Porphyrins and Hemoglobins Flashcards
True or False: Hemoglobin and myoglobin are chemically similar molecules that contain porphyrin groups.
True.
Porphyrins are cyclic structures comprised of four pyrrole rings joined by methane bridges
The chelation of ferrous iron to protoporphyrin produces what?
heme
Heme
the prosthetic group of hemoglobin responsible for binding oxygen
Porphyrias
a group of rare disorders that result from disturbances in heme synthesis
–
refers to a group of rare disorders that result from a buildup of natural chemicals called porphyrins in the body. Porphyrins are needed to make heme, a part of hemoglobin. Hemoglobin is a protein in red blood cells.
True or False: Analysis of porphyrins occurs in the lab
True
Hemoglobin molecules are designed to ___, ___, and release oxygen
bind, deliver
***Hemoglobinopathies
are disorders that have qualitative defects in the hemoglobin molecule
**Thalassemias
are quantitative defects in the production of hemoglobin molecules
is an inherited blood disorder that causes your body to have less hemoglobin than normal.
This is a simple heme protein found in cardiac and skeletal muscles
Myoglobin
These are chemical intermediates in the synthesis of hemoglobin myoglobin, and other respiratory pigments called cytochromes, what are they?
Porphyrins
Iron is chelated to what to form heme?
the porphyrin ring
Porphyrins are analyzed to aid in the diagnosis of what?
porphyrias: diseases that result from disturbances in heme synthesis
True or False: excess amounts of the intermediate compounds in blood, urine, or feces indicate a metabolic block in heme synthesis
True
–
So if you have too many intermediates, it means that something has gone wrong in the system
These reduces compounds that are intermediates in the biosynthesis of heme (the non-protein, iron-containing prosthetic group of hemoglobin).
They oxidized on exposure to air to porphyrins
Porphryinogens
Isomers
substitution of the pyrrole rings that make up the porphyrinogen macrocycle generate 4 isomers for every porphyrin compound
***Which types of isomers occur naturally?
-Type I and Type III
Which type of Isomer is the only one to form heme
Type III
In some disorders, which type of isomer in the non-functional state may be found in excess in body fluids and tissue?
-Type I isomers
Porphyrin are Photoactive due to extensive conjugation of the ___________ ring
tetrapyrrole
so that 4 sided ring
What color do Porphyrins show off?
Dark red color
due to strong absorbance in the visible region of the spectrum
Porphyrins absorb light of 400 nm wavelength and emit a characteristic orange-red fluorescence between ___________
600-650 nm
–
And a lot of time you will see that in urine
True or False: Aqueous solubility of porphyrins varies with the number of carboxylic acid substituents present in the partilcular porphyrin compounds
True
The solubility of porphyrins depends on how many groups it has.
**Uroporphyrin (Uro) has _____ carboxylic groups, so it’s soluble in what?
8
It is the most soluble in water! It’s excreted in urine.
Coproporphyrin (Copro) has ____ carboxylic acid constituents, intermediate solubility found in _____, _____ , ___
4, blood, urine, feces
Porphyrins with only ___ carboxyl groups are the least soluble
two
Why do we care about solubility?
Solubility influences what?
Solubility influences the type of specimen selected for measurement of particular porphyrin intermediates
–
If you have Porphyrin with only 2 carboxyl groups, you are not going to be able to look for it in the urine. It’s going to affect the sample your going to get for that issue.
True or False: All cells contain hemoproteins and can synthesis heme.
True.
but done mainly in bone marrow and liver
What are the two primary sites for heme synthesis?
The bone marrow and liver.
What is accomplished by a sequence of reactions catalyzed by 8 different enzymes?
Biosyntheis
*****One molecule of heme is produced from 8 molecules of what?
ALA - ( delta- aminolevulinic acid)
***In the last step of biosynthesis ( Syntheis of Heme) requires what to bind on to Protoporphyrin to get to heme?
Iron (Fe2+)
**If there is any issue with any of the steps of the Synthesis of Heme, it is due to what?
Porphyria
There is a Porphyria that correlates to every single one of those steps.
The first and the last 3 synthetic reactions in Biosynthesis requires how much energy?
Where do they take place? Where do the rest take place?
-a lot of energy
-They take place in the Mitochondria.
- The rest take place in the Cytosol.
In the synthesis of heme: each reaction is catalyzed by a particular ______.
enzyme
Heme formation is regulated by what kind of feedback?
negative
–
like most things in the body.
Control rate of heme in the liver is through regulation of which enzyme?
ALAS (delta-aminolevulinic acid) synthase
Increases in the pool of hepatic heme diminishes the production of what enzyme?
ALAS
–
because it is a negative feedback, so if you have a lot of heme, you don’t need that ALAS there to make more. But you get more production if heme is low.
ALAS production is increased when what is depleted/low?
heme
In bone marrow erythrocytes, do ALA control the rate of heme synthesis?
No, other enzymes in the pathway and the rate cellular iron uptake seem to control of the rate of heme synthesis.
Most diseases are ________ ____ with 50% reduction in activity of the affected enzyme
autosomal dominant
–
autosomal dominant is one of many ways that a trait or disorder can be passed down through families.
Excessive accumulation and excretion of intermediate compounds that produce particular symptoms are due to what?
Enzyme defects
–
So it’s where ever it was stopped on that cascade, is going to determine what kind of symptoms a person will have.
What are Neuropsychiatric symptoms?
What can they tell us about heme synthesis?
abdominal pain, nauseas, constipation, hypertension, PSYCHIATRIC symptoms, fever, paresthesia
These are excess of the early precursors in the pathway of heme synthesis (ALA, PBG)
–
paraesthesia is the term for sensation of tingling, during, pricking or prickling, skin-crawling, itching, “pins and needles” or numbness on or just underneath your skin.
What are the Pophryias that go along with Neuropsychiatric symptoms are
-ALA dehydrates deficiency porphyria (ADP)
-Acute intermittent porphyria (AIP)
What are typical Cutaneous symptoms of disorders of heme biosynthesis ?
What are the cause of Cutaneous symptoms?
photosensitivity, blisters, excess facial hair, hyperpigmentation
The cause is excess of porphyrin intermediates
What are the Porphyrias that are seen with Cutaneous symptoms?
-CEP (Congenital erythropoietic porphyria)
-PCT (Porphyria cutanea tarda)
-EPP (Erythropoietic portoporphyria)
-XLPP (E-linked protoporphyria )
With the Cutaneous symptoms, you get an increased fragility of light-exposed skin in which Porphyrias?
CEP and PCT
(congenital erythropoietic porphyria & Porphyria cutanea tarda)
**You get a Burning of light-exposed skin in which Porphyrias?
EPP and XLPP
(Erythropoietic protoporhyria and X-linked protoporphyria)
True or False: Photosensitizing effects of the porphyrins are attributed are attributed to absorption of light
True
–
So there is something going on in your skin. Fun fact this might be the reason for the legends of vampires, because if they go outside they burn .
True or False: Neurocutaneous symptoms have both neuro and cutaneous symptoms
True
Excess of both precursor compounds and porphyrin intermediates.
Which Porphyrias that have Neurocutaneous symptoms showing excess of BOTH precursor compounds and porphyrin intermediates?
-Hereditary coproporphyria (HCP)
-Variegate porphyria (VP)
These porphyrias are conditions with neurological symptoms and serous acute attacks.
acute porphyias
Usually the neurologiacal symptoms ones
Which acute porphyrias are responsible for neuroPSYCHiatric?
ADP, AIP
Which acute porphyrias are responsible for NEUROCUTANEous?
HCP, VP
Which porphyria is the most common acute porphyria?
AIP
(80% of individuals are asymptomatic)
Non-acute present as what conditions?
chronic conditions
mostly cutaneous
Non-acute present as chronic conditions (cutaneous) this includes
-CEP, PCT, EPP, XLPP
Which porphyria is the most common non-acute, present as chronic conditions?
PCT (Porphyria cutanea tarda)
Which non-acute present as chronic conditions porphyria is known for extreme photosensitivity ?
CEP
–
If you google it, you will find where sun light will actually destroy the skin, and can cause disfiguration of facial features fingers, nose, toes, ears, it will eat it away. You degrade based on the sun
What kind of porphyriurias acquired condition associated with increased excretion of urinary porphyrins?
Secondary Porphyriurias
Not due to inherited defect in heme synthesis, but it is caused by another disorder or toxin interfering with heme formation or metabolism
–
You are not genetically prone to it, but there is something else that is causing issues
True or False: Liver disease and heavy metal poisoning (lead) can cause excess urinary porphyrin excretion or if you have a inherited disorder of bilirubin metabolism
True
What are the initial testing of Acute porphyria?
-Inital testing - determination of PBG (Porphobilinogen) in urine
-If positive, measure the deficient enzyme PBG deaminase to identify AIP; analysis of fecal porphyrins to differentiate AIP, VP, and HCP
What are the initial testing of Cutaneous porphyria?
-Initial testing - measurement of porphyrins in random or timed urine
What different analytical methods are there available for Heme biosynthesis?
-Watson-Schwartz assay
-Liquid Chromatography (HPLC) with florescent detection
-Chomatographic Analysis
-Fecal HPLC with fluorescent detection
–
We don’t need to know which goes with which, but just know that they are available.
What is important when collecting sample for disorders of heme biosynthesis?
-Protect from light for it can degrade.
-Make sure unpreserved urine needs to be refrigerated (4 C) for 48 hrs like timed or random
-unpreserved urine is in freezer (-20 C) for several weeks (Send outs - depends on courier )
-Fecal can be frozen for several months
-Whole blood in EDTA is protected from light (for fridge )
–
1. Make sure you have the right kind of sample for what you are looking for.
What is the major role of Hemoglobin?
Major role is to transport oxygen to the tissue and CO2 transport back to the lungs
What is designed to to take up oxygen in areas of high oxygen tension and release of oxygen in areas of low oxygen tension
Hemoglobin
–
Drops off kids at the park
True or False: Hemoglobin is carried to all tissues of the body by erythrocytes.
True! RBCs!
This is one of the major buffering systems of the body
Hemoglobin
What is the structure of Hemoglobin
Large complex molecule roughly spherical 2 major parts:
- A central oxygen binding, non-protein heme molecule
-2 pairs of identical globin chains
The amino acid sequences of the global proteins vary and are the basis of fetal/adult chain nomenclature:
- alpha
- beta
- delta
- gamma
In adults the majority of Hgb in adults is
Hgb A or A1
–
(two alpha and two beta chains)
In less than 3% of normal adult hemoglobin is
Hgb A2
–
(two alpha and two delta chains)
In less than 1% of normal adult hemoglobin is
Hgb F
–
(Two alpha and two gamma chains)
***In Neonates, their hemoglobin is
Hgb F
(two alpha and two gamma chains)
Main Hgb during fetal life is what? It decreases to 60% at birth, and then decreases to 1% at 9 months of age
Hgb F
Genetic control of hemoglobin synthesis occurs in two areas..
- control of structure
- control of rate and quantity of production
Hemoglobinopathies occur when there is a defect in what?
structure of hemoglobin
Thalassemias occur when there is defect in what?
rate and quantity of production of hemoglobin
True or False: Each globin chain has its own genetic locus (own location), each individual chain is under genetic control and not the whole molecule
True
In most people the alpha gene is duplicated, so people will have
alpha1 and alpha2
–
where the A gene is identical and its that polypeptide chain in Hgb A, A2, and F
The non-alpha gene for the beta, delta, gamma chains are sufficiently close in genetic terms to be subjected to non-homologous crossover so in the result production of fused or hybrid globin chains such as…
- Hgb Lepore ( delta beta-globin chain)
- Hgb Kenya (gamma beta-globing chain
Hemoglobinopathies can be divided into 4 groups based on the genetics of the what chain production?
globin chain production
What are the 4 groups of hemoglobinopathies?
- Amino acid substitutions (e.g Hgbs S, C, D, E, O, G)
-Amino acid deletion (deletions of 3 or multiples of 3 nucleotides in DNA like Hgb Gun Hill)
-Elongated globin chains resulting from chain termination, frame shifts, or other mutations (eg Hgb Constant Spring)
-Fused or hybrid chains resulting from non-homologous c
What is the most common abnormalities group of hemoglobinopathies?
amino acid substitutions
Approx 2/3 of the hemoglobinopathies have an affect on which chain?
Beta chain
What is the definition of Thalassemias?
absent or diminished synthesis of one of the polypeptide chains of Hgb
What is the difference between alpha-thalassemia vs beta-thalassemia
-In alpha-thalassemia, alpha chain synthesis is absent or reduced.
-In Beta-thalassemia, beta-globin chain synthesis is absent or partially reduced
True or False: Hgb synthesis occurs in the immature RBCs in the bone marrow
True
What does normal synthesis of hemoglobin requires?
-Heme synthesis in the cell mitochondria
-Globin synthesis in the cytoplasm
-Adequate iron supply
Newly synthesized heme exits the mitochondria to where and to complex how many globin molescules?
-Newly synthesized heme exits the mitochondria to complex with 4 globin molecules in the cytoplasm
Iron gets transported to the what to be inserted into heme and to be able to bind to oxygen?
This is the last step
the developed RBCs
Where does degradation occurs?
Degradation occurs in either the blood vessels (intravascular hemolysis) or outside of the circulatory system in the phagocytic cells of the spleen, liver, and BM (extravascular hemolysis)
–
Depending on where they get degraded, depends the type of hemolysis
Lab measurement of hemoglobin degradation products helps to determine what?
-RBC destruction (hemolytic anemia)
What are the laboratory data and testing with Hemoglobin S?
-The most common hemoglobinopathy in the U.S.
-Microscopically - Sickle cells
-Testing: solubility test positive for both trait and disease
-Cellulose acetate with alkaline pH (Hgb S is between Hgb A and A2)
-Sickle cell trait (HbAS) or sickle cell disease (HbSS)
-Black African and African Americans have the highest incidence: 1 of 500 infants has the disease, 8-10% carry the trait
What are the laboratory data and testing with Hemoglobin C?
- Microscopically: target cells and crystalloid structures
-Testing: Cellulose acetate electrophoresis (Moves with Hgb A2, negative solubility test)
- 2-3% of African Americans of the U.S.
What are the laboratory data and testing with Hemoglobin SC?
-Most common mixed hemoglobinopathy
-One gene codes for ==
What are the laboratory data and testing with Hemoglobin E?
-Found in Asia, 20 million individuals
-Microscopically: homozygous form - microcytosis and target cells
-Testing:
—> Cellulose acetate: Hgb E moves with A2, C, and O
—> Citrate agar- Hgb E migrates with A
What are the laboratory data and testing with Hemoglobin D?
-Homozygous state is rare - usually found in Northwest India
-Microscopically: slight anisocytosis
-Testing:
—> Cellulose acetate - Hgb D migrates with Hgb S
—> Citrate agar - Hgb D migrates with A
A group of disease in which a defect causes reduced synthesis of one or more of the hemoglobin chains but the chains are structurally normal is known as ..
Thalassemias
A group of disease in which a defect causes reduced synthesis of one or more of the hemoglobin chains but the chains are structurally normal is known as ..
Thalassemias
In Thalassemias, Interaction among a large number of different molecular defects is the cause for what?
- decreased or absent globin chain synthesis
True or False: Any form of unbalanced production of globin chains causes the erythrocytes to be small, hypochromic and sometimes deformed.
True
This type of Thalassemia is heterozygous, clinically asymptomatic, and resembles iron deficiency
Thalassemia minor
This type of Thalassemia is usually lethal before birth or in childhood, early and continuous treatment allows survival into young adulthood with many complication.
Thalassemia major
How many principle types of different severity are there in alpha Thalassemias?
There are 4 principle types of different severity with deletions of 1-4 genes
- Hydrops fetalis - most clinically severe: total absence of alpha chain synthesis thus almost no oxygen transport to tissue, usually stillborn or die of hypoxia at birth
- Hemoglobin H diseases - alpha chain synthesis about 1/2 of B chain synthesis: moderate hemolytic anemia
- alpha thalassemia trait - two gene deletions; mild microcytic, hypochromic
- Silent carriers - missing only one gene, normal hgb production
Do Gene deletions cause Beta-Thalassemia?
No, mutations do
- Homozygous disease - thalassemia major or Cooley’s anemia
- Heterozygous disease - thalassemia minor
What are the 2 major subtypes of B Thalassemias?
- B+B chains produced in reduced amounts
- B0 is complete absence of B chains
This Beta Thalassemia is the most common; MCV low, sever anemia, basophilic stippling, target call, extreme poikilocytes, anisocytosis
Beta+ Thalassemia
This Beta Thalassemia is 10% homozygous, hgb low, severe anemia
B0 Thalassemia
This Beta Thalassemia is homozygous, crippling disease of CHILDHOOD; hypochromic, microcytic anemia; bone marrow compensates by expanding in size causing structural bone abnormalities
Thalassemia major
This is a heterozygous B-thalassemia, caused by inheritance of one thalassemia gene; RBC survival not shortened, usually asymptomatic, may have mild microcytic anemia, lab values resemble iron deficiency anemia
Thalassemia minor
True or False: Most hemoglobinopathies and thalassemias can be diagnosed by the use of: CBCs, blood smear evaluation, solubility test, and cellulose acetate electophresis.
True.
-Additonal tests that can be helpful: citrate agar electrophoresis, serum ferritin, newer techniques of HPLC, and isoelectric focusing (IEP)
-More complicated cases may use: alpha/Beta globin chain analysis, cation-exchange HPLC, or DNA technology testing
This test is a screening test for sickling hgbs
Solubility Test
-Based on the principle that sickling hemoglobin, in the deoxygenated state, is relatively insoluble and forms a precipitate when placed in a high-molarity phosphate buffer solution
-Cells are placed in a buffered solution and mixed. After 5 min if no sickling hemoglobin, you can read line through a card; if sickling hemoglobin is present, lines are not visible.
(You can’t see the lines of the paper thru the tube, because sickle cell does not dissolve the material)
-You can have false negative due to recent transfusions and infants with hgb F anemia
-You can’t differentiate between trait and disease
**What is the order of electrophoretic mobility of Cellulose Acetate Hemogloin Elecrophoresis from slowest to fastest?
-Crawl, Slow, Fast, Accelerated
-This is implemented in the pku’s for newborn screenings to reduce morbidity and mortality in fangs with sickle cell diesease
What are the analytical methods of Thalassemias
- Citrate Agar Elecrophoresis
- Hemoglobin A2 Quantitiation
- Acid Elution for Hemoglobin F
- Hemoglobin F Quantitation
True or False: Definitive diagnosis of some hemoglobinpathies and thalassemias that include combinations of genetic defects may required DNA analysis. Although, it is important to note special strength of DNA technology is in the prenatal diagnosis of thalassemia major
True
So if it is in the family, like the parent know that they are a carrier or both of them know, they would want to know/ do fetal screening tests to know if fetus has thalassemia major b/c that would influence their choices on treatments, how they would handle birth or their plans.
*** In Hemoglobin identification where is HgS located?
In between HbA2 and HgbF
*** In Hemoglobin identification where is HgS located?
In between HbA2 and HgbF
This heme protein is found in skeletal and cardiac muscle. It can reversibly bind to hemoglobin but only able to release under low oxygen tension, what is it?
Myoglobin
-Myoglobin acts as an oxygen carrier in the cytoplasm of the muscle cell
-Main role is to transport of oxygen form the muscle membrane to the mitochondria
-Extra reserve of oxygen to help exercising muscle maintain activity longer.
What is the clinical significance of Myoglobin?
-Damage to muscle will show increase levels in serum and urine
-Renal clearance is rapid - high concentrations may cause acute renal failure
-Myoglonin in urine will cross-react with hemoglobin on a dipstick causing a positive reaction
-Utility in Rhabdomylosis, myocardial infarction, and aid in diagnosis of hereditary progressive muscular dystrophy
What are the analystical methods for myoglobin?
-Fluorsence, chemiluinescence, immunochromatic
