Clinical Chemistry of Plasma Proteins:

Structure and function relationships and

Clinical significance
The concentration of globulins usually is calculate as follows:

Globulins (g/dL) = Total protein (g/dL) − Albumin (g/dL)

Globulins tend to be characterized as proteins that precipitate

in water and that re-dissolve when the salt concentration is raise
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• The total protein concentration of serum obtained from

healthy ambulatory adults is about 6.4 – 8.3 g/dL, and for
adults at bed rest, 6.0 – 7.8 g/dL fibrinogens1.0.3 sserum 110 sig plasms 1

• Plasma contains a protein concentration about 0.3 g/dL

higher because fibrinogen is remove from plasma when it is
clotted to form serum Soluble
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 ⑪19.

Positive acute-phase proteins

Proteins such as
1. α1-antitrypsin
2. α1-acid glycoprotein
3. Haptoglobin
4. Ceruloplasmin
5. Complement 4 (C4)
6. C3
7. Fibrinogen
8. C-reactive protein (CRP)
↳fanch increase.

9. Serum amyloid A
show increase concentrations in response to an APR an are known as
positive APPs
In extreme cases, concentrations of CRP an serum amyloid A have
been known to increase up to 1000-fold from a low initial baseline value
 • Measurement of APPs assists in detection of inflammation, and
sequential measurements of proteins such as CRP are used to
monitor the progress of the inflammation or its response to
Nastains5
treatment
*CBC level
WBCS15,100 infectins15.
shows WB ->
10:

js •<1Increase erythrocyte sedimentation rate (ESR) provides an

·159615 alternative means of measuring inflammatory responses.
50-18)
i•
-

Fibrinogen concentrations are considered to be a major

aggentials I

* determinant of ESR; therefore ESR probably reflects changes

95,284,55
* in fibrinogen concentration in the APR
inflammalsis

• In chronic infection, such as infection with hepatitis B or C and

i ESRI

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is. tuberculosis; immunoglobulins are often increased

• Concentrations of plasma proteins depend on their

1. rates of synthesis
2. extracellular distribution
3. rates of clearance
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Proteins and peptides substantially smaller than albumin are

cleared from the circulation by glomerular filtration unless
they are bound to larger carriers, such as small
apolipoproteins bound to lipoprotein particles or retinol-
binding protein bound to prealbumin
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largeproteins attach to small proteins to preventtheir escape asretinol binding protein caked)
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it to lipid.
ESR is a type of blood test that measures
how quickly erythrocytes (RBCs) settle at
the bottom of a test tube that contains a
blood sample. Normally, RBCs settle
relatively slowly. A faster-than-normal rate
may indicate inflammation in the body.
 Negative acute-phase proteins

• The concentration of other plasma proteins, including

1. Prealbumin (Transthyretin) it
plays major role inflammation, Tyrosine
a in attain
&
2. Albumin to
receptor initiatecascade thatmay excitatory:
be as

3. Transferrin -> bacterialiron 15156.0 if caba b

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4. Apolipoprotein A-I which is a part of HDL

5. Apolipoprotein B
6. α2-HS glycoprotein
9 neciprocal
ininsulin resistance,
7. Insulin-like growth factor I
8. Retinol-binding protein peration of liver.
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macrophage.
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are decreased and they are known as negative APPs

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• Changes in plasma protein concentrations are triggered by

interleukin-6 (IL-6) an other cytokines. decrease albuminsynthen it
*
Normal serum electrophoresis

- i01085

from changes in intensity of the small number of major bands and in the
overall distribution of proteins, it is often possible to interpret patterns as
consistent with particular disease processes, such as 1. acute inflammation,
2. nephrotic syndrome, or 3. chronic inflammation.
& Cellulate sucrose -> agarose

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Leukemia, multiple nyoloman" profiles obtained from
cellulose acetate (CAE) and
agarose (AGE)
electrophoresis.
A, Normal serum. B, Patient
serum containing a large M-
protein
C, Patient serum containing
a small monoclonal protein.
Arrows indicate the position
of the monoclonal proteins
Serum protein electrophoresis (SPE)
-for tumors

• the main current application of SPE is for the detection or

quantification of monoclonal immunoglobulins that occur in
disorders such as multiple myeloma
• Monoclonal immunoglobulins usually appear as sharp
bands in the γ- or β-region
• They differ from normal immunoglobulins, which migrate
as a diffuse zone, mainly in the γ-region, in terms of the
sequence and charge heterogeneity of polyclonal
immunoglobulins
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Serum Albumin
– Small globular protein (Mol. Mass 66.3 kDa)
– Most abundant protein in the plasma (~55% of Half
it's

life:19 days
protein mass) and all other body fluids in
plasma 2.5 w

– No carbohydrate
– High solubility
– High negative charge& of bec.

=>

synthesized by hepatocyte
Because of its high plasma concentration and medium size, albumin is the
major contributor to colloidal oncotic pressure (COP) in the vascular space

Albumin is the major protein component of most extravascular body fluids,

including 1) CSF, 2) interstitial fluid, 3) urine, and 4) amniotic fluid.
*

if hightraumal infection districtBBB.

1955855
1.02
 Albumin, (cont.) -> water soluble

->
itcreates counter pressure.

• Biochemistry and Function

– Synthesis: liver paranchymal cells
– Catabolism: by pinocytosis in all tissues
– Half-life: 15 – 19 days
– Normal range of plasma albumin is 3.5 – 5.2 g/dL
(35 – 52 g/L)
– Major function to maintain COP vascular and
extravascular Counter pressure =>

– Binds and transports many components & drugs

mainly cajugationsIliver I s

albumin(485j
h
– free fatty acids, bilirubin, calcium, thyroid and steroid
88815 -
hormones, drugs, and thiol-containing compounds
water soluble albuminsIsΔ
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 Albumin Clinical Significance

– Hyperalbuminemia: in dehydration "fuer, 5019.5;

/

bec, – Hypoalbuminemia gene defect

of cytokinse
as

thatinhibittheir
synthesis • Analbuminemia
bec. of histan• Inflammation (↑ capillary permeability, allowing more albumin to enter the
<-
&
pearmeability extravascular space, ↓hepatic synthesis in response to IL-6, ↑ catabolism)
increase, albumin

transmitted in • Hepatic disease (nutritional deficiency, loss in the extracellular space,

induced"
inhibition of synthesis by alcohol) hepatistic "may dug
be
amount to
high
extracellar.
• Urinary loss (normally, 99.9% of albumin in the glomerular ultrafiltrate is taken up by the
proximal tubules), Nonpathological increases in urine albumin excretion sometimes are observe with
of
bec. &
disease postural changes, strenuous exercise, an fever
nephron
or metabolic • Gastrointestinal loss bec. of wker, tumor...
->

defect. • Protein Energy malnutrition -> of malnutrition& starvation

in case

partal <-• Edema and Ascites

as vein

·defect or cirrhosis

• Burn injury: severe loss of albumin from wounds: epithelial losses,

accelerate catabolism, and the APR
 Albumin
– Only about 10 mg/day of albumin is normally excrete in urine
&a50*sueXcelis1855915ss
– Small increases in albumin excretion to >30 mg/ indicate early
stages of glomerular or tubular injury and risk of progression
to more severe kidney disease. This has been termed
microalbuminuria -> presence of albumin in wine -

• Laboratory Analysis -> marker so specific antibaly s809.88.15

– Dye binding technique BCG, BCP albums 5150 50 1051
crossman of sem 11 *

• Over-estimation of albumin in presence of fibrinogen and heparin

ELISA
In the nephrotic syndrome, the synthetic rate has been known to increase
to three fold above normal. The synthesis of albumin is controlled primarily
by COP an secondarily by protein intake.
& little neabsation.
hydrostatic
p
In the nephrotic syndrome, the kidney maintains some size selectivity. & lipoproh
Concentrations less than 200 kDa of protein such as albumin generally are albumin MW
->
so

substantially decreased, even though hepatic production is increased.

have
high
blood.
remain in

Concentrations of a dew very large proteins, such as α2-macroglobulin (AMG) and

apolipoprotein B–containing lipoproteins, are increased.
 Prealbumin or transthyhtim
& synthesied in liver, attach to retino by lil ratic

• Prealbumin is a nonglycosylated protein (molecular mass,

55 kDa) composed of four identical subunits
• It binds and transports ~10% of T3 and T4 hormones
(separately ‫( )ليسوا معا في نفس البروتين‬with 10-fold higher affinity toTE
than T4)
• Prealbumin is synthesized in the liver and to a lesser
extent in the choroid plexus of the central nervous
system, accounting for the relatively high concentration of
prealbumin in CSF
• Its synthesis is stimulated by
• glucocorticosteroid hormones Cortisol? ->

• Androgens there'sfluctuation "Androgens are precursor of estrogen

• many nonsteroidal anti-inflammatory drugs (NSAIDs), including
aspirin
 Prealbumin concentrations Transthyretin was found to
be more sensitive to
Concentrations fall in changes in acute
malnutrition than albumin,
1) inflammation and malignancy and its level reflects recent
dietary intake compared to
2) cirrhosis of the liver -> bec. it's synthe overall nutritional status
in liver
3) protein-losing diseases of the gut or kidneys
Prealbumin concentrations are often used as an indicator of protein nutrition because of its
relatively short half-life and high proportion of essential amino acids. It responds much more
rapidly to nutritional changes than of proteins such as albumin. if no protein source in diet.
1
consup 5060.0.09
It levels are increased in the central nervous system but not in the serum of patients with endogenous
depression or with Parkinson's disease (after adrenal medullary autotransplantation).
Estrogen descreases its level.
Zinic requires for its synthesis

Protein-bound hormones are biologically inactive and cannot be metabolized.

Thus, an increase in protein binding would tend to decrease hormone activity and plasma clearance and increase the half-life
of the hormone.
Free hormone is also responsible for negative feedback inhibition of hormone secretion.
Therefore, a sudden increase in hormone binding to plasma proteins would decrease negative feedback.
Protein binding of hormones does, however, provide a reservoir for the rapid replacement of free hormone.
 Retinol-binding protein (RBP) -> prealbumin disg
• Retinol-binding protein (RBP) is a small (21 kDa), monomeric
transport protein for all-trans-retinol, the active form of vitamin A
• It is synthesized by adipose tissue*
• RBP synthesis requires zinc, and retinol is required for its transport
out of the Golgi apparatus
• When circulating in the plasma, RBP is present in a 1:1 complex with
prealbumin, slowing glomerular filtration of RBP
• apoRBP (RBP without retinol)
• RBP has a half-life of only about 12 hours, but it is extended in renal
failure
• Decrease concentrations of RBP are seen primarily with
• 1) liver disease, 2) protein malnutrition, and 3) the APR
• Zinc deficiency is characterized by low serum concentrations of
both RBP and vitamin A
• Levels of serum (RBP4), secreted by fat and liver cells, are
increased in obesity and type 2 diabetes (T2D), hepatic insulin
resistance
• Retinol binding protein 4 was immunohistochemically localized in or
close to adipocytes within muscle and adipose tissue and in liver
stellate cells but not in hepatocytes
• Hepatocytes were identified as the principal source of circulating
RBP4 in mice

• References
• Retinol binding protein 4 abundance in plasma and tissues is related to body fat deposition in cattle.
Scientific Reports volume 9, Article number: 8056 (2019)
• Nora Klöting et al. Serum Retinol-Binding Protein Is More Highly Expressed in Visceral Than in
Subcutaneous Adipose Tissue and Is a Marker of Intra-Abdominal Fat Mass. Cell Metab . 2007
Jul;6(1):79-87
 High-density lipoprotein (HDL)
• high density because of its content of about 50%
protein and phospholipid as a major lipid component
• Apolipoprotein A-I (29 kDa) is the major protein
• High concentrations of HDL, usually measure as the
cholesterol in HDL or as apolipoprotein A-I, lower the
risk of cardiovascular disease
• Apolipoprotein A-I is a negative APP that is considered
to have anti-inflammatory properties

200-500 kDa
 Alpha1 –Acid glycoprotein (AAG)
(Orosomucoid)
• small protein (40 kDa, 45% carbohydrate (↑sialic acid))
• is a member of the lipocalin family of proteins that bind
lipophilic substances
• The major seromucoid fraction (↓ HClO4 Perchloric acid)

• Biochemistry and Function

– Synthesis: in liver
– Can bind to hepatic asialoglycoprotein receptors for degradation
– Binds and inactivates lipophilic hormones (progesterone)
– Binds and reduces bioavialability of drugs (propranolol,
quinidine, chlorpromazine, cocaine) with ↑ AAG, drug dose ↑

A glycoprotein of serum that is not coagulated by heat. Referring to a fluid with

properties of serum and mucus (mucoprotein in serum)
 Alpha1 –Acid glycoprotein (AAG) cont.
• Clinical significance
– [AAG] ↑ in APR especially in GI inflammation and
malignancy
– [AAG] ↑ by corticosteriods and other NSAIDs
– Estrogen ↓ its synthesis
– ↓ [AAG] in protein-losing syndrome, nephrotic
syndrome
• Laboratory consideration
– Immunochemical methods
– Electrophoresis and carbohydrate content
(It is visualize with the use of periodic acid –Schiff stain)
 Alpha1 Antitrypsin (AAT)
• AAT is a sepin (serine
protease inhibitor)

• Biochemistry and
Function
– Synthesis: in liver
– Highest protease
inhibitor in plasma
– Inactivates leukocyte
elastase (serine protease)
– Small size, so it can
diffuse into the tissues
 Emphysema is a lung condition that causes
AAT, cont. shortness of breath. The air sacs in the lungs
(alveoli) are ruptured
• Clinical significance
– ↑ [AAT] in APR and by estrogen (pregnancy and oralcontraceptives)
– Secondarily low in
• Neonatal respiratory distress syndrome (RDS)
• Sever pancreatitis
• Protein losing syndrome
– Its deficiency results in pulmonary emphysema ‫نقصه يؤدي‬
‫إلى‬

– Deficiency also associated liver cirrhosis, cholestasis,

hepatocellular carcinoma (hepatic metastasis)
 AAT, cont.

Laboratory consideration
– Major protein in α1–globulin band
– Glycoprotein, do not stain well in electrophoresis
– Some genetic variants are visible on electro. (5-8)
– Quantified by immunoturbidimetric, nephelometric
 Alpha1-Fetoprotein (AFP)
• Dominant plasma protein of embryonic life
• A protein produced in the fetal liver that is measured in
maternal serum for predicting risk of anencephaly, spina
bifida, and Down syndrome in the fetus
• Biochemistry and Function
– Albumin analog, 4% carbohydrate
– MW~70 kDa
– serum concentration of AFP serves as a tumor marker for
hepatocellular and germ cell carcinoma

Anencephaly: A birth defect, abnormal development of a brain, skull, and Scalp,

results from neural tube defect
 Alpha1-fetoprotein
• The 1st protein to appear in the plasma during the gestational life
• Elevated in adults in certain pathological conditions
• Biochemistry and Function
– Synthesized by fetal yolk sac and liver
– A band visible between AAT & ALB in fetalor newborn serum

• Measuring of AFP in maternal serum and amniotic fluid is useful for detecting some
serious fetal anomalies
• e.g., maternal serum AFP is elevated in 90% of cases of fetal open neural tube
defects and is low in cases of fetal Down syndrome and trisomy 18
• AFP measurement in non-pregnant patients may be used for monitoring certain cancers
 AFP, cont.
Clinical Significance
• ↑ in maternal serum and amniotic fluid indicate
possibility of open neural tube or abdominal wall defect
• ↑ in case of
– Multiple fetuses
– Fetomaternal bleeds
– Tumors
• ↓ Maternal serum concentrations during pregnancy with
fetal trisomy 18 or 21
 Alpha2 – macroglobulin (AMG)
• is a major plasma proteinase inhibitor
• Large protein (725 kDa), doesn’t diffuse to
extracellular fluid
• Biochemistry and Function
– Inhibit many proteinases (serine, cysteine, metal
ions in their catalytic site)
– Not APR
– Synthesized in parenchymal cells of liver
– α2-Macroglobulin binds to both inhibin and
activin
 Alpha2 –macroglobulin,cont.

• Clinical significance
– ↑ occurs with the effects of estrogen (females vs.
males)
– Concentration in infants and children 2-3X values
in adults (protective mechanism)
– ↑↑ in nephrotic syndrome
– ↓ in sever acute pancreatitis, and untreated prostate
carcinoma
Serine proteases (or serine
endopeptidases) are enzymes
that cleave peptide bonds in
proteins, in which serine
serves as the nucleophilic
amino acid at the (enzyme's)
active site
 Beta2 – Microglobulin (BMG)
• Low molecular weight (11.8 kDa) found on cell
surface of all cells
• Biochemistry and Function
– It is the light-chain  chain of the human leukocyte
antigen (HLAs)
– consists of single polypeptide chain with no
carbohydrate
– Filtered through glomerulus, <1 % excreted in
urine
Major Histocompatibility
complex (MHC) class I Small amounts are shed into plasma
by lymphocytes and tumor cells
(human leukocyte antigen
HLAs)

Cytotoxic T cell
• Endogenous
antigens are antigens found within the
cytosol of human cells such as viral
proteins, proteins from intracellular
bacteria, and tumor antigens

• MHC class I

• Exogenous
antigens are antigens that enter from
outside the body, such as bacteria,
fungi, protozoa, and free viruses

• MHC class II
 Beta2 – microglobulin (BMG), cont.
• Clinical Significance
‫ يتراكم البروتين في الدم ويتج ّمع‬،‫ لكن في حالة الفشل الكلوي‬،‫التخلص من هذا البروتين يحدث باستمرار‬
– In patients on long-term hemodialysis, it aggregates into
amyloid fibers that deposit in joint spaces, a disease known
as dialysis-related amyloidosis
lymphocytic leukemia: cancer of blood lymphocyte
– ↑ in multiple myeloma (cancer of plasma cell) and lymphoma
– ↑ in renal failure, neoplasm, inflammation (B-lymphocytes)
– Important in the evaluation of renal tubular function in heavy
metals exposure, and transplant
– Serial determination for monitor of B-lymphocyte tumors
(marker for blood cell cancer)
 Amyloid Proteins
• Amyloid proteins are a group of proteins that share the
common characteristic of the ability to form beta-pleated
sheets (fibrils), which are resistant to proteolysis
• Pathological extracellular deposits in many disorders named
Amyloidosis
– Primary amyloidosis
– Amyloidosis associated with multiple myeloma
– Secondary amyloidosis (inflammation)
– Amyloidosis associated with aging
– Familial amyloidosis

Amyloidosis is a build-up (deposit) of an insoluble abnormal protein called amyloid in

organs and tissues throughout the body
 Amyloid - Clinical findings
• Primary and associated with multiple myeloma: bone
marrow plasmacytosis – identical monoclonal light
chains (AL amyloidosis, Amyloid light-chain)
• Secondary: Amyloid A (AA); fragments of the N-
terminal of serum amyloid A (SAA) that is associated
with HDL
• SAA is acute-phase protein, deposits mainly in liver,
kidney, spleen  nephrotic syndrome,
hepatosplenomegaly
• SAA is normally soluble and its plasma concentration
is elevated during inflammation
 Amyloid - Clinical findings
• Senile amyloid (aging): deposits mainly in the
heart and may in the pancreas and brain (amyloid
plaque)
• Familial: mainly neurological, deposits in the
brain, and maybe in the kidney and blood
vessels, numerous forms of transthyretin
Examples of small proteins that accumulate in the blood in case of kidney failure are
(1) β2-microglobulin, (2) cystatin C, (3) Ig light chains, (4) complement factor D, and
(5) retinol-binding protein
 Ceruloplasmin (Cp)
• α2- protein
• Contains ~95% of serum Cu → blue color
• Biochemistry and Function
– Synthesized in the liver
– Cu is required for the normal folding of the
polypeptide, added by ATPase (intracellular process)
– ApoCp is synthesized in the absence of copper or ATPase
– Cp is catalysis of redox reactions, anti-oxidant
– Cp is vitally important in regulating the ionic state of iron,
oxidizing Fe2+ to Fe3+ ferrous/ferric to incorporate in transferrin
– Alb and transcuprein are the other Cu transport proteins
(α2M)
 Ceruloplasmin (Cp), cont.
Clinical Significance
• ↑ in APR (weak and late)
• ↑↑ by estrogen
• Primary deficiency (‫ )نقص البروتين‬results in symptoms resemble hereditary
hemochromatosis (inability to incorporate Fe into transferrin) (↑ Fe in tissue, ↓
Fe in serum, Normal Cu level)
• ↓ Cp secondary to lack of incorporation of Cu into ApoCp is more common than
primary deficiency –due to
1. Dietary Cu insufficiency is associated with→ neutropenia,
thrombocytopenia, ↓serum Fe, hypochromic, hypo-or normocytic anemia
2. Menkes disease (inability to transport Cu from GI epithelium to circulation)
3. Wilson’s disease (defective incorporation of Cu to developing Cp) total
body Cu ↑↑ → leads to a deposition in liver, brain, and iris
4. Genetic deficiency 2. ATP7A deficiency, copper-transporting ATPase 1 (Menkes ATPase)
 Chromosome; 13q14.3

Copper-transporting ATPase 2 (ATP7B)

By bile (90% gallbladder into intestine-faeces)

10% urine

Function: Anti-oxidant/ anti-free radical

Free radicals cause

 Cu can’t bind with Apoceruloplasmin,
unused ApoCp protein is then degraded

Apo

* Neural
cell death

Cu causes destruction of hepatocytes via forming of free radicals

In Wilson’s disease, a high blood Cu level causes

Copper deposits in the cornea

https://www.youtube.com/watch?v=Cr8R_bnKAtk
Treatment of Wilson disease aims to remove tissue copper through chelation
with agents such as penicillamine or trientene and inhibition of dietary
copper uptake with supplemental zinc
Which one of the following statements regarding laboratory diagnoses of Wilson's
disease is TRUE?

1. Serum ceruloplasmin level is low <20 mg/dL

2. Hepatic copper content is high > or =250 µg/g dry weight. (Normal < 50 µg/g)
3. A basal 24-hour urinary copper of more than 40 µg (>0.6 µmoles or >600 nmoles)
4. Total serum copper (which includes copper incorporated in ceruloplasmin) in WD is usually
decreased in proportion to the decreased ceruloplasmin in the circulation but serum non-
ceruloplasmin-bound copper concentration is elevated above 25 µg/dL in most
5. All of the above

Explanation :WD should be considered in any individual between the ages of 3 and 55 years with liver
abnormalities of uncertain cause. Age alone should not be the basis for eliminating a diagnosis of WD.
Diagnosis of WD is confirmed, if 2 of the following 3 are present:
• Serum ceruloplasmin level <20 mg/dL
• Presence of Kayser-Fleischer ring
• Hepatic copper content > or =250 µg/g dry weight. (Normal < 50 µg/g).

A basal 24-hour urinary copper of more than 40 µg (>0.6 µmoles or >600 nmoles) suggests WD.
24-hour urinary copper with Penicillamine challenge increases the sensitivity of the test
Total serum copper (which includes copper incorporated in ceruloplasmin) in WD is usually decreased in
proportion to the decreased ceruloplasmin in the circulation but serum nonceruloplasmin-bound copper
concentration is elevated above 25 µg/dL in most untreated patients (normal <15 µg/dL).
Which one of the following statements regarding treatment of Wilson Disease is false?

A. Adequacy of treatment can be monitored by ensuring nonceruloplasmin-bound

copper concentration in normal range, and 24-hour urinary copper repeatedly in the
range of 200 to 500 µg (3 to 8 µmoles) per day on treatment
B. Treatment is lifelong and should not be discontinued and should continue even if liver
transplant has been performed
C. Zinc is currently reserved for maintenance treatment after chelator has been used for
1-5 years but can be used as the first line for selected presymtomatic patients
D. Penicillamine or trientine promotes copper excretion by the kidneys
E. Penicillamine use is associated with numerous side effects i.e. sensitivity reactions,
nephrotoxicity, lupus like syndrome, bone marrow toxicity and hepatotoxicity

Explanation: Treatment is lifelong and should not be discontinued, unless a liver transplant has been performed.
Treatment should be initiated with a chelating agent in both symptomatic and asymptomatic or presymptomatic
patients identified through family screening. Zinc may be used as the first agent in presymptomatic patients. After
adequate treatment with a chelator (usually for 1-5 years), stable patients may be transitioned to treatment with
zinc. They will be clinically well, with normal serum aminotransferases and hepatic synthetic function,
nonceruloplasmin-bound copper concentration in normal range, and 24-hour urinary copper repeatedly in the
range of 200 to 500 µg (3 to 8 µmoles) per day on treatment
The advantages of long-term treatment with zinc include that it is more selective for removing copper than
penicillamine or trientine and is associated with few side effects.
Adequate studies regarding the timing of this change-over in treatment are not available. No matter how well a
patient appears, treatment should never be terminated indefinitely. Patients who discontinue treatment altogether
risk development of intractable hepatic decompensation.
 C-reactive protein (CRP) 23 kDa
• CRP is a substance found in the sera of acutely ill persons
that is able to bind the cell wall C-polysaccharide of
Streptococcus pneumoniae
• Is made by the liver and is an early positive APR (↑↑↑ most
dramatic increases in concentration in inflammatory
diseases)
• It has been used extensively as a marker of inflammation
• Can trigger the complement cascade to initiate
opsonization, phagocytosis and lysis of invading
organisms
• High concentrations after myocardial infarction, trauma,
infection, inflammation, surgery or neoplastic proliferation
CRP binds a variety of compounds in the presence of
Ca+2:
1) polysaccharides present in many bacteria, fungi, and
protozoans;
2) phosphorylcholine
3) phosphatidylcholines, such as lecithin
4) polyanions, such as nucleic acids
• CRP begins to rise within 6 to 12 hrs of the onset of the
stimuli, peaks within 48 hrs
• CRP concentration may increase more than 1000-fold
from a low initial baseline. Bacterial infection is a
stronger stimulus than viral infection
 Haptoglobin (Hp)
• Synthesized in the liver. Compose of four peptide chains
linked by disulfide bridges
• Is α2-glycoprotein that irreversibly combines with free plasma
hemoglobin (Hb dimers), preventing loss of iron through the
kidneys and protecting the kidneys from damage by Hb, while
making the hemoglobin accessible to degradative enzymes
(reticuloendothelial system clear Hp-Hb complex, macrophage CD163 receptor)
• Prevents iron-requiring bacteria (E.coli) to have access to Hb
iron, so it has a bacteriostatic effect
• Although homologous to serine proteases, it has lost all
essential catalytic residues and has no enzymatic activity
• Haptoglobin depletion is the most sensitive laboratory
indicator of hemolysis (especially intravascular)
• Binding of Hb by Hp prevents renal clearance of Hb and loss of iron
• It is a weak and late reacting APP
 Haptoglobin-Hemoglobin interaction
Hemopexin binds with the Heme part of Hb
hemoglobin

Haptoglobin dimerization

Biliary obstruction in the absence of severe hepatocellular

disease is associate with significantly increased Hp concentrations
hemoglobin
And most forms of liver disease are associated with decreased
amounts of Hp
 Fibrinogen (plasma level 1.5-3.0 g/L, depending on the method used)
• Fibrinogen (340 kDa), the terminal component of the coagulation
system
• It is a soluble fibrous protein that is produced by the liver and
found in blood plasma
• It aggregates to form a fibrous network (fibrin) when it is cleaved
by the protease thrombin (removing of small fibrinopeptides)
• Maintaining adequate concentrations of fibrinogen is important for
preventing bleeding
• Fibrinogen is a positive APP
• Low plasma concentrations occur as the result of extensive
bleeding or dysregulation of the coagulation system, such as
disseminate intravascular coagulation
• When tissue damage results in bleeding, fibrinogen is converted at
the wound into fibrin by the action of thrombin, a clotting enzyme
 Transferrin (TRF/Tf)
• Principal plasma transport protein for iron
• Plasma concentrations are regulated by availability of iron
• MW ~80 kDa and contains two specific high-affinity Fe (III)
binding sites
H+
• One molecule binds two ferric ions that physiologically
generated by oxidation of ferrous ion by Cp Fe++

• Transferrin flows through the blood until it finds a transferrin

receptor on the surface of a cell, forming vesicle
• The cell acidifies the inside of vesicle, which causes transferrin
to release its iron
• Then, the receptor and empty transferrin are recycled back to the
outside of the cell. Triggered by the neutral pH of the blood, the
receptor releases the empty transferrin, and it continues its job of
gathering iron
 Transferrin (TRF/Tf)
• Plasma concentrations are regulated primarily by availability of iron,
with TRF plasma concentrations rises with iron deficiency and falls with
adequacy of iron
• As with albumin, about one-half of the TRF exists outside the vascular
compartment in extracellular fluids

Iron metabolism
 Transferrin (TRF/Tf)
• Readily used for the differential diagnosis of anemia and
monitoring of treatment of iron deficiency anemia
• In case of Iron deficiency: ‫ ماذا يحصل لتركيز‬TNF
• TRF concentration is increased but the protein has low
saturated with iron
• In anemia of chronic disease instead of ‫ غير‬deficiency of iron,
TRF concentration may be normal or low, but the protein is
highly saturated with iron ‫يدل أن الخلل مش من نقص الحديد‬
• Failure to incorporate iron in erythrocytes: Normal concentration
(or low) but highly saturated
 Transferrin

Two domains.
Iron ions are
coordinated by
2 Tyr, 1 His, 1
Asp and an anion
(carbonate ion)

Siderophilin - a globulin in blood plasma that

carries iron. Beta globulin, transferrin
• In iron-overload states, such as hereditary hemochromatosis,
TRF concentration is normal, but iron saturation often exceeds
55%—considerably above reference values
• High concentrations of TRF are present in pregnancy and
during estrogen administration
• TRF is a negative APP, an low concentrations occur in
inflammation or malignancy
• Decreased synthesis occurs with chronic liver disease or
protein malnutrition, protein loss such as that seen in the
nephrotic syndrome or in protein-losing enteropathy, also
causes low concentrations
HH: is an autosomal recessive (genetic) disorder characterized by excessive intestinal
absorption of dietary iron, causing severe liver disease and other health problems
 Transferrin with 2 bound iron ions

Transferrin receptor

• TRF-Fe3+ complex transports iron to cells

for incorporation into cytochromes, Hb,
and myoglobin, and to storage sites, such
as the liver and the reticuloendothelial
system
• Every cell type has surface receptors for
TRF
• Ferritin is a protein that contains
iron and is the primary form of iron
stored inside of cells
• The small amount of ferritin that
is released and circulates in the
blood is a reflection of the total
amount of iron stored in the body
• Ferritin - iron-phosphorus-protein compound
formed when iron complexes with the protein
apoferritin
• It is a storage form of iron found primarily in the
bone marrow, spleen, and liver
(reticuloendothelial system)
• Small amounts can be found in the peripheral
blood proportional to that found in the bone
marrow.
• Can be used as a test for hereditary
hemochromatosis
• Apoferritin and hemosiderin bind with ferrous
iron and store it as a ferric state
• Hereditary haemochromatosis type 1 (HFE-related Hemochromatosis) is
a genetic disorder characterized by excessive intestinal absorption of dietary
iron, resulting in a pathological increase in total body iron stores

• Excess iron accumulates in tissues and organs, disrupting their normal function.
The most susceptible organs include the liver, heart, pancreas,

• the defect in the HFE gene, where a mutation puts the intestinal absorption of iron into
overdrive.
• Normally, HFE facilitates the binding of transferrin, which is iron's carrier protein in the
blood
• Transferrin levels are typically elevated at times of iron depletion
(low ferritin stimulates the release of transferrin from the liver)
• When transferrin is high, HFE works to increase the intestinal release of iron into the
blood
• When HFE is mutated, the intestines perpetually interpret a strong transferrin signal as
if the body were deficient in iron
• This leads to maximal iron absorption from ingested foods and iron overload in the
tissues
 Gamma band B
Fc
• IgG is the most common type of plasma immunoglobulins presents,
makes up 70% – 75% of total Ig

• Of this amount, 65% is extravascular and 35% is found in plasma

• Normal range of IgG 0.7 – 1.6 g/dL (7 – 16 g/L)

• IgG1 and IgG3 bind Fc receptors on phagocytic cells and cross the
placenta via receptor-mediated active transport

• IgG1 is the major neonatal Ig, with concentrations similar to maternal

concentrations

• Transport of maternal IgG into the fetal circulation causes lysis of fetal
RBCs (hemolytic disease of the newborn) react at 37ºC extravascular

• When the mother generates antibodies to fetal RBCs antigens (Rh+ve)

DCE
This specific transport of IgG is carried out by the neonatal Fc receptor
(FcRn) expressed on syncytiotrophoblast cells
• Immunoglobulin M (IgM) is produced at early stages of
B-cell development
• In the immature immune system of neonates, IgM is the
major immunoglobulin synthesized
• In adults, it accounts for only 5% – 10% of total
circulating Ig
• IgM as a membrane receptor molecule is monomeric, but
most of the serum IgM is a pentamer
• The high molecular weight of IgM (970 kDa) prevents its
ready passage into extravascular spaces
• IgM is not transported across the placenta and, therefore,
is not involved in hemolytic disease of neonates

Anti-A, Anti-B antibodies in serum of blood group type B and A, respectively are IgM
 IgA
• 10% to 15% of serum immunoglobulin is IgA, which contains 10%
carbohydrate an has a molecular weight of 160 kDa and a half-life of 6
days

• About 10% of IgA in serum is dimeric (IgA2) which is resistant to bacterial

destruction

• Secretory IgA is found in

• Tears
• Sweat
• Saliva
• Milk and colostrum
• gastrointestinal secretions
• bronchial secretions
• It is synthesized by plasma cells in the mucous membranes of
• the gut, bronchi, and in the ductules of the lactating breast
• Secretory IgA is more abundant than IgG in colostrum ‫ اللَّبأ‬and milk and
may help protect neonates from intestinal infection
• IgA also activates complement by the alternative pathway
NK cells activation: Antibodies bound to antigens on
virus-infected cells of the body are recognized by the
 Multiple myeloma Ag B cell

• Multiple myeloma: A cancer in which antibody-producing plasma cells

grow in an uncontrolled and malignant manner

• Paraprotein: A monoclonal immunoglobulin produced in excessive

amounts in disorders such as multiple myeloma, appear as sharp bands
in the γ- or β-region

• A single clone of plasma cells produces Ig molecules with a single

amino acid sequence. If the clone expands greatly, the concentration of
its monoclonal Ig in the patient’s serum may produce a discrete band on
electrophoresis, called M-protein

• Analysis of urine is helpful mainly in identifying patients with monoclonal

urinary light chains (Bence-Jones proteins)

• Immunoglobulin A (IgA) light chain amyloid A protein (AL amyloidosis) is

seen in multiple myeloma
• Bence-Jones protein - excessive immunoglobulin light chain in the urine.
TP: total protein
Chapter 44

• Hemolytic disease of the newborn (HDN) A disease of the fetus and newborn
caused by maternal antibody–mediated fetal erythrocyte destruction
• Human chorionic gonadotropin (hCG): A placental glycoprotein hormone that
stimulates the ovary to produce progesterone
• Eclampsia: Convulsions and coma occurring in a pregnant woman or a woman
who recently gave birth
• Preeclampsia A disorder of widespread vascular endothelial malfunction and
vasospasm that occurs after 20 weeks gestation and can present as late as 4 to
6 weeks postpartum. It is clinically defined by hypertension and proteinuria, with
or without pathologic Edema
• Prolactin A pituitary hormone that stimulates and maintains the secretion of
milk
• Spina bifida A congenital disorder caused by the incomplete closure of the
embryonic neural tube
• Dietary supplementation with folic acid around the time of conception ‫ الحمل‬has
long been known to reduce the risk of neural tube defects (NTDs) in the
offspring
• With HDN, fetal erythrocytes are destroyed. The resulting
anemia stimulates the fetal marrow and extramedullary
erythropoiesis in the liver and spleen to replace
destroyed RBCs
• Extramedullary erythropoiesis destroys hepatocytes and
leads to decreased production of serum albumin and
decreased oncotic pressure in the intravascular space
• When severe, these changes lead to congestive heart
failure and generalized fetal edema, a condition referred
to as hydrops fetalis (/ erythrobalstosis fetalis)
• Without therapeutic intervention, intrauterine demise
soon follows
Rhnull lack Rh antigens (DCE)
• Inhibins (A and B) are members of the transforming growth factor-β
(TGFβ) superfamily of proteins
• They and their closely related activins are proteins that suppress or
stimulate FSH secretion, respectively
• In the reproductive system, inhibin and activin subunits are expressed
in the 1) placenta, 2) granulosa cells of the ovary, and 3) Sertoli cells
of the testis

• Inhibin
• is a predictor of Down syndrome risk
• Monitoring of ovarian cancer
• disorders of ovulation
• early detection of viable pregnancy following IVF
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4167436/
Placenta secrete several hormones include
hCG (Human chorionic gonadotropin)
Estradiol 3
Progesterone

Prenatal screening is the process of identifying pregnancies

at sufficiently high risk of a serious birth defect, such as Down
syndrome, to warrant invasive diagnostic testing
• Fetal Lung Maturity
• Respiratory distress syndrome (RDS)
• The risk of RDS is inversely related to gestational age at the time of
birth
• Affected infants require supplemental oxygen and mechanical
ventilation to remain properly oxygenated
• The disorder is caused by a deficiency of pulmonary surfactant
• In healthy lungs, surfactant coats the alveolar epithelium and responds
to alveolar volume changes by reducing surface tension in the alveolar
wall during expiration
• When the quantity of surfactant is deficient, many of the alveoli collapse
on expiration and thereby overinflate the remaining airways
• The lungs become progressively noncompliant (rigid), and blood flowing
through the capillary beds of collapsed alveoli fails to oxygenate
Chapter 45: Newborn Screening and
Inborn Errors of Metabolism
PKU

Phenylketonuria (PKU) A disorder characterized by the

accumulation of phenylalanine in blood that results from the
absence of phenylalanine hydroxylase activity leading to
production of phenylketones that are excreted in urine

Phenylalanine does not present normally in urine

Aminoacidopathy Any one of group of inborn errors of
amino acid metabolism caused by
1) defective activity of an enzyme in the metabolic pathway
of one or more amino acids, or
2) a defect in a protein needed for transport of an amino
acid into or out of cells; usually detected by the presence
of increased concentrations of one or more amino acids
in blood or urine or both

Multiplex analysis: simultaneous assessment of multiple analytes

in a single sample
Inborn error of metabolism (IEM) Inherited disorder due to
deficiency of an enzyme or transporter impairing the
transformation of body chemicals
• Inborn errors of metabolism (IEM) are genetically determined
biochemical disorders affecting an individual’s ability to convert
nutrients or to use them for energy production

• They are caused by the impaired function of

• 1) enzymes
• 2) transporters, or
• 3) cofactors and result in accumulation of abnormal metabolites
(substrates) proximal to the metabolic block or by lack of necessary
products
• Aminoacidopathies in which the parent amino acid accumulates in
excess in blood and spills over into urine

• An example of an aminoacidopathy is phenylketonuria (PKU), a

disorder of phenylalanine metabolism caused in the majority of cases
by deficiency of phenylalanine hydroxylase (gene), the enzyme
responsible for the conversion of phenylalanine to tyrosine

• The greatly increased concentration of phenylalanine impairs brain

development and function

• Other examples include 1) maple syrup urine disease (MSUD), 2)

homocystinuria, and 3) tyrosinemia.
(MSUD) is autosomal recessive metabolic disorder characterized by deficiency of an
enzyme complex (branched-chain alpha-keto acid dehydrogenase)
People with this condition cannot break down the amino acids leucine, isoleucine, and valine. This
leads to a build-up of these chemicals in the blood
Homocystinuria is an inherited disorder of the metabolism of the amino acid methionine due to a deficiency
of cystathionine beta synthase or methionine synthase
Untreated PKU patients develop
1) microcephaly,
2) eczematous skin rash
3) “mousy” odor (due to accumulation of phenylacetate)
4) Severe mental retardation

The treatment of PKU includes a diet

1) low in protein and phenylalanine
2) Supplemented with tyrosine, minerals, vitamins, and other nutrients to
sustain normal growth
• The treatment should be continued for life
• Newborn screening leads to early detection and therapy of patients with PKU
with prevention of mental retardation.
• Ideally, treatment should start before 2 weeks of age.
 Amino acid catabolism

• The first step in the breakdown of amino acids is the

removal of the amino group, through a reaction known
as transamination
• Transamination is an exchange of functional groups
between any amino acid (except lysine, proline, and
threonine) and an α-keto acid
• Transamination reactions are catalyzed by specific
transaminases (also called aminotransferases)

• Requires Vit B6 as coenzyme

Hyperphenylalaninaemia (HPA)

Blood phenylalanine concentrations, PAH deficiency can be

classified into classic PKU (Phe >1200 μmol/L), mild PKU
(Phe = 600 -1200 μmol/L) and mild HPA, where blood Phe is
elevated above upper reference limit, but <600 μmol/L
The end