Degradation of proteins and amino acids by Caloramator proteoclasticus in pure culture and in coculture with Methanobacterium thermoformicicum Z245

This study investigated the degradation of proteins and amino acids by Caloramator proteoclasticus, an anaerobic thermophilic (55 °C) fermentative bacterium isolated from an anaerobic bioreactor. Experiments were performed in the presence and absence of Methanobacterium thermoformicicum Z245, a methanogen that can use both hydrogen and formate for growth. Higher production rates and yields of the principal fermentation products from gelatin were observed in methanogenic coculture. The specific proteolytic activity in coculture tripled the value obtained in pure culture. C. proteoclasticus fermented glutamate to acetate, formate, hydrogen and alanine. In methanogenic coculture, a shift towards higher amounts of acetate and hydrogen with no alanine production was observed. Extracts of glutamate-grown cells possessed high activities of β-methylaspartase, a key enzyme of the mesaconate pathway leading to acetate. The presence of two enzymes (alanine-α-ketoglutarate aminotransferase and NADH-dependent alanine dehydrogenase) usually involved in the biosynthesis of alanine from pyruvate was also detected. The fermentation of amino acids known to be oxidatively deaminated (leucine and valine) was improved in the presence of both methanogenesis and glycine, a known electron acceptor in the Stickland reaction. Culture conditions seem to be very important in the way C. proteoclasticus disposes of reducing equivalents formed during the degradation of amino acids. Received: 29 March 1999 / Received revision: 2 July 1999 / Accepted: 1 August 1999     

Thermophilic,anaerobic degradation of gelatin by Thermobacteroides proteolyticus

Summary The anaerobic thermophilic proteolysis of gelatin by Thermobacteroides proteolyticus (strain BT = ATCC 35245) was investigated. Gelatin was fermented within 5 to 7 days to mainly acetic acid, isobutyric acid, isovaleric acid, and ammonium. Carbon dioxide and hydrogen were produced as well. Maximal ammonification of organic-N was observed at an initial gelatin concentration greater than 2.7 and lower than 10 g/l. Gelatin degradation was not influenced when various amounts of yeast extract (0 to 1 g/l) or ammonium chloride (0 to 6 g/l) were added to the medium.     

Thermophilic anaerobic amino acid degradation: deamination rates and end-product formation

Anaerobic thermophilic degradation of several amino acids was studied in batch cultures using an inoculum from a steady-state semicontinuous enrichment culture. Experiments were done in the presence and absence of methanogenesis and known electron acceptors in the Stickland reaction. Methanogenesis was found to be crucial for the degradation of amino acids known to be oxidatively deaminated (leucine, valine and alanine). Other amino acids (serine, threonine, cysteine and methionine) were degraded under both methanogenic and non-methanogenic conditions. Degradation rates for these four amino acids were 1.3 to 2.2 times higher in cases where methanogenesis was active. The degradation rates of serine, threonine, cysteine and methionine were about twice as high as the rates of leucine, valine and alanine under methanogenic conditions. Inclusion of different electron acceptors, known to work in the Stickland reaction, did not enhance the degradation rates of any amino acid used nor did they alter the degradation patterns. Glycine was oxidatively deaminated to acetate, carbon dioxide, hydrogen and ammonium.     

Factors influencing the production of hydrogen by the purple non‐sulphur phototrophic bacterium Rhodopseudomonas acidophila KU001

Rhodopseudomonas acidophila KU001 was isolated from leather industry effluents and the effect of different cultural conditions on hydrogen production was studied. Anaerobic light induced more hydrogen production than anaerobic dark conditions. Growing cells produced more amounts of hydrogen between 96 and 144 h of incubation. Resting and growing cells preferred a pH of 6.0 ± 0.24 for hydrogen production. Succinate was the most preferred carbon source for the production of hydrogen while citrate was a poor source of carbon. Acetate and malate were also good carbon sources for hydrogen production under anaerobic light. Among the nitrogen sources, R. acidophila preferred ammonium chloride followed by urea for production of hydrogen_. L‐tyrosine was the least preferred nitrogen source by both growing and resting cells.     

Uptake of 15N-labelled alanine,ammonium and nitrate in Pinus sylvestris L. ectomycorrhiza growing in forest soil treated with nitrogen,sulphur or lime

The uptake of ¹⁵N-labelled alanine, ammonium and nitrate was studied in ectomycorrhizal morphotypes of intact Pinus sylvestris seedlings. PCR-RFLP analysis of the ITS-region of fungal rDNA was used to identify the morphotypes. Seedlings were grown in forest soil collected at an experimental site in southern Sweden. The treatments compared were a control, N fertilisation (600 kg N ha^-1 as urea), sulfur application (1200 kg S ha^-1) and lime application (6000 kg CaCO₃ ha^-1). The forest, which had been dominated by Picea abies, was clear-cut two years before the forest soil was sampled. Soil was also collected from an adjacent standing forest. The aim of the present study was to detect changes in the ectomycorrhizal communities in forest soils and relate these changes to the functional parameter of uptake of nitrogen from organic (alanine and protein) and inorganic (ammonium and nitrate) sources.Liming resulted in the detection of a morphotype not found in other samples, and one morphotype was only found in samples from the standing forest (the fungi in these two morphotypes could not be identified). All mycorrhizal root tips showed a higher ¹⁵N concentration after exposure to different nitrogen forms than non-mycorrhizal long roots. Uptake of¹⁵ N from a labelled solution of alanine or ammonium was higher (about tenfold) than uptake from a ¹⁵N-labelled solution of nitrate. Uptake of ammonium and alanine varied between 0.2 and 0.5 mg N g^-1 h^-1 and between 0.1 and 0.33 mg N g^-1 h^-1, respectively, among the different morphotypes.In seedlings grown in the control soil and in soil from standing forest, alanine and ammonium were taken up to a similar degree from a supply solution by all morphotypes, whereas ammonium uptake was higher than alanine uptake in seedlings grown in lime-treated soil (about twofold) and, to a lesser extent, in the nitrogen- and sulfur-treated soils. The higher ammonium uptake by morphotypes from the limed soil was confirmed in pure culture studies. In cases where ammonium was used as the N source, an isolate of the S. variegatus morphotype collected in the limed soil produced more biomass compared with isolates of S. variegatus collected in nitrogen- or sulphur-treated soil. One isolate of a silvery white morphotype produced about equal amounts of biomass on alanine and ammonium, whereas all S. variegatus isolated performed better with ammonium as their N source. Based on the results it is hypothesised that liming can induce a shift in the ectomycorrhizal community, favouring individuals that mainly utilise inorganic nitrogen over those that primarily utilise organic nitrogen.     

Induction of a metabolic switch in insect cells by substrate-limited fed batch cultures

Insect cell metabolism was studied in substrate-limited fed batch cultures of Spodoptera frugiperda (Sf-9) cells. Results from a glucose-limited culture, a glutamine-limited culture, a culture limited in both glucose and glutamine and a batch culture were compared. A stringent relation between glucose excess and alanine formation was found. In contrast, glucose limitation induced ammonium formation, while, at the same time, alanine formation was completely suppressed. Simultaneous glucose and glutamine limitation suppressed both alanine and ammonium formation. Although the metabolism was influenced by substrate limitation, the specific growth rate was similar in all cultures. Alanine formation must involve incorporation of free ammonium, if ammonium formation is mediated by glutaminase and glutamate dehydrogenase, as our data suggest. On the basis of the results, two possible pathways for the formation of alanine in the intermediary metabolism in insect cells are suggested. The cellular yield on glucose was increased 6.6 times during glucose limitation, independently of the cellular yield on glutamine, which was increased 50–100 times during glutamine limitation. The results indicate that alanine overflow metabolism is energetically wasteful and that glutamine is a dispensable amino acid for cultured Sf-9 cells. Preliminary data confirm that glutamine can be synthesised by the cells themselves in amounts sufficient to support growth.     

The influence of different nitrogen sources on the production of volatile compounds by Dipodascus aggregatus

Summary The yeast fungus Dipodascus aggregatus was grown aerobically on 9 different nitrogen sources and the production of volatile compounds determined by a gas chromatographic head-space technique. Excellent growth was supported by glutamine, aspartic acid, asparagine, (NH₄)₂-tartrate and NH₄H₂PO₄. Valine, leucine, and particularly isoleucine were utilized with a somewhat lower growth rate. Lysine was rapidly utilized after a prolonged lag phase.The highest production of volatile compounds was obtained from leucine and isoleucine. At least 20 volatile compounds were formed from each of them and many products were detected in high concentrations. Intermediate amounts of volatile compounds were produced from asparagine, the ammonium salts and valine, and low amounts from lysine, glutamine and aspartic acid.Ethyl acetate was a major product irrespective of the nitrogen source used. Regarding the pattern of volatile compounds produced, leucine, isoleucine and valine had much in common. Most of the volatile products formed from these amino acids contained a branched carbon chain and at least three high-boiling components eluted later than n-amyl acetate from the gas chromatographic column. The other six nitrogen sources could be grouped together. In general the same volatile compounds were formed from these sources, but the quantities of the individual compounds differed. Only one component eluted later than n-amyl acetate. No basic difference in production of volatile compounds was observed between the ammonium salts and -amino compounds like lysine and asparagine.     

Selenocysteine lyase activity in a cysteine-requiring mutant ofEscherichia coli K-12

Selenocysteine lyase activity was detected in crude extracts from a cysteine-requiring mutant ofEscherichia coli K-12. The level of activity was the same whether cells had been grown aerobically or anaerobically, with or without selenocysteine. Selenocysteine lyase catalyzes the conversion of selenocysteine to alanine and elemental Se, a reaction that is followed by a nonenzymatic reduction of the Se to hydrogen selenide. Both of these end products were identified in this study. With cysteine as the substrate, alanine and H₂S were formed, but only at levels 50% less than the products formed from selenocysteine. Selenocysteine lyase has been identified in a number of mammals and bacteria; its presence in a cysK mutant ofE. coli K-12 suggests a common route whereby hydrogen selenide, derived from selenocysteine, can then be assimilated into selenoproteins.     

Effect of carbon source on the production of oxalic acid and hydrogen peroxide by brown-rot fungus Poria placenta

Oxalic acid and hydrogen peroxide have been suggested to be essential in the degradation of wood carbohydrates by brown-rot fungi. The production of oxalic acid, hydrogen peroxide and endo-β-1,4-glucanase activity by the brown-rot fungus Poria placenta was studied on crystalline cellulose, amorphous cellulose and glucose media. Oxalic acid and hydrogen peroxide by P. placenta were clearly produced on culture media containing either crystalline or amorphous cellulose. Oxalic acid and hydrogen peroxide were formed simultaneously and highest amounts of oxalic acid (1.0 g l⁻¹) and hydrogen peroxide (39.5 μM) were obtained on amorphous cellulose after 3 weeks cultivation. On glucose medium the amounts were low. The endoglucanase activity was observed to increase during the cultivation and was most pronounced on glucose medium and thus indicated the constitutive characteristics of the brown-rot cellulases.     

Why Does Clostridium acetireducens Not Use Interspecies Hydrogen Transfer for Growth on Leucine?

Clostridium acetireducens is the first reported anaerobic bacterium that is dependent on acetate as an electron acceptor for growth on branched-chain amino acids and alanine. The fermentation pathway of leucine and its deamination product α-ketoisocaproate were studied in this organism. Addition of Methanobacterium formicicum to pure cultures of C. acetireducens stimulated the degradation of α-ketoisocaproate but not the degradation of leucine, indicating that the electrons produced during the oxidative deamination of leucine were not transferred to hydrogen. This conclusion is supported by the observed low NAD(P)H ferredoxin reductase activity. Not only acetate but also crotonate proved to be an appropriate electron sink for the regeneration of NAD(P)⁺ in this bacterium. Interestingly, C. acetireducens was shown to form polyhydroxybutyrate during growth on leucine plus acetate. Received: 2 December 1996 / Accepted: 4 March 1997     

Poduction of Biotin by Microbial Transformation of dl-cis-Tetrahydro-2-oxo-4-n-pentyl-thieno-(3,4-d)-imidazoline (dl-TOPTI)

The conditions for biotin production were investigated. Urea was a more effective nitrogen source than ammonium chloride and ammonium sulfate. About 60% conversion from dl-cis-tetrahydro-2-oxo-4-n-pentyl-thieno-(3,4-d)-imidazoline (dl-TOPTI) to biotinol and biotin occurred using Corynebacterium sp. B–321. Strain M–6318 which derived from B–321 as a mutant incapable of assimilating n-alkane produced large amounts of dl-biotin from dl-TOPTI. The inability of the microbe to assimilate n-alkane resulted in repression of biotin degradation. Maximum conversion (80%) was obtained by growing cultures of strain M–6318 in the constant presence of n-paraffin.     

Regulation by ammonium and glucose of Arthrobacter fluorescens alanine dehydrogenase

Alanine dehydrogenase in Arthrobacter fluorescens exhibited an allosteric behaviour and two K _m values for ammonium were estimated. In batch cultures at different ammonium concentrations and in continuous culture following an NH₄ ⁺ pulse, the level of ADH activity seems to be regulated by the ammonium concentration, high activities being observed when extracellular ammonium was in excess. The response to the growth rate of an ammonium-limited chemostat culture of A. fluorescens seems to indicate that alanine dehydrogenase and glutamine synthetase activities were inversely related. High activities of glutamate oxaloacetate transaminase and glutamate pyruvate transaminase have been found in crude extract of ammonium-limited cultures. From the results obtained in batch cultures grown at different glucose concentrations and in carbon-limited chemostat culture it appeared that the limitation by glucose influenced alanine dehydrogenase activity negatively. No glutamate dehydrogenase activity and no glutamate synthase activity could be detected with either NADH or NADPH as coenzymes.Abbreviations ADH alanine dehydrogenase - GS glutamine synthetase - GDH glutamate dehydrogenase - GOGAT glutamine oxoglutarate aminotransferase - GOT glutamate oxaloacetate transaminase - GPT glutamate pyruvate transaminase     

Influence of hydrogenothrophic methane formation on the thermophilic anaerobic degradation of protein and amino acids

Abstract Thermophilic (55°C) protein (peptone) degradation was studied in steady state, laboratory-scale reactors. Peptone was easily hydrolysed to amino acids under methanogenic conditions, and all amino acids were completely degraded to volatile fatty acids, carbon dioxide and ammonium. Under these conditions, amino acids known to be oxidatively deaminated were degraded more slowly than the other amino acids. Inhibition of methanogenesis by 2-bromoethanesulfonic acid led to the accumulation of hydrogen in the gas phase and to the immediate inhibition of both protein hydrolysis and the degradation of amino acids that are preferentially oxidatively deaminated. These effects resulted in lower concentrations of all volatile fatty acids except for butyrate and caproate, which increased in concentration. Interspecies hydrogen transfer appeared to be necessary for the complete degradation of alanine, phenylalanine, methionine, valine, leucine and isoleucine. α-Aminobutyrate also accumulated when methanogenesis was inhibited.     

Investigations of the anaerobic metabolism of the polychaete wormNereis diversicolor M.

Summary The anaerobic metabolism ofNereis diversicolor M. was studied during various periods of experimental anaerobiosis.The degradation of glycogen is shown to be the main source of anaerobic energy production. During first hours of anaerobiosis, aspartate, in addition to glycogen, is metabolized in considerable quantities.Five acids were found to accumulate as end-products: alanine, D-lactate, succinate, acetate and propionate (Table 2).Alanine is accumulated only during the first hours of anaerobiosis. The increase in alanine is correlated with a decrease in aspartate.D-Lactate is the main end-product during the first 24 h of anaerobiosis, and continues to be produced even during prolonged anaerobiosis. In accordance with lactate production,Nereis diversicolor possesses a high glycolytic capacity (Table 4).The major end-products of long term fermentation are propionate and acetate. In contrast to other end-products, these acids are excreted in substantial amounts.Abbreviations GAPDH glyceraldehydephosphate dehydrogenase, EC 1.2.1.12 - LDH lactate dehydrogenase, EC 1.1.1.27 - GOT aspartate aminotransferase, EC 2.6.1.1 - GPT alanine aminotransferase, EC 2.6.1.2 - MDH malate dehydrogenase, EC 1.1.1.37 Supported by Deutsche Forschungsgemeinschaft (Gr 456/5 and Gr 456/6)     

Methanol formation during lignin degradation by Phanerochaete chrysosporium

Summary Methanol formation during the degradation of synthetic lignin (DHP), spruce and birch milled wood lignin (MWL) by Phanerochaete chrysosporium Burds. was studied under different culture conditions. When 100-ml flasks with 15–20 ml volumes of culture media containing high glucose and low nitrogen concentrations were used the metabolism of methanol to formaldehyde, formic acid and CO₂ was repressed thereby facilitating methanol determination. In standing cultures with oxygen flushing the fungus converted up to 25% of the DHP-methoxyl groups to methanol and 0.5–1.5% to ¹⁴CO₂ within 22–24 h. Methanol formation from methoxyl-labelled DHP was strongly repressed by high nitrogen in the medium, by addition of glutamic acid and by culture agitation. These results indicate that methanol is formed only under ligninolytic conditions and during secondary metabolism. Methanol is most likely released both from the lignin polymer itself and from lignin degradation products. Methanol was also formed from MWL preparations with higher percentage yields produced from birch as compared to spruce MWL.Small amounts of methanol detected in cultures without lignin probably emanated from demethoxylation of veratryl alcohol synthesized de novo from glucose by the fungus during secondary metabolism. Catalase or superoxide dismutase added to the fungal culture prior to addition of lignin, did not decrease methanol formation. Horseradish peroxidase plus H₂O₂ in vitro caused 5–7% demethoxylation of O¹⁴CH₃-DHP in 22 h, while laccase gave smaller amounts of methanol (1.8%). Since addition of H₂O₂ gave similar results as peroxidase plus H₂O₂, it seems likely that the main effect of peroxidase demethoxylation emanates from the hydrogen peroxide.     

The accumulation of aspartate in the presence of ethanol in rat liver.

1. Isolated hepatocytes were used to establish the reasons for the accumulation of aspartate, previously observed when the isolated rat liver was perfused with ethanol in the presence of alanine or ammonium lactate. 2. The isolated cells did not form aspartate when incubated with alanine and ethanol, but much aspartate was formed on incubation with ammonium lactate and ethanol. 3. Urea was the main nitrogenous product on incubation with alanine, in contrast with the perfused liver, where major quantities of NH4+ are also formed. When the formation of urea was nullified by the addition of urease, alanine plus ethanol caused aspartate formation, indicating that aspartate formation depends on the presence of critical concentrations of NH4+. 4. The accumulated aspartate was present in the cytosol. Ethanol halved the content of 2-oxoglutarate in the cytosol and more than trebled that of glutamate in the mitochondria. 5. The findings support the assumption that 2-oxoglutarate formed by the mitochondrial aspartate aminotransferase is not translocated to the cytosol in the presence of ethanol and NH4+, because it is rapidly converted into glutamate, the dehydrogenation of ethanol providing the required NADH. Aspartate, however, is translocated to the cytosol and accumulates there because of the lack of stoicheiometric amounts of oxoglutarate.     

Biosynthesis of poly(γ-glutamic acid) from l-glutamic acid,citric acid,and ammonium sulfate in Bacillus subtilis IFO3335

Poly(-glutamic acid) (PGA) production in Bacillus subtilis IFO3335 was studied. When l-glutamic acid, citric acid, and ammonium sulfate were used as carbon and nitrogen sources, a large amount of PGA without a by-product such as a polysaccharide was produced. The time courses of cell growth, PGA, glutamic acid, and citric acid concentrations during cultivation were investigated. It was found that glutamic acid added to the medium was apparently not assimilated. It can be presumed that the glutamic acid unit in PGA is mainly produced from citric acid and ammonium sulfate. The PGA productivity was investigated at various concentrations of ammonium sulfate in the media, which caused the depression of cell growth, high productivity of PGA, and the production of PGA with a high relative molecular mass. The yield of PGA determined by gel permeation chromatography (GPC) reached approximately 20 g/l. This yield was the highest value for PGA production by B. subtilis IFO3335, suggesting that B. subtilis IFO3335 was a bacterium that could produce PGA effectively. Time courses relative to the molecular mass of PGA at various concentrations of ammonium sulfate were investigated. It was suggested that B. subtilis IFO3335 excreted a PGA degradation enzyme with the progress of cultivation and that PGA was degraded by this enzyme. Correspondence to: M. Kunioka     

Nitrogen deprivation results in photosynthetic hydrogen production in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

The unicellular green alga Chlamydomonas reinhardtii is able to use photosynthetically provided electrons for the production of molecular hydrogen by an [FeFe]-hydrogenase HYD1 accepting electrons from ferredoxin PetF. Despite the severe sensitivity of HYD1 towards oxygen, a sustained and relatively high photosynthetic hydrogen evolution capacity is established in C. reinhardtii cultures when deprived of sulfur. One of the major electron sources for proton reduction under this condition is the oxidation of starch and subsequent non-photochemical transfer of electrons to the plastoquinone pool. Here we report on the induction of photosynthetic hydrogen production by Chlamydomonas upon nitrogen starvation, a nutritional condition known to trigger the accumulation of large deposits of starch and lipids in the green alga. Photochemistry of photosystem II initially remained on a higher level in nitrogen-starved cells, resulting in a 2-day delay of the onset of hydrogen production compared with sulfur-deprived cells. Furthermore, though nitrogen-depleted cells accumulated large amounts of starch, both hydrogen yields and the extent of starch degradation were significantly lower than upon sulfur deficiency. Starch breakdown rates in nitrogen or sulfur-starved cultures transferred to darkness were comparable in both nutritional conditions. Methyl viologen treatment of illuminated cells significantly enhanced the efficiency of photosystem II photochemistry in sulfur-depleted cells, but had a minor effect on nitrogen-starved algae. Both the degradation of the cytochrome b ₆ f complex which occurs in C. reinhardtii upon nitrogen starvation and lower ferredoxin amounts might create a bottleneck impeding the conversion of carbohydrate reserves into hydrogen evolution.     

Pathways of glutamine metabolism in Spodoptera frugiperda (Sf9) insect cells: evidence for the presence of the nitrogen assimilation system, and a metabolic switch by 1H/15N NMR

1H/15N and 13C NMR were used to investigate metabolism in Spodoptera frugiperda (Sf9) cells. Labelled substrates ([2-15N]glutamine, [5-15N]glutamine, [2-15N]glutamate, 15NH4Cl, [2-15N]alanine, and [1-13C]glucose) were added to batch cultures and the concentration of labelled excreted metabolites (alanine, NH4+, glutamine, glycerol, and lactate) were quantified. Cultures with excess glucose and glutamine produce alanine as the main metabolic by-product while no ammonium ions are released. 1H/15N NMR data showed that both the amide and amine-nitrogen of glutamine was incorporated into alanine in these cultures. The amide-nitrogen of glutamine was not transferred to the amine-position in glutamate (for further transamination to alanine) via free NH4+ but directly via an azaserine inhibitable amido-transfer reaction. In glutamine-free media 15NH4+ was consumed and incorporated into alanine. 15NH4+ was also incorporated into the amide-position of glutamine synthesised by the cells. These data suggest that the nitrogen assimilation system, glutamine synthetase/glutamate synthase (NADH-GOGAT), is active in glutamine-deprived cells. In cultures devoid of glucose, ammonium is the main metabolic by-product while no alanine is formed. The ammonium ions stem both from the amide and amine-nitrogen of glutamine, most likely via glutaminase and glutamate dehydrogenase. 13C NMR revealed that the [1-13C] label from glucose appeared in glycerol, alanine, lactate, and in extracellular glutamine. Labelling data also showed that intermediates of the tricarboxylic acid cycle were recycled to glycolysis and that carbon sources, other than glucose-derived acetylCoA, entered the cycle. Furthermore, Sf9 cell cultures excreted significant amounts glycerol (1.9-3.2 mM) and ethanol (6 mM), thus highlighting the importance of sinks for reducing equivalents in maintaining the cytosolic redox balance.     

Sensitive flow-injection method with peroxyoxalate chemiluminescence detection combined with preparative high-performance liquid chromatography for determination of choline-containing phospholipids in human serum

A sensitive and rapid flow-injection analysis (FIA) of total choline-containing phospholipids (PLs) and a selective FIA method for the class assay of choline-containing PLs combined with preparative HPLC were described. The FIA method is based on peroxyoxaxalate chemiluminescence (PO-CL) detection of hydrogen peroxide enzymatically formed from choline-containing PL. The linear standard curves were obtained up to 1 nmol/20-μl injection (r>0.999) with the detection limits of 1.3–1.6 pmol at a signal-to-noise ratio of 2. The total amounts of choline-containing PLs in human serum were ranged from 1.63 to 3.19 mg/ml. The HPLC separation of choline-containing PLs was achieved with an aminopropyl-modified silica gel column using a mixture of acetonitrile-methanol-10 mM ammonium phosphate buffer pH 5.8 as eluent. The eluate corresponding to each choline-containing PL was collected, evaporated, dissolved in 0.1% Triton X-100 aqueous solution, and then injected into FIA system. The FIA method combined with preparative HPLC was applied to the assay of human serum.