Patent Application: US-77339401-A

Abstract:
a method of preserving bodily protein stores such as skeletal muscle mass in a catabolic patient involves the concomitant administration α - kg and / or α - kga and ammonium in amounts effective to preserving skeletal muscle . also disclosed is the combination of a first pharmaceutical composition comprising α - kg and / or α - kga in a pharmaceutically acceptable carrier and a second pharmaceutical composition comprising ammonium in a pharmaceutically acceptable carrier , in amounts effective to preserving skeletal muscle .

Description:
sixteen piglets of a mixed breed ( hampshire , yorkshire and swedish land race ), of both sexes , weighing 20 . 9 to 33 . 2 kg ( mean weight 25 . 6 kg ), were used in this study . food was withheld from the animals for twelve hours prior to the experiment , but they had free access to water . anaesthesia was induced by an intramuscular injection of xylazine 2 . 2 mg · kg − 1 , tiletamine 3 mg · kg − 1 + zolazepam 3 mg · kg − 1 and atropine 0 . 04 mg · kg − 1 . in addition , all animals were given an intravenous bolus of morphine 1 mg · kg − 1 and , following tracheotomy , pancuronium bromide 0 . 3 mg · kg − 1 . for continuation , an infusion of sodium pentobarbital 8 mg · kg − 1 · h − 1 and pancuronium bromide 0 . 26 mg · kg − 1 · h − 1 was started immediately after induction . during the experiment , the animals received 10 ml · kg − 1 of dextran 70 and 18 ml · kg − 1 · h − 1 of isotonic saline to maintain normovolaemia . after the last measurements , still under anaesthesia , the animals were killed with an overdose of potassium chloride . after induction of anaesthesia the animals were tracheotomised and ventilated through an 8 mm endotracheal tube ( mallincrodt laboratories , athlone , ireland ) with a servo ventilator 900c ( siemens - elema , solna , sweden ). ventilation was maintained at 25 breaths per minute with a positive end - expiratory pressure of 5 cm of h 2 o . tidal volume was adjusted to keep arterial pco 2 between 5 . 0 and 6 . 0 kpa . the inspired gas mixture during the surgical procedures was 30 % oxygen in nitrous oxide and 30 % oxygen in air during the experimental period . the body temperature was kept stable by means of a thermostat controlled heating pad and warmed intravenous infusions . after skin incisions , a catheter was introduced in the left external jugular vein and advanced to the right hepatic vein under fluoroscopic control . its position was confirmed by a contrast injection . the right external jugular vein was used for placement of a central venous catheter and a 7f pulmonary artery thermodilution catheter . an arterial catheter was inserted in a subclavian branch and advanced to a central position . through a small suprapubic incision , a urinary catheter was placed in the bladder . a cutdown was made to place an ultrasonic flow probe ( transonic systems inc ., ithaca , n . y ., usa ) around the femoral artery and to cannulate the femoral vein for blood sampling . arterial blood gases were analysed on an abl300 / osm3 system ( radiometer , copenhagen , denmark ). amino acid concentration were analysed in arterial , hepatic and femoral venous plasma on a pharmacia lkb biochrom 20 amino acid analyser ( pharmacia , uppsala , sweden ), using continuous flow ion exchange chromatography . the column eluate was mixed with ninhydrin reagent for amino acid detection . arterial and venous plasma urea and ammonium was analysed with routine enzymatic methods on a hitachi 717 automatic analyser ( hitachi ltd ., tokyo , japan ). blood glucose was analysed on a reflolux ® ii ( boehringer mannheim , germany ), using the glucose - oxidase / peroxidase reaction . arterial and hepatic venous blood was drawn for calculations of splanchnic blood flow . ecg , arterial and central venous pressure and cardiac output , were monitored and displayed on a sirecust 1281 ( siemens medical electronics inc ., danvers , mass ., usa ). cardiac output was measured with the thermodilution technique using 10 ml of iced normal saline as indicator . the mean value of at least three measurements was adopted . calculation of splanchnic blood flow was carried out according to the constant dye infusion technique ( 10 ). the dye , indocyanine green ( pulsion , medical systems , munich , germany ), was given intravenously at a rate of 0 . 17 mg · min − 1 , which gave a stable arterial concentration of indocyanine green . arterial and hepatic venous blood was drawn simultaneously and after centrifugation at 3000 rpm for 20 min , the indocyanine green plasma concentration was determined spectrophotometrically ( hitachi 101 , hitachi ltd ., tokyo , japan ) at a wavelength of 805 nm . splanchnic plasma flow was calculated according to a formula derived from fick &# 39 ; s principle : f p = i ( c a - c v ) arterial blood hematocrit ( erythrocyte volume fraction , evf ) was measured and the value added to the formula in order to calculate splanchnic blood flow : f b = i ( c a - c v )  ( 1 - evf ) where f p = splanchnic plasma flow ( ml · min − 1 ), f b = splanchnic blood flow ( ml · min − 1 ), i = indocyanine green infusion rate ( mg · min − 1 ), c a = arterial plasma concentration of indocyanine green ( mg · ml − 1 ), c v = hepatic venous plasma concentration of indocyanine green ( mg · ml − 1 ). femoral artery plasma flow was calculated as blood flow ( 1 - evf ). the turnover of amino acids , ammonium and urea were calculated as plasma flow times the arterio - venous plasma concentration difference with negative values indicating release and positive values uptake . after anaesthesia and surgical preparation the piglets were allowed a stabilisation period of one hour and randomly assigned to either group 1 or group 2 . blood sampling , measurements and pressure readings were made at 0 min ( baseline ), and at four different dosages in each group , after 60 ( dosage 1 ), 120 ( dosage 2 ), 180 ( dosage 3 ) and 240 min ( dosage 4 ). the timeline and interventions of the experiment is shown in fig1 . it should be pointed out that during the first hour of the study period ammonium chloride alone was administered in group 1 and α - kga alone in group 2 . the basal infusion rate of ammonium , aiming at a base excess ( be ) of − 6 mmol · l − 1 at the end of the experiment , was calculated according to the formula : nh 4 + ( mmol )= 0 . 3 ×( be +(− 6 ))× kg body weight , where be was the arterial base excess value at the baseline measurements . eight animals receiving an infusion of nh4cl mixed with normal saline , at a constant rate of 12 . 3 μmol · kg − 1 · min − 1 for 240 minutes , commencing after the baseline measurements ( 0 min ). an infusion of α - kga ( sigma chemical co , st louis , mo ., usa ), dissolved normal saline , was started after the 60 min measurement , at a rate of 2 . 85 μmol · kg − 1 · min − 1 during the first ( 60 - 120 min ), 5 . 7 μmol · kg − 1 · min − 1 during the second ( 120 - 180 min ) and 11 . 4 μmol · kg − 1 · min − 1 during the third ( 180 - 240 min ) hour of infusion . eight animals receiving a constant infusion of α - kga at a rate of 2 . 85 μmol · kg − 1 · min − 1 for 240 minutes , commencing after the baseline measurements ( 0 min ) and an infusion of nh 4 cl started after the 60 min measurement . during the first hour of infusion ( 60 - 120 min ) the rate was 12 . 7 μmol · min − 1 · kg − 1 , 25 . 5 μmol · kg − 1 · min − 1 during the second hour ( 120 - 180 min ) and 51 . 0 μmol · kg − 1 · min − 1 during the last hour ( 180 - 240 min ) of infusion . the data are presented as mean ± sem . due to the small sample sizes in the study groups we did not assume that the data were normally distributed . we therefore used nonparametric statistical tests . the wilcoxon signed rank test was used for paired comparisons within each group . correlation between variables was tested with the spearman rank correlation coefficient test . differences were considered statistically significant if p & lt ; 0 . 05 . the statistical calculations were performed with statview ® 5 . 0 ( sas institute inc ., cary , n . c ., usa ) computer software . blood gas and hemodynamic variables are presented in table 1 . 1 and 1 . 2 . in group 1 arterial ph decreased from baseline 7 . 46 ± 0 . 01 to 7 . 30 ± 0 . 01 ( p = 0 . 012 ) at the end of the experiment , and in group 2 from 7 . 47 ± 0 . 004 to 7 . 29 ± 0 . 01 ( p = 0 . 012 ). baseline arterial pco 2 in group 1 was 5 . 28 ± 0 . 07 kpa and 5 . 31 ± 0 . 06 kpa in group 2 and normoventilation was maintained for the whole experimental period . in group 1 , baseline cardiac output ( c . o .) was 140 ± 10 ml · min − 1 · kg − 1 , and splanchnic blood flow 51 ± 7 ml · min − 1 · kg − 1 , and it did not change significantly . in group 2 , however , c . o . decreased after 60 min compared to the baseline ( p = 0 . 025 ) c . o . of 157 ± 13 ml · min − 1 · kg − 1 , after which it resumed the baseline level . in group 2 , the baseline splanchnic blood flow of 53 ± 7 ml · min − 1 · kg − 1 was stable until the 180 min measurement when there was an increase ( p = 0 . 017 ) which was not present at the last measurement . in group 1 , baseline femoral artery blood flow was 6 . 0 ± 0 . 4 ml · min − 1 · kg − 1 and there was a significant decrease to 4 . 7 ± 0 . 4 ml · min − 1 · kg − 1 after 60 min ( p = 0 . 018 ) and it continued to decrease until 120 min ( p = 0 . 012 ) at which level it remained during the rest of the experiment . in group 2 the baseline value was 7 . 4 ± 0 . 8 ml · min − 1 · kg − 1 and there was a decrease after 120 min ( p = 0 . 012 ) which , as in group 1 , remained stable onwards to the end of the experiment . arterial and venous concentration data are shown in table 2 . 1 and 2 . 2 in group 1 , the arterial glutamine concentration was increased after 60 min ( p = 0 . 012 ) compared to 384 ± 34 μmol × l − 1 at baseline . glutamine concentration at the other dosages were not significantly different from baseline . in group 2 the baseline value was 397 ± 26 μmol × l − 1 and an initial decrease was seen after 60 min ( p = 0 . 017 ), and subsequently there were stepwise increasing glutamine concentrations for each dosage increment . interestingly , in group 2 , there was a significant correlation ( r s =− 0 . 77 ; p & lt ; 0 . 0001 ) between arterial glutamine concentration and arterial ph . no such correlation was seen in group 1 ( r s =− 0 . 28 ; p = 0 . 089 ). in group 1 the initial arterial concentration of glutamate was 181 ± 18 μmol × l − 1 and an increase was observed at 180 min ( p = 0 . 036 ) compared to the baseline value . in group 2 there was an elevated glutamate concentration after 120 , 180 and 240 min compared to the initial 241 ± 23 μmol × l − 1 . the arterial alanine concentration in group 1 was significantly lower ( p & lt ; 0 . 05 ) at all the studied dosages compared to the baseline value ( 503 ± 51 μmol × l − 1 ), and at 60 min compared to 240 min . in group 2 there was a significant decrease after 60 , 180 and 240 min compared to 506 ± 38 μmol × l − 1 at baseline , and also at 240 min ( p & lt ; 0 . 05 ) compared to 60 and 180 min . there was a marked increase in arterial arginine concentration in both groups . from 71 ± 6 μmol × l − 1 to 127 ± 8 μmol × l − 1 (+ 44 %, p = 0 . 012 ) in group 1 and 77 ± 8 μmol × l − 1 to 128 ± 6 μmol × l − 1 (+ 40 %, p = 0 . 017 ) in group 2 . α - ketoglutarate is a precursor of arginine and may as such be responsible for the 44 % ( group 1 ) and 40 % ( group 2 ) increase in arterial arginine concentration . also , it was reported in a human study by reaich et al . ( 20 ) that ammonium chloride induced acidosis was found to increase the plasma level of arginine and other amino acids but , in contrast to what is disclosed here , not of glutamine . there was a slight tendency for splanchnic uptake of arginine to switch to a net release after 180 min in group 2 while in group 1 no apparent effect on splanchnic turnover was observed . hind leg exchange data was similar to that of the splanchnic bed with a significant switch to release after 180 min that was not present at the final measurement . again , there was no effect on the hind leg uptake of arginine in group 1 . the arterial ammonium concentration in group 1 , 36 ± 2 μmol × l − 1 , increased after 60 min infusion to a level which was maintained for the rest of the study period . the baseline ammonium concentration in group 2 was 41 ± 4 μmol × l − 1 . the ammonium infusion started after the 60 min measurement and there was an increase after each change of the dose rate . in group 1 the arterial urea concentration did not change during the experiment , compared to 3 . 4 ± 0 . 4 μmol × l − 1 at baseline . in group 2 the arterial urea concentration at baseline , 2 . 9 ± 0 . 2 μmol × l − 1 , was increased to 4 . 3 ± 0 . 2 ( p = 0 . 012 ) after 240 min . compared to the 60 min value , the point when the ammonium infusion was started , the urea concentration increased with every new dosage . the baseline blood glucose level was 6 . 0 ± 0 . 4 and 5 . 7 ± 0 . 5 μmol × l − 1 in group 1 and group 2 , respectively . in group 1 there was a decrease after 180 and 240 min compared to baseline , and in group 2 there were no changes compared to baseline but a decrease at 180 and 240 min compared to the 60 min value . values are shown in table 3 . 1 and 3 . 2 . at baseline , splanchnic glutamine uptake was 2 . 5 ± 0 . 6 μmol · min − 1 kg − 1 in group 1 , and it was not affected by the different dose levels of the substrate infusion . in group 2 there was a splanchnic uptake at baseline of 2 . 6 ± 0 . 8 μmol · min − 1 · kg − 1 which was not significantly changed by the different dose rates . there was , however , a higher uptake after 180 ( p = 0 . 017 ) and 240 min ( p = 0 . 012 ) compared to the uptake at 120 min and at 240 min compared to 60 min . from hind leg skeletal muscle there was a net release of glutamine in both groups which was not affected by the different dose rates . there was a splanchnic release of 5 . 04 ± 0 . 49 μmol · min − 1 · kg − 1 glutamate in group 1 at baseline . the glutamate release at 240 min was significantly lower than was seen with all other dosages . in group 2 , the different dosages did not change the baseline glutamate release of 6 . 58 ± 0 . 52 μmol · min − 1 · kg − 1 . skeletal muscle turnover of glutamate at baseline presented a net uptake of 0 . 19 ± 0 . 03 μmol · min − 1 · kg − 1 in group 1 and 0 . 30 ± 0 . 05 μmol · min − 1 · kg − 1 in group 2 . in group 1 , the different dosages did not change the glutamate turnover , and in group 2 there was a greater uptake after 120 min compared to 180 min , but no differences compared to baseline . in both groups , there was a splanchnic uptake of alanine at baseline , 3 . 46 ± 0 . 89 and 4 . 24 ± 1 . 14 μmol · min − 1 · kg − 1 in group 1 and 2 , respectively , and it remained unaltered during the experimental period . in skeletal muscle there was a release of alanine , 0 . 19 ± 0 . 03 and 0 . 27 ± 0 . 13 μmol · min − 1 · kg − 1 , respectively , in group 1 and 2 . it was not altered by the different dose levels . there was a splanchnic uptake of arginine in both groups , 0 . 06 ± 0 . 21 μmol · min − 1 · kg − 1 and 0 . 39 ± 0 . 36 μmol · min − 1 · kg − 1 , respectively , at baseline ; no significant changes occurred during the study period . hind leg uptake of arginine was 0 . 01 ± 0 . 03 μmol · min − 1 · kg − 1 in group 1 and 0 . 09 ± 0 . 04 μmol · min − 1 · kg − 1 in group 2 . no effect on hind leg uptake was observed in group 1 whereas in group 2 a switch from uptake to release was seen after 180 min ( p & lt ; 0 . 05 ). group 1 demonstrated a net release of urea from the splanchnic region , and the baseline level 3 . 2 ± 1 . 3 μmol · min − 1 · kg − 1 , was not significantly changed during the study period . at 180 min there was , however , an increased release compared to the previous dose level ( p = 0 . 028 ). in group 2 there was a release of 6 . 0 ± 3 . 2 μmol · min − 1 · kg − 1 at baseline and no significant changes in splanchnic urea turnover compared to the baseline condition were recorded . at baseline there was a small splanchnic uptake of ammonium in both groups , 0 . 52 × 0 . 20 μmol · min − 1 · kg − 1 and 0 . 53 ± 0 . 22 μmol · min − 1 · kg − 1 , in group 1 and 2 , respectively . in group 1 there was a significant increase after 60 min which was maintained for the duration of the study period with a peak splanchnic uptake of 4 . 1 ± 1 . 0 μmol · min − 1 · kg − 1 after 180 min . group 2 presented an increased uptake after 120 min and for each dosage there was a concomitant increase in the splanchnic uptake . hind leg ammonium turnover in group 1 was near zero at baseline with only a small uptake of 0 . 02 ± 0 . 03 μmol · min − 1 · kg − 1 which was significantly increased after 60 , 120 and 180 min and then decreased to almost baseline level , 0 . 05 ± 0 . 09 μmol · min − 1 · kg − 1 , after 240 min . hind leg turnover in group 2 was similar to group 1 at baseline and it was significantly increased after 120 ( p = 0 . 012 ) and 240 ( p = 0 . 012 ) minutes compared to the baseline value . compared to the 60 min measurement ammonium uptake was higher at all of the ensuing dosages . significantly higher uptake was also seen at 240 min compared to the 120 min measurement . 1 . wernerman j , hammarqvist f , vinnars e . alpha - ketoglutarate and postoperative muscle catabolism . lancet 1990 ; 335 ( 8691 ): 701 - 3 . 2 . vinnars e , hammarqvist f , von der decken a , wernerman j . role of glutamine and its analogs in posttraumatic muscle protein and amino acid metabolism . jpen j parenter enteral nutr 1990 ; 14 ( 4 suppl ): 125s - 129s . 3 . le bricon t , cynober l , baracos v e . ornithine alpha - ketoglutarate limits muscle protein breakdown without stimulating tumor growth in rats bearing yoshida ascites hepatoma . metabolism : clinical and experimental 1994 ; 43 ( 7 ): 899 - 905 . 4 . blomqvist b i , hammarqvist f , von der decken a , wernerman j . glutamine and alpha - ketoglutarate prevent the decrease in muscle free glutamine concentration and influence protein synthesis after total hip replacement . metabolism : clinical and experimental 1995 ; 44 ( 9 ): 1215 - 22 . 5 . duranton b , schleiffer r , gosse f , raul f . preventive administration of ornithine alpha - ketoglutarate improves intestinal mucosal repair after transient ischemia in rats . crit care med 1998 ; 26 ( 1 ): 120 - 5 . 6 . de bandt j p , coudray - lucas c , lioret n et al . a randomized controlled trial of the influence of the mode of enteral ornithine alpha - ketoglutarate administration in burn patients . journal of nutrition 1998 ; 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71 ( 5 ): 487 - 96 . 13 . hannon j p , bossone c a , wade c e . normal physiological values for conscious pigs used in biomedical research . laboratory animal science 1990 ; 40 ( 3 ): 293 - 8 . 14 . jürgens p . new aspects on etiology , biochemistry , and therapy of portal systemic encephalopathy : a critical survey . nutrition 1997 ; 13 ( 6 ): 560 - 70 . 15 . lockwood a h , mcdonald j m , reiman r e et al . the dynamics of ammonia metabolism in man . effects of liver disease and hyperammonemia . journal of clinical investigation 1979 ; 63 ( 3 ): 449 - 60 . 16 . stein t p , leskiw m j , wallace h w . metabolism of parenterally administered ammonia . journal of surgical research 1976 ; 21 ( 1 ): 17 - 20 . 17 . almond m k , smith a , cohen r d , iles r a , flynn g . substrate and ph effects on glutamine synthesis in rat liver . biochem j 1991 ; 278 ( pt 3 ): 709 - 14 . 18 . atkinson d , bourke e . metabolic aspects of the regulation of systemic ph . am j physiol 1987 ; 252 ( 6 pt 2 ): f947 - 56 . 19 . atkinson d , camien m . the role of urea synthesis in the removal of metabolic bicarbonate and the regulation of ph . curr top cell regul 1982 ; 21 : 261 - 302 . 20 . reaich d , channon s m , scrimgeour c m , goodship t h . ammonium chloride - induced acidosis increases protein breakdown and amino acid oxidation in humans . american journal of physiology 1992 ; 263 ( 4 pt 1 ): e735 - 9 . 21 . roth e , karner j , roth - merten a et al . effect of alpha - ketoglutarate infusions on organ balances of glutamine and glutamate in anaesthetized dogs in the catabolic state . clin sci ( colch ) 1991 ; 80 ( 6 ): 625 - 31 . 22 . cynober l a . the use of alpha - ketoglutarate salts in clinical nutrition and metabolic care . curr opin clin nutr metab care 1999 ; 2 ( 1 ): 33 - 7 . 23 . häussinger d . hepatocyte heterogeneity in glutamine and ammonia metabolism and the role of an intercellular glutamine cycle during ureogenesis in perfused rat liver . eur j biochem 1983 ; 133 ( 2 ): 269 - 75 . table 1 . 1 dosages effects of α - ka and ammonium chloride on blood gas and hemodynamic variables in group 1 ( n = 8 ). baseline dosage 1 dosage 2 dosage 3 dosage 4 0 min 60 min 120 min 180 min 240 min arterial ph 7 . 46 ± 0 . 01 7 . 43 ± 0 . 01 a 7 . 39 ± 0 . 01 ab 7 . 35 ± 0 . 01 abc 7 . 30 ± 0 . 01 abcd base excess μmol · l − 1 3 . 9 ± 0 . 5 1 . 7 ± 0 . 3 a − 0 . 5 ± 0 . 3 ab − 2 . 9 ± 0 . 4 abc − 5 . 6 ± 0 . 4 abcd paco 2 kpa 5 . 28 ± 0 . 07 5 . 18 ± 0 . 06 5 . 37 ± 0 . 06 b 5 . 48 ± 0 . 07 ab 5 . 44 ± 0 . 07 c . o . ml · min − 1 · kg − 1 140 ± 10 138 ± 17 147 ± 17 157 ± 16 163 ± 17 spl . blood flow ml · min − 1 · kg − 1 51 ± 7 43 ± 5 44 ± 6 54 ± 5 b 49 ± 4 leg blood flow ml · min − 1 · kg − 1 6 . 0 ± 0 . 4 4 . 7 ± 0 . 4 a 3 . 9 ± 0 . 3 ab 3 . 5 ± 0 . 2 ab 3 . 4 ± 0 . 3 ab values are mean ± sem . statistical significance ( p & lt ; 0 . 05 ) according to the wilcoxon signed rank test is indicated by “ a ”— different from baseline ; “ b ”— different from 60 min ; “ c ”— different from 120 min ; “ d ”— different from 180 min . dose rates for group 1 : dosage 1 ( 0 - 60 min )— nh 4 + , 12 . 3 μmol · kg − 1 · min − 1 ; dosage 2 ( 60 - 120 min )— nh 4 + , 12 . 3 μmol · kg − 1 · min − 1 + α - ketoglutarate , 2 . 85 μmol · kg − 1 · min − 1 ; dosage 3 ( 120 - 180 min )— nh 4 + , 12 . 3 μmol · kg − 1 · min − 1 + α - ketoglutarate , 5 . 7 μmol · kg − 1 · min − 1 ; dosage 4 ( 180 - 240 min )— nh 4 + , 12 . 3 μmol · kg − 1 · min − 1 + α - ketoglutarate , 11 . 4 μmol · kg − 1 · min − 1 . table 1 . 2 dosages effects of α - ka and ammonium chloride on blood gas and hemodynamic variables in group 2 ( n = 8 ). baseline dosage 1 dosage 2 dosage 3 dosage 4 0 min 60 min 120 min 180 min 240 min arterial ph 7 . 47 ± 0 . 004 7 . 47 ± 0 . 01 7 . 42 ± 0 . 01 ab 7 . 37 ± 0 . 01 abc 7 . 29 ± 0 . 01 abcd base excess μmol · l − 1 5 . 0 ± 0 . 5 4 . 4 ± 0 . 3 1 . 7 ± 0 . 6 ab − 1 . 8 ± 0 . 4 abc − 6 . 3 ± 0 . 4 abcd paco 2 kpa 5 . 31 ± 0 . 06 5 . 27 ± 0 . 06 5 . 43 ± 0 . 09 b 5 . 42 ± 0 . 15 5 . 42 ± 0 . 11 c . o . ml · min − 1 kg − 1 158 ± 13 144 ± 10 a 151 ± 10 164 ± 11 185 ± 14 bcd spl . blood flow ml · min − 1 · kg − 1 53 ± 7 49 ± 6 50 ± 5 64 ± 5 abc 73 ± 11 c leg blood flow ml · min − 1 · kg − 1 7 . 4 ± 0 . 8 6 . 0 ± 0 . 5 a 4 . 7 ± 0 . 3 ab 4 . 1 ± 0 . 2 ab 4 . 3 ± 0 . 3 ab values are mean ± sem . statistical significance ( p & lt ; 0 . 05 ) according to the wilcoxon signed rank test is indicated by “ a ”— different from baseline ; “ b ”— different from 60 min ; “ c ”— different from 120 min ; “ d ”— different from 180 min . dose rates for group 2 : dosage 1 ( 0 - 60 min )— α - ketoglutarate , 2 . 85 μmol · kg − 1 · min − 1 ; dosage 2 ( 60 - 120 min )— α - ketoglutarate , 2 . 85 μmol · kg − 1 · min − 1 + nh 4 + , 12 . 7 μmol · kg − 1 · min − 1 ; dosage 3 ( 120 - 180 min )— α - ketoglutarate , 2 . 85 μmol · kg − 1 · min − 1 + nh 4 + , 25 . 5 μmol · kg − 1 · min − 1 ; dosage 4 ( 180 - 240 min )— α - ketoglutarate , 2 . 85 μmol · kg − 1 · min − 1 + nh 4 + , 51 . 0 μmol · kg − 1 · min − 1 . table 2 . 1 dosages effects of α - ka and nh 4 cl on arterial amino acid , nh 4 , urea and glucose concentrations in group 1 ( n = 8 ). baseline dosage 1 dosage 2 dosage 3 dosage 4 0 min 60 min 120 min 180 min 240 min glutamine μmol · l − 1 384 ± 34 450 ± 26 a 447 ± 34 455 ± 45 504 ± 52 glutamate μmol · l − 1 181 ± 18 191 ± 20 198 ± 17 209 ± 16 213 ± 18 alanine μmol · l − 1 503 ± 51 412 ± 44 a 368 ± 45 ab 356 ± 44 ab 344 ± 41 ab arginine μmol · l − 1 71 ± 6 80 ± 8 96 ± 8 ab 117 ± 7 abc 127 ± 8 abc total aa μmol · l − 1 3186 ± 167 3034 ± 139 2958 ± 140 3080 ± 166 3160 ± 165 nh 4 + μmol · l − 1 36 ± 2 135 ± 14 a 141 ± 13 a 145 ± 11 a 115 ± 22 a urea mmol · l − 1 3 . 4 ± 0 . 4 3 . 4 ± 0 . 3 3 . 5 ± 0 . 3 3 . 5 ± 0 . 2 3 . 6 ± 0 . 3 glucose mmol · l − 1 6 . 0 ± 0 . 4 5 . 5 ± 0 . 5 4 . 8 ± 0 . 6 4 . 6 ± 0 . 6 a 5 . 2 ± 0 . 5 a values are mean ± sem . statistical significance ( p & lt ; 0 . 05 ) according to the wilcoxon signed rank test is indicated by “ a ”— different from baseline ; “ b ”— different from 60 min ; “ c ”— different from 120 min ; “ d ”— different from 180 min . dose rates for group 1 : dosage 1 ( 0 - 60 min )— nh 4 + , 12 . 3 μmol · kg − 1 · min − 1 ; dosage 2 ( 60 - 120 min )— nh 4 + , 12 . 3 μmol · kg − 1 · min − 1 + α - ketoglutarate , 2 . 85 μmol · kg − 1 · min − 1 ; dosage 3 ( 120 - 180 min )— nh 4 + , 12 . 3 μmol · kg − 1 · min − 1 · h − 1 + α - ketoglutarate , 5 . 7 μmol · kg − 1 · min − 1 ; dosage 4 ( 180 - 240 min )— nh 4 + , 12 . 3 μmol · kg − 1 · min − 1 + α - ketoglutarate , 11 . 4 μmol · kg − 1 · min − 1 . table 2 . 2 dosages effect of α - ka and nh 4 cl on arterial amino acid , nh 4 , urea and glucose concentrations in group 2 ( n = 8 ). baseline dosage 1 dosage 2 dosage 3 dosage 4 0 min 60 min 120 min 180 min 240 min glutamine μmol · l − 1 397 ± 26 361 ± 24 z 451 ± 27 ab 521 ± 29 abc 621 ± 37 abcd glutamate μmol · l − 1 241 ± 23 264 ± 32 287 ± 28 ab 284 ± 25 a 296 ± 24 a alanine μmol · l − 1 506 ± 38 462 ± 34 436 ± 37 392 ± 32 a 353 ± 27 abd arginine μmol · l − 1 77 ± 8 75 ± 5 95 ± 7 b 112 ± 7 ab 128 ± 6 abcd total aa μmol · l − 1 3198 ± 167 3044 ± 158 3244 ± 178 3270 ± 154 3233 ± 119 nh 4 + μmol · l − 1 41 ± 4 43 ± 4 147 ± 10 ab 277 ± 29 abc 728 ± 135 abcd urea mmol · l − 1 2 . 9 ± 0 . 2 2 . 7 ± 0 . 2 2 . 9 ± 0 . 2 b 3 . 3 ± 0 . 2 bc 4 . 3 ± 0 . 2 abcd glucose mmol · l − 1 5 . 7 ± 0 . 5 5 . 6 ± 0 . 4 5 . 3 ± 0 . 4 4 . 9 ± 0 . 5 b 4 . 7 ± 0 . 4 b values are mean ± sem . statistical significance ( p & lt ; 0 . 05 ) according to the wilcoxon signed rank test is indicated by “ a ”— different from baseline ; “ b ”— different from 60 min ; “ c ”— different from 120 min ; “ d ”— different from 180 min . dose rates for group 2 : dosage 1 ( 0 - 60 min )— α - ketoglutarate , 2 . 85 μmol · kg − 1 · min − 1 ; dosage 2 ( 60 - 120 min )— α - ketoglutarate , 2 . 85 μmol · kg − 1 · min − 1 + nh 4 + , 12 . 7 μmol · kg − 1 · min − 1 ; dosage 3 ( 120 - 180 min )— α - ketoglutarate , 2 . 85 μmol · kg − 1 · min − 1 + nh 4 + , 25 . 5 μmol · kg − 1 · min − 1 ; dosage 4 ( 180 - 240 min )— α - ketoglutarate , 2 . 85 μmol · kg − 1 · min − 1 + nh 4 + , 51 . 0 μmol · kg − 1 · min − 1 . table 3 . 1 turnover effects for different dosages of a - ketoglutaric acid and ammonium chloride in group 1 ( n = 8 ). baseline dosage 1 dosage 2 dosage 3 dosage 4 0 min 60 min 120 min 180 min 240 min glutamine splanchinc 2 . 52 ± 0 . 60 2 . 59 ± 0 . 56 2 . 82 ± 0 . 32 3 . 50 ± 0 . 47 3 . 79 ± 0 . 54 hind leg − 0 . 04 ± 0 . 05 − 0 . 09 ± 0 . 04 − 0 . 10 ± 0 . 05 − 0 . 14 ± 0 . 04 − 0 . 10 ± 0 . 05 glutamate splanchinc − 5 . 04 ± 0 . 49 − 4 . 32 ± 0 . 28 − 4 . 33 ± 0 . 34 − 4 . 78 ± 0 . 46 − 3 . 60 ± 0 . 25 abcd hind leg 0 . 19 ± 0 . 03 0 . 22 ± 0 . 03 0 . 19 ± 0 . 03 0 . 17 ± 0 . 03 0 . 16 ± 0 . 03 alanine splanchinc 3 . 46 ± 0 . 89 2 . 22 ± 0 . 28 2 . 73 ± 0 . 58 3 . 14 ± 0 . 49 3 . 41 ± 0 . 73 hind leg 0 . 19 ± 0 . 03 − 0 . 13 ± 0 . 04 − 0 . 21 ± 0 . 14 − 0 . 11 ± 0 . 04 − 0 . 15 ± 0 . 05 arginine splanchinc 0 . 06 ± 0 . 21 − 0 . 23 ± 0 . 10 0 . 03 ± 0 . 15 0 . 11 ± 0 . 23 − 0 . 06 ± 0 . 22 hind leg 0 . 01 ± 0 . 03 0 . 01 ± 0 . 005 0 . 01 ± 0 . 02 0 . 01 ± 0 . 01 − 0 . 01 ± 0 . 02 total aa splanchinc 6 . 21 ± 2 . 54 3 . 93 ± 2 . 08 4 . 35 ± 0 . 98 5 . 41 ± 1 . 43 7 . 19 ± 2 . 64 hind leg − 0 . 01 ± 0 . 32 0 . 12 ± 0 . 23 0 . 09 ± 0 . 25 − 0 . 10 ± 0 . 16 − 0 . 10 ± 0 . 17 nh 4 + splanchinc 0 . 52 ± 0 . 20 3 . 09 ± 0 . 28 a 3 . 34 ± 0 . 19 a 4 . 10 ± 0 . 34 ab 2 . 76 ± 0 . 57 a hind leg 0 . 02 ± 0 . 01 0 . 22 ± 0 . 03 a 0 . 17 ± 0 . 02 a 0 . 13 ± 0 . 03 ab 0 . 05 ± 0 . 03 bcd fe (%)- nh 4 + splanchinc 37 ± 11 81 ± 4 a 77 ± 6 a 74 ± 6 a 65 ± 5 ab hind leg 12 ± 6 46 ± 3 a 40 ± 4 a 34 ± 3 a 8 ± 9 bcd urea splanchinc − 3 . 2 ± 1 . 3 − 3 . 2 ± 1 . 4 − 1 . 1 ± 1 . 0 − 6 . 4 ± 1 . 5 c − 2 . 2 ± 1 . 6 hind leg 0 . 3 ± 0 . 2 0 . 2 ± 0 . 2 0 . 6 ± 0 . 1 0 . 1 ± 0 . 2 − 0 . 1 ± 0 . 3 c values are mean ± sem . statistical significance ( p & lt ; 0 . 05 ) according to the wilcoxon signed rank test is indicated by “ a ”— different from baseline ; “ b ”— different from 60 min ; “ c ”— different from 120 min ; “ d ”— different from 180 min . dose rates for group 1 : dosage 1 ( 0 - 60 min )— nh 4 + , 12 . 3 μmol · kg − 1 · min − 1 ; dosage 2 ( 60 - 120 min )— nh 4 + , 12 . 3 μmol · kg − 1 · min − 1 + α - ketoglutarate , 2 . 85 μmol · kg − 1 · min − 1 ; dosage 3 ( 120 - 180 min )— nh 4 + , 12 . 3 μmol · kg − 1 · min − 1 · h − 1 + α - ketoglutarate , 5 . 7 μmol · kg − 1 · min − 1 ; dosage 4 ( 180 - 240 min )— nh 4 + , 12 . 3 μmol · kg − 1 · min − 1 + α - ketoglutarate , 11 . 4 μmol · kg − 1 · min − 1 . table 3 . 2 turnover effects for different dosages of a - ketoglutaric acid and ammonium chloride in group 2 ( n = 8 ). group 2 ( n = 8 ) baseline dosage 1 dosage 2 dosage 3 dosage 4 0 min 60 min 120 min 180 min 240 min glutamine splanchinc 2 . 63 ± 0 . 75 2 . 14 ± 0 . 31 2 . 13 ± 0 . 62 3 . 82 ± 0 . 61 c 5 . 48 ± 1 . 32 bc hind leg − 0 . 15 ± 0 . 11 − 0 . 09 ± 0 . 07 − 0 . 17 ± 0 . 04 − 0 . 24 ± 0 . 05 − 0 . 19 ± 0 . 07 glutamate splanchinc − 6 . 58 ± 0 . 52 − 5 . 83 ± 0 . 61 − 5 . 23 ± 0 . 67 − 5 . 94 ± 0 . 79 − 6 . 16 ± 1 . 61 hind leg 0 . 30 ± 0 . 05 0 . 25 ± 0 . 06 0 . 34 ± 0 . 05 0 . 27 ± 0 . 03 c 0 . 28 ± 0 . 05 alanine splanchinc 4 . 24 ± 1 . 14 4 . 11 ± 0 . 97 2 . 79 ± 0 . 68 3 . 65 ± 0 . 52 3 . 85 ± 0 . 84 hind leg − 0 . 27 ± 0 . 13 − 0 . 20 ± 0 . 09 − 0 . 17 ± 0 . 03 − 0 . 15 ± 0 . 04 − 0 . 15 ± 0 . 04 arginine splanchinc 0 . 39 ± 0 . 36 0 . 26 ± 0 . 22 0 . 03 ± 0 . 15 − 0 . 45 ± 0 . 41 − 0 . 04 ± 0 . 25 hind leg 0 . 09 ± 0 . 04 0 . 03 ± 0 . 02 0 . 02 ± 0 . 02 − 0 . 05 ± 0 . 04 abc 0 . 02 ± 0 . 04 d total aa splanchinc 7 . 58 ± 4 . 12 6 . 18 ± 2 . 26 2 . 75 ± 4 . 43 5 . 11 ± 2 . 98 9 . 84 ± 5 . 41 hind leg − 0 . 23 ± 0 . 82 − 0 . 36 ± 0 . 64 0 . 16 ± 0 . 28 − 0 . 11 ± 0 . 21 − 0 . 29 ± 0 . 35 nh 4 + splanchinc 0 . 53 ± 0 . 22 0 . 50 ± 0 . 21 3 . 72 ± 0 . 28 ab 9 . 73 ± 0 . 57 abc 19 . 58 ± 2 . 98 abcd hind leg 0 . 04 ± 0 . 03 0 . 01 ± 0 . 02 0 . 23 ± 0 . 03 ab 0 . 31 ± 0 . 04 abc 0 . 51 ± 0 . 14 abc fe (%)- nh 4 + splanchinc 27 ± 13 28 ± 10 70 ± 4 ab 77 ± 4 ab 56 ± 7 bd hind leg 8 ± 11 5 ± 7 43 ± 4 ab 35 ± 2 b 20 ± 3 bcd urea splanchinc − 6 . 0 ± 3 . 2 − 4 . 0 ± 1 . 2 − 3 . 0 ± 2 . 8 − 2 . 6 ± 3 . 3 5 . 3 ± 4 . 0 hind leg − 0 . 3 ± 0 . 6 − 0 . 2 ± 0 . 4 0 . 2 ± 0 . 4 0 . 5 ± 0 . 2 0 . 7 ± 0 . 2 b values are mean ± sem . statistical significance ( p & lt ; 0 . 05 ) according to the wilcoxon signed rank test is indicated by “ a ”— different from baseline ; “ b ”— different from 60 min ; “ c ”— different from 120 min ; “ d ”— different from 180 min . dose rates for group 2 : dosage 1 ( 0 - 60 min )— α - ketoglutarate , 2 . 85 μmol · kg − 1 · min − 1 ; dosage 2 ( 60 - 120 min )— α - ketoglutarate , 2 . 85 μmol kg − 1 · min − 1 + nh 4 + , 12 . 7 μmol · kg − 1 · min − 1 ; dosage 3 ( 120 - 180 min )— α - ketoglutarate , 2 . 85 μmol · kg − 1 · min − 1 + nh 4 − , 25 . 5 μmol · kg − 1 · min − 1 ; dosage 4 ( 180 - 240 min )— α - ketoglutarate , 2 . 85 μmol · kg − 1 · min − 1 + nh 4 + , 51 . 0 μmol · kg − 1 · min − 1 .