Inhibition of hepatic sterol and squalene biosynthesis in rats fed di-2-ethylhexyl phthalate

Di-2-ethylhexyl phthalate (DEHP), a commonly used plasticizer, was found to be an inhibitor of the biosynthesis of hepatic nonsaponifiable lipids in the rat. The addition of DEHP at levels of 0.5% or 1.0% to a stock diet of rats resulted in a decreased conversion of acetate-1-¹⁴C and mevalonate-5-³H into squalene, C₃₀ sterols, and C₂₇ sterols by liver minces or slices, in vitro. In studies conducted with 0.5% DEHP feeding from 2 to 11 days, the degree of inhibition was found to increase with the duration of DEHP feeding; the inhibition of³H-mevalonate conversion to squalene and sterols developed more slowly, being reduced to ca. 70% of control values in 11 days, whereas¹⁴C-acetate conversion was reduced to ca. 35% of control values during the same period.³H-mevalonate conversion to sterols and squalene was, however, found to be suppressable to the same extent as¹⁴C-acetate conversion when diets containing 1.0% DEHP were fed for 18 days. The inhibitory effect of dietary DEHP on sterol and squalene biosynthesis from¹⁴C-acetate and³H-mevalonate by rat liver preparations is unlikely to be accounted for by the negative feedback of cholesterol secondary to hepatic sterol accumulation since, in these studies, hepatic total lipid and hepatic total sterol levels were simialr in control and DEHP-fed rats.     

Tocopherol contents of lipoproteins from frozen plasma separated by affinity chromatography

We investigated whether freezing and storage of plasma altered α-tocopherol levels of whole plasma or the lipoprotein fractions derived from such plasma. Plasma from 24 men, at each of two collection periods, was frozen at −20°C for six weeks, then high-density lipoproteins (HDL) were separated from low- plus very low-density lipoproteins (LDL-VLDL) by heparin affinity chromatography. Whole plasma and the lipoprotein fractions were analyzed for α-tocopherol content and compared to counterparts from fresh plasma. Freezing and storage did not reduce α-tocopherol levels of plasma or the lipoprotein fractions. α-Tocopherol values from fresh and frozen plasma were highly correlated for both plasma (period 1, r=0.94; period 2, r=0.93) and the LDL-VLDL fractions (periods 1 and 2, r=0.97). Percent distribution of α-tocopherol between the two lipoprotein fractions was comparable for lipoproteins derived from fresh and frozen plasma. Under the storage conditions used in this study, plasma can be frozen for at least six weeks prior to lipoprotein fractionation with no detectable detrimental effects on α-tocopherol content of either plasma or lipoproteins.     

Inhibition of sterol biosynthesis in chlorella ellipsoidea by AY-99441

WhenChlorella ellipsoidea was grown in the presence of 4 ppm AY-9944, complete inhibition of Δ⁵-sterol biosynthesis was achieved. However total sterol production remained unaltered. As a result a number of sterols accumulated that appear to be intermediates in sterol biosynthesis. These sterols were described and identified as (24S)-5α-ergost-8(9)-en3β-ol, (24S)-5α-stigmast-8(9)-en-3β-ol, 4α-methyl-(24S)-5α-ergosta-8, 14-dien-3β-ol, 4α-methyl-(24S)-5α-stigmasta-8, 14-dien-3β-ol, 4α-methyl-(24S)-5α-ergost-8(9)-en-3β-ol and (24S)-4α-methyl-5α-stigmast-8(9)-en-3β-ol. The occurrence of these sterols inChlorella ellipsoidea is the first time they have been noted in biological material. The accumulation of these sterols in treated cultures indicates that AY-9944 is an extremely effective inhibitor of the Δ⁸→Δ⁷ isomerase and the Δ¹⁴ reductase of these plants. The occurrence of small amounts of other sterols in treated cultures has led to a proposed pathway for thebiosynthesis of sterols inChlorella ellipsoidea. Scientific Article No. A1775, Contribution No. 4565 of the Maryland Agricultural Experiment Station.     

Effects of cholestyramine and squalene feeding on hepatic and serum plant sterols in the rat

Hepatic and serum phytosterol concentrations were compared in the rat under basal conditions and during activated cholesterol and bile acid production due to squalene and cholestyramine feeding. Both treatments consistently decreased hepatic and serum levels of sitosterol and campesterol and, unlike esterified cholesterol, esterified plant sterols were not increased in liver during squalene feeding. Serum levels of phytosterols were decreased quite proportionately to those in the liver. The hepatic levels of sitosterol and campesterol closely correlated with each other, but not with cholesterol levels. The percentage esterification of both phytosterols was lower than that of cholesterol. The results indicate that activation of hepatic sterol production leads to depletion of hepatic plant sterols. It is suggested that poor esterification of plant sterols may contribute to this decrease.     

Stimulation of hepatic squalene and triglyceride synthesis by dimethylsulfoxide,in vitro

The incorporation of [¹⁴C] mevalonate and [¹⁴C] acetate into squalene by rat liver slices was increased over 7-fold by the presence of 5% dimethylsulfoxide (DMSO) in the incubation medium. The stimulation of squalene synthesis was dose-related over the concentration range of 1–5% DMSO and did not affect the incorporation of [¹⁴C] mevalonate, into the C₂₇-sterol fraction (cholesterol) but did increase (about 50%) incorporation into C₃₀-sterol (lanosterol) at a level, of 5% DMSO. The stimulation of squalene synthesis was observed under both anaerobic (N₂ atmosphere) and aerobic (ambient air or 95% O₂/5% CO₂) conditions and may represent a direct effect of DMSO on squalene synthetase. At a level of 5%, DMSO also stimulated 7-fold the incorporation of [¹⁴C] acetate into triglycerides by liver slices; this occurred without changes in incorporation into the phospholipid or free fatty acid fractions. The disproportionate increase in lipid labeling from [¹⁴C] acetate suggests that the effects of DMSO are not simply a matter of increasing [¹⁴C] acetate entry into the tissue.     

Inhibition of hepatic cholesterol biosynthesis by chloroquine

Chloroquine is shown to be a potent inhibitor of cholesterol biosynthesis by isolated rat hepatocytes. Half-maximal inhibition of cholesterogenesis occurs at ca. 10μM chloroquine. Chloroquine does not affect fatty acid synthesis by isolated hepatocytes. This suggests that chloroquine acts on the cholesterol biosynthetic pathway beyond the cytosolic acetyl-CoA branchpoint of cholesterol and fatty acid synthesis.     

Inhibition of hepatic lipogenesis by adenine nucleotides

Incubation of liver slices and isolated liver cells with adenosine cyclic-3',5'-monophosphate at concentrations which inhibit lipogenesis was found to expand the pool size of the noncyclic adenine nucleotides in the intact cells of the preparations. This observation led to studies which demonstrated that adenosine and adenosine-5'-monophosphate also inhibited lipogenesis and expanded the adenine nucleotide pool size. It is proposed but not proven that the increase in intracellular nucleotides produced by adenosine-5'-monophosphate, adenosine cyclic-3',5'-monophosphate, and adenosine may have an adverse effect upon the synthesis of fatty acids. Because of the expansion of the adenine nucleotide pool size, high concentrations of adenosine cyclic-3',5'-monophosphate should not be used to investigate the mechanism responsible for hormonal regulation of lipogenesis. As an added complication, exogenous adenosine-5'-monophosphate was found to produce a small but significant increase in the intracellular concentration of adenosine cyclic-3',5'-monophosphate of isolated liver cells. This effect also may be a factor in the inhibition of lipogenesis by adenosine-5'-monophosphate. Low concentrations of N6, O2'-dibutyryl adenosine cyclic-3',5'-monophosphate were found to inhibit lipogenesis without increasing the intracellular adenine nucleotide content of either liver slices or isolated liver cells. It is concluded that studies on the mechanism of glucagon regulation of lipogenesis should be carried out with glucagon or low concentrations of N6, O2'-dibutyryl adenosine cyclic-3',5'-monophosphate.     

Glycosphingolipids of human plasma lipoproteins

The content and structure of glycosphingolipids (GSL) in human plasma lipoproteins were studies. The quantitative distribution of the neutral GSL(Glc-Cer, Gal-Glc-Cer, Gal-Gal-Glc-Cer, and GalNAc-Gal-Gal-Glc-Cer) and the principal ganglioside (AcNeu-Gal-Glc-Cer) within the different lipoprotein classes was similar to that of whole plasma. The total amounts (μmol glucose/100 ml plasma) of GSL in the plasma lipoproteins of three normal subjects were VLDL (very low density lipoproteins) (trace to 0.46), LDL (low density lipoproteins) (1.08–1.48), HDL₂ (high density lipoproteins₂) (0.62–0.85), and HDL₃ (high density lipoproteins₃) (trace to 0.28). In subjects with Lp(a) lipoproteins, HDL₂ rather than HDL₃ contained most of the GSL in HDL. When the data were corrected for differences in the plasma concentrations of the lipoproteins, the total amounts of GSL(nmol glucose/mg lipoprotein cholesterol) were VLDL(trace to 21.20), LDL(11.70–15.36), HDL₂(8.50–9.10), and HDL₃(3.12). No GSL were detected in lipoprotein deficient plasma. Mass spectrometry of the trimethylsilyl derivatives of the GSL in LDL showed major fragment ions characteristic of their individual structural components. The elevated plasma levels of the GSL(2–18 fold), in a homozygote for familial hypercholesterolemia, resided in LDL which contained an absolute increase (per mg lipoprotein cholesterol) of GSL. Most, if not all, of the plasma GSL are associated with plasma lipoproteins and may have an important role in their biological functions.     

Inhibition of cholesterol synthesis by cyclopropylamine derivatives of squalene in human hepatoblastoma cells in culture

Two squalene derivatives, trisnorsqualene cyclopropylamine and trisnorsqualeneN-methylcyclopropylamine, were synthesized and tested for inhibition of lanosterol and squalene epoxide formation from squalene in rat hepatic microsomes, and for the inhibition of cholesterol syntheses in human cultured hepatoblastoma (HepG2) cells. Trisnorsqualene cyclopropylamine inhibited [³H]-squalene conversion to [³H]squalene epoxide in microsomes (IC₅₀=5.0 μM), indicating that this derivative inhibited squalene mono-oxygenase. Trisnorsqualenen-methylcyclopropylamine inhibited [³H]squalene conversion to [³H]lanosterol (IC₅₀=12.0 μM) and caused [³H]-squalene epoxide to accumulate in microsomes, indicating that this derivative inhibited 2,3-oxidosqualene cyclase. Cholesterol biosynthesis from [¹⁴C]acetate in HepG2 cells was inhibited by both derivatives (IC₅₀=1.0 μM for trisnorsqualene cyclopropylamine; IC₅₀=0.5 μM for trisnorsqualeneN-methylcyclopropylamine). Cells incubated with trisnorsqualene cyclopropylamine accumulated [¹⁴C]squalene, while cells incubated with trisnorsqualeneN-methylcyclopropylamine accumulated [¹⁴C]squalene epoxide and [¹⁴C]squalene diepoxide. The concentration range of inhibitor which caused these intermediates to accumulate coincided with that which inhibited cholesterol synthesis. The results indicate that cyclopropylamine derivatives of squalene are effective inhibitors of cholesterol synthesis, and that substitutions at the nitrogen affect enzyme selectivity and thus the mechanism of action of the compounds.     

Inhibition of hepatic lipogenesis by 2-tetradecylglycidic acid

2-Tetradecylglycidic acid (TDGA), a hypoglycemic agent, has been found to be a very effective inhibitor of de novo fatty acid synthesis by isolated hepatocytes. A comparison was made between the effectiveness of TDGA and 5-(tetradecyloxy)-2-furoic acid (TOFA), a hypolipidemic agent, on the metabolic processes of isolated hepatocytes. These compounds are structurally related and both inhibit fatty acid synthesis; however, they have opposite effects from each other on the oxidation and esterification of fatty acids. TDGA inhibits whereas TOFA stimulates fatty acid oxidation. TDGA stimulates whereas TOFA inhibits fatty acid esterification.     

Inhibition of malonaldehyde formation by antioxidants from ω3 polyunsaturated fatty acids

The inhibitory effect of α-tocopherol, β-carotene, 2″-O-glycosyl isovitexin (2″-O-GIV), and butylated hydroxytoluene (BHT) on malonaldehyde (MA) formation from ω3 polyunsaturated fatty acids (PUFA) was determined by gas chromatography. The levels of MA formed from 1 mg each of octadecatetraenoic acid (ODTA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) upon oxidation with Fenton's reagent were 29.8±1.5, 17.2±1.5, and 22.0±0.7 nmol, respectively. BHT was most effective toward protecting all three ω3 PUFA, whereas β-carotene did not exhibit any inhibitory effect. 2″-O-GIV inhibited MA formation from EPA and DHA by 56 and 43%, respectively, showing the second greatest inhibitory activity after BHT. α-Tocopherol inhibited MA formation from ODTA and DHA by 67 and 28%, respectively, but it did not show any activity toward EPA oxidation. The naturally occurring antioxidant, 2″-O-GIV, may be useful to prevent oxidation of ω3 PUFA.     

High performance reversed-phase chromatography of the triglycerides from human plasma lipoproteins

The triglycerides of human plasma lipoproteins were separated with high performance reversed-phase liquid chromatography. An octadecyl bonded 5-μ silica column was used with a mobile phase of acetonitrile/acetone. Individual triglyceride types and critical pairs may be easily separated and identified.     

Inhibition of LCAT in plasma from man and experimental animals by chlorpromazine

Chlorpromazine (CPZ), a major tranquilizer, was found to be a potent inhibitor of lecithin:cholesterol acyltransferase (LCAT, EC 2.3.1.43) in the plasma of normal man, rat, rabbit and dog in vitro. The inhibitory effect of CPZ reached 35–50% at 0.5 mM depending on species; dog plasma LCAT appeared to be somewhat more sensitive than that of the other species. In rats fed CPZ or lidocaine for 14 days (0.05% in the diet), there was no statistically significant change in total plasma cholesterol levels or the size of the plasma-free (unesterified) cholesterol pool. However, 5 hr after an intracardial injection of [¹⁴C] cholesterol, the percentage of plasma [¹⁴C] cholesterol that was esterified was significantly lower (ca. 6%, p<0.05) in the CPZ-treated group, suggesting that CPZ may also inhibit LCAT to some extent in vivo. The percentage of plasma [¹⁴C] cholesterol esterified in the lidocainetreated group was similar to control values and did not reflect its ability to inhibit LCAT in vitro.     

Sterol synthesis from biliary squalene in the jejunal mucosa of the rat in vivo

Because bile contains substantial amounts of cholesterol precursors, e.g., squalene and differnet methyl sterols, the fate of biliary squalene was studied by incubating isolated jejunal loops of the rat in vivo with bile containing³H-squalene and¹⁴C-cholesterol. After 90 min, no radioactivity was found in plasma lipids. Closer analysis of gut epithelium revealed that both labeled compounds were preferentially taken up by the villous cells. Biliary³H-squalene was absorbed almost completely and was further cyclized to free and esterified methyl sterols and cholesterol. Whereas squalene not cyclized to sterols stayed in the mucosa, the newly synthesized sterols were transferred to lumen. The lipid patterns of gut lumen and mucosal cells were quite different, suggesting that the transfer of the newly synthesized lipid to intestinal lumen was not due to the desquamation of epithelial cells alone. The results suggest that biliary cholesterol precursors can contribute to the cholesterol production of the jejunal villous cells bypassing the rate-limiting step of the cholesterol synthesis pathway, and to the “nonexchanging” fecal neutral sterols of the rat.     

Inhibition of malonaldehyde formation from lipids by an isoflavonoid isolated from young green barley leaves

The inhibitory effect of 2″-O-glycosylisovitexin (2″-O-GIV), isolated from young green barley leaves, on malonaldehyde (MA) formation from lipids was determined by gas chromatography. Two μmol of 2″-O-GIV inhibited MA formation from 0.2 mmol of squalene by almost 100% upon ultraviolet (UV) irradiation. Only 1 μmol of 2″-O-GIV inhibited 90% of MA formation from 0.2 mmol of ethyl linoleate upon UV irradiation. The effective quantities of 2″-O-GIV were much lower than those of either butylated hydroxytoluene or α-tocopherol. When ethyl linoleate, ethyl linolenate and ethyl arachidonate were oxidized by Fenton’s reagent in the presence of 2″-O-GIV or α-tocopherol, both compounds showed similar activity toward MA formation. Both compounds gave maximum inhibition at doses of 0.1–0.3 μmol for ethyl linoleate, 0.1–0.5 μmol for ethyl linolenate and 0.1–0.5 μmol for ethyl arachidonate.     

Inhibition and induction of bile acid synthesis by ketoconazole effects on bile formation in the rat

The effects of ketoconazole, an antimycotic agent, and metyrapone, an inhibitor of mixed function oxidases, on bile acid synthesis were compared in the rat bothin vitro andin vivo. In rat liver microsomes, ketoconazole was much more potent than metyrapone in inhibiting the activity of cholesterol 7α-hydroxylase, the rate-limiting enzyme in the synthesis of bile acids. The I₅₀ values were 0.42 μM and 0.91 mM for ketoconazole and metyrapone, respectively. Intraduodenal administration of ketoconazole caused a rapid, dose-dependent reduction of bile acid synthesis in eight-day bile diverted rats. A single dose of 50 mg/kg reduced bile acid synthesis to 5% of control value; the same dose of metyrapone caused a reduction to only 85%. Inhibition of bile acid synthesis by ketoconazole was followed by a marked overshoot. At 28 hr after injection of 50 mg/kg of the drug, formation of bile acids was stimulated maximally by 45% compared to control value and remained elevated for more than 20 hr thereafter. Synthesis of all primary bile acids was affected to the same extent. Cholesterol 7α-hydroxylase activity in livers of ketoconazole treated (30 mg/kg) rats with an intact enterohepatic circulation was increased by 70% at 16 hr after i.p. injection of the drug. During the very large decrease of biliary bile acid output with ketoconazole, bile flow rate was relatively increased, due to stimulation of the bile acid-independent fraction of bile flow. The latter effect can probably be explained as caused by biliary secretion of osmotically active metabolites of ketoconazole.     

Fatty acid and sterol synthesis by rat small intestine in vitro

Slices of rat jejunum were incubated with [2-¹⁴C]pyruvate, [1-¹⁴C]acetate, or [³H]H₂O to determine lipogenic activity. Under all conditions studied, pyruvate acted as a better precursor than acetate for fatty acid synthesis but not for the synthesis of sterol. Exogenous glucose significantly (P≤0.05) increased the conversion of both pyruvate and acetate to fatty acids. By contrast, fasting resulted in a decrease (p≤0.05) in lipogenic activity. The highest levels of lipogenesis were observed when [³H]H₂O + glucose at a concentration of 20 mM was used. From such experiments, the absolute rate of fatty acid synthesis in the tissue preparation was calculated: 734±54 nmoles acetyl units incorporated into fatty acids/g tissue/hr.     

Fractionation of squalene from amaranth seed oil

Amaranth seed oil was fractionated in a bench-scale short-path distillation unit to obtain fractions rich in squalene. Fractionations were conducted with degummed amaranth oil, alkali-refined amaranth oil, and simulated amaranth oil. Squalene concentration was increased about sevenfold with a squalene recovery of 76.0% in the distillate when degummed amaranth oil was fractionated at 180°C and 3 mtorr vacuum. Free fatty acids codistilled with squalene, lowering the squalene content of the distillate, and resulted in a semisolid distillate at room temperature. Alkali-refining was subsequently used to reduce the free fatty acid content before fractionation. A simulated oil (7% squalene/93% soybean oil) and alkali-refined amaranth oil were fractionated at three temperatures (160, 170, and 180°C) and five vacuum settings (10, 100, 200, 400, and 600 mtorr). The highest squalene recoveries from simulated oil and alkali-refined amaranth oil were 73.4 and 67.8%, respectively, both at 180°C and 100 mtorr, which translates to 12.1-and 9.2-fold increases in squalene concentration, respectively. The squalene recovery of the alkali-refined amaranth oil at 180°C was not significantly different at 10 mtorr vs. 100 mtorr. The results of this study can be used as a component to assess the economic feasibility of fractionating amaranth seed for starch, oil, meal, and squalene.     

硫酸铵和尿素抑制飞灰合成二(口恶)英

二(口恶)英(PCDD/Fs)排放制约了当前焚烧技术在垃圾处置和资源化利用领域的应用和推广。研究了硫酸铵和尿素在不同温度下对垃圾焚烧飞灰合成PCDD/Fs的抑制能力。实验结果表明:高温下(650℃)硫酸铵和尿素对PCDD/Fs合成具有更高的抑制效率,但高温下飞灰合成PCDD/Fs量远低于低温下(350℃)的生成量;PCDD/Fs的降低量均主要来自于PCDFs的减少,并且低温下TeCDF减少量最大,而高温下减少最多的同系物为OCDF。由于实际烟道中,高温处投入的抑制剂会进入低温段继续产生抑制作用,所以仍需要更多的研究正确评价抑制剂的抑制效果和最优使用温度。