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Biomedical subjects

B J Fuller

Publications and source records attributed to B J Fuller.

At least 109 records · Page 6Linked to original sources
PubMed1989

Biochemical consequences of reflushing hypothermically-stored liver with fresh cold perfusate. Studies on rat liver using 31P NMR spectroscopy.

The metabolic response of the rat liver to flushing and reflushing with Marshall's solution at pH 7.2 or pH 7.8 has been studied by 31P nuclear magnetic resonance spectroscopy. The changes in intracellular pH, inorganic phosphate, ATP and phosphomonoesters have been determined from the 31P spectra. We show that the intracellular pH at any stage of the flushing protocol is largely independent of the pH of the medium when using these solutions. However, we demonstrate that there are differences between the efficiency of the two solutions in respect of the rates of hydrolysis of ATP and accumulation of phosphomonoesters. There were also differences in the response of the livers upon reflushing--those livers reflushed at pH 7.2 resynthesized ATP from a lower initial concentration to achieve ATP concentrations similar to those restored in livers reflushed at pH 7.8. These trends were mirrored in the responses of the phosphomonoester peaks (which contain a contribution from AMP). We conclude that short-term control of liver metabolism during hypothermia is possible by use of solutions of different pH, but that for longer-term storage, other approaches may be necessary to maintain metabolic integrity.

Animals↗
PubMed1989

The application of nuclear magnetic resonance spectroscopy to assess viability in stored tissues and organs.

The use of nuclear magnetic resonance (NMR) spectroscopy to assess metabolic viability in organ preservation is discussed. A brief coverage of the physical principles involved and the biochemical information available from NMR spectroscopy is given. We also present the advantages and disadvantages of the method and outline the future possibilities of the technique in relation to organ preservation.

Animals↗
PubMed1989

The response of liver to lactobionate/raffinose (University of Wisconsin--UW) solution during hypothermic preservation: a study using 31phosphorus nuclear magnetic resonance.

31P nuclear magnetic resonance spectroscopy has been used to study rat livers following flushing with the University of Wisconsin (UW) lactobionate/raffinose solution (N. Jamieson, R. Sundberg, S. Lindell, J. Southard, and F.O. Belzer, Cryobiology 24, 573-574, 1987; M. Kalayoglu, H. Sollinger, R. Stratta, A. D'Alessandro, R. Hoffman, J. Pirsch, and F. O. Belzer, Lancet 1, 617-619, 1988). These studies have revealed that despite the improved storage properties that have been reported for this solution, hepatic ATP and ADP declined at a rate similar to that seen in the more commonly used Marshall's or Collins' solutions. However, there was a significant inhibition of the developing acidosis, such that by 5 hr postflush, the intracellular pH was 7.17 +/- 0.06 (mean +/- SD, n = 5) compared to 6.90 +/- 0.06 for Marshall's solution (4 hr postflush) and 6.94 +/- 0.04 for Collins' solution (4 hr postflush). This did not appear to be due to a buffering effect of the solution, as this was found to be relatively low, but was probably due to a modification of hepatic metabolism caused by the solution itself.

Animals↗
PubMed1989

Studies on cryoprotectant equilibration in the intact rat liver using nuclear magnetic resonance spectroscopy: a noninvasive method to assess distribution of dimethyl sulfoxide in tissues.

Nuclear magnetic resonance (NMR) spectroscopy was used in the study of rat livers following flushing with a clinically used preservation solution containing either 12 or 30% (v/v) Me2SO. The extent of equilibration of Me2SO in the tissue after 10-15 min of perfusion with Me2SO and again after subsequent washout with Me2SO-free medium was assessed by 1H NMR spectroscopy. 31P NMR spectroscopy was used to follow the changes in ATP, ADP, inorganic phosphate, and tissue pH. The data show that 1H NMR spectroscopy can be used as a sensitive and rapid method of assessing the equilibration and concentration of compounds such as Me2SO, since these compounds are likely to be present at concentrations greatly in excess of other constituents of the medium and will therefore give rise to strong, easily detected signals. At the same time, 31P NMR spectroscopy can be used to monitor the metabolic status of the tissue reflected in the levels of ATP, ADP, and inorganic phosphate, as well as being a noninvasive monitor of intracellular pH. The possibility of determining the tissue pH in the presence of solutes such as Me2SO is discussed.

Animals↗
PubMed1989

Protection against oxidative damage in cold-stored rabbit kidneys by desferrioxamine and indomethacin.

The storage of rabbit kidneys in hypertonic citrate solution at 0 degree C for 48-72 hr of cold ischemia resulted in oxidative damage to membranes as measured by the in vitro formation of two markers of lipid peroxidation (Schiff's base and thiobarbituric acid (TBA)-reactive material). This damage was further increased when the organs were autografted and reperfused for 60 min. The intravenous (iv) administration of desferrioxamine (a powerful iron-chelating agent) prior to the removal of the kidneys reduced the production of Schiff's bases and TBA-reactive material to low levels in the cortex of stored kidneys and decreased these measures of lipid peroxidation in the medulla by approximately 50%. Intravenous administration of indomethacin (a cyclooxygenase inhibitor) had no effect on the rate of lipid peroxidation in the renal cortex, but significantly reduced the formation of TBA-reactive material and Schiff's bases in the medulla of kidneys following storage for 72 hr. The existence of two separate pathways of lipid peroxidation (one iron-catalyzed and the other cyclooxygenase-catalyzed) in the medulla of stored kidneys was further confirmed when administration of desferrioxamine and indomethacin together resulted in significantly greater protection against lipid peroxidation than when these compounds were administered singly. The value of this combination of agents for protecting kidneys against the damage due to cold ischemia followed by reperfusion was further suggested by a trend toward improved long-term survival of the animals following replantation of the stored kidneys.

Animals↗
PubMed1989

The bioenergetics of mitochondria after cryopreservation.

The functional characteristics of rat liver mitochondria after cryopreservation with and without the addition of the cryoprotectant dimethyl sulfoxide (Me2SO) were evaluated. As criteria of functional integrity, polarographic measurements of substrate-linked oxygen consumption and luminescent assay of adenosine triphosphate (ATP) synthesis were considered before and after cryopreservation. The results demonstrated that mitochondrial damage after freezing was indicated by the polarographic studies but was not evident when ATP synthesis was considered. Me2SO present during cryopreservation was partially protective for mitochondrial substrate-linked oxygen consumption; however, simple exposure to and dilution from Me2SO effected some changes in mitochondrial function.

Adenosine Triphosphate↗
PubMed1989

Oxidative damage to kidney membranes during cold ischemia. Evidence of a role for calcium.

Storage of rabbit kidneys at 0 degrees C for periods of 72 hr after flushing with hypertonic citrate solution, or 24 hr when flushed with isotonic saline, resulted in significant increases in Schiff base and thiobarbituric acid-reactive markers of lipid peroxidation in vitro. The extent of lipid peroxidation was not significantly altered by addition of verapamil (100 microM), a Ca++ channel blocking agent, or calcium 1 mM (CaCl2) to the HCA storage solution. In contrast, verapamil significantly reduced the extent of lipid peroxidation in kidneys stored in saline solution, and a significant increase in oxidative damage occurred when CaCl2 was added to this storage solution. Thus the extent of lipid peroxidation in kidneys stored in saline was significantly mediated by extracellular Ca++, whereas in HCA this was probably chelated by the large excess of citrate (55 mM) in this medium that prevented, or at least slowed, its entry into the renal cells. Lipid peroxidation was however significantly increased in kidneys stored in both HCA and saline solutions by addition of the calcium ionophore A23187 (10 microM) or the polysaccharide dye ruthenium red (5 microM) that inhibits mitochondrial uptake of Ca++. This strongly suggested that altered intracellular Ca++ homeostasis during the storage period played an important role in the development of oxidative damage to kidneys stored in both these media.

Animals↗
PubMed1989

Allopurinol inhibits lipid peroxidation in warm ischaemic and reperfused rabbit kidneys.

Rabbit kidneys were subjected to 120 min of warm ischaemia or to 120 min of warm ischaemia followed by 60 min reperfusion with blood in vivo before being removed, homogenised and incubated at 37 degrees C for 90 min. Lipid extracts were obtained and monitored for Schiff base (fluorescence emission 400-450 nm, excited at 360 nm), thiobarbituric acid (TBA)-reactive material (emission 553 nm, excited at 515 nm) and diene conjugates (absorbance at 237 nm). Samples removed before incubation were assayed for reduced glutathione (GSH) and oxidised glutathione (GSSG) to provide an index of glutathione redox activity (GSH:GSSG). Allopurinol injected systemically i.v. (a) 15 mins before kidneys were clamped, (b) 15 mins before they were reperfused or (c) as two injections (before clamping and before reperfusion) significantly inhibited these biochemical markers of lipid peroxidation. Administration before reperfusion had a markedly more pronounced effect than when allopurinol was given before warm ischaemia only. It is concluded that allopurinol is probably effective because of its ability to inhibit xanthine oxidase and consequently lipid peroxidation during reperfusion rather than by preventing loss of purine nucleotides from hypoxic cells during ischaemia.

Allopurinol↗
PubMed1989

The effects of iron-mediated oxidative stress in isolated renal cortical brush border membrane vesicles at normothermic and hypothermic temperatures.

Experiments on renal cortical brush border membrane vesicles have been undertaken in order to assess the involvement of iron in oxidative stress at physiological temperatures and under conditions of hypothermia. A decrease in temperature stimulated iron-induced lipid peroxidation. The results are discussed in relation to the role of the oxidation state of the iron and iron(II)/iron(III) ratios in the initiation of peroxidative events.

Animals↗
PubMed1989

The importance of iron, calcium and free radicals in reperfusion injury: an overview of studies in ischaemic rabbit kidneys.

An overview of a series of experiments attempting to link iron and calcium redistribution and release of free fatty acids with falls in pH and adenine nucleotide levels during cold storage of rabbit kidneys is presented. The data reviewed strongly suggest that these events are inextricably linked to subsequent reperfusion injury. Circumstantial evidence incriminating iron was provided by experiments showing that iron chelation decreased reperfusion injury after warm (WI) and cold ischaemia (CI) in rat skin flap and rabbit kidney models. Evidence for a role for calcium was provided when it was found that a calcium channel blocking agent added to the saline flush solution before storage inhibited lipid peroxidation, whereas chemicals which caused release or influx of calcium into the cell exacerbated oxidative damage. Additional involvement of breakdown products of adenine nucleotides was suggested by the protection from lipid peroxidation afforded by allopurinol. Involvement of calcium-activated phospholipase A2 was strongly suggested by increases in free fatty acids during cold storage and both this increase and lipid peroxidation were inhibited by addition of dibucaine to the storage solution.

Animals↗
PubMed1988

Lipid peroxidation in hepatocyte cell cultures: modulation by free radical scavengers and iron.

Rat hepatocytes were isolated and then maintained in serum-free cell culture medium for 24 h. The amount of malondialdehyde (MDA) accumulated in the medium was assayed and used as a measure of lipid peroxidation. The activity of lactate dehydrogenase (LDH) and urea were measured in the medium and used as indicators of hepatocellular viability and function. The effects of iron; desferrioxamine mesylate (Desferal), an iron chelator; and mannitol, a hydroxyl free radical scavenger were investigated. The addition of iron, Fe2 resulted in a three-fold increase in the levels of MDA. Desferal inhibited the production of MDA and blocked the effect of Fe2+. Neither iron nor Desferal had any effect on LDH or urea levels. Mannitol had no effect on MDA or urea production, but caused a 4 to 8-fold increase in the LDH levels in the medium. The results show that iron is involved in the mechanism of lipid peroxidation in hepatocyte cultures but suggest that as a pathologic event lipid peroxidation is not expressed in terms of viability during the first 24 h of hepatocyte culture.

Animals↗
PubMed1988

Functional testing of hepatocytes following their recovery from cryopreservation.

Various tests of function have been suggested for assessing hepatocytes recovered from cryopreservation. In this study we have investigated hepatocyte attachment during tissue culture and cellular density in order to assess function and compared them with two classical dye exposure tests. The ability of hepatocytes to exclude trypan blue dye (TB) and metabolize fluorescein diacetate (FDA) was demonstrated. In populations of freshly prepared hepatocytes 88.07% were able to exclude TB and 87.31% were able to metabolize FDA. However in populations of hepatocytes recovered after cryopreservation using 1.5 M dimethyl sulfoxide as cryoprotectant only 33.44% were able to exclude TB and 31.59% able to metabolize FDA. Both of these tests gave the same estimate of functional ability. Density gradient centrifugation of hepatocytes on Percoll 400 (Pharmacia, Uppsala, Sweden) separated two populations of hepatocytes; one (density ca.1.07 g/ml Percoll) in which most of the cells were able to exclude TB and the second (density ca. 1.02 g/ml Percoll) in which they were stained blue. The dense population was highly enriched in dye-excluding hepatocytes: freshly prepared hepatocytes, 92.4%, and cryopreserved hepatocytes, 88.66%. When samples of these cells (2 x 10(6) dye-excluding cells per dish) were tested for their ability to attach to tissue culture dishes only 17.28% of the cryopreserved hepatocytes were able to attach compared to 55.28% of the freshly prepared cells. We conclude that cryopreservation of hepatocytes produces a population of cells which are not metabolically identical to a population of freshly prepared hepatocytes even though they appear to have the same buoyant density and dye-excluding capabilities.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Osmotic response of oocytes using a microscope diffusion chamber: a preliminary study comparing murine and human ova.

The hydraulic conductivity (Lp) of the plasma membrane determines how cells respond to the stresses of dehydration encountered during cryopreservation. We have used a microscope diffusion chamber which allows for direct real-time observation of the dynamic osmotic response of individual cells in microvolume suspension to compare the Lp of murine and human unfertilized ova. In this system, the response of an individual cell to the induced osmotic imbalance is documented via a series of photomicrographs or videotape; from these data the Lp can be computed. Donated human preovulatory oocytes were compared with macroscopically normal human ova, 43 hr after insemination, which had failed to fertilize (Ff) and with murine ova collected 13 hr post human chorionic gonadotropin injection. The permeability coefficients were 0.65 +/- 0.43, 0.84 +/- 0.39, and 0.36 +/- 0.07 micron3/micron2/atm/min. The results suggest that it may be possible to use Ff ova for experiments to design suitable cryopreservation procedures.

Animals↗
PubMed1988

Transplantation of isolated hepatocytes. A review of current ideas.

Over the past decade there has been an increase in interest in studies of transplantation of isolated hepatocytes. The survival of hepatocytes in sites distant from the normal hepatic circulation has provided insights into cell proliferation and phenotypic expression of liver-specific functions. The possibility of treating liver insufficiency in disease has also been investigated. The present review is intended to give an account of the development of methods for the study of hepatocyte transplantation, techniques for assessment of graft function and future possibilities for application of the technique.

Animals↗