Ductal carcinoma in situ of the breast arising in encapsulated mammary hamartoma†;†A case report.

Mammary hamartoma is benign lesion and relatively rare. 17 cases of breast cancer associated with a hamartoma had been previously documented in the literature. We describe herein a case of noninvasive ductal carcinoma of the breast arising in hamartoma in a woman of 60's. The discordance of images of the mass between mammogram and ultrasonogram can lead us to detect the carcinoma within the hamartoma in our case. J. Med. Invest. 67 : 368-371, August, 2020.

Influence of human serum albumin on the bile acid-mediated inhibition of liver microsomal type 1 11ß-hydroxysteroid dehydrogenase.

The influence of human serum albumin (HSA) on the bile acid-mediated inhibition of liver microsomal type 1 11ß-hydroxysteroid dehydrogenase (11ß-HSD1) was studied in vitro. A rat liver microsomal fraction was prepared, and the 11ß-HSD1 enzyme activity in the presence of various concentrations of bile acids and HSA was determined using hydrocortisone as the substrate. The products of the reaction were extracted and analyzed using high-performance liquid chromatography. The magnitude of the inhibition decreased with the addition of HSA in a dose-dependent manner. Four percent human albumin decreased the inhibitory effects of 100 µM chenodeoxycholic acid and lithocholic acid from 89.9 ± 5.6 to 54.5 ± 6.1% and from 83.8 ± 4.8 to 20.8 ± 4.2%, respectively. In contrast, ursodeoxycholic acid and deoxycholic acid showed no inhibitory effect on the enzyme activity in the presence of 4% human serum albumin, and the addition of 1% γ-globulin to the assay mixture in the presence of bile acids did not affect the enzyme activity. Our in vitro study showed that the addition of HSA ameliorated the inhibition of 11ß-HSD1 and that the magnitude of the change is dependent on the species of bile acid, presumably based on the numbers of hydroxyl groups. These results suggest that HSA seems to protect the bile acid-mediated inhibition of 11ß-HSD1 in the healthy subject. On the other hand, in the patients with obstructive biliary diseases, not only elevated serum bile acid but also the accompanying hypoalbuminemia is important to evaluate the pathophysiology of the bile acid-mediated inhibition of 11ß-HSD1 of the disease.

MR and US imaging for breast cancer patients who underwent conservation surgery after neoadjuvant chemotherapy: comparison of triple negative breast cancer and other intrinsic subtypes.

BACKGROUND: Neoadjuvant chemotherapy (NAC) is commonly utilized to treat operable breast cancer. The purpose of this study was to review the findings of ultrasonography (US) and magnetic resonance (MR) imaging in patients treated with breast conservation surgery (BCS) after NAC with a focus on intrinsic subtypes. METHODS: Eighty-six patients underwent BCS after NAC. The tumors were classified into four subgroups by receptor status. US and MR were performed before and after NAC. The tumor diameters in US and MR after NAC were examined for correlations with pathological tumor distances in the specimens from BCS after NAC. RESULTS: The correlation coefficient (r) of US to pathological tumor size was 0.3 in all tumors, 0.6 in HER2-type tumors, and 0.7 in triple negative breast cancers (TNBC). The correlation coefficient of tumor size in MR to pathological tumor size was 0.9 in TNBC, and other correlations were not statistically significant. CONCLUSIONS: The correlation between tumor size in MR and pathological tumor size in triple negative breast cancers corresponded best. This information is one of the clues to selecting patients for BCS after NAC.

Effects of bile acids on rat hepatic microsomal type I 11beta-hydroxysteroid dehydrogenase.

The aim of this study was to investigate the effect of various bile acids on hepatic type I 11beta-hydroxysteroid dehydrogenase (11beta-HSD1) activity in vitro. The rat liver microsome fraction was prepared and 11beta-HSD1 activity was assayed using cortisol and corticosterone as substrates for the enzyme reaction. The substrate and various concentrations of bile acids were added to the assay mixture. After incubation, the products were extracted and analyzed using high-performance liquid chromatography. All bile acids tested except deoxycholic acid and 7-keto bile acids inhibited the 11beta-HSD1 enzyme reaction to some degree. Ursodeoxycholic acid inhibited the activity less than cholic, chenodeoxycholic, and lithocholic acids. Deoxycholic acid and 7-keto bile acids did not inhibit, but enhanced the enzyme activity. Inhibitions of dehydrogenation by corticosterone were weaker than those by cortisol. Kinetic analysis revealed that the inhibition of 11beta-HSD1 was competitive. The inhibition of 11beta-HSD1 by bile acids depended on the three-dimensional structural difference in the steroid rings and the presence of the 7alpha-hydroxy molecule of the bile acids was important for the inhibition of rat hepatic 11beta-HSD1 enzyme activity. These results suggest that bile acid administration might modulate 11beta-HSD1 enzyme activity.

Biliary lipid output in the early stage of acute liver failure induced by 90% hepatectomy in the rat.

BACKGROUND: Differences in biliary lipid output were compared in rats after 70% or 90% hepatectomy (Hx) to evaluate a possible index of the early stage of acute liver failure. METHODS: Male Sprague-Dawley (SD) rats weighing 300 to 350 g were randomly divided into two groups for 70% Hx or 90% Hx, and animals were sacrificed at 0, 6, 24, and 48 h after Hx. Before sacrifice, a polyethylene tube was cannulated into the bile duct and bile was collected for 1 h. Outputs of total bile acids, phospholipid, and total cholesterol in serum and bile were determined. Biliary total cholesterol, bile acid concentrations, and bile acid component levels were determined using gas liquid chromatography. Hepatic microsomal cholesterol 7alpha-hydroxylase and sterol 12alpha-hydroxylase activities were also determined using high performance liquid chromatography. RESULTS: The 3-day survival rate after 90% Hx was 50%. In the 90% Hx group, the serum total bile acid concentration at each point was significantly higher than it was in the 70% Hx group. The bile flow rate and biliary outputs of cholesterol, phospholipid, and bile acids were significantly lower at 6 h after 90% Hx than after 70% Hx. Among bile acid species, cholic and chenodeoxycholic acid outputs into bile were significantly less at 6 h after 90% Hx. The activities of cholesterol 7alpha-hydroxylase and sterol 12alpha-hydroxylase were decreased after 90% Hx. CONCLUSIONS: Our results suggest that determinations of the bile flow rate and biliary lipid outputs are supposed to be useful for early detection of hepatic failure after extensive hepatectomy.

Species differences among various rodents in the conversion of 7alpha-hydroxycholesterol in liver microsomes.

Our previous study demonstrated that there are species differences among vertebrates in their conversion of 7alpha-hydroxycholesterol (7HC) to 7-ketocholesterol (7KC). To examine this further, we investigated the differences in the products of 7alpha-hydroxycholesterol in various species of male muroid rodents. Adult male Syrian hamsters (Mesocricetus auratus), dwarf hamsters (Phodopus rovolovskii), Djungarian hamsters (Phodopus sungorus), Chinese hamsters (Cricetulus griseus), rat-like hamsters (Tscherskia triton), and hispid cotton rats (Sigmodon hispidus) were used. Microsomal fractions were prepared from their livers, and the activities of the enzymes that participate in the dehydrogenation of 7alpha-hydroxycholesterol were determined by measuring the products using high-performance liquid chromatography. 7alpha-hydroxycholesterol was converted to both 7alpha-hydroxy-4-cholesten-3-one (7HCO) and 7-ketocholesterol in all of the hamsters tested. However, in the rat-like hamster and the hispid cotton rat, 7alpha-hydroxycholesterol was converted mostly to 7alpha-hydroxy-4-cholesten-3-one, as also observed in the rat (Rattus norvegicus). The results suggest that microsomal enzyme activity in the conversion of 7alpha-hydroxycholesterol to 7-ketocholesterol varies considerably, even within the subfamily Cricetinae and the family Muridae.

Bile acid biosynthesis during liver regeneration: enzyme activities of cholesterol 7alpha-hydroxylase and 3beta-hydroxy-delta5-C27-steroid dehydrogenase in rats.

The effects of two-thirds partial hepatectomy on bile acid metabolism have not been well established. This study investigated the changes in microsomal enzymes activities during liver regeneration. The cholesterol 7alpha-hydroxylase (7alpha-hydroxylase) and 3beta-hydroxy-delta5-C27-steroid dehydrogenase (3beta-dehydrogenase) activities in male Wistar rats were determined using high-performance liquid chromatography (HPLC). Cytochrome P450 (P450) and cytochrome b5 (b5) levels and NADPH cytochrome c reductase activities were also determined after hepatectomy. 7alpha-Hydroxylase activities were not reduced on days 3, 5, or 7 compared to those of sham-operated rats, but there was a significant decrease (by 60%) of 3beta-dehydrogenase activity compared to that of sham-operated groups (p <0.01) on days 3, 5, and 7 after hepatectomy. While 7alpha-hydroxylase activity had recovered by day 3 after hepatectomy, 3beta-dehydrogenase activity had not, even on day 7. These results suggest that the mechanisms of regulation of these 2 bile acid metabolizing enzymes are significantly different during liver regeneration.

Purification and characterization of 7beta-hydroxysteroid dehydrogenase from rabbit liver microsomes.

7beta-Hydroxysteroid dehydrogenase (7beta-HSD), a specific enzyme active in the metabolization of 7beta-hydroxycholesterol, was purified about 300-fold from male rabbit liver microsomes using ion exchange, hydroxylapatite, 2'5'ADP Sepharose 4B, and high-performance liquid chromatography on the basis of its catalytic activity. The specific activity of the purified enzyme was 276 nmol/min/mg protein. The molecular weight of the purified enzyme was 34,000. The preferred coenzyme was beta-NADP+. The optimum pH for oxidation was around 7.7 in potassium phosphate buffer, and 11.0 in glycine-NaOH buffer. The purified enzyme catalyzed the synthesis of not only 7beta-hydroxycholesterol but also corticosterone and hydrocortisone. Enzyme activities toward these three substrates accompanied all purification steps of 7beta-HSD. The amino acid sequence of the N-terminal of the purified enzyme showed that 7beta-HSD had sequence similarity to rabbit type I 11beta-hydroxysteroid dehydrogenase (11beta-HSD), indicating that 7beta-HSD may belong to the rabbit type I 11beta-HSD family and may play the same role in the metabolism of 11-hydroxysteroids and 7-hydroxysterols.

Species difference in cholesterol 7alpha-hydroxylase expression of rabbit and rat liver microsomes after bile duct ligation.

BACKGROUND: Bile duct ligation (BDL) produces a good animal model for investigation of the metabolic changes in obstructive jaundice. The aim of this study was to investigate the species difference in expression of cholesterol 7alpha-hydroxylase (7alpha-hydroxylase) in rabbits and rats after BDL. MATERIALS AND METHODS: Ten male New Zealand white rabbits weighing 2.5-3 kg and 12 male Wistar strain rats weighing 250-300 g were used. Half the animals underwent BDL, and half were sham operated (Sham). The animals were sacrificed on day 5 after operation. The livers were harvested, and levels of mRNA and 7alpha-hydroxylase activity were determined. Concentrations of serum bilirubin and bile acids were also measured. RESULTS: In BDL rats, the levels of mRNA were increased 30%, and 7alpha-hydroxylase activity was three times that of the Sham group. In BDL rabbits, however, these values were approximately 60 and 50% lower than the Sham group, respectively. Serum bile acid concentrations increased up to 13 times in BDL rabbits and 70 times in BDL rats over that of the Sham groups. Serum cholesterol and serum total bilirubin concentration also increased after BDL in both animals. CONCLUSION: These results suggest that there is a species difference in the expression of 7alpha-hydroxylase after BDL in rabbits and rats and the reason for this difference is most likely pretranslational regulation.

Assay method for mitochondrial sterol 27-hydroxylase with 7alpha-hydroxy-4-cholesten-3-one as a substrate in the rat liver.

Mitochondrial sterol 27-hydroxylase (EC 1.14.13.15) is an important enzyme, not only in the formation of bile acids from cholesterol intermediates in the liver but also in the removal of cholesterol by side chain hydroxylation in extrahepatic tissues. The enzyme has been assayed by complicated methods using radiolabeled substrates or deuterium-labeled tracers. These methods may be inaccurate for measuring enzyme activity, because the amount of electron-transferring proteins may be insufficient for maximal velocity. To solve this problem, after solubilization of the enzyme from rat liver mitochondria with n-octyl-beta-d-glucopyranoside (OGP), we measured the enzyme activity by incubating the solubilized enzyme with saturated amounts of electron-transferring proteins. In our assay system, using 7alpha-hydroxy-4-cholesten-3-one (HCO) as a substrate, we could easily measure the product, 7alpha,27-dihydroxy-4-cholesten-3-one, with HPLC monitoring absorbance at 240 nm. The product formation was proportionate to the time up to 5 min and the protein concentration up to 0.5 mg of protein/ml. The maximal velocity of the enzyme was 1.1 nmol/min/mg of protein, which was 4- to 16-fold higher than previously reported values. A simple and accurate assay method for sterol 27-hydroxylase in rat liver mitochondria is herein described.

Sulfhydryl groups responsible for extreme lability of human cholesterol 7alpha-hydroxylase identified by site-directed mutagenesis.

Cholesterol 7alpha-hydroxylase (cholesterol-NADPH oxidoreductase, EC 1.14.13.17, 7alpha-hydroxylating) is known to have extremely sensitive sulfhydryl group(s). It is believed that a cysteine residue that has a sulfhydryl group plays an important role in the decrease of this enzyme activity. The amino acid sequences of cholesterol 7alpha-hydroxylase of five different mammalian species, human, rat, rabbit, hamster and mouse, revealed that these mammalian species contain eight cysteine residues that are well conserved. To identify which cysteine residues are responsible for the extremely high lability, we used the technique of the site-directed mutagenesis. Eight mutated genes of human cholesterol 7alpha-hydroxylase in which one codon for a cysteine residue was changed to that for alanine were prepared and expressed in COS-1 cells. The protein mass and enzyme activity of cholesterol 7alpha-hydroxylse obtained from these eight mutated genes were determined. While all mutated genes expressed the enzyme mass, two mutated genes did not express protein capable of catalyzing 7alpha-hydroxylation of cholesterol: in one mutant a codon for the 7th cysteine residue (Cys 444) was substituted to that for alanine and in the other mutant a codon for the 8th cysteine residue (Cys 476) was changed similarly. These results suggest that the 7th and 8th cysteine residues are important for expression of the enzyme activity. Based on the fact that Cys 444 exists in the heme binding region, Cys 476 was suggested to be responsible for enzyme lability.

Half-life of cholesterol 7alpha-hydroxylase activity and enzyme mass differ in animals and humans when determined by a monoclonal antibody against human cholesterol 7alpha-hydroxylase.

A monoclonal antibody against human cholesterol 7alpha-hydroxylase was produced, and the half-life of the enzyme was studied. Both the activity and protein mass of the enzyme were measured at timed intervals during microsomal incubation. The enzyme activity dropped rapidly; the half-life was 1, 1.7 and 3h in humans, rats and rabbits, respectively. In contrast, the protein mass, measured by immunoblotting, declined slowly; its half-life was 5h in the human and 9h in the rat and rabbit enzymes. This suggests that there may be vulnerable sites responsible for the enzyme activity. Addition of dithiothreitol (DTT) restored the decreased activity considerably, indicating that at least one sulfhydryl group is involved in the vulnerability. These results show considerable decrement in cholesterol 7alpha-hydroxylase activity due to sulfhydryl groups.

Accumulation of 4- and 5-ene steroid sulfates in human breast cyst fluids.

Gross cystic disease of the breast is one of the most common diseases of adult females. Breast cyst fluid contains various steroid hormones. In order to obtain more information about the concentrations of 4- and 5-ene steroids in human breast cyst fluids, levels of pregnenolone sulfate (PREGS), pregnenolone (PREG), dehydroepiandrosterone sulfate (DHEAS) and dehydroepiandrosterone (DHEA) were determined by high-performance liquid chromatography (HPLC). A total of 35 human breast cyst fluid samples, obtained from 35 patients (28-54 years old) were analyzed. Cyst fluid electrolytes were simultaneously determined. Levels of PREGS (mean+/-S.D.) were 26.9+/-20.0 micromol/l (N=35) and of PREG were <0.1 micromol/l. Levels of DHEAS and DHEA were 89.1+/-111.7 micromol/l (N=35) and 0.3+/-0.2 micromol/l (N=35), respectively. Cyst fluids were divided into two groups (types I and II) according to their electrolyte ratio (K(+)/Na(+)). The cysts of the type I group (K(+)/Na(+) >1.5) contained significantly higher levels of PREGS (39.9+/-21.1 micromol/l) and DHEAS (133.2+/-87.9 micromol/l) than those of the type II group (K(+)/Na(+) <1.5), the mean levels of which were 19.8+/-16.2 micromol/dl for PREGS, and 36.3+/-29.0 micromol/dl for DHEAS (P<0.05). PREGS and DHEAS levels in the cysts were significantly correlated (r=0.49; P<0.01). Human breast cyst fluids contain high concentration of DHEAS and PREGS, especially in the cyst fluids containing high K(+)/Na(+) ratios.

A comparative study of the conversion of 7-hydroxycholesterol in rabbit, guinea pig, rat, hamster, and chicken.

The metabolism of epimeric 7-hydroxycholesterol was studied in vitro. 7Alpha-hydroxycholesterol or 7beta-hydroxycholesterol were incubated with rabbit, guinea pig, rat, hamster, and chicken microsomal suspensions and then extracted and analyzed using high-performance liquid chromatography (HPLC). 7Alpha-hydroxy-4-cholesten-3-one was the main product from 7alpha-hydroxycholesterol in the rabbit, guinea pig, and rat. A considerable amount of 7-ketocholesterol was also produced in the hamster and chicken. In all vertebrates, 7beta-hydroxycholesterol was converted only to 7-ketocholesterol in all vertebrates. 7Beta-hydroxy-4-cholesten-3-one was not detected. Reduction of 7-ketocholesterol was also studied in the rat and hamster. Whereas 7-ketocholesterol was converted to 7beta-hydroxycholesterol in the rat, it was converted to both 7alpha- and 7beta-hydroxycholesterol in the hamster. These results suggest that 7alpha-hydroxycholesterol is converted not only to 7alpha-hydroxy-4-cholesten-3-one but also to 7-ketocholesterol in the hamster and chicken. 7Beta-hydroxycholesterol was converted to 7-ketocholesterol in all vertebrates tested. The interconversion between 7alpha- and 7beta-hydroxycholesterol via 7-ketocholesterol was observed in the hamster in this in vitro study.

A simple, accurate, and sensitive assay method of dehydroepiandrosterone sulfate: application for quantitative determination in human breast cyst and duct fluids.

Radioimmunoassay (RIA) is the most prevalent method for measuring small amounts of hormones, peptides, and other compounds in human body fluids. The method, however, has several problems, such as cross reactions or non-specific reactions of the antibody used. In order to establish an improved method for assaying dehydroepiandrosterone sulfate (DHEAS) and cholesterol, which are the largest components of human breast cyst and duct fluids, we describe a simple, accurate, and sensitive method using high-performance liquid chromatography (HPLC). The samples were treated with cholesterol oxidase for quantitation of dehydroepiandrosterone (DHEA) and free cholesterol, and the respective oxidized substances, 4-androstene-3,17-dione and 4-cholesten-3-one, were extracted with n-hexane. The extracts were analyzed by straight phase HPLC. Effluents were monitored by measuring absorption at 240 nm, where a newly introduced chromophoric group, an alpha,beta-unsaturated ketone, showed intense absorption (epsilon = 16,000). When the total amount of DHEA (DHEAS plus DHEA) was measured, the sample had been solvolyzed by sulfatase beforehand. The amounts of DHEAS were quantified by comparing the amounts of DHEA before and after solvolysis. Levels of free cholesterol, DHEAS, and DHEA in human breast cyst fluids (n = 30) were 1.77 +/- 1.12 mmol/dl, 8.27 +/- 10.24 micromol/dl, and 0.02 +/- 0.02 micromol/dl (means +/- SD), respectively. The levels of sterol and steroid measured in breast duct fluids that were turbid, brown, dark green, or milky in color (n = 9) (mean levels, 3.20 +/- 2.97 mmol/dl for free cholesterol and 14.77 +/- 13.75 micromol/dl for DHEAS) were significantly (P < 0.01) higher than the levels in clear or serous breast fluids (n = 21) (mean levels, 0.14 +/- 0.13 mmol/dl for free cholesterol and 0.04 +/- 0.07 micromol/dl for DHEAS).