Search

Fluorescence spectroscopy provides high sensitivity in quantitative analysis. However, due to spectral interference, it is difficult to determine the individual components of fluorescent multi-component mixtures in such complicated and important body matrices as blood, urine and feces without any pre-separation. In this study, a simple and rapid approach based on non-linear variable-angle synchronous fluorescence spectrometry coupled with partial least squares analysis (NLVASF/PLS) was developed for the simultaneous determination of protoporphyrin IX (PP), uroporphyrin III (UP) and coproporphyrin III (CP). The detection limits were 0.18, 0.29 and 0.24 nmol L(-1) for protoporphyrin IX (PP), uroporphyrin III (UP) and coproporphyrin III (CP), respectively. The individual components of blood porphyrins were quantified, by this method, simultaneously in one scan with only about 30s. The recoveries of this method were above 80% in human whole blood samples. This method provided a potential tool for the determination of porphyrins in whole blood and the differential diagnosis of porphyria, especially for rapid routine screening of large number of samples., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

50 Patients with chronic renal failure undergoing hemodialysis with or without porphyria cutanea tarda (PCT)-like skin changes were investigated. The total porphyrin amount in erythrocytes, plasma and dialysate and the distribution of porphyrin metabolites in plasma and dialysate were measured. In plasma, the group of patients with skin changes (referred as PCU = porphyria cutanea uremica) showed significantly increased uroporphyrin levels as compared to the non-symptomatic group. In addition, significant differences concerning the ratio uro-/coproporphyrin in plasma were shown: non-symptomatic patients with 0.87, as opposed to the PCU group with 3.7. Considerable differences between the level of vitamin ingestion were identified between the groups. Patients with PCU took distinctly less vitamins C, E and B than patients without symptoms.

Background: Identification of porphyrias relies on the measurement of different porphyrins in urine, feces and plasma. Separation of porphyrin isomers is essential for the differential diagnosis of some porphyrias., Method: Separation of naturally occurring porphyrins was achieved on a Chromolith RP-18 column with fluorimetric detection using a methanol/ammonium acetate gradient mobile phase. Fecal and plasma porphyrins were extracted with acetonitrile and water at different pH values., Results: Eight porphyrins including protoporphyrin eluted within 20 min with good resolution of each of the I and III positional isomer pairs for standards, urine and plasma, and within 50 min for feces. Improvement of the extraction method for fecal and plasmatic porphyrins resulted in high recovery (up to 89%) and reliable quantification of protoporphyrin., Conclusions: The present RP-HPLC method is specific and efficient for routine analysis of porphyrins in human urine, feces and plasma.

The porphyrin compound in human body mainly includes hemoglobin, protoheme, protoporphyrin, coproporphyrin, uroporphyrin and so on. As we know, the metabolism of the porphyrin in human body depends on the physical conditions. It was found that the relative content of protoporphyrin (PPIX) and carotenoid in blood varied with the developing of the malignant tumor. A system, including Ti: sapphire laser, optical fiber delivery system, fluorescence probe, spectrometer CCD and computer, has been developed to examine the porphyrin in human superficial skin tissue without trauma. It was shown that the fluorescence spectrum of the porphyrin obtained from the superficial skin tissue of human earlobe has the same feature as that from the aqueous solution with PP IX.

A patient with porphyria cutanea tarda developed distinctive sclerodermoid changes with extensive dystrophic calcification and ulcerations in the sclerotic areas. In addition to these skin complications, over the course of 20 years other internal problems also appeared, such as diabetes mellitus, fatty cirrhosis and ocular inflammation. These severe clinical complications were related to omission of treatment and the alcohol abuse.

5-Aminolevulinate dehydrase (ALA-D) and porphobilinogen deaminase (PBG-D) are cytosolic enzymes involved in heme biosynthesis. ALA-D activity is altered both genetically and by the action of various environmental factors, including exposure to lead. The activity of PBG-D is reduced in acute intermittent porphyria. The aim of this work is to establish the 95% reference range of the erythrocytic activity of ALA-D and PBG-D in a control population. ALA-D activity limits were 15.8 and 50.2 nmol of PBG/ml of red blood cells (RBCS) per minute. For the activity of ALA-D restored by addition of zinc and dithiothreitol ("restored ALA-D"), these limits were 44.1 and 86.5 units. It has been found that the "restored ALA-D"/ALA-D ratio is very useful for the evaluation of lead toxicity, and its 95% reference range was between 1.22 and 3.06. It has been demonstrated that the best method for measuring erythrocytic PBG-D is using PBG, but not ALA, as substrate; its 95% reference range was between 20.9 and 63.2 nmol of uroporphyrin/ml of RBCs per hour. Knowledge of these reference range values in a control population constitutes the basis for an accurate diagnosis of heavy metal intoxication and acute intermittent porphyria.

Porphyria cutanea tarda (PCT) is a disorder of porphyrin metabolism that leads to massive overproduction and excretion of uroporphyrin. Most plasma porphyrins are bound to albumin and hemopexin. The aim of this work was to analyze the relationship between the concentrations of serum albumin and hemopexin, the levels of total, free and protein-bound plasma porphyrins and the urinary coproporphyrin and uroporphyrin excretion, in PCT patients at different stages of the disease. Urinary porphyrins showed a stronger correlation with total plasma porphyrin levels (r = 0.863) than with the free fraction of plasma porphyrins (r = 0.608). Patients considered in an active stage of PCT, have a higher mean level of total plasma porphyrins (8.80 micrograms/dl +/- 8.75) and a lower mean percentage of free plasma porphyrins (12.79% +/- 11.21) than those patients on remission (0.71 +/- 0.5 microgram/dl and 44.3 +/- 35.3%, respectively). 30% of patients showed hypohemopexinaemia, presumably due to hepatic damage. Despite the high affinity of this protein for porphyrins, no significant correlation was found between plasma porphyrin levels and hemopexin or albumin. It is concluded that (i) the kidney is not merely a passive filter for free plasma porphyrins and (ii) that the formation of hemopexin-porphyrin complex occurs when plasma porphyrins concentrations are increased (i.e. in those patients in an active stage of PCT).

Raised plasma uroporphyrin levels were found in all the 14 patients with end-stage renal disease studied, 7 of whom were on continuous ambulatory peritoneal dialysis (CAPD) and 7 on hemodialysis (HD). The elevation observed in the HD group was higher than that noted in the CAPD group; 18-fold in the former and 13-fold in the latter. The difference in uroporphyrin plasma levels between the two dialysis populations might be explained, at least partially, by the reduced activity of uroporphyrinogen decarboxylase (UROD), the enzyme which converts uroporphyrinogen to coproporphyrinogen. A decrease of 48% was noted in the HD group, whereas no change was observed in the CAPD group. A significant negative correlation (r = -0.37, p < 0.01) was found between the concentration of uroporphyrin in plasma and the activity of UROD. In view of the data shown, it is suggested that erythrocyte UROD activity should be interpreted with caution in HD patients suspected of having porphyria cutanea tarda.

In a considerable proportion of the patients with chronic renal failure, skin changes resembling porphyria cutanea tarda (PCT) develop some months to years after the onset of maintenance hemodialysis. This can be either real PCT, or secondary PCT, or PCT-like bullous dermatosis. In a minor proportion, real PCT can be diagnosed. In such cases, elevated total porphyrin levels with a predominance of uro- (I > III) and heptacarboxyl porphyrins (III > I) can be measured in the plasma (also in the urine, if not anuric), and fecal (perhaps urinary as well) isocoproporphyrin can be detected. The activity of the hepatic uroporphyrinogen decarboxylase (UD) is decreased in every type of PCT; in PCT-II, also that of the erythrocyte UD. In a higher proportion, secondary PCT (pseudo-PCT) develops. In this group, porphyrins are accumulated in the plasma due to the unsatisfactory renal function. Uro and hepta are the dominant fractions here as well, but alteration in the ratio of the uro isomers or the presence of isocoproporphyrin can not be expected. The UD activity is probably normal in every tissue. In 1% to 18% of the cases, PCT-like bullous dermatoses develop, but porphyrins are at normal levels in all compartments. The phototoxic agent here is other than porphyrin (e.g. nalidixic acid, furosemide, tetracycline, etc., or unknown). The authors review the knowledge on chronic hemodialysis-related PCT or the PCT-like bullous dermatoses: development of the above-mentioned conditions, clinical and morphological and biochemical features, difficulties in diagnosis, or the possibilities in therapy.

Patients with chronic renal failure, especially those receiving maintenance haemodialysis, have a number of dermatological alterations. Some of them are similar to those seen in porphyria cutanea tarda (PCT). Whereas early studies showed normal plasma porphyrin levels, a striking elevation of plasma porphyrins, and particularly of uroporphyrin, has recently been found. We measured the concentrations of porphyrins in the plasma and erythrocytes of 55 patients with chronic renal failure and receiving maintenance haemodialysis and also in 7 patients with PCT and 100 healthy volunteers. The mean concentration of porphyrins in plasma was 2.7-fold, and the maximum concentration, 6-fold the highest value measured in plasma of controls. The mean plasma concentration of uroporphyrin was 6-fold higher up to a maximum value of 37-fold the upper limit of the controls. The plasma porphyrin values of 3 of 7 patients with PCT were on the same high level as those measured in patients undergoing haemodialysis. The mean porphyrin concentration in the erythrocytes of haemodialysis patients were 1.5-fold the control values. Because of the known pathophysiological effect of uroporphyrin, especially its stimulation of the collagen synthesis and the activation of the complement system, we suppose that the porphyria cutanea tarda like skin lesions in patients with chronic renal failure are due to the highly increased uroporphyrin concentration.

Potential pitfalls in the determination of porphobilinogen deaminase activity, as well as ways of eliminating these sources of error and determining the activity accurately, are discussed. In addition to measurement of the accurate activity, the described method (a combination of incubation of homogenate with porphobilinogen and high-performance liquid chromatographic separation) can also be used to detect enzymic defects in the haem biosynthetic pathway, according to the pattern of accumulation of the various porphyrins.

A reversed-phase high-performance liquid chromatographic method is described for the determination of hydroxy and peroxy acid derivatives of uroporphyrin in the plasma of patients with congenital erythropoietic porphyria. The porphyrins were extracted from the plasma with 20% trichloroacetic acid-dimethyl sulphoxide (1:1, v/v). The supernatant after centrifugation was chromatographed on a Hypersil-ODS column by gradient elution with 9% (v/v) acetonitrile in 1 M ammonium acetate buffer (pH 5.16) (solvent A) and 10% (v/v) acetonitrile in methanol (solvent B) as the gradient mixture. The method was also suitable for the preparative isolation of the porphyrins.

The clinical value of a very rapid spectrofluorometric method for the determination of plasma uroporphyrin levels was studied during follow-up of 122 patients with porphyria cutanea tarda (PCT). Four-hundred and eight measurements were carried out within a 3-year period. In active PCT plasma uroporphyrin varied between 15 and 448 nmol/l (normal 0-1.4). A high correlation was seen between elevated levels of uroporphyrin in the plasma and urine in new cases (r = 0.72, n = 23) and relapses (r = 0.92, n = 37). A parallel course between these variables was noted during the treatment of 31 patients. The correlation was less pronounced (r = 0.42) in remission, i.e. in those cases with urinary uroporphyrin levels lower than 240 nmol/24 h. However, only in nine of 249 measurements taken in remission did plasma uroporphyrin exceed 15 nmol/l. In six of these cases a biochemical relapse had occurred at the next follow-up measurement as judged by an increase of uroporphyrin in the urine. It is suggested that treatment of new cases and relapses should continue until plasma uroporphyrin drops under 10 nmol/l. Values between 15 and 23 nmol/l in individuals already treated raise the suspicion of relapse and should be rechecked in the near future. Retreatment is necessary when the levels exceed 23 nmol/l. The use of the method is recommended as a simple and effective way for monitoring the progress of patients with PCT.

Reversed phase ion pair chromatography is a highly selective separation technique for the determination of free porphyrin carboxylic acids from human materials. Isocratic and gradient elution methods can be used to analyse porphyrin isomers and to establish porphyrin profiles for the biochemical diagnosis of porphyrias. Ion pair high performance liquid chromatography led to the discovery of the atypical isomers II and IV of uroporphyrin and coproporphyrin in human urine. Advantages and limitations of the ion pair technique are discussed.

Plasma porphyrins and free erythrocyte protoporphyrins were measured in sixty-two chronic hemodialysis patients. Plasma porphyrins were significantly elevated in these patients and overlapped the lower portion of the range observed in twenty-four patients with porphyria cutanea tarda. Free erythrocyte protoporphyrins were moderately elevated in four patients. Overall, these values were not statistically different from values for a control group of specimens from fifty ambulatory dermatology patients. However, they were, on average, lower than the normal values previously determined for the method used.

Seven patients undergoing treatment for cutaneous hepatic porphyria had serial measurements of their plasma uroporphyrin. In each case there was a progressive fall during treatment but even in remission the levels did not return to normal. There was a significant correlation between urinary and plasma uroporphyrin concentrations.

A 56-yr-old man presented with a photosensitive bullous dermatosis 1 yr after commencing hemodialysis. Urinary porphyrin levels were normal and fecal porphyrin levels only minimally increased, but the plasma uroporphyrin concentration was markedly elevated, being higher than that normally occurring in porphyria cutanea tarda. The high plasma uroporphyrin level was thought to result from the combined effects of increased porphyrin production and failure of adequate clearance by hemodialysis. Chronic alcohol ingestion, methyldopa therapy and elevated iron and aluminum levels may have contributed to the porphyrin overproduction. Charcoal hemoperfusion proved unhelpful in lowering the plasma uroporphyrin concentration.

Patients with porphyrias have varying degrees of photosensitivity, associated with elevated levels of porphyrins in plasma, erythrocyte, urine and/or feces. To investigate the role of complement in the pathogenesis of cutaneous lesions, varying amounts of uroporphyrin were added to normal human serum (0.1-10 microgram/ml), and the mixtures were then exposed to 405 nm irradiation. Such treatments result in the diminution of total hemolytic complement activity and hemolytic titers of C1, C4, C2, C3, and C5; furthermore, cleavage products of C3 and C5 were detected. Chemotactic activity for human polymorphonuclear leukocytes was generated that was inhibitable by incubation with anti-C5, but not with anti-C3 antisera. No chemotactic activity was generated in Mg++-EGTA treated serum nor in C4-deficient guinea pig serum. These data indicate that irradiation with 405 nm light of normal human serum containing uroporphyrin results in activation of the complement system via the classical pathway, and the generation of complement (C5)-derived chemotactic activity for human polymorphonuclear leukocytes.

The protoporphyrin accumulation observed in the red blood cells of patients with erythropoietic protoporphyria can be explained by decreased activity of the enzyme heme synthetase or by increased production of porphyrins in the affected cells. In literature experimental evidence both for a partial heme synthetase deficiency and for increased porphyrin biosynthesis has been presented. In a group of ten patients with erythropoietic protoporphyria the biosynthesis of porphyrins from delta-aminolevulinic acid and from glycine-succinic acid appeared to be normal in peripheral blood cells. These results are consistent with the partial heme synthetase deficiency previously found to be the basic defect of this disease.

.1 ml plasma was precipitated and extracted with 3 ml 0.61 mol/l trichloroacetic acid. After centrifugation the fluorescence intensity of the supernatant was measured using uroporphyrin as a standard. The porphyrins with a higher number of carboxylic groups (uroporphyrin and heptacarboxyporphyrin) were effectively extracted and the others, poorly or very poorly. Thus, the determined parameter was a mixture of chiefly highly carboxylated porphyrins. The results were expressed in nmol uroporphyrin/l. The coefficient of variation was 4%. No quenching of the fluorescence was established in higher plasma porphyrin concentrations. The range of the control group (n = 33) was 0-1.4 nmol/l. The data of the patients, needing treatment were within the interval of 17.9-448 nmol/l (n = 17). The correlation coefficient with the urinary uroporphyrin was 0.97. The values in the cases with remission (n = 74) ranged from 0 to 12.8 nmol/l. The method is particularly convenient for monitoring the effect of treatment in patients with Porphyria cutanea tarda, as well as for their long-term follow-up.

Rapid and reproducible assays for uroporphyrinogen III synthase (URO-S; EC 4.2.1.75) have been developed and used to determine the enzymatic activity in human erythrocytes and cultured lymphoid cells. In the coupled-enzyme assay, porphobilinogen was first converted to hydroxymethylbilane, the natural substrate for URO-S, by hydroxymethylbilane synthase which was conveniently obtained from heat-treated erythrocyte lysates. In the direct assay, synthetic hydroxymethylbilane was used as substrate. In both assays, the uroporphyrinogen reaction products were oxidized to their respective uroporphyrin isomers, which were then resolved and quantitated by reversed-phase high-pressure liquid chromatography. Both assays were optimized for pH, substrate concentration, and linearity with time and protein concentration. The mean URO-S activities in normal human erythrocyte lysates determined by the coupled-enzyme and direct assays were 7.41 +/- 1.35 and 7.64 +/- 1.73 units/mg protein, respectively. In normal human cultured lymphoid cells, the mean activities were 13.7 +/- 1.39 and 17.6 +/- 1.15 units/mg protein for the coupled-enzyme and direct assays, respectively. In four families with congenital erythropoietic porphyria, both assays reliably identified the markedly decreased URO-S activities in erythrocytes and cultured lymphoid cells from affected homozygotes and the half-normal activities in these sources from obligate heterozygotes. The coupled-enzyme assay was easier to perform and was suited for clinical diagnostic assays and for monitoring enzyme purification procedures, while the direct assay, which required substrate preparation and technical dexterity, was best for kinetic studies of URO-S.

About 3 years after commencing hemodialysis for chronic renal failure, a 39-year-old man developed cutaneous lesions of the face and hands which were compatible with porphyria cutanea tarda (PCT) clinically and histologically. There was no evidence of familial PCT, excessive alcohol consumption, iron overload, chronic liver disease. It was impossible to measure urinary porphyrins because of anuria. Fecal and erythrocytic porphyrins were within normal limits. Plasma porphyrins, however, were markedly elevated, and in an assay using high performance liquid chromatography, uroporphyrin accounted for 72% and 7-carboxyl porphyrin for 24%. Review of past reports of PCT associated with hemodialysis revealed that the most characteristic feature was a significant increase of plasma porphyrins, mainly uroporphyrin. This increase is difficult to explain by accumulation due to failure of adequate clearance. Participation of factors affecting uroporphyrinogen decarboxylase activity seems likely.

The rat hemidiaphragm was studied in vitro as a test system to evaluate the effects of heme precursors and the uroporphyrins upon neuromuscular excitability. Porphobilinogen and porphobilin had no effect on the resting miniature end-plate potential frequency, but the K(+)-augmented frequency was significantly reduced. Porphobilin and porphobilinogen gave 50% of their maximal effect at concentrations of 0.008 and 0.6 mug/ml, respectively; the effect increased with concentration. Uroporphyrin I at 0.05-1.0 mug/ml caused a 25% decrease in frequency, but the effect did not increase with concentration. At similar concentrations, uroporphyrin III was without effect. The concentrations of porphobilin and porphobilinogen effective in inhibiting the K(+) stimulation, which are several orders of magnitude lower than the effective concentrations of simple amino acids, are those which might reasonably be expected in the sera of patients with acute intermittent porphyria.