D. L. Wood

An Improved Copper Reagent for Quantitative Densitometric Thin-Layer Chromatography of Lipids

Abstract Eleven cupric compounds were screened to determine whether a different cupric anion might give greater and more uniform charring responses with lipid classes on TLC plates than cupric acetate. A procedure was developed which used 10% CuSO4 in 8% H3PO4 as a charring reagent. Improved reproducibility of charring was obtained by temperature-programmed heating of the thin layer plates from 30 to 180°C at 10 degrees per minute in the oven of a commercial gas-liquid chromatograph.

Two-Stage, One-Dimensional Thin Layer Chromatographic Method for Separation of Lipid Classes

Abstract A thin-layer chromatographic (TLC) technique was developed for routine analysis of lipid classes of blood, milk, tissue and egg yolk. This procedure provided rapid and reproducible separations suitable for in situ quantitation by densitometry The spotted TLC plate was subjected to two developments in one dimension to separate, in ascending order from the origin: phospholipids, monoglycerides, free fatty acids, cholesterol, 1,2-diglycerides, 1,3-diglycerides, triglycerides, and cholesterol esters. Development 1: chloroform: methanol: acetic acid (98:2:1) to 17.0 cm. Development 2: hexane: ethyl ether: acetic acid (94:6:0.2) to the top of the plate. After air drying, the plate was dipped into a solution of 3% cupric acetate in 8% phosphoric acid for 3 seconds, and heated at 130°C for 30 minutes to char the separated lipid classes. The chromatograms were scanned at 350 nm. Overloading of TLC plates with sample using the spotter resulted in the appearance of distorted kidney-shaped spots. A mathemati...

Escherichia coli induces apoptosis and proliferation of mammary cells.

Mammary cell apoptosis and proliferation were assessed after injection of Escherichia coli into the left mammary quarters of six cows. Bacteriological analysis of foremilk samples revealed coliform infection in the injected quarters of four cows. Milk somatic cell counts increased in these quarters and peaked at 24 h after bacterial injection. Body temperature also increased, peaking at 12 h postinjection. The number of apoptotic cells was significantly higher in the mastitic tissue than in the uninfected control. Expression of Bax and interleukin-1β converting enzyme increased in the mastitic tissue at 24 h and 72 h postinfection, whereas Bcl-2 expression decreased at 24 h but did not differ significantly from the control at 72 h postinfection. Induction of matrix metalloproteinase-9, stromelysin-1 and urokinase-type plasminogen activator was also observed in the mastitic tissue. Moreover, cell proliferation increased in the infected tissue. These results demonstrate that Escherichia coli-induced mastitis promotes apoptosis and cell proliferation. Cell Death and Differentiation (2001) 8, 808–816

Circadian and ultradian rhythms of body temperature and peripheral concentrations of insulin and nitrogen in lactating dairy cows

To investigate possible circadian and ultradian periodicities for peripheral insulin and urea in lactating dairy cows, integrated 15-min blood samples were taken sequentially over 48 hr from six cows. In addition, radiotelemetry measurements of body temperature were averaged over the same 15-min periods. Cows were housed in an environmental chamber at 19 degrees C with lights on 0700 to 2300 hr; fed daily at 0900 hr; and milked at 0800 and 2000 hr. For five of the six cows, body temperature showed a circadian rhythm peaking at 2323 hr with an amplitude of 0.34 degree C. For the sixth cow, body temperature was 180 degrees out-of-phase, peaking at 1230 hr with an amplitude of 0.12 degree C. Circadian rhythms for insulin and urea were consistent for all six cows peaking at 1743 hr with an amplitude of 0.74 ng/ml for insulin and at 1034 hr with an amplitude of 3.83 mM for urea. Body temperature and insulin also displayed episodic increases that often exceeded the amplitudes of circadian rhythms. For body temperature, a broad increase in spectral power was seen for periods between 100 and 175 min; time intervals between peaks averaged around 100 min. For insulin, power spectra for individual cows universally indicated rhythms with periods of approximately 45 and 80 min; time intervals between peaks averaged approximately 65 min. For urea, almost all spectral energy was confined to the 24-hr rhythm, although there was evidence of a low-amplitude, 60-min rhythm. In conclusion, when animals are acclimated to a rigidly controlled environment and frequent blood sampling is accomplished with minimal intervention, it is possible to detect rhythms inherent in the regulation of metabolic variables.

Quantitative Densitometry in Situ of Lipids Separated by thin Layer Chromatography

Abstract Operating parameters are described for a densitometric method to determine in situ eight lipid classes separated by thin layer chromatography. The separated lipids, visualized on the TLC plate by a cupric acetate-phosphoric acid charring method, were quantitatively determined by spectrodensitometry using the Shimadzu CS-910 Dual Wavelength TLC Scanner. Plates were scanned in either a linear scanning mode or in a zigzag scanning mode (flying spot). Reproducibility of a) sample application (spotting) and b) the lipid separation procedure was determined by scanning. Transmittance measurements yielded response areas that were 2.8 X higher than reflectance measurements. Operating parameters such as scanning direction, wavelength, single and dual-wavelength measurement, scanning speed, and slit geometry were studied. Optimal conditions were established for quantitative densitometry of lipids on thin layer plates.

Lipid Composition of Prepartum Human Mammary Secretion and Postpartum Milk

Changes in lipid composition of mammary secretions of five women were studied at two prepartum periods and compared with composition of colostrum, transitional, and mature human milk. Fat content was ∼1 g/dl during early (-42.0 days before parturition) and late (-9.5 days) prepartum periods and increased to 3–4 g/dl in colostrum (3.0 days post partum), transitional (7.2 days), and mature milk (56.2 days). Most of the lipid present was triglyceride either pre- (93%) or post- (97%) partum. All fat globule core lipids, with the exception of 1,3-diglycerides, increased from prepartum concentrations to levels usually found in milk. Prepartum secretions contained higher amounts of the membrane components, phospholipid (3.2 g/dl), cholesterol (2.3 g/dl), and cholesteryl ester (1.1 g/dl), which declined postpartum to 0.65, 0.37, and 0.09 g/dl, respectively. Thus, the content of core lipids exhibited an opposite pattern to the content of membrane lipids pre- and postpartum. With regard to synthesis of fatty acids, prepartum secretory mechanisms appeared to be very similar to those occurring postpartum since fatty acid composition of prepartum secretions closely resembled that of postpartum milk.

Comparison of the cholesteryl ester composition of human milk from preterm and term mothers.

Milk was obtained on postpartum days 2-3 (colostrum) and days 7, 21, 42, and 84 from mothers of 18 very premature (VPT, 26-30 weeks gestational age), 28 premature (PT, 31-36 weeks), and 6 term (T, 37-40 weeks) infants. Lipids were extracted in chloroform-methanol and analyzed by thin-layer (TLC) and gas liquid chromatography (GLC). Fatty acid composition of cholesteryl esters was determined by GLC after isolation of cholesteryl esters by preparative TLC and preparation of methyl esters of the constituent fatty acids. As lactation progressed, amounts of total cholesterol and cholesteryl esters declined. Cholesteryl esters decreased from about 5 mg/dl in colostrum to 1 mg/dl in mature milk. The cholesteryl esters of colostrum from mothers of premature infants were different in fatty acid composition from those of term infants. Proportions of medium-chain saturated fatty acids (12:0, 14:0, 16:0) of preterm colostrum (VPT and PT) were considerably lower than in term colostrum: 23% of total fatty acids versus 35%. Proportions of 18:3, 20:3, and 20:4 of VPT (5.6%) and PT (6.2%) colostrum were considerably higher than T colostrum (1.8%). The fatty acid composition of cholesteryl esters of VPT, PT, and T milk was relatively similar at all subsequent lactation periods. Fatty acids esterified with cholesterol in weight percents were as follows: 10:0, 0.7; 12:0, 2.6; 14:0, 2.3; 16:0, 11.4; 16:1, 5.0; 18:0, 8.8; 18:1, 32.9; 18:2, 30.6; 18:3, 1.7; 20:3, 0.9; and 20:4, 1.8. Unsaturated fatty acids contributed 73 wt % of fatty acids in cholesteryl esters, which is considerably higher than in milk triglycerides. The greatest difference occurred in 18:2 content, which was 30.6% in cholesteryl esters and only 13.0% of total fatty acids in milk. Results suggest that unsaturated fatty acids are associated preferentially with the cholesteryl ester fraction and that the fatty acid composition of cholesteryl esters differs from the composition of total milk lipid.

Vitamin e, cholesterol, and lipids during atherogenesis in rabbits.

Rabbits were fed diets including cholesterol and 10% butterfat to determine whether polyunsaturated butter (9% 18∶2) would be less atherogenic than normal saturated butter (3% 18∶2) when fed for 12 weeks. The cholesterol diets alone, 0.5% or 2%, produced aortic plaque development, and plasma cholesterol increased 20 times, lipids increased 10 times, and vitamin E increased 5 times. The inclusion of both fat and cholesterol in the diet produced a synergistic effect, doubling these values to 40 times for cholesterol, 20 times for lipids, and 10 times for vitamin E. The higher circulating levels of cholesterol caused increased tissue levels of cholesterol. With 2% cholesterol and fat, liver and aorta cholesterol increased 10 times, heart 4 times, and muscle cholesterol 2 times. The lower 0.5% dietary cholesterol load was successful in limiting the amount of tissue cholesterol increase. Liver, aorta, heart, and muscle levels of cholesterol were only about half the concentration attained when 2% cholesterol was fed. It was concluded that there were no differences in plasma or tissue cholesterol, vitamin E, or atherosclerosis attributable to the polyunsaturated nature of the diet. The 10% butterfat diets alone, whether saturated or unsaturated, did not induce aortic plaques and did not increase blood or tissue cholesterol, lipids, or vitamin E. Our results suggest that the lipid mobilizing effect is mediated by cholesterol, probably by conversion to bile acids and a stimulation in intestinal absorption.

Reduction of blood and liver cholesterol in the rat by straight and branched chain alkyl amines

The activities of a branched chain and several straight chain amines (C12 to C18 chain length), and the azasteroid 25-aza-5α-cholestane were compared with those of 20,25-diazacholesterol dihydrochloride, which is a potent hypocholesterolemic agent in the rat. These amines and azasteroids inhibit the Δ24-sterol reductase system in the tobacco hornworm,Manduca sexta (L.), and also block the conversion of C28 and C29 plant sterols to cholesterol, with a resulting accumulation of desmosterol. The effects of these compounds in the rat were determined on body weight gain, cholesterol, desmosterol, and lipid composition of blood, feces, liver, and epididymal fat pad weight. The two azasteroids and the branched chain amine, N,N-dimethyl-3, 7,11-trimethyldodecanamine, had the greatest effect, reducing total plasma lipids and plasma sterols to approximately 40–50% of the levels in control rats and produced a concomitant increase in plasma and liver desmosterol. The branched chain dodecanamine caused a reduction in both feed consumption and body weight gain. The branched and straight chain dodecanamines also severely reduced epididymal fat pad weight. Our results demonstrate that the simple azasteroid, 25-aza-5α-cholestane, is a more potent inhibitor of cholesterol biosynthesis than the diazasterol and that the Δ24-sterol reductase system in a mammal can be inhibited by simple, nonsteroidal, acyclic amines.

Effect of bovine growth hormone on incorporation of [14C]acetate into lipids by co-cultures of bovine mammary, liver, and adipose tissue explants.

Incorporation of [14C]acetate into lipids was measured in 24 hr co-cultures of mammary, liver and adipose tissue from Holstein cows at 53, 210 and 318 d of lactation in the presence or absence of bovine growth hormone. Little (less than 1%) of the labeled lipids appeared in the media relative to that incorporated into the tissue. In mammary tissue, incorporation of [14C]acetate was highest into triglycerides (16,298 cpm/mg mammary tissue), followed by phospholipids (1,887 cpm), free fatty acids (1,252 cpm), diglycerides (708 cpm), free cholesterol (360 cpm) and monoglycerides (93 cpm). Bovine growth hormone did not increase incorporation of [14C]acetate when mammary or adipose tissue were incubated separately. However, in the presence of liver and adipose tissue, bovine growth hormone significantly increased the incorporation of [14C]acetate into triglycerides, diglycerides, free fatty acids and free cholesterol by mammary tissue. These results suggest that bovine growth hormone acts on mammary tissue indirectly through liver and adipose tissue to increase lipid synthesis. This mechanism may play a role in the action of bovine growth hormone in vivo to increase milk and milk fat production.