PANCREATIC FUNCTION TESTS

Introduction/Overview
The gastrointestinal tract (GIT) is a tube, eight metres in length, beginning with the mouth and
ending with the anus. The major organs of the GIT include the stomach, the small and large
intestines, the pancreas, and the gall bladder, all of which are involved in the digestive process
that commences with the ingestion of food and water and culminates in the excretion of faeces.
The stomach, intestinal tract, and pancreas are closely related, both anatomically and
functionally, and symptoms such as diarrhoea or malabsorption, may be associated with
diseases or disorders of any of these organs. Advances in imaging techniques and improvements
in endoscopic procedures have led to enormous changes in the investigation of the GI and
pancreatic functions so that many laboratory tests, once considered important, have now been
superseded.

Pancreatic insufficiency is the inability of the pancreas to produce and/or transport enough
digestive juice/enzymes, to metabolize food in the intestine and allow its absorption. It typically
occurs as a result of chronic pancreatic damage. It is most frequently associated with cystic
fibrosis in children and with chronic pancreatitis in adults. It is less frequently but sometimes
associated with pancreatic cancer. In addition, disorders of the exocrine pancreas are frequently
associated with GI symptoms of malabsorption or diarrhoea because of its central role in the
absorption of carbohydrates, fats, and proteins. In this lecture, the pancreas, its exocrine
functions and disorders are discussed, and tests for assessing the exocrine pancreatic functions
are described.

The Pancreas
The adult pancreas is a pale yellowish gland, about 12 to 15 cm in length and weighs about 60 to
100 g. It lies across the posterior wall of the abdominal cavity. It is divided into a broad head
which is located in the duodenal curve, a body which lies behind the body of the stomach, and a
narrow tail which lies in front of the left kidney extending to the spleen. The gland consists of a
number of lobules which are made up of small alveoli lined with secretory cells. The secretion
from each cell is drained by a tiny duct which unites with other ducts and joins the main
pancreatic duct. The secretions are formed by the cells of the pancreatic acini and cells of the
intralobular ducts.

Functions of the Pancreas

The pancreas is a dual organ, having both endocrine (hormonal) and exocrine (digestive)
functions. The endocrine function of the pancreas is mainly the control of plasma glucose
concentration through the synthesis and secretion of the hormones insulin and glucagon by the
pancreatic islet tissues, first described by Langerhans. However, the main focus of this lecture is
on the exocrine functions of the pancreas, as the endocrine functions are discussed in details in
other lectures on carbohydrates metabolism.

Exocrine Functions of the Pancreas

The predominant exocrine functions of the pancreas are the production and secretion of
pancreatic juice, which is a colourless and odourless juice rich in enzymes and bicarbonate. The
composition of the secretion is divided into organic and inorganic constituents, both of which
assist in the digestive process. The thought, sight, and/or smell of food stimulate the production

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of pancreatic juice via the vagus, but the nervous phase is a minor one compared to the humoral
control.

The pancreatic juice secretion is controlled by two hormones which are secreted by the
intestines. The presence of acid chyme from the stomach activates the duodenum to produce
secretin, a polypeptide hormone, which stimulates the production by the pancreas, of a thin
watery fluid, high in bicarbonate but low in enzymes. The upper jejunum produces a hormone,
cholecystokinin (CCK) - pancreozymin, which stimulates the production of a viscous fluid low in
bicarbonate but rich in enzymes in response to the presence of products of digestion,
particularly proteins. The flow of pancreatic juice begins 5 minutes after a meal, reaches its peak
in 2–3 hours, and lasts 6–8 hours in all.

Composition of Pancreatic Juice

The inorganic constituent of pancreatic juice is the alkaline juice, which is mainly bicarbonate
used to render the acid chyme entering the duodenum alkaline. The chloride content rises and
falls inversely with the bicarbonate level, while the sodium and potassium levels are almost
identical with the level found in plasma. The alkaline reaction is all necessary for the effective
function of the pancreatic enzymes which are the organic constituents of the juice.

The gross and chemical characteristics of pancreatic juice include:

a. Volume per 24 hours: 500–800 mL, can be as high as 3,000 mL.
b. Specific gravity: 1.007 - 1.042.
c. Total solids: 1.5–2.5 g%.
d. Alkalinity: pH is 7.5–8.3.
e. Bicarbonate: 70–100 mmol/L.
f. Sodium: 100–150 mmol/L.
g. Potassium: 2–8 mmol/L.
h. Chlorides: 50–95 mmol/L.

Digestive Enzymes
A. Proteolytic Enzymes
1. Trypsin: The inactive trypsinogen secreted is activated by enterokinase or by trypsin itself.
Trypsin hydrolyzes proteins at peptide bonds.
2. Chymotrypsin: Two forms, A and B are secreted and are activated by trypsin. Its action is like
that of trypsin.
3. Collagenase: It digests collagen and is the one that initiates tissue destruction in necrotizing
pancreatitis.
4. Elastase: Digests elastin, which is the most resistant of all body proteins to lytic agents.

B. Peptidases
1. Carboxypeptidase: The active enzyme removes amino acids one by one from the carboxyl
ends of the peptide chains.
2. Aminopeptidase: The active enzyme removes amino acids one by one from the ends of the
peptide chains bearing the free amino groups.

C. Nucleases
Ribonuclease and deoxyribonuclease are secreted in probably more than one or perhaps several
forms; they hydrolyze the respective nucleic acids.

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D. Amylolytic Enzymes
Amylase: Alpha amylase attacks the alpha-1-4-glycosidic bonds of starches breaking them down
to the disaccharide maltose.

E. Lipolytic Enzymes
1. Lipase: It partially hydrolyzes neutral fats, splitting off one fatty acid at a time, thus forming
diglycerides and monoglycerides along with liberated free fatty acids.
2. Lecithinase (phospholipase): Phospholipases A and B act in succession. Both of these remove
fatty acids, the end products formed from lecithin and cephalin are glyceryl phosphoryl choline,
glyceryl phosphoryl ethanolamine, and glyceryl phosphoryl serine.

Disorders of Exocrine Pancreatic Functions

As earlier noted, the pancreas plays a central role in the digestive and absorptive processes for
fats, carbohydrates, and proteins. Absence of pancreatic secretions may, by impairing digestion,
therefore cause malabsorption. The exocrine disorders of pancreatic functions may be classified
into paediatric and adult disorders of the exocrine pancreas.

A. Paediatric Disorders
Pancreatic exocrine disorders in childhood can be due to a lot of factors which include disorders
of morphogenesis (e.g. annular pancreas, pancreas divisum, etc), inherited syndromes affecting
the pancreas (e.g. cystic fibrosis, Johnson Blizzard syndrome, etc), gene mutations leading to
pancreatic disease (e.g. hereditary pancreatitis; cationic trypsinogen gene mutations, trypsin
inhibitor gene mutations), pancreatic insufficiency syndromes (isolated enzyme deficiencies:
lipase, colipase, enterokinase), pancreatic insufficiency secondary to other disorders (e.g. celiac
disease), and acquired pancreatitis in childhood (idiopathic, drugs, viral, metabolic,
autoimmune, etc). However, the commonest amongst them is cystic fibrosis.

Cystic Fibrosis (Mucovicidosis)

Cystic fibrosis (CF), also called mucovicidosis, is an inherited autosomal recessive disorder with a
wide spectrum of clinical presentations, including chronic obstructive pulmonary disease and
pancreatic insufficiency. It is the commonest lethal genetic disorder of the Caucasian
population. It is a familial disease characterized by abnormal vicid secretion by the various
exocrine glands of the body, including the pancreas, salivary glands, peritracheal, peribronchial
and peribronchiolar glands, lacrimal glands, sweat glands, mucosal glands of small intestine and
even the bile ducts. However, CF primarily affects the pulmonary, GI, and reproductive organs.
Increased viscosity of the pancreatic secretions results in obstruction of the ducts of the gland
producing fibrosis of the gland. The cysts may also be found in the bronchi as well as the liver
and all produce the viscous secretions which are rich in glycoproteins.

CF is caused by a defect in the cystic fibrosis transmembrane conductance regulator protein

(CFTR), a protein that normally regulates electrolyte transport across epithelial membranes.
More than 1500 mutations of CFTR have been identified. Pancreatic insufficiency is present at
birth in 65% of infants with CF, and a further 15% develop it during infancy and early childhood.
The 20% who do not develop pancreatic insufficiency have a better prognosis and develop fewer
complications. The phenotypic expression of the disease is heterogeneous, ranging from
meconium ileus (failure to pass the first faeces after birth) and severe respiratory symptoms, to
no evidence of GI problems even in adulthood. Morbidity and mortality of the disease are most
related to mucous accumulation; recurrent infection with pathogens such as Pseudomonas

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aeruginosa, Staphylococcus aureus, Haemophilus influenza; and excessive inflammation in the
lungs. In childhood, the main clinical features are failure to thrive, malabsorption, and
steatorrhoea.

Sweat glands are also affected and the main biochemical finding is high sweat sodium and
chloride (often about twice the normal value), with their concentrations almost approaching
that of plasma. When affected by the disease therefore, even moderate sweating may cause
serious sodium and chloride loss. Although direct mutational analysis is available, it is not
informative in all cases, and a quantitative sweat chloride test remains the standard for
diagnostic testing. Sweat testing is often performed in conjunction with newborn screening
programme which may includes an immunoreactive trypsinogen assay (IRT) from a dried blood
spot, DNA testing, detection of albumin and trypsin in meconium, and measurement of
pancreatic elastase-1 in faeces

B. Adult Disorders
The major exocrine pancreatic disorders presenting in adult life are pancreatitis (acute or
chronic), and carcinoma of the pancreas.

1. Acute Pancreatitis
Acute pancreatitis is an abrupt inflammation of the pancreas, especially the acinar cells leading
to regurgitation of the pancreatic juice/enzymes into the blood stream. Acute pancreatitis
generally presents as acute abdomen with severe pain, as the pancreas becomes acutely
inflamed, and in severe cases haemorrhagic (acute haemorrhagic pancreatitis). It is also
associated with the release of pancreatic juice/enzymes into the retro peritoneal space. The
presence of pancreatic juice in the peritoneal cavity also causes severe abdominal pain and
shock. A viscous cycle is set up as more pancreatic cells are digested by the released enzymes. It
is most commonly idiopathic but may follow obstruction of the pancreatic ducts, or
regurgitation of bile along this duct. It is also known to be related to gallstones, alcoholism,
trauma, infection (mumps), renal transplantation, various metabolic disorders, e.g.
hyperlipidaemia, uraemia and hyperparathyroidism.

Typical plasma amylase activity increases about five-fold in acute pancreatitis, as such serum
amylase estimation has been widely used in the diagnosis of acute pancreatitis. Serum amylase
activity rises within hours following an episode. Values may return to normal within 5 days
following a mild oedematous attack. Persisting elevated values longer than this suggest
continuing necrosis or possible pseudocyst formation. The urine amylase activity rises promptly,
often within several hours of the rise in serum activity. Values over 1,000 units per hour (in
urine) or higher are seen, almost exclusively in patients with acute pancreatitis.

Other laboratory findings in acute pancreatitis are leucocytosis (up to 30,000/mm 3),
haemoconcentration (so raised haemoglobin), elevated serum levels of lecithinase A, trypsin
and deoxyribonuclease activity, falling serum calcium level, transient hyperglycemia, lipaemic
serum, and in alcohol-related pancreatitis, mild increase in bilirubin concentration and alkaline
phosphatase activity. "Resting “the gut by nasogastric aspiration is the biochemical basis of
management.

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2. Chronic Pancreatitis (Cirrhosis of Pancreas)
Chronic pancreatitis is an inflammatory disease characterized by persistent and progressive
destruction of the pancreas leading to destruction of both endocrine and exocrine functions. It
is also defined by irreversible pancreatic damage with histologic evidence of inflammation and
fibrosis. The major histological features are similar regardless of the cause. In Western
countries, the commonest cause is alcohol (60% to 90% of all cases); however there are clearly
other predisposing factors (e.g., smoking and diets high in fat and protein), because only 5% to
15% of heavy drinkers develop the disease. "Juvenile Tropical Pancreatic Syndrome” (described
by our own Professors Nwokolo and Oli in 1980) is a major cause of chronic pancreatitis in the
tropics. However, due to the functional reserve of the pancreas, symptoms do not develop until
an excessive progression of the disease or obstruction of the main pancreatic duct occurs.

Diagnosis as in the case of acute pancreatitis depends in part on determination of amylase

activity in serum and urine. Also, tests for malabsorption, serum carotenoid level estimation,
glucose tolerance test, three-day faecal fat determination, gross and microscopic examination of
stool, 13C-Triolein test, and D-xylose test can be done

3. Carcinoma of Pancreas
Carcinoma of the pancreas may be localized or generalized, that is, it may affect a part (head,
body, or tail) or the whole pancreas. In this disease, biochemical diagnosis is rarely useful.
Common clinical and biochemical features associated with the disease however, are obstructive
jaundice and very high alkaline phosphatase activity due to obstruction of the common bile duct
caused by tumour in the head of the pancreas. Serum amylase may be elevated but is of little
diagnostic importance.

Biochemical Implications of Pancreatic Diseases

Since the pancreas plays a central role in the digestive and absorptive processes; there are
therefore, various biochemical implications of pancreatic disease. The main effect is on fat
absorption which usually presents as steatorrhoea and malabsorption. Steatorrhoea is defined
as abnormal ill-formed stool containing excessive amount of fat. In pancreatic steatorrhoea, the
absorptive capacity is normal but fat cannot be digested because there is deficiency of digestive
enzymes. The daily volume, total wet and dry weight of faeces are increased. The faeces is pale,
bulky, greasy, often frothy as well as being offensive. It also often floats. Steatorrhoea, which is
part of the malabsorption syndrome, occurs early in pancreatic disease usually at the
preabsorption stage. It also manifests in various other conditions.

In all types of steatorrhoea, however, the failure to absorb fats leads to deficient intake of
energy. The implications of this are low plasma level of cholesterol, malabsorption of fat-soluble
vitamins (A, D, E, and K) leading to their deficiencies. Also the B group vitamins deficiencies
develop in cases where the intestinal flora is altered. There is resultant calcium malabsorption
due to malabsorption of vitamin D and excessive loss in stool as calcium soaps. This sometimes
produces hypocalcaemia and tetany. Both protein and carbohydrate absorption become
affected only in severe or prolonged disease. Indeed, malabsorption, and sometimes glucose
intolerance are the major presenting features in chronic pancreatitis. The defective
carbohydrate absorption presents with a typical flat glucose tolerance test (GTT) curve and
hypoglycaemia. There may also be protein maldigestion and malabsorption due to the loss of

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enzymes and of bile salts. Low albumin, due to the protein malnutrition, leads to negative
nitrogen balance.

Loss of pancreatic secretions either with bile and intestinal secretions, or through ileal fistula,
diarrhoea, or pancreatic fistula if prolonged may also result in metabolic acidosis arising from
the loss of electrolytes. Finally chronic pancreatic disease does not initially affect the islet tissues
and therefore, carbohydrate metabolism. However, as the disease progresses or is prolonged, or
if total pancreatomy is performed in cases of carcinoma of the pancreas, glucose tolerance
becomes impaired. Hyperglycaemia and glycosuria will therefore ensue.

Investigation of Exocrine Functions of the Pancreas

Biochemical tests used to measure exocrine function of the pancreas fall into two main
categories: invasive or noninvasive. A number of these tests are available to measure exocrine
function in the investigation of pancreatic insufficiency. Invasive tests require GI intubation to
collect pancreatic samples. Noninvasive tests (or “tubeless tests”) were developed to avoid
intubation, which is uncomfortable for the patient, time-consuming, and expensive. Noninvasive
tests are simpler and less expensive to perform, but in general they lack the clinical sensitivity
and specificity of the invasive tests, particularly for the diagnosis of mild pancreatic insufficiency.

However, it is important to recognize that laboratory tests have a limited clinical application in
the diagnosis of pancreatic disorders because of the complexity of the invasive tests or the
inadequate sensitivity and specificity for mild and moderate pancreatic disease of the
noninvasive tests. Of greater importance are the imaging procedures. These imaging techniques
include plain abdominal radiography, ultrasound, spiral computed tonography (CT), magnetic
resonance imaging (MRI), endoscopic ultrasound, and endoscopic retrograde
cholangiopancreatography (ERCP). These techniques continue to be developed that it is
projected that in the future, it may be possible to confidently diagnose pancreatic insufficiency
with the use of imaging techniques alone.

Nevertheless, laboratory tests still remain pivotal in the diagnosis of pancreatic diseases.
Histological and cytological examinations of pancreatic tissues and aspirates, as well as
haematological and immunological investigations, have immense contributions in the diagnosis
of pancreatic diseases too. Gross and microscopic examination of faecal specimen also play vital
roles in the diagnosis of pancreatic insufficiency.

A. Invasive Tests of Exocrine Pancreatic Function

Invasive tests include measuring the total volume of pancreatic juice, amount or concentration
of bicarbonate, and activities of pancreatic enzymes in duodenal contents. The enzyme most
commonly measured is trypsin, but amylase, lipase, chymotrypsin, and elastase are also
measured. These are accomplished by the use of stimulation tests such as the Lundh, secretin,
and secretin-CCK tests.

1. The Lundh Test

In this test, the response of the pancreas to a standard meal is assessed. It consists of giving a
standardized meal consisting of 5% protein, 6% fat, 15% carbohydrate, and 74% non-nutrient
fibre, as a physiological stimulus to the pancreas.

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Procedure
The procedure involves an overnight fast by the patient. The patient is thereafter intubated with
a single lumen tube positioned in the duodenum (fluoroscopically). Resting duodenal juice is
aspirated and discarded. The standard meal is then given to the patient orally. Duodenal juice is
aspirated for four 30-minutes period and samples collected and chilled in dry ice. The four
samples collected over the 2-hour periods can either be pooled and analyzed or analyzed
individually.

Interpretation
In normal individuals, the administration of the meal stimulates the production of pancreatic
juice of normal constituents and enzymes activity, in a pooled sample or in at least one of the
samples. In chronic pancreatic disease, the volume and enzymes activities are reduced.
Administration of the meal, however, prevents determination of the total enzyme and
bicarbonate output or secretory volume. Moreover, it provides inadequate stimulation in the
presence of mucosal diseases (e.g., celiac disease), in which hormone release from the duodenal
mucosa is impaired. In view of these limitations, the Lundh test is largely of historical interest.

2. Secretin-Cholecystokinin (CCK) Test

The secretin test is based on the principle that secretion of pancreatic juice and bicarbonate
output are related to the functional mass of the pancreatic tissue. Secretin stimulates the
secretion of pancreatic juice and bicarbonate, but stimulation of the secretion of pancreatic
enzymes is inconsistent. Addition of CCK (or a synthetic equivalent) stimulates the secretion of
pancreatic enzymes, giving a more complete assessment of pancreatic function than with
secretin alone.

Procedure
After an overnight fast, a double lumen tube, providing for separate aspiration of gastric and
duodenal contents, is passed into the duodenum fluoroscopically. Gastric contents are aspirated
and discarded, while all duodenal samples are collected and kept in ice to preserve enzyme
activity. After a 10-minute preliminary suction to empty the gut, two successive 10 minutes
samples of pancreatic juice are collected as resting controls from the duodenal tube. The patient
is then given IV one unit of (purified or synthetic porcine) secretin per kg of body weight.
Samples are collected over three successive 10-minute periods. After 30 minutes, CCK analogue
(CCK-8 or ceruletide) is also given intravenously and pancreatic secretion entering the
duodenum is collected at 15-minute intervals for 80 minutes. The aspirates are then examined
for volume, bicarbonate content, and enzyme activity.

Interpretation
The 10-minute control sample volume is usually 10 mL, with a pH of 7.5 and a bicarbonate
content of 25 mmol/L. After stimulation, there is an increase in the volume to about 2 ml/kg
body weight calculated over 1 hour (approximate total: 150 ml). An increase in bicarbonate
concentration to a peak greater than 20 mmol/L occurs. The pH increases to about 8.0.

The test is not employed for the diagnosis of acute pancreatic necrosis (it would be hazardous).
Patients with chronic pancreatitis are unable to secrete juice of high bicarbonate content (less
than 20 mmol/L), and little or no increase in alkalinity. As in the case of chronic pancreatitis, this
test may assist in diagnosis of pancreatic carcinoma. Tumours of head of pancreas tend to
depress the overall volume flow (lower limit of normal—2 mL per kg body weight per 80

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minutes). In carcinoma of body of pancreas, half the patients may show normal volume,
carcinoma of tail does not affect the volume. Cytological examination of aspirate may also be
helpful in the diagnosis of carcinoma, as are the results of enzyme assays

B. Noninvasive Tests of Exocrine Pancreatic Function

For many years, medical laboratory scientists and gastroenterologists have searched for the holy
grail of pancreatic function tests - the tubeless or noninvasive tests. The noninvasive tests are
based on the reduction in secretion of pancreatic enzymes with measurement of enzymes in
blood, faeces, or detection of products of their catalytic reaction, after oral administration of
synthetic substrates, in urine or in breath. Traditionally such tests as the estimation of plasma
enzymes, faecal fat estimation amongst others were employed. Later a variety of other
noninvasive tests (faecal chymotrypsin, N-benzoyl-L-tyrosyl-p-amino benzoic acid (NBT-PABA),
pancreolauryl, 13C-Mixed chain triglyceride absorption, xylose test, and faecal elastase-1) were
introduced, but none has adequate clinical sensitivity or reliably for detecting early pancreatic
disease. When malabsorption is present, such tests are of value in confirming or excluding
pancreatic disease. Considerable overlap often occurs between results observed in normal
individuals and those found in patients with pancreatic disorders, which is mainly due to the
large functional reserve of the pancreas.

1. Serum Enzymes
The determination of serum α-amylase and lipase plays an important role in the laboratory
diagnosis of pancreatic diseases; however, α-amylase is the far commoner parameter. These
tests however, are not very satisfactory as serum amylase activity usually show little change
from normal in pancreatic disease, except in acute pancreatitis, where it may be raised. Lipase
activity is not estimated routinely, but it can be of some value in that in acute pancreatitis, lipase
shows the greatest increase but its estimation alone is not sufficiently accurate to establish
diagnosis of acute pancreatitis. In chronic pancreatitis, the assay of amylase and lipase are of
less value, since parenchymal destruction leads to a substantial depression of the pancreatic
enzymes production resulting in low levels of both enzymes in blood.

i. Serum Amylase Estimation

There are two main methods available for estimating amylase activity: saccharogenic and
amyloclastic. The saccharogenic method determines the increase in reducing sugars released by
the enzyme from the starch molecule. Amyloclastic method measures the decrease in blue
colour given when iodine is added to starch after digestion by the enzyme. Other methods
available use dye marked starches which on hydrolysis release the dye fragments from the
substrate. These methods are simpler and follow zero-order kinetics over a wide range of
enzyme activity. However, only the amyloclastic method will be discussed briefly.

Amyloclastic Method of Amylase Estimation

A small amount of plasma or serum is incubated at 37oC with a solution containing 0.4 mg of
starch. The loss of blue colour which the starch gives with iodine is taken as a measure of the
extent to which the starch has been digested by the amylase. The result of this method was
initially reported in Somogyi amylase units, defined as the amount of amylase which will destroy
5 mg starch in 15 minutes. 1 Somogyi unit/100ml is equivalent to 1.85 IU/L.

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Somogyi quoted values of between 80 and 180 Somogyi units/100 ml (148 - 333 IU/L) are
normal, but this can vary from method to method. In acute pancreatitis, 1,000 Somogyi
units/100 mL (185 IU/L) can be expected. Amylase activity in blood (or in peritoneal fluid in
certain conditions) may also be raised to 1,000 Somogyi units in various (nonpancreatic)
disorders such as intestinal obstruction, strangulation, or perforation, following upper
abdominal surgery, ruptured ectopic pregnancy, mumps, renal insufficiency, and following
morphine administration. Values over 5,000 units however, suggest a diagnosis of acute
pancreatic necrosis. Occasionally the plasma enzyme activity in acute pancreatitis may not be
very high and usually falls rapidly as the enzyme is excreted in the urine. Consequently, a high
serum amylase activity is only a rough guide to the presence of acute pancreatitis, and normal
or only slightly raised values do not exclude the diagnosis.

ii. Lipase Estimation in Serum

Lipase is an esterase acting on ester linkages in triglycerides. Bile salts and calcium enhance its
activity. Lipase occurs predominantly in the pancreas, but small amounts are produced in the
gastric and small bowel mucosa. The classic method of serum lipase determination is that of
Cherry and Crandall using olive as substrate, overnight incubation (24 hours), and titration of
liberated fatty acids with sodium hydroxide, using phenolphthalein as indicator.

The method above takes a long time and if the report is to be given on emergency basis—a
rapid (20 minute incubation) specific turbidimetric method is available. The disadvantage of this
method is spuriously high results obtained in the presence of jaundice.

Interpretation
Values up to 1.5 units in serum are considered normal. In acute pancreatitis, serum lipase
activity rises slower than that of amylase, sometimes as late as 24 to 48 hours after onset, often
peaking on the fourth day. It may remain elevated longer than the serum amylase. Even though,
it is less sensitive than the serum amylase, it provides confirmatory evidence for the diagnosis
when positive. Elevation in patients with mumps strongly suggests significant pancreatic as well
as salivary gland involvement by the disease. Serum lipase estimation is of relatively little value
in the diagnosis of chronic pancreatitis, whereas it is elevated more often in patients with
pancreatic carcinoma than is serum amylase, although not with sufficient frequency to make it
of much value diagnostically.

2. Faecal Pancreatic Enzymes

i. Faecal Trypsin and Chymotrypsin
As trypsin and chymotrypsin are normally found in faeces, an inadequate secretion of pancreatic
enzymes should affect their faecal excretion. If the excretion of these proteolytic enzymes in
faeces is investigated, it may be a useful guide in the diagnosis of pancreatic insufficiency. For
instance, after the first three days of life, the faeces of babies and young children have a high
proteolytic activity, and thus these can be used in the screening for cystic fibrosis in the
newborn. Pancreatic chymotrypsin is almost completely digested during its passage through the
gut in adults, but the residual activity of the enzyme in faeces is stable for several days at 20oC.
The activities of these enzymes can be assessed either by measuring their proteolytic activities,
or using the X-ray film method, and other methods.

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Proteolytic Method
In the proteolytic method, gelatin is incubated with several dilutions of the faeces in sodium
hydrogen carbonate. If trypsin is present, gelatin is digested to water-soluble products, causing
liquefaction of the gelatin. The more enzymes there are in the faeces, the higher the dilution at
which liquefaction takes place.

The faeces of most normal newborn digest gelatin at a dilution of 1 in 100, whereas infants with
fibrocystic of the pancreas digest it only at a dilution of less than 1 in 50.

ii. Faecal Elastase

Pancreatic elastase-1 is a specific protease present in pancreatic juice. It is not degraded during
passage through the gut, and concentrations in faeces are five to six folds greater than those in
pancreatic juice. Measurement of pancreatic elastase-1 in faeces is considered to be a reliable
screening test for pancreatic insufficiency in infants over the age of two weeks with CF and in
older children at diagnosis of the disorder. The test also is used to detect the onset of pancreatic
insufficiency in those previously pancreatic sufficient. Faecal elastase is measured by ELISA assay
that uses two monoclonal antibodies specific to the human enzyme, and is currently considered
the noninvasive test of choice for assessing pancreatic insufficiency.

Faecal elastase-1 has been evaluated extensively both in CF and in adult pancreatic insufficiency;
and its use is recommended in both children and adults. Very low elastase-1 is seen in a wide
range of CFTR genotypes with undetectable levels in most homozygotes. Low faecal elastase
after 4 weeks of age is indicative of pancreatic insufficiency and provides supporting evidence
for a diagnosis of CF. Measurement of pancreatic elastase-1 in faeces also has high sensitivity for
the detection of severe and moderate chronic pancreatitis in adults. It has better sensitivity than
other tests for detecting mild chronic pancreatitis, and high sensitivity and high negative
predictive value for discriminating between diarrhoea of pancreatic and nonpancreatic origin.

3. Faecal Fat Estimation

Fat absorption has traditionally been assessed by measuring the excretion of fats in faeces. This
usually entailed 24-hour faecal collection for five consecutive days and measuring the quantity
passed and the fat content. Normal faecal fat is approximately 18 mmol/24 hrs but in
malabsorption, this quantity is increased.

Accurate collection and timing of specimen are difficult to achieve. The very unpleasant nature
of the test, coupled with the fact that, in the severe disease with obvious steatorrhoea,
quantifying faecal fat excretion adds nothing to the diagnosis. Moreover, the introduction of the
13
C-Triolein breath test has supplanted it. These have made the test virtually obsolete.

4. 13C-Triolein Breath Test

In this test, oral administration of 13C-labelled triglyceride followed by measurement of 13CO2 in
expired air by β-radiation counting, quantifies fat absorption. The test is based on the principle
that when 13C-labelled triglyceride is taken orally, digested and absorbed, some of the labels
show up in the breath as 13CO2. Since it is the specific activity of the expired CO2 that is
measured, a constant rate of CO2 production from all other sources must be assumed.

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The test is sensitive and the results correlate well with faecal fat assays. It is thus, a much
preferable alternative to unpleasant faecal fat quantitation of faeces in the laboratory.

5. Xylose Test
The xylose test is used in the differential diagnosis of pancreatic and intestinal malabsorption.
Xylose is a plant (pentose) sugar that like glucose is absorbed without digestion from the
jejunum. Metabolism of the absorbed xylose is very slow, but because it is freely filtered by the
glomeruli, most of it appears in the urine. In pancreatic disease, the absorption and excretion
are usually unaltered, but intestinal disease impairs xylose absorption and therefore reduces
excretion.

Procedure
After an overnight fast, the bladder is emptied and discarded. 5g of D-xylose dissolved in 200 mL
of water is given orally. Patient should drink at least 500 mL of water over the next 2 hours. All
urine passed (specifically at 2 and 5 hour) throughout the period (5 hours) of the test is
collected. As at 1 hour, blood is drawn and the xylose level estimated.

Interpretation
Normal serum level of xylose at 1 hours is greater than 1.3 mmol/L., and in urine normal xylose
excretion over a 5-hour period is >7 mmol/5hr. 23% (1.5 g) of the dose should be excreted
during the 5 hours. About 50% or more of the total excretion should occur during the first 2
hours. In pancreatic disease, the results should be normal. In mild intestinal malabsorption, the
5-hour excretion may be normal but delayed absorption is reflected in a 2- to 5-hour ratio of
<40%.

Limitations of the Xylose Test

1. Xylose is absorbed mostly from the upper small intestine and absorption may therefore be
normal despite significant ileal dysfunction, e.g. in Crohn's disease.
2. The urine test depends on accurate collection over a short time interval and therefore even
more significant errors may occur than in 24-hour collection.
3. Mildly impaired kidney function decreases glomerular filtration of xylose and may give falsely
high plasma concentration or falsely low results in the urine tests.
4. Both plasma concentrations and urine excretion depend partly on the volume of distribution
of the absorbed xylose. In oedematous or very obese patients, results may be falsely low.

6. N-Benzoyl-L-Tyrosyl-P-Amino BenzoicAcid (NBT-PABA) Tests

The N-benzoyl-L-tyrosyl-p-amino benzoic acid (NBT-PABA) test is based on the intestinal
hydrolysis, by chymotrypsin, of a synthetic tripeptide - N-benzoyl-L-tyrosyl-p-amino benzoic
acid. The tripeptide is administered orally together with a test meal to stimulate pancreatic
secretion. NBT is specifically hydrolyzed by chymotrypsin in the duodenum to release PABA. The
amount of PABA detected in serum or urine by high performance liquid chromatography is an
indirect measure of chymotrypsin activity in duodenal content. The test has low diagnostic
sensitivity and specificity for pancreatic insufficiency, and coupled with its complex nature, it is
not longer in use.

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7. Sweat Testing
Analysis of sweat for increased sodium and chloride concentration is used to confirm the
diagnosis of cystic fibrosis. The test can be performed using different approaches for stimulating
sweating, and measuring the sodium and chloride contents. Local stimulation can be achieved
either by subcutaneous injection of methacholine chloride or by pilocarpine iontophoresis. The
methacholine chloride technique is rarely used nowadays. The pilocarpine iontophoresis sweat
test is performed in three phases: sweat stimulation by pilocarpine iontophoresis on the skin,
collection of the sweat, and qualitative or quantitative analysis of sweat chloride and sodium.

Iontophoresis technique uses a small electrical current to deliver pilocarpine into the sweat
glands from the positive electrode (which is filled with 0.5% aqueous pilocarpine nitrate), while
an electrolyte solution (1% aqueous sodium nitrate) at the negative electrode completes the
circuit. The positive electrode is strapped to the flexor surface and the negative electrode on the
extensor surface of the forearm. After iontophoresis, sweat is collected onto preweighed gauze
pads, filter paper, Macroduct coils, or Nanoduct conductivity sensor cells, using techniques to
minimize evaporation and contamination. During collection, which lasts for 25 - 35 minutes, the
analyst must collect a sufficient amount of sample, and minimize skin reactions. Determination
of and adherence to a minimum sweat weight or volume are critical to obtain valid sweat testing
results. After collection, qualitative or quantitative tests can be done. A qualitative sweat test
represents a screening test for CF. Individuals who have positive or borderline results should
then undergo quantitative sweat chloride testing.

Interpretation
In normal children, the upper limit for sweat sodium is 70 mmol/L, while the upper limit for
chloride is 65 mmol/L. In adults, sodium concentration should not exceed 90 mmol/L. In adult
males values of sweat chloride up to 70 mmol/L and in females up to 65 mmol/L are normal. In
cystic fibrosis, the values for sweat sodium can vary from 80 - 150 mmol/L.

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