Porphyrins (Porphins) are cyclic tetrapyrol compounds formed by the linkage )). of four pyrrole rings through methenyl bridges (( HC In the reduced porphyrins (Porphyrinogens) the linkage of four pyrrole rings (tetrapyrol) through methylene bridges (( CH2 )) The characteristic property of porphyrins is the formation of complexes with the metal ion bound to nitrogen atoms of the pyrrole rings. e.g. Heme (iron porphyrin). Proteins which contain heme ((hemoproteins)) are widely distributed e.g. Hemoglobin, Myoglobin, Cytochromes, Catalase & Tryptophan pyrrolase. Natural porphyrins have substituent side chains on the eight hydrogen atoms numbered on the pyrrole rings. These side chains are: CH 1-Methyl-group (M)… (( 3 ))
2-Acetate-group (A)… (( CH2COOH ))
3-Propionate-group (P)… (( CH2CH2COOH ))
4-Vinyl-group (V)… (( CH CH2 ))
Porphyrins with asymmetric arrangement of the side chains are classified as type III porphyrins while those with symmetric arrangement of the side chains are classified as type I porphyrins. Only types I & III are present in nature & type III series is more important because it includes heme.
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Heme Biosynthesis Heme biosynthesis occurs through the following steps: 1-The starting reaction is the condensation between succinyl-CoA ((derived from citric acid cycle in the mitochondria)) & glycine, this reaction is a rate limiting reaction in the hepatic heme synthesis, it occurs in the mitochondria & is catalyzed by ALA synthase (Aminolevulinate synthase) enzyme in the presence of pyridoxal phosphate as a cofactor. The product of this reaction is α-amino-β-ketoadipate which is rapidly decarboxylated to form δ-aminolevulinate (ALA). 2-In the cytoplasm condensation reaction between two molecules of ALA is catalyzed by ALA dehydratase enzyme to form two molecules of water & one
2 molecule of porphobilinogen (PBG) which is a precursor of pyrrole. ALA dehydratase is a zinc-containing enzyme; it's inhibited by lead as occurred in lead poisoning. 3-Condensation of four molecules of PBG with loss of four molecules of ammonia lead to formation of linear tetrapyrrole called hydroxymethylbilane (HMB), this reaction is catalyzed by uroporphyrinogen I synthase (PBG deaminase, HMB synthase) enzyme. HMB cyclizes spontaneously to form uroporphyrinogen I or: 4- HMB converted to uroporphyrinogen III by the action of uroporphyrinogen III synthase enzyme, under normal conditions the uroporphyrinogen formed is almost exclusively the III isomer. Both of these uroporphyrinogens (I & III) contain A & P side chains & are colorless compounds, however, they readily reoxidized to their respective colored porphyrins (as in all porphyrinogens) by light with loss of six hydrogen atoms. 5-Uroporphyrinogen I & III are converted into coproporphyrinogen I & III by decarboxylation (loss of four CO2) of all of the acetate (A) groups to be converted into methyl (M) groups, this reaction is catalyzed by uroporphyrinogen decarboxylase enzyme. 6-Coproporphyrinogen III then enters the mitochondria to be converted into protoporphyrinogen III by oxidative decarboxylation of two (P) side chains into (V) side chains, this reaction is catalyzed by coproporphyrinogen oxidase enzyme, this enzyme acts only on coproporphyrinogen III, therefore, coproporphyrinogen I is not further progressed. Some times protoporphyrinogen III is also called protoporphyrinogen IX because it designated ninth in isomer. 7-Protoporphyrinogen III is oxidized into Protoporphyrin III ((loss of six hydrogen atoms), this reaction is catalyzed by protoporphyrinogen oxidase enzyme & it occurs in the mitochondria, however, this conversion can be induced by light in vitro. Some times protoporphyrin III is also called protoporphyrin IX. 8-The final step is the formation of heme by the incorporation of ferrous iron into protoporphyrin III in a reaction catalyzed by ferrochelatase (heme synthase) enzyme, this reaction occurs in the mitochondria. Heme synthesis occurs in most human cells with the exception of mature RBC because does not contain mitochondria, however, about 85% of heme synthesis occurs in erythroid precursors cells in the bone marrow to form hemoglobin & the majority of the remainder in the hepatocytes to form other hemoproteins mainly cytochrome P450.
3 ALA Synthase {ALAS} Two main forms: 1-Hepatic((ALAS 1)).. It is a regulatory (rate limiting) enzyme in the hepatic heme synthesis, heme acting as a negative regulator for this enzyme. Many drugs are porphyrogenic as barbiturates which can result in a marked increase of ALAS 1 activity by utilizing cytochrome P450 which in turn reduces the intracellular heme concentration lead to the activation of ALAS 1. Other factors can result in marked decrease of ALAS 1 activity as: A-Excess glucose which cause reduction of fat burn in which toxins are stored, therefore, excess glucose reduces toxin release so that the consumption of cytochrome P450 is reduced. B- Hematin (oxidized form of heme) leads to increase of intracellular heme concentration result in marked decrease of ALAS 1 activity. 2-Erythroid((ALAS 2)).. Its regulation is differ from that of ((ALAS 1)), therefore, it is not affected by drugs & does not undergoes feedback regulation by heme.
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Properties of Porphyrins 1-The various porphyrinogens are colorless, where as the various porphyrins are colored because it contains double bonds joining pyrrole rings which is absent in porphyrinogens, therefore, all porphyrins ((in 5% HCl)) but not porphyrinogens have a significant sharp absorbance band near 400 nm wavelength, this band is called Soret band ((discovered by the French physicist; Charles Soret))
2-When porphyrins is dissolved in strong mineral acids or in organic solvents & illuminated by ultraviolet light, they emit a strong red fluorescence because it contains double bonds joining pyrrole rings while porphyrinogens (because of absence of double bonds joining pyrrole rings) they have no fluorescence. 3-Coproporphyrins, uroporphyrins & protoporphyrins are of clinical importance because they are excreted in increased amounts in the porphyrias, these compounds when present in feces and/or urine; can be extracted & quantified using spectrophotometric methods. Generally uroporphyrins are present in urine; coproporphyrins are present in urine & feces while protoporphyrins are present in feces. 4-ALA & PBG can measured in urine by colorimetric method.
Porphyrias Porphyrias are group of disorders due to abnormalities of the heme biosynthesis pathway, they can be genetic or acquired. They are not prevalent; however, their importance lies in the differential diagnosis of certain clinical presentations as abdominal pain, neuropsychiatric & dermatological findings. Seven types of genetic porphyrias were found according to enzyme involved:
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Individuals with low activity of ALAS 2 develop anemia (not porphyria), inherited as X-linked condition. Low activity of ALA dehydratase is very rare while the remaining is more common. Generally porphyria is inherited as autosomal dominant except congenital erythropoietic porphyria is inherited as autosomal recessive. Clinical Features of Porphyria The clinical features of porphyria can result from a deficiency of metabolic products beyond the enzymatic block and/or from accumulation of metabolites before the block, the main clinical features are some or all of the followings: 1- Abdominal pain & neuropsychiatric presentations; It occurs mainly in acute intermittent porphyria. The exact biochemical cause of these symptoms has not been determined yet, however, probably may relate to elevated levels of ALA or PBG and /or deficiency of heme. 2- Photosensitivity; It occurs when porphyrinogens (as uroporphyrinogen, coproporphyrinogen & protoporphyrinogen) are accumulated ((as in porphyria cutanea tarda, hereditary coproporphyria, variegate porphyria & protoporphyria)) which undergo oxidation on exposure to light into corresponding porphyrins which are exited on exposure to visible light of wave length 400 nm, these excited porphyrins react with molecular oxygen to form oxygen radicals which damage lysosomes causing release of their lysosomal enzymes that cause photosensitivity.
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Classifications of Porphyrias *I-According to organs that are mostly affected: A-Erythropoietic porphyrias: heme synthesis in the bone marrow was affected as in congenital erythropoietic porphyria & protoporphyria. B-Hepatic porphyrias: heme synthesis in the liver was affected as in ALA dehydratase deficient porphyria, acute intermittent porphyria, porphyria cutanea tarda, hereditary coproporphyria & variegates porphyria.
*II-According to clinical presentations: A-Acute porphyrias: Patient presented with abdominal pain & neuropsychiatric symptoms. B-Cutaneous porphyrias: Patient presented with photosensitivity. C-Latent porphyrias: Patient is relatively symptoms-free between the attacks.
*III-According to cause: A-Genetic porphyrias: Inherited as autosomal dominant except congenital erythropoietic porphyria is inherited as autosomal recessive. B-Acquired porphyrias: as in lead poisoning can affect ALA dehydratase.
8 Diagnosis of Porphyrias 1-Clinical & family history. 2-Physical examination. 3-Laboratory tests include: A-Measure enzyme activity in blood. B-Urine test for uroporphyrins & coproporphyrins. C-Stool test for protoporphyrins. D-Urine test for ALA & PBG.
Treatment of Porphyrias 1-Gene therapy. 2-Avoid porphyrogenic drugs as barbiturates. 3- Administration of excess glucose & hematin that cause block of ALAS1. 4-Administration of β-carotene which is antioxidant to reduce free radicals formation, therefore, reduces photosensitivity. 5-Use of sunscreen that filter out visible light preventing the exposure of light with wave length 400 nm. 6-Sever pain is treated by analgesia.
Heme Catabolism Normally, senescent red blood cells & heme from other sources are engulfed by the cells of the reticuloendothelial system. The globins is recycled or converted into amino acids. Heme is converted into biliverdin ((green pigment)) by the action of endoplasmic reticulum enzyme complex known as microsomal heme oxygenase system through many sequential reaction steps involving reduction by NADPH, addition of oxygen with the release of ferric iron & CO ((only reaction in the body that is known to produce CO)). Finally by the action of biliverdin reductase biliverdin was converted into bilirubin ((yellow pigment)) which is transported to the liver for further metabolism. The daily bilirubin formation in human adult is about 250-350 mg, derived mainly from hemoglobin & to a less extent from other hemoproteins as cytochrome P450.
9 Bilirubin Transport of Bilirubin Bilirubin is mildly soluble in aqueous media (as in blood), however, its solubility is increased by binding to serum albumin. Each molecule of albumin has one high-affinity site & one low-affinity site for bilirubin binding. In 100 mL of blood, approximately 20-25 mg of bilirubin can be tightly bound to albumin at its high-affinity site, bilirubin in excess of this quantity can be bound only loosely on low-affinity site & thus can easily be detached & diffuse into tissues specially CNS causing kernicterus. A number of drugs such as antibiotics & other drugs compete with bilirubin for the high-affinity binding site on albumin. Thus, these compounds can displace bilirubin from albumin & so it has significant clinical effects.
Metabolism of Bilirubin The metabolism of bilirubin occurs through the following sequential steps: (1) Uptake of Bilirubin. (2) Conjugation of Bilirubin. (3) Secretion of Bilirubin into Bile.
(1) Uptake of Bilirubin In the liver, bilirubin is removed from albumin & taken up at the sinusoidal surface of the hepatocytes by a carrier-mediated system. Once bilirubin enters the hepatocytes, it binds to certain cytosolic proteins (Ligandin & protein Y), which help to keep it soluble prior to conjugation & to prevent it from the efflux back into the blood stream.
(2) Conjugation of Bilirubin Bilirubin is nonpolar & would persist in cells (e.g., bound to lipids) if not rendered water-soluble. Hepatocytes convert bilirubin to a polar form, which is readily excreted in the bile, by adding glucuronic acid molecules to it. This process is called conjugation. The conjugation of bilirubin is catalyzed by a specific enzyme called UDP-glucuronosyltransferase (uridine diphosphate- glucuronosyltransferase) or called Bilirubin-UGT (bilirubin-uridine diphosphate glucuronosyltransferase) which is mainly located in the endoplasmic reticulum, this enzyme transfer glucuronate from UDP-glucuronic acid to bilirubin forming bilirubin monoglucuronide which is an intermediate & is subsequently converted to the bilirubin diglucuronide.
10 Normally most of the bilirubin excreted in the human bile is in the form of bilirubin diglucuronide. However, when bilirubin conjugates exist abnormally high they become predominantly monoglucuronide. UDP-glucuronosyltransferase enzyme activity can be induced by a number of clinically useful drugs including phenobarbital.
(3) Secretion of Bilirubin into Bile Secretion of conjugated bilirubin into the bile occurs by an active transport mechanism, which is probably rate-limiting for the entire process of hepatic bilirubin metabolism. The protein involved in this process is called MRP-2 (multidrug resistance- like protein 2) or called multispecific organic anion transporter (MOAT) which is located in the canalicular membrane & handles a number of organic anions. The hepatic transport of conjugated bilirubin into the bile is induced by the same drugs that are capable of inducing the conjugation of bilirubin. Thus, the conjugation & excretion systems for bilirubin behave as a coordinated functional unit. As the conjugated bilirubin reaches the terminal ileum & large intestine, the glucuronides are removed by specific bacterial enzymes called β-glucuronidases & the bilirubin is subsequently reduced by the fecal flora to a group of colorless tetrapyrrolic compounds called urobilinogen. In the terminal ileum & large intestine a small fraction of the urobilinogen is reabsorbed & reexcreted through the liver to constitute the enterohepatic urobilinogen cycle while only very trace amount of urobilinogen appears in urine.
11 Under abnormal conditions particularly when excessive bilirubin is formed, urobilinogen may also be excreted in the urine in excess amount (excess urobilinoginuria). Normally, most of the colorless urobilinogen in the colon (in GIT urobilinogen is called stercobilinogen) are oxidized to a colored compound known as urobilin (in GIT urobilin is called stercobilin) & are excreted in the feces giving the brown color of stool.
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Reference Ranges of Serum Bilirubin In adult, the reference ranges of serum bilirubin are: Total Bilirubin = 0.3-1 mg/dL (5.1-17 µmol/L) Unconjugated (Indirect) Bilirubin = 0.2-0.7 mg/dL (3.4 -12 µmol/L) Conjugated (Direct) Bilirubin = 0.1-0.3 mg/dL (1.7 - 5.1 µmol/L) However, in newborn, the reference ranges is usually higher than that of the adult. 13 Hyperbilirubinemia Hyperbilirubinemia exists when serum total bilirubin concentration exceeds the upper limit of its reference range [1 mg/dL (17µmol/L)] & when it reaches a certain concentration (approximately 2.5-3 mg/dL); it diffuses into the tissues, which then become yellow. This condition is called jaundice or icterus. Hyperbilirubinemia may be due to:- 1- Overproduction of bilirubin above the capacity of the normal liver to metabolize it (Pre-hepatic hyperbilirubinemia) as in hemolysis. 2- Failure of a damaged liver to conjugate bilirubin produced in normal amounts (Hepatic hyperbilirubinemia). 3- Obstruction of the excretory ducts of the liver that preventing the excretion of bilirubin (Post-hepatic hyperbilirubinemia). Depending on the type of bilirubin present in plasma whether unconjugated (indirect) and/or conjugated (direct), hyperbilirubinemia may be classified as: 1- Unconjugated Hyperbilirubinemia (Retention Hyperbilirubinemia): Due to overproduction of unconjugated bilirubin lead to: A- Elevated level of serum unconjugated bilirubin (Indirect bilirubin) above its upper reference limit (in adult; 0.7 mg/dL). B- Since unconjugated bilirubin is hydrophobic, it can cross the blood-brain barrier into CNS (especially when serum unconjugated bilirubin level reach 20- 25 mg/dL) causing encephalopathy, this condition called kernicterus. C- Since unconjugated bilirubin is hydrophobic, it does not appear in urine. Accordingly, acholuric jaundice (absence of bilirubin in the urine) occurs. D- The increased production of bilirubin leads to increased production of urobilinogen which appears in the urine in large amounts (excess urobilinoginuria) while normally there are very traces amount of urobilinogen in the urine. 2-Conjugated Hyperbilirubinemia (Regurgitation Hyperbilirubinemia): Due to reflux of conjugated bilirubin into the bloodstream lead to: A- Elevated level of serum conjugated bilirubin (Direct bilirubin) above its upper reference limit (in adult; 0.3 mg/dL). B- Since conjugated bilirubin is hydrophilic, it cannot cross the blood-brain barrier so that there is no development of kernicterus. C- Since conjugated bilirubin is hydrophilic & cannot be excreted (due to biliary obstruction). It thus regurgitates from the liver & appears in the urine. Accordingly, choluric jaundice (presence of bilirubin in the urine) occurs, clinically the color of choluric urine is described as a tea-color. D- Since bilirubin has no access to the intestine (due to biliary obstruction) where it can be converted to urobilinogen, therefore, no urobilinogen (stercobilinogen) is found in the urine & feces, therefore, no urobilin (stercobilin) in the stool, the stool is clinically described as pale-stool.
14 3- Unconjugated & conjugated hyperbilirubinemia: Due to defect in both conjugation & secretion of bilirubin as occur mainly in hepatitis.
Examples of Unconjugated Hyperbilirubinemia:
1) - Neonatal (Physiologic) Jaundice. This transient condition is the most common cause of unconjugated hyperbilirubinemia in neonate. It results from an accelerated hemolysis around the time of birth & an immature hepatic system for bilirubin metabolism. Since the increased amount of bilirubin is unconjugated, it's capable of penetrating the blood-brain barrier when its concentration in plasma exceeds that which can be tightly bound by albumin (20–25 mg/dL) causing kernicterus. Treatment 1- Phenobarbital administration because it induce bilirubin-metabolizing system. 2- Exposure to blue light (phototherapy) promotes the hepatic excretion of unconjugated bilirubin by converting some of the unconjugated bilirubin to other derivatives that are excreted in the bile. 3- Exchange blood transfusion when plasma bilirubin exceeds that which can be tightly bound by albumin (20–25 mg/dL) to avoid kernicterus.
2) - Hemolytic Anemias. Unconjugated hyperbilirubinemia is usually slight (< 4 mg/dL).
3)- Viral and toxic hepatitis and cirrhosis. Unconjugated hyperbilirubinemia can result from toxin-induced liver dysfunction causing impaired conjugation such as that caused by chloroform, arsphenamines, carbon tetrachloride, acetaminophen, cirrhosis & Amanita mushroom poisoning.
Example of Conjugated Hyperbilirubinemia:
Extrahepatic obstruction: Blockage of the hepatic or common bile ducts, most often due to a gallstone or to cancer of the head of pancreas or spasm and stricture.
Example of Conjugated & Unconjugated Hyperbilirubinemia:
Intrahepatic obstruction: Microobstruction of intrahepatic biliary ductules by swollen damaged hepatocytes as may occur in infectious hepatitis and edema.
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