RESUMEN
Glycation and the subsequent formation of advanced glycation end products (AGEs) disrupt and impair the physiological functions of proteins. This study presents a comprehensive glycation site mapping of human serum albumin (HSA) utilizing liquid chromatography-tandem mass spectrometry (LC-MS/MS). Both in vitro glycation experiments and patient samples were investigated, exploring various enzymes, processing techniques, and their impacts on glycation site detection. A pilot study was conducted, analyzing sixteen serum samples, which spanned from healthy individuals to severe diabetic patients (with HbA1c values ranging from 5.7% to 18.1%). The aim was to comprehend the progression of glycation on various sites of HSA with increasing levels of glycation. Their glycated albumin levels (GA) spanned from 19.7% to 62.3%. Trypsin-mediated proteolytic digestion unveiled 12 glycation sites through direct in-solution digestion of whole serum. However, isolating albumin from serum enabled the identification of a higher number of glycation sites in each sample compared to direct serum digestion. Boronate affinity chromatography facilitated the segregation of less glycated albumin (LGA) from the more glycated albumin (MGA) fraction. Subsequent proteolytic digestion of both LGA and MGA samples revealed similar glycation sites. The MGA fraction exhibited a greater number of identified glycation sites, thereby elucidating which sites are particularly prone to glycation in highly glycated albumin samples. Changes in relative glycation levels were noted in the tryptic digests of albumin samples following the sample enrichment steps, as opposed to direct in-solution digestion of whole serum. Two enzymes, trypsin and Glu-C, were evaluated for efficacy in sequence coverage and glycation site analysis of HSA, with trypsin demonstrating superior efficiency over Glu-C.
Asunto(s)
Albúmina Sérica Glicada , Productos Finales de Glicación Avanzada , Albúmina Sérica Humana , Espectrometría de Masas en Tándem , Humanos , Productos Finales de Glicación Avanzada/química , Espectrometría de Masas en Tándem/métodos , Albúmina Sérica Humana/química , Glicosilación , Cromatografía Liquida/métodos , Albúmina Sérica/química , Albúmina Sérica/análisis , Albúmina Sérica/metabolismo , Proyectos Piloto , Diabetes Mellitus/sangre , Tripsina/química , Tripsina/metabolismoRESUMEN
Rapid identification of microbial pathogens "directly" from positive blood cultures (PBCs) is critical for prompt initiation of empirical antibiotic therapy and clinical outcomes. Towards higher microbial identification rates, we modified a published initial serum separator tubes-based MALDI-TOF-MS protocol, for blood culture specimens received at a non-hospital based standalone diagnostic laboratory, Bangalore, India: (a) "Initial" protocol #1: From 28 PBCs, identification= 39% (Gram-negative= 43%: Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa; Gram-positive: 36%: Enterococcus faecalis, Staphylococcus aureus, Staphylococcus haemolyticus); mis-identification= 14%; non-identification= 47%. (b) "Modified" protocol #2: Quality controls (ATCC colonies spiked in negative blood cultures) From 7 analysis, identification= 100% (Escherichia coli, Klebsiella pneumonia, Klebsiella oxytoca, Pseudomonas aeruginosa, Enterococcus faecalis, Staphylococcus aureus); From 7 PBCs, identification= 57%; mis-identification= 14%; non-identification= 29%. Microbial preparations of highest quality and quantity for proteomic analysis and separate spectra matching reference databases for colonies and PBCs are needed for best clinical utility.
Asunto(s)
Cultivo de Sangre , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Humanos , Cultivo de Sangre/métodos , India , Bacterias/aislamiento & purificación , Bacterias/clasificación , Bacteriemia/diagnóstico , Bacteriemia/microbiologíaRESUMEN
Aims: Cys34 albumin redox modifications (reversible "cysteinylation" and irreversible "di/trioxidation"), besides being just oxidative stress biomarkers, may have primary pathogenetic roles to initiate and/or aggravate cell, tissue, and vascular damage in diabetes. In an exploratory "proof-of-concept" pilot study, we examined longitudinal changes in albumin oxidation during diabetes therapy. Methods: Mass spectrometric analysis was utilized to monitor changes in human serum albumin (HSA) post-translational modifications {glycation [glycated albumin (GA)], cysteinylation [cysteinylated albumin (CA) or human non-mercaptalbumin-1; reversible], di/trioxidation (di/trioxidized albumin or human non-mercaptalbumin-2; irreversible), and truncation (truncated albumin)} during ongoing therapy. Four informative groups of subjects were evaluated [type 1 diabetes (T1DM), type 2 diabetes (T2DM), prediabetes-obesity, and healthy controls] at baseline, and subjects with diabetes were followed for a period up to 280 days. Results: At baseline, T2DM was associated with relatively enhanced albumin cysteinylation (CA% total) compared with T1DM (P = 0.004), despite comparable mean hyperglycemia (P values: hemoglobin A1c = 0.09; GA = 0.09). T2DM, compared with T1DM, exhibited selectively and significantly higher elevations of all the "individual" glycated cum cysteinylated ("multimodified") albumin isoforms (P values: CysHSA+1G = 0.003; CysHSA+2G = 0.007; and CysHSA+3G = 0.001). Improvements in glycemic control and decreases in albumin glycation during diabetes therapy in T2DM were not always associated with concurrent reductions of albumin cysteinylation, and in some therapeutic situations, albumin cysteinylation worsened (glycation-cysteinylation discordance). Important differences were observed between the effects of sulfonylureas and metformin on albumin molecular modifications. Conclusions: T2DM was associated with higher oxidative (cysteinylation) and combined (cysteinylation plus glycation) albumin molecular modifications, which are not ameliorated by improved glucose control alone. Further studies are required to establish the clinical significance and optimal therapeutic strategies to address oxidative protein damage and resulting consequences in diabetes.
Asunto(s)
Diabetes Mellitus Tipo 2 , Albúmina Sérica Glicada , Hipoglucemiantes , Oxidación-Reducción , Albúmina Sérica Humana , Humanos , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/metabolismo , Masculino , Persona de Mediana Edad , Femenino , Hipoglucemiantes/uso terapéutico , Albúmina Sérica Humana/metabolismo , Albúmina Sérica Humana/química , Glicosilación , Proyectos Piloto , Adulto , Albúmina Sérica/metabolismo , Estrés Oxidativo/efectos de los fármacos , Biomarcadores/sangre , Hemoglobina Glucada/metabolismo , Hemoglobina Glucada/análisis , Glucemia/metabolismo , Estudios de Casos y Controles , Anciano , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 1/metabolismo , Productos Finales de Glicación Avanzada/metabolismo , Procesamiento Proteico-Postraduccional , Metformina/uso terapéutico , Cisteína/metabolismoRESUMEN
Morbidity and mortality rates in patients with autosomal recessive, congenital generalized lipodystrophy type 4 (CGL4), an ultra-rare disorder, remain unclear. We report on 30 females and 16 males from 10 countries with biallelic null variants in CAVIN1 gene (mean age, 12 years; range, 2 months to 41 years). Hypertriglyceridemia was seen in 79% (34/43), hepatic steatosis in 82% (27/33) but diabetes mellitus in only 21% (8/44). Myopathy with elevated serum creatine kinase levels (346-3325 IU/L) affected all of them (38/38). 39% had scoliosis (10/26) and 57% had atlantoaxial instability (8/14). Cardiac arrhythmias were detected in 57% (20/35) and 46% had ventricular tachycardia (16/35). Congenital pyloric stenosis was diagnosed in 39% (18/46), 9 had esophageal dysmotility and 19 had intestinal dysmotility. Four patients suffered from intestinal perforations. Seven patients died at mean age of 17 years (range: 2 months to 39 years). The cause of death in four patients was cardiac arrhythmia and sudden death, while others died of prematurity, gastrointestinal perforation, and infected foot ulcers leading to sepsis. Our study highlights high prevalence of myopathy, metabolic abnormalities, cardiac, and gastrointestinal problems in patients with CGL4. CGL4 patients are at high risk of early death mainly caused by cardiac arrhythmias.
Asunto(s)
Lipodistrofia Generalizada Congénita , Proteínas de Unión al ARN , Humanos , Masculino , Femenino , Lipodistrofia Generalizada Congénita/genética , Lipodistrofia Generalizada Congénita/complicaciones , Lipodistrofia Generalizada Congénita/patología , Adolescente , Niño , Lactante , Preescolar , Adulto , Adulto Joven , Arritmias Cardíacas/genética , Arritmias Cardíacas/patología , Hipertrigliceridemia/genética , Hipertrigliceridemia/complicaciones , Hipertrigliceridemia/patologíaRESUMEN
BACKGROUND: Diabetes mellitus (DM) in children and adolescents is typically caused by type 1 DM, followed by type 2 DM and maturity-onset diabetes of the young (MODY). We report an unusual Asian Indian family in which three members presented with DM at ages 15, 20, and 30, but not fitting the typical clinical picture of type 1 DM, type 2 DM, or MODY. The primary objective was to elucidate the molecular genetic basis of DM in this family. METHODS: The proband, a 22-year-old man, had short stature, gray hair, osteoporosis, and markedly reduced subcutaneous fat on the body, especially on the extremities along with acanthosis nigricans, and developed myxoid malignant peripheral nerve sheath tumor. Detailed family history revealed multiple loops of consanguinity. The proband underwent whole-genome sequencing, and seven relatives underwent whole-exome sequencing. RESULTS: The proband and three additional family members were found to have the homozygous c.561A>G nucleotide variant of WRN RecQ-like helicase (WRN) gene consistent with the diagnosis of Werner's syndrome. The c.561A>G variant induces a new splicing site on exon 6 resulting in a truncated WRN protein, p.Lys187Trpfs*13. CONCLUSION: Our report brings to attention the onset of DM during childhood or early adulthood in patients with Werner's syndrome who typically develop type 2 DM around the age of 30-40 years. Presence of consanguinity among parents, dysmorphic features, and malignancy should prompt consideration of diagnosis of Werner's syndrome.
Asunto(s)
Diabetes Mellitus Tipo 1 , Diabetes Mellitus Tipo 2 , Osteoporosis , Síndrome de Werner , Masculino , Niño , Adolescente , Humanos , Adulto , Adulto Joven , Síndrome de Werner/diagnóstico , Síndrome de Werner/genética , ADN Helicasas/genética , Diabetes Mellitus Tipo 2/genéticaRESUMEN
Background: Albumin, the most abundant and physiologically vital serum protein, accumulates a range of chemical modifications, as consequence of encounters with large number of reactive molecules whose concentrations increase in serum under pathological conditions. Methods: In a "proof of concept" study, mass spectrometric analysis was utilized to quantitate albumin post-translational modifications (glycation, oxidation, and truncation; individual isoforms and total) in four informative subject groups [type 1 diabetes (T1DM), type 2 diabetes (T2DM), prediabetes-obesity and healthy; all with estimated glomerular filtration rate ≥60 mL/(min·m2)]. Besides glycated albumin (GA/mass spectrometry), glycated serum protein (GSP/nitro blue tetrazolium colorimetry), and glycated hemoglobin (HbA1c/high-performance liquid chromatography) were also measured. Results: A wide spectrum of albumin molecular modifications was identified in diabetes, with significant differences between T2DM and T1DM. Albumin glycation: GA correlated more strongly with HbA1c in T1DM, compared to T2DM. Higher albumin glycation isoforms (human serum albumin +3G/2G) were more stable and discriminative markers of mean glycemia. Albumin oxidation: T2DM, in comparison with T1DM, showed enhanced oxidative and dual (glycation plus oxidation) modifications, representing extreme molecular pathology. Albumin truncation: There was dramatic reduction ("deficiency") of truncated albumin isoforms in T2DM, and significant reduction in T1DM. Albumin truncation negatively correlated with severity of albumin glycation (mean glycemia) and albumin oxidation (cysteinylation). Possible mechanisms of insulin resistance, with associated increased free fatty acids binding to albumin, in stimulating albumin oxidation and inhibiting albumin glycation ("metabolic cross talks") are reviewed. Conclusions: Albumin molecular modifications in diabetes, together with significant differences between T2DM and T1DM, suggest possible role for insulin resistance in their genesis and consequent cell, tissue, and vascular dysfunction/damage. Albumin molecular fingerprinting can provide valuable insights into pathogenesis, diagnosis, monitoring, and future therapies for diabetes. Identification of biomarker battery ("albuminomics," "diabetomics") driven diverse "healthy," prediabetes, obesity, and T2DM phenotypes represents additional novel step toward precision medicine in diabetes and related disorders.