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High-performance liquid chromatography (HPLC) was used to separate, identify, and quantitate the trichothecin mycotoxin, T-2 (4 beta,15-diacetoxy-3 aopha-hydroxy-8 alpha [3-methyl-butyryloxy]-12,13-epoxy delta 9-trichothecin), and its metabolites in plasma and urine samples from cynomolgus monkeys treated with the toxin. A 15-min gradient elution system was developed to separate and measure radiolabeled T-2 mycotoxin and its metabolites. The HPLC technique for separating T-2 and its metabolites was compared with thin-layer chromatography. Samples from the in vitro metabolism of T-2 by plasma and urine were included as controls and as a measure of the toxin's stability in biological samples. Within 5 min, 22% of the plasma radiolabeled T-2 toxin was detected as metabolites after an intravenous administration of [3H] T-2 toxin to cynomolgus monkeys. By 24 h post-exposure, there was no parent T-2 toxin detected in plasma or urine. T-2 tetraol was the major metabolite detected in the plasma and urine of monkeys. Other metabolites observed in urine up to 5 days after exposure were 3'OH-T-2 and 3'OH-HT-2. We conclude that T-2 toxin was rapidly metabolized to more polar metabolites, which were eliminated in urine.

Tritiated saxitoxinol was used to obtain preliminary information on saxitoxin metabolism in the rat. Sublethal doses of tritiated saxitoxinol (18.9-microCi/kg; 3.8 micrograms/kg) were injected i.v. into each of six rats. Urine and fecal samples were collected up to 144 hr post-injection. Within 4 hr, 60% of injected radioactivity was excreted in urine. No radioactivity was found in feces. High performance liquid chromatography analyses of urine showed that saxitoxinol was not metabolized by the rats.

Rats were given an ip injection of T-2 mycotoxin (T-2), the T-2 metabolite, T-2 tetraol (tetraol), or cycloheximide. Serum, liver, heart, kidney, spleen, muscle, and intestine were collected at 3, 6, and 9 hr postinjection after a 2-hr pulse at each time with [14C]leucine and [3H]thymidine. Protein and DNA synthesis levels in rats were determined by dual-label counting of the acid-precipitable fraction of tissue homogenates. Rats given a lethal dose of T-2, tetraol, or cycloheximide died between 14 and 20 hr. Maximum inhibition of protein synthesis at the earliest time period was observed in additional rats given the same lethal dose of the three treatments and continued for the duration of the study (9 hr). With sublethal doses of T-2 or tetraol, the same early decrease in protein synthesis was observed but, in most of the tissues, recovery was seen with time. In the T-2-treated rats. DNA synthesis in the six tissues studied was also suppressed, although to a lesser degree. With sublethal doses, complete recovery of DNA synthesis took place in four of the six tissues by 9 hr after toxin exposure. The appearance of newly translated serum proteins did not occur in the animals treated with T-2 mycotoxin or cycloheximide, as evidenced by total and PCA-soluble serum levels of labeled leucine. An increase in tissue-pool levels of free leucine and thymidine in response to T-2 mycotoxin was also noted. T-2 mycotoxin, its metabolite, T-2 tetraol, and cycloheximide cause a rapid inhibition of protein and DNA synthesis in all tissue types studied. These results are compared with the responses seen in in vitro studies.

In this study, concentration-response parameters were determined for rats and guinea pigs systematically exposed to an aerosol of T-2 toxin. The LC50 for a 10-min exposure to T-2 toxin aerosol was 0.02 mg T-2/liter air for rats and 0.21 mg T-2/liter air for guinea pigs. Data from total T-2 deposition in rats and guinea pigs exposed to their respective LC50 aerosol concentration gave an LD50 of 0.05 mg T-2/kg body weight for the rat and 0.4 mg T-2/kg body weight for the guinea pig. These data show that inhaled T-2 toxin is approximately 20 times more toxic to the rat (0.05 mg T-2/kg body wt inhaled vs 1.0 mg T-2/kg body wt ip) and at least twice as toxic to the guinea pig (0.4 mg T-2/kg body wt inhaled vs 1-2 mg T-2/kg body wt ip) than ip administered T-2 toxin. Histopathologic examination of major organs in both the rat and guinea pig after respiratory exposure to T-2 toxin indicated that lesions were similar to those described after systemic administration of the toxin. Gross and microscopic alterations of respiratory tract tissue after T-2 aerosol exposure were minimal and could not account for the increase in toxicity.

Ribavirin has been shown to have broad-spectrum antiviral. To study its tissue distribution and disappearance rate, a single dose of 10 mg/kg which contained 10 microCi of [14C]ribavirin was injected intravenously into rhesus monkeys and intramuscularly into monkeys and rats. Except for peak plasma concentrations and the initial phases of the plasma disappearance and urine excretion curves, no significant difference was observed between plasma, tissue, or urine values for intramuscularly or intravenously injected monkeys. Plasma disappearance curves were triphasic; plasma concentrations of ribavirin were similar for both monkeys and rats. Rats excreted ribavirin in the urine more rapidly and to a greater extent (82% excreted in 24 h) than did monkeys (60% excreted in 72 h). In the rat, only 3% of the injected [14C]ribavirin was detected in expired CO2. Therefore, for both species, urine was the major route for the elimination of labeled ribavirin and its metabolites from the body. In monkeys, the amount of parent drug in blood cells increased through 48 h and remained stable for 72 h, whereas in rats, ribavirin decreased at a rate similar to the plasma disappearance curve. Concentrations of ribavirin at 8 h were consistently higher in monkeys than in rats for all tissues except the brain. Thus, these differences in blood cellular components and organ content and in urine excretion suggested that there was greater tissue retention of ribavirin in monkeys than in rats.

During caloric deprivation, the septic host may fail to develop ketonemia as an adaptation to starvation. Because the plasma ketone body concentration is a function of the ratio of hepatic production and peripheral usage, a pneumococcal sepsis model was used in rats to measure the complex metabolic events that could account for this failure, including the effects of infection on lipolysis and esterification in adipose tissue, fatty acid transport in plasma and the rates of hepatic ketogenesis and whole body oxidation of ketones. Some of the studies were repeated with tularemia as the model infection. From these studies, it was concluded that during pneumococcal sepsis, the failure of rats to become ketonemic during caloric deprivation was the result of reduced ketogenic capacity of the liver and a possibly decreased hepatic supply of fatty acids. The latter appeared to be a secondary consequence of a severe reduction in circulating plasma albumin, the major transport protein for fatty acids, with no effect on the degree of saturation of the albumin with free fatty acids. Also, the infection had no significant effect on the rate of lipolysis or release of fatty acids from adipose tissue. Ketone body usage (oxidation) was either unaffected or reduced during pneumococcal sepsis in rats. Thus, a reduced rate of ketone production in the infected host was primarily responsible for the failure to develop starvation ketonemia under these conditions. The liver of the infected rat host appears to shuttle the fatty acids away from beta-oxidation and ketogenesis and toward triglyceride production, with resulting hepatocellular fatty metamorphosis.

We describe a simple, rapid, and sensitive bioassay for the detection and quantitation of T-2 mycotoxin by using a protein synthesis assay in cultured cells. Increased sensitivity of the cells to the mycotoxin occurred with time up to ca. 60-min. Time and dose response curves show that an average of 10 to 20 ng of T-2 per ml was sufficient to cause 50% inhibition of protein synthesis in tissue culture cells. A wide range of tissue culture cells with varied type, tissue, and species sources and growth characteristics were tested by this system. All showed approximately the same sensitivity to the mycotoxin. A slight modification of the procedure was used for suspended cultures of mitogen-stimulated lymphocytes, which also showed an equal degree of sensitivity to the mycotoxin. By simply changing the labeled precursor, the inhibition of RNA, DNA, and protein synthesis by T-2 mycotoxin can be compared. Although T-2 mycotoxin had little effect on RNA synthesis, DNA and protein synthesis were equally inhibited. Because of its sensitivity and its capacity to quickly assay a large number of samples, this technique has been a valuable tool in screening samples for the presence of active toxin and has been used to help establish laboratory safety standards for the inactivation of T-2 mycotoxin by chemical agents. It is presently being used in studies of mycotoxin mechanism of action and approaches toward in vivo neutralization of the toxic effects of mycotoxins.

The catabolic effects of starvation alone, or starvation in the presence of pneumococcal sepsis, were compared in rats whose skeletal muscle protein had been tagged 14 days earlier with 14C-phenylalanine. In a fed rat, protein catabolism (as estimated by expired 14CO2) is not constant throughout the day but is highest during the dark hours. Starvation is associated with accelerated protein catabolism and a gradual loss of periodicity. Infection increases the rate of catabolism still further and results in a complete loss of periodicity.

4-Deoxyverrucarol (4) has been synthesized for use in studies for the preparation and development of monoclonal antibodies for trichothecenes. Both verrucarol (1) and anguidine (2) have been converted to deoxyverrucarol (DOVE) (4) by deoxygenation at C3 and at C3 and C4, respectively.

To determine spontaneous 24-h patterns of growth hormone (GH) plasma levels in unsedated and unrestrained nonhuman primates, a jacket and tethering system were used to study six cynomolgus monkeys. Hourly blood samples were collected, and body temperatures were recorded over 24-h periods. Measurements of GH were made on all samples. In one 24-h study cortisol levels were also measured as well to document a normal circadian rhythm. GH was released at mean intervals of 4.5 +/- 0.47 h (mean +/- SE) over the 24-h studies. There were no day-to-night differences in either the mean interval of GH release (day, 4.6 +/- 0.66 h; night, 4.4 +/- 0.51) or the mean GH values (day, 9.8 +/- 1.7 mU/l; night, 7.9 +/- 0.8). An apparent midday peak in GH in the 24-h studies followed feeding. As expected, body temperature was higher during the day than night, documenting a normal circadian rhythm. Plasma cortisol also showed a normal circadian variation with a low point midday and a progressive rise during the night in the one 24-h cycle in which it was measured. GH in unsedated, unrestrained cynomolgus monkeys was released in 4- to 5-h cycles both day and night without increased nighttime release. This contrasts sharply with the known nocturnal sleep release of GH seen in humans.

T-2 toxin produced significant coagulation abnormalities when administered parenterally to Hartley strain guinea pigs. The animals developed depressed activity of all coagulation factors except fibrinogen. Platelet aggregation in whole blood was depressed in response to ADP and collagen. The animals also exhibited an initial rise followed by a fall in hematocrit level, leukocytosis, and a decrease in platelet count. These changes were detectable within hours of toxin administration, reached a maximum at 24 hr, and returned to normal over the next 2 days. Pretreatment of animals with vitamin K1 had no effect on the activity of coagulation factors. The activated partial thromboplastin time of dilutions of plasma from animals given T-2 toxin with plasma from control animals revealed a pattern which pointed to a deficiency of coagulation factors as the principal cause of prolonged clotting times in treated animals. The presence of a weak circulating anticoagulant could not be ruled out. The addition of T-2 to plasma and blood of normal animals in a concentration of 1 microgram/ml had no effect on clotting times or platelet aggregation.

A model was developed in the rhesus monkey to determine if the marked wasting of body proteins associated with sepsis could be prevented by an intravenous supply of various nutritional substrates. All monkeys were given a basic infusion of 0.5 gm of amino acid nitrogen/kg body weight via an indwelling catheter in the jugular vein. Three groups were given diets with no added calories, 85 calories/kg from dextrose or 85 calories from lipid. In each group, six monkeys were inoculated with 3 x 10(8) Streptococcus pneumoniae and four with heatkilled organisms. In the monkeys infused with the amino acids alone, pneumococcal sepsis resulted in a fourfold increase in loss of body proteins compared with calorie-restricted controls. Addition of 85 calories/kg/day of either dextrose or lipid reduced body wasting associated with infectious disease. The calories from lipid were utilized bythe septic host as a source of energy, with a slightly reduced efficiency when compared with the isocaloric infusion of dextrose. The nitrogen sparing of the fat emulsion could not be accounted for by its glycerol content. Therefore, the septic monkey seemed to utilize fatty acids as an energy substrate. It appears that the carbohydrate calories tend to favor the synthesis of peripheral proteins (associated mainly with skeletal muscle), while lipid calories favor synthesis of visceral proteins such as plasma albumin and acute-phase proteins.

A jacket and tethering system was used to maintain chronic catheters in monkeys, which provided catheter access and manipulability without further restraint. Surgical placement of catheters and a temperature probe allowed for a common cutaneous exit and interface with the jacket and tether. Monkeys were fitted in a sterile leather or denim jacket which was attached to a sterilized flexible stainless steel cable. Through this conduit, an indwelling temperature probe, as well as catheters from the internal jugular and femoral veins, were attached to a swivel unit located on the upper portion of the cage. The internal jugular catheter was used for the continuous infusion of support solution. The catheter from the femoral vein was maintained with a heparin lock and used for serial blood sampling. Using this system, it was possible to obtain frequent blood samples and body temperature readings, and to administer a continuous intravenous infusion without chemical or excessive physical restraint. To date, 367 monkeys, 322 cynomolgus (Macaca fasicularis), 16 rhesus (Macaca mulatta), and 21 African green (Cercopithecus aethiops) have been studied using this procedure.

A proteinaceous secretion from phagocytizing polymorphonuclear leukocytes, termed "leukocytic endogenous mediator" (LEM), has been shown to have marked effects on hepatic amino acid transport and RNA and protein synthesis. A single injection of LEM results in a marked accumulation of labeled nonmetabolizable model amino acids in the liver of normal rats. The LEM-stimulated uptake of amino acids by liver was observed in adrenalectomized, hypophysectomized, thyroidectomized, or diabetic rats and could not be duplicated by pharmacological doses of a large variety of hormones. In addition, LEM stimulated an increased uptake of alpha-aminoisobutyric acid by isolated livers during their perfusion in vitro. LEM also stimulated an increased incorporation of orotic acid into hepatic RNA of intact rats, especially into the bound ribosomal fraction. This increased synthesis of RNA preceded an enhanced hepatic production of a number of the acute-phase plasma globulins. LEM did not stimulate the adenylate cyclase-cAMP system in liver and was not found to utilize this system as a second messenger. Thus, the effects of LEM in stimulating hepatic amino acid transport appear to be direct, without mediation by other hormones, and to be independent of cAMP. On the other hand, the ability of LEM to stimulate RNA and acute phase globulin synthesis in liver may require the presence of physiological quantities of hormones such as adrenal corticoids.

Infections or inflammatory states often cause significant increases in serum phenylalanine and the phenylalanine-tyrosine ratio. More than 95% of samples obtained during inflammatory diseases in man showed phenylalanine-tyrosine ratio increases greater than the maximum normal values. An increase in this ratio also occurred in monkeys with induced Rocky Mountain spotted fever, viral encephalitis, yellow fever, or pneumococcal and Salmonella infections, as well as in rats with pneumococcal and Salmonella infections, as well as in rats with pneumococcal, Salmonella or tularemia infections. A similar ratio increase occurred in rats inoculated with unpurified mediator substances (released by activated leukocytes) that appear to initiate many of the secondary metabolic phenomena associated with infection and/or inflammation. To identify responsible mechanisms, rats were given lethal doses of Streptococcus pneumoniae; serum phenylalanine and phenylalanine-tyrosine ratios increased significantly. Hepatic phenylalanine hydroxylase activities were slightly decreased when compared to noninfected controls. Infected and noninfected rats showed comparable oxidation rates for 14C-phenylalanine given with an oral phenylalanine load, as a pulse-oral dose, or as an intraperitoneal injection. After 8 hr, both infected and control rats had similar amounts of radioactivity in total body protein, but tissue distributions were markedly altered during pneumococcal sepsis. Serum proteins of infected rats contained almost twice as much total radioactivity as that found in controls, while the amount of labeled phenylalanine in skeletal muscle protein was significantly reduced in the infected group. Isolated muscles from infected rats released more phenylalanine and less tyrosine than control muscles. Infection-related increases in serum phenlalanine could not be explained by decreased hydroxylation or oxidation. Rather, the data were consistent with an increased flux of phenylalanine into serum, most likely as the result of increased skeletal muscle catabolism. Elevations in the serum phenylalanine-tyrosine ratio have potential value for estimating the presence of an inflammatory fisease and the catabolic state of a patient.

The ability of various adenosine analogs to inhibit cholera toxin activation of the intestinal epithelial cell adenylate cyclase-cyclic AMP system was investigated. After incubation of cells with cholera toxin for 6 hr, large increases in cellular cyclic AMP content were observed. Addition of 2', 5'-dideoxyadenosine during the last 30 min of this 6-hr incubation resulted in 70% reduction in elevated cyclic AMP content. Other analogs were not effective inhibitors. 2', 5'-Dideoxyadenosine was also a potent inhibitor of cholera toxin-activated intestinal cell adenylate cyclase activity with half-maximal inhibition occuring at 16 muM. NaF-stimulated cyclase was less susceptible to inhibition. The data suggest that inhibition by 2', 5'-dideoxyadenosine is due at least in part to direct inhibition of the cholera toxin-activated intestinal adenylate cyclase activity.

A model has been developed to measure the effects of dietary protein on daily fluctuations in the rate of endogenous amino acid oxidation in meal-fed and starved rats. In addition, N tau-methylhistidine and hydroxyproline were utilized to determine changes in the rate of degradation of myofibrillar and collagen proteins. In rats meal-fed a normal diet of 18% (w/w) casein, a diurnal response was observed in rate of oxidation of radioactive amino acids contained in endogenous labelled body protein, with a nadir 16--20 h and maximum 4--8 h after beginning the feeding. This observation in part may be related to alterations in flux of amino acids from non-hepatic tissues to site of oxidation in liver, as well as alterations in rates of amino acid oxidation after a protein meal. When meal-fed a 70% protein diet, the maximal rates of endogenous amino acid oxidation were significantly increased by 4--8 h after meal-feeding, with no change in fractional rates of degradation of myofibrillar- or collagen-protein breakdown. This could suggest increases in activities of enzymes involved in amino acid oxidation, in rats meal-fed 70% compared with 18% dietary protein. In contrast, meal-feeding of a protein-free diet muted the diurnal response in the rate of oxidation of endogenously labelled amino acids, which correlated with a decrease in the fractional rate of degradation of myofibrillar or collagen protein. Thus dietary protein is apparently responsible for the observed diurnal rhythm rhythms in the rate of amino acid oxidation, whereas carbohydrates tend to mute the response.

Experiments were conducted to study the acute inhalation toxicity of T-2 mycotoxin in both young adult and mature mice. For a 10-min aerosol exposure, the 24-hr LC50 of T-2 mycotoxin in young adult mice was 0.08 +/- 0.04 mg T-2/liter air and that for mature mice was 0.325 +/- 0.1 mg T-2/liter air. Deaths among mice exposed to the higher aerosol concentrations used in this study (i.e., 1.5 to 2.4 mg T-2/liter air) occurred in less than 5 hr. General clinical symptoms in these animals immediately postexposure were tremors, lethargy, stilted gait, and, in some animals, prostration. In experiments separate from the concentration-response studies, total deposition of T-2 aerosol and selective retention of T-2 in the respiratory tract and nasal turbinates were determined analytically from 3H-labeled T-2. When total deposition of T-2 was quantitated, there was excellent agreement between that amount of T-2 deposited and that amount of T-2 predicted from calculations based on aerosol size and animal minute volume. Based on the aerosol deposition data, the LD50 values of T-2 mycotoxins was 0.24 mg/kg for young adult mice and 0.94 mg/kg for mature mice. For mice, inhalation of T-2 mycotoxin is at least 10 times more toxic than systemic administration (LD50 approximately 4.5 mg/kg) and at least 20 times more toxic than dermal administration (LD50 greater than 10 mg/kg).

Acute clinical malaria caused by Plasmodium inui was diagnosed in an adult female cynomolgus monkey (Macaca fascicularis) 4 years after importation into the United States. Stress and immunosuppression associated with experimentation completed 2 weeks earlier may have contributed to the development of severe clinical disease. Clinical findings included severe regenerative anemia, hepatosplenomegaly, weakness, lethargy, weight loss, and anorexia. The infection was treated and successfully eliminated with chloroquine hydrochloride administered intramuscularly at a dose of 5 mg/kg base given at 0, 6, 24, 48, and 72 hours. Treatment also included a blood transfusion and intensive supportive care.

T-2 toxin, a mycotoxin produced by several strains of the genus Fusarium, has been implicated as a cause of serious illness in both animals and man. Hemorrhage is a feature of the syndromes which have been described. An LD20 dose of T-2 was administered im to adult cynomolgus monkeys. This resulted in prolongation of prothrombin and activated partial thromboplastin times and a decrease in multiple coagulation factors. These changes were detected within hours of toxin administration, were maximal at 24 hr, and returned to normal over the next 3 days. Fibrin-fibrinogen degradation products were not detected at any time point. Repeated phlebotomy produced a significantly greater increase in platelet count in control monkeys, which could be taken as evidence for an effect of toxin on platelet kinetics. In treated animals, the hematocrit level declined by about 10%, but a similar decrease occurred in control animals. The white blood cell count increased 4 to 5 times over pretreatment values. Despite the changes in multiple laboratory parameters, treated monkeys did not exhibit clinical evidence of hemorrhage. In three animals which died as a result of toxicosis, necropsy revealed mild petechial hemorrhage involving the colon and heart, as well as necrosis of lymphoid tissues.

Since recent studies have indicated that measurement in urine of the amino acid, 3-methylhistidine, accurately reflects the extent of muscle catabolism, and because it has been suggested that thyroid hormones may influence muscle breakdown, especially during fasting, the effect of T3 and reverse T3 (rT3) administration on the excretion of 3-methylhistidine was examined in obese subjects during fasting. The mean (+/- SE) 3-methylhistidine excretion in patients fed an egg protein diet (devoid of meat protein) was 256 +/- 35 mumoles/day and decreased to 190 +/- 14 mumoles/day during fasting. T3 administration (100 microgram/day x 5 days) increased 3-methylhistidine excretion to 304 +/- 37 mumoles/day during its ingestion and to 485 +/- 46 mumoles/day in the T3 posttreatment interval. T3 doses of 10 microgram every 4 hr (q4h) for the first 6 days of fasting also appeared capable of increasing 3-mehis excretion whereas 5 microgram T3 q4h administered during the first 6 days of fasting did not increase 3-mehis excretion. Reverse T3 administration (80 microgram q6h) during fasting was associated with a mean 3-methylhistidine of 130 +/- 13 mumoles/day, a value no higher than in patients fasted alone. These observations suggest that: (1) skeletal muscle catabolism decreases during fasting: and (2) pathophysiologic doses of T3 (60 microgram/day or more), but not reverse T3, enhance muscle catabolism during fasting.

Rat liver was quantitatively subfractionated into free ribosomal, bound ribosomal, nuclear, and soluble fractions to determine the effects of infection and endotoxin treatment on hepatic RNA production and distribution. A 4-h pulse label of [14C]orotic acid was used to monitor newly transcribed RNA; distribution was determined by measuring RNA content at various times after infection or endotoxin treatment. A significant increase in the rate of RNA synthesis was seen by 12 h and continued through 20 h in response to Streptococcus pneumoniae infection. During the peak hours of the RNA response, redistribution of RNA into the bound ribosomal fraction takes place at the expense of the free ribosomes. However, in Salmonella typhimurium and its endotoxin, more involvement of the free ribosoma fraction during the early stages of the infection was apparent. These data suggest that the hepatic RNA response takes place in two stages, an early "endotoxin" response, resulting in redistribution of cytoplasmic RNA into free ribosomes, and a later "infection" response, involving the mobilization of the bound ribosomes.

At 26 h after inoculation of rats with Diplococcus pneumoniae, the serum concentrations of 10 and 20 individual amino acids were lower than corresponding values observed in pair-fed controls. In contrast, only 2 of 20 serum amino acids were similarly decreased in rats inoculated with Salmonella typhimurium. Despite these serum differences, a greater accumulation of labeled non-metabolizable amino acids occurred in the livers of rats infected with S. typhimurium. These data suggested a greater increase in the flux of amino acids from muscle to liver in the rats infected with S. typhimurium as compared to those infected with D. pneumoniae. A similar increase in serum protein synthesis was observed in rats infected with D. pneumoniae or S. typhimurium. However, with the latter infection, a larger percentage of the amino acids appeared to be utilized as a source of energy in addition to their role as precursors of proteins.

With the onset of fever in volunteers exposed to virulent Salmonella typhii as part of a vaccine evaluation study, urinary excretion of kynurenine, acetyl-kynurenine and o-amino-hippurate significantly increased by N-methylnicotinamide decreased. Serum tryptophan concentration at that time was 116% of control in contrast to total serum amino acid values at 83% of control. A 3 gram oral tryptophan load given during illness further increased serum tryptophan concentration to 186% of control and enhanced excretion of the aforementioned catabolites. In addition, urinary anthranilate glucuronide, kynurenate and xanthurenate rose significantly; 3-hydroxykynurenine increased only slightly and N-methyl-nicotinamide was unaltered. Thus, though a tryptophan load aids in detection of alterations in tryptophan catabolism via the kynurenine pathway as a whole, it also distorts the pattern of metabolites detected, seemingly by causing more kynurenine to be shunted to the mitochondria to be acted upon by kynurenine 3-hydroxylase and kynurenine aminotransferase. In unloaded patients the increased urinary excretion of tryptophan metabolites during typhoid fever merely reflect the enhanced flux of amino acids from muscle to liver, akin to that noted in other infections, in excess of that needed for the synthesis of serum proteins or for other metabolic pathways.

The possible potentiation of an infection upon the metabolic consequences of trauma was tested in rats using a 2 X 2 block design which included control, femoral fracture, pneumococcal infection, and fracture plus infection groups. Infection introduced unique metabolic effects different from those of starvation, femoral fracture, or both together. Infection-induced effects included an accelerated conversion of 14C-alanine to glucose, higher serum haptoglobin, alpha2-macrofetoprotein, copper, and ceruloplasmin values, and lower serum iron, zinc, and transferrin concentrations. The first three of these infection-induced effects were diminished in rats with a femoral fracture. No measured effect of infection was increased in traumatized rats.

The effects of infection with Streptococcus pneumoniae, Francisella tularensis, and Venezuelan equine encephalitis virus as well as inflammatory stress induced by the administration of turpentine and endotoxin on plasma ketone bodies and insulin were studied in white rats. All of the infectious/inflammatory stresses caused a significant decrease in the ketonemia of fasting and an elevation of plasma insulin. When a pneumococcal infection was initiated in a diabetic rat, inhibition of fasting ketonemia did not occur. Similarly, pneumococcal infection in the hypophysectomized rat did not result in a noticeable depression of either fasting ketonemia or plasma FFA. The increase in circulating insulin appears to be closely correlated with the inhibition of fasting ketonemia noted in the infectious/inflammatory stress.

Cutaneous absorption and decontamination of [3H]T-2 mycotoxin using various treatment modalities incorporating water, detergent, sprays, and scrubbing of application sites were examined in the rat model at 5, 30, 60, and 1440 min (24 h) postexposure. Rats were killed immediately after treatment and radiolabeled T-2 remaining in full-thickness skin samples were determined. Absorption and decontamination were followed over time, and decontaminating treatment modalities were evaluated for efficacy. Less than 1% of the applied dose was absorbed in 5 min, and 50% was absorbed in 24 h. At 5 min, 99.5 +/- 0.05% of nonabsorbed (residual) [3H]T-2 was removed, and 58 +/- 5.2% of residual toxin was removed at 24 h with a 2.5% detergent/water spray. When treatment modalities were evaluated at 60 min, a 2.5% detergent/water scrub followed by a detergent/water spray produced optimal decontamination by removing 81 +/- 2.2% of residual toxin. All treatment modalities using detergent and/or water removed significant amounts of toxin (p less than or equal to .0001); a dry scrub was not efficacious. Treatment should be initiated as soon as possible after exposure for best results. However, the stratum corneum acts as a reservoir for the toxin, and decontamination should be carried out even if delayed several hours or days after exposure. Dermal absorption pharmacokinetics found in these studies are similar to those described for other low-molecular-weight compounds, and the decontamination results from T-2 toxin should be applicable to other, similar toxic substances.

T-2 toxin is a potent cytotoxic metabolite produced by the Fusarium species. The fate and distribution of 3H-labeled T-2 toxin were examined in male guinea pigs. Radioactivity was detected in all body tissues within 30 min after an im injection of an LD50 dose (1.04 mg/kg) of T-2 toxin. The plasma concentration of trichothecene molar equivalents versus time was multiphasic, with an initial absorption half-life equal to or less than 30 min. Bile contained a large amount of radioactivity which was identified as HT-2, 4-deacetylneosolaniol, 3'-hydroxy HT-2, 3'-hydroxy T-2 triol, and several more-polar unknowns. These T-2 metabolites are excreted from liver via bile into the intestine. Within 5 days, 75% of the total radioactivity was excreted in urine and feces at a ratio of 4 to 1. The appearance of radioactivity in the excreta was biphasic. Metabolic derivatives of T-2 excreted in urine were T-2 tetraol, 4-deacetylneosolaniol, 3'-hydroxy HT-2, and several unknowns. These studies showed a rapid appearance in and subsequent loss of radioactivity from tissues and body fluids. Only 0.01% of the total administered radioactivity was still detectable in tissues at 28 days. The distribution patterns and excretion rates suggest that liver and kidney are the principal organs of detoxication and excretion of T-2 toxin and its metabolites.

In volunteers experimentally infected with Salmonella typhi, serum iron and zinc concentrations became significantly depressed and there was a concomitant rise in serum copper before the onset of overt clinical illness. However, after several days of fever and the initiation of chloramphenicol therapy, serum iron and zinc concentrations significantly increased. Additional studies--in volunteers with typhoid fever treated with chloramphenicol, in a volunteer with typhoid fever receiving cefazolin and gentamicin, and in untreated rhesus monkeys infected with Salmonella typhimurium--provided evidence that the increase in serum iron concentration during the febrile phase was the result of chloramphenicol therapy, whereas the increase in serum zinc concentrations was a disease-related phenomenon. The importance of trace-metal monitoring during infectious disease and chemotherapy is discussed.

To investigate the effects of bacterial infection on glucose and alanine metabolism, a variety of studies were carried out in rat and monkey models. These included glucose turnover by a pulse-dose technique in infected rats; alanine and glucose production and utilization in control and septic monkeys; in vivo measurement of gluconeogenesis in rats, with and without an alanine load; the in vitro rate of glucose formation from various substrates by isolated liver perfusion and hepatic cells; and in vivo rates of oxidation of glucose labeled with 14C at the 1 or 6 carbon position. In rats, glucose turnover was markedly accelerated 24 hr after inoculation of either 10(4) or 10(7) Streptococcus pneumoniae. Glucose utilization and production were also accelerated during illness and early recovery from a pneumococcal infection in monkeys. The rates of gluconeogenesis as measured by either a pulse technique in rats or continuous infusion of labeled alanine in monkey were significantly elevated during pneumococcal septicemia. During the agonal stages (10(7) of the pneumococcal infection in rat, an alanine load resulted in a decreased rate of labeled glucose production and an increase in plasma glucose when compared to values of fasted control rats. However, early illness caused similar or increased rates of glucose production from alanine in vivo. Similar reduced rates of glucose formation were observed when the isolated livers or hepatocytes from rats during the agonal stages of infection were perfused with excess quantities of gluconeogenic substrates. Thus, in the rat, gluconeogenic capacity (ability to form glucose from excess substrates) appears to decrease only during the agonal stages of pneumococcal infection. During acute pneumococcal sepsis in the rhesus monkey, alanine production and utilization were significantly elevated and it was estimated that over 90% of the newly produced alanine was utilized for glucose synthesis. When arterial--venous differences were measured across the hindquarters, significantly more alanine was released, presumably from skeletal muscle of the septic monkey, compared to the recovery period or in the control groups. Thus, the increase in glucose synthesis in both rat and monkey appears to be correlated with substrate availability and kinetic rate, rather than gluconeogenic capacity of the liver. The major increase in glucose utilization during both S. pneumoniae and Francisella tularensis live vaccine strain (LVS) infections in rat was a progressive elevation in the rate of oxidation via the pentose phosphate shunt in the rat. Further, the rate of oxidation appeared to be correlated with the magnitude of the bacteremia, which is an indication of the severity of the infection...

Tularaemia in rats causes a doubling of the serum phenylalanine/tyrosine ratio and a more than twofold increase in muscle phenylalanine which cannot be accounted for by decreased hepatic phenylalanine hydroxylase (EC 1.14.16.1) activity, but appear to result from a generalized movement of amino acids from muscle to liver during infection.

Nineteen 12,13-epoxytrichothecene mycotoxins were tested for their relative capabilities to inhibit protein synthesis in Vero cells and rat spleen lymphocytes. Although the lymphocytes were generally more sensitive to the mycotoxins, good correlation existed between the relative potencies of the various trichothecenes in the two cell systems. The most potent mycotoxins (T-2, verrucarin A and roridin A) have acetyl side groups on, or a hydrocarbon chain between, carbons 4 and 15 of the basic ring structure. Loss of side groups from either of these positions or an isovaleryl group at carbon 8 resulted in reduced protein synthesis inhibition (T-2 to HT-2, neosolaniol or diacetoxyscirpenol). Any combination of loss from all three positions (T-2 triol, T-2 tetraol, 15-monoacetyl DAS, scirpentriol, fusarenon X and deoxynivalenol) further weakens their effect. Reduction of the hydroxyl groups to hydroxides, forming verrucarol and deoxyverrucarol, reduced their effectiveness by over a thousand-fold compared to the most potent mycotoxins. Addition of side groups resulted in reduced effectiveness only when an acetyl group was added to the carbon 3 position of T-2 (acetyl T-2) and deoxynivalenol (3-acetyl deoxynivalenol) or on substitution of an epoxide across the 9,10 carbons of diacetoxyscirpenol (beta-epoxide DAS). Effects of combining these and other mycotoxins were additive and showed no synergism or competition for binding to the active site. When in vitro effects of the mycotoxins were compared with results from whole animal lethality tests, several of the trichothecenes were weak inhibitors of protein synthesis in vitro but had in vivo toxicities similar to that of T-2 toxin. Thus, the in vitro cell response of a given trichothecene is not always an accurate predictor of toxicity in whole animals.

Leukocyte endogenous mediator (LEM), a low-molecular-weight peptide synthesized by monocytic cells during phagocytosis, has been implicated as the host's initiator of the protein metabolic response to infection and inflammation. To determine whether administration of LEM would alter protein kinetics, appearance and oxidation of plasma tyrosine as well as the rates of protein synthesis in liver and skeletal muscle were determined in fasted rats that received a 30-hour continuous infusion of either physiologic saline, LEM, or heat-inactivated LEM. The LEM was obtained from rabbit peritoneal exudate and the treatment solutions supplied 2.8 X 10(8) cell equivalents/100 g of body weight (BW) per day. Endogenous tyrosine oxidation increased from 4.0 +/- 0.4 mumol/100 g BW/h to 5.4 +/- 0.7 mumol/100 g BW/h in animals infused with heat-inactivated LEM and to 7.5 +/- 1.5 mumol/100 g BW/h in rats receiving LEM (P less than 0.01). Nonsecretory protein synthesis in the liver was greatest in rats administered LEM (2239 +/- 325 mg/d) when compared with control groups receiving physiologic saline (1122 +/- 195 mg/d) or heat-inactivated LEM (1374 +/- 62 mg/d; P less than 0.01), whereas skeletal protein synthetic rates were unchanged. Rates of muscle and collagen protein breakdown were estimated from the urinary excretion rate of Nt-methylhistidine and hydroxyproline, respectively, and their excretion rose by 30% (P less than 0.05) and 42% (P less than 0.05) with LEM administration. These results suggest that administration of LEM stimulates a mobilization of amino acids from peripheral tissues to support increased visceral protein anabolism while whole body amino acid oxidation is also enhanced. Since similar effects follow fever and infection, these results suggest that LEM may play an underlying role in the protein metabolic response to infection and inflammation.

Both bacterial and viral infections prevented or reversed the expected ketogenic response induced in rats by starvation. In contrast, ketogenesis was not inhibited by stresses such as aseptic femoral fracture or immobilization by screen restraint. Infection had no effect on mitochondrial enzymes of terminal respiration and fatty acid oxidation.

We describe the measurement of carnitine and short-chain and long-chain acylcarnitines in perchloric acid extracts of liver and muscle. [1-14C]Acetyl-CoA is used, and labeled acetylcarnitine is measured with a sensitivity of less than 1 to 50 nmol of L-carnitine per 50-microliter sample. Samples frozen at -20 degrees C lost no carnitine during several months. Analytical recovery of carnitine (5 nmol) added to 50-microliter perchloric acid extracts of liver was 103 +/- 2%. This method may be suitable for clinical applications such as screening patients with muscle and metabolic disorders.

An injection of unpurified leucocytice endogenous mediator into rats results in an increased incorporation of [6(-14)C]orotate into hepatic RNA, an increase in the concentration of RNA associated with the bound ribosomal fraction of liver, and increases in the concentrations in serum of acute-phase proteins such as alpha2-macrofoetoprotein and haptoglobin. If given 3 days after adrenalectomy or 7 days after hypophysectomy,, leucocyte factor did not induce the increase in RNA synthesis or alpha2-macrofoetoprotein concentrations but did stimulate an increase in serum haptoglobin. When hypophysectomized or adrenalectomized rats received daily subcutaneous injections of 0.5mg of cortisol, leucocyte factor again induced a significant increase in the synthesis of hepatic RNA and an increase in the concentration of serum alpha2-macrofoetoprotein. These observations suggest that leucocyte factor can regulate acute-phase-protein synthesis at several different sites, one or more of which requires permissive action of the glucocorticoid hormones. Futher, leucocyte factor will stimulate an increase rate of incorporation of orotate into hepatic ribosomes when added in vitro in the presence of cortisol to a liver-perfusion system. Thus the stimulatory effect of leucocyte factor may be directy on liver but may require the presence of other hormones to stimulate the incorporation of orotate into RNA.

An automated immunoprecipitin system has been utilized to quantitate the concentration of 10 specific proteins in the plasma of man. Values obtained by this technique are in agreement with the published concentrations for these specific plasma proteins. This technique was utilized to determine the sequential change s in 10 individual plasma proteins of volunteers exposed to Salmonella typhi. In those volunteers who developed typical typhoid fever, plasma concentrations of the acute phase proteins, alpha1-acid glycoprotein, alpha1-antitrypsin, and haptoglobin, as well as C3 complement were significantly increased with the onset of febrile illness. In contrast, the concentration of plasma albumin and tranferrin were depressed while plasma IgM became elevated during early convalescence from this infection. No significant changes were observed in the plasma concentrations of alpha2-macroglobulin, IgG, or IgA. In the exposed volunteers who did not become ill, the only significant change was a brief depression of alpha1-antitrypsin. During typhoid fever the patterns of change for individual plasma acute-phase globulins were different from those reported for patients with hepatitis, myocaridal infarction, or surgery.

The protein-sparing and ketogenic effect of postoperative infusion of maintenance solutions containing either 3.5% amino acids or 5% dextrose in 0.33 normal (N) saline was compared by nitrogen balance and urinary acetone determination in 31 patients. A specific muscle protein sparing effect was determined by quantitation of excretion of 3-methylhistidine in seven patients of the amino acid group and eight patients in the dextrose group. In addition, an extensive hormone and substrate profile was determined. It was found that amino acid infusions not only produce a less negative nitrogen balance but also significantly reduce excretion of 3-methylhistidine indicating specific preservation of the muscle compartment.

Serum samples from patients with many different kinds of infection were shown to contain a substance characteristic of leukocytic endogenous mediator. Generalized bacterial infections produced more leukocytic endogenous mediator than did localized ones; viral infections produced very little. In typhoid fever, mediator concentrations increased before the onset of illness.

Weanling (23-day-old) rats were fed on either a low-protein diet (6% casein) or a diet containing an adequate amount of protein (18% casein) for 28 days. Hepatic cells from animals fed on the deficient diet were characterized by markedly lower concentrations of protein and RNA in all cellular fractions as compared with cells from control rats. The bound rRNA fraction was decreased to the greatest degree, whereas the free ribosomal concentrations were only slightly less than in control animals. A good correlation was observed between the rate of hepatic protein synthesis in vivo and the cellular protein content of the liver. Rates of protein synthesis both in vivo and in vitro were directly correlated with the hepatic concentration of individual free amino acids that are essential for protein synthesis. The decreased protein-synthetic ability of the ribosomes from the liver of protein-deprived rats was related to a decrease in the number of active ribosomes and heavy polyribosomes. The lower ribosomal content of the hepatocytes was correlated with the decreased concentration of essential free amino acids. In the protein-deprived rats, the rate of accumulation of newly synthesized cytoplasmic rRNA was markedly decreased compared with control animals. From these results it was concluded that amino acids regulate protein synthesis (1) by affecting the number of ribosomes that actively synthesize protein and (2) by inhibiting the rate of synthesis of new ribosomes. Both of these processes may involve the synthesis of proteins with a rapid rate of turnover.

Markedly increased synthesis of alpha(2) and beta globulins and alpha(1), alpha(2), and beta glycoglobulins occurs during pneumococcal sepsis in the rat simultaneously with decreased albumin formation, diminished tritiated leucine incorporation into muscle protein, and enhanced excretion of nitrogen. This augmented synthesis of specific serum proteins does not become evident until fever and bacteremia develop, and it appears to be a fundamental aspect of host response to a proliferating bacterial infection in that it occurs even in rats fed a protein-deficient (6% protein) diet after weaning and before exposure to Diplococcus pneumoniae. Although amino acid catabolism, in general, appears to be increased during infection, tryptophan degradation via the kynurenine pathway, as assessed by measuring diazotizable urinary metabolites, changes little or is, at times, significantly less than in control animals. Coincidentally, functional tryptophan oxygenase activity decreases at 16 hr after exposure. Total tryptophan oxygenase activity, however, is unchanged.

The effect of a protein-free diet and starvation on the duration of the rat ileal crypt cell cycle time was studied by Quastler's technique of labeled mitoses. Rats were fed a protein-free diet for 3, 7, or 11 wk or were starved for 7 or 10 days. Progressive protein depletion resulted in a progressive lengthening of the cycle time (GT), due primarily to a lengthening of the synthetic phase (S) of the cycle. The presynthetic gap (G(1)) was the same as the control value after 3 wk and lower, but not significantly so, due to the large variability, after 11 wk. The duration of the postsynthetic gap (G(2)) plus mitotic phase (M) was not affected by the diet. As the dietary stress became more severe, the cell cycle also became more variable. Although the GT of rats starved for as long as 10 days was only slightly different from the control, the relative duration of the components of the cycle changed significantly. S and G(2) were longer in the starved animals while G(1) was of shorter duration.