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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.
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).
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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
|Nucleotide||Freeze blown no isoflurane||Freeze blown + isoflurane||p value|
|ATP||3.18 ± 0.19||3.27 ± 0.11||aNS|
|ADP||0.21 ± 0.03||0.20 ± 0.01||NS|
|AMP||0.030 ± 0.008||0.024 ± 0.006||NS|
|Citrate||0.180 ± 0.020||0.22 ± 0.05||NS|
|Isocitrate||0.010 ± 0.001||0.012 ± 0.003||NS|
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 intermediate||Microwave||Freeze blown||p value|
|Glu-6-phosphate||0.117 ± 0.021||0.139 ± 0.0176||aNS|
|Fru-1,6-bisphosphate||0.011 ± 0.002||0.007 ± 0.001||0.044|
|Phosphoenol pyruvate||0.013 ± 0.001||0.010 ± 0.001||NS|
|Dihydroxyacetone phosphate||0.040 ± 0.004||0.017 ± 0.003||0.002|
|3-Phosphoglycerate||0.023 ± 0.005||0.016 ± 0.001||0.005|
|Pyruvate||0.081 ± 0.007||0.085 ± 0.011||NS|
|l-Lactate||1.45 ± 0.17||1.44 ± 0.15||NS|
|Cyto free [NAD+]/[NADH] from the Lactate DH reaction||337 ± 53||341 ± 43||NS|
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
|Nucleotide||Microwave||Freeze blown||p value|
|ATP||2.25 ± 0.21||3.18 ± 0.19||0.01|
|ADP||0.52 ± 0.05||0.21 ± 0.03||0.0003|
|AMP||0.068 ± 0.008||0.030 ± 0.008||0.009|
|GTP||0.36 ± 0.03||0.35 ± 0.05||NS|
|GDP||0.040 ± 0.002||0.027 ± 0.005||0.04|
|P-Creatine||3.27 ± 0.17||3.41 ± 0.15||NS|
|Creatine||5.25 ± 0.22||5.81 ± 0.20||NS|
|[ATP]/([ADP] × [Pi]) from creatine kinase / Pi M−1||39 930 ± 2700||22 440 ± 1680||0.001|
|∆G′ ATP from creatine kinase kJ/mole ||−59.5 ± 0.17||−59.2 ± 0.17||NS|
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 metabolite||Microwave||Freeze blown||p value|
|Citrate||0.480 ± 0.070||0.180 ± 0.020||0.006|
|Isocitrate||0.010 ± 0.001||0.010 ± 0.001||aNS|
|α-Ketoglutarate||0.123 ± 0.007||0.149 ± 0.011||0.080|
|Succinate||0.081 ± 0.099||0.116 ± 0.007||0.010|
|Fumarate||0.091 ± 0.008||0.127 ± 0.010||0.03|
|Malate||0.219 ± 0.020||0.174 ± 0.008||0.07|
|Calc Oxaloacetate||0.019 ± 0.004||0.023 ± 0.004||NS|
|CoQ/CoQH2 from [Succ]/[Fum]||0.007 ± 0.001||0.006 ± 0.000||NS|
|[Citrate]/[Isocitrate]||53 ± 2.57||15 ± 0.74||0.001|
|Calculated Free [Mg2+] from [Cit]/[Isocit] (mM)||2.9 ± 0.48||0.53 ± 0.07||0.001|
|Free cytosolic [NADP+]/[NADPH] from isocitrate DH||0.019 ± 0.004||0.023 ± 0.004||NS|
|Free cytosolic [NADP+]/[NADPH] malic enzyme||0.022 ± 0.003||0.028 ± 0.004||NS|
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 values||Microwave||Freeze blown||p value|
|K′eq ATP hydrolysis M||244 000 ± 1800||388 000 ± 2300||0.0001|
|∆Go′ ATP hydrolysis kJ/mole||−32.09 ± 0.02||−33.28 ± 0.14||0.0000|
|K′GG-LDH-TPI M−1||1 217 000 ± 53 000||579 000 ± 36 000||0.0000|
|Free Cyto [ATP]/([ADP] × [Pi]) M−1 from [Mg2+]||131 400 ± 30 500||35 060 ± 4050||0.014|
|RT ln([ADP][Pi]/[ATP]) kJ/mole||−30.2 ± 0.6||−27.0 ± 0.3||0.001|
|∆G′ ATP hydrolysis (sum of lines 2 and 5 above) kJ/mole||−62.3 ± 0.6||−60.3 ± 0.3||0.013|
The cytosolic phosphorylation potential can be calculated from the measured concentrations of pyruvate, lactate, DHAP, and 3PG using the equation below:
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
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 ||Microwave||Freeze blown||p value|
|Aspartate||1.42 ± 0.13||1.47 ± 0.12||aNS|
|Glutamate||10.92 ± 0.90||9.46 ± 0.53||NS|
|Glutamine||6.40 ± 0.71||5.36 ± 0.47||NS|
|γ-amino butyric acid||1.44 ± 0.12||1.37 ± 0.29||NS|
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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.