Pharmacokinetics of omega-3 fatty acids in patients with severe sepsis compared with healthy volunteers: A prospective cohort study.

BACKGROUND: Pharmacokinetics (PK) of pharmaceuticals and pharmaconutrients are poorly understood in critically ill patients, and dosing is often based on healthy subject data. This might be particularly problematic with enteral medications due to metabolic abnormalities and impaired gastrointestinal tract absorption common in critically ill patients. Utilizing enteral fish oil, this study was undertaken to better understand and define PK of enteral omega-3 fatty acids (eicospentaenoic acid [EPA] and docosahexaenoic acid [DHA]) in critically ill patients with severe sepsis. MATERIALS AND METHODS: Healthy volunteers (n = 15) and mechanically ventilated (MV) adults with severe sepsis (n = 10) were recruited and received 9.75 g EPA and 6.75 g DHA daily in two divided enteral doses of fish oil for 7 days. Volunteers continued their normal diet without other sources of fish oil, and sepsis patients received standard enteral feeding. Blood was collected at frequent intervals during the 14-day study period. Peripheral blood mononuclear cells (PMBCs) and neutrophils were isolated and analyzed for membrane fatty acid (FA) content. Mixed linear models and t-tests were used to analyze changes in FA levels over time and FA levels at individual time points, respectively. PK parameters were obtained based on single compartment models of EPA and DHA kinetics. RESULTS: Healthy volunteers were 41.1 ± 10.3 years; 67% were women. In patients with severe sepsis (55.6 ± 13.4 years, 50% women), acute physiologic and chronic health evaluation (APACHE) II score was 27.2 ± 8.8 at ICU admission and median MV duration was 10.5 days. Serum EPA and DHA were significantly lower in sepsis vs. healthy subjects over time. PBMC EPA concentrations were generally not different between groups over time, while PBMC DHA was higher in sepsis patients. Neutrophil EPA and DHA concentrations were similar between groups. The half-life of EPA in serum and neutrophils was significantly shorter in sepsis patients, whereas other half-life parameters did not vary significantly between healthy volunteers and sepsis patients. CONCLUSIONS: While incorporation of n-3 FAs into PBMC and neutrophil membranes was relatively similar between healthy volunteers and sepsis patients receiving identical high doses of fish oil for one week, serum EPA and DHA were significantly lower in sepsis patients. These findings imply that serum concentrations and EPA and DHA may not be the dominant driver of leukocyte membrane incorporation of EPA and DHA. Furthermore, lower serum EPA and DHA concentrations suggest that either these n-3 FAs were being metabolized rapidly in sepsis patients or that absorption of enteral medications and pharmaconutrients, including fish oil, may be impaired in sepsis patients. If enteral absorption is impaired, doses of enteral medications administered to critically ill patients may be suboptimal.

Dietary saturated fat and monounsaturated fat have reversible effects on brain function and the secretion of pro-inflammatory cytokines in young women.

BACKGROUND: Previous literature suggests that a higher ratio of palmitic acid (PA)/oleic acid (OA) in the diet induces inflammation, which may result in deficient brain insulin signaling, and, secondarily, impaired physical activity, sleep efficiency, and cognitive functioning. OBJECTIVE: We hypothesized that lowering the typical dietary PA/OA would affect the activation of relevant brain networks during a working memory task and would also lower secretion of pro-inflammatory cytokines. DESIGN: In 12 female subjects participating in a randomized, cross-over trial comparing 3-week high PA diet (HPA) and low PA and a high OA diet (HOA), we evaluated functional magnetic resonance imaging (fMRI) using an N-back test of working memory, cytokine secretion by lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells (PBMC), and plasma cytokine concentrations. RESULTS: Brain activation during the HPA diet compared to the HOA diet was increased in regions of the basal ganglia including the caudate and putamen (p<0.005). In addition, compared to the HOA diet, during the HPA diet, the plasma concentrations of IL-6 (p=0.04) and IL-1ß (p=0.05) were higher, and there was a higher secretion of IL-18 (p=0.015) and a trend for higher IL-1ß secretion (p=0.066) from LPS-stimulated PBMCs. CONCLUSIONS: The HPA diet resulted in increased brain activation in the basal ganglia compared to the HOA diet as well as increased secretion of pro-inflammatory cytokines. These data provide evidence that short-term (2week) diet interventions impact brain network activation during a working memory task and that these effects are reversible since the order of the study diets was randomized. These data are consistent with the hypothesis that lowering the dietary PA content via substitution with OA also could affect cognition.

Lipidomic evidence that lowering the typical dietary palmitate to oleate ratio in humans decreases the leukocyte production of proinflammatory cytokines and muscle expression of redox-sensitive genes.

We recently reported that lowering the high, habitual palmitic acid (PA) intake in ovulating women improved insulin sensitivity and both inflammatory and oxidative stress. In vitro studies indicate that PA can activate both cell membrane toll-like receptor-4 and the intracellular nucleotide oligomerization domain-like receptor protein (NLRP3). To gain further insight into the relevance to human metabolic disease of dietary PA, we studied healthy, lean and obese adults enrolled in a randomized, crossover trial comparing 3-week, high-PA (HPA) and low-PA/high-oleic-acid (HOA) diets. After each diet, both hepatic and peripheral insulin sensitivities were measured, and we assessed cytokine concentrations in plasma and in supernatants derived from lipopolysaccharide-stimulated peripheral blood mononuclear cells (PBMCs) as well as proinflammatory gene expression in skeletal muscle. Insulin sensitivity was unaffected by diet. Plasma concentration of tumor necrosis factor-α was higher during the HPA diet. Lowering the habitually high PA intake by feeding the HOA diet resulted in lower secretion of interleukin (IL)-1ß, IL-18, IL-10, and tumor necrosis factor-α by PBMCs, as well as lower relative mRNA expression of cJun and NLRP3 in muscle. Principal components analysis of 156 total variables coupled to analysis of covariance indicated that the mechanistic pathway for the differential dietary effects on PBMCs involved changes in the PA/OA ratio of tissue lipids. Our results indicate that lowering the dietary and tissue lipid PA/OA ratio resulted in lower leukocyte production of proinflammatory cytokines and muscle expression of redox-sensitive genes, but the relevance to diabetes risk is uncertain.

Increased palmitate intake: higher acylcarnitine concentrations without impaired progression of ß-oxidation.

Palmitic acid (PA) is associated with higher blood concentrations of medium-chain acylcarnitines (MCACs), and we hypothesized that PA may inhibit progression of FA ß-oxidation. Using a cross-over design, 17 adults were fed high PA (HPA) and low PA/high oleic acid (HOA) diets, each for 3 weeks. The [1-(13)C]PA and [13-(13)C]PA tracers were administered with food in random order with each diet, and we assessed PA oxidation (PA OX) and serum AC concentration to determine whether a higher PA intake promoted incomplete PA OX. Dietary PA was completely oxidized during the HOA diet, but only about 40% was oxidized during the HPA diet. The [13-(13)C]PA/[1-(13)C]PA ratio of PA OX had an approximate value of 1.0 for either diet, but the ratio of the serum concentrations of MCACs to long-chain ACs (LCACs) was significantly higher during the HPA diet. Thus, direct measurement of PA OX did not confirm that the HPA diet caused incomplete PA OX, despite the modest, but statistically significant, increase in the ratio of MCACs to LCACs in blood.

Muscle-Specific Overexpression of PGC-1α Does Not Augment Metabolic Improvements in Response to Exercise and Caloric Restriction.

This study used mice with muscle-specific overexpression of PGC-1α, a transcriptional coactivator that promotes mitochondrial biogenesis, to determine whether increased oxidative potential facilitates metabolic improvements in response to lifestyle modification. MCK-PGC1α mice and nontransgenic (NT) littermates were fed a high-fat diet (HFD) for 10 weeks, followed by stepwise exposures to voluntary wheel running (HFD+Ex) and then 25% caloric restriction with exercise (Ex/CR), each for an additional 10 weeks with continued HFD. Running and CR improved weight and glucose control similarly in MCK-PGC1α and NT mice. Sedentary MCK-PGC1α mice were more susceptible to diet-induced glucose intolerance, and insulin action measured in isolated skeletal muscles remained lower in the transgenic compared with the NT group, even after Ex/CR. Comprehensive profiling of >200 metabolites and lipid intermediates revealed dramatic group-specific responses to the intervention but did not produce a lead candidate that tracked with changes in glucose tolerance irrespective of genotype. Instead, principal components analysis identified a chemically diverse metabolite cluster that correlated with multiple measures of insulin responsiveness. These findings challenge the notion that increased oxidative capacity defends whole-body energy homeostasis and suggest that the interplay between mitochondrial performance, lipotoxicity, and insulin action is more complex than previously proposed.

Dietary intake of palmitate and oleate has broad impact on systemic and tissue lipid profiles in humans.

BACKGROUND: Epidemiologic evidence has suggested that diets with a high ratio of palmitic acid (PA) to oleic acid (OA) increase risk of cardiovascular disease (CVD). OBJECTIVE: To gain additional insights into the relative effect of dietary fatty acids and their metabolism on CVD risk, we sought to identify a metabolomic signature that tracks with diet-induced changes in blood lipid concentrations and whole-body fat oxidation. DESIGN: We applied comprehensive metabolomic profiling tools to biological specimens collected from 18 healthy adults enrolled in a crossover trial that compared a 3-wk high-palmitic acid (HPA) with a low-palmitic acid and high-oleic acid (HOA) diet. RESULTS: A principal components analysis of the data set including 329 variables measured in 15 subjects in the fasted state identified one factor, the principal components analysis factor in the fasted state (PCF1-Fasted), which was heavily weighted by the PA:OA ratio of serum and muscle lipids, that was affected by diet (P < 0.0001; HPA greater than HOA). One other factor, the additional principal components analysis factor in the fasted state (PCF2-Fasted), reflected a wide range of acylcarnitines and was affected by diet in women only (P = 0.0198; HPA greater than HOA). HOA lowered the ratio of serum low-density lipoprotein to high-density lipoprotein (LDL:HDL) in men and women, and adjustment for the PCF1-Fasted abolished the effect. In women only, adjustment for the PCF2-Fasted eliminated the HOA-diet effect on serum total- and LDL-cholesterol concentrations. The respiratory exchange ratio in the fasted state was lower with the HPA diet (P = 0.04), and the diet effect was eliminated after adjustment for the PCF1-Fasted. The messenger RNA expression of the cholesterol regulatory gene insulin-induced gene-1 was higher with the HOA diet (P = 0.008). CONCLUSIONS: These results suggest that replacing dietary PA with OA reduces the blood LDL concentration and whole-body fat oxidation by modifying the saturation index of circulating and tissue lipids. In women, these effects are also associated with a higher production and accumulation of acylcarnitines, possibly reflecting a shift in fat catabolism.

Substituting dietary monounsaturated fat for saturated fat is associated with increased daily physical activity and resting energy expenditure and with changes in mood.

BACKGROUND: The Western diet increases risk of metabolic disease. OBJECTIVE: We determined whether lowering the ratio of saturated fatty acids to monounsaturated fatty acids in the Western diet would affect physical activity and energy expenditure. DESIGN: With the use of a balanced design, 2 cohorts of 18 and 14 young adults were enrolled in separate randomized, double-masked, crossover trials that compared a 3-wk high-palmitic acid diet (HPA; similar to the Western diet fat composition) to a low-palmitic acid and high-oleic acid diet (HOA; similar to the Mediterranean diet fat composition). All foods were provided by the investigators, and the palmitic acid (PA):oleic acid (OA) ratio was manipulated by adding different oil blends to the same foods. In both cohorts, we assessed physical activity (monitored continuously by using accelerometry) and resting energy expenditure (REE). To gain insight into a possible mood disturbance that might explain changes in physical activity, the Profile of Mood States (POMS) was administered in cohort 2. RESULTS: Physical activity was higher during the HOA than during the HPA in 15 of 17 subjects in cohort 1 (P = 0.008) (mean: 12% higher; P = 0.003) and in 12 of 12 subjects in the second, confirmatory cohort (P = 0.005) (mean: 15% higher; P = 0.003). When the HOA was compared with the HPA, REE measured during the fed state was 3% higher for cohort 1 (P < 0.01), and REE was 4.5% higher in the fasted state for cohort 2 (P = 0.04). POMS testing showed that the anger-hostility score was significantly higher during the HPA (P = 0.007). CONCLUSIONS: The replacement of dietary PA with OA was associated with increased physical activity and REE and less anger. Besides presumed effects on mitochondrial function (increased REE), the dietary PA:OA ratio appears to affect behavior. The second cohort was derived from a study that was registered at clinicaltrials.gov as R01DK082803.

A lipidomics analysis of the relationship between dietary fatty acid composition and insulin sensitivity in young adults.

Relative to diets enriched in palmitic acid (PA), diets rich in oleic acid (OA) are associated with reduced risk of type 2 diabetes. To gain insight into mechanisms underlying these observations, we applied comprehensive lipidomic profiling to specimens collected from healthy adults enrolled in a randomized, crossover trial comparing a high-PA diet to a low-PA/high-OA (HOA) diet. Effects on insulin sensitivity (SI) and disposition index (DI) were assessed by intravenous glucose tolerance testing. In women, but not men, SI and DI were higher during HOA. The effect of HOA on SI correlated positively with physical fitness upon enrollment. Principal components analysis of either fasted or fed-state metabolites identified one factor affected by diet and heavily weighted by the PA/OA ratio of serum and muscle lipids. In women, this factor correlated inversely with SI in the fasted and fed states. Medium-chain acylcarnitines emerged as strong negative correlates of SI, and the HOA diet was accompanied by lower serum and muscle ceramide concentrations and reductions in molecular biomarkers of inflammatory and oxidative stress. This study provides evidence that the dietary PA/OA ratio impacts diabetes risk in women.

Zinc-finger nuclease editing of human cxcr4 promotes HIV-1 CD4(+) T cell resistance and enrichment.

HIV-1-infected individuals can harbor viral isolates that can use CCR5, as well as CXCR4, for viral entry. To genetically engineer HIV-1 resistance in CD4(+) T cells, we assessed whether transient, adenovirus delivered zinc-finger nuclease (ZFN) disruption of genomic cxcr4 or stable lentiviral expression of short hairpin RNAs (shRNAs) targeting CXCR4 mRNAs provides durable resistance to HIV-1 challenge. ZFN-modification of cxcr4 in CD4(+) T cells was found to be superior to cell integrated lentivirus-expressing CXCR4 targeting shRNAs when CD4(+) T cells were challenged with HIV-1s that utilizes CXCR4 for entry. Cxcr4 disruption in CD4(+) T cells was found to be stable, conferred resistance, and provided for continued cell enrichment during HIV-1 infection in tissue culture and, in vivo, in peripheral blood mononuclear cell transplanted NSG mice. Moreover, HIV-1-infected mice with engrafted cxcr4 ZFN-modified CD4(+) T cells demonstrated lower viral levels in contrast to mice engrafted with unmodified CD4(+) T cells. These findings provide evidence that ZFN-mediated disruption of cxcr4 provides a selective advantage to CD4(+) T cells during HIV-1 infection.

Relative red blood cell enzyme levels as a clue to the diagnosis of pyruvate kinase deficiency.

Evaluation of two patients with transfusion dependent anemia revealed RBC pyruvate kinase to be 33% and 41% of the mean normal value, with normal or high values of other RBC enzymes. Parental PK activities were just below normal in three of four of the parents. Subsequent DNA analysis revealed both patients to be compound heterozygotes for PKLR gene mutations, two of which are previously undescribed. Borderline low pyruvate kinase activities with increased in other RBC enzyme activities should prompt consideration of measurement of parental enzyme activities, and confirmation by DNA analysis if available.

A new case of human atransferrinemia with a previously undescribed mutation in the transferrin gene.

Hereditary atransferrinemia is a very rare disorder characterized by microcytic anemia and iron overload. It has been reported in only 10 patients in 8 families. The molecular basis of atransferrinemia has been determined in only 3 human cases. We now report a new patient with this rare disorder, who is the first known case in Turkey, the 11th patient reported in the published literature and only the 4th case of human atransferrinemia characterized on a molecular basis. DNA analysis of the serum transferrin gene in the patient revealed a previously undescribed mutation in exon 4, a G-->A transition at cDNA 410(Cys137Tyr). A number of previously known polymorphisms and a previously undescribed mutation at IVS10(-23)C-->T, presumably a polymorphism, were also documented.

Human chitotriosidase polymorphisms G354R and A442V associated with reduced enzyme activity.

A common polymorphism in the chitotriosidase gene (CHIT1) exists in which a 24 bp duplication in exon 10 results in aberrant splicing and deletion of 87 nucleotides. In this study, the gene frequency was found to be 0.56 (n=2054) in subjects of Asian ancestry, 0.17 (n=984) in subjects of European ancestry and 0.07 (n=536) in subjects of African ancestry. Notably, the median enzyme activity in wild-type subjects (TT) was much higher in subjects of European ancestry (2.69 mU/ml, n=202 subjects), than subjects of African (2.57 mU/ml, n=230 subjects) (P<0.0001) and Asian ancestry (0.86 mU/ml, n=114 subjects) (P<0.0001). The question of why chitotriosidase deficiency exists at such a high frequency is a challenging one. We postulated that if there was a selective advantage for chitotriosidase deficiency then there would be polymorphisms that would be associated with reduced enzyme activity independent of the 24 bp duplication. We found that the G102S and the A442G polymorphisms found occurring in subjects of all ancestries were not significantly associated with a reduction of enzyme activity. In contrast, the G354R (P<0.0001) and the A442V (P=0.0013) polymorphisms occurring predominantly in subjects of African ancestry were significantly associated with reduced chitotriosidase activity. We also investigated the possibility that chitotriosidase deficiency was associated with tuberculosis or with atopy, including allergic rhinitis, contact dermatitis, food or drug allergies and asthma.

Genetic polymorphisms and susceptibility to lung disease.

Susceptibility to infection by bacterium such as Bacillus anthracis has a genetic basis in mice and may also have a genetic basis in humans. In the limited human cases of inhalation anthrax, studies suggest that not all individuals exposed to anthrax spores were infected, but rather, individuals with underlying lung disease, particularly asthma, sarcoidosis and tuberculosis, might be more susceptible. In this study, we determined if polymorphisms in genes important in innate immunity are associated with increased susceptibility to infectious and non-infectious lung diseases, particularly tuberculosis and sarcoidosis, respectively, and therefore might be a risk factor for inhalation anthrax. Examination of 45 non-synonymous polymorphisms in ten genes: p47phox (NCF1), p67phox (NCF2), p40phox (NCF4), p22phox (CYBA), gp91phox (CYBB), DUOX1, DUOX2, TLR2, TLR9 and alpha 1-antitrypsin (AAT) in a cohort of 95 lung disease individuals and 95 control individuals did not show an association of these polymorphisms with increased susceptibility to lung disease.