Porphyrias: diseases related to heme biosynthesis

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      Congenital or acquired disturbances of heme biosynthesis are called porphyrias. The reason why these disturbances may be congenital or acquired is that the same enzyme may be mutated or, in some cases, may be inhibited by exernal poisons, usually as a result of professional intoxications. The heme (iron protoporphyrin IX) is the prostethic group of hemoglobin and myoglobin; heme variants are present in many respiratory enzymes. The heme is so important that absence of its biosynthesis is incompatible with life. As a consequennce in porphyrias the biosynthesis is deranged but not blocked (as blockage would cause intrauterine death at an early stage of embrionic development rather than a congenital disease), and symptoms are due to accumulation of wrong products or precursors in the tissues. Heme precursors or wrong metabolytes accumulate mainly in the skin, causing cutaneous porphyrias and/or in the peripheral nerves, causing neurological porphyrias.

Audio: definition of porphyrias

      The main clinical symptom of cutaneous porphyrias is photosensytivity; the main symptom of neurological porphyrias is pain. The following table collects the different types of porphyrias, ordering them according to their main symptoms: neurological, mixed, and cutaneous.
DiseaseEnzymatic defectInheritance Symptoms
Acute Intermittent P.Uroporphyrinogen synthase Dominant Neu
Hereditary CoproporphyriaCoproporphyrinogen oxidase Dominant (Skin)/Neu
Variegate P.Protoporphyrinogen oxidase Dominant (Skin)/Neu
Congenital Erythropoietic P.Uroporphyrinogen III cosynthase Recessive Skin
P. cutanea tardaUroporphyrinogen decarboxylase Dominant or acquired Skin
Erythropoietic ProtoporphyriaFerrochelatase Dominant Skin
Symptoms: skin=major cutaneous lesions due to photosensitivity; Neu=neurological symptoms (mainly pain); (Skin)/Neu=minor cutaneous symptoms associated to major neurological symptoms
Pay attention not to confuse the similarly named congenital erythropoietic porphyria and erythropoietic PROTOporphyria!

Audio: classification of porphyrias

      In order to properly locate each disease along the heme biosynthesis pathway, and thus to identify the precursor(s) that accumulate, or the wrong metabolyte(s) that may be produced you may refer to the following picture that summarizes the complex sequence of reactions that, starting from glycine and succinyl-CoA, leads to heme biosynthesis. The process involves 8 main enzymes, and occurs partly in the mitochondrion and partly in the cytoplasm, thus membrane transporters are also necessary.
      The enzymes of heme biosynthesis have been named and renamed several times and some confusion exists in the literature. The first step of heme biosynthesis is the production of δ-aminolevulinic acid (ALA) from glycine and succinyl-CoA (from the Krebs cycle); the reaction is catalyzed by ALA synthaseand occurs in the mitochondrion. Two molecules of ALA are combined by the enzyme porphobilinogen synthase (also called ALA dehydratase) to yield porphobilinogen (PBG), a pyrrole derivative. Four molecules of PBG are deaminated and joined together to form hydroxymethyl bilane (HMB), a linear tetrapyrrole. The enzyme is HMB synthase (formerly called uroporphyrinogen synthase). HMB would spontaneously close to the tetrapyrrole ring of uroporphyriongen I, but is instead processed by uroporphyrinogen III cosynthase that inverts the orientation of ring D and closes the tetrapyrrole ring as uroporphyrinogen III (notice that the two propionate residues of pyrroles C and D are adjacent to each other in uroporphyrinogen III while thay are not in HMB). A series of decarboxylation and oxidation reaction convert uroporphyrinogen III to protoporphyrin IX to which iron is finally added by ferrochelatase.

Audio: heme biosynthesis

      It is important to recall that porphyrins and their precursors are poorly soluble in water, thus urinary excretion is usually insufficient, and accumulation in the tissues occurs. Indeed elimination of heme catabolytes: (i) requires degradation to biliverdin, and then to bilirubin, followed by conjugation to glucuronic acid to increase bilirubin's solubility; and (ii) is mainly via the faeces, where solubility is a minor concern. Our organism has scarce capability of disposing of heme precursors, as these are poor substrates for heme oxygenase, the enzyme that converts heme to biliverdin.

      Porphyrias are either genetic or acquired diseases. Genetic porphyrias are due to the patient inheriting a poorly functioning or unstable variant of one of the enzymes involved in the biosynthesis of the heme. Acquired porphyrias are usually due to intoxication with substances capable of inactivating the same enzymes (e.g. chronic lead intoxication; chronic alcholism). Depending on the enzyme affected, and the intermediate which is accumulated, one may distinguish between cutaneous and hepatic/neurological porphyrias.
      Two types of porphyrias are particularly noteworthy. Congenital erythropoietic porphyria, is due to a defect of uroporphyrinogen III cosynthase. This enzyme guarantees the appropriate orientation of the pyrroles in the biosynthesis of uroporphyrinogen III. In the absence of the enzyme, uroporphyrinogen synthase produces mostly uroporphyrinogen I, an isomer differing in the position of acetate and propionate side chains, that cannot be used in the heme biosynthesis and accumulates in the tissues. This is the most severe form of cutaneous porphyria. Congenital protoporphyria is interesting because it leads to accumulation of iron-free protoporphyrin IX that should be recognized as a substrate by heme oxygenase; however the substance accumulates not only in the erythrocytes, but also in tissues where it is not metabolyzed to biliverdin, e.g. in the liver, and this may lead to progressive liver failure.

      Most porphyrias are hereditary and dominant; thus, multiple cases occur among the patient's relatives, and the familial anamnesis provides important diagnostic clues.
      Cutaneous porphyrias are easily suspected on a purely clinical basis. Porphyrin precursors accumulate in the skin and, given their florescence properties, they harvest sun light and transfer the radiant energy to the surrounding cells causing damage of the DNA (e.g. dimerization of timine) and necrosis. The resulting dermatitis is severe, with extensive ulcerations. The patient becomes aware of his/her condition in the early infancy and avoids direct sunlight: he/she leaves home after sunset and uses extensive clothing.

      A cardinal sign of erythropoietic congenital porphyria is erythrodonthia, a reddish coloration of the teeth, due to accumulation of porphyrinogens in the teeth. Illumination with blue light reveals the reddish fluorescence of the teeth and is of diagnostic value (no other disease causes this phenomenon).

Audio: cutaneous porphyrias

      By contrast, neurological porphyrias are difficult to diagnose on clinical grounds alone. The chief manifestation is pain, due to peripheral neuropathy. Pain occurs suddenly in acute crises and is frequently misdiagnosed as an acute abdominal condition. Given the intensity of the syndrome, these patients often undergo repeated surgeries, because of suspected appendicitis, volvulus, biliary or urinary calculi, etc. Needless to say none of these conditions is the culprit, even though all of them may occasionally co-exist. The most dramatic cases are those caused by acute intermittent porphyria. Neurological porphyrias must be differentiated from: (i) acute abdominal conditions requiring surgery; and (ii) other non surgical conditions such as tabes dorsalis or the colica saturnina (in the course chronic lead poisoning).

      Diagnosis of porphyrias is based on the demonstration of the increased concentration of one of the heme precursors in the serum or in the urine. These compounds are strongly fluorescent, and in acidic medium (HCl), they react with benzaldehyde derivatives (Ehrlich reaction) to yield a characteristic purplish pigment. If the biological sample is positive to these simple test, identification of the specific porphyrin can be achieved by chromatography.
Laboratory diagnostic features of porphyrias
Porphyriaurine ALA and PBGurine porphyrinsfecal porphyrinsred blood cell porphyrins
Acute intermittent porphyria increased increased uroporphyrinogen normal normal
Hereditary coproporphyria increased increased coproporphyrinogen increased coproporphyrinogen normal
Variegate porphyria increased increased coproporphyrinogen increased coproporphyrinogen and protoporphyrin normal
Congenital erythropoietic porphyria normal increased uroporphyrinogen and coproporphyrinogen increased coproporphyrinogen increased uroporphyrinogen and coproporphyrinogen
Porphyria cutanea tarda normal increased uroporphyrinogen increased isocoproporphyrin normal
Erythropoietic Protoporphyria normal normal increased coproporphyrinogen increased uroporphyrinogena and coproporphyrinogen

      We remark that:
(i) in neurological porphyrias the urinary excretion of porphobilinogen (PBG) is increased (not so in cutaneous porphyrias). The normal value of PBG in the urine of healthy humans is <2.5 mg/die or <2 mg/L (in a random urine sample). During acute attacks of neurological porphyrias the patient may excrete >50 mg/die of PBG in the urine.
(ii) in cutaneous porphyrias, with the exception of porphyria cutanea tarda, the red blood cells contain porphyrinogens, which are absent in neurological porphyrias and in healthy subjects.
(iii) Gene sequencing may be carried out to confirm the diagnosis, but the biochemical laboratory tests should be first carried out, in order to reduce the number of genes to be sequenced.
      The differential diagnosis of cutaneous porphyrias is relatively simple: few diseases cause such severe damage of the skin exposed to sunlight. Dermatological diseases may be aggravated by exposure to sunlight, but most often are not limited only to the light-exposed areas, whereas skin lesions due to cutaneous porphyrias only affect exposed areas. Autoimmune diseases such as lupus erythematosus disseminatus or pemphigus may cause skin lesions in the light-exposed areas, but these are less severe than those observed in porphyrias, and are associated to other symptoms (e.g. renal malfunctiong) which are absent in porphyrias. Pellagra, caused by dietary deficiency of the vitamin niacin (B3, nicotinamide), or by genetic defects of triptophan metabolism, e.g by Hartnup's disease (nicotinamide can be produced during the metabolismo of triptophan; see the lecture on genetic defects of aminoacid metabolism), may cause severe skin lesions, aggravated by exposure to sunlight and coupled to neurological symptoms. Since both pellagra and porphyrias may have genetic causes and be present at birth or shortly thereafter, the differential diagnosis is important. The laboratory demonstration of heme precursors in the blood and urine confirms the diagnosis.

Audio: differential diagnosis of cutaneous porphyrias

      The differential diagnosis of neurological porphyrias is difficult, and many more common diseases may cause acute crises of abdominal pain. The first point the physician should address is whether the patient's symptoms indicate that peritoneal involvement is present (this usually suggests a surgical condition) or not (usually indicating a medical condition). An indicative diagnostic flow-chart is reported in the figure below.

Further readings
Phillips JD. Heme biosynthesis and the porphyrias. Mol Genet Metab. 2019; 128: 164-177. doi: 10.1016/j.ymgme.2019.04.008.
D.A. Bryant, C.N. Hunter and M.J. Warren Biosynthesis of the modified tetrapyrroles—the pigments of life J. Biol. Chem. 2020, 295, 6888-6925.
T Wiederholt, P Poblete-Gutiérrez, K Gardlo, G Goerz, K Bolsen, H.F. Merk, J. Frank Identification of Mutations in the Uroporphyrinogen III Cosynthase Gene in German Patients With Congenital Erythropoietic Porphyria Physiol Res. 2006; 55 Suppl 2: S85-92.

Questions and exercises:
1) Abnormally elevated concentration of porphobilinogen (PBG) and δ-aminolevulinic acid (ALA) in the urine suggests:
a neurological porphyria
a cutaneous porphyria
Congenital erythropoietic porphyria

2) Erythrodontia is characteristic of:
Acute intermittent porphyria
Variegate prophyria
Congenital erythropoietic porphyria

3) Most porphyrias are inherited; the exception is porphyria cutanea tarda, which may be inherited or acquired. Acquired cases may be due to:
Viral infections
Chronic metal poisoning

4) Neurological porphyrias may simulate:
Acute abdominal crises, possibly requiring surgery
The cutaneous photosensistivity of lupus erythematosus disseminatus
Palsies due to stroke

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Thank you Professor (lecture on bilirubin and jaundice).

The fourth recorded part, the one on hyper and hypoglycemias is not working.
Bellelli: I checked and in my computer it seems to work. Can you better specify
the problem you observe?

This Presentation (electrolytes and blood pH) feels longer than previous lectures
Bellelli: it is indeed. Some subjects require more information than others. I was
thinking of splitting it in two nest year.

Bellelli in response to a question raised by email: when we compare the blood pH
with the standard pH we do not mean to compare the "normal" blood pH (7.4)
with the standard pH. Rather we compare the actual blood pH of the patient, with
the pH of the same blood sample equilibrated under standard conditions.
Thus, if we say that standard pH is lower than pH we mean that equilibriation with
40 mmHg CO2 has caused absorption of CO2 and has lowered the pH with respect
to its value before equilibration.

(Lipoproteins) Is the production of leptin an indirect cause of type 2 diabetes since
it works as a stimulus to have more adipose tissue that produces hormones?
Bellelli: in a sense yes, sustained increase of leptin causes the hypothalamus to adapt
and to stop responding. Obesity ensues and this in turn may cause an increase in the
production of resistin and other insulin-suppressing protein hormones produced by the
adipose tissue. However, this is quite an indirect link, and most probably other factors
contribute as well.

(Urea cycle) what is the meaning of "dissimilatory pathway"?
Bellelli: a dissimilatory pathway is a catabolic pathway whose function is not to produce
energy, but to produce some terminal metabolyte that must be excreted. Dissimilatory
pathways are necessary for those metabolytes that cannot be excreted as such by the
kidney or the liver because they are toxic or poorly soluble. Examples of metabolytes
that require transformation before being eliminated are heme-bilirubin, ammonia,
sulfur and nitrogen oxides, etc.

Talking about IDDM linked neuropathy can be the C peptide absence considered a cause of it??
Bellelli: The C peptide released during the maturation of insulin, besides being an indicator
of the severity of diabetes, plays some incompletely understood physiological roles. For
example it has been hypothesized that it may play a role in the reparation of the
atherosclerotic damage of the small arteries. Thus said, I am not aware that it plays a direct
role in preventing diabetic polyneuropathy. Diabetic neuropathy has at least two causes: the
microvascular damage of the arteries of the nerve (the vasa nervorum), and a direct
effect of hyperglycemia and decreased and irregular insulin supply on the nerve metabolism.
Diabetic neuropathy is observed in both IDDM and NIDDM, and requires several years to
develop. Since the levels of the C peptide differ in IDDM and NIDDM, this would suggest
that the role of the C peptide in diabetic neuropathy is not a major one. If you do have
better information please share it on this site!

In acute intermitted porphyria and congenital erythropoietic porphyria why do the end product
of the affected enzymes accumulate instead of their substrate??
Bellelli: First of all, congratulations! This is an excellent question.
Remember that a condition is which the heme is not produced is lethal in the foetus; thus
the affected enzyme(s) must maintain some functionality for the patient
to be born and to come to medical attention. All known genetic defects of heme
biosynthesis derange but do not block this metabolic pathway.
Congenital Erythropoietc Porphyria (CEP) is a genetic defect of uroporphyrinogen
III cosynthase. This protein associates to uroporphyrinogen synthase (which is present
and functional in CEP) and guarantees that the appropriate uroporphyrinogen isomer is produced
(i.e. uroporphyrinogen III). In the absence of a functional uroporphyrinogen III
cosynthase other possible isomers of uroporphyrinogen are produced together with
uroporpyrinogen III, mostly uroporphyrinogen I. The isomers of uroporphyrinogen
that are produced differ because of the positions of propionate and acetate side chains,
and this in turn is due to the pseudo symmetric structure of porphobilinogen. Only
isomer III can be further used to produce protoporphyrin IX. Thus in the
case of CEP we observe accumulation of abnormal uroporphyrinogen derivatives, which, as
you correctly observed are the products of the enzymatic synthesis operated by
uroporphyrinogen synthase.
The case of Acute Intermittent Porphyria (AIP) is similar, although there may be variants
of this disease. What happens is that either the affected enzyme is a variant that does not
properly associate with uroporphyrinogen III cosynthase or presents active site mutations
that impair the proper alignement of the phoprphobilinogen substrates. In either case
abnormal isomers of uroporphyrinogen are produced, as in CEP.
Also remark that in both AIP and CEP we observe accumulation of the porphobilinogen
precursor: this is because the overall efficiency of the biosynthesis of uroporphyrinogens is
reduced. Thus: (i) less uroporphyrinogen is produced, and (ii) only a fraction of the
uroporphyrinogen that is produced is the correct isomer (uroporphyrinogen III).

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