Quantitative HPLC of pigments of irregularly coloured eggshells: application to aliquots of powdered shell from quail.

Twelve quail eggshells from farmed Coturnix coturnix japonica were separately ground to fine powder and two aliquots of each (average weights 13.86 mg and 51.90 mg) were extracted with formic acid. Biliverdin (38-284 pmol/mg) and protoporphyrin (841-1666 pmol/mg) were measured by HPLC. There was good agreement between the values for the corresponding samples and with those for two entire eggshells from the same source. The preparation of a homogenate as a powder from heterogeneously pigmented eggshells has the advantage that not all of the sample needs to be initially extracted for analysis and residual material can be stored in a stable form and used for repeat measurements and for longitudinal studies.

Isolation and characterization of free haem from the shell gland of quail and hen.

Free haem was isolated from the shell gland of the quail, Coturnix coturnix japonica, and of the fowl, Galinus domesticus, and characterized by HPLC-ESI-MS/MS. Quantification by HPLC gave values of 1.17-1.40 nmol/mg quail shell gland protein for haem, 1.66-2.17 nmol/mg protein for protoporphyrin and 0.25-0.40 nmol/mg protein for biliverdin. Possible implications of this previously unreported finding are discussed but they are not considered incompatible with the conclusion that all eggshell pigments are endogenously synthesized in the oviduct system.

Extraction and analysis of colourful eggshell pigments using HPLC and HPLC/electrospray ionization tandem mass spectrometry.

The literature on the pigments of avian eggshells is critically reviewed. Methods using methanolic sulfuric acid or hydrochloric acid to extract eggshell pigments are unsuitable to detect the occurrence of zinc protoporphyrin or zinc biliverdin because they demetallate these compounds. Extraction methods are described here using EDTA and acetonitrile-acetic acid or acetonitrile-dimethyl sulfoxide, which do not demetallate zinc protoporphyrin. Such extracts were prepared from eggshell of the common nighthawk, Chordeiles minor, and from another six bird species. Protoporphyrin and biliverdin were identified and fully characterized by HPLC/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS) in all samples, but none contained zinc protoporphyrin. The zinc complex of biliverdin, claimed to be an additional pigment responsible for eggshell background colours, was labile to EDTA and acid pH and if occurring naturally could not be extracted intact by the published or the modified protocols. An explanation is advanced for the exceptional report that all porphyrins from uroporphyrin to protoporphyrin were found in eggshells of the fowl Gallus domesticus.

Harderoporphyrin: a misnomer.

More than 30 years ago it was reported that rodent Harderian glands contained a tricarboxylic acid porphyrin, which the authors named Harderoporphyrin. The recent finding in rat Harderian glands of the porphyrin glycoconjugate, protoporphyrin-1-O-acyl-beta-xyloside as a major component led to scrutiny of earlier publications. It became apparent that the results were flawed and that the conclusions were unsustainable. The procedural artefacts which led to the errors are discussed and their bases are demonstrated experimentally. Harderoporphyrin as originally defined never existed.

Porphyrins in urine, plasma, erythrocytes, bile and faeces in a case of congenital erythropoietic porphyria (Gunther's disease) treated with blood transfusion and iron chelation: lack of benefit from oral charcoal.

Congenital erythropoietic porphyria is a rare genetic disorder in which deficiency of uroporphyrinogen III synthase results in excessive production of Type I porphyrins. The main clinical features are severe photodestruction of the skin and haemolytic anaemia. Treatment consists of shielding from light, blood transfusions and splenectomy, but is generally unsatisfactory. Previous studies have suggested that oral charcoal may be of benefit by binding porphyrins in the gut. A trial was therefore undertaken to evaluate this possibility. Porphyrins in urine, plasma and erythrocytes were measured by HPLC in a 23-year-old male patient with congenital erythropoietic porphyria, during an 8 week "run-in" period, and for a further 3 weeks when oral charcoal was given. Total urinary porphyrin excretion was 79-283 mumol/24 h consisting of 75% uroporphyrin I, 15% coproporphyrin I and smaller amounts of hepta-, hexa-, and pentacarboxylic porphyrins. Similar proportions were found in plasma and erythrocytes. During the first 24 h of charcoal administration a minor decrease in plasma and erythrocyte porphyrins was detected but this was not maintained during the remainder of the trial. In bile and faeces coproporphyrin I constituted approximately 95% of the porphyrins, with 2-3% coproporphyrin III and smaller amounts of pentaporphyrins I and III, but only trace amounts of uroporphyrin I. Oral charcoal was of no value in this case. Reasons are discussed in the context of biochemical differences between this patient with classical Gunther's disease and the similar clinical syndrome due to deficiency of uroporphyrinogen decarboxylase.

Drug treatment in acute porphyria.

The acute hepatic porphyrias are rare pharmacogenetic diseases inherited as autosomal dominant conditions of low penetrance. The genetic defect is a 50% deficiency of an enzyme of the haem biosynthetic pathway. Patients may develop 'neurovisceral attacks' which include severe abdominal pain, neuropsychiatric manifestations and potentially fatal respiratory paralysis. Attacks occur generally after puberty, are much commoner in females and may be precipitated by endogenous hormonal changes, dieting, alcohol, severe infections, and many drugs. Treatment includes analgesia, early administration of haem, and general supportive measures. Patients are at greater risk of a severe attack on first presentation since an abdominal emergency may be simulated and inappropriate medication, including that for general anaesthesia may exacerbate the crisis. The urine should be tested for raised porphobilinogen, which is pathognomonic of the acute attack, if there is the slightest doubt about diagnosis. The genotype of blood relatives of index cases must be determined so that carriers may avoid drug and other precipitants. Some drugs have been established as safe or unsafe by clinical use, but information about many drugs is not available or is based only on their properties in rodents or in tissue culture systems. The relevance of these to the human condition remains controversial, but drugs shown to be porphyrinogenic in animal systems should be avoided if there is a known safe alternative. Where it is essential to use a drug not known to be safe, close biochemical and clinical observation may warn of an impending attack.

Mannose binding protein gene mutations associated with unusual and severe infections in adults.

A defect in opsonisation can cause a common immunodeficiency. A mutation in mannose binding protein (MBP) caused by point mutations in the MBP gene will lead to such a defect. This type of syndrome can cause recurrent infections in infants between 6 and 18 months of age but is not generally believed to predispose to adult infections. We looked at 4 patients with severe and unusual infections in whom MBP gene mutations were the only identified cause of immunodeficiency and one patient with combined MBP and IgA deficiency. We analysed the MBP genotypes of all the patients in whom we suspected an immunodeficiency because of their clinical history. Infections seen were recurrent skin abscesses, chronic cryptosporidial diarrhoea, meningococcal meningitis with recurrent herpes simplex, and fatal klebsiella lobar pneumonia. Both sexes were affected and ages ranged from 15 to 56 years. Two patients were homozygous for codon 54 mutations, one patient had codon 52 and codon 54 mutations and was phenotypically homozygous, and two patients were heterozygous for codon 54 mutations. Individuals homozygous for MBP mutations are unusual in the general population (approximate frequency 0.3%). The occurrence of three homozygotes for MBP mutations among these five infected patients suggests that MBP deficiency may confer a life-long risk of infection.

Photosensitisation and photodynamic therapy of oesophageal, duodenal, and colorectal tumours using 5 aminolaevulinic acid induced protoporphyrin IX--a pilot study.

The first study of photodynamic therapy in the human gastrointestinal tract using 5 aminolaevulinic acid (ALA) induced protoporphyrin IX as the photosensitising agent is described. Eighteen patients with colorectal, duodenal, and oesophageal tumours were studied. After 30-60 mg/kg of ALA given orally, biopsy specimens of tumour and adjacent normal mucosa were taken 1-72 hours later. These specimens were examined by quantitative fluorescence microscopy for assessment of sensitisation with protoporphyrin IX. Ten patients were given a second dose of ALA a few weeks later and their tumours were treated with red laser light (628 nm). With 30 mg/kg ALA, the highest fluorescence values were detected in the duodenum and oesophagus, and the lowest in the large bowel. Doubling the ALA dose in patients with colorectal tumours gave protoporphyrin IX fluorescence intensities similar to those in patients with upper gastrointestinal lesions and improved the tumour:normal mucosa protoporphyrin IX sensitisation ratio. The treated patients showed superficial mucosal necrosis in the areas exposed to laser light. Six patients had transient rises in serum aspartate aminotransferases, two mild skin photosensitivity reactions, and five mild nausea and vomiting. In conclusion, photodynamic therapy with systemically administered ALA may be a promising technique for the treatment of small tumours and areas of dysplasia such as in Barrett's oesophagus.

Uptake of protoporphyrin and continuous spectrophotometric assay for magnesium chelatase in Rhodobacter spheroides.

Uptake of protoporphyrin was shown by Rhodobacter spheroides under anaerobic conditions in the dark. The process was not energy-dependent but required EGTA and was markedly stimulated by Methyl Viologen. Kinetic studies were consistent with a saturable process with Kd = 5-10 microM and Bmax. = 2.2 nmol of protoporphyrin bound/mg dry weight of cells. Bound protoporphyrin could be converted into magnesium protoporphyrin monomethyl ester under anaerobic conditions in the light or at low pO2 (6.3%) in the dark. This formed the basis of a sensitive continuous spectrophotometric assay for magnesium chelatase, which avoids the need to extract the product into organic solvent, and may facilitate the development of a cell-free system for magnesium chelatase in photosynthetic bacteria. It is proposed also that the uptake mechanism shown for exogenous protoporphyrin may indicate the existence of a ligand or carrier system for endogenously produced protoporphyrin essential for magnesium chelatase activity.

Sustained high plasma 5-aminolaevulinic acid concentration in a volunteer: no porphyric symptoms.

The pathogenesis of the acute porphyric attack is not known. One hypothesis is that porphyrin precursors, especially 5-aminolaevulinic acid (ALA), are toxic for neuronal tissue. This was tested by infusing ALA in a male volunteer after a loading dose at a rate of 50-80 mg h-1 for 92.5 h. During the experiment plasma ALA concentration was 9-11 mumol 1-l and porphobilinogen concentration 3-6 mumol 1-l which are the levels seen during acute attacks. Urinary excretion of these porphyrin precursors was also markedly increased. ALA infusion caused no subjective symptoms and no change in pulse rate, blood pressure, or autonomic nerve function or conduction velocity of peripheral nerves. Photosensitivity was not demonstrable. It is concluded that sustained high plasma ALA concentration does not cause porphyria-like symptoms.

Measurement of 5-aminolaevulinate synthase activity by gas-liquid chromatography with electron-capture detection.

A specific assay for 5-aminolaevulinate synthase activity is described, with a sensitivity comparable with that of radiochemical assays. It is based on measurement by g.l.c. with electron-capture detection of the pentafluorobenzyl ester of the ethyl acetoacetate pyrrole derivative of 5-aminolaevulinic acid, and of the corresponding compound from 6-amino-5-oxohexanoic acid used as internal standard. Enzyme activity has been measured in homogenates of rat liver, spleen, kidney and brain, and in human lymphocytes.