Temporal patterning of apical progenitors and their daughter neurons in the developing neocortex.

During corticogenesis, distinct subtypes of neurons are sequentially born from ventricular zone progenitors. How these cells are molecularly temporally patterned is poorly understood. We used single-cell RNA sequencing at high temporal resolution to trace the lineage of the molecular identities of successive generations of apical progenitors (APs) and their daughter neurons in mouse embryos. We identified a core set of evolutionarily conserved, temporally patterned genes that drive APs from internally driven to more exteroceptive states. We found that the Polycomb repressor complex 2 (PRC2) epigenetically regulates AP temporal progression. Embryonic age-dependent AP molecular states are transmitted to their progeny as successive ground states, onto which essentially conserved early postmitotic differentiation programs are applied, and are complemented by later-occurring environment-dependent signals. Thus, epigenetically regulated temporal molecular birthmarks present in progenitors act in their postmitotic progeny to seed adult neuronal diversity.

When a parent suffers ABI: Investigation of emotional distress in children.

PRIMARY OBJECTIVE: To investigate the type of emotional and behavioural impact that having a parent with a severe acquired brain injury (ABI) has on children during the first period of adjustment. METHODS AND PROCEDURE: The study involved 25 couples in which one of the spouses was affected by ABI, and their 35 children (3-14 years). The children attended three sessions with a psychologist aimed at identifying their spontaneous playing and relational behaviour by means of a grid created on the basis of ICD-10 criteria. Both members of each parental couple attended a session with the psychologist, and were administered the Dyadic Adjustment Scale, the 36-item Health Survey and the Caregiver Burden Inventory. RESULTS: 63% of the children showed signs of emotional suffering, the presence of which was underestimated by their parents on the basis of the psychologist's assessments. The variables that correlated most closely with the children's psychological condition were related to the quality of their parents' relationship. CONCLUSIONS: Our findings confirm the need for early interventions aimed at both parents and their children in order to investigate the children's emotional-affective situation, and favour an understanding of their discomfort by their parents.

(-)-Epigallocatechin-3-gallate downregulates Pg-P and BCRP in a tamoxifen resistant MCF-7 cell line.

We investigated the anticancer effect of EGCG treatment on a breast carcinoma cell line resistant to tamoxifen (MCF-7Tam cells). As there are no reports about the molecular mechanisms implicated in EGCG treatment of tamoxifen resistant breast carcinoma cells, we studied the effects of EGCG treatment on three plasma membrane proteins that are involved in the mechanism of drug-resistance: Multidrug Resistance Protein (MRP1), P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP). EGCG treatment (10-100 microg/ml for 24-72 hours) caused cell growth inhibition and dose-dependent apoptosis: after 100 microg/ml EGCG treatment for 24 hours, Bax expression increased and Bcl2 expression decreased (p<0.05). Coherently, Annexin V-FITC apoptosis assay detected a significant increase in labelled cells (p<0.05). EGCG did not affect MRP1: in contrast, 100 microg/ml EGCG administration caused P-gp decrease to 53% of control cells (p<0.001) and this effect was not due to downregulation of P-gp gene expression. EGCG induced P-gp decrease even when MG132, a strong proteasome inhibitor, was given together with EGCG to MCF-7Tam cells. EGCG treatment also inhibited BCRP activity: mRNA transcription and protein level did not change after treatment, but mitoxantrone test demonstrated a strong inhibition of BCRP activity (p<0.001). In conclusion, the present results showed that EGCG could down-regulate the activity of two molecules that play a key role in drug metabolism and transport and that are highly expressed in tamoxifen resistant breast carcinoma cells. The interaction of EGCG and drugs used in the therapy of estrogen sensitive breast carcinoma ought to be subject of studies and the potential use of EGCG in drug-resistant diseases ought to be better considered.

Somatostatin as a regulator of first-trimester human trophoblast functions.

We have tested the hypothesis that human early trophoblast is a target for somatostatin (SRIF) regulatory actions. We report for the first time that SSTR2A and 2B transcripts and proteins are present in first-trimester human chorionic villi and the trophoblast-derived HTR-8/SVneo and JAR cells. In both cell lines, SSTR are functional since SRIF inhibits cyclic AMP pathway, stimulates arachidonic acid release and enhances cell proliferation. Moreover, in HTR-8/SVneo cells, considered a good model of first-trimester EVT, SRIF also enhances migration. An involvement of the cyclic AMP pathway in mediating SRIF effects on proliferation and migration is suggested. Our data support the idea that SRIF regulates early trophoblast functions mainly through an interaction with SSTR2.

Growth inhibition and proapoptotic activity induction by IIF and valproic acid on RA-resistant leukemia cells.

All-trans retinoic acid (RA) induces complete remission in a high proportion of patients with acute promyelocytic leukemia (APL). Nevertheless, most of these patients develop RA resistance and relapse. In an attemp to mimic clinical conditions for the treatment of leukemia, a stably RA-resistant subclone of the human promyelocytic leukemia cell line HL60 (HL60-R) was developed to study the antiproliferative and proapoptotic effect of the retinoid IIF (6-OH-11-O-hydroxyphenantrene) in comparison with RA. Moreover whether the inhibitor of histone deacetylase (HDAC) activity, valproic acid (VPA), could enhance sensitivity to retinoids in HL60-R cells was evaluated. Finally, the effect of IIF on the expression of multidrug resistance-associated protein 1 (MRP1) and P-glycoprotein (P-gp) was evaluated. It was found that IIF strongly suppressed cell proliferation (as measured by growth curves) and induced apoptosis (as measured by DNA fragmentation and Annexin V detection assays), while RA was practically ineffective. The addition of VPA to IIF accentuated the antiproliferative effect of IIF alone and increased apoptosis; the combination of VPA with RA allowed growth arrest. Moreover IIF caused a reduction of transmembrane transporter expression, particularly of P-gp, as shown by Western blotting. Our results suggest that IIF may be useful in controlling the proliferation of RA-resistant leukemia cells, especially in combination with an HDAC inhibitor, such as VPA.

Inhibitory effects of retinoic acid and IIF on growth, migration and invasiveness in the U87MG human glioblastoma cell line.

Glioblastomas, the most malignant and prevalent brain tumors which remain incurable, are characterized by both extensive proliferation and invasive growth. We previously reported a remarkable antitumoral effect of the retinoid 6-OH-11-O-hydroxyphenantrene (IIF) on neuroblastoma, leukemia and colon carcinoma cells. In this study we examined the effect of IIF on proliferation, apoptosis and cell invasion in the human glioblastoma cell line U87MG, in comparison with all-trans-retinoic acid (RA). Our results showed that both retinoids induced cell growth inhibition and apoptosis in a dose- and time-dependent manner. We also demonstrated that the invasive ability of glioblastoma cells decreased after treatment with IIF or RA. Since cell invasion involves a complex system of tightly regulated proteases, matrix metalloproteinases (MMPs) and their specific inhibitors, tissue inhibitors of MMPs (TIMPs), we analysed the effect of IIF on MMP and TIMP expression in comparison with RA. Treatment with both retinoids resulted in a marked decrease of MMP2 and MMP9 expression and of lytic activity of MMP2. In addition, exposure to IIF led to enhanced expression of TIMP2. Collectively, our results demonstrated the effectiveness of both IIF and RA in inhibiting proliferation, cell migration, and the invasive potential of glioblastoma U87MG cells. Notably, the anticancer activity of IIF, on the whole, was more pronounced than that of RA. Therefore, these findings, besides providing further evidence that IIF may be a powerful tool in the development of cancer treatments, suggest that IIF may have therapeutic potential against the invasiveness of brain tumors.

A search for multidrug resistance modulators: the effects of retinoids in human colon carcinoma cells.

The development of multidrug resistance (MDR) is one of the major causes of failure in cancer therapy. The use of cell lines with acquired resistance to anticancer agents represents a very good tool for investigation into the possibility of reversal of MDR. In this study the ability of all-trans-retinoic acid (RA) and its derivative 6-OH-11-O-hydroxyphenanthrene (IIF; pat. WIPO W000 /117143) as antitumor agents was investigated in the human colon carcinoma cell line LoVo and in the counterpart resistant derivative LoVo/MDR cell line. Cell proliferation was measured by MTT assay, apoptosis was evaluated using DNA fragmentation and Annexin V detection assay. Retinoids suppressed cell proliferation in a time- and dose-dependent manner. Interestingly, IIF was significantly more effective than RA, particularly on LoVo/MDR cells. RA and IIF induced apoptosis in both cell lines, with IIF effect significantly higher than that of RA. Furthermore, on the basis that MDR phenotype is often caused by drug efflux due to overexpression of the membrane P-glycoprotein (P-gp), it was demonstrated that RA and IIF reduced P-gp synthesis in LoVo/MDR cells. Our results suggest that IIF could be a powerful tool in the development of colon carcinoma treatments, even when tumor cells present an MDR phenotype.

Retinoids and cancer: antitumor effect of ATRA and of a new derivative of retinoic acid, IIF, on colon carcinoma cell lines CaCo-2 and HT-29.

Vitamin A and its metabolic forms, like all-trans retinoic acid (ATRA), are used with promising results in the treatment of many tumors. Two major problems in the clinical use of retinoids are that the doses needed for successful treatment are often toxic, leading to "hypervitaminosis A syndrome" and that patients often develop drug resistance. In order to find compounds that can overcome these problems, many new derivatives of retinoids have been synthesized and tested. Here we present a study on the effect of a new derivative of retinoic acid, IIF (pat. WIPO W0 00/17143), on growth and differentiation of two colon carcinomna cell lines, CaCo-2 and HT-29, with different degrees of tumorigenicity, the second one being more undifferentiated. The effect of IIF was compared with that of ATRA, whose antitumoral action on colon cancer cells and other tumoral cells is widely described in the literature. Besides exerting a strong antiproliferative effect, even higher than that of ATRA, IIF induced cellular differentiation, as demonstrated by the appearance of morphological (domes and microvilli formation) and biochemical (alkaline phosphatase induction) markers. Therefore, these findings indicate the new retinoid IIF as a possible candidate in the treatment of colon cancer.

Contrasting effects of t10,c12- and c9,t11-conjugated linoleic acid isomers on the fatty acid profiles of mouse liver lipids.

The purpose of this study was to examine the effects of two purified isomers of CLA (c9,t11-CLA and t10,c12-CLA) on the weights and FA compositions of hepatic TG, phospholipids, cholesterol esters, and FFA. Eight-week-old female mice (n = 6/group) were fed either a control diet or diets supplemented with 0.5% c9,t11-CLA or t10,c12-CLA isomers for 8 wk. Weights of liver total lipids and those of individual lipid fractions did not differ between the control and the c9,t11-CLA groups. Livers from animals fed the t10, c12-CLA diet contained four times more lipids than those of the control group; this was mainly due to an increase in the TG fractions (fivefold), but cholesterol (threefold), cholesterol esters (threefold), and FFA (twofold) were also significantly increased. Although c9,t11-CLA did not significantly alter the weights of liver lipids when compared with the control group, its intake was associated with significant reductions in the weight percentage (wt% of total FAME) of 18:1n-9 and 18:1n-7 in the TG fraction and with significant increases in the weight percentage of 18:2n-6 in the TG, cholesterol ester, and phospholipid fractions. On the other hand, t10,c12-CLA intake was linked with a significant increase in the weight percentage of 18:1n-9 and a decrease in that of 18:2n-6 in all lipid fractions. These changes may be the result of alterations in the activity of delta9-desaturase (stearoyl CoA desaturase) and the enzymes involved in the metabolism of 18:2n-6. Thus, the two isomers differed not only in their effects on the weights of total liver lipids and lipid fractions but also on the FA profile of the lipid fractions.

Trans-10,cis-12 CLA increases liver and decreases adipose tissue lipids in mice: possible roles of specific lipid metabolism genes.

Although consumption of CLA mixtures has been associated with several health effects, less is known about the actions of specific CLA isomers. There is evidence that the t10,c12-CLA isomer is associated with alterations in body and organ weights in animals fed CLA, but the mechanisms leading to these changes are unclear. The purpose of this study was to determine the effects of two commonly occurring isomers of CLA on body composition and the transcription of genes associated with lipid metabolism. Eight-week-old female mice (n = 11 or 12/group) were fed either a control diet or diets supplemented with 0.5% c9,t11-CLA or t10,c12-CLA isomers or 0.2% of the peroxisome proliferator-activated receptor alpha (PPARalpha) agonist fenofibrate for 8 wk. Body and retroperitoneal adipose tissue weights were significantly lower (6-10 and 50%, respectively), and liver weights were significantly greater (100%) in the t10,c12-CLA and the fenofibrate groups compared with those in the control group; body and tissue weights in the c9,t11-CLA group did not differ from those in the control group. Livers from animals in the t10,c12-CLA group contained five times more lipids than in the control group, whereas the lipid content of the fenofibrate group did not differ from that in the control group. Although fenofibrate increased the mRNA for PPARalpha, t10,c12-CLA decreased it. These results suggest that PPARalpha did not mediate the effects of t10,c12-CLA on body composition. The CLA isomers and fenofibrate altered mRNA levels for several proteins involved in lipid metabolism, but the most striking difference was the reduction of mRNA for leptin and adiponectin in the t10,c12-CLA group. These initial results suggest that changes associated with energy homeostasis and insulin action may mediate the effects of t10,c12-CLA on lipid metabolism.

Effect of a new derivative of retinoic acid on proliferation and differentiation in human neuroblastoma cells.

Neuroblastoma, a tumor originating from the sympathetic nervous system, is the most common extracranial malignant solid tumor of childhood. Human neuroblastoma cells may differentiate in vitro under treatment with a variety of biological agents and drugs. Among these, retinoic acid (RA) is quite potent and its effectiveness as a therapeutic agent is now being evaluated in clinical trials. As its pleiotropic biological activities may produce side-effects limiting clinical use, it is important to find new compounds that present the same effectiveness together with few side-effects. In this study we have explored the action of IIF, (pat. WIPO W0 00/17143) a new derivative of RA, as a differentiation inducer in the human neuroblastoma cell line TS12. In the same cell line, we have also compared the effect of IIF with that of all trans RA (ATRA) and of 9 cis RA (9cRA), with respect to morphological and biochemical differentiation and growth inhibition. Treatment with IIF resulted in a strong inhibition of proliferation and in a marked induction of neuronal differentiation as revealed by neurite extension, increase of actylcholinesterase (AchE) specific activity and tyrosine hydroxylase (TH) expression. The results demonstrate the effectiveness of this new retinoid as a differentiation inducer on neuroblastoma cells TS12. Furthermore, the differentiation-promoter and antimitotic activities of IIF were on the whole more pronounced than those of ATRA and 9cRA. Therefore our study suggests the evaluation of the new retinoid IIF as a therapeutic approach in the treatment of neuroblastoma.

Retinoids and cancer: antitumoral effects of ATRA, 9-cis RA and the new retinoid IIF on the HL-60 leukemic cell line.

OBJECTIVE: To compare the antitumoral effects of all-trans retinoic acid (ATRA) and 9-cis retinoic acid (9-cis RA) with those of 5-OH,11-O-hydrophenanthrene (IIF), a new derivative of retinoic acid. MATERIALS AND METHODS: The effect of retinoids was tested on cell line HL-60. Cell differentiation and apoptosis were evaluated by morphological and biochemical analysis as BCL-2 protein and by DNA fragmentation assay. The ability to activate retinoic acid receptors (RAR) and/or retinoid X receptors (RXR) and to modulate gene expression was determined by transactivation assay. RESULTS: With cell line HL-60, the antiproliferative effect of IIF was stronger than that of ATRA and 9-cis RA. Following retinoid treatment, cells appeared to differentiate and apoptotic cells were observed. The appearance of DNA laddering and a decrease in the amount of BCL-2 protein confirmed apoptosis. IIF transcriptionally activated RXR-gamma more than RAR-alpha. CONCLUSION: The findings indicate that IIF transcriptionally activates RXR-gamma preferentially, induces apoptosis and has a more antiproliferative activity than ATRA and 9-cis RA on cell line HL-60.

Similar effects of c9,t11-CLA and t10,c12-CLA on immune cell functions in mice.

Published results regarding the effects of CLA on immune cell functions have ranged from stimulation to inhibition. In those studies, a mixture of CLA isomers were used, and food intake was not controlled. We have examined whether the discrepancies in the results of earlier studies may be due to the lack of controlled feeding and whether the two isomers of CLA may differ in their effects on immune cell functions. Three groups of C57BL/6 female mice were fed either a control, c9,t11-CLA-, or t10,c12-CLA (0.5 wt%)-supplemented diet, 5 g/d, for 56 d. At the end of the study, the number of immune cells in spleens, bone marrows, or in circulation; proliferation of splenocytes in response to T and B cell mitogens; and prostaglandin secretion in vitro did not differ among the three groups. Both CLA isomers significantly increased in vitro tumor necrosis factor alpha and interleukin (IL)-6 secretion and decreased IL-4 secretion by splenocytes compared to those in the control group. Thus, the two CLA isomers had similar effects on all response variables tested. The discrepancies among the results from previous studies did not seem to be caused by the differences in the isomer composition of CLA used.

The effect of conjugated linoleic acid on platelet function, platelet fatty acid composition, and blood coagulation in humans.

Despite extensive research on conjugated linoleic acid (CLA) showing multiple beneficial effects in animal models, little is known about the role of dietary CLA in human health. To investigate if the beneficial effects of CLA seen in animal models are relevant to humans, we conducted a study with 17 healthy female volunteers who lived in the Metabolic Research Unit of the Western Human Nutrition Research Center for 93 d. This paper reports only the results from this study that are related to the effects of CLA supplementation on blood coagulation, platelet function, and platelet fatty acid composition. Throughout the study, the subjects were fed a low-fat diet (30 en% fat, 19 en% protein, and 51 en% carbohydrate) consisting of natural foods with the recommended dietary allowances for all known nutrients. After a 30-d stabilization period, subjects were randomly assigned to either an intervention group (n = 10) whose diet was supplemented with 3.9 g/d of CLA or a control group (n = 7) who received an equivalent amount of sunflower oil consisting of 72.6% linoleic acid with no detectable CLA. Platelet aggregation was measured in platelet-rich plasma using adenosine diphosphate, collagen, and arachidonic acid agonists. No statistical difference was detected between the amount of agonist required to produce 50% aggregation of platelet-rich plasma before and after the subjects consumed the CLA, with the exception of a decrease in response to collagen. This decrease was found in both control and intervention groups with no significant difference between the groups, suggesting that both linoleic acid (sunflower oil) and CLA might have similar effects on platelet function. The prothrombin time, activated partial thromboplastin time, and the antithrombin III levels in the subjects were determined. Again, there was no statistically significant difference in these three parameters when pre- and post-CLA consumption values were compared. The in vivo bleeding times were also unaffected by CLA supplementation (10.4 + 2.8 min pre- and 10.2 + 1.6 min postconsumption). Platelet fatty acid composition was not markedly influenced by the consumption of dietary CLA, although there was a small increase in the amount of the 9 cis,11 trans-18:2 isomer normally present in platelets after feeding CLA for 63 days. In addition, small amounts of the 8 trans,10 cis-18:2 and the 10 trans,12 cis-18:2 isomers were detected in the platelets along with traces of some of the other isomers. Thus, when compared to sunflower oil, the blood-clotting parameters and in vitro platelet aggregation showed that adding 3.9 g/d of dietary CLA to a typical Western diet for 63 d produces no observable physiological change in blood coagulation and platelet function in healthy adult females. Short-term consumption of CLA does not seem to exhibit antithrombotic properties in humans.

The effect of conjugated linoleic acid on plasma lipoproteins and tissue fatty acid composition in humans.

Conjugated linoleic acid (CLA) has been suggested by some animal studies to possess antiatherogenic properties. To determine, in humans, the effect of dietary CLA on blood lipids, lipoproteins, and tissue fatty acid composition, we conducted a 93-d study with 17 healthy female volunteers at the Metabolic Research Unit of the Western Human Nutrition Research Center. Throughout the study, subjects were fed a low-fat diet [30 energy percent (en%) fat, 19 en% protein, and 51 en% carbohydrate] that consisted of natural foods with the recommended dietary allowances for all known nutrients. After a 30-d stabilization period, subjects were randomly assigned to either an intervention group (n = 10) supplemented daily with capsules containing 3.9 g of CLA or a control group (n = 7) that received an equivalent amount of sunflower oil. The CLA capsules (CLA 65%) contained four major cis/trans geometric isomers (11.4% 9 cis-,11 trans-18:2; 10.8% 8 trans-,10 cis-18:2; 15.3% 11 cis-,13 trans-18:2; and 14.7% 10 trans-,12 cis-18:2) and their corresponding cis/cis (6.74% total) and trans/trans (5.99% total) varieties in smaller amounts. Fasting blood was drawn on study days 30 (end of the stabilization period), 60 (midpoint of the intervention period), and 93 (end of the intervention period). Adipose tissue samples were taken on days 30 and 93. CLA supplementation for 63 d did not change the levels of plasma cholesterol, low density lipoprotein cholesterol, high density lipoprotein cholesterol, and triglycerides. The weight percentage of CLA in plasma increased from 0.28 +/- 0.06 to 1.09 +/- 0.31 (n = 10, P < 0.05) after the supplementation. The 9 cis-,11 trans-isomer was the most prominent variety followed by the 11 cis-,13 trans- and 10 trans-,12 cis-isomers in lesser amounts. CLA in adipose tissue was not influenced by the supplementation (0.79 +/- 0.18 to 0.83 +/- 0.19 wt%) (n = 10) and the 9 cis-,11 trans-variety was the only isomer present. Thus, contrary to findings from some animal studies, CLA does not seem to offer health benefits, in the short term, regarding the prevention of atherosclerosis in humans. CLA supplementation for 2 mon did not alter the blood cholesterol or lipoprotein levels of healthy, normolipidemic subjects. The supplementation did increase CLA in the plasma but only 4.23% of the ingested CLA was present in the plasma at any given time. No adverse effect of CLA supplementation was detected in this study.

Colocalization prostacyclin (PGI2) synthase--caveolin-1 in endothelial cells and new roles for PGI2 in angiogenesis.

In vascular cells, prostacyclin (PGI2) synthase (PGI2s) has been localized in the endoplasmic reticulum of endothelial cells and in the nuclear and plasma membrane of smooth muscle cells. In human umbilical vein endothelial (HUVE) cells, we detected the enzyme in abundant cytoplasmic vesicles apparently originating from the plasma membrane and similar to those stained by gold-albumin, which interacts with a caveolar receptor. This prompted us to try a direct confocal microscopy approach aimed at colocalizing gold-albumin, caveolin-1, and PGI2 synthase. Moreover, the staining of HUVE cells with an anti-BiP7Grp78 antibody (a marker of endoplasmic reticulum) shows a perinuclear localization, sharply separated from PGI2 synthase localization. The results indicate that more than 80% of the enzyme resides in cellular sites costaining with caveolin-1 antibody and gold-albumin. This evidence was confirmed by the demonstration that PGI2 synthase and caveolin-1 coimmunoprecipitate in HUVE cell lysates and that they are associated to detergent-insoluble membrane domains in the same low-density fractions of a sucrose gradient. In addition, depletion of cellular cholesterol by mevalonate and methyl-beta-cyclodextrin leads to the shift of PGI2 synthase and caveolin-1 to higher density fractions of the gradient. Biochemical evidence about colocalization was supported by the use of a fusion protein glutathione S-transferase (GST)/caveolin-1, which retained either PGI2s purified from ram seminal vesicles or PGI2s present in HUVE cell lysates. Binding of PGI2s to caveolin "scaffolding domain" and to C-terminal region was deduced by using full-length GST--Cav-1, GST--Cav 61--101, and GST C- and N-terminal fusion proteins. A double approach based on the usage of filipin as a specific caveolae-disrupting agent and antisense oligonucleotides targeting PGI2 synthase mRNA suggests that the production of PGI2 in caveolae is likely to be connected to the regulation of angiogenesis, at least in vitro.

Pulsed degenerate optical parametric oscillator based on a nonlinear-fiber Sagnac interferometer.

We report operation of an all-fiber degenerate optical parametric oscillator that employs a nonlinear-fiber Sagnac interferometer as a parametric amplifier. Synchronous pumping with 3.9-ps pulses at 1544 nm yields 0.83-ps output pulses. The wide bandwidth of the fiber parametric amplifier causes the oscillator to act as a pulse compressor. The output signal pulses exhibit improved spectral symmetry and a reduced time-bandwidth product compared with the pump pulses. Currently, the net group-velocity dispersion in the passive section of the fiber cavity limits the signal-pulse bandwidth and hence the minimum-obtainable pulse width. This experiment suggests the possibility of frequency conversion by operation of a similar pulsed parametric oscillator away from degeneracy.

The effect of dietary docosahexaenoic acid on platelet function, platelet fatty acid composition, and blood coagulation in humans.

The effect of dietary docosahexaenoic acid (DHA) in the absence of eicosapentaenoic acid (EPA) has been studied infrequently in humans under controlled conditions. This 120-d study followed healthy, adult male volunteers who lived in the metabolic research unit (MRU) of the Western Human Nutrition Research Center for the entire study. The basal (low-DHA) diet consisted of natural foods (30 en% fat, 15 en% protein, and 55 en% carbohydrate), containing < 50 mg/d of DHA, and met the recommended daily intake for all essential nutrients. The high-DHA (intervention) diet was similar except that 6 g/d of DHA in the form of a triglyceride containing 40% DHA replaced an equal amount of safflower oil in the basal diet. The subjects (ages 20 to 39) were within -10 to +20% of ideal body weight, nonsmoking, and not allowed alcohol in the MRU. Their exercise level was constant, and their body weights were maintained within 2% of entry level. They were initially fed the low-DHA diet for 30 d. On day 31, six subjects (intervention, group A) were placed on the high-DHA diet; the other four subjects (controls, group B) remained on the low-DHA diet. Platelet aggregation in platelet-rich plasma was determined using ADP, collagen, and arachidonic acid. No statistical differences could be detected between the amount of agonist required to produce 50% aggregation of platelet-rich plasma before and after the subjects consumed the high-DHA diet. The prothrombin time, activated partial thromboplastin time, and the antithrombin-III levels in the subjects were determined, and, again, there were no statistically significant differences in these three parameters when their values were compared before and after the subjects consumed the high-DHA diet. In addition, the in vivo bleeding times did not show any significant difference before and after the subjects consumed the high-DHA diet (9.4 +/- 3.1 min before and 8.0 +/- 3.4 min after). Platelets from the volunteers exhibited more than a threefold increase in their DHA content from 1.54 +/- 0.16 to 5.48 +/- 1.21 (wt%) during the DHA feeding period. The EPA content of the subjects' platelets increased from 0.34 +/- 0.12 to 2.67 +/- 0.91 (wt%) during the high-DHA diet despite the absence of EPA in the subjects' diets. The results from this study on blood clotting parameters and in vitro platelet aggregation suggest that adding 6 g/d of dietary DHA for 90 d to a typical Western diet containing less than 50 mg/d of DHA produces no observable physiological changes in blood coagulation, platelet function, or thrombotic tendencies in healthy, adult males.

The effect of dietary docosahexaenoic acid on plasma lipoproteins and tissue fatty acid composition in humans.

Normal, healthy male volunteers (n = 6) were fed diets [high docosahexaenoic acid-DHA] containing 6 g/d of DHA for 90 d. The stabilization (low-DHA) diet contained less than 50 mg/d of DHA. A control group (n = 4) remained on the low-DHA diet for the duration of the study (120 d). Blood samples were drawn on study days 30 (end of the stabilization period), 75 (midpoint of the intervention period), and 120 (end of the intervention period). Adipose tissue (AT) samples were taken on days 30 and 120. The plasma cholesterol (C), low density lipoprotein (LDL)-C and apolipoproteins (apo) [Al, B, and lipoprotein (a)] were unchanged after 90 d, but the triglycerides (TAG) were reduced from a mean value of 76.67 +/- 24.32 to 63.83 +/- 16.99 mg/dL (n = 6, P < 0.007 using a paired t-test) and the high density lipoprotein (HDL)-C increased from 34.83 +/- 4.38 mg/dL to 37.83 +/- 3.32 mg/dL (n = 6, P < 0.017 using a paired t-test). The control group showed no significant reduction in plasma TAG levels. Apo-E, however, showed a marked increase in the volunteers' plasma after 90 d on the high-DHA diet, from 7.06 +/- 4.47 mg/dL on study day 30 to 12.01 +/- 4.96 mg/dL on study day 120 (P < 0.002 using a paired t-test). The control subjects showed no significant change in the apo-E in their plasma (8.46 +/- 2.90 on day 30 vs. 8.59 +/- 2.97 on day 120). The weight percentage of plasma DHA rose from 1.83 +/- 0.22 to 8.12 +/- 0.76 after 90 d on the high-DHA diet. Although these volunteers were eating a diet free of eicosapentaenoic acid (EPA), plasma EPA levels rose from 0.38 +/- 0.05 to 3.39 +/- 0.52 (wt%) after consuming the high-DHA diet. The fatty acid composition of plasma lipid fractions--cholesterol esters, TAG, and phospholipid--showed marked similarity in the enrichment of DHA, about 10%, after the subjects consumed the high-DHA diet. The DHA content of these plasma lipid fractions varied from less than 1% (TAG) to 3.5% (phospholipids) at baseline, study day 30. EPA also increased in all plasma lipid fractions after the subjects consumed the high-DHA diet. There were no changes in the plasma DHA or EPA levels in the control group. Consumption of DHA also caused an increase in AT levels of DHA, from 0.10 +/- 0.02 to 0.31 +/- 0.07 (wt%) (n = 6, P < 0.001 using a paired t-test), but the amount of EPA in their AT did not change. Thus, dietary DHA will lower plasma TAG without EPA, and DHA is retroconverted to EPA in significant amounts. Dietary DHA appears to enhance apo-E synthesis in the liver. It appears that DHA can be a safe and perhaps beneficial supplement to human diets.