Börje Egestad

Recovery of malondialdehyde in urine as a 2,4-dinitrophenylhydrazine derivative analyzed with high-performance liquid chromatography

Malondialdehyde (MDA) in urine was measured as a 2,4-dinitrophenylhydrazine (DNPH) derivative using high-performance liquid chromatography (HPLC) for the analysis. MDA standard coeluted with a peak obtained from rat urine after i.p. administration of MDA standard. This peak was also the only peak containing 14C after injection of a [14C]MDA standard, and was shown by mass spectrometry to contain 1-(2,4-dinitrophenyl)pyrazole, the derivative formed when MDA is treated with DNPH. Depending on the amount given (0.3-5.5 mumol), the recovery (after 24 h sampling period) in urine was 0.7-2.6%. This apparent non-linear kinetics may relate to several factors, such as dose-dependent metabolism. However, the peak urinary concentration approached the expected plasma concentration and reproducible recovery data were obtained, suggesting that MDA was passively excreted in a reasonably stable form. These data indicate that monitoring MDA excretion in urine can give useful information about lipid peroxidation in vivo.

Fast atom bombardment mass spectrometry in the diagnosis of cerebrotendinous xanthomatosis

Urine from a patient with cerebrotendinous xanthomatosis (CTX) was extracted with a Sep-Pak C18 cartridge and the extract was analysed by fast atom bombardment mass spectrometry. The spectra indicated the presence of glucuronidated bile alcohols with four to seven hydroxyl groups. The method is simple and rapid and is suggested as an aid in the diagnosis of CTX with possible application to prenatal diagnosis.

Evidence for bile acid glucosides as normal constituents in human urine

A glucosyltransferase catalysing formation of bile acid glucosides was recently isolated from human liver microsomes. In order to investigate the potential occurrence of such bile acid derivatives in vivo, a method was devised for their isolation and purification from urine. Conditions were established with the aid of glucosides of radiolabelled, unconjugated glycine and taurine conjugated bile acids prepared enzymatically using human liver microsomes. Analysis by gas chromatography and mass spectrometry of methyl ester trimethylsilyl ether derivatives indicated the excretion of glucosides of nonamidated hyodeoxycholic, chenodeoxycholic, deoxycholic, ursodeoxycholic and cholic acids and of glycine and taurine conjugated chenodeoxycholic and cholic acids. Additional compounds were present giving mass spectral fragmentation patterns typical ofdi‐ and trihydroxy bile acid glycosides. Semiquantitative estimates indicated a total daily excretion of about 1 μmol.

Hepatic excretion and metabolism of polyethylene glycols and mannitol in the cat

The biliary clearances of fluid phase markers like erythritol and mannitol have been used to estimate canalicular bile flow. Larger fluid phase marker molecules like polyethylene glycol (PEG) 900 are excreted more extensively into bile, and it has been suggested that the biliary clearance of these give a more accurate measure of canalicular water flux than those of erythritol and mannitol. In this study, the biliary excretion of PEG 900 was compared with that of mannitol during choleresis induced by either sodium taurocholate or secretin. The biliary excretion of PEG 900 exceeded that of mannitol by a factor of 94. The biliary clearance of these markers was not influenced by secretin-induced choleresis. Using ion-exchange chromatography and fast atom bombardment mass spectrometry (FABMS) it was demonstrated that 26% of the PEG molecules are excreted into bile after oxidation to carboxylic acids, whereas sulphate conjugation is negligible. The majority of the PEG molecules (74%) were secreted unchanged, which supports the hypothesis of a mainly passive movement of PEG with the water flux into bile. FABMS showed an enrichment of larger PEG molecules in bile, which supports a previous finding that among differently sized PEGs the 1074-Da molecules have the highest biliary excretion.

Isolation of bile acid glucosides and N-acetylglucosaminides from human urine by ion-exchange chromatography and reversed-phase high-performance liquid chromatography

A method for the isolation, separation and analysis of glucosides and N-acetylglucosaminides of non-amidated bile acids and of glycine- and taurine-conjugated bile acid glucosides from normal human urine is described. Total bile acids were extracted from 24-h collections of urine by repetitive use of Sep-Pak C18 cartridges. After elution with 80% aqueous methanol, a group separation into non-amidated, glycine- and taurine-conjugated bile acids was performed by ion-exchange chromatography on Lipidex-DEAP. The glycosylated compounds were then separated from the corresponding non-glycosylated ones by high-performance liquid chromatography (HPLC) using a reversed-phase system with a linear methanol gradient. The glycosylated compounds isolated by HPLC were analysed by fast atom bombardment mass spectrometry and, after derivatization, by gas chromatography-mass spectrometry. Information about the sugar moieties of the bile acid glycosides was also obtained by treatment with different glycosidases.

Dolichol-like lipids with stimulatory effect on DNA synthesis: Substrates for protein dolichylation?

Substantial evidence has suggested that a nonsterol product of mevalonic acid (MVA) is essential for the initiation of DNA synthesis in mammalian cells. Several possible isoprenoid candidates have been suggested, but the identity of this compound still remains unknown. In this study we have isolated and purified MVA products from SV40‐transformed human fibroblasts and identified fractions with a growth‐stimulatory effect. The cells were labelled with [14C]MVA in the presence of inhibitors of 3‐hydroxy‐3‐methylglutaryl coenzyme A (HMG‐CoA) reductase. After lipid extraction, the [14C]MVA‐labelled lipids were subjected to high performance liquid chromatography and size‐exclusion chromatography, and the effect of the fractionated eluate on the DNA synthesis of arrested MVA‐depleted target cells was tested. Thereby we found a fraction of [14C]MVA‐labelled lipids with a substantial stimulatory effect on DNA synthesis. The chromatographic behavior suggested that the growth‐stimulating fractions contained dolichol‐20. This was confirmed by mass spectrometric analysis. Similar results were obtained when lipids from hepatocellular carcinoma cells and a sample from breast tumor were isolated and analyzed by the same procedure. The mechanisms by which these compounds induce DNA synthesis are unknown. Recent data obtained in our laboratory have provided evidence that dolichyl groups are covalently linked to tumor cell proteins, which implicates a new biological function for long‐chain polyisoprenoid alcohols (Hjertman et al. [1997] FEBS Lett 416:235–238). In this study we demonstrate that tumor cells containing dolichol‐like growth‐stimulatory lipids also contained dolichylated proteins. This raises the question whether the growth‐stimulatory dolichol‐like lipids serve as substrates for the dolichylation reaction. J. Cell. Biochem. 71:502–514, 1998.

Chromatographic fractionation and analysis by mass spectrometry of conjugated metabolites of bis(2-ethylhexyl)phthalate in urine

Mono(2-ethylhexyl)phthalate (MEHP), the primary metabolite of the plasticizer bis(2-ethylhexyl)phthalate (DEHP), was given to guinea pigs and mice and the methods for the isolation, separation and analysis of its metabolites in urine were developed. Following solid-phase extraction with octadecylsilane-bonded silica, individual metabolites were purified and separated using a combination of ion-exchange chromatography on lipophilic gels and reversed-phase high-performance liquid chromatography. Analysis of intact conjugates, as well as nonconjugated metabolites, was performed by fast atom bombardment mass spectrometry (FAB-MS) and, after derivatization, by gas chromatography-mass spectrometry. Enzymatic methods were used for further characterization. The study confirms glucuronidation as the major conjugation pathway for MEHP in the investigated species. Although less important quantitatively, glucosidation is shown to be an alternative conjugation pathway in mice. The methods developed were applied to a sample of urine from a hyperbilirubinemic newborn infant subjected to DEHP-exposure in conjunction with an exchange transfusion. It was demonstrated that metabolites of DEHP were excreted in amounts which could be analyzed by FAB-MS.

Glucuronidation of mono(2-ethylhexyl)phthalate : some enzyme characteristics and inhibition by bilirubin

A method for assaying mono(2-ethylhexyl)phthalate (MEHP) uridine diphosphate (UDP) glucuronyl transferase activity in microsomal preparations from guinea pig liver is described. The quantitation of the MEHP-glucuronide was performed by HPLC after direct injection of a sample of deproteinized incubation mixture. Solubilization of microsomal UDP-glucuronyltransferase activity was achieved by use of Lubrol, and optimal conditions for glucuronidation of MEHP were established. To investigate whether there is competition between MEHP and bilirubin for glucuronidation, inhibition experiments were performed with solubilized enzyme preparations. In these incubations addition of bilirubin decreased the formation of MEHP-glucuronide. No change in the maximal conversion rate (Vmax) was observed, indicating the occurrence of competitive inhibition. This observation may have implications in clinical situations where patients with hyperbilirubinemia are exposed to MEHP, e.g. in exchange transfusions in newborn infants.

Fast atom bombardment mass spectrometry and chemical analysis in determinations of acyl-blocked protein structures

Peptide generation and fast atom bombardment mass spectrometry in combination with conventional chemical analysis was used to identify the blocking group and establish the N‐terminal structure of six different proteins at the nanomole level. In this manner, the first terminal structures of three non‐mammalian alcohol dehydrogenases were determined, demonstrating the presence of N‐terminal acetylation in these piscine, amphibian, and avian enzymes. Similarly, two different yeast glucose‐6‐phosphate dehydrogenases and a minor variant of a human alcohol dehydrogenase were found to be acetylated. The exact end location of C‐terminal structures was also established. Together, the analyses permit the definition of terminal regions and blocking groups, thus facilitating the delineation of remaining structures.

Manual and automated enrichment procedures for biological samples using lipophilic gels

Aspects of the use of lipophilic gels in manual sample preparation procedures are reviewed. Neutral gels with a controlled hydrophobicity are used for sorbent extraction of non-polar and medium polarity compounds from biological fluids. Acidic amphiphilic compounds can be extracted as ion-pairs with decyltrimethylammonium ions. Solvent or detergent extracts of tissues or faeces can be mixed with hydrophobic gels for transfer of analytes from a solvent to a gel phase, permitting subsequent sample preparation in gel bed systems. Hydrophobic gels, alkyl-bonded silica and polystyrene matrices can be used in series for extraction of compounds with a wide range of polarities. Group fractionations are performed on neutral and ion-exchanging lipophilic gels to yield fractions of neutral, basic and acidic metabolites within selected polarity ranges. Selective isolation of phenolic acids on a strong anion exchanger, of ethynylic steroids on a strong cation exchanger in silver form and of oximes of ketonic steroids on a strong cation exchanger in hydrogen form is possible. A computerized system for automatic sample preparation is also described. It consists of an extraction bed, a cation-exchange column and an anion-exchange column. The pumps and switching valves are arranged so that the columns can operate in series or parallel for isolation of neutral, basic and acidic metabolites of amphiphilic compounds and for regeneration of the column beds. Fractions can be collected, or the effluent from the column beds can be diluted with water to permit sorption on a solid phase. The applicability of the automated method to the analysis of bile acids and metabolites of mono(2-ethylhexyl) phthalate is demonstrated.