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Proteins

in
Serum &
Urine
Proteins in Serum & Urine
Lecture outline
• About proteins
• Protein functions
• Proteins in plasma
• Protein metabolism
• Protein excretion
• Measure plasma proteins?
• Albumin
• Other proteins
• Acute phase proteins
• Proteins in the immune system
• Proteinuria
Proteins
 Complex, high molecular weight
measured in kiloDaltons
 Very long chains of amino acids joined
by peptide bonds
 Peptide – short chain of amino acids
 Proteins fold into unique 3-dimensional
structures determined by sequences of
amino acids
Proteins
 An amino acid:
NH2-CH(R)-COOH

 When amino acids are connected together to


make a polypeptide molecule, OH is removed
from the COOH group of one amino acid and H
from the NH2 group of the other to give:
H2O + NH2-CH(R)-CONH-CH(R)-COOH
Proteins
NH2-CH(R)-COOH + NH2-CH(R)-COOH

Condensation reaction ↓
H2O + NH2-CH(R)-CONH-CH(R)-COOH

 Join more amino acids together:

NH2-CH(R)-CONH-CH(R)-CONH-CH(R)-COOH
Proteins
 Primary structure: the amino acid sequence
 Secondary structure: highly patterned sub-structures
(alpha helix and beta sheet) or segments of chain that
assume no stable shape; formed by hydrogen bonding
 Tertiary structure: overall shape of a single protein
molecule; the spatial relationship of the secondary
structural motifs to one another; primarily formed by
hydrophobic interactions but hydrogen bonds, ionic
interactions, and disulfide bonds are usually involved too
 Quaternary structure: the shape or structure that results
from the union of more than one protein subunit, which
function as part of the larger assembly or protein
complex
Proteins
 Dietary protein ≠ proteins in cells and body
fluids
 Adequate dietary protein intake vital to provide
all necessary amino acids
 Dietary protein digestion ~~ pepsin ~~ in
stomach
 Proteolytic enzymes – protein-splitting enzymes
 Protein →→→→ small peptides →→→→ amino
acids ⇒ absorbed by musocal cells of small
intestines ⇒⇒ transported to liver
Proteins
 Thousands of proteins
 Plasma contains over 100 proteins
 Great diversity of physicochemical
characteristics and physiological roles
 Most have a role to play
 Some merely represent cellular or tissue
proteins shed into the circulation as a result
of degradative processes
 Some present in large amount but the
function is not known
Proteins - General Functions
 Transport ~ hemoglobin: O2 carrier in RBC;
myoglobin: O2 carrier in muscle cells
 Storage ~ ferritin stores iron in liver
 Movement ~ muscle contraction
 Structure ~ collagen; elastin; keratin
 Control of gene expression
 Growth substances ~ promote growth & regeneration
 Immune mechanisms ~ antibodies
 Clotting mechanisms ~ fibrinogen; thrombin
 Components of cell membranes
~ receptors on cell surfaces
 Hormones ~ insulin; growth hormone
 Enzymes
Proteins in plasma

 A mix of proteins differing in


origins and functions
 Amount of protein in vascular
compartment depends on:
 Balance between rate of
synthesis and rate of
catabolism
 Loss and relative distribution
between intra- and
extravascular (body tissues)
compartments
 Concentration depends on
relative amounts of protein and
water in vascular compartment
Proteins in plasma

 Albumin – 60%
Contribute to osmotic pressure
Help control fluid balance
Transport of substances, including
drugs
 Globulins – 35%
Antibodies
Transport
 Clotting proteins – 4%
 Regulatory proteins, e.g. enzymes – 1%
Proteins in plasma
Main functions
 Inflammatory response and infection control
Immunoglobulins + complement proteins
→ immune system
Acute-phase reactants → inflammatory
response
 Transport
Albumin and specific-binding proteins
 Control of extracellular fluid distribution
Water distribution between intra- and
extravascular compartments ← colloid
osmotic effect of albumin
Proteins - Metabolism
 Synthesis
 For most plasma proteins – in the liver
 Hepatocytes
 Proteins of the complement system – in
hepatocytes and macrophages
 Immunoglobulins – by B cells

 Distribution
 Pass continuously from vascular to the
extravascular space
Proteins - Metabolism
 Catabolism
 In most cells of the body
 The amino acids are used by tissues
 If contain CHO, then probably in the liver
 Some broken down in macrophages
 Low molecular weight proteins and protein
fragments produced by proteolysis are filtered
through the renal glomerulus and reabsorbed
and catabolized in the proximal tubules: 2 - 4
g/daily
Proteins - Excretion
 Most proteins are not excreted in appreciable
amount in urine
 Renal glomerulus filter cutoff ≅ molecular weight
= 60,000
 Compounds > 60,000 mwt are not filtered and
excreted
 Compounds < 60,000 mwt pass through
glomerulus into urine
 Detection of protein in urine can be of great
clinical significance
Why measure plasma proteins?
 Many diseases are associated with changes in the
 Concentration,

 Structure,

 Function of plasma proteins

 Sometimes these changes are the result of the


pathological processes of the disease
 But in some cases such as genetic deficiencies
they may be the cause
 At present their laboratory measurement only
provide clinically useful information in a limited
number of circumstances
Albumin
 Single polypeptide chain
 Molecular weight = 65,000
 Normal half life of circulating albumin in plasma
= 19 days
 Synthesized in the liver where it amounts to 30%
of hepatic protein synthesis
 Synthesis is sensitive to amino acid supply and
thus to nutritional status
 In protein depletion with adequate energy supply
– synthesis decreases by 60 %
Albumin

 Comprises of more than half of the total protein of


plasma (60%)
 Albumin acts as a binding protein for a wide range of
substances in plasma forming a reservoir to buffer
rapid changes in the free moeity
 Normal range of albumin concentration in human
blood: 3.5 - 5.0 g/dL
 Concentration of albumin in smaller intravascular
compartment is much higher because of the relative
impermeability of the blood vessel wall
 This concentration gradient across the capillary
membrane is important in maintaining plasma volume
Albumin changes
 Hyperalbuminaemia
 Dehydration

 Hypoalbuminaemia
 Expanded extravascular pool
 Protein loss
 Decreased synthesis
 Increased endogenous catabolism

 Takes 10 -14 days for albumin level to fall below


reference range due to relatively long half life
 Absolute rate of catabolism falls with decreasing
concentration
Albumin changes

 Consequence of hypoalbuminaemia
 Disruption of fluid distribution → oedema
 Disruption of binding functions
 Plasma albumin ↓↓ ⇒
 Binding capacity ↓↓ ⇒
 ↑↑↑ plasma free concentration of substances
 Toxicity
Albumin changes in
malnutrition
 Albumin synthesis is sensitive to dietary supply
of amino acids
 Inadequate dietary intake can cause
hypoalbuminaemia
 But 24 weeks of depletion of proteins and
energy sufficient to cause only 7% fall in plasma
albumin
 Plasma albumin level is not a very reliable
indicator of malnutrition
Albumin changes in disease

 Liver disease
 Albumin is frequently low
 Does not reflect hepatic cell mass as only 15 -
20% of hepatocellular mass is necessary to
synthesize normal amount of albumin
 In chronic liver disease fluid retention results
in expansion of extravascular pool
Albumin changes in disease
 Inflammation
 Any form of tissue damage, trauma, surgery, or
infection →→ increase in plasma acute phase
proteins and a fall in plasma albumin, transferin,
prealbumin and retinol binding proteins
 Plasma albumin falls 20% over 2 - 3 days
 Albumin synthesis falls by as much as 70% -
probably due to diversion of hepatic system to
making acute phase proteins rather than proteins
that are not involved in the inflammatory
response
Albumin changes in disease
 Nephrotic syndrome
 Glomerular damage may result in increased
permeability of the glomerular basement
membrane to plasma proteins
 May lead to very low concentration of albumin

 Gastrointestinal protein loss


 Gastrointestinal protein loss
 Normally 2-15 % protein degradation
 In many conditions may increase to 35% and
may result in hypoalbuminaemia
Albumin changes in disease
 Burns
 Fall in plasma albumin due to
 loss from superficial tissues

 decreased synthesis associated with acute


phase reaction

 Shifts in distribution from the intravascular to


extravascular compartment
Other proteins
 Prealbumin and retinol binding proteins
 Circulate in the plasma as a complex

 Retinol binding protein carries retinol and the

complex is stabilized by prealbumin


 Show decrease in plasma concentration in

association with both acute phase response and


decreased amino acid supply to the liver
 Retinol binding proteins is perhaps the most

sensitive indicator of nutritional deficiency


 Significant fall shown 24 hours after deprivation of

protein and energy


Other proteins
 Transferrin
 Iron transport protein

 Free iron is toxic – all iron is protein-bound:

transferrin, ferritin, haemosiderin, hemoglobin


 Considerable interchange between stored and

plasma iron
 Only 0.1% of total body iron circulates in

plasma: Fe3+ bound to transferrin


Transferrin
 Plasma iron estimation is rarely of clinical value
 Poor index of total body iron content
 Iron in plasma is of a very small proportion of total,
and is only a protein-bound transport fraction
 Plasma iron concentrations can alter rapidly –
representing shifts between plasma and stores,
NOT change in total body iron
Transferrin
 Plasma transferrin concentration rises:
 > 28th week of pregnancy
Physiologic
 In women taking certain oral contraceptives
 In patients treated with estrogens al
 Pathological changes in plasma transferrin
concentration:
 ↑↑ in iron deficiency
 ↓↓ in iron overload
 ↓↓ in chronic diseases associated with low plasma
iron concentrations
 ↓↓↓↓ in nephrotic syndrome ⇒ low plasma iron
concentration because low mwt transferrin + iron
lost in urine
 Transferrin concentration unchanged in acute illness
Other proteins
 Caeruloplasmin
 Cu transport

 Some plasma copper loosely bound to albumin

but most incorporated into caeruloplasmin


 Copper excretion → bile

 Defect in copper metabolism = Wilson’s

Disease
Wilson’s
Disease
Caeruloplasmin
 Wilson’s disease
 2 defects in copper metabolism
 Impaired biliary excretion leads to deposition in the
liver
 Deficiency of caeruloplasmin results in low plasma
copper concentrations; loose copper deposited in
tissues, filtered through glomeruli ⇒ urinary copper
excretion ↑↑
 Diagnosis:
 Most patients have low plasma caeruloplasmin and
copper concentration; high urinary copper excretion
 Treatment:
 D-penicillamine is a copper-chelating agent → reduce
tissue copper concentrations
Caeruloplasmin
 Low plasma Caeruloplasmin:
 May occur in the first few months of life due to
malnutrition
 In nephrotic syndrome due to urinary loss

 Raised plasma concentration:


 Found in active liver disease

 In women taking oral contraceptives during the last


trimester of pregnancy
 Non-specifically – when there is tissue damage due
to inflammation or neoplasia
 Non-specific ↑plasma caeruloplasmin may account for
the rare finding of ‘normal’ caeruloplasmin concentration
with Wilson’s disease
Other proteins
 Acute Phase Proteins: react to inflammation
 Inflammation results from any form of tissue damage

 Inflammation

Fever Leucocytosis Acute Phase Reaction


↑Antibody reaction ↑# of phagocytic cells ↑Synthesis
↑Plasma / tissue levels
of acute phase proteins
Acute phase proteins

 Any protein whereby plasma concentration


increases/decreases during inflammation
 Measured as a means of diagnosing and monitoring
a variety of inflammatory diseases
 Individual proteins may also be measured because
changes in their structure and concentration may
relate to other disease process
 Acute phase protein = acute phase reactant
Acute phase proteins
 Produced by liver in response to inflammation
 React as:
 Activators of other inflammatory pathways
 Inhibitors which control inflammatory response to
minimize damage to host tissue
 Scavengers
 C-reactive protein (CRP)
 Serum amyloid A (SAA)
 Fibrinogen
 Alpha 1-acid glycoprotein
C-Reactive Proteins (CRP)
 The proteins react with the C-polysaccahrides of
pneumoccocci ⇒ thus its name
 CRP react with polysaccharides released by damaged
tissues to become an activator of the complement
pathway
 Plasma concentration of CRP rises rapidly in response
to acute inflammation – as high as a thousand-fold
 Assay is particularly useful in the early detection of
acute infection
 Plasma CRP are not usually increased in viral
infections
α1-antitrypsin
 Major component of α 1 globulins
 Produced in liver
 Irritants or inflammation may release proteases
e.g. collagenases
 α 1-antitrypsin controls proteolytic action of
lysosomal enzymes – combine with and
inactivate trypsin or collagenases released from
leukocytes responding to inflammation
 Plasma α 1-antitrypsin ↑↑ 2 - 3 days after trauma
or acute infection
α1-antitrypsin

 An important factor in the homeostatic mechanism


modulating endogenous proteolysis within the
body which control the inflammatory response and
minimize damage to host tissues
 α1-antitrypsin Deficiency = A1AD
 Hereditary autosomal co-dominant
 Lack of α1-antitrypsin →→ uninhibited tissue
breakdown during inflammation
 Causes pulmonary emphysema in adults and
cirrhosis in children and young adults
α1-Antitrypsin
Deficiency
α1 Microglobulin
 One of the largest non-immunological proteins in
plasma (728 KD)
 Concentration comparable to α1-antitrypsin
 Concentration is up 10X in cases of nephrotic
syndrome when smaller proteins are lost
 May also be increased synthesis in nephrotic
syndrome
 Inactivates a wide range of proteases by complexing
with them and forming covalent bonds
 Complex removed from circulation by
reticuloendothelial system
 No known deficiency state reported
Haptoglobin
 Scavenger protein
 Produced by liver, secreted into blood
 Binds hemoglobin released by local in-vivo hemolysis
during inflammatory response, i.e. ‘free’ hemoglobin
 Concentration of ‘free’ hemoglobin usually low, but
level rises when RBC destroyed
 Haptoglobin + hemoglobin complex removed from
circulation by reticuloendothelial system
 In liver ⇒ components are recycled
 Haptoglobin is destroyed
 Hemoglobin is broken down to globin and heme and
then further to iron and bilirubin
Haptoglobin
 Haptoglobin rises in response to stress, infection,
tissue necrosis, acute inflammation by stimulation of
synthesis
 Levels fall as complex cleared from system
 Active destruction of RBC ⇒⇒ rate of haptoglobin
destruction by liver > rate of production ⇒⇒
haptoglobin level in blood ↓↓

 Migrates in the α2 region


Fibrinogen
 Plasma concentration increases during acute
phase reaction
 Most abundant of the coagulation factors
 Forms the fibrin clot
 Increases also with pregnancy, use of birth
control pill

 Absent from the normal serum but with plasma


migrates between the β and γ globulins
Proteins of immune system
 Immunoglobulins
 Synthesized by B lymphocytes

 Incorporated into cell membranes → antigen

receptors
 Upon exposure to specific antigens in the presence

of T-helper cells ⇒⇒ proliferation of B lymphocytes


 Differentiation of B lymphocytes ⇒ plasma cells

secreting immunoglobulins
Immunoglobulins
 IgG
 IgM
 IgA
 IgE
 IgD
Immunoglobulins

 In response to infection, each plasma cell


produces an immunoglobulin of a single class
 Chronic infections activate B lymphocytes and
cause polyclonal immunoglobulin response
 Also respond to allergy ⇒ plasma IgE ↑↑
Plasma immunoglobulins

IgG Bacterial infection


Raised in: autoimmune diseases, chronic active hepatitis, AIDS
IgM First line of defence; can be synthesized by fetus
Raised in: primary biliary cirrhosis, hemoprotozoans (malaria),
viral hepatitis, acute viral infections, intrauterine infection (if
detected at birth), AIDS
IgA Infections of GIT and respiratory tract; secretory
Raised in: Crohn’s disease, tuberculosis, bronchiectasis, AIDS
IgE Allergic response
Raised in: Type I hypersensitivity, allergies secondary to
immunodeficiencies, parasitic infections
Immunoglobulin deficiencies
 Reduction in plasma immunoglobulin concentrations ≡
detected as hypogammaglobulinaemia
 Diagnosis made only after measurement of individual
protein
 Deficiency of individual immunoglobulin ⇒ affects
specific function and distribution

 IgM deficiency – predisposes to septicaemia


 IgG deficiency – recurrent pyogenic infections of tissues
(lung, skin) by toxin-producing organisms, e.g.
staphylocci, streptococci
 IgE – symptomless or recurrent, mild respiratory tract
infections or GIT diseases
Transient immunoglobulin
deficiency
 Newborn infant derives IgG from mother ~ placenta
transfer over last 3 months of gestation
 Infant’s plasma IgG ↓ within first 3 – 6 months
 Infant’s plasma IgG then gradually ↑ ⇐ endogenous
IgG synthesis
 If onset of endogenous IgG synthesis delayed, infant
will have physiological hypogammaglobulinaemia
 May persist for several months

 Severe IgG deficiency →→ premature infants


Primary immunoglobulin deficiency
 Manifest within 6 months and 2 years of life – loss of maternal
antibodies

 Primary IgA deficiency


 Relatively common
 Incidence 1:500
 Familial or acquired
 Mostly symptom-free

 Bruton’s disease (infantile sex-linked agammaglobulinaemia)


 Only in males
 Almost complete absence of mature B cells and circulating
Ig
 Present with recurring bacterial infections at 8 - 9 mths
 Cellular immunity: T-cells normal ⇒ able to cope with viral
and fungal infections
Secondary immunoglobulin deficiency

 Low plasma Ig concentrations


 Malignant diseases – precipitated by chemo- /
radiotherapy
 Myelomatosis
 Nephrotic syndromes → loss of low mwt Ig
 Severe protein-losing states → increased
catabolism
Proteinuria
 Protein in urine
 Due to glomerular or tubular disease
 Massive proteinuria ⇒ almost always of glomerular
origin
 Can occur even when renal function normal – if ↑↑↑
↑ production of low mwt protein
 Usually asymptomatic ∴ detected by routine urine
test
 Protein ↑↑↑↑ in urine: nephrotic syndrome
 Water buildup in body → severe swelling: ankles,
fingers, around eyes; water around lungs →
breathlessness
Proteinuria
Nephrotic syndrome

 Characterised by proteinuria ≥ 5.0 g/day


 Low serum albumin
 Oedema
 Hyperlipidaemia
 Proteinuria is glomerular in type
Proteinuria causes
 Protein in the urine can be a marker of almost
any type of renal pathology → test to determine
cause of proteinuria
 Most common causes:
 Hypertension

 Infection

 Reflux nephropathy

 Diabetes

 Glomerulonephritis

 Minimal change nephritis


Proteinuria complications
 Most have minor renal disease, but some can
progress to renal failure
 Complications depend on the exact cause of
proteinuria in each case
 Commonly:
 Hypertension
 Increases risk of further renal disease, myocardial
infarct, stroke
 Controlled by drugs
 Elevated cholesterol levels
 In nephrotic syndrome
 Increased risk of myocardial infarct
Proteinuria treatment
 Underlying renal disease - drugs
 Water retention - reducing amount of
dietary salt and water, increase renal
production of urine
 Hypertension - reduce dietary salt, drugs
 Elevated cholesterol level - dietary control
(eating less fat) and/or cholesterol
lowering drugs
Protein Quantification
C-Reactive Proteins (CRP)
 Conditions that commonly lead to marked changes in CRP include infection,
trauma, surgery, burns, inflammatory conditions, and advanced cancer.
Moderate changes occur after strenuous exercise, heatstroke, and childbirth.
Small changes in CRP occur after psychological stress and in several psychiatric
illnesses.
 C-reactive protein is a test of value. Marked rises in CRP reflect the presence
and intensity of inflammation. An elevation in CRP, however, is not a telltale sign
pointing to just one disease.