Fish oil fatty acids impair VLDL assembly and/or secretion by cultured rat hepatocytes

We determined the effect of the two major fish oil fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), on VLDL assembly and secretion by cultured rat hepatocytes. The incorporation of [3H]glycerol into total triglyceride (cell plus media) was stimulated eight-fold when hepatocytes were incubated for 2 h with 1 mM EPA, DHA, or oleic acid (OA), suggesting that fish oil fatty acids stimulate hepatic triglyceride synthesis to an extent similar to OA. In contrast, mass quantitation of secreted triglyceride showed impaired triglyceride secretion with EPA and DHA compared to OA. During a 42-h time course, cells stimulated with EPA and DHA progressively accumulated triglyceride compared to cells stimulated with OA. To determine whether fish oil fatty acids impair very low density lipoprotein (VLDL) secretion, cells were labeled with [35S]methionine and the secretion of de novo synthesized apoB was measured. Compared to OA, EPA and DHA significantly impaired the secretion of both molecular weight forms of apoB. The cellular content of apoB was not altered by any of the fatty acids. The concordant decrease in the secretion of both triglyceride and apoB suggests that fish oil fatty acids impair VLDL assembly and/or secretion.     

Characteristics of human saliva proteome and peptidome

Recent studies on the characteristics of saliva proteome and peptidome greatly expanded our understanding of this biological fluid. Athough many scientists consider saliva to be an ideal biosubstrate in diagnosis of the human body state; currently, the research in this area is at the data accumulation stage. The physiology of saliva and salivary glands, as well as characteristics of interaction between the saliva proteins and the oral cavity microorganisms, has been insufficiently studied yet. The lack of standardization in collecting the saliva samples and in the proteome research protocols, and the requirements for sample representativeness introduce discrepancies in the results obtained by different researchers. Addressing these problems will allow the wide use of saliva proteome as a complex indicator of the functional state of the human body.     

Saliva proteome research: current status and future outlook

Human saliva harbours proteins of clinical relevance and about 30% of blood proteins are also present in saliva. This highlights that saliva can be used for clinical applications just as urine or blood. However, the translation of salivary biomarker discoveries into clinical settings is hampered by the dynamics and complexity of the salivary proteome. This review focuses on the current status of technological developments and achievements relating to approaches for unravelling the human salivary proteome. We discuss the dynamics of the salivary proteome, as well as the importance of sample preparation and processing techniques and their influence on downstream protein applications; post-translational modifications of salivary proteome and protein: protein interactions. In addition, we describe possible enrichment strategies for discerning post-translational modifications of salivary proteins, the potential utility of selected-reaction-monitoring techniques for biomarker discovery and validation, limitations to proteomics and the biomarker challenge and future perspectives. In summary, we provide recommendations for practical saliva sampling, processing and storage conditions to increase the quality of future studies in an emerging field of saliva clinical proteomics. We propose that the advent of technologies allowing sensitive and high throughput proteome-wide analyses, coupled to well-controlled study design, will allow saliva to enter clinical practice as an alternative to blood-based methods due to its simplistic nature of sampling, non-invasiveness, easy of collection and multiple collections by untrained professionals and cost-effective advantages.     

An Assay Method for Herpes Simplex Virus Type 1 Seroprevalence Survey—Detection of Antibody in Saliva by Avidin-Biotin Complex Enzyme-Linked Immunosorbent Assay (ABC-ELISA)

To determine whether the avidin-biotin complex enzyme-linked immunosorbent assay (ABC-E) is a potentially useful method for detection of herpes simplex virus type 1 (HSV-1) antibody in saliva, paired serum and saliva samples from 129 healthy individuals aged 18 to 25 years were collected simultaneously and subjected to a neutralization test (NT) for neutralizing antibody and also to an indirect ELISA (IE) and ABC-E for HSV-1 specific IgG detection. Compared with the results of NT, the sensitivities of the IE and ABC-E for serum were both 100% (45/45), and for saliva 82.2% (37/45) and 93.3% (42/45), respectively. The specificity of all these methods was 100% (84/84). With the same ABC-E method, a significant correlation (r=0.66, P < 0.001) between the OD-difference (d-OD) values of positive serum and saliva samples was observed. Furthermore, the consistency of ABC-E for salivary antibody detection was confirmed with the paired serum and saliva samples which were collected from four individuals followed up for eight months. It was clear that the ABC-E method for saliva can be used in place of the NT and ABC-E method for serum for seroprevalence studying of HSV-1 infection.     

Quantitative proteomic analysis of the fall armyworm saliva

Lepidopteran larvae secrete saliva on plant tissues during feeding. Components in the saliva may aid in food digestion, whereas other components are recognized by plants as cues to elicit defense responses. Despite the ecological and economical importance of these plant-feeding insects, knowledge of their saliva composition is limited to a few species. In this study, we identified the salivary proteins of larvae of the fall armyworm (FAW), Spodoptera frugiperda; determined qualitative and quantitative differences in the salivary proteome of the two host races—corn and rice strains—of this insect; and identified changes in total protein concentration and relative protein abundance in the saliva of FAW larvae associated with different host plants. Quantitative proteomic analyses were performed using labeling with isobaric tags for relative and absolute quantification followed by liquid chromatography-tandem mass spectrometry. In total, 98 proteins were identified (>99% confidence) in the FAW saliva. These proteins were further categorized into five functional groups: proteins potentially involved in (1) plant defense regulation, (2) herbivore offense, (3) insect immunity, (4) detoxification, (5) digestion, and (6) other functions. Moreover, there were differences in the salivary proteome between the FAW strains that were identified by label-free proteomic analyses. Thirteen differentially identified proteins were present in each strain. There were also differences in the relative abundance of eleven salivary proteins between the two FAW host strains as well as differences within each strain associated with different diets. The total salivary protein concentration was also different for the two strains reared on different host plants. Based on these results, we conclude that the FAW saliva contains a complex mixture of proteins involved in different functions that are specific for each strain and its composition can change plastically in response to diet type.     

Characterization of the human submandibular/sublingual saliva glycoproteome using lectin affinity chromatography coupled to multidimensional protein identification technology

In-depth analysis of the salivary proteome is fundamental to understanding the functions of salivary proteins in the oral cavity and to reveal disease biomarkers involved in different pathophysiological conditions, with the ultimate goal of improving patient diagnosis and prognosis. Submandibular and sublingual glands contribute saliva rich in glycoproteins to the total saliva output, making them valuable sources for glycoproteomic analysis. Lectin-affinity chromatography coupled to mass spectrometry-based shotgun proteomics was used to explore the submandibular/sublingual (SM/SL) saliva glycoproteome. A total of 262 N- and O-linked glycoproteins were identified by multidimensional protein identification technology (MudPIT). Only 38 were previously described in SM and SL salivas from the human salivary N-linked glycoproteome, while 224 were unique. Further comparison analysis with SM/SL saliva of the human saliva proteome, revealed 125 glycoproteins not formerly reported in this secretion. KEGG pathway analyses demonstrated that many of these glycoproteins are involved in processes such as complement and coagulation cascades, cell communication, glycosphingolipid biosynthesis neo-lactoseries, O-glycan biosynthesis, glycan structures-biosynthesis 2, starch and sucrose metabolism, peptidoglycan biosynthesis or others pathways. In summary, lectin-affinity chromatography coupled to MudPIT mass spectrometry identified many novel glycoproteins in SM/SL saliva. These new additions to the salivary proteome may prove to be a critical step for providing reliable biomarkers in the diagnosis of a myriad of oral and systemic diseases.     

Alteration of arachidonate levels in tick salivary glands by dietary modification of host blood lipids

Tick saliva contains prostaglandins of the 2-series, believed to facilitate bloodmeal acquisition. Because ticks cannot synthesize the prostaglandin precursor, arachidonic acid, investigations were undertaken to study the uptake, incorporation, and distribution of arachidonic acid in the salivary glands of the lone star tick in vitro and in vivo. Uptake of [³H]arachidonate by isolated salivary glands was reduced in the presence of low concentrations of arachidonic or eicosapentaenoic acids, but much higher, non-physiological concentrations of oleic and linoleic acids were required to inhibit [³H]arachidonate uptake. The incorporation of [³H]arachidonate into triglycerides increased at high concentrations of arachidonic or eicosapentaenoic acid, but not at any concentration of oleic or linoleic acid. Eicosatetraynoic acid greatly inhibited [³H]arachidonate uptake and increased intracellular unesterified [³H]arachidonic acid. Guinea pigs fed hydrogenated coconut oil, safflower/primrose oil, or fish oil exhibited altered blood lipids; notably increased levels of eicosapentaenoic acid when fed fish oil. Salivary gland lipids in ticks fed on these hosts were also altered. Ticks parasitizing fish oil-fed guinea pigs contained high levels of eicosapentaenoic acid with a 30% reduction in arachidonate levels. The results demonstrated that eicosapentaenoic acid in the host diet had profound effects on arachidonate assimilation by tick salivary glands, which could lead to altered prostaglandin content in tick saliva. © 1996 Wiley-Liss, Inc.     

The scientific exploration of saliva in the post-proteomic era: from database back to basic function

The proteome of human saliva can be considered as being essentially completed. Diagnostic markers for a number of diseases have been identified among salivary proteins and peptides, taking advantage of saliva as an easy-to-obtain biological fluid. Yet, the majority of disease markers identified so far are serum components and not intrinsic proteins produced by the salivary glands. Furthermore, despite the fact that saliva is essential for protecting the oral integuments and dentition, little progress has been made in finding risk predictors in the salivary proteome for dental caries or periodontal disease. Since salivary proteins, and in particular the attached glycans, play an important role in interactions with the microbial world, the salivary glycoproteome and other post-translational modifications of salivary proteins need to be studied. Risk markers for microbial diseases, including dental caries, are likely to be discovered among the highly glycosylated major protein species in saliva. This review will attempt to raise new ideas and also point to under-researched areas that may hold promise for future applicability in oral diagnostics and prediction of oral disease.     

Enrichment and identification of glycoproteins in human saliva using lectin magnetic bead arrays

Aberrant glycosylation of proteins is a hallmark of tumorigenesis and could provide diagnostic value in cancer detection. Human saliva is an ideal source of glycoproteins due to the relatively high proportion of glycosylated proteins in the salivary proteome. Moreover, saliva collection is noninvasive and technically straightforward, and the sample collection and storage is relatively easy. Although differential glycosylation of proteins can be indicative of disease states, identification of differential glycosylation from clinical samples is not trivial. To facilitate salivary glycoprotein biomarker discovery, we optimized a method for differential glycoprotein enrichment from human saliva based on lectin magnetic bead arrays (saLeMBA). Selected lectins from distinct reactivity groups were used in the saLeMBA platform to enrich salivary glycoproteins from healthy volunteer saliva. The technical reproducibility of saLeMBA was analyzed with liquid chromatography–tandem mass spectrometry (LC–MS/MS) to identify the glycosylated proteins enriched by each lectin. Our saLeMBA platform enabled robust glycoprotein enrichment in a glycoprotein- and lectin-specific manner consistent with known protein-specific glycan profiles. We demonstrated that saLeMBA is a reliable method to enrich and detect glycoproteins present in human saliva.     

Toward a standardized saliva proteome analysis methodology

The present study aimed the evaluation of saliva sample pre-treatment, in particular the sample clearance usually performed by centrifugation, to the contribution of salivary proteome and peptidome. Using in-gel and off-gel approaches, a large content of salivary proteins was detected in the pellet fraction that is usually discarded. In addition, chaotropic/detergent treatment in combination with sonication, before the centrifugation step, resulted in salivary complex disruption and consequently in the extraction of high amounts of proteins. Based on this data, we suggest the use of urea/detergent with sonication as a standard saliva sample pre-treatment procedure. We also described a procedure to extract salivary peptides which can be performed even after saliva sample treatment with chaotropic/detergents. In overall, we reported for the first time the contribution of the pellet fraction to the whole saliva proteome. iTRAQ analysis highlighted a higher number of different peptides as well as distinct quantities of each protein class when after sample treatment with urea and sonication, acetone precipitation followed by solubilization with acetonitrile/HCl was performed.     

Large-scale identification of proteins in human salivary proteome by liquid chromatography/mass spectrometry and two-dimensional gel electrophoresis-mass spectrometry

Human saliva contains a large number of proteins and peptides (salivary proteome) that help maintain homeostasis in the oral cavity. Global analysis of human salivary proteome is important for understanding oral health and disease pathogenesis. In this study, large-scale identification of salivary proteins was demonstrated by using shotgun proteomics and two-dimensinal gel electrophoresis-mass spectrometry (2-DE-MS). For the shotgun approach, whole saliva proteins were prefractionated according to molecular weight. The smallest fraction, presumably containing salivary peptides, was directly separated by capillary liquid chromatography (LC). However, the large protein fractions were digested into peptides for subsequent LC separation. Separated peptides were analyzed by on-line electrospray tandem mass spectrometry (MS/MS) using a quadrupole-time of flight mass spectrometer, and the obtained spectra were automatically processed to search human protein sequence database for protein identification. Additionally, 2-DE was used to map out the proteins in whole saliva. Protein spots 105 in number were excised and in-gel digested; and the resulting peptide fragments were measured by matrix-assisted laser desorption/ionization-mass spectrometry and sequenced by LC-MS/MS for protein identification. In total, we cataloged 309 proteins from human whole saliva by using these two proteomic approaches.     

Competitive Adsorption of Lecithin and Saliva at the O/W Interface in Relation to the Oral Processing of Lipid Continuous Foods

This research is relevant to oral processing of lipid continuous foods. During this first step of food digestion, lipid continuous foods such as chocolate or margarine phase invert into oil-in-water emulsions stimulated through the mechanical action of tongue and teeth in combination with the change in temperature and the high surface activity of salivary proteins. These are hypothesised to stabilise the newly formed interface in competition with surfactants or surface active molecules released from the food if present. Here competitive adsorption between mechanically stimulated human whole saliva (HWS) and lecithin dissolved in sunflower oil freed of interfacially active contaminants was investigated in-vitro using a pendant drop tensiometer for dynamic interfacial tension and interfacial rheological measurements. Initially, it was validated that the interfacial properties of HWS samples remained unaffected by frozen storage at ?80 °C during 6 weeks. Protein concentration affected the absolute values of interfacial tension and in particular the dilatational elastic modulus. Competitive adsorption studies revealed a mixed interface and it follows that emulsion stabilisation during oral processing involves both salivary proteins and lecithin present in the oil phase.     

Dietary supplementation of essential fatty acids in larval pikeperch (Sander lucioperca); short and long term effects on stress tolerance and metabolic physiology

The present study examined the effects of feeding pike perch larvae Artemia, enriched with either docosahexanoic acid (DHA), arachidonic acid (ARA), oleic acid (OA), olive oil (OO) or a commercial enrichment DHA Selco (DS) on tissue lipid deposition, stress tolerance, growth and development, and metabolic rate. There was higher tissue retention of ARA than DHA at comparable inclusion levels. No differences were observed between diets on the percentage contribution of ARA or DHA to the fatty acid profile of tissues (head and trunk). Total fatty acid content (mgg(-1)) was significantly higher in the head, reflecting its high content of neural tissue. Observations on larval erratic behaviour and mortality following exposure to salinity stress suggested that high inclusions levels of DHA had an alleviating effect, while ARA did not. Particularly larval groups reared for 16 days on diets enriched with OO and OA had mortality rates approaching 100% within two hours. Interestingly, this tendency, although not as pronounced, was also apparent in juvenile fish after 120 days of rearing on a common diet. Standard metabolic rate in larvae on an OO enriched diet was significantly elevated, but otherwise no groups had significant changes to their respiratory physiology. In addition to increased stress challenge sensitivity, early feeding with OA had long term impact on pike perch neural development indicated by a smaller brain size in juvenile fish. In conclusion, lack of DHA in the diet of pikeperch larvae suggests that this long chain polyunsaturated fatty acid is involved in processes that increase stress tolerance and that lack of dietary DHA in early larval stage caused increased stress sensitivity and long-term impaired neural development, while it does not appear to affect metabolic rate at rest.     

Salivary protein profiles and sensitivity to the bitter taste of caffeine

The interindividual variation in the sensitivity to bitterness is attributed in part to genetic polymorphism at the taste receptor level, but other factors, such as saliva composition, might be involved. In order to investigate this, 2 groups of subjects (hyposensitive, hypersensitive) were selected from 29 healthy male volunteers based on their detection thresholds for caffeine, and their salivary proteome composition was compared. Abundance of 26 of the 255 spots detected on saliva electrophoretic patterns was significantly different between hypo- and hypersensitive subjects. Saliva of hypersensitive subjects contained higher levels of amylase fragments, immunoglobulins, and serum albumin and/or serum albumin fragments. It also contained lower levels of cystatin SN, an inhibitor of protease. The results suggest that proteolysis occurring within the oral cavity is an important perireceptor factor associated to the sensitivity to the bitter taste of caffeine.     

The presence of 4-hydroxyphenylacetic acid in human saliva and the possibility of its nitration by salivary nitrite in the stomach

Human saliva contained 4-hydroxyphenylacetic acid (HPA) (2-10 microM) and nitrite (60-300 microM). HPA was nitrated to 4-hydroxy-3-nitrophenylacetic acid (NO2HPA) when HPA and sodium nitrite were mixed at pH 1.0. NO2HPA was also formed when saliva was incubated under acidic conditions. These results suggest that salivary HPA is nitrated to NO2HPA when saliva is swallowed into the stomach.     

Comparative Salivary Proteome of Hepatitis B- and C-Infected Patients

Hepatitis B and C virus (HBV and HCV) infections are an important cause of cirrhosis and hepatocellular carcinoma. The natural history has a prominent latent phase, and infected patients may remain undiagnosed; this situation may lead to the continuing spread of these infections in the community. Compelling reasons exist for using saliva as a diagnostic fluid because it meets the demands of being an inexpensive, noninvasive and easy-to-use diagnostic method. Indeed, comparative analysis of the salivary proteome using mass spectrometry is a promising new strategy for identifying biomarkers. Our goal is to apply an Orbitrap-based quantitative approach to explore the salivary proteome profile in HBV- and HCV-infected patients. In the present study, whole saliva was obtained from 20 healthy, (control) 20 HBV-infected and 20 HCV-infected subjects. Two distinct pools containing saliva from 10 subjects of each group were obtained. The samples were ultracentrifuged and fractionated, and all fractions were hydrolyzed (trypsin) and injected into an LTQ-VELOS ORBITRAP. The identification and analyses of peptides were performed using Proteome Discoverer1.3 and ScaffoldQ + v.3.3.1. From a total of 362 distinct proteins identified, 344 proteins were identified in the HBV, 326 in the HCV and 303 in the control groups. Some blood proteins, such as flavin reductase (which converts biliverdin to bilirubin), were detected only in the HCV group. The data showed a reduced presence of complement C3, ceruloplasmin, alpha(1)-acid glycoprotein and alpha(2)-acid glycoprotein in the hepatitis-infected patients. Peptides of serotransferrin and haptoglobin were less detected in the HCV group. This study provides an integrated perspective of the salivary proteome, which should be further explored in future studies targeting specific disease markers for HBV and HCV infection.     

Effects of fatty acids and growth hormone on liver fatty acid binding protein and PPARalpha in rat liver

The aim of this study was to investigate the interaction between long-chain fatty acids (LCFA) and growth hormone (GH) in the regulation of liver fatty acid binding protein (LFABP) and peroxisome proliferator-activated receptor-alpha (PPARalpha). Cultured rat hepatocytes were given oleic acid (OA; 500 microM) and GH (100 ng/ml) for 3 days. LFABP mRNA increased 3.6-fold by GH and 5.7-fold by OA, and combined incubation with GH and OA increased LFABP mRNA 17.6-fold. PPARalpha mRNA was decreased 50% by GH, but OA had no effect. Hypophysectomized (Hx) female rats were treated with L-thyroxine, cortisol, GH, and dietary fat for 7 days. PPARalpha mRNA levels were three- to fourfold higher in Hx than in normal female rats. GH decreased PPARalpha mRNA 50% in Hx rats. Dietary triglycerides (10% corn oil) increased LFABP mRNA and cytosolic LFABP about twofold but had no effect on PPARalpha mRNA in Hx rats. GH and dietary triglycerides had an additive effect on LFABP expression. Dietary triglycerides increased mitochondrial hydroxymethylglutaryl-CoA synthase mRNA only in the presence of GH. The diet increased serum triglycerides in Hx rats, and GH treatment prevented this increase. Addition of cholesterol to the diet did not influence LFABP levels but mitigated increased hepatic triglyceride content. In summary, these studies show that GH regulates LFABP expression independently of PPARalpha. Moreover, GH has different effects on PPARalpha-responsive genes and does not counteract the effect of LCFA on the expression of these gene products.     

Reversibility of n-3 fatty acid deficiency-induced alterations of learning behavior in the rat: level of n-6 fatty acids as another critical factor

Rats fed a semipurified diet supplemented with 3% (w/w) safflower oil [Saf, n-3 fatty acid deficient, high linoleic acid (18:2n-6)] through two generations exhibit decreased correct response ratios in a brightness-discrimination learning test compared with rats fed 3% perilla oil [Per, high alpha-linolenic acid (18:3n-3)]. This is associated with a decreased DHA (22:6n-3)-to-arachidonic acid (20:4n-6) ratio in brain lipids. In the first set of experiments, dietary oil was shifted from Saf to a mixture of 2.4% safflower oil plus 0.6% DHA after weaning (Saf-DHA), but all parameters measured in the learning test were essentially unchanged. Brain 22:6n-3 content of the Saf-DHA group reached that of the Per group but the levels of 20:4n-6 and docosatetraenoic acid (22:4n-6) did not decrease to those of the Per group at the start of the test. In the second set of experiments, dietary oil was shifted to a mixture of 0.6% safflower oil plus 1.2% oleic acid (OA) plus 1.2% DHA (Saf-OA-DHA group) with 18:2n-6 content comparable to that of the Per group. The Saf-OA-DHA group exhibited a learning performance similar to that of the Per group; brain 22:6n-3, 20:4n-6, and 22:4n-6 contents were also comparable to those of the Per group. These results indicate that the altered learning behavior associated with a long-term n-3 fatty acid deficiency is reversed by supplementing 22:6n-3 after weaning, when the levels of competing n-6 fatty acids in the diet and brain lipids are limited.     

Divergent effects of eicosapentaenoic and docosahexaenoic acid ethyl esters, and fish oil on hepatic fatty acid oxidation in the rat

The physiological activity of fish oil, and ethyl esters of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) affecting hepatic fatty acid oxidation was compared in rats. Five groups of rats were fed various experimental diets for 15 days. A group fed a diet containing 9.4% palm oil almost devoid of n-3 fatty acids served as a control. The test diets contained 4% n-3 fatty acids mainly as EPA and DHA in the form of triacylglycerol (9.4% fish oil) or ethyl esters (diets containing 4% EPA ethyl ester, 4% DHA ethyl ester, and 1% EPA plus 3% DHA ethyl esters). The lipid content of diets containing EPA and DHA ethyl esters was adjusted to 9.4% by adding palm oil. The fish oil diet and ethyl ester diets, compared to the control diet containing 9.4% palm oil, increased activity and mRNA levels of hepatic mitochondrial and peroxisomal fatty acid oxidation enzymes, though not 3-hydroxyacyl-CoA dehydrogenase activity. The extent of the increase was, however, much greater with the fish oil than with EPA and DHA ethyl esters. EPA and DHA ethyl esters, compared to the control diet, increased 3-hydroxyacyl-CoA dehydrogenase activity, but fish oil strongly reduced it. It is apparent that EPA and DHA in the form of ethyl esters cannot mimic the physiological activity of fish oil at least in affecting hepatic fatty acid oxidation in rat.