Clinical Chem proteins and peptides

20,000

In humans, how many genes encode peptides and proteins?

metabolism and the diagnosis of pathological and inherited disorders

Measurement of amino acids in physiological fluids assists with studies of?

Maintaining nitrogen balance in mammals.

What role does the side chain in amino acids play?

20-21

Most proteins contain how many amino acids

Nephelometry

Measures the light reflected at an angle by the aggregates

Turbid specimens such as lipemic sera may interfere with measurements.

What is the one limitation of these direct optical methods?

converting ammonia to urea

What is the urea cycle used for?

Transferring nitrogen and energy sources from muscle to liver

What is the alanine (Cori) cycle used for?

Glutamine and glutamic acid

Where does ammonia generation in the kidney stem from?

electrophoresis

– chromatography

–genetic analysis

– functional assays (coupled with quantitative analysis to identify changes in specific activity)

– mass spectrometry

Qualitative protein analysis reveals changes in the structure of proteins through?

Turbidimetry and nephelometry

Techniques widely used to assess high abundance proteins due to their speed and ease

These techniques assess the formation of aggregates when a reagent is added to lower protein solubility or when an antibody is added to a protein

Turbidimetry

Measures changes in absorbance caused by the formation of aggregates.

Some variants have increased binding affinities for thyroxine (T₄), leading to higher total serum T₄ (bound + free) levels while individuals remain euthyroid with normal thyrotropin concentrations.

What is unique about some albumin variants regarding thyroxine (T₄)?

Bovine or Human albumin

Used routinely to calibrate biuret methods, which react nearly equivalently with the peptide content of most proteins.

0.8 g/kg body weight (demand is higher during growth, pregnancy, lactation, states of protein loss, and disease involving protein breakdown).

What is the daily requirement for protein intake in adults?

α-Amino acids

What type of amino acids are found in proteins?

Kwashiorkor

Protein deficiency malnutrition leads to decreased serum albumin, edema, ascites, growth failure, immune deficiency and apathy.

Amino acids and they play a role in metabolism and neurotransmission.

What are the basic structural units of peptides and proteins?

Marasmus

Deficiency of both calories and protein (protein-calorie malnutrition), causes muscle wasting but lacks edema.

Albumin

Protein that is available in high purity, highly soluble and stable, well characterized, and contains no carbohydrate.

6.4 to 8.3 g/dL and 6.0-7.8 g/dL at bed rest.

What is the total protein concentration of serum obtained from healthy ambulatory adults?

Serine

A major source of one-carbon units transferred by tetrapyrrolic acid for purine synthesis and conversion of homocysteine to methionine.

0.3 g/dL higher (unless diluted with an anticoagulant such as citrate solution)

What protein concentration does plasma usually contain?

zone electrophoresis.

Plasma proteins are classified according to their mobility using?

direct spectrophotometric detection

Applications of capillary electrophoresis rely on_____ of proteins at a wavelength of about 200 to 225 nm, rather than staining of a solid support.

urea cycle

The liver is the primary site for which cycle?

aminoaciduria

Increased urinary excretion of amino acids.

Nutritional status, liver function, physiological changes, and therapeutic changes in steroid hormone concentrations.

What factors selectively affect plasma protein concentrations?

What is the acute-phase response (APR)?

A systemic response to infection, tissue injury, or inflammation that leads to fever, increased white blood cell count, and altered plasma protein concentrations.

It causes significant changes in protein levels, which are important markers for disease diagnosis and progression.

How does APR impact clinical measurements of plasma proteins?

They are transport proteins.

What are prealbumin and retinol-binding protein (RBP)?

RBP (retinol binding protein) is bound to prealbumin, which was named for its electrophoretic mobility.

How is RBP related to prealbumin?

A Non glycosylated protein with a molecular mass of 55 kDa, composed of four identical subunits forming a hollow core.

What is the molecular mass of prealbumin, and what is its structure?

It binds and transports thyroid hormones (T3 and T4) and retinol (vitamin A) by binding to retinol-binding protein. It also serves as a marker for nutritional status.

What is the function of prealbumin?

When prealbumin binds one hormone molecule, its affinity for binding a second hormone decreases, so only one site is typically occupied.

What is negative cooperativity in prealbumin?

It is mainly synthesized in the liver and, to a lesser extent, in the choroid plexus of the CNS.

Where is prealbumin synthesized?

Due to its synthesis in the choroid plexus of the central nervous system.

Why is prealbumin found in high concentrations in CSF?

Glycine

Which amino acid is the most abundant in healthy adults

Albumin is a nonglycosylated protein composed of 585 amino acids with a molecular weight of 66,438 Da.

What is the structure and molecular weight of albumin?

It is the most abundant plasma protein, making up slightly more than half of the total plasma protein mass from midgestation until death

What is the significance of albumin in plasma?

2-3 days

How many days should an individual follow a normal diet before collection for diagnosis of an inherited disorder of amino acid metabolism.

Albumin is the major contributor to COP, which helps retain fluid within the vascular space.

What is albumin’s role in colloidal oncotic pressure (COP)?

To help maintain intravascular volume in cases of fluid imbalance.

Why is albumin sometimes administered as an infusion?

Fluid moves into extravascular spaces, increasing the risk of edema.

What happens when albumin levels decrease?

Cerebrospinal fluid (CSF), interstitial fluid, urine, and amniotic fluid.

What body fluids contain albumin as a major protein component?

About 60% is in the extravascular space, though its concentration is higher in plasma.

Where is most of the body's albumin found?

Due to its high abundance of charged amino acids, giving it a net charge of about -12 at neutral pH.

Why is albumin highly soluble in water?

At normal concentrations, it contributes about 6 to 10 mmol/L to the anion gap, and low albumin levels lead to a decreased anion gap.

How does albumin contribute to the anion gap?

In the hepatic parenchymal cells of the liver.

Where is albumin synthesized?

The synthetic rate can increase up to threefold above normal.

How does albumin synthesis respond to nephrotic syndrome (a kidney disorder characterized by the excessive loss of protein in the urine (proteinuria))?

Primarily by colloidal osmotic pressure (COP) and secondarily by protein intake.

What controls the synthesis of albumin?

Through pinocytosis in all tissues.

How is albumin catabolized?

The neonatal IgG receptor selectively recycles these proteins from pinocytosed fluids, extending their half-life by two- to fourfold.

Why do albumin and IgG have an extended half-life?

15 to 19 days, while most other plasma proteins have half-lives of 7 days or less.

What is the usual half-life of albumin?

Maintenance of colloidal oncotic pressure (COP) and binding/transport of:

Free fatty acids

1. Bilirubin

2. Calcium

3. Thyroid and steroid hormones

4. Drugs

5. Thiol-containing compounds

6. buffer to regulate pH levels.

What are the main functions of albumin?

oligopeptides

Chains of up to five residues

polypeptides

Longer chains (6 to 30 residues)

protein

When the number of amino acids linked together exceeds 40, the chain is referred to as a.

Random coil

refers to segments that lack the beta sheet and alpha helix structures in secondary structure.

Tertiary structure

refers to the folding of the chain of amino acids into a three-dimensional structure, which may be stabilized by disulfide bonds between cysteine residue

secondary structure

is the specific organization of close segments of the polypeptide backbone into structures that are termed (1) α-helix, (2), β-sheet and (3) β-turn

creatine kinase with two subunits, lactate dehydrogenase with four subunits, hemoglobin with four subunits

What are examples of Quaternary structure?

Domains

Many proteins are organized as chains of smaller structural units and diversity in structure is achieved by assembly of these.

More than 80. Many of which have altered electrophoretic migration, resulting in so-called bisalbuminemia

How many genetic variants of albumin have been reported?

A condition where heterozygotes exhibit two bands for albumin on electrophoresis due to altered migration.

What is bisalbuminemia?

Bound drugs and metabolites.

What can affect the electrophoretic migration of albumin?

SDS-PAGE

high-resolution technique for separating proteins and estimating their molecular weight

Dehydration, prolonged tourniquet time, or specimen evaporation before analysis.

Under what conditions are increased concentrations of albumin observed?

They suggest issues with patient hydration or specimen handling.

What do high albumin concentrations indicate?

When parenchymal damage exceeds 50% loss of function

When does the liver lose its ability to maintain albumin concentration?

They include:

1. Nutritional deficiency

2. Increased distribution into the extravascular space

3. Direct inhibition of albumin synthesis by toxins such as alcohol

What additional mechanisms can lead to decreased albumin concentrations in liver disease?

It efficiently prevents proteins the size of albumin or larger from entering the urinary ultrafiltrate.

What is the role of the glomerular filtration barrier?

Only 1 to 2 g/d pass through, and 99.9% is taken up by the kidney's proximal tubules, where it is degraded.

How much albumin normally passes through the glomerular barrier daily?

About 10 mg/d of albumin.

What is the normal amount of albumin excreted in urine daily?

Microalbuminuria, defined as albumin excretion >30 mg/d, indicates early stages of glomerular or tubular injury and risk of progressing to more severe kidney disease.

What does microalbuminuria indicate, and at what level is it observed?

Inflammatory bowel disease, such as Crohn's disease and ulcerative colitis, is linked to increased gastrointestinal (GI) loss of albumin, which can be as significant as renal losses in the nephrotic syndrome.

What is the association between inflammatory bowel disease and albumin?

Albumin concentrations help detect and monitor protein nutritional status, though responses to dietary intake are slow due to albumin's long half-life.

How do albumin concentrations aid in monitoring protein nutritional status?

They cause severe albumin loss from wounds. Severely decreased albumin levels in massive burn injuries are due to epithelial losses, accelerated catabolism, and Acute Phase Reactants (APR).

How does burn injury affect albumin levels?

Edema and ascites are usually secondary to increased vascular permeability rather than hypoalbuminemia, as plasma albumin concentrations decrease due to redistribution into extravascular spaces.

What is the primary cause of edema and ascites?

They use dye-binding methods, which rely on a shift in the absorption spectrum of dyes such as bromcresol green (BCG) or bromcresol purple (BCP) upon albumin binding.

How do most clinical laboratories measure albumin in plasma or serum?

The dyes have a higher affinity for albumin than other proteins, providing partial specificity for albumin measurement.

Why are dye-binding methods effective for measuring albumin?

Bromocresol purple (BCP) is slightly more specific for albumin and generally yields lower values than bromocresol green (BCG), particularly in patients with kidney disease.

Which dye is more specific for albumin, and how do its results compare to BCG?

α-Fetoprotein (AFP)

What is the most abundant plasma protein in early embryonic life?

AFP has a molecular mass of ~70 kDa and is homologous to albumin but contains about 4% carbohydrate.

What is the molecular mass of α-Fetoprotein (AFP), and how does it compare to albumin?

AFP is usually present at very low concentrations after the neonatal period, requiring highly sensitive immunoassays for detection.

What happens to AFP levels after the neonatal period?

AFP is a tumor marker for hepatocellular carcinoma and germ cell carcinoma.

What is AFP used as a tumor marker for?

Maternal serum AFP levels are analyzed as indicators of fetal trisomy 18 (Edward’s syndrome) and trisomy 21 (Down’s syndrome).

How is maternal AFP concentration used in prenatal screening?

AMG levels increase, while most other plasma proteins decrease due to protein loss exceeding production, except for very large proteins

How does AMG behave in nephrotic syndrome compared to other plasma proteins?

The liver increases protein synthesis, but because AMG is a large protein, it is better retained by the impaired glomerular filtration barrier.

Why does AMG increase in nephrotic syndrome?

Severe acute pancreatitis and advanced prostate carcinoma.

In which conditions are decreased plasma concentrations of AMG commonly observed?

AMG is measured using immunoturbidimetry or immunonephelometry.

How is AMG quantified in the lab?

AMG is a major α2-globulin, especially in newborns and after in vivo hemolysis, it plays a role as one of the proteins separated during the process.

What is AMG's role in serum zone electrophoresis?

It is an α2-globulin that contains 95% of total serum copper, which gives it its color

What type of globulin is ceruloplasmin (Cp), and what gives it a blue color?

When Cp levels are elevated (e.g., during pregnancy) or yellow plasma pigments decrease, the plasma may appear greenish.

Why might plasma have a greenish tint?

It is synthesized primarily by the liver, and copper is essential for the normal folding of its polypeptide chain.

Where is ceruloplasmin synthesized, and why is copper important in this process?

Cp catalyzes redox reactions, specifically the oxidation of Fe²⁺ (ferrous) to Fe³⁺ (ferric).

What is the primary physiological role of ceruloplasmin?

Cp helps regulate iron’s ionic state, oxidizing Fe²⁺ to Fe³⁺, which allows transferrin to incorporate and transport iron.

Why is ceruloplasmin important for iron metabolism?

It is the major plasma transport protein for iron and its responsible for most of the total iron-binding capacity (TIBC) of plasma.

What is transferrin (TRF), also called siderophilin, and what is its primary function?

in the liver and usually migrates in the β1-region on routine clinical electrophoresis.

Where is transferrin (TRF) primarily synthesized, and where does it migrate on serum electrophoresis?

They are regulated by iron availability—they increase with iron deficiency and decrease when iron levels are adequate.

How are plasma transferrin (TRF) concentrations regulated?