We have investigated the in vitro hamster hepatic microsomal metabolism of the amino-azaheterocyc... more We have investigated the in vitro hamster hepatic microsomal metabolism of the amino-azaheterocycle, 2-amino-1-benzylbenzimidazole (ABB). Three major metabolites were isolated and structurally characterized, using a combination of off-line HPLC, in conjunction with both electron ionization and fast atom bombardment ionization tandem mass spectrometry. ABB was shown to be debenzylated to afford 2-aminobenzimidazole (AB), as well as N- and C-oxidized to give 1-benzyl-N2-hydroxyaminobenzimidazole (BHB) and 2-amino-1-benzyl-hydroxybenzimidazole, respectively. The possible reasons for formation of the exocyclic hydroxylamine BHB are discussed. Furthermore, ABB is proposed as a suitable model compound for investigating parameters that control formation of toxic hydroxylamines derived from amino-azaheterocycles.
Aqueous mixtures of formaldehyde and glutathione react to form a variety of cyclized adducts in a... more Aqueous mixtures of formaldehyde and glutathione react to form a variety of cyclized adducts in addition to S-hydroxymethylglutathione. The adducts are in labile equilibrium with each other and are not readily separated. The structures of two of the other major adducts were determined by concerted application of 13C-1H two-dimensional chemical-shift correlation, fast-atom-bombardment mass spectrometry and tandem mass spectrometry to the adduct mixtures in aqueous solution.
Eosinophils are important effector cells in defense against helminth infection and in allergic di... more Eosinophils are important effector cells in defense against helminth infection and in allergic diseases. To identify novel eosinophil proteins, large scale sequencing of a cDNA library prepared from interleukin-5-stimulated umbilical cord precursor cells was performed, and the major genes expressed by maturing eosinophils were determined. This resulted in the identification of a cDNA with 64% identity to human prepro-major basic protein (hprepro-MBP). This cDNA was designated hprepro-MBP homolog (hprepro-MBPH). Interestingly, the calculated pI values for hMBPH and hMBP differed by >100-fold, with pI values of 8.7 and 11.4, respectively. Given this pronounced basicity difference, the homolog transcript's abundance (1.1%), and MBP's critical role in eosinophil biological activity, we further characterized the homolog. Reverse transcription-polymerase chain reaction detected transcription of hprepro-MBPH in bone marrow only, and this result was confirmed by analysis of a large cDNA data base (electronic Northern). hMBPH was isolated from human eosinophil granule lysates, and its identity was verified by amino acid sequencing and by mass spectrometry. Analyses of the biological activities showed that hMBPH had effects similar to hMBP in cell killing and neutrophil (superoxide anion production and interleukin-8 release) and basophil (histamine and leukotriene C 4 release) stimulation assays, but usually with reduced potency. Overall, this novel homolog's unique physical properties indicated that the high net positive charge of hMBP is important but not essential for biological activity.
Pyrazoloacridine (PZA) is an experimental antitumor agent presently under investigation for treat... more Pyrazoloacridine (PZA) is an experimental antitumor agent presently under investigation for treatment of solid tumors on the basis of its unique mechanism of action and selectivity for human solid tumor xenograft in mice. Using capillary electrophoresis coupled with electrospray ionization mass spectrometry, we have identified three oxidative PZA metabolites, 9-desmethyl-PZA, N-demethyl-PZA, and PZA N-oxide. The cytochrome P450 (CYP) isoforms involved in PZA metabolism were characterized by studies with CYP chemical inhibitors, correlation of marker activities for selected CYPs with formation of the metabolites using a human liver panel, and PZA metabolism by cDNA-expressed CYPs. 9-Desmethyl-PZA formation was catalyzed by CYP1A2, whereas N-demethyl-PZA formation was catalyzed by CYP3A4. PZA N-oxide formation was catalyzed by flavin monooxygenase (FMO) rather than CYP, as determined by studies with chemical inhibitors of FMO and metabolism by cDNA-expressed human flavin monooxygenase...
ABSTRACT. Objective. To determine the chemical structure of a contaminant, X 1 , previously found... more ABSTRACT. Objective. To determine the chemical structure of a contaminant, X 1 , previously found in eosinophilia myalgia syndrome case-implicated 5-hydroxytryptophan (5-OHTrp), and also present in over-thecounter (OTC) commercially available 5-OHTrp. Methods. Case-implicated 5-OHTrp as well as 6 OTC samples were subjected to accurate mass HPLCmass spectrometry and HPLC-electrochemical detection, and reacted with reduced glutathione. Peak X 1 was subsequently subjected to HPLC-tandem mass spectrometry (MS/MS), as well as the resulting nucleophilic glutathione product. All these data were compared with analysis carried out under identical conditions on authentic 4,5-tryptophan-dione (Trp-4,5D). Results. Based on accurate mass, tandem mass spectrometric analysis, and comparision with authentic standard compound analysis, X 1 was determined to be 4,5-tryptophan-dione, a putative neurotoxin. The presence of X 1 in OTC samples varied from 0.5 to 10.3% of the amount of Trp-4,5D present in...
The eosinophilia-myalgia syndrome (EMS) outbreak that occurred in the USA and elsewhere in 1989 w... more The eosinophilia-myalgia syndrome (EMS) outbreak that occurred in the USA and elsewhere in 1989 was caused by the ingestion of Showa Denko K.K. (SD) L-tryptophan (L-Trp). "Six compounds" detected in the L-Trp were reported as case-associated contaminants. Recently the final and most statistically significant contaminant, "Peak AAA" was structurally characterized. The "compound" was actually shown to be two structural isomers resulting from condensation reactions of L-Trp with fatty acids derived from the bacterial cell membrane. They were identified as the indole C-2 anteiso (AAA-343) and linear (AAA-343) aliphatic chain isomers. Based on those findings, we utilized a combination of on-line HPLC-electrospray ionization mass spectrometry (LC-MS), as well as both precursor and product ion tandem mass spectrometry (MS/MS) to facilitate identification of a homologous family of condensation products related to AAA-343 and AAA-343. We structurally characteriz...
The eosinophilia-myalgia syndrome (EMS) outbreak of 1989 that occurred in the USA and elsewhere w... more The eosinophilia-myalgia syndrome (EMS) outbreak of 1989 that occurred in the USA and elsewhere was caused by the ingestion of L-tryptophan (L-Trp) solely manufactured by the Japanese company Showa Denko K.K. (SD). Six compounds present in the SD L-Trp were reported to be case-associated contaminants. However, "one" of these compounds, Peak AAA has remained structurally uncharacterized, despite the fact that it was described as "the only statistically significant (p=0.0014) contaminant". Here, we employ on-line microcapillary-high performance liquid chromatography-electrospray ionization mass spectrometry (LC-MS), and tandem mass spectrometry (MS/MS) to determine that Peak AAA is in fact two structurally related isomers. Peak AAA1 and Peak AAA2 differed in LC retention times, and were determined by accurate mass-LC-MS to both have a protonated molecular ion (MH(+)) of mass 343.239 Daltons (Da), corresponding to a molecular formula of C21H30N2O2, and possessing ei...
ligands involved in positive selection would be low-affinity nonstimulatory ligands (Lo and Spren... more ligands involved in positive selection would be low-affinity nonstimulatory ligands (Lo and Sprent, 1986). Several Department of Pathology and Kaplan Cancer Center 2 Department of Cell Biology reports suggested that subthreshold densities of a stimulatory peptide/MHC ligand were capable of promoting
Alcoholism: Clinical & Experimental Research, 1999
Background The conversion of S-methyl-NJV-diethyldithiocarbamate (MeDDC) to MeDDC sulfine is the ... more Background The conversion of S-methyl-NJV-diethyldithiocarbamate (MeDDC) to MeDDC sulfine is the first step after methylation in the metabolic pathway of disulfiram, an alcohol deterrent, to its ultimate active metabolite. Various isoforms of CYP450 have recently been shown to catalyze this reaction, but the involvement of flavin monooxygenase (FMO) in this metabolism in humans has not been evaluated. In this study we examined the ability of recombinant human F M 0 3 in insect microsomes to metabolize MeDDC, and investigated the relative roles of FMO and CYP450 in the metabolism of MeDDC in human liver microsomes. Methods: HPLC-mass spectrometrywas used to identify the products of MeDDC formed by human liver rnicrosomes and by recombinant human FM03. MeDDC metabolism in human liver microsomes was studied by using either heat inactivation to inhibit FMO, or N-benzylimidazole (NBI) or antibodies to the CYP450 NADPH reductase to inhibit CYP450. Results: We confirmed by HPLC-mass spectrometry that MeDDC sulfine was the major product of MeDDC formed by human liver microsomes and by FM03. Recombinant F M 0 3 was an efficient catalyst for the formation of MeDDC sulfine (5.3 ? 0.2 nmol/min/mg, mean ? SEM, n = 6). Inhibition studies showed MeDDC was metabolized primarily by CYP450 in human liver microsomes at pH 7.4, with a 10% contribution from FMO (total microsomal activity 3.1 2 0.2, n = 17). In the course of this work, methyl p-tolyl sulfide (MTS), sulfoxidation of which is used by some investigators as a specific probe for FMO activity, was found to be a substrate for both FMO and CYP450 in human liver microsomes. Conclusions: Our results prove that MeDDC sulfine is the major product of MeDDC oxidation in human liver microsomes, MeDDC is a good substrate for human FM03, and MeDDC is metabolized in human liver microsomes primarily by CYP450. We also showed that use of MTS sulfoxidation as an indicator of FMO activity in microsomes is valid only in the presence of a CYP450 inhibitor, such as NBI.
Ten reactive metabolites of five polycyclic aromatic hydrocarbons and styrene were investigated t... more Ten reactive metabolites of five polycyclic aromatic hydrocarbons and styrene were investigated to determine the generality of ester adduct formation with human hemoglobin in the form of RBC and hydrolysis to the corresponding tetrahydrotetrols or dihydrodiols. No exceptions were noted among the compounds tested, which included the anti-diol epoxides of benzo[a]pyrene (BaP), chrysene, and benz[a]anthracene; the syn-diol epoxide of BaP; a mixture of syn- and anti-diol epoxides of benzo[e]pyrene; and epoxides of styrene, benzo[e]pyrene, BaP, and cyclopenta[c,d]pyrene. A test of the propensity of the simplest benzylic epoxide, styrene oxide, to form esters that hydrolyze via a BAL1 mechanism was performed. Hydrolysis of styrene oxide-adducted hemoglobin in H2(18)O at neutral pH yielded 18O incorporation results that suggest this mechanism of hydrolysis is operant to a minor degree in styrene oxide-hemoglobin ester adducts. A method was developed for the isolation and quantification of ...
In this work we describe the use of a modular multidimensional chromatography-tandem mass spectro... more In this work we describe the use of a modular multidimensional chromatography-tandem mass spectrometry approach for rapid identification of proteins. In particular we highlight the use of a strong cation exchange cartridge in conjunction with a membrane postconcentration cartridge and nano-HPLC on-line with tandem mass spectrometry to characterize the eosinophil granule organelle proteome. Details are provided of the analytical approach we have developed and we discuss some of the advantages compared with previously reported analyses, as well as providing some specific examples of novel proteins identified.
We are all perplexed that current medical practice often appears maladroit in curing our individu... more We are all perplexed that current medical practice often appears maladroit in curing our individual illnesses or disease. However, as is often the case, a lack of understanding, tools and technologies are the root cause of such situations. Human individuality is an often-quoted term but, in the context of human biology, it is poorly understood. This is compounded when there is a need to consider the variability of human populations. In the case of the former, it is possible to quantify human complexity as determined by the 35,000 genes of the human genome, the 1-10 million proteins (including antibodies) and the 2000-3000 metabolites of the human metabolome. Human variability is much more difficult to assess, since many of the variables, such as the definition of race, are not even clearly agreed on. In order to accommodate human complexity, variability and its influence on health and disease, it is necessary to undertake a systematic approach. In the past decade, the emergence of a...
The human vitamin D receptor (VDR) and retinoid X receptor-(RXR ) modulate gene activity by formi... more The human vitamin D receptor (VDR) and retinoid X receptor-(RXR ) modulate gene activity by forming homodimeric or heterodimeric complexes with specific DNA sequences and interaction with other elements of the transcriptional apparatus in the presence of their known ...
Background: Congenital disorders of glycosylation (CDG) are autosomal recessive disorders that pr... more Background: Congenital disorders of glycosylation (CDG) are autosomal recessive disorders that produce increased serum carbohydrate-deficient transferrin (CDT) isoforms. Methods to resolve CDT from fully glycosylated transferrin (Trf) have been based on a neutral shift in the isoelectric focusing (IEF) pattern or on a reduction in the negative charge, allowing resolution by anion-exchange chromatography. Our purpose was to develop a method of resolution and relative quantification of Trf isoforms using online immunoaffinity liquid chromatography–mass spectrometry (LC-MS). Methods: Serum (25 μL) was diluted with 100 μL of water before application to an immunoaffinity column that sequestered Trf isoforms. Trf isoforms were eluted from the immunoaffinity column, concentrated on a C4 column, eluted from the C4 column, and introduced into the mass spectrometer. Analysis of the Trf isoforms was entirely automated and completed in <10 min per sample. Results: The LC-MS method demonstrat...
The current paradigm of modern healthcare is a reactive response to patient symptoms, subsequent ... more The current paradigm of modern healthcare is a reactive response to patient symptoms, subsequent diagnosis and corresponding treatment of the specific disease(s). This approach is predicated on methodologies first espoused by the Cnidean School of Medicine approximately 2500years ago. More recently escalating healthcare costs and relatively poor disease treatment outcomes have fermented a rethink in how we carry out medical practices. This has led to the emergence of "P-Medicine" in the form of Personalized and Precision Medicine. The terms are used interchangeably, but in fact there are significant differences in the way they are implemented. The former relies on an "N-of-1" model whereas the latter uses a "1-in-N" model. Personalized Medicine is still in a fledgling and evolutionary phase and there has been much debate over its current status and future prospects. A confounding factor has been the sudden development of Precision Medicine, which has cu...
We have investigated the in vitro hamster hepatic microsomal metabolism of the amino-azaheterocyc... more We have investigated the in vitro hamster hepatic microsomal metabolism of the amino-azaheterocycle, 2-amino-1-benzylbenzimidazole (ABB). Three major metabolites were isolated and structurally characterized, using a combination of off-line HPLC, in conjunction with both electron ionization and fast atom bombardment ionization tandem mass spectrometry. ABB was shown to be debenzylated to afford 2-aminobenzimidazole (AB), as well as N- and C-oxidized to give 1-benzyl-N2-hydroxyaminobenzimidazole (BHB) and 2-amino-1-benzyl-hydroxybenzimidazole, respectively. The possible reasons for formation of the exocyclic hydroxylamine BHB are discussed. Furthermore, ABB is proposed as a suitable model compound for investigating parameters that control formation of toxic hydroxylamines derived from amino-azaheterocycles.
Aqueous mixtures of formaldehyde and glutathione react to form a variety of cyclized adducts in a... more Aqueous mixtures of formaldehyde and glutathione react to form a variety of cyclized adducts in addition to S-hydroxymethylglutathione. The adducts are in labile equilibrium with each other and are not readily separated. The structures of two of the other major adducts were determined by concerted application of 13C-1H two-dimensional chemical-shift correlation, fast-atom-bombardment mass spectrometry and tandem mass spectrometry to the adduct mixtures in aqueous solution.
Eosinophils are important effector cells in defense against helminth infection and in allergic di... more Eosinophils are important effector cells in defense against helminth infection and in allergic diseases. To identify novel eosinophil proteins, large scale sequencing of a cDNA library prepared from interleukin-5-stimulated umbilical cord precursor cells was performed, and the major genes expressed by maturing eosinophils were determined. This resulted in the identification of a cDNA with 64% identity to human prepro-major basic protein (hprepro-MBP). This cDNA was designated hprepro-MBP homolog (hprepro-MBPH). Interestingly, the calculated pI values for hMBPH and hMBP differed by >100-fold, with pI values of 8.7 and 11.4, respectively. Given this pronounced basicity difference, the homolog transcript's abundance (1.1%), and MBP's critical role in eosinophil biological activity, we further characterized the homolog. Reverse transcription-polymerase chain reaction detected transcription of hprepro-MBPH in bone marrow only, and this result was confirmed by analysis of a large cDNA data base (electronic Northern). hMBPH was isolated from human eosinophil granule lysates, and its identity was verified by amino acid sequencing and by mass spectrometry. Analyses of the biological activities showed that hMBPH had effects similar to hMBP in cell killing and neutrophil (superoxide anion production and interleukin-8 release) and basophil (histamine and leukotriene C 4 release) stimulation assays, but usually with reduced potency. Overall, this novel homolog's unique physical properties indicated that the high net positive charge of hMBP is important but not essential for biological activity.
Pyrazoloacridine (PZA) is an experimental antitumor agent presently under investigation for treat... more Pyrazoloacridine (PZA) is an experimental antitumor agent presently under investigation for treatment of solid tumors on the basis of its unique mechanism of action and selectivity for human solid tumor xenograft in mice. Using capillary electrophoresis coupled with electrospray ionization mass spectrometry, we have identified three oxidative PZA metabolites, 9-desmethyl-PZA, N-demethyl-PZA, and PZA N-oxide. The cytochrome P450 (CYP) isoforms involved in PZA metabolism were characterized by studies with CYP chemical inhibitors, correlation of marker activities for selected CYPs with formation of the metabolites using a human liver panel, and PZA metabolism by cDNA-expressed CYPs. 9-Desmethyl-PZA formation was catalyzed by CYP1A2, whereas N-demethyl-PZA formation was catalyzed by CYP3A4. PZA N-oxide formation was catalyzed by flavin monooxygenase (FMO) rather than CYP, as determined by studies with chemical inhibitors of FMO and metabolism by cDNA-expressed human flavin monooxygenase...
ABSTRACT. Objective. To determine the chemical structure of a contaminant, X 1 , previously found... more ABSTRACT. Objective. To determine the chemical structure of a contaminant, X 1 , previously found in eosinophilia myalgia syndrome case-implicated 5-hydroxytryptophan (5-OHTrp), and also present in over-thecounter (OTC) commercially available 5-OHTrp. Methods. Case-implicated 5-OHTrp as well as 6 OTC samples were subjected to accurate mass HPLCmass spectrometry and HPLC-electrochemical detection, and reacted with reduced glutathione. Peak X 1 was subsequently subjected to HPLC-tandem mass spectrometry (MS/MS), as well as the resulting nucleophilic glutathione product. All these data were compared with analysis carried out under identical conditions on authentic 4,5-tryptophan-dione (Trp-4,5D). Results. Based on accurate mass, tandem mass spectrometric analysis, and comparision with authentic standard compound analysis, X 1 was determined to be 4,5-tryptophan-dione, a putative neurotoxin. The presence of X 1 in OTC samples varied from 0.5 to 10.3% of the amount of Trp-4,5D present in...
The eosinophilia-myalgia syndrome (EMS) outbreak that occurred in the USA and elsewhere in 1989 w... more The eosinophilia-myalgia syndrome (EMS) outbreak that occurred in the USA and elsewhere in 1989 was caused by the ingestion of Showa Denko K.K. (SD) L-tryptophan (L-Trp). "Six compounds" detected in the L-Trp were reported as case-associated contaminants. Recently the final and most statistically significant contaminant, "Peak AAA" was structurally characterized. The "compound" was actually shown to be two structural isomers resulting from condensation reactions of L-Trp with fatty acids derived from the bacterial cell membrane. They were identified as the indole C-2 anteiso (AAA-343) and linear (AAA-343) aliphatic chain isomers. Based on those findings, we utilized a combination of on-line HPLC-electrospray ionization mass spectrometry (LC-MS), as well as both precursor and product ion tandem mass spectrometry (MS/MS) to facilitate identification of a homologous family of condensation products related to AAA-343 and AAA-343. We structurally characteriz...
The eosinophilia-myalgia syndrome (EMS) outbreak of 1989 that occurred in the USA and elsewhere w... more The eosinophilia-myalgia syndrome (EMS) outbreak of 1989 that occurred in the USA and elsewhere was caused by the ingestion of L-tryptophan (L-Trp) solely manufactured by the Japanese company Showa Denko K.K. (SD). Six compounds present in the SD L-Trp were reported to be case-associated contaminants. However, "one" of these compounds, Peak AAA has remained structurally uncharacterized, despite the fact that it was described as "the only statistically significant (p=0.0014) contaminant". Here, we employ on-line microcapillary-high performance liquid chromatography-electrospray ionization mass spectrometry (LC-MS), and tandem mass spectrometry (MS/MS) to determine that Peak AAA is in fact two structurally related isomers. Peak AAA1 and Peak AAA2 differed in LC retention times, and were determined by accurate mass-LC-MS to both have a protonated molecular ion (MH(+)) of mass 343.239 Daltons (Da), corresponding to a molecular formula of C21H30N2O2, and possessing ei...
ligands involved in positive selection would be low-affinity nonstimulatory ligands (Lo and Spren... more ligands involved in positive selection would be low-affinity nonstimulatory ligands (Lo and Sprent, 1986). Several Department of Pathology and Kaplan Cancer Center 2 Department of Cell Biology reports suggested that subthreshold densities of a stimulatory peptide/MHC ligand were capable of promoting
Alcoholism: Clinical & Experimental Research, 1999
Background The conversion of S-methyl-NJV-diethyldithiocarbamate (MeDDC) to MeDDC sulfine is the ... more Background The conversion of S-methyl-NJV-diethyldithiocarbamate (MeDDC) to MeDDC sulfine is the first step after methylation in the metabolic pathway of disulfiram, an alcohol deterrent, to its ultimate active metabolite. Various isoforms of CYP450 have recently been shown to catalyze this reaction, but the involvement of flavin monooxygenase (FMO) in this metabolism in humans has not been evaluated. In this study we examined the ability of recombinant human F M 0 3 in insect microsomes to metabolize MeDDC, and investigated the relative roles of FMO and CYP450 in the metabolism of MeDDC in human liver microsomes. Methods: HPLC-mass spectrometrywas used to identify the products of MeDDC formed by human liver rnicrosomes and by recombinant human FM03. MeDDC metabolism in human liver microsomes was studied by using either heat inactivation to inhibit FMO, or N-benzylimidazole (NBI) or antibodies to the CYP450 NADPH reductase to inhibit CYP450. Results: We confirmed by HPLC-mass spectrometry that MeDDC sulfine was the major product of MeDDC formed by human liver microsomes and by FM03. Recombinant F M 0 3 was an efficient catalyst for the formation of MeDDC sulfine (5.3 ? 0.2 nmol/min/mg, mean ? SEM, n = 6). Inhibition studies showed MeDDC was metabolized primarily by CYP450 in human liver microsomes at pH 7.4, with a 10% contribution from FMO (total microsomal activity 3.1 2 0.2, n = 17). In the course of this work, methyl p-tolyl sulfide (MTS), sulfoxidation of which is used by some investigators as a specific probe for FMO activity, was found to be a substrate for both FMO and CYP450 in human liver microsomes. Conclusions: Our results prove that MeDDC sulfine is the major product of MeDDC oxidation in human liver microsomes, MeDDC is a good substrate for human FM03, and MeDDC is metabolized in human liver microsomes primarily by CYP450. We also showed that use of MTS sulfoxidation as an indicator of FMO activity in microsomes is valid only in the presence of a CYP450 inhibitor, such as NBI.
Ten reactive metabolites of five polycyclic aromatic hydrocarbons and styrene were investigated t... more Ten reactive metabolites of five polycyclic aromatic hydrocarbons and styrene were investigated to determine the generality of ester adduct formation with human hemoglobin in the form of RBC and hydrolysis to the corresponding tetrahydrotetrols or dihydrodiols. No exceptions were noted among the compounds tested, which included the anti-diol epoxides of benzo[a]pyrene (BaP), chrysene, and benz[a]anthracene; the syn-diol epoxide of BaP; a mixture of syn- and anti-diol epoxides of benzo[e]pyrene; and epoxides of styrene, benzo[e]pyrene, BaP, and cyclopenta[c,d]pyrene. A test of the propensity of the simplest benzylic epoxide, styrene oxide, to form esters that hydrolyze via a BAL1 mechanism was performed. Hydrolysis of styrene oxide-adducted hemoglobin in H2(18)O at neutral pH yielded 18O incorporation results that suggest this mechanism of hydrolysis is operant to a minor degree in styrene oxide-hemoglobin ester adducts. A method was developed for the isolation and quantification of ...
In this work we describe the use of a modular multidimensional chromatography-tandem mass spectro... more In this work we describe the use of a modular multidimensional chromatography-tandem mass spectrometry approach for rapid identification of proteins. In particular we highlight the use of a strong cation exchange cartridge in conjunction with a membrane postconcentration cartridge and nano-HPLC on-line with tandem mass spectrometry to characterize the eosinophil granule organelle proteome. Details are provided of the analytical approach we have developed and we discuss some of the advantages compared with previously reported analyses, as well as providing some specific examples of novel proteins identified.
We are all perplexed that current medical practice often appears maladroit in curing our individu... more We are all perplexed that current medical practice often appears maladroit in curing our individual illnesses or disease. However, as is often the case, a lack of understanding, tools and technologies are the root cause of such situations. Human individuality is an often-quoted term but, in the context of human biology, it is poorly understood. This is compounded when there is a need to consider the variability of human populations. In the case of the former, it is possible to quantify human complexity as determined by the 35,000 genes of the human genome, the 1-10 million proteins (including antibodies) and the 2000-3000 metabolites of the human metabolome. Human variability is much more difficult to assess, since many of the variables, such as the definition of race, are not even clearly agreed on. In order to accommodate human complexity, variability and its influence on health and disease, it is necessary to undertake a systematic approach. In the past decade, the emergence of a...
The human vitamin D receptor (VDR) and retinoid X receptor-(RXR ) modulate gene activity by formi... more The human vitamin D receptor (VDR) and retinoid X receptor-(RXR ) modulate gene activity by forming homodimeric or heterodimeric complexes with specific DNA sequences and interaction with other elements of the transcriptional apparatus in the presence of their known ...
Background: Congenital disorders of glycosylation (CDG) are autosomal recessive disorders that pr... more Background: Congenital disorders of glycosylation (CDG) are autosomal recessive disorders that produce increased serum carbohydrate-deficient transferrin (CDT) isoforms. Methods to resolve CDT from fully glycosylated transferrin (Trf) have been based on a neutral shift in the isoelectric focusing (IEF) pattern or on a reduction in the negative charge, allowing resolution by anion-exchange chromatography. Our purpose was to develop a method of resolution and relative quantification of Trf isoforms using online immunoaffinity liquid chromatography–mass spectrometry (LC-MS). Methods: Serum (25 μL) was diluted with 100 μL of water before application to an immunoaffinity column that sequestered Trf isoforms. Trf isoforms were eluted from the immunoaffinity column, concentrated on a C4 column, eluted from the C4 column, and introduced into the mass spectrometer. Analysis of the Trf isoforms was entirely automated and completed in <10 min per sample. Results: The LC-MS method demonstrat...
The current paradigm of modern healthcare is a reactive response to patient symptoms, subsequent ... more The current paradigm of modern healthcare is a reactive response to patient symptoms, subsequent diagnosis and corresponding treatment of the specific disease(s). This approach is predicated on methodologies first espoused by the Cnidean School of Medicine approximately 2500years ago. More recently escalating healthcare costs and relatively poor disease treatment outcomes have fermented a rethink in how we carry out medical practices. This has led to the emergence of "P-Medicine" in the form of Personalized and Precision Medicine. The terms are used interchangeably, but in fact there are significant differences in the way they are implemented. The former relies on an "N-of-1" model whereas the latter uses a "1-in-N" model. Personalized Medicine is still in a fledgling and evolutionary phase and there has been much debate over its current status and future prospects. A confounding factor has been the sudden development of Precision Medicine, which has cu...
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