Size of lipid microdroplets effects results of hepatic arterial chemotherapy with an anticancer agent in water-in-oil-in-water emulsion to hepatocellular carcinoma.

We have initially prepared a new drug delivery system for hepatocellular carcinoma (HCC). Using sonication and a detergent, iodinated poppy seed oil (IPSO) can be mixed with an aqueous solution of epirubicin to make a water-in-oil emulsion. The water-in-oil emulsion is further passed through a microporous glass membrane and split into saline to make a long-term inseparable water-in-oil-in-water emulsion (W/O/W) that consists of IPSO microdroplets. To investigate the effect of the size of IPSO microdroplets on the efficacy of injection chemotherapy with W/O/W in patients with HCC, 32 HCC patients were randomly assigned and treated with W/O/W of small IPSO microdroplets (30 micrometers in diameter) containing 60 mg of epirubicin (n = 16, group A) or W/O/W of large IPSO microdroplets (70 micrometers) containing the same amounts of epirubicin (n = 16, group B). Effects were assessed by measuring the percentage of decline of the alpha-fetoprotein (AFP) level in a week from the AFP level immediately before the treatment. The decline was significantly larger in group B (50.5 +/- 19.8, mean +/- S.D.) compared with group A (18.9 +/- 33.1; p <.005). The size of IPSO microdroplets injected into the hepatic artery determines the decrease of serum AFP levels of the patients with HCC.

A predominant increase of arterial beta-hydroxybutyrate concentration during partial hepatectomies in patients with impaired indocyanine green clearance test.

BACKGROUND: It has been reported that a concentration ratio of two ketone bodies, [acetoacetate]/[beta-hydroxybutyrate], reflects the redox state in the liver mitochondria, but there are few reports which examined each ketone body concentration during hepatectomy. Materials and Methods. (1) Ketone body levels in the radial artery and the hepatic vein were measured simultaneously during hepatectomies in 12 patients. (2) Arterial ketone body levels were measured during hepatectomies (at three points) in 73 patients. RESULTS: (1) Both ketone body levels in the radial artery well reflected those in the hepatic vein (P < 0.001). (2) There was no change in the arterial acetoacetate level. The level of beta-hydroxybutyrate increased significantly from 30.4 to 76.5 micromol/L (P < 0.005) only in the group of 25 patients with seriously impaired indocyanine green clearance test. CONCLUSIONS: Ketosis during hepatectomy was caused by increased beta-hydroxybutyrate, and this phenomenon was observed only in patients with seriously impaired liver function.

Diagnosis of the first Japanese patient with 3-oxo-delta4-steroid 5beta-reductase deficiency by use of immunoblot analysis.

UNLABELLED: A 3-oxo-delta4-steroid 5beta-reductase (5beta-reductase) deficiency is difficult to diagnose because severe liver damage can result in a similar pattern of metabolite excretion. We investigated the usefulness of immunoblot analysis for diagnosis of 5beta-reductase deficiency and quantitatively analysed urinary bile acids by gas chromatography-mass spectrometry in a 5-month-old Japanese boy with severe neonatal cholestasis associated with hypertyrosinaemia. A liver sample was examined by immunoblot analysis using monoclonal antibodies against 5beta-reductase. Urinary 3-oxo-delta4 bile acids accounted for 88.3% of total bile acids, 5alpha-bile acids for 0.9%, and primary bile acids for 9.1%. Immunoblot analysis of the liver tissue showed an indistinct band of 5beta-reductase. CONCLUSIONS: These findings suggest that this patient had a secondary 5beta-reductase deficiency due to severe liver damage, even though 3-oxo-delta4 bile acids constituted more than 70% of total urinary bile acids. However, the patient may possibly have had an inherited 5beta-reductase deficiency.

7 alpha hydroxylation of 25-hydroxycholesterol in liver microsomes. Evidence that the enzyme involved is different from cholesterol 7 alpha-hydroxylase.

Rat, pig and human liver microsomes were found to catalyze 7 alpha-hydroxylation of 25-hydroxycholesterol. In contrast to cholesterol 7 alpha-hydroxylase activity, the 7 alpha-hydroxylase activity towards 25-hydroxycholesterol in rat liver was not stimulated by cholestyramine treatment. After transfection with cDNA for human cholesterol 7 alpha-hydroxylase, COS cells showed a significant activity towards cholesterol but not towards 25-hydroxycholesterol. During purification of cholesterol 7 alpha-hydroxylase from pig liver microsomes, about 99% of the 7 alpha-hydroxylase activity towards 25-hydroxycholesterol and 27-hydroxycholesterol was clearly separated from 7 alpha-hydroxylase activity for cholesterol. The small amount of 25-hydroxycholesterol 7 alpha-hydroxylase activity retained in a partially purified preparation of cholesterol 7 alpha-hydroxylase was not inhibited by addition of cholesterol, indicating that the oxysterol binding site is different from the cholesterol binding site, presumely due to the presence of two different enzymes. It is concluded that different enzymes are involved in 7 alpha-hydroxylation of cholesterol and 7 alpha hydroxylation of side-chain-oxidized cholesterol in rat, pig and human liver. Inhibition experiments with a partially purified fraction of the oxysterol 7 alpha-hydroxylase from pig liver gave results consistent with the contention that the same enzyme is responsible for 7 alpha hydroxylation of both 25-hydroxycholesterol and 27-hydroxycholesterol. It has been suggested that cholesterol 7 alpha-hydroxylase can preferentially use oxysterols, in particular 25-hydroxycholesterol, as substrates and by this means inactivate important physiological regulators of cholesterol homeostasis. Such a mechanism would explain the unique property of the liver to resist down-regulation of the low-density-lipoprotein receptor [Dueland, S., Trawick, J.D., & Davies, R.A. (1993) J. Biol. Chem. 267, 22695-22698]. The present results do not support the contention that the important coupling between cholesterol 7 alpha-hydroxylase activity, the low-density-lipoprotein receptor activity and hydroxymethylglutaryl coenzyme A reductase activity in liver cells is due to inactivation of 25-hydroxycholesterol or 27-hydroxycholesterol by the action of cholesterol 7 alpha-hydroxylase.

Cloning and expression of cDNA of human delta 4-3-oxosteroid 5 beta-reductase and substrate specificity of the expressed enzyme.

The enzyme delta 4-3-oxosteroid 5 beta-reductase (3-oxo-5 beta-steroid: NADP+ oxidoreductase and 4,5 beta-dihydrocortisone: NADP+ delta 4-oxidoreductase) catalyzes the reduction of the delta 4 double bond of bile acid intermediates and steroid hormones carrying the delta 4-3-one structure in the A/B cis configuration. Human delta 4-3-oxosteroid 5 beta-reductase cDNA was isolated from a liver cDNA library by cross hybridization with a previously cloned rat cDNA, which was used as a probe [Onishi, Y. Noshiro, M., Shimosato, T. & Okuda, K.-I. (1991) FEBS Lett. 283, 215-218]. DNA sequence analysis of a hybridization-positive clone predicted the human delta 4-3-oxosteroid 5 beta-reductase to contain 326 amino acids. The amino acid sequence of the human delta 4-3-oxosteroid 5 beta-reductase had 79% overall identity to the rat enzyme sequence. It also showed 54% and 50% overall identity with rat 3 alpha-hydroxysteroid dehydrogenase and human aldose reductase, respectively. RNA blotting analysis demonstrated the existence of a single delta 4-3-oxosteroid 5 beta-reductase mRNA of approximately 2.7 kb in human liver. Transfection of the cDNA into COS cells resulted in the expression of an active enzyme with a high activity toward the bile acid intermediates 7 alpha,12 alpha-dihydroxy-4-cholesten-3-one and 7 alpha-hydroxy-4-cholesten-3-one. In addition, the expressed enzyme showed a small but significant 5 beta-reduction activity toward 11 beta,17 alpha,21-trihydroxy-delta 4-pregnene-3,20-dione (cortisol) and 17 beta-hydroxy-delta 4-androsten-3-one (testosterone) whereas no activity was observed toward delta 4-pregnene-3,20-dione (progesterone) or delta 4-androstene-3-17-dione (androstenedione). The substrate specificity of the human enzyme is considerably narrower than that of the rat enzyme, and the enzyme seems to be more important for bile acid biosynthesis than for metabolism of steroid hormones.