Establishment of modern examining procedures for diagnosis of porphyrias

Reversed-phase high-performance liquid chromatography together with fluorescence detection is suitable for the determination of urinary porphyrin carboxylic acid isomers, and Copro I and III, as well as Uro I and III porphyrin isomers are separated simultaneously.     

Analysis of porphyrin carboxylic acids in biological fluids by high-performance liquid chromatography

A method for the rapid and sensitive fluorometric analysis of porphyrin carboxylic acids by reverse-phase high-performance liquid chromatography is described. Separation of free porphyrin carboxylic acids was carried out with a microparticulate octadecylsilane column with elution by a gradient of methanol in phosphate buffer containing tetrabutylammonium hydroxide. Separation and quantitation of di-, tri-, tetra-, penta-, hexa-, hepta-, and octacar-boxylic porphyrins was achieved within 25 min at picomolar concentrations. The method is also capable of separating the type I and type III isomers of tetracarboxylic through hexacarboxylic porphyrins. By using a stopped flow technique, one can record fluorescence excitation and emission spectra of porphyrin carboxylic acids. This method is directly applicable to biological fluids such as urine, plasma, red cell lysates, or medium or extracts from cell culture.     

High-pressure liquid chromatography of plasma free acid porphyrins

The separation and quantitation of plasma free acid porphyrins by high-pressure liquid chromatography and fluorescence is described. Porphyrins were extracted from plasma in a simple manner with a recovery >90%. They were separated by high-pressure liquid chromatography on a silica gel (10 μm) column, using a gradient of acetone:dilute acetic acid. Resolution of seven free acid porphyrin standards including coproporphyrins I and III, but not uroporphyrins I and III, was achieved in 12 min at picomolar concentrations. Plasma of patients with erythropoietic protoporphyria displayed protoporphyrin. Uroporphyrin was the only porphyrin found in plasma of eight patients with porphyria cutanea tarda. Normal plasma contained small amounts of uroporphyrin and/or traces of protoporphyrin.     

Preparation and Enantioselectivity Binding Studies of a New Chiral Cobalt(II)porphyrin‐Tröger's Base Conjugate

A new bis[cobalt(II)porphyrin]‐Tröger's base conjugate was studied as a potential receptor for methyl esters of several amino acids. The conjugate was prepared as racemate, and then resolved via preparative high‐performance liquid chromatography (HPLC) on a chiral column. The high affinity to lysine, histidine, and proline methyl esters was found by complexation studies followed by UV‐Vis spectroscopy. The studies of pure enantiomers, followed by UV‐Vis and electronic circular dichroism spectroscopy, revealed the highest enantioselectivity for lysine methyl ester. Chirality 26:361–367, 2014. © 2014 Wiley Periodicals, Inc.     

Method for analysis of dopamine sulfate isomers by high performance liquid chromatography

Conjugated dopamine occurs in the tissues and fluids of many species, and much of this is thought to occur as dopamine sulfate. This paper describes the development and use of a method utilizing reversed-phase paired-ion high performance liquid chromatography to separate and quantitate each of the two naturally occurring dopamine sulfate isomers. Use of the method permitted demonstration of dopamine-3-0-sulfate in human urine from drug-free control subjects. It was found that this compound accounted for 73.1 ± 27% of the total daily conjugated dopamine excretion in the four subjects studied.     

Different patterns of hepatic microsomal enzyme activity produced by administration of pure hexachlorobiphenyl isomers and hexachlorobenzene

Three hexachlorobiphenyl isomers, 2,2′,4,4′,5,5′-hexachlorobiphenyl (I), 2,2′,3,3′,4,4′-hexachlorobiphenyl (II) and 2,2′,3,4,4′,5′-hexachlorobiphenyl (III), have been administered to rats and the effects of these three compounds upon hepatic microsomal drug metabolism and upon hepatic porphyrins have been studied. Comparisons have been made with hexachlorobenzene and a commercial polychlorinated biphenyl mixture, Aroclor 1254. From measurements of activities of microsomal drug oxidations in vitro, the durations of pharmacological actions of certain drugs in vivo and spectral shifts associated with cytochrome P-450 it is shown that the three pure hexachlorobiphenyl isomers initially produce changes in hepatic microsomal activity which resemble those seen after treatment with phenobarbitone (PB). In contrast, following chronic feeding of the isomers, compounds II and III but not I produce a pattern of hepatic microsomal enzyme activity which shows some characteristics of the 3-methylcholanthrene (3-MC) and some characteristics of the phenobarbitone classes of inducer. Also, compounds II and III, but not I, cause accumulation in the liver of porphyrins containing either seven or eight carboxyl groups. These two responses are similar to those observed following hexachlorobenzene treatment and suggest that a relationship may exist between the mixed pattern of enzyme induction and the onset of hepatic porphyrin accumulation.     

High-performance liquid chromatographic identification of eight constitutional disaccharides from heparan sulfate isomers digested with heparitinases

Identification with specific heparan sulfate-lyases, heparitinase I and heparinase of the constitutional unsaturated disaacharide (ΔDi-S_HS) derived from heparan sulfate (HS) isomers and heparin was achieved using high-performance liquid chromatography (HPLC) with a sulfonated styrene-divinylbenzene copolymer. Eight ΔDi-S_HS products derived from HS isomers were identified. Enzymatic digestion with heparitinase I and heparinase converts heterogeneous sulfated HS isomers and heparin into different ΔDi-S_HS. The practical application of these enzymes was examined using specific enzymes and HPLC. In a patient with Hurler syndrome, eight individual Δi-S_HS were identified in urinary HS isomers.     

Molecular, immunological, enzymatic and biochemical studies of coproporphyrinogen oxidase deficiency in a family with hereditary coproporphyria.

A 27-year-old woman who had recurrent pain in renal bed since 1998 with increasing character, was stationary admitted. The patient showed dark urine, complained of hair loss and took since 1994 a hormonal oral contraceptive. No photosensitivity was observed. Determinations of urinary porphyrin metabolites in 1998 revealed a porphyria cutanea tarda like excretion pattern with elevations of uro- (1767 nmol/24 hr, normal <29 nmol/24 hr) and heptacarboxyporphyrin (568 nmol/24 hr; normal <4 nmol/24 hr). Follow-up studies in feces showed the characteristics of a hereditary coproporphyria with dominance of coproporphyrin isomer III (total= 1470 nmol/g, isomer III= 93%), (normal: <37 nmol/g, isomer III = 25-35%). The excretion of porphyrin precursors (delta-aminolevulinic acid and porphobilinogen) was increased by taking an ethinylestradiol-cyproteronacetate-preparation, but acute and/or chronic manifestations were not observed. Coproporphyrinogen oxidase activity was decreased to 35% in the patient (normal=138+/-21 pkat/g protein; x+/-s), whereas the activity of red cell uroporphyrinogen decarboxylase was normal. Her mother and both sisters could be verified as heterozygous gene carriers of hereditary coproporphyria by their urinary and fecal excretion parameters and because of reduced coproporphyrinogen oxidase activity up to 50%. The father was normal with respect to his genotype. Molecular analysis revealed a hitherto unknown mutation with the transversion of a cytosine to thymine at nucleotide position 854 in exon 4 of the coproporphyrinogen oxidase gene. The gene defect was confirmed by DGGE in the mother and her three daughters. The investigation of the immunological nature of the defective coproporphyrinogen oxidase gene from the whole family revealed decreased concentrations of coproporphyrinogen oxidase protein in the patient, her mother and her two sisters.     

High-performance liquid chromatographic analysis of porphyrins and their isomers with radial compression columns

Porphyrin methyl esters and the isomers of uroporphyrin and heptacarboxylic porphyrin were separated by high-performance liquid chromatography. Isocoproporphyrin was also separated from coproporphyrin. By slight modifications to the solvent mixture, the separation of all biological polycarboxylic porphyrins was achieved. These separations were made possible through the high efficiency of 10- or 5-μm particle-size Radial-PAK cartridges, which have been used in the separation of porphyrins in various excreta and tissues in a number of porphyrias.     

Separation of porphyrin isomers by high-performance liquid chromatography.

A reversed-phase gradient elution system is described for the simultaneous separation of the type I and type III isomers of 8-, 7-, 6-, 5- and 4-carboxylated porphyrins and isocoproporphyrins. The method, adaptable for isocratic and stepwise separation of individual groups of isomers, is also suitable for preparative isolation of pure porphyrins. The analyses of porphyrin isomers in the urine and faeces of porphyric patients are examples of applications.     

液相色谱/质谱联用法分析不同年龄鼠皮肤中I型、III型胶原蛋白相对含量

哺乳动物皮肤真皮中胶原蛋白含量约为70%,主要为是I型、III型胶原蛋白,本实验利用稀酸溶解和酶法提取了大鼠皮肤中的总胶原蛋白,将胶原蛋白粗提品在60℃变性后用胰蛋白酶进行降解,液相色谱/质谱联用法分析了两种胶原蛋白的特征多肽,利用特征多肽比较了不同生长期大鼠皮肤中I型和III型胶原蛋白相对含量。结果表明,大鼠皮肤中的III型胶原蛋白的相对含量随生长期延长逐渐降低,而I型胶原蛋白的相对含量逐渐升高,8周后两种胶原蛋白的比例趋于稳定。本实验结果表明使用高效液相色谱/质谱联用法分析组织中的胶原蛋白类型及其动态变化具有可行性,为更好的临床应用提供了实验基础。     

Identification of porphyrins produced from isopropanol by Arthrobacter hyalinus

When Arthrobacter hyalinus was grown on isopropanol, a large amount of red pigment was accumulated in the culture broth. The pigment was isolated from the culture broth. With thin layer chromatography, FD mass, IR, ¹H-NMR, ¹³C-NMR, and absorption spectra methods it was found that the red pigments were composed of type III varieties of coproporphyrin, penta carboxyl porphyrin, hexa carboxyl porphyrin, hepta carboxyl porphyrin and uroporphyrin, and some type I uroporphyrin.     

Horse heart ferricytochromec: Conformation and heme configuration of low ionic strength acidic forms

Resonance Raman, absorption and circular dichroism spectroscopic studies of the stable forms of horse heart ferricytochromec in thepH range 6–0.8 and at the lowest possible ionic strengths, in water, and at 30°C are reported. The neutralpH form, state III, changes to the acidicpH form, state I, through a three-step process: state III ? state IIIa ? state II ? state I, with pKa's of 3.6±0.3, 2.7±0.2, and 1.2±0.2, depending on the monitoring probe, respectively. State IIIa ferricytochromec is like state III (i.e., with the Met-80-sulfur-iron linkage and a closed heme crevice) but with a higher degree of folding and a slightly larger porphyrin core. State II ferricytochromec is an unfolded form with an open heme crevice and no Met-80-sulfur-iron linkage. The heme iron is high-spin and hexacoordinated with weak ligand-field groups, water, and nitrogen of the protonated/hydrogen-bonded imidazole of the His-18 residue at the axial positions. The state I form also lacks the Met-80-sulfur-iron linkage and has an open heme crevice like the state II form; however, it is less unfolded and has a high-spin pentacoordinated heme iron, with the nitrogen of the imidazole of His-18 as the axial ligate, which is out of the porphyrin plane by about 0.45 Å.     

Chromatographic separation and spectroscopic characterization of the e/z isomers of acrivastine

A reverse phase high performance liquid chromatography (HPLC) method has been developed for the separation of two geometric isomers of Acrivastine using crude reaction mixture. The resolution between two isomers was found more than 2.9. The geometric isomers have been isolated by preparative HPLC and characterized by spectroscopic techniques, such as NMR, infrared, and MS. The developed method has been validated for the determination of Z‐isomer in Acrivastine. The limit of detection and limit of quantification of the Z‐isomer were 0.05 and 0.2 μg/ml, respectively. The developed method is precise, linear, accurate, rugged and robust for its intended use. Chirality, 2011. © 2011 Wiley‐Liss, Inc.     

Chromatographic quantitation of territrems A,B, and C,tremorgenic mycotoxins from Aspergillus terreus

This paper describes assay procedures for territrems A, B, and C by thin layer chromatography (TLC)-fluorodensitometry and reverse phase high performance liquid chromatography (HPLC).     

Porphyrin biosynthesis from porphobilinogen by duck blood hemolysate

Summary The formation of porphyrins from porphobilinogen by a duck blood hemolysate was examined. The system was found to form mainly protoporphyrin IX and hemin, and accumulated lesser amounts of uroporphyrins, heptacarboxylic porphyrin, and coproporphyrins. By storage at –20° the accumulation of uroporphyrins and heptacarboxylic porphyrin was increased. Both porphyrins were mainly the type III isomers. By addition of dithiothreitol the porphyrin pattern reversed to the original one formed by the fresh hemolysate. Addition of a number of amines also inhibited the decarboxylating system without affecting the original isomer distribution among the porphyrins. Addition of Fe²⁺ (3mm) did not affect the porphyrin pattern or the isomer distribution. Addition of Pb²⁺ (2.5mm) partially inhibited the decarboxylating system, whereas at higher concentrations (4mm) it increased the decarboxylation rate of the heptacarboxylic porphyrin. The obtained results are discussed in relation to porphyrin accumulation in porphyria cutanea tarda and in acquired hepatic porphyrias.Dedicated to professorLuis F. Leloir on the occasion of his 70th birthday.     

High Performance Liquid Chromatography of Hydroperoxides Formed by Autoxidation of Vegetable Oils

Trilinoleoylglycerol (TL) was autoxidized at 37°C in the dark. Monohydroperoxides (MHP) obtained from the oxidized products were analyzed by high performance liquid chromatography (HPLC). Several peaks which appeared in the chromatogram were identified by infrared (IR), gas chromatography mass spectrometry (GC-MS) and enzymatic hydrolysis. Some positional and geometrical isomers of their hydroperoxy fatty acid components were separated using both absorption and reversed phase systems. Furthermore, 1-hydroperoxylinoleoyl-2,3-dilinoleoyl-glycerol and 1,3-dilinoleoyl-2-hydroperoxylinoleoylglycerol were partly separated by HPLC using an absorption system. MHP obtained from autoxidized corn oil, safflower oil and soybean oil were separated into some peaks by HPLC, although resolution into the individual isomers was incomplete. When oxidized oils were subjected to HPLC analysis directly, a linear relationship was observed between the peak areas of MHP and peroxide value in the range of 10 ~ 50 meq/kg.     

Triamminemonoaquocobalt(III) complexes of pyrophosphate and adenosine diphosphate (ADP)

Cobalt(III)H2O(NH3)3 pyrophosphate has been shown by proton and 31P nuclear magnetic resonance (NMR) to be a facial bidentate complex. Cobalt(III)H2O(NH3)3 adenosine diphosphate has been resolved into lambda and delta isomers by chromatography on cycloheptaamylose. Both the lambda and delta forms are a pair of isomers that are not separated by cycloheptaamylose, reverse phase high-pressure liquid chromatography (HPLC), or cation exchange chromatography. These isomers presumably represent syn- and anti-arrangement of coordinated water and adenosine.     

Fluorescent nucleotide triphosphate substrates for snake venom phosphodiesterase

The procedure described utilizes a crude cell-free extract from the yeast Saccharomyces cerevisiae as enzymatic source for the synthesis of coproporphyrin III from [¹⁴C]δ-aminolevulinic acid with a high yield of conversion (?60%). Both specific radioactivity and total radioactivity of coproporphyrin III can be adjusted fairly well. This procedure is not time consuming for yeast acellular extracts or porphyrin ester preparations. The acellular extracts can be stored frozen (?30°C) for at least 1 year without loss of enzymatic activity. The same procedure can be used for [¹⁴C]protoporphyrin preparation.