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It has been widely reported that the Harderian gland, present in most vertebrates, accumulates high levels of porphyrins, particularly protoporphyrin. The present study describes the extraction, identification and characterization of a group of hitherto unreported protoporphyrin glycoconjugates in the rat Harderian gland using HPLC, capillary electrophoresis, on-line HPLC/electrospray ionization MS and tandem MS. The major glycoconjugate was identified as protoporphyrin-1-O-acyl beta-xyloside with a smaller amount of protoporphyrin-1-O-acyl beta-glucoside also detected. In the Harderian glands studied, 50-70% of the porphyrins present were in the form of protoporphyrin glycoconjugates. This is the first reported occurrence of glycoconjugates of porphyrins in Nature and suggests that previous studies have wrongly identified the major porphyrin in the Harderian gland as the unconjugated protoporphyrin.

Homology models of cytochrome P450 2D6 (CYP2D6) have identified serine 304 as an active-site residue and implicated a putative role for this residue in substrate enantioselectivity and the differential inhibition of enzyme activity by the diastereoisomers quinine and quinidine. The role of serine 304 in selectivity is thought to be achieved through a preferential hydrogen-bond interaction between the hydroxyl group of the residue and one of the stereoisomers of each ligand. We have tested this hypothesis by substituting serine 304 with alanine, a non-hydrogen-bonding residue, and compared the properties of the wild-type and mutant enzymes in microsomes prepared from yeast cells expressing the appropriate cDNA-derived enzyme. The Ser(304)Ala substitution did not alter the enantioselective oxidation of metoprolol; the O-demethylation reaction remained R-(+)-enantioselective (wild-type, R/S, 1.7; mutant, R/S, 1.6), whereas alpha-hydroxylation remained S-(-)-enantioselective (wild-type and mutant, R/S, 0.7). Similarly, the selective oxidation of the R-(+) and S-(-) enantiomers of propranolol to the major 4-hydroxy metabolite was identical with both wild-type and mutant forms of the enzyme (R/S 0.9), although the formation of minor metabolites (5-hydroxy and deisopropylpropranolol) did show some slight alteration in enantioselectivity. The differential inhibition of enzyme activity by quinine and quinidine was also identical with both forms of CYP2D6, the IC(50) values for each enzyme being approx. 10 microM and 0.1 microM for quinine and quinidine, respectively. The kinetics of formation of alpha-hydroxymetoprolol and 4-hydroxydebrisoquine by wild-type and the Ser(304)Ala mutant was also very similar. However, modest changes in the regioselective oxidation of metoprolol and debrisoquine were observed with the Ser(304)Ala mutant. The regio- and enantioselective oxidation of an analogue of metoprolol, in which the hydroxyl group attached to the chiral carbon was replaced by a methyl moiety, was again identical with both wild-type and Ser(304)Ala mutant. However, the observed selectivity was the reverse of that observed with metoprolol. Collectively, these data indicate that Ser(304) is unlikely to be a key ligand-binding residue, although the residue may indeed be located in the active-site cavity. The reversal of selectivity with the methyl analogue of metoprolol indicates that the hydroxyl group attached to the chiral centre of ligands, such as metoprolol, is important in defining the enzyme's selective properties, and that a hydrogen-bonding residue, other than Ser(304), may be involved in this interaction. Current homology models of the active site of CYP2D6 that predict a hydrogen-bond interaction between Ser(304) and specific ligands will need to be re-evaluated, and other candidate residues capable of such an interaction nominated and tested by site-directed mutagenesis studies.

The isomeric compositions of the heptacarboxylic, hexacarboxylic and pentacarboxylic porphyrinogens formed by incubation of porphobilinogen with human red-cell haemolysates have been analysed and compared with those derived from incubation with chemically prepared uroporphyrinogen III as substrate. The results indicated that when supplied with an excess (3.7 microM) of exogenous uroporphyrinogen III, uroporphyrinogen decarboxylase utilized the substrate at random and a mixture of isomers was produced; whereas with uroporphyrinogen III generated enzymically from porphobilinogen as substrate a clockwise decarboxylation sequence was observed, resulting in the formation of intermediates mainly with the ring-D, rings-AD and rings-ABD acetate groups decarboxylated. Using [14C]uroporphyrinogen III as substrate at low concentrations (0.01-0.5 microM) also led to preferential decarboxylation of the ring-D acetate group. It was concluded that the order of uroporphyrinogen III decarboxylation is substrate-concentration-dependent, and under normal physiological conditions enzymic decarboxylation is most probably orderly and clockwise, starting at the ring-D acetate group.

The isomeric composition of type-III heptacarboxylic porphyrinogens derived from decarbosylation of uroporphyrinogen III by erythrocyte uroporphyringogen decarboxylase was analysed by h.p.l.c. with electrochemical detection. All four possible isomers were identified, and there were little differences in the proportion of isomers formed by erythrocytes from normal subjects and from patients with sporadic porphyria cutanea tarda. The results provide conclusive evidence that the normal decarboxylation pathway is random in nature, and the fourth isomer only increases when enzyme abnormality is found.

An h.p.l.c. method is described for the assay of protoporphyrinogen oxidase activity in rat liver. A relatively pure protoporphyrinogen IX substrate was obtained by selectively removing any protoporphyrin IX unreduced by sodium amalgam on a small disposable cartridge packed with a strong anion-exchanger. The protoporphyrin IX formed was extracted with dimethyl sulphoxide/methanol (3:7, v/v) containing mesoporphyrin as the internal standard for separation and quantification by reversed-phase chromatography. The Km for protoporphyrinogen was 9.5 +/- 1.6 microM, and the enzyme activities were 0.59 +/- 0.11 nmol of protoporphyrin IX produced/min per mg of mitochondrial protein and 33.5 +/- 2.7 nmol protoporphyrin IX produced/min per g of liver tissue homogenate. The method is applicable to the determination of enzyme activity in small amounts of human liver biopsy.

A reversed-phase gradient elution system is described for the simultaneous separation of the type I and type III isomers of 8-, 7-, 6-, 5- and 4-carboxylated porphyrins and isocoproporphyrins. The method, adaptable for isocratic and stepwise separation of individual groups of isomers, is also suitable for preparative isolation of pure porphyrins. The analyses of porphyrin isomers in the urine and faeces of porphyric patients are examples of applications.

A reversed-phase h.p.l.c. system, with an ODS-Hypersil column with acetonitrile or methanol in ammonium acetate buffer as mobile phase, is described for the separation of uro-and copro-porphyrinogen isomers. The porphyrinogens are detected amperometrically with sensitivity comparable with that of the fluorescent detection of porphyrins. The effects of pH, buffer concentration and organic modifiers on retention and resolution were studied. The method is suitable for both analytical and preparative separation of porphyrinogens.

A high-performance-liquid-chromatographic method is developed for the simultaneous determination of hydroxymethylbilane synthase and uroporphyrinogen III synthase activity in erythrocytes. Effective separation of uroporphyrin I and III isomers allows the accurate quantification of individual isomers and the total uroporphyrin concentration. Total uroporphyrin production is used to calculate hydroxymethylbilane synthase activity, and the amount of uroporphyrin III formed represents the activity of uroporphyrinogen III synthase. Normal ranges are established for the two enzymes.

A reversed-phase h.p.l.c. system is described for the separation of the four type-III heptacarboxylic porphyrinogen isomers. The effects of buffer concentration, pH and type and proportion of organic modifier in the mobile phase on retention and resolution of isomers were studied. Optimum separation on an ODS-Hypersil column was by elution with a ternary mobile phase of acetonitrile, methanol and 1 M-ammonium acetate, pH 5.16 (7:3:90, by vol.). Isomer identification was based on a comparison of their retention times with those of authentic standards, and was further confirmed by h.p.l.c. analysis of the characteristic mixture of three pentacarboxylic porphyrins formed after partial decarboxylation of individual isomers in 0.3 M-HCl at 160 degrees C.

An h.p.l.c. method was developed for the assay of coproporphyrinogen oxidase activity in rat liver. The protoporphyrinogen IX formed is completely oxidized to protoporphyrin IX for separation and quantification by reversed-phase chromatography with mesoporphyrin as the internal standard. The Km of coproporphrinogen oxidase is 1.01 +/- 0.23 microM. The activities are 4.07 +/- 0.40 nmol of protoporphyrin IX/h per mg of mitochondrial protein and 224 +/- 19 nmol of protoporphyrin IX/h per g of liver tissue homogenate. The method is sensitive enough for measuring enzyme activity in small amounts of human tissue from needle biopsy.

A reversed-phase h.p.l.c. system is described for the separation of all five naturally occurring pentacarboxylic porphyrinogen isomers. The compounds are detected electrochemically with high sensitivity. The peaks are positively identified by h.p.l.c. analysis of the pentacarboxylic porphyrinogens from reduction of pentacarboxylic porphyrins prepared by partial decarboxylation of hexa- and hepta-carboxylic porphyrin III of known structures. The resolution of pentacarboxylic porphyrinogens is superior to that of the porphyrins and the method is applicable to the small-scale preparative isolation of pure isomers.

A new porphyrin, meso-hydroxyuroporphyrin I, was isolated from the urine of a patient with congenital erythropoietic porphyria. The structure was characterized as the methyl ester, ethyl ester and acetoxy derivatives by fast-atom-bombardment m.s., by conversion into uroporphyrin I and by chemical synthesis.

A reversed-phase system is described for the simultaneous isocratic separation of coproporphyrin I, II, III and IV isomers. The retention behaviour of coproporphyrin I and III is studied in detail. The method is suitable for both analytical and semi-preparative separation.