T Laboratory and Instrumental Methods
FUNCTIONAL STUDY OF THE LIVER
Not all functions are affected in an involved liver; the dysfunctions are not simultaneous, and their degree is different. The reserves of the liver are significant: preservation of 20 per cent of the functioning parenchyma is sufficient to maintain vital activity of experimental animals. The regenerative power of the liver is great too. Therefore, certain hypofunction of the liver cannot bear substantial effect on the patient's condition: the affected liver is able to maintain the main vital processes in the new conditions.
faeces
unbound bilirubin bound bilirubin
Fig. 94. Secretion of bilirubin in norm (/), in haemolytic jaundice (2); in parenchymatous jaundice (3), and in obstructive jaundice (4).,
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Early in the 20th century, van den Bergh noted different reaction of jaundice serum with sulphodiazo reagent in jaundice of various aetiology. The serum of a patient with obstructive jaundice turned red immediately after the diazo reagent was added, while the serum of a patient with haemolytic jaundice turned red only after alcohol was added. The first reaction was called "direct" and the second "indirect". Unbound bilirubin gives indirect reaction while bilirubin glucuronide gives a direct reaction. Depending on the number of molecules (one or two) of glucuronic acid added to the bilirubin molecule, mono- or diglucuronide of bilirubin is formed.
The blood of healthy people contains only free liver pigment. In diseases attended by disordered or perverted bound-bilirubin excretion with bile, the bound bilirubin enters blood and both pigments then become involved in the circulation. (They can be determined separalety.) Van der Bergh's qualitative test gives only tentative information: if the reaction is indirect, it may be considered that unbound bilirubin alone is present in the blood; if the reaction is direct, the proportion of both pigments in the blood is unknown because the positive direct reaction masks the presence of any quantity of unbound bilirubin. The bilirubin fractions are now determined separately. The same diazo reagents (as in qualitative tests) are used in most techniques. Diazo reagent I is prepared by dissolving 5 g of sulphanylic acid and 15 ml of strong hydrochloric acid in distilled water, which is then added to make 1 litre. Diazo reagent II is a 0.5 per cent solution of sodium nitrite. (The diazo mixture consists of 10 ml of solution I and 0.25 ml of solution II.)
Qualitative test: 0.25 ml of the diazo mixture is added to 0.5 ml of serum. If the mixture turns red sooner than in 1 min, the reaction is quick direct to indicate the presence of bound bilirubin in the serum. If the colour changes slowly (within 1—10 min), the bound bilirubin content is relatively small; the reaction is considered to be slow direct. If the solution turns red in more than ten minutes, the direct reaction is considered to be negative. If it is necessary to make sure that the yellow colour of the serum depends on the presence of bilirubin, a double quantity of alcohol is added, the solution is passed through a filter, and the diazo mixture is added to the filtrate: pink colour develops (indirect reaction).
There are many methods for quantitative determination of the bilirubin fractions. Some of them are based on the reaction of bilirubin with the diazo reagent in the presence of caffeine (which is used in a common Jen-drassik test), methyl alcohol, and some other reagents which act like a catalyst. The total amount of both fractions contained in the serum treated with the catalyst can be determined. Only bound pigment is determined in
another portion of the serum containing no catalyst. The amount of free bilirubin can be found by subtracting the bound fraction from the total bilirubin content of the blood. Other methods for separate determination of bilirubin fractions (chemical, chromatographic) are more complicated.
Unbound bilirubin is insoluble in water and cannot be excreted by the kidneys. When bound with glucuronic acid it becomes soluble and can be determined in the urine of patients with subhepatic and hepatic jaundice. Only bound bilirubin (bilirubin glucuronide) is excreted into the bile ducts. A small portion of bilirubin is reduced to urobilinogen in the large bile ducts and the gall bladder (especially if they are affected by inflammation) and farther in the intestine. Urobilinogen is resorbed in the upper portion of the small intestine and delivered to the liver with the blood of the portal vein. A healthy liver completely traps it and oxidizes, but an affected liver cannot perform this function and urobilinogen passes into blood and is excreted with urine as urobilin. Urobilinuria is an early and very sensitive sign of liver dysfunction. The greater portion of bilirubin is reduced in the intestine to stercobilinogen. Its main portion is excreted with faeces in which it is converted into stercobilin to give faeces its normal colour (upon exposure to air and light). A small portion of stercobilinogen is absorbed in the lower portions of the large intestine, bypasses the liver, and enters the general circulation through the haemorrhoidal veins to be excreted by the kidneys. Normal urine always contains traces of stercobilinogen which converts into stercobilin on exposure to air and light.
Most reactions by which products of bilirubin reduction are discovered in urine give similar results with both urobilin and stercobilin, although these two compounds differ in their chemical structure and physical properties. Methods of their separation are relatively complicated. They are therefore determined together and designated by a common name of urobilinoids.
Urobilin content of the urine increases not only in the presence of hepatic insufficiency, but also in increased haemolysis. In this case, due to release of considerable quantity of haemoglobin, greater amounts of bilirubin are produced and liberated into the intestine. Increased production of stercobilin intensifies its excretion with urine. In obstructive jaundice, when bile is not supplied to the intestine, stercobilin is absent from faeces and the urine is free from urobilin casts. Bilirubin excretion with bile and stercobilin content of faeces decrease in hepatic jaundice, while urobilin casts of urine increase. Their normal ratio is from 10:1 to 20:1, but in pathology it decreases significantly, and is as low as 1:1 in severe liver affections.
Stercobilin content of faeces in haemolytic jaundice exceeds significantly urobilin excretion in the urine. Their ratio increases to 300:1-500:1.
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The ratio between the bilirubin reduction products in faeces and urine is much more informative for differentiation of various types of jaundice than the absolute content of each substance.
Carbohydrate metabolism. With participation of the enzyme systems, liver cells synthesize glycogen and deposit it; glycogenolysis and glyconeogenesis also occur here. The normal sugar content of blood is maintained also by some other organs and systems, such as the pancreas, the pituitary-adrenal system, etc. In this connection, the blood sugar content with the fasting stomach changes only in very severe liver affections, and therefore functional tests can only reveal carbohydrate hypofunction of the liver. The glucose tolerance test is not effective because the blood glucose content depends not only on the above-mentioned organs but also on the condition of the vegetative nervous system, on the glycogen stores in the liver and muscles, and some other factors.
The galactose tolerance test is an effective method to assess the liver function. Galactose is not assimilated by the bodily tissues or organs except the liver; its blood content does not depend on hormones. The patient is given to drink a solution of 40 g of galactose in 200 ml of water, and galactose excretion in the urine is then determined. Normally maximum 3 g of galactose are excreted in 4 hours. The renal function and the absorption power of the intestine may affect galactose excretion in the urine, and the determination of galactose in the blood is therefore more reliable. The maximum rise of the blood sugar occurs in 30-60 minutes with the normal liver function. The maximum level does not exceed 150 per cent of the initial one, which is restored in two hours. If the liver function is inadequate, the sugar level is higher, while the decrease in the galactose level in the blood is slower.
The glycogenolysis function is determined by the adrenaline test. The patient is given subcutaneously 1 ml of a 0.1 per cent adrenaline solution. In healthy subjects this injection increases the blood sugar level by at least 50 per cent. If the liver is affected, no rise is observed.
The role of the liver in the protein metabolism is very important: the liver synthesizes and retains proteins. Amino acids, polypeptides of food, and products of breakdown of tissue proteins are delivered into the liver with blood where they are catabolized, detoxicated, and the unused breakdown products are removed. Some amino acids are deaminated and reaminated. The released ammonia is converted by the liver into less toxic urea. Amino acids (both produced by the liver and carried from outside) are used by the liver to build proteins of its own tissue and also blood proteins: albumin, globulins, (alpha, beta, and to a certain extent gamma globulins), fibrinogen, prothrombin, heparin, and certain enzymes. The liver synthesizes also compounds of proteins with lipids (lipoproteins) and
I
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carbohydrates (glycoproteins). The disorder in the protein-synthesizing function of the liver is revealed by studying the proteins of the blood plasma or serum. This dysfunction has its effect not only on the total protein content but also on the ratio of its different fractions, which is more important diagnostically: the upset protein ratio (dysproteinaemia) is characteristic of most liver pathologies.
Paper electrophoresis is widely used in clinic now. Depending on the size and shape of molecules, their charge, and some other factors, various proteins move at different velocities toward the positive electrode. Various protein fractions concentrate on different portions of paper during electrophoresis; proteins are then revealed by staining. The content of each separate fraction is determined by colour intensity. Proteins of the blood plasma are separated into five main fractions: albumins, alpha^ alpha2, beta, and also gamma globulins (see Table 4). Electrophoresis in other media (agar, starch gel, etc.) separates proteins into greater quantity of fractions.
Table 4
Fraction
Normal Proteinogram
average
Normal percentage
variation
60.9 39.1
4.2
8.2
11.5
15.2
1.2-2
56.3-68.8
32.0-43.0
3.0- 5.8
6.9-10.5
7.3-12.5 12.8-19.2
Albumins Globulins:
alpha j
alpha2
beta
gamma A-G ratio
The A-G ratio most frequently decreases in liver diseases. This occurs mainly due to the decrease in the albumin content (their upset synthesis). In patients with acute inflammation of the liver (acute hepatitis) the content of alpha2 globulins in the blood plasma increases, while in chronic hepatitis the gamma globulin content increases probably due to accumulation of antibodies which move during electrophoresis with gamma globulins. The total serum protein content often increases as well. The total protein content decreases sharply (at the expense of albumins) in patients with liver cirrhosis; the content of gamma globulins, however, increases markedly.
During electrophoresis, fibrinogen migrates together with gamma globulins and cannot be detected separately. For quantitative determination of fibrinogen, it is precipitated from plasma by calcium chloride with
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subsequent weighing of the washed and dried precipitate, or by determining protein in the precipitate after its dissolution. Fibrinogen is synthesized in the liver and its content of plasma therefore decreases significantly if the liver is seriously affected. This affects blood coagulation. The normal fibrinogen content of plasma is from 200 to 400 mg/100 ml (2-4 g/1 or 8—13 mg/ml; clot weight).
The total plasma protein content is determined mostly by the refrac-tometric method; if a refractometer is not available, chemical methods can be used (Kjeldahl method, biuret test, nephelometric method, etc.).
The ratio of the protein fractions can also be determined by im-munoelectrophoresis, ultracentrifuging, etc. In addition to direct determination of the ratio of the protein fractions, dysproteinaemia can also be determined by simple methods. These are the so-called protein sedimentation (flocculation) tests. In the presence of dysproteinaemia (especially in decreased albumin content) the equilibrium of the colloidal blood system becomes upset. This disturbance can be revealed by adding electrolyte to the blood serum in the concentration that would not alter normal serum but in dysproteinaemia would cause cloudiness or precipitation of flakes (protein flocculation). The presence of paraproteins (pathological proteins) can also be detected in the blood. These tests include reactions with corrosive sublimate (Takata-Ara test, Grinsted and Gross sublimate tests), zinc sulphate, cadmium sulphate, Lugol's iodine solutions, etc. In another group of flocculation tests the stability of a colloidal solution is upset by adding a small portion of dys- or paraproteinaemic serum (thymol, colloidal gold test, etc.).
The thymol turbidity test is based on determination of turbidity of a colloidal thymol reagent caused by adding 1/60 volume of serum. The test is positive mostly in the increased blood serum beta-lipoprotein content. It is always positive in virus hepatitis and diffuse affections of the liver. It is negative in obstructive jaundice.
In the presence of significant excess of globulins, and especially of fibrinogen, the formol-gel test becomes positive (the serum converts into a gel from the addition of formaldehyde).
Dozens of sedimentation tests have been proposed. They all are nonspecific and are positive not only in the presence of liver diseases but also in multiple myeloma, collagenosis, etc. These tests reveal dysproteinaemia more easily than electrophoresis.
Prothrombin (factor II of blood coagulation) is synthesized in the liver with participation of vitamin K. The cause of hypoprothrombinaemia is either upset synthesis of prothrombin by the hepatocytes or vitamin K deficiency (vitamin K is fat-soluble and is delivered to the liver from the intestine). In the presence of obstructive jaundice, when absorption of fats
and vitamin K is deranged due to the obstructed delivery of bile acid to the intestine, the synthesis of prothrombin in the liver decreases and the blood prothrombin content decreases as well. In order to reveal the cause of hypoprothrombinaemia, a test with parenteral administration of vitamin K is used. If the blood prothrombin content increases after administration of vitamin K, the prothrombin synthesizing function of the liver is normal. This test helps differentiate between obstructive and parenchymatous jaundice. Prothrombin is determined by the rate of coagulation of recalcified plasma in the presence of excess thromboplastin.
Amino acids, urea, residual nitrogen and ammonia are the products of protein decomposition which have a certain diagnostic importance. The total blood content of amino acids increases only in severe affections of the liver with impairment of its deaminating and urea-forming functions (otherwise rather stable). The condition for the increase in residual nitrogen of blood is a simultaneous renal dysfunction. Increased residual nitrogen occurring in renal insufficiency alone differs from that occurring in hepatorenal insufficiency by the main component of residual nitrogen. This is urea in renal insufficiency and amino acids in hepatorenal dysfunction. Separate determination of amino acids of the blood by chromatographic methods does not give reliable diagnostic information and the labour-consuming chromatographic procedure is not thus justified. Determination of leucine and tyrosine crystals that appear in the urine sediment in the presence of acute dystrophy of the liver is of certain diagnostic importance.
The blood ammonia content increases when the liver is unable to detox-icate ammonia delivered from the intestine (by synthesizing urea). Accumulation of ammonia in the blood produces a toxic effect on the central nervous system. Hyperammoniaemia is therefore a forerunner of hepatic coma.
The importance of the liver is especially great in fat metabolism. The liver performs the decisive role in the synthesis and splitting of fats, phospholipids, and cholesterol, in esterification and liberation of cholesterol, and in maintaining constant cholesterol content of blood. Blood lipids change their concentration in liver affections. Normal blood serum cholesterol content is 3.9-5.2 mmole/1 (150-200 mg/100 ml). The cholesterol concentration decreases in patients with severe forms of acute and chronic hepatitis and cirrhosis of the liver. Cholesterol increases in most obstructive jaundices. The activity of alkaline phosphatase usually increases simultaneously. But these quantitative shifts are not always demonstrative. The degree of esterification of cholesterol (their combination with fatty acids), which occurs mostly in the liver, is more informative diagnostically. Normally 60-70 per cent of blood cholesterol is ester-
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bound, but this percentage decreases in liver affections due to the decreased esterase activity. The decrease is proportional to the degree of liver dysfunction. The ratio of the ester-bound cholesterol to the total blood cholesterol (normally 0.6-0.7) is called esterification coefficient. When markedly decreased, this coefficient is a poor prognostic sign.
The blood phospholipid content in the presence of liver pathology changes mainly in the same way as the content of cholesterol. The blood content of lipoprotein fractions is also affected by liver diseases.
Certain blood microelements are important diagnostically. The most informative are iron and copper. Both elements are contained in the serum as metal proteids, i.e. compounds with proteins, in which they are present in microgram quantities. These metals are also present in the liver, which serves as a depot for them. Iron is deposited in the liver as ferritin, an iron-protein complex, that is a reserve of iron used for the synthesis of haemoglobin in the bone marrow. Another iron compound is haemosiderin, the product of haemoglobin decomposition, which is accumulated in the liver in increased haemolysis and also in some diseases. Transferrin (the transport protein) which carries iron from the liver to the bone marrow is also synthesized in the liver. Iron that is not bound in haemoglobin is also determined in the blood serum for diagnostic purposes: its content increases significantly (2—3 times) in acute hepatitis; in chronic hepatitis and cirrhosis the increase is less significant, while it does not change (or even decreases) in obstructive jaundice.
Copper is contained in the blood as an oxidative enzyme ceruloplasmin; it is also contained in the liver as a copper-containing protein hepatocuprein. The blood serum copper content slightly increases in hepatitis, and the increase is pronounced in obstructive jaundice. The iron to copper ratio is always decreased in obstructive jaundice and is mostly increased in parenchymatous affections of the liver.
Study of liver enzymes. The liver cells contain numerous enzymes regulating metabolic processes in the liver. Affection of hepatocytes causes an increased excretion into the blood of some enzymes, while the synthesis of other enzymes decreases. Changes in activity of enzymes in the blood serum, which are a sensitive and quick response to liver affections, are widely used for diagnostic purposes. Some of these enzymes are produced not only by the liver but also by some other organs. But changes occurring in some enzymes during liver pathology are so constant that their determination becomes of great practical value. These are transaminase, aldolase, alkaline phosphatase, cholinesterase, lactic dehydrogenase (see Table 12).
Determination of isoenzymes of certain enzymes mentioned above gives more reliable diagnostic information. Isoenzymes are group of enzymes
that have similar catalytic action but differ in the structure of their proteins. During electrophoresis in starch gel, an isolated enzyme is subdivided into isoenzymes due to their different electrophoretic mobility.
A spectrum of certain isoenzymes is typical for affection of separate organs. For example, of the five isoenzymes of lactic dehydrogenase, the fifth fraction (LDG5) is regularly increased in chronic hepatitis and cirrhosis of the liver, while the increase in the LDG! is characteristic of myocardial infarction. Determination of isoenzymes of aldolase, aspartate aminotransferase, leucine aminopeptidase, and of some other enzymes is also important.
Changes in the activity of organospecific enzymes, i.e. the enzymes characteristic only of liver cells, are even more informative: their activity changes only in pathology of the liver. These enzymes are ornithine car-bamoyl transferase and arginase taking part in the synthesis of urea, sor-bitol dehydrogenase catalysing oxidation of sorbitol to fructose, guanine deaminase catalysing conversion of guanine to xanthine, quinine oxidase oxidizing quinine, etc.
Transaminases are the enzymes catalysing the transfer of the amino group from amino acids to keto acids. Among them diagnostically important are aspartate aminotransferases (AsAT, glutamino-oxalo-acetic acid transaminase) and alanine aminotransferase (A1AT, glutaminopyruvic transaminase). Although their increasing activity is a non-specific sign because it is observed in liver diseases and generally in their diffusion from injured tissues (e.g. myocardium, kidneys, pancreas), their activity may nevertheless be very high in myocardial infarction and hepatitis. A1AT dominates in hepatitis, and AsAT in myocardial infarction. The main importance of the test is that activity of both transaminases increases significantly during non-icteric period of acute hepatitis (Botkin's disease). This facilitates its early diagnosis and also identification of non-icteric forms of hepatitis.
The content of aldolase (fructose 1,6-phosphate aldolase) increases markedly in the blood serum of patients with liver diseases. Hyperaldolasaemia regularly attends epidemic hepatitis and the test for aldolase is therefore obligatory for diagnosis of the disease.
Alkaline phosphatase is the enzyme hydrolysing esters of phosphoric acid. It is mostly formed outside the liver but is excreted by this organ. The increase in the activity of the blood alkaline phosphatase is especially pronounced in obstructive jaundice, due to malignant tumour, and also in in-trahepatic cholestasis, and biliary cirrhosis. In patients with the affected liver parenchyma, the activity of this enzyme increases moderately.
Serum cholinesterase (pseudocholinesterase) splits acetylcholine and other choline esters. It is formed in the cells of the liver parenchyma. Its
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determination is of great prognostic importance: the lower the activity of pseudocholinesterase in hepatitis, the more severe is the course of the disease.
Detoxicating function of the liver. Blood of the portal vein supplied from the gastro-intestinal tract contains various toxic substances for which the liver imposes a barrier. These substances are retained by the liver and almost completely detoxicated by the enzymes responsible for oxidation, reduction, deamination, hydrolysis, methylation, combination with sulphuric acid, glucuronic acid, and with glycine. These reactions give less toxic or more readily soluble substances that can be excreted in the bile or urine. Ammonia, for example, is converted into less toxic urea, free bilirubin combines with glucuronic acid to turn into a less toxic and water-soluble compound that can be excreted in the bile and urine. Phenols, in-doles, ketones, alcohols, sulpha preparations, amidopyrine, camphor, and morphine are detoxicated mainly by combining with glucuronic or sulphuric acid; sodium benzoate combines with glycine; santonin oxidizes to oxysantonin; metals react with nucleoproteins. Stellar reticuloen-dotheliocytes retain and phagocytise microbes.
Sodium benzoate test. Sodium benzoate is given per os or intravenously. It combines with glycine in the liver to give hippuric acid which is excreted in the urine. The detoxicating function of the liver is assessed by the percentage of urine-excreted sodium benzoate in the form of hippuric acid. If the liver parenchyma is affected, synthesis of hippuric acid is disturbed and its excretion is slowed. The test has some disadvantages: it precipitates in obstructive jaundice, tumours, fevers (positive test), and requires normal renal function.
Excretory function of the liver. Among substances to be excreted from the body are water-soluble compounds that are excreted mainly by the kidneys, and water-insoluble or protein-bound compounds that are excreted by the liver. The normal hepatic excretory capacity is restricted. In significantly intensified haemolysis, a healthy liver cannot excrete all bilirubin from the blood where it thus accumulates. Parenchymal affections decrease the excretory capacity of the liver to cause, for example, bilirubinaemia, which however is not an obligatory symptom. In order to reveal the disordered excretory function of the liver, especially in non-icteric affections, tests are used with administration into the blood of substances that should be excreted with bile.
Bilirubin test is most informative: after an intravenous injection of 50 mg of bilirubin, its content in the blood is determined in 5 minutes and in 4 hours. If excretory function of the liver is normal the pigment concentration decreases in 4 hours to 15 per cent of its concentration as determined in 5 minutes after the injection. The test is not however used universally because of expensiveness of the preparation and uselessness of the test in the presence of jaundice.
The bromsulphthalein test is one of the most specific. Brom-sulphthalein is given intravenously (5 mg/kg body weight) and the first specimen of blood is taken in 3 minutes (when the concentration of the preparation in the blood attains its maximum). Another specimen is taken in 45 minutes after the injection. Concentration of bromsulphthalein is determined colorimetrically: it turns red-violet when alkali is added. If the liver function is adequate, the preparation concentration in the blood in 45 minutes does not exceed 5 per cent of its initial concentration, which is assumed to be 100 per cent. The stain can be detected in the bile in 15 minutes after the injection. The test is very sensitive: even insignificant hepatic dysfunction that cannot be detected by other methods is revealed by the bromsulphthalein test.
Indocyanine green test is based on the same principle. The preparation is given intravenously in a dose of 0.5 mg/kg. Normally, not more than 4 per cent of the injected stain remains in the blood in 20 minutes. The test is more sensitive than that with bromsulphthalein.
The number of tests for the liver function is great. But clinicists are not satisfied by merely stating this or that type of metabolic disorder without relating them to the provoking changes occurring in the liver. There is therefore a tendency to unite groups of pathologically altered test results into syndromes characteristic of various pathologies. For example, when bile drainage is disordered, the concentration of cholesterol, bile acids, bound bilirubin, alkaline phosphatase, and copper in the blood increases. The combination of these positive tests gives the syndrome of cholestasis. The syndrome of hepatocyte insufficiency is characterized by decreased concentration in the blood of substances synthesized by hepatocytes: serum albumins, cholesterol, prothrombin, etc. Inflammatory changes in the liver are characterized by an increased content of various globulin fractions produced by reticulohistiocytary elements of the liver. This is revealed by a number of positive protein sedimentation tests (the inflammatory syndrome). These syndromes can bring the physician closer to understanding of prevailing pathological processes occurring in the liver.
STUDY OF DUODENAL CONTENTS
Duodenal contents are studied for determining the bile composition, which in turn is necessary to diagnose affections of the gall bladder and bile ducts, and also to estimate the function of the pancreas.
Technique. Duodenal contents are obtained by probing the duodenum by an elastic rubber tube 3—5 mm in diameter. The oval bulb at the end of the tube opens into its lumen. The overall length of the tube is about 150
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cm.
The tube bears a mark at a distance of 45 cm from its distal end
(the distance to the stomach); next marks follow at 70 and 80 cm
lengths.
The procedure is carried out on a fasting stomach. The patient sits with the mouth slightly open. The tube is placed in the mouth so that the bulb is at the root of the tongue and the patient is asked to make swallowing movements. The operator should only slightly promote the independent movement of the tube. If the patient attempts to vomit, he is recommended to breathe deeply through the nose. In rare cases the throat and the upper oesophagus are anaesthetized. When, according to the marks, the tube reaches the stomach, its position is verified by aspiring the stomach contents with a syringe which is inserted into the outer end of the tube: extraction of a slightly turbid acid fluid shows that the tube is inside the stomach. The fluid may be coloured yellow by the duodenal contents, but the reaction remains acid. The patient is now placed on his right side so that the tube bulb would be directed (by gravity) toward the pylorus. A soft pad is placed under the patient's pelvis. The patient continues swallowing the tube to the mark of 70 cm. Breathing should be through the mouth. The tube end passes the pylorus and enters the duodenum in about 60—90 minutes (sometimes even later). The outer end of the tube is lowered into a test tube in the stand placed on a low stool at the head-end of the bed. Sometimes the tube passes the pylorus in a shorter time if the patient walks slowly about for 15-20 minutes and continues the swallowing movements. When the tube is swallowed to the mark of 70 cm, the patient lies on his right side. If the bulb has reached the duodenum, yellow alkaline fluid is gathered in the test tube. If the common bile duct is obstructed (pronounced jaundice) the duodenal contents are colourless and the reaction is alkaline. In order to check the position of the tube end (if no juice is discharged from the probe) air can be forced into the tube by a syringe. If the probe is inside the stomach, the patient feels the stream of the injected air, and bubbling can be heard. If the tube is in the duodenum, the patient does not feel anything and no sounds are heard. The position of the tube can most accurately be established by X-rays. The correct position of the tube bulb is between the descending and the lower horizontal portions of the duodenum. If the tube is stopped before the pylorus, the patient is given to drink a warm solution (2—3 g) of sodium hydrocarbonate in 10 ml of water.
The normal duodenal contents discharged from the tube (the first phase of examination) is golden-yellow, slightly viscous, clear, and opalescing. If it contains gastric juice, it becomes turbid from precipitating bile acids and cholesterol. This portion is designated by the letter A. This is a mixture of bile, pancreatic and intestinal secretion. Their proportion in the mixture is unknown and the diagnostic value of this fluid is therefore low. Bile A is
collected for 10-20 minutes. An agent stimulating contraction of the gall bladder is then given through the tube. This is usually a warm solution of magnesium sulphate (25-50 ml of 25-33 per cent solution). Less frequently this is vegetable oil, egg yellow, 10 per cent sodium chloride solution, 30-40 ml of a 40 per cent glucose solution or 40 per cent sorbitol solution, and also hormones (cholecystokinin or pituitrin) which are given sub-cutaneously.
Following the administration of the stimulant into the duodenum, the Oddi sphincter contracts and excretion of bile is discontinued. This is the second phase. Normally it continues 4—6 minutes following the administration of magnesium sulphate and about 10 minutes after administration of olive oil. The phase is elongated if the tone of the Oddi sphincter is increased and shortened in its hypotonia. Next follows the third phase, the excretion of golden-yellow contents of the bile duct and the neck of the gall bladder (portion At). The fourth phase is evacuation of the gall bladder, which is attended by discharge of thicker dark-yellow, brown or olive bile. It is greenish when congested or if the gall bladder is inflamed. Portion B is the bile of the gall bladder, whose secretion is associated with positive Meltzer-Lyon reflex: contraction of the gall bladder concurrent with relaxation of the bladder sphincter and Oddi sphincter. Bladder bile (B bile) is a kind of concentrated liver bile. The wall of the gall bladder has selective absorbability: the sodium ion and water are absorbed especially actively. Ions of potassium, calcium, and chlorine are absorbed much slower. As a result, the content of bile acids and their salts increases 5—8 times, and that of bilirubin and cholesterol 10 times compared with their content in the hepatic bile (C bile). Epithelium of the gall bladder secretes mucin whose concentration in B bile is from 1 to 4 per cent. In accordance with the capacity of the gall bladder, the amount of secreted B bile is 30-60 ml during 20—30 minutes. The bladder reflex may sometimes be absent in healthy subjects after administration of magnesium sulphate, but it usually appears in repeated examinations, or after giving vegetable oil, pituitrin, or atropine (subcutaneously). The appearance of the reflex after giving pro-caine or atropine indicates spasm of the sphincter and the absence of organic obstacles. Persistent absence of the bladder reflex is observed in cholelithiasis, cirrhosis of the gall bladder, obstruction of the bile duct with a stone or an inflammatory process in its mucosa, in contractile dysfunction of the gall bladder, etc. Excretion of very thick dark bile or ample amounts of bile indicates its congestion in dyskinesia of the bile ducts. Intensification of colour alone indicates haemolysis (excess secretion of bilirubin).
After B bile excretion discontinues, C bile (hepatic bile) is delivered from the tube. This is the fifth phase of the examination. The golden-
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1 J
yellow C bile is considered to be hepatic though it also contains admixtures of duodenal juice. Five-minute portions are collected separately during the entire examination. This fractional duodenal probing is used to determine the properties of the contents, volumes of separate portions of the bile system, and the tone of its sphincters. All the three portions of bile are studied by microscopic, chemical and sometimes bacteriological methods.
Microscopy of duodenal contents should be carried out immediately after collection of each portion. Leucocytes are decomposed more slowly but their breakdown is still very rapid. If the sample of bile cannot be examined immediately corrosive sublimate or a 10 per cent formaldehyde should be added (with warming up). But these reagents distort the cells and kill lamblia. Flakes of mucus are pipetted from the bile and transferred onto an object glass. The remaining liquid is centrifuged and the precipitate (as well as the flakes) is examined in native preparations.
Untill recently, the presence of leucocytes in bile was given great diagnostic importance. Their presence in B bile was considered as a diagnostic sign of cholecystitis, and in C bile of cholangitis. If the leucocytes were impregnated with bile (coloured by bilirubin), this was considered a proof of their genesis from the gall bladder. At present, many investigators believe that accumulations of round cells in bile are actually altered and rounded nuclei of intestinal epithelium. Their combination with bilirubin depends probably not on the place of their origin but on the thickness of mucous coat that protects them. Diagnostic importance can therefore only be given to the presence of leucocytes in bile after their identification (by peroxidase staining). Epithelium can be quite informative provided it is well preserved and its properties can be indicative of the site of its origin: fine prisms originate from the bile ducts, elongated columnar cells with oblong nuclei originate from bile passages; large cells with a large round nucleus and vacuolized cytoplasm are attributed to the mucosa of the gall bladder; large epithelial cells with a round nucleus, accounting for the expanded lower third of the cell, and with a thickened cuticle, belong to the duodenum. The cells can easily be identified in the native preparation by phase-contrast microscopy.
The presence of tumour cells in bile is of great diagnostic importance. Microscopy of native preparations only in rare cases can reveal them. Histological study of consolidated duodenal precipitate is more informative.
Discovery of cholesterol crystals and brown grains of calcium bilirubinate are of importance. They can be found in small quantities in healthy subjects but large amounts suggest cholelithiasis.
Discovery of parasites in bile is of great significance. Lamblia in-testinalis occur frequently; the eggs of liver fluke or Chinese fluke, eggs of
duodenal wryhead and also larvae of intestinal Strongyloides stercoralis are found less frequently.
Some chemical constituents of bile are determined. These are bilirubin, cholesterol, bile acids, and protein. It is not the total bilirubin of bile that is important, but rather bilirubin proportion in C and B bile, which characterizes the concentration capacity of the gall bladder. The normal bilirubin content in B bile is 3.4-6.8 mmol/1 (200-400 mg/100 ml) and of C bile, 0.17-0.34 mmol/1 (10-20 mg/100 ml). Decreasing concentration of bilirubin in the gall bladder can depend on bile dilution with inflammatory exudation. Concentration of bilirubin is determined by the icterus index: bile is diluted to match its colour with that of a standard solution of potassium dichrornate. The degree of dilution indicates "units of bilirubin". Cholesterol is determined as in the blood. A bile contains about 0.5 mmol/1 (20 mg/100 ml), B bile about 2.6-23.4 mmol/1 (100-900 mg/100 ml), and C bile 2-2.6 mmol/1 (80-100 mg/100 ml) of cholesterol. Protein is absent from the normal bile. Its presence (proteincholia) indicates inflammation.
Bile acids are determined colorimetrically using the Pettenkofer test (and its modifications) based on the interaction between bile acids and sucrose in the presence of sulphuric acid and subsequent development of cherry-red colour. There are more accurate, though more complicated methods for determining bile acids. These are chromatographic, luminescence and other methods. Decreasing the cholate to cholesterol ratio below 10 in bile (the cholatocholesterol coefficient) indicates predisposition to formation of bile stones.
The ability of the liver to excrete foreign substances together with bile (stains, medicines, iodine compounds, salts of heavy metals) is used for diagnostic purposes. Patency of bile ducts is determined by the speed of excretion with bile of bromsulphthalein given intravenously. If the concentrating capacity of the gall bladder is impaired, it is difficult to differentiate between A, B and C biles by colour. A methylene blue test (chromodiagnostic probing) is then used. The reagent is reduced in the liver to a colourless leucobase, but it again oxidizes in the gall bladder and its colour reappears. The patient is given 0.15 g of methylene blue in a capsule in the evening and common probing is performed in the morning. If blue bile is discharged after giving magnesium sulphate, this indicates that it is B bile.
Bacteriological study of bile is only of relative importance because it is difficult to establish the origin of the cultured flora (it may originate in the mouth, the intestine, or the bile ducts). But if the same flora is found in repeated studies of the same portion of bile, it can be suggested that the found microbes may originate in the bile ducts.
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X-RAY
STUDY
Survey roentgenoscopy and roentgenography of the liver and gall bladder are not diagnostically important because the increased density due to consolidated liver tissue can hardly be differentiated from shadows of the other abdominal organs. Therefore only in rare cases (usually in thin and asthenic patients) it is possible to determine the lower border of the liver, its position, configuration, and the size of the liver and the spleen. In some cases X-ray examination can reveal inclusions in the liver tissue (calcified echinococcal cysts, tuberculous foci), in the gall bladder and bile passages (stones containing much calcium salts).
Various X-ray techniques have been proposed during the past decade to study the liver vessels with contrast substances. Splenoportography is most widely used now. This is the method by which the splenic vein and portal vein with its intrahepatic ramifications can be determined with contrast substances and with serial radiography. The patient is given local anaesthesia and the spleen is punctured in the 8th and 9th intercostal spaces in the left midaxillary line to administer 40—50 ml of a contrast substance (70 per cent solution of cardiotrast or triombrin). Series of X-ray pictures are then taken in 2, 5, 10, 20, 35, and 45 seconds, which are used for combined examination of the portal circulation and the condition of the bile-secretion system. A splenoportogram gives a distinct picture of branching veins. Their section and the pattern of branching can be used to judge about intra- and extrahepatic causes of portal hypertension, the development of collateral circulation, the character of extension and the degree of pathology of the liver (cirrhosis, primary and metastatic tumours, cysts). Splenoportography is especially indicated in cases of portal cirrhosis of the liver with ascites, when the patient is offered an operation for placing a portocaval anastomosis to bypass a part of blood outflow from the portal vein into the inferior vena cava and to decrease the degree of portal hypertension. The presence of portal hypertension can be established indirectly by contrast X-ray study of the oesophagus (using barium meal). It reveals varicosity of the oesophageal veins.
The arterial system of the liver is examined by coeliacography, the method based on the administration of a contrast substance into the coeliac artery through a catheter, which is usually passed into the coeliac artery through the femoral and then abdominal artery, and then the abdominal aorta. This method reveals foci of liver affection (primary and metastatic tumours, cysts, and abscesses).
All X-ray studies should be carried out only for special indications with proper consideration of all possible contraindications (acute diseases of the liver, haemorrhagic diathesis, hypersensitivity to iodine preparations, etc.).
Fig. 95. Cholecystogram with gall stones.
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can be seen, their number and size corresponding to the number and size of the stones (Fig. 95). If the shadow of the bladder is free from stones, the next stage of examination is begun: a cholecystokinetic (usually 10 ml of raw egg yellow) is given to the patient. The preparation provokes contraction and evacuation of the gall bladder. Series of pictures taken at regular intervals are used to assess the motor function of the gall bladder (by the time of its evacuation and the size of the maximum contracted gall bladder).
A contrast substance, bilignost, is given in cholangiography (30-40 ml of a 20 per cent solution, a slow intravenous injection). If the patient's condition is normal, radiographs taken 5-10 minutes after the injection show large intra- and extrahepatic bile ducts and the gall bladder (provided the bile duct is patent). Cholangiography reveals not only the shadow of the gall bladder and areas of rarified density in the presence of stones, but also gives information on the position, calibre, and patency of the intra- and extrahepatic bile ducts. Cholangiography is used to study the intra- and extrahepatic bile ducts (e.g. in patients with removed gall bladder) and also the gall bladder in patients in whom the shadow of the bladder is not determined by cholecystography.
Advances in endoscopic technique have made it possible to work out a method of endoscopic (retrograde) cholangiopancreatography. An iodine-containing contrast substance is administered into the common bile duct and the pancreatic ducts (by catheterization of the major duodenal papilla during duodenofibroscopy) which is followed by radiography. This method is used to determine the presence of stricture of the common bile duct, its compression from outside, and to reveal stones in it.
RADIOISOTOPE METHODS OF STUDY
Radioisotope studies of the function and structure of the liver are based on tracing the distribution and motion of radioactive substances inside the human body. Short-lived isotopes are usually used in clinical practice to label certain mineral and organic substances that are selectively absorbed by various cells of the liver tissue.
The following preparations are mostly used now: rose bengal (dichlortetraiodofluorescein) labelled with 13II, which is captured by the liver hepatocytes, and the colloidal solution of gold 198Au, captured by the reticulohistiocytic cells of the liver, spleen, and bone marrow.
Radioisotope hepatography is performed with rose bengal labelled with 131I. Its sterile solution (from 15 to 20 /xCi) is given intravenously in 0.5-0.9 ml of sterile isotonic solution of sodium chloride. The liver function is then examined by a radiometric apparatus whose scintillation
\
transmitters are fixed over the heart region (to determine stain withdrawal from the blood; blood clearance), over the right lobe of the liver (to determine stain accumulation and withdrawal), and the central part of the abdomen (to control stain discharge via the bile passages to the intestine). Changes in radioactivity over these areas are recorded for 60-90 minutes, and in some cases (in obstructive jaundice, various forms of liver cirrhosis) the time of examination can be prolonged to 24-72 hours. The results are presented graphically as hepatograms (Fig. 96).
In healthy subjects, the half-period clearance (the time of blood clearance from 50 per cent stain absorbed by the liver) is from 10 to 15 minutes. During the first two minutes following injection of the stain, radioactivity of the liver increases sharply to characterize the condition of its blood flow. Further absorption of the stain by the liver is slow. The time of maximum accumulation of the stain in the liver is normally 16-22 minutes. The time during which half of the stain is released from the liver to the gall bladder and the small intestine (period of half-clearance) varies between 75 and 110 minutes. Not more than 2.5 per cent of the initial amount of the preparation remains in the liver in 24 hours. Radiohepatography can thus assess blood circulation in the liver, the absorption-excretion function of the liver, and patency of the bile ducts.
fa jo Why 2o\ 7? ifr y\k ' Himi nl in 11 i: 111 i
Fig. 96. Accumulation and excretion of rose bengal. a—in norm; b—in hepatitis.
I
n
liver pathology, the rate and the degree of absorption and discharge
of rose bengal decreases. If polyhedral cells are affected, the
absorption process is especially affected. The secretory function of
the liver is predominantly affected in inflammation and especially
in impaired patency of the bile ducts.
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I I
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Fig.
97. Scanogram of a normal liver.
Scanning is a graphic registration of distribution of labelled compounds in the liver (hepatoscanogram). The patient is given intravenously rose bengal labelled with 131I (3 fid/kg) in 0.8-1 ml of isotonic sodium chloride solution (or labelled with 198Au). Scanning is carried out in 30 minutes after the injection.
A scanogram of a healthy subject (Fig. 97) shows distinct borders of the liver and diffuse distribution of the radioactive substance in it. In diffuse affection of the liver (chronic hepatitis, cirrhosis) its contours are irregular and indistinct, the liver shadow is uneven and spotty: areas with normal absorption of the isotope alternate with large areas of decreased radioactivity which indicates dysfunction of the polyhedral cells of the liver (Fig 98). Focal affections of the liver (primary and metastatic cancer, echinococcal cysts) can be seen on a scanogram as defects of absorption of the radioactive substances, i.e. as foci of diffuse thinned shadows. Scanning with radioactive colloidal gold is used to determine total activity of the entire reticulohistiocytic system and the mesenchymal function of the liver and spleen. In healthy people 198Au is accumulated mainly in the liver as compared with the spleen. Absorption of the colloidal gold in an enlarged spleen increases in cirrhosis of the liver (Fig. 98).
Fig. 98. Scanogram in portal liver cirrhosis.
Colour scanning has been used in recent years. Scanograms more vividly show different colouration of various zones due to different accumulation of the isotope in the organs. Scanograms are interpreted quantitatively by special gamma-chambers which record simultaneously radioactivity over the entire organ (without moving the detector over the examined region). The time of examination is thus shortened. Computerized tomography is used in cases where scanning fails to give sufficient information for a correct diagnosis. This, however, is a more complicated method.
ECHOGRAPHY
Echography is widely used in hepatology. Ultrasound can be used to assess the condition of the liver tissue, to detect cysts (almost in 90 per cent of cases), abscesses and tumours of the liver (almost in 80 per cent of cases). Successive use of radioisotope scanning of the liver and echohepatography improves accuracy of diagnosis and facilitates differentiation of focal affections of the liver. Ultrasound helps to perform sighting biopsy of the liver and to differentiate between cirrhosis, hepatitis, and fatty degeneration, and to assess the extent of liver affection. This method can be used to reveal liver affections in a comparatively early stage
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of
the process. Ultrasound is used to study the porta hepatis, e.g. to
detect dilated and twisted portal vein in portal hypertension.
Examination of the spleen establishes its position, reveals possible
enlargement (which may be an indirect sign of liver cirrhosis), and
determines the structure of this organ.
Echography is especially useful to diagnose diseases of the gall bladder. Position of the gall bladder, the presence of stones in it, and the condition of its walls can be assessed. True, ultrasound is not so effective as cholecystography in revealing cholelithiasis, but it is especially advantageous in cases when cholecystography proves ineffective or intravenous cholecystography becomes impossible due to hyperbilirubinaemia and jaundice which are contraindications to these examinations, or due to allergy to contrast substances, or general grave condition of the patient. Meteorism and much subcutaneous fat (in disorders of fat metabolism) impair accuracy of echographic diagnosis, but these factors interfere also with cholecystographic study of the gall bladder.
A two-dimensional scanning method (B-scanning) can be used to study the common bile duct and sometimes to establish the cause of its obstruction (stones, tumour). Echography is used to diagnose obstruction of the gall bladder by a stone, dropsy or empyema of the gall bladder which arise in such obstructions, and also cancer of the gall bladder, which occurs not infrequently.
PUNCTURE BIOPSY OF THE LIVER
Puncture biopsy is used to take specimens of liver tissue from a patient for histochemical examinations (electron microscopy) and also to study liver enzymes. Puncture biopsy helps in cases where diagnosis of diffuse liver affections is difficult. The procedure is only carried out for special indications. Two methods of biopsy are used: "blind" and sighting (under control of laparoscope). The former method is used in diffuse affections, and the latter in focal affections.e.g. in suspected cancer.
Technique. The patient lies on his back without pillow, slightly turning to his left side with the right arm behind the head. The skin is properly treated and anaesthetized at the site of puncture. A 1 or 2 per cent procaine solution is used for anaesthesia (2-3 ml). Using a stilette, the skin is cut to the depth of 2—4 mm in the region of the 9th interspace, in the anterior axillary line. This is the broadest zone of liver dullness. A special Menghini needle (or its modification according to Bluger and Sinelnikova) with a plunger inside it is introduced into the punctured skin. The needle is connected to a syringe containing a few millilitres of isotonic sodium chloride solution. After puncturing the skin, part of the isotonic solution is
discharged from the syringe in order to expell pieces of skin and subcutaneous fat from the needle. The needle is then introduced by a swift movement perpendicularly to the skin surface into the liver and its tissue is sucked in by the syringe. The obtained sample is quickly placed in a fixing solution and then given the necessary examination. After the biopsy is over, the patient should lie on his right side for an hour and remain in bed during 24 hours. Complications of this procedure are quite rare.
LAPAROSCOPY
Laparoscopy (peritoneoscopy) is endoscopic examination of the abdominal cavity by a special optical instrument called laparoscope. Laparoscopy is now used not only to examine the abdominal organs but also to perform sighting biopsy, to take colour pictures, and to carry out cholangiography (introduction of contrast substances into the gall bladder and bile passages, with subsequent radiography). Laparoscopy is done in hospitals after thorough examination of the patient.
Fig. 99. Introducing a laparoscope into the abdomen.
Technique. The patient is placed on the operating table. The site of the puncture is treated by the common technique (usually in the median line, 3-4 cm below the navel, or in the anterolateral parts of the mesogastric region, anteriorly of the abdominal rectus muscles; Fig. 99). In order to ensure higher safety and better conditions for examination of the abdominal
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organs,
pneumoperitoneum is first placed (oxygen, carbon dioxide or nitrous
oxide are injected into the abdomen through a blunt-pointed
needle). In cases with ascites, the fluid is removed. After
infiltrative anaesthesia with a 0.25 per cent procaine solution, a
1-cm cut is made by a scalpel at a needed site and a trocar of the
laparoscope is passed into the cut. The optical part of the
laparoscope is then passed inside the trocar. The abdominal organs
are inspected according to a predetermined plan. Depending on the
object of examination (stomach, liver, gall bladder, etc.) the
patient assumes the appropriate position on the operating table.
After laparoscopy is over, air (gas) is released from the abdominal
cavity, the trocar is removed, and the wound is sutured. The patient
should remain in bed for three days. (The air remaining in the
abdomen is absorbed within several days).
Anterosuperior and inferior surfaces of the liver can be examined during laparoscopy. The size of the liver, its colour, the character of the surface, the condition of the liver edge, and its consistency, and also a considerable part of the gall bladder are determined by laparoscopy. Laparoscopy should be done strictly for special indications. It is used in cases where other techniques fail to diagnose the disease. Laparoscopy is especially valuable in diagnosis of focal affections of the liver (tumour, cyst), in establishing the cause of ascites of unknown aetiology (cirrhosis, cancer of the liver, tuberculous peritonitis, etc.), in establishing the character of jaundice (differentiation of obstructive jaundice from hepatic one, and location of possible site and character of obstruction), in cases suspected for cancer of the gall bladder, and in splenomegaly of dubious origin.
Laparoscopy is contraindicated in severe diseases of the cardiovascular system, of the lungs, in haemorrhagic diathesis, grave forms of anaemia, and some other conditions.