Arterial ketone body ratio as a parameter of hepatic mitochondrial redox state during and after hemorrhagic shock.

This study was aimed at clarifying the usefulness of the arterial ketone body ratio (AKBR), which reflects hepatic mitochondrial redox state and closely correlates with hepatic energy production, for understanding the degree of hepatic mitochondrial damage and the extent of the deterioration in hepatic energy metabolism during or shortly after hemorrhagic shock. Changes in the AKBR of 33 trauma victims who were admitted to our institute in hemorrhagic shock with a systolic pressure lower than 70 mmHg were measured until the patient recovered with the restoration of AKBR to the normal range (> or = 1.0) or until the patient died. During hemorrhagic shock the AKBRs were highly decreased, indicating deteriorated hepatic function. With successive fluid resuscitation the AKBR quickly recovered in 15 surviving patients from an initial value of 0.26 +/- 0.03 toward normal within hours, indicating that hepatic mitochondria are functioning normally. The AKBR recovered to a normal value of 1.10 +/- 0.06 on day 2 (p < 0.001). In 18 expired patients, AKBR did not recover to normal range, even though some of the patients recovered from the shock state. On the other hand, AST, ALT, LDH, and prothrombin time on day 2 were not significantly different from the values on admission, and the changes during the interval were not unidirectional even in the surviving patients, providing no information on the current functional state of the liver. Measurement of AKBR during and shortly after hemorrhagic shock provides timely and accurate information about liver function.

The white IRIS leukocyte differential analyzer for rapid high-precision differentials based on images of cytoprobe-reacted cells.

Combining unique cytoprobe, rapid hemacyte fractionation, and novel color image analysis, The White IRIS (TWI) extends automated intelligent microscopy to leukocyte differentiation. TWI provides flow cytometry precision and microscopical resolution to review specimens flagged by hematology analyzers with differential capabilities or to complement other analyzers without these capabilities. The system includes compartments for closed sampling, rapid leukocyte-rich plasma preparation, cytoprobe-induced metachromasia, and collection and color analysis of leukocyte images, and presents the results as a single-view 500-cell differential on a 20-in. (50-cm) touch-screen monitor. Method correlations for the five mature cell types averaging r > 0.90 were obtained with a prototype system. Classification of normal and abnormal specimens showed 95% agreement with a reference method without any undetected significant morphologic abnormality. False-positive and false-negative rates of 7.27% and 3.53%, respectively, exceeded performance of current commercial systems. Case studies demonstrate the ease and speed with which unusual pathologies and leukemias can be observed and interpreted.

Redox state in liver mitochondria in acute copper sulfate poisoning.

A patient with acute copper sulfate poisoning was found to have maintained a relatively oxidized hepatic mitochondrial redox state in spite of his being in refractory shock. The mechanism underlying this unexpected clinical observation was investigated. The study involved the allocation of male Wistar rats into three groups; copper sulfate-treated (intraperitoneal injection), hemorrhagic shock, and control. Since the copper sulfate-treated rats developed severe hypovolemic shock, hypovolemia was induced in the hemorrhagic shock group to mimic the time course of blood pressure reduction in the copper sulfate-treated group. The control rats were injected intraperitoneally with a saline solution. The hepatic energy charge and the arterial ketone body ratio (AKBR), which reflects the hepatic mitochondrial redox state, of each group of rats was determined. The hepatic energy charge of the copper sulfate-treated rats decreased significantly, reflecting severe hypotension and copper sulfate-induced hepatic damage. This decrease was greater than that observed in the rats subjected to hemorrhagic shock. Despite the profound shock and the markedly decreased hepatic energy charge, the AKBR in copper sulfate-treated rats did not decrease compared with the control group, while the AKBR in rats with induced hemorrhagic shock did decrease significantly. These observations, together with in vitro studies, suggest that the relatively oxidized redox state of liver mitochondria in the copper sulfate-treated rats, notwithstanding the severe shock state of these animals, may be the result of direct oxidation of NADH by copper sulfate.

Imaging of human tumor xenografts in nude mice with radiolabeled monoclonal antibodies. Limitations of specificity due to nonspecific uptake of antibody.

To determine if in vivo binding specificities of monoclonal antibodies to tumor nodules would reflect in vitro antibody specificities as determined by radioimmunoassay, two monoclonal antibodies were selected for imaging of human tumor xenografts in nude mice. By in vitro radioimmunoassay, antibody 436 binds to the M20 melanoma cell line, but not to the P3 carcinoma cell line; antibody 44 binds to P3 but not to M20. Both antibodies are IgG1 isotypes. Nude mice bearing an M20 melanoma in one flank and a P3 carcinoma in the other were injected intravenously with 5 to 25 micrograms of each antibody labeled with either I-125 or I-131; in separate animals the labels were reversed. Animals were imaged daily with a scintillation camera equipped with a pinhole collimator. On day 6 the animals were sacrificed, and binding of the antibodies to the tumors and normal tissue were compared. Antibody 436 had up to a two-fold binding advantage in vivo for the melanoma, whereas antibody 44 had up to a two-fold binding advantage for the carcinoma, thus confirming the in vitro specificities of each. However, imaging on day 4 and computer analysis of percent radioactivity in the tumors showed that tumor images were related directly to tumor size and relatively uninfluenced by antibody specificity. Thus, even though antibody specificity can be demonstrated by differential tissue counting, imaging of tumor deposits appears to involve a nonspecific phenomenon that is largely dependent upon tumor weight.

Further purification of neutrophil migration inhibition factor from T lymphocytes (NIF-T): evidence that NIF-T and leukocyte inhibitory factor (LIF) are immunologically distinct.

Neutrophil migration inhibition factor from T lymphocytes (NIF-T) purified by antibody affinity chromatography and gel filtration chromatography was radioiodinated and identified as a 26,000-MW protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). NIF-T was identified by elution of biological activity from gel fractions and selective adsorption of a radioiodinated mediator by HL60 cells differentiated in the presence of dimethyl sulfoxide (DMSO) to develop receptors for NIF-T. A goat neutralizing antibody for NIF-T neutralized and immunoprecipitated migration inhibition activity in the conditioned medium from Mo T-lymphoblast cells and human peripheral blood lymphocytes (PBL) cultured with concanavalin A (Con A), but not from RPMI 1788 B-lymphoblast cells with leukocyte migration inhibitory factor (LIF) activity. These studies distinguish NIF-T both chemically and immunologically from LIF.

The varying tissue distribution of L-glutamic acid labelled at three different sites.

Methods using 5 and 6 enzymes were devised to synthesize L-[11C]glutamic acid (GA), labeled on the carboxyl group of either the alpha-(AGA) or gamma-(GGA) carbon atom. The distribution of the 11C-labeled AGA or GGA in rabbits was compared with that of L-[13N]glutamic acid. The results show that AGA was rapidly decarboxylated with loss of the 11C-label, and that GGA was also decarboxylated, but to a lesser degree. Thus, the radiolabel distribution may not reflect the distribution of the original compound. The results also demonstrate that positron labeled pharamaceuticals may be rapidly synthesized via complex enzymatic pathways.

Glycosaminoglycans of some mouse mastocytomas.

The glycosaminoglycan fractions of four murine mastocytomas (AB-CBF1-MCT-1, AB-CXBG-MCT-1, AB-CXBI-MCT-1, and Furth), were isolated and characterized using microelectrophoresis, degradation with specific mucopolysaccharidases, paper chromatography and, in some instances, ion-exchange chromatography. Intramuscular and ascites tumors and tumors cultured in vitro were used. The glycosaminoglycans were labeled with [35S]sulfate and , in a few instances, with D-[6-3H]glucosamine. The glycosaminoglycan fraction obtained from the AB-CBF1-MCT-1 mastocytoma cultured in vitro consisted predominantly of dermatan sulfate-like material. On the other hand, the major components of the glycosaminoglycan fractions of the AB-CXBG-MCT-1 and AB-CXBI-MCT-1 and Furth tumors were observed to behave like heparin or heparan sulfate. In most of the mastocytoma samples examined, chondroitin 4-sulfate-like components were found to be present in smaller amounts, even in samples derived from tumor cells cultured in vitro. A component exhibiting a keratan sulfate-like behavior was detected in fractions obtained from the solid CXBG, CXBI and Furth tumors.

Enzymatic sulfation of Triton X-100.

Triton X-100 increased incorporation of [35S]sulfate into lipid-soluble substances when it was incubated with the high-speed supernatant enzymes of bovine or sheep adrenal cortex or rat liver, kidney or brain. Thin-layer chromatographic comparison of these compounds with chemically synthesized standards indicated that sulfate derivatives of Triton X-100 oligomers were formed in the liver and adrenal supernatant enzyme preparations. Further evidence for Triton X-100 sulfation by these enzymes was obtained by the use of [3H]Triton X-100 and purified Triton X-100 oligomers. No discernible sulfation of Triton X-100 occurred in kidney or brain high-speed supernatant enzyme preparations. A series of [35S]sulfate-containing substances, which closely resembled the homologous series of [35S]sulfate-labeled Triton X-100 oligomers formed by adrenal cortex enzymes, were formed in detergent-free adrenal incubates. Their chromatographic behavior suggests that they are not steroid sulfates. The kidney supernatant enzyme fraction was shown to contain steroid sulfotransferase activity; sulfation of cholesterol, dehydroepiandrosterone, pregnenolone, cholic acid and lithocholic acid was stimulated by the presence of Triton X-100.

Stimulation of fibroblast biosynthetic activity by serum of patients with pretibial myxedema.

Skin fibroblasts from the shoulder and lower extremities of normal individuals, as well as from patients with pretibial myxedema (PTM) were grown in culture. When cells reached the monolayer stage, they were labeled with 3H-glucosamine and tested for hyaluronic acid synthesis in the presence of either serum from PTM patients or normal human serum. All the fibroblasts from the pretibial area synthesized 2 to 3 times more hyaluronic acid when incubated with PTM sera than when incubated in normal human serum. Fibroblasts cultured from skin of the back or prepuce did not respond to PTM sera. This heat-stable, protease-sensitive, and dialyzable, fibroblast-stimulating factor is not a 7S gamma-globulin. The enhanced sensitivity to PTM sera exhibited by fibroblasts from the lower extremities may explain why the lesions in this disease are restricted primarily to that area.

Proteoglycans of soluble fraction of mouse mastocytoma.

Proteoglycans have been isolated from a high speed supernatant fraction of a mouse mastocytoma by procedures which should minimize alteration of the native protein-polysaccharide molecule. The methods used include in vivo labeling proteoglycans with 35S-sulfate, 3H-leucine and 3H-lysine, centrifugation of the tumor homogenate at 105,000 g, cetylpyridinium fractionation of the supernatant, and further purification of some of the fractions obtained by DEAE-cellulose column chromatography, gel filtration on Sepharose 4B and cellulose acetate electrophoresis. Two major sulfated proteoglycans were obtained, one containing keratan sulfate-like material (KSP-S), the other a heparin-like polymer (HP-S). The presence in HP-S of a compound similar to heparin was confirmed by its digestibility with flavobacterium heparinase. HP-S contained about 4 per cent protein. Glycine was the predominant amino acid, and serine did not appear to be involved in the peptide-carbohydrate linkage. The proteoglycan present in HP-S appeared to be homogeneous when examined using cellulose acetate electrophoresis. KSP-S was found to contain sialic acid and its protein content was significantly higher than that of HP-S. Glutamic and aspartic acids were the most abundant amino acids in KSP-S.