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Comparison of zinc protoporphyrin and free erythrocyte protoporphyrin in whole blood.

The recent change in emphasis from blood lead to erythrocyte protoporphyrin as the primary diagnostic test for screening for childhood lead poisoning has made it desirable to develop the quantitative relationship between the two protoporphyrins, free erythrocyte protoporphyrin (FEP) and zinc protoporphyrin (ZP), that are now widely used for screening purposes. ZP was found to be equal to 1.4 EP, thus permitting the measurement of either parameter, and its conversion to the other.

Child, Preschool↗

Separation of Mg-Protoporphyrin IX and Mg-Protoporphyrin IX Monomethyl Ester Synthesized de novo by Developing Cucumber Etioplasts.

High pressure liquid chromatography was used to demonstrate that chelation of Mg(2+) into protoporphyrin IX precedes methylation in isolated greening etioplasts from cucumber (Cucumis sativus L. var. Beit Alpha) cotyledons. Mg-protoporphyrin IX synthesized in vitro from protoporphyrin IX, Mg(2+), and ATP or exogenous Mg-protoporphyrin IX could serve as substrates for the methylation step. In either case, S-adenosylmethionine was the methyl donor and could not be replaced by ATP plus methionine.

Journal Article↗

The relationship between zinc protoporphyrin (ZPP) and "free" erythrocyte protoporphyrin (FEP) in lead-exposed individuals.

The relationship between zinc protoporphyrin (ZPP) and total erythrocyte protoporphyrin, measured as "free" erythrocyte protoporphyrin (FEP), was determined in 194 adult subjects with different occupational and non-occupational lead exposures. Furthermore, the ZPP-FEP comparison was considered with respect to the dose-effect relationship of ZPP and FEP with blood lead (PbB) for males and females, respectively. Bilirubin (Bil.) interferences in ZPP analysis were taken into account. A very close and highly significant relationship (r = 0.962, P < 0.001) was established between ZPP and FEP values. A significant correlation (P < 0.001) between log ZPP or log FEP and PbB (males r = 0.767 and 0.718; females r = 0.525 and 0.405) was also found. It was established, by both in vitro and in vivo studies, that Bil. interferes with the ZPP fluorescence readings; the relationship between "false" positive ZPP concentrations and Bil. concentrations (in vitro r = 0.987, in vivo r = 0.903) was highly significant (P < 0.001). A small but highly significant (r = 0.948, P < 0.001) influence of increased carboxyhemoglobin (COHb) concentrations on the decrease in hematofluorometer ZPP readings, due to inadequate oxygenation of the blood, was found. The results obtained confirm the usefullness of ZPP determinations using hematofluorometers for surveillance of increased lead absorption but stress that the interfering effect of Bil., and to a lesser extent of COHb, cannot be ignored.

Adult↗

Red cell zinc protoporphyrin and protoporphyrin by HPLC with fluorescence detection.

A high performance liquid chromatographic (HPLC) method was developed for the determination of zinc protoporphyrin (ZnPP) and protoporphyrin (PP) in whole blood. After adding the blood to dilute acetic acid, ZnPP and PP were extracted with dimethyl sulfoxide-acetone containing mesoporphyrin as internal standard. Following evaporation of the acetone, the haemin-free extract was analysed by HPLC. ZnPP and PP were separated on a reversed-phase column and quantitated by measuring fluorescence peak areas. The extraction method is simple, and applicable to batch analysis, and the HPLC separation is rapid and repoducible. The coefficient of variation for ZnPP was 5.6% and 3.3% for total red cell porphyrin levels of 3.5 and 10.2 mumol per litre RBC respectively. Results are discussed in patients with erythrohepatic protoporphyria, lead exposure, iron-deficiency and nonspecifically elevated total red cell porphyrins.

Anemia, Hypochromic↗

Zinc protoporphyrin as an indicator of lead exposure: precision of zinc protoporphyrin measurements.

In lead-exposed workers many factors affect the relationship between the levels of lead and of zinc protoporphyrin (ZPP) in blood. When the zinc protoporphyrin level is used to assess the lead in the blood level, the analytical accuracy of the ZPP and the PbB determinations should be known. Also the variability of lead exposure over time is an important parameter of this assessment. The absolute and relative precision of ZPP measurements with two brands of ZPP meters (AVIV and ESA) were compared. The absolute precision of the ZPP measurements is not constant for the AVIV meter, but is constant for the ESA meter. The relative precision for both brands of meters decreases with increasing ZPP levels. Between the AVIV and the ESA meters, a significant difference in response, irrespective of the kind of anticoagulant used, was observed. The regression equations between PbB and ZPP levels were studied. Different factors can affect the relationship between PbB and ZPP, e.g. variability in lead exposure, the time-lag between the increase of PbB and ZPP, and the precision and accuracy of PbB and ZPP measurements.

Humans↗

High-performance liquid chromatographic determination of protoporphyrin and zinc protoporphyrin in blood.

Zinc protoporphyrin and protoporphyrin free acid in blood were determined by high-performance liquid chromatography on a C18 column. Results for 63 human blood samples obtained through a lead poisoning detection program compared favorably with the widely-used ethyl acetate--acetic acid extraction determination. Blood from 16 rats which had been maintained on water heavily spiked with chloroform or bromodichloromethane and blood from a lead-poisoned cow were examined by this procedure.

Animals↗

A direct and simultaneous detection of zinc protoporphyrin IX, free protoporphyrin IX, and fluorescent heme degradation product in red blood cell hemolysates.

Fluorescence emission of free protoporphyrin IX (PPIX, em. approximately 626 nm), zinc protoporphyrin IX (ZPP, em. approximately 594 nm) and fluorescent heme degradation product (FHDP, em. approximately 466 nm) are identified and simultaneously detected in mouse and human red cell hemolysates, when excited at 365 nm. A novel method is established for comparing relative FHDP, PPIX and ZPP levels in hemolysates without performing red cell porphyrin extractions. The ZPP fluorescence directly measured in hemolysates (F(365/594)) correlates with the ZPP fluorescence obtained from acetone/water extraction (R(2) = 0.9515, P < 0.0001). The relative total porphyrin (ZPP and PPIX) fluorescence obtained from direct hemolysate fluorescence measurements also correlates with red blood cell total porphyrins determined by ethyl acetate extraction (Piomelli extraction, R(2) = 0.88, P < 0.0001). These fluorescent species serves as biomarkers for alterations in Hb synthesis and Hb stability.

Animals↗

Free erythrocyte protoporphyrin, zinc protoporphyrin and blood lead in newly re-exposed smelter workers: a prospective study.

The relationship between blood lead and free erythrocyte protoporphyrin (FEP) or zinc protoporphyrin (ZPP) was evaluated prospectively for 50 weeks in a group of workers who were removed from occupational lead exposure for 10 weeks. FEP or ZPP continued to fall in spite of a rising blood lead upon re-exposure. Although statistically significant relationships between square root FEP or ZPP and blood lead were found, wide confidence limits prevent meaningful predictions of blood lead from FEP or ZPP for an individual. Blood lead is a better index of exposure than FEP or ZPP, especially when exposure is not stable.

Environmental Exposure↗

A comparison of protoporphyrin IX and protoporphyrin IX dimethyl ester as a photosensitizer in poorly differentiated human nasopharyngeal carcinoma cells.

Protoporphyrin IX dimethyl ester (PME), a dimethyl esterification of protoporphyrin IX (PpIX), exhibits higher intracellular uptake into NPC/CNE2 cells, a poorly differentiated human nasopharyngeal carcinoma, than does PpIX. Phototoxicity studies reveal PME to be a more potent photosensitizer than is PpIX, at the early and late incubation time points. Correlating phototoxicity with subcellular localization indicates that PME is a more potent photosensitizer when its primary target of photodamage is mitochondria. Also, additional targeting of lysosome enhances phototoxicity.

Apoptosis↗

Phototoxicity of protoporphyrin IX, diarginine diprotoporphyrinate and N,N-diphenylalanyl protoporphyrin toward human fibroblasts and keratinocytes in vitro: effect of 5-methoxypsoralen.

The phototoxicity of two new porphyrin photosensitizers, diarginine diprotoporphyrinate (PP(Arg)2) and N,N-diphenylalanyl protoporphyrin (PP(Phe)2), and the synergistic effect of 5-methoxyposralen (5-MOP) have been studied in comparison with that of protoporphyrin IX (PPIX). Under ultraviolet-A (UV-A) irradiation (lambda=365 nm), the phototoxicity of the porphyrins toward cultured human fibroblasts and keratinocytes decreases in the order: PPIX > PP(Arg)2 > PP(Phe)2. A synergistic effect of 5-MOP on the phototoxicity of PPIX, PP(Arg)2 and PP(Phe)2 has been observed. The combination of PPIX, PP(Arg)2 and PP(Phe)2 with 0.1-0.5 microM 5-MOP significantly potentiates the phototoxicity of the three porphyrins. The most effective potentiation was observed with the water-soluble PP(Arg)2 and 5-MOP concentrations lower than 0.75 microM. Above this 5-MOP concentration this potentiation is abolished. The intracellular concentration of PPIX and PP(Phe)2 is independent of the presence of 5-MOP. On the other hand, the intracellular content of PP(Arg)2 is decreased in a concentration-dependent manner by the psoralen. Illumination with red light, not absorbed by 5-MOP, leads to a weak potentiation of the PP(Arg)2 phototoxic effect in the presence of 5-MOP, suggesting that dark interaction of 5-MOP with cell membranes aggravated by porphyrin photosensitization is involved in the observed phenomena. The results are tentatively explained by differences in hydrophobicity and molecular structures of the examined photosensitizers. PPIX, which is barely soluble in water, has a significantly higher affinity for cell membranes and simultaneously exerts a stronger phototoxic effect than PP(Arg)2 whose solubility in water is high. On the other hand, the weak phototoxicity of PP(Phe)2 could be explained by the steric hindrance brought by the phenylalanyl substituents on the pyrrole ring. The loss in the PP(Arg)2 cell content probably explains the inhibition of the synergistic effect of 5-MOP on the PP(Arg)2 phototoxicity at high 5-MOP concentration. This study suggests that PP(Arg)2 in combination with 5-MOP might reveal a strong phototoxic effect when applied to skin cancer treatment.

5-Methoxypsoralen↗

[Dose-effect relationships between blood lead and free erythrocyte protoporphyrin or zinc protoporphyrin].

Studies on dose-effect relationships between blood lead (PbB) and free erythrocyte protoporphyrin (FEP) or zinc protoporphyrin (ZPP) in 583 workers (male 326, female 257) was conducted. Good dose-effect relationships and S-shaped curves between PbB and logFEP, logZPP or log (ZPP/Hb) were established. Sectioned linear regression showed that linear correlation was obtained while PbB between 0.48 (10 micrograms/dl) and 3.36 mumol/L (70 micrograms/dl) (male) or 0.48 (10 micrograms/dl) and 2.88 mumol/L (60 micrograms/dl) (female). However, while PbB below 0.48 mumol/L (10 micrograms/dl), there was no linear correlation. The increasing of FEP, ZPP slowed down remarkably as PbB were beyond 3.36 mumol/L (70 micrograms/dl) (male) or 2.88 mumol/L (60 micrograms/dl) (female). Therefore, the degree of lead poisoning can't be classified by the values of FEP, ZPP among plumbism.

Dose-Response Relationship, Drug↗

Investigation of protoporphyrin IX standard materials used in acid-extraction methods, and a proposed correction for the millimolar absorptivity of protoporphyrin IX.

Erythrocyte protoporphyrin (EP) has been used for more than 30 years as an indicator of lead intoxication, iron deficiency, and porphyrias. Recently, numerous analytical problems associated with various EP methods have been reported, including a lack of consensus among investigators regarding the best calibration material or analytical procedure. We investigated commercially available protoporphyrin IX (PPIX) standard materials and measured the millimolar absorptivity (m epsilon) of these materials, focusing on variables affecting the determination of their absorptivities. Among the five forms of PPIX available, PPIX dimethyl ester, when hydrolyzed to PPIX free acid, gave the most consistent and reproducible results. This work confirmed our earlier observations, made on more than 600 separate occasions during 12 years, that the m epsilon of PPIX free acid in 1.5 mol/L HCl at the Soret maximum is 297 +/- 1.3 L.mmol-1.cm-1, 19% higher than the arbitrary value of 241 L.mmol-1.cm-1 generally accepted by most investigators but based on unpublished data. We propose that the m epsilon of 297 L.mmol-1.cm-1 for PPIX be adopted and that PPIX dimethyl ester be used for the calibration of acid-extraction methods. A detailed protocol for the preparation and verification of PPIX from the dimethyl ester is available upon request.

Chromatography, High Pressure Liquid↗

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.

Chromatography, High Pressure Liquid↗

Simultaneous liquid-chromatographic determination of zinc protoporphyrin IX, protoporphyrin IX, and coproporphyrin in whole blood.

We describe a method for simultaneously measuring concentrations of coproporphyrin, zinc protoporphyrin IX, and protoporphyrin IX in whole blood by liquid chromatography, with use of reversed-phase ion-pair system, fluorometric detection, and internal standardization. Each analysis requires 10 microL of whole blood and 15 min total analysis time. Analytical recovery ranged from 84 to 92%, day-to-day precision (CV) from 5 to 12%. Uroporphyrin, though not studied in detail, can also be detected by this method.

Chromatography, Liquid↗

Antioxidative effect of protoporphyrin and increase of glutathione in protoporphyrin-administered rat liver.

The effect of protoporphyrin (PP) administration on the activities of enzymes related to and/or involved in lipid peroxidation and on the content of reduced glutathione (GSH) was investigated in rat liver. PP, at an intravenous dose of 20 mg/kg, increased GSH content, caused a weak suppression of NADPH-cytochrome c reductase activity and a slight increase of gamma-glutamyl transpeptidase activity 24 h after dosing, but had no effect on the activities of other enzymes such as xanthine oxidase, superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glutathione S-transferase, gamma-glutamylcysteine synthetase or glutathione synthetase. Treatment of rats with diethyl maleate following PP injection resulted in the disappearance of antioxidative action of PP. Furthermore, sinusoidal, but not canalicular, efflux of hepatic GSH was decreased by the PP treatment. The increase of liver GSH content by PP treatment due to the decrease of sinusoidal efflux of GSH from the liver, thus would be involved in the exertion of antioxidative action of PP.

Animals↗

Interaction of hemopexin with Sn-protoporphyrin IX, an inhibitor of heme oxygenase. Role for hemopexin in hepatic uptake of Sn-protoporphyrin IX and induction of mRNA for heme oxygenase.

Sn-protoporphyrin IX (SnPP), an inhibitor of heme oxygenase and a potential therapeutic agent for neonatal hyperbilirubinemia, is bound tightly by hemopexin. The apparent dissociation constant (Kd) at pH 7.4 is 0.25 +/- 0.15 microM, but estimation of the Kd for the SnPP-hemopexin complex is hampered by the fact that at physiological pH SnPP exists as monomers and dimers, both of which are bound by hemopexin. SnPP is readily displaced from hemopexin by heme (Kd less than 1 pM). The hemopexin-SnPP interaction, like that of heme-hemopexin, is dependent on the histidine residues of hemopexin. However, as expected from the differences in the coordination chemistries of tin and iron, the stability of the histidyl-metalloporphyrin complex is lower for SnPP-hemopexin than for mesoheme-hemopexin. Nevertheless, when SnPP binds to hemopexin, certain of the ligand-induced changes in the conformation of hemopexin which increase the affinity of the protein for its receptor are produced. Binding of SnPP produces the conformational change in hemopexin which protects the hinge region of hemopexin from proteolysis, but SnPP does not produce the characteristic increase in the ellipticity of hemopexin at 231 nm that heme does. Competition experiments confirmed that human serum albumin (apparent Kd = 4 +/- 2 microM) has a significantly lower affinity for SnPP than does hemopexin. Appreciable amounts of SnPP (up to 35% in adults and 20% in neonates) would be bound by hemopexin in the circulation, and the remainder of SnPP would be associated with albumin due to the latter's high concentration in serum. Essentially no non-protein-bound SnPP is present. Importantly, SnPP-hemopexin binds to the hemopexin receptor on mouse hepatoma cells with an affinity comparable to that of heme-hemopexin and treatment of the hepatoma cells with SnPP-hemopexin causes a rapid increase in the steady state level of heme oxygenase messenger RNA. These results show that hemopexin participates in the transport of SnPP to heme oxygenase and in its regulation by SnPP.

Animals↗

Iron protoporphyrin IX (hemin) but not tin or zinc protoporphyrin IX can stimulate gene expression in K562 cells from enhancer elements containing binding sites for NF-E2.

Many genes whose transcription is erythroid-specific contain enhancer or promoter elements that bind the transcription factor NF-E2. Hemin induction increases the expression of globin genes in the human erythroleukemia cell line K562, and increases the expression of reporters gene regulated by an enhancer elements containing binding sites for NF-E2. The failure of metalloporphyrins other than hemin to stimulate the transient expression of a CAT reporter gene linked to an enhancer element containing a binding site for NF-E2 was correlated with their failure to induce benzidine-positive K562 cells and increase the steady-state level of gamma-globin mRNA. This study suggests that elevated levels of zinc protoporphyrin IX found in the anemia of chronic disease, iron deficiency, and lead poisoning may contribute to a decrease in globin gene expression by interfering with the transcriptional activity of enhancer elements containing binding sites for NF-E2.

Base Sequence↗