William Mogilevsky

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.

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.

Plasma desorption characterization of glucuronide and glutathione conjugates derived from bilirubin

Abstract Plasma desorption was used for the first successful analysis by mass spectrometry of the underivatized mono- and diglucuronides of bilirubin. The method was also extended to characterize three novel metabolites formed by Gunn rats from the dimethyl diester of bilirubin. Based on mass spectrometry, chromatography and chemical degradation, these structures are proposed to be isomeric glutathione conjugates of dimethyl diesters of epoxybilirubins. Five measurements of mass of the M + H + ions of one of the glutathione conjugates made over 17 months had an average value of 936.8 u and a standard deviation of ±0.5 u.

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.

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.

ISOLATION OF THE ACTIVATOR (A) OF BILIRUBIN GLUCURONYL TRANSFERASE (BGt) ROM NORMAL (JJ) AND HOMOZYGOUS (jj) GUNN RATS

Lubrol solubilized microsomes of liver from JJ and jj rats were salt fractionated and the 60% (NH4)2 SO4 precipitate was redissolved, dialyzed and further fractionated by DEAE cellulose chromatography (DE-52). After initial protein elution with 10 mM PO4 buffer, pH8.0, a linear salt gradient from 0-0.5M KCl was begun. Three separate elution fractions (F1, F2, F3) were collected with BGt activity for bilirubin (B). The DE-52, F3 fractions from both JJ and jj rats activated Che BGt activity of the F1 fraction of JJ rats 5-10 fold (n=20). The A was separated from the BGt in F3 by Sephacryl-300 (S-300) sieving into two molecular weight fractions BGt≅230 kD and A≅60 kD. Further purification of the BGt from F1 and F3 of JJ liver was performed by affinity chromatography. After adsorption on UDP-hexanolamine Sepharose 4-B, the purified BGt was eluted by 5mM UDPGA which showed by polyacrylamide gel electophoresis (PAGE) a subunit couplet of 52 and 54 kD. This purified BGt was activitated 5-8 fold by the A from the S-300 eluate of both JJ and jj microsomes. The subunit size of A by PAGE is similar to its size after S-300 sieving (≅60 kD). It is heat labile, has no BGt activity, is precipitated by TCA, inactivated by alkylation but not by trypsin or iodoacetamide. The A has no effect on the purified transferase (Gt) for p-nitrophenol. Thus in addition to separate Gts in microsomes additional cofactors appear important in determining their substrate specificity. The A increases the formation of both the mono- and diglucuronides of B by BGt.

EFFECTS OF ANESTHESIA ON BILE PIGMENT EXCRETION

Anesthetic agents can alter hepatic glucuronidation. Under diethyl ether (E), pentobarbital (P) or ketamine (K) (n=5 each), rats received bile duct and venous catheters. Basal bile was collected for 300 min without further anesthesia. Bilirubin diglucuronide (DG) was quantified by HPLC, total bilirubin excretion (μg/100 gm body wt/min) by Van den Bergh analysis and bile flow (μ1/100 gm body wt/min) by weight.E anesthesia is associated with a reversible suppression of DG formation and total bilirubin excretion. Flow and excretion are variable with K. P provided the most uniform excretion data.

719 AN ALTERNATIVE INTERPRETATION OF THE ENZYME DEFICIENCY IN THE GUNN RAT

Bilirubin glucuronyl transferase activity (BGT) is negligible in jaundiced (jj) Gunn rats, with intermediate and higher levels in heterozygous (Jj) and outbred (JJ) rats, respectively. This could be explained either by a defective enzyme or structural defects in the endoplasmic reticulum in which the enzyme is compartmented. If the latter interpretation is correct, similarly segregating activities might be expected among other microsomal enzymes. The activity of aminopyrine demethylase (AD), a hepatic microsomal mixed-function oxidase, was assayed in male jj, Jj, JJ Gunn and Wistar (Wi) rats. BGT (μg bilirubin conjugated/30 min/mg protein N) and AD (nmoles CH2O produced/min/mg protein) are shown below (mean±SD). Results: jj AD < Jj, JJ or Wi AD (p < .05). F-test demonstrated AD variance in Jj > jj or JJ rats (p < .05). A correlation (r=.81) exists between BGT and AD (p < .01) in JJ rats, but not in Jj rats. The data suggests a microsomal membrane defect in enzyme compartmentation may be the primary abnormality in the Gunn rat and possibly in Crigler-Najjar Syndrome.