Contributions of basic neurochemistry towards a novel concept of epilepsy

Epilepsy is an ancient disorder which treatment over the centuries has been guided by preconceptions regarding its origin. The major improvements in epilepsy management came following the discovery of the EEG and the development of seizure suppressing agents. These advances in diagnosis and anticonvulsant therapy have further ingrained the conviction that epilepsy is a disease of neurons. Evidence presented here is intended to support a different point of view which suggests that the metabolic modifications in epileptogenic tissue denote subtle alterations in the anatomical and biochemical relationship between neurons and their glial envelopes. As a result the extracellular environment of these cells contain higher than normal levels of glutamic acid. This creates an unnatural functional connectivity between neurons so that they establish abnormal synchronous activity between them and become hyperexcitable due to the depolarizing milieu. To compensate for these biochemical changes it is suggested that some thought might be given to epilepsy management by metabolic manipulation. The measures should be directed specifically towards improving the ability of glia to remove glutamic acid from the extracellular milieu. Two obvious possibilities are to enhance glial glutamine synthesis and to improve the interstitial wash-out of glutamic acid in epileptogenic epicenters. Such a therapy would anticipate to gradually diminish seizure incidence and susceptibility without, however, having a direct action on convulsive episodes per se. The approach must be considered an adjunct to current epilepsy treatment and not a substitute for the use of anticonvulsants.Special Issue Dedicated to Dr. Abel Lajtha.     

Decomposition of polyurethane in a garbage landfill leakage water and by soil microorganisms

Summary The decomposition of polyurethane, measured gravimetrially or using infrared spectrophotometry, was found to be more complete in polyurethane based on polyester and only very small in polyurethane based on polyether. In the presence of clay minerals the decomposition was inhibited. If positive, the decomposition of polyurethane followed the sequence: remaining free isocyanatesurea and amide groupsurethane groupsisocyanuric acid rings.     

Guanosine enhances glutamate uptake in brain cortical slices at normal and excitotoxic conditions

1. The effect of guanosine on L-[2,3-³H]glutamate uptake was investigated in brain cortical slices under normal or oxygen–glucose deprivation (OGD) conditions.2. In slices exposed to physiological conditions, guanosine (1–100 M) stimulated glutamate uptake (up to 100%) in a concentration-dependent manner when a high (100 M) but not a low (1 M) concentration of glutamate was used.3. In slices submitted to OGD, guanosine 1 and 100 M also increased 100 M glutamate uptake (38 and 70%, respectively).4. The increasing of glutamate and taurine released to the incubation medium in cortical slices submitted to OGD were significantly attenuated by the presence of guanosine in the incubation medium.5. Guanosine prevented the increase in propidium iodide incorporation into cortical slices induced by OGD, indicating a protective role against ischemic injury.6. These results support the hypothesis of a protective role for guanosine during brain ischemia, possibly by activating glutamate uptake into neural cells.     

Sulfur amino acid metabolism in the developing rhesus monkey brain: Interrelationship of taurine and glutamate

The relationship of taurine to glutamate, and to other amino acids, has been examined in the occipital lobe of the developing rhesus monkey. During development taurine decreases in concentration (4.96 mol/g in fetus to 1.52 mol/g in adult) while glutamate increases (7.92 mol/g in fetus to 11.26 mol/g in adult). When the concentration of taurine is plotted against that of glutamate in fetal, neonatal and adult animals there is a significant correlation in the fetal (p<0.01) and adult (p<0.01) but not in the neonatal occipital lobe samples. This correlation in both fetal and adult brain is specific for these two amino acids. Subcellular fractionation studies further indicate that this relationship may be of special importance in nerve endings.This paper is dedicated to Dr. Derek Richter on his seventy-fifth birthday.     

Effects of L-Glutamate Transport Inhibition by a Conformationally Restricted Glutamate Analogue (2S,1'S,2'R)-2-(Carboxycyclopropyl)Glycine (L-CCG III) on Metabolism in Brain Tissue In Vitro Analysed by NMR Spectroscopy

(2S,1'S,2'R)-2-(Carboxycyclopropyl)glycine (L-CCG III) was a substrate of Na⁺-dependent glutamate transporters (GluT) in Xenopus laevis oocytes (IC₅₀ 13 and 2 M for, respectively, EAAT 1 and EAAT 2) and caused an apparent inhibition of [³H]L-glutamate uptake in mini-slices of guinea pig cerebral cortex (IC₅₀ 12 M). In slices (350 M) of guinea pig cerebral cortex, 5 M L-CCG III increased both the flux of label through pyruvate carboxylase and the fractional enrichment of glutamate, GABA, glutamine and lactate, but had no effect on total metabolite pool sizes. At 50 M L-CCG III decreased incorporation of ¹³C from [3-¹³C]-pyruvate into glutamate C4, glutamine C4, lactate C3 and alanine C3. The total metabolite pool sizes were also decreased with no change in the fractional enrichment. Furthermore, L-CCG III was accumulated in the tissue, probably via GluT. At lower concentration, L-CCG III would compete with L-glutamate for GluT and the changes probably reflect a compensation for the missing L-glutamate. At 50 M, intracellular L-CCG III could reach > 10 mM and metabolism might be affected directly.     

The Free Radical Antioxidant Vitamin E Protects Cortical Synaptosomal Membranes from Amyloid β-Peptide(25-35) Toxicity But Not from Hydroxynonenal Toxicity: Relevance to the Free Radical Hypothesis of Alzheimer's Disease

Amyloid -peptide (A) is a key factor in the neurotoxicity of Alzheimer's disease (AD). Recent research has shown that A-mediated neurotoxicity involves free radicals and that A peptides can initiate multiple membrane alterations, including protein oxidation and lipid peroxidation, eventually leading to neuronal cell death. Research also has emphasized the role of 4-hydroxynonenal (HNE), a downstream product of lipid peroxidation, in being able to mimic some of the effects of A peptides. In the current investigation, electron paramagnetic resonance (EPR) studies of spin labeled cortical synaptosomal membrane proteins has been employed to study conformational changes in proteins, spectrophotometric methods have been used to measure protein carbonyl content, and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for mitochondrial function has been used to study the effect of vitamin E on samples that were treated with A or HNE. The free radical dependence of -amyloid-associated toxicity was confirmed by the ability of the free radical scavenger vitamin E to prevent the toxic effects of A. In contrast, HNE was still toxic in the presence of vitamin E. These results support our A-associated free radical model for neurotoxicity in AD brain and are discussed with reference to potential therapeutic strategies for AD.     

Excitatory Sulfur-Containing Amino Acid-Induced Release of [3H]GABA from Rat Olfactory Bulb

The effect of L-cysteine sulfinic acid (CSA) and L-homocysteic acid (HCA) on the release of tritiated -amino butyric acid ([³H]GABA), from the external plexiform layer (EPL) of the rat olfactory bulb, was compared with that of glutamate. These amino acids induced release of GABA was strongly inhibited by the glutamate uptake blocker, pyrrolidine-2,4-dicarboxylate (2,4,PDC) (50 M), while it was not inhibited by the specific GABA uptake blockers nipecotic acid (0.5 mM) or NO-711 (5M). Only the HCA induced GABA release was 60% inhibited by -alanine (0.5 mM), a glial GABA uptake blocker and 78% by the NMDA receptor antagonist 2-amino-5-phosphonopentanoic acid (AP-5) (100 M). The non-NMDA receptor antagonists 6-cyano-2,3-dihydroxy-7-nitro-quinoxaline (CNQX) up to 500 M had no effect on HCA or CSA stimulated GABA release. These results bring evidence for an excitatory role of HCA and CSA together with glutamate on GABAergic neuronal or glial elements, in the olfactory bulb. This role could be mediated through the reversal of the glutamate or/and the glial GABA transporter and through the activation of a NMDA type receptor.     

Action of L-Acetylcarnitine on Different Cerebral Mitochondrial Populations from Cerebral Cortex

The maximum rate (V_max) of some mitochondrial enzymatic activities related to the energy transduction (citrate synthase, -ketoglutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, cytochrome oxidase) and amino acid metabolism (glutamate dehydrogenase, glutamate-pyruvate-transaminase, glutamate-oxaloacetate-transaminase) was evaluated in non-synaptic (free) and intra-synaptic mitochondria from rat brain cerebral cortex. Three types of mitochondria were isolated from rats subjected to i.p. treatment with L-acetylcarnitine at two different doses (30 and 60 mg·kg^–1, 28 days, 5 days/week). In control (vehicle-treated) animals, enzyme activities are differently expressed in non-synaptic mitochondria respect to intra-synaptic light and heavy ones. In fact, -ketoglutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, glutamate-pyruvate-transaminase and glutamate-oxaloacetate-transaminase are lower, while citrate synthase, cytochrome oxidase and glutamate dehydrogenase are higher in intra-synaptic mitochondria than in non-synaptic ones. This confirms that in various types of brain mitochondria a different metabolic machinery exists, due to their location in vivo. Treatment with L-acetylcarnitine decreased citrate synthase and glutamate dehydrogenase activities, while increased cytochrome oxidase and -ketoglutarate dehydrogenase activities only in intra-synaptic mitochondria. Therefore in vivo administration of L-acetylcarnitine mainly affects some specific enzyme activities, suggesting a specific molecular trigger mode of action and only of the intra-synaptic mitochondria, suggesting a specific subcellular trigger site of action.     

Metabolic manipulation of neural tissue to counter the hypersynchronous excitation of migraine and epilepsy

Very prominent in the large biochemical data bank on epilepsy, is the almost universal finding that a familial or environmental predisposition towards epilepsy, as well as the earliest signs preceding other forms of hypersynchronous excitation, coincide with an altered glutamate metabolism. Hence, it has become increasingly apparent that glutamate occupies a central position in the development of epilepsy or in the onset of a migraine incident. The importance of glutamate is explained by a variety of functions in the CNS: as a dominant constituent of many proteins, by its intermediary role in linking energy metabolism to that of many other amino acids, and as the virtually exclusive precursor of GABA. Moreover, glutamate serves as the primary substrate in ammonia detoxification and the product, glutamine, actively participates in CSF water homeostasis. Finally, by its direct electrophysiological and metabolic actions on neurons and glia, via at least four distinct types of receptor proteins, glutamate is implicated in a number of critical mechanisms of information. These include neuronal excitatory modulation, intracellular Ca²⁺ redistribution, and key metabolic (phosphorylation) mechanisms. The phenomena, when exaggerated due to excessive extracellular glutamate levels, may cause pathological effects such as hypersynchrony-epilepsy, Spreading Depression-migraine, high internal Ca²⁺-damage, impaired phosphorylation/dephosphorylation-necrosis, among others. Not surprising therefore that severe epilepsy may eventually cause CNS cytoarchitectual and metabolic damage, or conversely, that neural tissue trauma not infrequently gives rise to epilepsy many years later. Both conditions are associated with a persistent, excessive leakage or release of glutamate into the extracellular milieu. An electrophysiological and neurochemical commonality between migraine and epilepsy has also been noted. A specific nutritional supplement is proposed, consisting of a combination of taurine (100 mg),l-leucine (75 mg), (acetyl-)l-carnitine (25 mg), and zinc gluconate (10 mg) which is aimed at, principally, normalizing internal Ca²⁺ ionization, and removal of extracellular glutamate by glial amidation to glutamine.Special issue dedicated to Dr. Claude Baxter.     

Effect of γ-Decanolactone on Glutamate Binding in the Rat Cerebral Cortex

Epilepsy is one of the most common neurological disorders. Even though antiepileptic drugs can afford a reasonably satisfactory treatment for 80% of diagnosed patients, chronic intractable epilepsy still affects a significant number of people and more effective and less harmful antiepileptic drugs are needed. Previous studies have shown that -decanolactone has dose-dependent sedative effects, including hypnotic, anticonvulsant and hypothermic properties in mice. The present study reports an inhibitory effect of -decanolactone on glutamate binding (96.8% with 5 mM) in rat cortex membranes. The non competitive nature of glutamate binding inhibition as a neurochemical correlate of the anticonvulsant activity of -decanolactone may be a relevant mode of action for further drug development.     

Guanylyl Cyclase Participates in the Mechanism of Glutamatergic Modulation of Acetylcholine Non-Quantum Release in the Rat Neuromuscular Junction

It was shown that glutamate (10 M to 1 M) suppresses in a dose-dependent manner the non-quantum release of acetylcholine from rat motor nerve endings; the release intensity was estimated by the H effect. The action of glutamate was completely eliminated by the blockade of guanylyl cyclase by 1 M ODQ. An increase in the intracellular cGMP concentration by 1 M dibutyryl-cGMP reduced the H effect in a similar manner as glutamate did.     

In vivo effects of insulin and bis(maltolato)oxovanadium (IV) on PKB activity in the skeletal muscle and liver of diabetic rats

In this study, the in vivo effects of insulin and chronic treatment with bis(maltolato)oxovanadium (IV) (BMOV) on protein kinase B (PKB) activity were examined in the liver and skeletal muscle from two animal models of diabetes, the STZdiabetic Wistar rat and the fatty Zucker rat. Animals were treated with BMOV in the drinking water (0.75–1 mg/ml) for 3 (or 8) weeks and sacrificed with or without insulin injection. Insulin (5 U/kg, i.v.) increased PKB activity more than 10fold and PKB activity more than 3fold in both animal models. Despite the development of insulin resistance, insulininduced activation of PKB was not impaired in the STZdiabetic rats up to 9 weeks of diabetes, excluding a role for PKB in the development of insulin resistance in type 1 diabetes. Insulin-induced PKB activity was markedly reduced in the skeletal muscle of fatty Zucker rats as compared to lean littermates (fatty: 7fold vs. lean: 14fold). In contrast, a significant increase in insulinstimulated PKBa activity was observed in the liver of fatty Zucker rats (fatty: 15.7fold vs. lean: 7.6fold). Chronic treatment with BMOV normalized plasma glucose levels in STZdiabetic rats and decreased plasma insulin levels in fatty Zucker rats but did not have any effect on basal or insulininduced PKB and PKB activities. In conclusion (i) in STZdiabetic rats PKB activity was normal up to 9 weeks of diabetes; (ii) in fatty Zucker rats insulininduced activation of PKB (but not PKB) was markedly altered in both tissues; (iii) changes in PKB activity were tissue specific; (iv) the glucoregulatory effects of BMOV were independent of PKB activity.     

Effect of gamma irradiation on membranes of normal and pathological erythrocytes (beta-thalassemia)

Summary The influence of ionizing radiation on the membrane of human normal erythrocytes has extensively been studied and a variety of effects including changes in the cation fluxes [3, 9] or in non-electrolytes permeability [5, 6, 11], in membrane fluidity, in peroxidation of unsaturated lipids as well as chemical composition or structural modifications [4, 7, 8] has been observed.However, only few studies deal with the effects of ionizing radiation on pathological red blood cells.In this work, we have investigated by means of electron spin resonance (ESR) spectroscopy the effects of⁶⁰Co-radiation on the normal and homozygous-thalassemic human erythrocyte membranes [2, 13].Presented in part at the VII Congress of the Società Italiana di Biofisica Pura ed Applicata Lipari (Italy) 1985     

Effects of D‐mannoheptulose upon D‐glucose metabolism in tumoral pancreatic islet cells

D[³H]mannoheptulose was recently reported to be poorly taken up by tumoral pancreatic islet cells of the RINm5F and INS1 lines. We have now investigated the effects of Dmannoheptulose upon Dglucose metabolism in these two cell lines. Dmannoheptulose (1.0–10.0 mM) only caused a minor decrease of Dglucose metabolism in RINm5F cells, whether at low (1.1 mM) or higher (8.3 mM) Dglucose concentration. A comparable situation was found in INS1 cells examined after more than 20 passages. In both cases, however, the hexaacetate ester of Dmannoheptulose (5.0 mM) efficiently inhibited Dglucose metabolism. In the INS1 cells, the relative extent of the inhibitory action of Dmannoheptulose upon Dglucose metabolism increased from 12.4 ± 2.6 to 38.3 ± 3.8% as the number of passages was decreased from more than 20 to 13–15 passages, the latter percentage remaining lower, however, than that recorded in INS1 cells also examined after 13–15 passages but exposed to Dmannoheptulose hexaacetate (66.9 ± 2.2%). These findings when compared to our recent measurements of D[³H]mannoheptulose uptake, reinforce the view that the entry of the heptose into cells and, hence, its inhibitory action on Dglucose metabolism are dictated by expression of the GLUT2 gene.     

Microphotometric determination of enzyme activities in type-grouped fibres of reinnervated rat muscle

Summary Activities of succinate dehydrogenase (SDH), glycerolphosphate oxidase (GP-OX), cytochrome oxidase (CYT-OX) and lactate dehydrogenase (LDH) were determined microphotometrically in single, actomyosin-ATPase typed (Guth and Samaha 1970) fibres within cross-sections of normal and reinnervated rat tibialis anterior muscles. SDH and GP-OX activities displayed pronounced scattering and large overlaps existed between -, -, and -fibres of normal muscle. Coefficients of variation were in the range of 16–40% for GP-OX and SDH in the different fibre populations. Enzyme activity determinations in typegrouped -, -, and -fibres of reinnervated muscle showed much less scattering than in normally distributed -, -, and -fibres of control muscles. Coefficients of variation were in the range of 10–13% for SDH, GP-OX, CYT-OX and LDH. The experimental error of the kinetic microphotometric measurement of enzyme activities in situ is in the range of 10% (Reichmann and Pette 1982). Our results therefore suggest a high degree of metabolic similarity or homogeneity of typed-grouped muscle fibres and thus support the assumption that type-groped fibres are homogeneous and correspond to regularly assembled motor units.     

Structural identification of oligosaccharides produced by Zymomonas mobilis levansucrase

Summary The chemical structure of oligosaccharides produced by Zymomonas mobilis levansucrase (EC 2.4.1.10) was determined using enzymatic hydrolysis, mass spectroscopy, and ¹³C-NMR spectroscopy. The major oligosaccharide (98% of total oligomers) produced from transfructosylation reactions with -sucrose was identified as 1-kestose (O--D-glucopyranosyl-(12)-O--D-fructofuranosyl-(12)--D-fructofuranoside).     

Amyloid Beta Peptide Impaired Carbachol but not Glutamate-Mediated Phosphoinositide Pathways in Cultured Rat Cortical Neurons

Signal transduction systems, including cholinergic pathways, which are likely to be of pathophysiological significance are altered in Alzheimer's disease (AD). Muscarinic cholinergic receptors are linked to the hydrolysis of phosphoinositide, involving the production of inositol 1,4,5-trisphosphate [Ins (1,4,5)P₃] and the mobilization of cytosolic free calcium concentrations ([Ca²⁺]_i). Effects of amyloid peptide (A) on these signals prior to neuronal degeneration were examined in cultured rat cortical cells. A increased the release of lactate dehydrogenase (LDH) in a concentration-dependent manner, however, it was blocked by B27 supplement. Prolonged exposure to a sublethal dose of A 25–35 or 1–42 disrupted carbachol-mediated release of Ins(1,4,5)P₃ and [Ca²⁺]_i, which was inhibited in media supplemented with B27 or the antioxidant vitamin E. In order to determine the specificity of the effect of A, various agonists glutamate or KCl but not bradykinin which utilize the phosphoinositide cascade were investigated. Our results indicated that A did not affect the stimulation of glutamate or KCl-mediated production of Ins(1,4,5)P₃ or cause elevation in [Ca²⁺]_i. Furthermore, metabotropic agonist trans-1-amino-cyclopentane-1,3,-dicarboxylate (ACPD) elevated calcium level was not inhibited by A pretreatment. Taken together, the results demonstrate that a sublethal dose of A selectively impaired cholinergic receptor-mediated signal transduction pathways, and antioxidant or B27 supplement attenuated this effect of A. Alterations of cholinergic signaling by prolonged exposure to A could be involved in cortical neurodegeneration that occurs in AD. Because functional loss of cholinergic pathways is an important aspect of AD, the differences in susceptibility of these two types of receptors prior to other signs of A action is important and requires further investigation.     

Biosynthesis of (1,3)(1,4)-β-glucan and (1,3)-β-glucan in barley (Hordeum vulgare L.)

Mixed membrane preparations from the coleoptiles and first leaves of young barley (Hordeum vulgare L. cv. Triumph) plants catalysed the synthesis of 55% methanol-insoluble labelled material from UDP[U-¹⁴C]glucose, the main components of which were identified as (1,3)(1,4)-- and (1,3)--D-glucans. The membrane preparations also catalysed the transformation of UDP-glucose into labelled low-molecular-weight products, mainly glucose (by phosphatase action), glucose-1-phosphate (by phosphodiesterase action) and glyco(phospho)lipids (by glycosyltransferase action). The formation of (1,3)(1,4)--glucans, (1,3)--glucans, and the other reactions competing for UDP-glucose, were monitored simultaneously and quantitatively by a novel procedure based on enzymatic analysis, thin-layer chromatography and digital autoradiography. Thus it was possible (i) to optimise conditions to obtain (1,3)(1,4)--glucan synthesis or (1,3)--glucan synthesis in isolation, and (ii) to study the influence of temperature, pH, cofactors, substrate concentration etc. on the (1,3)(1,4) and (1,3)--glucan synthesis reactions even when both occurred together. The synthesis of both -glucans was optimal at 20°C. In Tris-HCl buffer, the pH optima for (1,3)(1,4)--glucan synthesis and (1,3)--glucan synthesis were pH 8.5 and pH 7.0, respectively. Both glucan-synthesis reactions required Mg²⁺: (1,3)--glucan synthesis was optimal at 2 mM, whereas (1,3)(1,4)--glucan synthesis continued to increase up to 200 mM Mg²⁺, when the ion was supplied as the sulphate. (1,3)--Glucan synthesis was Ca²⁺ dependent and this dependence could be abolished by proteinase treatment. The K _m with respect to UDP-glucose was 1.5 mM for (1,3)--glucan synthesis and approximately 1 mM for (1,3)(1,4)--glucan synthesis. The (1,3)(1,4)--glucan formed in vitro had the same ratio of trisaccharide to tetrasaccharide structural blocks irrespective of the experimental conditions used during the synthesis: its enzymatic fragmentation pattern was indistinguishable from that of barley endosperm (1,3)(1,4)--glucan. This indicates either a single synthase enzyme, which is responsible for the formation of both linkage types, or two enzymes which are very tightly coupled functionally.Abbreviations G4G4G3G Glc(1,4)Glc(1,4)Glc(1,3)Glc (-linked) - UDP-Glc uridine-5-diphosphate glucose We are grateful to the Commission of the European Communities for the award of Training Fellowships to Christine Vincent and Martin Becker.     

Intracerebroventricular Guanine-Based Purines Protect Against Seizures Induced by Quinolinic Acid in Mice

Acute and chronic administration of the nucleoside guanosine have been shown to prevent quinolinic acid (QA) and -dendrotoxin-induced seizures, as well as to impair memory and anxiety in rats and mice. In this study, we investigated the effect of i.c.v. administration of guanine-based purines (GTP, GDP, GMP, and guanosine) against seizures induced by the NMDA agonist and glutamate releaser quinolinic acid in mice. We also aimed to study the effects of the poorly hydrolysable analogs of GTP (GppNHp and GTPS) and GDP (GDPS) in this seizure model. QA produced seizures in 100% of mice, an effect partially prevented by guanine-based purines. In contrast to GTP (480 nmol), GDP (320–640 nmol), GMP (320–480 nmol) and guanosine (300–400 nmol), the poorly hydrolysable analogs of GTP and GDP did not affect QA-induced seizures. Thus, the protective effects of guanine nucleotides seem to be due to their conversion to guanosine. Altogether, these findings suggest a potential role of guanine-based purines for treating diseases involving glutamatergic excitotoxicity.     

The North Sea: source or sink for nitrogen and phosphorus to the Atlantic Ocean?

Annual nitrogen and phosphorus budgets for the whole North Sea taking into account the most recent data available were established. The area considered has a total surface of approximately 700,000km² and corresponds to the definition by OSPARCOM (Oslo and Paris Commission) with the exclusion of the Skagerrak and Kattegat areas. Input and output fluxes were determined at the marine, atmospheric, sediment and continental boundaries, and riverine inputs based on river flows and nutrient concentrations at the river–estuary interface were corrected for possible estuarine retention. The results showed that the North Sea is an extremely complex system subjected to large inter-annual variability of marine water circulation and freshwater land run-off. Consequently, resulting total N (TN) and P (TP) fluxes are extremely variable from 1 year to another and this has an important influence on the budget of these elements. Total inputs to the North Sea are 8870±4860kTNyear^–1 and 494±279kTPyear^–1. Denitrification is responsible for the loss of 23±7% of the TN inputs while sediment burial is responsible for the retention of only of 2±2% of the TP input. For TN, due to the large variability on marine and estuarine fluxes, and to the uncertainty related to the denitrification rate, it was concluded that the North Sea could either be a source (1930kTNyear^–1) or a sink (1700kTNyear^–1) for the waters of the North Atlantic Ocean. For TP it was concluded that the North Sea is mostly a source (–4 to 52kTPyear^–1) for the waters of the North Atlantic Ocean.