Identification of an N-acetylated microsomal glutathione S-transferase by mass spectrometry.

Microsomal glutathione S-transferase (mGST) was purified to homogeneity from male Sprague-Dawley rat liver, as determined by SDS-PAGE. Removal of Triton X-100 and further separation by reversed phase HPLC revealed two proteins, mGST 1 and mGST 2, in a 1:3 ratio. Analysis of mGST 1 and mGST 2 by electrospray ionization mass spectrometry determined their molecular weights to be 17,354.2 +/- 6.6 and 17,397.9 +/- 6.6, respectively. mGST 1 was in close agreement with the calculated molecular weight of 17,348, as predicted by the previously reported cDNA sequence. Cyanogen bromide digestion and peptide mapping by fast atom bombardment mass spectrometry (FAB-MS) localized the mass increase to the N-terminal peptide, 1-7. FAB-tandem mass spectrometry of this peptide in conjunction with Edman reactions on the intact protein demonstrated the N-terminal alanine to be acetylated.

Bilirubin levels in subarachnoid clot and effects on canine arterial smooth muscle cells.

BACKGROUND AND PURPOSE: Previous studies have suggested that bilirubin is a potential contributor to cerebral vasospasm. The purpose of this investigation was to determine whether bilirubin accrues in subarachnoid clot, whether its vasoconstrictive effect could involve a direct action on arterial smooth muscle cells, and, if so, whether bilirubin affects their Ca2+ uptake. METHODS: Subarachnoid clots were analyzed for bilirubin using high-performance liquid chromatography. The length and 45Ca2+ uptake of vascular smooth muscle cells enzymatically dissociated from canine carotid arteries were measured before and after exposure to bilirubin solution. Additional experiments were conducted on cultured smooth muscle cells from canine basilar artery and on ATP-depleted cardiac myocytes. RESULTS: Mean +/- SE bilirubin concentration in experimental clot was 263 +/- 35.7 mumol/L. Vascular smooth muscle cells exposed to bilirubin showed progressive shortening (P < .01) and an increased uptake of 45Ca2+ (P < .001). Contraction was prevented by Ca(2+)-free media but not by verapamil. Experiments with heart myocytes showed that bilirubin caused an increased uptake of 45Ca2+ but not of [14C]sucrose. CONCLUSIONS: The results indicate that bilirubin accrues in subarachnoid clot, that it exerts a direct constrictive effect on arterial smooth muscle cells, and that this effect is associated with an increased uptake of Ca2+. Studies on heart myocytes suggest that the Ca2+ uptake induced by bilirubin could be due to a selective increase in membrane permeability to Ca2+.

Hemin: levels in experimental subarachnoid hematoma and effects on dissociated vascular smooth-muscle cells.

Although hemin is known to exert toxic effects on a variety of cell types, its possible participation in the genesis of cerebral vasospasm has received little attention. The authors measured the concentration of hemin in experimental subarachnoid clot and studied its effects on the morphology and 45Ca++ uptake of vascular smooth-muscle cells dissociated from canine carotid artery. Craniectomies were performed in five dogs under general anesthesia, and 3 to 5 ml of autologous whole blood was deposited in the supraclinoid subarachnoid compartment. The concentration of hemin recovered by Folch extraction from clotted material removed 7 days after surgery was 390 +/- 247 microM (mean +/- standard error of the mean). Mean vascular smooth-muscle cell length after 40 minutes of exposure to 50 microM hemin was 37.3 +/- 1.2 microns (control 51.6 +/- 1.6 microns) (p < 0.01). The mean percent permeation of 45Ca++, measured by a dual label technique, of cells exposed to hemin was 200.9% +/- 23% (control 102.9% +/- 4.3%) (p < 0.01). These findings indicate that hemin accrues in subarachnoid hematoma, that it exerts a constrictive effect on vascular smooth-muscle cells, and that this effect is associated with an increased uptake of Ca++. This study demonstrates that hemin should be included in the list of potential agents that participate in the development of cerebral vasospasm.

Identification of glutathione conjugates of the dimethyl ester of bilirubin in the bile of Gunn rats.

The Gunn rat, which is deficient in the UDP-glucuronosyltransferase for bilirubin, promptly excreted polar conjugates of the dimethyl ester of bilirubin in bile after intravenous infusion of this ester. The conjugates proved to be monoglutathione thioether adducts of the vinyl groups of the parent tetrapyrrole. High performance liquid chromatographic analysis of the conjugates as their dipyrrolic azosulfanilates demonstrated that only one of the dipyrroles of each tetrapyrrole was conjugated. The nonconjugated dipyrrole eluted as either the methyl endo- or exovinyl azodipyrrole. The amino acid composition of the pigments was consistent with that of a monoglutathione conjugate. NMR spectroscopy of the two major pigments demonstrated the loss of the proton signals of the C-18 vinyl group, indicating it to be the site of conjugation. Cation fast atomic bombardment tandem mass spectrometry demonstrated a molecular ion, [M + H]+, of m/z 937, which fragmented with a loss of 307 atomic mass units, consistent with glutathione. A molecular ion of m/z 807 was observed for the conjugate treated with gamma-glutamyltranspeptidase, consistent with the loss of glutamate. The mass spectrometry data indicated that the conjugates also contained a functional group whose mass was equivalent to hydroxyl, suggesting initial formation of an epoxide, which then reacts with glutathione. Pretreatment of the rat with 2,3,7,8-tetrachlorodibenzo-p-dioxin to induce cytochrome P-450 resulted in a 6-fold increase of the biliary excretion of the glutathione conjugates. Such induction also resulted in the excretion of a glutathione conjugate of bilirubin itself.

In vitro formation of glutathione conjugates of the dimethylester of bilirubin.

Rat hepatic microsomes catalyzed the formation of two distinct glutathione conjugates of bilirubin dimethylester (DMB). The two conjugates were identical to those isolated from the bile of Gunn rats infused with DMB. The microsomal reaction was dependent on NADPH, oxygen and glutathione and was inhibited by nitrogen and the cytochrome P450 inhibitors metyrapone, 1-benzyl-imidazole, and alpha-naphthoflavone. Conjugate formation was inducible with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) but not phenobarbital pretreatment. The rate of formation of conjugates was not affected by washings of the microsomal pellet or by the presence of superoxide dismutase and/or catalase. Cation fast atom bombardment mass spectrometry (FAB/MS) of the conjugates indicated a molecular ion of 937 atomic mass units (amu). Fragmentation revealed a loss of 307 amu, consistent with glutathione, and a residual mass of 629 amu suggesting a hydroxylated derivative of DMB (612 amu). Cation FAB/MS/MS of conjugates formed in vitro under an atmosphere of oxygen-16 and oxygen-18 demonstrated the incorporation of molecular oxygen by a difference of 2 amu in the respective molecular ions. Our results suggest that DMB is oxidized by the cytochrome P450 IA gene family to an epoxide intermediate which is then subsequently conjugated with glutathione.

High-performance liquid chromatographic analysis of bile pigments as their native tetrapyrroles and as their dipyrrolic azosulfanilate derivatives.

A reversed-phase high-performance liquid chromatographic (HPLC) analysis of bile pigments is described that provides baseline separation of the major bilirubin conjugates found in bile. The advantage of the technique is that the bile pigments can be analyzed directly as their native tetrapyrroles without prior solvent extractions or derivatization. The use of ammonium acetate in place of sodium salts permits preparative isolation and lyophilization of the pigments for mass spectroscopy. The derivatization of the pigments as their dipyrrolic azosulfanilates with subsequent HPLC analysis demonstrates baseline separation of the endo- and exovinyl azodipyrroles and allows identification of that half of the tetrapyrrole which contains the conjugate in the instances of monoglycosides.

Lymphangioendothelioma of the liver in a neonate.

The case report describes the clinical features, imaging findings, and pathology of a lymphangioendothelioma that almost entirely replaced the liver parenchyma in a neonate. No other organs were involved.

Alpha 1-antitrypsin deficiency and the PiMS phenotype: case report and literature review.

We describe a premature infant with cholestatic liver disease and protease inhibitor MS phenotype. This infant demonstrated an abnormally low serum alpha 1-antitrypsin concentration. Liver histologic studies revealed diastase-resistant, periodic acid-Schiff-positive globules inside hepatocytes. Immunoperoxidase staining for alpha 1-antitrypsin was positive. Electron microscopy showed amorphous material in the dilated lumina of the endoplasmic reticulum. These findings are characteristic of alpha 1-antitrypsin deficiency. We suggest that this usually nonpathologic phenotype resulted in cholestatic liver disease because of the cumulative effect of several cholestatic conditions.

Isolation of an activator of bilirubin glucuronyltransferase from normal and jaundiced Gunn rats.

A microsomal activator of the UDP-glucuronyltransferase for bilirubin has been isolated from lubrol solubilized and salt fractionated liver microsomes. The activator has been partially purified by anion exchange and molecular sieving chromatography and found to have a molecular weight of about 60 kDa. The activator is present in liver from normal and bilirubin UDP-glucuronyltransferase deficient Gunn rats. When tested with purified UDP-glucuronyltransferase for bilirubin it accelerated the conjugation rate 10 fold but with the purified UDP-paranitrophenol transferase the rate of conjugation was increased only 1.5 times.

Bilirubin conjugate excretion and bilirubin uridine diphosphoglucuronyltransferase activity in nonjaundiced homozygous and heterozygous Gunn rats.

High-performance liquid chromatography was used to analyze the composition of bilirubin conjugates in bile from adult male and female outbred normal (JJ) and heterozygous (Jj) Gunn rats. Bile was examined directly without prior derivatization or extraction. Significantly higher bilirubin diglucuronide and lower bilirubin monoglucuronide (both C-8 and C-12 isomers) excretion in JJ rats was demonstrated. Bilirubin monoglucuronide C-8/C-12 ratios were similar in both genotypes, as was the percentage of bilirubin monoglucuronide monoglycoside diester. Hepatic bilirubin uridine diphosphoglucuronyltransferase activity showed a statistically significant positive correlation with the relative amount of bilirubin diglucuronide present in bile and a significant negative correlation with the amount of bilirubin monoglucuronide. The relative percentage of bilirubin monoglucuronide vs. diglucuronide in bile allows an indirect assessment of hepatic bilirubin uridine diphosphoglucuronyltransferase activity.

Effects of anesthetic agents on bile pigment excretion in the rat.

Anesthesia-induced alterations in bilirubin conjugation were studied. Rats were fitted with bile duct and jugular vein catheters while anesthetized with diethyl ether, ketamine or pentobarbital. As anesthesia abated, bile was collected for the next 5 hr and analyzed for flow rate, total bilirubin excretion and bilirubin glucuronide composition. The high-performance liquid chromatography method used allowed direct analysis of bile without derivatization or extraction. Ether anesthesia was associated with a reversible suppression of diglucuronide formation and total bilirubin excretion, with reciprocal monoglucuronide changes. Bile flow and pigment excretion were variable with ketamine. Pentobarbital provided the most uniform excretion data, although the ratio of C-8:C-12 monoglucuronide varied with all drugs. These data are consistent with recently reported drug-induced alterations in hepatic uridine diphosphoglucuronic acid concentration and support the hypothesis that alterations in this substrate concentration are capable of influencing rates of hepatic glucuronide formation.

The reversal of bilirubin-induced intestinal secretion by agar.

Agar binds bilirubin in vitro and lowers serum bilirubin concentrations by interrupting the enterohepatic circulation and increasing the fecal excretion of bilirubin. Nonconjugated bilirubin has been reported to induce secretion of sodium and water by the small intestine of perfused hamsters. We investigated the possibility that agar could prevent the secretory effect of bilirubin on hamster gut by sequestering it and reducing its enterocyte exposure. The small intestine of hamsters was luminally perfused in vivo with 0.5 mM bilirubin either alone or simultaneously with 0.15 g% agar. Control perfusions demonstrated luminal absorption of water and sodium in the absence of bilirubin. The inclusion of bilirubin resulted in secretion of sodium and water. The addition of agar to the bilirubin-infused animals resulted in net absorption of sodium and water comparable to that of controls. An additional agar-containing control perfusion demonstrated apparent secretion of sodium and water.

Enteral administration of agar as an effective adjunct to phototherapy of neonatal hyperbilirubinemia.

Phototherapy increases the biliary excretion of unconjugated bilirubin. In this form, bilirubin would be subject to enterohepatic circulation, and the true efficacy of phototherapy would be blunted. We tested the hypothesis that sequestration of lumenal unconjugated bilirubin by enteral agar administration would enhance the efficacy of phototherapy in jaundiced infants. Fifty-two infants were studied, 21 control and 31 agar-supplemented. The birth weights, sex distribution, and postnatal age at onset of phototherapy did not differ between the two groups of infants; pre- and post-phototherapy bilirubin concentrations also did not differ between the groups. The bile acid concentrations and bilirubin saturation indices were also similar. The rate of declination of the plasma bilirubin concentrations after 24 h of phototherapy was greater and significantly more uniform in the agar-supplemented infants (-1.59 +/- 2.3 versus -2.51 +/- 1.44). Stool frequencies were greater in control infants (5.5 versus 4.3 per 24 h) whereas fecal bilirubin excretions were greater in agar-supplemented infants during the second day of phototherapy (1.32 versus 3.29 mg . kg-1 . 24 h-1). Agar supplementation reduced the duration of phototherapy by 23% (37.6 +/- 3.2 versus 48.1 +/- 5.0 h).