Quantification of Phospholipids Classes in Human Milk

Phospholipids are integral constituents of the milk fat globule membranes and they play a central role in infants’ immune and inflammatory responses. A methodology employing liquid chromatography coupled with evaporative light scattering detector has been optimized and validated to quantify the major phospholipids classes in human milk. Phospholipids were extracted using chloroform and methanol and separated on C18 column. Repeatability, intermediate reproducibility, and recovery values were calculated and a large sample set of human milk analyzed. In human milk, phospholipid classes were quantified at concentrations of 0.6 mg/100 g for phosphatidylinositol; 4.2 mg/100 g for phosphatidylethanolamine, 0.4 mg/100 g for phosphatidylserine, 2.8 mg/100 g for phosphatidylcholine, and 4.6 mg/100 g for sphingomyelin. Their relative standard deviation of repeatability and intermediate reproducibility values ranging between 0.8 and 13.4 % and between 2.4 and 25.7 %, respectively. The recovery values ranged between 67 and 112 %. Finally, the validated method was used to quantify phospholipid classes in human milk collected from 50 volunteers 4 weeks postpartum providing absolute content of these lipids in a relatively large cohort. The average content of total phospholipids was 23.8 mg/100 g that corresponds to an estimated mean intake of 140 mg phospholipids/day in a 4-week old infant when exclusively breast-fed.     

Analysis of Fluorescent Ceramide and Sphingomyelin Analogs: A Novel Approach for in Vivo Monitoring of Sphingomyelin Synthase Activity

A novel sensitive high‐performance liquid chromatography‐fluorescence detection (HPLC‐FLD) method was developed for real‐time monitoring of relative sphingomyelin synthase (SMS) activity based on the measurement of a fluorescent ceramide (Cer) analog and its metabolite, a fluorescent sphingomyelin (CerP Cho) analog, in plasma. Analyses were conducted using HPLC‐FLD following a protein precipitation procedure. The chromatographic separations were carried out on an Agilent C18 RP column (150 × 4.6 mm, 5 μm) based on a methanol—0.1 % trifluoroacetic acid aqueous solution (88:12, by vol) elution at a flow‐rate of 1 mL/min. The limit of quantification in plasma was 0.05 μM for both the fluorescent Cer analog and its metabolite. Significant differences in the fluorescent Cer analog and its metabolite concentration ratio at 5 min were found between vehicle control group and three D2 (a novel SMS inhibitor) dose groups (P < 0.05). Dose‐dependent effects (D2 doses: 0, 2.5, 5, 10 mg/kg) were observed. Our method could be used to detect relative SMS activity in biochemical assays and to screen potential SMS inhibitors in vivo. D2 was found to be a potent SMS inhibitor in vivo, and may have a potential antiatherosclerotic effect, which is under further study. D609 was also selected as another model SMS inhibitor to validate our newly developed method.     

Phospholipidomic Analysis Reveals Changes in Sphingomyelin and Lysophosphatidylcholine Profiles in Plasma from Patients with Neuroborreliosis

In recent years, the number of patients suffering from Lyme Disease (LD) has significantly increased. The most dangerous manifestation of LD is neuroborreliosis associated with invasion of the central nervous system by Borrelia burgdorferi. Phospholipids (PL) and their metabolites are involved in inflammation, which plays a dominant, but still unclear, role in the pathogenesis of neuroborreliosis. We analyzed the plasma PL profiles of neuroborreliosis patients (n = 8) and healthy volunteers (n = 8) using a lipidomic approach. Significant increases in the lysophosphatidylcholines LysoPtdCho 16:0 and LysoPtdCho 18:2 were observed. The plasma of neuroborreliosis patients appeared to have an increased relative abundance of sphingomyelin CerPCho d18:1/24:1 and a decrease in CerPCho d18:0/18:0. Principal components analysis of the relative abundances of all PL class species distinguished between neuroborreliosis patients and healthy subjects. This is the first report comparing PL classes and their molecular species in neuroborreliosis patients and healthy subjects.     

Analysis of Molecular Species Profiles of Ceramide-1-phosphate and Sphingomyelin Using MALDI-TOF Mass Spectrometry

Ceramide‐1‐phosphate (C1P) is a potential signaling molecule that modulates various cellular functions in animals. It has been known that C1P with different N‐acyl lengths induce biological responses differently. However, molecular species profiles of the C1P in animal tissues have not been extensively examined yet. Here, we developed a method for determination of the molecular species of a C1P using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry with Phos‐tag, a phosphate capture molecule. The amounts of total C1P in skin, brain, liver, kidney and small intestine of mice were determined to be 344, 151, 198, 96 and 90 pmol/g wet weight, respectively. We found a C1P species having an α‐hydroxypalmitoyl residue (h‐C1P, 44 pmol/g wet weight) in mouse skin. The h‐C1P was detected only in the skin, and not other tissues of mice. The same analysis was applied to sphingomyelin after conversion of sphingomyelin to C1P by Streptomyces chromofuscus phospholipase D. We found that molecular species profiles of sphingomyelin in skin, kidney and small intestine of mice were similar to those of C1P in corresponding tissues. In contrast, molecular species profiles of sphingomyelin in liver and brain were quite different from those of C1P in these tissues, indicating selective synthesis or degradation of C1P in these tissues. The method described here will be useful for detection of changes in molecular species profiles of C1P and sphingomyelin.     

Inhibition of Endothelial Lipase Activity by Sphingomyelin in the Lipoproteins

Endothelial lipase (EL) is a major determinant of plasma HDL concentration, its activity being inversely proportional to HDL levels. Although it is known that it preferentially acts on HDL compared to LDL and VLDL, the basis for this specificity is not known. Here we tested the hypothesis that sphingomyelin, a major phospholipid in lipoproteins is a physiological inhibitor of EL, and that the preference of the enzyme for HDL may be due to low sphingomyelin/phosphatidylcholine (PtdCho) ratio in HDL, compared to other lipoproteins. Using recombinant human EL, we showed that sphingomyelin inhibits the hydrolysis of PtdCho in the liposomes in a concentration‐dependent manner. While the enzyme showed lower hydrolysis of LDL PtdCho, compared to HDL PtdCho, this difference disappeared after the degradation of lipoprotein sphingomyelin by bacterial sphingomyelinase. Analysis of molecular species of PtdCho hydrolyzed by EL in the lipoproteins showed that the enzyme preferentially hydrolyzed PtdCho containing polyunsaturated fatty acids (PUFA) such as 22:6, 20:5, 20:4 at the sn‐2 position, generating the corresponding PUFA‐lyso PtdCho. This specificity for PUFA‐PtdCho species was not observed after depletion of sphingomyelin by sphingomyelinase. These results show that sphingomyelin not only plays a role in regulating EL activity, but also influences its specificity towards PtdCho species.     

Decreases in Phospholipids Containing Adrenic and Arachidonic Acids Occur in the Human Hippocampus over the Adult Lifespan

One of the biggest risk factors for developing Alzheimer's disease is advanced age. Despite several studies examining changes to phospholipids in the hippocampus during the pathogenesis of Alzheimer's disease, little is known regarding changes to phospholipids in this region during normal adult aging. This study examined the phospholipid composition of the mitochondrial and microsomal membranes of the human hippocampus from post‐mortem tissue of neurologically normal subjects aged between 18 and 104 years. Many of the age‐related changes found were in low‐to‐moderately abundant phospholipids in both membrane fractions, with decreases with age being seen in many phospholipids containing either adrenic or arachidonic acid. The most abundant phospholipid of this type was phosphatidylethanolamine 18:0_22:4, which decreased in both the mitochondrial and microsomal membranes by approximately 20 % from ages 20 to 100. Subsequent decreases with age were seen in total adrenic and arachidonic acid in the phospholipids of both membrane fractions, but not in either fatty acid specifically within the phosphatidylethanolamine class. Increases with age were seen in the hippocampus for mitochondrial phosphatidylserine 18:0_22:6. This is the first report of changes to molecular phospholipids of the human hippocampus over the adult lifespan, with this study also providing a comprehensive profile of the phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine phospholipids of the human hippocampus.     

Apoptotic Effects of Dietary and Synthetic Sphingolipids in Androgen-Independent (PC-3) Prostate Cancer Cells

Stress-induced activation and metabolism of plasma membrane sphingolipids results in intracellular ceramide accumulation and has been shown to induce apoptosis in human prostate cancer cells. This effect has been observed using synthetic ceramide analogs, such as C6-ceramide; however, the effects of naturally-occurring sphingolipids, such as C18-ceramide and sphingomyelin (CerPCho), on apoptosis and prostate cancer cell proliferation have not been examined. The results of the present study demonstrate that natural (CerPCho, C18-ceramide) and synthetic (C6-ceramide) sphingolipids reduced PC-3 cell proliferation by 15 ± 1.8, 17 ± 2.5, and 46 ± 2.1%, respectively (P < 0.05). These reductions in proliferation were due, in part, to increased cellular apoptosis. Treatment of PC-3 cells with CerPCho and C18-ceramide significantly increased apoptosis by 3.0 ± 0.8 and 3.6 ± 0.6%, respectively, compared to the untreated control, while the synthetic C6-ceramide significantly increased apoptosis by 55.7 ± 0.4%. C6-ceramide-induced apoptosis was associated with cell cycle arrest in the G₂/M phase, decreased extracellular signal-regulated kinase (ERK1/2) signaling and activation of the cell cycle regulatory protein, retinoblastoma (pRb). Treatment of PC-3 cells with C18-ceramide and CerPCho did not alter cell cycle distribution, pRb or ERK1/2 activation. Taken together, these results suggest that natural and synthetic sphingolipids induce apoptosis in PC-3 cells via distinct signaling mechanisms and potencies.     

Comprehensive Quantitation Using Two Stable Isotopically Labeled Species and Direct Detection of <Emphasis Type="Italic">N</Emphasis>-Acyl Moiety of Sphingomyelin

Sphingomyelin (ceramide‐phosphocholine, CerPCho) is a common sphingolipid in mammalian cells and is composed of phosphorylcholine and ceramide as polar and hydrophobic components, respectively. In this study, a qualitative liquid chromatography‐electrospray ionization tandem mass spectrometry (LC–ESI–MS/MS/MS) analysis is proposed in which CerPCho structures were assigned based on product ion spectra corresponding to sphingosylphosphorylcholine and N‐acyl moieties. From MS/MS/MS analysis of CerPCho, we observed product ion spectra of the N‐acyl fatty acids as [RCO₂]^? ions as well as sphingosylphosphorylcholine. A calibration curve for CerPCho was constructed using two stable isotopically labeled CerPCho species and then used to quantify the CerPCho species in HeLa cells as a proof‐of‐principle study. The present study proposes an accurate method for quantifying and assigning structures to each CerPCho species in crude biologic samples by LC–ESI–MS/MS/MS analysis.     

Canola Proteins Used as Co‐Emulsifiers with Phospholipids Influence Oil Oxidability,Enzymatic Lipolysis,and Fatty Acid Absorption in Rats

The objective of this study is to formulate and characterize oil‐in‐water emulsions with plant‐derived ingredients only, that is, proteins extracted from canola oil bodies, used as co‐emulsifiers with a canola lecithin, and to assess their suitability for food applications. Using the protein extract increases the chemical stability of rapeseed oil emulsions toward oxidation, based on the delay in conjugated diene formation under accelerated storage conditions, and favors pancreatic lipase activity. Bioaccessibility of rapeseed fatty acids is compared in lymph‐duct‐cannulated rats fed oil or emulsion. Fatty acid absorption by the intestine is increased by 78% when the oil is emulsified with canola proteins as co‐emulsifier: 28.7 mg mL^?1 versus 16.1 mg mL^?1 for oil (p < 0.05). In vitro lipolysis results are in overall agreement with fatty acid absorption in vivo. Practical Applications: Results obtained for rapeseed oil emulsified with canola proteins and phospholipids suggest that increased bioaccessibility of n‐3 polyunsaturated fatty acids could be offered in vegan food products.     

Maternal Dietary Fat Affects Milk Fatty Acid Profile and Impacts on Weight Gain and Thermogenic Capacity of Suckling Rats

We aimed to assess the effects of maternal supplementation with the main fat sources used in the human Western diet (olive oil, butter, margarine) on milk FA composition and on plasma FA profile of offspring, and to determine whether it may influence body-weight-gain (BWG) and adiposity of offspring during the suckling period. Wistar rats were supplemented with the different fat sources from day 14 of gestation and throughout lactation. Olive oil-supplemented dams showed the highest proportion of oleic-acid in milk, with no changes in plasma. Their offspring also showed the highest proportion of this FA in plasma, lower BWG during the suckling period, and higher levels of UCP1 in brown adipose tissue (BAT) at weaning. Margarine-supplemented dams showed the highest percentage of PUFA in milk, and a similar tendency was found in plasma of their offspring. Butter-supplemented dams displayed higher proportion of saturated FA (SFA) in milk compared to other fat-supplemented dams, but lower than controls. Control offspring also showed higher proportion of SFA in plasma and greater BWG during the suckling period than fat-supplemented groups. Significant correlations were found between the relative content of some milk FA and BWG of offspring, in particular, oleic-acid levels correlated negatively with BWG and positively with UCP1 levels. These results show that maternal dietary source of fat affects milk FA composition and circulating FA profile, as could be expected, but also BWG and thermogenic capacity of offspring during the suckling period. An effect of oleic-acid stimulating BAT thermogenic capacity of suckling pups is proposed.     

Temporal Changes of Phospholipids Fatty Acids and Cholesterol in Breast Milk and Relationship with Diet

Phospholipids (PLs) and cholesterol in human milk (HM) are affected by lactation, and differential lipids are closely related to maternal diet. The contents of PLs and cholesterol in Chinese HM are quantified by gas chromatography and high performance liquid chromatography, respectively, and the relationship between differential lipids and the maternal diet is obtained by Pearson. The result shows that SFA, MUFA, and polyunsaturated fatty acid (PUFA) are not affected by lactation and geography for total fatty acids, but almost all sn‐2 fatty acids are influenced by geography and remain unchanged during lactation. Most SFAs show absolute sn‐2 selectivity and the majority of MUFAs and PUFAs are esterified at the sn‐1 position. Cholesterol (13.8–22.6 mg per 100 g milk) and 25‐hydroxycholesterol (0.45–1.01 mg per 100 g milk) increase significantly and remain constant during lactation, respectively, and they are affected by regions. In addition, the differential lipids (22:1n‐9, C9:0, trans‐PUFA, 22:4n‐6, etc.) of PLs are closely related to the maternal diet. PLs and cholesterol content differ from western research and infant formula, which will help to design an infant formula that is more suitable for Chinese babies in the future. Practical Application: Compared with PLs and cholesterol in western countries and infant formula, the specificity of Chinese HM can more accurately target the development of formulas suitable for the growth of Chinese infants. At the same time, according to the influence of the mother?s diet on the composition of HM, it is more reasonable to guide the diet of the mother.     

Phospholipid,Oleic Acid Micelles and Dietary Olive Oil Influence the Lutein Absorption and Activity of Antioxidant Enzymes in Rats

This study reports on the results of repeated gavages and dietary feeding of lutein dispersed either in phospholipids or fatty acid micelles or vegetable oils and the effects on lutein bioavailability and antioxidant enzymes in rats. For the gavage study, rats (n = 5/group) were intubated with lutein solubilized either in oleic acid (OLA, 18:1n-9) or linoleic acid (LNA, 18:2n-6) or phosphatidylcholine (PC) or lysophosphatidylcholine (LPC) or no phospholipid (NoPL) micelles for 10 days. For the dietary study, rats (n = 5/group) were fed a diet containing fenugreek leaf (lutein source), either with olive (OO) or sunflower (SFO) or groundnut (GNO, control) oil or l-α-lecithin (PL) for 4 weeks. The gavage study showed that the plasma, liver and eye lutein levels in OLA and LPC groups were higher by 23.9, 20.8 and 25.5% and 16.1, 28.5 and 14.0% than LNA and PC groups, respectively. The dietary study showed the plasma (35.0 and 43.5%) and eye (18.5 and 37.0%) lutein levels in OO were higher than SFO and GNO groups. The plasma and eye lutein levels in the PL group were higher by 20 and 31.3% than in the control. It is evident that OO and PL modulate lutein absorption, which in turn modulates antioxidant enzymes and fatty acids in plasma and tissues compared to SFO. Hence, selection of the fat source may be vital to enhancing the lutein bioavailability.     

Profiling and Imaging of Phospholipids in Brains of Abcd1‐Deficient Mice

ABCD1 is a gene responsible for X‐linked adrenoleukodystrophy (X‐ALD), and is critical for the transport of very long‐chain fatty acids (VLCFA) into peroxisomes and subsequent β‐oxidation. VLCFA‐containing lipids accumulate in X‐ALD patients, although the effect of ABCD1‐deficiency on each lipid species in the central nervous system has not been fully characterized. In this study, each phospholipid and lysophospholipid species in Abcd1‐deficient mice brains were profiled by liquid chromatography‐mass spectrometry. Among the phospholipid and lysophospholipid species that are significantly more enriched in Abcd1‐deficient mice brains, VLCFA were present in 75, 15, 5, 4, and 1 species of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, lysophosphatidylcholine, and lysophosphatidylethanolamine, respectively. Most VLCFA were incorporated at the sn‐1 position of phosphatidylcholine and phosphatidylethanolamine. Among the phospholipid species that are significantly less enriched in Abcd1‐deficient mice brains, odd‐numbered saturated or mono‐unsaturated fatty acyl moieties are contained in all phosphatidylcholine species. In addition, a number of phosphatidylglycerol, phosphatidylinositol, and phosphatidylserine species contained highly unsaturated fatty acyl moieties. Intriguingly, 44:1 phosphatidylcholine with VLCFA was mainly distributed in the gray matter, such as the cortex, but not in the white matter in the cerebrum and cerebellum. These results show that ABCD1‐deficiency causes metabolic alternation of long‐chain fatty acids and VLCFA. Moreover, our results imply a molecular mechanism for the incorporation of saturated or monounsaturated VLCFA into the sn‐1 position of phospholipids, and also indicate that the distribution of phospholipids with VLCFA may correlate with the development of X‐ALD.     

Lymphatic Absorption and Deposition of Various Plant Sterols in Stroke-Prone Spontaneously Hypertensive Rats,a Strain Having a Mutation in ATP binding cassette transporter G5

ATP binding cassette transporter G5 (ABCG5) and ATP binding cassette transporter G8 (ABCG8) have been suggested to transport absorbed plant sterols and cholesterol from enterocytes to the intestinal lumen and from hepatocytes to bile. It has been thought that mutations of ABCG5 or ABCG8 cause the deposition of plant sterols in the body. In the present study, lymphatic absorption of various plant sterols and their deposition in various tissues was investigated in stroke-prone spontaneously hypertensive rats (SHRSP), having a mutation in Abcg5 and depositing plant sterols in the body. The order of lymphatic 24-h recovery of plant sterols was as follows: campesterol > sitosterol > brassicasterol > stigmasterol = sitostanol. When SHRSP were fed a diet containing one of the plant sterols, the depositions of campesterol and sitosterol were comparatively higher than those of brassicasterol, stigmasterol and sitostanol. Highly positive correlations were obtained between lymphatic recovery of plant sterols and their levels in plasma, liver, adipose tissue and heart. The tendency of differential absorption of plant sterols to the lymph in SHRSP was similar to that in normal Wistar rats previously reported by us (Hamada et al. Lipids 41:551–556, 2006). These observations suggest that differential absorption of various plant sterols is kept in SHRSP in spite of a mutation in Abcg5.     

规整填料在化肥工业吸收塔上的应用

介绍规整料与新型分布器的特性，及其在中、小型化肥厂的脱硫、脱碳、铜洗等工艺中应用的生产数据和成功范例。     

原子吸收法测定石灰石中的元素

文章详细介绍了用原子吸收光谱法测定石灰石中的Ca、Mg、Fe、K、Na、Mn的测定方法和注意事项,并用石灰岩国家标准物质进行验证,结果表明,该方法简便、快速、准确。     

Dietary Crude Lecithin Increases Systemic Availability of Dietary Docosahexaenoic Acid with Combined Intake in Rats

Crude lecithin, a mixture of mainly phospholipids, potentially helps to increase the systemic availability of dietary omega‐3 polyunsaturated fatty acids (n‐3 PUFA), such as docosahexaenoic acid (DHA). Nevertheless, no clear data exist on the effects of prolonged combined dietary supplementation of DHA and lecithin on RBC and plasma PUFA levels. In the current experiments, levels of DHA and choline, two dietary ingredients that enhance neuronal membrane formation and function, were determined in plasma and red blood cells (RBC) from rats after dietary supplementation of DHA‐containing oils with and without concomitant dietary supplementation of crude lecithin for 2–3 weeks. The aim was to provide experimental evidence for the hypothesized additive effects of dietary lecithin (not containing any DHA) on top of dietary DHA on PUFA levels in plasma and RBC. Dietary supplementation of DHA‐containing oils, either as vegetable algae oil or as fish oil, increased DHA, eicosapentaenoic acid (EPA), and total n‐3 PUFA, and decreased total omega‐6 PUFA levels in plasma and RBC, while dietary lecithin supplementation alone did not affect these levels. However, combined dietary supplementation of DHA and lecithin increased the changes induced by DHA supplementation alone. Animals receiving a lecithin‐containing diet also had a higher plasma free choline concentration as compared to controls. In conclusion, dietary DHA‐containing oils and crude lecithin have synergistic effects on increasing plasma and RBC n‐3 PUFA levels, including DHA and EPA. By increasing the systemic availability of dietary DHA, dietary lecithin may increase the efficacy of DHA supplementation when their intake is combined.     

H2O2吸收SO2的降膜传质系数模型的建立

为中小型燃煤锅炉烟气脱硫提出一种以列管式并流降膜塔为脱硫反应器,H2O2溶液为脱硫剂的简便高效、无二次污染的脱硫技术.建立了气、液相传质系数模型,通过实验数据回归了降膜管传质关联式.对气、液相传质系数模型进行验证,结果显示,得出的计算结果与实验数据基本相符,这表明建立脱硫模型对基本设计参数的确定具有一定的指导意义.