Frederick C. Phillips

Genetically obese MMTV-TGF-α/Lepob Lepob female mice do not develop mammary tumors

Elevated body weight is a risk factor for postmenopausal breast cancer and is associated with increased incidence of spontaneous and chemically induced mammary tumors (MTs) in rodents. In this study, genetically obese LepobLepob female mice that overexpress human TGF-α (transforming growth factor-alpha) were used to assess the role of body weight on oncogene-induced MT development in comparison to lean counterparts. MMTV (mouse mammary tumor virus)-TGF-α and Lep strain mice were crossed to produce TGF-α/Lep+Lep+ (homozygous lean), TGF-α/Lep+Lepob (heterozygous lean) and TGF-α/LepobLepob (homozygous obese) genotypes. Body weights were determined weekly and mice palpated for the presence of MTs until 104 weeks of age. Despite their significantly higher body weight, obese TGF-α/LepobLepob mice failed to develop MTs. MTs were detected between 48 and 104 weeks of age for 26/39 TGF-α/Lep+Lepob mice and for 19/38 TGF-α/Lep+Lep+ mice between 67 and 104 weeks of age. Although MT incidence was not statistically different between the lean groups, age of MT detection tended to be younger for TGF-α/Lep+Lepob mice (p < 0.09). There were significant effects of both genotype and MTs on final body weight, that is, TGF-α/Lep+Lepob mice weighed more than homozygous lean mice, and mice with MTs weighed more than those without MTs. TGF-α/LepobLepob mice are not a good model to evaluate the effect of body weight on MT development possibly due to leptin deficiency. However, the finding that increased body weight is associated with increased oncogene-induced MT development within the normal weight range provides experimental support for the role of body weight in breast cancer.

Leptin Receptor-Deficient MMTV-TGF-α/LeprdbLepr db Female Mice Do Not Develop Oncogene-Induced Mammary Tumors

Being overweight is a risk factor for postmenopausal breast cancer and is associated with an increased incidence and shortened latency of spontaneous and chemically Induced mammary tumors in rodents. However, leptin-deficient obese Lepob Lepob female mice have reduced incidences of spontaneous and oncogene-induced mammary tumors. Of interest, leptin enhances the proliferation of human breast cancer cell lines in which leptin receptors are expressed, which suggests that leptin signaling plays a role in tumor development. We evaluated oncogene-induced mammary tumor development in obese MMTV-TGF-α/Leprdb Leprdb mice that exhibit a defect in OB-Rb, which is considered to be the major signaling isoform of the leptin receptor. Lepr and MMTV-TGF-α mice were crossed, and the offspring were genotyped for oncogene expression and the determination of Lepr status. Lean MMTV-TGF-α/Lepr+ Lepr+ (homozygous) and MMTV-TGF-α/Lepr+ Leprdb (heterozygous) mice and obese MMTV-TGF-α/Leprdb Leprdb mice were monitored until age 104 weeks. Body weights of MMTV-TGF-α/Leprdb Leprdb mice were significantly heavier than those of the lean groups. No mammary tumors were detected in MMTV-TGF-α/LeprdbLeprdb mice, whereas the incidence of mammary tumors in MMTV-TGF-α/Lepr+ Lepr+ and MMTV-TGF-α/Lepr+ Leprdb mice was 69% and 82%, respectively. Examination of mammary tissue whole mounts indicated an absence of duct formation and branching for MMTV-TGF-α/Leprdb Leprdb mice. Both age at mammary tumor detection and tumor burden (tumors/mouse and tumor weights) were similar for the lean genotypes. Serum leptin levels of MMTV-TGF-α/Leprdb Leprdb mice were 12-20-fold higher than levels of lean mice. Thus, despite elevated serum leptin levels, leptin receptor-deficient MMTV-TGF-α/Leprdb Leprdb mice do not develop mammary tumors. This study provides additional evidence that leptin and its cognate receptor may be involved in mammary tumorigenesis.

Quantitative analysis of triglyceride species of vegetable oils by high performance liquid chromatography via a flame lonization detector

A method for the quantitative analysis of triglyceride species composition of vegetable oils by reversed-phase high performance liquid chromatography (RP-HPLC) via a flame ionization detector (FID) is described. Triglycerides are separated into molecular species via Zorbax chemically bonded octadecylsilane (ODS) columns using gradient elution with methylene chloride in acetonitrile. Identification of species is made by matching the retention times of the peaks in the chromatogram with the order of elution of all of the species that could be present in the sample on the basis of a random distribution of the fatty acids and comparision of experimental and calculated theoretical carbon numbers (TCN). Quantitative analysis is based on a direct proportionality of peak areas. Differences in the response of individual species were small and did not dictate the use of response factors. The method is applied to cocoa butter before and after randomization, soybean oil and pure olive oil.

Reactions of dimethyl sulfoxide with sulfonate esters of fatty alcohols. I. Synthesis of higher saturated and unsaturated fatty aldehydes

Long-chain saturated fatty aldehydes (C10 to C18), as well as the C18 unsaturated aldehydes (oleyl, linoleyl, and linolenyl), were synthesized in good yields by the selective oxidation of the sulfonate esters of the corresponding alcohols with dimethyl sulfoxide in the presence of sodium bicarbonate. Chromatographic procedures for the isolation of the pure aldehydes from the reaction mixtures are described. The purity of the aldehydes was ascertained by thin-layer chromatography, melting points of their 2,4-dinitrophenyl hydrazones, infrared spectra and other physical methods.

Quantitative analysis of lipid classes by liquid chromatography via a flame ionization detector

A method is described for the direct quantitative analysis of the lipid classes of mammalian tissue lipids using high performance liquid chromatography (HPLC) with a flame ionization detector (FID). The lipid is extracted from the tissue with chloroform/methanol after deactivation of hydrolytic enzymes and removal of nonlipid substances by extraction with hot dilute acetic acid (0.05N). Separation of the lipid classes is performed with a column (45 cm × 0.2 cm id) of 8 μm silica (Spherisorb, Phase Sep, Hauppague, NY) treated with concentrated ammonium hydroxide at a solvent flow rate of 0.5 ml/min, which requires a pressure of ca. 900 psi. Cholesteryl esters (CE) and triglycerides (TG) are eluted first with Skellysolve B/methylene chloride (1∶1, v/v); cholesterol (CH) is eluted with chloroform/methylene chloride (1∶2, v/v) and the phospholipids with methanol containing 6% ammonium hydroxide added to the latter solvent in a linear gradient. The neutral lipids are eluted in ca. 12 min and the phospholipids in an additional 30 min. The relative amount of each lipid class was determined from standard curves of the peak areas obtained according to response factors using erucyl alcohol as an internal standard. The method was applied to samples of kidney, liver and serum of rats. Duplicate analyses were generally within ca. 1.0% and good agreement was obtained in the analysis of the lipid classes of Azolectin and liver mitochondria lipid compared to thin layer chromatography (TLC) via photodensitometry of charred spots or phosphorus analysis of recovered phospholipids.

Two-dimensional reaction thin-layer chromatography in the analysis of phosphatide plasmalogens.

Abstract A two-dimensional reaction thin-layer chromatography procedure on micro- samples of mixtures of alkenyl acyl and diacyl phosphatides has been described by which it is possible to determine simultaneously their plasmalogen content, the fatty acid composition of invidual analogs and the fatty aldehyde composition of the alkenyl acyl analog.

Analysis of triglyceride species by high-performance liquid chromatography via a flame lonization detector.

The analysis of triglyceride species by high performance liquid chromatography (HPLC) with a flame ionization detector (FID) and reversed-phase chromatography using chemically bonded octadecyl silane (ODS) Zorbax columns and gradient or isocratic solvent elution with methylene chloride/acetonitrile is described. Triglycerides containing acyl groups of critical pairs,trans and positional isomers, as well as mixtures of even and odd chain lengths are separated. Identification of triglycerides is made on the basis of retention times compared with equivalent and theoretical carbon numbers, and comparison with chromatograms of reference triglyceride mixtures. The methodology is demonstrated by fractionizing the triglycerides of olive oil under different chromatographic conditions using single and coupled conventional 250×4.6 mm columns and a short 80×6.2 mm column for fast separations.

Metabolic effects of coconut, safflower, or menhaden oil feeding in lean and obese Zucker rats.

The aim of the present investigation was to study the effects of fish oil feeding in obese Zucker rats to establish its suitability as an animal model of hyperlipidaemia, and to understand the possible mechanism of fish oil-induced perturbations in cell metabolism. Lean and obese Zucker rats were fed on diets containing 180 g coconut, safflower, or menhaden oil/kg for 10 weeks. Body-weights and food intakes of lean coconut (LC), safflower (LS), and menhaden (LM) groups were similar. Obese menhaden (OM) rats had lower food intakes and body-weights compared with obese coconut (OC) and obese safflower (OS) groups, but values for all obese rats were higher than those for lean rats. Liver weights were higher in obese compared with lean rats, but on a percentage body-weight basis menhaden oil rats had higher values within genotype. Serum cholesterol and triacylglycerol levels were lower in the OM group compared with the OC and OS groups, and in the LM group compared with the LC group. Glucose and insulin levels were highest in OS rats followed by OC and OM rats and then the lean rats. Serum triiodothyronine and thyroxine were lower in OM rats compared with OC and OS rats. Liver mitochondrial state 3 rates with glutamate-malate and succinate were lower; mitochondrial beta-oxidation was unaffected and peroxisomal beta-oxidation was higher in menhaden oil rats compared with both coconut and safflower oil rats. In general, consumption of menhaden oil lowered hepatic malic enzyme (EC 1.1.1.38, 1.1.1.40), glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and glutathione peroxidase (EC 1.11.1.9) activities and elevated long-chain fatty acyl-CoA hydrolase (EC 3.1.2.2) activity when compared with the two other diets. It is concluded that obese Zucker rats do respond like human subjects to fish oil feeding but not to vegetable oils. The hypolipidaemic effect of fish oil appears to be mediated through a lowering of lipogenic enzymes, glucose-6-phosphate dehydrogenase and malic enzyme.

Restoration of fertility in young obese (Lep(ob) Lep(ob)) male mice with low dose recombinant mouse leptin treatment.

OBJECTIVE: We investigated the effects of low-dose leptin treatment on restoration of fertility in young adult male leptin deficient obese mice.EXPERIMENTAL DESIGN AND RESULTS: MMTV-TGF-α Lepob Lepob mice (8–10 weeks old) were treated with recombinant mouse leptin. In experiment 1, four mice (5 µg/g body weight leptin followed by 2.5 µg/g) lost weight and impregnated females (number of pregnancies/number of females, 3/6, 5/6, 5/10, 4/10). In experiment 2, Leptin-Obese (2.5 µg/g) and Control-Lean mice weighed significantly less than Control-Obese mice. Epididymal pad weights of Control-Obese mice were the heaviest, followed by those of Leptin-Obese mice, and Control-Lean mice were the lightest. Testes weight was greater in Control-Lean vs Control-Obese mice. Leptin-Obese mice had testes weight not significantly different from either control group. Four of five Leptin-Obese mice impregnated females (4/10, 5/10, 2/10, 5/12, 0/10).CONCLUSIONS: These results indicate that low-dosage mouse recombinant leptin treatment restored fertility to young Lepob Lepob male mice. Although body weights of Leptin-Obese mice were similar to those of lean age-matched mice, epididymal fat pad weights were heavier.

Extraction and analysis of lipids from immature soybeans

A procedure is described for the extraction of lipids from immature soybeans that eliminates artifact formation and provides complete recovery of the lipid, including highly polar glycolipids free of nonlipid substances. The method involves pretreatment and extraction of the beans with hot dilute (0.25%) acetic acid, followed by chloroform-methanol extraction. Pretreatment and extraction of the tissue with hot dilute acetic acid destroys hydrolytic enzymes and removes organic-soluble, nonlipid substances that contaminate extracts obtained by chloroform-methanol extraction. Application of the method to immature soybeans confirmed that phosphatidic acid was largely an artifact of freezing and thawing of the beans, and phosphatidylmethanol was produced via transphosphatidylation of phosphatidylcholine and phosphatidylethanolamine upon extraction with chloroform-methanol.