Comparisons of recycled manure solids and wood shavings/sawdust as bedding material-Implications for animal welfare, herd health, milk quality, and bedding costs in Swedish dairy herds.

Increasing shortages and costs of common bedding materials have led dairy farmers in Sweden to consider using recycled manure solids (RMS), which are readily available and low cost, as an alternative bedding material. The main risks are effects on udder health and milk quality, but RMS could also affect animal welfare and claw health. The advantages and disadvantages of using RMS bedding have not been fully investigated, and findings in other countries cannot be directly applied to Swedish conditions and climate. This observational cross-sectional study investigated the use of RMS as bedding, regarding associations with certain aspects of animal welfare, herd health, milk quality, and bedding costs in Swedish dairy herds. Thirty-four dairy farms using RMS or wood shavings/sawdust (each n = 17) were compared. Each farm was visited 2 times during the housing period from 2020 to 2021, once from October to December and once from March to May. Dairy barns were observed, animal welfare was assessed, and freestall dimensions were measured. Farm owners were interviewed about housing system characteristics, herd performance, and herd management. Data on milk production and herd health were obtained from the Swedish official milk recording scheme for the indoor period from October to March. The prevalence of claw disorders and abnormal claw conformation were collected from the national claw health database for the period from October to May. On each farm visit, composite samples of unused bedding outside the barn and used bedding material from the freestalls, respectively, were taken for total bacterial count and DM analysis. Samples of bulk tank milk for determination of total bacterial count were taken in connection to the visits. In addition, samples of unused and used bedding material and manure from alleys for analysis of 3 Treponema species associated with digital dermatitis (DD) were gathered and analyzed. Total bacterial count was significantly higher in unused (8.50 log10 cfu/g) and used RMS bedding (9.75 log10 cfu/g) than in wood shavings/sawdust (used 4.74; unused 8.63 log10 cfu/g), but there were no significant differences in bulk milk total bacterial count (median 4.07 vs. 3.89 log10 cfu/mL) or SCC (median 243,800 vs. 229,200 cells/mL). The aspects of animal welfare assessed did not differ significantly between the 2 bedding systems, whereas the prevalence of total claw disorders (25.9% vs. 38.0% of trimmed cows), dermatitis (6.9% vs. 16.2% of trimmed cows) and sole ulcers (2.0% vs. 4.0% of trimmed cows) were significantly lower in the RMS herds. Treponema spp. were not detected in unused RMS material, but all RMS herds had presence of DD recorded at foot trimming. An economic assessment based on the interview results and price level from winter 2021 revealed that the costs of RMS bedding varied with amount of RMS produced. Thus, RMS is a potential alternative bedding material for dairy cows in Sweden and can be a profitable option for large dairy herds. However, the high level of total bacteria in the material requires attention to bedding and milking routines as well as regular monitoring of herd health.

Incidence of udder cleft dermatitis (UCD) in dairy cows and risk factors for transitions to UCD.

Udder cleft dermatitis (UCD) is a common skin condition in Swedish dairy cows, affecting the anterior parts of the udder. The main objective of this study was to investigate incidence rate and duration of UCD in a 1-yr longitudinal study. Other objectives were to investigate risk factors for transitions from being healthy to having mild or severe UCD, and from having mild UCD to having severe UCD, and associations between UCD and clinical mastitis, somatic cell count (SCC) and hock lesions. Seven herds were included in the study and visited 9 times each at 6-wk intervals. At the visits, mild and severe UCD lesions, hock lesions, udder conformation traits, and hygiene scores were registered for each cow milked in the milking parlor. Information on breed, parity, days in milk (DIM), results from test milkings (milk production, SCC, and urea level), and veterinary treatments was also obtained. A UCD case was defined as one or several consecutive observations of UCD. The incidence and duration of UCD were described. Mixed-effect logistic regression models were used to analyze associations between potential risk factors and transitions to any type of UCD. Separate risk factor analyses were performed for transitions to mild and severe UCD. Associations with SCC, mastitis, and hock lesions were also analyzed with mixed-effect logistic regression models. The mean overall incidence of new UCD cases for all visits and herds was 0.5 cases per cow-year at risk. Risk factors associated with a higher risk of a transition to any type of UCD and mild UCD were breed (Swedish Red vs. Swedish Holstein), an indentation or fold at the fore udder attachment, and increasing DIM. In addition, a low milk urea level was associated with a lower risk of transition to any type of and mild UCD. Cows with previous mild UCD and high-yielding cows had increased risk for a transition to severe UCD. Cows that had an observed transition to severe UCD had an increased risk of veterinary-treated clinical mastitis within 6 wk after the UCD observation. No associations were found between UCD and SCC or hock lesions. The median observed duration of a UCD case was 12 wk, but most cases did not have an observed start or end during the study period. The observed duration of cases including severe UCD was longer than for cases involving only mild UCD. The high incidence and often long duration of UCD emphasize the need for preventive measures and treatment strategies.

Mild and severe udder cleft dermatitis-Prevalence and risk factors in Swedish dairy herds.

Udder cleft dermatitis (UCD) is an inflammatory skin condition affecting the anterior parts of the udder of dairy cows. The lesions may present as mild or severe skin lesions and have been associated with mastitis and digital dermatitis. The full etiology and pathogenesis are not understood and no large-scale studies have investigated prevalence and risk factors. Therefore, the main objectives of the study were to investigate the prevalence of mild and severe UCD in Swedish dairy herds and to identify risk factors associated with such lesions. We also wanted to investigate risk factors for all cases of UCD and to determine whether UCD increases the risk for mastitis and culling. A random sample of 100 freestall dairy herds were included in the study, and each herd was visited once. Cows were registered as having no, mild, or severe UCD. Additional cow and herd data were obtained via observations, interviews, and the Swedish Official Milk Recording Scheme. The data were analyzed using logistic regression models to identify risk factors for mild and severe UCD. In total, data from 3,479 cows in 99 herds were analyzed. The prevalence of mild and severe UCD was 19 and 9%, respectively. Lesions were found in 98 of 99 herds but the within-herd prevalence of mild (0-43%) and severe (0-33%) UCD varied notably between herds. Breed (Swedish Red compared with Swedish Holstein), certain udder conformation traits, and higher parity were risk factors associated with increased risk of UCD. In addition, cows with hock lesions and cows in herds with high incidence of culling due to hoof and leg diseases had a higher risk for mild UCD. More days in milk and high milk yield were cow-related risk factors associated with severe UCD. Three housing-related factors (shorter cubicles, mattress as cubicle base, and cubicles installed before 2001 compared with 2001-2005), a high incidence of veterinary-treated clinical mastitis and culling due to udder diseases, and a low incidence of culling of first-parity cows in early lactation were herd-related risk factors associated with increased risk for severe UCD. In addition, cows in herds with a high proportion of heifers older than 17 mo that were not inseminated were associated with lower risk of all UCD. Finally, UCD was not associated with the outcomes milk somatic cell count, veterinary-treated clinical mastitis, or culling in the multivariable analyses. The etiology of UCD is most likely multifactorial, involving udder conformation traits and other cow-related risk factors as well as herd-related risk factors. The high prevalence of severe UCD lesions in Swedish dairy cows emphasizes the need for preventive measures and efficient treatments.

Lack of evidence for elevated breakdown rate of skeletal muscles in weight-losing, tumor-bearing mice.

Protein degradation was measured as tyrosine release rate from proteins of extensor digitorum longus (EDL) muscles and as urinary excretion of 3-methylhistidine in freely fed adult nongrowing C57BL/6J mice with sarcomas, to study protein degradation in cancer-induced wasting of skeletal muscles. Whole muscle protein breakdown rate was unchanged, whereas protein synthesis was depressed, leading to an increased net degradation of skeletal muscles with loss of soluble, myofibrillar, and collagen proteins. Starvation for 24 hours elevated whole muscle protein breakdown in mice with and without sarcomas. Subsequent refeeding for 24 hours normalized the degradation. Adaptation to anorexia in pair-fed controls was achieved by a decrease in muscle protein turnover evaluated by urinary excretion of 3-methylhistidine over 5 days. The measurement of "catabolic decrease" of muscle protein breakdown protected the muscle mass in mice without tumors, but it was ineffective in tumor-bearing animals. The unchanged rate of breakdown of proteins in whole EDL muscles from tumor-bearing mice was accompanied by increased maximum cathepsin D activity and by elevated autolytic activity at acid pH in some muscles. Therefore, cathepsin D activity and net protease activities did not reflect whole muscle protein degradation in tumor-induced malnutrition. The results demonstrate that wasting of skeletal muscles in experimental cancer was not dependent on increased degradation but was dependent on depressed protein synthesis.

Energy metabolism in nongrowing mice with sarcoma.

The time course of energy metabolism has been studied in weight-stable and nongrowing mice with a transplantable methylcholanthrene-induced sarcoma. Daily oxygen consumption and carbon dioxide production were measured in relation to the tumor growth from the time of tumor implantation. The time course of energy dynamics was related to the end-state changes in body composition. Freely fed sarcoma-bearing mice decreased their whole-body energy expenditure in proportion to the tumor growth. This was due to the accompanying anorexia. The alteration in oxygen consumption and carbon dioxide production was continuously evident 24 hr/day in sarcoma-bearing mice. The tumor-bearing mice lost body fat and had decreased respiratory quotient, while pair-fed controls maintained their body composition, and their respiratory quotients agreed with the food respiratory quotient. Loss of body lipids in freely fed sarcoma-bearing mice reflected a negative energy balance, accompanied with increased fat oxidation, while maintenance of body composition in pair-fed controls reflected a decreased metabolic rate. Sarcoma-bearing mice showed a significantly higher energy expenditure in relation to their food intake compared to that of pair-fed controls. Estimates of partition of oxygen uptake in sarcoma-bearing mice support that both the host and the tumor account for the elevated energy expenditure. This study has confirmed a small but significantly increased energy expenditure in sarcoma-bearing mice, which was continuously present 24 hr/day in spite of unlimited availability of food. This illustrates the fatal outcome of experimental cancer.

Evaluation of anorexia as the cause of altered protein synthesis in skeletal muscles from nongrowing mice with sarcoma.

The importance of decreased food intake as the mechanism behind altered protein metabolism in skeletal muscle in cancer was evaluated. A methylcholanthrene-induced sarcoma (MCG 101) transplanted in weight-stable and nongrowing mice (C57BL/6J) was used as the tumor-animal model. Three study groups with appropriate control groups were used: sarcoma-bearing mice; pair-fed mice; and starved mice. The synthesis of myofibrillar and sarcoplasmic proteins was decreased in sarcoma-bearing mice. This was correlated to decreased content of RNA in the muscles and caused a net loss of muscle tissue was measured by dry weight of skeletal muscles. The incorporation rate of amino acids into myofibrillar and sarcoplasmic proteins was decreased to the same extent in the pair-fed mice as that in the sarcoma-bearing mice. This probably reflected decreased protein synthesis, since the radioactivity (dpm/mg) did not differ significantly in the crude transfer RNA fraction between the groups. Separation of soluble proteins from muscle tissue by means of ion-exchange chromatography showed that the pattern of decreased protein synthesis was not tumor specific when compared to muscle affected by starvation. The decrease in protein synthesis was more or less selective, since the synthesis of basic proteins was considerably decreased and was influenced more than were neutral and acidic proteins in both cancer and starvation. Anorexia of a tumor-bearing host is a sufficient trigger to induce decreased protein synthesis in skeletal muscles, but other factors may also be of quantitative importance.

Reutilization of amino acid carbons in relation to albumin turnover in nongrowing mice with sarcoma.

Reutilization of amino acid carbons was evaluated in relation to increased turnover of albumin in tumor-bearing mice. A methylcholanthrene-induced sarcoma (MCG 101) was used in nongrowing mice (C57BL/6J). Sarcoma-bearing mice developed hypoalbuminemia, but pair-fed controls did not. The hypoalbuminemia was caused by increased albumin degradation rate, measured by injection of Na214CO3, and by exponentially increased deposition of albumin into the tumor compartment. The fractional synthesis rate of albumin was doubled in tumor-bearing mice compared with controls. The translational capacity of albumin synthesis evaluated in vitro was maintained in tumor host livers. The recycling of [14C]leucine carbons was almost extinguished in plasma albumin of sarcoma-bearing mice, while that of control mice contributed to 30 to 40% of the total leucine carbon flux in turned over albumin. The recycling of arginine carbons was also different when measured after simultaneous injection of [guanido-14C]arginine and [2,3-3H]arginine. The hepatic pool of free leucine was increased by 22% in tumor-bearing mice. It is concluded that increased albumin degradation in cancer may be a disordered event and is earlier and of initially greater quantitative importance than is altered synthesis of albumin for the development of hypoalbuminemia in experimental cancer.

Protein synthesis in liver tissue under the influence of a methylcholanthrene-induced sarcoma in mice.

Amino acids are incorporated at increased rates into hepatic proteins in tumor-bearing humans and animals. In this study, we hoped to elucidate whether this is an expression of increased hepatic protein synthesis or altered isotope dilution in the precursor pool(s). Liver tissue from sarcoma-bearing mice (MCG 101) showed increased specific activities of arginine and leucine in hepatic proteins after i.p. injection of these precursors. The specific radioactivity of leucine in the free amino acid incorporation rate into proteins was also found in incubated liver slices and in a cell-free system of incubated free and membrane-attached polysomes. The increased amino acid incorporation was the net result of increased as well as decreased relative turnover of various hepatic proteins. The hepatic content of RNA was increased, but hepatic DNA and protein content was unchanged in tumor-influenced livers. Increased amino acid incorporation into hepatic proteins in tumor-bearing animals and also probably in cancer patients is due to a net increased hepatic protein synthesis, probably not confined to acute-phase reactants only.

Determination of fentanyl in human plasma and fentanyl and norfentanyl in human urine using LC-MS/MS.

Fentanyl, a potent analgesic drug, has traditionally been used intravenously in surgical or diagnostic operations. Formulations with fentanyl in oral transmucosal delivery system and in transdermal depot-patch have also been developed against breakthrough pain in cancer patients. In this report, LC-MS/MS methods to determine fentanyl in human plasma as well as fentanyl and its main metabolite, norfentanyl, in human urine are presented together with validation data. The validation ranges were 0.020-10.0 and 0.100-50.0 ng/ml for fentanyl in plasma and urine, respectively, and 0.102-153 ng/ml for norfentanyl in urine. Liquid-liquid extraction of the compounds fentanyl, norfentanyl and the deuterated internal standards, fentanyl-d5 and norfentanyl-d5 from the matrixes was applied and separation was performed on a reversed phase YMC Pro C18-column followed by MS/MS detection with electrospray in positive mode. The inter-assay precision (CV%) was better than 4.8% for fentanyl in plasma and 6.2% and 4.7% for fentanyl and norfentanyl, respectively, in urine. The ruggedness of the methods, selectivity, recovery, effect of dilution and long-term stability of the analytes in plasma and urine were investigated. Effect of haemolysis and stability of fentanyl in blood samples were also studied. The methods have been applied for the determination of fentanyl in plasma samples and fentanyl/norfentanyl in urine samples taken for pharmacokinetic evaluation after a single intra-venous (i.v.) dose of 75 microg fentanyl.

Cholera toxin adjuvant promotes a balanced Th1/Th2/Th17 response independently of IL-12 and IL-17 by acting on Gsα in CD11b⁺ DCs.

Despite an extensive literature on the mechanism of action of cholera toxin (CT), we still lack critical information about how the toxin acts as an adjuvant and, especially, which dendritic cells (DCs) are the target cells. Although a T helper type 2 (Th2)-skewing effect of CT is most commonly reported, effective priming of Th17 cells as well as suppression of Th1 responses are well documented. However, the ability of CT to block interferon regulatory factor 8 (IRF8) function and interleukin (IL)-12 production in DCs, which blocks CD8α DC and Th1 cell development, is inconsistent with priming of Th1 and CD8 T cells in many other reports. This prompted us to investigate the adjuvant effect of CT in wild-type, IL-12p40-/-, Batf3-/-, and IL-17A-/- mice and in mice that selectively lack the Gsα target protein for CT adenosine diphosphate (ADP)-ribosylation in DCs. We found that CT promoted Th1 priming independently of IL-12, and whereas Th2 and also Th17 responses were augmented, the gut IgA responses did not require IL-17A. Adjuvanticity was intact in Batf3-/- mice, lacking CD8α(+) DCs, but completely lost in mice with Gsα-deficient CD11c cells. Thus, our data demonstrate that the adjuvant effect requires Gsα expression in CD11b(+) DCs, and that priming of mucosal IgA and CD4 T cells appears unbiased and is independent of IL-12 and IL-17A.

Metabolic response of simultaneous versus sequential intravenous administration of amino acids and energy substrates to rats.

Three groups of rats were maintained on total intravenous nutrition for ten days. Group SA and SB were infused sequentially (2 X 12 h periods per day), SA received amino acids (AA) during the night and carbohydrates (CHO) + FAT during the day. The SB group received nutrients in the opposite order. A control group received a mixed solution simultaneously for 24 h/day. The sequentially fed groups showed a lower weight gain (2.4 +/- 0.4, 2.6 +/- 0.2 vs 4.9 +/- 0.3 g/day), nitrogen balance (95 +/- 7, 95 +/- 6 vs 139 +/- 7 mg/day) and nitrogen utilization (69 +/- 3, 67 +/- 3 vs 87 +/- 3%) compared with the control group. Administration of energy substrate in the SA and SB was a stronger denominator for O2 consumption and changes in RQ than the periods of physical activity. Control animals did not show any diurnal variations in O2 and RQ. Glucose, FFA and insulin were higher with CHO + FAT administration compared to AA infusion or simultaneous AA/CHO/FAT administration. In conclusion, the results suggest that simultaneous administration of a mixture of AA/CHO/FAT is preferable for whole body nitrogen economy during TPN.

Human fetal and adult liver metabolism of ethylmorphine. Relation to immunodetected cytochrome P-450 PCN and interactions with important fetal corticosteroids.

The N-demethylation of ethylmorphine was studied in liver microsomes from human fetuses and adult patients as well as from human fetal adrenals and kidneys. Unexpectedly the reaction was catalysed at the same rate in fetal (42.3-1277.4 pmol/mg/min in 11 individuals) and adult microsomes (414-1617.8 pmol/mg/min in two individuals), which also had similar values of the apparent Km (1.50, 1.72 mM respectively) and Vmax (1.33, 1.81 nmol/mg/min respectively) in studies of the enzyme kinetics. There was a close correlation (r = 0.96) between the semiquantitative immunoblotting assessment of cytochrome P-450 HL-p in fetal liver microsomes (with the use of a monoclonal antibody against pregnenolone-16-alpha-carbonitrile induced rat hepatic cytochrome P-450) and the catalytic activity. The fetal adrenal microsomal N-demethylation was only 11-30% of the hepatic activity when compared within three fetuses in which such a comparison was possible. No activity was measurable in the kidneys. Two drugs that are believed to be substrates of the cytochrome P-450 HLp were tested as inhibitors of the ethylmorphine N-demethylation in human fetal and adult liver microsomes and in rat liver microsomes. Midazolam was a potent inhibitor (100% at 0.4 mM) of the reaction in all specimens, whereas cyclosporin A inhibited the reaction clearly only in adult liver microsomes. Endogenous steroids of importance in the fetal circulation were also tested as inhibitors. Progesterone and dehydroepiandrosterone inhibited the reaction by 75-80% at a concentration of 0.4 mM, whereas pregnenolone and 17-alpha-hydroxyprogesterone were almost devoid of inhibitory potency. These results are of interest in the discussion about the physiological role of the human fetal cytochrome P-450 HLp which has an unprecedented relative abundance in the liver.

Gluconeogenesis in tumor-influenced hepatocytes.

The growth of a tumor leads to alterations in host carbohydrate metabolism. In this study we examined gluconeogenic capacity and amino acid transport in tumor-influenced and control rat hepatocytes. Serum glucose level decreased with increasing tumor burden and a significant correlation (r = -0.80) was observed. Hepatic glycogen content was similar in both groups after an overnight fast. Endogenous glucose production was 27% higher in tumor-influenced hepatocytes. The presence of 10mM of alanine led to 72% stimulation of gluconeogenesis in tumor-influenced hepatocytes as compared to 48% stimulation in control hepatocytes. The same trends were present when lactate was used as a substrate. Alanine transport into the cells was increased in tumor-influenced hepatocytes by 55% +/- 5% at a physiologic level of substrate. In conclusion, gluconeogenesis from alanine and lactate is significantly increased in tumor-influenced hepatocytes despite decreased serum glucose levels. This is associated with increased gluconeogenic capacity and accelerated alanine transport.

Protein synthesis in skeletal muscle during starvation and refeeding: comparison of data from intact muscle and muscle biopsy material.

The intact extensor digitorum longus (EDL) preparation in rat is a well-documented model for assessing protein synthesis in skeletal muscle. Human muscle biopsy material has also been used, but the extent to which biopsy material is representative for evaluation of muscle protein synthesis has not been established. Therefore, the aim of this study was to compare protein synthesis in intact muscle and in muscle biopsy material simultaneously in rats. The animals (70 g) were divided into three groups: fed (n = 22), starved for 36 hours (n = 22), and refed for 24 hours (n = 19). Protein synthesis and RNA content were measured in each group. Protein synthesis was determined as the incorporation of 14-C-phenylalanine into muscle protein in the intact EDL muscle from one leg and in a muscle biopsy from the contralateral EDL muscle. The incorporation of 14-C-phenylalanine was linear over time in both preparations, but was consistently lower in the muscle biopsy compared with the intact muscle. The relative change in incorporation, in % of that obtained in the fed state, showed a decrease in incorporation after 36 hours of starvation, in both intact muscle and in muscle biopsy material, 33% +/- 10% and 42% +/- 6%, respectively. After 24 hours of refeeding, an overshoot in protein synthesis was seen, to 136% +/- 6% in the intact muscle and to 133% +/- 6% in the muscle biopsy, as compared with the fed state. The RNA content decreased during the starvation period from 21.6 +/- 0.7 to 14.5 +/- 0.4 mg RNA/g protein.(ABSTRACT TRUNCATED AT 250 WORDS)

N-acetyl-L-tyrosine and N-acetyl-L-cysteine as tyrosine and cysteine precursors during intravenous infusion in humans.

The usefulness of N-acetyl-L-tyrosine (NAT) and N-acetyl-L-cysteine (NAC) as tyrosine and cysteine precursors during intravenous infusion was investigated in humans. Plasma levels and urinary excretion of NAT, tyrosine, NAC, and total cysteine were determined, and the site of deacetylation was examined by measuring the splanchnic and renal balances. Eleven healthy volunteers were given 5 g of either NAT or NAC as a 4-hour intravenous infusion. Plasma levels of NAT and NAC increased rapidly, accompanied by a 25% increase in tyrosine levels and a 35% decrease in total cysteine. Urinary excretion of NAT and NAC in 4 hours accounted for 56% and 11% of the infused amount, respectively. No net production of tyrosine or cysteine was found from the splanchnic area, but from the kidneys there was a small release of both tyrosine (10 +/- 3 mumol/min) and cysteine (64 +/- 3 mumol/min). We conclude that under these conditions the usefulness of NAT and NAC as precursors for the corresponding amino acids in humans is not apparent.

Whole-body tyrosine flux in relation to energy expenditure in weight-losing cancer patients.

Whole-body tyrosine flux was measured in seven weight-losing cancer patients and compared with that of seven noncancer patients with malnutrition. L[U-14C] tyrosine was infused intravenously (IV) after an overnight fast under resting conditions and flux rates, oxidation, and incorporation into proteins of tyrosine were calculated from plateau values of specific activity of tyrosine in plasma and of labeled expired carbon dioxide. Rates of protein synthesis were calculated from the flux rate of tyrosine after subtracting the proportion oxidized. Simultaneous measurements of whole-body carbon dioxide production and oxygen uptake were also performed in each subject. Cancer patients had elevated whole-body tyrosine flux, protein synthesis, and energy expenditure when expressed in relation to body weight and whole-body potassium while the differences in tyrosine kinetics became of borderline significance when expressed in relation to energy expenditure. Tyrosine incorporation into whole-body proteins corresponded to a synthesis rate of 2.70 +/- 0.17 protein/kg/d in cancer patients and 2.18 +/- 0.17 in control patients (P less than 0.025). The results show that elevated protein synthesis in weight-losing cancer patients may explain not more than one third of the elevated energy expenditure. Therefore, other systems that utilize energy must increase in activity.

Utilization of intravenously administered N-acetyl-L-glutamine in humans.

L-glutamine is too unstable for inclusion in solutions for parenteral nutrition, but its acetylated analogue, N-acetyl-L-glutamine is not. The purpose of this three-part study was to investigate the utilization of intravenously (IV) administered acetylglutamine in humans. In study 1, nine healthy postabsorptive subjects were given 9.4 g acetylglutamine IV during four hours. In study 2, five healthy subjects were studied on two occasions following an overnight fast. They were given 9.4 g of acetylglutamine or an equivalent amount of glutamine as part of a total parenteral nutrition (TPN) regimen during 7.2 hours. A control group of five subjects was given the same TPN regimen, but without acetylglutamine or glutamine. The nutrient solution included glucose, amino acids, and a fat emulsion, supplying 9.4 g nitrogen and 6,300 kJ in a total volume of 1.8 L. In study 3, four patients were studied the day after major surgery. They were given the same TPN regimen as in study 2, containing 9.4 g acetylglutamine, during 7.2 hours. Plasma concentrations and urinary excretion of acetylglutamine and glutamine were measured in all three studies, and so were splanchnic and renal exchange of acetylglutamine and glutamine in study 1. In study 1, the plasma concentration of glutamine rose from 594 +/- 28 mumol/L to 728 +/- 26 mumol/L (P less than .001), whereas plasma levels of acetylglutamine exceeded 1,000 mumol/L in all subjects at the end of infusion. The eight-hour urinary excretion of acetylglutamine and glutamine corresponded to 18% of the infused amount of acetylglutamine.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulatory effect of interleukin-1 upon hepatic metabolism.

The liver plays an important role in the acute-phase response to sepsis and injury, and host survival often depends upon an adequate hepatic response. Many of the metabolic sequelae to sepsis and injury are mediated by interleukin-1. This study was undertaken to investigate the impact of interleukin-1 upon hepatic metabolism and whether this mediator acted directly upon the liver. Interleukin-1 (5 rabbit pyrogen dose units) was administered to male Fisher F344 rats (175 to 200 g), and hepatocytes were isolated at three time periods; 2 to 4, 6 to 10, and 12 to 14 hours following an intraperitoneal injection. Alanine transport, gluconeogenesis, nonsecretory protein synthesis, and oxygen consumption were measured simultaneously in freshly isolated hepatocytes. Interleukin-1 stimulated initial rates of alanine uptake over a four-minute period. Peak stimulation of gluconeogenesis occurred at six to ten hours (0.52 +/- .14 v 0.08 +/- .01 nmol alanine converted/10(6) cells/min, P less than 0.05); nonsecretory protein synthesis was significantly stimulated at 12 to 14 hours (2.1 +/- .7 v 0.7 +/- 0.1 nmol valine converted/10(6) cells/min, P less than 0.05). These enhanced metabolic processes were associated with an increased oxygen consumption, with peak oxygen utilization occurring at six to ten hours (69 +/- 2 v 25 +/- 7 nmol of oxygen consumed 10(6) cells/min, P less than 0.05). In order to examine if interleukin-1 exerted its effect directly upon the liver, hepatocytes from normal rats were incubated in vitro with this mediator for two hours. Under these experimental conditions, interleukin-1 did not reproduce the stimulatory effect obtained following in vivo administration.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of morphine, morphine-3-glucuronide and morphine-6-glucuronide in human serum by solid-phase extraction and liquid chromatography-mass spectrometry with electrospray ionisation.

A high-performance liquid chromatographic (HPLC) method for the simultaneous determination of morphine and two of its metabolites, morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G), in serum is described. The compounds are extracted from serum using Sep-Pak light C18 solid-phase extraction cartridges, separated on an ODS C18 analytical column (100 x 4.6 mm I.D.) and detected by electrospray ionisation mass spectrometry. The separation was achieved by running a linear gradient from 4 to 70% acetonitrile with formic acid added as modifier. The flow-rate in the column was 1.0 ml/min. After the column, the eluate was subjected to a 1:50 split, with 20 microliters/min delivered to the mass spectrometer and 980 microliters/min delivered to waste. The compounds were detected in the mass spectrometer by selected-ion monitoring for m/z 286.2 for morphine and 462.2 for M3G and M6G. The spray voltage was 2.4 kV and the sampling cone was set at 40 V. The compounds have been quantified in serum over a concentration range of 2.9-60 nmol/l (0.84-17 ng/ml) for morphine, 11-1080 nmol/l (5.0-500 ng/ml) for M3G and 4.3-220 nmol/l (2.0-100 ng/ml) for M6G using external standardisation. Intra-assay and inter-assay precision were in the range of 2.4-9.0% for all compounds. The major advantage with the present LC-MS method was the shorter analysis time, 10 min per sample compared to 45 min per sample with our previous LC method with dual detectors. The LC-MS method has proved to have both the selectivity and sensitivity needed for pharmacokinetic studies.

The international harmonisation of guidelines in the future: a viewpoint from the industry.

In the area of safety, 10 completely new toxicology guidelines have been produced up to 1997 under the umbrella of the ICH. Surveys on the use and impact of the ICH guidelines in the pharmaceutical industry in three regions, Europe, the USA and Japan, showed that approximately 80% of the guidelines were utilised and, as expected, the guidelines which had been in place longest were the ones most often used. The work on the different guidelines has initiated a number of specific investigations such as studies on the duration of dosing of male rats to detect adverse effects on male fertility and the duration of chronic toxicity studies in non-rodents. Evaluation of data from various databases on long-term carcinogenicity studies showed that the mouse studies had had very little influence on the outcome of the regulatory decision about the human carcinogenic risk of a specific compound. An important role for the ICH organisation in the future should be to see that changes and amendments to current guidelines are made as soon as they are warranted by the progress of toxicological science. The decision to accept the Common Technical Document as an official ICH topic is important for the industry and will help the industry to reduce the period between drug discovery and regulatory acceptance. In the area of safety there are still topics which could benefit from harmonisation. Such topics are safety pharmacology, clinical pathology, immunotoxicology, juvenile toxicity studies, statistical methods in certain types of toxicity studies, and recommendations for additional short- to medium-term carcinogenicity studies.