Porphyrins and Hemoglobin Flashcards
chemical intermediates of hemoglobin, myoglobin,
cytochromes, and enzymes (catalase and peroxidase)
Porphyrins
What causes red color of hemoglobin?
presence of alternating double bonds of porphyrins allows the structure to absorb light
All porphyrins are macrocyclic structures consisting of a _________ ring linked by______ bridges.
tetrapyrrole, methyl
Porphyrins also fluoresce reddish-pink at ____ nm
400
Four ____ atoms chelate metal atoms (usually iron, competes with zinc)
▪ Iron chelates with protoporhyrin IX to form heme
Nitrogen
Iron in heme can also bind to globin proteins to form ________ &
____________.
HEMOGLOBIN &
MYOGLOBIN
fights against reactive oxygen species
catalase
hemoglobin, myoglobin, and
cytochromes all contain…
heme
Heme synthesis occurs in all cells, but primarily
____________(erythroidprecursors) and ______.
bone marrow, liver
Iron chelates with ________________ to form heme
protoporhyrin IX
Enzymes in heme synthesis localized in…
mitochondria and cytoplasm
what are the Mitochondrial enzymes involved with heme synthesis?
1) ALA synthase, 2) copropophyinogen oxidase, 3)
protoporhyrinoghen oxidase, and 4) ferriochelatase
What are the Cytoplasmic enzymes involved with heme synthesis?
1) ALA dehydrogenase, 2) PBG deaminase, 3)
Uroporphyrinogen III synthetase, and 4) Uroporphyrinoghen decarboxylase
ALA & PBG are ________soluble
water
precursors to porphyrins
▪ Porphyrinogens
these spontaneously oxidize (with light oxygen or oxidizing agents) to form three major porphyrins:
Porphyrinogens
three major porphyrins…
- Uroporphyrin [URO]: excreted in urine
- Coproporphyrin [CORPO]: excreted in urine and/or feces
- Protoporphyrin [PROTO]: excreted in feces
porphyrin that is excreated in urine?
Uroporphyrin [URO]
porphyrin excreted in urine and/or feces
Coproporphyrin [CORPO]
porphyrin that is excreted in feces?
Protoporphyrin [PROTO]
▪Porphyria can be inherited or caused by…
drugs, liver disease, anemias, or toxic
metals that cause disturbances in heme synthesis and porphyrin metabolism
How many enzymes are involved in heme synthesis?
8
Deficiency in seven of the eight enzymes involved in heme synthesis
! Leading to a distinct form of porphyria
Lack of enzyme activity –> decrease in formation and excretion of porphyrin or
accumulation of its __________.
precursors
Symptoms of Neurological porphyria?
Constipation, nausea, vomiting, severe
abdominal pain, tachycardia, hypertension,
sweating and urinary retention,
*neurotoxicity, psychiatric disturbances,
Cutaneous lesions, hyperpigmentation
Neurological porphyria:
Acute disturbances of porphyrin synthesis that allow accumulation of early precursors (____ and _____)
ALA, PBG
excess accumulation of all three porphyrias may cause…
photosensitivity, cutaneous lesions, and hyperpigmentation
As porphyrin molecules absorb light in
skin, electrons become excited and
eventually decay to ground state
-transferring energy to molecular oxygen
-activated oxygen species
Cutaneous porphyria
If ALA and PBG are elevated it is mostly likely what type of porphyria?
neurological porphyria
What type of porphyria causes the most skin damage?
protoporphyrin (not secreted in urine)
-Most common porphyria
-barbiturates and sulfonamides
-increased Urinary ALA, PBG, URO
Acute intermittent porphyria
Urine may turn red or black after exposure to air due to spontaneous polymerization
of PBG to porphyrin
Urine may turn red or black after exposure to air due to spontaneous polymerization
of PBG to porphyrin
NO photosensitivity with Acute intermittent porphyria, why?
URO won’t be accumulated in the tissue
Variegate porphyria is due to issues with what enzyme?
protopophyrinogen oxidase
major symptom of Variegate porphyria?
Photosensitivity
Urine: ALA URO; Feces: PROTO, COPRO
Variegate porphyria:
Photosensitivity, but less than Variegated porphyria
Hereditary coproporphyria
Urine: ALA, URO; Feces: COPRO
Hereditary coproporphyria
Porphyria cutanea tarda is due to an issue with what enzyme?
uroporphyrinogen decarboxylase
Hepatoerythropoietic porphyria
Porphyria cutanea tarda
Feces: Isocoproporphyrine, Urine:URO
Porphyria cutanea tarda
Protoporphyria is an issue with what enzyme?
Ferrochelatase (partial)
RBC, plasma, feces: PROTO, Urine: normal porphyrin
Erythropietic Protoporphyria
Protoporphyria
Congenital erythropoietic porphyria is due to an issue with what enzyme?
Uroporphyrinogen III synthase
Feces: porphyrins (Corpo, prorp), RBC, urine: increased URO I and COPRO I
Congenital erythropoietic porphyria
Erythrodontia (reddish-brown discoloring of teeth):
Disfigurement and tendency to avoid daylight
Erythrodontia (reddish-brown discoloring of teeth):
Disfigurement and tendency to avoid daylight
Unknown enzyme defect, probably coproporphyrinogen
oxidase
Erythropoietic coproporphyria
increased RBC coproporphyrin & fecal COPRO (key finding )
Erythropoietic coproporphyria
acquired porphyria (secondary disorders) are a result of…
toxins or drugs
How is acquired porphyria distinguished from true porphyrias?
only urinary ALA excretion is increased
but PBG remains normal.
Lead poisoning inhibits….
inhibits ALA dehydratase and ferrochelatase
clinically presents with increased RBC Zn-protoporphyrins and increased urinary COPRO.
Lead poisoning
decreased iron in blood —-> __________ protoporphyrin
increased
most common cause of increased RBC porphyrins
Iron deficiency
What are the analytes in the disease of porphyrin metabolism
Porphobilinogen (PBG)
Urinary ALA
Fecal porphyrins
Urine porphyrins
Red cell porphyrins
Enzyme assays
Molecular genetics
Screening method of choice for PBG is __________.
Watson-Schwartz
____________ facilitates the movement of O2 from blood to muscle and serves
as a reserve O2 supply
Myoglobin
Consists of a single protein chain & one heme group
▪ Located in the muscle [skeletal & cardiac]
▪ Detection method: Immunoassay
Myoglobin
in general, Serum & Urine myoglobin: elevated due to…
Muscle damage
Historically, it was used to monitor Severe muscle tissue damage in crush injuries
and used to aid in diagnosis and monitoring of Muscle dystrophy
Myoglobin
is used to diagnose Acute MYOCARDIAL INFARCTIONS (MI)
Myoglobin
myoglobin is used to detect SUCCESSFUL _____________following tPA therapy in AMI
case
REPERFUSION
Can also be elevated in cases of open heart surgery, exhaustive exercise,
shock or severe renal failure
Myoglobin
It is small and filters readily into the urine.
▪ Urine will turn red-dark brown.
✦ Dipstick method
Myoglobin in urine
Free plasma Hb: __________ in intravascular hemolysis
increases
-Heart valve damage
- Complement fixation
Hb (α-chain) + Haptoglobbin
-Transported to the liver
-Cleared
Uncleared Free plasma Hb is oxidized to…
metHb
In IDA,
iron =
ferritin =
TIBC =
decreased
decreased
increased
myocardial infarction:
after ____ hours, myoglobin is no longer useful. (used only for acute!)
24
myocardial infarction:
What peaks at 16 hours and is back to normal in 2 days?
CK-MB
myocardial infarction:
What marker is widely used for chest pain? stays elevated for a week.
Troponin I
Uncleared Free plasma Hb is oxidized to _________.
metHb
“methemoglobinemia”
over ____% of total Hb
30
▪ At high concentrations, metHb results in…
hypoxia and cyanosis
Form of Hb in which Fe2+ ! Fe3+
▪Found in oxygenated or deoxygenated form
▪ It does NOT reversibly bind to the O2
Methemoglobin (metHb)
In normal individuals, ________________ will reduce Fe3+ to Fe2+
so that metHb is less than 1% of total Hb.
NADH metHb reductase*
- NADH metHb reductase
- Reverse NADPH metHb reductase
- Antioxidant Vitamins: Ascorbic acid (Vit C)
- **Glutathione (GSH)
Acquired _______ in normal individuals after exposure to agents such as
nitrites, nitrates, sulfonamides, aniline dyes and pyridium.
MetHb
*these oxides irons exceed the body’s capacity to reduce iron
Individuals with hereditary HbM have persistent levels of metHb which can vary. What does this lead to?
- Leading to the NADH metHb reductase deficiency 2. AA substitution in globin chain (heme pocket) Tyr ! His
Is there hemolytic anemia with acquired MetHb?
NO
How is MetHb assessed?
Spectrophotometry, HPLC, MS
MetHb is detected by reading absorbance at 630 nm.
SulfHb is the result of sulfur binding to hemoglobin. Is it reversible?
No
What can cause sulfhemoglobin?
Caused by exposure to sulfonamides or TNT in some individuals
Phenacetin, acetanilid (FDA prohibited painkiller)
SulfHb causes apoxia and cyanosis, but rarely causes __________ formation
Heinz body
CarboxyHb is the result of ______________ exposure.
carbon monoxide (CO)
Complex is greatly favored and binding is reversible, but very slow.
Usually caused by exposure to automobile exhaust, industrial pollutants, tobacco smoke, etc.
Hyperbolic treatment can displace CO from hemoglobin
CarboxyHb is the result of carbon monoxide (CO) exposure.
Primarily Functions of hemoglobin?
Transport O2 from lungs to tissues.
Acts as pH buffer
hemoglobin is a tetramer that can carry ___ oxygens.
4
what are the Four globin units?
2 α’s (α or ζ) and 2 β’s (ε, γ, β, or δ)
Hb composition for adults?
HbA(α2 β2), HbA2 (α2 δ2)
Fetal Hb composition?
HbF (α2γ2)
Each hemoglobin composition has its unique affinity for ____
O2
Embryonic hemoglobin composition?
Gower 1 (ζ2 ε2), Gower 2 (α2 ε2), Hb Portland (ζ2 γ2)
What are the buffers in the body?
-bicarbonate
-phosphorous
-hemoglobin
-albumin (minor but still important)
How many different hemoglobin defects are there?
over 700
Hemoglobin:
Defects in structure produce a group of diseases called…
Defect in rate of synthesis or amount of α or β-globin is called…
hemoglobinopathies
“thalassemia”
Structural abnormalities of hemoglobin are divided into what four groups?
- AA substitutions
: common ones (e.g. HbM, HbS, HbC, HbD, HbE, HbO, HbG, etc) - AA deletion
(e.g. Hb GunHill) - Lack of protein chain termination signal or frame shift:
: Elongated globulin chains (e.g. Hb Constant Spring) - Fused or hybrid chains (Hb Lepore or Hb Kenya)
heptoglobin helps recycle…
iron
Sickle cell mutation. Intracellular crystals of deoxygenated
HbS form causing cells to adapt a sickle shape
what mutation causes this?
HbS (β6 glu –> val)
Sickle cell disease similar to HbS. Complications seem to be
less frequent than with HbS
what mutation causes this?
HbC (β6 glu —-> lys)
Second most common abnormality in the world. “Thalassemictype” expression of β causes microcytic cells & normochromic anemia.
what mutation causes this?
HbE (β26 glu —> lys)
No anemia, but O2 affinity is higher than normal
what mutation causes this?
HbD (β121glu —-> gly)
Thalassemia of α-chains results in the tetramerization of __________________.
β chains
What is Hb (Bart’s)?
γ4 tetramerization
Diminished α-chains synthesis: excess γ chain synthesis**
What is HbH?
β4 tetramerization.
Diminished α-chain synthesis: excess β chain synthesis**
________ is glycosylated hemoglobin
Typically due to excess carbohydrates in patients.
Useful for monitoring DM patients.
HbA1c***
The elevation of GHb occurs about ___ wks after sustained elevation in blood glucose
3-4
What measures how well diabetes is being managed in the last month?
Hemoglobin A1c
Hemoglobin A1c:
- Good diabetic control: ___%
- Fair diabetic control : ____%
- Poor diabetic control : ____%
7, 10, 13-20
Hemoglobin A1c:
normal range for non DM….
pre DM range…
4-6
5.7-6.4
what are the Hemoglobin Methods?
Electrophoresis – KNOW THESE PATTERNS***
Molecular
Chemical
HPLC
Immunological
Electrophoresis:
Protein migration is from __________ to __________.
cathode (-) , anode (+)
In the Cellulose Acetate (pH 8.4), Hbs are _________ charged
negatively
In Citrate Agar (pH 6.0), some Hbs are ___________ charged
positively
What is the pH for Citrate Agar and Cellulose Acetate?
Citrate- pH 6
Cellulose Acetate- pH 8.4
Electrophoresis:
Where is it loaded for cellulose acetate?
Where is it loaded for citrate agar?
negative end
middle
What are the methods for hemoglobin?
-Molecular (DNA sequencing)
-Chemical
-HPLC (Column chromatography can be used to quantify A2 in β- thalassemias)
DNA sequencing is not useful for indicating….
severity of disease
Column chromatography can be used to quantify A2 in β- thalassemias.
A2 elutes from DEAE column first, elution of other Hb’s requires higher ionic strength
buffer.
Elutes measured at 415nm and A2 is expressed as % of total Hb.
HPLC
