Accurate and sensitive quantitation of glucose and glucose phosphates derived from storage carbohydrates by mass spectrometry

Young, Lyndsay E.A.; Brizzee, Corey O.; Macêdo, Jéssica K. A.; Murphy, Ray; Contreras, Christopher; DePaoli‐Roach, Anna A.; Roach, Peter J.; Gentry, Matthew S.; Sun, Ramon C.

doi:10.1016/j.carbpol.2019.115651

Cited by 29 publications

(23 citation statements)

References 51 publications

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“…The average total glycogen content measured in muscle of 5 month-old WT and Epm2b -/mice is within the range of what has previously been published for skeletal muscle in WT mice (12,22,23,31,37,38) with the exception of a recent publication which, using GC/MS following a novel extraction protocol, demonstrated muscle glycogen contents less than 10% of all previous findings (26). Glycogen accumulation in muscle of 9-12 month-old LD mice was previously clearly detected, but changes in total muscle glycogen content were mostly undetectable in mice younger than 5 months (22,37).…”

Section: Quantification Of Inert α-Glucans In Muscle Of Ld Micesupporting

confidence: 84%

“…Extraction from pulverized tissue in hot 30% potassium hydroxide has been widely used for glycogen quantification (21)(22)(23). Other extraction procedures include homogenization in cold 10% trichloroacetic acid (24)(25)(26) or in Tris-based glycogen isolation buffer (12,27). Extraction in a suitable buffer allows separation of soluble and insoluble glycogen, followed by glycogen quantification without further purification of the glycogen by repeated ethanol precipitations (12), which are otherwise required to remove excess amounts of potassium hydroxide or trichloroacetic acid.…”

Section: Introductionmentioning

confidence: 99%

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Sensitive quantification of α-glucans in mouse tissues, cell cultures, and human cerebrospinal fluid

Nitschke

Petković

Ahonen

et al. 2020

Journal of Biological Chemistry

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The soluble α-polyglucan glycogen is a central metabolite enabling transient glucose storage to suit cellular energy needs. Glycogen storage diseases (GSDs) comprise over 15 entities caused by generalized or tissue-specific defects in enzymes of glycogen metabolism. In several, e.g. in Lafora disease caused by the absence of the glycogen phosphatase laforin or its interacting partner malin, degradation-resistant abnormally structured insoluble glycogen accumulates. Sensitive quantification methods for soluble and insoluble glycogen are critical to research, including therapeutic studies, in such diseases. This paper establishes methodological advancements relevant to glycogen metabolism investigations generally, and GSDs. Introducing a pre-extraction incubation method, we measure degradation-resistant glycogen in as little as 30 mg skeletal muscle or a single hippocampus from Lafora disease mouse models. The digestion-resistant glycogen correlates with the disease-pathogenic insoluble glycogen and can readily be detected in very young mice where glycogen accumulation has just begun. Secondly, we establish a high-sensitivity glucose assay with detection of ATP depletion, enabling 1) quantification of α-glucans in cell culture using a medium-throughput assay suitable for assessment of candidate glycogen synthesis inhibitors, and 2) discovery of α-glucan material in healthy human cerebrospinal fluid, establishing a novel methodological platform for biomarker analyses in Lafora disease and other GSDs.

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Section: Quantification Of Inert α-Glucans In Muscle Of Ld Micesupporting

confidence: 84%

Section: Introductionmentioning

confidence: 99%

Sensitive quantification of α-glucans in mouse tissues, cell cultures, and human cerebrospinal fluid

Nitschke

Petković

Ahonen

et al. 2020

Journal of Biological Chemistry

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“…Citrate and malate showed direct labeling of TCA intermediates, which are used to generate amino acids like aspartate, and as such they displayed similar fractional labeling patterns ( Figure 5 g–i). Finally, using our previously established method of glycogen quantitation [ 33 , 34 ], we observed dynamic glycogen enrichment in the liver and brain. Liver glycogen enrichment peaked at 30min, followed by a slow but steady decline until 4 h post gavage, while a similar pattern was observed in brain ( Figure S2, Supplementary Materials ).…”

Section: Resultsmentioning

confidence: 99%

Oral Gavage Delivery of Stable Isotope Tracer for In Vivo Metabolomics

et al. 2020

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Stable isotope-resolved metabolomics (SIRM) is a powerful tool for understanding disease. Advances in SIRM techniques have improved isotopic delivery and expanded the workflow from exclusively in vitro applications to in vivo methodologies to study systemic metabolism. Here, we report a simple, minimally-invasive and cost-effective method of tracer delivery to study SIRM in vivo in laboratory mice. Following a brief fasting period, we orally administered a solution of [U-13C] glucose through a blunt gavage needle without anesthesia, at a physiological dose commonly used for glucose tolerance tests (2 g/kg bodyweight). We defined isotopic enrichment in plasma and tissue at 15, 30, 120, and 240 min post-gavage. 13C-labeled glucose peaked in plasma around 15 min post-gavage, followed by period of metabolic decay and clearance until 4 h. We demonstrate robust enrichment of a variety of central carbon metabolites in the plasma, brain and liver of C57/BL6 mice, including amino acids, neurotransmitters, and glycolytic and tricarboxylic acid (TCA) cycle intermediates. We then applied this method to study in vivo metabolism in two distinct mouse models of diseases known to involve dysregulation of glucose metabolism: Alzheimer’s disease and type II diabetes. By delivering [U-13C] glucose via oral gavage to the 5XFAD Alzheimer’s disease model and the Lepob/ob type II diabetes model, we were able to resolve significant differences in multiple central carbon pathways in both model systems, thus providing evidence of the utility of this method to study diseases with metabolic components. Together, these data clearly demonstrate the efficacy and efficiency of an oral gavage delivery method, and present a clear time course for 13C enrichment in plasma, liver and brain of mice following oral gavage of [U-13C] glucose—data we hope will aid other researchers in their own 13C-glucose metabolomics study design.

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“…Even if glucose phosphates are structurally similar, G1P and G6P are involved in the processes of glycogen degradation and synthesis, respectively. Oppositely, G2P or G3P residues cannot be confirmed by biochemical enzymatic assays as there is no known enzyme with specific activity towards these two phosphates [ 9 , 10 , 11 ]. Actually, glucose-2-phsophate itself was used for studying phosphate stability or hydrolysis rates [ 8 , 12 , 13 ], or for identification of the hydrolyzed monomeric forms of glycogen to understand the metabolism of the phosphate in this biopolymer [ 9 , 14 ].…”

Section: Resultsmentioning

confidence: 99%

Esters of Glucose-2-Phosphate: Occurrence and Chemistry

Zhang

Ahmar

et al. 2020

Molecules

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Phosphodiesters of glucose-2-phosphate (G2P) are found only in few natural compounds such as agrocinopine D and agrocin 84. Agrocinopine D is a G2P phosphodiester produced by plants infected by Agrobacterium fabrum C58 and recognized by the bacterial periplasmic binding protein AccA for being transported into the bacteria before cleavage by the phosphodiesterase AccF, releasing G2P, which promotes virulence by binding the repressor protein AccR. The G2P amide agrocin 84 is a natural antibiotic produced by the non-pathogenic Agrobacterium radiobacter K84 strain used as a biocontrol agent by competing with Agrobacterium fabrum C58. G2P esters are also found in irregular glycogen structures. The rare glucopyranosyl-2-phophoryl moiety found in agrocin 84 is the key structural signature enabling its action as a natural antibiotic. Likewise, G2P and G2P esters can also dupe the Agrobacterium agrocinopine catabolism cascade. Such observations illustrate the importance of G2P esters on which we have recently focused our interest. After a brief review of the reported phosphorylation coupling methods and the choice of carbohydrate building blocks used in G2P chemistry, a flexible access to glucose-2-phosphate esters using the phosphoramidite route is proposed.

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Accurate and sensitive quantitation of glucose and glucose phosphates derived from storage carbohydrates by mass spectrometry

Cited by 29 publications

References 51 publications

Sensitive quantification of α-glucans in mouse tissues, cell cultures, and human cerebrospinal fluid

Sensitive quantification of α-glucans in mouse tissues, cell cultures, and human cerebrospinal fluid

Oral Gavage Delivery of Stable Isotope Tracer for In Vivo Metabolomics

Esters of Glucose-2-Phosphate: Occurrence and Chemistry

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