Peritoneal protein losses and cytokine generation in automated peritoneal dialysis with combined amino acids and glucose solutions.

OBJECTIVES: Protein-energy malnutrition as a consequence of deficient protein intake frequently occurs in peritoneal dialysis (PD) patients. Previously, we showed that peritoneal dialysate containing a mixture of amino acids (AA) and glucose has anabolic effects. However AA-dialysate has been reported to increase intraperitoneal protein and AA losses and the release of proinflammatory cytokines (interleukine-6 (IL-6) and tumor necrosis factor alpha (TNFalpha)). We investigated the effect of AA plus glucose (AAG) solutions on peritoneal protein losses and cytokine generation. METHODS: In 6 patients on standard automated peritoneal dialysis (APD) 12 APD sessions of 6 cycles each were performed during the night using dialysate containing 1.1% AA plus glucose or glucose alone as control. Protein losses and TNFalpha and IL-6 concentrations were measured in dialysates separately collected from nightly cycling and daytime dwell. RESULTS: The 24 hour-protein losses with AAG (median 6.7 g, range 4.7-9.4 g) were similar to control dialysate (median 6.0 g, range 4.2-9.2 g). Daytime dialysate IL-6 levels were higher after nightly AAG dialysis than after control dialysis (142 pg/ml and 82 pg/ml, respectively, P<.05). TNFalpha concentrations were very low. CONCLUSION: Nightly APD with amino acids containing dialysate was associated with an increase in peritoneal IL-6 generation during the day. The addition of AA to standard glucose dialysis solutions did not induce a significant increase of peritoneal protein losses.

Identification of melanoma inhibitory activity and other differentially expressed messenger RNAs in human melanoma cell lines with different metastatic capacity by messenger RNA differential display.

The differential display technique was used to identify mRNAs differentially expressed in human melanoma cell lines with different metastatic capacity. We report the isolation of nine different clones, of which four were uniquely expressed in the highly metastatic human melanoma cell line MV3, whereas the other five clones were uniquely expressed in the poorly metastatic human melanoma cell line 530. The differences in expression identified by differential mRNA display were confirmed by Northern blot analyses. DNA sequencing followed by computer search analyses indicated that of the nine differentially expressed clones, five represented novel gene products. The other four were histocompatibility antigen HLA-DR, laminin B2, melanoma inhibitory activity (MIA), and tissue inhibitor of metalloproteinases 3. MIA was also identified in RNA from human melanoma metastasis lesions in a comparison by differential display with pooled human nevi. Northern blot analysis confirmed MIA mRNA expression in nonmetastasizing melanoma cell lines and in melanoma metastasis lesions, while expression was absent in highly metastasizing cell lines and pretumor stages. In the 11 metastasis lesions examined, MIA mRNA expression was apparently inversely correlated with pigmentation.

Induction of the catalytic protein of (Na+ plus K+)-ATPase in the salt gland of the duck.

The (Na+ plus K+)-ATPase activities in salt gland homogenates increased 3- to 4-fold after saline treatment of ducks for 3 weeks. The ATPase was purified to a specific activity of 460 and 1015 mumol Pi/mg protein per h, respectively, in control and saline-treated ducks. The catalytic protein was identified on polyacrylamide electrophoresis gels by phosphorylating the enzyme with (32P)ATP. The molecular weight of the protein was estimated to be 98 000. The amount of catalytic unit increased commensurately with the enzyme activity after saline treatment. It is therefore concluded that the increased enzyme activity is due to a de novo enzyme synthesis and is not an activation effect. Phospholipid concentration in the salt gland tissue increased 1.7-fold after the saline treatment. Significant increases occurred in the percentage of the total phospholipids as phosphatidylserine and sphingomyelin. In the partially purified (Na+ plus K+)-ATPase preparation, the percentage composition of phosphatidylserine and phosphatidylethanolamine increased after saline treatment.

Rat desmin gene structure and expression.

The isolation of two partial genomic clones and a near full length cDNA clone encoding the rat intermediate filament protein desmin is reported. Desmin is a differentiation marker for all types of muscle cells. The nucleotide order of the coding region and 0.1 kb of 5'-flanking sequences of the rat desmin gene has been determined. One genomic clone encompasses exons I-III and approx. 12 kb of 5'-flanking sequences, while the other clone contains exons VII-IX and about 12 kb of 3'-flanking sequences. Northern analysis of RNA from different organs reveals that, as expected, desmin is expressed in striated, heart and smooth muscle cells containing tissues; among other tissues, lung displays relatively high expression levels, while desmin mRNA is barely detectable in spleen, kidney and liver. S1 mapping reveals that the same transcription initiation site is used in all desmin mRNA containing tissues.

memA/DRS, a putative mediator of multiprotein complexes, is overexpressed in the metastasizing human melanoma cell lines BLM and MV3.

memA was isolated by subtractive hybridization in which the mRNA repertoire was compared in a panel of human melanoma cell lines with different metastasizing potential. Expression of memA mRNA is elevated in the highly metastasizing human melanoma cell lines and derived xenografts, as compared with the non-metastasizing ones. In a collection of human tumor cell lines and melanoma metastasis lesions, memA mRNA expression could be detected in the A-431 (epidermoid carcinoma), HT-1080 (fibrosarcoma), JEG-3 and JAR (choriocarcinomas) cell lines and in three out of 11 melanoma metastasis lesions. The distribution of memA mRNA in a collection of healthy human organs is also tissue restricted. Sequence analysis revealed that the MEMA protein is identical with a 160 kDa nuclear 'domain rich in serines' (DRS) protein occurring free in the nucleoplasm and in U2-ribonucleoprotein structures. MEMA is also homologous to pinin, a 140 kDa protein associated with the desmosome-intermediate filament complex, and to a 32 kDa porcine neutrophilic protein that was copurified with components of the NADPH-oxidase enzyme complex. The encoded amino acid sequence predicts that the MEMA protein has three coiled-coil domains, one glycine loop domain, is very hydrophilic and contains regions rich in glutamine/proline, glutamic acid and serine residues.

Kirromycin-induced modifications facilitate the separation of EF-Tu species and reveal intermolecular interactions.

A simplified method for the separation of a kirromycin-sensitive tufB-encoded elongation factor Tu (EF-TuBs) from a kirromycin-resistant tufA product (EF-TuAr) was obtained by exploiting the specific increase of negative [corrected] charges induced by the antibiotic, resulting in a retarded elution of kirromycin-bound EF-TuBs on ionic chromatography. The kirromycin-free EF-TuBs is active in poly(Phe) synthesis and shows similar properties to EF-TuAsBs. As expected for these two distinct species, the dissociation of the EF-TuArBs.GTP complex in the presence of kirromycin shows a biphasic curve; in contrast, a monophasic GTP dissociation rate was found for a combination of two mutated EF-Tu species, EF-TuArBo, revealing the existence of intermolecular interactions. These observations prove for the first time the existence of cooperative phenomena between EF-Tu species in vitro, as suggested earlier by in vivo experiments.

nmd, a novel gene differentially expressed in human melanoma cell lines, encodes a new atypical member of the enzyme family of lipases.

nmd, a novel gene, was isolated by applying the differential mRNA display method to human melanoma cell lines with different metastatic capacity. In a panel of 17 other human tumor cell lines, nmd RNA expression could only be detected at low levels in T24 (bladder carcinoma) and Caco-2 (colon adenocarcinoma). Furthermore, it was found in placenta and liver, but not in skin, colon, spleen, lung, muscle, prostate and kidney. Sequence analysis classified the nmd gene product as a new member of the enzyme family of lipases (almost 30% identity in amino acid sequence with other human lipases). Active site residues of lipases were conserved in NMD, but NMD lacks the regulatory lid domain, which controls entry to the active site in classical lipases. A similar deletion was earlier reported by others in the guinea pig pancreatic (phospho)lipase GPLRP2 and the phospholipase A1 from hornet venom (DolmI).

Body composition in growth hormone-deficient adults.

Body composition in a group of growth hormone (GH)-deficient adults was compared with a control group matched for age, sex, body height, body weight, and body mass index by using bioelectrical impedance and deuterium oxide-dilution methods and hydrodensitometry. The body-fat percentages of GH-deficient adults were higher despite comparable weight, height, age, and body mass index. Body impedance was higher in the GH-deficient adults after correction for differences in height and fat-free mass. As a consequence, prediction formulas for body composition from body impedance developed in normal subjects cannot be applied to GH-deficient subjects. The higher body impedance in the GH-deficient subjects can be ascribed to a smaller amount of extracellular water in these subjects than in control subjects.

Whole-body and segmental bioelectrical-impedance analysis in patients with cirrhosis of the liver: changes after treatment of ascites.

Bioelectrical impedance analysis (BIA) is a simple technique for determining body water and calculating body composition. It has been validated in healthy control subjects but not in patients with liver disease. We examined the ability of BIA to detect changes in total body water (TBW) due to removal of ascites. In 12 cirrhotic patients, BIA of the whole body and of body segments was performed before and after treatment of ascites with paracentesis (n = 12) and diuretics (n = 2). TBW changes predicted by BIA, by using two prediction equations, were significantly less than body weight changes (51% and 45% of the weight loss). BIA of body segments showed highly significant changes in both the trunk and the leg and small changes in the arm. These data indicate that BIA of the whole body is not a suitable technique for monitoring fluid changes in cirrhotic patients with ascites. Changes in BIA of body segments may be due to mobilization of edema after the removal of ascites.

Transformation-defective adenovirus 5 E1A mutants exhibit antioncogenic properties in human BLM melanoma cells.

Adenoviral E1 A proteins exhibit a strong tumor-suppressive activity in human tumor cells. However, E1 A is capable of transforming rodent and human cells in cooperation with other oncoproteins, such as activated RAS. Thus, the therapeutic use of wild-type E1A harbors the principal risk of enhancing tumor malignancy. This prompted us to construct E1A 13S cDNA-derived mutants that were unable to transform baby mouse kidney cells in cooperation with E1B and to test their tumor-suppressive activity in BLM human melanoma cells. Anchorage-independent growth in soft agar was reduced for those cell lines expressing the E1AdelCR2 mutant, which lacks the entire conserved region 2 (CR2) sequences, or for cells expressing the E1AcR3Ex2 mutant, which contains CR3 plus exon 2 sequences. In contrast, cell lines expressing the entire E1A wild-type (E1AWT) or only the exon 2 sequences (E1AEx2) grew like the parental BLM cells. Moreover, inoculation of nude mice with BLM cells or cells expressing E1AEx2 revealed large tumors after 2 weeks. In contrast, tumors derived from E1AdelCR2- or E1ACR3Ex2-expressing cells exhibited a substantial delay in tumor growth accompanied by a loss of E1A expression in the outgrown tumors. Cell lines expressing E1AWT showed an intermediate phenotype. Thus, expression of CR3 plus exon 2 sequences is sufficient to enhance both the antioncogenic properties and the therapeutic safety of E1A in our system.

Effects of the glucosidase inhibitor acarbose in patients with liver cirrhosis.

In this preliminary study, we examined the effects of acarbose and placebo together with a standardized breakfast on blood glucose levels, on breath hydrogen excretion and on plasma insulin and glucagon levels; in addition, the effects on fasting blood levels of metabolites were studied following an evening meal with acarbose or placebo. Acarbose significantly reduced blood glucose levels in 10 patients with alcoholic cirrhosis following a meal containing 100 g of carbohydrate. There were no significant changes in plasma insulin after breakfast but glucagon levels were increased at 1 h after the meal. Breath hydrogen excretion did not change significantly. Acarbose given with a late evening snack reduced fasting beta-hydroxybutyrate levels the next morning in these cirrhotic patients.

Lipolysis and lipid oxidation in weight-losing cancer patients and healthy subjects.

Increased lipolysis has been suggested as one of the possible mechanisms underlying cancer cachexia. The study aim was to assess whether lipolysis is increased in weight-losing cancer patients, considering their differences in food intake and body composition. Sixteen healthy subjects and 18 cancer patients with different tumor types and a weight loss of at least 5% in the previous 6 months were included in the study. Food intake was recorded for 4 days. After an overnight fast, [1,1,2,3,3-2H5]glycerol was infused to determine the rate of appearance (Ra) of glycerol as a measure of whole-body lipolysis, and [1-13C]palmitic acid was infused to determine the Ra of palmitate as a measure of adipocyte fatty acid release. Palmitate oxidation was determined by measuring 13CO2 enrichment in breath samples, and body composition was measured by bioelectrical impedance analysis. After adjustment for energy intake, whole-body lipolysis was significantly higher in cancer patients versus healthy subjects (6.46 +/- 0.63 and 4.67 +/- 0.46 micromol/kg +/- min, respectively, P < .05). The difference in adipocyte fatty acid release did not reach statistical significance. The rate of palmitate oxidation was also significantly higher in patients than in healthy subjects (1.15 +/- 0.10 and 0.93 +/- 0.07 )micromol/kg x min, respectively, P < .05). No differences in body composition were observed between groups. In conclusion, whole-body lipolysis (as measured by the Ra of glycerol) and palmitate oxidation were elevated in weight-losing cancer patients, but fatty acid release was not significantly different.

Measuring plasma fibrinogen levels in patients with liver cirrhosis. The occurrence of proteolytic fibrin(ogen) degradation products and their influence on several fibrinogen assays.

In patients with liver cirrhosis the fibrinogen molecule is under constant attack of various proteolytic enzymes, which might affect results of the different assay systems for fibrinogen. We therefore studied the measurement of fibrinogen in the plasma of patients with mild, moderate and severe cirrhosis of the liver. Fibrinogen levels were measured with the Clauss method (functional fibrinogen); an enzyme immuno assay (EIA) for HMW + L MW fibrinogen; and an assay that measures the total clottable fibrinogen. With all three methods we found normal or slightly increased fibrinogen levels in patients with mild or moderate cirrhosis, whereas patients with severe cirrhosis had decreased levels. No evidence was found for increased partial fibrinogen proteolysis, resulting in increases of LMW'-fibrinogen in cirrhotic patients. We further observed that fibrinogen degradation products levels increased slightly with the severity of the disease, but were still in the normal range in patients with severe cirrhosis. This indicates a very low level of primary fibrinolysis. Fibrin degradation products levels increased much stronger, which points to intravascular coagulation. The levels of the fibrin degradation products remained below the level where they are expected to influence the Clauss assay. In patients with liver cirrhosis the measurement of plasma fibrinogen levels with the three studied methods give comparable results. We suggest to apply the Clauss assay in cirrhotic patients because of this and because it has good reproducibility and because the test is cheap, quick and easy to perform.

Habitual pattern of food intake in patients with liver disease.

Patients with liver disease are frequently undernourished. We determined the habitual pattern of energy intake and intake of protein, fat, carbohydrate and alcohol in patients with liver disease. 20 Patients differing with respect to cause, duration and severity of liver disease reported their habitual food intake using a 7-day food record. Control subjects formed a representative sample of the Dutch population. The results showed that patients with liver disease, had a significantly decreased daily energy intake (both expressed in absolute values and per kg body mass), compared with control subjects. Total fat intake was reduced in patients, whereas total intake of protein, carbohydrate and alcohol was similar to the control group. In the first 4 hours after rising, energy and protein intake were significantly increased in the patient group. During the evening the consumption of protein was lower in patients with liver disease than in control subjects. These findings are probably functional in order to minimize episodes of catabolism.

Minimum protein requirements in liver cirrhosis determined by nitrogen balance measurements at three levels of protein intake.

Nitrogen balance at three levels of protein intake was measured in eight patients with cirrhosis of the liver; moreover, at each level of protein intake, the effects on nitrogen balance of branched-chain amino-acid enriched protein and natural protein were compared. From these nitrogen balance data, minimum protein requirements were calculated by linear regression analysis. The patients were in a negative nitrogen balance on a 40 g protein diet (-0.75 +/- 0.15 gN.), and in positive nitrogen balance on 60 g (+1.23 +/- 0.22 gN.) or 80 g of protein per day (+2.77 +/- 0.20 g N.). Their mean minimum protein requirement (48 +/- 5 g of protein/day or 0.75 g/kg/day) is higher than expected in healthy people; the safe level of protein intake (mean + 2 sd) is 58 g per day or 1.2 g/kg/day. Nitrogen balances and protein requirements were not different on branched-chain amino-acid enriched diets. The physical condition of the patients improved when they came into positive nitrogen balance; the higher rates of protein intake were well tolerated without onset of encephalopathy. We conclude that protein requirements are elevated in cirrhosis of the liver; diets supplying less than 60 g of protein per day should not be prescribed in long term treatment of cirrhotic patients.

Eicosapentaenoic acid ethyl ester supplementation in cachectic cancer patients and healthy subjects: effects on lipolysis and lipid oxidation.

BACKGROUND & AIMS: Recent reports suggest that weight loss in cachectic cancer patients may be inhibited by supplementation of the n-3 fatty acid eicosapentaenoic acid (20:5n-3; EPA), presumably due to inhibition of lipolysis. The aim of the present double-blind, randomized trial was to assess whether short-term oral EPA ethyl ester (EE) supplementation inhibits lipolysis and lipid oxidation in weight-losing cancer patients and in healthy subjects. METHODS: Seventeen weight-losing, cancer patients of different tumor types, and 16 healthy subjects were randomized to receive EPA-EE (6 g/d) or placebo (oleic acid (OA)-EE; 6 g/d) for seven days. At baseline (day 0) and during supplementation (days 2 and 7) whole-body lipolysis and palmitic acid release were measured in the overnight fasting state using [1, 1, 2, 3, 3-(2)H(5)]glycerol and [1-(13)C]palmitic acid. Palmitate oxidation was determined by measuring(13)CO(2)enrichment in expired breath. RESULTS: No significant effects of EPA-EE on whole-body lipolysis, palmitic acid release, or palmitate oxidation were detected in cancer patients nor in healthy subjects in comparison with OA-EE. EPA-EE supplementation reduced plasma-free fatty acid and triacylglycerol concentrations significantly in healthy subjects but not in cancer patients. CONCLUSION: We conclude that supplementation of EPA-EE does not significantly inhibit lipolysis or lipid oxidation in weight-losing cancer patients or in healthy subjects during short-term supplementation when using OA-EE as a placebo supplement.

The hemodynamic and arterial blood gas response to asphyxiation: a canine model of pulseless electrical activity.

OBJECTIVE: Asphyxiation is a time-honored animal model for producing pulseless electrical activity cardiac arrest. To date, there has not been a detailed description of the hemodynamic and arterial blood gas response to asphyxiation in a large number of animals. Our objective was to describe a single laboratory's experience with a standardized canine model of asphyxial pulseless electrical activity arrest. METHOD: Design--Data from 4 separate research protocols using a standardized asphyxial model were retrospectively reviewed. Setting--Resuscitation research laboratory. Participants--169 mixed-breed dogs. Interventions--Each animal was anesthetized and instrumented for hemodynamic monitoring. The endotracheal tube was clamped and hemodynamic data was monitored. Following loss of aortic fluctuations by thoracic aortic catheter, animals remained in pulseless electrical activity for up to 20 min. Hemodynamic data was measured continuously and arterial blood gases were sampled intermittently. RESULTS: Following endotracheal tube clamping, there was a characteristic increase in heart rate and systolic blood pressure. The heart rate peaked at 2-3 min following clamping, while the systolic blood pressure peaked at 7 min. Both heart rate and systolic blood pressure then steadily decreased until loss of aortic fluctuations. Loss of aortic fluctuations occurred 11.4 +/- 2.4 min following clamping. Following loss of aortic fluctuations, the heart rate steadily decreased. Arterial blood gases during asphyxiation and pulseless electrical activity arrest showed profound hypoxemia with hypercarbia (pH 7.03 +/- 0.07; Pco2 93 +/- 19; Po2 12 +/- 7 at loss of aortic fluctuation). CONCLUSIONS: In this canine asphyxial model of pulseless electrical activity, a characteristic hemodynamic pattern of mild tachycardia-hypertension-bradycardia-hypotension was produced. Arterial blood gases reflect a profound hypoxemia and respiratory acidosis.

Evaluation of an esophageal Doppler probe for the identification of experimental pseudo-electromechanical dissociation: a preliminary study.

STUDY OBJECTIVE: To determine the effectiveness of an esophageal doppler device to non-invasively detect experimental pseudo-electromechanical dissociation (pseudo-EMD). DESIGN: Prospective, controlled, laboratory investigation using an asphyxial canine cardiac arrest model and a newly-developed esophageal flat-flow probe doppler unit. INTERVENTIONS: Mongrel dogs (20) were instrumented for hemodynamic monitoring. The esophageal doppler probe was placed in the distal esophagus of each animal. Electromechanical dissociation (EMD) was induced by clamping the endotracheal tube. MEASUREMENTS AND MAIN RESULTS: A period of pseudo-EMD was defined as the time where cardiac contractility was present, measured by a micromanometer tipped thoracic aortic catheter, without concurrent femoral pulses by palpation. The pseudo-EMD period could be produced consistently in all 20 animals. The characteristic doppler flow sounds were easily heard using the esophageal device in all animals. The time from endotracheal tube clamping until loss of femoral pulses was 622 +/- 96 s; until loss of radial artery doppler signals was 616 +/- 92 s; until loss of esophageal doppler signals was 728 +/- 88 s; and until loss of aortic fluctuations by thoracic aortic catheter was 728 +/- 82 s. The times to loss of esophageal doppler sounds and loss of aortic fluctuations were not significantly different. However, they were significantly longer than the time to loss of femoral pulses (P < 0.02). CONCLUSIONS: The canine asphyxial EMD model can be used for short experimental studies of pseudo-EMD. Pseudo-EMD can be consistently and non-invasively detected with this esophageal doppler device. The device is as reliable as a micromanometer tipped aortic arch catheter in detecting pseudo-EMD. The doppler device could potentially be useful in improving recognition of near cardiac arrest in pre-hospital and emergency department settings. Further research on the utility of this device in other models of low-flow states should be performed.

The efficacy of atropine in the treatment of hemodynamically unstable bradycardia and atrioventricular block: prehospital and emergency department considerations.

OBJECTIVE: To determine the efficacy of atropine therapy in patients with hemodynamically compromising bradycardia or atrioventricular block (AVB) in the prehospital and emergency department settings. DESIGN: Retrospective review of prehospital, emergency department, and hospital records. PARTICIPANTS: Prehospital patients with hemodynamically compromising bradycardia or AVB with evidence of spontaneous circulation who received atropine as delivered by emergency medical services personnel (advanced life support level). SETTING: Urban/suburban fire department-based emergency medical service system with on-line medical control serving a population of approximately 1.6 million persons. DEFINITIONS: Hemodynamic instability was defined as the presence of any of the following: ischemic chest pain, dyspnea, syncope, altered mental status, and systolic blood pressure less than 90 mmHg. Bradycardia was defined as sinus bradycardia, junctional bradycardia, or idioventricular bradycardia (grouped as bradycardia) while AVB included first-, second- (types I and II), or third-degree (grouped as AVB). The response that occurred within one minute following each dose of atropine was defined as none, partial, complete, or adverse. MAIN RESULTS: Of 172 patients meeting entry criterion complete data was available for 131 (76.1%) and constitutes the study population. The mean age was 71 years. Fifty-one percent were female. Forty-five patients had AVB and 86 bradycardia. Patients with AVB were more likely to have a presenting systolic blood pressure less than 90 mmHg than those with bradycardia. In the 131 patients, responses to atropine were as follows: 26 (19.8%) = partial, 36 (27.5%) = complete, 65 (49.6%) = none, and 4 (2.3%) = adverse. Patients presenting with bradycardia (compared to AVB) more commonly: (1) received a single dose of atropine; (2) a lower total dose of atropine in the prehospital interval; (3) were more likely to arrive in the ED with a normal sinus rhythm; and (4) were less likely to receive additional atropine or isoproterenol in the ED. Those patients who achieved normal sinus rhythm over the total course of care were likely to have achieved that rhythm during the prehospital interval. There was no difference between groups in the likelihood of leaving the ED with a normal sinus rhythm achieved during the ED interval. Acute myocardial infarction was more common in patients presenting with AVB (55.5%) than with bradycardia (23.2%, P = 0.001). CONCLUSIONS: Approximately one-half of patients who received atropine in the prehospital setting for compromising rhythms had either a partial or complete response to therapy. Adverse responses were uncommon. Those patients who presented with hemodynamically unstable bradycardia to EMS personnel responded more commonly to a single dose and a lower total dose of atropine compared to similar patients with AVB. Those patients who achieve normal sinus rhythm by ED discharge were likely to have achieved it during the prehospital interval.