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.

Pharmacological properties of kryptopyrrole and its oxidation products on isolated sciatic nerve of rat and on guinea-pig ileum.

1 Kryptopyrrole (2, 4-dimethyl, 3-ethylpyrrole) inhibited conduction in rat sciatic nerve by a local anaesthetic action. 2 Tone and both spontaneous and electrically-induced contractions of guinea-pig ileum were also inhibited by kryptopyrrole. The concentration of kryptopyrrole required for 50% inhibition of a maximum twitch tension (ID50) was 0.085 mM. 3 Oxidation products of kryptopyrrole with chromatographic properties similar to those of urinary constituents reported in schizophrenia and hepatic porphyrias had little or no effect at similar concentrations. 4 Dose-response curves to exogenous acetylcholine in guinea-pig ileum were shifted to the right by kryptopyrrole, with loss of parallelism and reduction in the maximum contraction. 5 Acetylcholine overflow from ileal segments at rest and during electrical stimulation was reduced by kryptopyrrole. 6 These results on ileal segments are consistent with kryptopyrrole having both a post-junctional site of action, presumably directly on the muscle, and a pre-junctional site reducing the output of acetylcholine from the myenteric plexus. 7 The significance of these findings is discussed in relation to a possible clinical pathological role for these compounds.

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.

Urine concentration of 3-ethyl-5-hydroxy-4,5-dimethyl-delta 3-pyrrolin-2-one ('mauve factor') is not causally related to schizophrenia or to acute intermittent porphyria.

1. 3-Ethyl-5-hydroxy-4,5-dimethyl-delta 3-pyrrolin-2-one (HPL, 'mauve factor') was determined quantitatively in the urine of schizophrenic, general medical and porphyric subjects by a sensitive gas/liquid-chromatographic method using a nitrogen-specific detector. 2. A comparative evaluation with previously used methods for HPL was made and some problems of specificity are discussed. 3. The concentration of HPL in early morning and spot smaples of urine from 146 subjects with schizophrenia was not greater than that in 42 general medical patients, contrary to previous reports. 4. Of the three patients with acute intermittent porphyria, two excreted HPL. One subject, studied over a 2 year period, did so intermittently in a manner unrelated to her attacks of porphyria. 5. It is concluded that the urine content of HPL is unlikely to be causally related to schizophrenia or to the clinical manifestations of acute intermittent porphyria.

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.

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.

Magnesium protoporphyrin chelatase activity in Rhodopseudomonas spheroides. Studies with whole cells.

1. Whole cells of Rhodopseudomonas spheroides grown under semi-anaerobic conditions in the light incorporated magnesium into exogenous protoporphyrin when incubated with EDTA or the related chelators EGTA, N-(2-hydroxyethyl)-ethylenediamine-NN'N'- triacetate and trans-1,2-diaminocyclohexanetetra-acetate. 2. The reaction was demonstrated under anaerobic conditions in the light or at low oxygen partial pressure in the dark. Partial pressures of oxygen greater than 15% inhibited the reaction. 3. Cells grown under pure oxygen were completely inactive, but on adaptation to growth under low oxygen partial pressure (O(2)+N(2), 5:95) the development of activity paralleled the synthesis of bacteriochlorophyll. 4. The reaction with normal cells did not require protein synthesis, but cells that had lost their activity by being illuminated in Mg(2+)-deficient medium did not recover it in the absence of protein synthesis. 5. The product of the reaction was magnesium protoporphyrin monomethyl ester. 6. Evidence is presented that insertion of magnesium is obligatorily coupled with methylation and it is concluded that the reaction is dependent on a multienzyme complex.

Control of magnesium-protoporphyrin chelatase activity in Rhodopseudomonas spheroides. Role of light, oxygen, and electron and energy transfer.

1. Magnesium-protoporphyrin chelatase activity, previously shown in whole cells of Rhodopseudomonas spheroides, could not be demonstrated in cell-free extracts prepared in different ways, although spheroplasts retained moderate activity. Slight activity was detected also in whole cells of Rhodospirillum rubrum. 2. The effects on the activity of the enzyme of inhibitors of electron and energy transfer were studied in whole cells of Rps. spheroides. Amytal, rotenone, azide and cyanide inhibited at low pO(2) in the dark but not under anaerobic conditions in the light. Antimycin A and 2-heptyl-4-hydroxyquinoline N-oxide, as well as uncouplers and oligomycin, inhibited under all environmental conditions. 3. The effects on magnesium chelatase activity of intermediates of the tricarboxylic acid cycle, of thenoyltrifluoroacetone, of a number of artificial electron donors or acceptors, of various quinones and of the oxidation-reduction indicator dyes Benzyl Viologen and Methyl Viologen are described. 4. It was concluded that electron transport between a b-type and a c-type cytochrome as well as associated energy conservation and transformation reactions were essential for activity. There was also a specific requirement for ATP. 5. Exogenous protoporphyrin and magnesium protoporphyrin monomethyl ester were incorporated into bacteriochlorophyll or late precursors by whole cells. 6. Evidence is presented that the insertion of magnesium was the only step inhibited by oxygen in the biosynthetic pathway between protoporphyrin and bacteriochlorophyll.

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.

Determination of delta-aminolaevulinic acid in biological fluids by gas-liquid chromatography with electron-capture detection.

A derivative of delta-aminolaevulinic acid (AmLev), 2-methyl-3-acetyl-4-(3-propionic acid pentafluorobenzyl ester)pyrrole, with favourable properties for g.l.c. with electron-capture detection, was synthesized. Less than 1 pg could be detected on the column. 6-Amino-5-oxohexanoic acid formed the analogous derivative under similar conditions and was used as the internal standard in the development of a highly sensitive and specific assay for AmLev. The method has been applied to peripheral-venous and umbilical-cord plasma and to cerebrospinal fluid of normal and porphyric subjects.

delta-Aminolaevulinic acid in plasma, cerebrospinal fluid, saliva and erythrocytes: studies in normal, uraemic and porphyric subjects.

delta-Aminolaevulinic acid (ALA) was determined by g.l.c. with electron-capture detection. Normal plasma level was 92 nmol/l (SD = 39, n = 89, range 24-270 nmol/l). ALA was undetectable in 35 of 53 samples of normal cerebrospinal fluid (limit of assay 2 nmol/l). The mean of the other 18 samples was 19 nmol/l (SD = 10, range 6-36 nmol/l). Salivary ALA was generally only 10-30% of the plasma level in normal and porphyric subjects. Erythrocytes of normal and porphyric subjects contained no detectable ALA and were impermeable to its entry. ALA clearance correlated closely with that of creatinine, consistent with it being excreted by glomerular filtration with limited tubular reabsorption. In chronic renal failure, serum ALA was elevated to a maximum of three to four times the normal, but its urinary excretion was reduced, in keeping with lessened production. In two cases of acute intermittent porphyria with overwhelming neuropathy the maximum plasma levels of ALA were 9 and 12 mumol/l. Haematin infusion decreased the ALA levels but without obvious clinical benefit. Limited neurological recovery occurred without major reduction in plasma levels of ALA. One subject's attack was precipitated by pregnancy. The neonate was apparently normal, despite high levels of ALA in maternal plasma throughout gestation and a high level of ALA in the cord blood. The observations described here do not support the view that ALA may be directly neurotoxic.

The 'mauve factor' of schizophrenia and porphyria, 5-hydroxyhaemopyrrole lactam, has low pharmacological potency on guinea-pig ileum.

5-Hydroxyhaemopyrrole lactam, the 'mauve factor' reported in the urine of schizophrenics and porphyrics was found to inhibit electrically-stimulated contractions of guinea-pig ileum only at high concentrations (ID50 = 8.5 mM). This low potency makes it unlikely that the compound can account for neurotoxic effects in human porphyria.

Long term spironolactone and the adrenal cortex in essential hypertension.

In view of recent evidence that spironolactone may inhibit synthesis of corticosteroids by a direct effect on the adrenal cortex, adrenocortical function was studied in eight patients with essential hypertension who had been treated with spironolactone from 3 months to 14 years. Their 24 h renal excretion of adrenocorticoid metabolites and the responses of cortisol, aldosterone and 18-hydroxy-11 -deoxycorticosterone (18-OH-DOC) to an incremental infusion of tetracosactrin (1-24 ACTH) were compared with those in eight patients with recently diagnosed essential hypertension who had received no spironolactone. The spironolactone-treated group had a significantly higher excretion of aldosterone, whilst the excretion of other adrenocorticoid metabolites did not differ. The same group also required less tetracosactrin to stimulate a detectable rise of plasma cortisol and 18-OH-DOC, they had greater plasma 18-OH-DOC responses at all infusion rates and, at the lowest infusion rates, had greater aldosterone responses. These results indicate that long-term spironolactone therapy does not inhibit adrenocortical function and may have some stimulatory effects.

The effect of adenosine triphosphate on porphyrin excretion and on glycine metabolism in Rhodopseudomonas spheroides.

1. ATP, GTP, CTP and UTP at concentrations of 1mm markedly decrease the amount of coproporphyrin excreted by Rhodopseudomonas spheroides illuminated in a medium containing glycine, succinate and fumarate. 2. The effect of ATP is decreased if ethionine is also added to the medium. 3. Evidence is presented showing that ATP is taken up by the organisms from the medium. 4. ATP is shown to have a marked effect on the utilization of glycine. In the presence of ATP the incorporation of the methylene carbon atom of glycine into the fatty acid moieties of the phospholipids is greatly increased, and more of the carboxyl carbon atom is lost, probably as carbon dioxide. 5. ATP has little effect on the utilization of succinate or fumarate. 6. The possible significance of these results with regard to the control by ATP of porphyrin synthesis and excretion and glycine metabolism is discussed.