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Clinical Chemistry 1 > Proteins > Flashcards

Proteins Flashcards

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1
Q

What two processes reflect the resulting protein concentration?

A

Protein concentrations (plasma or serum) reflect balance between anabolism and catabolism.

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2
Q

What body fluids are typically measured for their protein content?

A

Serum, urine and CSF total protein are measured in the lab.

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3
Q

What are the reference ranges for total protein for plasma, urine and CSF?

A

Plasma: 60 - 80 g/L
Urine: < 0.14 g/L
CSF 0.20-0.40 g/L

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4
Q

Where are most proteins synthesized?

A

Most proteins at synthesized in the liver, with the exception that immunoglobulins are produced by lymphocytes.

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5
Q

What protein represents the largest fraction and by ~ how much?

A

Albumin is the largest fraction of total protein at ~60%.

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6
Q

Where is albumin synthesized?

A

Liver

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7
Q

What functions does albumin serve (5)?

A
  1. Main transporter of insoluble organic anions.
  2. Binding of toxic heavy metal ions,
  3. Transport of excess quantities of poorly soluble hormones.
  4. Maintenance of serum osmotic pressure, and
  5. Provision of a reserve store of protein.
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8
Q

What does the glomerular basement membrane do?

A

Ultrafilter allows only small molecules to pass through.

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9
Q

What occurs if the glomerular basement membrane is damaged?

A

Albumin increases in the urine. Degree of damage reflected in size of urinary proteins.

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10
Q

What is proteinuria?

A

Presence of protein, usually albumin, in urine. Also known as: albuminuria.

  • Proteins pass through glomerulus of kidney.
  • Not re-absorbed by renal tubules.
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11
Q

What is the implications of finding protein in urine?

A

Small amount so protein in normal urine does not always indicate renal disease. - Need to investigate cause.
- Could be the result of strenuous exercise for example.

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12
Q

What is the test of choice when screening for albuminuria?

A

Random urine to creatinine ratio is the test of choice when screening for albuminuria. Early indication of glomerular dysfunction.

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13
Q

What is microabluminuria?

A

Small excretion of albumin in urine.

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14
Q

What is overtnephropathy?

A

Persistent high levels of albumin in urine.

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15
Q

What types of reference ranges are there for albumin?

A

Plasma (33-45 g/L)
Urine (24 hours) < 30 mg/day
Urine (Random) Male < 2.0 mg/mmol creatinine.
Urine (Random) Female < 2.8 mg/mmol creatinine.

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16
Q

What disease states result in an increase in CSF proteins?

A

Meningitis, hemorrhage, tumors, Multiple Sclerosis, diabetes, etc.

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17
Q

What condition could cause a decrease in CSF proteins?

A

Decrease in CSF proteins could be related to a leakage from the CNS.

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18
Q

What are some testing methods used for total protein analysis on CSF?

A

Chemistry analyzer and electrophoresis.

Purpose: To identify specific proteins.

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19
Q

What is oligoclonal banding?

A

Oligoclonal banding is representative of the presence of inflammation. Appearance of multiple bands in gamma region on electrophoresis of CSF that are not present in serum is indicative of local synthesis of immunoglobulins (IgG).

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20
Q

What disease state does one expect to see oligoclonal banding appear in the electrophoresis analysis that is not present in serum?

A

Multiple Sclerosis.

It is typical for MS to have two discrete oligoclonal bands in the CSF.

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21
Q

What is the reference range and critical value for total proteins of CSF?

A

Reference range: 0.20-0.40 g/L
Critical value of CSF Protein is > 1.9 g/L
Too high of CSF protein is life threatening.

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22
Q

What does alpha1-antitrypsin do?

A
  1. Inhibition of neutrophil elastase.

2. Released during infection but causes damage if not inhibited.

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23
Q

When and why is maternal AFP screening performed?

A

Maternal AFP screen is looking for alpha1-fetoprotein between 15 and 20 weeks of gestation.
Elevated levels may indicate spina bifida or neural tube defects.
Low levels may indicate Trisomy 21 or Trisomy 18.

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24
Q

Where does alpha1-fetoprotein (AFP) come from?

A

Synthesized in fetus and decreased after birth.

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25
Q

What does the protein haptoglobin do?

A
  1. Functions to bind hemoglobin.

2. Allows iron to be recycled.

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26
Q

What conditions are increased and decreased levels of haptoglobin related to?

A

Increased: Inflammatory conditions.
Decreased: Red cell breakdown.

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27
Q

What is the purpose of the ceruloplasmin protein?

A
  1. Binds 90% of serum copper.
  2. Enzymatic activities.
    It is an acute phase protein.
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28
Q

What conditions are the increased and decreased levels of ceruloplasmin protein related to?

A

Increased: Inflammation.
Decreased: Wilson’s disease (if not bound to copper it degrades; excess copper is deposited in tissues like the liver).

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29
Q

What function does the protein transferrin serve?

A

Binds iron for transport to storage sites like the liver.

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30
Q

What is the function of lipoproteins?

A

Transports cholesterol, triglycerides, and phospholipids in the bloodstream (HDL and LDL).

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31
Q

What is the function of complement proteins?

A

Participates in immune reaction.

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32
Q

What type of disease state are complement proteins associated with?

A

Linked to inflammatory disease.

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33
Q

What is the function of the protein fibrinogen?

A

Functions to form a fibrin clot when activated by thrombin.

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34
Q

What disease condition is the protein C-Reactive Protein associated with?

A

Increased in inflammatory disease.

Note: There is a debate about measuring CRP as it is released for many diseases and hence is non-specific, but still requested at times.

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35
Q

Where is the protein myoglobin found?

A

Found in striated skeletal or cardiac muscles. It is a heme protein.

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36
Q

What happens to myoglobin when there is damage to the muscle?

A

It is released with damage.

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37
Q

What is troponin a complex of?

A

Complex of three proteins that function to regulate cardiac muscle contraction:

  1. Troponin T (cTnT)
  2. Troponin I (cTnI)
  3. Troponin C (TnC)
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38
Q

What is the troponin complex proteins sensitive indicators of?

A

The three proteins are often measured separately for a myocardium infarction. They are sensitive indicators of even small amounts of cardiac damage, in particular Troponin I.

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39
Q

When are newborn screening card specimens taken and why?

A
  1. They are collected no sooner than 24 hrs after birth and aim to be collected no later than up to 5 days of age.
  2. The reason for no sooner than 24 hrs is so that the baby’s own body is given a chance to support itself (just out of Mom). If later than 5 days then if the baby had one of the conditions of concern it could start to be detrimental to the baby.
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40
Q

What types of diseases are screened for in Manitoba’s Newborn screening program?

A
  1. Metabolic and endocrine dysfunction that can be caused by enzyme defects, abnormal metabolism of proteins/carbs/fats, gene defects, etc.
  2. Disease found w/in population shortly after birth that can be treated.
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41
Q

What are the impacts of a block in the metabolic pathway for an infant?

A
  1. Block in metabolic pathway –> results in build-up of substance before the block.
  2. Deficiency of products after the block
    • -> abnormal metabolism.
  3. Symptoms may include:
    a) failure to thrive.
    b) Abnormalities in features, hair or skin.
    c) Behavioral and learning problems.
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42
Q

What enzyme is lacking in the disease PKU and what was it suppose to do?

A
  1. Absence of phenylalanine hydroxylase (PAH).

2. Normally converts phenylalanine to tyrosine.

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43
Q

What is the result if phenylalanine hydroxylase (PAH) enzyme is not present?

A
  1. If absent there is a build up of phenylalanine and low levels of tyrosine. This results in toxic accumulations in the brain causing significant and permanent damage, such as learning and development challenges.
  2. Can be managed if identified early with a diet low in phenylalanine.
44
Q

What enzyme is lacking in the condition Glutaric Acidemia - type 1 (GA-1)? What is the result?

A
  1. Glutaryl-CoA dehydrogenase enzyme is lacking.
  2. Glutaric acid, that is a product from Tryptophan + Lysine, after the breakdown of amino acids, cannot be further broken down as the Glutaryl-CoA dehydrogenase enzyme is lacking so it builds up in the body along with other harmful substances.
  3. The final result is health problems (details are not on the slide). When it works properly there is energy and growth.

See Slide 37 for diagram.

45
Q

What are aminoacidopathies?

A

There are many other aminoacidopathies listed on the slide (we don’t need to memorize those). Explanation of what an aminoacidopathies is below:

Inborn errors of metabolism (IEMs) are a group of inherited metabolic disorders which are caused by mutations in the specific genes that lead to impaired proteins or enzymes production. Different metabolic pathways are perturbed due to the deficiency or lack of enzymes. IEMs have been classified into different categories and one class of IEMs, characterized by the physiological disturbances of amino acids is called as aminoacidopathies.

Sources: https://pubmed.ncbi.nlm.nih.gov/29094226/

46
Q

What are light measuring methods for testing of proteins?

A
  1. Refractometry
  2. Turbidimetric Methods
  3. Nephlelometric Methods
47
Q

What do nephlelometric methods work best with for protein measurements?

A

Works best for small aggregates formed by antigen: antibody reactions.

48
Q

What is tested by immunoturbidimetric methods?

A

Microalbuminuria

49
Q

What is protein Electrophoresis?

A

Separation of proteins in a buffer on a porous medium under influence of an electrical field.

50
Q

What types of porous support mediums are used in protein electrophoresis and some of their pros or features?

A
  1. Cellulose acetate:
    - Usually used on serum
    - Fast separation, and
  2. Agarose:
    - Most common, e.g. 1% agarose gel.
    - Pore size allows separation based on charge and molecular size of molecule.
51
Q

Where to the + charged amino acids and - charged amino acids move towards in protein electrophoresis?

A

+ charged amino acids (cations) move towards the - electrode.
- charged amino acids (anions) move towards the + electrode.

52
Q

Do amino acids at their pI migrate?

A

No, they do not.

53
Q

With protein electrophoresis, what process steps are taken after migration?

A 
After migration:
1. Gels are fixed.
2. Stained
3. Washed to remove excess stain.
4. Protein bands are visualized.
Amino acids are identified as separate bands.
54
Q

Where is the sample placed in protein electrophoresis?

A

In the middle of the gel (not like DNA electrophoresis).

55
Q

What factors can affect electrophoresis?

A
  1. Time
  2. Voltage or current.
  3. Buffer.
  4. Gel or support medium.
  5. Particle shape and size.
  6. pH
  7. Temperature
  8. Charge.
56
Q

What are cations?

A

Positive ions. (The t in cation is like +).

57
Q

What are anions?

A

Negative ions. (Anions is like onions and who in the family likes onions but me!).

So I am a neg ion, an anion, but I try to be positive so I migrate to the positive electrode!.

58
Q

What factors affect the rate of migration of proteins?

A

Rate of migration:

  1. Directly proportional to net charge of the protein in solution.
  2. Greater the net charge the greater the mobility.
  3. Inversely proportional to the size of the protein and viscosity of the buffer.
59
Q

What happens if your time of migration for protein electrophoresis is not optimized?

A

If time not optimized:
Too much time = decreased resolution.
Decrease time = lack of separation.

60
Q

What happens in protein electrophoresis if the voltage is too high?

A

Since V = IR, if the voltage is too high you will produce heat from resistance in gel although you will increase the migration rate. The heat in turn will denature the proteins. BAD if voltage too high!

61
Q

What happens in protein electrophoresis if the voltage is decreased from optimal?

A

There will be decrease migration.

62
Q

What is the purpose of the buffer in protein electrophoresis?

A
  1. Solution buffered to maintain a constant pH (resist pH changes).
  2. Ions in buffer maintain electrical flow.
  3. Buffers are selected so pH gives optimum net charge for maximum separation.
63
Q

What does the buffer contain?

A

The buffer contains an acid and conjugate base.

64
Q

What happens if ions in buffers are too high in concentration?

A

Higher ionic concentration = less mobility (ionic cloud hinders movement).

65
Q

What pH range and types of buffer is typically used in protein electrophoresis?

A
  1. Use buffers with pH between 7 and 9.
    Net negative charge (anions) of proteins at pH 8.6 causing them to migrate towards the positive terminal (anode).
  2. Barbital or tris-boric acid-EDTA buffers are commonly used.
66
Q

What qualities are desired in the support medium?

A
  1. Biochemically inert (neutral).
  2. Concentration optimized (not too high or low) for pore size as this affects migration in relation to the size and shape of molecule being separated.
67
Q

What are the application steps for loading a specimen onto an agarose gel?

A
  1. An applicator template containing specimen application slots is applied to the gel.
  2. A few microliters of sample are applied across each slot.
  3. After samples are allowed to diffuse into the gel, the applicator template is removed.

Note: You may want to compare this to lab notes to add some more explanation here.

68
Q

Describe the steps for loading the agarose gel into the electrophoresis chamber and running gel?

A
  1. Place gel in electrophoresis cell containing 2 separate buffer compartments. Note: Each compartment contains an electrode (anode or cathode) which later becomes electrically charged.
  2. The electrodes are connected to a power supply and an electric field is applied for a time period specified in the procedure.
69
Q

What does the gel sit on in the electrophoresis chamber?

A

Gel sits on a gel bridge.

Alternatively a cooled bridge for high resolution electrophoresis.

70
Q

What is each end of the gel in contact with while in the electrophoresis chamber?

A

The gel is in contact with buffer in the 2 compartments.

Each compartment contains an electrode (anode or cathode) which later becomes electrically charged.

71
Q

After the gel has completed electrophoresis what is the next step?

A

Fixation and drying.

72
Q

What kind of solution is used for fixing the protein electrophoresis gel? What does this do?

A
  1. Acid solution in a bath (container).
  2. Functions of acid solution fixation:
    a) Denatures the proteins causing them to fix to the proteins in the gel.
    b) Removes gel buffer components, most importantly SDS, which may interfere in the staining process.
    c) Acidic environment increases interactions with stains.
73
Q

After fixation what is the next important step?

A

After fixation, the gel is then dried before going through the staining process.

74
Q

How many stains and what kind of stains are used in the staining process?

A

Multi-step procedure using stains in the:
a) Amido Black family.
b) Coomassie Brilliant Blue family.
This stains amino groups of proteins.

75
Q

What can use see now after staining that you couldn’t before?

A

You can now visualize and identify the different bands that you were unable to see up to this point.

76
Q

After staining what is needed to be done again?

A

Dry the agarose gel in a gel dryer.

Dried gel can be stored indefinitely.

77
Q

How is the dried gel interpreted?

A

By direct visualization of the stained bands and by densitometry.

78
Q

What is densitometry?

A
  1. It is a measurement method used following protein separation & visualization.
  2. A densitometer uses spectrophotometry to scan the gel and draw a pattern referred to as an electropherogram.
79
Q

How are values for the different protein fractions estimated?

A
  1. Total protein value is given to the instrument.
  2. Area under each peak is measured.
  3. Proteins fractions are a % of the total protein
  4. The thicker/denser band/broader band = increased % of that fraction.
80
Q

What is an example of a machine that can automate the entire process?

A

Hydrasys

Sample application, migration and staining.

81
Q

What is wick flow? How do you prevent?

A
  1. Evaporation of solvent from support medium. Heat generated during electrophoresis causes buffer to be drawn into support from both ends.
  2. Results in even pattern.
  3. Affects rate and length of migration.
  4. Correct: Using a lid or cover to prevent evaporation or using a cooling system.
82
Q

What is electroendosmosis?

A
  1. Proteins take on negative charge with pH 8-9 forming a negative ion cloud that migrates towards anode.
  2. Positive ions from the buffer form a positive ion cloud migrating towards the cathode.
  3. Tension between these ion clouds moving in opposite directions causes movement of sample macromolecules.
  4. Slows migration of some proteins in sample or can make them immobile or push them back towards the cathode.
    DON’T GET NICE SEPARATION!
83
Q

How do you correct/prevent electroendosmosis from happening?

A

Use support media that lacks agaropectin.

84
Q

What happens if voltage is off, too high or too low?

A

No voltage: No migration.
Too High: Quick migration, but may cause too much heat and denature proteins.
Too Low: Slow migration, less separation. Gel had to read.

85
Q

What happens if buffer is too acidic or too basic?

A

Too Acidic: Proteins will have a net + charge, & migrate towards cathode (negative electrode) and diffuse bands will result.

Too Basic: Proteins will have net - charge, migrate towards anode (+ electrode) and bands will be indistinct and stuck together.

Note: She says the terms cathode & anode can switch whether they are referring to + or -. That seems weird so I wonder why she says that. See slide 6.

86
Q

What if the time in electrophoresis is too little or too much what happens?

A

Too Little time: Sample won’t be separated enough.

Too Much time: Samples can run all the way towards the end of your gel making them hard to interpret.

87
Q

What occurs in protein electrophoresis if there is too much electric heat generated?

A

Heat can cause buffer to evaporate –> affect rate and length of migration.
Too hot: wick flow.
Too cold:???

88
Q

On lab protein slides 20 to 23, cover issues noted and state what you think the problem is.

A

Activity.

89
Q

What are the main groups of serum protein electrophoresis (SPE)?

A 
Classic 5 zone pattern of:
1 a) Pre-albumin
b) Albumin
2. alpha-1 globulin
3. alpha-2 globulin
4. B globulin (B = beta)
5. gamma globulin
90
Q

What are the reference ranges of each of the basic protein fractions?

A

1 Albumin = 35 - 50 g/L or 53-65%

  1. alpha-1 globulin = 1-3g/L or 2.5-5%
  2. alpha-2 globulin = 6-10 g/L or 7-13%
  3. B globulin (B = beta) = 7-11 g/L or 8-14%
  4. gamma globulin = 8-16g/L or 12-22%
91
Q

What is the albumin / globulin (A/G) ratio? How is it determined?

A

Total protein and albumin are measured
Globulin result is calculated by subtracting total protein from albumin
Globulins = total protein – albumin
3.69 = 6.6 – 2.91
Divide albumin concentration by globulin concentration
A/G = albumin/globulin
0.79 = 2.91/3.69

92
Q

What is the meaning of an increased A/G?

A

Increased A/G

Decreased globulin synthesis

93
Q

What is the meaning of a decreased A/G?

A

Decreased A/G
↓ Albumin ↑ globulins
↓ Albumin due to decreased protein synthesis by liver due to pathology or renal loss of proteins due to disease
↑ globulins chronic inflammatory disease, rheumatoid arthritis, Multiple Myeloma

94
Q

How are the electrophoretic patterns used?

A
  1. Different diseases produce different electrophoretic patterns.
  2. A diagnosis cannot be made from an electrophoresis result alone
  3. The result must be interpreted in the context of other clinical findings
95
Q

What happens if plasma is used for SPE?

A

Protein electrophoresis uses serum samples. The inadvertent use of plasma results in a band (or peak on the electrophoregram) between the β and γ regions due to the presence of fibrinogen.

96
Q

What occurs if there is free hemoglobin in a hemolyzed sample?

A

Free hemoglobin causes a small band in the alpha-2 region.

97
Q

Why do CSF and urine samples need to be concentrated first?

A

Because they have very low levels of proteins in those body fluids.

98
Q

What is the purpose of immunofixation?

A

Enables one to identify the specific immunoglobulin that is related to the monoclonal peak in the gamma region of the electrophoregram.

99
Q

What are antibodies comprised of that gives them their designations?

A 
Heavy chain (α, δ, ε, γ, or μ) which gives it is designation (IgA, IgD, IgE, IgG, or IgM)
Light chains (κ or λ) adding to its variety for recognizing an array of potential non-self invaders
100
Q

What body fluids can immunofixation be performed on?

A

Can be done on various body fluids.

101
Q

What is immunofixation?

A

Reaction of specific antiserum to precipitate individual proteins.
Note: Non-specific precipitation used in normal protein electrophoresis

102
Q

What is done following precipitation in immunofixation?

A

Following precipitation of the protein of interest, other proteins are washed out of gel prior to staining to visualize the precipitated band(s).

103
Q

What is high resolution electrophoresis (HRE)? What is its advantage?

A

High Resolution Electrophoresis (HRE):
Uses high voltage (>250V)
System is cooled to eliminate over heating
Uses high ionic strength buffer
Advantage: 12 bands can be separated rather that the routine 5

104
Q

What is isoelectric focusing (IEF)? What is it typically used on?

A

Isoelectric Focusing (IEF):
Gel has different pHs
Polyanions and polycations in gel to create pH gradient
Migration stops where pH is equal to pI of protein
Creates a high resolution
Used for CSF samples

105
Q

What is capillary electrophoresis (CE)? What is its advantage?

A

Capillary Electrophoresis (CE)
Narrow silica capillary used instead of gel
Higher voltage – heat dissipates from silica capillary easily
Polyacrylamide inside
One end is in the cathode and the other end is in the anode
Size and charge of protein determines which emerges and is detected
Easily automated, quick, and efficient

Clinical Chemistry 1 (11 decks)

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