Composition of bovine milk lipids

Recent literature on the composition of milk lipids is reviewed and discussed. Many additional exotic fatty acids, mainly branched chain, keto, hydroxy and isomers containing double bonds, have been identified, often with the aid of mass spectrometry. It is estimated that ca. 500 fatty acids have been found in milk lipids. Components of lipid classes have been isolated and their structure determined, e.g., glucosyl and lactosyl ceramide, sphingomyelin, ether lipids, etc. The fatty acid composition of “protected” milk is discussed. This milk, obtained from cows fed polyunsaturated oils encapsulated with formaldehyde-treated casein, contains about five times more linoleic acid than regular milk. The future of research on milk lipids is promising, as there are still many intriguing problems of separation and identification. One of eight papers presented in the symposium “Milk Lipids,” AOCS Meeting, Ottawa, September 1972. Contribution No. 528, Storrs Agricultural Experiment Station, University of Connecticut, Storrs.     

Neutral glycolipids of human and bovine milk

The neutral glycolipids of milk, a small fraction of the total lipids, are of potential biological importance. The simultaneous quantitation of the simple (less than five sugars) glycosphingolipids of human milk samples was achieved by high-pressure liquid chromatography. The samples, representing various stages of lactation, parity of the nursing child, and age of the mother, contained similar glycolipid patterns, but with varying individual glycolipid concentrations. The cerebrosides are major glycosphingolipids of human milk: the non-hydroxylated fatty acid (NFA)-containing species are present at 1.8 μM, and the hydroxylated and/or short-chain fatty acid-containing species (HFA) are present at 1.7 μM; NFA lactosylceramide is present at 931 nM. The cerebrosides appear to be primarily galactosylceramides (galactocerebrosides); glucosylceramides (glucocerebrosides) are a minor component. Globotriaosylceramide (Gb₃) is found at 50 nM and 73 nM for the NFA and HFA species, respectively, while globoside (Gb₄) is found at 45 nM and 46 nM for the NFA and HFA species. Bovine milk glycosphingolipids differ from those of human milk, with bovine milk containing mainly NFA glucosylceramide (8 μM) and NFA lactosylceramide (17 μM); bovine milk contains little Gb₃ or Gb₄. Based on a paper presented at the Symposium on Milk Lipids held at the AOCS Annual Meeting, Baltimore, MD, April 1990.     

Structure of bovine milk fat triglycerides

The triglycerides of bovine milk fat globules were isolated and separated into short, medium and long chain lengths by thin-layer chromatography. The molecular weight distribution and the fatty acid composition of the component triglycerides was then separately determined by gas chromatography following argentation-thin-layer and preparative gas chromatography. Some 38 triglyceride types (28% of total), of which there could be up to 6 isomers, were specifically identified and quantitatively estimated. The quantitative estimates for the rest of the milk fat triglycerides were limited to much more complex glyceride groups. The results confirm the earlier claim that butyric and caproic acids occur in milk fat almost exclusively in combination with medium and long chain fatty acids. Presented in part at the AOCS Meeting, Philadelphia, October, 1966.     

The phytanic acid content of the lipids of bovine tissues and milk

In three steers which were given grass silage for six months, the content of phytanic acid (i.e. 3,7,11,15-tetramethylhexadecanoic acid) in plasma lipid increased to about 8% of the total fatty acids, whereas after this time the proportion in the total fatty acids of liver and heart lipids was about 1%, and only 0.1% in those of kidney lipids; the acid was present in trace amounts in adipose-tissue triglycerides and was apparently absent from brain lipids. In eight lactating cows which were given grass silage for about 3 months, the content of phytanic acid in the total long chain fatty acids of milk and of plasma was 0.7% and 13%, respectively. In the plasma lipids of both steers and lactating cows, phytanic acid constituted a substantial proportion of the total fatty acids of the triglycerides and phospholipids; the acid was present in lowest proportion in the cholesteryl esters.     

Vaccenic acid metabolism in the liver of rat and bovine

Hepatic metabolism of vaccenic acid (VA), especially its conversion into CLA, was studied in the bovine (ruminant species that synthesizes CLA) and in the rat (model for nonruminant) by using the in vitro technique of liver explants. Liver tissue samples were collected from fed animals (5 male Wistar rats and 5 Charolais steers) and incubated at 37°C for 17 h under an atmosphere of 95% O₂/5% CO₂ in medium supplemented with 0.75 mM of FA mixture and with 55 μM [1-¹⁴C]VA. VA uptake was about sixfold lower in bovine than in rat liver slices (P<0.01). For both species, VA that was oxidized to partial oxidation products represented about 20% of VA incorporated by cells. The chemical structure of VA was not modified in bovine liver cells, whereas in rat liver cells, 3.2% of VA was converted into 16∶0 and only 0.33% into CLA. The extent of esterification of VA was similar for both animal species (70–80% of incorporated VA). Secretion of VA as part of VLDL particles was very low and similar in rat and bovine liver (around 0.07% of incorporated VA). In conclusion, characteristics of the hepatic metabolism of VA were similar for rat and bovine animals, the liver not being involved in tissue VA conversion into CLA in spite of its high capacity for FA desaturation especially in the rat. This indicates that endogenous synthesis of CLA should take place exclusively in peripheral tissues.     

Efficiency of transfer of polyunsaturated fats into milk

Polyunsaturated milk has been produced by feeding cows safflower oil enclosed in a casein coat protected with formaldehyde (SOC-F) or formaldehyde-treated soybean (SB) preparations. The efficiency of transfer of dietary 18∶2 ranged from 17 to 42% for various lots of SOC-F and was only 2–8% for SB (per cent transfer=18.2 in milk fat per dietary 18∶2×100). The 18∶2 content of the milk fat increased from basal levels of 2–3% of total fatty acids to 35% with certain SOC-F levels and 7% with SB. Major compensatory changes were noted in 14∶0 and 16∶0 fatty acids. Blood cholesterol, triglycerides and nonesterified fatty acids all increased markedly as cows were fed increasing amounts of SOC-F. There was no increase in cholesterol in the milk. Presented at the AOCS Meeting, Los Angeles, April 1972.     

Triglyceride composition of bovine milk fat with elevated levels of linoleic acid

The effect of increasing the linoleic acid (18∶2) content of milk fat on the composition and structure of the triglycerides (TG) was investigated. Protected sunflower seed supplement was added to the diet of a cow grazing on pasture, and the structure and composition of the milk fat compared with the milk fat from its monozygous twin which had been fed a control diet. The relative proportions of TG fractions of high, medium, and low molecular weight in the milk fat with elevated levels of 18∶2 (15.5% 18∶2) were 43.0, 19.5, and 37.5 moles %, respectively, compared with 36.1, 19.7, and 44.2 moles %, respectively, in the milk fat from the cow fed the control diet. Separation of these three TG fractions of each milk fat into TG classes with different levels of unsaturation showed that the milk fat with elevated levels of 18∶2 contained higher proportions of diene, triene, and tetraene TG and correspondingly lower proportions of saturated and, to a lesser extent, monoene TG. The saturated and monoene TG from the two milk fats had similar fatty acid compositions. However, the diene TG of the 18∶2-rich milk fat included high proportions of the combination of 18∶2 with two saturated fatty acids (FA) which are minor constituents of normal milk fats. Likewise, the triene TG reflected the presence of 18∶2 in combination with 18∶1 and a saturated FA.     

Fatty acids of bovine milk glycolipids and phospholipids and their specific distribution in the diacylglycerophospholipids

Milk lipids were fractionated by silicic acid column chromatography and preparative thinlayer chromatography (TLC). Ceramide monohexoside (CMH), ceramide dihexoside (CDH), phosphatidyl ethanolamine (PE), phosphatidyl choline (PC), phosphatidyl serine (PS), and sphingomyelin (Sph) were isolated, and the purity of each was checked by infrared spectroscopy and TLC. The diacylphospholipids were hydrolyzed with phospholipase A and the products separated by TLC. Fatty acid methyl esters were prepared from the various fractions and analyzed by gas chromatography. The glycolipids, CMH and CDH, and Sph contained large amounts of long-chain saturated fatty acids, especially C_22:0, C_23:0, and C_24:0, PE, PS, and PC contained C₁₀-C₂₂ normal and branched-chain saturated fatty acids, and C₁₅-C₂₀ unsaturated fatty acids (mainly monoenes). The distributions of saturated acids between the α′- and β-positions were respectively: PE, 46 and 11%; PS, 65 and 19%; and PC, 72 and 53%. PC was exceptional in that there was 10.8% myristic acid in the β-position and only 5.6% in the α′-position. PE and PS were similar in composition except that in PE oleic acid was evenly distributed, and in PS was largely in the β-position. In general, PC was much more saturated than PE or PS, and there was no overall pattern governing the specific distribution of the fatty acids in the three diacylphospholipids. Comparison with PC from other bovine tissues and from egg lecithin showed that fatty acids are located much less specifically in milk phospholipids than in PC from other sources. Presented at the AOCS Meeting, Houston, Texas, April, 1965.     

Structure of bovine milk fat triglycerides: II. Long chain lengths

The long chain triglycerides of bovine milk fat were isolated by thin layer chromatography, and their chemical structure determined by combined thin layer and gas liquid chromatography, and a stereospecific analysis of a molecular distillate of butteroil of comparable composition. The milk fat fraction (39% of total) contained C₈–C₂₀ fatty acids which were distributed among the glycerides of 40–56 acyl carbon atoms in a manner not unlike that found for the same acids in the short chain triglycerides. Although individual triglycerides were not identified, the specific distribution of the fatty acids could best be accounted for by assuming a common pool of long chain 1,2-diglyceride precursors from which the bulk of both short and long chain triglycerides are synthesized by a stereospecific introduction of C₄–C₁₈ fatty acids in position 3 of sn-glycerol. This hypothesis is compatible with the results of stereospecific analyses of the short and long chain fractions and of the total butteroil. It is supported by the nonrandom distributions demonstrated for the molecular weights of the milk fat triglycerides of different degrees of saturation. Presented in part at the AOCS meeting, Philadelphia, October, 1966.     

玉米赤霉烯酮单克隆抗体的制备及鉴定

采用玉米赤霉烯酮(ZEN)与羧甲基羟胺半盐酸盐在吡啶中反应,生成玉米赤霉烯酮肟(ZENO),用活性酯法将ZENO与牛血清白蛋白(BSA)的氨基相连合成ZEN人工抗原;并进行了紫外吸收鉴定;以合成的BSA偶联物(ZEN-BSA)为免疫抗原,免疫Balb/c小鼠,经细胞融合,采用非竞争/竞争ELISA两步筛选法获得能够稳定分泌抗体的杂交瘤细胞株1C7.抗体1C7灵敏度(IC50)达0.61μg/L,与T-2毒素、呕吐毒素(DON)、黄曲霉毒素(AFT)的交叉反应率均＜0.1％.本研究为研发粮油产品中玉米赤霉烯酮特异性免疫分析技术及产品奠定了重要基础.     

Destruction of zearalenone in contaminated corn

Several chemical and physical treatments were investigated as possible methods for destroying zearalenone in contaminated corn. An ammoniation process which significantly lowers aflatoxin levels had no effect on zearalenone contamination in yellow corn. Also, treatments of propionic acid, acetic acid, hydrochloric acid, sodium bicarbonate and hydrogen peroxide failed to reduce toxin levels. High-temperature treatment (150 C) had no effect on zearalenone. Formaldehyde, in vapor form from paraformaldehyde crystals or in aqueous solutions, destroyed significant quantities of zearalenone in naturally contaminated yellow corn meal and in spiked corn grits and animal feed. Samples treated with aqueous formaldehyde must be dried at 50 C or more to cause effective destruction of zearale-none. Levels as high as 10 ppm zearalenone in animal feed and 8 ppm in ground corn were reduced to less than 0.5 ppm with formal-dehyde. Ammonium hydroxide and formaldehyde partially des-troyed zearalenone in highly contaminated ground corn. Levels as high as 33.5 ppm were reduced to 12 ppm by 3% ammonium hydroxide and to 2.1 ppm by 3.7% formaldehyde. No treatment used in this study significantly reduced zearalenone levels in whole-kernel corn. Presented at the AOCS meeting, San Francisco, April 1979.     

Determination of the conjugated linoleic acid-containing triacylglycerols in New Zealand bovine milk fat

Reversed-phase high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection at 233 nm was used to separate, quantify, and identify the triacylglycerols (TAG) of milk fat that contain conjugated linoleic acid (CLA). The absorbance at 233 nm was substantially due to CLA-TAG (chromatography of some representative TAG devoid of CLA, such as tripalmitin and triolein, showed poor responses at 233 nm, 1/800th that of CLA-TAG). A CLA molar extinction coefficient at 233 nm of 23 360 L mol⁻¹ cm⁻¹ and an HPLC UV response factor were obtained from a commercially available cis-9, trans-11-CLA standard. This molar extinction coefficient was only 86% reported literature values. Summation of all chromatographic peaks absorbing at 233 nm using the corrected response factor gave good agreement with independent determinations of total CLA by gas chromatography and UV spectrophotometry. This agreement allowed quantification of individual CLA-TAG peaks in the HPLC separation of a typical New Zealand bovine milk fat. Three CLA-containing TAG, CLA-dipalmitin, CLA-oleoyl-palmitin and CLA-diolein, were prepared by interesterification of tripalmitin with the respective fatty acid methyl esters and used to assign individual peaks in the reversed-phase chromatography of total milk fat, of which CLA-oleoyl-palmitin was coincident with the largest UV peak. Band fractions from argentation thin-layer chromatography of total milk fat were similarly employed to identify five predominant CLA-TAG groups in total milk fat: CLA-disaturates, CLA-oleoyl-saturates, CLA-vaccenyl-saturates, CLA-vaccenyl-olein, and CLA-diolein.     

Regiospecific analysis of fractions of bovine milk fat triacylglycerols with the same partition number

Bovine milk fat was fractionated using preparative reversed-phase high-performance liquid chromatography. The conditions consisted of two successive linear gradients of acetonitrile and tert-butylmethylether, followed by a final isocratic mixture of the two eluants, leading to triacylglycerols grouped by their partition number (PN). Fractions corresponding to partition numbers 32 to 50 were isolated and analyzed for fatty acid distribution between sn-1,3 and sn-2 positions by Grignard degradation. Results showed that the fatty acid distribution in milk fat triacylglycerols is nonrandom. The distribution of short-chain fatty acids, stearic (predominantly at sn-1,3 position) and palmitic (predominantly sn-2 position), did not change with triacylglycerol size. Medium-chain fatty acids were predominantly located at sn-2 position, but their proportion at this position decreased with triacylglycerol size. Oleic acid distribution was also size-dependent in that it was located in high proportions at sn-2 position in smaller triacylglycerols and vice versa. Results also showed that the sn-2 position was more unsaturated than sn-1,3 position in the PN range from 32 to 40, but it was more saturated in triacylglycerols with higher PN.     

Insights into Comparative Modeling of VHH Domains

In the particular case of the Camelidae family, immunoglobulin proteins have evolved into a unique and more simplified architecture with only heavy chains. The variable domains of these chains, named V_HHs, have a number of Complementary Determining Regions (CDRs) reduced by half, and can function as single domains making them good candidates for molecular tools. 3D structure prediction of these domains is a beneficial and advantageous step to advance their developability as molecular tools. Nonetheless, the conformations of CDRs loops in these domains remain difficult to predict due to their higher conformational diversity. In addition to CDRs loop diversity, our earlier study has established that Framework Regions (FRs) are also not entirely conformationally conserved which establishes a need for more rigorous analyses of these regions that could assist in template selection. In the current study, V_HHs models using different template selection strategies for comparative modeling using Modeller have been extensively assessed. This study analyses the conformational changes in both CDRs and FRs using an original strategy of conformational discretization based on a structural alphabet. Conformational sampling in selected cases is precisely reported. Some interesting outcomes of the structural analyses of models also draw attention towards the distinct difficulty in 3D structure prediction of V_HH domains.     

离子型表面活性剂与牛血清白蛋白作用的比较研究

采用荧光光谱、紫外吸收光谱、动态光散射和Zeta电位法对比研究了十二烷基硫酸钠(SDS)和十二烷基三甲基溴化铵(DTAB)与牛血清白蛋白(BSA)的相互作用机理。结果表明：SDS和DTAB均能猝灭BSA内源荧光,298 K时的猝灭常数Ksv分别为2.64×104和304.21 L/mol。同步荧光光谱和三维荧光光谱显示SDS和DTAB对BSA的构象产生了影响。SDS对BSA荧光猝灭机理为动静联合猝灭机制;热力学计算表明,SDS与BSA的结合过程中,静电力起主导作用,并且能与BSA形成SDS/BSA超分子复合物;DTAB对BSA荧光猝灭机理为动态猝灭,作用力主要是疏水作用。SDS和DTAB与BSA的平均结合距离分别为2.77和4.73 nm。综合结合常数、粒径和Zeta电位等变化,在相同条件下具有较大电荷密度和较小体积极性头基的SDS与BSA具有更强的结合作用。     

Comparative molecular dynamics simulation studies of salmon and bovine trypsins in aqueous solution

The flexibility and conformational behaviour of salmon and bovinetrypsins were modelled with a 300 ps molecular dynamics simulationin aqueous solution. Trajectories from both trypsins were analysedto eventually detect differences in mobility that could explainobserved variations in stability and activity. The simulationswere performed at 300 K with all the acidic groups deprotonatedand the basic groups protonated. The radius of gyration, theoverall r.m.s. deviation from the starting structure as a functionof time, together with the r.m.s. deviation from the startingstructures as a function of residue number, demonstrated thatthe simulations were stable and representative of the X-raystructures of both enzymes. Isotropic Debye-Waller factors werecalculated from the fluctuations for mainchain atoms and werein good agreement with experimental values. The overall dynamicproperties of the two enzymes were similar. Based on the present300 ps molecular dynamics simulation, it cannot be concludedthat either of the two enzymes is more 'flexible' than the other.However, there are clearly differences in mobility on a moredetailed level and for particular regions.     

玉米赤霉烯酮抗独特型单抗的制备及特异性分析

目的制备并鉴定玉米赤霉烯酮(Zearalenone,ZEN)抗独特型单抗Ab2β-1D5。方法将ZEN单抗1G4与载体蛋白KLH偶联作为免疫原,免疫BALB/c小鼠,取免疫小鼠脾细胞,与小鼠骨髓瘤细胞SP2/0融合后,以ZEN单抗Fab片段作为包被抗原,间接ELISA法筛选阳性杂交瘤细胞。经小鼠腹腔注射杂交瘤细胞,制备腹水型单抗,并经Protein G亲和层析柱进行纯化。间接ELISA法检测ZEN抗独特型单抗的抗体效价及特异性;间接竞争ELISA法检测ZEN抗独特型单抗类型、灵敏度及其与ZEN毒素间的相关性。结果共获得6株稳定分泌ZEN抗独特型单抗的杂交瘤细胞株,腹水型抗体效价为1∶1.2×105～1∶2.0×105;6株单抗均为β型抗独特型抗体(Ab2β),其中Ab2β-1D5抗体灵敏度最高,对ZEN的IC50值达10.09 ng/ml,其与ZEN毒素呈线性相关(r=0.990);ZEN抗独特型单抗与莱克多巴胺、重金属铅、铬及虾过敏原单抗均无交叉反应,特异性良好。结论已成功制备玉米赤霉烯酮抗独特型单抗,该抗体与ZEN间存在"内影像"关系,可以替代ZEN毒素标准品,用于建立ZEN的无毒免疫学检测技术。     

Comparative study of the molecular species of chloropropanediol diesters and triacylglycerols in milk fat

In an effort to establish the origin of the fatty acid esters of 3-chloropropanediol, which recently have been isolated in small amounts from goat milk, we compared the molecular species composition of the chlorohydrin diesters and of goat milk triacylglycerols. The chloropropanediol diesters were found to be composed of molecular species containing C₁₀−C₁₈ fatty acids and corresponded closely in carbon number to those calculated for the long chain sn-1,2-diacyl-glycerol moieties of goat milk triacylglycerols. The molecular species of goat milk total triacylglycerols contained C₄−C₁₈ fatty acids. It is suggested that triacylglycerols and chloropropanediol diesters are derived from the same pool of long chain fatty acids. A molecular distillate of bovine milk fat did not contain chloropropanediol diesters, while the available samples of human milk fat were shown to contain alkyldiacylglycerols as the major components of a neutral lipid fraction corresponding in polarity to the chloropropanediol diesters.     

Influence of elevated levels of linoleic acid on the thermal properties of bovine milk fat

The thermal properties of bovine milk fat containing 15.5% linoleic acid have been compared with those of milk fat containing a normal level (1.8%) of linoleic acid in order to examine the influence of altered triglyceride (TG) composition on their physical characteristics. The total TGs of 18∶2-rich milk fat melted over the range −38 to 30 C compared with the range −33 to 34 C for control milk fat. Polymorphism exhibited by the high mol wt TGs of control milk fat was absent in the same fraction of 18∶2-rich milk fat. Similarly, the complex melting thermogram of the low mol wt TGs of control milk fat and its obvious polymorphic behavior contrasted with the single broad melting peak of the low mol wt TGs of 18∶2-rich milk fat. This solid miscibility in the 18∶2-rich milk fat could be a consequence of the lower proportion of saturated TGs or the presence of high proportions of diene and triene TGs containing 18∶2 instead of monoene and diene TGs containing 18∶1.