Sin Man Lam

Extensive characterization of human tear fluid collected using different techniques unravels the presence of novel lipid amphiphiles

The tear film covers the anterior eye and the precise balance of its various constituting components is critical for maintaining ocular health. The composition of the tear film amphiphilic lipid sublayer, in particular, has largely remained a matter of contention due to the limiting concentrations of these lipid amphiphiles in tears that render their detection and accurate quantitation tedious. Using systematic and sensitive lipidomic approaches, we validated different tear collection techniques and report the most comprehensive human tear lipidome to date; comprising more than 600 lipid species from 17 major lipid classes. Our study confers novel insights to the compositional details of the existent tear film model, in particular the disputable amphiphilic lipid sublayer constituents, by demonstrating the presence of cholesteryl sulfate, O-acyl-ω-hydroxyfatty acids, and various sphingolipids and phospholipids in tears. The discovery and quantitation of the relative abundance of various tear lipid amphiphiles reported herein are expected to have a profound impact on the current understanding of the existent human tear film model.

Meibum Lipid Composition in Asians with Dry Eye Disease

Background Previous lipidomic analyses of the human meibum had largely focused on individuals from non-Asian populations, despite the higher prevalence of dysfunctional tear syndrome (DTS) observed across Asia. Information pertaining to the alterations in lipid profiles in relation to DTS onset and progression is also lacking and warrants comprehensive experimental analysis. Methodologies/Principal Findings We examined the meibum lipidome of 27 DTS patients and 10 control subjects for a total of 256 lipid species from 12 major lipid classes, including cholesteryl ester (CE), wax ester (WE), triacylglyceride (TAG), (O-acyl)-ω-hydroxy fatty acid (OAHFA), glycerophospholipids (phosphatidylcholine, PC; phosphatidylethanolamine, PE; phosphatidylinositol, PI; phosphatidylglycerol, PG) and sphingolipids (sphingomyelin, SM; ceramide, Cer; glucosylceramide, GluCer; dihexosylceramide, DihexCer). Neutral lipids were analysed using high-performance liquid-chromatography coupled with mass spectrometry (HPLC/MS) and tandem mass spectrometry (MS/MS) was used for the qualitative and quantitative analysis of polar lipid species. DTS patients were classified into three severity groups (i.e. mild, moderate and severe) based on the ocular surface disease index (OSDI). A significantly lower level of TAG (p<0.05) was observed in patients under the moderate category compared to the mild category. Notably, a number of OAHFA species displayed consistently decreasing levels that correlate with increasing disease severity. An attempt was also made to investigate the changes in meibum lipid profiles of DTS patients compared to normal individuals classified based on OSDI score. Several unsaturated TAG and PC species were found at significantly higher levels (p<0.05) in patients than controls. Conclusion The current study presents, for the first time, a comprehensive lipidome of meibum from individuals of an Asian ethnicity, which can potentially offer new insights into the higher prevalence of DTS observed amongst Asian populations. This study also represents an attempt towards identification of lipid species in meibum which could serve as marker for DTS.

Lipidomics as a Principal Tool for Advancing Biomedical Research

Lipidomics, which targets at the construction of a comprehensive map of lipidome comprising the entire lipid pool within a cell or tissue, is currently emerging as an independent discipline at the interface of lipid biology, technology and medicine. The diversity and complexity of the biological lipidomes call for technical innovation and improvement to meet the needs of various biomedical studies. The recent wave of expansion in the field of lipidomic research is mainly attributed to advances in analytical technologies, in particular, the development of new mass spectrometric and chromatographic tools for the characterization and quantification of the wide array of diverse lipid species in the cellular lipidome. Here, we review some of the key technical advances in lipidome analysis and put forth the applications of lipidomics in addressing the biological roles of lipids in numerous disease models including the metabolic syndrome, neurodegenerative diseases and infectious diseases, as well as the increasing urgency to construct the lipidome inventory for various mammalian/organism models useful for biomedical research.

Comparative Plasma Lipidome between Human and Cynomolgus Monkey: Are Plasma Polar Lipids Good Biomarkers for Diabetic Monkeys?

Background Non-human primates (NHP) are now being considered as models for investigating human metabolic diseases including diabetes. Analyses of cholesterol and triglycerides in plasma derived from NHPs can easily be achieved using methods employed in humans. Information pertaining to other lipid species in monkey plasma, however, is lacking and requires comprehensive experimental analysis. Methodologies/Principal Findings We examined the plasma lipidome from 16 cynomolgus monkey, Macaca fascicularis, using liquid chromatography coupled with mass spectrometry (LC/MS). We established novel analytical approaches, which are based on a simple gradient elution, to quantify polar lipids in plasma including (i) glycerophospholipids (phosphatidylcholine, PC; phosphatidylethanolamine, PE; phosphatidylinositol, PI; phosphatidylglycerol, PG; phosphatidylserine, PS; phosphatidic acid, PA); (ii) sphingolipids (sphingomyelin, SM; ceramide, Cer; Glucocyl-ceramide, GluCer; ganglioside mannoside 3, GM3). Lipidomic analysis had revealed that the plasma of human and cynomolgus monkey were of similar compositions, with PC, SM, PE, LPC and PI constituting the major polar lipid species present. Human plasma contained significantly higher levels of plasmalogen PE species (p<0.005) and plasmalogen PC species (p<0.0005), while cynomolgus monkey had higher levels of polyunsaturated fatty acyls (PUFA) in PC, PE, PS and PI. Notably, cynomolgus monkey had significantly lower levels of glycosphingolipids, including GluCer (p<0.0005) and GM3 (p<0.0005), but higher level of Cer (p<0.0005) in plasma than human. We next investigated the biochemical alterations in blood lipids of 8 naturally occurring diabetic cynomolgus monkeys when compared with 8 healthy controls. Conclusions For the first time, we demonstrated that the plasma of human and cynomolgus monkey were of similar compositions, but contained different mol distribution of individual molecular species. Diabetic monkeys exhibited decreased levels of sphingolipids, which are microdomain-associated lipids and are thought to be associated with insulin sensitivity. Significant increases in PG species, which are precursors for cardiolipin biosynthesis in mitochondria, were found in fasted diabetic monkeys (n = 8).

Adipose-Specific Knockout of Seipin/Bscl2 Results in Progressive Lipodystrophy

Berardinelli-Seip congenital lipodystrophy type 2 (BSCL2) is the most severe form of human lipodystrophy, characterized by an almost complete loss of adipose tissue and severe insulin resistance. BSCL2 is caused by loss-of-function mutations in the BSCL2/SEIPIN gene, which is upregulated during adipogenesis and abundantly expressed in the adipose tissue. The physiological function of SEIPIN in mature adipocytes, however, remains to be elucidated. Here, we generated adipose-specific Seipin knockout (ASKO) mice, which exhibit adipocyte hypertrophy with enlarged lipid droplets, reduced lipolysis, adipose tissue inflammation, progressive loss of white and brown adipose tissue, insulin resistance, and hepatic steatosis. Lipidomic and microarray analyses revealed accumulation/imbalance of lipid species, including ceramides, in ASKO adipose tissue as well as increased endoplasmic reticulum stress. Interestingly, the ASKO mice almost completely phenocopy the fat-specific peroxisome proliferator–activated receptor-γ (Pparγ) knockout (FKO-γ) mice. Rosiglitazone treatment significantly improved a number of metabolic parameters of the ASKO mice, including insulin sensitivity. Our results therefore demonstrate a critical role of SEIPIN in maintaining lipid homeostasis and function of adipocytes and reveal an intimate relationship between SEIPIN and PPAR-γ.

Lipidomic analysis of human tear fluid reveals structure-specific lipid alterations in dry eye syndrome

As current diagnostic markers for dry eye syndrome (DES) are lacking in both sensitivity and specificity, a pressing concern exists to develop activity markers that closely align with the principal axes of disease progression. In this study, a comprehensive lipidomic platform designated for analysis of the human tear lipidome was employed to characterize changes in tear lipid compositions from a cohort of 93 subjects of different clinical subgroups classified based on the presence of dry eye symptoms and signs. Positive correlations were observed between the tear levels of cholesteryl sulfates and glycosphingolipids with physiological secretion of tears, which indicated the possible lacrimal (instead of meibomian) origin of these lipids. Notably, we found wax esters of low molecular masses and those containing saturated fatty acyl moieties were specifically reduced with disease and significantly correlated with various DES clinical parameters such as ocular surface disease index, tear breakup time, and Schirmers I test (i.e., both symptoms and signs). These structure-specific changes in tear components with DES could potentially serve as unifying indicators of disease symptoms and signs. In addition, the structurally-specific aberrations in tear lipids reported here were found in patients with or without aqueous deficiency, suggesting a common pathology for both DES subtypes.

The brain lipidomes of subcortical ischemic vascular dementia and mixed dementia

Despite its importance as the leading cause of vascular dementia, the primary pathogenic mechanisms in subcortical ischemic vascular dementia (SIVD) have remained elusive. Because of the lack of approved therapeutic agents for SIVD, there is a pressing need to identify novel therapeutic targets. Comparative lipidomic analyses of SIVD and mixed dementia (i.e., SIVD and Alzheimers disease, MixD) may also confer new insights pertaining to the possible interaction between neurodegenerative and vascular mechanisms in the pathogenesis of dementia. Liquid chromatography coupled to mass spectrometry was used to comprehensively analyze the lipidomes of white and gray matter from the temporal cortex of nondemented controls, SIVD, and MixD subjects. Detailed molecular profiles highlighted the pathologic relevance of gray matter sphingolipid fatty acyl chain heterogeneity in dementia. In addition, the levels of sulfatides and lysobisphosphatidic acids were progressively increased in the temporal cortex gray matter from control to SIVD to MixD. White matter phospholipid profiles indicated possible adaptive mechanisms (i.e., increased unsaturation) to chronic ischemia in SIVD and elevated membrane degradation in MixD.

Longitudinal changes in tear fluid lipidome brought about by eyelid-warming treatment in a cohort of meibomian gland dysfunction.

Meibomian gland dysfunction (MGD) is a leading cause of evaporative dry eye and ocular discomfort characterized by an unstable tear film principally attributed to afflicted delivery of lipids to the ocular surface. Herein, we elucidated longitudinal tear lipid alterations associated with disease alleviation and symptom improvement in a cohort of MGD patients undergoing eyelid-warming treatment for 12 weeks. Remarkably, eyelid-warming resulted in stark reductions in lysophospholipids (P < 0.001 for lyso-plasmalogen phosphatidylethanolamine, lysophosphatidylcholine, and lysophosphatidylinositol), as well as numerous PUFA-containing diacylglyceride species in tears, accompanied by significant increases in several PUFA-containing phospholipids. These changes in tear lipidomes suggest that eyelid-warming leads to diminished activity of tear phospholipases that preferentially target PUFA-containing phospholipids. In addition, treatment led to appreciable increases (P < 0.001) in O-acyl-ω-hydroxy-FAs (OAHFAs), which are lipid amphiphiles critical to the maintenance of tear film stability. Longitudinal changes in the tear lipids aforementioned also significantly (P < 0.05) correlated with reduced rate of ocular evaporation and improvement in ocular symptoms. The foregoing data thus indicate that excess ocular surface phospholipase activity detrimental to tear film stability could be alleviated by eyelid warming alone without application of steroids and identify tear OAHFAs as suitable markers to monitor treatment response in MGD.

Effects of a high-fat, high-cholesterol diet on brain lipid profiles in apolipoprotein E ε3 and ε4 knock-in mice

Apolipoprotein E (ApoE) is important in facilitating the transport of lipids (cholesterol, phospholipids, and sulfatides) and plays a fundamental role in normal lipid metabolism. High cholesterol levels increases the risk of developing Alzheimers disease. In this study, we investigated the effects of a high-fat high cholesterol (HFHC) diet on brain lipid profiles in 95 young and aged APOE ε3 and ε4 knock-in mice to determine whether diet leads to altered brain levels of a number of glycerophospholipids, sphingolipids, cholesterol precursors, cholesterol, cholesterol oxidation products, and cholesterol esters. The results in this study revealed significant changes in lipid levels. The HFHC-enriched diet influenced the levels of cholesterol esters. A sharp increase in cholesterol ester levels, particularly in the aged APOE ε4 diet-enriched group, might be suggestive of abnormal acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT) activity and/or levels. Age exerts appreciable effects on the brain lipidome, especially with regard to polar lipid species.

Metabolomics, a Powerful Tool for Agricultural Research

Metabolomics, which is based mainly on nuclear magnetic resonance (NMR), gas-chromatography (GC) or liquid-chromatography (LC) coupled to mass spectrometry (MS) analytical technologies to systematically acquire the qualitative and quantitative information of low-molecular-mass endogenous metabolites, provides a direct snapshot of the physiological condition in biological samples. As complements to transcriptomics and proteomics, it has played pivotal roles in agricultural and food science research. In this review, we discuss the capacities of NMR, GC/LC-MS in the acquisition of plant metabolome, and address the potential promise and diverse applications of metabolomics, particularly lipidomics, to investigate the responses of Arabidopsis thaliana, a primary plant model for agricultural research, to environmental stressors including heat, freezing, drought, and salinity.