Comparative study of phototoxicity of protoporphyrin IX synthetic and extracted from ssp Rattus novergicus albinus rats toward murine melanoma cells.

Protoporphyrin IX (PpIX) is a precursor of heme synthesis and is known to be an active photosensitizer and precursor of photosensitizers applied in photodynamic therapy (PDT) and photodynamic diagnostics (PDD). On irradiation with visible light, PpIX undergoes phototransformation, producing photoproducts which may also be phototoxic and increase its efficacy. The mechanism of PpIX phototransformation depends on environmental characteristics and can be different in vitro and in vivo. In this paper, we present a comparative study of the photoactivity of synthetic PpIX and PpIX extracted from the Harderian gland of ssp Rattus novergicus albinus rats, along with their photoproducts toward murine B16F-10 melanoma cells. It was observed that when irradiated with visible light the endogenous PpIX demonstrates photocytotoxicity ten times higher than the synthetic PpIX. The photoproduct of endogenous PpIX also possesses phototoxicity, though slightly lower than that of PpIX itself. The rate of cell internalization for both endogenous PpIX and its photoproduct was eightfold greater than that obtained for the synthetic porphyrin. This difference might result from a complexation of the native PpIX with some amphiphilic compounds during its synthesis within the Harderian glands, which facilitates the cell uptake of PpIX. Fluorescence microscopy images show that both endogenous and synthetic porphyrins are localized after uptake predominantly in the mitochondrial region of cells.

Compositional analysis of endogenous porphyrins from Helicobacter pylori.

Bacteria able to accumulate porphyrins can be inactivated by visible light irradiation thanks to the photosensitizing properties of this class of aromatic pigments (photodynamic therapy, PDT). Since the bacterial resistance to antibiotic is growing, PDT is becoming a valid alternative. In this context, the pathogen Helicobacter pylori (Hp) is a suitable target for PDT since it spontaneously produces and accumulates porphyrins. It is then important to understand the spectroscopic behavior of these endogenous species to exploit them as photosensitizers, thus improving the results given by the application of PDT in the treatment of Hp infections. In this work we extracted porphyrins from both a laboratory-adapted and a virulent strain of Hp, and we performed spectroscopic and chromatographic experiments to collect information about the composition and the spectrophotometric features of the extracts. The main components of the porphyrin mixtures were identified and their relative contribution to the global red fluorescence was examined.

Structural analysis of haemin demetallation by L-chain apoferritins.

There are extensive structural similarities between eukaryotic and prokaryotic ferritins. However, there is one essential difference between these two types of ferritins: bacterioferritins contain haem whereas eukaryotic ferritins are considered to be non-haem proteins. In vitro experiments had shown that horse spleen apoferritin or recombinant horse L chain apoferritins, when co-crystallised with haemin, undergoes demetallation of the porphyrin. In the present study a cofactor has been isolated directly from horse spleen apoferritin and from crystals of the mutant horse L chain apoferritin (E53Q, E56Q, E57Q, E60Q and R59M) which had been co-crystallised with haemin. In both cases the HPLC/ESI-MS results confirm that the cofactor is a N-ethylprotoporphyrin IX. Crystal structures of wild type L chain horse apoferritin and its three mutants co-crystallised with haemin have been determined to high resolution and in all cases a metal-free molecule derived from haemin was found in the hydrophobic pocket, close to the two-fold axis. The X-ray structure of the E53Q, E56Q, E57Q, E60Q+R59M recombinant horse L-chain apoferritin has been obtained at a higher resolution (1.16Å) than previously reported for any mammalian apoferritins. Similar evidence for a metal-free molecule derived from haemin was found in the electron density map of horse spleen apoferritin (at a resolution of 1.5Å). The out-of-plane distortion of the observed porphyrin is clearly compatible with an N-alkyl porphyrin. We conclude that L-chain ferritins are capable of binding and demetallating haemin, generating in the process N-ethylprotoporphyrin IX both in vivo and in vitro.

Identification of specific inhibitors for a trypanosomatid RNA editing reaction.

Several mitochondrial mRNAs of the trypanosomatid protozoa are edited through the post-transcriptional insertion and deletion of uridylates. The reaction has provided insights into basic cellular biology and is also important as a potential therapeutic target for the diseases caused by trypanosomatid pathogens. Despite this importance, the field has been hindered by the lack of specific inhibitors that could be used as probes of the reaction mechanism or developed into novel therapeutics. In this study, an electrochemiluminescent aptamer-switch was utilized in a high-throughput screen for inhibitors of a trypanosomatid RNA editing reaction. The screen identified GW5074, mitoxantrone, NF 023, protoporphyrin IX, and D-sphingosine as inhibitors of insertion editing, with IC(50) values ranging from 1 to 3 µM. GW5074 and protoporphyrin IX are demonstrated to inhibit at or before the endonuclease cleavage that initiates editing and will be valuable biochemical probes for the early events of the in vitro reaction. Since protoporphyrin IX and sphingosine are both naturally present within the trypanosomatids, their effectiveness as in vitro inhibitors is also suggestive of the potential for in vivo modulatory roles.

Double-resonance spectroscopy of the jet-cooled free base and Cu(II) complex of protoporphyrin IX.

The excited-state dynamics of porphyrins, and related compounds, impact on their applications as photosensitizers for tumor-targeting drugs and solar cells. Many researchers have examined the influence of non-planar distortions in the ground-state geometry on the properties of photoexcited states. We have identified the added importance of conformational changes in the excited state, relative to the initial geometry, on the resulting decay pathways. The ground-state structure and photodynamics of free-base and Cu(ii) complexes of protoporphyrin IX, laser desorbed into a cold supersonic expansion, have been investigated using infrared ion-dip spectroscopy combined with density-functional theory calculations. The vibrational bands associated with the N-H stretching mode of the free base are broader in the first electronically excited state, accessed via the Q band of protoporphyrin IX, than the corresponding bands in the ground-electronic state. This is attributed to rapid intersystem crossing in the excited state promoted by extension of the N-H bonds. Our calculations show that the stretching modes are highly anharmonic, which suggests the likelihood that other conformational changes are also taking place in the excited state.

Application of 5-aminolevulinic acid and its derivatives for photodynamic therapy in vitro and in vivo.

Photodynamic therapy (PDT) with 5-aminolevulinic acid (ALA) is the most widely used form of PDT in clinical practice. Topical application of ALA leads to overproduction of the endogenous photosensitizer protoporphyrin IX (PpIX). ALA-PDT is efficient treatment of superficial skin lesions, but not for thicker lesions. The main reason for this is suboptimal penetration of ALA molecules through cellular membranes and through stratum corneum of intact skin. Different approaches (formulations, mechanical and physical penetration enhancers, ALA derivatives) are currently used to increase the penetration. The content and distribution of ALA intracellularly and in tissues is difficult to measure, but PpIX content, on a relative scale, can be easily measured by fluorimetric assays.

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.

A new method for isolating physiologically active Mg-protoporphyrin monomethyl ester, the substrate of the cyclase enzyme of the chlorophyll biosynthetic pathway.

Mg-protoporphyrin monomethyl ester (MPE) is a biosynthetic intermediate of chlorophyll and converted by MPE cyclase to protochlorophyllide. Limited availability of MPE has so far hampered cyclase research. In a new, simplified, method MPE was prepared from freeze dried bchE mutant Rhodobacter capsulatus DB575 cells by extraction with acetone/H(2)O/25% NH(3). Isolated MPE was identified by absorption and fluorescence spectroscopy, and its purity was analyzed by HPLC. The extracted MPE was dried and redissolved in buffered DMSO and its substrate activity is shown by enzymatic cyclase assays. A linear time course was observed for MPE conversion to protochlorophyllide by enzymes from barley etioplasts. Our innovation of freeze drying the R. capsulatus cells before extraction provides a high yield method for MPE, which is significantly faster and more reproducible than previous extraction methods.

Transient erythropoietic protoporphyria associated with chronic hepatitis and cirrhosis in a cohort of German shepherd dogs.

Over the course of one year, slight jaundice and ascites suggestive of chronic liver disease occurred in 17 German shepherd dogs from one breeding colony. Blood analyses, performed twice with a six-month interval, revealed elevated serum activities of liver enzymes in 13 dogs. In addition, four young adult German shepherd dogs that showed severe ascites, slight jaundice and increased serum liver enzyme activities were referred for further evaluation. Because of their poor prognosis these four dogs were euthanased. There were no signs of photosensitivity. Postmortem examinations revealed macronodular darkened livers, which were characterised histopathologically by cirrhosis associated with aggregates of brown pigments showing a striking orange birefringence in polarised light. Ultrastructurally, the crystalline pigments were typical of protoporphyrins. High-performance liquid chromatographic analysis of liver samples revealed very high levels of protoporphyrins (mean 9550 nmol/g wet liver, reference value 0.41 nmol/g wet liver) and low activities of ferrochelatase (mean 0.274 mmol/mg protein/hour, reference value 0.684 nmol/mg protein/hour). Twenty-six months after the onset of the hepatopathies, the clinical condition of the 13 surviving dogs had improved and their serum liver enzyme activities were normal. The clinical histories and pedigree analyses were not in concordance with an inherited form of protoporphyria. There was no known history of exposure to toxic substances or drugs. The findings are in accordance with a transient erythropoietic protoporphyria associated with hepatic complications, presumably caused by exposure to a porphyrinogenic, ferrochelatase-inhibitory substance of unknown origin.

In situ assessment of porphyrin photosensitizers in Propionibacterium acnes.

Porphyrins are known to be efficient photosensitizer molecules and the combined action of light and porphyrins in Propionibacterium acnes have a lethal action on the cells. Identification and quantification of in situ porphyrins in P. acnes have been done using an integrating sphere connected to an ordinary absorption spectrophotometer, and the amounts of porphyrins in the cells were quantified by measuring scattering free absorption spectra of the cell suspensions. The concentration of porphyrins in P. acnes cells were increased in either of two ways; by the addition of delta-aminolevulinic acid (ALA), which lead to the formation of coproporphyrin III under the incubation conditions used in these experiments, or by the addition of protoporphyrin IX (PPIX) to the cell suspension. In the latter case, PPIX molecules are taken up by the cells in a membrane-mediated uptake mechanism, and accumulate in the cells either on a monomeric or a particular aggregate form. The fraction of porphyrins on aggregate form increased with increasing PPIX additions. In the case of ALA induced porphyrin production, only monomeric porphyrins were stored in the cells. In both cases, the cells have a limited binding capacity of monomeric porphyrins, which is estimated to be 3 x 10(5) molecules/cell, or one porphyrin molecule to every 100st lipid molecule in the cell membrane.

Extraction of protoporphyrin disodium and its inhibitory effects on HBV-DNA.

AIM: To explore an ideal method for extracting protoporphyrin disodium (PPN) from unanticoagulated animal blood, and to study the inhibitory effects of PPN on HBV-DNA duplication and its cytotoxicity to 2.2.15 cell strain. METHODS: Protoporphyrin methyl ester and other intermediate products were prepared with protoheme separated from protein hydrolysates of coagulated animal blood, which were finally made into PPN and detected quantitatively with an ultraviolet fluorescent analyzer. Ten microg/ml, 20 microg/ml, 40 microg/ml, 80 microg/ml and 160 microg/ml of PPN-aqueous solution were added into culture medium for 2.2.15 cells respectively. Eight days later, the drug concentration in supernatant from the culture medium was detected when inhibition rate of HBeAg, cell survival rate when inhibition rate of HBeAg was 50% (ID50), and when survival cells in experimental group were 50% of those in control group (CD50), and the therapeutic index (TI) was also detected. PPN with different concentration of 10 microg/ml, 20 microg/ml, 40 microg/ml, 80 microg/ml and 160 microg/ml was respectively mixed and cultivated with HepG2 2.2.15 cell suspension, and then the inhibition of PPN against HBV-DNA was judged by PCR. RESULTS: The extract of henna crystal was identified to be PPN. When the concentrations of PPN were 160 microg/ml and 80 microg/ml, the inhibition rates of HBeAg were 89.8% and 82.4%, and the cell survival rates were 98.7% and 99.2%. CONCLUSION: It is suggested that PPN can be extracted from unanticoagulated animal blood. PPN can inhibit HBV-DNA expression and duplication in vitro, and has no cytotoxicity to liver cells. Further study and application of PPN are warranted.

Identification of human hepatic cytochrome P450 sources of N-alkylprotoporphyrin IX after interaction with porphyrinogenic xenobiotics, implications for detection of xenobiotic-induced porphyria in humans.

Porphyrinogenicity of certain xenobiotics depends upon mechanism-based inactivation of specific cytochrome P450 (P450) enzymes, followed by formation of N-alkylprotoporphyrin IX (N-alkylPP). Examination of the porphyrinogenicity of xenobiotics in animals and extrapolation of the results to humans is associated with ambiguity due, in part, to differences between P450 enzymes. The goal of this study was to develop an in vitro test for the detection of N-alkylPPs, produced in human liver after administration of xenobiotics found to be porphyrinogenic in animals. This goal was achieved using fluorometry to detect N-alkylPP formation following mechanism-based inactivation by porphyrinogenic xenobiotics of single cDNA-expressed human P450 enzymes in microsomes prepared from baculovirus-infected insect cells (Supersomes) and in human liver microsomes. The following combinations of P450 enzymes were major sources of N-alkylPPs in Supersomes: CYP3A4 [3-[(arylthio)-ethyl]sydnone (TTMS)]; CYP1A2 and 2C9 [3,5-diethoxycarbonyl-1,4-dihydro-2,6-dimethyl-4-ethylpyridine (4-ethyl DDC)]; and CYP2C9, 2D6, and 3A4 [allylisopropylacetamide (AIA)]. Whereas similarities were found between results with human enzymes in Supersomes and their rat orthologs in rat liver microsomes, some differences were found. The results with TTMS and AIA, but not with 4-ethyl DDC, were the same in individual human enzymes expressed in Supersomes and human liver microsomes. We conclude that some differences exist between human liver P450 enzymes and their rat P450 orthologs in liver microsomes. It would therefore be prudent when dealing with xenobiotics in which porphyrinogenicity depends upon N-alkylPP formation to supplement animal data with studies using human P450 enzymes.

Isolation of regioisomers of N-alkylprotoporphyrin IX from chick embryo liver after treatment with porphyrinogenic xenobiotics.

Several porphyrinogenic xenobiotics cause mechanism-based inactivation of cytochrome P450 (P450) isozymes with concomitant formation of a mixture of four N-alkylprotoporphyrin IX (N-alkylPP) regioisomers, which have ferrochelatase inhibitory properties. To isolate the four regioisomers of N-methylprotoporphyrin IX (N-methylPP), 3,5-diethoxycarbonyl, 1-4-dihydro-2,4,6-trimethylpyridine (DDC) was administered to untreated, beta-naphthoflavone-, phenobarbital-, and glutethimide-pretreated 18-day-old chick embryos. Separation of the N-methylPP regioisomers by high pressure liquid chromatography (HPLC) revealed no marked difference in the regioisomer pattern among the different treatments. After administration of griseofulvin, allylisopropylacetamide (AIA), or 1-[4-(3-acetyl-2,4,6-triemethylphenyl)-2,6-cyclohexanedionyl]-O-ethyl propionaldehyde oxime (ATMP) to untreated and glutethimide-pretreated 18-day-old chick embryos, an N-alkylPP was isolated after AIA administration only. This finding strengthened previous reports of the species specificity of N-alkylPP formation with griseofulvin and ATMP. A series of dihydropyridines, namely 4-ethylDDC, 4-hexylDDC, and 4-isobutylDDC were administered to untreated and glutethimide-pretreated 18-day-old chick embryos and hepatic N-alkylPPs were isolated and separated by HPLC into regioisomers. The regioisomer patterns obtained did not support a previous proposal of masked regions above both rings B and C in the heme moieties of the P450 isozymes responsible for N-alkylPP formation. However, the data support the hypothesis of a partially masked region above ring B alone. The regioisomer patterns were in agreement with results previously obtained in rats showing that the percentage of Nc and (or) ND regioisomers in the regioisomer mixture increases as the length and bulk of the 4-alkyl substituent of a DDC analogue increase. Differences in the regioselectivity of heme N-alkylation may be due to intrinsic chemical features of DDC analogues themselves or to differences in the P450 isozymes inactivated.

Measurement of erythrocyte protoporphyrin concentration by double extraction and spectrofluorometry.

Quantification of the level of erythrocyte protoporphyrin can be used as a screening assay for lead exposure. In this method, porphyrins and heme compounds are extracted from whole blood using ethyl acetate, and porphyrins are then separated from heme by back-extraction with HCl solution. The extracted porphyrins are then quantified using a spectrofluorometer calibrated with protoporphyrin IX standard solutions.

Isolation and characterization of protoporphyrin glycoconjugates from rat harderian gland by HPLC, capillary electrophoresis and HPLC/electrospray ionization MS.

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.

N-Methylprotoporphyrin is a more potent inhibitor of recombinant human than of recombinant chicken ferrochelatase.

The potency of N-methylprotoporphyrin IX (N-methylPP) as a ferrochelatase (FC) inhibitor has been previously studied using crude chick embryo liver FC preparations. However, interactions between N-methylprotoporphyrin IX (N-methylPP) and impurities in the enzyme preparation may have compromised the results. The first objective of this study was to compare the potency of N-methylPP as an inhibitor of purified chicken FC and crude chick embryo liver FC. The EC(50) values of N-methylPP previously observed in crude chick embryo liver FC was 2.9 x 10(-3) nmol/mg protein, and with purified recombinant chicken FC was 2.07 x 10(-3) nmol/mg protein. The difference in EC(50) values was not statistically significant, and we conclude that interactions between N-methylPP and impurities in crude enzyme preparations did not affect the estimation of potency of N-methylPP. The second objective of this study was to compare the potency of N-methylPP between purified human and chicken FC. The EC(50) value of N-methylPP observed in the purified human FC preparation was 1.7 x 10(-6) nmol/mg protein (chicken FC 2.07 x 10(-3) nmol/mg protein). Thus, the potency of N-methylPP was much higher with purified human FC than with purified chicken FC. Because the porphyrinogenicity of several xenobiotics involves N-alkylprotoporphyrin IX formation, results on drug-induced porphyria obtained with avian species may underestimate the potential porphyrinogenicity in humans.

Isolation of N-vinylprotoporphyrin IX after hepatic cytochrome P450 inactivation by 3-[(arylthio)ethyl]sydnone in chick embryos pretreated with phenobarbital, glutethimide, dexamethasone, and beta-naphthoflavone: differential inhibition of ferrochelatase by N-vinylprotoporphyrin regioisomers.

Several xenobiotics caused hepatic porphyrin accumulation through mechanism-based inactivation of cytochrome P450(P450) and heme alkylation. Loss of iron from the alkylated heme results in formation of an N-alkylporphyrin, which is a potent inhibitor of ferrochelatase. N-Vinylprotoporphyrin IX (N-vinylPP) was identified in chick embryo liver after in ovo administration of 3-[(arylthio)ethyl]sydnone (TTMS). Pretreatment of chick embryos with beta-naphthoflavone, which causes a 90-fold increase in P450 1A levels, did not increase the formation of N-vinylPP after TTMS administration, showing that the heme moiety of P450 1A does not contribute to the formation of N-vinylPP. Increased amounts of N-vinylPP were isolated from dexamethasone-, phenobarbital-, and glutethimide-pretreated chick embryos, and it is possible that P450 2H and/or a P450 3A-like isozyme contributes to the formation of N-vinylPP. The ring B-substituted (NB) regioisomer of N-vinylPP constituted the lowest percentage of the total regioisomers (9-13%) in untreated and drug-induced chick embryos, thus supporting the concept that ring B of heme is occluded by a protein residue in the P450 active site. Previously, the finding that the NB regioisomer of N-ethylprotoporphyrin IX had one fifth the potency of the ring A-substituted (NA) regioisomer as a ferrochelatase inhibitor led to a proposal that an A-C ring tilt was important in N-alkylprotoporphyrins for ferrochelatase inhibition. The finding in the present study that the NA and NB regioisomers of N-vinylPP are equipotent does not support the above proposal. The ring C-substituted (NC) and ring D-substituted (ND) regioisomers of N-vinylPP had low potency.

Determination of the structure of an N-substituted protoporphyrin isolated from the livers of griseofulvin-fed mice.

Feeding mice with griseofulvin, a widely used anti-fungal agent which induces protoporphyria as a side-effect, leads to the formation in the liver of two green pigments which have been shown to be porphyrin adducts. In this work, the major porphyrin adduct isolated from the livers of griseofulvin-fed mice has been characterized structurally using one- and two-dimensional NMR spectroscopy. The adduct was shown to be an N-alkylated protoporphyrin IX in which the whole of griseofulvin (less a hydrogen atom) is attached to a pyrrole ring nitrogen of the porphyrin. It was shown that the drug-to-porphyrin linkage is an an -O-CH2-Npyrrole = linkage, to either the 4- or 6-position of ring a of griseofulvin. In an attempt to identify which pyrrole nitrogen is involved in this linkage, the 1H spectra of the free base and zinc complex of the adduct were compared with the corresponding spectra of the four regioisomers of N-methylprotoporphyrin. These comparisons indicated that the adduct isolated from the livers of griseofulvin-fed mice is either the NC or the ND regioisomer, although a clear distinction between these two could not be made on the available evidence. The mechanism of formation of the adduct and its relation to griseofulvin-induced protoporphyria are discussed.

Formation of N-methyl protoporphyrin in chemically-induced protoporphyria. Studies with a novel porphyrogenic agent.

1-[4-(3-Acetyl-2,4,6-trimethylphenyl)-2,6-cyclohexanedionyl]-O-eth yl propionaldehyde oxime (for short ATMP) is a novel porphyrogenic agent causing hepatic protoporphyria in the mouse. Mice given a single dose of the drug showed 24 h later a 70% inhibition of liver ferrochelatase and marked accumulation of protoporphyrin. These changes were not seen in similarly treated rats, guinea pigs, hamsters or chick embryos. A green pigment was isolated from the liver of mice treated with ATMP and identified by its electronic absorption spectrum and chromatographic properties on HPLC as N-methyl protoporphyrin. The ATMP pigment markedly inhibited the enzyme ferrochelatase in vitro, thus supporting its identification as N-methyl protoporphyrin. Two inhibitors of liver cytochrome P450, compound SKF 525-A and piperonyl butoxide, when given before ATMP, afforded protection against ATMP-induced porphyria and production of N-methyl protoporphyrin, suggesting a role of cytochrome P450 in the induction of the metabolic disorder. The most likely interpretation for these findings is therefore that ATMP is metabolized in the mouse to a reactive species, which in turn alkylates the haem moiety of liver cytochrome P450, thus producing N-methyl protoporphyrin. This inhibits ferrochelatase and, as a secondary response, protoporphyrin accumulates. This pathway of metabolism to the postulated reactive metabolite presumably does not occur to a significant extent in the other species examined and hence is the likely basis for the species difference in protoporphyria.

Thin-layer chromatographic separation of the ferrochelatase-inhibitory ring A and ring B regioisomers of N-ethylprotoporphyrin from a mixture of the four regioisomers.

When N-alkylprotoporphyrins are prepared synthetically or biologically, a mixture of four regioisomers is obtained. For our studies, separation of the potent ferrochelatase-inhibitory ring A (NA) and ring B (NB) regioisomers from the ring C (NC) and ring D (ND) regioisomers of low potency is required. Previously this separation required two successive high-performance liquid chromatography procedures. We now report that the separation of the zinc complexes of the NA and NB regioisomers from the NC and ND regioisomers can be achieved by a rapid and inexpensive thin-layer chromatography procedure.