reflection of either biochemical or pharmacological impairment of urea cycle function or fairly extensive hepatic damage. Clinical signs of hyperammonaemia occur at concentrations >60 mmol/L and include anorexia, irritability, lethargy, vomiting, somnolence, disorientation, asterixis, cerebral oedema, coma and death; appearance of these findings is generally proportional to free ammonia concentration. Causes of hyperammonaemia include genetic defects in the urea cycle and disorders resulting in significant hepatic dysfunction. Ammonia plays an important role in the increase in brain water which occurs in acute liver failure. Measurement of the blood ammonia concentration in the evaluation of patients with known or suspected hepatic encephalopathy can help in diagnosis and assessing the effect of treatment. Valproic acid can induce hyperammonaemic encephalopathy as one of its adverse neurological effects.

Amylase

The pancreas and salivary glands are the main producers of serum amylase. The serum amylase concentration rises within the first 24 h of an attack of pancreatitis and then declines to normal over the following week. Although a number of abdominal and extra-abdominal conditions including loss of bowel integrity through infarction or perforation, chronic alcoholism, post-operative states and renal failure can result in a high amylase activity, in patients with the clinical picture of severe upper abdominal symptoms the specificity and sensitivity of an amylase level over 1000 IU/L in the diagnosis of pancreatitis is over 90%. There is a lack of prognostic significance of absolute values of amylase as values are directly related to the degree of pancreatic duct obstruction and inversely related to severity of pancreatic disease.

Troponins

Cardiac troponin I (cTnI) and cardiac troponin T (cTnT) are regulatory proteins that control the calcium-mediated interaction between actin and myosin in cardiac muscle. They are the preferred biomarker for myocardial necrosis as they have near absolute myocardial tissue specificity as well as high clinical sensitivity. The major international cardiac societies have introduced an international definition and classification for myocardial infarction which depends on the detection of a rise and/or fall (≥20%) of troponin with at least one value above the 99th percentile of the upper reference limit (URL) plus at least one of the following: (i) symptoms of ischaemia, or (ii) ECG evidence or imaging evidence of ischaemia. Sampling of cTn at two time points, usually admission and 12 h from worst pain, is usually needed, although if it is entirely clear that there has been a myocardial infarction, particularly in a late presentation, a second sample may not be needed. They contribute significantly to stratification of individuals with acute coronary syndromes, either alone or in combination with admission ECG or a predischarge exercise stress test. The decision as to whether to monitor cTnI or cTnT in a given laboratory is a balance between cost, availability of automated instrumentation and assay performance in which there is not yet standardisation between laboratories. Cardiac troponins offer extremely high tissue specificity and sensitivity but do not discriminate between ischaemic and non-ischaemic mechanisms of myocardial injury, such as myocarditis, cardiac surgery and sepsis. New, more sensitive assays are being developed, although studies will be required to define the clinical significance of minor releases of cTn and its relation to cardiac tissue viability.

Creatine kinase (CK)

CK is an enzyme which is present in relatively high concentrations in heart muscle, skeletal muscle and in brain in addition to being present in smooth muscle and other tissues. Levels are markedly increased following shock and circulatory failure, myocardial infarction and muscular dystrophies. Less marked increases have been reported following muscle injury, surgery, physical exercise, muscle cramp, an epileptic fit, intramuscular injection and hypothyroidism. The most important adverse effects associated with statins are myopathy and an increase in hepatic transaminases, both of which occur infrequently. Statin-associated myopathy represents a broad clinical spectrum of disorders, from mild muscle aches to severe pain and restriction in mobility, with grossly elevated CK levels. In rhabdomyolysis, a potentially life-threatening syndrome resulting from the breakdown of skeletal muscle fibres as a result of ischaemic crush injury, for example, large quantities of CK are measurable in the blood with the level of CK in the blood predicting the developments of acute renal failure. Medications and toxic substances that increase the risk of rhabdomyolysis are shown in Table 6.3.

Table 6.3 Medications and toxic substances that increase the risk of rhabdomyolysis

HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; LSD, lysergic acid diethylamide; MDMA, methylene dioxymethamphetamine.

CK has two protein subunits, M and B, which combine to form three isoenzymes, BB, MM and MB. BB is found in high concentrations in the brain, thyroid and some smooth muscle tissue. Little of this enzyme is present in the serum, even following damage to the brain. The enzyme found in serum of normal subjects is the MM isoenzyme which originates from skeletal muscle.

Cardiac tissue contains more of the MB isoenzyme than skeletal muscle. Following a myocardial infarction there is a characteristic increase in serum CK activity. Although measurement of activity of the MB isoenzyme was used in the past to detect myocardial damage, cardiac troponin measurement is now the preferred biomarker.

Lactate dehydrogenase (LD)

Lactate dehydrogenase has five isoenzymes (LD1–LD5). Total LD activity is rarely measured because of the lack of tissue specificity. Levels of activity are elevated following damage to the liver, skeletal muscle and kidneys, in both megaloblastic and immune haemolytic anaemias, and in intravascular haemolysis such as occurs in thrombotic thrombocytopenic purpura and paroxysmal nocturnal haemoglobinuria. In lymphoma, a high LD activity indicates a poor prognosis. Elevation of LD1 and LD2 occurs after myocardial infarction, renal infarction or megaloblastic anaemia; LD2 and LD3 are elevated in acute leukaemia; LD3 is often elevated in some malignancies; and LD5 is elevated after damage to liver or skeletal muscle.

RBC count

RBCs are produced in the bone marrow by the process of erythropoiesis. One of the major stimulants of this process is erythropoietin, produced mainly in the kidney. Immature erythroblasts develop into mature erythrocytes which are then released into the circulation:

Normally, only reticulocytes and non-nucleated mature erythrocytes are seen in the peripheral blood.

The lifespan of a mature red cell is usually about 120 days. If this is shortened, as for instance in haemolysis, the circulating mass of red cells is reduced and with it the supply of oxygen to tissues is decreased. In these circumstances, red cell production is enhanced in healthy bone marrow by an increased output of erythropoietin by the kidneys. Under normal circumstances red cells are destroyed by lodging in the spleen due to decreasing flexibility of the cells. They are removed by the reticuloendothelial system.

A high RBC (erythrocytosis or polycythaemia) indicates increased production by the bone marrow and may occur as a physiological response to hypoxia, as in chronic airways disease, or as a malignant condition of red cells such as in polycythaemia rubra vera.

Reticulocytes

Reticulocytes are the earliest non-nucleated red cells. They owe their name to the fine net-like appearance of their cytoplasm which can be seen, after appropriate staining, under the microscope and contains fine threads of ribonucleic acid (RNA) in a reticular network. Reticulocytes normally represent between 0.5% and 1.0% of the total RBC and do not feature significantly in a normal blood profile. However, increased production (reticulocytosis) can be detected in times of rapid red cell regeneration as occurs in response to haemorrhage or haemolysis. At such times the reticulocyte count may reach 40% of the RBC. The reticulocyte count may be useful in assessing the response of the marrow to iron, folate or vitamin B₁₂ therapy. The count peaks at about 7–10 days after starting such therapy and then subsides.

Mean cell volume (MCV)

The MCV is the average volume of a single red cell. It is measured in femtolitres (10⁻¹⁵ L). Terms such as ‘microcytic’ and ‘macrocytic’ are descriptive of a low and high MCV, respectively. They are useful in the process of identification of various types of anaemias such as caused by iron deficiency (microcytic) or vitamin B₁₂ or folic acid deficiency (megaloblastic or macrocytic).

Packed cell volume (PCV)

The PCV or haematocrit is the ratio of the volume occupied by red cells to the total volume of blood. It can be measured by centrifugation of a capillary tube of blood and then expressing the volume of red cells packed in the bottom as a percentage of the total volume. It is reported as a fraction of unity or as a percentage (e.g. 0.45% or 45%). The PCV is calculated nowadays as the product of the MCV and RBC. The PCV often reflects the RBC and will, therefore, be decreased in any sort of anaemia. It will be raised in polycythaemia. It may, however, be altered irrespective of the RBC, when the size of the red cell is abnormal, as in macrocytosis and microcytosis.

Mean cell haemoglobin (MCH)

The MCH is the average weight of haemoglobin contained in a red cell. It is measured in picograms (10⁻¹² g) and is calculated from the relationship:

The MCH is dependent on the size of the red cells as well as the concentration of haemoglobin in the cells. Thus, it is usually low in iron-deficiency anaemia when there is microcytosis and there is less haemoglobin in each cell, but it may be raised in macrocytic anaemia.

Mean cell haemoglobin concentration (MCHC)

The MCHC is a measure of the average concentration of haemoglobin in 100 mL of red cells. It is usually expressed as grams per litre but may be reported as a percentage. The MCHC will be reported as low in conditions of reduced haemoglobin synthesis, such as in iron-deficiency anaemia. In contrast, in macrocytic anaemias the MCHC may be normal or only slightly reduced because the large red cells may contain more haemoglobin, thus giving a concentration approximating that of normal cells. The MCHC can be raised in severe prolonged dehydration. If the MCHC is low, the descriptive term ‘hypochromic’ may be used (e.g. a hypochromic anaemia) whereas the term ‘normochromic’ describes a normal MCHC.

Haemoglobin

The haemoglobin concentration in men is normally greater than in women, reflecting in part the higher RBC in men. Lower concentrations in women are due, at least in part, to menstrual loss.

Haemoglobin is most commonly measured to detect anaemia. In some relatively rare genetic diseases, the haemoglobinopathies, alterations in the structure of the haemoglobin molecule can be detected by electrophoresis. Abnormal haemoglobins which can be detected in this manner include HbS (sickle haemoglobin in sickle cell disease) and HbA₂ found in β-thalassaemia carriers.

Platelets (thrombocytes)

Platelets are formed in the bone marrow. A marked reduction in platelet number (thrombocytopenia) may reflect either a depressed synthesis in the marrow or destruction of formed platelets.

Platelets are normally present in the circulation for 8–12 days. This is useful information when evaluating a possible drug-induced thrombocytopenia, since recovery should be fairly swift when the offending agent is withdrawn.

A small fall in the platelet count may be seen in pregnancy and following viral infections. Severe thrombocytopenia may result in spontaneous bleeding. A reduced platelet count is also found in disseminated intravascular coagulation, which manifests clinically as severe haemorrhages, particularly in the skin and results in rapid consumption of clotting factors and platelets.

An increased platelet count (thrombocytosis) occurs in malignancy, inflammatory disease and in response to blood loss.

White blood cell (WBC) count

White cells (leucocytes) are of two types: the granulocytes and the agranular cells. They are made up of various types of cells (Fig. 6.4) with different functions and it is logical to consider them separately. A haematology profile often reports a total white cell count and a differential count, the latter separating the composition of white cells into the various types.

Fig. 6.4 Types of white cells.

Erythrocyte sedimentation rate (ESR)

The ESR is a measure of the settling rate of red cells in a sample of anticoagulated blood, over a period of 1 h, in a cylindrical tube.

In youth, the normal value is less than 10 mmol/h, but normal values do rise with age. The Westergren method, performed under standardised conditions, is commonly used in haematology laboratories. The ESR is strongly correlated with the ability of red cells to aggregate into orderly stacks or rouleaux. In disease, the most common cause of a high ESR is an increased protein level in the blood, such as the increase in acute-phase proteins seen in inflammatory disease. Proteins are thought to affect the repellent surface charges on red cells and cause them to aggregate into rouleaux and hence the sedimentation rate increases. Although some conditions may cause a low ESR, the test is principally used to monitor inflammatory disease. The ESR may be raised in the active phase of rheumatoid arthritis, inflammatory bowel disease, malignant disease and infection. The ESR is non-specific and, therefore, of little diagnostic value, but serial tests can be helpful in following the progress of disease, and its response to treatment.

C-reactive protein (CRP)

CRP, named for its capacity to precipitate the somatic C-polysaccharide of streptococcus pneumoniae, was the first acute-phase protein to be described. This non-specific acute-phase response occurs in animals in response to tissue damage, infection, inflammation and malignancy. Production of CRP is rapidly and sensitively upregulated, in hepatocytes, under the control of cytokine (IL-6) originating at the site of pathology. It recognises altered self and foreign molecules, as a result of which it activates complement and generates pro-inflammatory cytokines and activation of the adaptive immune system.

Serum concentrations rise by about 6 h, peaking around 48 h. The serum half-life is constant at about 19 h, so serum level is determined by synthesis rate, which therefore reflects the intensity of the pathological process stimulating this, and falls rapidly when this ceases. CRP values are not diagnostic, however, but can only be interpreted in knowledge of all other clinical and pathological results. In most diseases, the circulating value of CRP reflects ongoing inflammation or tissue damage more accurately than do other acute-phase parameters such as serum viscosity or the ESR. Drugs reduce CRP values by affecting the underlying pathology providing the acute-phase stimulus.

Coagulation

Coagulation is the process by which a platelet and fibrin plug is formed to seal a site of injury or rupture in a blood vessel. The current model of a ‘coagulation network’ differs from the previous popular cascade scheme. It proposes that blood coagulation is localised on the surfaces of activated cells in three overlapping steps: initiation, amplification and propagation. Coagulation is initiated when a tissue factor (TF) bearing cell is exposed to blood flow, following either damage of endothelium such as by perforation of a vessel wall or activation by chemicals, cytokines or the inflammatory process. The formation of a clot then involves a complex interaction between platelets and factor VIII bound to von Willebrand factor which leave the vascular space and adhere to collagen and other matrix components at the site of injury. The coagulation process is amplified when enough thrombin is generated on or near the TF bearing cells to trigger full activation of platelets and coagulation co-factors on the platelet surface. It ends with the generation of sufficient thrombin, to clot fibrinogen. To prevent inappropriate propagation of the thrombus, the process is controlled by naturally occurring anticoagulants and the fibrinolytic system, the final effector of which is plasmin which cleaves fibrin into soluble degradation products. The interaction between TF and factor VII is the most important in the initiation of coagulation and many of the coagulation reactions occur on the surface of cells (particularly platelets). The cellular model of normal haemostasis is shown in Fig. 6.5. Despite the complexity of this model, the basic coagulation tests can still be interpreted in relation to the ‘intrinsic’, ‘extrinsic’, and ‘final common pathway’ components of the traditional and previously held cascade (Fig. 6.6). The extrinsic pathway can be considered to consist of the factor VIIa/TF complex working with the factor Xa/Va complex and the intrinsic pathway to consist of factor XIa working with the complexes of factors VIIIa/IXa and factors Xa/Va. The extrinsic pathway operates on the TF bearing cell to initiate and amplify coagulation with the intrinsic pathway operating on the activated platelet surface to produce the burst of thrombin to form and stabilise the fibrin clot.

Fig. 6.5 Normal haemostasis; the cellular model.

Fig. 6.6 Coagulation network.

One stage PT

Measuring the PT is the most commonly used method for monitoring oral anticoagulation therapy. The PT is responsive to depression of three of the four vitamin K dependent factors (factors II, VII and X). The PT is measured by adding calcium and thromboplastin (a phospholipid-protein extract of tissue that promotes the activation of factor X by factor VIII) to citrated plasma.

International Normalised Ratio (INR)

The results of the test are commonly expressed as a ratio of the PT time of the patient compared with that of the normal control. This is known as the INR, a system used to standardise reporting worldwide.

The ISI is the international sensitivity index and represents the responsiveness of a given thromboplastin to the reduction of vitamin K dependent clotting factors and is allocated to commercial preparations of thromboplastin to standardise them. More responsive thromboplastins have lower ISI values.

The target value varies according to the indication for the anticoagulant, but for most, including for thrombo-embolic prophylaxis in atrial fibrillation, is 2.5. For some indications including recurrent deep vein thrombosis and pulmonary embolism whilst on warfarin, the target is higher at 3.5.

The most common use of the PT and INR is to monitor oral anticoagulant therapy, but the PT is also useful in assessing liver function because of its dependence on the activity of clotting factors I, II, V, VII and X which are produced in the liver. It may be prolonged also by deficiency in vitamin K, and consumptive coagulopathy. Obstructive jaundice may decrease the absorption of vitamin K and thereby increase PT. PT will respond to parenteral administration of vitamin K, which is ineffective when jaundice is caused by decreased functioning liver mass.

Activated partial thromboplastin time (APTT)

The APTT is the most common method for monitoring unfractionated heparin therapy.

A thromboplastic reagent is added to an activator such as activated silicone or kaolin. If the activator is kaolin, the test may be referred to as the PTTK (partial thromboplastin time kaolin) or the KCCT (kaolin–cephalin clotting time). Cephalin is a brain extract supplying the thromboplastin.

The mixture of thromboplastin and activator is mixed with citrated plasma to which calcium is added, and the time for the mixture to clot is recorded. The desirable APTT for optimal heparin therapy is between 1.5 and 2.5 times the normal control.

Low molecular weight heparins are effective and safe for the prevention and treatment of venous thromboembolism, and because they provide more predictable anticoagulant activity than unfractionated heparin it is usually not necessary to monitor the APTT during treatment. Laboratory monitoring using an anti-factor Xa assay may be of value in certain clinical settings, including patients with renal insufficiency, and use of fractionated heparin for prolonged periods in pregnancy or in newborns and children.

D-dimers

D-dimers are degradation products of fibrin clots, formed by the sequential action of three enzymes, thrombin, factor VIIIa and plasmin which degrades cross-linked fibrin to release fibrin degradation products and expose the D-dimer antigen. D-dimer assays measure an epitope on fibrin degradation products using monoclonal antibodies. As each has its own unique specificity, there is no standard unit of measurement or performance and clinicians need to be aware of that of their institution. Levels of D-dimers in the blood are raised in conditions associated with coagulation and are used to detect venous thromboembolism, although they are influenced by the presence of co-morbid conditions such as cancer, surgery and infectious diseases. D-dimer measurement has been most comprehensively validated in the exclusion of venous thromboembolism in certain patient populations and in the diagnosis and monitoring of coagulation activation in disseminated intravascular coagulation. More recently, assays are being used in the prediction of the risk of VTE recurrence. Diagnosis of DVT or PE should include a clinical probability assessment as well as D-dimer measurements. In patients with a low clinical probability of pulmonary embolism, a negative quantitative D-dimer test result effectively excludes PE. In suspected DVTs, D-dimer measurements combined with a clinical prediction score and compression ultrasonography study have a high predictive value.

Xanthochromia

Xanthochromia is a yellow discolouration of cerebrospinal fluid caused by haemoglobin catabolism. It is thought to arise within several hours of subarachnoid haemorrhage (SAH) and can help to distinguish the elevated red cell count observed after traumatic lumbar puncture from that observed following SAH, particularly if few red cells are present. Spectrophotometry to detect the presence of both oxyhaemoglobin and bilirubin, which both contribute to xanthochromia following SAH, is used, although some hospitals rely on visual inspection.

Iron, transferrin and iron binding

Iron is necessary for the functioning of all mammalian cells, but is particularly important in cells producing haemoglobin and myoglobin. Iron circulating in the serum is bound to transferrin. It leaves the serum pool and enters the bone marrow where it becomes incorporated into haemoglobin in developing red cells. Serum iron levels are extremely labile and fluctuate throughout the day and, therefore, provide little useful information about iron status.

Transferrin, a simple polypeptide chain with two iron binding sites, is the plasma iron binding protein which facilitates its delivery to cells bearing transferrin receptors. Measurement of total iron binding capacity (TIBC), from which the percentage of transferrin saturation with iron may be calculated, gives more information. Saturation of 16% or lower is usually taken to indicate an iron deficiency, as is a raised TIBC of greater than 70 μmol/L.

Ferritin is an iron storage protein found in cell cytosol. It acts as a depot, accepting excess iron and allowing for mobilisation of iron when needed. Serum ferritin measurement is the test of choice in patients suspected of having iron deficiency anaemia.

In normal individuals, the serum ferritin concentration is directly related to the available storage iron in the body. The serum ferritin level falls below the normal range in iron deficiency anaemia, and its measurement can provide a useful monitor for repletion of iron stores after iron therapy. Ferritin is an acute-phase protein and levels may be normal or high in the anaemia of chronic disease, such as occurs in rheumatoid arthritis or chronic renal disease.

Iron balance is regulated by hepcidin, a circulating peptide hormone, which aims to provide iron as needed, whilst avoiding excess iron promoting formation of toxic oxygen radicals.

Iron overload causes high concentrations of serum ferritin, as can liver disease and some forms of cancer. Genetic iron overload results from mutations in molecules which regulate hepcidin production or activity.

Vitamin B₁₂ and folate

In the haematology literature, B₁₂ refers not only to cyanocobalamin but also to several other cobalamins with identical nutritional properties. Folic acid, which can designate a specific compound, pteroylgutamic acid, is also more commonly used as a general term for the folates. Deficiency of cobalamin can result both in anaemia, usually macrocytic, and neurological disease, including neuropathies, dementia and psychosis. Folate deficiency produces anaemia, macrocytosis, depression, dementia and neural tube defects.

Liver disease tends to increase B₁₂ levels, and they may be reduced in folate deficient patients: malabsorption of B₁₂ may result from long-term ingestion of antacids such as proton-pump inhibitors or H₂-receptor antagonists or biguanides (metformin). Serum folate levels tend to increase in B₁₂ deficiency, and alcohol can reduce levels. RBC folate is a better measure of folate tissue stores.

Current assays analyse total B₁₂ concentration, only a small percentage of which is metabolically active. Varying test sensitivities and specificities result from the lack of a precise ‘gold standard’ for the diagnosis of cobalamin deficiency. In the future, new assays for the active component which is carried on holotranscobalamin may be of greater relevance if their clinical usefulness can be established.