Erucic Acid-Rich Yellow Mustard Oil Improves Insulin Resistance in KK-Ay Mice.

Obesity is a major risk factor for some metabolic disorders including type 2 diabetes. Enhancement of peroxisome proliferator-activated receptor (PPAR) γ, a master regulator of adipocyte differentiation, is known to increase insulin-sensitive small adipocytes. In contrast, decreased PPARγ activity is also reported to improve insulin resistance. We have previously identified erucic acid as a novel natural component suppressing PPARγ transcriptional activity. In this study, we investigated the effect of erucic acid-rich yellow mustard oil (YMO) on obese/diabetic KK-Ay mice. An in vitro luciferase reporter assay and mesenchymal stem cell (MSC) differentiation assay revealed that 25 µg/mL YMO significantly inhibited PPARγ transcriptional activity and differentiation of MSCs into adipocytes but promoted their differentiation into osteoblasts. In KK-Ay mice, dietary intake of 7.0% (w/w) YMO significantly decreased the surrogate indexes for insulin resistance and the infiltration of macrophages into adipose tissue. Furthermore, 7.0% YMO increased bone mineral density. These results suggest that YMO can ameliorate obesity-induced metabolic disorders.

Erucic acid derived from rosemary regulates differentiation of mesenchymal stem cells into osteoblasts/adipocytes via suppression of peroxisome proliferator-activated receptor γ transcriptional activity.

Osteoporosis is associated with increase in fat tissue in bone marrow in humans. Mesenchymal stem cells in bone marrow are induced to differentiate into osteoblasts rather than adipocytes by the stimulation of peroxisome proliferator-activated receptor (PPAR) γ antagonists. PPARγ antagonists are expected to be useful to prevent osteoporosis by regulating the lineages of mesenchymal stem cells in bone marrow, as well as the prevention of obesity. In this study, we explored natural components suppressing PPARγ transcriptional activity in rosemary. Separation of active fraction of rosemary extract by repeated high performance liquid chromatograph and PPARγ luciferase reporter assay identified erucic acid, one of the monounsaturated fatty acids, as an active component. Twenty-five-micrometer erucic acid significantly decreased PPARγ luciferase activity and enhanced the differentiation of mouse-delivered C3H10T1/2 cells into osteoblasts rather than adipocytes. Furthermore, 25-µM erucic acid significantly decreased the expression of adipocyte marker genes, while accelerating osteoblast marker genes. In conclusion, erucic acid is a novel natural component derived from rosemary regulating mesenchymal stem cell differentiation via suppression of PPARγ transcriptional activity.

A model-based approach to assess the exposure-response relationship of Lorenzo's oil in adrenoleukodystrophy.

AIMS: X-linked adrenoleukodystrophy (X-ALD) is a peroxisomal disorder, most commonly affecting boys, associated with increased very long chain fatty acids (C26:0) in all tissues, causing cerebral demyelination and adrenocortical insufficiency. Certain monounsaturated long chain fatty acids including oleic and erucic acids, known as Lorenzo's oil (LO), lower plasma C26:0 levels. The aims of this study were to characterize the effect of LO administration on plasma C26:0 concentrations and to determine whether there is an association between plasma concentrations of erucic acid or C26:0 and the likelihood of developing brain MRI abnormalities in asymptomatic boys. METHODS: Non-linear mixed effects modelling was performed on 2384 samples collected during an open label single arm trial. The subjects (n = 104) were administered LO daily at ~2-3 mg kg(-1) with a mean follow-up of 4.88 ± 2.76 years. The effect of erucic acid exposure on plasma C26:0 concentrations was characterized by an inhibitory fractional Emax model. A Weibull model was used to characterize the time-to-developing MRI abnormality. RESULTS: The population estimate for the fractional maximum reduction of C26:0 plasma concentrations was 0.76 (bootstrap 95% CI 0.73, 0.793). Our time-to-event analyses showed that every mg l(-1) increase in time-weighted average of erucic acid and C26:0 plasma concentrations was, respectively, associated with a 3.7% reduction and a 753% increase in the hazard of developing MRI abnormality. However, the results were not significant (P = 0.5344, 0.1509, respectively). CONCLUSIONS: LO administration significantly reduces the abnormally high plasma C26:0 concentrations in X-ALD patients. Further studies to evaluate the effect of LO on the likelihood of developing brain MRI abnormality are warranted.

Lorenzo's oil inhibits ELOVL1 and lowers the level of sphingomyelin with a saturated very long-chain fatty acid.

X-linked adrenoleukodystrophy (X-ALD) is a peroxisomal disorder caused by impaired degradation of very long-chain fatty acids (VLCFAs) due to mutations in the ABCD1 gene responsible for VLCFA transport into peroxisomes. Lorenzo's oil, a 4:1 mixture of glyceryl trioleate and glyceryl trierucate, has been used to reduce the saturated VLCFA level in the plasma of X-ALD patients; however, the mechanism by which this occurs remains elusive. We report the biochemical characterization of Lorenzo's oil activity toward elongation of very long-chain fatty acid (ELOVL) 1, the primary enzyme responsible for the synthesis of saturated and monounsaturated VLCFAs. Oleic and erucic acids inhibited ELOVL1, and, moreover, their 4:1 mixture (the FA composition of Lorenzo's oil) exhibited the most potent inhibitory activity. The kinetics analysis revealed that this was a mixed (not a competitive) inhibition. At the cellular level, treatment with the 4:1 mixture reduced the level of SM with a saturated VLCFA accompanied by an increased level of SM with a monounsaturated VLCFA, probably due to the incorporation of erucic acid into the FA elongation cycle. These results suggest that inhibition of ELOVL1 may be an underlying mechanism by which Lorenzo's oil exerts its action.

[The hypolipidemic and liver protective effect of (Z)-N-(2-hydroxyethyl) docos-13-enamide on hyperlipidemic golden hamsters].

This study is to observe preventive effect of (Z)-N-(2-hydroxyethyl) docos-13-enamide on hyperlipidemia and fatty liver of golden hamsters. Hyperlipidemic golden hamsters fed with high-fat diet was administered orally with (Z)-N-(2-hydroxyethyl) docos-13-enamide (10, 20 and 40 mg x kg(-1)) for 5 weeks. Levels of serum and hepatic lipid content, liver histology, hepatic MDA and SOD levels, serum ALT and AST levels were evaluated in golden hamsters. (Z)-N-(2-Hydroxyethyl) docos-13-enamide has a hypolipidemic effect, and could reduce hepatic lipid content, serum ALT and AST levels, hepatic MDA level, increase hepatic SOD activity. (Z)-N-(2-Hydroxyethyl) docos-13-enamide plays an important role in reducing serum lipid, restraining hepatic fatty deposition and protecting liver to get rid of peroxidation injury of hyperlipidemic golden hamsters. The exact lipid-lowering mechanism of (Z)-N-(2-hydroxyethyl) docos-13-enamide needs further investigation.

A new pyrimidine alkaloid from the roots of Tadehagi triquetrum (L.) H.Ohashi.

Tadehagi triquetrum (L.) H.Ohashi, also known as Desmodium triquetrum (Fabaceae) is the most important plant in the herbal remedies. The present study focus on the isolation, in-silico and in-vitro studies of the two alkaloids C1 (5-(4-[(methylcarbamoyl) amino]-2-oxopyrimidin-1(2H)-yl) tetrahydrofuran-2-yl) methyl methyl carbamate is novel alkaloid and C2 13-Docosenamide is a known alkaloid. The chemical structures of compounds have been elucidated based on comprehensive techniques like GCMS, IR and NMR. In order to know the molecular mechanisms for the two compounds, in silico molecular docking study has been performed. Both compounds have shown perfect binding affinity to the enzymes TNF α, IL-4, IL-13 and 5 LOX Enzyme. The compounds also exhibited comparable G-scores and Glide energy values in comparison with the standard dexamethasone. In addition both the compounds have been tested for in vitro antioxidant assay by using ABTS and DPPH method and the results were compared with standard ascorbic acid.

Preventing neurodegeneration in the Drosophila mutant bubblegum.

The Drosophila melanogaster recessive mutant bubblegum (bgm) exhibits adult neurodegeneration, with marked dilation of photoreceptor axons. The bubblegum mutant shows elevated levels of very long chain fatty acids (VLCFAs), as seen in the human disease adrenoleukodystrophy (ALD). In ALD, the excess can be lowered by dietary treatment with "Lorenzo's oil," a mixture of unsaturated fatty acids. Feeding the fly mutant one of the components, glyceryl trioleate oil, blocked the accumulation of excess VLCFAs as well as development of the pathology. Mutant flies thus provide a potential model system for studying mechanisms of neurodegenerative disease and screening drugs for treatment.

Ligands of the mitochondrial 18 kDa translocator protein attenuate apoptosis of human glioblastoma cells exposed to erucylphosphohomocholine.

BACKGROUND: We have previously shown that the anti-neoplastic agent erucylphosphohomocholine (ErPC3) requires the mitochondrial 18 kDa Translocator protein (TSPO), formerly known as the peripheral-type benzodiazepine receptor (PBR), to induce cell death via the mitochondrial apoptosis pathway. METHODS: With the aid of the dye JC-1 and cyclosporin A, applied to glioblastoma cells, we now investigated the significance of opening of the mitochondrial permeability transition pore (MPTP) for ErPC3-induced apoptosis in interaction with the TSPO ligands, PK 11195 and Ro5 4864. Furthermore, we measured cytochrome c release, and caspase-9 and -3 activation in this paradigm. RESULTS: The human glioblastoma cell lines, U87MG, A172 and U118MG express the MPTP-associated TSPO, voltage-dependent anion channel and adenine nucleotide transporter. Indeed, ErPC3-induced apoptosis was inhibited by the MPTP blocker cyclosporin A and by PK 11195 and Ro5 4864 in a concentration-dependent manner. Furthermore, PK 11195 and Ro5 4864 inhibited collapse of the mitochondrial membrane potential, cytochrome c release, and caspase-9 and -3 activation caused by ErPC3 treatment. CONCLUSIONS: This study shows that PK 11195 and Ro5 4864 inhibit the pro-apoptotic function of ErPC3 by blocking its capacity to cause a collapse of the mitochondrial membrane potential. Thus, the TSPO may serve to open the MPTP in response to anti-cancer drugs such as ErPC3.

Erucamide from Radish Leaves Has an Inhibitory Effect Against Acetylcholinesterase and Prevents Memory Deficit Induced by Trimethyltin.

In this study, we investigated a potent acetylcholinesterase inhibitor that was isolated from radish leaf (Raphanus sativus L.) extracts. Through sequential fractionation of radish leaf extract, the active constituent was identified as cis-13-docosenamide (erucamide). To validate the potency, erucamide derived from radish leaves was supplemented in diets and then fed to trimethyltin (TMT)-exposed mice. Specifically, mice had free access to a control diet or diets containing different concentrations of erucamide for 3 weeks, followed by an injection of TMT (2.5 mg/kg body weight). Our results showed that pretreatment of mice with erucamide (20 and 40 mg/kg body weight per day) significantly attenuated the TMT-induced learning and memory deficits that were assessed by Y-maze and passive avoidance tests. These findings suggest that radish leaves, and possibly its isolated erucamide, may have preventive effects against memory deficits related to Alzheimer's disease by modulation of cholinergic functions.

Erucic acid, a component of Lorenzo's oil and PPAR-δ ligand modifies C6 glioma growth and toxicity of doxorubicin. Experimental data and a comprehensive literature analysis.

BACKGROUND: PPAR-δ is a transcription factor which has crucial roles in stimulating oligodendroglial differentiation and myelination and its activation was also shown to differentiate malignant C6 glioma cells into oligodendrocytes. OBJECTIVE: One of the ligands of PPAR-δ is erucic acid (EA), an edible omega-9 fatty acid consumed more by Asian populations and exists highly in Chinese womens milk. There exist epidemiological evidence that pediatric brain tumor incidence is among the lowest in the Chinese population. EA is also an ingredient of Lorenzo's oil used against adrenoleukodystrophy, a pediatric demyelinating disease. EA was inappropriately assumed as a strong cardiotoxin based on Spanish oil syndrome, caused by toxic-aniline dye refined rapeseed oil. In this study, we studied whether EA is capable to block growth of C6 glioma cells and modify cardiotoxicity of doxorubicin. MATERIALS AND METHODS: We studied effects of EA on the 3-dimensional appearance of the adherent cells, soft agar colony formation and S-phase in the 3-dimensional spheroids in C6 glioma cell cultures. We also investigated the effects of EA on hepatic and cardiac toxicity of doxorubicin. RESULTS: EA decreased in vitro growth of C6 glioma cells at therapeutically achievable concentrations. EA effects were more prominent in 3D-assays (soft agar colonies and spheroids) and induced cell fusions in monolayer cultures. EA decreased S-phase inhibitory potency of doxorubicin (DOX), yet augmented its efficacy to induce a senescent morphology (as assessed by scanning electron microscopy) in monolayer and to increase iNOS and eNOS expression in spheroids. In our study, EA reduced DOX-induced necrosis in mice heart and liver and induced healthier morphology of heart mitochondria (as assessed by transmission electron microscopy); yet intercalated disks (ID) were more disturbed with DOX + EA. CONCLUSIONS: Both the antitumor and cardiac effects of EA may associate with the cell-to-cell contact mechanisms. Combining systemic EA with intrathecal DOX-chemotherapy via Ommaya reservoirs may reduce DOX concentrations in systemic circulation, hinder toxic interactions with EA and induce selective kill of glioma cells.

Antidepressant and anxiolytic-like behavioral effects of erucamide, a bioactive fatty acid amide, involving the hypothalamus-pituitary-adrenal axis in mice.

Erucamide (Era) is a bioactive fatty acid amide, which is similar to the classical endocannabinoid analogue oleoylethanolamide (OEA). In the present study, we hypothesized that Era may regulate the central nervous system and may have the potential to antagonize depression and anxiety. Therefore, we investigated the antidepressant and anxiolytic effects of Era in animal models in comparison with fluoxetine (Fxt). Fifty mice were randomly divided into 5 groups, and treated with a vehicle (0.3% methyl cellulose, 20mL/kg, p.o.), Era (5, 10, 20mg/kg, p.o.), or Fxt (20mg/kg, p.o.) for 7days. Immobility was used to evaluate depressive-like behavior in the forced swimming test (FST) and tail suspension test (TST). Animal activity and exploratory behavior as well as anxiety-like behaviors were measured in open field test (OFT) and elevated plus-maze test (EPMT) in mice. Additionally, serum adrenocorticotrophic hormone (ACTH) and corticosterone (CORT) levels were determined using the ELISA method, and the total anti-oxidative capacity (T-AOC) was detected by ultraviolet spectrophotometry. Our data showed that Era (5, 10, or 20mg/kg) induced a significant reduction in mouse immobility time in the TST and FST compared to the normal control group (vehicle group). The positive control, Fxt (20mg/kg group), also induced a significant change in immobility time in the TST and FST compared to the control (vehicle) group. In the OFT, compared with the control group, Fxt (20mg/kg) and Era (5, 10, or 20mg/kg) did not significantly change the locomotive activity (locomotive time, immobility time, or locomotive distance) in mice, but Fxt (20mg/kg) and Era (10, or 20mg/kg) significantly increased the percentage of time spent and squares visited in the OFT central area. In regards to the EPMT, the data showed that Fxt (20mg/kg) and Era (10, 20mg/kg) significantly increased the ratio of time spent and entries in open arms, but did not significantly change the total locomotive distance (including open arms and closed arms) compared to the control group. Biochemical tests found that after 7days of drug treatment, compared with the control group, ACTH and CORT serum levels in mice were significantly decreased, although T-AOC levels did not significantly change. In conclusion, Era (dose range of 5-20mg/kg) administered orally may alleviate depression- and anxiety-like behaviors in mice, and the antidepressant and anti-anxiety effects of Era may be related to the regulation of the hypothalamus-pituitary-adrenal axis (HPA).

Increased cytotoxicity of ionizing radiation in combination with membrane-targeted apoptosis modulators involves downregulation of protein kinase B/Akt-mediated survival-signaling.

BACKGROUND AND PURPOSE: The membrane-targeted apoptosis modulators erucylphosphocholine (ErPC) and erucylphosphohomocholine (ErPC3) induce apoptosis in highly apoptosis resistant malignant glioma cell lines and enhance radiation-induced cell death and eradication of clonogenic tumor cells in vitro. Aim of the present study was to elucidate molecular mechanisms of combined action. MATERIALS AND METHODS: Induction of apoptosis was evaluated by determination of nuclear morphology (fluorescence microscopy), alteration of mitochondrial function and caspase-activation (flow cytometry, Western blot). Activity of protein kinase B (PKB/Akt) and key downstream effectors involved in apoptosis regulation was verified by Western blot analysis using activation-specific antibodies. RESULTS: Increased cytotoxicity of the combination was linked to a more efficient activation of the intrinsic apoptosis pathway with increased damage of the mitochondria and caspase-activation. Moreover, activity of the survival kinase PKB/Akt was downregulated upon treatment with ErPC/ErPC3 alone or in combination with ionizing radiation. Inhibition of PKB/Akt was associated with decreased phosphorylation and thus activation of the pro-apoptotic Bcl-2 protein Bad as well as dephosphorylation of the transcription factor FOXO3A (FKHRL1) that may be responsible for the observed increased expression of the pro-apoptotic Bcl-2 protein Bim. CONCLUSIONS: Our data suggest a role for inhibition of PKB/Akt-mediated anti-apoptotic signaling in increased efficacy of the combination.

The effect of Lorenzo's oil on oxidative stress in X-linked adrenoleukodystrophy.

X-linked adrenoleukodystrophy (X-ALD) is a peroxisomal disorder biochemically characterized by the accumulation of very long chain fatty acids (VLCFA), particularly hexacosanoic acid (C(26:0)) and tetracosanoic acid (C(24:0)), in tissues and biological fluids. Although patients affected by this disorder predominantly present central and peripheral demyelination as well as adrenal insufficiency, the mechanisms underlying the brain damage in X-ALD are poorly known. The current treatment of X-ALD with glyceroltrioleate (C(18:1))/glyceroltrierucate (C(22:1)) (Lorenzo's oil, LO) combined with a VLCFA-poor diet normalizes VLCFA concentrations, but the neurological symptoms persist or even progress in symptomatic patients. Considering that free radical generation is involved in various neurodegenerative disorders and that in a previous study we showed evidence that oxidative stress is probably involved in the pathophysiology of X-ALD symptomatic patients, in the present study we evaluated various oxidative stress parameters, namely thiobarbituric acid reactive species (TBA-RS) and total antioxidant reactivity (TAR) in plasma, as well as the activities of the antioxidant enzymes catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) in erythrocytes from symptomatic and asymptomatic X-ALD patients and verified whether LO treatment and a VLCFA restricted diet could change these parameters. We observed a significant increase of plasma TBA-RS in symptomatic and asymptomatic X-ALD patients, reflecting induction of lipid peroxidation even before the disease was manifested. In addition, LO treatment did not alter this profile. Furthermore, plasma TAR measurement of X-ALD patients was not different from that of controls. Similarly, the antioxidant enzyme activities CAT, SOD and GPx were not altered in erythrocyte from X-ALD patients as compared to controls. We also examined the in vitro effects of hexacosanoic acid (C(26:0)) and tetracosanoic acid (C(24:0)) alone or combined with oleic (C(18:1))/erucic (C(22:1)) acids on various oxidative stress parameters in cerebral cortex of young rats, namely chemiluminescence, TBA-RS, TAR, CAT, SOD and GPx in order to investigate whether those fatty acids were able to induce oxidative stress. We found that there was a significant increase of TBARS and of chemiluminescence in rat cerebral cortex exposed to C(26:0)/C(24:0), and that the addition of C(18:1)and C(22:1) to the assays did not prevent this effect. Furthermore, TAR measurement was not altered by C(26:0) and C(24:0) acids in rat cerebral cortex. Taken together, our results indicate that lipid peroxidation occurs in X-ALD and that LO treatment does not attenuate or prevent free radical generation in these patients. Therefore, it may be presumed that antioxidants should be considered as an adjuvant therapy for X-ALD patients.

Short communication: Eicosatrienoic acid and docosatrienoic acid do not promote vaccenic acid accumulation in mixed ruminal cultures.

Previous research found that docosahexaenoic acid (C22:6n-3) was a component of fish oil that promotes trans-C18:1 accumulation in ruminal cultures when incubated with linoleic acid. The objective of this study was to determine if eicosatrienoic acid (C20:3n-3) and docosatrienoic acid (C22:3n-3), n-3 fatty acids in fish oil, promote accumulation of trans-C18:1, vaccenic acid (VA) in particular, using cultures of mixed ruminal microorganisms. Treatments consisted of control, control plus 5 mg of C20:3n-3 (ETA), control plus 5 mg of C22:3n-3 (DTA), control plus 15 mg of linoleic acid (LA), control plus 5 mg of C20:3n-3 and 15 mg of linoleic acid (ETALA), and control plus 5 mg of C22:3n-3 and 15 mg of linoleic acid (DTALA). Treatments were incubated in triplicate in 125-mL flasks, and 5 mL of culture contents was taken at 0 and 24 h for fatty acid analysis by gas-liquid chromatography. After 24 h of incubation, the concentrations of trans-C18:1 (0.87, 0.88, and 0.99 mg/culture), and VA (0.52, 0.56, and 0.62 mg/culture) were similar for the control, ETA, and DTA cultures, respectively. The concentrations of trans-C18:1 (5.51, 5.41, and 5.36 mg/culture), and VA (4.78, 4.62, and 4.59 mg/culture) were also similar between LA, ETALA, and DTALA cultures, respectively. These data suggest that C20:3n-3 and C22:3n-3 are not the active components in fish oil that promote VA accumulation when incubated with linoleic acid.

The memory-enhancing effect of erucic acid on scopolamine-induced cognitive impairment in mice.

Erucic acid is a monounsaturated omega-9 fatty acid isolated from the seed of Raphanus sativus L. that is known to normalize the accumulation of very long chain fatty acids in the brains of patients suffering from X-linked adrenoleukodystrophy. Here, we investigated whether erucic acid enhanced cognitive function or ameliorated scopolamine-induced memory impairment using the passive avoidance, Y-maze and Morris water maze tasks. Erucic acid (3mg/kg, p.o.) enhanced memory performance in normal naïve mice. In addition, erucic acid (3mg/kg, p.o.) ameliorated scopolamine-induced memory impairment, as assessed via the behavioral tasks. We then investigated the underlying mechanism of the memory-enhancing effect of erucic acid. The administration of erucic acid increased the phosphorylation levels of phosphatidylinositide 3-kinase (PI3K), protein kinase C zeta (PKCζ), extracellular signal-regulated kinase (ERK), cAMP response element-binding protein (CREB) and additional protein kinase B (Akt) in the hippocampus. These results suggest that erucic acid has an ameliorative effect in mice with scopolamine-induced memory deficits and that the effect of erucic acid is partially due to the activation of PI3K-PKCζ-ERK-CREB signaling as well as an increase in phosphorylated Akt in the hippocampus. Therefore, erucic acid may be a novel therapeutic agent for diseases associated with cognitive deficits, such as Alzheimer's disease.

Lipid metabolism in Chinese hamster V79-R membranes composed of unusual phospholipid molecular species.

The relationship between the inhibition of cell growth and the changes in phospholipid metabolism in the presence of erucic acid was studied in Chinese hamster V79-R cells. 1. The addition of erucic acid to the medium inhibited cell growth. The degree of inhibition by erucic acid at a given concentration was dependent on cell density. 2. Exogenous erucic acid was incorporated into cellular phospholipids to form new phospholipid molecular species, which were identified to be the erucoyl/oleoyl, erucoyl/gondoyl and erucoyl/erucoyl species. 3. Synthesis of phosphatidylcholine and phosphatidylethanolamine in endoplasmic reticulum was reduced by erucic acid. Erucic acid had no effect on membrane flow of phospholipids from endoplasmic reticulum to plasma membrane. 4. The specific activity of sn-glycerol-3-phosphate acyltransferase in the membrane fraction from the cells supplemented with erucic acid was lower than that from the control cells. The reduction of phospholipid synthesis was attributed to the decrease in sn-glycerol-3-phosphate acyltransferase activity.

Evidence of accumulation of ceramides containing [14C]nervonic acid in the rat lung following injection of [14C]erucic acid.

A mixture of albumin-bound [14C]erucate and [3H]oleate was injected into rats fed a stock pellet diet containing 4% by weight of lipid. Chylomicrons containing the same labelled fatty acids were also injected into rats fed diets containing 15% by weight of rapeseed oil (48% of erucic acid), canbra oil (less than 5% of erucic acid) or ground nut oil (no erucic acid). Lung lipids were analyzed at various times after injection. In all cases, except in the rapeseed oil diet group, 14C radioactivity of lung 'monoacylglycerol' was ten times higher than 3H radioactivity. More than 85% of this 14C radioactivity was found in nervonic acid (24 : 1). It was shown by TLC and GLC analysis that 85-90% of the 14C radioactivity of this fraction was in ceramides (N-acyl-4-sphingenine). Ceramides containing [14C]nervonic acid disappeared from the lung with time and their incorporation with time into sphingomyelin was also observed. The absence of accumulation of 3H and 14C (18 : 1) in ceramides showed that oleic acid was not incorporated into sphingomyelin in the same way as nervonic acid. In the rapeseed oil diet group, there was no accumulation of 14C radioactivity in ceramides and conversion of erucic acid into nervonic acid was less, and into oleic acid more, than in other diet groups indicating a possible enzyme adaptation.

Evidence for a negative modulating effect of erucic acid on the peroxisomal beta-oxidation enzyme system and biogenesis in rat liver.

In male rats fed a 30 cal% high-erucic acid (22:1 (13) (cis] rapeseed oil diet for 4 weeks a transient, small increase in peroxisomal beta-oxidation was observed in liver homogenates and isolated peroxisomes after approximately 1 week. Morphometric analysis revealed a progressive decrease in the average size of the liver peroxisomes (by approx. 20%), as well as their volume fraction (by more than 40%). A negative dose-response was observed when peroxisomal beta-oxidation was determined in animals fed rapeseed oil diets with a variable content of erucic acid. When erucic acid was given as subcutaneous injections the peroxisomal beta-oxidation decreased, in both liver homogenates and isolated peroxisomes. In contrast to recent proposals, our findings indicate that the amount of erucic acid cannot account for the small increase in peroxisomal beta-oxidation observed in the liver of rats adapted to a diet containing 30 cal% rapeseed oil with a high content of this fatty acid. In fact, by the selected criteria erucic acid seems to have a negative modulating effect on both the peroxisomal beta-oxidation enzyme system and biogenesis.

Failure of dietary erucic acid to impair oxidative capacity or APT production of rat heart mitochondria isolated under controlled conditions.

1. Male, 8-week old rats were fed Purina Rat Chow for semisynthetic diets containing 20% by weight of rapeseed oil or corn oil for 3 days. 2. The hearts from the animals fed the three diets were analyzed for total lipid, phospholipid, free fatty acids, cholesterol esters, tri-, di- and monoacylglyerols. There was a seven-fold increase in the levels of triacylglycerols in the hearts of rats fed rapeseed oil diet compared to the levels in the hearts of animals fed the other two diets. Smaller increases in the content of other neutral lipid fractions were also observed. 3. Heart mitochondria from the three groups of animals were isolated under controlled conditions in the presence or absence of heparin. The rats of oxidation of different substrates and of ATP synthesis by these mitochondria were compared. 4. Mitochondria isolated in the absence of heparin from rapeseed oil-fed rats had much lower rates of oxidation and ATP synthesis than mitochondria isolated similarly from rats fed the other two diets. 5. With mitochondria freshly isolated in the presence of heparin, no significant differences in rates of oxidation or ATP synthesis were found among the three groups of animals. 6. It is concluded that, when properly isolated, mitochondria from rapeseed oil-fed rats are functionally intact with respect to oxidation and energy-coupling capacity.

Studies on the mechanism of the inhibitory effects of erucylcarnitine in rat heart mitochondria.

1. The mechanism of the inhibitory effect of erucylcarnitine on palmityl-carnitine oxidation in rat heart mitochondria was studied. 2. Erucylcarnitine inhibited in the same time the oxidation of [U-14-C]-palmitylcarnitine and the total rate of oxygen uptake. Other acylcarnitines competed as well for the oxidation with radioactive palmitylcarnitine, but they were well oxidized themselves, so that the total oxygen uptake did not decrease. 3. The presence of erucylcarnitine did not change the distribution pattern of Krebs cycle intermediates derived from [U-minus 14 C] palmitylcarnitine except that succinate/malate ratio increased. 4. The presence of erucylcarnitine did not lead to the formation of any beta-oxidation cycle intermediates from [U-minus 14 C] palymitylcarnitine. The formation of beta-hydroxy-palmityl derivative when rotenon was included into the incubation medium, decreased in the presence of erucylcarnitine. 5. It is postulated, that the inhibited entrance of palmityl groups into the beta-oxidation cycle is due to the fact that erucylcarnitine and palmitylcarnitine behave as substrate-competitive inhibitors for long chain acyl-CoA dehydrogenase. 6. There was observed a latency of 1-2 min in the effect of erucylcarnitine on the palmitylcarnitine oxidation, which seems to correspond to the time required for the formation of high amounts of intramitochondrial erucyl-CoA. 7. Erucylcarnitine inhibited the total oxygen uptake with long, medium and short chain acylcarnitines, pyruvate and alpha-ketoglutarate as substrates, while the oxidation of succinate was not affected. 8. Sequestration of free CoA in the form of very slowly metabolized erucyl-CoA is proposed as the partial explanation of the observed inhibitory effects of erucylcarnitine on the oxidation of CoA-dependent substrates (alternatively to the inhibition at the level of acyl-CoA dehydrogenases in case of acylcarnitines).