High performance liquid chromatography–tandem mass spectrometry for the determination of bile acid concentrations in human plasma

We report a sensitive and robust method to determine cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA), ursodeoxycholic acid (UDCA), and their taurine- and glycine-conjugate concentrations in human plasma using liquid chromatography–tandem mass spectrometry. Activated charcoal was utilized to prepare bile acid-free plasma, which served as the biological matrix for the preparation of standard and quality control samples. Plasma sample preparation involved solid-phase extraction. A total of 16 bile acids and 5 internal standards were separated on a reverse column by gradient elution and detected by tandem mass spectrometry in negative ion mode. The calibration curve was linear for all the bile acids over a range of 0.005–5 μmol/L. The extraction recoveries for all the analytes fell in the range of 88–101%. Intra-day and inter-day coefficients of variation were all below 10%. A stability test showed that all the bile acids were stable in plasma for at least 6 h at room temperature, at least three freeze–thaw cycles, in the −70 °C or −20 °C freezer for 2 months, and also in the reconstitution solution at 8 °C for 48 h. Comparison of the matrix effect of bile acid-free plasma with that of real plasma indicated that the charcoal purification procedure did not affect the properties of charcoal-purified plasma as calibration matrix. This method has been used to determine the bile acid concentrations in more than 300 plasma samples from healthy individuals. In conclusion, this method is suitable for the simultaneous quantification of individual bile acids in human plasma.     

Substitution of dietary oleic acid for myristic acid increases the tissue storage of α-linolenic acid and the concentration of docosahexaenoic acid in the brain, red blood cells and plasma in the rat

Various strategies have been developed to increase the cellular level of (n-3) polyunsaturated fatty acids in animals and humans. In the present study, we investigated the effect of dietary myristic acid, which represents 9% to 12% of fatty acids in milk fat, on the storage of α-linolenic acid and its conversion to highly unsaturated (n-3) fatty acid derivatives. Five isocaloric diets were designed, containing equal amounts of α-linolenic acid (1.3% of dietary fatty acids, i.e. 0.3% of dietary energy) and linoleic acid (7.0% of fatty acids, i.e. 1.5% of energy). Myristic acid was supplied from traces to high levels (0%, 5%, 10%, 20% and 30% of fatty acids, i.e. 0% to 6.6% of energy). To keep the intake of total fat and other saturated fatty acids constant, substitution was made with decreasing levels of oleic acid (76.1% to 35.5% of fatty acids, i.e. 16.7% to 7.8% of energy) that is considered to be neutral in lipid metabolism. After 8 weeks, results on physiological parameters showed that total cholesterol and low-density lipoprotein-cholesterol did not differ in the diets containing 0%, 5% and 10% myristic acid, but were significantly higher in the diet containing 30% myristic acid. In all the tissues, a significant increasing effect of the substitution of oleic acid for myristic acid was shown on the level of both α-linolenic and linoleic acids. Compared with the rats fed the diet containing no myristic acid, docosahexaenoic acid significantly increased in the brain and red blood cells of the rats fed the diet with 30% myristic acid and in the plasma of the rats fed the diet with 20% myristic acid. Arachidonic acid also increased in the brain of the rats fed the diet with 30% myristic acid. By measuring Δ6-desaturase activity, we found a significant increase in the liver of the rats fed the diet containing 10% of myristic acid but no effect at higher levels of myristic acid. These results suggest that an increase in dietary myristic acid may contribute in increasing significantly the tissue storage of α-linolenic acid and the overall bioavailability of (n-3) polyunsaturated fatty acids in the brain, red blood cells and plasma, and that mechanisms other than the single Δ6-desaturase activity are involved in this effect.     

Simple,sensitive and rapid HPLC–MS/MS method for the determination of cepharanthine in human plasma

A rapid, sensitive and specific method for the determination of cepharanthine in human plasma using high performance liquid chromatography coupled with tandem mass spectrometry (HPLC–MS/MS) was described. Cepharanthine and the internal standard (I.S.), telmisartan, were extracted from human plasma by methanol to precipitate the protein. A centrifuged upper layer was then evaporated and reconstituted with 100 μL methanol. Chromatographic separation was performed on an AGILENT XDB-C₈ column (150 mm × 2.1 mm, 5.0 μm, Agilent, USA) using a gradient mobile phase with 1 mmol/L ammonium acetate in water with 0.05% formic acid and methanol. Detection and quantitation was performed by MS/MS using electrospray ionization (ESI) and multiple reaction monitoring (MRM) in the positive ion mode. The most intense [M+H]⁺ MRM transition of cepharanthine at m/z 607.3 → 365.3 was used for quantitation and the transition at m/z 515.5 → 276.4 was used to monitor telmisartan. The calibration curve was linear within the concentration range of 0.5–200.0 ng/mL (r = 0.9994). The limit of quantification (LOQ) was 0.5 ng/mL. The extraction recovery was above 81.1%. The accuracy was higher than 92.3%. The intra- and inter-day precisions were less than 9.66%. The method was accurate, sensitive and simple and was successfully applied to a pharmacokinetic study after single intravenous administration of 50 mg cepharanthine in 12 healthy Chinese volunteers.     

A rapid gas chromatographic method for the determination of poly-β-hydroxybutyric acid in microbial biomass

Summary The gas chromatographic method for the determination of poly--hydroxybutyric acid (PHB) consists of a mild acid or alkaline methanolysis of poly--hydroxybutyric acid directly without previous extraction of PHB from the cells; this is followed by gas chromatography of the 3-hydroxybutyric acid methylester. The method is characterized by high accuracy and excellent reproducibility, permitting determinations as low as 10^–5 g/l. Only 4 h is required from sampling from the fermenter till completion of the PHB determination.     

Metabolic engineering of microorganisms for the production of multifunctional non-protein amino acids: γ-aminobutyric acid and δ-aminolevulinic acid

Gamma-aminobutyric acid (GABA) and delta-aminolevulinic acid (ALA), playing important roles in agriculture, medicine and other fields, are multifunctional non-protein amino acids with similar and comparable properties and biosynthesis pathways. Recently, microbial synthesis has become an inevitable trend to produce GABA and ALA due to its green and sustainable characteristics. In addition, the development of metabolic engineering and synthetic biology has continuously accelerated and increased the GABA and ALA yield in microorganisms. Here, focusing on the current trends in metabolic engineering strategies for microbial synthesis of GABA and ALA, we analysed and compared the efficiency of various metabolic strategies in detail. Moreover, we provide the insights to meet challenges of realizing industrially competitive strains and highlight the future perspectives of GABA and ALA production.     

Human β2-glycoprotein I: molecular analysis of DNA and amino acid polymorphism

In this study, we describe a two-allelic RsaI restriction fragment length polymorphism identified by Southern blot analysis and by allele-specific polymerase chain reaction amplification for the human ₂-glycoprotein I (₂-I; apolipoprotein H=APOH) gene. This polymorphism, which segregates in a co-dominant fashion, leads to a valine-leucine amino acid exchange at amino acid position 247. The allele frequency has been established in 34 unrelated parents of the Centre d'Etude du Polymorphisme Humain family panel and was found to be 0.76 for valine and 0.23 for leucine. The Val-Leu polymorphism described in this study does not correlate with the four isoelectric focusing alleles previously described, indicating that other variants are responsible for this polymorphism.     

How does displacement of albumin-bound tryptophan cause sustained increases in the free tryptophan concentration in plasma and 5-hydroxytryptamine synthesis in brain?

Models of tryptophan catabolism and binding to serum albumin are presented to explain the observed effect of displacement of tryptophan from albumin on the concentrations of free and bound tryptophan and on the rate of 5-hydroxytryptamine (5-HT) synthesis from tryptophan in the brain. A rapid rate of dissociation of tryptophan from albumin (compared to the transit time of tryptophan through the liver) and a large fractional extraction of the free pool of tryptophan during passage through the liver are shown to be necessary factors in determining the effects observed. Because of the low fractional extraction of free tryptophan in the brain, the synthesis of 5-HT will be dependent only upon the free pool of tryptophan. Dissociation of tryptophan from albumin only causes a sustained increase in 5-HT synthesis in the brain because of the effect that this dissociation has on hepatic tryptophan catabolism and thereby on the free pool of tryptophan.     

Variation of amino acid concentrations in the medium of HU β-IFN and HU IL-2 producing cell lines

There are different requirements for the nutrient medium of various mammalian cell lines. We have determined the behaviour of the amino acid concentrations in the medium of two growing cell lines used for producing human interleukin 2 and human interferon constitutively. The experiments are based on a fermentation process with a bubble free cell culture aeration system with porous moving membranes, which allows production of high cell densities without foaming. We found interesting alterations in which the actual amino acid ratios are able to trigger consumption and production of a particular component depending on the supply of other possible replacements. Such data indicate the complicated biochemical network of synthesis, conversion and transport phenomena. Finally, we demonstrated the influence of product synthesis upon the amino acid requirements using as an example transformed hu IL-2- and hu -IFN-producing mouse L-cells.Abbreviations CTLL Cytotoxic T Cell Line - DME Dulbeccos Modified Eagle - FCS Foetal Calf Serum - Hepes N(2-Hydroxyethyl)piperazine-N'-2-ethanesulfonic acid - hu -IFN human -Interferon - hu IL-2 human Interleukin 2 - THF Tetrahydrofuran     

Comparison of derivatization and chromatographic methods for GC–MS analysis of amino acid enantiomers in physiological samples

GC–MS analysis of fluorinated and non-fluorinated chloroformate and anhydride derivatives of amino acid (AA) enantiomers on two different chiral columns was compared for the direct quantification of free l- and d-AAs in human serum and urine in a single analytical run. Best sensitivity was achieved with pentafluoropropionic anhydride/heptafluorobutanol derivatives separated on a Chirasil-l-Val column. However, the occurrence of racemization during derivatization precluded accurate quantification of AA enantiomers. Derivatization with methyl chloroformate/methanol and separation on an Rt-γDEXsa column did not exhibit racemization and yielded ten baseline separated racemates of proteinogenic AAs with resolution values greater than 2.4. However, protein and peptide hydrolysis occurred in serum and urine during the highly exothermal derivatization reaction under alkaline conditions. Removing serum proteins by precipitation before derivatization and performing the reaction at neutral pH enabled the determination of accurate free AA enantiomer concentrations. Accuracy of quantification was validated by an established nonchiral GC–MS method for AA analysis. Reliable quantification was achieved using stable-isotope labeled l-AAs as internal standards. Limits of detection (LOD) and lower limits of quantification (LLOQ) for the d-AAs were in the range of 3.2–446 nM and 0.031–1.95 μM, respectively. Relative standard deviations (N = 6) for the measurement of AAs in urine and serum ranged from 0.49–11.10% to 0.70–3.87%, respectively. The method was applied to the analysis of urine from 19 patients with renal insufficiency. In comparison to healthy probands, D-ratios of Ala, Val, Pro, Thr, Asp, and Asn were significantly increased.     

Serum amino acid profiles and dopamine in schizophrenic patients and healthy subjects: Window to the brain?

Summary Altered dopamine turnover has been postulated as underlying cause for schizophrenia. This is partially inferred from pharmacological studies and from changes in serum dopamine and dopamine metabolite levels. It is not clear whether the serum amino acid precursors' availability and neurotransmitter-mediated hormonal release could be indicative of the neurotransmitter turnover. We speculate in this context that the profile of serum amino acids and neurotransmitters reflects differences of neurotransmitter activity in the central nervous system and may be considered in a broad sense window to the brain.We analyzed basal serum amino acids (including monoamine precursors), and monoamines in schizophrenic patients after a drug holiday of 3 or more days, and in healthy subjects.Asparagine, phenylalanine, and cystine were higher and tyrosine, tryptophan, and the ratio of tryptophan to competing amino acids lower in schizophrenic patients than in healthy subjects (P < 0.05). Dopamine was increased in schizophrenic patients compared to healthy subjects.We speculate that these results sustain the notion for dopamine overactivity in schizophrenia, which might be caused by altered amino acid precursor availability.Presented at the 2nd International Congress on Amino Acids and Analogues, Vienna, Austria August 5–9, 1991     

Analytical tools for the analysis of β-carotene and its degradation products

Abstract

β-Carotene, the precursor of vitamin A, possesses pronounced radical scavenging properties. This has centered the attention on β-carotene dietary supplementation in healthcare as well as in the therapy of degenerative disorders and several cancer types. However, two intervention trials with β-carotene have revealed adverse effects on two proband groups, that is, cigarette smokers and asbestos-exposed workers. Beside other causative reasons, the detrimental effects observed have been related to the oxidation products of β-carotene. Their generation originates in the polyene structure of β-carotene that is beneficial for radical scavenging, but is also prone to oxidation. Depending on the dominant degradation mechanism, bond cleavage might occur either randomly or at defined positions of the conjugated electron system, resulting in a diversity of cleavage products (CPs).

Due to their instability and hydrophobicity, the handling of standards and real samples containing β-carotene and related CPs requires preventive measures during specimen preparation, analyte extraction, and final analysis, to avoid artificial degradation and to preserve the initial analyte portfolio. This review critically discusses different preparation strategies of standards and treatment solutions, and also addresses their protection from oxidation. Additionally, in vitro oxidation strategies for the generation of oxidative model compounds are surveyed. Extraction methods are discussed for volatile and non-volatile CPs individually. Gas chromatography (GC), (ultra)high performance liquid chromatography (U)HPLC, and capillary electrochromatography (CEC) are reviewed as analytical tools for final analyte analysis. For identity confirmation of analytes, mass spectrometry (MS) is indispensable, and the appropriate ionization principles are comprehensively discussed. The final sections cover analysis of real samples and aspects of quality assurance, namely matrix effects and method validation.     

Development and validation of a liquid chromatography–mass spectrometry method for the determination of verticinone in rat plasma and its application to pharmacokinetic study

We have developed and validated a sensitive liquid chromatography–electrospray ionization-mass spectrometric (LC–ESI-MS) method for the quantification of verticinone, a major active constituent from Fritillaria hupehensis Hsiao et KC Hsia., in rat plasma. Verticinone and the internal standard (IS), hupehenine, were extracted from plasma samples by a simple liquid–liquid extraction with ethyl acetate after being alkalified by 1 M ammonia hydroxide. Chromatographic separation was achieved on a C₁₈ column using a gradient elution program with methanol and water as the mobile phase. The detection was performed by selected ion monitoring (SIM) mode via positive electrospray ionization (ESI) interface. The lower limit of quantification (LLOQ) was 0.1 ng/mL. The calibration curves were linear (r² > 0.998) over the concentration range of 0.1–200 ng/mL. Within- and between-run precision was less than 6.5% and accuracy was within ±10.7%. The validated method was applied to the pharmacokinetic study of verticinone in rats after a single oral administration of 1 mg/kg.     

Salivary chenodeoxycholic acid and its glycine-conjugate: Their determination method using LC–MS/MS and variation of their concentrations with increased saliva flow rate

Measurement of steroid levels in saliva has been proposed as a new laboratory tool for characterizing steroid metabolism, but it is not known whether the salivary levels of bile acids can be measured with accuracy and if so, whether such measurements provide information that is of clinical value. We developed and validated a sensitive and specific liquid chromatography–electrospray ionization-tandem mass spectrometric (LC–ESI-MS/MS) method for the quantification of chenodeoxycholic acid (CDCA) and glycochenodeoxycholic acid (GCDCA), representative primary non-amidated and glycine-conjugated bile acids, in whole saliva. We also examined whether the salivary bile acid concentrations were dependent on the saliva flow rate, because this is a very important aspect in a discussion of the utility of salivary diagnostics. Saliva was deproteinized with ethanol and purified using a Strata-X cartridge. Bile acids were converted to their hydrazide derivatives using 2-hydrazinopyridine, and subjected to LC–MS/MS. Quantification was based on selected reaction monitoring using characteristic transitions, and deuterated CDCA and GCDCA were used as internal standards. This method allowed the reproducible and accurate quantification of the salivary bile acids using a 200-μl sample and the limits of quantification for CDCA and GCDCA were 25 and 50 pg/ml, respectively. Using this method, the effect of increased saliva flow rate by gum-chewing on the salivary concentrations of CDCA and GCDCA was determined. The salivary level of GCDCA was significantly decreased by gum-chewing, whereas the concentration of CDCA remained constant. These results indicate that there is a good possibility that saliva may be a clinical tool for non-amidated bile acid testing.     

Differences in circadian time course and level for the plasma concentrations of glucose,free fatty acids and amino acids between ad lib‐eating and fasting rabbits

Abstract

The entrained rhythm in food approaches, representative of the circadian fluctuation in locomotor and food intake activity was recorded from 12 rabbits eating ad libitum during exposure to an LD 12:12 h‐regimen. They were also subjected to 53 h‐sessions with the same LD alternation, in which blood samples were taken at 2 h‐intervals. During these sessions 6 of them were permitted to eat freely whereas the others were food‐deprived. It appeared that in eating rabbits plasma levels are higher for glucose and a‐amino nitrogen but lower for free fatty acids and, further, that eating specimens exhibit marked circadian fluctuations in the levels of glucose and FFA which are closely correlated with, and probably a consequence of, the circadian rhythm in the amounts of food actually taken up in the course of the sessions. In fasting rabbits, however, the same plasma parameters show rhythms with quite another time course, that can be attributed to the circadian fluctuations in (locomotor) activity and endocrine balance.     

Regional distribution of adenosine uptake in guinea-pig brain slices and the effect of some inhibitors: Evidence for nitrobenzylthioinosine-sensitive and insensitive sites?

The accumulation of [2-³H]adenosine was measured in slices prepared from 7 regions of the guinea-pig central nervous system. There was a similar level of uptake in forebrain regions (cerebral cortex, striatum, hippocampus and midbrain), a lower level in the cerebellum, with lowest uptake in the pons-medulla and spinal cord. Uptake in all regions was strongly inhibited by the nucleoside transport inhibitor dipyridamole and by 5-iodotubercidin, an adenosine kinase inhibitor. The activity of adenosine kinase was similar in crude supernatants prepared from 8 regions of the guinea-pig and rat brain, with the exception of the spinal cord (lower activity than other regions in the guinea-pig CNS) and olfactory bulb (higher activity than other regions in the rat CNS). 5-Nitrobenzylthioinosine (NBMPR) and related thiopurines produced about 50% inhibition of adenosine uptake into guinea-pig cerebral cortex slices at 200 nM but increasing the concentration did not produce significant further inhibition. [³H]NBMPR has been proposed as a useful tight-binding ligand for nucleoside transport sites in various tissues but it is suggested that the distribution of such binding sites in different regions of the CNS may not directly reflect the adenosine uptake capacity of these regions1. Data suggest that there may be NBMPR-sensitive and -insensitive sites. Results confirm those of previous studies which suggest that intracellular adenosine kinase plays an important part in the uptake of adenosine in guinea-pig brain. The relatively homogeneous distribution of adenosine uptake activity in the brain contrasts with the heterogeneous distribution of A1-adenosine receptors in the CNS.     

Urinary amino acid analysis: A comparison of iTRAQ®–LC–MS/MS,GC–MS,and amino acid analyzer

Urinary amino acid analysis is typically done by cation-exchange chromatography followed by post-column derivatization with ninhydrin and UV detection. This method lacks throughput and specificity. Two recently introduced stable isotope ratio mass spectrometric methods promise to overcome those shortcomings. Using two blinded sets of urine replicates and a certified amino acid standard, we compared the precision and accuracy of gas chromatography/mass spectrometry (GC–MS) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) of propyl chloroformate and iTRAQ^® derivatized amino acids, respectively, to conventional amino acid analysis. The GC–MS method builds on the direct derivatization of amino acids in diluted urine with propyl chloroformate, GC separation and mass spectrometric quantification of derivatives using stable isotope labeled standards. The LC–MS/MS method requires prior urinary protein precipitation followed by labeling of urinary and standard amino acids with iTRAQ^® tags containing different cleavable reporter ions distinguishable by MS/MS fragmentation. Means and standard deviations of percent technical error (%TE) computed for 20 amino acids determined by amino acid analyzer, GC–MS, and iTRAQ^®–LC–MS/MS analyses of 33 duplicate and triplicate urine specimens were 7.27 ± 5.22, 21.18 ± 10.94, and 18.34 ± 14.67, respectively. Corresponding values for 13 amino acids determined in a second batch of 144 urine specimens measured in duplicate or triplicate were 8.39 ± 5.35, 6.23 ± 3.84, and 35.37 ± 29.42. Both GC–MS and iTRAQ^®–LC–MS/MS are suited for high-throughput amino acid analysis, with the former offering at present higher reproducibility and completely automated sample pretreatment, while the latter covers more amino acids and related amines.