Impact of fertilisation practices on soil respiration, as measured by the metabolic index of short-term nitrogen input behaviour.

The main objective of this study was the evaluation of the impact of different sources of organic waste (used as an N source) on soil quality (as measured by CO(2) release) and N transformation processes (available inorganic N forms) in a short-term field study of an almond tree plantation. Three compost types were used as organic fertilisers: EC compost constituted from organic agriculture farm (vegetables and manure), SC compost formed from sewage sludge and pruning waste composted, and XC compost comprised a mixture of composted sewage sludge plus slurry and manure from an intensive pig farm. The two compost doses were compared according to N content, and a high dose (H), corresponding to 210 kg N ha(-1), and a low dose (L), equivalent to 105 kg N ha(-1), were used. In addition, an N rate corresponding to 130 kg N ha(-1), which resulted from the supplementation of NPK mineral fertiliser with compost application at a low dose (mixed fertilisation), was compared in a parallel study. Generally, almost all organically treated soils demonstrated an improvement in the levels of C, N and P, compared to controls (unfertilised soils). In addition, the nitrate content increased, predominating over ammonium content, with the highest values in the soils with the low dose application of SC. Furthermore, soil respiration improved in organically treated soils, which showed different responses according to the organic-exogenous source of the incorporated matter. In contrast, a mineral supplement promoted a decrease in biological activity and resulted in lower CO(2) production in soils with XC and mineral fertiliser. Contrary to the organically treated soil, in soils with mix fertilisation the NH(4)(+)-N was the primary available form of nitrogen. However, the application of SC plus mineral fertiliser to soil caused a positive effect on CO(2) emissions compared to the control soil. Soil respiration behaviour was closely related to the form of inorganic N available in the soils due to the fertilisation practice type (organic or mixed), where both parameters seemed to depend on the mobilisations of cations (Na(+) and Ca(2+)) to the soil solution.

Pyrenemethyl laurate, a new fluorescent substrate for continuous kinetic determination of lipase activity.

A fluorescent acyl derivative of pyrenemethanol, pyrenemethyl laurate, was synthesized and used for the determination of several lipases by a continuous kinetic assay. The influence of the physical parameters of the substrate (pyrenemethyl laurate) and its hydrolysis product (pyrenemethanol), on the fluorescence emission was studied. The hydrolysis of pyrenemethyl laurate could be monitored directly in a spectrofluorometer because of the very high monomeric emission of pyrenemethanol at about 375 nm, whereas an aqueous dispersion of pyrenemethyl laurate emitted at 475 nm ('excimeric'). Pyrenemethyl laurate was hydrolyzed by gastric lipase, cellular lipases of haemopoietic cells, and the bacterial lipase of Rhizopus arrhizus.

Metabolism of pyrenedecanoic acid in Epstein-Barr virus-transformed lymphoid cell lines from normal subjects and from a patient with multisystemic lipid storage myopathy.

A lymphoid cell line has been established from a patient with multisystemic lipid storage myopathy and showed a major triacylglycerol storage, whereas the content of other neutral lipids and phospholipids was in the normal range. The metabolism of the triacylglycerols has been investigated in this lymphoid cell line from multisystemic lipid storage myopathy as well as in control cells through pulse-chase experiments using 10-(1-pyrene)decanoic acid (P10), a fluorescent fatty acid derivative, as precursor. After 1 h incubation, the uptake of P10 was not significantly different in multisystemic lipid storage myopathy and control lymphoid cells. The amount of fluorescent lipids synthesized by the lymphoid cells was proportional to the concentration of P10 in the culture medium. After 24 h incubation, at any extracellular concentration of P10, the content of P10-labelled triacylglycerols was much higher in multisystemic lipid storage myopathy cells than in controls. Chase experiments showed an impairment in the rate of degradation of biosynthesized triacylglycerols in multisystemic lipid storage myopathy lymphoblasts compared to controls with time of chase (the ratio P10-triacylglycerols/P10-phospholipids increased in mutant cells while it decreased in normal cells). Elsewhere, no enzyme deficiency of the neutral triacylglycerol lipase activity, has been found in multisystemic lipid storage myopathy lymphoid cells.

Independence of triacylglycerol-containing compartments in cultured fibroblasts from Wolman disease and multisystemic lipid storage myopathy.

The functional relationship between the two subcellular compartments involved in catabolism of triglycerides, i.e. lysosomes and lipid-containing cytoplasmic vacuoles, has been investigated using cultured fibroblasts from patients affected with two different genetic lipid (triacylglycerol) storage disorders: Wolman disease and multisystemic lipid storage myopathy. As shown by metabolic studies in intact cultured cells, lysosomal degradation of exogenous labelled triacylglycerols (incorporated into lipoproteins and internalized via the apo B/E receptor pathway) was blocked in Wolman cells, whereas catabolism of endogenously biosynthesized triacylglycerols was in the normal range. In contrast, in fibroblasts from multisystemic lipid storage myopathy, the degradation of endogenous triacylglycerols was blocked, whereas that of exogenous triacylglycerols (i.e. from lipoproteins) was normal. This comparative study demonstrates that the lysosomal and cytoplasmic compartments are functionally independent. Enzymatic studies allows one to discriminate clearly between 3 lipases and 2 carboxylesterases the role of which is discussed.

Diffuse storage of vegetal wax hydrocarbons of dietary origin. Pathologic and chemical findings in a case.

A 55-year-old man died suddenly, after presenting with unstable angina and an asymptomatic micronodular pulmonary pattern. Autopsy revealed storage of a crystallized fatty substance in the lymph nodes, liver, spleen, adrenal glands, and lungs. In the latter organ, the deposition formed foreign body granulomas, accounting for the radiographic appearance. Mass spectrometry identified the fatty substance as straight-chain saturated hydrocarbons (n-alkanes) of carbon-29 and carbon-31 atoms, which are naturally present in the cuticular wax of many vegetals. The case history and the elimination as causal agent of products manipulated in the patient's work led to the conclusion that the storage was due to excessive consumption of apples, and to a lesser degree of Brussels sprouts. We present the light-microscopic findings and the principal biochemical results. Pathogenic mechanisms are described. As far as we are aware, this is the only observation of vegetal alkane storage to be described to date.

Pyrene-methyl lauryl ester, a new fluorescent substrate for lipases: use for diagnosis of acid lipase deficiency in Wolman's and cholesteryl ester storage diseases.

Fluorescent pyrene-methyl lauryl ester (PMLes) was synthesized and used for the determination of cellular lipase activities in lymphoblasts and fibroblasts from normal subjects and from patients affected with Wolman's or cholesteryl ester storage diseases (both exhibiting a deficiency of the lysosomal acid lipase). The hydrolysis of PMLes by acid lipase could be followed directly in a spectrofluorometer; this was possible because of the very high fluorescence emission of pyrene-methanol at 378 nm (monomeric form) in aqueous medium, whereas the substrate has practically no monomeric emission at 378 nm but emits only at 475 nm (excimeric form) in the experimental conditions used: this property permitted us to use PMLes as a fluorogenic substrate. In an alternative procedure, the enzymatic reaction could be determined after partition of the reaction mixture in a biphasic system of heptane and aqueous ethanol; the residual undegraded substrate partitioned into the upper heptane phase and the fluorescence of the product (i.e. pyrene-methanol) was read in the lower aqueous-ethanolic phase, at 378 nm. PMLes was hydrolyzed in extracts of normal lymphoblasts and fibroblasts by at least two lipases, one acidic lipase (pH 4.0) and a second more neutral enzyme (pH 6.5). The acidic lipase activity was practically absent in lymphoblasts and fibroblasts from Wolman's or cholesteryl ester storage diseases. This demonstrates that the fluorescent PMLes is hydrolyzed by the lysosomal acid lipase and can be used as a very sensitive fluorogenic substrate which permits direct recording of product formation and is suitable for the enzymatic diagnosis of either of these diseases.

Hydrolysis of fluorescent pyrenetriacylglycerols by lipases from human stomach and gastric juice.

Fluorescent triacylglycerols containing pyrenedecanoic (P10) and pyrenebutanoic (P4) acids were synthesized and their hydrolysis by lipases from human gastric juice and stomach homogenate was investigated. The existence in stomach homogenate of four different lipolytic enzymes hydrolyzing fluorescent triacylglycerols is suggested by the comparison of various enzymatic properties: acyl chain length specificity, heat inactivation and effect of detergents (Triton X-100 and taurocholate), serum albumin, diethyl-para-nitrophenyl phosphate (E600) and other inhibitors. (1) The acid pH4-lipase hydrolyzes P10-triacylglycerols but not P4-triacylglycerol and exhibited the characteristic properties of the lysosomal lipase: the maximal activating effect of detergents occurs at relatively high concentrations (the substrate/detergent optimal molar ratios were 1:5 and 1:25 for triacylglycerols/taurocholate and triacylglycerols/Triton X-100, respectively); its activity was strongly inhibited by para-chloromercuribenzoate (2.5 mmol/l), but was not significantly affected by serum albumin and E600 (10(-2) mmol/l). (2) The neutral pH7-lipase hydrolyzes P10-triacylglycerols but not P4-triacylglycerol. It is resistant to E600 and heat-stable, similarly to the acid pH4-lipase, but it is well discriminated from the acid enzyme by its substrate/detergent optimal molar ratios (1:2 and 1:3 for triacylglycerols/taurocholate and triacylglycerols/Triton X-100, respectively), whereas higher detergent concentrations, optimal for the acid lipase, are strongly inhibitory for the neutral enzyme. (3) The pH5-lipase present in gastric juice as well as in stomach homogenate exhibited properties obviously discriminating it from the other lipolytic enzymes from stomach homogenate: broad substrate specificity for P10- as well as P4-triacylglycerols, activation by low concentrations of amphiphiles (with optimal ratios triacylglycerols/taurocholate, triacylglycerols/taurocholate and triacylglycerols/phosphatidylcholine around 1:1, 1:3 and 1:0.1, respectively), heat-lability, strong activation by serum albumin and inhibition by E600 (10(-2) mmol/l). This pH5-lipase is the sole lipolytic enzyme present in gastric juice and is probably identical with the well-known 'gastric' lipase. (4) A pH7.5-enzyme is characterized by its specificity for P4-triacylglycerols, its heat-lability at 50 degrees C and its strong inhibition by E600 (10(-2) mmol/l).

Relative fluorescence of normal and acid lipase-deficient cultured fibroblasts following administration of pyrene decanoic acid.

Skin fibroblasts, derived from normal individuals or patients with Wolman's disease (an autosomal recessive disorder due to acid lysosomal lipase deficiency) were incubated with the fluorescent fatty acid, pyrene-decanoic acid (P10). Measurements of the fluorescence intensities of the total lipid extracts indicated that equal quantities of P10 were incorporated into both cell types. The fluorescence emitted by the intact cells was subsequently recorded in a fluorescence microscope equipped with a microdetector unit, which permitted determination of the fluorescence emitted by the intact cell or by specific regions thereof. The fluorescence intensities emitted by the lipidotic cells exceeded those of their normal counterparts 2- and 5-fold when comparing the entire cells or the perinuclear region, respectively. The cells were then subjected to subcellular fractionation and an analysis of the fractions revealed that up to 85-90% of the fluorescence of the lysosome-mitochondrial pellet was derived from free pyrenedecanoic acid; the latter contributed only 15-18% to the fluorescence of the homogenate or the cytosol. There was no difference in the fluorescence of the lipid extracts from the respective fractions of the lipidotic or normal cells. However, the fluorescence emitted by the intact lysosome-mitochondrial fraction of the lipidotic cells exceeded that of its normal counterpart 2.5-fold. These data suggest that the increased fluorescence intensity of the intact lipidotic cells resulted from a higher quantum yield of free P10 molecules solubilized in the hydrophobic environment of their neutral lipid-containing storage granules.

Use of a fluorescent radiolabeled triacylglycerol as a substrate for lipoprotein lipase and hepatic triglyceride lipase.

A fluorescent radiolabeled triacylglycerol has been synthesized by using a fluorescent fatty acid (pyrene decanoic acid) and a radiolabeled oleic acid. This analog of the natural substrate, 1(3)pyrene decanoic-2,3 (1,2)-dioleoyl-sn-glycerol, has been tested as substrate for determining lipoprotein lipase and hepatic triacylglycerol lipase activities in post-heparin plasma. Optimal conditions for the determination of the two post-heparin plasma lipases were similar to those using radiolabeled triolein. Using this substrate, both post-heparin lipases exhibited their characteristic properties (pH optimum and effect of inhibitors) and attacked external ester bonds (1 or 3) containing pyrene decanoic and oleic acids at a similar rate.

[Acid lipases and acid cholesterol esterases: Wolman's disease and cholesteryl ester storage disease]. / Lipases et cholestérol estérases acides: maladie de Wolman et "cholesteryl ester storage disease" (polycorie cholestérolique de L'adulte).

In the first part of the review are reported the properties of mammalian acid lipases and cholesterol esterases. Lysosomal acid lipase differs from the other acid or neutral lipases by its subcellular localization and a large substrate specificity on natural lipids, triglycerides and cholesteryl esters and on semi-synthetic or synthetic coloured or fluorescent substrates; the enzymatic activity of acid lipase depends on the presence of detergents and phospholipids and the structural properties are well known. In vivo, lysosomal acid lipase hydrolyses neutral lipids from exogenous origin (lipoprotein). The second part is an updated review on the diseases caused by hereditary acid lipase deficiency: Wolman's disease occurring in the first months of life and fatal before the age of one year and Cholesteryl Ester Storage Disease, a more benign form with normal lifespan. Both diseases are characterized by massive storage of neutral lipids. Molecular and metabolic pathways, new diagnostic tools used for the diagnosis and experimental cellular model systems are reviewed and discussed.

A new human pathology with visceral accumulation of long-chain n-alkanes; tissue distribution of the stored compounds and pathophysiological hypotheses.

This report deals with a new human disorder characterized by the accumulation of plant long-chain n-alkanes in viscera of a human patient. Lipid analysis of tissues from an adult male after sudden death (affected with diffuse visceral granuloma containing lipophilic crystallized material) showed the presence of abnormal compounds identified as long-chain n-alkanes with 29 (n-nonacosane), 31 (n-hentriacontane) and 33 carbons (n-tritriacontane). Study of n-alkane distribution in patient tissues showed a major accumulation in lumbo-aortic lymph nodes, adrenal glands, lung (the highest levels were found in lung granulomas) and liver; significantly lower amounts were detected in myocardium and kidney, whereas no detectable level was found in brain. On the basis of the structural composition and of the tissue distribution of the accumulated n-alkanes, their dietary (plant) origin and the pathophysiological mechanism of the storage are discussed.

Cholesterol esterases from normal and FEC hamster liver.

1. Synthetic cholesteryl esters with various acyl chain length (C2-C18) are hydrolysed by several enzymes in hamster liver. 2. The comparison of effect of inhibitors, divalent cations, detergents, pH and substrate specificity allows discrimination between four enzymes hydrolyzing cholesteryl esters, which are characterized by their enzymatic properties, two cholesterol esterases (resistant to E600) hydrolyzed medium- and long-chain cholesteryl esters, whereas short-chain cholesteryl esters were hydrolyzed by two different carboxylesterases (dramatically inhibited by E600). 3. The acid cholesterol esterase (identical to the lysosomal lipase) exhibited a pH optimum at pH 5.0 and is activated by 1 mM taurocholate. 4. The alkaline cholesterol esterase (pH optimum 7.5) is not very sensitive to the tested effectors. 5. Both acid and alkaline carboxylesterases (pH optima 5.5 and 7.5), were characterized by their strict dependence on divalent cations (Mn2+ or Mg2+). 6. The acid carboxylesterase was inhibited by increasing concentrations of Triton X-100, whereas the alkaline carboxylesterase was dramatically activated by 2 g/l Triton X-100. 7. No significant difference was observed in activities of cholesterol esterases or carboxylesterases between normal and FEC hamster livers.

Biochemical and ultrastructural features of human fibroblasts cultured from a new variant of type 3 lipid storage myopathy.

A new variant of multisystemic lipid storage myopathy (type 3) has been identified. Human cultured fibroblasts present a major triacylglycerol storage whereas other neutral lipids and phospholipids are in the normal range. When feeding the cells in the presence of radiolabelled oleic acid we observed an accumulation of radiolabelled triacylglycerols demonstrating the endogenous biosynthesis of the stored triacylglycerols. After a 72-hr chase period, no degradation of radiolabelled triacylglycerols was observed. Histochemical examination of multisystemic lipid storage myopathy skin fibroblasts showed a massive accumulation of neutral lipids (stained by the fluorescent probe Nile Red) in cells grown in medium supplemented with 10% fetal calf serum. These cytoplasmic vacuoles were not obviously membrane-surrounded as shown by electron microscopy.

Extracellular origin of the lipid lysosomal storage in cultured fibroblasts from Wolman's disease.

The experiments reported here allowed us to compare the metabolism of neutral lipids from extracellular origin (lipoproteins) and endogenous origin (triacylglycerol biosynthesis induced by feeding cells with high levels of free fatty acid) in normal and acid-lipase-deficient fibroblasts (Wolman's disease). When the cells were grown in hyperlipemic-rich medium, a major neutral lipid storage appeared in normal as well as in acid-lipase-deficient cells; this storage disappeared rapidly in normal cells during the 'chase', whereas in Wolman cells, the storage of cholesteryl esters and triacylglycerols remained unchanged, or only decreased very slowly. When the cells were fed with high levels of radiolabelled oleic acid, a major accumulation of radiolabelled triacylglycerols was observed. These cytoplasmic triacylglycerols were similarly degraded in normal and Wolman fibroblasts during the 'chase' period. From these results it was concluded that the neutral lipids stored in lysosomes of Wolman fibroblasts are only of extracellular origin (lipoproteins), whereas triacylglycerols biosynthesized by the cells do not participate in this accumulation. Therefore, both cellular compartments involved in triacylglycerol metabolism (lysosomes containing exogenous lipids and cytoplasmic granules of endogenously biosynthesized triacylglycerols) are strictly independent.

Metabolism of 1-pyrenedecanoic acid and accumulation of neutral fluorescent lipids in cultured fibroblasts of multisystemic lipid storage myopathy.

The lipid metabolism in cultured fibroblasts from multisystemic (type 3) lipid storage myopathy and controls has been studied through pulse-chase experiments using 1-pyrenedecanoic acid as precursor. The uptake of 1-pyrenedecanoic acid was not significantly different in multisystemic lipid storage myopathy and control fibroblasts. The amount of fluorescent lipids synthesized by the cells was proportionally increasing with rising 1-pyrenedecanoic acid concentration in the culture medium. The proportion of the various fluorescent lipids does not significantly vary between 17 to 67 nmol/ml. But a 1-pyrenedecanoic acid concentration higher than 70-100 nmol/ml seems to be severely toxic for the cells. When incubated for 24 h in the presence of 1-pyrenedecanoic acid, at any concentration, the neutral lipid content (triacylglycerols, diacylglycerols and cholesterol esters) of cultured multisystemic lipid storage myopathy fibroblasts was higher than that of controls (around 600% of controls). Chase experiments showed that the biosynthesized triacylglycerols were not degraded in multisystemic lipid storage myopathy cells, but on the contrary were increased, probably by acylation of fluorescent fatty acids liberated from phospholipid turnover. In normal fibroblasts all the cellular fluorescence disappeared after 5 days chase and 1-pyrenedecanoic acid was recovered (as free 1-pyrenedecanoic acid) in the culture medium. In contrast, in multisystemic lipid storage myopathy fibroblasts, 40% of the fluorescence was remaining in the cells after 5 days chase; it was contributed by fluorescent triacylglycerols, which appeared as strongly fluorescent cytoplasmic vesicles. This probably results from a defect of the cytoplasmic catabolism of triacylglycerols which are accumulated in a cytoplasmic compartment independent of the lysosomal compartment (since the acid lysosomal lipase is not deficient in the multisystemic lipid storage myopathy cells). Finally, these results suggest a practical diagnostic application of 1-pyrenedecanoic acid, which can be used to differentiate multisystemic lipid storage myopathy from normal cultured fibroblasts.

Metabolism of neutral lipids in cultured fibroblasts from multisystemic (or type 3) lipid storage myopathy.

The lipid metabolism in cultured fibroblasts from multisystemic (type 3) lipid storage myopathy (MLSM) and controls has been studied through pulse-chase experiments using radiolabelled oleic acid and acetate precursors. The uptake of radiolabelled oleic acid by MLSM fibroblasts was slightly higher than in controls but did not seem to be the primary defect of the multisystemic lipid storage myopathy. The uptake of radiolabelled acetate was quite similar in MLSM and in control cells. During short-time pulse periods, using either radiolabelled oleic acid or acetate as precursors, we observed no significant difference in lipid composition between MLSM and controls. In contrast, pulse experiments using radiolabelled oleic acid as precursor showed a major accumulation of radiolabelled triacylglycerols in MLSM (around 1000% of controls); no significant increase of other neutral or polar lipids was noticed. A similar triacylglycerol storage was observed by using radiolabelled acetate as precursor, but in this case the difference between MLSM and controls was more pronounced; we also observed in MLSM cells a higher amount of polar lipids which can be due to an increased rate of fatty acid biosynthesis (from radiolabelled acetate). Chase experiments, after pulse by low concentration of exogenous radiolabelled oleic acid or acetate, showed similar features: the biosynthesized triacylglycerols were not at all degraded in MLSM, but on the contrary increased, probably by accumulation of radiolabelled triacylglycerols newly synthesized from radiolabelled fatty acids liberated during the phospholipid turnover. Similarly, the triacylglycerol storage induced by high doses of fatty acids was not degraded in MLSM cells, in contrast to control cells. This suggested that the triacylglycerols synthesized in the presence of low and high levels of fatty acids were accumulated in only one subcellular cytoplasmic compartment without relationship with the lysosomal compartment, since these cells were not deficient in acid lysosomal lipase. The more probable hypothesis is a deficiency of the cytoplasmic catabolism of triacylglycerols in MLSM cells.