Inherited predisposition to breast carcinoma. Results of first round examination of 537 women at risk.

Five hundred and thirty seven women at risk for breast carcinoma were identified. Family history was detailed and each woman given genetic counselling. Diagnostic examination for breast carcinoma was performed at the major hospitals of Norway, and included physical examination by expert surgeon, mammography and/or ultrasonography, and fine needle aspiration cytology when appropriate. Altogether 8 carcinomas and 5 cases of atypical hyperplasia were found, compared with 1.6 and 0.3 expected, respectively, from population studies. The finding exceeded the expected numbers described by autosomal dominant inheritance. In addition we found one carcinoma in situ. It is concluded that the methods employed are suitable to identify and examine women at risk for breast carcinoma. It is suggested that atypical hyperplasia may be the precancerous lesion, and should be treated as such.

Inherited breast carcinoma--prospective findings in 1,194 women at risk.

According to preset criteria, 1,194 women at risk for inherited breast carcinoma were selected and subjected to examination. Six hundred and three women were examined once, 591 were followed for a mean of 1.8 years. Twenty infiltrating cancers (median age 49 years) and 16 precancers (median age 44 years) were found, demonstrating that breast carcinoma continued to occur in the selected families as expected under the hypothesis of dominant inheritance. At first round, 14 (1.2%) infiltrating carcinomas and a total of 22 (1.8%) cancers or precancers were found. Incidence rates of 0.58% pr. year for infiltrating cancers, and 1.04% pr. year for cancer or precancer were calculated. This confirms the tentative conclusions that were drawn in our previous report. These are the first prospective reports documenting how to delineate a high risk group for premenopausal breast cancer, and how to diagnose cancer at an early stage. All but two affected women had cancer without lymph node metastasis. Although a longer observation time is needed, we cautiously conclude that the results are in keeping with our aim of providing safety for those at risk. Clinical use of predictive genetic testing may be implemented within these families.

Retinol and retinyl esters in parenchymal and nonparenchymal rat liver cell fractions after long-term administration of ethanol.

Chronic ethanol consumption reduces the liver retinoid store in man and rat. We have studied the effect of ethanol on some aspects of retinoid metabolism in parenchymal and nonparenchymal liver cells. Rats fed 36% of total energy intake as ethanol for 5-6 weeks had the liver retinoid concentration reduced to about one-third, as compared to pair-fed controls. The reduction in liver retinoid affected both the parenchymal and the nonparenchymal cell fractions. Plasma retinol level was normal. Liver uptake of injected chylomicron [3H]retinyl ester was similar in the experimental and control group. The transport of retinoid from the parenchymal to the nonparenchymal cells was not found to be significantly retarded in the ethanol-fed rats. Despite the reduction in total retinoid level in liver, the concentrations of unesterified retinol and retinyl oleate were increased in the ethanol fed rats. Hepatic retinol esterification was not significantly affected in the ethanol-fed rats. Since our study has demonstrated that liver uptake of chylomicron retinyl ester is not impaired in the ethanol-fed rat, we suggest that liver retinoid metabolism may be increased.

Binding protein for vitamin D and its metabolites in rat mesenteric lymph.

A protein with high affinity for vitamin D3 and 25-hydroxyvitamin D3 in rat mesenteric lymph has been studied. Mesenteric lymph was collected after duodenal instillation of radiolabeled vitamin D3 and 25-hydroxyvitamin D3. As previously described, approximately 10% of vitamin D3 (S. Dueland, J.I. Pedersen, P. Helgerud, and C.A. Drevon, J. Biol. Chem. 257: 146-150, 1982) and 95% of 25-hydroxyvitamin D3 (S. Dueland, J.I. Pedersen, P. Helgerud, and C.A. Drevon, Am. J. Physiol. 245 (Endocrinol. Metab. 8): E463-E467, 1983) recovered in mesenteric lymph were associated with the alpha-globulin fractions. The radioactive vitamin D3 recovered in the lymph fraction with d greater than 1.006 (free of chylomicrons) coeluted with purified rat serum binding protein for vitamin D and its metabolites (DBP) from an antirat DBP column. The results obtained by immunoblotting after sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that this protein in mesenteric lymph had molecular weight and immunological properties identical with purified serum DBP. Purified serum DBP labeled with 125I was injected intravenously and mesenteric lymph was collected. in lymph, suggesting that DBP may be transferred from blood to mesenteric lymph and that plasma and lymph DBP may have a similar origin.

Chain-shortening of erucic acid and microperoxisomal beta-oxidation in rat small intestine.

The ability of rat small intestine to chain-shorten C22:1 fatty acids was investigated. Radioactive chain-shortened products, mainly C18:1, were demonstrated in intestinal-lymph lipids after intraluminal injection of [14-14C]erucic acid. Chain-elongation to C24:1 was also observed. Adaptation to a diet containing C22:1 fatty acids (partially hydrogenated-marine-oil diet) slightly increased the percentage of chain-shortened products. Microperoxisomal beta-oxidation activity, measured as CN(-)-insensitive palmitoyl-CoA-dependent NAD+ reduction, was detected in a microperoxisome-enriched fraction from mucosal scrapings. This activity was increased 1.9-fold by a soya-bean-oil diet, and 2.7-fold by a diet containing partially hydrogenated marine oil.

Clearance of acetyl low density lipoprotein by rat liver endothelial cells. Implications for hepatic cholesterol metabolism.

We have studied the hepatic uptake of human [14C] cholesteryl oleate labeled acetyl low density lipoprotein (LDL). Acetyl-LDL injected intravenously into rats was cleared from the blood with a half-life of about 10 min. About 80% of the injected acetyl-LDL was recovered in the liver after 1 h. Initially, most of the [14C]cholesterol was recovered in liver endothelial cells (about 60%). Some radioactivity (about 15%) was also recovered in the hepatocytes, while the Kupffer cells and stellate cells contained only small amounts of the label (less than 5%). About 1 h after injection, radioactivity started to disappear from endothelial cells and appeared instead in hepatocytes. Radioactivity subsequently declined in hepatocytes as well. After a lag phase of 4 h, significant amounts of radioactivity were recovered in bile. The in vitro uptake and hydrolysis of [14C]cholesteryl oleate-labeled acetyl-LDL were saturable in isolated rat liver endothelial cells. Native LDL does neither affect the uptake nor the hydrolysis of acetyl-LDL. Ammonia and monensin reduced the hydrolysis of acetyl-LDL in isolated liver endothelial cells. Furthermore, monensin at concentrations above 10 microM completely blocked the binding of acetyl-LDL to the liver endothelial cells, suggesting that the receptor for acetyl-LDL is trapped inside the cells. The liver endothelial cells may be involved in the protection against atherogenic lipoproteins, e.g. liver endothelial cells may mediate uptake of cholesterol from plasma and transfer of cholesterol to the hepatocytes for further secretion into the bile.

Lymphatic absorption and transport of retinol and vitamin D-3 from rat intestine. Evidence for different pathways.

The lymphatic absorption and transport of retinol and vitamin D-3 from rat intestine has been studied. When rats were cannulated in the intestinal lymph duct and given an intraduodenal bolus of [3H]retinol and 14C-labelled vitamin D-3, 14C-labeled vitamin D-3 appeared later in the intestinal lymph than [3H]retinol and the rate of absorption of vitamin D-3 was still maximal at a time when that of retinol had declined. Both vitamins were absorbed via the lymphatic route in association with chylomicrons. Almost all the retinol was esterified, while vitamin D-3 appeared in the chylomicrons as free vitamin D-3. In vitro incubations and in vivo studies using hepatectomized and normal rats showed that the retinyl ester was a relatively nonexchangeable component of the chylomicrons and their remnants. Hence, all the vitamin A followed the remnants in their clearance from plasma. In contrast, significant amounts of vitamin D-3 were transferred from the chylomicrons to other plasma fractions. Therefore, only a fraction of this vitamin may be removed in association with the chylomicron remnants.

Microsomal esterification of retinol in human liver.

Recent work has shown that esterification of retinol in microsomes from rat liver, mammary gland and small intestine and from human small intestine is catalyzed by an acyl CoA: retinol acyl transferase (ARAT). The current study demonstrates ARAT activity in human liver microsomes. At optimal incubation conditions the rate of retinyl ester formation due to ARAT (0.37 +/- 0.31 nmole ester formed X mg microsomal protein-1 X minute-1, mean +/- SD, n = 6) suggests that the enzyme is of physiological importance.

Absorption, distribution, and transport of vitamin D3 and 25-hydroxyvitamin D3 in the rat.

The absorption of [14C]vitamin D3 and 25-[3H]hydroxyvitamin D3 into the mesenteric lymph and portal vein after simultaneous instillation in rat duodenum has been studied. The appearance of both isotopes in the mesenteric lymph and portal vein during the first 2 h was negligible. After 2-4 h the isotopes appeared both in the lymph and portal vein. The appearance of 25-[3H]hydroxyvitamin D3 in the mesenteric lymph was faster than that of [14C]vitamin D3. Total recovery in the lymph during 24 h for [14C]vitamin D3 and 25-[3H]hydroxyvitamin D3 was 15.2 and 14.3%, respectively. While 80% of vitamin D3 in mesenteric lymph was recovered in the chylomicrons (d less than 1.006 g/ml), 94% of 25-hydroxyvitamin D3 was recovered in the protein fraction with d greater than 1.21 g/ml. Seventy-one percent of 25-hydroxyvitamin D3 in the protein fraction was associated with the alpha-globulins. Both vitamin D3 and 25-hydroxyvitamin D3 that were recovered in the chylomicrons (d less than 1.006 g/ml) were transferred in vitro to the lymph fraction with d greater than 1.006 g/ml. Less than 12% of 25-hydroxyvitamin D3 was recovered in the liver after intravenous injection of mesenteric lymph labeled with 25-[3H]hydroxyvitamin D3. Previously, under similar conditions 40% of administered vitamin D3 was recovered in the liver.

Transport of cholesterol.

.ur current model for cholesterol transport is summarized in Figure 10. In this figure we have put together the various steps in cholesterol transport that were described previously in this review. Under normal conditions, cholesterol metabolism and transport are well regulated. If the transport system is overloaded for a long time, however, hypercholesterolemia caused mainly by increased plasma LDL may develop in several species, including humans. Under such circumstances reverse transport of cholesterol may also fail, giving rise to deposits of cholesterol. Tissue macrophages may be responsible for this lipid accumulation, because receptor-mediated (adsorptive) endocytosis of lipoprotein-associated cholesterol in these cells is not under negative-feedback control. The deposits are mainly found in tissues poorly supplied with blood and lymph: the skin, tendons, the cornea, and arteries. Overload of cholesterol transport may be the result of too much fat and cholesterol in the diet, giving rise to cholesterol-rich lipoproteins from the gut and to increased production of liver (formula; see text) VLDL, which in humans ends up as LDL. In many individuals, however, no hypercholesterolemia is seen, even after eating large amounts of a "western" diet for decades; others may develop increased LDL on a relatively "prudent" diet. Obviously many of the factors and mechanisms in cholesterol transport are influenced by genetic factors. Although studies of several inborn errors of lipid metabolism have given information about some mechanisms, the quantitatively more important differences in genetic patterns, which determine whether or not a western diet will result in hyperlipidemia, are not well known. Perhaps studies of different forms of apoB and apoE and of HDL subgroups and hyper-alpha-lipoproteinemia will explain why certain individuals develop hypercholesterolemia and premature atherosclerosis. All the recent information related to cholesterol metabolism and transport gives rise to new questions. There are many problems of interest for future research: What are the metabolic differences between the apoB produced in the liver and that produced in the gut? To what extent is the protein moiety of LDL modified in the plasma of blood and lymph and in interstitial tissue? Are such modifications important to whether LDL uptake goes through the classic LDL pathway or through the macrophage (i.e., scavenger?) pathway? Are some changes in apoB important for liver recognition of LDL?(ABSTRACT TRUNCATED AT 400 WORDS)

Plasma clearance, transfer, and distribution of vitamin D3 from intestinal lymph.

The clearance of vitamin D3 from plasma after intravenous injection to rats of intestinal lymph labeled in vivo with radioactive vitamin D3 has been studied. The half-life of the injected vitamin D3 was about 6 min. In double-label experiments the half-life was the same for vitamin D3 as for vitamin A, which is removed from the circulation as chylomicron remnants. Fractionation of the blood plasma according to the density showed that 2 min after the injection of the lymph 30-95% of the radioactive vitamin D3 was recovered in plasma fractions with higher densities than chylomicron remnants (d greater than 1.019 g/ml). The percentage of vitamin D3 in the chylomicron fraction (d less than 1.006 g/ml) of the injected lymph was 66-95%. Taking this into account it could be calculated that the amount of radioactive vitamin D3 transferred from chylomicrons or chylomicron remnants to plasma with d greater than 1.019 g/ml within 2 min varied from 26 to 72%. The transferred radioactivity was recovered in a protein fraction with chromatographic mobilities identical to the binding protein for vitamin D and its metabolites. During the first 13 min after injection the amount of radioactivity recovered in the liver was less than 10%, but from 15 to 55 min the amount increased to about 50% of the injected dose. These findings suggest that the transfer of vitamin D3 from chylomicrons to the alpha-globulin fraction previously observed in vitro is of importance for the in vivo handling of vitamin D3.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of diets on acyl-CoA:cholesterol acyltransferase and on acyl-CoA:retinol acyltransferase in villous and crypt cells from rat small intestinal mucosa and in the liver.

Cholesterol and retinol are both esterified with long-chain fatty acid within the mucosal cells of the small intestine. The reactions are catalyzed by microsomal acyl-CoA:cholesterol and acyl-CoA:retinol acyltransferases (EC 2.3.1.26, and EC 2.3.1.-, respectively). To gain more insight into the physiological importance of these acyltransferases, they were studied in villous and crypt cells from rats either fasting or on diets which varied in fat and cholesterol content. Both enzymes had a higher activity in villous than in crypt cells. The activities in villous cells varied with feeding and fasting and the composition of diet when the animals were killed postprandially. Acyl-CoA:cholesterol acyltransferase activity went up upon cholesterol feeding whereas retinol acyltransferase in the mucosa was reduced by high-fat diets. The liver cholesterol acyltransferase activity varied with diet, it increased with both cholesterol and fat feeding, whereas retinol acyltransferase activity remained relatively constant. The results obtained suggest that different diets are of importance for cholesterol and retinol acyltransferase activities both in the intestinal mucosa and in the liver. The variation in activities of the two acyltransferases suggests that they may be different enzymes.

Retinol esterification by microsomes from the mucosa of human small intestine. Evidence for acyl-Coenzyme A retinol acyltransferase activity.

The mechanism of the intestinal esterification of retinol has been obscure. Recently, an acyl-Coenzyme A (CoA):retinol acyltransferase (ARAT) was found in rat intestinal microsomes, and experiments were therefore conducted to determine whether a corresponding enzyme exists in human small intestine. When microsomes were incubated with [3H]retinol and palmitoyl-CoA, or retinol and [1-14C]palmitoyl-CoA, radioactive retinyl palmitate was formed as identified by alumina column chromatography and reverse-phase high-pressure liquid chromatography. Heating the microsomes for 30 min at 60 degrees C resulted in loss of activity. The esterification was negligible without exogenous acyl-CoA and markedly stimulated by palmitoyl-, oleoyl-, and stearoyl-CoA in concentrations up to 20 microM. The acyl-CoA was successfully replaced by an acyl-CoA generating system, but not by unactivated palmitate (2.5-200 microM). The assay was dependent on the presence of albumin with optimum activity at 2-10 mg/ml. The optimal retinol concentration was 20-30 microM and pH approximately 7.4. The esterifying activity was completely inhibited by 8 mM of taurocholate and to 90% by 1 mM of 5,5'-dithiobis(2-nitrobenzoic acid). Activity was found throughout the small intestine. In jejunum the rate of retinol esterification was: 3.44 +/- 2.24 nmol [3H]retinyl ester formed . mg microsomal protein-1 . min-1 (mean +/- SD, n = 12). The corresponding activity in whole homogenates of biopsies were 1.17 +/- 0.28 (n = 8). It is concluded that human small intestine contains a microsomal acyl-CoA:retinol acyltransferase. Due to its high activity in vitro this enzyme is likely to be responsible for the intestinal esterification of retinol.

In vivo uptake of chylomicron [3H]retinyl ester by rat liver: evidence for retinol transfer from parenchymal to nonparenchymal cells.

We have studied hepatic uptake of chylomicron retinyl ester. Chylomicrons were obtained from intestinal lymph of rats that were given retinol in groundnut oil by intraduodenal injection. When lymph was injected intravenously into normal rats, the radioactivity was cleared from blood with t1/2 approximately equal to 10 min. Retinyl ester was taken up initially by the liver, which, after 30 min, contained 80-90% of the radioactivity injected. Initially, most of the radioactivity was in hepatocytes, but after 30 min it disappeared from these cells and reappeared in nonparenchymal liver cells. After 2 hr these cells contained more radioactivity than the hepatocytes. When lymph was injected into vitamin A-deficient rats or rats given vitamin A in the form of retinoic acid, the plasma clearance and initial hepatic uptake of radioactivity were similar to that found in control animals. However, the nonparenchymal cells in these animals did not accumulate radioactivity. The current data suggest that vitamin A (in chylomicron remnants) is taken up initially by hepatocytes and then is released from these cells and delivered mainly to nonparenchymal liver cells in normal animals. In vitamin A-deficient rats, the vitamin is transferred from the hepatocytes to extrahepatic tissues.

The effect of feeding and fasting on the activity of acyl-CoA: cholesterol acyltransferase in rat small intestine.

The purpose of the present study was to test if the microsomal acyl-CoA:cholesterol acyltransferase (ACAT) in rat small intestine is regulated under physiological conditions. Two previously described in vitro assays were used, both based on the esterification of endogenous cholesterol with exogenous acyl-CoA, performed or generated during the incubation. The important and consistent finding with rats on normal diet was an increase in ACAT activity with fasting and a decrease with feeding. Independent of the assay used, the ratio between ACAT activity in night-fasted and night-fed animals was about 2 (P less than 0.005). When the fasting period was extended to 36 h a corresponding difference was found whether the ACAT assay was based on preformed [1-14C]oleoyl- or [1-14C]palmitoyl-CoA as acyl-donor (P less than 0.05). The microsomal content of unesterified cholesterol was higher in fasted than fed animals, suggesting that availability of this substrate might be a factor in the regulation of rat intestinal ACAT activity.

Evidence that reverse cholesterol transport occurs in vivo and requires lecithin-cholesterol acyltransferase.

The transport of cholesterol from extrahepatic tissues into plasma (reverse cholesterol transport) and the possible requirement for lecithin:cholesterol acyltransferase was examined in the rat. One hour after removal of the liver plasma cholesterol ester concentrations were significantly increased by 20%, whereas free cholesterol concentrations were unchanged. The lecithin:cholesterol acyltransferase inhibitor, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) was administered to eviscerated rats. It inhibited plasma lecithin:cholesterol acyltransferase activity by 90% which in turn totally prevented the increase in plasma cholesterol ester concentrations. In addition, heat-inactivated plasma from DTNB-treated eviscerated rats was 50% more reactive toward a standard source of lecithin:cholesterol acyltransferase compared to plasma from control or untreated eviscerated rats. These data suggest that in the rat a reactive lecithin:cholesterol acyltransferase substrate is formed extrahepatically. Together with lecithin:cholesterol acyltransferase, this reactive substrate removes cholesterol from peripheral tissues.

Acyl CoA:retinol acyltransferase in rat small intestine: its activity and some properties of the enzymic reaction.

The present study was conducted to examine whether the intestinal esterification of retinol could be due to a microsomal acyl-CoA transferase. When the 'microsomal fraction' of rat mucosa was incubated with [3H]retinol and palmitoyl-CoA or oleoyl-CoA, [3H]retinyl esters were formed as identified by alumina column chromatography and reverse phase high-pressure liquid chromatography (HPLC). Unlabeled retinol and [1-14C]palmitoyl-CoA yielded retinyl[1-14C]palmitate. The esterifying activity was lost when microsomes were heated at 60 degrees C for 30 min. Only negligible activity was observed without exogenous acyl-CoA while 10-20 muM gave optimum activity provided that 2-5 mg/ml of albumin was present. Replacement of acyl-CoA by palmitate gave no esterification, indicating that the activity was not a reversed hydrolase reaction. Optimum pH was 7.1-7.6 and optimal concentration of retinol was 15 muM. With palmitoyl-CoA, the formation of retinyl ester was 1.00 +/- 0.26 nmol . mg protein-1 . min-1 (x +/- SD, n = 4) in rats killed postprandially versus 2.06 +/- 0.66 (n = 5) after 36 hr of fasting. Oleoyl-CoA gave lower activity: 0.52 +/- 0.14 and 1.41 +/- 0.36, respectively. The variation with feeding and fasting was significant (P less than 0.05) and corresponded to that of the intestinal acyl-CoA:cholesterol acryltransferase (ACAT). Inhibition of retinol esterification was observed with taurocholate and the thiol-blocking agent 5,5'-dithiobis (2-nitrobenzoic acid). The data show that rat intestinal microsomes catalyze the formation of retinyl esters by an acyl-CoA:retinol acyltransferase with several properties in common with ACAT located in the same subcellular fraction.