Because of its dire consequences, great efforts have been made  to determine the best tools for the early and accurate diagnosis of acute myocardial infarction (AMI). The World Health Organization has established three criteria for thå diagnosis of AMI:

1.     History: The history is typical if acute, severe, and prolonged chest pain is present.

2.     ECG changes.

3.     Serum cardiac markers: Unequivocal change consist­ing of serial enzyme/protein changes, with an initial rise and subsequent fall of serum concentrations.

Because a single, diagnostic laboratory test that will quickly and accurately assess cardiac function does not exist, a combination of cardiac markers is required, the search for a cardiac marker that would be useful in eval­uating many types of heart conditions continues; the fol­lowing features would be required for an ideal marker:

    The marker should be absolutely heart specific to al­low reliable diagnosis of myocardial damage in the presence of skeletal muscle injury.

    The marker should be highly sensitive to detect even minor heart damage.

    The marker should be able to differentiate reversible from irreversible damage.

    In acute myocardial infarction, the marker should al­low monitoring of reperfusion therapy and estimation of infarct size and prognosis; 

    The marker should bå stable and the measurement rapid, easy to perform, quantitative, and cost effective.

    The marker should not be detectable in patients who do not have myocardial damage

The diagnosis of the acute coronary syndrome

The diagnosis of myocardial infarction has for the past two decades been based on WHO criteria which comprise a typical history of chest pain, the presence of diagnostic ECG abnormalities and a rise in biochemical markers. The presence of two or more of these three has defined the diagnosis. In recent years, this long-established definition has been overtaken by the advent of the tro­ponins, which are more sensitive biochemical markers. Many studies have shown that troponins are released in some patients without conven­tional ECG changes of infarction, in particular ele­vation of the ST segment and T wave inversion. These patients are found to be at increased risk of subsequent cardiac events. This has given rise to the concept of 'acute coronary syndromes', which comprise unstable angina with or without a rise in troponin, non-ST segment elevation myocar­dial    infarction    and    ST    segment    elevation myocardial infarction. Troponin measurements can thus stratify patients into different risk cate­gories and therefore contribute to planning treat­ment and determining the need for more investigation, such as angiography.

However, the use of troponin measurement potentially reveals biochemical changes that would not previously have been detected in patients with chest pain. This has major implications in the defi­nition of myocardial infarction, and of course fc i the patients who may now be given this diagnosis, but who would formerly have been reassured that they had not had an infarct. Expert bodies are ye: to reach an international consensus, with some suggesting that a diagnosis of myocardial infarction should be given to all patients with any rise in tro­ponin, and others reserving this definition for patients whose rise exceeds an arbitrary cut-off level and who have ECG changes.

Biochemical tests in myocardial infarction and ischaemia

After myocardial infarction, a number of intracellular proteins are released from the damaged cells The proteins of major diagnostic interest include:

  Troponin I and troponin T;

  Enzymes, such as CK, CK-MB, AST and LDH (or 'heart-specific' LDH);


  Ischaemia modified albumin.

The troponins and CK will be considered in the greatest detail, since they are the most widely established biochemical indices of myocardial damage, with the troponins largely taking the place of the enzymes in recent years. Myoglobin is also a sensitive index of myocardial damage, and it rises very rapidly after the event. However, it is non-specific, since it is raised following any form of muscle damage. Ischaemia modified albumin is a new biochemical marker of myocardial ischaemia.



Time-course of changes

After a myocardial infarction, the time-course of plasma biochemical markers always follows the same general pattern (Figure 11.1). After an initial lag' phase of at least 3 h, during which levels remain normal, they rise rapidly to a peak between 18 and 36 h, and then return to normal at rates that depend on the half-life of each marker m plasma. The biphasic response of troponins with a rapid rise and prolonged elevation, and the rapid rise and fall of CK and CK-MB activity should be particularly noted.

Thrombolytic therapy

In  patients treated with thrombolytic agents, the general pattern of plasma marker changes shown in  Figure  11.1  is  slightly modified.  Following successful thrombolytic therapy (e.g. with streptokinase), there is a 'washout' of markers from the infarcted area, and levels rise rapidly to reach an early peak, at 10-18 h.


Optimal times for blood sampling

In many patients, two samples should be taken. A sample taken on admission, if elevated, will make the required diagnosis, but if not elevated will not rule out the diagnosis since insufficient time may have elapsed for a significant CK or troponin rise to have occurred. A second sample can then be taken between 6 and 12 h after the onset of symptoms (Table 11.1).



Except for the occasional patient seen for the first time 2 days or more after the episode, in whom troponin (or LD) measurements might still be useful, it is very rarely of any value to take sam­ples for plasma markers after 48 h from the onset of symptoms that suggest a diagnosis of myocar­dial infarction.

Selection of tests

Plasma troponin and CK measurements are requested most often early after the episode of chest pain. At a later stage total LD or 'heart-specific' LD may be requested, particularly if more than 36 h since the episode of chest pain, but in practice this has largely been supplanted by the measurement of troponin.


The troponin complex is exclusively present in striated muscle fibres and regulates the calcium-mediated interactions of actin and myosin.  It comprises equimolar quantities of the structurally unrelated proteins troponin T, troponin I and troponin C. Troponin T binds tropomyosin; troponin I is an inhibitory protein and troponin C is responsible for binding calcium. Three distinct isoforms of troponin T and I exist, one each in slow twitch and fast twitch fibres of skeletal muscle, and one in cardiac muscle. These cardiac-specific forms of troponin can be recognised and measured in the plasma. There are 2 isoforms of troponin C, one found in slow twitch and car­diac muscle, and one in fast twitch muscle, so there is not a troponin C species unique to myocardium. In human heart the cardiac-specific troponin T and troponin I are largely insoluble, but 3-5 % exists as a soluble cytoplasmic pool. Following cardiac myocyte necrosis, this soluble fraction probably accounts for the early rapid release of troponin into the circulation, and the slower release of the insolu­ble fraction accounts for the prolonged plateau of troponin release. The existence of the cardiac-specific isoforms of these troponins makes them the most specific of all the biochemical markers for cardiac damage. Under normal circumstances there is no cardiac troponin T or I detectable in the circulation by currently available assays, so any detectable rise is of significance, contributing to these tests' high sensitivity. However, troponin may also sometimes be elevated in renal failure, severe heart failure and acute pulmonary embolism In general, although the initial rise in care . troponins after myocardial infarction occurs a about the same time as CK and CK-MB, this rise continues for longer than for most of the enzymes because of continuing release of 'insoluble' protein from the infarcted muscle. Because of the inherent sensitivity of these tests for myocardial damage, increases are seen in some patients with angina as described above, as well as those with infarcts. Cardiac troponin measurements are par­ticularly useful in excluding the  diagnosis  of myocardial damage, particularly after 12 h following chest pain or other symptoms, and in patient: who are likely to have concurrent cardiac and skeletal muscle damage.

Cardiac myosin light chains, (MLC) are also involved
with muscle contractions. They were first thought to be
unique myocardial proteins, but recent research has de­
termined that MLC is no more specific for cardiac injury
than CK-MB determinations. Like the troponins, MLC is
released from reversibly ischemic tissue.


Creatine kinase (CK)

Creatine kinase (CK) is a cytosolic enzyme involved in the transfer of energy in muscle metabolism. It is a dimer comprised of two subunits (the B, or brain form, and the M, or muscle form), resulting in three CK isoenzymes. The CK-BB (CK1) isoenzyme is of brain origin and only found in the blood if the blood-brain barrier- has been breached. CK-MM (CK3) isoenzyme accounts for most of the CK activity in skeletal muscle, whereas CK-MB (CK2) has the most specificity for cardiac muscle, even though it accounts for only 3-20 % of total CK activity in the heart.

As a marker of early AMI, total CK shows sensi­tivity of only about 40 % and specificity of only 80 %.

Skeletal muscle has a very high total CK content over 98 % normally comprises CK-MM, less than 2 % CK-MB. CK-MB may rise to 5-15 % in some patients with muscle disease, and also in athlete in training.

1 Cardiac muscle also has a high CK content. It comprises 70-80 % CK-MM and 20-30 % CK-MB. As a general rule, cardiac muscle is the only tissue with more than 5 % CK-MB.

2 Other organs, such as brain, contain less CK often CK-BB. However, CK-BB rarely appears in plasma  and  is  not  of  diagnostic  importance.
Plasma normally contains more than 95 % of its
CK as CK-MM.

Plasma CK is valuable in the diagnosis of myocardial infarction and some muscle disease;. Increases, sometimes large, may occur after trauma or surgical operations, intramuscular injections, in comatose patients, diabetic ketoacidosis, acute renal failure and hypothyroidism and after prolonged muscular exercise, especially in unfit individuals.

Creatine kinase and muscle disease

Plasma CK, AST, LD and ALT activities may be increased in muscle disease. However, plasma total CK is usually the measurement of choice, irrespec­tive of the aetiology of the disorder, since it is increased in the greatest number of cases and shows the largest changes.

Muscular dystrophy In Duchenne-type dystrophy, high plasma CK activity is present from birth, before the onset of clinical signs. During the early clinical stages of the disease, very high activities ire usually present, but these tend to fall as the Terminal stages of the disease are reached. Smaller CK increases are present in other forms of muscular dystrophy. About 75 % of female carriers of the Duchenne dystrophy gene have small increases in plasma CK activity.

Malignant hyperpyrexia This is a rare but serious dis­order, characterised by raised body temperature, convulsions and shock following general anaesthesia. Many of the patients show evidence of myopathy. Extremely high plasma CK activities are seen in the acute, postanaesthetic stage, but smaller increases often persist and can also be detected in the relatives of affected patients. Preoperative screening of plasma CK is not a reli­able way of detecting patients liable to develop malignant hyperpyrexia, and should be limited to those patients with a family history of anaesthetic deaths or of malignant hyperpyrexia.

Miscellaneous muscle diseases CK is variably increased in various myopathies, including that due to alcohol. It is also raised in polymyositis.

Neurogenic muscle disease Plasma CK activity is usu­ally normal in peripheral neuritis, poliomyelitis and motor neurone disease.


CK-MB isoenzyme

CK-MB is a more sensitive and specific test for myocardial damage than total CK. However, its transient rise and the lack of reliability of some methods have weighed against its general introduc­tion as the first-line test for myocardial infarction in all laboratories. Its use has been largely over­taken by the widespread availability of troponin measurement. If troponin measurement is not readily available, plasma CK-MB measurements (preferably by a mass measurement method) may help in the following circumstances:

1 When very early evidence of infarction is required (less than 8 h from the onset of symptoms).

2 When investigating post-operative or traumatised patients for suspected myocardial infarc­tion.  In  these  patients,  plasma  CK-MB  (and troponin)  remains normal in the absence of myocardial damage, whereas total CK and LD activities are often increased in plasma for non-cardiac causes.

3 In patients suspected of having had a second infarct within a few days of the first. It is easier to show that a second rise in plasma enzyme activity has occurred if the activity of the enzyme that is being measured rises and falls rapidly after the previous incident involving myocardial damage (Figure 11.1).

4 When concurrent release of CK-MM is likely (e.g. after an intramuscular injection).

Ischaemia modified albumin

At the time of admission after chest pain due to cardiac ischaemia, not all patients who are des­tined to develop ECG changes or elevated troponins will have done so, since insufficient time will have passed. This means that at this time the sensitivity of these tests may be as low as 50 %. In addition, some patients with cardiac ischaemia will not develop elevated troponin levels because no myocardial necrosis occurs, and may have nor­mal or uninterpretable ECGs. There is, therefore, a need for an early and sensitive marker of cardiac ischaemia, and the measurement of 'ischaemia modified albumin' may find a role here.

Normally the N-terminus of albumin strongly binds transition metals such as copper, cobalt or nickel. In patients with myocardial ischaemia the structure of the N-terminus is altered in such a way that it can no longer bind these metals, prob­ably as a result of exposure to reactive oxygen species generated during ischaemia and/or reper-fusion. This can be measured as a reduction in albumin's ability to bind cobalt.

This test has shown early promise in the identi­fication of patients with chest pain due to cardiac ischaemia, with earlier diagnosis and greater sensi­tivity than either ECG or troponin. However, it is not yet in general clinical use, and its precise role in patient management is yet to be established.

The diagnosis of heart failure

Heart failure is a complex clinical condition in which the heart's ability to pump is compromised by one or more of a number of underlying con­ditions, commonly ischaemic heart disease, but also heart valve abnormalities. The prognosis is poor if untreated, with a two-year survival rate of under 50 %.

The diagnosis of heart failure can be difficult, especially since the usual presenting symptoms such as breathlessness or ankle swelling are com­mon and can be due to many different conditions. Physical examination is neither sensitive nor spe­cific for heart failure, even in expert hands, with incorrect diagnoses in up to 50 % of patients. The definitive diagnosis is best made by echocardiography, but access to this may be limited or delayed.

1.     B natriuretic peptide (BNP) is a neurohormone secreted by cardiac myocytes in response to volume expansion and pressure overload, and plays a role in circulatory homeostasis. In heart failure the level of BNP increases, enabling differ­entiation of cardiac and pulmonary causes of breathlessness. It has an evolving role in the diag­nosis of heart failure in both primary care and in the emergency setting, since it costs considerably less than echocardiography, and the result can be available much more rapidly.

Laboratory and point of care assays for BNP and for    the    inactive    peptide    N-terminal-proBNP (NT-proBNP) are available, and provide qualitively similar information. Their accuracy is greatr in patients with more severe disease and poorest in  those already receiving treatment. Levels rise with age, so age-related cut-offs should be used.

A number of other conditions can cause elevated BNP levels, but in a patient, who is not on heart failure treatment, if levels are below the cut-off level then heart failure is highly unlikely, and the patient should be investigated for other conditions. If the level is elevated the patient should proceed to further assessment, including echocar­diography. The introduction of this strategy has the potential to speed up accurate diagnosis of heart failure, and to save money by restricting the use of echocardiography to those patients mos likely to benefit from its use.

2.     Glycogen Phosphorylase Isoenzyme BB (GPBB)

GPBB is a glycolytic enzyme that plays an essential role in the regulation of carbohydrate metabolism by mobi­lizing glycogen. It is not specific for cardiac tissue, but it is significantly more sensitive than CK, CK-MB mass, myoglobin, and TnT in patients with AMI during the first 3-4 hours after the onset of chest pain. In most AMI pa­tients, GPBB increases between 1 and 4 hours from chest pain onset and returns to within the reference level within 1-2 days.

3.     Heart Fatty Acid-Binding Protein

Heart fatty acid-binding protein (H-FABP) is a low-molecular-weight protein found in large quantities in the cytoplasm of myocardial and muscle cells that is involved in fatty acid metabolism and lipid homeostasis. Although H-FABP is not cardiac-specific, die H-FABP content of skeletal muscle is only 10-30 % of that found in cardiac muscle, whereas the skeletal muscle content of myoglo­bin is approximately twice that of cardiac tissue. Thus, H-FABP is expected to be a more sensitive and specific marker than myoglobin for use in die early detection of myocardial injury. It increases rapidly upon cellular damage, usually within 2-4 hours, peaks within 5-10 hours, and returns to normal within 24-36 hours after onset of chest pain. The magnitude of die increase in plasma H-FABP has also demonstrated a good correlation with the size of the infarction.

4.     Carbonic Anhydrase (CA) Isoenzyme III

CA is a soluble enzyme that catalyzes the hydration of carbon dioxide to bicarbonate and a proton and is in­volved in pH regulation, transport of ions, water and electrolyte balance, and metabolism of carbohydrates, urea, and lipids. There are seven CA isoenzymes with a wide range of tissue distribution but a major site of CA activity is skeletal muscle. CAIII is not found in cardiac muscle and, therefore, can be used to differentiate be­tween skeletal muscle and cardiac muscle damage when performed in conjunction with a more heart-specific analyte such as myoglobin. In the patient with actual or possible coexisting skeletal muscle injury, TnT or TnI would still be the markers of choice for confirming or ex­cluding myocardial damage.


Markers of Inflammation and Coagulation Disorders

1.     High sensitivity C - reactive protein (Hs-CRP)

Studies have evaluated several acute-phase proteins as potential markers for cardiovascular risk assessment, and there is evidence that C-reactive protein (CRP) is a reli­able predictor of acute coronary syndrome risk. CRP is an acute-phase reactant produced primarily by the liver. It is stimulated by interleukin-6 and increases rapidly with inflammation. The plasma concentration of CRP is determined mainly by its synthesis rate and, provided normal liver function exists; is a sensitive marker for on­going chronic inflammation that is not affected by is chemic injury. It rises significantly in response to injury, infection, or other inflammatory conditions-end is not present in appreciable amounts in healthy individuals. Although it is a nonspecific response to inflammation, its presence indicates an inflammatory process within the body. The increases in CRP have been proven to be minimal in acute coronary syndromes,  often remaining within the established reference range. Reliable, automated high sensitivity assays for CRP (hs-CRP) have been developed that allow detection of the small creases of CRP often seen in cardiac disease.

Epidemiologic data document a positive association between hs CRP and the prevalence of coronary artery disease. Elevated baseline levels of hs-CRP are correlate with higher risk of future cardiovascular morbidity an mortality among those with and without clinical evidence of vascular disease. In patients with established vascular disease, each standard deviation increase in| baseline hs-CRP is associated with a 45 % increase in relative risk of nonfatal myocardial infarction or sudden cardiac death over 2 years of follow-up.

hs-CRP also demonstrates prognostic capacity in those who do not yet have a diagnosis of vascular disease. A mild elevation of baseline levels of hs-CRP among apparently healthy individuals is associated with higher  long-term risk for future cardiovascular events. This predictive capacity offers patients the ability to receive treat­ment to reduce inflammation and, thus, their risk.

2.     Fibrinogen

Fibrinogen is a soluble glycoprotein produced in the liver and involved in platelet aggregation and coagulation. It is also  acute-phase protein produced in response to inflammation. A relationship has been established between elevated  levels of fibrinogen and risk of cardiovascular disease and may serve as a marker of long-term prognosis. Including measurement of fibrinogen in screening for cardio-vascular risk may be valuable in identifying persons who may benefit from aggressive preventive strategies.

3.     D- Dimer

is the end product of the ongoing process of thrombus formation and dissolution that occurs at the site active plaques in acute coronary syndromes. Because this process precedes myocardial cell damage and release protein contents, it can be used for early detection.  It remains elevated for days so it may be an easily able physiologic marker of an unstable plaque even when the troponins or CK-MB are not increased, potentially identifying high-risk patients who otherwise might be missed. D-Dimer lacks specificity for cardiac damage as it is increased in other conditions that cause thrombosis. Elevations of D-Dimer have been shown to be useful in predicting risk for future cardiac events.


Patient-Focused Cardiac Tests

It is widely accepted that early diagnosis of patients with AMI will result in less cardiac tissue damage, fewer com­plications, reduced hospital length of stay, and faster re­covery. Also, treatment options, such as thrombolytic therapy or angioplasty, that can prevent further damage to the heart must be administered in a timely fashion. Typically, 90 % of patients admitted to the hospital re­quire biochemical testing to confirm or exclude AMI. The American College of Cardiology and the American Heart Association recommend that the initial patient evaluation be performed within 20 minutes of arrival to the emergency department (ED) and that the optimum turnaround time (TAT) from patient arrival to the avail­ability of test results for cardiac markers should be less than 30 minutes. The National Academy of Clinical Biochemistry Standards of Laboratory Practice recom­mends that cardiac marker results should be available within 1 hour of sampling. The pressure to comply with these standards is great, as well as difficult to achieve, in large institutions with busy EDs. Point-of-care (ÐÎÑ) testing for cardiac markers is one strategy to reduce turnaround time and the recent development of devices for performing whole blood cardiac assays at the patient's bedside has made it feasible to meet these strict guidelines.

The Role of the Laboratory in Monitoring Heart Disease

The laboratory's role in monitoring heart function prima­rily involves measuring the effects of the heart on other organs, such as the lungs, liver, and kidney. Arterial blood gases measure the patient's acid-base and oxygen status and are used to determine the respiratory acidosis and elevated carbon dioxide levels that are often seen in patients with heart disease. The patient with edema will develop electrolyte and osmolality changes as a result of fluid retention and ionic redistribution. Decreased cardiac output results in sodium retention by the kidneys, but also causes increased fluid retention; therefore, the serum sodium generally stays within the reference ranges may be slightly decreased. Serum electrolyte determi-nations, including sodium, potassium, chloride, and calcium, are important to monitor diuretic and drug therapy patients with heart disease.

Elevations of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) are often seen in patients with chronic right ven-tricular failure  and the -γ-glutamyltransferase (GGT) value may be twice the value of the upper limit of the normal range in congestive heart failure, suggesting liver

congestion and damage.

Lipid evaluation will assess risk for coronary artery disease. Maintenance of near-normal HDL-cholesterol, LDL-cholesterol, and triglyceride levels is highly recommended for cardiac patients. Determination of a lipoprotein similar to  LDL, called lipoprotein (a), may also be indicated as it is an independent risk factor associated with development of premature coronary artery and vascular disease.

The patient who has secondary heart failure due to thyroid dysfunction can be identified by a highly sensitive thyroid-stimulating hormone assay. The laboratory is also invaluable for monitoring therapeutic drugs following the diagnosis of heart disease.

The routine complete blood count is important for detecting anemia and infection. Hemolysis may indicate additional testing for hemoglobinuria and myoglobinuria,

indicators of cardiovascular damage and myocardial disease. An increase in white blood cells may indicate pericarditis, endocarditis, or valvular infections. If kidney dysfunction has occurred as a result of die heart disease, anemia due to decreased production of renal erythropoi­etin may develop.

An infection associated with pericarditis, endocarditis, and valvular problems would be identified by blood cul­tures. Cultures of specimens from the pericardium and endocardium might also be performed if a pericardial in­fection is suspected.

The laboratory's role during the treatment of heart dis­ease may also extend to providing blood components when surgical intervention is needed. Bypass grafts, cor­recting valvular defects, and other surgical procedures to correct heart failure may involve the use of blood com­ponents during the surgery and the patient's recovery.

Cardiovascular risk factors

Many factors are associated with or cause increased cardiovascular risk. These can pragmatically be divided into those which cannot be influ­enced and those which can be influenced and reduction of which has been demonstrated to reduce risk. Those that cannot be influence; include a family history of premature vascular disease, age and pre-existing vascular disease. Those whose modification has an established role include cigarette smoking and hypertension (not considered further here), diabetes.  Lipid metabolism and the hyperlipi­daemias are covered in some detail below followed by a description of how cardiovasculr risk is assessed and treated. Other novel biochem­ical markers of vascular risk are briefly described.


Lipids act as energy stores (triglycerides) and as important structural components of cells (cholesterol and phospholipids). They also have specialised functions (e.g. as adrenal and sex hormones). The main lipids, being insoluble in water, are transported in plasma as particulate complexes with proteins, the lipoproteins. From the clinical viewpoint, it is the strong relationship between plasma lipid levels and the incidence of ischaemic vascular disease, particuirly of the coronary arteries, that is of major importance. In the following section, we outline:

1The biochemistry of the main body lipids.

2The mechanisms for lipid transport in plasma.

3The importance of lipids, and other factors, in the pathogenesis of arterial disease.

4       The role of plasma lipid measurements in the management of hyperlipidaemia and in cardiovas­cular risk reduction.


This is a steroid that is present in the diet, but it is mainly synthesised in the liver and small intestine, the rate-limiting step being catalysed by ß-hydroxy-ß-methylglutaryl-coenzyme A (HMG-CoA) reductase. Cholesterol is a major component of cell membranes, and acts as the substrate for steroid hormone formation in the adrenals and the gonads. It is present in plasma mainly esterified with fatty acids. The body cannot break down the sterol nucleus, so cholesterol is either excreted unchanged in bile or converted to bile acids and then excreted. Cholesterol and bile acids both undergo an entero-hepatic circulation.


These are fatty acid esters of glycerol, and are the main lipids in the diet. They are broken down in the small intestine to a mixture of monoglycerides, fatty acids and glycerol. These products are absorbed, and Triglycerides are resynthesised from them in the mucosal cell. Most of these exogenous triglycerides pass into plasma as chylomicrons.

Endogenous triglyceride synthesis occurs in the .:ver from fatty acids and glycerol. The triglyc­erides synthesised in this way are transported as YLDL.

Fatty acids

These are mostly straight-chain monocarboxylic acids. They are mainly derived from dietary or tissue triglyceride, but the body can also synthesise most of them, apart from certain polyunsaturated (essential) fatty acids. Fatty acids act as an alterna­tive or additional energy source to glucose.


These have a structure similar to triglycerides, but a polar group (e.g. phosphorylcholine) replaces one of the three fatty acid components. The pres­ence of both polar and nonpolar (fatty acid) groups gives the phospholipids their characteristic detergent properties. Phospholipids are mainly synthesised in the liver and small intestine; they are important constituents of cells, and are often present in cell membranes.


Cholesterol and its esters, triglycerides and phos­pholipids are all transported in plasma as lipoprotein (Table 11.2) particles. Fatty acids are transported bound to albumin.


Lipoprotein particles comprise a peripheral enve­lope, consisting mainly of phospholipids and free cholesterol (which each have both water-soluble polar and lipid-soluble nonpolar groups) with some apolipoproteins, and a central nonpolar core (mostly triglyceride and esterified cholesterol). The mole­cules in the envelope are distributed in a single layer in such a way that the polar groups face out towards the surrounding plasma, while the nonpolar groups face inwards towards the lipid core in which the insoluble lipids are carried. Most lipoproteins are assembled in the liver or small intestine.

Five main types of lipoprotein particle can be recognised:

1  Chylomicrons are the principal form in which dietary triglycerides are carried to the tissues.

2  Very low density lipoproteins (VLDL) are triglyceride-rich particles that form the major route whereby endogenous triglycerides are carried to the tissues from the liver and, to a lesser extent, from then small intestine.

3  Intermediate-density lipoproteins (IDL or 'VLDL remnants') are particles formed by the removal of triglycerides  from  VLDL  during  the  transition from VLDL to LDL.

4  Low-density lipoproteins (LDL) are cholesterol- rich particles, formed from IDL by the removal of more triglyceride and apolipoprotein. Increased plasma [LDL cholesterol], and hence plasma [total cholesterol], is positively correlated with the inci­dence of ischaemic heart disease.

 5 High-density lipoproteins (HDL) are of two main types, HDL2 and HDL3. They act as a means whereby cholesterol can be transported from peripheral cells to the liver, prior to excretion. Increased plasma [HDL cholesterol] is negatively correlated with the incidence of ischaemic heart disease, presumably explained by its role in trans­porting cholesterol from the periphery.

A sixth type of lipoprotein particle, Lp(a), is synthesised in the liver and has about the same lipid composition as LDL (see below). The physio­logical role of Lp(a) is not known. Plasma [Lp(a)] is positively associated with the incidence of ischaemic heart disease, independently of other lipoprotein fractions. The effect may be due to competition between Lp(a) and plasminogen for endothelial cell receptors, thereby inhibiting thrombolysis.

Enzymes involved in lipid transport

Four enzymes of relevance to clinical disorders need to be described:

1  Lecithin cholesterol acyltransferase  trans­fers  an  acyl  group  (fatty  acid  residue)   from lecithin to cholesterol,  forming a cholesterol ester. In plasma, this reaction probably takes place exclusively on HDL, and may be stimulated by apoA-I.

2  Lipoprotein lipase is attached to tissue capillary endothelium and splits triglycerides (present in chylomicrons and VLDL) to glycerol and free fatty
acids. Its activity increases after a meal, partly as a result of activation by apoC-II, which is present on the surface of triglyceride-bearing lipoproteins.

3  Hepatic lipase has an action similar to that of lipoprotein lipase.

4  Mobilising lipase, present in adipose tissue cells, controls the release of fatty acids from adipose tis­sue into plasma. It is activated by catecholamines, growth hormone and glucocorticoids (e.g. cortisol), and inhibited by glucose and by insulin.

Metabolism of plasma lipoproteins

The above description of the lipoproteins and apolipoproteins is an oversimplification, and the following points should be emphasised:

1  Plasma lipids and apolipoproteins exist in a dynamic state. There is interchange of lipids both between different lipoprotein particles and with tissues.

2  There is considerable variation in the size and composition of individual lipoprotein particles within each lipoprotein class.

Chylomicron metabolism

Chylomicrons (Figure 11.2) are formed in the ntestinal mucosa after a fat-containing meal, and reach the systemic circulation via the thoracic

duct. They then transfer apoA to HDL and acquire apoC and apoE from HDL. The apoC-II activates lipoprotein lipase in the tissues, and triglycerides are progressively removed from the hydrophobic core of the chylomicrons. As the size of the parti­cles decreases, the more hydrophilic surface components (apoC, unesterified cholesterol and phospholipid) transfer to HDL. The triglyceride-poor chylomicron remnants are taken up by the liver, where they are catabolised.


VLDL and IDL metabolism

Most VLDL is secreted into plasma by the hepatocytes ('endogenous' VLDL), but some originate from the intestinal mucosa ('exogenous' VLDL) (Figure 11.2). Hepatic VLDL synthesis is increased whenever there is increased hepatic triglyceride synthesis, for example when there is increased transport of fatty acids to the liver, or after a large carbohydrate-containing meal.

When first produced, VLDL consists mainly of triglycerides and some unesterified cholesterol, with apoB100 and lesser amounts of apoE. ApoC-II is then acquired, mainly from HDL, and triglyc­erides are removed from the VLDL 'core' in a man­ner analogous to that for chylomicrons. The residual particles are known as 'VLDL remnants', or IDL, which are either rapidly converted to LDL or removed from the circulation to the liver.

LDL metabolism

LDL probably all arises from VLDL metabolism in man. The LDL particles are rich in cholesterol esters, probably derived from HDL; apoB100 is the only apolipoprotein. LDL is removed from the cir­culation by two processes; one regulated, the other unregulated.

The regulated mechanism involves the binding of LDL to specific apoB100 receptors present on the 'sur­face pits' of hepatocytes and other peripheral tis­sue cells. The entire LDL particle is incorporated into the cell by invagination of the cell mem­brane. Inside the cell, the particle fuses with lyso-somes; apoB is then broken down and the cholesterol esters are hydrolysed, thereby making unesterified cholesterol available to the cell. The size of the intracellular cholesterol pool regulates:

1 The rate of cholesterol synthesis in the cell, through the effect of cholesterol on HMG-CoA reductase.

2 The number of LDL-apoB receptors on the cell surface.

The unregulated mechanism involves receptor-independent mechanisms of cholesterol uptake by cells; these are present particularly in macro-phages. These mechanisms are brought into oper­ation especially when plasma [cholesterol] is increased.

HDL metabolism

This heterogeneous group of particles (HDL2, HDL3 etc.) is formed in the liver and intestinal mucosa. The HDL particles then undergo fairly complex exchanges of lipid and protein with other plasma lipoproteins. However, the main point to note is that free cholesterol in tissues transfers to HDL in plasma. The cholesterol is then esterified by LCAT and transferred to LDL, which, in turn, is mainly taken up by the liver. Thus, HDL forms the principal route whereby cholesterol can return from peripheral tissues to the liver.

Investigation of plasma lipid abnormalities

Most laboratories measure plasma [total choleserol], [HDL cholesterol] and [triglycerides]. Further tests to characterise the lipoprotein abnormalities may be indicated in a few patients. The investiga­tions are mainly of value in the investigation and management of ischaemic vascular disease.

Plasma total cholesterol

Diet, for example, recent meals, does not affect plasma [cholesterol] much in the short term. This means that random [cholesterol] can be measured to assess cardiovascular risk. Plasma [cholesterol] is affected by both within-individual and between-individual factors. However, these tend to be long-term effects, as follows:

Diet The amount and the composition of dietary fat affect plasma [cholesterol]. In particular, those fats containing mainly polyunsaturated fatty acids, such as those in fish and vegetable oils, tend to lower plasma [cholesterol], whereas those fats containing mainly saturated fatty acids, such as animal fat and butter, tend to raise plasma [cho­lesterol]. Dietary fibre may have a small effect in lowering plasma [cholesterol]. The consumption of one to three units of alcohol per day causes a significant rise in plasma [HDL cholesterol]. Dietary cholesterol intake has relatively little effect on plasma [cholesterol].

Exercise Regular exercise tends to cause a rise in plasma [HDL cholesterol] and a small fall in plasma [total cholesterol].

Age In developed countries, plasma [cholesterol] rises with age. This is probably related to diet.

Sex In pre-menopausal women, plasma [total cholesterol] is lower than in men, and plasma [HDL cholesterol] is higher. These differences disappear after the menopause.

Race It is likely that the marked racial differences, with particularly high plasma [cholesterol] in north Europeans, are mainly due to dietary and environ­mental factors rather than genetic differences.

Numerous studies have shown that the inci­dence of ischaemic heart disease is directly corre­lated with plasma [cholesterol] (Figure 11.3), even within the 'reference range'. There is no clear cut­off between values for normal risk and increased risk, although risk rises particularly rapidly above about 6.5 mmol/L. Because of this association, it is inappropriate to employ reference ranges for plasma [cholesterol] in the usual way, as these imply health without increased risk of disease. Instead, it seems more appropriate to define a desirable concentration (e.g. below 5 mmol/L).

Plasma [total cholesterol] is a rather unsatisfac­tory measurement, since it represents the sum of the various ways in which cholesterol is trans­ported in plasma. In fact, although raised plasma [LDL-cholesterol] is associated with an increased risk of ischaemic  heart  disease,  raised plasma

[HDL-cholesterol] is associated with a decreased risk of ischaemic heart disease and seems to have a protective effect (Figure 11.3).

Plasma triglycerides

Plasma [triglycerides] also show variations with age and sex, but more especially with diet. There is, in addition, a very considerable within-individual variation, which makes interpretation of a single result difficult.

Plasma low density lipoprotein

Plasma [LDL] can be measured by ultracentrifuga-tion, but this is not a practical technique for rou­tine clinical laboratory use. The following formula can be used to calculate [LDL]:

[LDL cholesterol] = [total cholesterol] - [HDL cholesterol] - [triglyceride]/2.2

where all measurements are in mmol/L. This for­mula is valid only if [triglyceride] is less than 4.5 mmol/L.

LDL exists in a range of sizes and densities. There is evidence that the small dense subtrac­tions of LDL are particularly atherogenic. There are no readily available routine laboratory meth­ods for examining LDL subfractions, but surrogate markers for the presence of these small dense LDL species are low or low-normal [HDL] and high or high-normal [triglyceride]. This combination is sometimes referred to as an 'atherogenic lipid pro­file'. Measurement of apolipoprotein B levels may provide similar information.

Specimen collection

It is important to collect specimens for plasma lipid and lipoprotein studies under the appropri­ate conditions:

1  The patient should have been leading a normal life in terms of diet (including alcohol consump­tion) and exercise for at least the previous fortnight.

2  Blood specimens should be collected after an overnight fast of 10-14 h, if triglyceride measure­ments are to be performed.

3  Venous stasis should be minimal.

Results of plasma lipid and lipoprotein investiga­tions can be misleading in specimens collecte;-during or within a few weeks after a serious illness (e.g. a myocardial infarction or a major operation > They often then show reduced plasma [cholesteroL and sometimes hypertriglyceridaemia. However, specimens collected within 24 h of a myocardia. infarction may be used to determine the need for subsequent cholesterol-lowering treatment.

Routine investigations

One or more of the following investigations should be requested in patients suspected to be at increased risk of ischaemic heart disease or of a lipid disorder:

1 Plasma [cholesterol]. This may be sufficient, if decisions about secondary prevention of cardio­vascular disease are to be made.

2 Plasma  [HDL-cholesterol],  if plasma [choles­terol] is raised or if additional risk factors are pre­sent. This can be used to calculate the [cholesterol]:[HDL  cholesterol]   ratio,   which  correlates  well with cardiovascular risk and which is used in a number of methods developed to calculate overall cardiovascular risk in making decisions about primary   prevention   of   cardiovascular   risk. It is also needed for the diagnosis of hyper-a-lipoproteinaemia.

3 Plasma [fasting triglycerides]. This is a weak independent risk factor for cardiovascular disease, and a  risk factor for acute pancreatitis if greater than
10 mmol/L, and is used in the calculation of [LDL].

Specialised investigations

A large number of specialised investigation, including ultracentrifugation, apolipoprotein and enzyme studies and molecular genetic studies may occasionally be helpful.

Content, mmole/l

Ideal level

Good level

Border level



Total cholesterol

< 5,2


5,2 – 6,2

Ø    6,2

Cholesterol of LDL (β cholesterol)

Male < 1,23

Female < 1,63


1,23 – 3,34

3,35 – 4,45

Male > 4,45

Female > 4,32


Cholesterol of LDL (α cholesterol)

Ø    1,55

1,0 – 1,55

0,9 – 1.0

< 0,9


< 1,71

1,71 – 2,35

2,36 – 4,5

Ø    4,5


The probability of the development and progression of atherosclerosis







The primary hyperlipoproteinaemias

The causes of hyperlipoproteinaemia (Table 11.3) are complex, and different disease mechanism can   give   rise   to   similar   lipid   patterns.


  The approach adopted here will be primarily descrip­tive, based on the observed lipid abnormalities Increased plasma lipid concentrations may be due to:

1 . Genetic factors.

2 . Environmental factors.

3   A combination of the above.

4  Other diseases (secondary).

Primary hypercholesterolaemia

In about 95 % of patients with primary hypercholes­terolaemia, the abnormality is due to a combination of dietary factors and a number of yet to be identi­fied genetic abnormalities in handling cholesterol. In the minority of patients who have familial hypercholesterolaemia, there is a specific genetic defect in the production or nature of high-affinity tissue apoB100 receptors (or in the structure of the apoB100) itself, so that it is not recognised by the normal receptor). Heterozygotes have about 50 % of normal receptor activity, and homozygotes have no receptor activity. Many heterozygotes have tendon xanthomas, and over 50 % will have symptoms of coronary artery disease by the fourth or fifth decade. In homozygotes, heart disease often presents in the second decade. Plasma [cho­lesterol] is usually raised to 8-15 mmol/L in het­erozygotes, and is even higher in homozygotes.



Familial hypertriglyceridaemia

This group of conditions is associated with defects either in the production or in the catabolism of VLDL. Plasma [triglycerides] and [VLDL] are increased but, whereas plasma [cholesterol] is often also moderately increased, plasma [HDL] is often reduced. Patients have an increased risk of ischaemic heart disease.

In some patients, there is chylomicronaemia in addition to increased plasma [VLDL]. This pattern may be brought on by alcohol excess, and is also seen in diabetics. These patients may have erup­tive xanthomas and attacks of acute pancreatitis.

Familial combined hyperlipidaemia

This disorder is difficult to classify, and the method of inheritance is unclear. Even in the same family, the gene does not always express itself in the same way, as there may be increased plasma [LDL] only, increased plasma [VLDL] only, or increases in both. The incidence of ischaemic heart disease is three to four times greater than in the general population.

Remnant hyperlipoproteinaemia

This is an uncommon disorder characterised clini­cally by cutaneous xanthomas and a high risk of premature ischaemic heart disease. In the plasma, there is an increase in cholesterol-rich but other­wise VLDL-like particles; these are probably IDL (i.e. 'VLDL remnants'). Both plasma [cholesterol] and [triglycerides] are increased; plasma [LDL] is decreased.

This disorder is probably due to a combination of factors. There is abnormal conversion of VLDL to LDL. This is usually associated with the apoE2/2 genotype. However, since as many as 1% of nor­mal individuals have this genotype, whereas the incidence of remnant hyperlipoproteinaemia is only about 1 in 5000, an additional factor must be present.

Remnant hyperlipoproteinaemia responds well to treatment with fibric acid derivatives (e.g. fenofibrate), so its recognition is important. Ultracentrifuge studies provide the definitive means of confirming the diagnosis.

Lipoprotein lipase deficiency

This is a rare autosomal recessive disorder causing hypertriglyceridaemia and chylomicronaemia. The incidence of ischaemic heart disease and acute pancreatitis is increased; eruptive xanthomas often occur. The primary defect is deficiency of either lipoprotein lipase or its activator, apoC-II.

Treatment involves restriction of normal dietary fat and replacement by means of triglycerides containing fatty acids of medium chain-length (C8-C11); these are less prone to lead to chylomicron formation.

Other inherited defects

Hyper-α-lipoproteinaemia is an inherited abnormal­ity, giving rise to increased plasma [HDL] and mildly increased plasma [cholesterol]. Patients have a reduced incidence of ischaemic heart disease. The only importance of hyper-a-lipoproteinaemia is that no treatment is required for the raised plasma [cholesterol].

Secondary hyperlipidaemia

Probably less than 20 % of cases of hyperlipi­daemia are secondary to other disease. Patterns of abnormality tend to vary, even within a single dis­ease; plasma [cholesterol] or [triglycerides], or both, may be affected.

Hypercholesterolaemia is often a marked feature of hypothyroidism and of the nephrotic syndrome; in these two disorders, there is increased plasma [LDL].

It also occurs in cholestatic jaundice, but in this condition there is an accumulation of abnormal discoid particles rich in phospholipid and unesterified cholesterol, and an additional abnornormal lipoprotein - lipoprotein X - is detectable. Coronary artery disease tends to develop in those patients with long-standing secondary hyperlipidaemia

Hypertriglyceridaemia,  secondary to other dis­ease, is most commonly due to diabetes mellitus or to excessive alcohol intake. It may also occur in chronic renal disease and in patients on oestrogtr therapy,   including   women   taking   oestroger containing oral contraceptives.

The effects of alcohol on plasma lipids are com­plex. Regular drinking of small amounts increase plasma [HDL] without affecting other lipoproter particles. Some heavy drinkers develop hypertriglyceridaemia due to increased plasma [VLDL], possib. as a result of increased direction of fatty acid metab­olism into triglyceride synthesis in the liver.

The hyperlipidaemia secondary to diabetes mellitus is also complex. Increased plasma [VLDL] i the usual finding, but often plasma [LDL] is ah increased, whereas plasma [HDL] is reduced.

The primary hypolipoproteinaemias

Three rare familial diseases require brief mention Their recognition has helped with the under­standing of normal lipoprotein metabolism.

Tangier disease is due to an increased rate : apoA-I catabolism. Only traces of HDL are detectable in plasma, and plasma [LDL choles­terol] is also reduced. Cholesterol esters accumu­late in the lymphoreticular system, probably due to excessive phagocytosis of the abnormal chylomicrons and VLDL remnants that result from the apoA-I deficiency.

Abetalipoproteinaemia is associated with a com­plete absence of apoB. The lipoproteins that nor­mally contain apoB in significant amounts (i.e chylomicrons, VLDL, IDL and LDL) are absent from plasma. Plasma [cholesterol] and [triglyc-erides] are very low.

Hypobetalipoproteinaemia is due to decrease; synthesis of apoB. Plasma [VLDL] and [LDL; although reduced, are not absent.

Secondary hypolipidaemia

Greatly reduced plasma [cholesterol] occurs when­ever hepatic protein synthesis is depressed, as in protein-energy malnutrition (e.g. kwashiorkor in children), severe malabsorption or some forms of chronic liver disease.

Other biochemical cardiovascular risk factors or markers

Very high levels of homocysteine, up to 50-fold normal, are seen in homocystinuria, an inborn error of metabolism due to deficiency of the enzyme cystathionine (3-synthase. Patients develop ocular, skeletal and vascular problems, with increased arterial and venous thrombotic events at an early age, and a markedly increased mortality.

Much lesser elevations in homocysteine levels are associated with an increased risk of cardiovascular disease, with patients in the upper quartile having twice the risk of patients in the lowest quartile, pos­sibly through mechanisms involving endothelial damage and the promotion of thrombosis. However, the precise relation remains unclear, and raised [homocysteine] may yet prove to be simply a marker of increased vascular risk rather than a causative risk factor. Homocysteine levels are strongly influenced both by genetic factors and by diet. Folic acid and vitamins B6 and B12 are involved in the catabolic pathways of homocysteine, and deficiencies of these vitamins can cause elevation of [homocysteine] and supplementation can cause its reduction. So far there are no controlled trials of the effect of supplementation of these vitamins on the development or recurrence of cardiovascular dis­ease. Because of this the precise role of [homo­cysteine] measurement remains controversial. However, a possible role is developing for its mea­surement in individuals with a personal or family history of cardiovascular disease in the absence of the conventional well-established risk factors such as hypercholesterolaemia or hypertension. Finding a high [homocysteine] under these circumstances provides a possible explanation, and can reinforce advice to ensure that the diet contains adequate amounts of folic acid and vitamins B6 and B12.

Patients with CRP, an inflammatory marker, at the high end of the normal range (measured with a highly sensitive assay, hsCRP) have 1.5 to 4 times the cardiovascular risk of those with CRP at the low end of the normal range. This has been demonstrated both in apparently healthy individuals and in patients with estab­lished vascular disease. The origin of this increased CRP can be speculated to be endothelial inflammation in association with atherosclerotic plaques. Treatment of patients with statins results in a reduction of hsCRP. The role of hsCRP mea­surement is not yet established, and it is not yet recommended for widespread use. It may however complement use of conventional major risk fac­tors, especially in patients at moderate risk of vas­cular disease, in whom the finding of an elevated hsCRP might reinforce the need for treatment.


Calculation of cardiovascular risk and its treatment by lipid lowering

It is possible to lower plasma [LDL cholesterol] by dietary and other lifestyle means, but the most effective therapy, usually leading to reductions of up to 30% or more, is achieved by HMG-CoA reductase inhibitors ('statins')- It is this class of drug that has mainly been used in the clinical tri­als mentioned below.

It is conventional to consider cardiovascular risk reduction under the subdivisions of 'secondary pre­vention' (where the patient has established vascular disease, and the goal is to prevent recurrence); and 'primary prevention' (where the patient has no overt vascular disease, and the goal is to prevent its development). Multiple sets of guidelines have been published, differing to a greater or lesser extent in detail. In more recent times, greater consensus has been achieved on the division into primary and sec­ondary prevention; on the risk factors that should be treated; the concentration of primary prevention on those at greatest overall vascular risk; and the therapeutic options available. However, the precise thresholds and targets for treatment continue to evolve. The most recent British guidelines are those published by the British Hypertension Society  which introduce some changesin how risk might be calculated, with a move from coronary heart disease risk to cardiovascular risk, a reduction in age bands, treatment of virtually all patients who have diabetes and stipulate stricter treatment thresholds and targets. They have gener­ated controversy because of the large numbers of the population who will fall within the treatment categories, and the costs involved.

In patients with previous myocardial infarction or with angina, trial results are conclusive. Pre­existing vascular disease is the most potent risk factor for the development of further vascular dis­ease, and the size of the population requiring treatment is relatively limited. Cholesterol reduc­tion by about 25 % reduces all-cause mortality by 30% and cardiac events by over 40 %. This means that treatment of these patients is the first priority, and decisions in secondary prevention are rela­tively straightforward. Lifestyle interventions to discontinue smoking, adopt a healthy diet and to take exercise are important, and rigorous control of blood pressure, diabetes and lipids is recom­mended. Current guidelines suggest that virtually all patients with established vascular disease should be treated with lipid-lowering drugs, irres­pective of baselise cholesterol levels.

In primary prevention, large-scale clinical trials have shown that cholesterol lowering (by an aver­age 20 %) in hyperlipidaemic men can reduce cardiovascular death and nonfatal myocardial infarction by about 30%. The available evidence strongly supports the concept that those who might benefit most from treatment are those at greatest overall absolute risk. Someone with multi­ple modestly elevated risk factors may be at a greater risk than someone with a single markedly elevated risk factor. This means that there is a need for a means of calculating overall risk. The guide­lines achieve this by the use of charts which strat­ify risk on the basis of sex, age, smoking, blood pressure and the [cholesterol: HDL cholesterol] ratio (Figure 11.4, see plate section). A computer program to calculate this risk is also available. An important caution is that these calculations do not apply to patients with inherited dyslipidaemias. An absolute risk derived from these charts of 20% or more for the development of cardiovascular disease over the next 10 years is sufficient to justi: treatment. The total cholesterol target is less tha: 4.0 mmol/L (LDL less than 2.0 mmol/L).



Normal and pathologic biochemistry of lung

Only recently has the lung been recognized as an important metabolic organ rather than just a tissue for passive gas exchange. A major reason for the delay in appreciation of the metabolic role of the lung is related to its structure and ana-tomic relationships. The lung, although filling most of the thoracic cavity, actually comprises only 1% of the body weight, and approximately 30 % of that weight is due to contained blood. Further, the blood flow to the lung comprises the entire cardiac output making it the most richly perfused organ in the body. Because of the high blood flow in relation to metabolizing tissue mass, arteriovenous differences of most metabolites cannot be measure easured across the lung in situ. Consequently, it has been necessary to develop in vitro models for study of lung metabolism. One model that has been extensively used is the isolated perfused lung preparation. Perfusion of the lung with artificial media removes the red cells from the pulmonary capillaries and results in tissue with a completely white appearance. Additional models to study lung metabolism are tissue slices and preparations of subcellular organelles.

Measurements with the isolated perfused lung preparation or lung slices have shown oxygen up-take in the range of 30-150 jA/min-g dry weight, depending on species and preparation. There-fore, lung tissue has significant O2 consumption although values are low compared with the metabolically very active organs. For example, dog lung oxygen uptake per unit weight is only 10-20 % of the oxygen uptake of canine heart, kidney, thyroid, and brain. The oxygen uptake of the lung is greater, however, than that of resting skeletal muscle, intestine, and many other metabolically less active tissues. Actually, the lung can be considered an average organ in terms of O2 utilization, since its oxygen  uptake of the organism, and this is approximately the contribution of the lung to total body weight. On the other hand, it should be noted that the lung represents a heterogeneous collection of cell types, and it is likely that some components of the lung, e.g., the type II granular pneumocytes, have considerably higher oxygen uptake than the mean for the whole lung.

What are the substrates utilized by the lung for its metabolic requirements? Although intact lungs and lung subcellular organelles can oxidize fatty acids, glucose probably serves as the major oxidizable substrate under usual conditions. Approximately half of the glucose was converted to lactate and pyruvate. The "reason" for the high rate of production of these three-carbon compounds has not been defined. One postulated explanation relates to the presence of numerous cells in the lung with relatively sparse mitochondria and, therefore, limited citric acid cycle activity. Additional possibilities include limited activity of mitochondrial H+ shuttle mechanisms or mitochondrial pyruvate dehydrogenase. In any case, the high lactate production under control conditions was probably not due to cellular hypoxia since the lung was being ventilated with 95 % 02, the perfusate L/P ratio was within a normal range (i.e., 5-10), and the lung responded briskly to inhibitors of oxidative metabolism with change in redox state. Approximately one fourth of the glucose carbons utilized by the perfused lungs are oxidized to CO2. There are also active pathways for incorporation of glucose carbons into tissue components including proteins, nucleic acids, polysaccharides (chiefly glycogen), and other unidentified components. Finally, a small but significant fraction of glucose carbons is used for synthesis of lipids, including the fatty acid as well as glyceride-glycerol moieties.

The next question to explore is whether oxidative metabolism is required in order to maintain normal energy stores of the lung tissue. In-sight into this problem can be obtained by measurement of changes in lung tissue adenine nucleotide content during inhibition of oxidative metabolism, or uncoupling of oxidative phosphoryla-tion. During control perfusion, ATP content of the lung per unit weight is comparable to values observed in other aerobic tissues able to values observed in other aerobic tissues and the ATP/ADP ratio is approximately 8.5.

What are the metabolic processes in the lung that are energy-dependent? Certainly the lung has no physiologic process that requires large expenditures of energy such as occurs with cardiac muscle contraction, renal transport, or maintenance of ionic gradients in nerve tissue. Energy utilization, however, is required for functioning of several lung systems. For example, lung clearance depends on bronchial ciliary activity and phagocytosis by alveolar macrophages, both of which are energy dependent. During anoxia, there is cessation of ciliary beating and inhibition of particle phagocytosis. Secretion by bronchial glands and constriction of tracheobronchial smooth muscle are other processes that presumably are energy-dependent. Synthesis of dipalmitoyl lecithin, a major component of the lung surfactant system, requires a supply of ATP.

The actual secretion of surfactant is also probably an energy-requiring process analogous to cellular secretion elsewhere. Finally, energy is required for cell transport processes.


The most dramatic metabolic function of the lung and the one that has been studied to the greatest extent relates to the synthesis of lung surfactant. The lung surfactant is contained in the extracellular alveolar lining layer that coats the epithelial surface of the lung alveoli. The presumed physiologic function of the surfactant system is to maintain the surface tension at the interface between the alveolar surface and the air spaces at low levels and thereby promote alveolar stability. The extracellular material can be obtained for study by alveolar lavage with saline or other physiologic solution. The material so obtained contains approximately 75° phospholipids, 15% neutral lipids, and 10 % protein. Dipalmitoyl lecithin (dipalmitoyl phosphatidyl-choline) accounts for approximately 40 % of the Dipalmitoyl lecithin (dipalmitoyl phosphatidyl-choline) accounts for approximately 40 % of the total solids in the surfactant fraction. Synthetic dipalmitoyl lecithin (DPL) manifests similar surface active properties to the crude surfactant fraction so that the physiologic properties of the surfactant are thought to be related chiefly to the presence of DPL. An additional 25 % of the surfactant material is comprised of lecithins containing unsaturated fatty acids. The physiologic role of this fraction of the surfactant is not known but may be important as an aid to spreading or other-wise influencing dispersion of the dipalmitoyl lecithin fraction. The role of the protein in surfactant is also incompletely understood. Current evidence suggests that the protein is specific to the lung and that surfactant is secreted as a lipoprotein complex.

What is the source of surfactant and what are the pathways involved in its production? It now seems clear that surfactant is synthesized in the lung and subsequently secreted onto the alveolar surface. Most studies that have investigated surfactant production have focused on pathways of dipalmitoyl lecithin synthesis although, as noted above, DPL is only one component of the surfactant fraction. These studies have shown that DPL is synthesized chiefly by the classical pathway involving phosphatidic acid (formed from glycerol-3-phosphate and palmitate) and CDP-choline (formed from CTP and choline). Considerable effort is currently being directed to determine the factors that are important in control of this path-way in the lung. Based on experiments, the current concept is that surfactant synthesis is a major metabolic activity of the lung. Substrates for surfactant synthesis are transferred from the circulation or alveolar space to type II epithelial cells where the lipid and protein components are synthesized, presumably in the endoplasmic reticulum and Golgi organelles. These components are then packaged in lamellar bodies for storage and subsequently released on-to the alveolar surface. Considerable work remains in order to define factors controlling synthesis, release, and turnover of surfactant.

Disorders of Surfactant Synthesis

Shortly after appreciation of the importance of surfactant in respiratory physiology, surfactant deficiency was found in lungs of infants with respiratory distress syndrome (RDS). Investigations into possible mechanisms have resulted in increased understanding of the development and maturation of the surfactant system. One key finding is that the lung surfactant system matures relatively late in the course of fetal development. In the monkey (gestation period 170 days), significant amounts of lecithin are not present in the alveolar lining material until gestation is approximately 80 % complete. Appearance of lecithin on the alveolar surface correlates with a sharp increase of lecithin in the lung hemogenate and the presence of lamellar bodies in the type II alveolar cells. Since respiratory distress syndrome in the newborn is associated with prematurity, the concept has arisen that RDS represents birth of the fetus before complete maturation of the lung surfactant system. Recently, several groups have investigated potential mechanisms to accelerate the normal rate of maturation of the surfactant system. One promising method is administration of adrenocorticosteroid hormones which do appear to accelerate maturation and protect infants delivered prematurely against development of RDA. Several of the enzymes in the pathways of phospholipid synthesis undergo induction with steroid treatment which might account for the accelerated maturation. This area is under active investigation at the moment and promises great potential benefit in terms of the prevention of a common cause of infant mortality.

Metabolism of Hormones and Xenobiotics

Another important role for the lung that has recently been extensively investigated is concerned with the metabolism of xenobiotics and endogenous hormones. Through these pathways, the lungs are able to exert a major influence on bodily homeostasis. As one example of hormone metabolism, lungs rapidly clear serotonin (5-HT) from blood or other media perfusing the pulmonary circulation. This amine can be taken up against a concentration gradient by a mechanism that shows saturation kinetics, is dependent on external sodium, and can be blocked by specific competitive inhibitors, inhibitors of oxidative metabolism. After uptake, serotonin is metabolized by a monoamine oxidase (MAO) to 5-hydroxy-indole-acetic acid. The rate of metabolism does not significantly influence the rate of uptake oxidase (MAO) to 5-hydroxy-indole-acetic acid. The rate of metabolism does not significantly influence the rate of uptake. These characteristics suggest that uptake of serotonin by the lung occurs by active transport and that transport is the rate limiting step in serotonin clearance. Autoradiographic studies have demonexclusively into the pulmonary endothelium. The mechanism for amine transport shows specificity, since histamine, another vasoactive compound, is not taken up nor metabolized by lung to any significant extent. As another example of

specificity, norepinephrine is taken up and metabolized whereas epinephrine is not.

Uptake and metabolism is only one mechanism by which the lung transforms vasoactive compounds. An additional pattern is operative with respect to conversion of angiotensin I to angiotensin II (resulting in formation of a potent vasoactive compound) or hydrolysis of peptide bonds in brandykinin (resulting in a loss of biological activity). The responsible peptidase enzyme (converting enzyme) is either the same for both reactions or represents a group of closely related enzymes. Converting enzyme activity is present on the luminal surface of the pulmonary endothelium, so that hydrolysis occurs at the membrane surface and active transport into the cell is not required. A more complex relationship can be seen with several of the prostaglandins since the lung is an important site for uptake and metabolism as well as synthesis, storage, and release.

In addition to contact with endogenous agents, the lung is exposed to a wide variety of potentially toxic substances delivered either through in-halation or via the circulation. A major pathway for detoxification of foreign components such as

drugs is through hydroxylation in the liver by cytochrome P-450-linked reactions. Recently, this pathway has also been demonstrated in isolated lung microsomes. These organelles from lung contain, on a weight basis, approximately 25 % of the

cytochrome P-450 activity of microsomes isolated from liver.


Sputum is a pathological secret that is formed in the case of respiratory diseases and lung and released during coughing or spitting. In healthy people, excluding smokers, singers and teachers, sputum is not released. Sputum is not homogeneous. It consists of mucus (secret of mucosa of the respiratory tract), pus, blood, serous fluid, fibrin.  All these elements can be present in sputum or only some of them.

Composition and properties of sputum depend on the nature of the pathological process in the respiratory organs.

Laboratory investigation of sputum has great diagnostic value to detect inflammatory and destructive processes in the lungs and airways, as well as to identify the pathogen agent of diseases ( in case of pulmonary tuberculosis). Laboratory investigation of sputum helps to determine the degree of pathological process and its severity.

Rules of sputum collection

Very often sputum is collected for the overall clinical investigation, investigation under the microscope for its morphology. Often sputum is examined for detection of mycobacterium tuberculosis.

In the hospital sputum is collected and examined often. In ambulatory patients with chronic diseases of the respiratory  tract sputum should be examined periodically, at least 1-2 times a month.  Investigation on mycobacterium tuberculosis also is performed periodically. In clinical analysis should be sent fresh material, collected in a clean glass dish that prevents projects its oxidation and drying. Patients prefer to gather sputum in individual pocket spittoon graduated with dark glass, which covers twist. This dish is easy to clean and disinfect, it is convenient for both the patient and for sending material in the laboratory.

Sputum is collected in the morning before eating. In the nasopharynx and oral cavity saliva and nasal secret are mixed with sputum so that interfere with the investigation. To prevent these before collection of  sputum  the patient should thoroughly rinse the mouth and pharynx with boiled water. During collection of  sputum, the patient should not pollute the outer edges and walls of glass dish.

Preferably as soon as possible investigate the sputum, which is collected, if there is no such possibility, sputum should keep in refrigerator or a cool place.

Often sputum is infected material, so after investigation to decontaminate it, filling the 5 % solution chloramine, or 5 % solution of carbolic acid and kept at least 4 hours. Laboratory dishes, spittoons, work place also are disinfected with the 5 % solution of chloramine. Metal spatulas and needles are  disinfected over the burner flame.

Clinical sputum analysis includes the study of physical properties, microscopic and bacterioscopic investigation. Chemical investigation of  is of little diagnostic value, usually limited by microchemical reaction to hemosyderin.

Physical properties of sputum

In laboratory investigated sputum is transfered in a petri dish, on black and white background and physical properties are determined.

Amount of sputum can vary from small (2-5 ml), for example in case of acute bronchitis, bronchial asthma, the catarrh of the upper respiratory tract, to a rather large (200 - 300 ml or more), which is the most typical for diseases accompanied by the formation of cavities in the respiratory organs (abscess, gangrene, bronchiectasis).

Amount of sputum is determined in a glass graduated container. If the collected sputum leave to stand, then after a while it can be distributed in layers. In the case of bronchiectasis, gangrene of lung, rot bronchitis sputum is divided into three layers: upper - foam (mucus), medium - serous (with opalescence) and bottom - crumbly, close-grained (pus). In patients with pulmonary tuberculosis lower layer of sputum is clotted, because consists of large clots covered with mucus. In the case of lung abscess sputum during standing is divided into two layers: the upper - serous and lower - purulent, yellow color.

a.     Two-layers sputum: the upper – serous layer and bottom – pus.

b.    Three-layers sputum: upper – colorless foam (mucus), medium - serous fluid and bottom - pus


Smell. Fresh sputum is odorless. Rot smell is typical of an abscess, gangrene of lung, as well as rot bronchitis, when rot flora is joined to sputum. Evil-smelling odor of sputum can be in patient with  the decomposition of lung tissue (cancer).

The character of sputum is determined by its composition:

1) Mucus sputum - a colorless, vitreous, viscous - released in the early stages of bronchitis and bronchial asthma;

2) Purulent sputum - without admixture of mucus occurs very rarely, for example, when empyema breaks in the lumen of the bronchus or in patients with bronchiectasis;

3) Mucous-purulent or purulent-mucous sputum occurs in case of most inflammation in the lungs, bronchi and trachea, cloudy viscous mass, which closely mixed pus and mucus;

4) Bloody sputum contains blood clots, released in case of pulmonary tuberculosis, tumors (cancer lungs).

5) Serous sputum - liquid, foamy - often is observed during pulmonary edema. 

The color depends on the nature of sputum. Gray or grayish-yellow is observed in mucous-purulent, yellowish-gray - in purulent-mucous sputum. Yellow is asthmatic sputum through accumulation of eosinophils in its clots. Bloody sputum is red, brown, rusty or crimson.  Brown (chocolate) or brown sputum becomes due to destruction of hemosiderin. Presense of  bilirubin can dye it in yellow or green color. Black color - whether caused by coal dust.

Consistence of sputum depends on its nature and can be:

1. Viscous - the presence of mucus;

2. Sticky or moderately sticky - the presence of pus;

3. Liquid - the presence of blood or serous fluid;

4. Gelatinous - the presence of fibrin and mucus.

Viscous mucus is found in the case of lobar pneumonia, viscous - in case of chronic bronchitis, bronchopneumonia, bronchiectasis, lung abscess, liquid - in case of pulmonary hemorrhage, edema of  the lungs, gelatinous - in case of allergic bronchitis, bronchial asthma.

Form. There is sputum grainy, clotty and piece-like. Normally, mucus is clotty (clots of mucus) piece-like or mixed form. Clotted- piece-like form - the characteristic feature of severe pneumonia with destruction or lung cancer when mucus contained pieces of lung tissue. In case of enhanced desquamation of the epithelium of the alveoli in pneumonia, when the sputum contains a large number of casts of the alveoli, sputum has granular (grainy) form.

Pathological components of sputum that are visible with the naked eye:

Kurshman’ spirals- whitish, winding, spiral –like mucous formations, sharply differentiated from other mucous sputum, have diagnostic value in case of bronchial asthma;

Fibrinous clots - look like whitish- red balls, elastic consistence, consist  of mucus and fibrin. Occur in sputum in case of fiber bronchitis, lobar pneumonia;

Rice-like bodies (Koch lenses) – not transparent tight greenish-white formation with cheesy consistency, there size like head of pins. They are formed in caverns and contain products of lipid decomposition, detritus, fiber, cholesterol crystals and a large number of mycobacteria tuberculosis. They are found in sputum in case of cavernous tuberculosis of lungs;

Ditryh Tubes - purulent clots of whitish-gray or yellow-gray color size from pinhead to a bean with sharp odor. They consists of products of cell decomposition, detritus, crystals of fatty acids and bacteria. They are found in sputum in case of bronchiectasis, lung gangrene;

Necrotic pieces of lung tissue can appear in the sputum during the destructive processes in lung tissue (gangrene of lung and lung abscess). They have black co lor, various size;

Pieces of lung tumors are found in sputum in case of tumors in the lungs and bronchi. They look like, grayish, brown or bloody formations of different size, often not larger than the pin head;

In the sputum also it is possible to find eggs and larvae of worms, hooks of ehinokok and scraps of his shell.

Foreign bodies usually are found in sputum accidently. They can include: seeds, berries, cereal ears, coins, needles, etc. Foreign bodies may be in the bronchi during years. Sometimes there is inflammation around the foreign body.

Microscopic examination: morphology of the sputum elements

For microscopic examination of sputum, first of all native preparations are used. Usefulness of investigation depends on the correct preparing and number of revised native preparations. To prepare native preparations (at least two or three) all suspicious pieces and formations, other than mucus and impurities are selected from sputum and transferred on the a glass slide, covered with a coverage lenses. Preparation has to be thin and not go beyond covering lenses.

Elements of sputum microscopy are divided into four groups: cellular, fibrous, crystalline and flora.

Cellular elements

Leukocytes - round cells, the size of 10-15 microns, grainy - neutrophils. They are constantly detected in sputum, in mucous - single in the field of view, in purulent or mucous-purulent - completely cover the field of view.

Eosinophils - round cells, darker colored than neutrophils, have dense cytoplasm, with granules. They are constantly detected in sputum in case of bronchial asthma and other allergic diseases.

Red blood cells - round cells, greenish-yellow color, there diameter is smaller than leukocytes, grain free. Isolated red blood cells are constantly detected in sputum. The presence of a large number of RBC is characteristic of bloody sputum. Under the influence of rot processes red blood cells are destroyed. 

Flat epithelium looks like flat colorless cells, 10 times greater than the leukocytes; has a round or polygonal shape and a small round nucleus located in the centre of the  cells. This is desquamated epithelium of the mucous membranes of the oral cavity and nasopharynx. Some squamous cells as always detected in the sputum,  in large numbers - if sputum is  mixed with saliva, so there is no  diagnostic value.

Cylindrical ciliary epithelium looks like elongated cells expanded at one end and narrow at the other (form of  wineglass). At the expanded end of the cells are placed cilia, and in the narrower end - oval nucleus. These cells are placed in groups.  In the fresh material can be seen active movement of cilia. Very often cylindrical epithelium modifies: losing cilia, changing the shape of cells (becoming triangular or  round).

The detection in sputum ciliary cylindrical epithelium, which lining the mucosa of the trachea and bronchi, shows damage of these organs (asthma, bronchitis, tracheitis, etc.).

Alveolar macrophages - cells of reticulo-histiotsytic origin, freely moving to the place of inflammation and are capable of phagocytosis. They have oval or round shape, size, twice or three times larger than white blood cells, the nucleus is located eccentrically bean-like or round form, in the cytoplasm are inclusions (dark brown particles). Macrophages absorb dust, leukocytes, erythrocytes, bacteria, etc. In the case of chronic inflammatory processes  macrophages often undergo fatty degeneration. Cytoplasm of such cells filled with fat droplets (granular balls).

Alveolar macrophages in sputum occur as clusters in patients with pneumonia, bronchitis, professional  lung diseases and in smokers. Macrophages also are detected in sputum of  patients with cancer, actinomycosis, pulmonary tuberculosis.

Syderophages - are alveolar macrophages containing hemosiderin in the form of golden-yellow color inclusions. The old name of these cells - "cells of heart defects," as they appear in the sputum in case of stasis in the lungs, which is typical for heart disease, myocardial infarction. To differentiate syderophages  from alveolar macrophages containing dust particles and nicotine, should make the reaction to Prussian blue, which is positive in syderophages (hemosiderin contained in syderophages is  painted in blue or blue-green color).

Tumor atypical cells are found in sputum in case of tumors in the lungs and bronchi. Features of malignancy cells are their size polymorphism, violation of nuclear-cytoplasmic ratio in the direction of increasing the nucleus, the presence of hyperchromatic nuclei, changing shape of the nucleus, the presence in it nucleoli of irregular shape, mitosis of the cells. These cells are arranged separately or as clusters (complexes) with no clear boundaries. Polymorphism of the cells size and  nuclei shape, randomly placing the cells in the complex - is characteristic feature  of malignancy.

Fiber formations

Elastic fibers are elements of the connective tissue, they are detected in sputum in case of pathological processes such as destroying of  lung tissue (lung abscess, tuberculosis, cancer, etc.). Identification of elastic fibers is important for differentiating lung abscess from lung gangrene. As with cavernous tuberculosis, and for lung abscess in sputum elastic fibers are present; in the case of gangrene elastic fibers are absent due to rapid destruction.

Elastic fibers in the native preparation look like shiny, double, fibrous strands that sometimes are packed in bunches, often they follow the structure of the alveoli. In the case of pulmonary tuberculosis in clusters and fragments of elastic fibers Mycobacterium tuberculosis is detected. In the case of lung abscess elastic fibers look like clusters among the pus together with hematoidyn crystals.

Coral-like fibers in sputum are present in case of  chronic lung diseases such as cavernous tuberculosis. In the cavernous cavity elastic fibers are covered with fatty acids and alkalis and became coarser, like sea coral.

Calx fiber - this elastic fibers are impregnated with calx salts. They lose their elasticity, become fragile, brittle and take the form of dotted lines, that composed of separate, gray rods that refract light. They are found in sputum in the amorphous mass of calx salt and drops of fat, which indicates the presence in the lungs calx caseous decomposition that is characteristic for pulmonary tuberculosis.

Ehrlich Tetrada – these are elements, which get into a sputum from calx primary tuberculous center. The composition of Ehrlich tetrady includes four elements:

                1) Calx elastic fibers

                2) Calx detritus

                3) Crystals of  cholesterol

                4) Mycobacterium tuberculosis.

Kurshman’ spirals- whitish, winding, spiral –like mucous formations, sharply differentiated from other mucous sputum, have diagnostic value in case of bronchial asthma


Crystal formations

Charcot-Leyden crystals have the form of colorless, transparent, elongated rhombus of various sizes. Typically, they are detected in sputum containing eosinophils. The formation of Charcot-Leyden crystals is associated with the break down of eosinophils, that’s why very often fresh sputum does not contain these crystals, they formed it in 24-48 hours. They are detected in sputum in patients with bronchial asthma,  eosinophilic bronchitis, worms invasion of lungs.

Crystals of hematoydin have the form of rhombus, stars, needles, collected in bundles of golden-yellow color. They are formed   due to the collapse of hemoglobin in oxygen-free environment (hemorrhage in the necrotic tissue). In preparations of sputum they are placed  on the detritus and elastic fibers, indicating the breakdown of lung tissue. Crystals of hematoydin have  be distinguished from grains of hemosyderin - golden-yellow inclusions in the cytoplasm of alveolar macrophages, giving a positive reaction to Prussian blue.

Cholesterol crystals have the form of colorless rectangular with cut corners; situated separately or superimposed on each other. They are form due to the collapse of fatty-rebirth cells, staing sputum in the cavities and are placed on the detritus. They are detected in sputum in patients with purulent processes in the lung (abscess), tuberculosis, tumors.

Crystals of fatty acids have the form of grayish formations with niddle shape, which are situated on the detritus and bacterias.  They are formed in case of staing sputum in cavities. They are detected in sputum in patients with lung abscess, bronchiectasis, tuberculosis.

Micropreparation of sputum. Flat epithelium, cylindrical  epithelium, WBC.

Micropreparation of sputum. Alveolar macrophages, flat epithelium, cylindrical  epithelium WBC

Micropreparation of sputum. Alveolar macrophages, which contain in the cytoplasm inclusions of hemosiderin dark-blue color

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Micropreparation of sputum. Alveolar macrophage



Microflora. In stained preparations of sputum it is possible to find  various microorganisms, which in small quantities always are present in the airways of healthy humans. In particularly adverse conditions  (cooling, decreased resistance, this flora becomes pathogenic and causes disease. The most important diagnostic significance has detection Mycobacterium tuberculosis in sputum, which is agent of pulmonary tuberculosis. Mycobacterium tuberculosis - acid and alcohol resistant microorganisms. Due to  Tsile-Nielsen Mycobacterium tuberculosis colored in red color (thin, slightly curved rods of different lengths, placed together or separately), and all other organisms have blue color.

In the sputum it is possible to detect these microorganisms: streptococci, staphylococci, pneumococci, Klebsiella in Gram stained preparations. Bacteria that are colored by Gram stain, are called gram-positive (blue) and those that are not painted - Gram-negative (red).  Streptokoky, staphylococci, diphtheritic rod and so on are gram-positive.  Klebsiella, typhoid rods, catarrhal micrococcuses et al. - gram-negative.


Acute bronchitis. At the onset of the disease is excreted small amount of mucous, viscous sputum. Further increasing the amount of sputum. It becomes mucous-purulent. During microscopic studies is detected a lot  of cylindrical epithelium, leukocytes, sometimes - red blood cells.

Chronic bronchitis. As a rule, is excreted a lot of mucous-purulent sputum, often with streaks of blood. Microscopically is detected a large number of leukocytes, erythrocytes, cylindrical epithelium, alveolar macrophages, many different microorganisms.

Micropreparation of sputum. Inflammation. Alveolar macrophages, leukocytes.

Bronchial asthma. It is excreted a small amount of  mucous, viscous, glassy - like sputum. Kurshman’ spirals can be detected macroscopically. Microscopic studies is particularly characterized by detection of eosinophils and cylindrical epithelium. Charcot-Leyden crystals also are present.

Micropreparation of sputum of patient with Bronchial asthma. Kurshman’ spirals,  eosinophils, Charcot-Leyden crystals.

Bronchiectasis. It is excreted a lot of purulent sputum (in the morning to 1 liter) grayish-green color. After settling sputum is divided into three layers: mucous, serous and purulent. Macroscopically Ditryh Tubes can be detected. Microscopically a large number of white blood cells, crystals of fatty acids, sometimes crystals of hematoydin and cholesterol, different microflora are detected.

Lobar pneumonia. At the onset of disease small amount of viscous rusty sputum is excreted. As the disease progresses  sputum is secreted more, it becomes mucous-purulent. Rusty sputum frequently contains clots of fibrin and altered blood, which gives it a brown color. Microscopically red blood cells, grains of hemosyderin, crystals of hematoydin, a small amount of white blood cells, many pneumococci are identified et the beginning of disease. At the end of the disease amount of leukocytes increases and red blood cells decreases, a lot of alveolar macrophages are identified.

Lung abscess. At the time of breakthrough abscess in bronchus is excreted a lot of sputum (600 ml). After settling the sputum becomes two-layer. Microscopically it is possible to find many white blood cells, elastic fibers, scraps of lung tissue, crystals of fatty acids and cholesterol, different flora.

Tuberculosis of the lungs. The amount of sputum depends on the stage of the disease. In the presence of cavities in the lungs amount of sputum can be significant. Sputum is mucous-purulent, often mixed with blood. Macroscopically in sputum can be detected rice-like bodies (Koch lenses), consisting of the elements of decomposed lung tissue. Under the microscope elastic fibers, crystals of fatty acids, hematoydin are identified. Ehrlich Tetrada – these are elements, which get into a sputum from calx primary tuberculous center. The composition of Ehrlich tetrady includes four elements: 1) Calx elastic fibers, 2) Calx detritus, 3) Crystals of  cholesterol, 4) Mycobacterium tuberculosis.

Lung cancer. The amount  of sputum may be different. In case of breakup of tumors - is significant. It is mucous-purulent-bloody. During macroscopic study can be found fragments of lung tissue. Microscopically atypical cells and their complexes are detected.

Micropreparation of sputum of patient with lung cancer. Tumor cells.




Micropreparation of sputum of patient with lung cancer. Tumor cells.



The closed cavities of the body — namely, the pleural, pericardial, and peritoneal cavities—are each lined by two membranes referred to as the serous membranes. One membrane lines the cavity wall (parietal membrane), and the other covers the organs within the cavity (visceral membrane). The fluid between the membranes is called serous fluid, and it provides lubrication between the parietal and visceral membranes. Lubrication is necessary to prevent the friction between the two membranes that occurs as a result of movement of the enclosed organs. An example of this movementis the expansion and contraction of the lungs. Normally, only a small amount of serous fluid is present, because production and reabsorption take place at a constant rate. Serous fluids are formed as ultrafiltrates of plasma, with no additional material contributed by the mesothelial cells that line the membranes. Production and reabsorption are subject to hydrostatic and colloidal (oncotic) pressures from the capillaries that serve the cavities and the capillary permeability. Under normal conditions, colloidal pressure from serum proteins is the same in the capillaries on both sides of the membrane. Therefore, the hydrostatic pressure in the parietal and visceral capillaries causes fluid to enter between the membranes. The filtration of the plasma ultrafiltrate results in increased oncotic pressure in the capillaries that favors reabsorption of fluid back into the capillaries. This produces a continuous exchange of serous fluid and maintains the normal volume of fluid beween the membranes. The slightly different amount of positive pressure in the parietal and visceral capillaries creates a small excess of fluid that is reabsorbed by the lymphatic capillaries located in the membranes.

Disruption of the mechanisms of serous fluid formation and reabsorption causes an increase in fluid between the membranes. This is termed an effusion. Primary causes of effusions include increased hydrostatic pressure (congestive heart failure), decreased oncotic pressure (hypoproteinemia), increased capillary permeability (inflammation and infection), and lymphatic obstruction (tumors)

Pathological causes of effusions

1. Increased capillary hydrostatic pressure

Congestive heart failure

Salt and fluid retention

2. Decreased oncotic pressure

Nephrotic syndrome

Hepatic cirrhosis


Protein-losing enteropathy

3. Increased capillary permeability

Microbial infections

Membrane inflammations


4. Lymphatic obstruction

Malignant tumors, lymphomas

Infection and inflammation

Thoracic duct injury



Formation of Pleural Fluid



Pleural Cavity

Effusion of Serous Fluid

It is the disruption of the mechanism of serous fluid formation and reabsroption causes an increase in fluid between the membranes

Effusion of Serous Fluid


1. Increased Hydrostatic Pressure

• Congestive heart failure pressure

2. Decreased Colloid Pressure

• Hypoproteinemia

• Increased capillary permeability (inflammation and infection)

• Lymphatic obstruction (tumors)

Collection of Serous Fluid

Fluid is collected by needle aspiration (100 mL) from the respective cavities

1. Thoracentesis for pleural cavity

2. Pericardiocentesis for pericardial cavity

3. Paracentesis for peritoneal cavity





Classification of Effusion

1. Transudates


They produced because of a systemic disorder that disrupts the balance in the regulation of fluid filtration and reabsorption as the change in hydrostatic pressure created by congestive heart failure or the hypoproteinemia associated with the

nephrotic syndrome.

2. Exudates


They are produced by conditions that directly involve the membranes of the particular cavity, including infections and malignancies.

Normal mesothelial cells at 40X. Notice groups and singly distributed cells with central or (occasionally) eccentrically placed round nuclei.  Nucleoli are prominent.

This is a cluster of inflammatory cells (PMN’s and macrophages) at 40X.

Sheet of normal mesothelial cells from a pelvic washing at 40X. Notice their small nuclei and honeycombed pattern over most of the group and the variation that exists at the 5:00 position.

Reactive mesothelial cells at 40X from a peritoneal washing specimen.  Cells are slightly larger with increased nucleolar prominence.


Pleural Fluid

1. It is obtained from the pleural cavity, located between the parietal pleural membrane lining the chest wall and visceral pleural membrane covering the lungs

2. Pleural effusions can be transudative or exudative origin

3. Procedures are helpful when analyzing pleural fluid

• For Exudates, if

• Pleural Fluid Cholesterol > 60 mg/dL or

• Pleural Fluid/Serum Cholesterol Ratio > 0.3

• Pleural Fluid/Serum Total Bilirunbin Ratio > 0.6

If at least one of the following three criteria is present, the fluid is virtually always an exudate

• If none is present, the fluid is virtually always a transudate

• Pleural fluid protein/serum protein ratio greater than 0.5.

• Pleural fluid LDH/serum LDH ratio greater than 0.6.

• Pleural fluid LDH greater than two thirds the upper limits of normal of the serum LDH

Adenocarcinoma is the most common malignancy found in the serous cavities.  These are characteristics shared by most adenocarcinomas.  However, further criteria can be specified when describing tumors from specific primaries.  For example, an adenocarcinoma originating from the ovary is identifiable by its prominent eosinophilic nucleoli, large, round vesicular nuclei and highly vacuolated cytoplasm.

Common metastatic lesions:

Pleura: Lung, breast, ovary

Peritoneum: Pancreas, liver, colon, cervix, ovary, kidney, bladder

Peritoneal fluid containing metastatic endometrial carcinoma at 60X with abundant psammoma bodies.   Notice cell ball arrangement and delicate cytoplasm with large vacuoles.

Peritoneal fluid containing metastatic ovarian adenocarcinoma at 10X.

40X view of metastatic breast adenocarcinoma




40X adenocarcinoma in pleural fluid.


These are some general criteria that may be used to differentiate between adenocarcinoma and malignant mesothelioma.  The collagen core that may be present at the center of groups of mesothelial cells usually stains a faint green with the pap stain. Also, orangeophilic cells with pyknotic nuclei, reminiscent of squamous cells, may be seen in malignant mesotheliomas and probably represent degenerated mesothelial cells.


Malignant mesothelioma

May have two cell population

Single cell population

Lacy cytoplasm

Dense cytoplasm

Groups have smooth borders

Groups are hobnailed with “windows” between cells

Nucleus is peripheral

Nucleus is central

Mucin may be present

Collagen core may be present


Melanoma in a pleural fluid at 60x.  This specimen shows abundant melanin pigment.

Multiple myeloma in ascites at 40X.


Pericardial Fluid

1. Normally, only a small amount (10-50 mL) of fluid is found between the pericardial serous membranes.

2. Pericardial effusions are result primarily of changes in the permeability of the membranes due to infection (pericarditis), malignancy, trauma, or metabolic disorders as uremia.

3. Presence of pericardial effusion is expected when cardiac compression is noted during the physician’s examination.


Peritoneal Fluid

1. Accumulations of fluid in the peritoneal fluid cavity is called ascites, and the fluid is commonly referred to as ascitic fluid rather than peritoneal fluid

2. Hepatic disorder, such as cirrhosis, are frequent causes ascetic transudative fluids

3. Bacterial infections (peritonitis) are most frequent causes of ascitic exudative fluids

Ascitic Transudates vs Exudates

1. Differentiation between ascitic fluid transudates and exudates is more difficult that for pleural and pericardial effusions

2. Serum/ascites albumin gradient is recommended over the fluid/serum total protein and LDH ratios for detection for the transudates of hepatic origin

3. A difference (gradient) of 1.1 or greater suggests a transudates effusion of hepatic origin, and lower gradients are associated with exudative effusions

4. Example:

Serum albumin = 3.8 mg/dL

Fluid albumin = 1.2 mg/dL

Gradient 3.8 – 1.2 = 2.6 then indicating hepatic effusion