Early in the 17th Century in the City of London two men pondered the same conundrum of life almost coincidentally, although their vantage points and personal circumstances could scarcely have been more disparate. John Donne, arguably the greatest love poet in the English language and later a respected theologian, preacher and Dean of St. Paul's Cathedral (despite his hedonic youth), mused on human frailty in one of his elegiac compositions on the death of 15-year old Elizabeth, the daughter of his patron Sir Robert Drury.
“Why is grass green, or why is blood red, Are mysteries which none have reached unto. In this low form, poor soul What wilt thou do?”1
A little over a mile from St. Paul's, charged with treason and incarcerated in the Tower of London, languished Sir Walter Raleigh — writer, poet, swashbuckling explorer and onetime court favorite of Elizabeth I.2 While in prison, Raleigh began his ambitious History of the World,3 the completion of which, like its author, was cut short by his beheading in 1618. In his preface to the History, Raleigh too deliberated on the mystery of the colors of life.
“But man to cover his ignorance in the least of things, who can not give true reason for the grass under his feet, why it should be green rather than red, or any other colour?”3
The answer to the riddle of green and red that neither Donne nor Raleigh could have been expected to fathom, earned for Hans Fischer the Nobel Prize for Chemistry in 1930, “for his researches into the constitution of hæmin and chlorophyll and especially for his synthesis of hæmin”, as set forth in the citation to the prize.4 Both hæmin (the oxidized ferric form of heme [hæm], also called hematin [hæmatin]) and chlorophyll are members of a family of vivid compounds, the modified tetrapyrroles or porphyrins, which Sir Alan Battersby dubbed the “pigments of life”5, 6 and which Nature uses in numerous roles in the biosphere among the broad gamut of its varied life forms.7Porphura, the origin of the word porphyrin, and all its related variations, comes from πóρφνρoς (porphuros) the Greek for purple. Ultimately porphura, the term for the purple-mollusc, was derived from the Semitic name for the precious purple dye8 that the Minoans in 1750BCE Crete9 and notably the Phoenicians in Tyre and Sidon two to three hundred years later, extracted in great secrecy by boiling in water8 the mucus of the hypobranchial gland of marine molluscs of the purpura and murex families. All of this was described in great detail for posterity by Pliny the Elder.10 About 12,000 molluscs were processed to yield merely a gram of Tyrian purple11 that was scarcely enough to dye a toga,12 but it was used nonetheless to adorn the robes of kings, emperors and high priests in the Ancient World.7, 11 From 10th Century Constantinople in the age of the Byzantine Emperor Constantine VII, who was nicknamed Porphyrogenitos, being “born in the purple” became synonymous with being royal, centuries before purple was ever thought of, ironically, as a Royal Malady.
In organisms as diverse as bacteria and humans, and in all manner of animals and vegetables in between, aminolevulinic acid (ALA) dehydratase dimerizes ALA to form porphobilinogen, the monopyrrole building block of the pyrroles. Four porphobilinogen molecules coalesce in a stepwise manner to create ring-closed fully reduced tetrapyrroles, the colorless porphyrinogens that on oxidation yield the corresponding red-purple fluorescing porphyrins, which some have described as disguised octamers of ALA. Uroporphyrinogen III, the key intermediate, is then processed either by oxidative transformations or by C-methylations depending on the organisms, and many other steps besides,4 to give rise to various porphinoid pigments6, 7 that surpass by their abundance in all spheres of life the anthrocyanins, the flavones, the ubiquitous carotenes and many other chromatic compounds that give life its rich hues. Each of the four natural macrocycle porphyrins entraps at its center a unique metal ion that mediates its participation in the manifold chemical reactions that are essential to terrestrial existence. Chlorophyll, the green photosynthesizing pigment of Donne's and Raleigh's grass, other higher plants and algae, contains magnesium. Chlorophyll metabolism, the most visible sign of life on earth, can be seen from outer space as it goes through its familiar cycles of synthesis and degradation with the seasons.13, 14 Yellow Coenzyme F430 of methane-forming bacteria, distorts itself to accommodate nickel, whereas bright red cobalamin that animals crave but cannot make, contains cobalt held by a most unusual so-called corrin-nucleotide ligand that is crucial to the cobalamin-catalyzed production of succinyl coenzyme A and methylation of homocysteine. And, of course, iron, the metal of the sword and the headman's axe, nestles in its ferrous form in the center of protoporphyrin IX to give the red heme of blood and the cytochromes their unique power to harness and transport or activate the otherwise toxic oxygen that chlorophyll has bestowed on the Earth for some two billion years.
It has been reasoned15, 16 that porphyrins are pervasive vitally, because in these molecules evolution has discovered an astonishingly simple synthetic process that produces compounds befitting electron transfer, and suitable for chelating transition metals and enhancing their catalytic power by several orders of magnitude. Yet simplicity is no guarantee against error. The recognition that heme biosynthesis in animals begins when glycine condenses with succinate (under the influence of the rate-determining mitochondria-bound ALA synthase) emanated from David Shemin's pioneering 1944 demonstration that the 66g of 15N-labelled glycine he ingested appeared in heme extracted from his own blood.17 This was followed by studies on the incorporation of succinate18, 19 and a lifetime of seminal publications from his group at Columbia University in New York City.20, 21 Although heme is produced by one of the most conserved pathways known, managed by the sequential action of 8 dedicated enzymes located in the cytosol or mitochondria of liver, bone marrow and other tissues,22 inherited and acquired defects or porphyrinopathies do happen that lead to the accumulation of the neuro- and dermatotoxic intermediates that are responsible for the diseases known collectively as the porphyrias.23–27 Precisely which intermediates of heme biosynthesis build up in the porphyrias to intoxicate nervous tissue and/or the skin, to spill into the blood, urine and/or stool, and to undergo non-enzymatic metabolism further, depends on the site of the enzyme defect in the porphyrin pathway and the dominant organ wherein the enzymatic fault lies. It is difficult for the average mortal to commit to memory the Byzantine intricacies of porphyrinology that are indispensable for understanding the pathogenesis and manifestations of the porphyrias, and essential for diagnosis and for devising therapeutic strategies. Fortunately for us, several excellent comprehensive reviews of all of these topics have recently appeared23–29 that will reward careful study in a bright light and with a well-rested state of mind free of distractions. This collection of articles should provide a readily accessible vade-mecum to porphyrin greenhorns.
It is worth remembering, however, that although the porphyrias are diseases of aberrant heme synthesis, the enzyme defects are generally only partial and thus a deficiency of hemoproteins per se is not a regular manifestation of the porphyrias. Rather, when the genetic or acquired enzyme defect is restricted to the liver, an increase in the demand for heme (such as due to loss of heme or induction of cytochrome synthesis) or the occurrence of another event that upregulates the activity of the hepatic isoform 1 of ALA synthase (such as fasting, starvation or induction by drugs and other xenobiotics), leads to an increased throughput of porphyrin precursors. In turn, these rogue metabolites precipitate neurovisceral attacks of abdominal pain, neuropathy, constipation, vomiting, psychiatric disturbances and the like, which are clinical hallmarks of the acute hepatic porphyrias. Since the small heme pool in hepatocytes is itself a key downregulator of ALA synthase there, an adequate supply of heme can abrogate the acute porphyric chain reaction. This all contrasts with the erythropoietic porphyrias, in which the enzyme defects predominate in erythroid tissue. Isoform 2 of ALA synthase in developing red cells is susceptible to the cellular supply of iron but not to the same inducements as its hepatic counterpart, and especially not to inhibition by heme. In these cutaneous porphyrias, therefore, the supply of porphyrin intermediates is fairly constant and so skin disease activity, i.e., solar urticaria, acute photosensitivity, fragility, and vesiculobullous eruptions, depends instead on the extent of sunlight ultraviolet illumination. Phototoxicity is provoked especially by exposure to the restricted peak spectral absorption and fluorescence excitation ultraviolet wavelengths of the porphyrins, known as the Soret band (≈400nm)30 that its discoverer, the pioneer Swiss spectroscopist Jaques Louis Soret, called the “great” ultraviolet band when working with oxyhemoglobin in the 1880s. A decade later, British physiologist Arthur Gamgee found that Soret's band also applied to other hemoglobin states, as well as to hematin and the porphyrins.31
The history of the porphyrias has certainly been colorful and remains so to this day, with clinical and chemical threads that at first ran separately and later were interwoven to create a rich tapestry of scientific lore. Some authorities32, 33 attributed to J. H. Schultz, a medical student in Germany, the first clinical description of porphyria in a case that he published in his doctoral thesis34 for the University of Greifswald, the alma mater of Billroth and Bismark. A contrary view was held by Claude Rimington, the respected 1950s British porphyrin biochemist who had elucidated the structure of porphobilinogen.35 Hippocrates' primacy in clinical porphyria was favored by Rimington in an article that he published posthumously.36 While we cannot know for certain who was right, it seems entirely plausible that both contestants were good choices. Schultz's 33 year old weaver with “pemphigus leprosus”34 had endured skin sensitivity from infancy, was found to have splenomegaly, and passed wine-red urine37 (porphyrinuria), typical of congenital erythropoietic porphyria in which red urine may be noticed shortly after birth.38 In contrast, Hipprocates' patient may have had an acute hepatic porphyria as she suffered frequent great pains, delirium, agitation, spasms, sweating and coma before her menses, and she also passed dark urine.39
The chemical studies that led to the discovery of the porphyrias started simply enough in 1841 when Johann Joseph von Scherer described an iron-free residue that he had extracted from blood using concentrated sulfuric acid.40 Soon thereafter, Dutch organic chemist Gerardus Johannes Mulder, the man who isolated glycine from gelatin and first coined the term “protein”, called Scherer's purple-red extract “iron-free hematin”.41 It was not until 20 years had passed that the somewhat controversial Johann Ludwig Wilhelm Thudichum — physician, gallstone chemist and neuroscientist, and later otorhinologist living in London — recognized the “splendid blood-red” fluorescence of Scherer's hematin, which he purified and called “cruentine”.42 In 1871, the great physiological chemist Ernst Felix Immanuel Hoppe-Seyler called the purple substance found in iron-free hematin hemato-porphyrin, in other words “purble-blood”,43 which he, like Thudichum before him, thought erroneously was the precursor of bilirubin. It was only later that porphyrin was identified as a derivative of hemin.44 The clinical chemistry of the porphyrias was inaugurated by the demonstration that Schultz's patient had pigment in his urine that was similar to Hoppe-Seyler's hematoporphyrin.34, 45 Porphyrins were thought then to be exclusively breakdown products of hemoglobin. The suggestion was made, with exceptional perspicacity 60 years ahead of its time, that the pigment responsible for the weaver's red urine resulted from an error in the biosynthesis of hemoglobin.45 The urinalysis approach to the porphyrias was boosted next by Charles Alexander MacMunn's discovery of a dark pigment that resembled hematoporphyrin, in the urine of a patient with acute rheumatism.46, 47 MacMunn was a remarkable general practitioner living in Wolverhampton in the English Midlands, who had conducted detailed and extensive spectroscopic analyses of many biological materials and whose original discovery of the cytochromes using spectroscopy48 was, because of Hoppe-Seyler's objections, overlooked for 40 years until their re-discovery by David Keilin.49 From this time on, the chemical and clinical strands of porphyric history became intertwined, courtesy the drug industry and the talents of legendary clinical investigators and chemists. From the 1880s through the turn of the 20th Century, sedative drugs were introduced widely, like Sulphonal (sulfonmethane) and the barbiturates, which turned out to be potent inciters of porphyria and, as Röhl, Warren and Hunt remarked, “produced a large influx of porphyric patients whose urine provided a chemical soup of porphyrins for chemists to study”.50 The clinical impact and scientific insights that stemmed from this pharmaceutical accident are exemplified in the lucid report by acclaimed Dutch physician and chemist Barend Joseph Stokvis, of the first documented case of Sulphonal-induced porphyria.51 His account of the unfortunate woman's red urine, paralysis and death, and of his investigations, is a model of astute clinical observation and innovative chemical analysis, followed by animal experimentation to discover the mechanism of the disease. Thus, by the beginning of the 20th Century the cardinal features of neurovisceral porphyria had been recognized. In 1912, Friedrich Meyer-Betz, Chief Physician in Königesberg, exposed his left arm and the right side of his face to sunlight during a tram ride, 2 days after self-injecting with hematoporphyrin. The ensuing severe acute phototoxic reaction of erythema, edema and a blistering rash gave unequivocal proof for all to see, in a published photo opportunity,52 the photosensitizing dermatotoxicity of the porphyrins, which is typical of the cutaneous forms of the disease. A tragic accidental counterpart to this phenomenon occurred 40 years later in Turkey. An epidemic of skin blisters and facial hypertrichosis broke out a among hundreds of children from poor peasant families,53, 54 who had eaten bread made from seed wheat that was intended for planting and had been sprayed with hexachlorobenzene. These “monkey children”, as they were called, had severe porphyria cutanea tarda due to inhibition, by the fungicide and its metabolites, of uroporphyrinogen decarboxylase, the 5th enzyme of the porphyria pathway octet.
The role-call of investigators who pursued the purple with a passion on both sides of “The Pond” in the 20th Century is long and illustrious but, regrettably, to do them justice would take more space than is available in this brief essay. However, two men deserve special mention, Hans Günther and Jan Waldenström. Both were associates of Nobel Laureate Hans Fischer, who did more than putter in the purple when he was not dabbling in the green and the red. Based on his personal experience in the early 1900s with a large cohort of porphyric patients, Günther devised the first classification of the disorders that he called “Die Hämatoporphyrie”.55, 56 He stratified the patients into those who were afflicted from birth (congenital), those suffering from an acute toxic reaction due to drugs, and patients with acute “genuine” symptoms, meaning those who developed acute attacks spontaneously. Günther was credited as having realized early on that these porphyric patients had what came to be called “Inborn Errors of Metabolism”.57, 58 As with so many sagas in the chronicles of porphyria, that of the link between Hans Günther and Hans Fischer was hardly mundane. Fischer's interest in the chemistry of the pyrroles was sparked when he isolated two different pigments, which he called uroporphyrin59 and coproporphyrin,60 from the urine and stools, respectively, of Günther's prized patient, Mathias Petry. Günther had diagnosed Petry with a rare skin disorder from birth that he called hematoporphyria congenita,55 later changed by Schmid, Schwartz, and Watson to porphyria erythropoietica61 and now known as congenital erythropoietic porphyria (or more aptly by the eponym Günther's disease). Fischer soon had unencumbered access to virtually unlimited supplies of Petry's precious products, as he recruited him to his laboratory as a technician and generous donor of porphyric urine and stool samples. Little wonder that with his ingenuity and a unique supply of “reagents”, Fischer made inestimable contributions to the field of porphyrin chemistry. The tale finally took a macabre turn when Petry died in 1925, and Fischer conducted on him an extensive biochemical and pathological autopsy, through which he identified the bone marrow and not the liver as the culprit in his disease.62 Jan Gösta Waldenström from Sweden, spent a most productive year in Fischer's laboratory, before returning to Uppsala to make his mark internationally in hematology and in disorders of the plasma proteins.63 Waldenström succeeded in contracting the name of the porphyric disorders to “Porphyria”, and in separating some of the cases of chronic porphyria that only showed photosensitivity many years after birth, into a new class that he called porphyria cutanea tarda. He showed that acute porphyria in Sweden was inherited as an autosomal dominant disease58, 64 and that the detection of urinary porphobilinogen was virtually a pathognomonic diagnostic tool. Sadly for the mythology of porphyria, it turns out that the popular legend that Einar Wallqvist, Arthur Engel and Waldenström found porphyric kindreds in Lapland or when skiing in Arjeploj, by tracking their red spots of urine in the snow,65 is lamentably apocryphal.
Perhaps because the porphyrias are mysterious and mystifying, often frightening, even grotesque and occasionally fatal, they have intrigued physicians as diverse as Hippocrates and Freud.66 Prophryia has also attracted both scientific whimsy and lay curiosity, as well as serious scientific endeavor. Under the rubric of whimsical science, must be considered the assertions that the demise of the Etruscans,67 the Fall of the Roman Empire68 and the ill-fate of the Franklin expedition to map the Northwest Passage,69 was a result of plumbism-induced acute porphyria, like the lead poisoning that leaches out from toxic decanters and Toby jugs.70–73 Lay preoccupation with the illnesses of the rich and famous has been no less active in the realm of porphyria that seems to have a predilection for artists and writers, if the experience of van Gogh,74, 75 Heinrich Heine76 and Isabel Allende77 are representative. What does seem clear, however, is that the mythology of werewolves and vampires deserves only scorn and has no place in the legend of porphyrins,78 being particularly hurtful to true sufferers of the cutaneous forms of the disease. The most celebrated and most contentious question of porphyria in great personalities started in 1966 when two British psychiatrists, Ida Macalpine and her son Richard Hunter, postulated with irksome authoritarianism that the madness of King George III, which had precipitated the Regency crisis (see Fig. 1) indirectly led to the loss of the American colonies for Great Britain and ended in his death in delirium, was due to acute intermittent porphyria.80 Even though Macalpine and Hunter later revised their diagnosis to variegate porphyria,81 their confident assertion that the porphyria gene was expressed from Mary Queen of Scots in the 16th Century to the decendents of Queen Victoria, was met with opposition from experts in the field. Correspondence was first couched in the genteel language of science but ended in vitriol in the pages of the journals, as reviewed with vehemence and fervor by one of the major opponents, Geoffrey Dean,82 countered gently but no less persuasively by Röhl, Warren and Hunt.83 Now that passions have settled, it seems likely that the retrospective diagnosis of variegate porphyria was correct because the medical history given by the physicians attending the King was consistent with acute attacks of porphyria and two descendants seem to have been diagnosed correctly by experts in the field too,83 the absence of Royal genetic analysis notwithstanding. A fascinating codicil to the Royal Malady was published last year by Cox and colleagues,84 who performed sophisticated metal analysis on a hair from the King, and found evidence of high concentrations of arsenic, which physic the King's attending physicians undoubtedly used to the full. Since arsenic interferes with heme metabolism, it may well have contributed to the King's unusually severe and prolonged bouts of illness.
After this romp through the history of porphyria, it might seem daunting to have to select a single publication to highlight as this month's Landmark in Hepatology. Yet it is relatively simple because of the results of an experiment performed a little over 30 years ago that led to the introduction of effective therapy for the treatment of severe acute intermittent porphyria, derived from a knowledge of the pathogenesis of the disease and based on animal experimentation.85 Herbert Bonkovsky and his colleagues at the National Institutes of Health, under the direction of Cecil Watson, treated a woman with end-stage acute intermittent porphyria with intravenous hematin and showed conclusively a profound suppression of porphyrin precursors, porphobilinogen and ALA, which were grossly elevated before therapy.86 This result confirmed indirectly in vivo the feedback inhibition of ALA synthase by heme. The patient did not survive but the data served as proof of principle that led to the introduction of lyophilized hematin in the United States and heme arginate in Europe and other parts of the world, for patients who fail standard therapy with intravenous glucose or when the attacks are severe. Oddly enough, there are still no extensive randomized controlled trials showing efficacy. However, experts in the field agree that heme administration, preferably in the arginate form because of better stability, fewer side-effects and better therapeutic benefits, is the treatment of choice for a severe attack of acute intermittent porphyria.87 Bonkovsky et al.'s study is a landmark because it showed for the first time the feasibility of treatment for this genetic disease based on knowledge gained over a century or more of inspiration, ingenuity and innovation by scores of investigators around the world.