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N,N-dimethylpiperidinium (mepiquat) is a new process-induced compound formed from natural constituents during the cooking process. Mepiquat was first found in coffee and cereal products, but its formation mechanism in coffee is still unclear. In the current study, Arabica and Robusta coffee beans were roasted at different temperatures (215, 220, and 230 °C) to study the effect of roasting process on mepiquat formation. The highest mepiquat content, 1,020 µg/kg, was found in dark roast (230 °C) Indonesia Wahana, while 430 µg/kg of mepiquat was detected in medium roast (220 °C) Vietnam Robusta. At the same roasting temperature, higher level of mepiquat was observed in Arabica than in Robusta. In both species, substances related to mepiquat formation, including betaine, choline, trigonelline, lysine, carnitine, pipecolic acid (PipAc), pipecolic acid betaine (PipBet), were also detected. The lysine-based Maillard reaction and decarboxylation in Arabica and Robusta promoted mepiquat formation through the degradation of choline and trigonelline, and the formation of intermediate products. Results from both the model system and selected commercial beans showed that choline and trigonelline had a significant correlation (P < 0.01) with mepiquat formation in Arabica. PRACTICAL APPLICATION: Mepiquat is considered as a new process-induced compound resulting from typical roasting conditions, but its formation mechanism in coffee is still unclear. This work demonstrates the formation mechanism of mepiquat by many precursor substances contained in Arabica and Robusta. It is very important to figure out how mepiquat is ''naturally" present in daily diets, especially in those processed at high temperatures., (© 2020 Institute of Food Technologists®.)

Pyridoxine-dependent epilepsy (PDE) is often characterized as an early onset epileptic encephalopathy with dramatic clinical improvement following pyridoxine supplementation. Unfortunately, not all patients present with classic neonatal seizures or respond to an initial pyridoxine trial, which can result in the under diagnosis of this treatable disorder. Restriction of lysine intake and transport is associated with improved neurologic outcomes, although treatment should be started in the first year of life to be effective. Because of the documented diagnostic delay and benefit of early treatment, we aimed to develop a newborn screening method for PDE. Previous studies have demonstrated the accumulation of Δ 1 -piperideine-6-carboxylate and α-aminoadipic semialdehyde in individuals with PDE, although these metabolites are unstable at room temperature (RT) limiting their utility for newborn screening. As a result, we sought to identify a biomarker that could be applied to current newborn screening paradigms. We identified a novel metabolite, 6-oxo-pipecolate (6-oxo-PIP), which accumulates in substantial amounts in blood, plasma, urine, and cerebral spinal fluid of individuals with PDE. Using a stable isotope-labeled internal standard, we developed a nonderivatized liquid chromatography tandem mass spectrometry-based method to quantify 6-oxo-PIP. This method replicates the analytical techniques used in many laboratories and could be used with few modifications in newborn screening programs. Furthermore, 6-oxo-PIP was measurable in urine for 4 months even when stored at RT. Herein, we report a novel biomarker for PDE that is stable at RT and can be quantified using current newborn screening techniques., (© 2019 SSIEM.)

Capillary electrophoresis (CE) instrument was used for the generation of a robust and reliable nanoreactor for enzymatic assays in the context of antithrombotic drug screening. The activity of the screened molecules was monitored in a quick and fully automated fashion using only few nanoliters of reactants. To achieve this goal, the targeted enzyme (thrombin) and the chromogenic substrate with or without the screened inhibitor were injected as separate plugs. The mixing of the reactants was then realized using electrophoretically mediated microanalysis (EMMA) or fast transverse diffusion of laminar flow profiles (TDLFP) procedure. The latest provided better mixing performance and was chosen to investigate the inhibitory potency of a fragment library. This very straightforward and fast CE activity assay showed results in good accordance with a previously developed CE affinity assay that confirms the potential of CE at the early stages of drug discovery, providing not only an efficient nanoscale bioreactor but also a selective and integrated separation device., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Spiny restharrow root (Ononis spinosa L.) and its preparations are mainly used for the treatment of urinary infections or bladder stones in numerous countries. Spiny restharrow root is rich in isoflavonoids (formononetin, calycosin and pseudobaptigenin), pterocarpans (medicarpin and maackiain) and dihydroisoflavonoids (onogenin and sativanone), which metabolites are present as glucosides, glucoside malonates, glucoside acetates and free aglycones in the root. The in-depth analysis of tandem mass spectrometric (MS) and high-resolution MS (HR-MS) data revealed the presence of nitrogen-containing compounds in the root extracts. An ion-exchange-based purification and a preparative-scale reversed phase chromatographic isolation procedure was developed for the characterization of these new natural products. For the unambiguous identification of the isolated compounds NMR experiments were carried out. The thorough characterization confirmed the presence of six piperidin-2-yl-acetic acid (homopipecolic acid) esters of isoflavonoid glucosides. This is the first report of homopipecolic acid esters isolated from higher plants., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Following a previous microbial inoculation, plants can induce broad-spectrum immunity to pathogen infection, a phenomenon known as systemic acquired resistance (SAR). SAR establishment in Arabidopsis thaliana is regulated by the Lys catabolite pipecolic acid (Pip) and flavin-dependent-monooxygenase1 (FMO1). Here, we show that elevated Pip is sufficient to induce an FMO1-dependent transcriptional reprogramming of leaves that is reminiscent of SAR. In planta and in vitro analyses demonstrate that FMO1 functions as a pipecolate N-hydroxylase, catalyzing the biochemical conversion of Pip to N-hydroxypipecolic acid (NHP). NHP systemically accumulates in plants after microbial attack. When exogenously applied, it overrides the defect of NHP-deficient fmo1 in acquired resistance and acts as a potent inducer of plant immunity to bacterial and oomycete infection. Our work has identified a pathogen-inducible L-Lys catabolic pathway in plants that generates the N-hydroxylated amino acid NHP as a critical regulator of systemic acquired resistance to pathogen infection., (Copyright © 2018 Elsevier Inc. All rights reserved.)

An accurate, rapid, and cost-effective methodology for enzyme assay is highly demanded to screen the effect of compounds on target at the molecular level. Thrombin (EC 3.4.21.5) and factor Xa (FXa, EC 3.4.21.6) have been identified as the critical targets for the development of potential drugs with anticoagulant activity. In this study, a rapid, sensitive and accurate assay based on UHPLC-MS/MS method has been developed for inhibitor screening against thrombin and factor Xa simultaneously. For thrombin and factor Xa, the Michaelis-Menten constants (K m ) were calculated to be 6.14 and 57.27 μM, respectively. The inhibition constants (K i ) for two known inhibitors, argatroban and rivaroxaban, were determined to be 16.23 and 0.41 nM, respectively. The assay was further validated through the determination of a high Z' factor value of 0.89. Finally, the developed assay was applied to screen a chemical library against two enzymes. Three hit compounds belonging to a class of sulfated polysaccharides were identified and their targets of inhibition action were further evaluated. The results indicated that the dual-target assay by UHPLC-MS/MS analysis could be used as a reliable method for screening anticoagulant agents., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Argatroban (ARGA), used as intravenous anticoagulant drug, has been reported to photodegrade under light exposure, requiring specific precautions at handling, storage and administration. Thus, for the first time, aqueous ARGA photodegradation under aerobic conditions has been described in terms of photoproducts, phototransformation processes and potential implications. ARGA significant photoproducts were successfully separated and characterized by gradient reversed-phase liquid chromatography coupled with high-resolution multistage mass spectrometry (LC/HR-MS n ). Hitherto still not available in literature, ARGA in-depth fragmentation study was conducted so as to thoroughly sort out the main mechanisms specific to the molecule and therefore, to propose a fragmentation pattern relevant to the identification of ARGA related substances. Thereafter, in view of the structural characteristics of the photoproducts formed, ARGA photodegradation pathways could be worked out, showing that whether by direct photolysis or through photosensitization, the methyltetrahydroquinoline nitrogen and that of guanidine group would be mainly involved in photolysis initiation reactions, through one-electron oxidation along with proton loss. Desulfonation, cyclisation affording compounds of diazinane type, and/or rearrangements with transfer of the methyltetrahydroquinoline group toward the guanidine function were observed accordingly. Having a good insight into ARGA photodegradation pathways allows for consistent measures in view of mitigating or avoiding the drug decay and the related potential effects., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The occurrence of pipecolic acid betaine (homostachydrine) and its biosynthetic precursor N-methylpipecolic acid was detected for the first time in green coffee beans of Robusta and Arabica species. The analyses were conducted by HPLC-ESI tandem mass spectrometry and the metabolites identified by product ion spectra and comparison with authentic standards. N-methylpipecolic acid was found at similar levels in green coffee beans of Robusta and Arabica, whereas a noticeable difference of homostachydrine content was observed between the two green coffee bean species. Interestingly, homostachydrine content was found to be unaffected by coffee bean roasting treatment because of a noticeable heat stability, a feature that makes this compound a candidate marker to determine the content of Robusta and Arabica species in roasted coffee blends. To this end, a number of certified pure Arabica and Robusta green beans were analyzed for their homostachydrine content. Results showed that homostachydrine content was 1.5±0.5mg/kg in Arabica beans and 31.0±10.0mg/kg in Robusta beans. Finally, to further support the suitability of homostachydrine as quality marker of roasted blends of Arabica and Robusta coffee beans, commercial samples of roasted ground coffee blends were analyzed and the correspondence between the derived percentages of Arabica and Robusta beans with those declared on packages by manufacturers was verified., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Objectives: Elevated levels of pipecolic acid (PA), α-aminoadipic semialdehyde (AASA) and its cyclic form Δ1-piperideine-6-carboxylate (P6C) are characteristic of pyridoxine dependent epilepsy (PDE), a rare disorder of inborn error of metabolism. Recent studies showed the effectiveness of dietary therapy in PDE patients and emphasized the importance of the assessment of these metabolites for monitoring treatment efficacy. The objective of this study was to develop a robust and sensitive method for simultaneous quantification of AASA-P6C and PA in plasma and urine., Design and Methods: Plasma and urine samples were derivatized with 3N HCl in n-butanol (v/v) and injected onto ACQUITY BEH-C18 column. A gradient of water/methanol containing 0.1% formic acid was used for the chromatographic separation of AASA, P6C and PA. The analytes' concentrations were calculated using their calibration curves and the sum of AASA and P6C (AASA-P6C) was calculated. To evaluate the clinical utility of this test, samples from unaffected controls and patients with confirmed PDE were analyzed., Results: The performance characteristics of the assay as well as sample stability and interferences were determined. The intra- and inter- assay CVs were ≤2.9% and ≤10.9% for AASA-P6C, and ≤3.3% and ≤12.6% for PA, respectively. Reference ranges for AASA-P6C and PA in plasma and urine were established. Comparison of values obtained from unaffected controls and PDE patients showed high clinical sensitivity and specificity of the assay., Conclusions: This novel method for the simultaneous quantification of AASA-P6C and PA in plasma and urine can be used in a clinical laboratory setting for the diagnosis and monitoring of patients with PDE., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Robust immunity requires basal defense machinery to mediate timely responses and feedback cycles to amplify defenses against potentially spreading infections. AGD2-LIKE DEFENSE RESPONSE PROTEIN 1 (ALD1) is needed for the accumulation of the plant defense signal salicylic acid (SA) during the first hours after infection with the pathogen Pseudomonas syringae and is also upregulated by infection and SA. ALD1 is an aminotransferase with multiple substrates and products in vitro. Pipecolic acid (Pip) is an ALD1-dependent bioactive product induced by P. syringae. Here, we addressed roles of ALD1 in mediating defense amplification as well as the levels and responses of basal defense machinery. ALD1 needs immune components PAD4 and ICS1 (an SA synthesis enzyme) to confer disease resistance, possibly through a transcriptional amplification loop between them. Furthermore, ALD1 affects basal defense by controlling microbial-associated molecular pattern (MAMP) receptor levels and responsiveness. Vascular exudates from uninfected ALD1-overexpressing plants confer local immunity to the wild type and ald1 mutants yet are not enriched for Pip. We infer that, in addition to affecting Pip accumulation, ALD1 produces non-Pip metabolites that play roles in immunity. Thus, distinct metabolite signals controlled by the same enzyme affect basal and early defenses versus later defense responses, respectively.

Stochastic and potentiometric microsensors based on porphyrins and polymeric surfactants such as polysodium N-undecanoyl-L-leucylvanilate and polysodium N-undecanoyl-L-vanilate were developed for enantioselective assay of pipecolic acid. The matrices used for the design of the stochastic sensors were diamond paste and graphite paste, while the matrix used for the design of potentiometric sensors was carbon paste. The response characteristics of the microsensors were determined for the enantiomers of pipecolic acid. The response characteristics, selectivity, and enantioselectivity studies proved that the proposed microsensors can be used for clinical enantioanalysis of pipecolic acid in biological fluids, e.g., urine and whole blood., (Copyright © 2012 Wiley Periodicals, Inc.)

The presence of pipecolic acid and pipecolic acid betaine, also known as homostachydrine, is herein reported for the first time in Citrus genus plants. Homostachydrine was found in fruits, seeds, and leaves of orange, lemon, and bergamot (Citrus bergamia Risso et Poit). As homostachydrine was not commercially available, as a comparative source, extracts of alfalfa leaves ( Medicago sativa L.) were used, in which homostachydrine is present at high concentration. Then, the results where confirmed by comparison with an authentic standard synthesized and purified starting from pipecolic acid. The synthesized standard was characterized by a ESI-MS/MS study using a 3D ion-trap mass spectrometer. When subjected to MS/MS fragmentation in positive ion mode, homostachydrine, unlike its lower homologue proline betaine (also known as stachydrine), showed a pattern of numerous ionic fragments that allowed unambiguous identification of the compound. For the quantitation in the plant sources, high sensitivity and specificity were achieved by monitoring the transition (158 → 72), which is absent in the fragmentation patterns of other major osmolytes commonly used as markers for studies of abiotic stress. As for the metabolic origin of homostachydrine, the occurrence in citrus plants of pipecolic acid leads to the hypothesis that it could act as a homostachydrine precursor through direct methylation.

A simple and highly sensitive high-performance liquid chromatography method for the determination of pipecolic acid in mouse brain areas was developed. After homogenization of brain and pretreatment with o-phthalaldehyde, the pipecolic acid and (2S,3S)-3-methylpyrrolidine-2-carboxylic acid (internal standard) were derivatized with 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride at 70 °C for 15 min in basic medium (pH 9.0). The fluorescent derivatives of pipecolic acid and internal standard were separated on a reversed-phase column by stepwise elution using acetic acid (30 mM)-acetonitrile at 50 °C and detected by fluorescence measurement at 316 nm (excitation) and 403 nm (emission). The detection limit (signal-to-noise ratio=3) of pipecolic acid was 13 fmol per injection. The recovery was about 106.7%. The precision (relative standard deviation) was 3.2%.

A new, highly sensitive chemiluminescence method for measurement of pipecolic acid in various substances such as human serum, cow's milk, beer, and apple juice has been developed. The method is based on reverse-phase ion-pair high performance liquid chromatographic separation and subsequent tris(2,2'-bipyridine)ruthenium(III) chemiluminescence detection. It was confirmed that imino acids show strong chemiluminescence upon mixing with tris(2,2'-bipyridine)ruthenium(III). A calibration graph, based on a standard pipecolic acid solution, was linear over the range 5.0x10(-9)M to 2.0x10(-5)M and the detection limit was 24fmol (signal-to-noise ratio=3). This highly sensitive and selective determination method can be applied to selected samples without purification or pre-concentration procedures. Compared to the previous HPLC methods, the proposed method is easier, more sensitive, and time-saving.

The structures of neoefrapeptins A to N, peptides with insecticidal activity, were elucidated. They showed a close similarity to efrapeptin. However, all neoefrapeptins contained the very rare amino acid 1-amino-cyclopropane-carboxylic acid and some of them also contained (2S,3S)-3-methylproline. The neoefrapeptins are the first case, in which these amino acids are found as building blocks for linear peptides. They were identified by comparison of the silylated hydrolyzate to reference material by GC/MS (EI-mode). The sequence was elucidated using mass spectrometry (ESI+ mode). Full scan spectra showed two fragments in high yield, even under mild ionization conditions. MS/MS spectra of these two fragments yielded fragment rich spectra from which the sequence of the compounds was determined almost completely. The proteolytic cleavage with the proteinase papain yielded products that allowed to prove the rest of the sequence and the identity of the C-terminus to efrapeptin. The proteolytic cleavage products allowed furthermore to determine the position of the isobaric amino acids, pipecolic acid and 3-methylproline in neoefrapeptin F, as well as the location of R-isovaline and S-isovaline. Papain digestion was such established as a tool for structure elucidation of peptides rich in alpha,alpha-dialkylated amino acids. CD spectra suggested a 3(10) helical structure for neoefrapeptins A and F.

Monocrystalline natural diamond, L-amino acid oxidase (L-AAOD), D-amino acid oxidase (D-AAOD), and paraffin oil were used for the design of the modified diamond paste. The technique used for the direct voltammetric assay was differential pulse voltammetry (DPV) with applied potential pulse amplitude of 25 mV vs. Ag/AgCl. Using the new amperometric biosensors L-pipecolic acid (L-PA) and D-pipecolic acid (D-PA) were determined reliably from serum samples at 700 and 200 mV vs. Ag/AgCl, respectively, with low limits of detection.

Background: Pipecolic acid, a metabolite of lysine, is found in human physiological fluids and is thought to play an important role in the central inhibitory gamma-aminobutyric acid system. However, it is unclear whether plasma D- and L-pipecolic acid originate from oral food intake or intestinal bacterial metabolites., Methods: We analyzed the contents of D- and L-pipecolic acid in several processed foods including dairy products (cow's milk, cheese and yogurt), fermented beverages (beer and wine) and heated samples (beef, bovine liver, bread and tofu) to clarify the relationship between plasma D- and L-pipecolic acid and dietary foods., Results: Our study revealed that some of the samples contained high concentrations of total pipecolic acid, and a higher proportion of L- than D-isomers. The other samples also showed high proportions of L-pipecolic acid. It was also shown that there is no significant change in the ratio of the D-isomer before and after heat treatment. The heat treatments could not cause the racemization of pipecolic acid in this study., Conclusion: These findings suggest that plasma pipecolic acid, particularly the D-isomer, does not originate from direct food intake and that D- and L-pipecolic acid can possibly be derived from intestinal bacterial metabolites., (Copyright 2003 S. Karger AG, Basel)

A novel procedure to specifically quantify low amounts of pipecolic acid and structurally related compounds in several types of biological materials has been characterized. From crude extracts of various types of biological material, the first step was to clear all low-molecular-weight compounds containing primary amino groups by a treatment of nitrous acid. Using a microwave-assisted reaction, the remaining substances containing secondary amino groups were then derivatized with ninhydrin and made soluble in glacial acetic acid. The derivatives produced were resolved by reverse-phase HPLC and detected by spectrophotometry at 570nm. This procedure allowed more rapid determination of pipecolic acid since microwave heating shortened the time needed for derivatization compared with heating at 95 degrees C in a water bath. The complete analysis of the chromogens for pipecolic acid and related substances was achieved in 20min. Under such conditions, the detection threshold for pipecolic acid was about 20pmol. The suitability of the technique was assessed in various biological matrices known to contain significant amounts of this amino acid. The data obtained are in accordance with those available in the literature. To our knowledge, this is the first method using the ninhydrin reaction in a precolumn, microwave-assisted derivatization procedure for detection and determination of heterocyclic alpha-amino acids.

A high-performance liquid chromatography method for the simultaneous determination of pipecolic acid (Pip) and lysine (Lys), a precursor of Pip, in the rumen liquor and plasma of ruminant animals was established. Samples of rumen liquor and plasma were deproteinized with 50% acetonitrile and derivatized with a fluorescent agent 9-fluorenylmethyloxy carbonyl chloride (Fmoc-Cl). Chromatographic separation was achieved on a TSK gel ODS-80TM column using a reversed-phase gradient elution system. For the gradient elution, two mobile phases, A and B, were needed, both commonly consisted of: 5 mM L-proline, 2.5 mM cupric sulfate and 6.5 mM ammonium acetate. Mobile phase B additionally contains 50% (v/v) acetonitrile. The pH of both mobile phases was adjusted to 7.0. Derivatized Pip and Lys were detected on a fluorescent detector at excitation and emission wavelengths of 260 and 313 nm, respectively. The calibration curves were linear within the range 0 to 1 mM (r>0.999). The average recoveries for Pip and Lys were 95.9+/-1.8 and 93.2+/-2.5% in rumen liquor and 98.3+/-1.4 and 97.5+/-1.3% in plasma, respectively. The limits of detection for Pip and Lys were 0.6 and 0.7 microM in rumen liquor and 0.01 and 0.05 microM in plasma. The assay has acceptable precision, relative standard deviation (RSD) for reproducibility (within-day and day-to-day variation) were less than 5.2% for aqueous (5.0 microM Pip and Lys), MB9 (5.0 microM Pip and Lys), plasma (7.1 microM Pip and 85.6 microM Lys) and rumen liquor (28.4 microM Pip and 10.2 microM Lys) samples. The levels of Pip and Lys in faunated goats, determined from three animals over a period of two days sampling, were found to be 36.8+/-18.1 and 14.6+/-2.8 microM in rumen liquor, and 7.3+/-2.5 and 137.3+/-38.0 microM in plasma at 1 h after feeding. This is the first report on the normal levels of Pip in the rumen liquor and plasma of faunated goat.

Pipecolic acid, a metabolite of lysine, is found in human physiological fluids. It is known that plasma pipecolic acid levels are elevated in patients with Zellweger syndrome, a genetic disorder, and chronic liver diseases. However, it is uncertain if this acid originates directly from food intake or from mammalian or intestinal bacterial enzyme metabolism. To characterize the relationship between plasma pipecolic acid and diet, we analyzed the contents of pipecolic acid in 17 edible plants and changes in plasma and urinary pipecolic acid levels following soybean juice ingestion by 4 healthy volunteers. Our study revealed that some of the plants contained high concentrations of total pipecolic acid, and a higher portion of L-isomer than D-isomer. Loading tests demonstrated that plasma levels and urinary excretion of D-isomer increased significantly 2 h after soybean juice ingestion. Plasma lysine levels showed a similar increase to that of D-isomer. These findings suggest that plasma pipecolic acid, particularly the D-isomer, originates mainly from the catabolism of dietary lysine by intestinal bacteria rather than by direct food intake and that D- and L-isomer may have different mechanisms of metabolism. Moreover, these findings may be important for clarifying the pathogenesis of peroxisomal disorders.

Our chemical studies on Cycas circinalis seeds from Guam has provided two new nonprotein amino acids, N-(3'-one-5'-methyl)-hexylalanine and leucine betaine. N-methylisoleucine, previously reported as a component of naturally occurring peptides, has been isolated as a free amino acid from the seeds of Phaseolus vulgaris (pinto bean), together with S-methylcysteine, pipecolic acid and a dipeptide, gamma-glutamyl-leucine.

Quantitative analysis of the following peroxisomal metabolites is reported: very long-chain fatty acids (VLCFA), pipecolic acid, bile acid intermediates, phytanic and pristanic acid, in plasma, urine, cerebrospinal fluid (CSF), blood spots collected at neonatal screening and amniotic fluid. An overview is given of the concentrations of these metabolites in body fluids from control subjects and all patients investigated so far in this laboratory. The method of choice is gas chromatography -- mass spectrometry (GC-MS) with electron capture detection, combined with the use of stable-isotope-labelled internal standards.

An enzyme fraction with catalytic activities for the biosynthesis of the pipecolic acid containing cyclopeptolide SDZ 90-215 was partially purified and characterized from the genus Septoria. The crude cell homogenate was subjected to polyethyleneimine precipitation, ammonium sulfate precipitation and FPLC gel filtration. The denatured enzyme shows an apparent molecular mass of 1.2 MDa in 3% SDS-PAGE. Peptolide SDZ 90-215 synthetase catalyzes the ATP-PPi exchange reaction dependent on all substituent amino and hydroxy acids. SDZ 90-215 synthetase synthesizes the peptolide in vitro when incubated together with D-lactic acid, all constitutive amino acids in their N-unmethylated form, ATP, magnesium chloride and S-adenosyl-L-methionine. The yield of SDZ 90-215 is higher when O-methyl-L-tyrosine instead of L-tyrosine is used, indicating that O-methylation of tyrosine is not carried out by the synthetase.

Plasma, urine, cerebrospinal fluid (CSF), and amniotic fluid were examined to determine whether free D-amino acids were present and if so at what levels. It was found that D-amino acids exist in all physiological fluids tested, but that their level varied considerably. The lowest levels of D-amino acids were usually found in amniotic fluid or CSF (almost always < 1% of the corresponding L-amino acid). The highest levels were found in urine (usually tenth percent to low percent levels). Pipecolic acid seemed to be different from the other amino acids tested in that it was excreted primarily as the D-enantiomer (often > 90%). Correspondingly high levels of D-pipecolic acid were not found in plasma. Some of the trends found in this work seemed to be analogous to those found in a recent rodent study.

Accurate estimation of in vivo turnover rates of collagen is complicated by amino acid reutilization. It was previously shown that the ideal, non-recycling tracer was [18O]hydroxyproline synthesized in vivo. The analytical method for measuring turnover rates with [18O]hydroxyproline must include analyte quantitation for pool size determination and isotope ratio measurement for determining levels of label incorporation. For ease of use and widest availability, a benchtop gas chromatograph-mass spectrometer in the electron-impact ionization mode was chosen. Here we present a versatile procedure for hydroxyproline derivatization that is well suited for routine, large-scale determination of analyte concentrations and relative levels of 18O incorporation.

A procedure for the simultaneous analysis of brain pipecolic acid, proline, gamma-aminobutyric acid and glycine--amino acids with potent inhibitory actions on the central nervous system--was developed. The identification and quantification of the amino acids were performed with a gas chromatographic--mass spectrometric--computer system using deuterium-labelled amino acids as the internal standards. After separation of the amino acids by high-performance liquid chromatography, the methyl ester heptafluorobutyryl derivatives were prepared. The lower limit of quantification for this method is at the picomole level. The usefulness of this chromatographic procedure has been demonstrated by measurement of trace amounts of pipecolic acid in rat brain.

A sensitive and accurate stable isotope dilution assay was developed for the measurement of pipecolic acid in body fluids using electron capture negative ion mass fragmentography. The method utilizes [2H11]pipecolic acid as the internal standard. Sample preparation consisted of derivatization in aqueous solution (pH 11.5) of the amine moiety with methyl chloroformate to the N-methylcarbamate, followed by acidic ethyl acetate extraction (pH 2) and further derivatization of the carboxyl moiety to the pentafluorobenzyl ester. Normal values have been determined in cerebrospinal fluid (mean means = 0.041 mumol/l, range 0.010-0.120 mumol/l), in plasma of at term infants (age less than 1 wk, means = 5.73 mumol/l, range 3.75-10.8 mumol/l; age greater than 1 wk, means = 1.46 mumol/l, range 0.70-2.46 mumol/l), in urine of at term infants (age less than 6 mth, means = 32.5 mumol/g. creat., range 9.81-84.5 mumol/g. creat; age greater than 6 mth, means = 6.35 mumol/g. creat., range 0.15-13.6 mumol/g. creat.) and in amniotic fluid (means = 4.65 mumol/l, range 2.24-8.40 mumol/l). The utility of the method was demonstrated for the pipecolic acid quantification in these biofluids of patients with peroxisomal disorders. As affected fetuses with infantile Refsum's disease and Zellweger syndrome showed no significant elevation of pipecolic acid in their surrounding amniotic fluids, the measurement of pipecolic acid in amniotic fluid seemed not to be useful for prenatal diagnosis in these disorders.

A rapid and sensitive method for the quantitative determination of pipecolic acid (PA), one of the three cyclic secondary imino acids present in mammalian brain is described. The quantification and identification of PA are accomplished in rat and mouse brain using high performance liquid chromatography with electrochemical detection (LCEC) and nipecotic acid (NPA) as an internal standard. The cyclic imino acids are derivatized with 2,4-dinitrofluorobenzene (DNFB) to dinitrophenyl derivatives. The remaining time for LCEC analysis is less than 30 min and the limit of sensitivity is in the lower picomole range. The levels of PA found in rat and mouse brain are comparable to those reported using gas chromatography/mass spectrometry. The regional distribution of PA shows higher concentrations of PA in hypothalamus, pons-medulla oblongata and cerebellum. The present results demonstrate that LCEC is sensitive enough to determine endogenous levels of PA in mg amounts of rodent brain tissue. Due to its simplicity and rapidity, the technique represents an alternative to existing methods. This method can also be used for determination of PA in CSF, blood or urine of hyperpipecolic patients.

Pipecolic acid (PA) excretion from normal newborn, preterm and/or small-for-dates infants has been determined and correlated with gestational age, sex and age after birth. An effect of fetal sex was not detectable. Only small-for-dates infants with a gestational age of greater than 34-37 weeks had a lower urinary excretion of PA than the appropriate-for-dates infants with the same gestational age. Preterm infants had a higher excretion of PA than term neonates. PA excretion of infants decreases with age after birth. This higher excretion in "younger" infants can be explained by a higher serum concentration and less efficient tubular reabsorption. PA level in serum and urine remains a valuable tool for the confirmation of the clinical diagnosis of Zellweger syndrome when gestational age and age after birth are taken into consideration. No PA was detected in serum or urine of four children suffering from hyperthyroidism.

In Streptomyces lactamdurans, the precursor of the alpha-aminoadipoyl side-chain of cephamycin C is L-lysine. In this regard, streptomycetes differ strikingly from the fungi, which produce alpha-aminoadipic acid during the synthesis, rather than the breakdown, of L-lysine. Studies using a cell-free system showed that an aminoadipic acid. The product of this reaction was trapped and subsequently purified by ion-exchange chromatography. Thin-layer chromatography, spectrophotometry, and amino acid oxidase digestion studies identified the reaction product as L-1-piperideine-6-carboxylate, implying enzymatic removal of the epsilon amino group of L-lysine. This enzymatic activity (E.C. 2.6.1.36; L-lysine: 2-oxoglutarate 6-aminotransferase) is highly unusual and was previously conclusively demonstrated only in the genus Flavobacterium. In S. lactamdurans, the specific activity of this enzyme reaches a peak early in the fermentation (approximately 20 h) and decreases as the antibiotic begins to appear.

The regional distribution of pipecolic acid (PA) in newborn mouse brain, measured by a new sensitive high performance liquid chromatography with electrochemical detection (HPLC-EC) method, shows a two-fold difference among various areas. Diencephalon, olfactory bulb and anterior telencephalon show the highest PA levels, while the lowest PA levels are seen in mesencephalon and rhombencephalon. The pattern of regional distribution of PA is identical to the regional accumulation in brain of the newborn seen by us following i.p. injections of D,L-[3H]PA9. The highest levels of PA are seen in both brain and serum during the perinatal period of development. Pipecolic acid levels decrease in brain and serum at one day of age and reach adult values within two weeks postnatal. The brain/serum PA ratio (2.9-3.5) during the perinatal period declines gradually after birth to adult values (0.7-0.8) at 30 days. The liver and kidney follow the same pattern with higher levels of PA seen during the perinatal period; however, these levels decreased rapidly to adult levels within one week postnatal. Following injections (250 mg/kg, i.p. and s.c. in the adult and newborn, respectively), D,L-PA accumulates for up to 24 h in the newborn mouse brain. In adult, the cerebral concentration of PA increases rapidly and reaches its peak level in 5-10 min. It remains relatively constant up to 5 h and then declines slowly to 24 h. Pipecolic acid levels in serum show essentially the same pattern of accumulation between adult and newborn mice with some quantitative differences.(ABSTRACT TRUNCATED AT 250 WORDS)

A method is described for the quantitative assay of pipecolic acid in biological fluids using column chromatography, with acid ninhydrin for color development. The technique permits measurement of plasma pipecolic acid in normal individuals with blood samples of 1 ml. The mean value in 29 pediatric patients, aged 4 months to 17 years, hospitalized with a variety of diagnoses was 2.1 mumol/1 +/- 1.6. Newborn infants had slightly elevated plasma levels with a mean of 12 mumol/1 +/- 5.6. Among the diagnostic categories, patients with liver disease were distinctive in commonly having levels at the upper limits of normal. In some cases, the levels were exceedingly high, reaching 78 mumol/1 in one fatal case.

Imino acid and related alicyclic amine concentrations in blood and urine of mammals including humans were concurrently determined by a selected ion monitoring technique. Nanomole levels of proline and pipecolic acid, and pyrrolidine and piperidine as well, were found in human urine. Proline levels but not pipecolic acid levels were higher in blood of humans than in urine. Pyrrolidine and piperidine levels in blood of humans were picomole levels and much lower than those in urine. Similar tendencies were also recognized when these 4 compounds were analyzed using animal blood and urine, although the levels were generally higher in animals than in humans. Significantly high concentrations of the imino acids and the amines were found also in animal semen.

A method for the analysis of 5-hydroxypipecolic acid and 4-hydroxyproline in mammalian brain and blood is reported. The identification and quantification of the two hydroxyimino acids were accomplished with gas chromatography/mass spectrometry including a selected ion-monitoring technique following HPLC prepurification. The lower limit of detection for the method is 2 to 10 pmol. The amounts of 5-hydroxypipecolic acid and 4-hydroxyproline in blood were 20 to 30 pmol/ml and 3 to 6 nmol/ml, respectively. Their concentrations in the rabbit whole brain were determined to be 5 and 120 pmol/g, respectively.