CCML240 Primary disorders, proteins, SPE and IFE

what are the characteristics of osteoporosis

1. low bone mass and deterioration of the bone

2. bones become more porous and thin and more likely to break

why is osteoporosis seen is post menopausal women

As estrogen decreases bone loss accelerates

- estrogen regulates bone remodeling so after the decrease osteoclasts are no longer restrained and bone resorption exceeds formation

what is the pathophysiology of osteoporosis

Decrease in bone density leads to brittle bones

1. bone resorption (osteoclasts) exceeds new bone formation (osteoblasts)

2. bones diminished in overall quantity as they contain normal matrix and mineralization but remain vulnerable to fractures

Causes of osteoporsis

1. Vitamin D deficiency

2. insufficient dietary calcium

3. insensitivity to PTH

4. decreased estrogen production with increased osteoclastic activity

what does vit D do

regulates intestinal uptake of Ca & P, renal reabsorption of Ca & mobilization of Ca to bone

What is the bone mineral densitometry test

A diagnostic for osteoporosis

1. X ray is directed at a small area where Denser bones absorb the X ray beam while osteoporosis bones show a decreased xray absorption

2. releases a Tscore that compares bone density with a healthy 30yr individual - the more negative the score the greater severity and greater risk of fracture

What is osteomalacia/rickets

Abnormal mineralization of bone where more and more bones are formed made up of collagen matrix without a mineral cover resulting in soft bones that tend to bend and crack

what cause osteomalacia/rickets

1. Vitamin D deficiency

2. Can occur secondary to gastrointestinal, liver or kidney diseases

what can osteomalacia result in

secondary hyperparathyroidism

what can vit D deficiency lead to

1. Impaired intestinal absorption of Ca and P

2. reduced Ca absorption leading to increased PTH

3. increased PTH leads to increase loss of Ca from the bone, Decrease serum P due to increased urinary excretion

4. poor delivery of minerals to the bone resulting in defective mineralization and deposition of uncalcified bone matrix resulting in soft and pliable bones

What are the general roles of proteins

All proteins have some physiological or biological functions

1. transport of molecules

2. hormones and receptors

3. catalytic enzymes

4. structural

5. nutritional

where is albumin synthesized

in the liver

what controls albumin synthesis

By Colloidal osmotic pressure and secondarily by protein intake (essential AA)

what is colloid osmotic pressure

pressure exerted by plasma proteins and tendency to pull water into circulation

1. plasma proteins cannot cross through capillaries they are mainly there to prevent fluids from moving out

2. when plasma proteins are low there is a reduction is osmotic pressure and an increase in water moving out of the capillaries which results in excess fluids in tissues called edema

what is the function of albumin

1. Maintenance of COP

2. Maintenance of acid-base balance

3. Binds and transports

4. Metabolism/regulation/detoxification

5. Source of endogenous amino acids

How does albumin maintain COP

If plasma albumin levels change, albumin in extravascular spaces will re-equilibrate with plasma to compensate

How does albumin maintain acid-base balance

Can bind or release excess hydrogen ions as needed to balance excess acidic or alkaline conditions

What does albumin bind and transport

Large number of compounds, particularly those with poor solubility in plasma

What does albumin metabolize, regulate, detoxify

1. free fatty acids

2. bilirubin

3. phospholipids

4. calcium

5. metallic ions

6. hormones

7. amino acids

8. drugs

How is albumin a source of endogenous amino acids

It transport AA to tissues or it can get catabolized to provide AA

What is the clinical significance of LOW albumin levels

Associated with Liver disease, malnutrition, GI loss and renal disease (high not important)

What is present in A1 globulins

-a1 -antitrypsin

-a1 -acid glycoprotein (aka orosomucoid)

What is present in A2 globulins

haptoglobin and ceruloplasmin

What is present in B globulins

transferrin, C4, and C3

What is present in Y globulins

IgG, IgA, and IgM

What is the function of globulin

1. Regulate/deactivate hormones

2. protease inhibitors

3. transport of metallic ions

4. detoxification of substances released from damaged tissue

5. participate in classical complement pathway

What is acute phase reaction

Inflammatory cells, red cells in response to injury and local neutrophils, granulocytes and macrophages.

These WBCs secrete cytokines such as Interleukins, IL1,6,8 and a

Liver responds by producing specific acute phase proteins

When does acute phase reaction occur

Occurs when levels of certain acute-phase proteins increase in response to inflammation

what is the clinical significance of Alpha1 anti-trypsin (AAT)

Major component of A1 globulin fraction

1. a protease inhibitor that neutralizes the protease neutrophil ELASTASE (damages structural protein)

SPE shows lack of a1 globulin band

When is elastase released

Released by neutrophils during phagocytosis, results in loss of elastin in lung tissue

When are elevated levels of elastase seen

Associated with acute phase reaction, and genetic deficiencies

What is the clinical significance of A1 acid glycoprotein (AAG orosomucoid)

May regulate immune responses as AAG is a lipocalin that binds lipophilic substances

What is the clinical significance of haptoglobin (HAP)

-Binds free hemoglobin to prevent loss of hemoglobin and iron into the urine

-Elevated levels are associated with burns, nephrotic syndrome

-Decreased levels associated with bleeding

what is the clinical significance of ceruloplasmin (CER)

A glycoprotein bound to 6-8 atoms of copper that represents 90% of serum copper

What does a deficiency of CER lead to

Wilson's disease

what is wilsons diease

An inherited disorder where free copper deposits into the skin, cornea, liver, and brain resulting in cirrhosis and neurologic damage

what is the clinical significance of transferrin

A molecule that transports iron

- its level correlates inversely with plasma iron

-elevated levels associated with IDA

-Decreased levels associated with inflammation and rbc failure

what is the clinical significance of C3 and C4

These complements are major mediators of inflammation and facilitate eradication of antigens

-Elevated levels are associated with acute phase reaction

-Decreased levels associated with malnutrition, DIC and diseases

What is the clinical significance of C-reactive protein (CRP)

Initiates opsonizatoin and encourages phagocytosis

- is an early maker of ARP (acute phase reaction)

What is the clinical significance of IgG

autoimmune disease, hepatic disease

What is the clinical significance of IgA

skin, gut, respiratory infections, cirrhosis

What is the clinical significance of IgM

viral infections, blood infections (malaria), acute hepatitis

What is the clinical significance of IgD

is a surface receptor for antigen on B lymphocytes; primary function unknown

What is the clinical significance of IgE

brings about histamine release from mast cells

what is polyclonal increase in gammaglobulins

mixture of antibodies

-chronic infection, autoimmune disease, hepatocellular disease, parasitic infestations

What is monoclonal increase in gammaglobulins

single class of antibody e.g. Multiple myeloma

What is oligoclonal increase in gammaglobulins

a few "monoclonal" proteins of different specificities

what is the clinical significance of fibrinogen

A large glycoprotein that forms fibrin clots in the presence of thrombin

What is the A/G ratio

A calculation of serum albumin compared to serum globulin

-used to determine if globulin production is disproportionate to albumin, since total protein may be normal

What does a decreased A/G ratio mean

Increase in globulins or decrease in albumin

What does an increase A/G ratio mean

Decrease in globulins

What is the significance of an increase in total protein

Albumin is never increased so an increase in TP reflects increases in multiple globulins

-multiple myeloma

-chronic infection

-dehydration

what is the significance of decreased total protein

characterized primarily by decrease in albumin from a variety of things with inflammation being the most common

quantitative measurement of total protein

-spectrophotometric, automated

-serum, urine, CSF

immunoassay/scattered light

automated rate nephelometry for measuring specific globulins

electrophoresis (SPE)

manual or automated

-based on molecular size and charge

what is the biuret method

Cupric ions react similarly to Biuret and form a colored chelate with peptide bonds, once 2 or more peptide bonds react with Cu ions in alkaline solution it forms a pink to reddish violet product

The intensity of the color is inversely proportional to the # of peptide bonds

What does CuSO4 do in the biuret reagent

Source of Cu that reacts with peptide bonds

What does NaOH do in the biuret method

Provides alkaline medium

What does NaK tartrate do in the biuret method

Chelating agent to complex cupric ions and maintain their solubility in alkaline solution

What does KI do in the biuret method

Provides iodine as an antioxidant and prevents separation of cuprous oxide

What are the sources of errors in biuret method

1. NH4 ions interfere

2. Hemolyzed samples should be avoided

3. Lipemic samples add turbidity

What is the albumin dye binding method

At a constant pH, dissociated and undissociated forms of dye have different colors when bound to albumin

-uses bromocresol green or bromocresol purple

sources of error in dye binding method

1. Anything that interferes with binding of dye to albumin OR the complex itself OR the detection of the complex

what is rate nephelometry

A more sensitive method needed to measure individual proteins as globulins are in much lower concentration

-Measures forward scatter light at an angle less than 90

-antigen-antibody complex formed causes increased in light scatter that is measured over time

Rate/change of light scatter proportional to concentration of specific protein

What is the SPE in nephrotic syndrome

Decrease albumin

Increase A2 Globulin

Decrease Y globulin

What is the SPE in acute phase proteins

Increase A1 globulin

Increase A2 globulin

What is the SPE of severe hepatic disease

Decreased albumin

Decrease A1 globulin

Decrease A2 globulin

Decrease B globulin

Increase Y globulin

What is the SPE of liver Cirrhosis

Increase in Y globulins with a B-Y bridge

What is the SPE in hypogammaglobulinemia

Decrease to absent Y globulins

What is the SPE of monoclonal gammopathy

Increase in Y globulins to produce a M spike

What is the SPE in polyclonal gammopathy

Increase in Y globulins

Monoclonal gammopathies

1. narrow spike or band in β-γ region

2. homogeneous production of Ig's

3. indicative of malignancy or primary disorder

Polyclonal gammopathies

1. broad increase in γ region

2. numerous clones of plasma cells

3. heterogeneous mixture of Ig's produced

4. secondary disease states e.g. chronic liver disease,

5. rheumatoid arthritis, chronic infections

What must you do if there is an abnormality in gamma region

1. Identify Ig by class

2. Determine presence of free light chains

3. Identify oligoclonal bands

What causes polyclonal decrease (hypogammaglobulinemia)

1. Ig deficiency

2. protein loss

3. inherited defects in synthesis

4. toxins

What causes polyclonal increase (hypergammaglobulinemia)

1. normal response to infection

2. chronic bacterial infections

3. primary liver disease

What causes monoclonal increase

1. occurs when single clone of plasma cell undergoes unrestricted growth

2. indicative of malignancy

What is multiple myeloma (plasma cell myeloma)

Proliferation of malignant plasma cells that produce IgG in bone marrow

-CRAB criteria - very important to distinguish from MGUS

What is the CRAB critera

hypercalcemia, renal insufficiency, anemia, lytic bone lesions

What is MGUS

Monoclonal Gammopathy of Undetermined Significance (MGUS)

-a stable level of M protein in serum or urine without clinical features of MM

What is waldenstrom macroglobulinemia

Plasma cell malignancy producing IgM

What is Bence jones protein

Presence of free monoclonal K or theta light chains in urine or serum, if secreted from plasma cells its known as light chain disease

What is biclonal gammopathy

When 2 separate monoclonal proteins are produced simultaneously in the same patient

What are oligoclonal bands

Multiple, narrow usually faint, discrete bands in gamma zone visible in both serum and CSF and is a supportive diagnosis of MS

What is immunofixation electrophoresis (IFE)

A 2 stage procedure that combines the principals of SPE and immunoprecipitation

What are the steps in immunofixation electrophoresis

Step 1: Proteins in serum, urine or CSF separated using HighResolution Protein Electrophoresis

Step 2: Antiseras are added and react with their complimentary heavy or light chain forming insoluble Ag-Ab complexes ➢3 heavy chain antisera: anti-IgG, -IgA, -IgM ➢2 light chain antisera: anti-kappa, -lambda

Step 3: Stain for visualization of bands ➢Acid blue, Acid purple