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. 2020 Jan 30:178:112884.
doi: 10.1016/j.jpba.2019.112884. Epub 2019 Sep 30.

Combinatory high-resolution microdissection/ultra performance liquid chromatographic-mass spectrometry approach for small tissue volume analysis of rat brain glycogen

Khaggeswar Bheemanapally  1 , Mostafa M H Ibrahim  1 , Karen P Briski  2
Affiliations

Combinatory high-resolution microdissection/ultra performance liquid chromatographic-mass spectrometry approach for small tissue volume analysis of rat brain glycogen

Khaggeswar Bheemanapally et al. J Pharm Biomed Anal. .

Abstract

Cyto-architectural diversity of brain structures emphasizes need for analytical tools for discriminative investigation of distinctive neural structures. Glycogen is the major energy reserve in the brain. There is speculation that brain utilization of this fuel source may affect detection of hypoglycemia. To evaluate sex-specific regulation of glycogen mass and mobilization in the glucose-sensory ventromedial hypothalamic nucleus (VMN), current research coupled UHPLC-electrospray ionization mass spectrometric (LC-ESI-MS) analysis capabilities with novel derivatization protocols for high-sensitivity measurement of glucose and glycogen in small-volume neural tissue samples. This work also sought to demonstrate utility of pairing this approach with optimized Western blot methods for measurement of glycogen metabolic enzyme protein expression. Here, high-resolution micropunch dissection tools for discriminative isolation of VMN tissue were used in conjunction with newly developed glycogen analytical methods and an experimental treatment paradigm for intra-cranial hindbrain-targeted administration of estrogen receptor-alpha (ERα) or -beta (ERβ) receptor antagonists to address the hypothesis that estradiol activates one or both hindbrain ER populations to exert sex-specific regulatory effects on VMN glycogen mass and hypoglycemia-associated mobilization. Outcomes validate a novel multi-analytical platform for investigation of in vivo sex-dimorphic regulation of glycogen metabolism in precisely-defined brain elements under conditions of energy balance versus imbalance. This combinatory approach will facilitate ongoing efforts to elucidate effects of acute versus chronic hypoglycemia on glycogen metabolism in characterized brain glucose-sensory loci and determine effects local glycogen mass and/or mobilization adaptions on sensory monitoring and signaling of recurring hypoglycemia in each sex.

Keywords: 1-Phenyl 3-methyl 5-pyrazolone; Estrogen receptor; Glycogen; Insulin-induced hypoglycemia; Ventromedial hypothalamic nucleus; Western blot.

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Conflict of interest statement

Conflict of Interest Statement

The authors declare that they do not have any conflicts of interest.

Figures

Figure 1.
Figure 1.
Glucose reacts with 1-phenyl-3-methyl-5-pyrazolone in mild alkaline conditions, forms glucose-PMP derivative. This derivative can be detected in negative mode at m/z 510.2.
Figure 2.
Figure 2.. Optimization of Chromatography Conditions for D-(+)-Glucose-PMP.
A) Representative total ion current (TIC) chromatogram of D-(+)-Glucose-PMP 12.48μg/mL in C18 column, Inj. Volume: 10μL, showing very low chromatographic sensitivity at retention time 2.1 min compared to PMP alone (large PMP peak shown in box at upper right-hand corner) at 10 min. B) Extracted D-(+)-Glucose-PMP showed very low chromatographic area at 2.1 min and poor resolution. C) Extracted PMP has very high chromatographic area at retention time 10 min and can be detected easily over D-(+)-Glucose-PMP in C18 column, indicating unsuitability of this column for identification of D-(+)-Glucose-PMP. D) TIC chromatogram of D-(+)-Glucose-PMP 50μg/mL in NH2P-40 3E column, Inj. Vol: 10μL, shows D-(+)-Glucose-PMP detection with distinguishing chromatographic peak at retention time 3 min in NH2P-40 3 E column. E) Extracted chromatogram of D-(+)-Glucose-PMP in NH2P-40 3E column at m/z 510.2 shows sele.ctive D-(+)-Glucose-PMP at retention time 3.5 min.
Figure 3.
Figure 3.. TIC Chromatogram of D-(+)-Glucose-PMP 50μg/mL, Inj.Vol: 10μL.
Effects of vaporizer temperature (VT), ion transfer tube temperature (ITT), sheath gas pressure (SGP), aux gas pressure (AGP), and sweep gas pressure (SWGP), described in Methods 1–9, are shown in corresponding TIC D-(+)-Glucose-PMP chromatograms 1–9.
Figure 4.
Figure 4.. Preliminary Optimization of Mass Spectrometric Parameters for D-(+)-Glucose-PMP in NH2P-40 3E column at m/z 510.2.
Extracted chromatograms 1–9 of D-(+)-Glucose-PMP 50μg/mL, Inj.Vol: 10μL. Mass spectrometric parameters of Method 9 show highest D-(+)-Glucose-PMP peak area in NH2P-40 3E column using Method 9.
Figure 5.
Figure 5.. Optimization of SGP and AGP Parameters for D-(+)-Glucose-PMP.
A) At AGP 2, SGP 50 psig yielded highest peak area at vaporizing temperature (VT) 200°C compared to 14.7, 25, or 75 SGP, respectively. SGP 50 psig caused a poor response at VT 50°C. Most chromatograms showed split peaks. B) At AGP 5, a distinct chromatographic peak without splits were observed at SGP 25 psig, VT 150°C, though peak area was medium when compared to SGP 75 psig and 14.7 psig. C) At AGP 10, SGP 50 and 75 psig, peak area degrades at ITT and 150°C VT; most data had split peaks and were not selected for further analyses.
Figure 6.
Figure 6.. TIC Chromatogram, Inj. Vol: 2μL.
AGP 4.6 psig, SGP 25 psig, SWGP 0.5 psig, VT 150°C, and ITT 150°C were selected for the analysis of D-(+)-Glucose-PMP. Representative chromatograms here show lack of D-(+)-Glucose-PMP background from standard (at left) or hydrolyzed glycogen (at right) when inj. vol. less than or equal to 2μL.
Figure 7.
Figure 7.. Extracted chromatograms at m/z 510.2, Inj. Vol: 2μL.
Extracted chromatograms show peaks with highest and least D-(+)-Glucose-PMP area from standard (at left) and hydrolyzed glycogen (at right) with retention time 5 min. Different concentrations were selected to generate a linear equation for glucose and glycogen.
Figure 8.
Figure 8.. Internal standard D-(+)-Glucose-PMP (13C6) lacks stability [M-H], m/z 515.2.
Mass spectrometric parameters of AGP 4.6 psig, SGP 25 psig, SWGP 0.5 psig, VT 150°C, and ITT 150°C selected for the analysis of internal standard (IS) 13C6-D-(+)-Glucose-PMP. A high IS concentration of 4mg/mL was selected, but failed to show a good response at 0 h and was destabilized after 7 h.
Figure 9.
Figure 9.. LC-ESI-MS analysis of derivatized VMN tissue glucose.
A coronal section through the brain at the level of the VMN is illustrated in Panel 8.A.1; hypothalamic area containing the VMN is enclosed within the rectangular box. That same area is enlarged in Panel 8.A.2 to depict the positioning of the hollow punch tool for selective harvesting of VMN tissue. Panels 8.B and 8.C show representative Negative Mode TIC and extracted chromatograms, respectively, of microdissected VMN tissue D-(+)-Glucose-PMP. Abbreviations: V3: third ventricle; ARH; arcuate hypothalamic nucleus; VMHc,dm,vl: central, dorsomedial, and ventrolateral parts of the ventromedial hypothalamic nucleus; AHNp,c,a: posterior, central, and anterior parts of the anterior hypothalamic nucleus; mpd, pv: medial parvicellular part (dorsal zone), periventricular parts of the PVH; PVi: intermediate periventricular hypothalamic nucleus; LHA: lateral hypothalamic area; ZI: zona incerta; fx: fornix; MEin: median eminence, internal lamina; MEex: median eminence, external lamina; TU: tuberal nucleus.
Figure 10.
Figure 10.
D-(+)-glucose-PMP. Molecular mass: 510.21.
Figure 11.
Figure 11.. Effects of Caudal Fourth Ventricular (CV4) Administration of the ERα Antagonist 1,3-Bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole dihydrochloride (MPP) or ERβ Antagonist 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol (PHTPP) Ventromedial Hypothalamic Nucleus (VMN) Glycogen Metabolic Enzyme Protein Expression during Insulin-Induced Hypoglycemia (IIH) in Female Versus Male Rats.
Micropunch-dissected VMN tissue was obtained from groups of male and estradiol – implanted ovariectomized female rats pretreated by CV4 administration of MPP, PHTPP, or vehicle prior to sc insulin (INS) injection for Western blot analysis of glycogen synthase (GS) [Panel A, male; Panel B, female] or glycogen phosphorylase-muscle type (GPmm) [Panel C, male; Panel D, female]. Data depict mean normalized protein optical density (O.D.) values ± S.E.M. for vehicle-pretreated animals injected sc with vehicle- (white bars; n=6) or INS (gray bars; n=6) or INS-injected rats pretreated with MPP (cyan bars; n=6) or PHTPP (purple gray bars; n=6). *p<0.05; **p<0.01; **p<0.001.
Figure 12.
Figure 12.
Glucose-PMP selective ion monitoring mass chromatogram and mass spectrum.
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