scholarly journals GC-MS Discrimination of Citrulline from Ornithine and Homocitrulline from Lysine by Chemical Derivatization: Evidence of Formation of N5-Carboxy-ornithine and N6-Carboxy-lysine

Molecules ◽  
2021 ◽  
Vol 26 (8) ◽  
pp. 2301
Author(s):  
Svetlana Baskal ◽  
Alexander Bollenbach ◽  
Dimitrios Tsikas
Keyword(s):  
Amino Acids ◽  
Biological Samples ◽  
Methyl Esters ◽  
Gas Chromatography Mass Spectrometry ◽  
Reaction Products ◽  
Internal Standards ◽  
In Situ Preparation ◽  
Synthetic Amino Acids ◽  
Carboxylic Groups

Derivatization of amino acids by 2 M HCl/CH3OH (60 min, 80 °C) followed by derivatization of the intermediate methyl esters with pentafluoropropionic anhydride (PFPA) in ethyl acetate (30 min, 65 °C) is a useful two-step derivatization procedure (procedure A) for their quantitative measurement in biological samples by gas chromatography-mass spectrometry (GC-MS) as methyl ester pentafluoropropionic (PFP) derivatives, (Me)m-(PFP)n. This procedure allows in situ preparation of trideutero-methyl esters PFP derivatives, (d3Me)m-(PFP)n, from synthetic amino acids and 2 M HCl/CD3OD for use as internal standards. However, procedure A converts citrulline (Cit) to ornithine (Orn) and homocitrulline (hCit) to lysine (Lys) due to the instability of their carbamide groups under the acidic conditions of the esterification step. In the present study, we investigated whether reversing the order of the two-step derivatization may allow discrimination and simultaneous analysis of these amino acids. Pentafluoropropionylation (30 min, 65 °C) and subsequent methyl esterification (30 min, 80 °C), i.e., procedure B, of Cit resulted in the formation of six open and cyclic reaction products. The most abundant product is likely to be N5-Carboxy-Orn. The second most abundant product was confirmed to be Orn. The most abundant reaction product of hCit was confirmed to be Lys, with the minor reaction product likely being N6-Carboxy-Lys. Mechanisms are proposed for the formation of the reaction products of Cit and hCit via procedure B. It is assumed that at the first derivatization step, amino acids form (N,O)-PFP derivatives including mixed anhydrides. At the second derivatization step, the Cit-(PFP)4 and hCit-(PFP)4 are esterified on their C1-Carboxylic groups and on their activated Nureido groups. Procedure B also allows in situ preparation of (d3Me)m-(PFP)n from synthetic amino acids for use as internal standards. It is demonstrated that the derivatization procedure B enables discrimination between Cit and Orn, and between hCit and Lys. The utility of procedure B to measure simultaneously these amino acids in biological samples such as plasma and urine remains to be demonstrated. Further work is required to optimize the derivatization conditions of procedure B for biological amino acids.

scholarly journals Analysis of bilirubin and bilirubin mono- and di-conjugates. Determination of their relative amounts in biological samples

1980 ◽  
Vol 185 (1) ◽  
pp. 115-128 ◽  
Author(s):  
N Blanckaert
Keyword(s):  
Biological Samples ◽  
Methyl Esters ◽  
Dimethyl Ester ◽  
Crystalline Form ◽  
Reaction Products ◽  
Reaction Conditions ◽  
Normal Rat ◽  
Novel Method ◽  
Rat Bile

1. A novel method for determination of the relative amounts of unconjugated bilirubin and sugar mono- and di-conjugates of bilirubin in biological samples, including serum, is described and illustrated by its application to the analysis of bilinoids in rat bile. 2. The method is based on specific conversion of the carbohydrate conjugates of bilirubin into the corresponding mono- or di-methyl esters by base-catalysed transesterification in methanol. Under the selected reaction conditions, unconjugated biliru-in remains intact and no dipyrrole exchange in the bilinoids is detectable; transesterification of bilirubin mono- or di-glucuronide is virtually complete (approx. 99%), and sponification is negligible (less than 1%); recovery of the pigments is approx. 95%. 3. The reaction products bilirubin and its methyl esters are separated by t.l.c. and determined spectrophotometrically; the two isomeric bilirubin-IX alpha monomethyl esters are separated and therefore can be determined individually. 4. Reference bilirubin mono- and di-methyl esters have been synthesized and characterized, and the two isomers of bilirubin-IX alpha monomethyl ester and bilirubin dimethyl ester were obtained individually, in crystalline form. 5. With this new method, virtually all bilinoids (over 99%) in normal rat bile have been found to be conjugated, with diconjugates (71%) predominating. A significantly increased proportion of monoconjugates is present in bile collected from heterozygous Gunn rats or from normal rats that were refused with large amounts of bilirubin.

Vascular perfusion: a novel means of studying oviduct function

1985 ◽  
Vol 248 (5) ◽  
pp. E624-E632 ◽  
Author(s):  
H. J. Leese ◽  
S. M. Gray
Keyword(s):  
Amino Acids ◽  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
Bovine Serum ◽  
Vascular Perfusion ◽  
Ovarian Artery ◽  
In Situ Preparation ◽  
Luminal Perfusion ◽  
Oviduct Fluid

An in situ preparation for the combined vascular and luminal perfusion of the rabbit oviduct has been developed. Medium 199, gassed with 5% CO2 in O2 and supplemented with heparin, antibiotics, and 2.5% wt/vol dialyzed bovine serum albumin was infused into the ovarian artery at a rate of 1 ml/min. Krebs Ringer bicarbonate medium was recirculated through the lumen at a rate of 50 microliter/min. The ovary was perfused together with the oviduct, and the preparation is viable for up to 3 h. Equal concentrations of pyruvate, lactate, glucose, and sucrose added to the vascular medium were transported at different rates into the lumen, as was a physiological mixture of amino acids. A proportion of the lactate entering the lumen was synthesized within the oviduct from vascular glucose. When glucose and pyruvate were omitted from the vascular medium, their appearance and that of lactate in the lumen was barely detectable, suggesting that these oviduct fluid components are mainly derived from the blood. The oviduct maintained a steady transmural potential difference of 5.9 mV (lumen negative). With vascular perfusion alone, oviduct fluid entered the oviduct lumen at a rate of 16.8 microliter/h. In oviducts taken from rabbits 3 days postovulation, there was a general decrease in the vascular to lumen flux of all nutrients measured. Preliminary work has shown that the preparation may be used to study ovulation, ovum pickup and transport, and fertilization.

scholarly journals Two-Step Derivatization of Amino Acids for Stable-Isotope Dilution GC–MS Analysis: Long-Term Stability of Methyl Ester-Pentafluoropropionic Derivatives in Toluene Extracts

Molecules ◽  
2021 ◽  
Vol 26 (6) ◽  
pp. 1726 ◽  
Author(s):  
Svetlana Baskal ◽  
Alexander Bollenbach ◽  
Dimitrios Tsikas
Keyword(s):  
Amino Acids ◽  
Methyl Ester ◽  
Stable Isotope ◽  
Internal Standards ◽  
Term Stability ◽  
Long Term Stability ◽  
Endogenous Amino Acids ◽  
Derivatives Of

Analysis of amino acids by gas chromatography-mass spectrometry (GC–MS) requires at least one derivatization step to enable solubility in GC–MS-compatible water-immiscible organic solvents such as toluene, to make them volatile to introduce into the gas chromatograph and thermally stable enough for separation in the GC column and introduction into the ion-source, and finally to increase their ionization by increasing their electronegativity using F-rich reagents. In this work we investigated the long-term stability of the methyl esters pentafluoropropionic (Me-PFP) derivatives of 21 urinary amino acids prepared by a two-step derivatization procedure and extraction by toluene. In situ prepared trideuteromethyl ester pentafluoropropionic derivatives were used as internal standards. GC–MS analysis (injection of 1 µL aliquots and quantification by selected-ion monitoring of specific mass fragments) was performed on days 1, 2, 8, and 15. Measured peak areas and calculated peak area ratios were used to evaluate the stability of the derivatives of endogenous amino acids and their internal standards, as well as the precision and the accuracy of the method. All analyses were performed under routine conditions. Me-PFP derivatives of endogenous amino acids and their stable-isotope labelled analogs were stable in toluene for 14 days. The peak area values of the derivatives of most amino acids and their internal standards were slightly higher on days 8 and 15 compared to days 1 and 2, yet the peak area ratio values of endogenous amino acids to their internal standards did not change. Our study indicates that Me-PFP derivatives of amino acids from human urine samples can easily be prepared, are stable at least for 14 days in the extraction solvent toluene, and allow for precise and accurate quantitative measurements by GC–MS using in situ prepared deuterium-labelled methyl ester as internal standard.

The effect of base particle size in complementary feedingstuffs using early maillard reaction products on the performance of post weaning piglets

2007 ◽  
Vol 2007 ◽  
pp. 79-79
Author(s):  
M. J. Hutchinson ◽  
M .E .E McCann ◽  
V Beattie
Keyword(s):  
Amino Acids ◽  
Maillard Reaction ◽  
Animal Feed ◽  
Maillard Reaction Products ◽  
Reaction Products ◽  
Chemical Catalysis ◽  
Advantages And Disadvantages ◽  
Feed Industry ◽  
Synthetic Amino Acids

The animal feed industry produces various complementary feedstuffs with high levels of crude protein (CP) and synthetic amino acids designed to provide optimum nutrition to the post weaned pig. The use of synthetic amino acids has both advantages and disadvantages and other ways of delivering amino acids are of interest to the feed industry. One possible way of delivering amino acids is through the Maillard reaction; this is the chemical catalysis of the amine group of an amino acid to the carboxyl group of a sugar giving Maillard reaction products (MRP). These molecules occur during cooking, but have been shown to have a variety of other applications (Namiki, 1996) In this study, lysine (Lys), methionine (Met) and threonine (Thr) where chemically reacted with sugar molecules to give in vitro early Maillard Reaction Products (MRP). The aim of this study was to assess the effect of a solution of these MRPs with particle sizes of cereal base in a complementary feedingstuff (Matan XL, Devenish Nutrition) on overall diet performance.

The Kinetics of Inhibition of the Action of Thrombin by the Fibrinopeptides Formed during the Clotting of Fibrinogen

1966 ◽  
Vol 16 (01/02) ◽  
pp. 277-295 ◽  
Author(s):  
A Silver ◽  
M Murray
Keyword(s):  
Amino Acids ◽  
Competitive Inhibition ◽  
Amorphous Material ◽  
Peptide Bond ◽  
Initial Reaction ◽  
Reaction Products ◽  
Coagulation Mechanism ◽  
Kinetics Of ◽  
Kinetics Of Inhibition ◽  
Burk Plot

SummaryVarious investigators have separated the coagulation products formed when fibrinogen is clotted with thrombin and identified fibrinopeptides A and B. Two other peaks are observed in the chromatogram of the products of coagulation, but these have mostly been dismissed by other workers. They have been identified by us as amino acids, smaller peptides and amorphous material (37). We have re-chromatographed these peaks and identified several amino acids. In a closed system of fibrinogen and thrombin, the only reaction products should be fibrin and peptide A and peptide B. This reasoning has come about because thrombin has been reported to be specific for the glycyl-arginyl peptide bond. It is suggested that thrombin also breaks other peptide linkages and the Peptide A and Peptide B are attacked by thrombin to yield proteolytic products. Thrombin is therefore probably not specific for the glycyl-arginyl bond but will react on other linkages as well.If the aforementioned is correct then the fibrinopeptides A and B would cause an inhibition with the coagulation mechanism itself. We have shown that an inhibition does occur. We suggest that there is an autoinhibition to the clotting mechanism that might be a control mechanism in the human body.The experiment was designed for coagulation to occur under controlled conditions of temperature and time. Purified reactants were used. We assembled an apparatus to record visually the speed of the initial reaction, the rate of the reaction, and the density of the final clot formed after a specific time.The figures we derived made available to us data whereby we could calculate and plot the information to show the mechanism and suggest that such an inhibition does exist and also further suggest that it might be competitive.In order to prove true competitive inhibition it is necessary to fulfill the criteria of the Lineweaver-Burk plot. This has been done. We have also satisfied other criteria of Dixon (29) and Bergman (31) that suggest true competitive inhibition.

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