Variation in the human lipidome associated with coffee consumption as revealed by quantitative targeted metabolomics

The effect of coffee consumption on human health is still discussed controversially. Here, we report results from a metabolomics study of coffee consumption, where we measured 363 metabolites in blood serum of 284 male participants of the Cooperative Health Research in the Region of Augsburg study population, aged between 55 and 79 years. A statistical analysis of the association of metabolite concentrations and the number of cups of coffee consumed per day showed that coffee intake is positively associated with two classes of sphingomyelins, one containing a hydroxy‐group (SM(OH)) and the other having an additional carboxy‐group (SM(OH,COOH)). In contrast, long‐ and medium‐chain acylcarnitines were found to decrease with increasing coffee consumption. It is noteworthy that the concentration of total cholesterol also rises with an increased coffee intake in this study group. The association observed here between these hydroxylated and carboxylated sphingolipid species and coffee intake may be induced by changes in the cholesterol levels. Alternatively, these molecules may act as scavengers of oxidative species, which decrease with higher coffee intake. In summary, we demonstrate strong positive associations between coffee consumption and two classes of sphingomyelins and a negative association between coffee consumption and long‐ and medium‐chain acylcarnitines.     

Lipid rafts in the bovine milk fat globule membrane revealed by the lateral segregation of phospholipids and heterogeneous distribution of glycoproteins

This study reveals the lateral organisation of the milk fat globule membrane (MFGM). Using confocal laser scanning microscopy (CLSM) and a lipid soluble molecule, an exogenous phospholipid and two lectins as fluorescent probes we located triacylglycerols in the core of fat globules and investigated the organisation of the polar lipids and glycoproteins of the MFGM, in situ in milk. Lipid rafts corresponding to the lateral segregation of sphingolipids in liquid-ordered phases surrounded by liquid-disordered domains composed by the glycerophospholipids were observed in the MFGM. These lipid rafts which correspond to rigid sphingolipid-rich domains have a circular shape at room temperature. CLSM experiments revealed that glycoproteins and glycolipids are heterogeneously distributed around fat globules and that they are not located in the lipid rafts. The characterisations performed by in depth thin sectioning of fat globules and in dynamic as a function of time revealed chemical and structural heterogeneities in the MFGM. Schematic 3D and 2D representations of the MFGM are proposed and discussed. The physiological and nutritional consequences of the lateral organisation of polar lipids and glycoproteins in the MFGM are discussed but remain to be elucidated.     

The potential of sphingomyelin as a chemopreventive agent in AOM-induced colon cancer model: wild-type and p53+/- mice

A protective effect of sphingolipids on colorectal cancer (CRC) has been reported in certain mouse strains. It is unknown if sphingolipids are protective in a p53 deficiency mouse model of CRC. This study investigated the effect of sphingomyelin (SM) on intestinal sphingomyelinase (SMase) activity, colonic epithelial biology and azoxymethane (AOM)-induced CRC. Groups of wild-type (C57BL/6J) and p53+/- mice were fed 0.1% SM diet for 4 wk, administered a single AOM injection and then killed 6 h later to measure apoptosis and proliferation. Separately, both mouse types were fed 0.05% SM diet, administered three AOM injections and killed 33-38 wk later to measure tumour formation. SM significantly increased SMase activity and reduced proliferation (p < 0.05) in wild-type and p53+/- mice. SM did not regulate baseline apoptosis, apoptotic response to AOM or apoptosis in tumours, nor did it restore defective apoptosis in p53+/- mice. There was a nonsignificant trend to reduced tumour incidence with SM in wild-type (p = 0.15) and p53+/- (p = 0.12) mice. In conclusion, while increasing intestinal SMase activity and suppressing proliferation, SM did not promote any form of apoptosis and failed to achieve significant protection in these mice. Further investigation to understand the variable effect of SM in preventing CRC is warranted.     

The functional role of sphingomyelin in cell membranes

Sphingomyelins (SM) constitute an important class of phospholipids in the membranes of most eukaryotic cells. In mammalian tissues, SM usually constitute 2–15% of the total organ phospholipid, but certain tissues such as brain, peripheral nervous tissue and ocular lenses have even higher SM contents. Typical properties of SM include their low degree of unsaturation, an asymmetric molecular structure, and their extensive hydrogen‐bonding properties. These features are all very important for the structural role of SM in biological membranes. SM interact favorably with cholesterol (and other sterols) and there is an established co‐localization of SM and cholesterol in the plasma membranes of cells and at the surface of lipoprotein particles. Together they form SM/sterol‐rich domains that often are more ordered than the surrounding phase in biological membranes. The growing body of evidence regarding their favorable interaction with sterols indicates that the functional role of SM per se is largely related to being a regulator of cholesterol distribution within cellular membranes and cholesterol homeostasis in cells. Together with other sphingolipids, SM also have an important functional role as precursors of sphingolipid signaling molecules, extensively reviewed elsewhere and not to be discussed here in more detail.     

Sphingomyelinase C from Streptomyces sp. A9107: Unusual primary structure for bacterial sphingomyelinase C

A sphingomyelinase C (SMase) was identified in the culture supernatant of Streptomyces sp. A9107 (S-SMase). Although S-SMase seems to be a typical bacterial SMase, the primary structure of S-SMase was unusual for known bacterial SMase. The gene was functionally overexpressed in the culture medium of recombinant Rhodococcus erythropolis.     

Decreased Serum Levels of Sphingomyelins and Ceramides in Sickle Cell Disease Patients

Limited data are available on the serum levels of different sphingomyelin (CerPCho) and ceramide (CER) species in sickle‐cell disease (SCD). This study was aimed at identifying the levels of C16–C24 CerPCho and C16–C24 CER in serum obtained from SCD patients and controls. Circulating levels of neutral sphingomyelinase (N‐SMase) activity, ceramide‐1‐phosphate (C1P), and sphingosine‐1‐phosphate (S1P) were also determined. Blood was collected from 35 hemoglobin (Hb)A volunteers and 45 homozygous HbSS patients. Serum levels of C16–C24 CerPCho and C16–C24 CER were determined by an optimized multiple reaction monitoring (MRM) method using ultrafast liquid chromatography (UFLC) coupled with tandem mass spectrometry (MS/MS). Serum activity of N‐SMase was assayed by standard kit methods, and C1P and S1P levels were determined by enzyme‐linked immunosorbent assay. A significant decrease was observed in the serum levels of C18–C24 CerPCho in patients with SCD compared to controls. No significant difference was found in C16 CerPCho levels between the two groups. Very‐long‐chain C22–C24 CER were significantly decreased in SCD, while long‐chain C16–C20 CER levels showed no significant difference between SCD patients and controls. Significant positive correlation was found between the serum total cholesterol levels and C18–C24 CerPCho and C22–C24 CER in SCD patients. Patients with SCD had significantly elevated serum activity of N‐SMase as well as increased circulating levels of C1P and S1P compared to controls. The decrease in serum levels of C18–C24 CerPCho in patients with SCD was accompanied by decreased levels of C22–C24 CER. Future studies are needed to understand the role of decreased CerPCho and CER in the pathophysiology of SCD.     

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.     

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.