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.     

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%.     

Urine and faecal porphyrin profiles by reversed-phase high-performance liquid chromatography in the porphyrias

A novel reversed-phase system is described for the simultaneous separation of type I and type III isomers of uro-, heptacarboxylic-, hexacarboxylic -, pentacarboxylic - and copro -porphyrins and the dicarboxylic meso- and proto-porphyrins. The porphyrins were resolved on a Hypersil -SAS column eluted with 10% (v/v) acetonitrile in 1 mol/l ammonium acetate, pH 5.16 (solvent A) and 10% (v/v) acetonitrile in methanol (solvent B) with a linear gradient from 100% A (0%B) to 35% A (65%B) in 30 min followed by isocratic elution at 65% B for a further 10 min. The method is simple and reproducible and has been applied to the analysis of porphyrins in urine and faeces from patients with acute intermittent, variegate, hereditary copro , congenital erythropoietic, erythrohepatic proto and symptomatic porphyrias.     

On accuracy and precision of a HPLC method for measurement of urine porphyrin concentrations.

We studied the accuracy and precision of a HPLC method for determination of porphyrins in urine. A commercial standard solution appeared to contain less porphyrins than indicated by the manufacturer, since calibration resulted in lower concentrations of uroporphyrin and coproporphyrin: 16% and 8%, respectively. Coefficients of variation for the measurement of uro-, hepta-, copro I and copro III porphyrins in samples of patients with and without porphyria were often much less than 15%. Comparison of measurements with and without calibrated standards revealed differences for uroporphyrin and coproporphyrin of 27% and 5%, respectively. Recovery of added uroporphyrin and coproporphyrin was 99%. The main cause of the variability in test results was apparently the improperly calibrated standard solutions. The precision of porphyrin measurements was not influenced by the type of porphyria.     

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.     

The stability of uric acid in ammonium hydroxide

We examined the stability of uric acid in dilute aqueous ammonium hydroxide solution by mass spectrometry. Uric acid decomposes in ammonium hydroxide even as dilute as 15 mmol/L when the mole ratio of ammonium hydroxide to uric acid is 50:1. There are at least four products of the decomposition, two of which have been identified as allantoin and urea. The slope of the decomposition curve indicates that uric acid is destroyed at an initial rate of 2-3% per hour. In ammonium hydroxide at a concentration of 1 mmol/L and a mole ratio of ammonium hydroxide to uric acid of less than or equal to 3.4, uric acid is not detectably decomposed. Evidently, any method for determination of uric acid that involves treating the analyte with ammonium hydroxide before analysis may destroy it. Therefore, a published method described as being "definitive" for uric acid (J Clin Chem Clin Biochem 1985; 23:129-35) could produce incorrect results because it involves storing the uric acid in 15 mmol/L ammonium hydroxide at a mole ratio of ammonium hydroxide to uric acid of greater than 120:1.     

Improved micromethod for assay of serum angiotensin converting enzyme

We describe conditions for determining angiotensin converting enzyme (EC 3.4.15.1) in serum, by liquid chromatography. Serum (10 microL) is assayed with the artificial substrate hippuryl-glycyl-glycine (30 mmol/L) in a 50 mmol/L "HEPES" buffer solution with a high salt content (300 mmol of NaCl and 400 mmol of Na2SO4 per liter), at pH 8.0. The resulting enzymic activity is ninefold that of the currently popular spectrophotometric assay of Cushman and Cheung as modified by Lieberman (Am. J. Med. 59: 365-372, 1975). The hippuric acid end product is separated from the substrate by reversed-phase liquid chromatography and measured spectrophotometrically at 228 nm. o-Methyl hippuric acid is used as internal standard. The mean value for 100 normal control subjects was 317 (SD 96) nmol of hippuric acid released per milliliter of serum per minute. The enzyme activity is greater in newborns (p less than 0.05) and has a tendency to decrease with age. This partly automated method, which is optimized with regard to activity and detection, can be used in clinical routine.     

Spectrophotometric quantification of total urinary porphyrins as a screening test for porphyrias: Threshold value revisited

Objectives

The quantification of total urinary porphyrins based on spectrophotometry or spectrofluorimetry is a key screening test in cutaneous or mixed porphyria, performed before the quantitative fractionation of porphyrin isomers by chromatography. The aim of the present study was to determine the best threshold value for a spectrophotometric screening test and to estimate its diagnostic performances.

Design and methods

Data from samples sent to the laboratory between January 2006 and July 2013 from patients with a suspicion of cutaneous or mixed porphyria were retrospectively collected. The final diagnosis was based on the clinical presentation and biochemical results performed on appropriate specimens. Control samples were obtained from 91 non-porphyria patients and 58 healthy individuals and patient samples were obtained from 38 patients with symptomatic porphyria. The sensitivity and specificity of the spectrophotometric screening test were calculated at different cutoff values expressed in nmol/L and nmol/mmol creatinine using receiver operator curves.

Results and conclusion

A threshold of 18.8 nmol/mmol creatinine was considered as the best cutoff value for the screening test, achieving a sensitivity of 100% and a specificity of 93.2%.     

Automated determination of urinary creatinine without sample dilution: theory and practice

The rate of the Jaffé reaction depends on the concentration of sodium hydroxide; the pseudo-first-order rate constant of the reaction, at 37 degrees C in 10 mmol/L picrate solution, is 0.004 mmol/L. We formulated an automated method to determine urinary creatinine directly without manual sample dilution. The conditions are as follows: 10 mmol/L picrate and 60 mmol/L sodium hydroxide (final concentrations); ratio of sample to final volume, 1:41; temperature, 37 degrees C; wavelengths of measurement, 500 or 510 nm; interval of measurement, 30 to 90 s; and mode of measurement, kinetic. Determinations of creatinine in patients' samples by the new method compared favorably with those obtained with the AutoAnalyzer and aca. The run-to-run CVs were 3.6% or less, and the method was accurate for concentrations of creatinine up to 3000 mg/L. We recommend this method as a good replacement for the AutoAnalyzer or aca methods.     

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.     

Single stable reagent (Arsenazo III) for optically robust measurement of calcium in serum and plasma.

We describe a method based on a single stable reagent for the determination of calcium in serum and plasma with use of Arsenazo III, 200 mumol/L in 50 mmol/L 1,4-piperazinediethanesulfonic acid (PIPES) buffer. The method showed significant positive interference in plasma at pH less than 6.6 because of the precipitation of fibrinogen, which was eliminated by increasing the pH to 6.8. The assay showed no interference from as much as 600 mumol of bilirubin and 12 g of hemoglobin per liter when applied in a simple monochromatic procedure at 660 nm. The standard curve for calcium was linear from 0 to 5.0 mmol/L. Addition of Intralipid at concentrations greater than 3 g/L demonstrated positive interference, which could be eliminated by using a 700-nm blanking wavelength. The procedure showed good agreement with all-method mean values from two external quality-control schemes.     

Enzymatic assay of inorganic phosphate with use of sucrose phosphorylase and phosphoglucomutase.

We developed a new enzymatic method for the assay of inorganic phosphate (Pi) by using sucrose phosphorylase (SP; EC 2.4.1.7) and phosphoglucomutase (PGM; EC 5.4.2.2). Pi is transferred to sucrose by SP, producing alpha-D-glucose 1-phosphate (G1P) and alpha-D-fructose. G1P is transphosphorylated by PGM in the presence of alpha-D-glucose 1,6-bisphosphate to form alpha-D-glucose 6-phosphate, which is oxidized by NAD+ and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) to form 6-phosphogluconate (6PG) and NADH. Finally, the oxidation of 6PG by NAD+, catalyzed by 6-phosphogluconic dehydrogenase (EC 1.1.1.44), yields D-ribulose 5-phosphate and NADH. Thus two molecules of NADH are formed for each molecule of Pi, and the reaction is monitored at 340 nm. The Km values of SP for Pi and sucrose were 4.44 and 5.31 mmol/L, respectively. The best buffer was 1,4-piperazinediethanesulfonic acid (PIPES) at 50 mmol/L and pH 6-7. Implementing this method with a Cobas-Bio centrifugal analyzer allowed us to measure Pi accurately and precisely.     

Rapid analysis for methylglyoxal bis(guanylhydrazone) by reversed-phase ion-pair liquid chromatography

We describe a "high-performance" reversed-phase ion-pair liquid-chromatographic procedure for measuring methylglyoxal bis(guanylhydrazone) (MGBG) in plasma, urine, and bone-marrow leukocytes. Specimens of plasma and bone-marrow leukocytes are deproteinized with perchloric acid, then neutralized with KOH. Urinary MGBG is isolated by liquid-solid extraction in a C18 Sep-Pak. The chromatographic system consists of a 45 X 4.6 mm (i.d.) octadecylsilyl (C18, 5-microns particle) column and a mobile phase consisting of methanol/sodium acetate buffer (200 mmol/L, pH 4.5), 2/3, by vol. The acetate buffer also contains 20 mmol of 1-octanesulfonate and 40 mg of sodium azide per liter. The column effluent is monitored at 283 nm. At a flow rate of 3.0 mL/min, MGBG is eluted in 1.67 min. The detection limit is 20 nmol/L, and peak height varies linearly with concentration from 0.02 to 40 mumol/L. Analytical recovery exceeds 99%. Within-day CVs ranged from 0.9% to 2.9%, between-day CVs from 4.2% to 6.2%.     

Effects of porphyrins on proteins of cytosol and plasma. In vitro photo-oxidation and cross-linking of proteins by naturally occurring and synthetic porphyrins

We examined the photodynamic effects of porphyrins, known photosensitizers, on proteins of cytosol and plasma that bind them and are implicated in their transport. Their susceptibility to photodecomposition by porphyrins was found to be higher than that of proteins with low or no affinity for tetrapyrroles. Inhibition of porphyrin binding by the addition of equimolar amounts of heme had no effect, indicating that protein photodecomposition may be induced, in part, by free or nonspecifically bound porphyrins. HBP, a heme-binding Z protein of liver cytosol, exhibited the highest susceptibility of all proteins tested, including glutathione S-transferases, albumin, hemopexin, and apotransferrin. HBP was extensively photo-oxidized, as evidenced by a decrease in its antigenicity and electrophoretic mobility, and it was cross-linked by naturally occurring porphyrins as well as by the synthetic tin-protoporphyrin and hematoporphyrin derivative. The water-soluble singlet oxygen scavengers L-histidine (50 mmol/L) and sodium azide (100 mmol/L) completely prevented the photodynamic effects of uroporphyrin (100 mumol/L) on HBP. Hydroxyl radical scavengers such as manitol and benzoate were partially effective, whereas water-insoluble singlet oxygen scavengers such as beta-carotene were totally ineffective. Preferential inhibition of cross-linking over other photodynamic effects of uroporphyrin was consistent with previous reports that cross-linking occurs subsequently to amino acid oxidation.     

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.     

Assay of carboxypeptidase N activity in serum by liquid-chromatographic determination of hippuric acid

We describe conditions for determining carboxypeptidase N (EC 3.4.17.3) activity by liquid chromatography. Serum (10 microL) is mixed with the artificial substrates hippuryl-L-arginine (30 mmol/L) and hippuryl-L-lysine (100 mmol/L) in 50 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer solution at pH 8.2 and 7.8, respectively. The hippuric acid product is separated from the substrate in less than 2 min by reversed-phase "high-performance" liquid chromatography and measured spectrophotometrically. o-Methyl hippuric acid is used as internal standard. By this method, optimized for activity and sensitivity of detection, carboxypeptidase N activities are 60-fold greater than those by another procedure (J Chromatogr 266:173-177, 1983). The mean value for 80 normal control subjects was 74.8 (SD 10.3) nmol of hippuric acid released per milliliter of serum per minute for hippuryl-L-arginine substrate, 378 (SD 55) for hippuryl-L-lysine substrate. The sensitivity and precision of the method make it suitable both for routine clinical determinations and as a reference procedure.     

Testosterone measured by 10 immunoassays and by isotope-dilution gas chromatography-mass spectrometry in sera from 116 men,women, and children

BACKGROUND: Commercially available testosterone immunoassays give divergent results, especially at the low concentrations seen in women. We compared immunoassays and a nonimmunochemical method that could quantify low testosterone concentrations. METHODS: We measured serum testosterone in 50 men, 55 women, and 11 children with use of eight nonisotopic immunoassays, two isotopic immunoassays, and isotope-dilution gas chromatography-mass spectrometry (ID/GC-MS). RESULTS: Compared with ID/GC-MS, 7 of the 10 immunoassays tested overestimated testosterone concentrations in samples from women; mean immunoassay results were 46% above those obtained by ID/GC-MS. The immunoassays underestimated testosterone concentrations in samples from men, giving mean results 12% below those obtained by ID/GC-MS. In women, at concentrations of 0.6-7.2 nmol/L, 3 of the 10 immunoassays gave positive mean differences >2.0 nmol/L (range, -0.7 to 3.3 nmol/L) compared with ID/GC-MS; in men at concentrations of 8.2-58 nmol/L, 3 of the 10 immunoassays tested gave mean differences >4.0 nmol/L (range, -4.8 to 2.6 nmol/L). CONCLUSION: None of the immunoassays tested was sufficiently reliable for the investigation of sera from children and women, in whom very low (0.17 nmol/L) and low (<1.7 nmol/L) testosterone concentrations are expected.     

Assay of total and free propranolol in plasma by liquid chromatography with fluorometric detection

We report a liquid-chromatographic method for assay of total and free propranolol and its metabolite, propranolol glycol, in plasma. An ultrafiltration device is used to separate the free drugs from the protein-bound drugs. The total and free drugs are isolated from plasma or ultrafiltrate by liquid-solid extraction on a Waters C18 SEP-PAK and the drugs are quantified on a C18 mu-Bondpak radial-compression column. The mobile phase consists of equal parts of methanol and a solution of potassium 50 mmol/L phosphate and 2.5 mmol/L 1-pentane-sulfonic acid, pH 5.0. Detection was by fluorescence, with excitation at 296 nm and emission at 338 nm. Time required for sample preparation for total-propranolol assay is 40 min per eight samples. Sample preparation for free-propranolol assay requires an additional 1 h per eight samples. Chromatography requires 15 min per sample. The extraction recovery is approximately 75% and the assay curve is linear from 5 to 400 micrograms/L. The run-to-run CV is 4% for total propranolol, 15% for free propranolol.     

Measurement of plasma uroporphyrin during treatment and long-term follow-up of patients with Porphyria cutanea tarda

.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.     

A modification of the kinetic determination of pancuronium bromide based on its inhibitory effect on cholinesterase

A modification of the existing spectrophotometric kinetic method for the determination of pancuronium bromide (PCBr), based on pooled human serum cholinesterase (ChE, EC 3.1.1.8 acylcholine acylhydrolase) inhibition, was developed. Butyrylthiocholine iodide (concentration 1.667 mmol/L) was used as substrate and determination was performed at pH 7.6. Essential basic kinetic parameters were also determined: Michaelis-Menten's constant KM=0.33 mmol/L, maximal reaction rate Vmax=42.29 micromol/L min, inhibition constant KI=0.34 micromol/L, and IC50=0.235 micromol/L. Linear dependence between the reaction rate and the inhibitor concentration exists in PCBr concentration range 8.29-265.28 nmol/L, which corresponds to the real sample concentrations from 0.166 to 5.306 micromol/L. The method detection limit was established to be 1.86 nmol/L and the quantification limit was 6.18 nmol/L. Precision of the method was tested for three pancuronium concentrations (16.58, 99.48, and 198.96 nmol/L). The relative standard deviation (RSD) was in the range 0.78-5.13%. Accuracy was examined by the standard addition method. The influence of substances usually present in serum and urine on the reaction rate was determined. The method developed was applied for PCBr determination in spiked serum and urine samples and in the urine taken during surgery. The method was proven to have good sensitivity, accuracy, and precision and can be considered suitable for clinical practice.