Chemistry Lecture 10: LMU

Heme Synthesis Porphyrias Oxygen Dissociation Curve
Porphyrin Structure & Function
-A central ring structure consisting of four pyrrole units joined together
-This structure is often described as a tetrapyrole to emphasize its four-rings within a ring pattern
--This gives Hgb its red color
-The porrphyrin ring has four nitrogen atoms at the center (like prongs of a diamond ring) that chelates iron
-Free porphyrins have no biological function of their own
Metal Chelation
-The arrangement of the four nitrogen atoms in the center of the porphyrin ring enables porphyrins to chelate various metal ions, including iron
__________ inserts Fe2+ (ferrous) iron into the ___________ ____ complex
-Protoporphyrin IX

--This new complex is called heme
Optical properties
-Concentrated porphyrin solutions are dark red to purple in color
-The Soret band:
--Porphyrins show a very strong abs near the 400 nm range
--When exposed to light near the 400 nm wavelength, porphyrins display a characteristic orange-red fluorescence in the 550-650 nm range
In humans, there are 3 major porphyrins:
-Uroporphyrin (URO)
-Coproporphyrin (COPRO)
-Protoporphyrin (PROTO)
Heme Synthesis:
-occurs to the greatest extent in the bone marrow and the liver
-8 steps in the process, each of which is catalyzed by a different enzyme
Heme Synthesis: Step 1
-ALA Synthase catalyzes the formation of ALA from glycine and succinyl CoA
--Aminolevulinic acid
--5'-aminolevulinic acid
--delta-aminolevulinic acid
-ALA synthase is the rate-limiting step in the formation of heme and is controlled tightly by a number of mechanisms
HS: Step 2
-Porphobilinogen synthase forms porphobilinogen from two molecules of ALA by removing water during the reaction
-Two molecules of ALA form porphobilinogen (PBG)
HS: Step 3
-Hydroxymethylbilane synthase, formally called uroporphyrinogen-I synthase, combines 4 molecules of PBG to form hydroxymethylbilane
HS: Step 4
-Uroporphyrinogen-III synthase catalyzes the conversion of hydroxymethylbilane to uroporphyrinogen
HS: Step 5
-Uroporphyrinogen decarboxylase decarboylates uroporphyrinogen to coproporphyrinogen
HS: Step 6
-Coproporphyrinogen oxidase catalyzes the oxidation of coproporphyrinogen to protoporphyrinogen
HS: Step 7
-Protoporphyrinogen oxidase oxidizes protoporphyrinogen to protoporphyrin
HS: Step 8
-Ferrochelatase inserts Ferrous iron (Fe 2+) into protoporphyrin IX to form heme
--ferrochelatase is specific fro ferrous iron (Fe 2+) and it can't place ferric iron (Fe 3+) into the protoporphyrin ring
--It can place Zn 2+ into the protoporphyrin to form zinc protoporphyrin (ZPP). (Zinc competes with iron)
Heme Synthesis
Diagnosis of Porphyrias
-The excretion of excess porphyrins and their precursors is the basis for diagnosing the prophyrias.
--URO: Most soluble; excreted almost entirely in the urine
---URO: urine
--PROTO: Found most often in stool but can be found in uring
--PROTO: poop
--COPRO: Both urine and stool
---COPRO: can do either
--ALA (delta-aminolevulinic acid) and PBG (porphobilinogen) excreted in the urine
Types of Porphyrias
-Neurological Porphyrias
--Acute Intermittent Porphyria
--Variegate Porphyria
--Hereditary Coproporphyria
-Cutaneous Porphyrias
--Porphyria Cutanea Tarda
--Congenital Erythropoietic Porphyria
Neurological (Neuropsychiatric) Porphyrias
-During the acute neurological attack phase, PBG is excreted in the urine in marked excess.
-If an acute porphyric neurologic attack is suspected, analysis should include a porphobilinogen screen
**Watson-Schwartz Screen**
-The method of choice for the diagnosis of a protracted attack of porphyria is a HPLC analysis or porphobilinogen on a 24-hour urine specimen
Ehrlich's Tube Test Review
-Addition of Ehrlick's reagent to urine containing uronilinogen producesa cherry-red color, as so the Ehlrich's-reactive compounds
-Serves as the basis of the Waston-Schwartz screening test for porphobilinogen
Watson-Schwartz Differentiation Test
-Differentiates urobilinogen from porphobilinogen
-After the addition of Ehrlich's reagent, the specimen is divided into two tubes: Butanol & Chloroform
-Urobilinogen is soluble is both chloroform & butanol:
--Clear urine layers
--Red Chloroform & red butanol layers

-Porphobilinogen is insoluble in both:
--Red urine layers
--Clear chloroform & clear butanol layers
Neurological/Psychiatric Forms of Porphyria
-Abdominal Pain
-Chest Pain
Acute Intermittent Porphyria
-Enzyme defect: Hydrozymethylbilane synthase, formally called uroporphyrinogen-I synthase
--Analysis of RBCs for uroprophyrinogen-I-synthetase detects AIP
-Most common type of neurological porphyrias

Lab Findings:
-High ALA & PBG concentration in the urine during attacks
-High URO
-Normal PROTO
Variegate Porphyria
-Enzyme defect: Protoporphyrinogen oxidase
-Photosensitvity and skin lesions
-Common among South African whites

Lab Findings:
-Urine: High ALA & PBG, High URO
-Stool: High PROTO & COPRO
Hereditary coproporphyria
-Enzyme defect: Coproporphyrinogen oxidase
-Photosensitivity & Skin lesions

Lab Findings:
-Urine: High ALA & PBG, High URO
-Stool: High COPRO

The detection of carriers of hereditary coproporphyria should include analysis of a fresh morning urine for ALA (delta-aminolevulinic acid)
Porphyria Cutanea Tarda
-Most common porphyrin associated with photosensitvity
--Skin disorders generally appears in middle-age
-Enzyme defect: Uroporphyrinogen decarboxylase
-Fragile skin, blister formation, thickening, and scarring of sun-exposed skin, and areas of hyperpigmentation

Lab Findings:
-Urine: High URO
-Stool: Presence of isocoproporphyrin, an isomer of COPRO, is distinctive for this porphyria
-Enzyme defect: Ferrochelatase, the lase enzyme is heme synthesis
-Photosensitive, itching, burning, redness of skin

Lab Findings:
-Urine porphyrins are normal
-Stool: Fecal PROTO is usually increased
Congenital Erythropoietic Porphyria
-Worst prognosis of all porphyrias
-Enzyme defect: Uroporphyrinogen-III synthase (decarboxylase)
-Usually presents in early childhood with extreme photosensivity
-Reddish-brown teeth due to porphyrin deposition
-Hemolytic anemia & enlargement of the spleen can develop

Lab Findings:
-Urine is pink or red from massive amounts of URO and COPRO
-RBCs contain large amounts of URO & COPRO and fluoresce when examined under UV light
-Fecal PROTO are increased:
--Fecal porphyrin analysis by thin-layer chromatography will reveal erythrocytic protoporphyria
Tips for Porphyrin Case Studies:
1) Is the ALA and PBG elevated in the urine?
-Yes: Neurological porphyria
--The random urine PBG is the primary test for neurological porphyrias:

-No: Cutaneous Porphyria
*Porphyria cutanea tarda
*Congenital erythropoietic porphyria

2) Is isocoproporphyrin present?
-Yes: Porphyria cuntanea tarda
Neurological Porphyrias
-HIGH ALA, PBG, URO: Acute intermittent porphyria

-HIGH ALA, PBG, URO, PROTO, COPRO: Variegate porphyria

-HIGH ALA, PBG, URO, COPRO: Hereditary coproporphyria
Cutaneous porphyrias all have normal:
Cutaneous Porphyrias

-HIGH PROTO: Protoporphyria

-HIGH URO, COPRO, PROTO WITH A PINK/RED URINE: Congenital erythropoietic porphyria
Secondary Disorders of Porphyrin Metabolism
Lead Poisoning:
-Lead paint chips, "moonshine"
-Lead inhibits two enzymes in heme synthesis
-Diagnosis: Whole blood lead concentration

Iron Deficiency:
-Iron is not readily available for heme synthesis
-Diagnosis: Serum iron, TIBC, and ferritin
Lab Test for the Detection of the Porphyrias
-Neurological Porphyrias
*Random urine PBG as a screen
*24 hours urine as a quantitative test

-Acquired "Secondary" Porphyria (Lead Poisoning)
*Whole blood lead test
Basic Terminology
Hgb: A red colored oxygen carrying protein found in RBCs/ Combination of heme, globin, and iron

Anemia: Low concentrations of Hgb in the blood

Heme: An iron-containing porphyrin derivative that gives hemoglobin its red color

Globin: The polypeptide chains without the heme of Hgb
Basic structure & function of Hgb
- Contains two pairs of different globin chains
- Each chain carries an iron-containing prophyrin derivative called heme in which one iron atom is bound in the center of porphyrin ring
- Normal mammalian Hgb contains two pairs of chains:
*Two alpha
*Two non-alpha (beta,gamma,delta, etc)
Hgb Sythesis
-Hgb Synthesis is normally stimulated by tissue hypoxia (low oxygen content)
- Hypoxia stimulates the kidneys to produce EPO, which stimulates the production of Hgb and RBCs
- As EPO increases, the bone marrow is stimulated to produce and release increased amounts of RBCs. When the RBC reaches normal, EPO is "shut-off" (negative feedback loop)
- Androgens and thyroid hormones also enhance erythropoiesis
-Normal: 5-36 mlU/mL
-Glycoprotein hormone produced by the kidneys that regulates erythropoiesis
-Morning values are higher than afternoon values because of dinural rhythm of secretion
-Neg feedback loop

-High EPO levels:
*anemias, renal tumors, pregnancy, 2nd polycythemia
-Low EPO levels;
*renal failure, primary polycythemia
Hgb-Oxygen Dissociation Curve
-To fullfill its function, Hgb must specifically bind oxygen molecules with high affinity, transport them, and unload them at the tissues
-Hgb's affinity for oxygen is not a constant but rather is dependent upon several factors:
*2,3 DPG
Summary of the Hgb-Oxygen Dissociation Curve
-Temp: increased temp causes decreased affinity and right shift. Vice Versa
-pH: decreased pH, decreased affinity, right shift, vice-versa
-pCO2: increased pCO2, decreased affinity, right shift, vice versa
-2,3 DPG: increased concentration, decreased affinity, right shift, vice-versa
The Bohr Effect
-Hgb's affinity for oxygen varies with pH
-In the lungs:
*Four molecules of oxygen are taken up, and two protons are released
-At the tissues;
*Two protons are taken up for every four molecules of oxygen released
-This reciprocal action between protons and oxygen is known as the Bohr Effect
-It is essential in oxygen transport and the buffering carbon dioxide