Reversal of ammonia coma in rats by L-dopa: a peripheral effect

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1 Gut, 1979, 2, Reversal of ammonia oma in rats by L-dopa: a peripheral effet L. ZV1, W. M. DOZAK, AND R. F. DRR From the Department of Mediine, Hennepin ounty Medial enter and Minneapolis Veterans Hospital, University of Minnesota, Minneapolis, Minnesota, USA SUMMARY Ammonia oma was produed in rats within 1 to 15 minutes of an intraperitoneal injetion of 1.7 mmol NH41. This oma was prevented with 1-68 numol L-dopa given by gastri intubation 15 minutes before the ammonium salt injetion. The effet of L-dopa was orrelated with a derease in blood and brain ammonia, an inrease in brain dopamine, and an inrease in renal exretion of ammonia and urea. ntraventriular infusion of dopamine suffiient to raise the brain dopamine to the same extent did not prevent the ammonia oma nor affet the blood and brain ammonia onentrations. Bilateral nephretomy eliminated the benefiial effet of L-dopa on blood and brain ammonia and the ammonia oma was not prevented. Thus, the redution in blood and brain ammonia, and the prevention of ammonia oma after L-dopa, an be aounted for by the peripheral effet of dopamine on renal funtion rather than its entral ation. These results provide a reasonable explanation for the benefiial effets observed in some enephalopathi patients reeiving L-dopa. The arousal effet of L-dopa in hepati oma has been observed repeatedly sine the original observations (Parkes et al., 197). The arousal response is observed in approximately one-half ofpatients treated and is temporary in all but a few (Fisher et al., 1976). The benefiial effet of L-dopa has generally been presumed to be a entral effet of its derivative, dopamine, and has been ited as evidene supporting the false neurotransmitter hypothesis of the aetiology of hepati oma (Fisher and Baldessarini, 1971). We studied the effet of L-dopa in experimental ammonia oma in rats, thinking that there might be an assoiation between the arousal effet of L-dopa and the brain ammonia onentration. We found, indeed, that L-dopa in suffiient dosage prevented ammonia oma, and that the presene or absene of oma was related to the brain ammonia but not the brain dopamine onentration. We also observed that the redution in brain and blood ammonia after L-dopa was assoiated with an inrease in renal exretion of ammonia and urea, and that nephretomy eliminated the effets of L-dopa on oma and on the blood and brain ammonia onentrations. 'Address for reprint requests: Dr Leslie Zieve, Hennepin ounty Medial enter, 71 Park Ave. S., Minneapolis, MN 55415, USA. Reeived for publiation 19 June 1978 Methods XPRMNTAL PRODUR Healthy male Sprague-Dawley rats weighing g were used. One set of non-fasting animals was divided into four groups onsisting of five or six rats per group. The animals of the first group reeived only an intraperitoneal injetion of a omaproduing dose of NH41, 1.7 mmol. oma ourred in all animals within 1 to 15 minutes as judged by their failure to roll over when plaed upon their baks and their lak of response to a painful stimulus. Blood was drawn for ammonia determination within two to four minutes after onset of oma (12 to 18 minutes after injetion of the NH4l). ah animal was exsanguinated, the head deapitated immediately thereafter, and the brain quikly extrated from the skull and froen in liquid nitrogen, to be saved for later analysis for ammonia, dopamine, and noradrenaline. The whole proess of getting and freeing the brain took less than two minutes. The animals of the seond group reeived L-dopa, 1.68 mmol by intubation 15 minutes before they reeived 1.7 mmol NH4l intraperitoneally. oma did not our in any of these rats, though two beame lethargi. Blood was drawn for ammonia determination 17 to 2 minutes after the injetion of NH41. 28

2 L-Dopa in ammonia oma The remainder of the proedure was similar to that for the first group. The rats of the third group reeived L-dopa, 1.68 mmol, by intubation like those of the seond group. n addition, they were injeted intraperitoneally with 15 mg benseraide, a dopa dearboxylase inhibitor, 15 minutes before they reeived the intraperitoneal injetion of 1.7 mmol NH41. Three of these rats beame omatose, two lethargi, and only one remained alert. Blood was drawn for ammonia determination 16 to 17 minutes after the injetion of NH41. The remainder of the proedure was similar to that for the first group. The rats of the fourth group reeived 14 gg dopamine through a annula plaed in the lateral ventrile of the erebrum as desribed previously (Zieve and Olsen, 1977). The infusion of dopamine ourred over a period of 3 minutes before the intraperitoneal injetion of 1-7 mmol NH4. oma ourred in all animals within seven to 12 minutes, and blood was drawn for ammonia determination three to four minutes later. The exsanguination and taking of the brain was similar to that for the first group. The urinary exretion of ammonia and urea was studied in a seond set of non-fasting rats of similar sie. Two groups were formed and housed individually in metabolism ages. One group (six rats) reeived 5 ml water and 15 minutes later a oma produing dose of NH41 (1-7 mmol) intraperitoneally. The rats were killed 2 minutes after the ammonia injetion. The bladder was flushed three times with water and the bladder washings ombined with the metabolism age washings and analysed for ammonia and urea. A seond group of seven rats was treated exatly the same way, exept that eah reeived 1.68 mmol L-dopa by intubation 15 minutes before the injetion of NH41. A third set of 34 rats that had fasted for 24 hours was subdivided into four groups. The first two groups were treated the same as the orresponding groups of the first set of rats and served as ontrols for the third and fourth groups of rats, whih were nephretomised two hours before reeiving NH4 or L-dopa and NH4 in idential fashion to the first two groups. oma ourred faster in the nephretomised rats, about six minutes after the injetion of NH4. Blood was drawn for ammonia determinations within two to three minutes of onset of oma and the brain taken as before. ANALYTAL MTHODS Blood and urine ammonia were determined promptly after olletion by the proedure of Reinhold and hung (1961). Brain ammonia was measured similarly after homogenisation of a froen piee. Brain 2 dopamine and noradrenaline were measured by the proedure of Refshauge et al. (1974) as previously desribed by Zieve and Olsen (1977). Urine and blood urea were measured by the proedure of Mather and Roland (1969). Results Figure 1 gives results of blood and brain ammonia 2 to " O 16k 12 F 8-4F 2 X 16 ) _ m- 4 All 4A 3-2L 2L A F U L s pil-, t... ;h 6.1 i-: All i.3 w- NH NH4 NH4 NH4 L-Dopo L-Dopo DAv Bens. Fig. 1 ffet of various treatment ombinations on the blood and brain ammonia onentrations and on the prodution of oma. The number of rats in eah of the four groups is shown at the base of the upper set of bars. The bars show the means SM. The dashed line extensions of the vertial line on the third bar of the lower set represent the range of values in this ase. = oma, A = alert, L = lethargi, Bens = benseraide, DA, = intraventriular dopamine. Diagonal lines in bars indiate oma; dots indiate alertness; dots overlaid with widely spaed diagonal lines indiate lethargy. Horiontal arrows along ordinate indiate average normal values of the variables measured. 29

3 3 nitrogen measurements (heneforth referred to simply as blood or brain ammonia). As shown in the upper setion of the figure, the average blood ammonia onentration within a few minutes of the onset of oma was 1424 nmol/ml, equivalent to approximately 24,g/dl. For referene, the average blood ammonia in six normal uninjeted rats of equivalent weight was 71 nmol/ml as shown by the arrow on the ordinate. The blood ammonia was redued signifiantly (t = 2.5, p <.5) by approximately 25 % when 1 '68 mmol L-dopa, a large dose, was given by gastri intubation 15 minutes before the intraperitoneal injetion of NH4l, and oma was prevented as shown by the seond bar. The dopa dearboxylase inhibitor benseraide prevented this benefiial effet of L-dopa to a signifiant degree as shown by the third bar. A large dose of benseraide, 5 mg/kg, was required to produe this effet. Only one of six rats remained alert and three beame omatose. Diret instillation of dopamine into the erebral ventrile, unlike oral L-dopa, did not prevent oma and had little effet on the blood ammonia. The effets of these manipulations on brain ammonia were similar. The redution in brain ammonia by L-dopa was approximately 3% (t = 2.8, p <.5), and oma was prevented though a dose of NH4l that normally produed oma was given. Benseraide reversed the effet of L-dopa in three of six rats, though the average brain ammonia for the group did not return to the level of the first group. n this instane, the range of values observed is indiated by the dashed vertial line. As with blood ammonia, instillation of dopamine diretly into the erebral ventrile had no effet on the brain ammonia and did not prevent ammonia oma. As an be seen in Fig. 2, brain dopamine and noradrenaline did not hange signifiantly from the normal (shown by arrows) after the injetion of a oma-produing dose of NH41. When the oral dose of L-dopa was added brain dopamine inreased by 4% (t = 4.2, p <.1), while the brain noradrenaline remained unhanged. Benseraide prevented the rise in brain dopamine after L-dopa. The intraventriular instillation of 1.4 gg dopamine also aused a rise in brain dopamine of approximately 4% but, as seen previously, oma was not prevented. n Fig. 3 the alterations in brain ammonia and brain dopamine are ontrasted in relation to the presene and absene of oma after the injetion of 1-7 mmol NH4l. Only the brain ammonia rose and fell onsistently with the presene and absene of oma. Brain dopamine inreased signifiantly after intraventriular instillation of dopamine, as it did 1a;. P ' L ' a..._ 6vv l6q 12 8 L. Zieve, W. M. Doiaki, and R. F. Derr gnr-fl 4,'S l a] [P J. -i:..-k& NH4 NH4 NH4 NH4 L-Dop L-Dopo DAy Bens. Fig. 2 fet of various treatment ombinations on the brain dopamine and noradrenaline onentrations and on the prodution of oma. Where appliable, the remainder of the legend is the same as for Fig. 1. v All 4A 3 All 2L 2L A All 4A All 16-2L - ) 4-- D NH4 NH4 NH4 L-Dopa DAV Brain NH3 X Brain DA Fig. 3 Relative hanges in brain ammonia and brain dopamine in response to NH, plus oral L-dopa or intraventriular dopamine. Bars show means ± SM.

4 L-Dopa in ammonia oma after oral L-dopa, but oma was not prevented and the brain ammonia did not fall. This lak of effet of intraventriular dopamine on the brain ammonia in ontrast to that of oral L-dopa, suggested to us that the benefiial effet of the L-dopa on ammonia oma might be due to its effet on ammonium ion availability. We therefore studied the effet of L-dopa on the renal exretion of ammonia and urea after a loading dose (1-7 mmol) of NH41. n Fig. 4 are shown the effets of L-dopa on N N h-(._ 4, U X12 'or L-Dopo L-Dope x13 Fig. 4 ffet of L-dopa on urinary exretion of ammonia and urea after the oma-produing dose of NH4l. Bars show means ± SM. Number of animals shown at base ol bars. urinary ammonia and urea after the oma-produing dose of NH41. The mean ammonia exretion inreased by 44% and urea exretion by 114%. The variability of ammonia exretion was great, preventing statistial signifiane (t = 1.7, p >.1) for this inrease, however, the median inrease was 54%. The inrease in urea exretion was highly signifiant (t = 5-85, p <.1). f the results of Fig. 4 indiate a renal basis for the oma-preventing effet of L-dopa, nephretomised rats given the same doses of NH4l and L-dopa should remain in oma. That this is the ase is shown in Fig. 5 whih gives the blood and brain ammonia onentration in a group of nephretomised rats and another ontrol group run at the same time. The ontrols show the same redution (27%) in blood (t = 4.16, P < 1) and brain (t = 3.4, P <.2) ammonia onentrations on pretreatment with L-dopa as observed in the first set of rats. The nephretomised rats show no effet of L-dopa on the ourrene of oma or on the onentrations of blood and brain ammonia. The blood ammonia values in the nephretomised rats were about 35% higher than in the ontrols, as -o m ~'18 12 ' 6~~~~~ NH4 NH4 NH4 NH4 L-Dopo L-Dopa ontrol Nephretomy Fig. 5 ffet of L-dopa on the prodution of oma and on blood and brain ammonia onentrations in nephretomised rats after the oma-produing dose of NH4l. Where appliable, the remainder of the legend is the same as for Fig. 1. might be expeted. The blood urea nitrogen in the ontrols after injetion of the oma-produing dose of NH41 was 41 ± 1.5 mg/dl, and in the rats two hours after nephretomy 65 ± 3. mg/dl. L-dopa had no signifiant effet on these values. Disussion The data of this report demonstrate that ammonia oma an be reversed by L-dopa and that this effet is probably related to a renal effet of dopamine rather than the build-up of dopamine in the brain. This study has impliations for the observations of the L-dopa effet in hepati oma. t has been assumed that the arousal effet of L-dopa in hepati oma is a onsequene of an inrease in brain dopamine that presumably ours (Parkes et al., 197, Fisher et al., 1976). This thought has been used in support of the false neurotransmitter hypothesis of pathogenesis of hepati oma (Fisher and Baldessarini, 1971). The inrease in brain dopamine after ingestion of L-dopa was probably real and the orrelation of this inrease with improvement in the enephalopathy was likely, as we demonstrated in our rats with ammonia-indued 31

5 32 L. Zieve, W. M. Doiaki, and R. F. Derr oma. However, the simultaneous ourrene of other effets from the inrease in systemi dopamine after L-dopa ingestion was ignored. Two of the striking effets of dopamine are on renal funtion and on the irulation. Dopamine inreases renal blood flow and glomerular filtration. t also inreases ardia output and dereases peripheral resistane. n 1964, MDonald et al. studied the ardiovasular and renal effets of an intravenous infusion of dopamine at a rate of 4-5,g/kg/min. Renal blood flow inreased by an average of 57 % in nine normal people and by 48 % in seven with ongestive heart failure. orresponding inreases in sodium exretion were three- and fourfold. The ardia output in the normal subjets inreased by 48 % and the peripheral resistane dereased by 33 %. n another study of the effet of a single 1-2 g dose of L-dopa in seven patients with Parkinson's disease, Goldberg and his olleagues (Finlay et al., 1971) found the average renal blood flow inreased by 2%, the exretion of potassium inreased by 4%, and the exretion of sodium inreased by 15%. There is thus every reason to antiipate that another renal effet of L-dopa or dopamine would be inreased exretion of ammonia and urea among other substanes. Thus our rats given an exess of NH¼4 responded to the L-dopa they reeived with inreased renal exretion of ammonia and urea, suffiient to redue the blood and brain ammonia by 25-3 %. This modest redution in ammonia onentration was apparently enough to bring the animals below the oma level for this substane. When the kidneys were removed the benefiial effet of L-dopa on the blood and brain ammonia was eliminated and the rats were not proteted from the oma-produing dose of NH41. We suggest that this renal effet may be the basis for the benefiial results seen with L-dopa in hepati enephalopathy. We are indebted to arolyn Lyftogt, Kay Draves, and Sandra Lindblad for exellent tehnial assistane. We also thank Dr W.. Sott of Hoffman- LaRohe n. for a generous gift of benseraide. Referenes Finlay, G. D., Whitsett, T. L., uinell,. A., and Goldberg, L.. (1971). Augmentation of sodium and potassium exretion, glomerular filtration rate and renal plasma flow by levodopa. New ngland Journal of Mediine, 284, Fisher, J.., and Baldessarini, R. J. (1971). False neurotransmitters and hepati failure. Lanet, 2, Fisher, J.., Funovis, J. M., Falao, H. A., and Wesdorp, R. 1.. (1976). L-Dopa in hepati oma. Annals of Surgery, 183, MDonald, R. H., Goldberg, L.., MNay, J. L., and Tuttle,. P., Jr. (1964). ffets of dopamine in man: augmentation of sodium exretion, glomerular filtration rate, and renal plasma flow. Journal of linial nvestigation, 43, Mather, A., and Roland, D. (1969). The automated thiosemiarbaide-diaetyl monoxime method for plasma urea. linial hemistry, 15, Parkes, J. D., Sharpstone, P., and Williams, R. (197). Levodopa in hepati oma. Lanet, 2, Refshauge,., Kissinger, P. T., Dreiling, R., Bland, L., Freeman, R., and Adams, R. N. (1974). New high performane liquid hromatographi analysis of brain ateholamines. Life Sienes, 14, Reinhold, J. G., and hung,.. (1961). Formation of artifatual ammonia in blood by ation of alkali. ts signifiane for the measurement of blood ammonia. linial hemistry, 7, Zieve, L., and Olsen, R. L. (1977). an hepati oma be aused by a redution of brain noradrenaline or dopamine? Gut, 18,