LAB: Amino Acids & Proteins Flashcards
It regulates the distribution of amino acids.
Liver
In the stomach, this breaks down proteins into peptides.
Pepsin
Amino acid absorption occurs from the ________ into the blood stream
small intestine
In what organ does the enzymes break peptides into amino acids?
Small intestine
Enumerate the enzymes secreted by the stomach cells
- Pepsin
- Hydrochloric acid (HCl)
Enumerate the enzymes secreted by the acinar cells of pancreas
- Trypsin
- Chymotrypsin
- Elastase
- Sodium bicarbonate
Enumerate the enzymes secreted by the intestinal cells
- Secretin
- Cholecystokinin (CCK)
Based on chemical properties, what are the amino acids that are aromatic
- Phenylalanine
- Tyrosine
- Tryptophan
Based on chemical properties, what are the amino acids that are negatively charged
- Aspartic acid
- Glutamatic acid
Based on nutritional requirements, what are the essential amino acids
- Isoleucine
- Leucine
- Lysine
- Methionine
- Phenylalanine
- Threonine
- Tryptophan
- Valine
Based on nutritional requirements, what are the semi- essential amino acids
- Arginine
- Histidine
Based on chemical properties, what are the amino acids that are positively charged
- Lysine
- Arginine
- Histidine
Based on nutritional requirements, what are the non-essential amino acids
- Alanine
- Asparagine
- Aspartic acid
- Cysteine
- Glutamic acid
- Glutamine
- Glycine
- Hydroxylysine
- Hydroxyproline
- Proline
- Serine
- Tyrosine
What are the stop codons
- UAA
- UAG
- UGA
What is the start codon
AUG
Refers to the number, type, and sequence of amino acids in the polypeptide chain
Primary structure
Refers to commonly formed arrangements stabilized by hydrogen bonds between nearby amino acids within the protein
Secondary structure
Refers to the overall shape,
or conformation, of the protein molecule
Tertiary structure
The shape or structure that results from the interaction of more than one protein molecule, or protein subunits, referred to as a multimer, that functions as a single unit.
Quaternary structure
Are a class of inborn errors of metabolism in which an enzyme defect inhibits the body’s ability to metabolize certain amino acids
Aminoacidopathies
May also be analyzed to aid in the diagnosis of select neurotransmitter disorder
Cerebrospinal fluid (CSF)
Blood samples for amino acid analysis should be drawn after how many hours of fasting?
6 to 8 hours
For amino acid analysis, the sample should be collected in a _________ tube and the plasma removed from the cells within _______ of collection.
heparinized and 2 hours, respectively
For quantitation of urinary amino acid what sample is preferred?
a well-mixed aliquot from a first morning collection
If testing is to be delayed, samples can be refrigerated up to _______or frozen for up to ______.
24 hours and 1 month, respectively
This may be used to quantitate amino acids and their metabolites in the patient sample
high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS)
Considered to have higher specificity and greater sensitivity allowing for detection of lower concentrations of the amino acid(s) and an earlier diagnosis
MS/MS methods
Performed to aid in the diagnosis and detection of carrier status in families with an inborn error of metabolism
Genetic assays using DNA analysis or DNA analysis
The pH at which an amino acid or protein has no net charge is known as
isoelectric point (pI)
When the pH is greater than the pI, the protein has a _________________, and when the pH is less than the pI, the protein has a _________________.
net negative charge and net positive charge, respectively
Proteins differ in their pI values, but most occur in the pH range of
5.5 to 8.0
Suppresses proteolysis
Insulin
Stimulates protein synthesis
Growth Hormone
Stimulates protein degradation in muscles
Glucocorticoids
Increases protein deposition in tissues
Sex Hormones
Inhibits proteolysis in fasting state, but stimulates protein synthesis in fed state
Insulin-like Growth Factor-1
Promotes uptake of amino acids in the liver
Glucagon
Increases protein deposition in tissues
Sex Hormones
Increases production of gluconeogenic amino acids
Catecholamines
Increases the basal metabolic rate
Thyroxine
Are proteins that catalyze biochemical reactions. They are normally found intracellularly and are released into the bloodstream when tissue damage occurs, making enzyme measurements an important diagnostic tool
Enzymes
Are chemical messenger proteins that control the action(s) of specific cells or organs. They directly affect growth and development, metabolism, sexual function, reproduction, and behavior
Hormones
Are proteins that are produced by B cells (lymphocytes) in the bone marrow. They mediate the humoral immune response to identify and neutralize foreign antigens
Immunoglobulins or antibodies
Are fibrous proteins that provide structure to many cells and tissues throughout the body, such as muscle, tendons, and bone matrix
Structural proteins
Serve as reservoirs for metal ions and amino acids so they can be stored without causing harm to the cell and released when needed
Storage proteins
Routine analysis of blood specimens will typically include measurement of
- Measurement of total protein and albumin
- Albumin-to-globulin (A/G) ratio
The relationship between pH, pKa, and charge for individual amino acids can be described by the
Henderson-Hasselbalch equation
Cite the Henderson-Hasselbalch equation
pH = pKa + log [conjugate base]/ [conjugate acid]
Indicator of nutrition; binds thyroid hormones and retinol- binding protein
Prealbumin or transthyretin
Binds bilirubin, steroids, fatty acids; major contributor to oncotic pressure
Albumin
FUNCTION OF PROTEINS: Maintenance of water distribution between cells and tissue, interstitial compartments, and the vascular system of the body
Osmotic force
FUNCTION OF PROTEINS: Participation as buffers to maintain pH
Acid-base balance
FUNCTION OF PROTEINS: participation in coagulation of blood
Hemostasis
FUNCTION OF PROTEINS: Tissue nutrition
Energy
FUNCTION OF PROTEINS: Metabolic substances
Transport
FUNCTION OF PROTEINS:
Connective tissue
Structure
Acute-phase reactant; protease inhibitor
α1-Antitrypsin
Principal fetal protein; elevated levels indicate risk for spina bifida
α1-Fetoprotein
Acute-phase reactant; transport of drugs and hormones
α1-Acid glycoprotein (orosomucoid)
Lipid transport (HDL)
α1-Lipoprotein
Transports vitamin D and binds actin
Gc-globulin
Acute-phase reactant, oxidase activity; contains copper
Ceruloplasmin
Transports lipids (primarily VLDL triglyceride)
Pre-β lipoprotein
Component of human leukocyte antigens (HLA)
β2-Microglobulin (B2M)
Acute-phase reactant; may be related to immune responses
α1-Acid glycoprotein (orosomucoid)
Transports iron
Transferrin
Binds heme
Hemopexin
Transports lipids (primarily LDL cholesterol)
β-Lipoprotein
Precursor to fibrin
Fibrinogen
Acute-phase reactant; motivates phagocytosis in inflammatory disease
C-Reactive Protein (CRP)
Antibodies (in secretions)
Immunoglobulin A
Antibodies (early response)
Immunoglobulin M
Antibodies (reagins, allergy); promotes release of histamine (allergies)
Immunoglobulin E
Reference range for Total Protein - Serum, Plasma:
Adult: 6.5-8.3 g/dL (65-83 g/L)
Reference range for Albumin- Serum, Plasma:
Adult: 3.5-5.5 g/dL (35-55 g/L)
Reference range for Albumin-Globulin Ratio (A/G) - Serum, Plasma:
Adult: 1.1-1.8
Enumerate the other proteins
- Troponins
- Natriuretic peptides
- Myoglobins
- Fibronectins
- Cystatin-C
They are considered the gold standard for diagnosis of acute coronary syndrome (ACS), in which the blood supply to the heart muscle is suddenly impeded
Cardiac troponin (cTn) which consists of troponin C (TnC), troponin I (cTnI), and troponin T (cTnT)
Neurohormones that affect body fluid homeostasis, through natriuresis and diuresis, and blood pressure, through decreased angiotensin II and norepinephrine synthesis
Natriuretic Peptides
Used to help predict the short-term risk of premature delivery
Fetal fibronectin (fFN)
A proteolytic fragment of type I collagen crosslinked at the N- and C-terminal ends of the molecule and formed during bone resorption
Cross-linked C-telopeptide (CTX)
A low-molecular-weight protease inhibitor produced by all nucleated cell. Used to evaluate glomerular function in individuals for whom creatinine measurements may be misleading, such as patients with cirrhosis, obesity, malnutrition, or who have a reduced muscle mass
Cystatin C
Refers to conditions in which the serum or plasma total protein concentration is below the reference range
Hypoproteinemia
An increase in total plasma proteins, is not generally associated with a specific disease state, but rather, it is more likely the result of dehydration
Hyperproteinemia
Cite atleast two applications of molecular size
- Gel chromatography
- Ultracentrifugation
- Gradient pore electrophoresis
- Mass spectrometry
Cite atleast two applications of solubility
- Protein precipitation
- Turbidimetry
- Nephelometry
Cite atleast two applications of Charge
- Serum protein electrophoresis
- Isoelectric focusing
- Immunofixation electrophoresis
- Ion exchange chromatography
Cite atleast two applications of Molecular Interactions
- Affinity chromatography
- Immunoassays
- Immunoelectrophoresis
- Immunofixation
- Dye-binding
For testing other proteins, plasma samples should be collected using a?
plasma separator tube
For protein analysis, cite the physical methods under the quantitative type
- Dye binding
- Direct Absorbance Measurements
- Protein Ligands
- Precipitation
- Mass Spectrometry
Collected in a serum separator tube and are preferred for protein analyses due to the presence of fibrinogen in plasma
Serum
For protein analysis, cite the activity measurements under the quantitative type
- Binding protein
- Protease inhibitors
- Complement factors
- Coagulation factors
Enumerate the quantitative methods for protein analysis
- Physical methods
- Activity measurements
- Immunoassays
Acid digestion is used to convert protein nitrogen into ammonium ions, which are then quantified
Kjeldahl Method
Enumerate the qualitative methods for protein analysis
- Electrophoresis
- Chromatography
- Genetic Analysis
- Functional Assays
- Mass Spectrometry
Principle: Dye binds to protein causing a spectral shift in the absorbance maximum of the dye
Dye binding
Principle: Formation of violet-colored chelate between
Cu2+ ions and peptide bonds
Biuret Method
Principle: Proteins in solution absorb UV light with maximum absorbances of 200 and 280 nm
Ultraviolet (UV)
absorption
Principle: Formation of aggregates by protein precipitation or antibody-binding that affect light scatter
Turbidimetry & Nephelometry
Principle: Migration of proteins based on their density and charge under the influence of an electric field
Protein electrophoresis
Used in automated protein measurements and to quantitate proteins separated by electrophoresis
Dye binding
Routine method; requires at least two peptide bonds and an alkaline medium
Biuret method
Immunoturbidimetric and immunonephelometric methods used to quantitate specific proteins
Turbidimetry & Nephelometry
Limited utility for protein mixtures due to variable absorption characteristics; potential use in measurement of specific fractions after separation by another method
Ultraviolet (UV) absorption
Used to separate and quantify protein fractions in serum, urine, and cerebrospinal fluid
Protein electrophoresis
In Kjeldahl method, to correct for the average nitrogen content of protein, you multiply with?
6.25
Transerythrin is stimulated by hormones:
- Glucocorticosteroids
- Androgens
- NSAIDS (Aspirin)
Synthesized in the liver and lesser extent from CSF (choroid plexus)
Prealbumin or Transerythrin
Prealbumin Diagnosis:
- Inflammation and Malignancy
- Cirrhosis of the liver
- Protein-losing diseases of the gut or kidneys
- Familial euthyroid hyperthyroxinemia
Reference interval for Prealbumin
20-40 mg/dL
Laboratory tests used for prealbumin
- Serum electrophoresis
- Immunoturbidimetric or Immunonephelometric
The major protein component of the most extravascular body fluids
Albumin
Albumin is increased in:
- Dehydration
- Prolonged tourniquet application
Albumin is decreased in:
- Analbuminemia
- Inflammation
- Hepatic disease
- Urinary loss
- Gastrointestinal loss
- Protein- energy malnutrition (Marasmus)
- Burn injury
- Edema and ascites
Reference interval for Albumin
35-52 g/L or 3500-5200 mg/dL
The most widely used methods for determining albumin are
bromocresol green (BCG) or bromocresol purple (BCP)
Enumerate the disadvantages of Kjeldahl method
- Time-consuming
- Inconvenient
- Impractical for routine use
What dyes are used to stain protein after electrophoresis?
- Bromophenol blue
- Ponceau S
- Amido black
- Lissamine green
- Coomassie brilliant blue
Laboratory tests for Albumin
- Dye binding
- Serum electrophoresis
- Immunoturbidimetry, immunoelectrophoresis, and immunofixation electrophoresis
- Nephelometry
- Isoelectric focusing
This separates proteins on the basis of their electric charge and density across a support
media
Electrophoresis
What are the support medias are used in electrophoresis
Agarose gel or cellulose acetate
This show a dramatic decrease in the relative amounts of albumin, al -globulins, B-globulins, and y-globulins and an apparent increase in the a2 -globulin fraction
Nephrotic syndrome
Decreased or absent a1- globulin fraction is associated with a1 -antitrypsin deficiency, as a1 -antitrypsin accounts for approximately 90% of the a1 -globulin fraction
Alpha-1 Antitrypsin deficiency
Normal except for alpha2-globulin
Acute phase proteins
Defective gamma globulins which results to defective or absent immunoglobulins; common to HIV patients
Hypogammaglobulinemia
Increase results in all y-globulin
Polyclonal gammopathies
Increase results in a single homogenous spike (M protein) in the y-globulin region
Monoclonal gammopathies
Increases in fast-moving γ-globulins, such a IgA, prevent resolution of the β- and γ-globulin bands, resulting in a β–γ bridge
Liver cirrhosis
Shows a relative decrease in albumin, α1 -globulins, α2- globulins, and β-globulins, with a relative increase in γ-globulin
Severe hepatic damage
IDENTIFY THE DISEASE based on TPAG levels:
- Total Protein- normal, decrease
- Albumin- decrease
- Globulin- increase
- Hepatic damage
Cirrhosis B-Y bridging
Hepatitis ↑ y- globulins
Obstructive jaundice ↑ a2, B-globulins - Burns, trauma
- Infection
Acute ↑ a1-, a2- globulins
Chronic ↑ a1, a2, y- globulins
IDENTIFY THE DISEASE based on TPAG levels:
- Total Protein- decrease
- Albumin- decrease
- Globulin- normal
- Malabsorption
- Inadequate diet
- Nephrotic syndrome ↑ a2-, B-globulins, ↓ y-globulins
IDENTIFY THE DISEASE based on TPAG levels:
- Total Protein- decrease
- Albumin- normal
- Globulin- decrease
Immunodeficiency syndromes
IDENTIFY THE DISEASE based on TPAG levels:
- Total Protein- decrease
- Albumin- decrease
- Globulin- decrease
Salt retention syndrome
IDENTIFY THE DISEASE based on TPAG levels:
- Total Protein- increase
- Albumin- increase
- Globulin- increase
Dehydration
IDENTIFY THE DISEASE based on TPAG levels:
- Total Protein- increase
- Albumin- normal
- Globulin- increase
- Multiple myeloma
- Monoclonal and polyclonal gammopathies
Rapid, easy to use; unequal sensitivity for individual proteins
Turbidimetric methods
(sulfosalicylic acid, trichloroacetic acid, or benzethonium chloride)
This urine protein method is accurate
Biuret
This urine protein method is very sensitive
Folin-Lowry
Initial biuret reaction; oxidation of tyrosine, tryptophan, and histidine residues by phenol reagent; measurement of resultant blue color
Folin-Lowry
This urine protein method is used in automated methods
Dye binding (pyrogallol red)
Reference Ranges for Urine Protein Analyses:
Total Protein—Urine, 24-hour
Adult: <150 mg/24 hours
Reference Ranges for Urine Protein Analyses:
Protein/Creatinine Ratio- Urine, 24-Hour
Adult: ≤0.114 mg/mg creatinine
Reference Ranges for CSF Protein Analyses:
Total Protein - CSF
- 0–7 days: 40–120 mg/dL
- 8 days–1 month: 20–40 mg/dL
- > 1 month: 14–45 mg/dL
Reference Ranges for CSF Protein Analyses:
Albumin- CSF
0-35 mg/dL
Reference Ranges for CSF Protein Analyses:
CSF- Serum Albumin Ratio
2.7- 7.3
Reference Ranges for CSF Protein Analyses:
IgG INDEX
0.26–0.70
IgG index formula
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