Prototype application of a robot in the clinical laboratory enabling fully automated quantification of fecal porphyrins.

Unpleasant specimens, sensitive analytes, and a lengthy chromatographic procedure were the main reasons we implemented fecal porphyrin analysis with a laboratory robot. We describe the system in detail and compare it with the same technique performed manually. The day-to-day variation of assays of standards was lower with the robot than with the manual operation: 8% (CV) for coproporphyrin I and 11% for protoporphyrin IX. We repeatedly analyzed a specimen from a healthy volunteer and determined that the specimen contained (in nmol/g dry wt) 7.1 (SD 0.7) for coproporphyrin I, 3.0 (SD 0.4) for coproporphyrin III, and 44.4 (SD 4.3) for protoporphyrin IX. Upper reference limits as measured in 20 healthy volunteers were 20 nmol/g dry wt for coproporphyrin I, 12 nmol/g for coproporphyrin III, and 80 nmol/g for protoporphyrin IX. We also present characteristic chromatograms for samples from various different porphyrias that exhibit abnormal fecal porphyrin excretion. Calculations of return on investment show that the robot, working at full capacity, is a profitable tool.     

Effects of haem infusion on biliary secretion of porphyrins, haem and bilirubin in man

Abstract. Employing a continuous bile collection, we measured the bile secretion of porphyrins, haem (iron protoporphyrin IX regardless of oxidation state) and bilirubin in five healthy subjects. The baseline values for the flow of porphyrins in the bile were: 4·7 pL 1·9 nmol/h uroporphyrin, 27·3 pL 3·8 nmol/h coproporphyrin and 39·2 pL 11·7 nmol/h protoporphyrin. Bile haem flow was 59·7 pL 12·6 nmol/h, and that of bilirubin 23·8 pL 8·2 μmol/h. Following haem injection (6·4 μmol/kg) the flow of protoporphyrin but not of the other porphyrins was reduced, and the bile haem flow increased (232 pL 109·5 nmol/h), while the flow of bilirubin did not increase significantly. A few patients with representative porphyrias showed the expected increase in copro- and protoporphyrin in the bile. The patient with coproporphyria exhibited a bile flow of coproporphyrin of 1470 pL 133 nmol/h and of protoporphyrin of 334 pL 29 nmol/h; haem infusion significantly reduced the bile flow of both porphyrins (to 649 pL 101 for copro- and 215 pL 36 nmol/h for protoporphyrin). The patient with protoporphyria had an increased protoporphyrin flow, yet haem infusion caused no reduction in protoporphyrin flow (106 pL 7 after v. 81·4 pL 13 nmol/h before haem).
In conclusion, we found that haem and porphyrins are normal constituents of bile, and that injected haem appears in bile. Bile bilirubin did not rise within 12 h after haem infusion a finding which warrants further investigation.     

Direct quantification of coproporphyrins and uroporphyrins in urine by derivative synchronous fluorescence spectroscopy

The second-derivative synchronous fluorescence spectroscopic technique is applied to the simultaneous and direct assay of coproporphyrins and uroporphyrins in human urine. This technique resolves the overlapping conventional spectra, obviating the need for pre-analysis sample separation techniques, and measurements can be made in a single scan. The amplitudes of the derivative peaks are linearly related to uroporphyrin (0.4 to 300 micrograms/L) and coproporphyrin (0.4 to 250 micrograms/L) concentrations. The detection limit for both porphyrins is 0.1 microgram/L. Analytical recoveries range between 98 and 101%. Within- and between-assay CVs are reported. Results for 24-h urine specimens correlated well with those obtained by an extraction-ion-exchange chromatography combined method. The proposed method is inexpensive and requires no sophisticated detection equipment.     

Plasma fluorescence scanning and fecal porphyrin analysis for the diagnosis of variegate porphyria: precise determination of sensitivity and specificity with detection of protoporphyrinogen oxidase mutations as a reference standard

BACKGROUND: Variegate porphyria (VP) is the autosomal dominant disorder associated with deficiency of the enzyme protoporphyrinogen oxidase (PPOX). Plasma fluorescence scanning has been reported to be a more sensitive test for VP than traditional fecal chromatography. Previous comparisons of these techniques predated identification of the PPOX gene. We assessed these techniques in a large group of patients characterized for VP at the DNA level. METHODS: We evaluated all patients for whom the genotype and a plasma scan or fecal porphyrin result were available. Mutations were detected by restriction digest analysis. Plasma fluorescence scanning was conducted according to published methods. Fecal porphyrins were identified and quantified by thin-layer chromatography. RESULTS: Plasma fluorescence scanning was assessed in 679 patients (205 with VP who were carriers of a PPOX mutation, either with disease symptoms or asymptomatic) and fecal analysis in 473 (190 with VP). Sensitivity and specificity of both tests were higher in adults than in children and higher for adults with disease symptoms than for asymptomatic carriers. In a direct comparison in 168 adults (73 with VP), plasma scanning was significantly more sensitive than fecal porphyrin analysis [sensitivity, 0.96 (95% confidence interval, 0.89-0.99) vs 0.77 (0.66-0.85)]. Fecal coproporphyrin [area under the curve, 0.87 (0.83-0.90)] was a better predictor of VP than protoporphyrin [0.80 (0.76-0.84)]. CONCLUSIONS: Plasma scanning is a more sensitive and specific test for VP than fecal porphyrin analysis. Neither test is sensitive in children, and both are less sensitive in asymptomatic carriers than in symptomatic cases. DNA analysis therefore remains the preferred method for the identification of carriers, particularly in children.     

Hereditary coproporphyria in Germany: clinical-biochemical studies in 53 patients

OBJECTIVES: To describe the biochemical and clinical features in hereditary coproporphyria (HCP). DESIGN AND METHOD: Within the last 20 years, we investigated 53 patients (male:female = 1:2.5; age = 8-86 years) suffering from HCP. We describe the characteristic levels of urine, and fecal porphyrins and their precursors in hereditary coproporphyria and present the clinical features. Especially, we measured the coproporphyrin isomers I and III. RESULTS AND CONCLUSION: The group of hereditary coproporphyria patients exhibited a significantly higher (p<0.0001) excretion of urinary porphyrin precursors, delta-aminolevulinic acid (median = 84 micromol/24 h) and porphobilinogen (median = 39 micromol/24 h), as compared to controls (delta-aminolevulinic acid: 22 micromol/24 h, porphobilinogen: 3 micromol/24 h; median, n = 20). The median of coproporphyrin in urine (1315 nmol/24 h) and feces (1855 nmol/g) were enhanced 12- and 168-fold, as compared to healthy subjects (urinary coproporphyrin: 106 nmol/24 h, fecal coproporphyrin: 11 nmol/g; median, n = 20). During therapy on one female patient, with IV application of heme arginate, a considerable decline of porphyrin precursors and porphyrin excretion was observed. The examination of urinary and fecal coproporphyrin isomers I and III revealed an excessive elevation of the coproporphyrin isomer III of 87% in urine and 94% in feces, respectively (normal: urinary isomer III = 69-83% and fecal isomer III = 25-40%). In feces the increase of isomer III caused an inversion of the physiologic coproporphyrin isomer III:I ratio that could be recognized in all various stages in hereditary coproporphyria and in children. Acute attacks of hereditary coproporphyria are accompanied by an acute polysymptomatic clinical syndrome, and this is associated with high levels of urinary porphyrin precursors. On review of our patients, the highest percentage had abdominal pain (89%), followed by neurologic (33%), psychiatric (28%), cardiovascular (25%), and skin symptoms (14%).     

Rapid procedure for fecal porphyrin assay

Hydrochloric acid extraction of feces in the presence of ether yields an extract suitable for spectrophotometric estimation of total porphyrin and for further separation by "high-performance" liquid chromatography (HPLC) or thin-layer chromatography. A total porphyrin reference interval of less than 200 nmol/g dry weight of feces was established from data on 106 normal subjects on an unrestricted diet. Total fecal porphyrin values in 11 porphyria cutanea tarda patients were considerably higher than given by the widely used Rimington method (respective means, 652 and 239 nmol/g dry weight). Our HPLC method for separation of porphyrin methyl esters on a silica column, with quantification by fluorescence, is described. HPLC separations performed on 23 porphyria cutanea tarda patients gave the following mean proportions of total fecal porphyrins: dicarboxylics 21%, coproporphyrin 9%, isocoproporphyrins 28%, pentacarboxylporphyrin 9%, hexacarboxylporphyrin 11%, heptacarboxylporphyrin 18%, and uroporphyrin 4%.     

Stool porphyrins determined by high pressure liquid chromatography and by fractional hydrochloric acid--ether extraction

When stool copro- and protoporphyrin were quantitated by spectrophotometry, after fractional hydrochloric acid-ether extraction, up to 30% of the protoporphyrins were recovered in the coproporphyrin extract. In disorders with elevated stool protoporphyrin excretion (e.g. erythropoietic protoporphyria), this method therefore may give falsely elevated coproporphyrin values. When the stool porphyrins were determined by high pressure liquid chromatography there was no carry-over of protoporphyrin to coproporphyrin. The recoveries were 82 and 87%, and the coefficients of variation 5.6 and 3.1% for proto- and coproporphyrin, respectively. Moreover, in specimens containing a more complex mixture of porphyrins, a complete separation of the C2-C8 porphyrins and porphyrin isomers was obtained in a single run. High pressure liquid chromatography should therefore be considered the method of choice to obtain a quantitative profile of stool porphyrins in the routine laboratory.     

A rapid and accurate spectrofluorometric method for quantification and screening of urinary porphyrins

We describe a fluorescence method for screening and quantifying urinary porphyrins. New and effective approaches are used to oxidize prophyrinogens, correct the baseline, and ensure that uroporphyrin (uro) and coproporphyrin (copro) are equally detected, mole for mole. No preliminary purification is required. A 45-microL aliquot of urine is oxidized with 3 mmol/L iodine in 3 mol/L HCl to convert porphyrinogens to porphyrins, and then decolorized with 5 mL of 0.45 mmol/L sodium thiosulfate. An excitation scan is done from 350 nm to 440 nm, monitoring emission at 650 nm. Total porphyrin content is determined at the isosbestic point for uro and copro, and the mole fractions of uro and copro are estimated from the wavelength of the signal maximum. There is no interference from protein, glucose, bilirubin, or hemoglobin in high concentration. The limit of detection is less than 30 nmol/L and linearity is maintained up to 3200 nmol/L. Recoveries and precision are excellent. This is a rapid, sensitive screen for porphyrinuria as well as an accurate and precise quantitative method. We compared the method with existing methods and discuss some shortcomings common to many of them.     

Quantification of urinary porphyrins by liquid chromatography after oxidation of porphyrinogens

A quantitative "high-performance" liquid-chromatographic method is described for determining porphyrins in human urine. Porphyrinogens in urine are first converted to the corresponding porphyrins by oxidation with iodine. Uroporphyrin, hepatacarboxylic acid porphyrin, hexacarboxylic acid porphyrin, pentacarboxylic acid porphyrin, and coproporphyrin I and III isomers are then separated on a reversed-phase column and measured by fluorometry. Analysis for the six porphyrins is complete within 24 min, including reconditioning for the next sample. The detection limit (twice the signal/noise ratio) for each porphyrin was 1 nmol/L for urine (25 fmol per 50-microL injection). Mean analytical recovery of each porphyrin ranged from 85% to 91%, within-day CVs from 1.4% to 7.3%. Normal reference intervals for porphyrins were established by assaying urine samples from 75 healthy subjects. Significant sex-related differences in coproporphyrin I and III isomers were evident when the values were expressed as nanomoles per gram of creatinine. Coproporphyrin isomer ratio was estimated for utility in the diagnosis of porphyrinurias.     

Quantitative fluorometric screening test for fecal porphyrins

We describe a fluorometric method for screening and quantifying porphyrins in stool. A small sample of stool is extracted with concentrated HCI and is diluted 200-fold in 3 mol/L HCI before analysis. An excitation scan is done from 350 to 450 nm, monitoring emission at 603 nm. Total porphyrin is estimated at the isosbestic point for coproporphyrin and protoporphyrin (402.5 nm). Monitoring emission at 603 nm eliminates interference from chlorophyll, obviating the need for extraction with ether. The position of the excitation peak gives some indication of the nature of the porphyrins in the stool. The acid extract can be injected directly into an HPLC system for fractionation studies. Our method correlates well with the spectrophotometric method developed by Lockwood et al. (Clin Chem 1985;31:1163-7). However, in our method, the sample is easier to process and the assay has higher sensitivity than their assay. The reference interval for porphyrin in healthy individuals by the fluorometric method is less than 300 nmol/g dry weight. We can detect as little as 1 nmol of porphyrin per gram (dry weight) of stool. Results of the method vary linearly with stool porphyrin concentrations as great as 4000 nmol/g dry weight. The within-run imprecision of the method is 3%.     

Improvement in HPLC separation of porphyrin isomers and application to biochemical diagnosis of porphyrias.

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.     

Erythrocyte protoporphyrins in hepatitis C viral infection

OBJECTIVES: Protoporphyrin is the immediate precursor of the heme molecule. Due to a spillover from hemaotopoietic tissue it is regularly found in small amounts in erythrocytes and excreted into the bile. In hereditary erythropoietic protoporphyria excess protoporphyrin accumulates and can cause severe liver damage both by crystallization and induction of oxidative stress. The aim of this investigation was to study protoporphyrin concentrations in other liver disorders. DESIGN AND METHODS: Erythrocyte protoporphyrin and zinc protoporphyrin concentrations were studied in 50 patients with chronic hepatitis C infection and various degrees of liver damage. High-performance liquid chromatographic analysis with fluorescence detection was used. RESULTS: Erythrocyte protoporphyrin was increased in 32% of the patients studied; in 12 patients up to two-fold higher than the maximum of the reference range, in 4 up to three-fold higher (median concentration 98 nmol/L, interquartile range 68-142; maximum 379 nmol/L (reference range: <125 nmol/L)). In 6 of the 10 patients in the subgroup with signs of severe liver dysfunction (decreased serum albumin and prolonged thromboplastin time, elevated serum bilirubin), protoporphyrin was elevated. Erythrocyte zinc protoporphyrin was increased in 7 cases out of all 50 studied; in these seven cases, erythrocyte protoporphyrin was also elevated (median concentration of zinc protoporphyrin in the whole study group: 232 nmol/L, interquartile range 182-342; maximum 827 nmol/L (reference range <464 nmol/L). CONCLUSIONS: Elevated erythrocyte protoporphrin levels are frequently found in patients with advanced chronic hepatitis C infection. Because protoporphyrin is well known to be hepatotoxic, these findings warrant further investigation.     

Factors influencing fluorescence spectra of free porphyrins

We recorded fluorescence excitation and emission spectra of uro- and coproporphyrin under different experimental conditions, to see how these conditions influence quantifications based on measurement of fluorescence intensity. We found that, for bands alpha and beta of the emission spectra and the main peak of the excitation spectra, fluorescence depends on pH and is minimal near pH 5 and near pH 7-7.5 for copro- and uroporphyrin, respectively. For band gamma of the emission spectra there was a constant decrease of fluorescence with increasing alkalinity of the solution. The intensity of porphyrin fluorescence also depends on ionic strength, reaching sharp maxima at 0.1 mol/L (for uroporphyrin) and 1 mol/L (for coproporphyrin). The organic mixture ethyl acetate:acetic acid (4:1 by vol), commonly used to extract porphyrins from biological samples, markedly diminishes the fluorescence of both porphyrins as compared with the same concentration of each porphyrin in aqueous acidic solvent. Furthermore, when we measured different ratios of uro:copro mixture at three distinct pHs and buffers, we found that at pH 10.5 (in carbonate buffer) the measured units of fluorescence depend only on total porphyrin concentration and not on the composition of the mixture.     

Precision and accuracy of a HPLC method for measurement of fecal porphyrin concentrations.

We studied precision and accuracy of a HPLC method for determination of porphyrins in feces. A commercial standard solution appeared to contain less coproporphyrin (15%) than stated by the manufacturer. The between-batch coefficients of variation were often below 15% and were higher than the within-batch coefficients. The precision of porphyrin measurements was not influenced by the type of porphyria. Recoveries of added coproporphyrin and protoporphyrin were 90% and 108%; coefficients of variation were 6% and 19%, respectively.     

Seasonal variation of serum bone GLA protein

Variegate porphyria (VP) is an inherited metabolic disease that results from the partial deficiency of protoporphyrinogen oxidase. In this communication we have used DNA technology in the diagnosis of VP and compared the results with the biochemical and clinical data. To date, we have diagnosed 107 VP patients using either biochemical or DNA techniques or both. In addition, in 106 family members the diagnosis of VP could be excluded. The sensitivity and specificity of the biochemical screening for VP were studied among 38 family members. These individuals were either asymptomatic (n=19) or had experienced occasional skin symptoms (n=13), acute attacks (n=5) or both (n=1). The sensitivity of urinary and fecal coproporphyrin analysis was 48% and 52%, respectively. The sensitivity of urinary uroporphyrin analysis was 71% and for fecal protoporphyrin 77%. Plasma fluorescence was sensitive in symptomatic patients even in remission, but resulted in false negatives in four asymptomatic patients with normal excretion of porphyrins in the urine. In our series of mutation screening, many new asymptomatic patients were identified, and this demonstrated that DNA analysis is the only reliable way to screen (a)symptomatic patients facilitating correct treatment and proper genetic counselling of family members at risk. Biochemical analyses (e.g. plasma fluorescence, fecal protoporphyrins, urinary copro- and uroporphyrins, porphobilinogen and delta-aminolevulinic acid) are essential when the diagnosis of VP is confirmed at the symptomatic phase.     

Diagnosis of variegate porphyria--hard to get?

Variegate porphyria (VP) is an inherited metabolic disease that results from the partial deficiency of protoporphyrinogen oxidase. In this communication we have used DNA technology in the diagnosis of VP and compared the results with the biochemical and clinical data. To date, we have diagnosed 107 VP patients using either biochemical or DNA techniques or both. In addition, in 106 family members the diagnosis of VP could be excluded. The sensitivity and specificity of the biochemical screening for VP were studied among 38 family members. These individuals were either asymptomatic (n = 19) or had experienced occasional skin symptoms (n = 13), acute attacks (n = 5) or both (n = 1). The sensitivity of urinary and fecal coproporphyrin analysis was 48% and 52%, respectively. The sensitivity of urinary uroporphyrin analysis was 71% and for fecal protoporphyrin 77%. Plasma fluorescence was sensitive in symptomatic patients even in remission, but resulted in false negatives in four asymptomatic patients with normal excretion of porphyrins in the urine. In our series of mutation screening, many new asymptomatic patients were identified, and this demonstrated that DNA analysis is the only reliable way to screen (a)symptomatic patients facilitating correct treatment and proper genetic counselling of family members at risk. Biochemical analyses (e.g. plasma fluorescence, fecal protoporphyrins, urinary copro- and uroporphyrins, porphobilinogen and delta-aminolevulinic acid) are essential when the diagnosis of VP is confirmed at the symptomatic phase.     

Plasma porphyrins in the porphyrias.

BACKGROUND: As an aid in the diagnosis and management of porphyria we have developed a method to fractionate and quantify plasma porphyrins and have evaluated its use in various porphyrias. METHODS: We used HPLC with fluorometric detection to measure plasma concentrations of uroporphyrin I and III, heptacarboxyl III, hexacarboxyl III, pentacarboxyl III, and coproporphyrin I and III. We studied 245 healthy subjects, 32 patients with classical porphyria cutanea tarda (PCT), 12 patients with PCT of renal failure, 13 patients with renal failure, 8 patients with pseudoporphyria of renal failure, 3 patients with acute intermittent porphyria, 5 patients with variegate porphyria, 5 patients with hereditary coproporphyria, and 4 patients with erythropoietic protoporphyria. RESULTS: Between-run CVs were 5.4-13%. The recoveries of porphyrins added to plasma were 71-114% except for protoporphyrin, which could not be reliably measured with this technique. Plasma porphyrin patterns clearly identified PCT, and its clinical sensitivity equaled that of urine porphyrin fractionation. The patterns also allowed differentiation of PCT of renal failure from pseudoporphyria of renal failure. CONCLUSIONS: The assay of plasma porphyrins identifies patients with PCT and appears particularly useful for differentiating PCT of renal failure from pseudoporphyria of renal failure.     

Rapid Changes in Human Right Heart Blood Temperature at Variations in Venous Return

Abstract

Quantitative determinations were made of coproporphyrin (CP) and protoporphyrin (PP) in the feces from 26 patients with porphyria acuta intermittens (PAI), from 26 patients with porphyria cutanea tarda (PCT), and from 32 workers in the lead industry.

In about 4/5 of the PAI-patients the fecal excretion of CP was slightly increased, while almost half of them had a slightly increased excretion of PP. The ratio between PP and CP was normal, namely 6:1.

of the patients with PCT, all showed an increased excretion of CP. Three-fifths of them also showed an increased excretion of PP. The excretion of CP was higher than among the patients with PA1 and the ratio PP/CP was about 1.

Fecal excretion of porphyrins in the lead-workers was largely normal.

The difference in the excretion patterns of porphyrins and their precursors in the urine and feces in PAI, PCT and in lead intoxication is elucidated.     

A high-speed liquid-chromatographic method for measuring urinary porphyrins

We describe a rapid quantitative and qualitative "high-performance" liquid-chromatographic (HPLC) method for measuring porphyrins in urine. Direct injection of acidified, filtered urine onto a 3-micron (particle size) 3-cm-long reversed-phase column fully resolves uroporphyrin, hepta-, hexa-, and pentacarboxylic acid porphyrins, and coproporphyrin. Instrument response is linearly related to concentration over the range 25 to 300 nmol/L. The method provides data essential for the differential diagnosis of porphyric states, including porphyria variegata and porphyria cutanea tarda. This relatively inexpensive method requires a run time of only 8 min per sample, making it particularly suitable for routine use in the clinical chemistry laboratory.     

Fecal coproporphyrin isomers in hereditary coproporphyria.

To see whether the fecal coproporphyrin III:coproporphyrin I (CIII:CI) ratio (determined by HPLC) would be suitable for screening patients at risk of hereditary coproporphyria (HC), we compared such ratios with the lymphocyte coproporphyrinogen oxidase (EC 1.3.3.3) activities (COOX) in 38 subjects from one large family and two smaller families with HC. The CIII:CI ratio was normal (less than 1.3) in adults with normal COOX (greater than 180 nmol/g of protein per hour) and high (greater than 2) in those with low COOX. Results were difficult to interpret in six of 10 children, who had borderline or low COOX but normal fecal CIII:CI ratios. Five subjects with low COOX and abnormal fecal CIII:CI ratios had normal fecal total porphyrin, indicating that the latter investigation alone is inadequate for family studies. The sample for determining the fecal CIII:CI ratio is easier to obtain and the assay is technically less demanding than COOX. We found the fecal CIII:CI ratio suitable for investigation of adults in a family study, but its usefulness in children needs to be established.