Tissue Phthalate Levels Correlate With Changes in Immune Gene Expression in a Population of Juvenile Wild Salmon.

Phthalates have detrimental effects on health and have been shown to dysregulate the immune system of mammals, birds, and fish. We recently reported that di(2-ethylhexyl) phthalate exposure reduces the abundance and inhibits the proliferation of rainbow trout (Oncorhynchus mykiss) IgM(+) B lymphocytes and expression of secreted immunoglobulin heavy-chain mu transcripts in an in vitro culture system. We proposed that phthalates act as immunomodulators by modifying the normal B cell-activation pathways by accelerating B cell differentiation while suppressing plasmablast expansion, thus resulting in fewer IgM-secreting plasma cells. This hypothesis was tested here in an in vivo field study of juvenile Dolly Varden (Salvelinus malma) from a plastic-polluted lake in the Gulf of Alaska. Fish tissues were analyzed for both phthalate levels using liquid chromatography-coupled tandem mass spectrometry and for changes in immune gene expression using reverse transcriptase-real time polymerase chain reaction. Results showed that fish with higher tissue levels of di(2-ethylhexyl) phthalate, di(n-butyl) phthalate, and/or dimethyl phthalate expressed significantly fewer secreted and membrane-bound immunoglobulin heavy-chain mu and Blimp1 transcripts in their hematopoietic tissue. This suggests that in vivo uptake of phthalates in fish changes the expression of B cell-specific genes. Chronic exposure to phthalates likely dysregulates normal B-lymphoid development and antibody responses in salmonids and may increase susceptibility to infection. Given the conserved nature of B-lineage cells in vertebrate animals, other marine species may be similarly affected by chronic phthalate exposure.

Fatty acids linked to cardiovascular mortality are associated with risk factors.

BACKGROUND: Although saturated fatty acids (FAs) have been linked to cardiovascular mortality, it is not clear whether this outcome is attributable solely to their effects on low-density lipoprotein cholesterol (LDL-C) or whether other risk factors are also associated with FAs. The Western Alaskan Native population, with its rapidly changing lifestyles, shift in diet from unsaturated to saturated fatty acids and dramatic increase in cardiovascular disease (CVD), presents an opportunity to elucidate any associations between specific FAs and known CVD risk factors. OBJECTIVE: We tested the hypothesis that the specific FAs previously identified as related to CVD mortality are also associated with individual CVD risk factors. METHODS: In this community-based, cross-sectional study, relative proportions of FAs in plasma and red blood cell membranes were compared with CVD risk factors in a sample of 758 men and women aged ≥35 years. Linear regression analyses were used to analyze relations between specific FAs and CVD risk factors (LDL-C, high-density lipoprotein cholesterol, triglycerides, C-reactive protein, systolic blood pressure, diastolic blood pressure, heart rate, body mass index, fasting glucose and fasting insulin, 2-hour glucose and 2-hour insulin). RESULTS: The specific saturated FAs previously identified as related to CVD mortality, the palmitic and myristic acids, were adversely associated with most CVD risk factors, whereas unsaturated linoleic acid (18:2n-6) and the marine n-3 FAs were not associated or were beneficially associated with CVD risk factors. CONCLUSIONS: The results suggest that CVD risk factors are more extensively affected by individual FAs than hitherto recognized, and that risk for CVD, MI and stroke can be reduced by reducing the intake of palmitate, myristic acid and simple carbohydrates and improved by greater intake of linoleic acid and marine n-3 FAs.

Di(2-ethylhexyl) phthalate inhibits B cell proliferation and reduces the abundance of IgM-secreting cells in cultured immune tissues of the rainbow trout.

Plasticizer di(2-ethylhexyl) phthalate (DEHP) and its active metabolite MEHP have important immunotoxic effects in mammalian species, including inhibition of cell proliferation, inflammation inhibition, lowering of the antibody response, and apoptosis. Virtually nothing is known about the potential detrimental effects of DEHP/MEHP on the teleost immune system, although phthalates are a likely threat to fish health. Here we investigated whether short-term in vitro DEHP exposure would affect B lineage cells in the rainbow trout, using cultured immune tissues. Cell culture conditions, evidence of cellular incorporation of DEHP, and possible effects of DEHP on immune genes were first established using the mouse pre-B cell line PD31 and data confirmed a dose-dependent cellular uptake of DEHP using liquid chromatography-coupled ion trap mass spectrometry. Effects of in vitro DEHP exposure on trout B cell proliferation were tested by flow cytometry. Significant, dose-dependent inhibition was evident in both anterior and posterior kidney cultures after 24 h exposure to ≥4 µM DEHP. DEHP-induced cell death was not significant for the range of DEHP tested. Further, the abundance of IgM-secreting plasmablasts and plasma cells was significantly reduced after in vitro exposure of ≥16 µM DEHP for 2 or 7 days. Finally, in vitro DEHP exposure significantly lowered the levels of secreted HCmu transcripts in a dose-dependent manner. B lineage cells from posterior kidney were more sensitive to effects of in vitro DEHP exposure than those from anterior kidney. Together, the data support a model where DEHP modifies the normal B cell activation pathways in rainbow trout, promoting B cell differentiation while suppressing plasmablast expansion, resulting in fewer IgM-secreting plasma cells. Insufficient production of protective antibody make fish more susceptible to infection, and increases their risk for disease and mortality in polluted waters.

Variants in CPT1A, FADS1, and FADS2 are Associated with Higher Levels of Estimated Plasma and Erythrocyte Delta-5 Desaturases in Alaskan Eskimos.

The delta-5 and delta-6 desaturases (D5D and D6D), encoded by fatty acid desaturase 1 (FADS1) and 2 (FADS2) genes, respectively, are rate-limiting enzymes in the metabolism of ω-3 and ω-6 fatty acids. The objective of this study was to identify genes influencing variation in estimated D5D and D6D activities in plasma and erythrocytes in Alaskan Eskimos (n = 761) participating in the genetics of coronary artery disease in Alaska Natives (GOCADAN) study. Desaturase activity was estimated by product: precursor ratio of polyunsaturated fatty acids. We found evidence of linkage for estimated erythrocyte D5D (eD5D) on chromosome 11q12-q13 (logarithm of odds score = 3.5). The confidence interval contains candidate genes FADS1, FADS2, 7-dehydrocholesterol reductase (DHCR7), and carnitine palmitoyl transferase 1A, liver (CPT1A). Measured genotype analysis found association between CPT1A, FADS1, and FADS2 single-nucleotide polymorphisms (SNPs) and estimated eD5D activity (p-values between 10(-28) and 10(-5)). A Bayesian quantitative trait nucleotide analysis showed that rs3019594 in CPT1A, rs174541 in FADS1, and rs174568 in FADS2 had posterior probabilities > 0.8, thereby demonstrating significant statistical support for a functional effect on eD5D activity. Highly significant associations of FADS1, FADS2, and CPT1A transcripts with their respective SNPs (p-values between 10(-75) and 10(-7)) in Mexican Americans of the San Antonio Family Heart Study corroborated our results. These findings strongly suggest a functional role for FADS1, FADS2, and CPT1A SNPs in the variation in eD5D activity.

Heart rate is associated with markers of fatty acid desaturation: the GOCADAN study.

OBJECTIVES: To determine if heart rate (HR) is associated with desaturation indexes as HR is associated with arrhythmia and sudden death. STUDY DESIGN: A community based cross-sectional study of 1214 Alaskan Inuit. METHODS: Data of FA concentrations from plasma and red blood cell membranes from those ≥35 years of age (n = 819) were compared to basal HR at the time of examination. Multiple linear regression with backward stepwise selection was employed to analyze the effect of the desaturase indexes on HR, after adjustment for relevant covariates. RESULTS: The Δ(5) desaturase index (Δ(5)-DI) measured in serum has recently been associated with a protective role for cardiovascular disease. This index measured here in plasma and red blood cells showed a negative correlation with HR. The plasma stearoyl-CoA-desaturase (SCD) index, previously determined to be related to cardiovascular disease (CVD) mortality, on the other hand, was positively associated with HR, while the Δ(6) desaturase index (Δ(6)-DI) had no significant effect on HR. CONCLUSION: Endogenous FA desaturation is associated with HR and thereby, in the case of SCD, possibly with arrhythmia and sudden death, which would at least partially explain the previously observed association between cardiovascular mortality and desaturase activity.

A modified method for determining tannin-protein precipitation capacity using accelerated solvent extraction (ASE) and microplate gel filtration.

The protein precipitation assay used by Robbins et al., (1987) Ecology 68:98-107 has been shown to predict successfully the reduction in protein availability to some ruminants due to tannins. The procedure, however, is expensive and laborious, which limits its utility, especially for quantitative ecological or nutritional applications where large numbers of assays may be required. We have modified the method to decrease its cost and increase laboratory efficiency by: (1) automating the extraction by using Accelerated Solvent Extraction (ASE); and (2) by scaling and automating the precipitation reaction, chromatography, and spectrometry with microplate gel filtration and an automated UV-VIS microplate spectrometer. ASE extraction is shown to be as effective at extracting tannins as the hot methanol technique. Additionally, the microplate assay is sensitive and precise. We show that the results from the new technique correspond in a nearly 1:1 relationship to the results of the previous technique. Hence, this method could reliably replace the older method with no loss in relevance to herbivore protein digestion. Moreover, the ASE extraction technique should be applicable to other tannin-protein precipitation assays and possibly other phenolic assays.

Chlorinated fatty acids in lipid class fractions from cardiac and skeletal muscle of Chinook salmon.

Analysis of chlorinated fatty acids (CFA) in tissues can be difficult because of their low concentrations. This task becomes even more difficult when samples are from organisms living in remote locations with very little exposure to environmental contamination. Therefore, enrichment of CFA is necessary prior to analysis. In this study, CFA were enriched from fractionated lipid classes of cardiac and skeletal muscle of Chinook salmon (Oncorhynchus tshawytscha) to determine CFA distribution among lipid classes and tissue types and to demonstrate the sensitivity of this method to detect CFA at trace concentrations. The lipids extracted from cardiac and skeletal muscle of O. tshawytscha were separated into fractions containing TAG, FFA, and phospholipids. After transesterification, the FAME derivatives from each lipid class were analyzed by GC with a halogen-selective detector (XSD) to determine the concentrations of dichlorostearic acid and dichloropalmitic acid. Other chlorinated compounds detected by GC-XSD were analyzed by GC-MS. CFA were observed in all lipid classes in both cardiac and skeletal muscle tissues. However, the highest concentrations of CFA were found in the phospholipids of both tissue types, about 1-2 mg/g lipid. It was also shown that dichloropalmitic acid concentrated in cardiac phospholipids whereas dichlorostearic acid was found primarily in the phospholipids of skeletal tissue. CFA concentrations in TAG and FFA fractions were below 150 mg/g lipid. This study demonstrates a small-scale approach to the study of CFA at trace concentrations and their distribution among lipid classes.

Eskimos have CHD despite high consumption of omega-3 fatty acids: the Alaska Siberia project.

OBJECTIVES: The thirty-year-old hypothesis that omega-3 fatty acid (FA) may "reduce the development of thrombosis and atherosclerosis in the Western World" still needs to be tested. Dyerberg-Bang based their supposition on casual observations that coronary atherosclerosis in Greenlandic Inuit was 'almost unknown' and that they consumed large amounts of omega-3 FAs. However, no association was demonstrated with data. STUDY DESIGN: Cross-sectional study. METHODS: 454 Alaskan Eskimos were screened for coronary heart disease (CHD), using a protocol that included ECG, medical history, Rose questionnaire, blood chemistries, including plasma FA concentrations, and a 24-hour recall and a food frequency questionnaire assessment of omega-3 FA consumption. RESULTS: CHD was found in 6% of the cohort under 55 years of age and in 26% of those > or = 55 years of age. Eskimos with CHD consume as much omega-3 FAs as those without CHD, and the plasma concentrations confirm that dietary assessment. CONCLUSIONS: Average daily consumption of omega-3 FAs among Eskimos was high, with about 3-4 g/d reported, compared with 1-2 g/d used in intervention studies and the average consumption of 0.2 g/d by the American population. There was no association between current omega-3 FA consumption/blood concentrations and the presence of CHD.

Omega-3 fatty acids improve glucose tolerance and components of the metabolic syndrome in Alaskan Eskimos: the Alaska Siberia project.

OBJECTIVES: To test the hypothesis that the unusually low prevalences of insulin resistance (IR), metabolic syndrome (MS) and diabetes (DM) in Alaskan Eskimos, compared to American Indians, is related to the traditional Eskimo diet, high in C20-C22 omega-3 fatty acids (FAs). To determine if the relatively low blood pressures, low serum triglycerides and high HDL cholesterol levels in Eskimos result from high omega-3 FA consumption. STUDY DESIGN: Cross-sectional study. METHODS: We measured plasma FA concentrations in 447 Norton Sound Eskimos (35-74 years of age) and screened for DM, CHD and associated risk factors. A dietary assessment (24-hr recall) was obtained for comparison the day before the blood sampling. RESULTS: Plasma omega-3 FA concentrations were highly correlated with dietary omega-3 FAs and HDL levels and inversely correlated with plasma levels of insulin, 2-h insulin (OGTT), HOMI-IR, 2-h glucose (OGTT), triglyceride levels and diastolic blood pressure. CONCLUSIONS: High consumption of omega-3 FAs positively affects components of the MS, insulin sensitivity and glucose tolerance. This finding suggests that high consumption of C20-C22 omega-3 FAs protects against the development of the MS and glucose intolerance.

A successful diabetes prevention study in Eskimos: the Alaska Siberia project.

OBJECTIVES: To test the efficacy of a simple intervention method to reduce risk factors for type 2 diabetes (DM) and cardiovascular disease (CVD) in Alaskan Eskimos. STUDY DESIGN: The study consisted of 1) a comprehensive screening for risk factors of 454 individuals in 4 villages, 2) a 4-year intervention and 3) a repetition of the screening in year 5 to test the efficacy of the intervention. METHODS: Personal counseling (1hr/year) stressed the consumption of more traditional foods high in omega-3 fatty acids and less of certain specific store-bought foods high in palmitic acid, which was identified as being associated with glucose intolerance. RESULTS: The intervention resulted in significant reductions in plasma concentrations of total cholesterol (p = 0.0001), LDL cholesterol (p = 0.0001), fasting glucose (p = 0.0001), diastolic blood pressure (p = 0.0007) and improved glucose tolerance (p = 0.0006). This occurred without loss of body weight. Sixty percent of the participants had improved glucose tolerance; only one of the 44 originally identified with impaired glucose tolerance (IGT) developed DM during the study. CONCLUSIONS: Dramatic improvements of risk factors for DM and CVD were achieved in the intervention by primarily stressing the need for changes in the consumption of specific fats. The results suggest that fat consumption is an important risk factor for DM.

Gas chromatographic quantification of fatty acid methyl esters: flame ionization detection vs. electron impact mass spectrometry.

The determination of FAME by GC is among the most commonplace analyses in lipid research. Quantification of FAME by GC with FID has been effectively performed for some time, whereas detection with MS has been used chiefly for qualitative analysis of FAME. Nonetheless, the sensitivity and selectivity of MS methods advocate a quantitative role for GC-MS in FAME analysis-an approach that would be particularly advantageous for FAME determination in complex biological samples, where spectrometric confirmation of analytes is advisable. To assess the utility of GC-MS methods for FAME quantification, a comparative study of GC-FID and GC-MS methods has been conducted. FAME in prepared solutions as well as a biological standard reference material were analyzed by GC-FID and GC-MS methods using both ion trap and quadrupole MS systems. Quantification by MS, based on total ion counts and processing of selected ions, was investigated for FAME ionized by electron impact. Instrument precision, detection limits, calibration behavior, and response factors were investigated for each approach, and quantitative results obtained by each technique were compared. Although there were a number of characteristic differences between the MS methods and FID with respect to FAME analysis, the quantitative performance of GC-MS compared satisfactorily with that of GC-FID. The capacity to combine spectrometric examination and quantitative determination advances GC-MS as a powerful alternative to GC-FID for FAME analysis.

Quantitative analysis of perchlorate in extracts of whole fish homogenates by ion chromatography: comparison of suppressed conductivity detection and electrospray ionization mass spectrometry.

The perchlorate anion (ClO4-) is an anthropogenic contaminant of increasing concern in water supplies, and has been shown to disrupt thyroid activity. Most perchlorate analyses are currently carried out by ion chromatography (IC) with suppressed conductivity detection (SCD). While this procedure has been demonstrated to provide acceptable performance for analysis of water samples, the determination of perchlorate in high-conductivity aqueous extracts of plant or animal material is not readily accomplished by IC-SCD unless lengthy cleanup protocols are applied. With the addition of electrospray ionization mass spectrometry (ESI-MS) to IC, it was hypothesized that the interference imposed by various ionic species could be significantly reduced without the need for purification; however, the analysis of perchlorate in relatively unpurified extracts of biologically derived homogenates by IC-ESI-MS has not previously been described in the literature. The research presented here represents a comparison of the capabilities of IC-SCD and IC-ESI-MS to detect perchlorate in reagent water and in crude extracts of perchlorate-exposed fish (threespine stickleback, Gasterosteus aculeatus). ESI-MS was found to compare favorably to SCD for the detection of perchlorate in deionized water, and to exceed SCD performance in perchlorate analysis of fish-derived extracts.