A State Space Transformation Can Yield Identifiable Models for Tracer Kinetic Studies with Enrichment Data

Tracer studies are analyzed almost universally by multicompartmental models where the state variables are tracer amounts or activities in the different pools. The model parameters are rate constants, defined naturally by expressing fluxes as fractions of the source pools. We consider an alternative state space with tracer enrichments or specific activities as the state variables, with the rate constants redefined by expressing fluxes as fractions of the destination pools. Although the redefinition may seem unphysiological, the commonly computed fractional synthetic rate actually expresses synthetic flux as a fraction of the product mass (destination pool). We show that, for a variety of structures, provided the structure is linear and stationary, the model in the enrichment state space has fewer parameters than that in the activities state space, and is hence better both to study identifiability and to estimate parameters. The superiority of enrichment modeling is shown for structures where activity model unidentifiability is caused by multiple exit pathways; on the other hand, with a single exit pathway but with multiple untraced entry pathways, activity modeling is shown to be superior. With the present-day emphasis on mass isotopes, the tracer in human studies is often of a precursor, labeling most or all entry pathways. It is shown that for these tracer studies, models in the activities state space are always unidentifiable when there are multiple exit pathways, even if the enrichment in every pool is observed; on the other hand, the corresponding models in the enrichment state space have fewer parameters and are more often identifiable. Our results suggest that studies with labeled precursors are modeled best with enrichments.     

Multiple apolipoprotein kinetics measured in human HDL by high-resolution/accurate mass parallel reaction monitoring

Endogenous labeling with stable isotopes is used to study the metabolism of proteins in vivo. However, traditional detection methods such as GC/MS cannot measure tracer enrichment in multiple proteins simultaneously, and multiple reaction monitoring MS cannot measure precisely the low tracer enrichment in slowly turning-over proteins as in HDL. We exploited the versatility of the high-resolution/accurate mass (HR/AM) quadrupole Orbitrap for proteomic analysis of five HDL sizes. We identified 58 proteins in HDL that were shared among three humans and that were organized into five subproteomes according to HDL size. For seven of these proteins, apoA-I, apoA-II, apoA-IV, apoC-III, apoD, apoE, and apoM, we performed parallel reaction monitoring (PRM) to measure trideuterated leucine tracer enrichment between 0.03 to 1.0% in vivo, as required to study their metabolism. The results were suitable for multicompartmental modeling in all except apoD. These apolipoproteins in each HDL size mainly originated directly from the source compartment, presumably the liver and intestine. Flux of apolipoproteins from smaller to larger HDL or the reverse contributed only slightly to apolipoprotein metabolism. These novel findings on HDL apolipoprotein metabolism demonstrate the analytical breadth and scope of the HR/AM-PRM technology to perform metabolic research.     

Isolation of plasma albumin by ethanol extraction is inappropriate for isotope ratio measurements during the acute phase response

Isolation of high-purity albumin from plasma is essential to study albumin kinetics in vivo with tracer techniques. Because of its simplicity ethanol extraction has been repeatedly used for albumin purification. However, it cannot be excluded that this single-step procedure completely prohibits contamination by other proteins, especially those known to be produced at an accelerated rate during the acute phase response. In the present study, we wanted to examine the reliability of ethanol extraction in different clinical conditions and to study the effects of potential impurities on albumin enrichment during stable isotope tracer studies. SDS-PAGE revealed a contaminating protein band at about 25,000 Da in healthy subjects and postoperative patients during the acute phase response, but not in critically ill patients. According to densitometry about 8% of proteins after ethanol extraction were contaminants. To examine potential contaminant effects on tracer enrichment 1-[13C]-leucine was given to healthy subjects and postoperative patients. Blood samples were taken after various amounts of time, and albumin enrichments (tracer/tracee ratios) were determined from isotope ratios obtained by mass spectrometry. Irrespective of the magnitude of tracer enrichment, postoperative tracer/tracee ratios were significantly higher (on average +10%) in samples exclusively analysed by ethanol extraction than in samples which had undergone additional electrophoretic purification. No significant effect of the contaminant was seen in healthy subjects. N-terminal protein sequencing revealed contaminants to mainly consist of apolipoprotein A-1. Its physiology and pathophysiology may sufficiently explain its variable effects of albumin enrichment. Our findings suggest that exclusive ethanol extraction is inappropriate for albumin isolation in tracer studies performed during the acute phase response. Ethanol extraction may also not be advisable in all other situations known to be associated with a rise in apolipoprotein A-1 turnover.     

Incorporation of a stable isotopically labeled amino acid into multiple human apolipoproteins

Procedures are presented for the separation and determination of the isotopic enrichment of multiple human apolipoproteins labeled in vivo with a stable isotope amino acid. The isotopic enrichments of plasma lysine and plasma apolipoproteins were monitored for 16 days after a single intravenous dose of [4,4,5,5-2H4]lysine (5 mg/kg body weight). The use of a multiply deuterated amino acid enabled the measurement of isotopic enrichments above background over the entire 16-day time course in all proteins. Individual apolipoproteins were separated on a specially designed gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis system cast in a conventional slab gel apparatus which resolved apoB-100, apoE, apoA-I, apoA-II, apoC-I, apoC-II, apoC-III-1, and apoC-III-2 on a single gel. After staining with Coomassie blue, proteins bands (containing 5 to 30 micrograms of individual apolipoprotein) were excised from the gel. Amino acids were recovered from hydrolyzed gel slices, derivatized, and analyzed by gas chromatography-mass spectrometry for determination of lysine isotopic enrichments. The utility of the method is demonstrated using examples of apolipoproteins B-100, A-I, A-II, C-I, C-II, and C-III from either total plasma d less than 1.21 g/ml lipoproteins or selected lipoprotein subfractions. Lysine isotopic enrichments of proteins were generally determined with a precision of better than 5%. The isotopic enrichment profiles were consistent with literature reports of apolipoprotein metabolic kinetics based on the use of radioiodinated apolipoproteins. The procedures outlined can be used to separate and measure the isotopic enrichment of virtually any apolipoprotein from any chosen lipoprotein fraction. Thus, these procedures should find wide application in the study of apolipoprotein metabolic kinetics.     

Chenodeoxycholic acid pool size determination from children using isotope dilution mass spectrometry

An isotope dilution mass spectrometry method is described for determining chenodeoxycholic acid pool size in children. The stable isotopically labeled tracer, (11,12-2H2) chenodeoxycholic acid, was administered orally to children, and the enrichment of bile was measured by selected ion monitoring gas chromatography mass spectrometry. The level of (11,12-2H2)chenodeoxycholic acid enrichment found in the patient samples was in the range of 0.5 to 5%. Data are presented illustrating the duplication of this method in two independent laboratories using standard quadrupole mass spectrometers. This procedure provides the clinician with a non-radioactive method for determining chenodeoxycholic acid pool size which is especially beneficial in studies involving children and pregnant women.     

Total-body creatine pool size and skeletal muscle mass determination by creatine-(methyl-d3) dilution in rats

There is currently no direct, facile method to determine total-body skeletal muscle mass for the diagnosis and treatment of skeletal muscle wasting conditions such as sarcopenia, cachexia, and disuse. We tested in rats the hypothesis that the enrichment of creatinine-(methyl-d(3)) (D(3)-creatinine) in urine after a defined oral tracer dose of D(3)-creatine can be used to determine creatine pool size and skeletal muscle mass. We determined 1) an oral tracer dose of D(3)-creatine that was completely bioavailable with minimal urinary spillage and sufficient enrichment in the body creatine pool for detection of D(3)-creatine in muscle and D(3)-creatinine in urine, and 2) the time to isotopic steady state. We used cross-sectional studies to compare total creatine pool size determined by the D(3)-creatine dilution method to lean body mass determined by independent methods. The tracer dose of D(3)-creatine (<1 mg/rat) was >99% bioavailable with 0.2-1.2% urinary spillage. Isotopic steady state was achieved within 24-48 h. Creatine pool size calculated from urinary D(3)-creatinine enrichment at 72 h significantly increased with muscle accrual in rat growth, significantly decreased with dexamethasone-induced skeletal muscle atrophy, was correlated with lean body mass (r = 0.9590; P < 0.0001), and corresponded to predicted total muscle mass. Total-body creatine pool size and skeletal muscle mass can thus be accurately and precisely determined by an orally delivered dose of D(3)-creatine followed by the measurement of D(3)-creatinine enrichment in a single urine sample and is promising as a noninvasive tool for the clinical determination of skeletal muscle mass.     

Determination of protein replacement rates by deuterated water: validation of underlying assumptions

H(2)O administration has recently been proposed as a simple and convenient method to measure protein synthesis rates. (2)H(2)O administration results in deuterium labeling of free amino acids such as alanine, and incorporation into proteins of labeled alanine can then be used to measure protein synthesis rates. We examined first whether during (2)H(2)O administration plasma free alanine enrichment is a correct estimate of the enrichment in the tissue amino acid pools used for protein synthesis. We found that, after (2)H(2)O administration, deuterium labeling in plasma free alanine equilibrated rapidly with body water, and stable enrichment values were obtained within 20 min. Importantly, oral administration of (2)H(2)O induced no difference of labeling between portal and peripheral circulation except for the initial 10 min after a loading dose. The kinetics of free alanine labeling were comparable in various tissues (liver, skeletal muscle, heart) and in plasma with identical plateau values. We show next that increased glycolytic rate or absorption of unlabeled amino acids from ingested meals do not modify alanine labeling. Calculated synthesis rates of mixed proteins were much higher (20- to 70-fold) in plasma and liver than in muscle and heart. Last, comparable replacement rates of apoB100-VLDL were obtained in humans by using the kinetics of incorporation into apoB100 of infused labeled leucine or of alanine labeled by (2)H(2)O administration. All of these results support (2)H(2)O as a safe, reliable, useful, and convenient tracer for studies of protein synthesis, including proteins with slow turnover rate.     

Oxidation inhibits amyloid fibril formation of transthyretin

The role of amino acid side chain oxidation in the formation of amyloid assemblies has been investigated. Chemical oxidation of amino acid side chains has been used as a facile method of introducing mutations on protein structures. Oxidation promotes changes within tertiary contacts that enable identification of residues and interactions critical in stabilizing protein structures. Transthyretin (TTR) is a soluble human plasma protein. The wild-type (WT) and several of its variants are prone to fibril formation, which leads to amyloidosis associated with many clinical syndromes. The effects of amino acid side chain oxidations were investigated by comparing the kinetics of fibril formation of oxidized and unoxidized proteins. The WT and V30M TTR mutant (valine 30 substituted with methionine) were allowed to react over a time range of 10 min to 12 h with hydroxy radical and other reactive oxygen species. In these timescales, up to five oxygen atoms were incorporated into WT and V30M TTR proteins. Oxidized proteins retained their tetrameric structures, as determined by cross-linking experiments. Side chain modification of methionine residues at position 13 and 30 (the latter for V30M TTR only) were dominant oxidative products. Mono-oxidized and dioxidized methionine residues were identified by radical probe mass spectometry employing a footprinting type approach. Oxidation inhibited the initial rates and extent of fibril formation for both the WT and V30M TTR proteins. In the case of WT TTR, oxidation inhibited fibril growth by approximately 76%, and for the V30M TTR by nearly 90%. These inhibiting effects of oxidation on fibril growth suggest that domains neighboring the methionine residues are critical in stabilizing the tetrameric and folded monomer structures.     

Mass kinetics of apolipoprotein A-I in interstitial fluid after administration of intravenous apolipoprotein A-I/lecithin discs in humans

Apolipoprotein kinetics are customarily determined by modeling time curves of specific radioactivity or isotopic enrichment in plasma after intravenous infusion of radiolabeled lipoproteins or stable isotope-enriched amino acids. However, this provides no information on the fractional rate of transfer of the apolipoprotein from plasma to interstitial fluid (k(p-if)) or its mean residence time in interstitial fluid (MRT(if)). To determine these parameters for a pharmacologic dose of exogenous apolipoprotein A-I (apoA-I) given intravenously as apoA-I/lecithin discs, we measured apoA-I in plasma and prenodal leg lymph in five healthy men before, during, and after a 4 h infusion at 10 mg/kg/h. ApoA-I concentrations in plasma and lymph were modeled by linear compartmental models (SAAM II version 1.1), using lymph albumin to adjust for the effects of variations in lymph flow rate. k(p-if) averaged 0.75%/h (range, 0.33-1.32), and MRT(if) averaged 29.1 h (14.1-40.0). Neither parameter was correlated with the distribution volume (57-105 ml/kg) or the fractional elimination rate (1.44-2.91%/h) of apoA-I, determined by modeling plasma apoA-I concentration alone. Although used here to study the mass kinetics of apoA-I, if combined with infusion of a tracer, analysis of lymph could also expand the modeling of endogenous apolipoprotein kinetics.     

The determination of specific radioactivity of proteins eluted intact from polyacrylamide gels, utilizing a fluorescamine assay

The methodology described permits the measurement of the specific radioactivity of diverse proteins resolvable by separatory techniques using cylindrical polyacrylamide gels. Following separation, the proteins are electroeluted; eluted protein is quantitated in the microgram range using a fluorescamine assay, while the major portion of the recovered sample is used for radioactivity measurement. These procedures have been adapted for use in tracer studies of protein metabolism. Their utility in kinetic investigations is demonstrated with data on the time course of changing specific radioactivities of human plasma albumin and apolipoprotein B labeled in vivo with a [3H]leucine tracer.     

Oxazolinone derivative of leucine for GC-MS: a sensitive and robust method for stable isotope kinetic studies of lipoproteins

Stable isotope labeled amino acids are commonly used as endogenous tracers to study the metabolism of lipoproteins. The determination of isotopic enrichment of particular amino acids in apolipoproteins is carried out by gas chromatography mass spectrometry (GC-MS). This report describes a robust and sensitive derivative for analysis of d3-leucine by GC-MS and its utility in studying the metabolism of human lipoproteins. The trifluoromethyloxazolinone (oxazolinone) derivative of leucine was formed in a rapid single step procedure using a mixture of trifluoroacetic anhydride (TFAA) and trifluoroacetic acid (TFA). Analysis of the oxazolinone by negative ion chemical ionization GC-MS gave excellent sensitivity and precision, which enabled accurate determination of low levels of isotopic enrichment from small amounts of protein. For example, enrichments between 0.05% and 100% in 100 pg leucine can be measured with a coefficient of variation of <3%. To demonstrate the utility of this procedure, we measured d3-leucine enrichment in apolipoprotein B (apoB) isolated from VLDL and LDL as well as apoA-I isolated from HDL by gel electrophoresis and western blotting. The derivatization procedure gave excellent enrichment data from a single intravenous bolus dose of 5 mg/kg, from which the fractional catabolic rate and production rate of the lipoproteins were calculated. In conclusion, the oxazolinone derivative provides a robust and simple procedure for the sensitive analysis of isotopic enrichment for metabolic studies of human lipoproteins.     

Transthyretin is a metallopeptidase with an inducible active site

TTR (transthyretin) was found recently to possess proteolytic competency besides its well-known transport capabilities. It was described as a cryptic serine peptidase cleaving multiple natural substrates (including β-amyloid and apolipoprotein A-I) involved in diseases such as Alzheimer's disease and atherosclerosis. In the present study, we aimed to elucidate the catalytic machinery of TTR. All attempts to identify a catalytic serine residue were unsuccessful. However, metal chelators abolished TTR activity. Proteolytic inhibition by EDTA or 1,10-phenanthroline could be reversed with Zn2+ and Mn2+. These observations, supported by analysis of three-dimensional structures of TTR complexed with Zn2+, led to the hypothesis that TTR is a metallopeptidase. Site-directed mutagenesis of selected amino acids unambiguously confirmed this hypothesis. The TTR active site is inducible and constituted via a protein rearrangement resulting in ~7% of proteolytically active TTR at pH?7.4. The side chain of His88 is shifted near His90 and Glu92 establishing a Zn2+-chelating pattern HXHXE not found previously in any metallopeptidase and only conserved in TTR of humans and some other primates. Point mutations of these three residues yielded proteins devoid of proteolytic activity. Glu72 was identified as the general base involved in activation of the catalytic water. Our results unveil TTR as a metallopeptidase and define its catalytic machinery.     

Determination of kinetic parameters of apolipoprotein B metabolism using amino acids labeled with stable isotopes.

The use of amino acids labeled with stable isotopes represents a relatively new approach for determining kinetic parameters of apolipoprotein metabolism; thus, several aspects of experimental protocols need to be defined. The aims of the present study were to determine whether a) different amino acid tracers or b) different methods of tracer administration affected apolipoprotein (apo) B kinetic parameters obtained by multicompartmental modeling, and c) to compare very low density lipoprotein (VLDL)-apoB metabolic parameters determined by multicompartmental modeling with those estimated by linear regression or by monoexponential analysis. [1-13C]leucine and [15N]glycine were given either as bolus injections or as primed constant infusions. A bolus of one amino acid was administered simultaneously with a primed constant infusion (8 h) of the other amino acid into four healthy normolipidemic subjects (age 23.0 +/- 1.4 yr; BMI 20.9 +/- 0.9 kg.m-2). VLDL-, intermediate density lipoprotein (IDL)-, and low density lipoprotein (LDL)-apoB enrichments were followed over 110 h. For subsequent analysis these values were converted to tracer/tracee ratios. Using the multicompartmental model, the fractional catabolic rate (FCR) for VLDL-apoB was estimated to be 0.36 +/- 0.09 h-1 after the administration of the tracer as a primed constant infusion and 0.35 +/- 0.07 h-1 when the tracer was administered as a bolus. The values for VLDL-apoB production were 14.6 +/- 6.5 mg.kg-1.d-1 and 14.1 +/- 5.4 mg.kg-1.d-1, respectively. The corresponding values for LDL-apoB were 0.027 +/- 0.016 h-1 (0.026 +/- 0.018 h-1) for the FCR and 10.5 +/- 3.7 mg.kg-1.d-1 (10.4 +/- 3.8 mg.kg-1.d-1) for the production following administration of the tracer as a primed constant infusion and a bolus, respectively. Approximately 47% of VLDL-apoB ultimately reached the LDL fraction via the VLDL-IDL-LDL pathway. Thirty-five percent of LDL-apoB did not originate from this cascade pathway, but was shunted from a rapidly turning over VLDL compartment directly into the LDL fraction. While there was some variation between individuals, VLDL-apoB and LDL-apoB parameters derived from the bolus and the primed constant infusions showed no significant differences and were closely correlated. Metabolic parameters were also independent of the two amino acids tested. Although values for FCRs of VLDL-apoB obtained from linear regression (0.36 +/- 0.19 h-1) or monoexponential analysis (0.50 +/- 0.36 h-1) did not differ significantly from those obtained by the multicompartmental model, there was considerable variation and no significant correlation in a given individual.(ABSTRACT TRUNCATED AT 400 WORDS)     

Connective tissue growth factor (CTGF) promotes activated mesangial cell survival via up-regulation of mitogen-activated protein kinase phosphatase-1 (MKP-1)

Destabilization of the tetrameric fold of TTR (transthyretin) is important for aggregation of the protein which culminates in amyloid fibril formation. Many TTR mutations interfere with tetramer stability, increasing the amyloidogenic potential of the protein. The vast majority of proposed TTR fibrillogenesis inhibitors are based on in vitro assays with isolated protein, limiting their future use in clinical assays. In the present study we investigated TTR fibrillogenesis inhibitors using a cellular system that produces TTR intermediates/aggregates in the medium. Plasmids carrying wild-type TTR, V30M or L55P cDNA were transfected into a rat Schwannoma cell line and TTR aggregates were investigated in the medium using a dot-blot filter assay followed by immunodetection. Results showed that, in 24 h, TTR L55P forms aggregates in the medium, whereas, up to 72 h, wild-type TTR and V30M do not. A series of 12 different compounds, described in the literature as in vitro TTR fibrillogenesis inhibitors, were tested for their ability to inhibit L55P aggregate formation; in this system, 2-[(3,5-dichlorophenyl) amino] benzoic acid, benzoxazole, 4-(3,5-difluorophenyl) benzoic acid and tri-iodophenol were the most effective inhibitors, as compared with the reference iododiflunisal, previously shown by ex vivo and in vitro procedures to stabilize TTR and inhibit fibrillogenesis. Among these drugs, 2-[(3,5-dichlorophenyl) amino] benzoic acid and tri-iodophenol stabilized TTR from heterozygotic carriers of V30M in the same ex vivo conditions as those used previously for iododiflunisal. The novel cellular-based test herein proposed for TTR fibrillogenesis inhibitor screens avoids not only lengthy and cumbersome large-scale protein isolation steps but also artefacts associated with most current in vitro first-line screening methods, such as those associated with acidic conditions and the absence of serum proteins.     

Identification of S-sulfonation and S-thiolation of a novel transthyretin Phe33Cys variant from a patient diagnosed with familial transthyretin amyloidosis

Familial transthyretin amyloidosis (ATTR) is an autosomal dominant disorder associated with a variant form of the plasma carrier protein transthyretin (TTR). Amyloid fibrils consisting of variant TTR, wild-type TTR, and TTR fragments deposit in tissues and organs. The diagnosis of ATTR relies on the identification of pathologic TTR variants in plasma of symptomatic individuals who have biopsy proven amyloid disease. Previously, we have developed a mass spectrometry-based approach, in combination with direct DNA sequence analysis, to fully identify TTR variants. Our methodology uses immunoprecipitation to isolate TTR from serum, and electrospray ionization and matrix-assisted laser desorption/ionization mass spectrometry (MS) peptide mapping to identify TTR variants and posttranslational modifications. Unambiguous identification of the amino acid substitution is performed using tandem MS (MS/MS) analysis and confirmed by direct DNA sequence analysis. The MS and MS/MS analyses also yield information about posttranslational modifications. Using this approach, we have recently identified a novel pathologic TTR variant. This variant has an amino acid substitution (Phe --> Cys) at position 33. In addition, like the Cys10 present in the wild type and in this variant, the Cys33 residue was both S-sulfonated and S-thiolated (conjugated to cysteine, cysteinylglycine, and glutathione). These adducts may play a role in the TTR fibrillogenesis.     

2H- and13C-labeled amino acids generated by obligate methylotrophs Biosynthesis and MS monitoring

Summary Biosynthetic preparation of²H- and¹³C- labeled amino acids was studied using a leucine-producing mutant of the obligate methylotroph,Methylobacillus flagellatum. The strain was cultivated in various media containing¹³C- or²H-analogs of methanol. The total protein from each experiment was subjected to acid hydrolysis and converted into a mixture of dansyl amino acid methyl esters. The samples of excreted leucine were converted into methyl esters of dansyl and benzyloxycarbonyl derivatives. Electron impact mass spectrometry was performed to detect stable isotope enrichment of the amino acids. According to the mass spectrometric analysis it is feasible to use methylotrophic microorganisms for the preparation of²H- and¹³C- analogs of amino acids by labeled methanol bioconversion; the excreted amino acids can be convenient for express analysis as an indicator of isotopic enrichment of the total protein. The data obtained testified to a high efficiency of dansyl derivatization for mass spectrometric analysis of complex amino acid mixtures.     

Basic studies on metabolic steady state. Linearity of a metabolic tracer system

Summary In dogs the changes in the plasma concentration following the injection and/or infusion of labeled glucose reveal the labeled glucose system to be a linear system. Based on a simple chemical model it was shown that the rate of removal from the system of any tracer injected as single impulse can be described by a first order chemical reaction, even in systems from which the tracee is removed by a process of a higher order, provided the tracee is in the steady state and the concentration of tracer in the plasma is low compared to that of the tracee. The validity of the commonly used formulae for the calculations of the rates of disappearance from systems in a steady state is based on this first order process.     

Preparation of CO2 from blood and protein-bound amino acid carboxyl groups for quantification and 13C-isotope measurements

Measurements of protein and amino acid metabolism in man using stable isotopes and selected ion monitoring gas chromatographic/mass spectrometric techniques are limited by the requirement of relatively high levels of labelling for adequate precision (greater than 0.05 at % excess). We describe here a means of extending the scope of such studies by measurement of lower levels of enrichment achieved in gaseous CO2 derived from whole blood or protein-bound amino acids following the administration of tracer amounts of appropriately labelled substrates. Construction and operation of a novel glass vacuum line required for this work are described in detail and specific applications relevant to clinical investigations are outlined. Measurements of both the total amount of CO2 and its 13C enrichment are performed in an isotope-ratio mass spectrometer which provides acceptable levels of accuracy and reproducibility for both measurements (+/- 0.1% and +/- 0.0001 at % excess respectively).     

Lysine residues direct the chlorination of tyrosines in YXXK motifs of apolipoprotein A-I when hypochlorous acid oxidizes high density lipoprotein

Oxidized lipoproteins may play an important role in the pathogenesis of atherosclerosis. Elevated levels of 3-chlorotyrosine, a specific end product of the reaction between hypochlorous acid (HOCl) and tyrosine residues of proteins, have been detected in atherosclerotic tissue. Thus, HOCl generated by the phagocyte enzyme myeloperoxidase represents one pathway for protein oxidation in humans. One important target of the myeloperoxidase pathway may be high density lipoprotein (HDL), which mobilizes cholesterol from artery wall cells. To determine whether activated phagocytes preferentially chlorinate specific sites in HDL, we used tandem mass spectrometry (MS/MS) to analyze apolipoprotein A-I that had been oxidized by HOCl. The major site of chlorination was a single tyrosine residue located in one of the protein's YXXK motifs (where X represents a nonreactive amino acid). To investigate the mechanism of chlorination, we exposed synthetic peptides to HOCl. The peptides encompassed the amino acid sequences YKXXY, YXXKY, or YXXXY. MS/MS analysis demonstrated that chlorination of tyrosine in the peptides that contained lysine was regioselective and occurred in high yield if the substrate was KXXY or YXXK. NMR and MS analyses revealed that the N(epsilon) amino group of lysine was initially chlorinated, which suggests that chloramine formation is the first step in tyrosine chlorination. Molecular modeling of the YXXK motif in apolipoprotein A-I demonstrated that these tyrosine and lysine residues are adjacent on the same face of an amphipathic alpha-helix. Our observations suggest that HOCl selectively targets tyrosine residues that are suitably juxtaposed to primary amino groups in proteins. This mechanism might enable phagocytes to efficiently damage proteins when they destroy microbial proteins during infection or damage host tissue during inflammation.     

Application of liquid chromatography-mass spectrometry to measure the concentrations and study the synthesis of short chain fatty acids following stable isotope infusions

A new method involving zinc sulphate deproteinization was developed to study short chain fatty acids (SCFA) production in the colon and subsequent occurrence of SCFA in blood. SCFA were baseline separated in a 30 min cycle using ion-exclusion chromatography and detected by mass spectrometry. Concentrations could be measured down to 10 microM and isotopomeric distributions could be assessed, enabling the conduction of tracer studies to study changes in SCFA synthesis. The applicability of the method was tested in an extensively characterized pig model yielding portal SCFA concentrations ranging from 70 microM (butyric acid) to 150 microM (propionic acid) to 440 microM (acetic acid) prior to butyrate tracer infusion, reaching butyric acid isotopic steady state within 2 h.