Freeze-clamping

The ratio [NAD+]/[NADH] appears to be maintained at a relatively constant value and in equilibrium with a series of different reduced and oxidized substrate pairs. Thus, it was observed that in the cytoplasm of rat liver cells, the dehydrogenations catalyzed by lactate dehydrogenase, sn-glycerol 3-phosphate dehydrogenase, and malate dehydrogenase are all at equilibrium with the same ratio of [NAD+]/[NADH].166 In one experiment rat livers were removed and frozen in less than 8 s by "freeze-clamping" (Section L,2) and the concentrations of different components of the cytoplasm determined167 the ratio [NAD+] / [NADH] was found to be 634, while the ratio of [lactate]/[pyruvate] was 14.2. From these values an... 

Fig. 4.5 (A) Protocols for non-preconditioned and preconditioned hearts. Arrows indicate times of freeze-clamping. (B) cAMP content of hearts during sustained ischemia. P < 0.05 versus non-PC.

At the end of the experiment, the rats are terminally anesthetized, the abdomen is opened, and terminal blood collection is performed from the vena cava caud. or the aorta abdominalis for determination of metabolic blood parameters and insulin. A part of the liver is freeze clamped immediately as well as a part (about 1 gr) of skeletal muscle (e.g. M. gastrocne-micus). The frozen tissue is stored in liquid nitrogen for subsequent determinations of intrahepatic and intramuscular concentrations of glycogen, glucose-6-phosphate (G6P) and ATR In liver samples hepatic... 

Nonequilibrium reactions can be detected by determining metabolite concentrations in the tissue of interest. Conventionally, a tissue sample is rapidly frozen by compression between metal plates that have been cooled to 77 K by immersion in liquid nitrogen (freeze-clamping). This procedure rapidly halts any enzymatic processes that might alter the metabolite concentrations the concentrations can then be determined by enzymatic or chemical assays. Recently, 31P-NMR spectroscopy has shown considerable value in measuring the concentrations of such metabolites as ATP, ADP, AMP, phosphate, and phosphocreatine in living cells or tissues. 

The cytosolic concentrations of pyruvate and lactate can be obtained from the results of freeze-clamping experiments. Hence, from the known [H + ] of the cytosol and the known equilibrium constant (Ke)... 

Fig. 2. ESR spectra of (A) ground heart tissue and (B) chopped heart tissue. Heart tissues were freeze-clamped at liquid N2 following aerobic perfusion. Details are given in ref. [75] (reproduced...

Fig. 3. ESR spectra of control, (A) aerobically perfused, (B) ischemic, (C) postischemic aerobically reperfused, and (D) postischemic anaerobically reperfused heart, freeze-clamped with the frozen tissue chopped prior to recording of spectra. Reproduced from ref. [75], with permission.

Davies, S.P., Carling, D., Munday, M.R., and Hardie, D.G., 1992, Diurnal rhythm of phosphorylation of rat liver acetyl-CoA carboxylase by the AMP-activated protein kinase, demonstrated using freeze-clamping. Effects of high fat diets. Eur. J. Biochem. 203 615-623. 

However, the number of reports are few due to the heavy reliance for its quantitation on a bioassay involving Ca2+ release from sea urchin egg and other microsomes. The procedure involves several steps. The first is to freeze-clamp tissues to prevent metabolism of cADPR by cADPR hydrolases (see Section... 

In one experiment rat livers were removed and frozen in less than 8 s by "freeze-clamping" (Section L,2) and the concentrations of different components of the cytoplasm determined the ratio [NAD+] / [NADH] was found to be 634, while the ratio of [lactate]/[pyruvate] was 14.2. From these values an... 

Fractionation of freeze-clamped, freeze-dried tissue in organic solvents [31]. 

Previous studies with purified RuBisCO have shown that the functioning forms of RuBisCO in the primary carboxylase reaction can be deduced from the curve of the progress of the second reaction in the presence of 0.5 to 1.0 mM RuBP (Fig. 2) [1,2]. The leaves of spinach and radish were freeze clamped at liq. N2 -temperature after reaching steady photosynthesis in an atmosphere containing various concentrations... 

The amount of 5-phospho-a D-ribosyl pyrophosphate (PRPP) in chicken liver was measured using the procedure of LaLanne and Henderson (9). The soluble purine base levels were determined using freeze-clamped liver that was extracted with perchloric acid, centrifuged and then boiled to hydrolyze the soluble nucleotides to the free bases. The bases were separated and quantitated on a Partisil pressure liquid chromatography column (Whatman). 

Measurements of metabolites in freeze-clamped tissue samples, taken at various time intervals from perfused livers of fed rats (Table I), revealed that the concentration of ATP decreased... 

The metabolite concentrations, expressed as mol per g of tissue wet weight, were determined in freeze clamped biopsies that were taken after the addition of 25 mM ethanol to the perfusion medium, at the times indicated. Values shown are means S.E.M. of 3 to 4 experiments. c<-G-P = a-glycerol-P. 

Fig. 2. Influence of 2.5 x 10 M coformycin on the catabolism of the adenine nucleotides induced by ethanol in the perfused liver of a fed rat. The purine catabolites in the medium were determined by thin-layer chromatography. The concentration of ATP was measured in freeze-clamped biopsies. Further explanations are given in the text.

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