Simultaneous quantification of four hormone therapy drugs by LC-MS/MS: Clinical applications in breast cancer patients.

INTRODUCTION: Aromatase inhibitors such as anastrozole, letrozole, exemestane and selective estrogen down-regulator (SERD) fulvestrant are used mostly to treat breast cancer estrogen receptor positive in post-menopausal women. These drugs are given either through the oral route or by intramuscular injection. They have shown great inter-individual variability with a risk of cardiometabolic disorders. Hence the importance of their therapeutic drug monitoring not only for exposure-efficacy but also exposure-toxicity. We describe here a LC-MS/MS method for the simultaneous quantification of anastrozole, letrozole, exemestane and fulvestrant in human plasma. MATERIAL AND METHODS: Plasma samples were prepared by a single-step protein precipitation. The liquid chromatography system was paired with a triple quadrupole mass spectrometer. Quantification were achieved in Multiple Reactions Monitoring mode and the electrospray ionization was in positive mode. RESULTS: The method demonstrated consistent analytical performance across various parameters, including linearity, specificity, sensitivity, matrix effect, upper and lower limits of quantification, extraction recovery, precision, accuracy, hemolysis effect, dilution integrity, and stability under different storage conditions, in accordance with established guidelines. The analysis time for each run was 4 min. Calibration curves exhibited linearity within the 1-100 ng/mL range, with correlation coefficients > 0.99 for the four analytes. Plasma concentrations from 42 patients were integrated into the selected calibration. Stability assessments indicated that the four drugs remained stable at - 20 °C for three months, 15 days under refrigeration, up to 7 days at room temperature, and after three freeze-thaw cycles. CONCLUSION: We have developed and validated this quantitative method for therapeutic drug monitoring of those four hormone therapy drugs:anastrozole, letrozole, fulvestrant and exemestane. This method can be also used for future clinical pharmacokinetics /pharmacodynamics studies.

Determination of plasma concentration of Belimumab by LC-MS/MS: Method development, validation, and clinical application.

INTRODUCTION: Belimumab is a monoclonal antibody against B cell activating factor (BLyS). This monoclonal antibody (mAb) has been shown to be effective in reducing disease activity in patients with systemic lupus erythematosus (SLE). Belimumab is available in two forms as a lyophilized powder for intravenous (IV) use, or single-dose syringe for subcutaneous (SC) use. The aim of this study was to develop and validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantitation of belimumab in human serum. MATERIAL AND METHODS: All analyses relied on nano-surface and molecular-orientation limited (nSMOL) proteolysis coupled with LC-MS/MS. Quantifications was performed in multiple reactions monitoring (MRM) mode, and electrospray ionization was conducted in positive mode. RESULTS: Belimumab was quantified with signature peptide QAPGQGLEWMGGIPFGTAK and normalized using P14R. The total run time per assay was 10 min. Linearity was measured from 5 to 800 µg/mL (r² > 0.995). Accuracy and precision based on three quality control levels range from 11.2 - 9.51 % and 1.24 - 13.12 % respectively. The carryover was less than 7 %. In all, 87 patient samples were processed (65, IV; 22, SC). Mean concentration of belimumab was significantly higher for SC (93.0 ± 74.0 µg/mL) than for IV (67.4 ± 38.9 µg/mL) administration. CONCLUSION: We have developed the first method of belimumab quantification combining LC-MS/MS and nSMOL proteolysis. It can be used for future clinical pharmacokinetic studies of belimumab and for investigating the relationship between belimumab concentration, efficacy, and toxicity in SLE patients.

Plasma lysosphingolipids in GRN-related diseases: Monitoring lysosomal dysfunction to track disease progression.

GRN mutations are among the main genetic causes of frontotemporal dementia (FTD). Considering the progranulin involvement in lysosomal homeostasis, we aimed to evaluate if plasma lysosphingolipids (lysoSPL) are increased in GRN mutation carriers, and whether they might represent relevant fluid-based biomarkers in GRN-related diseases. We analyzed four lysoSPL levels in plasmas of 131 GRN carriers and 142 non-carriers, including healthy controls and patients with frontotemporal dementias (FTD) carrying a C9orf72 expansion or without any mutation. GRN carriers consisted of 102 heterozygous FTD patients (FTD-GRN), three homozygous patients with neuronal ceroid lipofuscinosis-11 (CLN-11) and 26 presymptomatic carriers (PS-GRN), the latter with longitudinal assessments. Glucosylsphingosin d18:1 (LGL1), lysosphingomyelins d18:1 and isoform 509 (LSM18:1, LSM509) and lysoglobotriaosylceramide (LGB3) were measured by electrospray ionization-tandem mass spectrometry coupled to ultraperformance liquid chromatography. Levels of LGL1, LSM18:1 and LSM509 were increased in GRN carriers compared to non-carriers (p < 0.0001). No lysoSPL increases were detected in FTD patients without GRN mutations. LGL1 and LSM18:1 progressively increased with age at sampling, and LGL1 with disease duration, in FTD-GRN. Among PS-GRN carriers, LSM18:1 and LGL1 significantly increased over 3.4-year follow-up. LGL1 levels were associated with increasing neurofilaments in presymptomatic carriers. This study evidences an age-dependent increase of ß-glucocerebrosidase and acid sphingomyelinase substrates in GRN patients, with progressive changes as early as the presymptomatic phase. Among FTD patients, plasma lysoSPL appear to be uniquely elevated in GRN carriers, and thus might serve as suitable non-invasive disease-tracking biomarkers of progression, specific to the pathophysiological process. Finally, this study might add lysoSPL to the portfolio of fluid-based biomarkers, and pave the way to disease-modifying approaches based on lysosomal function rescue in GRN diseases.

Plasma GM2 ganglioside potential biomarker for diagnosis, prognosis and disease monitoring of GM2-Gangliosidosis.

GM2-Gangliosidosis are a group of inherited lysosomal storage pathologies characterized by a large accumulation of GM2 ganglioside in the lysosome. They are caused by mutation in HEXA or HEXB causing reduced or absent activity of a lysosomal ß-hexosaminidase A, or mutation in GM2A causing defect in GM2 activator protein (GM2AP), an essential protein for the activity of the enzyme. Biochemical diagnosis relies on the measurement of ß-hexosaminidases A and B activities, which is able to detect lysosomal enzyme deficiency but fails to identify defects in GM2AP. We developed a rapid, specific and sensitive liquid chromatography-mass spectrometry-based method to measure simultaneously GM1, GM2, GM3 and GD3 molecular species. Gangliosides were analysed in plasma from 19 patients with GM2-Gangliosidosis: Tay-Sachs (n = 9), Sandhoff (n = 9) and AB variant of GM2-Gangliosidosis (n = 1) and compared to 20 age-matched controls. Among patients, 12 have a late adult-juvenile-onset and 7 have an infantile early-onset of the disease. Plasma GM2 molecular species were increased in all GM2-Gangliosidosis patients (19/19), including the patient with GM2A mutation, compared to control individuals and compared to patients with different other lysosomal storage diseases. GM234:1 and GM234:1/GM334:1 ratio discriminated patients from controls with 100% sensitivity and specificity. GM234:1 and GM234:1/GM334:1 were higher in patients with early-onset compared to those with late-onset of the disease, suggesting a relationship with severity. Longitudinal analysis in one adult with Tay-Sachs disease over 9 years showed a positive correlation of GM234:1 and GM234:1/GM334:1 ratio with age at sampling. We propose that plasma GM2 34:1 and its ratio to GM3 34:1 could be sensitive and specific biochemical diagnostic biomarkers for GM2-Gangliosidosis including AB variant and could be useful as a first line diagnostic test and potential biomarkers for monitoring upcoming therapeutic efficacy.

Efficacy and pharmacokinetics of betaine in CBS and cblC deficiencies: a cross-over randomized controlled trial.

BACKGROUND: Betaine is an "alternate" methyl donor for homocysteine remethylation catalyzed by betaine homocysteine methyltransferase (BHMT), an enzyme mainly expressed in the liver and kidney. Betaine has been used for more than 30 years in pyridoxine non-responsive cystathionine beta-synthase (pnrCBS) and cobalamin C (cblC) deficiencies to lower the hyperhomocysteinemia, although little is known about the optimal therapeutic dosage and its pharmacokinetic in these patients. AIMS: We compared 2 betaine doses (100 mg/kg/day vs. 250 mg/kg/day) in children affected by pnrCBS or cblC deficiencies. We also measured the pharmacokinetics parameters after a single dose of betaine (100 or 250 mg/kg) in these patients. METHODS: We conducted a prospective, randomized, crossover clinical trial with blinded evaluation. The primary outcome was the equivalence of total plasma homocysteine (tHcy) concentrations upon one-month oral treatment with betaine at 100 versus 250 mg/kg/day. RESULTS: Eleven patients completed the study (5 pnrCBS and 6 cblC). tHcy concentrations were equivalent after a one-month treatment period for the two betaine dosages. Multivariate analysis showed a significant effect of betaine dose on methionine (Met) (p = 0.01) and S-adenosylmethionine (SAM) concentrations (p = 0.006). CONCLUSIONS: Our analysis shows that there is no overt benefit to increasing betaine dosage higher than 100 mg/kg/day to lower tHcy concentrations in pnrCBS and cblC deficiencies. However, increasing betaine up to 250 mg/kg/d could benefit cblC patients through the increase of methionine and SAM concentrations, as low Met and SAM concentrations are involved in the pathophysiology of this disease. In contrast, in pnrCBS deficiency, betaine doses higher than 100 mg/kg/day could be harmful to these patients with pre-existing hypermethioninemia. TRIAL REGISTRATION: Clinical Trials, NCT02404337. Registered 23 May 2015-prospectively registered, https://clinicaltrials.gov .

Simple and accurate quantitative analysis of cefiderocol and ceftobiprole in human plasma using liquid chromatography-isotope dilution tandem mass spectrometry: interest for their therapeutic drug monitoring and pharmacokinetic studies.

OBJECTIVES: Cefiderocol and ceftobiprole are new generation cephalosporin antibiotics that exhibit high inter-individual plasma concentration variability that potentially impact their efficacy or toxicity. The aim of this study was to develop and validate a selective, simple, and fast UPLC-MS/MS method for simultaneous quantification of cefiderocol and ceftobiprole in human plasma to enable their therapeutic drug monitoring (TDM) and support PK and PK/PD studies, in particular in critically ill patients. METHODS: After a simple and fast single-step protein precipitation, cefiderocol and ceftobiprole were separated on a Waters Acquity UPLC BEH C18 column by linear gradient elution; with subsequent detection by Shimadzu MS 8060 triple quadrupole tandem mass spectrometer in a positive ionization mode. RESULTS: Analysis time was 5 min per run. The analytical performance of the method in terms of specificity, sensitivity, linearity, precision, accuracy, matrix effect (ME), extraction recovery (ER), limit of quantification, dilution integrity, and stability of analytes under different conditions met all criteria for a bioanalytical method for the quantification of drugs. The calibration curves were linear over the range of 1-200 mg/L for cefiderocol and 0.5-100 mg/L for ceftobiprole with a linear regression coefficient above 0.995 for both. CONCLUSIONS: A simple, fast, and selective liquid chroma-tography-tandem mass spectrometry method was developed and validated for the simultaneous quantification of cefiderocol and ceftobiprole. This new method was successfully applied to the measurement of plasma concentration of cefiderocol and ceftobiprole in critically ill patients and showed good performance for their therapeutic monitoring and optimizing antibiotic therapy.

ShiF acts as an auxiliary factor of aerobactin secretion in meningitis Escherichia coli strain S88.

BACKGROUND: The neonatal meningitis E. coli (NMEC) strain S88 carries a ColV plasmid named pS88 which is involved in meningeal virulence. Transcriptional analysis of pS88 in human serum revealed a strong upregulation of an ORF of unknown function: shiF, which is adjacent to the operon encoding the siderophore aerobactin. The aim of this work is to investigate the role of shiF in aerobactin production in strain S88. RESULTS: Study of the prevalence of shiF and aerobactin operon in a collection of 100 extra-intestinal pathogenic E. coli strains (ExPEC) and 50 whole genome-sequenced E. coli strains revealed the colocalization of these two genes for 98% of the aerobactin positive strains. We used Datsenko and Wanner's method to delete shiF in two S88 mutants. A cross-feeding assay showed that these mutants were able to excrete aerobactin meaning that shiF is dispensable for aerobactin excretion. Our growth assays revealed that the shiF-deleted mutants grew significantly slower than the wild-type strain S88 in iron-depleted medium with a decrease of maximum growth rates of 23 and 28% (p < 0.05). Using Liquid Chromatography-Mass Spectrometry, we identified and quantified siderophores in the supernatants of S88 and its shiF deleted mutants after growth in iron-depleted medium and found that these mutants secreted significantly less aerobactin than S88 (- 52% and - 49%, p < 0.001). CONCLUSIONS: ShiF is physically and functionally linked to aerobactin. It provides an advantage to E. coli S88 under iron-limiting conditions by increasing aerobactin secretion and may thus act as an auxiliary virulence factor.

A new AMPK activator, GSK773, corrects fatty acid oxidation and differentiation defect in CPT2-deficient myotubes.

Carnitine palmitoyl transferase 2 (CPT2) deficiency is one of the most common inherited fatty acid oxidation (FAO) defects and represents a prototypical mitochondrial metabolic myopathy. Recent studies have suggested a pivotal role of adenosine monophosphate-activated protein kinase (AMPK) in skeletal muscle plasticity and mitochondrial homeostasis. Thus, we tested the potential of GSK773, a novel direct AMPK activator, to improve or correct FAO capacities in muscle cells from patients harboring various mutations. We used controls' and patients' myotubes and studied the parameters of FAO metabolism, of mitochondrial quantity and quality and of differentiation. We found that AMPK is constitutively activated in patients' myotubes, which exhibit both reduced FAO and impaired differentiation. GSK773 improves or corrects several metabolic hallmarks of CPT2 deficiency (deficient FAO flux and C16-acylcarnitine accumulation) by upregulating the expression of CPT2 protein. Beneficial effects of GSK773 are also likely due to stimulation of mitochondrial biogenesis and induction of mitochondrial fusion, by decreasing dynamin-related protein 1 and increasing mitofusin 2. GSK773 also induces a shift in myosin heavy chain isoforms toward the slow oxidative type and, therefore, fully corrects the differentiation process. We establish, through small interfering RNA knockdowns and pharmacological approaches, that these GSK773 effects are mediated through peroxisome proliferator-activated receptor gamma co-activator 1-alpha, reactive oxygen species and p38 mitogen-activated protein kinase, all key players of skeletal muscle plasticity. GSK773 recapitulates several important features of skeletal muscle adaptation to exercise. The results show that AMPK activation by GSK773 evokes the slow, oxidative myogenic program and triggers beneficial phenotypic adaptations in FAO-deficient myotubes. Thus, GSK773 might have therapeutic potential for correction of CPT2 deficiency.

Mitochondrial trifunctional protein deficiency in human cultured fibroblasts: effects of bezafibrate.

Mitochondrial trifunctional protein (MTP) deficiency caused by HADHA or HADHB gene mutations exhibits substantial molecular, biochemical, and clinical heterogeneity and ranks among the more severe fatty acid oxidation (FAO) disorders, without pharmacological treatment. Since bezafibrate has been shown to potentially correct other FAO disorders in patient cells, we analyzed its effects in 26 MTP-deficient patient fibroblasts representing 16 genotypes. Overall, the patient cell lines exhibited variable, complex, biochemical profiles and pharmacological responses. HADHA-deficient fibroblasts showed markedly reduced alpha subunit protein levels together with decreased beta-subunit abundance, exhibited a -86 to -96% defect in LCHAD activity, and produced large amounts of C14 and C16 hydroxyacylcarnitines. In control fibroblasts, exposure to bezafibrate (400 µM for 48 h) increased the abundance of HADHA and HADHB mRNAs, immune-detectable alpha and beta subunit proteins, activities of LCHAD and LCKAT, and stimulated FAO capacities, clearly indicating that MTP is pharmacologically up-regulated by bezafibrate in human fibroblasts. In MTP-deficient patient fibroblasts, which were found markedly FAO-deficient, bezafibrate improved FAO capacities in six of 26 (23%) cases, including three cell lines heterozygous for the common c1528G > C mutation. Altogether, our results strongly suggest that, due to variable effects of HADHA and HADHB mutations on MTP abundance and residual activity, improvement of MTP deficiency in response to bezafibrate was achieved in a subset of responsive genotypes.

Stilbenes and resveratrol metabolites improve mitochondrial fatty acid oxidation defects in human fibroblasts.

BACKGROUND: Inborn enzyme defects of mitochondrial fatty acid beta-oxidation (FAO) form a large group of genetic disorders associated to variable clinical presentations ranging from life-threatening pediatric manifestations up to milder late onset phenotypes, including myopathy. Very few candidate drugs have been identified in this group of disorders. Resveratrol (RSV) is a natural polyphenol with anti-oxidant and anti-inflammatory effects, recently shown to have beneficial metabolic properties in mice models. Our study explores its possible effects on FAO and mitochondrial energy metabolism in human cells, which are still very little documented. METHODS: Using cells from controls and from patients with Carnitine Palmitoyl Transferase 2 (CPT2) or Very Long Chain AcylCoA Dehydrogenase (VLCAD) deficiency we characterized the metabolic effects of RSV, RSV metabolites, and other stilbenes. We also focused on analysis of RSV uptake, and on the effects of low RSV concentrations, considering the limited bioavailability of RSV in vivo. RESULTS: Time course of RSV accumulation in fibroblasts over 48 h of treatment were consistent with the resulting stimulation or correction of FAO capacities. At 48 h, half maximal and maximal FAO stimulations were respectively achieved for 37,5 microM (EC50) and 75 microM RSV, but we found that serum content of culture medium negatively modulated RSV uptake and FAO induction. Indeed, decreasing serum from 12% to 3% led to shift EC50 from 37,5 to 13 microM, and a 2.6-3.6-fold FAO stimulation was reached with 20 microM RSV at 3% serum, that was absent at 12% serum. Two other stilbenes often found associated with RSV, i.e. cis- RSV and piceid, also triggered significant FAO up-regulation. Resveratrol glucuro- or sulfo- conjugates had modest or no effects. In contrast, dihydro-RSV, one of the most abundant circulating RSV metabolites in human significantly stimulated FAO (1.3-2.3-fold). CONCLUSIONS: This study provides the first compared data on mitochondrial effects of resveratrol, its metabolites, and other natural compounds of the stilbene family in human cells. The results clearly indicate that several of these compounds can improve mitochondrial FAO capacities in human FAO-deficient cells.

The ssbL gene harbored by the ColV plasmid of an Escherichia coli neonatal meningitis strain is an auxiliary virulence factor boosting the production of siderophores through the shikimate pathway.

The ability to capture iron is a challenge for most bacteria. The neonatal meningitis Escherichia coli strain S88 possesses several iron uptake systems, notably including siderophores. Transcriptional analysis of the ColV plasmid pS88 has shown strong induction of a previously undescribed gene with low identity to three E. coli chromosomal genes encoding phospho-2-dehydro-3-deoxyheptonate aldolases involved in aromatic amino acid and catecholate/phenolate siderophore biosynthesis through the shikimate pathway. Here, we investigated the role of this gene, ssbLp (ssbL carried on the plasmid), in siderophore biosynthesis and, consequently, in S88 virulence. We constructed an S88 mutant designated S88 ΔssbLp, which exhibited reduced growth under low-iron conditions compared to the wild-type strain. Liquid chromatography-mass spectroscopy analysis of culture supernatants showed that the mutant secreted significantly smaller amounts of enterobactin, salmochelin SX, and yersiniabactin than the wild-type strain. The mutant was also less virulent in a neonatal rat sepsis model, with significantly lower bacteremia and mortality. Supplementation with chorismate, the final product of the shikimate pathway, restored the wild-type phenotype in vitro. In a collection of human extraintestinal E. coli isolates, we found that ssbL was present only in strains harboring the iro locus, encoding salmochelins, and was located either on the chromosome or on plasmids. Acquisition of the iro locus has been accompanied by acquisition of the auxiliary gene ssbL, which boosts the metabolic pathway essential for catecholate/phenolate siderophore biosynthesis and could represent potential therapeutic targets.

Methylation metabolites in amniotic fluid depend on gestational age.

OBJECTIVE: Methylation metabolism is essential for fetus development. However, normative data for amniotic fluid (AF) concentrations of methylation metabolites at different gestational ages are lacking. We aimed to determine in AF reference values of 14 intermediates involved in methylation. METHODS: Two hundred sixty-eight AFs sampled between 14 and 39 weeks of gestation were retrospectively selected in our AF bank. Next, we measured methionine (Met)-cycle intermediates [S-adenosyl Met (AdoMet), S-adenosyl-l-homocysteine (AdoHcy), total Hcy, Met, and methyl malonic acid] and methyl donors and methyl acceptors (betaine, dimethylglycine, sarcosine, free and total choline, free and total ethanolamine, creatine, and guanidinoacetate) by liquid chromatography coupled with tandem mass spectrometry. RESULTS: Reference ranges according to gestational age were determined for each parameter. Strong correlations between metabolites directly connected in their metabolic pathway and between total Hcy and betaine were observed. CONCLUSION: Methionine, an essential amino acid required for protein synthesis, is the only parameter that dramatically decreases with gestational age. The AdoMet/AdoHcy ratio exponentially increases from 25 weeks of gestation, which could reflect increasing methylation capacities. The negative correlation between betaine and total Hcy together with a constant betaine to dimethylglycine ratio during gestation suggests that betaine may be used as a methyl donor during fetal life.

Mouse studies to shape clinical trials for mitochondrial diseases: high fat diet in Harlequin mice.

BACKGROUND: Therapeutic options in human mitochondrial oxidative phosphorylation (OXPHOS) diseases have been poorly evaluated mostly because of the scarcity of cohorts and the inter-individual variability of disease progression. Thus, while a high fat diet (HFD) is often recommended, data regarding efficacy are limited. Our objectives were 1) to determine our ability to evaluate therapeutic options in the Harlequin OXPHOS complex I (CI)-deficient mice, in the context of a mitochondrial disease with human hallmarks and 2) to assess the effects of a HFD. METHODS AND FINDINGS: Before launching long and expensive animal studies, we showed that palmitate afforded long-term death-protection in 3 CI-mutant human fibroblasts cell lines. We next demonstrated that using the Harlequin mouse, it was possible to draw solid conclusions on the efficacy of a 5-month-HFD on neurodegenerative symptoms. Moreover, we could identify a group of highly responsive animals, echoing the high variability of the disease progression in Harlequin mice. CONCLUSIONS: These results suggest that a reduced number of patients with identical genetic disease should be sufficient to reach firm conclusions as far as the potential existence of responders and non responders is recognized. They also positively prefigure HFD-trials in OXPHOS-deficient patients.

Measurement of free and total sialic acid by isotopic dilution liquid chromatography tandem mass spectrometry method.

The measurement of urine sialic acid (N Acetylneuraminic Acid: Neu5Ac) is useful for screening sialic acid storage disorders. We developed a new LC MS/MS method for the determination of a sialic acid. Urine samples were analyzed, after an HCl n-Butanol derivatization step, by a reverse phase based high-performance liquid chromatography method using 1,2,3-(13)C(3) N-Acetyl-D-neuraminic Acid ((13)C-Neu5Ac) as an internal standard. Selective detection was performed by tandem mass spectrometry using an electrospray source operating in positive ionization mode employing multiple reactions monitoring to monitor N-Acetylneuraminic Acid and the internal standard. The transitions m/z 366→330 and 369→333 for Neu5Ac and (13)C-Neu5Ac were respectively monitored. The limit of the method quantification was 1.40 µM of N-Acetylneuraminic Acid and the calibration curve showed a good linearity up to 1000 µM. The within assay precision and accuracy of the method ranged from 3.22 to 5.95% and 98.69 to 109.18%, respectively and the between assay precision and accuracy ranged, respectively, from 5.15 to 7.65% and 96.14 to 102.30%. The method can be applied for the determination of N-Acetylneuraminic Acid concentrations in urine and other biological fluids (e.g., amniotic and peritoneal fluids).

CABC1 gene mutations cause ubiquinone deficiency with cerebellar ataxia and seizures.

Coenzyme Q(10) (CoQ(10)) plays a pivotal role in oxidative phosphorylation (OXPHOS) in that it distributes electrons between the various dehydrogenases and the cytochrome segments of the respiratory chain. Primary coenzyme Q(10) deficiency represents a clinically heterogeneous condition suggestive of genetic heterogeneity, and several disease genes have been previously identified. The CABC1 gene, also called COQ8 or ADCK3, is the human homolog of the yeast ABC1/COQ8 gene, one of the numerous genes involved in the ubiquinone biosynthesis pathway. The exact function of the Abc1/Coq8 protein is as yet unknown, but this protein is classified as a putative protein kinase. We report here CABC1 gene mutations in four ubiquinone-deficient patients in three distinct families. These patients presented a similar progressive neurological disorder with cerebellar atrophy and seizures. In all cases, enzymological studies pointed to ubiquinone deficiency. CoQ(10) deficiency was confirmed by decreased content of ubiquinone in muscle. Various missense mutations (R213W, G272V, G272D, and E551K) modifying highly conserved amino acids of the protein and a 1 bp frameshift insertion c.[1812_1813insG] were identified. The missense mutations were introduced into the yeast ABC1/COQ8 gene and expressed in a Saccharomyces cerevisiae strain in which the ABC1/COQ8 gene was deleted. All the missense mutations resulted in a respiratory phenotype with no or decreased growth on glycerol medium and a severe reduction in ubiquinone synthesis, demonstrating that these mutations alter the protein function.

Prenyldiphosphate synthase, subunit 1 (PDSS1) and OH-benzoate polyprenyltransferase (COQ2) mutations in ubiquinone deficiency and oxidative phosphorylation disorders.

Coenzyme Q10 (CoQ10) plays a pivotal role in oxidative phosphorylation (OXPHOS), as it distributes electrons among the various dehydrogenases and the cytochrome segments of the respiratory chain. We have identified 2 novel inborn errors of CoQ10 biosynthesis in 2 distinct families. In both cases, enzymologic studies showed that quinone-dependent OXPHOS activities were in the range of the lowest control values, while OXPHOS enzyme activities were normal. CoQ10 deficiency was confirmed by restoration of normal OXPHOS activities after addition of quinone. A genome-wide search for homozygosity in family 1 identified a region of chromosome 10 encompassing the gene prenyldiphosphate synthase, subunit 1 (PDSS1), which encodes the human ortholog of the yeast COQ1 gene, a key enzyme of CoQ10 synthesis. Sequencing of PDSS1 identified a homozygous nucleotide substitution modifying a conserved amino acid of the protein (D308E). In the second family, direct sequencing of OH-benzoate polyprenyltransferase (COQ2), the human ortholog of the yeast COQ2 gene, identified a single base pair frameshift deletion resulting in a premature stop codon (c.1198delT, N401fsX415). Transformation of yeast Deltacoq1 and Deltacoq2 strains by mutant yeast COQ1 and mutant human COQ2 genes, respectively, resulted in defective growth on respiratory medium, indicating that these mutations are indeed the cause of OXPHOS deficiency.

Monolysocardiolipins accumulate in Barth syndrome but do not lead to enhanced apoptosis.

Barth syndrome (BTHS) is an X-linked recessive disorder that is biochemically characterized by low cellular levels of the mitochondrial phospholipid cardiolipin (CL). Previously, we discovered that the yeast disruptant of the TAZ ortholog in Saccharomyces cerevisiae not only displays CL deficiency but also accumulates monolysocardiolipins (MLCLs), which are intermediates in CL remodeling. Therefore, we set out to investigate whether MLCL accumulation also occurs in BTHS. Indeed, we observed MLCL accumulation in heart, muscle, lymphocytes, and cultured lymphoblasts of BTHS patients; however, only very low levels of these lysophospholipids were found in platelets and fibroblasts of these patients. Although the fatty acid composition of the MLCLs was different depending on the tissue source, it did parallel the fatty acid composition of the (remaining) CLs. The possible implications of these findings for the two reported CL remodeling mechanisms, transacylation and deacylation/reacylation, are discussed. Because MLCLs have been proposed to be involved in the initiation of apoptosome-mediated cell death by the sequestration of the proapoptotic protein (t)BH3-interacting domain death agonist (Bid) to the mitochondrial membrane, we used control and BTHS lymphoblasts to investigate whether the accumulation of MLCLs results in higher levels of apoptosis. We found no differences in susceptibility to death receptor-mediated apoptosis or in cellular distribution of Bid, cytochrome c, and other parameters, implying that MLCL accumulation does not lead to enhanced apoptosis in cultured BTHS lymphoblasts.

Development and validation of a liquid chromatographic/tandem mass spectrometric assay for the quantitation of nucleoside HIV reverse transcriptase inhibitors in biological matrices.

Besides liquid chromatographic (LC)/UV methods adapted to therapeutic drug monitoring, there is still a need for more powerful techniques that can be used for pharmacological research and clinical purposes. We developed an LC method coupled with tandem mass spectrometry (MS/MS) to separate, detect and quantify with high sensitivity the nucleoside analogues used in multitherapies (zidovudine, stavudine, zalcitabine, didanosine, lamivudine and abacavir) in plasma and in the intracellular medium. We worked on two essential issues: (i) the need to use two ionization modes in order to achieve the best sensitivity, which leads to the optimization of the chromatographic separation of drugs detected in the positive ionization mode and drugs detected in the negative ionization mode, and (ii) the need to optimize the extraction step in order to enhance sample recovery. The peripheral blood mononuclear cells were lysed in Tris buffer-MeOH. A clean-up procedure was performed by solid-phase extraction only for plasma samples. The LC separation was carried out on a Zorbax Stable Bond C(18) column followed by MS/MS analysis after electrospray ionization in either the negative or positive mode. The positive ionization mode was applied at the beginning of the run to detect zalcitabine and lamivudine, then the ionization mode was changed to negative for the detection of didanosine, stavudine, internal standard and zidovudine. The calibration range for all the analytes was 0.5-200 ng ml(-1). The recoveries were between 64 and 90%, with coefficients of variation (CVs) lower than 15%. The inaccuracy (bias) was +/-15% with CVs always lower than 12%. The analytes were stable at room temperature and in the extraction solvent for at least 24 h, after storage at -80 degrees C for 3 months, after three freeze-thaw cycles and in the injection solvent after 48 h at 4 degrees C. Together with the measurement of intracellular triphosphorylated metabolites thanks to the powerful plasma and intracellular assay method for intact drugs, it is possible to describe the behaviour of nucleoside analogues against HIV through plasma pharmacokinetics, cell membrane diffusion including drug transport involvement, and also the intracellular metabolism.

Peroxisome proliferator activated receptor delta (PPARdelta) agonist but not PPARalpha corrects carnitine palmitoyl transferase 2 deficiency in human muscle cells.

Type 2 carnitine palmitoyl transferase (CPT2) is involved in the transfer of long-chain fatty acid into the mitochondria. CPT2-deficient patients carry gene mutations associated with different clinical presentations, correlating with various levels of fatty acid oxidation (FAO) and residual CPT2 enzyme activity. We tested the hypothesis that pharmacological stimulation of peroxisome proliferator-activated receptors (PPAR) can stimulate FAO in CPT2-deficient muscle cells. Accordingly, we show that a 48-h treatment of CPT2-deficient myoblasts by bezafibrate restored FAO in patient cells. Specific agonists of PPARdelta (GWdelta 0742), and, to a lower extent, PPARalpha (GWalpha 7647) also stimulated FAO in control myoblasts. However, when tested in CPT2-deficient myoblasts, only the delta-agonist was able to restore FAO, whereas the alpha-agonist had no effect. GWdelta 0742 increased CPT2 mRNA levels, whereas no change in CPT2 transcripts was found in response to GWalpha 7647. Bezafibrate and GWdelta 0742 increased residual CPT2 activity and normalized long-chain acylcarnitine production by deficient cells. Finally, CPT1-B mRNA was also stimulated after PPAR agonist treatment, and this likely takes part in drug-induced increase of FAO in control muscle cells. In conclusion, this study clearly suggests that PPARs could be therapeutic targets for correction of inborn beta-oxidation defects in human muscle. Furthermore, these data also illustrate a selective control of beta-oxidation enzyme gene expression by PPARdelta, with no contribution of PPARalpha.