Porphyrias are a group of diseases that are clinically and genetically heterogeneous and originate mostly from inherited dysfunctions of specific enzymes involved in heme biosynthesis.
Porphyrias | ADP/AIP/HCP | VP | PCT/HEP/CEP | EPP/XLP |
---|---|---|---|---|
Plasma peak (nm) | 618–622 | 626–628 | 618–620 | 632–636 |
The quantification of ALA and PBG forms the first line of laboratory testing for acute porphyria in the event of potentially life-threatening acute neurovisceral attacks [42]. In specialized porphyria diagnostic laboratories, ALA and PBG are commonly quantified after purification, from a spot urine sample, using two commercially available anion-exchange and cation-exchange columns (ClinEasy® Complete Kit for ALA/PBG in Urine, Recipe GmbH, Munich, Germany; ALA/PBG by Column Test, Bio-Rad Laboratories, Hermes, CA). This approach enables the selective purification of ALA and PBG, which removes the sample matrix, thereby preventing possible interferences from other compounds [43]. In several certified European laboratories (EPNET), the preferred choice is the ClinEasy® Complete Kit for ALA/PBG in Urine of Recipe for the quantification of both ALA and PBG in the urine. Briefly, urine samples are passed through two overlapping columns – the top column containing an anion exchange resin adsorbs PBG, and the ALA passes through this top column and is subsequently retained by the cation exchange column at the bottom. The adsorbed PBG is eluted from the top column using acetic acid and mixed with Ehrlich’s reagent, upon which the solution develops a purple color, whose intensity is proportional to the amount of the metabolite in the solution. The retained ALA is eluted using sodium acetate, and after derivatization with acetyl-acetone at 100 °C, it is converted into a monopyrrole, which is the measurable form capable of reacting with Ehrlich’s reagent. The absorbance of both PBG and ALA is measured against the blank reagent at 533 nm using a UV-VIS spectrophotometer. Metabolite concentrations are calculated through comparison to the appropriate calibrators (Urine Calibrator Lyophil RECIPE GmbH, Munich, Germany). The results are validated using normal and pathological controls (ClinChek Urine Control L1, L2, RECIPE GmbH, Munich, Germany) reconstituted in high purity water and stored in single-dose aliquots at –20 °C until use. The concentration values are expressed as µmol/mmol creatine; ALA and PBG are considered over the normal limits if the concentration values are >5 and >1.5 µmol/mmol creatine, respectively. A high level of ALA and a normal level of PBG indicate either the rare ALAD deficiency porphyria or the more common heavy-metal intoxication and hereditary tyrosinemia type I caused by the inhibition of ALAD by lead and succinyl-acetone, respectively [44;45]. High levels of up to 20–50 times the normal values of both the metabolites establish the diagnosis of an acute attack, while moderate ALA and PBG levels could indicate VP and HCP [6]. The majority of the patients affected by acute porphyria after the symptomatic period may revert completely, while a few might become clinically asymptomatic along with persistent moderate increments in the heme precursors.
Over the last decades, the technique of liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been employed in numerous clinical biochemistry applications. In comparison to traditional methods, LC-MS/MS has major advantages of higher analytical sensitivity, specificity, and diagnostic reliability. About porphyria, the LC-MS/MS technique has been applied successfully for the simultaneous quantification of ALA and PBG in urine and plasma samples [46-49].
Notably, significantly lower levels of urinary ALA and PBG could be measured using MS-based methods, particularly for healthy individuals, confirming that these methods had a higher selectivity compared to the colorimetric ones [43]. The difference is probably due to the presence of contaminant molecules in the urine, which react with Ehrlich’s reagent. Recently, LC-MS/MS measurements of a large number of healthy subjects established the upper limit of the normal values (ULN) of ALA and PBG as 1.47 and 0.137 µmol/mmol creatinine, respectively [50], and it was reported that during an acute attack, these values could reach 40 and 55 µmol/mmol creatinine, respectively [48]. Moreover, sample preparation using a solid-phase extraction (SPE) system allows the detection of concentrations as low as 0.05 µM [49]. Therefore, measurements of porphyrin precursors in plasma and tissue samples have become achievable. Floderus et al. quantified the plasma levels of ALA and PBG in 10 asymptomatic AIP patients and reported the mean concentrations to be 1.7 and 3.1 µmol/L, respectively, which were significantly higher than those of the healthy subjects (0.38 and <0.12 µmol/L, respectively) [46]. However, during an acute attack of porphyria, ALA and PBG concentrations may increase dramatically, up to 13 µmol/L [47].
Since the variation over the time of the plasma concentrations of ALA and PBG is reported to be highly correlated with urinary concentrations [46], these measurements may contribute to the monitoring of AIP patients during the course of an acute attack [51], particularly if the patients are in an anuric state. Plasma ALA and PBG levels could also be useful in evaluating the safety and the pharmacokinetic effects of the existing or future therapies in AHP patients [52]. However, not all hospitals are equipped with LC-MS/MS, restricting this technique to a few specialized centers.
The differential diagnosis of porphyria relies on the measurements of porphyrins and relative isomers in urine, feces and plasma. As plasma porphyrin separation find a practical application only in patient with renal failure, this topic is not treated in this review.
Although various methods have been developed for the analysis of urine porphyrins, reverse-phase high-pressure liquid chromatography coupled with fluorescence detection (FLD-HPLC) has become the gold standard method for this purpose [53-55]. The success of this technique is attributed to its capacity to separate the physiologically relevant porphyrins as free acids and resolve type I and type III porphyrin isomers simultaneously [56]. Since the fluorescence detection enhances the specificity of analytic method, matrix extraction procedures are not required and acidic urine samples could be directly injected.
Typical chromatographic runs are performed on C18-bonded silica stationary phase using a linear gradient elution system from 10% (v/v) acetonitrile in 1M ammonium acetate, pH 5.16 (phase A) and 10% acetonitrile in methanol (phase B). Porphyrins containing from two to eight carboxylic groups, including the resolution of type I and type III isomers could be achieved in less of 30 minutes. The excitation and emission wavelength ranges used are usually around 395–420 nm and 580–620 nm, respectively. Direct standardization is obtained by comparison to chromatographic runs of suitable calibration standard that are now commercially available (RECIPE GmbH, Munich, Germany; Chromsystems GmbH, Gräfelfing, Germany). Total urine porphyrin measurement is calculated as sum of chromatographic fractions and the single fractions as relative percentages. As spot urine samples are commonly used, results should be normalized on creatinine concentration.
In normal subjects, the total porphyrins excretion is found below 35 nmol/mmol creatinine, COPRO predominate on URO while hepta-, hexa-, and penta-carboxyl porphyrins are present only in small amounts. Moreover, relative concentrations of COPRO isomer III are higher than COPRO isomer I.
In porphyric patients, the excretion of urinary porphyrins varies in relation to the enzymatic defect underlying the particular type of porphyria and with respect to the disease stage [57]. URO, both the type I and type III isomers, and hepta- type III are evidently raised in PCT and HEP since the type I isomers of URO and COPRO are detected in CEP almost exclusively. In acute hepatic porphyrias (AIP, VP, and HCP), porphyrins excretion is extremely variable from normal values in asymptomatic phase to very high values observed during the acute exacerbation of disease. In AIP, a pattern of marked elevation of URO I and III isomers, along with less pronounced elevations of COPRO III and I, is detected, while HCP and VP demonstrate a marked elevation of COPRO III. In EPP, total urine porphyrins are normal however, abnormal chromatographic profiles are frequently observed. Particularly, higher relative amounts of COPRO I are commonly observed as a consequence of potential hepatic implications. In fact, increased levels of porphyrins excretion may also occur in several physio-pathological conditions including hereditary hyperbilirubinemias, toxic syndromes or liver diseases [58].
Recently, protocols using mass spectrometry (LC-MS/MS) to separate and detect porphyrins have also been reported to facilitate the clinical diagnosis of porphyria [48]. However, the low prevalence of this equipment in hospitals restricts the application of such protocols to the identification and characterization of unknown porphyrins in research.
Two different systems may be employed for the analysis of fecal porphyrins. First, the total fecal porphyrins can be quantified using a spectrophotometric [58;59] or fluorimetric [60] method and then separated by HPLC to obtain porphyrin patterns [61]. Second, total fecal porphyrins can be calculated as the sum of fractions following HPLC analysis [62]. The former approach is considered more suitable for routine use in clinical analysis, while the latter is technically more correct and, therefore, allows quantification with higher reliability [63].
In the widely employed method reported by Lockwood et al., porphyrins are extracted from a small sample of feces in the aqueous acid phase using the solvent partition [59]. Briefly, 25–50 mg of feces are processed by sequential addition of 1 mL of concentrated HCl to dissolve the organic matrix, 3 mL of diethyl ether to eliminate the contaminants – chlorophylls and carotenoid pigments, and 3 mL water to avoid any alteration in protoporphyrin. The hydrochloric acid extract is then analyzed by performing a spectrophotometric scan between 370 nm and 430 nm, which includes the Soret region. After background signal subtraction, the absorbance measured at the maximum peak is used for calculating the total porphyrin content. It is also necessary to separately evaluate the water percentage in the feces sample to report the result as nmol/g dry weight (total fecal porphyrin normal value < 200 nmol/g dry weight) [59]. Although no commercial reference material is available, an internal quality control (ICQ) could be prepared from the patient’s specimens to check the day-today reproducibility. The obtained hydrochloric acid extracts may be directly injected into HPLC systems for subsequent characterization using the same protocol as the one used for urine porphyrins chromatography. Calibration solution is prepared adding appropriate concentrations of meso- and proto-porphyrin to the urine calibrators.
The fecal excretion of porphyrins increases in hepatic PCT, HCP and VP, in erythropoietic CEP and HEP and sometimes in EPP and XLP. The detection of the specific patterns of fecal porphyrins allows the differentiation of these enzymatic disorders that otherwise share similar clinical presentation and overlapping biochemical characteristics. Feces samples from healthy subjects, as well as from porphyria patients, contain varying amounts of dicarboxylic porphyrins, particularly deutero-, pempto-, and meso-porphyrins, in addition to protoporphyrin. These dicarboxylic porphyrins lack a diagnostic relevance, depending on the gut microflora [64] and even on the diet [65]. Moreover, gastrointestinal bleeding may interfere with feces analysis, causing anomalous increase in the levels of protoporphyrin and the dicarboxylic porphyrins derived from it [66]. Tricarboxylic porphyrins remain generally undetected, although the presence of harderoporphyrin in feces [67] played a major role in the identification of a variant of homozygous HCP (harderoporphyria). Uroporphyrin is excreted prevalently in the urine and may be detected in feces only in trace amounts.
Fecal porphyrins profiles of PCT and HEP are the most complex, with a wide range of peaks. It is possible to recognize COPRO I isomer, which always prevails over COPRO III, although increasing signals corresponding to epta-, esa-, and penta-porphyrins, with the prevalence of isomer III, are also observed. Since UROD is the enzyme that catalyzes the sequential decarboxylation of uroporphyrin to coproporphyrin, its activity deficit leads to the accumulation of all porphyrin intermediates in PCT. In HEP, the activity of UROD is severely compromised and the porphyrins containing a higher number of carboxylic groups are represented more. PCT chromatographic profiles are also characterized by the presence of isocoproporphyrin and its derived metabolites hydroxy-, keto-, deethyl-, dehydro-isocoproporphyrin. These molecules, are commonly identified as diagnostic sign of symptomatic PCT [64] nevertheless diagnoses based on the presence of isocoproporphyrin in the feces are prone to be erroneous.
Chromatographic profiles of fecal porphyrins of AIP and ADP patients are similar to those of healthy subjects. On the other hand, the fecal porphyrin chromatograms of HCP patients are easily recognizable by the presence of a prominent peak corresponding to COPRO III. Peaks for both PROTO and COPRO III are elevated in the chromatographic profiles of VP patients. In particular conditions, such as hepatitis or drug consumption, which inhibit the UROD activity, HCP and VP patients may exhibit fecal porphyrin patterns quite similar to those of PCT patients, including the isocoproporphyrin series. Since the relative abundance of COPRO III is always higher compared to isomer I in VP and HCP, while the opposite is true for PCT, the ratio between the coproporphyrin isomers serves as a suitable diagnostic parameter [68;69]. The fecal porphyrins pattern in CEP contains an elevated peak corresponding to COPRO I. Finally, the chromatograms of EPP and XLP patients present an elevated PROTO peak.
Initially, to assay total erythrocyte porphyrins, fluorometric methods involving the acid extraction procedure that converted ZnPP to its metal-free form PPIX were used [70]. Later, when it became possible to detect ZnPP in the whole blood samples without prior acid extraction, hematofluorometry methods were developed for this assessment [71;72]. Finally, the application of high-performance liquid chromatography (HPLC) coupled with fluorometric detection to separate and quantify unchelated PPIX and ZnPP simultaneously commenced [73;74]. A method using derivative variable-angle synchronous fluorescence (DVASF) for determining PPIX and ZnPP simultaneously in whole blood sample, avoiding the spectral compensation factor for PPIX and the chromatographic separation has been also reported [75]. A simple, rapid, and specific HPLC method is here described. Whole blood samples are collected in vacutainer tubes containing the anticoagulant K3EDTA and then stored at –20 °C in the dark. At the time of analysis, samples are thawed to room temperature and mixed well, followed by the dilution of 60 µL aliquots in 200 µL of lysing solution (4% aqueous formic acid) and the extraction of porphyrins using 900 µL acetone. An aliquot of the extracted porphyrins is injected directly into the HPLC system, using a Chromsystem C-18 column (Chromsystems GmbH, Gräfelfing, Germany) for chromatographic for separations. ZnPP and PPIX are eluted in isocratic conditions (90% methanol in aqueous 1% acetic acid solution) at 42 °C and subsequently detected using fluorescence excitation/emission wavelengths of 400 nm/620 nm (1–7 min) and 387/633 nm (7–15 min), respectively. In order to quantify PPIX and ZnPP, homemade calibration curves are used, and the analytes are observed to be linear in the concentration ranges of 1.5 - 50 µg/dL and 2 - 100 µg/dL, respectively. The results are reported as the sum of PPIX and ZnPP peaks, and the concentrations in the blood are expressed as both µg/g Hb and relative percentage of each porphyrin. The normal level for the sum is <3 µg/g Hb and normal percentage ranges are ZnPP 80 – 90 % and PPIX 10 - 20%. Erythrocyte porphyrins are abnormally increased in EPP, XLP, CEP, and HEP, although the percentage of each porphyrin differs among these disorders. It is noteworthy that elevated erythrocyte protopophyrins along with normal ZnPP support the diagnosis of the classical form of EPP, while an elevation in both components with balanced percentages occurs in the XLP. High levels of erythrocyte porphyrins also occur in the case of exposure to lead from both environmental and occupational conditions [76], in iron deficiency [77], as well as in sideroblastic anemia, all of which are conditions associated with elevated Zn PP. In CEP and HEP the predominant porphyrins may be uroporphyrins or ZnPP depending on phenotype expression.
The entry is from https://doi.org/10.3390/diagnostics11081343
This entry is adapted from the peer-reviewed paper 10.3390/diagnostics11081343