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Heme & Hb

Acute Intermittent Porphyria

Inherited - autosomal dominant trait.
PBG-deaminase (UPG-I-synthase) is deficient (Enzyme -3 in heme synthesis)
Secondary increase in activity of ALA synthase due to the non effective end-product inhibition.
The levels of ALA and PBG are elevated in blood and urine.
As ALA & PBG are colorless compounds, urine is colorless when voided
But the color is increased on standing due to photo-oxidation of PBG to porphobilin.
Hence, urine samples for PBG estimation should be freshly collected and transported in dark bottles.
Porphyrins are not excreted or elevated in blood; so there is no photosensitivity.
Symptoms appear intermittently and they are quite vague. Hence, it is at times called the “little imitator”.
Most commonly, patients present with acute abdominal pain and vomiting.
The patients often land up with the surgeon as a case of acute abdomen and on several instances exploratory laparotomies had been done.
A preoperative ultrasonography (USG) may exclude conditions like appendicitis and intestinal obstruction.
No sex difference in gene carriage, but acute attacks occur with five times more frequency in women than in males.
Women have less severe manifestations before menarche and after menopause.
Thus, it is evident that the female sex hormones have a stimulatory effect on ALA synthase.
An attack is precipitated by starvation and symptoms are alleviated by a high carbohydrate diet.
Drugs like barbiturates, which are known to induce ALA synthase, can precipitate an attack.
Another group of patients presents with neuropsychiatric manifestations, fluctuating blood pressure (BP) and may have neurological manifestations like sensory and motor disturbances, confusion, and agitation.
Some patients may present with psychiatric problems and may be treated accordingly.

Vasudevan, DM; S., Sreekumari; Vaidyanathan, Kannan. Textbook Of Biochemistry For Medical Students (p. 365). Jaypee Brothers Medical Publishers.

2. Laboratory Diagnosis of Porphyria

Laboratory diagnosis is mainly based on detection of increased amounts of porphyrins and their precursors in urine and blood.
A fresh sample of urine protected from sunlight.
UV fluorescence is the best technique.
Under ultraviolet light; porphyrins in urine, will emit strong red fluorescence.
The presence of PBG in urine is detected by Watson Schwartz test, using Ehrlich’s reagent. Here, the persistent presence of a pink color after chloroform extraction of the color produced by urobilinogen (UBG) indicates a positive test for increased excretion of PBG in urine.
The diagnostic importance of the presence of heme precursors in urine is shown in Table 20.6.
Porphyrins and Absorption Bands In porphyrins (uroporphyrin, coproporphyrin, and protoporphyrin), the four pyrrole rings are joined by methenyl (-CH=) bridges and they are colored compounds. The iron atom in the center of porphyrin is hexavalent and bonded to the four pyrrole nitrogens by coordinate valencies. The double bonds are resonating and therefore keep shifting their position. When the Fe++ in heme gets Fe+++ form, hematin is formed, which loses the property of carrying the oxygen.
Heme is red in color, but hematin is dark brown. When Hb or porphyrin solutions are viewed through a spectroscope, the absorbed wavelengths are seen as dark bands.
All porphyrins will have an absorption band near 400 nm; this distinguishing band is called the Soret band, after its discoverer.
Table 20.7 shows the absorption bands of porphyrins.

Disease and enzyme defect Laboratory findings
ALAD-porphyria; (ALA dehydratase deficiency) (Enzyme 2) Urine ALA ++ Urine PBG normal RBC HMBS normal
AIP (HMBS or PBG Deaminase (enzyme 3) Urine ALA ++ Urine PBG ++ Plasma porphyrins normal
HCP; Coproporphyrinogen oxidase deficiency, enzyme 5 Urine ALA normal Urine PBG ++ Plasma porphyrins normal
VP; Protoporphyrinogen oxidase deficiency (Enzyme 6) Urine ALA normal Urine PBG +++ Urine porphyrins ++ Plasma porphyrins ++

Vasudevan, DM; S., Sreekumari; Vaidyanathan, Kannan. Textbook Of Biochemistry For Medical Students (p. 366). Jaypee Brothers Medical Publishers. Kindle Edition.

Regulation of Heme Synthesis

Major site of heme synthesis: erythroid cells 85% & heme is incorporated into Hb.
In Liver 15% & depends on metabolic needs & on the need for hemoproteins especially cytochromes P450 (CYP 450) required from drug metabolism and detoxification.
Hepatic ALA synthase is regulated by repression mechanism.
1. Heme inhibits the synthesis of ALA synthase by acting as a corepressor.
2. ALA synthase is allosterically inhibited by hematin.
3. Excess of free heme, the Fe++ is oxidized to Fe+++ (ferric),and forms hematin.
The compartmentalization of the enzymes of heme synthesis makes the regulation easier.
1. The rate-limiting enzyme is in the mitochondria.
2. The steps 1, 5, 6, and 7 are taking place inside mitochondria,
3. while the steps 2, 3, and 4 are in cytoplasm.
4. The rate of transport of ALA to the cytoplasm is influenced by hematin levels in cells.
Effect of drugs and poisons
1. drugs like barbiturates induce heme synthesis.
2. Barbiturates require the heme containing CYP 450 for their metabolism.
3. Two-thirds of the total heme synthesized is used for CYP 450 production.
The steps catalyzed by ferrochelatase and ALA dehydratase are inhibited by lead.
High cellular concentration of glucose prevents induction of ALA synthase.
1. Administration of glucose relieves the acute attack of porphyrias.
PBG deaminase and ferrochelatase also have a controlling effect on heme synthesis.
The ALAS has both erythroid and nonerythroid (hepatic) forms.
1. Erythroid form is called ALAS2; it is not induced by the drugs that affect ALAS1.
2. Erythroid form is not subject to feedback inhibition by heme.

Vasudevan, DM; S., Sreekumari; Vaidyanathan, Kannan. Textbook Of Biochemistry For Medical Students (p. 363). Jaypee Brothers Medical Publishers. Kindle Edition.

Steps in heme synthesis

Heme synthesis occurs partly in the mitochondria and partly in the cytoplasm. The process begins in the mitochondria because one of the precursors is found only there. Since this reaction is regulated in part by the concentration of heme, the final step (which produces the heme) is also mitochondrial. Many of the intermediate steps are cytoplasmic. Notice in the diagram of the pathway that there is a branch with no apparent useful endproduct.

delta-aminolevulinic acid synthase (ALA synthase).

The substrates are
1. succinyl CoA (from the tricarboxylic acid cycle)
2. glycine (from the general amino acid pool)
3. An essential cofactor is pyridoxal phosphate (vitamin B-6).
  1. The reaction is sensitive to nutritional deficiency of this vitamin.
  2. Drugs which are antagonistic to pyridoxal phosphate will inhibit it. Such drugs include
    1. penicillamine
    2. isoniazid (isonicotinic acid hydrazide)
The reaction occurs in two steps.
1. Condensation of succinyl CoA and glycine to form enzyme-bound alpha-amino-beta-ketoadipate.
2. Decarboxylation of alpha-amino-beta-ketoadipate to form delta-aminolevulinate.
This is the rate-limiting reaction of heme synthesis in all tissues, and it is therefore tightly regulated.
There are two major means of regulating the activity of the enzyme.
1. The first is by regulating the synthesis of the enzyme protein. This is important because its half life is only about one hour.
2. Enzyme synthesis is repressed by heme and hematin.
  1. It is stimulated by
    1. barbiturates (as a result, these drugs exacerbate certain porphyrias).
    2. steroids with a 4,5 double bond, such as testosterone and certain oral contraceptives. This double bond can be reduced by two different reductases to form either a 5-alpha or a 5-beta product. Only the 5-beta product affects synthesis of ALA synthase. Since the 5-beta reductase appears at puberty, some porphyrias are not manifested until this age.
3. The second control is feedback inhibition by heme and hematin, presumably by an allosteric mechanism.
4. Hence, heme has a dual role in decreasing its own rate of synthesis.
The product of the reaction, ALA, diffuses into the cytoplasm, where the next several steps of heme synthesis occur.

2. delta-aminolevulinic acid dehydratase (ALA dehydratase)

The ALA dehydratase reaction occurs in the cytoplasm; the product is porphobilinogen .
The substrates are two molecules of ALA.
The reaction is a condensation to form porphobilinogen, the first pyrrole.
Two molecules of water are released. The asymmetry of the reaction relative to the two molecules of substrate results in the pyrrole ring having two different substituent groups:
1. acetic acid
2. propionic acid.
ALA dehydratase is a -SH containing enzyme.
It is very susceptible to inhibition by heavy metals, especially lead.
increased urinary excretion of its substrate is a good indicator of lead poisoning.

3. uroporphyrinogen I synthase and uroporphyrinogen III cosynthase

Production of uroporphyrin III requires two enzymes. Uroporphyrinogen I synthase and uroporphyrinogen III cosynthase
The substrates are four molecules of porphobilinogen.
The first reaction is catalyzed by uroporphyrinogen I synthase.
1. The porphobilinogen molecules lose their amino groups.
2. A linear tetrapyrrole with alternating acetic acid and propionic acid groups is produced. This linear molecule cyclizes slowly (nonenzymatically) to yield uroporphyrinogen I. Without the second reaction (below), the heme synthesis pathway would end with porphyrinogens of the I series, which have no known function.
The second reaction is catalyzed by uroporphyrinogen III cosynthase.
1. This enzyme rapidly converts the alternating linear tetrapyrrole to the cyclic uroporphyrinogen III (pronounce), which has the substituents of its IV ring reversed: AP AP AP PA. This is the physiologically useful product.

4. Uroporphyrinogen decarboxylase

The physiologically significant substrate is uroporphyrinogen III.
The product is coproporphyrinogen III, which is transported back to the mitochondria, where the remainder of heme synthesis occurs.
The substituent pattern in the coproporphyrinogen is MP MP MP PM.
Uroporphyrinogen decarboxylase also acts on uroporphyrinogen I, yielding coproporphyrinogen I. Coproporphyrinogen I has no known function, and its formation is thought to be a blind pathway.

5. coproporphyrinogen III oxidase

The mitochondrial enzyme, coproporphyrinogen III oxidase, catalyzes the next reaction.
The substrate is coproporphyrinogen III.
The reaction is conversion of the propionic acid groups of rings I and III to vinyl groups. We now have the final substituent pattern of MV MV MP PM
The product is protoporphyrinogen IX.
1. (Some naming systems would call this protoporphyrinogen III to preserve the logic of the nomenclature, but "protoporphyrinogen III" is a departure from a time-honored tradition of referring to this and subsequent compounds by the number "IX.")

6. protoporphyrinogen IX oxidase

Protoporphyrinogen IX oxidase converts the methylene bridges between the pyrrole rings to methenyl bridges. Resonance of double bonds around the entire great ring, with its resulting stabilization, is now possible.

7. ferrochelatase

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