Synthesis and separation by thin-layer chromatography of bilirubin-IX isomers. Their identification as tetrapyrroles and dipyrrolic ethyl anthranilate azo derivatives.

Procedures for the synthesis, separation and determination of structure of the bilirubin-IX isomers are described. 1. The four biliverdin-IX isomers were prepared by oxidative cleavage of haemin and were separated as their dimethyl esters. The individual esters were reduced with NaBH4, and the bilirubin esters obtained were subjected to alkaline hydrolysis yielding the corresponding bilirubin-IX isomers. 2. The bilirubin-IX isomers were structurally characterized (a) at the tetrapyrrolic stage by mass spectrometry of their trimethylsilyl derivatives and (b) by formation and structural analysis of their dipyrrolic ethyl anthranilate azo derivatives. 3. The absorption spectrum of bilirubin-IX alpha differed strikingly from the spectra of the other isomers. The presence of a pronounced shoulder around 453 nm in the spectrum of bilirubin-IXbeta allows easy differentiation from bilirubin-IXdelta. Methylation of the carboxyl groups largely eliminates the spectral differences between the IXalpha- and non-alpha isomers. 4. The bilirubin-IX isomers are conveniently separated by t.l.c. Detection and unequivocal identification is possible on a micro-scale by (a) t.l.c. with respect to reference compounds and (b) subsequent formation and t.l.c. of the more stable ethyl anthranilate azopigments. 5. Pronounced differences in polarity, i.e. solvent distribution, between the bilirubin-IX isomers indicate that a re-evaluation of conclusions reached previously with regard to the presence in, or absence from, biological fluids of some isomers and their relative amounts is needed.     

Application of a rapid and efficient h.p.l.c. method to measure bilirubin and its conjugates from native bile and in model bile systems. Potential use as a tool for kinetic reactions and as an aid in diagnosis of hepatobiliary disease.

We have developed an extremely rapid and efficient reverse-phase h.p.l.c. method for the measurement of bilirubin and its conjugates in human bile and in model bile systems. Our method involves the use of a Perkin-Elmer 3 mu C18 column and a methanol/sodium acetate/aq. ammonium acetate buffer system. Three isomers of bilirubin diglucuronide (BDG), two isomers of bilirubin monoglucuronide (BMG), three isomers of unconjugated bilirubin (UCB) and minor conjugates containing glucose and xylose were separated in 12 min. Initial quantification of BDG and BMG was based on the use of the ethyl anthranilate azo derivative of bilirubin (AZO UCB); however, the standard curves for BDG, BMG and UCB were similar enough to permit quantification to be later based on the UCB standard curve only, thereby simplifying the quantification process. Routine direct injection of 6 or 10 microliter of crude undiluted or diluted (1:1) bile sample was sufficient for analysis. The method was helpful in diagnosing biliary-tract obstruction in a newborn and a partial deficiency state of bilirubin conjugation (Crigler-Najjar syndrome) in a 10-year-old male. When the method was applied to biles of patients both with and without gallstones, levels of UCB were less than 2% of total pigment, consistent with previous reports. Because of its speed and efficiency, this method has the potential for a broad range of applications including enzymic, kinetic and bile sample analyses.     

Single-step purification and h.p.l.c. analysis of glutathione transferase 8-8 in rat tissues.

GSSG selectively elutes two GSH transferases from a mixture of rat GSH transferases bound to a GSH-agarose affinity matrix. One is a form of GSH transferase 1-1 and the other is shown to be GSH transferase 8-8. By using tissues that lack this form of GSH transferase 1-1 (e.g. lung), GSH transferase 8-8 may thus be purified from cytosol in a single step. Quantitative analysis of the tissue distribution of GSH transferase 8-8 was obtained by h.p.l.c.     

Isolation of four forms of acetone-induced cytochrome P-450 in chicken liver by h.p.l.c. and their enzymic characterization.

The purpose of this study was to purify and characterize the forms of cytochrome P-450 induced in chicken liver by acetone or ethanol. Using high performance liquid ion-exchange chromatography, we were able to isolate at least four different forms of cytochrome P-450 which were induced by acetone in chicken liver. All four forms of cytochrome P-450 proved to be distinct proteins, as indicated by their N-terminal amino acid sequences and their reconstituted catalytic activities. Two of these forms, also induced by glutethimide in chicken embryo liver, appeared to be cytochromes P450IIH1 and P450IIH2. Both of these cytochromes P-450 have identical catalytic activities towards benzphetamine demethylation. However, they differ in their abilities to hydroxylate p-nitrophenol and to convert acetaminophen into a metabolite that forms a covalent adduct with glutathione at the 3-position. Another form of cytochrome P-450 induced by acetone is highly active in the hydroxylation of p-nitrophenol and in the conversion of acetaminophen to a reactive metabolite, similar to reactions catalysed by mammalian cytochrome P450IIE. Yet the N-terminal amino acid sequence of this form has only 30-33% similarity with cytochrome P450IIE purified from rat, rabbit and human livers. A fourth form of cytochrome P-450 was identified whose N-terminal amino acid sequence and enzymic activities do not correspond to any mammalian cytochromes P-450 reported to be induced by acetone or ethanol.     

Digestion of chlorophylls and carotenoids by the marine protozoan Oxyrrhis marina studied by h.p.l.c. analysis of algal pigments

Algal pigments were measured, by reverse-phase h.p.l.c., duringgrazing experiments with the protozoan Oxyrrhis marina Dujardinon Rhodomonas sp. Chlorophylls and carotenoids were degradedto colourless residues; the rate of degradation of pigmentswas highest in the light. Very little chlorophyll a (5%) wasdegraded to phaeophytin and none to phaeophorbide during grazing.This suggests that phaeopigment concentrations cannot be usedas a measure of algal mortality due to grazing when heterotrophicProtozoa are a component of the grazer community.     

A novel metal-dye detection system permits picomolar-range h.p.l.c. analysis of inositol polyphosphates from non-radioactively labelled cell or tissue specimens.

A novel complexometric dye- and transition-metal-based post-column detection system for polyanions, called 'metal-dye detection' has been developed. This technique, combined with a new h.p.l.c. separation protocol, permits a direct highly-isomer-selective determination of bis- to poly-phosphorylated non-radioactively labelled compounds in the picomolar range, a sensitivity hitherto unknown for these substances. The application of the technique in the quantitative microanalysis of inositol polyphosphates from milligram amounts of cells or tissue specimens is described. The technique promises to answer hitherto unresolved questions about the role of inositol phosphates, especially those in intact tissues, which are not readily amenable to analysis by radioisotopic techniques.     

Improved assay method for activity of thyroid peroxidase-catalysed coupling of iodotyrosine residues of thyroglobulin utilizing h.p.l.c. for analysis of iodothyronines.

The coupling of iodotyrosine residues of thyroglobulin (Tg) catalysed by thyroid peroxidase (TPO) has scarcely been studied with respect to the TPO of abnormal human thyroid glands. The present paper proposes a rapid and convenient assay method applicable for determining the coupling activity of a sample of less than 500 mg from each patient's thyroid. The main characteristics of the method are as follows: (i) mitochondrial/microsomal fractions of thyroid glands were treated with sodium cholate plus trypsin, and the supernatants obtained by ultracentrifugation were directly used for the assay of coupling and peroxidase activity of TPO; (ii) the formation of iodotyrosine residues catalysed by TPO was performed by using chemically iodinated Graves'-disease Tg containing 41 iodine atoms per molecule and with a high iodotyrosine and a low iodothyronine content; (iii) newly synthesized iodothyronine residues (thyroxine, 3,5,3'-tri-iodothyronine, and 3,3',5'-tri-iodothyronine) were analysed by h.p.l.c. after hydrolysis of Tg with proteinases and extraction of iodothyronines with ethyl acetate.     

Estimation of polyhydroxy alcohols in fermentation broths. 2. Quantitative analysis based on t.l.c/f.i.d

A simple and rapid method for the quantitative analysis of polyhydroxy alcohols in fermentation broths has been developed. The method is based on thin layer chromatography (t.l.c.) coupled with a flame ionization detector (f.i.d.) and has been found to be useful in the screening of cultures for fermentative glycerol production.     

A study of the metabolism of [U-14C]3-methyl-2-oxopentanoate by rat liver mitochondria using h.p.l.c. with continuous on-line monitoring of radioactive intact acyl-coenzyme A intermediates.

A reverse-phase h.p.l.c. system for the resolution of the acyl-CoA intermediates of the degradation of 3-methyl-2-oxopentanoate is described. The validation of a method for the measurement of radioactive intermediates produced by the incubation of [U-14C]3-methyl-2-oxopentanoate with rat liver mitochondrial fractions is described. The absence of bicarbonate caused the accumulation of [14C]propionyl-CoA. The accumulation of [14C]2-methylbutyryl-CoA was observed in incubations with mitochondrial fractions derived from riboflavin-deficient animals. Studies of the accumulation of labelled intermediates with time suggest that there is regulation of the pathway of isoleucine degradation at methylmalonyl-CoA mutase, as suggested for valine [Corkey, Martin-Requero, Walajtys-Rode, Williams & Williamson (1982) J. Biol. Chem. 257, 9668-9676]. These studies demonstrate that h.p.l.c. with on-line continuous radiochemical detection is a powerful method for the investigation of the control of intermediary metabolism.     

Measurement of the acyl-CoA intermediates of beta-oxidation by h.p.l.c. with on-line radiochemical and photodiode-array detection. Application to the study of [U-14C]hexadecanoate oxidation by intact rat liver mitochondria.

The quantitative isolation of acyl-CoA esters of chain length C2-C17 from mitochondrial incubations and their analysis by reverse-phase radio-h.p.l.c. is described. Photodiode-array detection was used to characterize 2-enoyl-CoA esters. The chromatographic behaviour of all 27 intermediates of the beta-oxidation of hexadecanoyl-CoA is documented. Only C16, C14 and C12 intermediates were detected in uncoupled mitochondria oxidizing [U-14C]hexadecanoyl-CoA in the presence of fluorocitrate and carnitine, providing evidence for some organization of the enzymes of beta-oxidation [Garland, Shepherd & Yates (1965) Biochem. J. 97, 587-594; Sumegi & Srere (1984) J. Biol. Chem. 259, 8748-8752]. Rotenone increased concentrations of 3-hydroxyacyl-CoA and 2-enoyl-CoA esters and inhibited flux. These experiments provide the first direct unambiguous measurements of acyl-CoA esters in intact respiring rat liver mitochondrial fractions.