N omega-amino-L-arginine, an inhibitor of nitric oxide synthase, raises vascular resistance but increases mortality rates in awake canines challenged with endotoxin.

Inhibitors of nitric oxide synthase (NOS) have been reported to increase mean arterial pressure in animal models of sepsis and recently have been given to patients in septic shock. However, controlled studies to determine the effects of these agents on cardiovascular function and survival in awake animal models of sepsis have not been reported. To examine the therapeutic potential of NOS inhibition in septic shock, we challenged canines with endotoxin (2 or 4 mg/kg i.v.) and treated them with either normal saline or N omega-amino-L-arginine (10 or 1 mg/kg/h), the most specific inhibitor available for the isoform of NOS implicated in septic shock. Endotoxemic animals treated with N omega-amino-L-arginine (n = 11) had higher systemic and pulmonary vascular resistance indices (SVRI and PVRI, p less than or equal to 0.033) and decreased heart rates (p = 0.009), cardiac indices (CI, p = 0.01), oxygen delivery indices (p = 0.027), and oxygen consumption indices (p = 0.046) compared with controls (n = 6). Moreover, N omega-amino-L-arginine increased mortality rates after endotoxin challenge (10 of 11 vs. 1 of 6 controls, p = 0.005). Administration of L-arginine did not improve survival or alter the cardiopulmonary effects of N omega-amino-L-arginine, which suggests that inhibition of NOS may not have been competitive. In normal animals, N omega-amino-L-arginine alone (n = 3) increased SVRI (p = 0.0008) and mean arterial pressure (p = 0.016), and decreased CI (p = 0.01) compared with saline-treated controls (n = 3), but, at the high dose, also produced neuromuscular rigidity and seizure-like activity that was not apparent in the endotoxemic model. Thus, the mortality rate from endotoxemia increased either because of NOS inhibition per se or because of properties unique to N omega-amino-L-arginine, or both.

The third component of complement protects against Escherichia coli endotoxin-induced shock and multiple organ failure.

We investigated whether the third component of complement (C3) is involved in the pathophysiology of endotoxic shock, and if it is involved, whether it plays a protective role or whether it mediates shock and multiple organ failure. In a prospective, controlled investigation, six Brittany spaniels that were homozygous for a genetically determined deficiency of C3 (C3 deficient, < 0.003% of normal serum C3 levels) and six heterozygous littermates (controls, approximately 50% of mean normal serum C3 level) were given 2 mg/kg of reconstituted Escherichia coli 026:B6 acetone powder as a source of endotoxin, intravenously. All animals were given similar fluid and prophylactic antibiotic therapy, and had serial hemodynamic variables obtained. After E. coli endotoxin infusion, C3-deficient animals had higher peak levels of endotoxin and less of a rise in temperature than controls (P < 0.05). During the first 4 h after E. coli endotoxin infusion, C3-deficient animals had significantly greater decreases in mean central venous pressure and mean pulmonary artery pressure than controls (P < 0.02). During the first 48 h after E. coli endotoxin infusion, C3-deficient animals had significantly greater decreases in mean arterial pH, left ventricular ejection fraction, and mean pulmonary capillary wedge pressure, and greater increases in mean arterial lactate, arterial-alveolar O2 gradient, and transaminases (aspartate aminotransferase and alanine aminotransferase) than controls, (all P < 0.05). After E. coli endotoxin infusion, C3-deficient animals compared to controls had significantly less of a decrease in mean C5 levels (P < 0.01), but similar (P = NS) increases in circulating tumor necrosis factor levels, bronchoalveolar lavage neutrophils, and protein, and similar (P = NS) decreases in blood leukocytes and platelets. Two of six C3-deficient animals and two of six controls died. In summary, after intravenous infusion of E. coli endotoxin, canines with C3 deficiency have decreased endotoxin clearance and worse E. coli endotoxin-induced shock and organ damage. Thus, the third component of the complement system plays a beneficial role in the host defense against E. coli endotoxic shock.

Instrumentation in clinical chemistry.

Major advances in instrumentation have revolutionized the clinical chemistry laboratory during the past two decades. This article focuses on some of the more recent developments in instrumentation for clinical chemistry in the areas of general chemistry, immunoassays, urinalysis, electrophoresis, chromatography, and trace metal analyses.

Hematopoiesis in the grey collie dog: studies of the regulation of erythropoiesis.

Hematopoiesis in the grey collie dog undergoes periodic fluctuations which involve reticulocytes, granulocytes, platelets, lymphocytes, and monocytes. This syndrome is inherited in an autosomal recessive manner and can be transmitted or abolished by appropriate bone marrow transplantation experiments, thus demonstrating this to be a primary marrow defect. Investigation of humoral regulation in this setting indicates that serum erythropoietin (ESF) also undergoes cyclic fluctuation and that shortly after the increase and peak in serum ESF levels recognizable red cell precursors appear in the marrow. Erythropoiesis in the grey collie is reciprocally related to the blood O2 carrying capacity. With phlebotomy, ESF activity and reticulocytes increase but continue to cycle, while hypertransfusion eliminates reticulocyte production completely. Neither phlebotomy nor hypertransfusion alter the underlying cycle time (11-12 days) nor influence the peaks of peripheral blood granulocytes. Thus, in these experiments, no direct evidence of competition between reticulocyte and granulocyte production is observed. In vitro studies of canine hemoglobin synthesis fail to demonstrate evidence of an inhibitor to ESF. These results indicate that periodic fluctuation of serum ESF is an integral part of the grey collie syndrome and are most consistent with some form of feedback regulation of ESF production.

Changes in human serum amyloid A and C-reactive protein after etiocholanolone-induced inflammation.

Secondary amyloidosis is a complication of diseases characterized by recurrent acute inflammation. In this study, a standardized stimulus which induced fever and inflammation was given to six normal subjects (19-24 yr old) to follow the fluctuation in concentration of serum amyloid A (SAA), the precursor of the secondary amyloid fibril protein. After a single intramuscular injection of etiocholanolone (0.3 mg/kg), blood samples were drawn twice a day for 12 days for determination of SAA by solid phase radioimmunoassay. From a base line of <100 mug/ml, the SAA concentration began rising within 12 h to a maximum value at about 48 h of 1,350-1,800 mug/ml in three males and 380-900 mug/ml in three females and returned to base line by 4-5 days. The SAA response showed a similar time response to C-reactive protein (CRP), a well-documented acute phase protein which was assayed semiquantitatively by capillary tube precipitin reaction. CRP, but not SAA, showed a quantitative correlation with the amount of fever induced by etiocholanolone. One subject exhibited a second rise in SAA and CRP concentrations after acute over-indulgence with alcohol, suggesting that acute liver damage may have caused an acute phase reaction. Thus, a controlled episode of fever and inflammation produced a prompt and prolonged elevation of SAA and CRP concentrations. Unlike SAA, CRP has not been implicated in the pathogenesis of amyloidosis, although its relationship to the P component of amyloid has recently been established.

Granulocyte transfusion therapy of experimental Pseudomonas pneumonia.

Pseudomonas pneumonia was produced in dogs with radiation-induced leukopenia. Treatment of this infection with either gentamicin alone or gentamicin plus daily granulocyte transfusion was compared in a randomized controlled trail. The dogs receiving granulocytes plus gentamicin survived significantly longer than those treated with gentamicin alone (P < 0.05). The Pseudomonas immunotype which was inoculated into the dogs were recovered at autopsy from none of the granulocyte-transfused dogs, whereas seven or eight of the dogs treated with gentamicin alone had the inoculated Pseudomonas immunotype in the area of induced pneumonia at autopsy. As measured by the limulus test, the granulocyte-transfused dogs also did not have endotoxemia as frequently as the dogs given only gentamicin (P < 0.05). This controlled study establishes that transfused granulocytes can favorably alter the course of experimental Pseudomonas pneumonia and suggests that granulocyte transfusion may be a useful therapy in serious bacterial infections of leukopenic subjects.

Role of endotoxemia in cardiovascular dysfunction and mortality. Escherichia coli and Staphylococcus aureus challenges in a canine model of human septic shock.

Using different types of bacteria and a canine model simulating human septic shock, we investigated the role of endotoxin in cardiovascular dysfunction and mortality. Either Escherichia coli (a microorganism with endotoxin) or Staphylococcus aureus (a microorganism without endotoxin) were placed in an intraperitoneal clot in doses of viable or formalin-killed bacteria. Cardiovascular function of conscious animals was studied using simultaneous radionuclide heart scans and thermodilution cardiac outputs. Serial plasma endotoxin levels were measured. S. aureus produced a pattern of reversible cardiovascular dysfunction over 7-10 d that was concordant (P less than 0.01) with that of E. coli. Although this cardiovascular pattern was not altered by formalin killing (S. aureus and E. coli), formalin-killed organisms produced a lower mortality and less myocardial depression (P less than 0.01). S. aureus, compared to E. coli, produced higher postmortem concentrations of microorganisms and higher mortality (P less than 0.025). E. coli produced significant endotoxemia (P less than 0.01), though viable organisms (versus nonviable) resulted in higher endotoxin blood concentrations (P less than 0.05). Significant endotoxemia did not occur with S. aureus. Thus, in the absence of endotoxemia, S. aureus induced the same cardiovascular abnormalities of septic shock as E. coli. These findings indicate that structurally and functionally distinct microorganisms, with or without endotoxin, can activate a common pathway resulting in similar cardiovascular injury and mortality.

Tyrphostin AG 556 improves survival and reduces multiorgan failure in canine Escherichia coli peritonitis.

Tyrosine kinase-dependent cell signaling is postulated to be a pivotal control point in inflammatory responses initiated by bacterial products and TNF. Using a canine model of gram-negative septic shock, we investigated the effect of tyrosine kinase inhibitors (tyrphostins) on survival. Animals were infected intraperitoneally with Escherichia coli 0111: B4, and then, in a randomized, blinded fashion, were treated immediately with one of two tyrphostins, AG 556 (n = 40) or AG 126 (n = 10), or with control (n = 50), and followed for 28 d or until death. All animals received supplemental oxygen, fluids, and antibiotics. Tyrphostin AG 556 improved survival times when compared to controls (P = 0.05). During the first 48 h after infection, AG 556 also improved mean arterial pressure, left ventricular ejection fraction, cardiac output, oxygen delivery, and alveolar-arterial oxygen gradient compared to controls (all P < or = 0.05). These improvements in organ injury were significantly predictive of survival. Treatment with AG 556 had no effect on clearance of endotoxin or bacteria from the blood (both P = NS); however, AG 556 did significantly lower serum TNF levels (P = 0.03). These data are consistent with the conclusion that AG 556 prevented cytokine-induced multiorgan failure and death during septic shock by inhibiting cell-signaling pathways without impairing host defenses as determined by clearance of bacteria and endotoxin.

Detection of endotoxin in cerebrospinal and joint fluids by limulus assay.

Cerebrospinal fluid (CSF) and joint fluid from 81 patients were studied for the presence of endotoxin using the limulus assay. All fluids that grew Gram-negative organisms had a positive limulus test. However, one sample of CSF and one of joint fluid, both very purulent, that contained Gram-positive organisms also gave positive limulus assays. All culturally negative fluids had a negative limulus assay. Thus, a positive limulus assay with CSF or joint fluid is indicative of bacterial infection probably caused by Gram-negative organisms and can be an extremely useful diagnostic adjunct.

Magnesium measures across the menstrual cycle in premenstrual syndrome.

The purpose of this study was to evaluate blood magnesium (Mg) measures across the menstrual cycle in women with premenstrual syndrome (PMS) and control women. Longitudinal determinations of plasma, red blood cell (RBC) and mononuclear blood cell (MBC) Mg were made in 26 women with prospectively confirmed PMS and in a control group of 19 women. Data were analyzed using analysis of variance with repeated measures and Spearman rank correlations. Significant diagnostic group effects were observed for RBC and MBC Mg concentrations (p < 0.05). These effects reflected lower Mg concentrations in PMS patients at each sampling time. No significant effects were observed for either plasma Mg or MBC Mg content, nor were there significant time by diagnosis effects for any of the measures. Consistent with earlier studies, we found decreased RBC Mg concentrations and additionally observed decreased MBC Mg concentrations in women with PMS. However, neither of these relative deficits were confined to the luteal phase.

Effects of etiocholanolone-induced fever on plasma antipyrine half-lives and metabolic clearance.

The plasma half-life and metabolic clearance rate of antipyrine, a drug metabolized by hepatic microsomal enzymes, were determined in 33 normal volunteers during a basal state and during fever induced with a single intramuscular injection of etiocholanolone. Of the 14 normal volunteers who achieved significant fever (fever index greater than 50), in 11 plasma antipyrine half-life was prolonged after a single oral dose of 10 mg/kg and antipyrine metabolic clearance rate was decreased. There was no significant change of these mean values in 19 normal volunteers who failed to develop significant fever (fever index smaller than 50). Therefore, under the conditions of this study plasma antipyrine half-life was prolonged, probably due to impaired hepatic metabolism, during etiocholanolone-induced fever, although no correlation was observed between the magnitude of fever and the extent to which plasma antipyrine half-life was prolonged. Failure to obtain such a correlation may be attributable to the very small range of temperature elevation, extending from 37.9 degrees C to 39.2 degrees C, in the group of 14 subjects achieving significant etiocholanolone-induced fever (fever index greater than 50). A higher dose of antipyrine (18 mg/kg) suppressed induction of fever by etiocholanolone; antipyrine is the only orally administered drug thus far shown to be effective in repressing etiocholanolone-induced fever.

Endotoxin-induced platelet activation in human whole blood in vitro.

The effect of purified bacterial endotoxin was studied on human platelets in vitro. In adding up to 1 microgram/mL of a highly purified endotoxin, we found neither aggregation nor ATP release in heparinized or citrated human platelet-rich plasma. On the other hand; endotoxin at concentrations as low as a few ng/mL (as may be found in septic patients) caused platelet aggregation in both heparinized and citrated human whole blood, as monitored by change in impedance, free platelet count, and size. Unlike collagen, the platelet aggregation with endotoxin occurred after a long lag phase, developed slowly, and was rarely coupled with measurable release of ATP. The platelet aggregating effect of endotoxin was dose-dependent and modified by exposure of the endotoxin to ionizing radiation. Thus, the activation of human platelets by "solubilized" endotoxin in plasma requires the presence of other blood cells. We propose that the platelet effect is mediated by monocytes and/or neutrophils stimulated by endotoxin.

Predicting space and personnel in academic clinical pathology.

This report compares the results from two surveys of the resources of academic clinical pathology departments. In 1971, the Academy of Clinical Laboratory Physicians and Scientists (ACLPS) obtained data from 25 academic clinical pathology departments on actual workload variables (census, procedures per year, admissions per year, and outpatient visits per year), along with estimates of ideal space and ideal staffing. A study conducted by NIH obtained data from 31 academic clinical pathology departments for 1976 for the same workload variables, along with actual and ideal space and actual staffing. Regression analyses were performed with space, technical personnel, and doctoral personnel as the dependent variables and patients per day, admissions per year, and numbers of procedures per year as the independent variables. For each study, admissions per year and patients per day were statistically significant predictors of both departmental space and technical personnel. The slope of each significant regression from the ACLPS study was greater than the comparable significant slope from the NIH study. These data provide a basis for comparing resources among academic clinical pathology departments, assessing current resources, and projecting future needs.

Pseudomonas aeruginosa compared with Escherichia coli produces less endotoxemia but more cardiovascular dysfunction and mortality in a canine model of septic shock.

We investigated the effects of two different Gram-negative bacteria and radiation-induced leukopenia on endotoxemia, cardiovascular abnormalities, and mortality in a canine model of septic shock. Serial hemodynamics were measured in conscious dogs using radionuclide heart scans and thermodilution cardiac output catheters. Plasma endotoxin concentrations were determined with a chromogenic Limulus amebocyte lysate assay. Viable Pseudomonas aeruginosa or Escherichia coli implanted intraperitoneally produced concordant hemodynamic patterns of septic shock (p less than 0.01). Endotoxin concentrations were more than tenfold lower in dogs infected with P aeruginosa compared with E coli (p less than 0.0001). Despite lower endotoxin levels, P aeruginosa-infected dogs had a higher mortality (p less than 0.01), more severe hypotension (p less than 0.05), and greater depression of the left ventricular ejection fraction (p less than 0.05) than dogs with E coli sepsis. A nonlethal E coli challenge combined with leukopenia (induced by a nonlethal dose of radiation) resulted in a mortality of 60 percent (p less than 0.01) without greater cardiovascular dysfunction or higher endotoxin concentrations. These findings suggest that bacterial products other than endotoxin and host-related factors may be important contributors to the toxicity, cardiovascular instability, and mortality of Gram-negative septic shock. Quantitative determinations of plasma endotoxin are unlikely to correlate with the clinical severity of septicemia in heterogeneous patient populations infected with different Gram-negative organisms.

Endotoxemia in human septic shock.

To evaluate the incidence, pattern and clinical importance of endotoxemia in septic shock, frequent, serial endotoxin determinations were made prospectively in patients with shock. Detectable endotoxin occurred in 43 of 100 patients with septic shock, but in only one of ten patients with shock due to nonseptic causes. During septic shock, endotoxemia frequently occurred in the absence of Gram-negative bacteremia. Using a logistic regression model, multiple organ failure occurred 10.3 times more frequently and depression of left ventricular ejection fraction (less than or equal to 45 percent) 4.8 times more frequently in endotoxemic patients. In patients with positive blood cultures, endotoxemia was associated with a high mortality. We conclude that endotoxemia occurs frequently in septic shock and is associated with severe manifestations of this syndrome, including cardiac depression and multiple organ failure. This study suggests that endotoxin is an important mediator of septic shock and supports efforts to develop anti-endotoxin therapies for treating patients with this disease.

A program for residency training in clinical pathology.

The goals, organization, components, instruction approaches, and the continuing assessment of a clinical pathology residency program are described. This program emphasizes training by rotation in the services of the clinical laboratory, application and interpretation of laboratory data for patient care, laboratory management, teaching conferences, and research and development.

Magnesium: the fifth but forgotten electrolyte.

Magnesium (Mg) is the second most abundant intracellular cation and is a cofactor in more than 300 enzymatic reactions involving energy metabolism and protein and nucleic acid synthesis. Ionized Mg is the physiologically active form of the element. Protein-bound and chelated Mg buffer the ionized pool. Approximately half the total Mg in the body is present intracellularly in soft tissue, and the other half is present in bone. Less than 1% of the total body Mg is present in blood. However, the majority of our clinical laboratory information comes from the determination of total Mg in serum. Currently, the clinical laboratory evaluation of Mg status is limited primarily to the total serum Mg concentration and a 24-hour urinary excretion. Instrumentation to determine ionized Mg in serum (ion-selective electrode) and in soft tissue (nuclear magnetic resonance spectroscopy) should be available in the near future. Magnesium may be a factor in the treatment of acute myocardial infarction and the rate of atherosclerosis. Chronic changes of Mg status, that may be latent, are poorly understood and require a better knowledge of ionized Mg metabolism.

Considerations for the design of a new laboratory.

The purpose of this article is to relate the process of designing a new Clinical Pathology Department laboratory at the National Institutes of Health and the assessment of the resultant open laboratory design based on the response of the laboratory staff to two questionnaires. The responses indicated acceptance and approval of the large open area, work area design, carpeted floor, and laboratory environment. It is the authors' hope that this information will be of help to other pathology departments who are involved in designing or remodeling a new or existing laboratory.

Workload, space, and personnel of clinical chemistry laboratories in teaching hospitals.

The resources needed to operate a clinical chemistry laboratory are known only as rough approximations. In order to gather more information about these resources, a 13-page questionnaire was completed by 50 pathology departments of which 38 departments provided adequate information about their clinical chemistry section to permit adequate data analysis. This study provides the results for resource and workload factors, and significant regression analyses among these factors for the whole clinical chemistry section and the subsections of general chemistry, urinalysis, endocrinology, enzymology, lipid analysis, and toxicology. These data provide a basis for evaluating the current needs of clinical chemistry and for making projections for the future.

Departmental resources and statistics of academic pathology.

The resources needed to operate an academic pathology department have not been evaluated for several years. Fifty academic pathology departments in this country returned a 14-page questionnaire that included information about procedures, personnel, space, and patient load. These data were summarized using means, percentiles, ratios, and linear regression analyses. The average academic pathology department performed more than 5,000 procedures for 1,200 inpatients and outpatients per day and produced 42 publications per year. The results of this study may assist members of the academic pathology community in assessing current and future needs.