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Keywords:

  • anoxic changes;
  • biochemical thermodynamics;
  • freeze blowing;
  • intermediate metabolites;
  • neurotransmitters;
  • phosphorylation potential

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Rapid inactivation of metabolism is essential for accurately determining the concentrations of metabolic intermediates in the in vivo state. We compared a broad spectrum of energetic intermediate metabolites and neurotransmitters in brains obtained by microwave irradiation to those obtained by freeze blowing, the most rapid method of extracting and freezing rat brain. The concentrations of many intermediates, cytosolic free NAD(P)+/NAD(P)H ratios, as well as neurotransmitters were not affected by the microwave procedure. However, the brain concentrations of ATP were about 30% lower, whereas those of ADP, AMP, and GDP were higher in the microwave-irradiated compared with the freeze-blown brains. In addition, the hydrolysis of approximately 1 μmol/g of ATP, a major in vivo Mg2+-binding site, was related to approximately five-fold increase in free [Mg2+] (0.53 ± 0.07 mM in freeze blown vs. 2.91 mM ± 0.48 mM in microwaved brains), as determined from the ratio [citrate]/[isocitrate]. Consequently, many intracellular properties, such as the phosphorylation potential and the ∆G' of ATP hydrolysis were significantly altered in microwaved tissue. The determinations of some glycolytic and TCA cycle metabolites, the phosphorylation potential, and the ∆G' of ATP hydrolysis do not represent the in vivo state when using microwave-fixed brain tissue.

Abbreviations used
3 PG

3-phosphoglycerate

DEE

diethyl ether

DHAP

dihydroxy acetone phosphate

PCA

perchloric acid

PEP

phosphoenol pyruvate

TIM

triose phosphate isomerase

TMCS

trimethylchlorosilane

To accurately assess the in vivo metabolite concentrations, rapid inactivation of intracellular metabolism is critical to prevent changes in metabolite concentrations resulting from the anoxic state. A number of methods to preserve the in vivo state have been attempted, including cervical dislocation followed by ‘rapid’ removal of brain and freezing, decapitation into liquid N2 (Richter and Dawson 1948), funnel freezing (Ponten et al. 1973) which involves opening the cranial cavity of anesthetized animals and pouring liquid nitrogen via a funnel to freeze the contents in situ. None of these techniques achieves rapid inactivation of metabolism. Extracting brain following cervical dislocation takes at least several seconds, exposing the highly glycolytic brain tissue to anoxia for the duration and significantly altering metabolite levels. Decapitation into liquid nitrogen is even less satisfactory as the freezing occurs in a non-uniform manner, beginning from the outer layers following a changing temperature gradient toward the central parts of brain, and can require up to 80 s to freeze (Jongkind and Bruntink 1970). It also creates the practical difficulty of removing the frozen brain from the skull without thawing. Funnel freezing requires animals to be anesthetized, which itself causes significant alterations in brain metabolism (Brunner et al. 1971). Furthermore, because of the thickness of brain tissue, it takes several seconds for brain to freeze completely.

More recently, microwave irradiation has been more widely used for obtaining tissue samples from which metabolite concentrations are then determined (Schmidt et al. 1971; Delaney and Geiger 1996). The primary mechanism of microwave fixation is the rapid heating of brain to about 80°C, which inactivates many enzymatic reactions, although non-thermal effects of microwaves on chemical reactions have also been reported (Hoz et al. 2005). The rate of heating depends on a number of parameters, including the power and duration (Kobayashi et al. 1989) of microwave exposure as well as the age of the animal (Miller and Shamban 1977), which affects the size of the organ and the thickness of the skull. By optimizing the duration of heating, it is possible to maintain tissue integrity, whereby discrete brain regions may undergo biochemical analyses. Previous studies have reported that the redox state is maintained by microwaving (Miller and Shamban 1977). Enzymes, however, vary in their resistance to microwave-induced denaturation. Slow or incomplete inactivation of several metabolic reactions may confound the determination of associated metabolites. Freeze blowing is a method by which the cranial cavity is ejected under a pressurized stream of air and the brain tissue spread thinly over aluminum discs at liquid N2 temperature, so that chemical reactions are stopped in microseconds. Previous studies have reported lower ATP and higher ADP concentrations in microwave-treated brains compared with freeze-blown brains (Veech et al. 1973; Miller and Shamban 1977). An important regulator of equilibrium constants of many intracellular reactions is the free [Mg2+]. While the typical concentration of total Mg2+ in cells is between 5–15 mM, most of this is bound to various negatively charged metabolites, proteins, and DNA. Only about 10% exists as free [Mg2+]. ATP is a primary species that binds intracellular Mg2+ ions and maintains the levels of intracellular free [Mg2+] to usually about 1/10 of the total intracellular Mg2+ concentration. ATP hydrolysis releases the Mg2+ bound to the phosphate groups and thereby increases the level of intracellular free [Mg2+]. The intracellular free [Mg2+] can be determined by the ratio of [citrate]/[isocitrate], which is sensitive to changes in free [Mg2+] (Veloso et al. 1973). Alterations in the concentration of free [Mg2+] cause large changes in the free energy of a number of reactions, most particularly the 3-phosphoglycerate kinase (Bergman et al. 2010), creatine kinase, and other important phosphate energy-dependent reactions (Veech et al. 1994).

The aim of this study was to compare the effects of microwave irradiation and freeze blowing on ATP hydrolysis, the levels of free [Mg2+], and subsequent changes to the in vivo reactions. Accordingly, we exposed adult rat brains to either freeze blowing or focused high-energy microwave irradiation and measured a variety of intermediary metabolites that may be potentially sensitive to hypoxia or alterations in the free [Mg2+]. In addition, as animals that are microwaved are anesthetized with isoflurane, whereas in the freeze-blowing procedure they are not a subset of animal brains, exposed to isoflurane prior to freeze blowing, were analyzed for adenosyl nucleotides and citrate and isocitrate concentrations, both determinants in calculating the free [Mg2+] (Veloso et al. 1973).

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

All experiments were reviewed and approved by the NIAAA/NIH Animal Care and Use Committee (ACUC) and University of North Dakota Animal Care and Use Committee. Fifteen adult male Sprague–Dawley rats weighing approximately 300 g were obtained from Charles River Laboratories (Wilmington, MA, USA). The animals were housed individually and maintained on a standard rat diet. Animals were acclimatized for 5 days before being killed.

Microwave procedure

Rats were anesthetized with isoflurane before microwaving and heads were fixed with a restrainer. Rats were killed with a 6-kW head-focused high-energy microwave irradiation system (Cober Electronics, Norwalk, CT, USA), set to 70% of maximal power for a duration of 1.3 s. Brains were immediately excised and placed on a block of dry ice. Brain samples were kept frozen and shipped overnight on dry ice to NIH, where the samples were stored at −80°C until analyzed. Brains were cleanly cleaved along the mid sections hemisphere while frozen, and half of the brain was ground to a powder in liquid nitrogen with a mortar and pestle to obtain a fine powder. Brains from five rats were obtained using microwave.

Freeze-blowing procedure

An additional five animals each were killed by brain freeze blowing without any anesthesia (n = 5) and a subset of animals (n = 5) under anesthesia. For anesthesia, animals were kept in a chamber infused with 2.5% isoflurane for 3–5 min to achieve deep anesthesia. Animals were held securely in a Plexiglas chamber. The heads were aligned and held in position using stereotaxic positioning device and inhaled isoflurane throughout this period. The freeze-blowing apparatus consisted of two solenoid-operated mounts which rapidly impaled two sharp, hollow steel probes into the cranial cavity. Vertical and horizontal positioning of the probes was achieved by rack and pinion gears, and a jet of air (25 lb/in2) ejected the cranial contents as a thin layer in between hollow aluminum discs pre-cooled in liquid N2. The description of the freeze-blowing apparatus can be found in (Veech et al. 1973).

Perchloric acid extraction

Frozen tissue samples (~100 mg) were extracted in four volumes of 3.6% perchloric acid (PCA). The PCA solutions were kept frozen in plastic tubes containing 0.5-mm glass beads. Brain tissue was added to the frozen PCA and the tubes shaken for 20 s at 5000 beats per min in a Mini Beadbeater (Biospec Products, Bartlesville, OK, USA). This thawed the PCA and extracted the samples in PCA. Samples were then placed on ice. The samples were centrifuged at 9300 g in an Eppendorf centrifuge for 10 min at 4°C. About 340 μL of the supernatant was taken and neutralized by the addition of 3 M KHCO3 using a 0.05% solution of phenol red (5 μL) as indicator. The potassium perchlorate was removed by centrifuging at 9300 g for 15 min at 4°C.

Labeled internal standards

Labeled internal standards were obtained either commercially or prepared within the laboratory using published procedures. The labeled internal standards, 13C-glutamate, 13C-aspartate, 13C-glutamine, and 2H-4-amino γ-butyric acid were purchased from CDN Isotopes (Quebec, Canada). 13C- 15N-labeled nucleotides and13C-lactate, 13C-citrate, 13C-fumarate, 13C-succinate, and 13C-malate were obtained from Sigma Chemical Co. (St. Louis, MO, USA). 13C-phosphoenol pyruvate and 13C-pyruvate were purchased from Cambridge Isotope Labs (Cambridge, MA, USA). 13C-glucose-6-phosphate and 13C-1,6 bisphosphate-fructose were obtained from Omicron Biochemicals (South Bend, IN, USA). 13C-dihydroxy acetone phosphate, 13C-3-phosphoglycerate, 13C-α-ketoglutarate, and 13C-isocitrate were synthesized in laboratory. 13C-dihydroxy acetone phosphate was synthesized from 13C-glycerol using glycerokinase and glycerophosphate oxidase reactions, and purified on anion-exchange column (Fessner 1997). 13C-3PG was synthesized from the 13C-DHAP using Triose phosphate isomerase (TIM), GAPDH, and AsO4- (Kappel 1983). LDH reaction was utilized to refurbish any NADH formed and catalase was used to remove any hydrogen peroxide generated. At the end of the reaction, enzymes were precipitated by PCA and the 13C-3PG purified using anion-exchange chromatography. Fractions containing 13C-3PG were pooled, acidified to pH 1.2 with conc. HCl and lyophilized. The powder was washed three times with diethyl ether (DEE). The DEE was pooled and evaporated in a rotary evaporator. The purified 13C-3PG was then dissolved in deionized water and stored at −80°C until further analysis. 13C-isocitrate was synthesized and purified from 13C-α-ketoglutarate using a previously published procedure (Ehrlich and Colman 1987). Purity of the synthesized standards was determined using GC–MS analysis and validated using pure commercially available non-labeled analogues.

Chloroform–methanol extraction

Brain tissue samples were extracted using a modified chloroform–methanol extraction procedure for determination of adenosyl and guanosyl nucleotides using capillary electrophoresis mass spectrometry method described below. Approximately 50 mg of frozen whole brain tissue was added to a frozen (−80°C) solution of water : chloroform : methanol (300 : 400 : 200 μL) containing labeled nucleotide internal standards purchased from ICN Isotopes (Pointe-Claire, Quebec) in two-fold excess of the concentrations of the analytes in the tissue in 2-mL polypropylene screw-capped tubes with 40–50 small glass beads. Samples were shaken two times on Mini Beadbeater for 30 s until a homogeneous suspension was obtained. Samples were placed on ice until centrifugation at 4°C in an Eppendorf benchtop centrifuge at 2000 g for 10 min to allow the chloroform and water layers to separate. The water layer was removed and filtered through an Amicon Microcon Ultracel YM-10 filter centrifuge tube at 20000 rcf. The filtrate (about 300 μL) was used in CE-MS analysis (described below).

Chemical derivatization procedures

Procedures for the determination of citrate, isocitrate, α-ketoglutarate, succinate, fumarate, malate, lactate, and pyruvate have been described elsewhere (Pawlosky et al. 2010) and briefly related here. The organic acids were analyzed as their tertiary butyl dimethylsilyl ether derivatives (TBDMS) using GC–MS in the electron impact mode and quantified using the 13C-labeled internal standards for each analyte. The N-methyl-N-(tert-butylmethylsilyl) trifluoroacetamide (MTBSTFA) with 1% tert-butyldimethylchlorosilane (TBDMCS) reagent was purchased from Pierce Chemical Co. (Rockford, IL, USA). The N,O-bis(trimethylsilyl) trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS) reagent was purchased from Pierce Chemical Co.

The phosphorylated compounds glucose-6-phosphate (G-6-P), fructose di-phosphate (Fru-D-P), dihydroxy acetone phosphate (DHAP), 3-phosphoglycerate (3PG), and phosphoenol pyruvate (PEP) were analyzed as their tri-methylsilyl ether derivatives (TMS) using GC–MS in the electron impact mode and quantified using the 13C-labeled internal standards for each analyte.

Gas chromatography–mass spectrometry

Aliquots (15 μL) of perchloric acid brain extracts were prepared for GC–MS analysis as described above. The 13C-labeled internal standards were added in two-fold excess of the concentrations of the individual analytes in brain tissue to the neutralized PCA extracts and evaporated to dryness under a stream of nitrogen. Samples were immediately reacted with 5 μL of either of the two silylating reagents in 15 μL of acetonitrile in 1.5-mL screw-capped glass vials and heated to 60°C for 5 min. In case of phosphorylated compounds, the samples were reacted overnight at 60°C to achieve complete derivatization. Samples were analyzed on an Agilent 5973 quadrupole GC–MS (Agilent, Wilmington, DE, USA). One microliter of the sample solutions was injected onto a 250 μm × 30 m capillary DB-1 (Agilent) column in split-less injection mode using helium as the carrier gas. The injector temperature was set at 270°C and the transfer line at 280°C. The GC oven temperature was programmed from 80 to 325°C at 15°C/min. The mass spectrometer was operated in the electron impact mode (70 eV), and the quadrupole mass analyzer scanned for ions which corresponded to a loss of 15 mass units (-CH3) from the molecular ion and the base peak of each analyte and its corresponding 13C-labeled internal standard using selected ion monitoring. Analytes eluted the column between 10 and 20 min following injection, and the ratio of peak area counts of the 13C-labeled internal standard to that of the analyte were used to quantify its concentration.

Capillary electrophoresis–mass spectrometry (CE–MS)

Sample extracts were analyzed on an Agilent capillary electrophoresis–ion trap mass spectrometer (Ultra) using an Agilent 1100 series binary pump to deliver a make-up flow of 50 mM ammonium acetate in methanol : water (50 : 50) to the electrospray ionization tip (Agilent) according to published procedures (Pawlosky et al. 2010). Samples were ionized in either the positive or negative ion ionization modes. Anions and cations were analyzed on 50 μm × 100 cm dynamically cationic- or anionic-coated capillary columns using either positive (+30kV) or negative (−30kV) electrokinetic injections. The capillary column was cooled to 20°C using thermostatic controlled capillary cassette and the electrospray needle was set orthogonal to the mass spectrometer inlet. The CE electrolyte used for separation was 50 mM ammonium acetate (pH 9.0). The column was flushed successively between runs with acetic acid (pH 3.4) and ammonium acetate before injection. The applied voltage was set at −30 kV. The binary pump was set to deliver 8 μL of 5 mM ammonium acetate in water : methanol (50 : 50) as a sheath liquid to the capillary tip. The mass spectrometer was scanned throughout 300–1000 mass range to accommodate analysis of the various nucleotides. Analytes were quantified using the ratio of peak area counts of the 13C-labeled internal standard pseudo molecular ion to that of the analyte.

Calculation of free [Mg2+] and cytosolic phosphorylation potential

Free [Mg2+] was calculated from the measured values of citrate and isocitrate (Veloso et al. 1973) using the following equation:

  • display math

where, inline image is the ratio of measured citrate to isocitrate.

The cytosolic phosphorylation potential ([ATP]/([ADP) × [Pi])) was calculated by the following method (Bergman et al. 2010):

  • display math

where K′GG-LDH-TPI is defined as

  • display math

K′GG-LDH-TPI for intracellular pH of 7.2 and the calculated free [Mg2+] was calculated from Kionic GG-LDH-TPI using the f functions, which define the changes in the equilibrium constant with ionic strength (Bergman et al. 2010). Briefly,

  • display math

K′ionic GG-LDH-TPI was calculated using previously published K′ GG-LDH-TPI data for given pH and ionic strength using the f functions as previously defined (Bergman et al. 2010).

Data analysis and statistical treatment

Data analyses were conducted using Microsoft Excel program. Data are presented as mean ± standard error of five samples. Two-tailed Student's t-test was used to assess the significance of difference.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The microwave-treated brains were wholly intact having complete structural integrity, whereas freeze-blown brains were compacted as a frozen disc of brain tissue. Powders from either set of brain tissue produced by grinding under liquid N2 were indistinguishable and appeared pinkish white.

To determine whether isoflurane anesthesia exerted some independent effect on the concentration of brain nucleotides or determinants of free [Mg2+], a subset of rats were anesthetized prior to freeze blowing and metabolite concentrations were compared to tissue samples from animals not receiving the anesthetic. There were no differences in the concentration of the adenosyl nucleotides or in the citrate and isocitrate concentrations in brains of animals with or without exposure to isoflurane (Table 1). Thus, brief exposure (5 min) to isoflurane prior to microwave irradiation is not likely to have had an effect on nucleotide cleavage or in free [Mg2+].

Table 1. Concentrations of measured total brain adenine nucleotides and citrate and isocitrate in freeze-blown brains in animals with and without isoflurane
NucleotideFreeze blown no isofluraneFreeze blown + isofluranep value
  1. a

    NS indicates not significant.

  2. Values are reported in μmoles/g wet weight ± SEM, where = 5 in each group.

  3. The significance of the difference between the means as judged by Student's t-test is reported.

ATP3.18 ± 0.193.27 ± 0.11aNS
ADP0.21 ± 0.030.20 ± 0.01NS
AMP0.030 ± 0.0080.024 ± 0.006NS
Citrate0.180 ± 0.0200.22 ± 0.05NS
Isocitrate0.010 ± 0.0010.012 ± 0.003NS

Consistent with previous studies, the phosphorylated glycolytic intermediates Fru-DP, DHAP, and 3PG were elevated in the microwaved brains compared with the freeze-blown tissue (Table 2) (Miller and Shamban 1977). However, the concentrations of pyruvate and L-lactate were not different between the groups, indicating that the microwave irradiation was effective in preventing post-mortem anoxic changes.

Table 2. Concentrations of measured total brain glycolytic intermediates and the cytosolic redox potential in microwaved versus freeze-blown brains
Glycolytic intermediateMicrowaveFreeze blownp value
  1. a

    NS indicates not significant.

  2. Values are reported in μmoles/g wet weight ± SEM, where = 5 in each group.

  3. The significance of the difference between the means as judged by Student's t-test is reported.

Glu-6-phosphate0.117 ± 0.0210.139 ± 0.0176aNS
Fru-1,6-bisphosphate0.011 ± 0.0020.007 ± 0.0010.044
Phosphoenol pyruvate0.013 ± 0.0010.010 ± 0.001NS
Dihydroxyacetone phosphate0.040 ± 0.0040.017 ± 0.0030.002
3-Phosphoglycerate0.023 ± 0.0050.016 ± 0.0010.005
Pyruvate0.081 ± 0.0070.085 ± 0.011NS
l-Lactate1.45 ± 0.171.44 ± 0.15NS
Cyto free [NAD+]/[NADH] from the Lactate DH reaction337 ± 53341 ± 43NS

Importantly, the concentration of ATP was 30% lower in the microwaved brains, whereas the concentration of ADP and AMP were higher (Table 3) compared with freeze-blown brains. Lower concentrations of brain ATP under microwave irradiation have also been reported by other investigators (Veech et al. 1973; Miller and Shamban 1977). Although, the total amount of GTP was not different between the groups, GDP was significantly greater in microwave-irradiated tissue. The phosphocreatine (PCr) content of the brains of the two groups was similar, indicating that the microwave irradiation was effective in inactivating brain creatine kinase, as has been previosly reported (Nelson 1973).

Table 3. Concentrations of measured total brain adenine, guanine, and creatine nucleotides in microwaved versus freeze-blown brains
NucleotideMicrowaveFreeze blownp value
  1. Values are reported in μmoles/g wet weight ± SEM, where = 5 in each group.

  2. The significance of the difference between the means as judged by Student's t-test is reported.

  3. NS indicates not significant.

ATP2.25 ± 0.213.18 ± 0.190.01
ADP0.52 ± 0.050.21 ± 0.030.0003
AMP0.068 ± 0.0080.030 ± 0.0080.009
GTP0.36 ± 0.030.35 ± 0.05NS
GDP0.040 ± 0.0020.027 ± 0.0050.04
P-Creatine3.27 ± 0.173.41 ± 0.15NS
Creatine5.25 ± 0.225.81 ± 0.20NS
[ATP]/([ADP] × [Pi]) from creatine kinase / Pi M−139 930 ± 270022 440 ± 16800.001
∆G′ ATP from creatine kinase kJ/mole −59.5 ± 0.17−59.2 ± 0.17NS

The concentrations of the Krebs cycle intermediates were also differentially affected by microwave-irradiation compared with the freeze-blowing procedure (Table 4). The citrate concentration was 1.7-fold greater in microwave-irradiated brains, whereas the isocitrate content was not different between the two groups (Table 4). Citrate is a major intracellular metabolite binding Mg2+ (Veloso et al. 1973). Hydrolysis of ATP to ADP and AMP by microwave irradiation resulted in the liberation of free [Mg2+] into the intracellular space, thereby raising the free [Mg2+], as determined from the ratio of citrate to isocitrate (Veloso et al. 1973), from a typical value of 0.53 mM in freeze-blown brain to a value of 2.9 mM in microwave-treated brains (Table 4, line 10).

Table 4. Concentrations of measured total brain Krebs cycle metabolites and the calculated Free [Mg2+] in microwaved versus freeze-blown brains
Krebs cycle metaboliteMicrowaveFreeze blownp value
  1. a

    NS indicates not significant.

  2. Values are reported in μmoles/g wet weight ± SEM of = 5 in each group.

  3. The significance of the differences between the means as judged by Student's t-test is reported.

Citrate0.480 ± 0.0700.180 ± 0.0200.006
Isocitrate0.010 ± 0.0010.010 ± 0.001aNS
α-Ketoglutarate0.123 ± 0.0070.149 ± 0.0110.080
Succinate0.081 ± 0.0990.116 ± 0.0070.010
Fumarate0.091 ± 0.0080.127 ± 0.0100.03
Malate0.219 ± 0.0200.174 ± 0.0080.07
Calc Oxaloacetate0.019 ± 0.0040.023 ± 0.004NS
CoQ/CoQH2 from [Succ]/[Fum]0.007 ± 0.0010.006 ± 0.000NS
[Citrate]/[Isocitrate]53 ± 2.5715 ± 0.740.001
Calculated Free [Mg2+] from [Cit]/[Isocit] (mM)2.9 ± 0.480.53 ± 0.070.001
Free cytosolic [NADP+]/[NADPH] from isocitrate DH0.019 ± 0.0040.023 ± 0.004NS
Free cytosolic [NADP+]/[NADPH] malic enzyme0.022 ± 0.0030.028 ± 0.004NS

Consistent with earlier reports, the brain concentrations of α-ketoglutarate were not different between the two treatment procedures (Miller and Shamban 1977). However, the concentrations of both succinate and fumarate were about 30% lower in microwave-irradiated samples (Table 4). As the succinate/fumarate ratio was similar using either method, the calculated free [CoQ]/[CoQH2] ratios (Bergman et al. 2010) were also no different between the two groups. Also, the concentrations of malate and calculated oxaloacetate (Table 4) were similar in both groups of brains. The free cytosolic [NADP+]/[NADPH] calculated from the components of the isocitrate dehydrogenase reaction were similar in both sets of brain tissues, having a typical value of about 0.02. Moreover, the values of the free [NADP+]/[NADPH] ratios calculated from the components of the malic enzyme reactions agreed with those values obtained from the isocitrate dehydrogenase reaction (Krebs and Veech 1969).

As a result of the nearly five-fold elevation in the free [Mg2+] in the microwave-treated brains the K'eq of ATP hydrolysis decreased from 388 000 to 244 000 M (Table 5). This also decreased the ∆Go′of ATP hydrolysis from −33.3 kJ/mole in the freeze-blown tissue to −32.1 kJ/mole in the microwaved samples.

Table 5. Derived nucleotide ratios in microwaved versus freeze-blown brains
Calculated valuesMicrowaveFreeze blownp value
  1. Values are reported in μmoles/g wet weight ± SEM of = 5 in each group.

  2. The significance of the difference between the means as judged by Student's t-test is reported.

K′eq ATP hydrolysis M244 000 ± 1800388 000 ± 23000.0001
∆Go′ ATP hydrolysis kJ/mole−32.09 ± 0.02−33.28 ± 0.140.0000
K′GG-LDH-TPI M−11 217 000 ± 53 000579 000 ± 36 0000.0000
Free Cyto [ATP]/([ADP] × [Pi]) M−1 from [Mg2+]131 400 ± 30 50035 060 ± 40500.014
RT ln([ADP][Pi]/[ATP]) kJ/mole−30.2 ± 0.6−27.0 ± 0.30.001
∆G′ ATP hydrolysis (sum of lines 2 and 5 above) kJ/mole−62.3 ± 0.6−60.3 ± 0.30.013

The cytosolic phosphorylation potential can be calculated from the measured concentrations of pyruvate, lactate, DHAP, and 3PG using the equation below:

  • display math(1)

The equilibrium constant K′GG-(TPI+LDH) must be adjusted for the substrate binding of [Mg2+] (Bergman et al. 2010). From the calculation of the cytosolic phosphorylation potential above, the ∆G′ of ATP hydrolysis can be determined in tissue using the following equation

  • display math(2)

The increase in calculated free [Mg2+] in the microwave-irradiated tissue elevated the equilibrium constant K′GG-(TPI+LDH) by over two-fold (1 217 000 in the microwaved tissue vs. 579 000 in the freeze-blown tissue, Table 5). The level of DHAP in the microwaved samples was more than twice of that in the freeze-blown tissue (Table 2), whereas the concentration of 3-PG was only about 40% greater, reflecting an increase in the combined KG+G constant. The free cytosolic phosphorylation potential, [ATP]/([ADP] × [Pi]) calculated from eqn 1 and the ∆G′ of phosphorylation, calculated from eqn 2 above were therefore higher in the microwaved tissue, leading to an elevation of the total ∆G′ of ATP hydrolysis in the tissue from −60.3 kJ/mol in the freeze-blown brains to about −62.3 kJ/mol in the microwaved brains (line 6, Table 5).

The concentrations of four major neurotransmitters (aspartate, glutamate, glutamine, and γ-amino butyric acid) in the tissues were similar in both freeze-blown and microwave-treated tissues (Table 6).

Table 6. Concentrations of neurotransmitters: aspartate, glutamate, glutamine, and γ-amino butyric acid (GABA) in microwaved versus freeze-blown brains
Analyte MicrowaveFreeze blownp value
  1. a

    NS indicates not significant.

  2. Values are reported in μmoles/g wet weight ± SEM, where = 5 in each group.

  3. The significance of the difference between the means as judged by Student's t-test is reported.

Aspartate1.42 ± 0.131.47 ± 0.12aNS
Glutamate10.92 ± 0.909.46 ± 0.53NS
Glutamine6.40 ± 0.715.36 ± 0.47NS
γ-amino butyric acid1.44 ± 0.121.37 ± 0.29NS

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We compared two state-of-the-art methods to prepare rat brains for subsequent metabolite analysis. The microwave technique offers the advantage to measure certain metabolites from different brain regions. Our results show that microwave irradiation does not affect the estimations of cytosolic or mitochondrial redox states. The concentrations of several neurotransmitters were also similar between the two methods, suggesting that microwave irradiation may be suitable when determining regional concentrations of these metabolites. In addition, as most enzymes are denatured by microwaves, this implies that microwaved tissue cannot be used to determine differences in enzyme activities. Freeze blowing rapidly freezes the cranial contents into a thin disc between two aluminum discs pre-cooled in liquid nitrogen. The advantage of this method is that the speed at which tissue is frozen preserves the in vivo concentrations of metabolites. However, it is not possible to conduct regional brain analyses and the technique cannot be used to determine intracellular calcium because of sample contamination with bone fragments. This technique allows one to measure both the metabolite levels and enzyme activities. However, as the enzymes remain active, it is necessary to keep the sample below −20°C prior to extraction.

Our investigation encompassed a broad range of metabolites from glycolytic and the TCA cycle pathways through high-energy nucleotides and various neurotransmitters. In addition, we explored the consequences of increased ATP hydrolysis on intracellular free [Mg2+] and ensuant thermodynamic parameters. Our results indicate that ATP hydrolysis and release of Mg ions into intracellular spaces in the microwaved brains affect equilibrium constants of certain metabolic reactions and grossly distort the determination of cytosolic phosphorylation potential.

It is common to anesthetize animals prior to the microwave procedure, which may alter the concentration of metabolties in tissues. Studies have shown that prolonged anesthesia significantly reduces brain glucose metabolism (Biebuyck and Hawkins 1972) and the metabolic rate (as determined by oxygen uptake) (Brunner et al. 1975). However, isoflurane anesthesia for a 5-min period did not affect the concentrations of cerebral nucleotides or the citrate and isocitrate concentrations in freeze-blown tissue. Even long-term exposure to high concentrations of isoflurane did not reduce cerebral ATP levels as reported in a previous study (Newberg et al. 1983). Therefore, the reduced ATP levels in the microwaved brains presented here were likely because of microwave effects and not anesthesia.

Previous studies have reported lower ATP levels in the brains of rats treated with microwaves, compared with literature values from freeze-blown brains (Veech et al. 1973; Miller and Shamban 1977). The cause of this reduction may include direct effect of microwaves on ATP (Sun et al. 1988), or incomplete deactivation of adenylate kinase (Nelson 1973). The decrease in [ATP] in the microwaved brains in this study was on the order of 0.93 μmol/g tissue, whereas the increase in the calculated intracellular free [Mg2+] was about 2.4 mM. Having a stoichiometry of unity, the increase in the calculated free [Mg2+] was greater than that which could be accounted for by the release of Mg2+ from the hydrolysis of ATP alone, indicating the existence of another potential source of bound Mg2+. Although, some of the released free [Mg2+] may subsequently bind to either ADP or citrate, the binding constants for these associations are an order of magnitude less than that of ATP (Veech et al. 1994).

Increased free [Mg2+] had further effects on other intracellular reactions. One reaction intimately tied to the intracellular free [Mg2+] is the aconitase reaction that affects the citrate:isocitrate ratio (Veloso et al. 1973). The elevation of free [Mg2+] in the microwave-treated group was associated with about three-fold elevation of citrate, but had no affect on the concentration of isocitrate—a metabolite closely associated with the redox state of the NADP system. The increased citrate:isocitrate ratio also indicated that citrate synthase, aconitase, or IDH may not be rapidly inhibited by microwave irradiation. Specifically, microwave irradiation has been shown to increase the binding of the chaperone, alpha-crystalline, to citrate synthase, stabilizing the protein and maintaining enzyme activity compared with direct thermal denaturation (George et al. 2008). Thus, microwaves may also modulate enzyme activity through enhancement of protein–protein interactions. Also, heat-resistant enzymes may partially retain their activity following microwave irradiation, especially if the exposure is brief. Microwaved brains retained about 10% of the adenylate kinase activity (myokinase) following irradiation (Nelson 1973). Similarly, brains of mice irradiated for 0.4 s retained significant glutaminase activity. No measurable activity, however, was detected in brains irradiated for 0.8 s (Kobayashi et al. 1989). As animals were irradiated for 1.3 s in this study, glutaminase activity appeared to be eliminated as the concentrations of glutamate and glutamine in microwaved and freeze-blown brains were simliar. The other two neurotransmitters, aspartate and γ-amino butyric acid, were also of similar concentration in the freeze-blown and microwaved brains. These results suggest that microwaved tissue may be appropriate when measuring regional neurotransmitter levels.

The higher concentrations of the glycolytic intermediates Fru-1,6-bisphosphate, DHAP, and 3-PG was most likely owing to the increased free [Mg2+] in the microwaved brains, which is in accord with the effect of magnesium ion concentrations on the equilibrium constants of these reactions. The measured lactate and pyruvate concentrations and the derived cytosolic free [NAD+]/[NADH] ratios, however, were no different between groups, which is also in accord with the lack of sensitivity that free [Mg2+] has on the dehydrogenase equilibrium constants, or perhaps that LDH is rapidly deactivated by microwave (Nelson 1973).

Results from this study demonstrate that for brain tissue in particular, a comprehensive metabolic profile should include a determination of free magnesium in the analysis to accurately describe the energy state of the system, and to account for alterations of concentrations of metabolites from the in vivo state. Wider use of methods which measure the free [Mg2+] in tissue should be explored and an alternate means for its determination developed.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The authors would like to thank Professor Eric Murphy for the use of the microwave apparatus. This publication was made possible by grants to JDG from the NCRR, a component of the NIH (2P20RR017699), and from NINDS (R01NS065957). The authors have no conflict of interest to report.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References