Medium & long-chain acylcarnitine's relation to lipid metabolism as potential predictors for diabetic cardiomyopathy: a metabolomic study.

BACKGROUND: Acylcarnitine is an intermediate product of fatty acid oxidation. It is reported to be closely associated with the occurrence of diabetic cardiomyopathy (DCM). However, the mechanism of acylcarnitine affecting myocardial disorders is yet to be explored. This current research explores the different chain lengths of acylcarnitines as biomarkers for the early diagnosis of DCM and the mechanism of acylcarnitines for the development of DCM in-vitro. METHODS: In a retrospective non-interventional study, 50 simple type 2 diabetes mellitus patients and 50 DCM patients were recruited. Plasma samples from both groups were analyzed by high throughput metabolomics and cluster heat map using mass spectrometry. Principal component analysis was used to compare the changes occurring in the studied 25 acylcarnitines. Multivariable binary logistic regression was used to analyze the odds ratio of each group for factors and the 95% confidence interval in DCM. Myristoylcarnitine (C14) exogenous intervention was given to H9c2 cells to verify the expression of lipid metabolism-related protein, inflammation-related protein expression, apoptosis-related protein expression, and cardiomyocyte hypertrophy and fibrosis-related protein expression. RESULTS: Factor 1 (C14, lauroylcarnitine, tetradecanoyldiacylcarnitine, 3-hydroxyl-tetradecanoylcarnitine, arachidic carnitine, octadecanoylcarnitine, 3-hydroxypalmitoleylcarnitine) and factor 4 (octanoylcarnitine, hexanoylcarnitine, decanoylcarnitine) were positively correlated with the risk of DCM. Exogenous C14 supplementation to cardiomyocytes led to increased lipid deposition in cardiomyocytes along with the obstacles in adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) signaling pathways and affecting fatty acid oxidation. This further caused myocardial lipotoxicity, ultimately leading to cardiomyocyte hypertrophy, fibrotic remodeling, and increased apoptosis. However, this effect was mitigated by the AMPK agonist acadesine. CONCLUSIONS: The increased plasma levels in medium and long-chain acylcarnitine extracted from factors 1 and 4 are closely related to the risk of DCM, indicating that these factors can be an important tool for DCM risk assessment. C14 supplementation associated lipid accumulation by inhibiting the AMPK/ACC/CPT1 signaling pathway, aggravated myocardial lipotoxicity, increased apoptosis apart from cardiomyocyte hypertrophy and fibrosis were alleviated by the acadesine.

Mitochondrial bioenergetics deregulation caused by long-chain 3-hydroxy fatty acids accumulating in LCHAD and MTP deficiencies in rat brain: a possible role of mPTP opening as a pathomechanism in these disorders?

Long-chain 3-hydroxylated fatty acids (LCHFA) accumulate in long-chain 3-hydroxy-acyl-CoA dehydrogenase (LCHAD) and mitochondrial trifunctional protein (MTP) deficiencies. Affected patients usually present severe neonatal symptoms involving cardiac and hepatic functions, although long-term neurological abnormalities are also commonly observed. Since the underlying mechanisms of brain damage are practically unknown and have not been properly investigated, we studied the effects of LCHFA on important parameters of mitochondrial homeostasis in isolated mitochondria from cerebral cortex of developing rats. 3-Hydroxytetradecanoic acid (3 HTA) reduced mitochondrial membrane potential, NAD(P)H levels, Ca(2+) retention capacity and ATP content, besides inducing swelling, cytochrome c release and H2O2 production in Ca(2+)-loaded mitochondrial preparations. We also found that cyclosporine A plus ADP, as well as ruthenium red, a Ca(2+) uptake blocker, prevented these effects, suggesting the involvement of the mitochondrial permeability transition pore (mPTP) and an important role for Ca(2+), respectively. 3-Hydroxydodecanoic and 3-hydroxypalmitic acids, that also accumulate in LCHAD and MTP deficiencies, similarly induced mitochondrial swelling and decreased ATP content, but to a variable degree pending on the size of their carbon chain. It is proposed that mPTP opening induced by LCHFA disrupts brain bioenergetics and may contribute at least partly to explain the neurologic dysfunction observed in patients affected by LCHAD and MTP deficiencies.

13-Methyltetradecanoic acid induces mitochondrial-mediated apoptosis in human bladder cancer cells.

OBJECTIVE: 13-Methyltetradecanoic acid (13-MTD), a saturated branched-chain fatty acid purified from soy fermentation products, is known to induce apoptosis in many types of human cancer cells. This study was designed to investigate the molecular mechanisms involved in 13-MTD-induced apoptosis in human bladder cancer cells. METHODS AND MATERIALS: MTT assay was used to investigate the potential effects of 13-MTD on the growth and viability of human bladder cancer cells. To find out whether anti-proliferation and cell death were associated with apoptosis, we used flow cytometry to quantify the extent of apoptosis and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay to measures DNA degradation of apoptotic cells. The proteins involved in the 13-MTD induced apoptosis were examined using Western blot. RESULTS: We show that 13-MTD inhibits cellular proliferation and viability in human bladder cancer cells, which has been attributed to apoptosis. 13-MTD down-regulates Bcl-2 and up-regulates Bax. This promotes mitochondrial dysfunction, leading to the release of cytochrome c from the mitochondria to the cytoplasm, as well as the proteolytic activation of caspases. Moreover, 13-MTD down-regulates AKT phosphorylation and activates phosphorylation of p38 and c-Jun N-terminal kinase (JNK). Up-regulating AKT phosphorylation and down-regulating JNK and P38 phosphorylation could attenuate the13-MTD-induced apoptosis. CONCLUSION: Taken together, these data indicate that 13-MTD induces mitochondrial-mediated apoptosis through regulation of the AKT and MAPK pathways, suggesting 13-MTD is a potential candidate for treatment of human bladder cancer.

Diets high in palmitic acid (16:0), lauric and myristic acids (12:0 + 14:0), or oleic acid (18:1) do not alter postprandial or fasting plasma homocysteine and inflammatory markers in healthy Malaysian adults.

BACKGROUND: Dietary fat type is known to modulate the plasma lipid profile, but its effects on plasma homocysteine and inflammatory markers are unclear. OBJECTIVE: We investigated the effects of high-protein Malaysian diets prepared with palm olein, coconut oil (CO), or virgin olive oil on plasma homocysteine and selected markers of inflammation and cardiovascular disease (CVD) in healthy adults. DESIGN: A randomized-crossover intervention with 3 dietary sequences of 5 wk each was conducted in 45 healthy subjects. The 3 test fats, namely palmitic acid (16:0)-rich palm olein (PO), lauric and myristic acid (12:0 + 14:0)-rich CO, and oleic acid (18:1)-rich virgin olive oil (OO), were incorporated at two-thirds of 30% fat calories into high-protein Malaysian diets. RESULTS: No significant differences were observed in the effects of the 3 diets on plasma total homocysteine (tHcy) and the inflammatory markers TNF-α, IL-1ß, IL-6, and IL-8, high-sensitivity C-reactive protein, and interferon-γ. Diets prepared with PO and OO had comparable nonhypercholesterolemic effects; the postprandial total cholesterol for both diets and all fasting lipid indexes for the OO diet were significantly lower (P < 0.05) than for the CO diet. Unlike the PO and OO diets, the CO diet was shown to decrease postprandial lipoprotein(a). CONCLUSION: Diets that were rich in saturated fatty acids prepared with either PO or CO, and an OO diet that was high in oleic acid, did not alter postprandial or fasting plasma concentrations of tHcy and selected inflammatory markers. This trial was registered at clinicaltrials.gov as NCT00941837.

Identification of light-independent inhibition of human immunodeficiency virus-1 infection through bioguided fractionation of Hypericum perforatum.

BACKGROUND: Light-dependent activities against enveloped viruses in St. John's Wort (Hypericum perforatum) extracts have been extensively studied. In contrast, light-independent antiviral activity from this species has not been investigated. RESULTS: Here, we identify the light-independent inhibition of human immunodeficiency virus-1 (HIV-1) by highly purified fractions of chloroform extracts of H. perforatum. Both cytotoxicity and antiviral activity were evident in initial chloroform extracts, but bioassay-guided fractionation produced fractions that inhibited HIV-1 with little to no cytotoxicity. Separation of these two biological activities has not been reported for constituents responsible for the light-dependent antiviral activities. Antiviral activity was associated with more polar subfractions. GC/MS analysis of the two most active subfractions identified 3-hydroxy lauric acid as predominant in one fraction and 3-hydroxy myristic acid as predominant in the other. Synthetic 3-hydroxy lauric acid inhibited HIV infectivity without cytotoxicity, suggesting that this modified fatty acid is likely responsible for observed antiviral activity present in that fraction. As production of 3-hydroxy fatty acids by plants remains controversial, H. perforatum seedlings were grown sterilely and evaluated for presence of 3-hydroxy fatty acids by GC/MS. Small quantities of some 3-hydroxy fatty acids were detected in sterile plants, whereas different 3-hydroxy fatty acids were detected in our chloroform extracts or field-grown material. CONCLUSION: Through bioguided fractionation, we have identified that 3-hydroxy lauric acid found in field grown Hypericum perforatum has anti-HIV activity. This novel anti-HIV activity can be potentially developed into inexpensive therapies, expanding the current arsenal of anti-retroviral agents.

Effect of dietary cholesterol, trans and saturated fatty acids on serum lipoproteins in non-human primates.

Nine cynomolgus monkeys were rotated randomly through four dietary treatments with each treatment lasting 6 weeks. A wash-out period of 4 weeks was maintained between each dietary rotation. The animals were fed diets containing 32% energy fat derived from palm olein (POL), lauric-myristic-rich oil blend (LM), American Heart Association (AHA) rich oil blend and hydrogenated soybean oil blend (trans). Diets were fed with (phase 1) or without (phase 2) the addition of dietary cholesterol (0.1%). In phase 1, when animals were fed without dietary cholesterol, plasma total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) was significantly raised and high-density lipoprotein cholesterol (HDL-C) was significantly depressed by the trans diets relative to all other dietary treatments. The resulting LDL-C/HDL-C ratio was also significantly increased. The LM diet increased TC significantly relative to the AHA diet while LDL-C was significantly increased compared to both POL and AHA. Apolipoprotein (apo) B was not affected significantly by these dietary treatments. Apo A1 was significantly increased by POL relative to all other dietary treatments. The trans diet reduced apo A1 and the resulting apo B/A1 ratio was increased significantly by trans relative to all other dietary treatments. Addition of 0.1% dietary cholesterol to these diets almost doubled the plasma TC and LDL-C in all dietary treatments. However, HDL-C was only marginally higher with the addition of dietary cholesterol. The LM + C (cholesterol added) diet resulted in the highest TC and LDL-C that was significant compared to all other dietary treatments. Trans + C increased TC compared to POL + C and AHA + C diets while increases in the LDL-C did not attain significance. The addition of dietary cholesterol did not affect HDL-C between treatments whereas plasma triglycerides were significantly increased by the trans + C diet relative to all other treatments. Both the trans + C and LM + C diets increased apo B and decreased apo A1 relative to the POL + C and AHA + C diets. The resulting apo B/A1 ratio was similarly altered. These results affirm that the lauric + myristic acid combination, along with trans fatty acids, increased lipoprotein-associated coronary heart disease risk factors compared to either POL or AHA.

Accumulation of a polyisoprene-linked amino sugar in polymyxin-resistant Salmonella typhimurium and Escherichia coli: structural characterization and transfer to lipid A in the periplasm.

Polymyxin-resistant mutants of Escherichia coli and Salmonella typhimurium accumulate a novel minor lipid that can donate 4-amino-4-deoxy-l-arabinose units (l-Ara4N) to lipid A. We now report the purification of this lipid from a pss(-) pmrA(C) mutant of E. coli and assign its structure as undecaprenyl phosphate-alpha-l-Ara4N. Approximately 0.2 mg of homogeneous material was isolated from an 8-liter culture by solvent extraction, followed by chromatography on DEAE-cellulose, C18 reverse phase resin, and silicic acid. Matrix-assisted laser desorption ionization/time of flight mass spectrometry in the negative mode yielded a single species [M - H](-) at m/z 977.5, consistent with undecaprenyl phosphate-alpha-l-Ara4N (M(r) = 978.41). (31)P NMR spectroscopy showed a single phosphorus atom at -0.44 ppm characteristic of a phosphodiester linkage. Selective inverse decoupling difference spectroscopy demonstrated that the undecaprenyl phosphate group is attached to the anomeric carbon of the l-Ara4N unit. One- and two-dimensional (1)H NMR studies confirmed the presence of a polyisoprene chain and a sugar moiety with chemical shifts and coupling constants expected for an equatorially substituted arabinopyranoside. Heteronuclear multiple-quantum coherence spectroscopy analysis demonstrated that a nitrogen atom is attached to C-4 of the sugar residue. The purified donor supports in vitro conversion of lipid IV(A) to lipid II(A), which is substituted with a single l-Ara4N moiety. The identification of undecaprenyl phosphate-alpha-l-Ara4N implies that l-Ara4N transfer to lipid A occurs in the periplasm of polymyxin-resistant strains, and establishes a new enzymatic pathway by which Gram-negative bacteria acquire antibiotic resistance.

In vitro antiviral activities of myristic acid analogs against human immunodeficiency and hepatitis B viruses.

A group of myristic acid analogs, designed as alternative substrates for N-myristoyltransferase (NMT), were evaluated against human immunodeficiency virus (HIV), hepatitis B virus (HBV) and duck hepatitis B virus (DHBV) in vitro. Antiviral potency was increased when S or O was substituted for -CH2- in myristic acid and selectivity was affected by the presence and position of the heteroatoms and phenyl groups. A correlation was established among anti-HIV activity, Log P and Log D7.4 and between anti-HIV activity and carbonyl-heteroatom interatomic distances in the myristoyl analogs. 12-Thioethyldodecanoic acid 6 was moderately active (EC50 = 9.37 microM) against HIV-infected T4-lymphocytes (CEM-SS cell line), and it exhibited in vitro activity (EC50 = 17.8 microM) against HBV-producing 2.2.15 cell cultures derived from a human hepatoblastoma cell line (Hep G2). 12-Methoxydodecanoic acid 1 exhibited in vitro activity (EC50 = 20-30 microM) against hepatitis B in the HBV DNA-transfected 2.2.15 cell line. At a concentration of 10 microg/ml, none of the fatty acids significantly inhibited the replication of DHBV in infected hepatocytes.

Platelet lysis and functional perturbation by 13-methyl myristate. The major fatty acid in Flavobacterium ranacida.

Flavobacterium ranacida consisted of 75% of 13-methyl myristate in total fatty acids. The acid at > 60 microM caused the lysis of gel-filtered platelets (GFP) in both time- and concentration-dependent manners. Scanning electron microscopy showed that: 1). GFP in 40 microM of the acid changed the morphology to speculate discoid shape at 15 sec, and to ellipsoids after 30 sec; and 2), the cells gradually swelled to spherical forms as the concentration of the acid increased. At nonlytic concentration, the acid inhibited platelet responses to various agonists with differential concentrations. The order of inhibitory potency was U46619 > low dose collagen > ADP-fibrinogen > phorbol ester > high dose collagen. The results demonstrated that 13-methyl myristate exhibited both cell lytic activity and perturbation on membrane function.

Dietary myristic, palmitic, and linoleic acids modulate cholesterolemia in gerbils.

Previous studies with cebus monkeys and review of published human data indicated that 85% to 90% of the variation in plasma cholesterol (TC) could be explained on the basis of dietary myristic (14:0) and linoleic (18:2) acid intake in the absence of cholesterol, and that 16:0 contributed to cholesterolemia as dietary cholesterol was increased. Because monkeys are a limited resource, a more convenient, sensitive model was sought for investigating these dietary fatty acid and plasma lipid relationships. Accordingly, this report describes the results of multiple regression analysis of the TC response to dietary fatty acids based on 319 young, male Mongolian gerbils fed a total of 59 purified diets supplying about 40% energy as fat from single or blended fat sources. Gerbils (6-16 animals per dietary group) were fed purified diets (21 with 0.01 to 0.08% cholesterol and 38 cholesterol-free) for 4-week periods. When cholesterol-free diets were fed, dietary 14:0 and 18:2 together accounted for 89% of the observed variation in TC. Although 14:0 consumption increased TC in a linear manner, the independent ability of 18:2 to lower cholesterol was nonlinear and exhibited a threshold effect at 5-6% dietary energy, above which further lowering of TC was less remarkable. In gerbils consuming cholesterol-supplemented diets, 87% of the observed variation in TC could be accounted for by a regression equation that included 14:0, palmitic acid (16:0), and a log function of 18:2 plus dietary cholesterol itself. These results demonstrate the applicability of gerbils for such studies and confirm previous observations in monkeys and humans that dietary 14:0 and 18:2 are the main fatty acids modulating plasma cholesterol under normocholesterolemic circumstances (i.e., when consuming low-cholesterol diets and lipoprotein metabolism is normal) whereas 16:0 also appears modestly hypercholesterolemic when LDL receptors are compromised (i.e., when dietary cholesterol or certain metabolic factors have encumbered lipoprotein metabolism).

Influence of fatty acid anilides present in toxic oils on the metabolism of exogenous arachidonic acid in cultured human endothelial cells.

The effect of fatty acid anilides (FAA) on the exogenous arachidonic acid (AA) metabolism and toxicity of isolated human endothelial cells was studied to clarify their possible role in the etiology of toxic oil syndrome. Confluent cells were incubated with and without linoleic acid anilide (LAA), oleic acid anilide (OAA) and two unrelated samples for 2-24 h prior to the addition of [l-14C]AA alone or with calcium ionophore A-23187. The eicosanoids produced were analyzed by RP-HPLC. A dual stimulatory and inhibitory effect on the conversion of exogenous AA as a function of preincubation time with anilides (100 and 1000 microM) was observed. Treated cells significantly increased (1-3-fold) the production of the main cyclooxygenase-derived prostanoids (6-keto-PGF1 alpha and PGF2 alpha) formed by these cells, with a maximum stimulatory effect after 2-3 h, only when AA was used alone. However, afterwards a time- and dose-dependent decrease in prostanoid formation was observed with LAA (P < 0.05 at 24 h), either in the absence or presence of ionophore A-23187 in the incubation mixture. This inhibitory effect on cyclooxygenase was not observed with OAA, which still stimulate after 24 h of treatment. The changes in prostanoid synthesis were not followed with a parallel release in the lactate dehydrogenase activity in the medium (except with unrelated samples). Moreover, anilide treatment increased the appearance of cytosolic lipid droplets or vacuoles after 2 and 5 h of contact with LAA and OAA, respectively. From these results, it was suggested that anilides impair prostanoid synthesis in endothelial cells; their stimulatory effect could be explained by an unspecific effect on cell membrane, not related to cell toxicity and the inhibitory effect by an inhibition of the cyclooxygenase activity. These observations further contribute to our understanding of the possible role of anilides in the etiology of the toxic oil syndrome.

Dietary palmitic and oleic acids exert similar effects on serum cholesterol and lipoprotein profiles in normocholesterolemic men and women.

To compare the effects of dietary palmitic acid (16:0) vs oleic acid (18:1) on serum lipids, lipoproteins, and plasma eicosanoids, 33 normocholesterolemic subjects (20 males, 13 females; ages 22-41 years) were challenged with a coconut oil-rich diet for 4 weeks. Subsequently they were assigned to either a palm olein-rich or olive oil-rich diet followed by a dietary crossover during two consecutive 6-week periods. Each test oil served as the sole cooking oil and contributed 23% of dietary energy or two-thirds of the total daily fat intake. Dietary myristic acid (14:0) and lauric acid (12:0) from coconut oil significantly raised all the serum lipid and lipoprotein parameters measured. Subsequent one-to-one exchange of 7% energy between 16:0 (palm olein diet) and 18:1 (olive oil diet) resulted in identical serum total cholesterol (192, 193 mg/dl), low-density lipoprotein cholesterol (LDL-C) (130, 131 mg/dl), high-density lipoprotein cholesterol (HDL-C) (41, 42 mg/dl), and triglyceride (TG) (108, 106 mg/dl) concentrations. Effects attributed to gender included higher HDL in females and higher TG in males associated with the tendency for higher LDL and LDL/HDL ratios in men. However, both sexes were equally responsive to changes in dietary fat saturation. The results indicate that in healthy, normocholesterolemic humans, dietary 16:0 can be exchanged for 18:1 within the range of these fatty acids normally present in typical diets without affecting the serum lipoprotein cholesterol concentration or distribution. In addition, replacement of 12:0 + 14:0 by 16:0 + 18:1, but especially 16:0 or some component of palm olein, appeared to have a beneficial impact on an important index of thrombogenesis, i.e., the thromboxane/prostacyclin ratio in plasma.

A new differentiated cell line (Dif 5) derived by retinoic acid treatment of F9 teratocarcinoma cells capable of extracellular matrix production and growth in the absence of serum.

Treatment of F9 teratocarcinoma cells with all trans retinoic acid (RA) causes them to differentiate into two or three morphologically distinct cell types. Whereas the majority of these retinoid-derived cells exhibit properties resembling parietal endoderm, a small percentage of this differentiated cell population manifests properties distinct from the parietal endoderm cell type. The isolation and partial characterization of such a non-parietal endoderm cell line (Dif 5) derived from F9 cells following prolonged (44 days) exposure to 1 microM retinoic acid are described. Unlike the retinoid-induced parietal endoderm-like cell population, which exhibits a dramatic, characteristic morphological change upon treatment with 8-bromo cAMP, Dif 5 cells do not show any morphological change with exposure to this cAMP analog. Dif 5 cells synthesize and deposit an extracellular matrix consisting of several components of Reichert's membrane (fibronectin, laminin, and type IV collagen). This new cell line does not synthesize alpha-fetoprotein but does secrete plasminogen activator. An interesting property of these cells is their ability to grow in the absence of serum or other hormonal supplements. Yet the Dif 5 cells do exhibit density-dependent inhibition of growth. Unlike the parent F9 cells or parietal yolk sac (PYS-2) cells, these cells do possess specific cell surface receptors for epidermal growth factor (EGF). The growth-arrested Dif 5 cells can be reinitiated to proliferate by the addition of fetal calf serum (FCS) or EGF. The properties of Dif 5 cells determined fail to fulfill all the characteristics described for either parietal or visceral endodermal cells. This raises the possibility that Dif 5 cells might represent an endodermal cell type which is intermediate in differentiation to either parietal or visceral endoderm but which lacks the biochemical signal to complete this stage of differentiation. This new Dif 5 cell line should be of considerable value in studying the modulation of growth requirements and extracellular matrix formation during early embryonic development.

Histological changes in rabbits after application of medicaments and cosmetic bases. II.

A further study of the topical effects of certain pharmaceutical and cosmetic bases on rabbit skin is reported. Cetyl alcohol, myristic acid, castor oil and sorbitol were applied in fixed doses daily for 30 days and their irritant activity was assessed. The macroscopic and microscopic changes were on the whole minimal. Castor oil alone produced some macroscopic alterations in the form of slight erythema and edema, and microscopic changes consisting of acanthosis, disorganization of the basal layer and slight infiltration of the dermis.