Simultaneous quantification of erythrocyte zinc protoporphyrin and protoporphyrin IX by liquid chromatography

A simple, rapid, specific, and sensitive isocratic "high-performance" liquid-chromatographic procedure is described for measuring protoporphyrin (PPIX) and zinc protoporphyrin (ZPP) in erythrocytes. A 30-microL whole-blood sample is treated with a solution of formic acid, deproteinized with acetone, and centrifuged. A 20-microL aliquot of the supernate is injected into a system consisting of a stationary phase of mu-Bondapak C18 and a mobile phase of acetone, methanol, water, and formic acid. ZPP and PPIX are detected fluorometrically (lambda ex = 417 nm, lambda em = 635 nm) within 6 min. The range of linearity extends beyond 10 mg/L for ZPP and 580 micrograms/L for PPIX. The detection limits for ZPP and PPIX are 11.9 micrograms/L (6.93 pg) and 2.55 micrograms/L (1.485 pg), respectively. The precision for ZPP and PPIX determinations averaged 2.86 and 5.59%, respectively, for within-day CVs and 4.98 and 8.14, respectively, for among-day CVs. Analytical recoveries averaged 97.2% for ZPP and 101.5% for PPIX. Interferences in the form of fluorescent quenching of ZPP and PPIX by hemin are avoided by chromatographic separation. We also used this method to determine the purity of commercially prepared ZPP, and compared the results obtained with this method with those from an extraction method.     

Effect of protoporphyrin IX on ursodeoxycholate-induced hypercholeresis in the rat

1. It is known that the perfusion of rat livers with solutions containing protoporphyrin IX induces a decrease in bile flow which is not due to inhibition of bile acid secretion but rather to decreased electrolyte transport into bile. By contrast, ursodeoxycholate induces hypercholeresis, partly due to a marked stimulation of biliary bicarbonate secretion. The aim of the present work was to investigate the effect of protoporphyrin IX on ursodeoxycholate-induced choleresis in anesthetized male Wistar rats. 2. Protoporphyrin IX infusion at rates of 10, 20 and 40 micrograms min-1 100 g-1 body weight into the jugular vein induced a dose-dependent inhibitory effect on bile flow as well as on bile acid and electrolyte secretion. The lowest infused rate only induced slight and non-significant changes in spontaneous bile formation and functional variables such as glycaemia, packed cell volume, blood pH, PCO2, PO2 and bicarbonate concentration, and in hepatic carbonic anhydrase activity. It was thus considered as a subtoxic dose. 3. Sodium taurocholate was infused (0.5 mumol min-1 100 g-1 body weight) over the second hour of the lowest dose of protoporphyrin IX infusion. In these rats, no significant changes in bile flow or bile acid and electrolyte secretion were observed as compared with animals receiving sodium taurocholate plus saline solution. 4. Bile acid secretion induced by ursodeoxycholate infusion (1 mumol min-1 100 g-1 body weight) was similar both in rats receiving ursodeoxycholate plus saline solution and in animals infused with this bile acid over the second hour of the lowest dose of protoporphyrin IX infusion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of bias in hematofluorometer measurements of protoporphyrin in screening programs for lead poisoning

Values for erythrocyte protoporphyrin (EP), measured in our laboratory after extraction with ethyl acetate-acetic acid, were compared with hematofluorometer measurements made in 21 other laboratories. We found that: (a) for samples of patients' blood, hematofluorometer results were 11 to 28% lower than the extraction-based values, depending on the concentration of EP and the mathematical model used; (b) hematofluorometers had mean errors of 0 to 3% for federal proficiency-testing samples; (c) there were no performance differences between fresh and shipped blood for the six laboratories that were analyzing both; (d) a hematofluorometer with a 20% low bias at an EP concentration of 500 micrograms per liter of whole blood (by the extraction method) will not detect about a third of the children whose EP concentration exceeds that cutoff value; and (e) at this same cutoff value for EP, the extraction tests detects about 45% of children whose blood lead exceeds 300 micrograms/L, whereas a 20% low-bias hematofluorometer detects only about 37%.     

5-Aminolevulinic acid-induced protoporphyrin IX for the detection of gastrointestinal dysplasia

The detection of gastrointestinal dysplasia is an unresolved problem. Fluorescence detection of these premalignancies after exogenous application of 5-aminolevulinic acid, which is converted to protoporphyrin IX and accumulates selectively in tumors, is an interesting approach. Illumination with light of appropriate wavelength allows the discrimination and detection of gastrointestinal neoplasia either by spectroscopy or fluorescence endoscopy because of a typical red fluorescence of protoporphyrin IX. Surveillance of patients with Barrett's esophagus and ulcerative colitis might benefit from this new technique.     

Accumulation of protoporphyrin IX from delta-aminolevulinic acid in bovine skin fibroblasts with hereditary erythropoietic protoporphyria. A gene-dosage effect

Bovine skin fibroblasts accumulated protoporphyrin IX when incubated in culture with the porphyrin-heme precursor, delta-aminolevulinic acid (ALA). Fibroblasts from cattle homozygous for erythropoietic protoporphyria (EPP) and with the clinical symptoms of the disease accumulated approximately sixfold greater amounts of protoporphyrin IX than cells from normal control animals. Cells from obligatory heterozygous animals, which are clinically normal, accumulated an intermediate level of protoporphyrin IX. When these cells were incubated with ALA and CaMg EDTA, all types of cells accumulated approximately the same amount of protoporphyrin IX (approximately 500 nmol/mg protein), suggesting that ferrochelatase activity was equally low after inhibition by treatment with CaMg EDTA in all cells. Thus the ratio of protoporphyrin IX accumulation from ALA in cultures treated with CaMg EDTA compared with controls treated with ALA alone was greatest in normal cells, least in EPP cells, and intermediate in the heterozygote cells. These findings suggest that the amount of protoporphyrin IX accumulation from ALA reflects the extent of deficiency of ferrochelatase and is proportional to the dosage of abnormal EPP gene in cultured fibroblasts. Similarly, stimulation of porphyrin accumulation by CaMg EDTA reflects diminished ferrochelatase activity in these cells. Thus, the results of this study demonstrate the usefulness of estimating protoporphyrin IX formation from ALA for the detection of an EPP gene defect in cultured bovine skin fibroblasts.     

Separation of protoporphyrins and related compounds by reversed-phase liquid chromatography.

We describe the separation of protoporphyrin and related porphyrins by reversed-phase "high-performance" liquid chromatography, with fluorometric detection. We used the method to demonstrate that acid hydrolysis of the dimethyl ester of protoporphyrin IX is complete in 2 to 3 h and is followed by the acid-catalyzed conversion of protoporphyrin IX to a chemical species that chromatographic evidence indicates to be hematoporphyrin IX. In addition, the method was used to evaluate the purity of a commercial preparation of protoporphyrin IX and was also demonstrated to have the sensitivity and specificity needed for measuring the protoporphyrin IX content of whole-blood.     

Observational study of erythrocyte protoporphyrin screening test for detecting low lead exposure in children: impact of lowering the blood lead action threshold

We examined a retrospective sample of 1800 children on whom both erythrocyte protoporphyrin (EP) and blood lead (BPb) measurements were taken. The primary objective was to ascertain whether EP is a cost-effective screening test for low but increased BPb concentrations and to establish the optimal thresholds. The data did not provide evidence of an EP threshold at low BPb concentrations; however, the data did show a significant age effect. A subset of 500 children for whom both EP and hematocrit data were available showed no correlation between those variables. Age-specific operating characteristic curves, total error, and cost analyses are presented. The latter sets bounds on the relative cost of EP testing, above which only BPb determination should be performed. The implications of these findings are discussed in light of impending changes in U.S. federal guidelines for preventing lead poisoning in young children.     

Molar absorptivities of bilirubin (NIST SRM 916a) and its neutral and alkaline azopigments

Three laboratories in the U.S. and two in the Netherlands determined molar absorptivities (epsilon) of Standard Reference Material (SRM) 916a Bilirubin from the National Institute of Standards and Technology. In caffeine reagent the average epsilon values were 50,060 and 48, 980 L.mol-1.cm-1 at 432 and 457 nm, respectively. The epsilon value of the blue azopigment, obtained with the Reference Method for total serum bilirubin, was 76,490 L.mol-1.cm-1 at 598 nm. When the addition of alkaline tartrate was omitted, the molar absorptivity of the red azopigment was 56,600 L.mol-1.cm-1 at 530 nm.     

Erythrocyte protoporphyrin in the detection of iron deficiency.

Any decrease in the availability of iron for incorporation into the heme moieties of hemoglobin results in an increase in the erythrocyte protoporphyrin concentration. Our aim was to compare protoporphyrin concentrations, determined spectrophotometrically, with body iron stores, as assessed from the amount of iron demonstrable by Prussian blue staining of bone marrow aspirates. The mean protoporphyrin concentration (175 mu-g/dl) in the erythrocytes of a group of patients with markedly decreased stainable marrow iron or no iron was significantly greater (P less than .001) than the mean concentration (76 mu-g/dl) in a comparable group with adequate bone marrow iron stores, except in the presence of certain interfering conditions. These results suggest that the erythrocyte protoporphyrin test may be a useful addition to the methods now available for assessing disorders of heme synthesis, the most common of which is iron deficiency.     

Light-induced protoporphyrin release from erythrocytes in erythropoietic protoporphyria.

The photohemolysis of normal erythrocytes incubated with protoporphyrin is reduced in the presence of albumin. When globin is added to normal erythrocytes loaded with protoporphyrin, protoporphyrin is bound to globin. During irradiation protoporphyrin moves from globin to the erythrocyte membrane and photohemolysis is initiated. Erythrocytes in patients with erythropoietic protoporphyria contain large amounts of protoporphyrin bound to hemoglobin. Upon irradiation of these cells in the absence of albumin, 40% of protoporphyrin and 80% of hemoglobin is released after 240 kJ/m2. The released protoporphyrin is hemoglobin bound. In contrast, when albumin is present only 8% of hemoglobin is released whereas protoporphyrin is released to 76%. The released protoporphyrin is albumin bound. A hypothesis for the release of erythrocyte protoporphyrin in erythropoietic protoporphyria without simultaneous hemolysis is proposed. Upon irradiation protoporphyrin photodamages its binding sites on hemoglobin, moves through the plasma membrane, and is bound to albumin in plasma.     

The rapid determination of erythrocyte porphyrins using reversed-phase high performance liquid chromatography

The extraction, separation and quantification of free acid erythrocyte porphyrins are described. The porphyrins are extracted from whole blood using 3:1 ethyl acetate/acetic acid (v/v) and separated using the reversed-phase mode of high performance liquid chromatography (RPLC). The compounds are detected on-line spectrofluorometrically using an excitation wavelength of 404 nm and an emission cut-off filter of 550 nm. Excellent resolution of the porphyrin free acids is achieved in < 6 min. The analytical recoveries for protoporphyrin IX and zinc protoporphyrin IX were > 90% with relative standard deviations for day-to-day analysis under 6%.     

A vehicle for photodynamic therapy of skin cancer: influence of dimethylsulphoxide on 5-aminolevulinic acid in vitro cutaneous permeation and in vivo protoporphyrin IX accumulation determined by confocal microscopy.

Topical application of 5-aminolevulinic acid (5-ALA) followed by light irradiation is a new concept of photodynamic therapy (PDT) of skin cancers. 5-ALA is a prodrug that can be converted by the heme biosynthetic pathway into protoporphyrin IX, an effective photosensitizer. In the present work we propose the enhancement of 5-ALA-induced protoporphyrin IX accumulation by dimethylsulphoxide (DMSO) and ethylenediamine-tetraacetic acid disodium salt (EDTA). The presence of 20% DMSO (w/w) in oil-in-water emulsions increased the in vitro permeation of 5-ALA through hairless mouse skin. In vivo studies demonstrated a significant increase in the amount of protoporphyrin IX extracted from healthy hairless mouse skin after 3 h treatment with an oil-in-water emulsion containing 10% 5-ALA (w/w), 3% EDTA (w/w) and 20% DMSO (w/w). By confocal scanning laser microscopy imaging, an observed increase in red fluorescence, at 476 nm excitation and emission detected longer than 590 nm, in skin that had received this treatment, was attributed to protoporphyrin IX accumulation. Although no effect of EDTA on short-term protoporphyrin IX accumulation in skin was detected, this chelator could protect 5-ALA from decomposition during prolonged topical administration. The results obtained indicate that association of 5-ALA, EDTA and 20% DMSO may enhance the delivery of 5-ALA to the skin in the topical PDT.     

Rapid and simultaneous determination of coproporphyrin and protoporphyrin in feces by derivative matrix isopotential synchronous fluorescence spectrometry

BACKGROUND: Measurement of fecal porphyrins is important in the diagnosis of porphyria, but conventional methods to measure them have drawbacks. We explored the use of derivative matrix isopotential synchronous fluorescence (MISF) spectrometry for the measurement of coproporphyrin and protoporphyrin. METHODS: The MISF scanning route was selected based on information from the three-dimensional fluorescence spectrum, which was a combination of the contour line of protoporphyrin via a detection point of coproporphyrin and that of coproporphyrin via a detection point of protoporphyrin. Derivative technique eliminated the constant interfering signals. MISF was used to measure porphyrins in stools from 2 pregnant women and 20 healthy volunteers. RESULTS: The coproporphyrin and protoporphyrin spectra were resolved with almost no mutual interference. The amplitudes of the derivative peaks were linearly related to the concentrations of coproporphyrin up to 310 nmol/L and protoporphyrin up to 590 nmol/L. The detection limits for coproporphyrin and protoporphyrin were 1.2 and 1.7 nmol/L, respectively. The within-run imprecision (CV; n = 6) was 2.2% at 175 nmol/L for coproporphyrin and 2.3% at 500 nmol/L for protoporphyrin. Bland-Altman analysis indicated no significant differences between the proposed MISF method and conventional spectrophotometry or fluorimetry. Mean (SD) recoveries of porphyrins added to fecal samples were of 98 (7)% for coproporphyrin and 102 (4)% for protoporphyrin. CONCLUSIONS: This technique provides spectral resolution of coproporphyrin and protoporphyrin, obviating the need for chromatographic separation, and measurements can be made in a single scanning. The method also appears suitable for routine testing of large numbers of samples.     

Characterization of protoporphyrin in red blood cells of patients with erythropoietic protoporphyria.

It was investigated whether the protoporphyrin that can be extracted from red blood cells of erythropoietic protoporphyria (E.P.P.) patients is present in the cells as free molecules or protein-bound. With isoelectric focusing and with starch gel electrophoresis it could be shown that virtually all protoporphyrin in the erythrocytes is protein-bound. It is very likely that the protoporphyrin is bound to hemoglobin at heme-binding sites. This was indicated by several observations: 1. With isoelectric focusing the protoporphyrin-protein complex is focused at a pH only slightly higher than the isoelectric point of hemoglobin. 2. With chromatography on Sephadex columns it appeared that hemoglobin and the protopotphyrin-protein complex have the same molecular weight. 3. A Heme-protoporphyrin exchange occurred when the heme-globin bond was labialized by conversion to hemiglobin. The resulting protoporphyrin-hemoglobin complex had the same electrophoretic mobility with starch gel electrophoresis as the protoporphyrin-protein complex, extracted from red blood cells of E.P.P. patients.     

Erythropoietic protoporphyria: two populations of reticulocytes, with and without protoporphyrin

Erythrocytes from patients with erythropoietic protoporphyria contain large amounts of protoporphyrin. The photosensitivity experienced by these patients is assumed to be due to a leakage of protoporphyrin from the erythrocytes and transfer to the skin, where protoporphyrin acts as a photosensitizer. The leakage of protoporphyrin from the erythrocytes has been offered as an explanation for the great variety in protoporphyrin content observed among erythrocytes in this disease. Based on density gradient separation of red cells, it has been concluded that all reticulocytes and young erythrocytes contain large amounts of protoporphyrin. From our results, density gradient centrifugation is not suitable for age separation of red cells from patients with erythropoietic protoporphyria. By developing a new method for isolation of reticulocytes and applying flow cytometry to determine protoporphyrin content in individual cells, it was observed that two populations of reticulocytes were present in patients with erythropoietic protoporphyria, one with and the other without protoporphyrin. The half-life of protoporphyrin in red cells was found to be 12–14 days, in contrast to 1–2 days described previously, suggesting a slower release of protoporphyrin from the red cells than previously anticipated.     

The myxoma poxvirus protein,M11L,prevents apoptosis by direct interaction with the mitochondrial permeability transition pore

M11L, an antiapoptotic protein essential for the virulence of the myxoma poxvirus, is targeted to mitochondria and prevents the loss of mitochondrial membrane potential that accompanies cell death. In this study we show, using a cross-linking approach, that M11L physically associates with the mitochondrial peripheral benzodiazepine receptor (PBR) component of the permeability transition (PT) pore. Close association of M11L and the PBR is also indicated by fluorescence resonance energy transfer (FRET) analysis. Stable expression of M11L prevents the release of mitochondrial cytochrome c induced by staurosporine or protoporphyrin IX (PPIX), a ligand of the PBR. Transiently expressed M11L also prevents mitochondrial membrane potential loss induced by PPIX, or induced by staurosporine in combination with PK11195, another ligand of the PBR. Myxoma virus infection and the associated expression of early proteins, including M11L, protects cells from staurosporine- and Fas-mediated mitochondrial membrane potential loss and this effect is augmented by the presence of PBR. We conclude that M11L regulates the mitochondrial permeability transition pore complex, most likely by direct modulation of the PBR.     

Photodynamic therapy based on 5-aminolevulinic acid and its use as an antimicrobial Agent

Exogenous 5-aminolevulinic acid (ALA) is taken up directly by bacteria, yeasts, fungi, and some parasites, which then induces the accumulation of protoporphyrin IX (PPIX). Subsequent light irradiation of PPIX leads to the inactivation of these organisms via photodamage to their cellular structures. ALA uptake and light irradiation of PPIX produced by host cells leads to the inactivation of other parasites, along with some viruses, via the induction of an immune response. ALA-mediated PPIX production by host cells and light irradiation result in the inactivation of other viruses via either the induction of a host cell response or direct photodynamic attack on viral particles. This ALA-mediated production of light-activated PPIX has been extensively used as a form of photodynamic therapy (PDT) and has shown varying levels of efficacy in treating conditions that are associated with microbial infection, ranging from acne and verrucae to leishmaniasis and onychomycosis. However, for the treatment of some of these conditions by ALA-based PDT, the role of an antimicrobial effect has been disputed and in general, the mechanisms by which the technique inactivates microbes are not well understood. In this study, we review current understanding of the antimicrobial mechanisms used by ALA-based PDT and its role in the treatment of microbial infections along with its potential medical and nonmedical applications.     

Erythropoietic protoporphyria: a quantitative determination of erythrocyte protoporphyrin in individual cells by flow cytometry

A fraction of the erythrocytes from patients with erythropoietic protoporphyria contains large amounts of protoporphyrin. By extraction methods, only a mean value of the protoporphyrin content in erythrocytes is obtained. In this study, flow cytometry was used to determine the protoporphyrin content in each individual erythrocyte. The mean erythrocyte protoporphyrin fluorescence, calculated from flow cytometric data, correlated linearly to the mean cellular protoporphyrin concentration measured by ethyl acetate/acetic acid extraction. This indicates that flow cytometry gives a reliable quantitative determination of protoporphyrin in individual cells. Erythrocytes from patients with erythropoietic protoporphyria had a markedly skewed distribution of protoporphyrin. In the most fluorescent erythrocytes (protoporphyrin concentration 3.3 mM), there was about one protoporphyrin molecule for every sixth haeme molecule. This might explain the slight anaemia in some of these patients.     

Screening with zinc protoporphyrin for iron deficiency in non-anemic female blood donors

Iron-depleted donors are at increased risk of developing anemia; if these donors could be identified by a screening test, iron supplementation or decreased donation frequency could be considered. Tests to determine serum ferritin, blood hemoglobin, and erythrocyte (Erc)-zinc protoporphyrin concentrations were examined in 679 consecutive female blood donors to identify donors with non-anemic iron deficiency. The test to determine serum ferritin is expensive and slow, whereas the two latter tests are rapid and less costly and could therefore be used for screening. Women in the fertile age groups had the lowest average serum ferritin values. In all, 93 women (13.7%) had depleted iron stores, as indicated by serum ferritin concentrations less than 14 micrograms/L. In these women, a much better correlation was found between Erc-zinc protoporphyrin and serum ferritin (rs = -0.49, P less than 0.001) than between blood hemoglobin and serum ferritin (rs = 0.31, P less than 0.01). These findings suggest that measurement of Erc-zinc protoporphyrin is superior to that of blood hemoglobin in identifying donors with non-anemic iron deficiency.     

Response of various indices of iron status to acute iron depletion produced in menstruating women by low iron intake and phlebotomy

We investigated response sensitivities of indices of iron status to controlled iron depletion and repletion in 11 premenopausal women. The women were depleted of storage iron (as reflected by serum ferritin) through a combination of a low-iron diet and phlebotomy. They then consumed a diet containing 13.7 mg of iron per 2000 kcal, supplemented with either ascorbic acid or placebo (for 5 1/2 weeks) and a daily 50-mg iron supplement (for the subsequent 17 days). The relative sensitivities of different indices for detecting iron depletion were as follows: ferritin greater than % transferrin saturation greater than plasma iron greater than hemoglobin greater than hematocrit greater than zinc protoporphyrin (ZnPP) and erythrocyte protoporphyrin (EP). Ascorbic acid treatment during repletion, before iron supplementation, significantly (P less than 0.05) affected changes in hemoglobin, ZnPP, ZnPP/heme, and EP/heme. Changes in heme synthesis evidently do not occur until iron stores are depleted and, conversely, during iron repletion hematopoiesis must be satisfied before iron stores, as reflected by ferritin, increase. Thus, the use of only one index of iron status is of limited value for detecting iron depletion.