Docosahexaenoic acid (DHA) has many unique physiological functions such as promoting the development of brain and retina in infants. Therefore, it is widely applied to food, pharmacy, breeding and other industries. To obtain engineered strains of Aurantiochytrium limacinum SR21 suitable for industrial application with increased lipid and DHA production, we designed a simple, fast, accurate and high-throughput screening method based on Nile red staining of oil droplets. First, ultraviolet C (UVC) mutagenesis was used to generate a random mutant library of A. limacinum. Second, screening conditions were optimized including staining conditions of Nile red and the sorting criterion. Thereby, three putative high-lipid mutants (D03432, D05106 and D01521) were selected from the mutant library containing 3 648 mutated clones. The three mutants grew faster and produced higher amounts of lipids and DHA compared to wild type (WT). In 100 mL cultures, the lipid content of D03432 and D05106 mutants reached 64.74% and 63.13% of dry cell weight respectively, whereas the wild strain exhibited only 43.19%. DHA yield in these two mutants were even 2.26-fold and 2.37-fold higher than that of the wild strain. Experiment with 5 L fermentor further confirmed that D03432 and D05106 mutants had better performance than the wild strain on DHA yield (45.51% and 66.46% more than that of the wild strain, respectively), and demonstrated their high potential for industrial application. This work not only generated several high-DHA content mutants with high potential for industrial use, but also provided vital guidance for high-throughput screening of lipid hyper-accumulating mutants in other oil-producing microorganisms.
Bioreactors ; Docosahexaenoic Acids ; Mutagenesis ; Staining and Labeling ; Stramenopiles

The fatty acid composition of spermatozoa has been shown to be important for their function, and L-carnitine is crucial for fatty acid metabolism. Its levels in the seminal plasma positively correlate with semen quality, whereas high body mass index (BMI) is associated with both reduced semen quality and altered sperm fatty acid composition. Here, we examined the associations between free seminal L-carnitine levels and sperm fatty acid composition as well as BMI. Semen samples were collected and analyzed from 128 men with unknown fertility status and with BMI ranging from 19 kg m^-2 to 63 kg m^-2. Sperm fatty acid composition was assessed by gas chromatography, while free seminal L-carnitine analysis was performed using high-performance liquid chromatography. Multiple linear regression analysis showed a positive correlation of free seminal L-carnitine levels with the amount of sperm palmitic acid (β = 0.21; P = 0.014), docosahexaenoic acid (DHA; β = 0.23; P = 0.007), and total n-3 polyunsaturated fatty acids (β = 0.23; P = 0.008) and a negative correlation of free seminal L-carnitine levels with lignoceric acid (β = -0.29; P = 0.001) and total n-6 polyunsaturated fatty acids (β = -0.24; P = 0.012) when adjusted for covariates. There was no relationship between free seminal L-carnitine levels and BMI. Since free seminal L-carnitine levels are associated with semen quality, the absence of a correlation with BMI suggests that reduced semen quality in obese men is independent of seminal L-carnitine.
Carnitine ; Docosahexaenoic Acids ; Fatty Acids ; Humans ; Male ; Semen ; Semen Analysis ; Sperm Count ; Sperm Motility ; Spermatozoa

Docosahexenoic acid (DHA) is an important polyunsaturated fatty acid which is beneficial to human health. Compared with the DHA derived from fish oil, DHA by microbial production possesses many advantages, and has a bright prospect. In this article, we reviewed strains, metabolic pathway, key enzymes and mechanism of lipid accumulation for microbial production of DHA. Those information would be greatly helpful for further improving DHA production by metabolic engineering.
Docosahexaenoic Acids ; biosynthesis ; Eukaryota ; metabolism ; Fermentation ; Industrial Microbiology ; methods ; Metabolic Engineering ; methods

OBJECTIVETo examine the effect of docosahexaenoic acid (DHA) deficiency in brain on spatial learning and memory in rats.

METHODSSprague Dawley rats were fed with an n-3 fatty acid deficient diet for two generations to induce DHA depletion in brain. DHA in seven brain regions was analyzed using the gas-liquid chromatography. Morris water maze (MWM) was employed as an assessing index of spatial learning and memory in the n-3 fatty acid deficient adult rats of second generation.

RESULTSFeeding an n-3 deficient diet for two generations depleted DHA differently by 39%-63% in the seven brain regions including cerebellum, medulla, hypothalamus, striatum, hippocampus, cortex and midbrain. The MWM test showed that the n-3 deficient rats took a longer time and swam a longer distance to find the escape platform than the n-3 Adq group.

CONCLUSIONThe spatial learning and memory in adult rats are partially impaired by brain DHA depletion.

Animals ; Brain ; metabolism ; Docosahexaenoic Acids ; metabolism ; Maze Learning ; physiology ; Memory ; physiology ; Rats ; Rats, Sprague-Dawley

OBJECTIVE: To investigate the levels of serum inflammatory cytokines and Resolvin D1 (RvD1) and their association with pathological staging of colon cancer. METHODS: Clinical data of 50 colon cancer patients (colon cancer group) admitted to the General Surgery Department of Zhongshan Hospital of Fudan University from January to December 2016 and 5 ml of whole blood specimen were collected at admission. During the same period, 50 healthy volunteers were enrolled (healthy volunteer group). Inclusion criteria for the colon cancer group: colon cancer diagnosed by preoperative colonoscopy and pathology; no recent enteral or parenteral nutrition support treatment or use of oral nutrition preparation; age ≤85 years; no surgical contraindications by preoperative evaluation; no history of taking fish oil-related preparations; no radiotherapy or chemotherapy before surgery. Healthy volunteer group enrollment criteria: no history of malignant tumors; no organ with organic lesions detected by the healthy examination center of our hospital; detection indicators in normal reference range; no administration of fish oil-related preparations; age ≤ 85 years. Serum inflammatory factors(IL-1β, IL-6, IL-10 and TNF-α) concentrations were detected by chemiluminescence immunoassay; serum RvD1 concentration was measured by enzyme-linked immunosorbent assay. The levels of inflammatory factors and RvD1 were compared between the two groups, and their associations with TNM staging of colon cancer patients were analyzed. RESULTS: There were no significant differences in age, gender and nutrition-related indicators between the two groups (all P>0.05). There were 31 males and 19 females in the healthy volunteer group with age of (61.8±11.6) years. There were 23 males and 27 females in the colon cancer group with age of (65.4±12.4) years. According to the 7th edition of the American Cancer Society TNM staging criteria, 10 cases were stage I, 13 cases stage II, 17 cases stage III, and 10 cases stage IV. Compared with healthy volunteer group, colon cancer group had higher serum IL-1β [(3.89±0.24)×10 μg/L vs.(1.55±0.37)×10 μg/L, t=37.52, P<0.01], higher IL-6 [(129.14±3.07)×10 μg/L vs.(51.46±3.14)×10 μg/L, t=125.08, P<0.01], higher IL-10 [(100.59±8.69)×103 μg/L vs.(27.57±4.77)×10 μg/L, t=52.09, P<0.01] and higher TNF-α [(114.31±4.43)×10 μg/L vs.(41.04±5.27)×10 μg/L, t=75.25, P<0.01], while lower RvD1 [(34.19±1.93)×10 μg/L vs.(77.76±1.02)×10 μg/L, t=140.56, P<0.01], all the differences were statistically significant. Subgroup analysis revealed that concentrations of IL-6, IL-1β, IL-10 and TNF-α gradually increased with the advancement of TNM staging (P<0.01). In stage III, concentrations of IL-6, IL-1β, and IL-10 were the highest, TNF-α concentration was the highest in stage IV. RvD1 concentration gradually decreased with the advancement of TNM staging(P<0.01). CONCLUSIONS Compared with healthy volunteers, the levels of serum inflammatory cytokines in colon cancer patients increase significantly while the level of RvD1 decreases significantly. Both are associated with higher TNM stage of colon cancer.
Aged ; Colonic Neoplasms ; blood ; immunology ; pathology ; Cytokines ; blood ; Docosahexaenoic Acids ; blood ; Female ; Humans ; Male ; Middle Aged

Compared with cultured fish, the fish oil of ocean wild fish contains much more Eicosapntemacnioc acid (EPA), Docosahexenoic acid (DHA), fat-soluble vitamin. To improve the utility value of oacean wild fish, small hairtail was used as raw material to investigate the technology of extracting fish oil with enzyme. The variables to affect the efficiency of extraction, extracting and centrifugation were selected as temperature, reaction time and pH value. Optimal technology conditions were determined by the response surface method: The liquid/solid ratio is 6, pH 7.3, enzyme amount of 1000 u/g raw material, agitation speed of 200 r/min, enzymolysis under 45 degrees C for 90 min. The optimum extraction conditions were as follows: 100 mL extractant (every 20 g surimi), pH4.0, extracted under 40 degrees C for 25 min. The optimal centrifuge conditions were: centrifuge speed of 3000 r/min (1865 g), centrifuged for 10 min. The oil extraction efficiency was 79.9%. This study developed the traditional technology of fish oil extraction, and improved the protection of the active components.
Animals ; Docosahexaenoic Acids ; analysis ; Eicosapentaenoic Acid ; analysis ; Enzymes ; metabolism ; Fish Oils ; isolation & purification ; Fishes ; metabolism ; Oceans and Seas ; Technology, Pharmaceutical ; methods

BACKGROUND/OBJECTIVES: Docosahexaenoic acid (DHA), an n-3 long chain polyunsaturated fatty acid (LCPUFA), is acquired by dietary intake or the in vivo conversion of α-linolenic acid. Many enzymes participating in LCPUFA synthesis are regulated by peroxisome proliferator-activated receptor alpha (PPARα). Therefore, it was hypothesized that the tissue accretion of endogenously synthesized DHA could be modified by PPARα. MATERIALS/METHODS: The tissue DHA concentrations and mRNA levels of genes participating in DHA biosynthesis were compared among PPARα homozygous (KO), heterozygous (HZ), and wild type (WT) mice (Exp I), and between WT mice treated with clofibrate (PPARα agonist) or those not treated (Exp II). In ExpII, the expression levels of the proteins associated with DHA function in the brain cortex and retina were also measured. An n3-PUFA depleted/replenished regimen was applied to mitigate the confounding effects of maternal DHA. RESULTS: PPARα ablation reduced the hepatic Acox, Fads1, and Fads2 mRNA levels, as well as the DHA concentration in the liver, but not in the brain cortex. In contrast, PPARα activation increased hepatic Acox, Fads1, Fads2 and Elovl5 mRNA levels, but reduced the DHA concentrations in the liver, retina, and phospholipid of brain cortex, and decreased mRNA and protein levels of the brain-derived neurotrophic factor in brain cortex. CONCLUSIONS: LCPUFA enzyme expression was altered by PPARα. Either PPARα deficiency or activation-decreased tissue DHA concentration is a stimulus for further studies to determine the functional significance.
Animals ; Brain ; Brain-Derived Neurotrophic Factor ; Clofibrate ; Docosahexaenoic Acids ; Fatty Acid Desaturases ; Liver ; Mice ; Peroxisomes ; PPAR alpha ; Retina ; RNA, Messenger

OBJECTIVETo investigate the effect of docosahexaenoic acid (DHA) on invasiveness of aflatoxin B1 (AFB1)-induced hepatocellular carcinoma cells in vitro.

METHODSHepG2.2.15 cells were exposed to different concentrations of AFB1 and DHA plus AFB1. The cell migration and invasion were assessed using wound-healing and Transwell assay, and flow cytometry was used to analyze the cell cycle changes. The ultrastructural changes of the cells were observed by transmission electron microscopy.

RESULTSCompared with the control group, the cells exposed to2 µmol/L AFB1 showed obviously enhanced migration and invasion with decreased cell ratio in G1/G1 phase and increased cell ratio in G2/M phase but no changes in S phase cells; transmission electron microscopy revealed the presence of multiple nucleoli and significantly increased mitochondria and Golgi apparatus in the exposed cells. Compared with AFB1-exposed cells, the cells treated with DHA and AFB1 showed decreased migration and invasion abilities, and the G1/G1 phase cells increased and G2/M phase cells decreased significantly; ultrastructurally, the cells contained single nucleoli with decreased mitochondria and vacuolization occurred in the cytoplasm.

CONCLUSIONDHA can significantly inhibit AFB1-induced enhancement of cell migration and invasion in hepatocellular carcinoma cells in vitro.

Aflatoxin B1 ; pharmacology ; Carcinoma, Hepatocellular ; pathology ; Cell Cycle ; Cell Movement ; drug effects ; Docosahexaenoic Acids ; pharmacology ; Golgi Apparatus ; Hep G2 Cells ; Humans ; Liver Neoplasms ; pathology ; Mitochondria ; Neoplasm Invasiveness

DHA (22:6n-3) is a Ω-3 polyunsaturated fatty acid with 22 carbon atoms and 6 double bonds, which has important biological functions in human body. Human and other mammals synthesize only limited amounts of DHA, more requirements must be satisfied from food resources. However, the natural resources of DHA (Mainly deep-sea fish and other marine products) are prone to depletion. New resources development is still insufficient to satisfy the growing market demand. Previous studies have revealed that the mammals can increase the synthesis of DHA and other long-chain polyunsaturated fatty acids after transgenic procedures. In this study, mammalian cells were transfected with Δ6, Δ5 desaturase, Δ6, Δ5 elongase, Δ15 desaturase (Isolated from nematode Caenorhabditis elegans) and Δ4 desaturase (Isolated from Euglena gracilis), simultaneously. Results show that the expression or overexpression of these 6 enzymes is capable of conversion of the o-6 linoleic acid (LA, 18:2n-6) in DHA (22:6n-3). DHA content has increased from 16.74% in the control group to 25.3% in the experimental group. The strategy and related technology in our research provided important data for future production the valuable DHA (22:6n-3) by using genetically modified animals.
Animals ; Caenorhabditis elegans ; enzymology ; Cells, Cultured ; Docosahexaenoic Acids ; chemistry ; Euglena gracilis ; enzymology ; Fatty Acid Desaturases ; biosynthesis ; Linoleic Acid ; chemistry ; Mammals ; Transfection

This study was carried out in adult ferret cardiomyocytes to investigate the effects of the n-3 polyunsaturated fatty acids (PUFAs) on voltage-gated K(+) currents. We report that the two outward K(+) currents: the transient outward K(+) current (I(to)) and the delayed rectifier K(+) current (I(K)), are both inhibited by the n-3 PUFAs, while the inwardly rectifying K(+) current (I(K1)) is unaffected by the n-3 PUFAs. Docosahexaenoic acid (C22:6n-3, DHA) produced a concentration dependent suppression of I(to) and I(K) in adult ferret cardiomyocytes with an IC(50) of 7.5 and 20 micromol/L, respectively; but not I(K1). In addition, eicosapentaenoic acid (C20:5n-3, EPA) had the effects on the three K(+) channels similar to DHA. Arachidonic acid (C20:4n-6, AA) at 5 or 10 micromol/L, after an initial inhibitory effect on I(K), caused an activation of I(K),AA which was prevented by pretreatment with indomethacin, a cyclooxygenase inhibitor. Monounsaturated and saturated fatty acids, which are not antiarrhythmic, lack the effects on these K(+) currents. Our results demonstrate that the n-3 PUFAs inhibit cardiac I(to) and I(K) with much less potency compared to their effects on cardiac Na(+) and Ca(2+) currents as we reported previously. This inhibition of the cardiac ion currents by the n-3 PUFAs may contribute to their antiarrhythmic actions.
Animals ; Arachidonic Acid ; pharmacology ; Docosahexaenoic Acids ; pharmacology ; Dose-Response Relationship, Drug ; Eicosapentaenoic Acid ; pharmacology ; Ferrets ; Myocytes, Cardiac ; drug effects ; metabolism ; Potassium Channels, Voltage-Gated ; metabolism