Hepatic microsomal composition studies in the Gunn rat.

The homozygous jaundiced (jj) Gunn rat exhibits hepatic microsomal enzyme activities which vary from markedly decreased to normal when compared with the non-jaundiced (JJ) Gunn rat. In order to determine if an alteration in microsomal lipid might be related to these observations, cholesterol, phospholipid, fatty acid and fluorescence polarization determinations were carried out in Gunn rats of both genotypes. Significant differences in microsomal palmitic, stearic and arachidonic acid composition were present, but these were not striking. Fluorescence polarization data best fit a two-phase linear model for both genotypes with no significant differences in breakpoint temperatures. In jj rats, the anisotrophy parameter ((r0/r)-1)-1 was significantly greater than that seen in JJ rats at both 25 and 37 degrees C, indicating a decreased membrane fluidity in the jaundiced animals. Alterations in enzyme microenvironment due to subtle changes in lipid composition may be related to the different enzyme activities observed in Gunn rats.

Essential fatty acid deficiency after hepatic portoenterostomy for biliary atresia.

Gas chromatography was used to determine the fatty acid composition of total lipids extracted from plasma and erythrocytes of five patients who had received an hepatic portoenterostomy for treatment of extrahepatic biliary atresia. Three patients, including one with successful surgery, demonstrated evidence of essential fatty acid deficiency, including decreased levels of linoleic and arachidonic acids with concomitant increases in palmitoleic and oleic acids. In two of these patients, the ratio of 5,8 11-eicosatrienoic acid to arachidonic acid ("triene/tetraene") exceeded 0.3, diagnostic of essential fatty acid deficiency. Even patients with successful hepatic portoenterostomy are at risk to develop essential fatty acid deficiency.

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.

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.

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.

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.

The in vitro and in vivo photoreactivity of bilirubin: I. Laser-defined wavelength dependence.

Monochromatic light was provided by a continuous wave Argon ion laser. We chose to study the in vitro effects of light at 457.9, 465.8, 476.5, 488.0, 501.7, and 514.5 nm as representative of a reasonably evenly spaced sampling across the blue-green spectrum. The in vivo experiments were conducted at 457.9, 476.5, 488.0, and 514.5 nm. In vitro light at 488.0 nm appeared to be more effective than the others studied. After 24 h of irradiance, the in vivo decline in serum bilirubin concentration produced by light at 488.0 nm was one-and-one-half, two, and four times as effective as light at 457.9, 476.5 and 514.5 nm, respectively. By 48 h of exposure, the declines produced by light at 457.9 nm and 488.0 nm are significantly superior to that at 476.5 nm and 514.5 nm, but they do not differ from one another.

Increased osmotic fragility of erythrocytes in chronically jaundiced rats after phototherapy.

Littermate homozygous (jj) and heterozygous (Jj) Gunn rats, were irradiated with blue fluorescent light for 18 hours continuously. The incident irradiance was 1.5 mWatts/cm2 in the 420--480 nm band pass. The influence of the irradiance on circulating erthrocytes was studied by testing their osmotic fragility before and after the irradiance. The non-jaundiced, Jj, animals did not exhibit any increase in the osmotic fragility of their erythrocytes. The osmotic fragility of the erythrocytes from jaundiced, jj, animals was the same as the Jj animals prior irradiance. However, the fragility of the erythrocytes from the jj animals was significantly increased after the 18 hours of irradiance. The results indicated that the photodynamic action of bilirubin may be present in vivo.