Endoscopic measurements of canine colonic mucosal blood flow using hydrogen gas clearance.

We have examined the feasibility of hydrogen (H2) clearance for endoscopic measurements of colonic mucosal blood flow in anesthetized dogs. In 6 animals, measurements of H2 clearance did not differ significantly in different regions of the sigmoid colon and they were highly reproducible (p less than 0.001) on different days. In a total of 12 dogs, measurements of H2 clearance correlated closely with those obtained using radioactive microspheres under resting conditions and, in 4 dogs, during infusion of vasopressin (slope = 0.94, p less than 0.001). In 8 dogs, ligation of the major arteries supplying the sigmoid colon resulted in an acute 60% decrease in sigmoid mucosal blood flow (p less than 0.001); however, in 5 animals that survived the procedure, mucosal blood flow returned nearly to control levels as early as 3 days after operation. Endoscopic H2 clearance thus appears to be feasible for measuring mucosal blood flow in the colon. Serial measurements of H2 clearance may prove useful in characterizing the role of mucosal blood flow in the pathogenesis of various forms of human colonic disease.

Protective effect of exogenous phospholipid on aspirin-induced gastric mucosal injury.

The protective effects of exogenous phospholipid on aspirin-induced gastric mucosal injury were examined in a canine chamber model which provided two separate segments of mucosa supplied by a single vascular pedicle. In each dog, one segment was treated with a suspension of surface-active phospholipid, similar in composition to that normally present in the gastric mucosa, whereas the other segment served as the control. Pretreatment of the test segments significantly prevented aspirin-induced disruption of the mucosal barrier as evidenced by an increase in potential difference and a decrease in acid back-diffusion and sodium ion and potassium ion flux. These findings were associated with a marked reduction in the degree of mucosal injury. Our results support the recent hypothesis that surface-active phospholipid plays an important role in gastric mucosal defense against the damaging effects to luminal acid.

Insulin effects on methotrexate polyglutamate synthesis and enzyme binding in cultured human breast cancer cells.

Previous studies have demonstrated that insulin augments methotrexate transport and enhances its cytotoxicity to human breast cancer cells. We therefore investigated the effects of insulin on methotrexate polyglutamate synthesis and binding to dihydrofolate reductase (DHFR) in two human breast cancer cell lines, MCF-7 and MDA-MB-231. Cells were exposed to 2 microM [3H]MTX and varying insulin concentrations for the desired time before determination of the polyglutamate content by high-performance liquid chromatography (HPLC). DHFR-bound drug was separated from free intracellular drug by chromatography on DEAE-Sephacel minicolumns prior to HPLC analysis. Incubation of MCF-7 cells with 2.5 nM insulin for 48 h before exposure to 2 microM [3H]MTX for a further 24 h resulted in a significant increase in both total drug and total polyglutamates compared with control cells. Increasing the insulin concentration in the medium yielded further increases in polyglutamylation so that at 250 nM insulin and above total polyglutamates were increased by 64% compared with control cells. Further evaluation of the effects of physiologic insulin levels on polyglutamate synthesis revealed that 2.5 nM insulin caused an increase in the net glutamylation rate for each polyglutamate derivative during the final 12 h of a 24 h exposure to MTX. Analysis of the effects of insulin on polyglutamate binding to DHFR revealed that exposure to 2.5 nM insulin resulted in the preferential binding of higher polyglutamates to DHFR. In MDA-231 cells, a breast cancer cell line with a poor capacity for polyglutamate synthesis, insulin exposure resulted in an increase in the cellular accumulation of each polyglutamate derivative, with the greatest proportionate increases occurring in the cellular levels of higher polyglutamates. These data suggest that insulin augmentation of MTX polyglutamate synthesis may account for its previously observed ability to enhance MTX cytotoxicity.

Simultaneous determination of cytosine arabinoside, its nucleotides and metabolites by ion-pair high-performance liquid chromatography.

Currently available high-performance liquid chromatographic assays for cytosine arabinoside (ara-C) and its metabolites suffer from two major shortcomings: inability to resolve both ara-C and its nucleotides in a single chromatographic step and/or inadequate sensitivity to allow quantitation of intracellular cytosine arabinofuranoside-5'-triphosphate (ara-CTP) without the use of radiolabelled drug. In this paper, we describe a new ion-pairing high-performance liquid chromatographic assay for ara-C in biological samples that can separate ara-C from its nucleotides, metabolites, and naturally occurring ribonucleotides in a single chromatographic step with a lower limit of quantitation of 5 pmol for ara-C and 10 pmol for ara-CTP. Examples of the utility of this assay are shown in studies of intracellular pharmacokinetics of ara-C in cultured human breast cancer cells and in analysis of plasma nucleoside levels in patients receiving high-dose thymidine chemotherapy. We conclude that this assay provides a rapid and versatile system that can be applied to the study of both cellular and plasma nucleoside pharmacokinetics.

Tropolone: a lipid solubilizing agent for cationic metals.

Lipid soluble agents which chelate radioactive cations have several potential uses in nuclear medicine including: brain imaging, labeling of blood elements, and identifying fatty infiltration of organs. A tropolone-gallium complex has been characterized by the determination of in vitro partition ratios correlated with in vivo organ distribution in the rat. Partition ratios were determined for gallium-67 citrate, indium-114m chloride, and iron-59 chloride cations complexed with tropolone in chloroform + water, octanol + water, olive oil + water, and olive oil + plasma two-phased systems. Tropolone proved to be highly effective in the lipid solubilization of these metal cations. Distribution studies in animals of these cations complexed with tropolone demonstrated an increased concentration of these cation complexes in tissues of high lipid content when compared with appropriate controls.

The effect of hypoxia on monoamine levels in discrete regions of aged rat brain.

Aged rats were exposed to 10% oxygen for 1, 13, and 36 hr. Norepinephrine levels in cerebral cortex, hypothalamus, hippocampus, midbrain, cerebellum, pons-medulla and dopamine levels in striatum were determined after each exposure. While there was no significant change in monoamine levels in brain regions after 2 hr, norepinephrine concentration in hypothalamus and midbrain decreased significantly after 13 hr of hypoxia. After 36 hr in a hypoxic environment, levels of the monoamines in brain regions were similar to the controls. This would suggest NE metabolism is most vulnerable to hypoxia in two regions of the aged brain. The precise mechanism of these changes is unknown, but they suggest both a vulnerability and an adaptive recovery of central adrenergic metabolism by the aged brain under hypoxia.

Aging effect on the noradrenaline content of rat brain microvessels.

Microvessels were isolated by sucrose gradient centrifugation and molecular seiving from forebrains of aged and young adult male Sprague-Dawley rats. Determination of noradrenaline levels in preparations obtained from both groups of animals indicated that the level of NA in cerebral microvessels isolated from aged animals (120 /+- 28 pg NA/mg protein) was significantly reduced when compared with the corresponding value from control (young adult) animals (226 /+- 35 pg NA/mg protein). This decrease in NA may indicate a deficiency in the neural control of the microcirculation and predispose to metabolic and functional disturbances of brain tissue.

An ultrafiltration technique for labeling red blood cells with Tc-99m.

This method automates the preparation of autologous Tc-99m labeled red blood cells utilizing the Amicon on-line column eluate concentrator to separate the plasma from the red blood cells. The red blood cells were pre-tinned with stannous diphosphonate and continuously recirculated over a 0.6 mu filter until all of the plasma was removed and the red blood cells remained suspended in a solution of 0.9% sodium chloride. Once the plasma has been removed the red blood cells are incubated with Tc-99m pertechnetate. The above Tc-99m red blood cells were compared to Tc-99m red blood cells produced in a similar manner except that centrifugation was used to separate the red blood cells from the plasma. Both preparations had a tagging efficiency of 98% or greater and rat distribution studies demonstrate that both preparations are equally stable as an in vivo intravascular agent.