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I. Infusino et al.
cholecystitis, and various intra-abdominal events cause an
increase in serum pancreatic amylase activity of up to four
times the URL and sometimes even higher.
In renal insufciency, serum amylase activity is
increased in proportion to the degree of renal impairment.
Hyperamylasemia also occurs in multiple myeloma,
pulmonary neoplastic diseases, and serous and mixed ovarian carcinomas, with increases even >50 times the URL.In
1% of the population, the presence of macroamylases,
which can cause hyperamylasemia, can be detected in the
serum. These macroamylases are complexes composed of
amylase (S-type amylase) and IgG or IgA.These macroamylases cannot be ltered by renal glomeruli because of their
large size (>200 kDa) and are thus retained in plasma,
where their presence, typically stable over time, can
increase measurable amylase activity by two to eight times
the URL. No clinical signicance is associated with this
biological manifestation.
Reference Intervals
In apparently healthy adults, pancreatic amylase accounts for
∼40–50% of total serum amylase activity. The reference
interval is 13–51U/L.The activity of the pancreatic form is
not demonstrable in the serum of most children <6months of
age, then slowly increasing to adult concentrations around
5years of age; this reects postnatal development of the exocrine pancreatic function. Consequently, the use of this
enzyme for the diagnosis of suspected acute pancreatitis in
young children should be avoided.
of osteoblastic cells because they attempt to rebuild bone
that is being resorbed by uncontrolled osteoclast activity.
Values of 10–25 times the URL are not unusual, and, in general terms, the magnitude of the increase reects the extent
of the disease. It is, however, worth commenting that, for a
correct clinical setting, it is mostly sufcient to measure total
ALP activity for the assessment of bone turnover and monitoring of Paget’s disease.
In vitamin D deciency (osteomalacia and rickets), BAP
concentrations two to four times higher than the URL may
be observed, slowly returning to baseline with treatment.
Primary and secondary hyperparathyroidism is associated
with mild to moderate increases in serum BAP, in which the
degree of the increase reects the presence and degree of
skeletal involvement. BAP may be slightly increased in
osteoporosis, but subjects with osteoporosis are not clearly
distinguishable from healthy controls. Very high concentrations of BAP are present in patients with osteogenic bone
cancer. Increased BAP indicates bone metastases in 70% of
prostate cancers and its measurement has been recommended
by the European Association of Urology guidelines for its
staging. The Kidney Disease: Improving Global Outcomes
(KDIGO) guidelines for the evaluation and management of
chronic kidney disease recommend assessing associated
mineral and bone disorders by measuring total ALP activity
along with calcemia, phosphatemia, and parathyroid hormone (PTH) in adults with glomerular ltration rate (GFR)
<45mL/min/1.73m
ing the specic measurement of BAP instead of total ALP
was considered inadequate by the guideline.
2
. The level of evidence for recommend-
Bone Enzymes
Bone enzymes are direct products of activated osteoblasts
(bone ALP [BAP]) and osteoclasts (tartrate-resistant acid
phosphatase [TR-ACP]).
Alkaline Phosphatase (Bone Isoform)
The bone, liver, and kidney isoforms of ALP have posttranslational modications of the product of the same gene and
are differentiated according to their carbohydrate content.
Bone alkaline phosphatase (BAP) is produced by osteoblasts: the enzyme is therefore an excellent indicator of
global bone formation activity.
Clinical Signicance
Advantages of using serum BAP concentrations as markers
of bone formation in clinical practice include low biological
variability and no inuence by renal dysfunction. Among
bone diseases, the highest BAP concentrations are found in
Paget’s disease (osteitis deformans) as a result of the action
Reference Intervals
The reference interval for adult men of serum BAP concentration does not change with age (7.5–26.1μg/L), whereas
menopause increases BAP concentrations in women (URL:
22.7μg/L in women of childbearing age and 31.6 μg/L in
postmenopausal women). Prepubertal BAP concentrations
are six to seven times higher than in healthy adults.
Acid Phosphatase (Tartrate-Resistant
Isoform5b)
The term acid phosphatase (ACP) includes all phosphatases
with optimal activity below a pH of 7.0. ACP is expressed in
lysosomes, cell organelles present in every cell except erythrocytes; in addition, high concentrations of ACP are found in
the prostate, bone (osteoclasts), spleen, platelets, and erythrocytes. Lysosomal and prostatic enzymes are strongly
inhibited by tartrate ions, which, however, do not inhibit
erythrocyte and bone isoenzymes. Most of the low activity of
serum ACP (nonhemolysate) is tartrate-resistant (TR-ACP)
and originates primarily from osteoclasts. The activity of this
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fraction is increased physiologically in growing children and
pathologically under conditions of increased osteolysis and
bone remodeling.
A gene on chromosome 19 encodes for TR-ACP
expressed in osteoclasts and macrophages of other tissues,
such as alveolar macrophages and Kupffer cells (ACP type
5). Isoenzyme 5 consists of two structurally related isoforms
that differ in their carbohydrate content: TR-ACP 5a, which
is derived primarily from macrophages and dendritic cells,
and TR-ACP 5b, a more specic marker of osteoclastic
activity.
Clinical Signicance
TR-ACP 5b is a potentially useful marker in conditions characterized by a marked osteolytic component. Slight or moderate increases in TR-ACP 5b serum levels occur in Paget’s
disease, hyperparathyroidism with skeletal involvement, and
in the presence of malignant bone invasion. TR-ACP 5b
shows relatively small dynamic changes compared to other
markers of bone resorption (e.g., those derived from type I
collagen catabolism). This could be attributable to the fact
that the enzyme is released into the sealed microenvironment
of osteoclasts rather than directly into the circulation.
However, unlike blood concentrations of bone resorption
markers derived from mature type I collagen, TR-ACP 5b is
not affected by renal dysfunction.
Reference Intervals
In the serum of apparently healthy adults, the reference interval of TR-ACP 5b activity is 1.5–4.5U/L.Children show a
higher activity (3.4–9.0U/L).
Other Enzymes
Lactate Dehydrogenase
Lactate dehydrogenase (LDH) is an enzyme that, through
hydrogen transfer, catalyzes the oxidation of L-lactate to pyruvate with the mediation of NAD+ as a hydrogen acceptor. The
enzyme has a molecular weight of 134kDa and is composed
of four peptide chains of two types (M and H), each under
separate genetic control. The structures of LDH-M and
LDH-H are expressed, respectively, from loci localized on
chromosomes 11 and 12. The subunit compositions of the ve
isoenzymes, in descending order of anodic mobility in an
alkaline medium, are: LDH-1 (HHHH; H
H3M), LDH-3 (HHMM; H2M2), LDH-4 (HMMM; HM3), and
LDH-5 (MMMM; M4). A sixth different isoenzyme of LDH,
LDH-X, composed of 4 X subunits, is present in postpubertal
human testes. A seventh LDH, termed LDH-6, has been identied in the sera of critically ill patients.
), LDH-2 (HHHM;
4
LDH activity is present in many human cells and tissues.
The concentration of enzymes in tissues ranges from 1500 to
5000 times that found physiologically in serum. Thus, the
release of the enzyme from even a small amount of damaged
tissue signicantly increases the observed serum LDH activity. Different tissues show different isoenzyme compositions.
In the heart, kidney, and erythrocytes, LDH-1 and LDH-2
isoenzymes predominate, while LDH-4 and LDH-5 isoenzymes predominate in the liver and skeletal muscle.
Clinical Signicance
Because of its wide tissue distribution, increases in serum
LDH occur in a variety of clinical conditions, including
myocardial infarction, hepatitis, and hemolysis. However,
the measurement of LDH in the serum is relevant only in
hematology and oncology.
Hemolytic anemia signicantly increases serum LDH
concentrations. Marked increases in LDH activity, up to 50
times the URL, are observed in megaloblastic anemias. The
latter usually result from a deciency of folic acid or vitamin B12 and causes the weakening of the erythrocyte precursor cell in the bone marrow (ineffective erythropoiesis),
leading to the release of large amounts of LDH. These
increases quickly return to baseline values after appropriate
treatment.
For monitoring purposes, LDH is relevant in predicting
disease activity in leukemias and the probability of survival
in Hodgkin’s disease and non-Hodgkin’s lymphomas.
Patients with malignant disease often show increases in
serum LDH; up to 70% of patients with liver metastases and
20–60% of patients with other metastases (e.g., lymph
nodes) show increased LDH activity. Increased LDH-1 is
observed in germ cell tumors (∼60% of cases), such as teratoma, testicular seminoma, and dysgerminoma of the ovary.
The percentage of patients with increased LDH depends on
the stage of the disease. LDH also appears to be a useful
predictor of outcome in patients with nonseminomatous
germ cell tumors of the testis, melanoma, and pulmonary
microcytoma.
Increases in LDH are observed in hepatopathies, but their
clinical use in a liver prole appears very limited because
they do not add signicant information to the determination
of transaminases. Finally, measurement of LDH in pleural
uid (better if in combination with serum LDH) helps to distinguish between exudative (pleural uid/LDH serum LDH
>0.6) and transudative effusions (ratio <0.6).
The presence of macro-LDH due to the formation of an
autoantibody–enzyme complex that leads to a persistent
increase in the amount of circulating enzyme is estimated to
occur in <1 person each 10,000. Its presence should be
checked in suspected individuals to avoid further in-depth
investigation or unnecessary treatment.
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I. Infusino et al.
Reference Intervals
The reference interval of LDH in adult Caucasian subjects is
125–220 U/L, whereas Asian individuals show slightly
higher values. The reference intervals are markedly higher in
children, with a gradual decrease observed throughout the
childhood period.
Cholinesterase (Pseudocholinesterase)
Two interrelated enzymes can hydrolyze acetylcholine. The rst
is acetylcholinesterase, also called true cholinesterase. This is
found in erythrocytes, lungs and spleen, nerve endings and brain
gray matter. It is responsible for the hydrolysis of acetylcholine
released at nerve endings to mediate the transmission of nerve
impulses across the synapse. The degradation of acetylcholine
is necessary for the depolarization of the nerve so that it can be
repolarized in the next impulse conduction event. The other
enzyme is acetylcholine acetylhydrolase (CHE), also called
pseudocholinesterase, serum cholinesterase, or butyrylcholinesterase, which is found in liver, pancreas, heart, and serum.
Although CHE activity in the human body is ∼3 times greater
than acetylcholinesterase activity, its exact biological role is still
unknown. Of clinical interest are atypical (genetic) variants of
the enzyme characterized by decreased activity toward acetylcholine and other substrates, which are found in the serum of a
small number of apparently healthy people.
The gene on chromosome 3 that controls CHE synthesis
can exist in many allelic forms. Four of the most common
forms are designated as Eu, Ea, Ef, and Es. These four allelic
genes can be combined into one normal and nine abnormal
genotypes. The most common (normal) genotype is designated as EuEu or UU (“usual”). The Ea gene is designated as
an atypical gene; sera from subjects homozygous for this
gene (EaEa or AA) are weakly active toward most CHE substrates and show increased resistance to inhibition of enzymatic activity by dibucaine. The Ef (uoride-resistant) gene
gives rise to a weakly active enzyme but with increased resistance to inhibition by uoride. The Es (silent) gene is associated with the absence of the enzyme or the presence of a
protein with minimal or no catalytic activity. Abnormal
enzymes are fewer effective catalysts than the common form;
the afnity of the enzymes for substrates and that for competitive inhibitors, such as dibucaine or uoride, is reduced.
This gives rise to the characteristic dibucaine- or uorideresistant properties of the genetic variants, which can be
exploited in their characterization. The homozygous forms,
AA and FF, are found in 0.3–0.5% of the white population.
Clinical Signicance
Table 10.3 shows the indications for the measurement of
serum CHE activity.
Table 10.3 Indications for measuring serum cholinesterase activity
To assess the liver function (e.g., in monitoring patients after liver
transplantation)
To evaluate a possible acute poisoning by organophosphorus
insecticides
To identify subjects with atypical forms of enzyme, who are at risk
of prolonged responses to some muscle relaxants (e.g.,
succinylcholine) used in anesthesia
Measurement of serum CHE activity can serve as a sensitive indicator of liver synthesis capacity. In the absence of
genetic causes or known inhibitors, any decrease in CHE
activity reects an impairment of hepatic synthesis of the
enzyme.
Organophosphorus compounds that inhibit CHE activity
include many insecticides, such as parathion, sarin, and tetraethylpyrophosphate. Agricultural and chemical workers
may be subject to poisoning by inhalation of these substances
or by direct contact. Both cholinesterases are inhibited, but
the activity of the serum enzyme falls more rapidly than that
of the erythrocyte enzyme.
Succinylcholine and mivacurium, myorelaxant drugs
used in surgical procedures as an aid for endotracheal intubation, are hydrolyzed by CHE and their pharmacological
effect normally persists enough to meet the needs of the
surgical procedure. In subjects with low enzyme activities
or in those with a weakly active variant, drug degradation
does not occur rapidly enough, and the patient may enter a
prolonged period of respiratory muscle paralysis (apnea),
requiring mechanical ventilation until the effects of the
drug gradually wear off. Preoperative screening of CHE
activity has been proposed to identify individuals in whom
succinylcholine administration may lead to complications;
however, in many countries, this screening is not recommended, as all individuals undergoing surgery are considered potentially at risk. The phenotypes most susceptible to
apnea after succinylcholine administration are AA, AS, FF,
FS, SS, AF, and, to some extent, UA.Measurements of total
CHE activity and determination of “dibucaine number” and
“uoride number” are necessary for the complete characterization of CHE variants. The dibucaine number and uoride number indicate the percentage inhibition of enzyme
activity in the presence of standard concentrations of these
two compounds. Mutation genotyping can conrm alterations in the CHE gene.
Reference Intervals
The reference intervals for CHE are method dependent. At
birth, CHE activity is below adult values by ∼50%. It
increases during the next 3–6years until it exceeds adult values of ∼30%. From the fth year of life, the activity begins
to decrease until it stabilizes at adult values with puberty.
The signicant decrease in CHE (∼30%) during pregnancy
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93
Table 10.4
serum cholinesterase phenotypes
Phenotype Interval DN Interval FN
UU
UF 72–26
UA 48–72
AF 45–59 <44
FF 64–69 <44
AA <35
a
Redundant for the attribution of the phenotype AA
Values of dibucaine (DN) and uoride (FN) for various
≥77 ≥55
≥53
≥44
a
and puerperium is explained by the hemodilution inherent in
this condition.
Based on the determination of dibucaine and uoride
numbers, the values characterizing the various CHE phenotypes were established (Table10.4).
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Liver: FromBiochemistry toClinical
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Biochemistry
MarcelloCiaccio, LuisaAgnello, andAnnaMariaCiaccio
11
Introduction
Generally, liver function is studied by evaluating parameters
that allow the discrimination of the main clinical pictures,
which will then be investigated by specialized second- and
third-level laboratory tests and imaging investigations. This
chapter describes the main clinical pictures that can affect
the liver and the relative laboratory tests that allow their correct classication.
Jaundice
The term jaundice, from the Greek ίκτερος (yellow), refers
to the yellowish discoloration of the skin, sclerae, and
mucous membranes, caused by the deposition of bilirubin.
The latter is the product of the catabolism of heme, the prosthetic group, i.e., the nonprotein part of various proteins,
including hemoglobin, myoglobin, and enzymes belonging
to the cytochrome P450 family, catalases, and peroxidases.
In this chapter, the metabolism of bilirubin is initially
described to allow a better understanding of the classication
of jaundice. Subsequently, the various classications of jaundice are presented with a description of the main causes.
M. Ciaccio (*) · L. Agnello
Department of Biomedicine, Neurosciences and Advanced
Diagnostics, Institute of Clinical Biochemistry, Clinical Molecular
Medicine and Clinical Laboratory Medicine, and Department of
Laboratory Medicine, University Hospital “P.Giaccone”,
Palermo, Italy
e-mail: marcello.ciaccio@unipa.it
A. M. Ciaccio
Department of Health Promotion, Mother and Child Care, Internal
Medicine and Medical Specialties (ProMISE) “G.D’Alessandro”,
University of Palermo, Palermo, Italy
Bilirubin Metabolism
Every day an adult produces about 250–400mg of bilirubin,
most of which (>80%) comes from the catabolism of hemoglobin and a small amount from the hepatic catabolism of
other hemoproteins (proteins containing the heme group). In
particular, the bilirubin produced by the catabolism of hemoglobin can be divided into a hemo-cateretic portion (70%),
which comes from the physiological destruction of red blood
cells in the reticuloendothelial system of the spleen, and an
erythropoietic portion (30%) from ineffective erythropoiesis
in the bone marrow (catabolism of heme molecules not used
in erythropoiesis and destruction of erythroblasts, reticulocytes, and newly formed red blood cells).
Bilirubin is formed by the sequential catalytic degradation of heme mediated by two enzymes, heme oxygenase and
biliverdin reductase. Heme oxygenase initiates the opening
of the porphyrin ring of heme, leading to the formation of the
green pigment, known as biliverdin, which, subsequently,
will be reduced by biliverdin reductase to a yellow-orange
pigment, namely thebilirubin (Fig.11.1).
The bilirubin synthesized, called indirect or unconjugated, is poorly soluble in water at physiological pH and,
being liposoluble, is responsible for the toxic effects of bilirubin. This form of bilirubin circulates in the plasma bound
to proteins, mainly albumin and, to a much lesser extent, to
high-density lipoproteins; the binding to lipoproteins
becomes signicant in severe hypoalbuminemia. Binding to
albumin keeps bilirubin in the vascular space, thus preventing its deposition in extra-hepatic tissues, including the
brain, and minimizing its ltration at the glomerular
level.Albumin is also vital in transporting indirect bilirubin
to the sinusoidal surface of hepatocytes, where the pigment
dissociates from albumin and enters the hepatocytes by facilitated diffusion. Defects in genes encoding for bilirubin
transporters may result in hyperbilirubinemia. The passage
of bilirubin across the membrane of the sinusoidal surface of
© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023
M. Ciaccio (ed.), Clinical and Laboratory Medicine Textbook, https://doi.org/10.1007/978-3-031-24958-7_11
95
96
Reticuloendothelial system
Large
FecesUrine
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Fig. 11.1 Bilirubin
metabolism. (Copyright
EDISES 2021. Reproduced
with permission)
Hemoglobin
Heme oxygenase
Biliverdin
Biliverdin
Indirect bilirubin
reductase
Indirect Bilirubin-Albumin
Blood
Urobilinogen
M. Ciaccio et al.
Indirect bilirubin
UGT1A1
Direct bilirubin
Liver Gallbladder Small intestine
hepatocytes is bidirectional. In order to prevent the escape of
bilirubin from hepatocytes, bilirubin, as well as other organic
anions, is bound to the cytosolic protein glutathione
S-transferase (GST), also known as ligandin, which mediates
its transport to the endoplasmic reticulum (ER). In the ER,
indirect bilirubin undergoes a process of glucuronidation
that converts it to a water-soluble form, the direct bilirubin
(Fig.11.1). The glucuronidation of bilirubin, as well as of
various compounds, both endogenous (steroid hormones,
thyroid hormones, and catecholamines) and exogenous
(drugs, toxins, carcinogens), is mediated by a family of
enzymes known as uridine diphosphate glucuronosyltransferases (UGT). The resultingglucuronides are water-soluble
and, therefore, readily excreted in bile and urine.
Glucuronidation is one of the most important detoxication
mechanisms in our body. Among isoforms of the UGT family, only UGT1A1 is physiologically crucial in bilirubin
metabolism. Following glucuronidation, mono- and diglucuronide bilirubin molecules are formed and directed
toward the apical canalicular membrane and then transported
into the bile canaliculus. Conjugated bilirubin, also called
direct bilirubin, is secreted by active transport against a concentration gradient mediated by an ATP-dependent pump,
known as MRP-2 (Multidrug Resistance Protein) or ABCC2, expressed on the membrane of the biliary canaliculi of
hepatocytes. Most of the bilirubin (approximately 80%)
excreted in bile is present in the form of diglucuronide. In
subjects with reduced glucuronidation activity, the bilirubin
Kidney
Urobilinogen
Urobilinogen
Direct
bilirubin
Direct
bilirubin
Bilirubin
Urobilinogen
Stercobilinogen
diglucuronide rate decreases, and the bilirubin monoglucuronide rate increases.Inhibitors of hepatic UGT1A1 may
be present in breast milk, leading to neonatal jaundice, or in
maternal plasma and cross the placental barrier, reaching the
fetus (Lucey Discroll syndrome). A UGT1A1 deciency can
be found in newborns, in patients with chronic hepatitis, and
in patients with some hereditary disorders, such as Gilbert’s
syndrome or Crigler–Najjar syndrome. Biliary excretion of
conjugated bilirubin may be impaired in several acquired
conditions, such as viral or alcoholic hepatitis and cholestasis in pregnancy, or congenital conditions, such as Dubin–
Johnson syndrome and Rotor syndrome.
Conjugated bilirubin, produced at the hepatic level, after
a brief stay in the gallbladder, reaches through the bile in the
small intestine where, being water-soluble, it is not absorbed
through the membrane of the epithelial cells. In the intestine,
beta-glucuronidase enzymes mediate a process inverse to the
hepatic one, removing glucuronic acid from bilirubin.
Bilirubin is further metabolized by enzymes produced by the
bacterial ora that mediate its reduction to urobilinogen
(Fig.11.1). A small fraction of urobilinogen is reabsorbed
and returns to the circulation to be subsequently picked up in
the liver or eliminated inurine, where urobilinogen undergoes further oxidation to urobilin. Urobilin is the molecule
that gives urine its characteristic golden-yellow or strawyellow color. Most of the urobilinogen (80%) in the colon is
further processed by the bacterial ora into stercobilinogen
and eliminated inthe feces, where it undergoes further oxi-
intestine
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97
Table 11.1 Reference range of bilirubin
Total bilirubin <1mg/dL
Nonconjugated bilirubin <0.8mg/dL
Conjugated bilirubin <0.3mg/dL
dation to stercobilin. Stercobilin gives the characteristic
color to the feces.
Classication ofJaundice
Jaundice is the clinical sign of hyperbilirubinemia. Normal
values of total bilirubinemia are <1mg/dL,of which the conjugated form accounts for less than 5%(Table 11.1).
Total bilirubinemia values between 2 and 3mg/dL result
in sub-jaundice, characterized by a yellowish discoloration
of the sclerae; values >3mg/dL result in a clinically manifest
jaundice condition characterized by a yellowish discoloration of the sclerae, skin, and mucous membranes. Jaundice
and asymptomatic hyperbilirubinemia represent common
clinical conditions that may be caused by a variety of disorders, which include increased bilirubin synthesis, impaired
bilirubin conjugation, and biliary obstruction, and hepatic
inammation. Jaundice may represent the rst and only sign
of liver disease; therefore, its evaluation is of critical
importance.
Various classications of jaundice have been proposed.
The following are the main ones.
According to traditional classication, jaundice can be
distinguished based on the site of the cause into:
• Prehepatic: it results from the hyperproduction of biliru-
bin and it ischaracterized by an increase in the indirect
bilirubin form
• Hepatocellular: it results fromparenchymal lesions of the
liver that impair the hepatic metabolism of bilirubin, lead-
ing toan increase in total bilirubinemia
• Posthepatic: it results from intra- and/or extra-hepatic
biliary obstruction, with an increase in the direct form
According to a clinical biochemistry classication based
on the form of altered bilirubin, jaundice is divided into three
main categories:
• Jaundice characterized by a predominant increase in
unconjugated bilirubin, which may be due to three patho-
physiological mechanisms: increased production of bili-
rubin; altered uptake; or altered conjugation of bilirubin
in the liver. The physiological jaundice of the newborn is
a classic example in which all three pathophysiological
mechanisms are present.
• Jaundice characterized by increased circulating levels of
conjugated bilirubin, mainly due to biliary obstruction.
• Jaundice characterized by increased circulating levels of
both conjugated and unconjugated bilirubin due to hepatocellular disease.
Finally, according to a pathophysiological classication,
jaundice is distinguished as:
• Increased production of bilirubin
• Bilirubin uptake defect
• Bilirubin conjugation defect
• Defective bilirubin excretion
• Mechanical obstruction of the intra- and/or extra-hepatic
biliary tract
Jaundice duetoincreased Bilirubin production
Overproduction of bilirubin is the result of excessive catabolism of heme derived from hemoglobin that may be due to
hemolytic anemia, extravasation of blood into tissues, or
dyserythropoiesis (characterized by altered incorporation of
hemoglobin in erythrocytes resulting in the degradation of a
large fraction of unincorporated hemoglobin).
These forms of jaundice are characterized by an increase
in indirect bilirubinemia due to the inability of the liver to
metabolize excess bilirubin.
Jaundice duetoBilirubin Uptake
andConjugation Defect
These forms of jaundice are characterized by a normal production of bilirubin, butthe liver is unable tofully metabolize, leading to an increase in indirect bilirubinemia.
Alterations in the release of bilirubin to the liver and in bilirubin internalization in hepatocytes result in reduced hepatic
uptake. Congestive heart failure or portosystemic shunt
(spontaneous collaterals in subjects with cirrhosis or surgical
shunts) reduce hepatic blood ow and bilirubin release to
hepatocytes, resulting in mainly indirect hyperbilirubinemia.
In some patients with cirrhosis, direct plasma contact with
hepatocytes may be impaired by capillarization of sinusoidal
endothelial cells (loss of fenestrae), resulting in further
reduced bilirubin uptake. Other causes of altered bilirubin
uptake include Gilbert’s syndrome and administration of
various drugs, such as rifamycin, avaspidic acid, and cholecystogram agents; drug-induced defects usually resolve
within 48hours of discontinuation of therapy.
Congenital Defects
Reduced or absent UGT1A1 enzyme activity is a major
cause of jaundice due to bilirubin conjugation defects and
can be detected in several acquired or inherited disorders,
such as Gilbert’s syndrome and Crigler–Najjar’s syndrome
types I and II.UGT1A1 activity is, moreover, modulated by
various hormones; for example, thyroid hormones and ethi-
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nylestradiol inhibit bilirubin glucuronidation. Conversely,
the combination of progestin and estrogenic steroids results
in an increase in enzyme activity. Bilirubin glucuronidation
may also be inhibited by some antibiotics (e.g., novobiocin
or gentamicin at serum concentrations above therapeutic levels) and in chronic persistent hepatitis, advanced cirrhosis,
and Wilson’s disease.
Gilbert’s syndrome is a benign hereditary disorder transmitted in an autosomal dominant trait, characterized by a
defect in bilirubin uptake associated with a glucuronidation
decit due to alterations in the gene encoding for the enzyme
UGT1A1. From an epidemiological point of view, Gilbert’s
syndrome is quite common, with a prevalence that varies
between 4% and 16% in various populations. Patients with
Gilbert’s syndrome have moderately elevated levels of
unconjugated bilirubin, with total bilirubin levels generally
<3 mg/dL. Some associated diseases or physiological or
paraphysiological events, such as stress, fasting, menstrual
cycle, or asthenia, may determine an increase in total bilirubinemia, generally not exceeding 6mg/dL.Other than jaundice, patients are typically asymptomatic, and no treatment
is required.
Crigler-Najjar syndrome, also known as “congenital nonhemolytic jaundice with glucuronyltransferase deciency” is
a rare autosomal recessive disorder of bilirubin metabolism.
Depending on the severity of the disease, two forms can be
distinguished.
• Type I: It results from mutations (deletions, insertions,
missense mutations, etc.) in one of the ve exons of the
gene encoding for the enzyme UGT1A1, which are asso-
ciated with an altered protein characterized by a complete
loss or a strong reduction in catalytic activity. Affected
patients have severe jaundice and neurological impair-
ment due to bilirubin encephalopathy, which can result in
permanent neurological sequelae. Frequently, subjects
affected by type I die in the neonatal period due to kernic-
terus (bilirubin encephalopathy). The hallmark of type I
syndrome is the presence of pure unconjugated hyperbili-
rubinemia, with levels ranging from 20 to 25mg/dL up to
50mg/dL.
• Type II: It isknown as “Arias syndrome” and it is phenotypically similar to type I. However, the unconjugated
hyperbilirubinemia is usually lower than type I, with a concentration ranging from 8 to 18mg/dL.Patients commonly
survive to adulthood without neurological alterations. The
disease is due to mutationsin any of the ve exons of the
gene encoding for UGT1A1 but compared with the type I
syndrome, which can be caused by a wide variety of alterations, the type II syndrome is due only to point mutations
resulting in the substitution of single amino acid, with consequent reduction, but not abrogation, of the catalytic activity of the enzyme. The residual catalytic activity is
responsible for the partial conjugation of bilirubin and the
reduced severity of hyperbilirubinemia.
The differential diagnosis between Crigler–Najjar syn-
drome type I and type II is based on hyperbilirubinemia levels (lower in type II) and age of onset (late for type II). In
addition, administration of phenobarbital may support the
differential diagnosis because in most type IIpatients, but
not type I, phenobarbital (60–120mg for 14days) can reduce
bilirubinemia levels by 25%. Table 11.2 summarizes the
main features of Gilbert’s syndrome and Crigler–Najjar’s
syndrome types I and II.
Jaundice duetoDefective Excretion ofBilirubin
or Mechanical Obstruction oftheIntra- and/or
Extra-hepatic Biliary Tracts
These forms of jaundice are characterized by a normal production of bilirubin, which is taken up and conjugated in the
liver but is not regularly excreted, leading to an accumulation
of circulatingdirect bilirubin. Since direct bilirubin is watersoluble, it is ltered through glomerulus and eliminated
inurine. In these forms of jaundice, therefore, there is direct
hyperbilirubinemia and bilirubinuria. The altered excretion
may be due to intra- and/or extra-hepatic cholestasis, the
causes of which are summarized in Tables 11.3 and 11.4.
Table 11.2 Characteristics of Gilbert syndrome and Crigler–Najjar syndrome type I and II
Hepatic histology Normal Normal Normal
Bilirubinemia 20–50mg/dL <20mg/dL <3mg/dL
Liver function Normal Normal Normal
Bile Usually pale, it contains small
Hepatic activity of UGT1A1 Absent Markedly reduced Reduced
Effect of phenobarbital on
bilirubinemia
Inheritance Autosomal recessive Autosomal recessive Autosomal dominant
Prevalence Rare Rare Common
Prognosis Neurological alterations Usually benign, neurologic
Crigler–Najjar syndrome type I Crigler–Najjar syndrome type II Gilbert syndrome
amounts of unconjugated bilirubin
None Reduction Reduction
Increased proportion of bilirubin
monoglucuronide
impairment is rarely present
Increased proportion of bilirubin
monoglucuronide
Benign
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Table 11.3 Causes of jaundice from intra-hepatic cholestasis
Acute hepatocellular
injury
Chronic hepatocellular
injury
Multifactorial Total parental nutrition
Various Hypotension/hypoxemia/heart failure
Hereditary/endocrine Benign recurrent intra-hepatic cholestasis
Inltrative/
granulomatous
Table 11.4
Cholangiopathies and other
disorders involving the bile ducts
Extrinsic causes Pancreatitis (acute and
Causes of jaundice from extra-hepatic cholestasis
Viral hepatitis
Steatosis and/or alcoholic hepatitis
Nonalcoholic steatohepatitis
Drugs
Primary sclerosing cholangitis
Primary biliary cholangitis
Drugs
Hepatitis (viral, alcoholic, autoimmune)
Cirrhosis due toany cause
Systemic infection
Postoperative
Sickle cell anemia
Organ transplant (rejection; venoocclusive disease; graft-versus-host
disease)
Budd–Chiari syndrome
Parasitic infection (Clonorchis sinensis;
Fasciola hepatica)
Benign familial intra-hepatic cholestasis
Cholestasis associated with low
phospholipid levels
Dubin–Johnson syndrome
Rotor syndrome
Thyrotoxicosis
Alagille syndrome
Disorders of the metabolism of
carbohydrates, lipids, or bile acids
Caroli’s disease
Pregnancy
Protoporphyria
Amyloidosis
Lymphoma
Sarcoidosis
Tuberculosis
Cholelithiasis
Biliary stenosis after invasive
procedure
Cholangiocellular carcinoma
Primary sclerosing cholangitis
AIDS cholangiopathy
Cytomegalovirus
Cryptosporidium spp.
HIV
Cyst of the choledochus
Dysfunction of the sphincter of
Oddi
Parasitic infections
Ascaris lumbricoides
Histiocytosis X
chronic)
Pancreatic cancer
Portal adenopathy (metastasis,
tuberculosis)
Periampullary carcinoma
Periampullary diverticulum
Mirizzi syndrome
Neonatal Jaundice
In newborns, jaundice is a relatively frequent condition due
to several mechanisms. Almost all newborns have total bilirubinemia values >1 mg/dL. As bilirubinemia increases,
neonatal jaundice is observed. First, it affects the face and
then progresses cranio-caudally to the trunk and extremities.
Approximately 60% of term infants and 80% of preterm
infants have physiologic jaundice, which is the result of the
different neonatal metabolism of bilirubin, characterized by:
• Increased production of bilirubin: hemocateretic (shorter
erythrocyte half-life) and hepatic share.
• Insufcient metabolic capacity of the liver: reduced levels
of ligandin, glucuronic acid and UGT1A1. Specically,
UGT1A1 activity in term infants, at 7days after birth, is
approximately 1% of that of adults and does not reach
adult levels until 14weeks of age.
• Insufcient development of intestinal bacterial ora,
resulting in insufcient production of bilinogens.
All these conditions result in an increase in unconjugated
bilirubinemia. In Caucasian and African-American term
infants, the mean peak total plasma bilirubinemia is 7–9mg/
dL and is reached between 48 and 96 hours of life. EastAsian infants may have total bilirubinemia values up
to10–14mg/dL between 72 and 120hours of life.
Generally, neonatal jaundice resolves within the rst
2weeks after birth, depending on the maturation of the bilirubin clearance system. Infants with severe hyperbilirubinemia,
dened as total bilirubinemia >25mg/dL, are at high risk of
developing bilirubin-induced neurologic dysfunction due to the
passage of indirect bilirubin across the blood–brain barrier.
Infants with clinically relevant hyperbilirubinemia should
undergophototherapy, which exploits the ability of radiation
in the visible spectrum to convert bilirubin into isomers that
can be rapidly eliminated hepatically and renally.
Diagnosis
The diagnostic approach to a jaundiced patient is based on
careful anamnesis, physical examination, and laboratory data.
Although evaluation is usually not urgent, in some cases,
jaundice may represent a medical emergency, such as ascending cholangitis or fulminant liver failure.
From a laboratory point of view, the initial assessment is
based on the measurement of circulating levels of:
• Total bilirubin
• Alkaline phosphatase
• Aminotransferase
• Prothrombin time (PT)
• Albumin
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Total bilirubinemia
<1.2 mg/dL
STOP
Fig. 11.2 Reex test for the evaluation of bilirubin. (Copyright
EDISES 2021. Reproduced with permission)
≥1.2 mg/dL
Fractional
bilirubin
Aminotransferases (alanine aminotransferase [ALT] and
aspartate aminotransferase [AST]), alkaline phosphatase,
and bilirubin are biochemical markers of liver injury; albumin and PT are markers of hepatocellular function.
Some laboratories use a reex test (Fig.11.2) for bilirubinemia evaluation, where total bilirubinemia is determined
rst; if values are >1.2mg/dL, direct bilirubinemia is determined. The value of indirect bilirubinemia is obtained with
the following formula:
Indirect bilirubinTotal bilirubinDirect bilirubi
The result of the laboratory investigations allows an initial differential diagnosis among the possible causes of jaundice (Table11.5).
An increase in indirect bilirubinemia could be due to
hemolytic anemia, medications that impair hepatic uptake of
bilirubin, or inherited disorders, such as Gilbert’s syndrome
and Crigler–Najjar’s syndrome. Direct hyperbilirubinemia,
on the other hand, maybe due to biliary obstruction, intrahepatic cholestasis, hepatocellular injury, or hereditary disorders, such as Rotor’s syndrome or Dubin–Johnson’s
syndrome. Notably, both syndromes are characterized by
direct hyperbilirubinemia in the absence of other altered
liver tests; normal levels of alkaline phosphatase support the
differential diagnosis between these disorders and alterations
associated with biliary obstruction.
Aminotransferases are generally normal in subjects with
jaundice due to prehepatic causes, whereas levels increase in
thecase of jaundice due to posthepatic causes and, above all,
to hepatic alterations. In addition, the pattern of aminotransferase alteration could point to a specic cause; for example,
alcoholic hepatitis is characterized by a disproportionate
increase in AST compared to ALT, with an AST/ALT ratio
>2. Similar to aminotransferases, circulating levels of alkaline phosphate will be normal in subjects with jaundice due to
prehepatic causes. However, they will be increased in subjects with jaundice due to hepatic and, above all, posthepatic
alterations. In particular, an increase in alkaline phosphatase
associated with normal or slightly increased aminotransferase
values suggestsbiliary obstruction or intrahepatic cholestasis.
Table 11.5
Total bilirubin
Direct bilirubin (conjugated) N
Indirect bilirubin
(non-conjugated)
Aminotransferase
Alkaline phosphatase N
Albumin N
Differential diagnosis of jaundice
Prehepatic Hepatic Posthepatic
↑ ↑ ↑
↑ ↑↑
↑ ↑ N/↑
N/↑ ↑↑↑ ↑
N/↑ ↑↑↑
↓ N/↓
An increase in alkaline phosphatase could also be seen in
granulomatous liver diseases, such as tuberculosis or sarcoidosis; these conditions could be characterized by jaundice. Increased alkaline phosphatase could result from
increased extra-hepatic release, particularly in the bone.
Extra-hepatic disorders do not cause jaundice, except in rare
cases, such as bone tumors metastasizing to the liver.
Measuring the serum canalicular enzyme γ-glutamyltransferase
activity could help conrm the hepatic origin of alkaline
phosphatase. Finally, an increased value of the prothrombin
time that is corrected by the administration of vitamin K suggests an altered intestinal absorption of fat- soluble vitamins,
compatible with obstructive jaundice (postepathic); on the
other hand, an increase in the prothrombin time that is not
corrected by vitamin K suggests the presence of moderatesevere hepatocellular disease with impaired synthesis function, especially if associated with hypoalbuminemia.
Based on the anamnesis, physical examination, and laboratory, an etiological diagnosis can be made, which can be
conrmed with further investigations, such as liver imaging
tests if biliary obstruction or intrahepatic cholestasis is suspected or serological tests if viral hepatitis or primary biliary
cholangitis is suspected (Table11.6).
Hepatitis
The term hepatitis refers to the inammation of the liver. It
can be due to various causes, mainly infection with hepatotropic viruses, alcohol abuse, drug intake, or alterations in
the immune system. Hepatitis may occur in an acute or
chronic form when it lasts for more than 6months.
Viral Hepatitis
Viral hepatitis is an inammatory process of the liver due to
viral infection.
Although viral hepatitis is similar from clinical biochemistry and morphological point of view, it differs from an etiological (different viruses responsible for infection),
epidemiological (different distribution and frequency of
infection and disease), and immunopathogenic point of view.
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