Tohoku J. exp. Med., 1982, 137, 213-220
`
`Diurnal Fluctuation of Blood Ammonia Levels
`in Adult-Type Citrullinemia
`
`YosHiaki YAgmMaA. Takasat Hrrasawa and TaKEYORI
`SAHEKI*
`
`Clinie of Internal Medicine, Iwaki-Kyoritsu General Hospital,
`Iwaki 972 and *Department of Biochemistry, Tokai University
`School of Medicine, Isehara 259-11
`
`Yautma, Y., Hrrasawa. T. and Sanent, T. Diurnal Fluctuation of Blood
`Ammonia Levels
`in Adult-Type Citrullinemia.
`Tohoku J. exp. Med..
`1982,
`137 (2), 213-220 ——A 48-year-old man, who was diagnosed as adult-type
`citrullinemia by quantitative estimation cf urea cycle enzymes in the liver,
`showed a regular nocturnal rise of blood ammcnia level.
`In order to elucidate the
`mechanism of diurnal fluctuation of blood ammonia level, the patient was put into
`fasting state for four days. The blood ammonia level rose at the night of the
`first fasting day even though no food was taken, then it decreased gradually and
`reached the lowest
`level
`in the morning of the third fasting day.
`Intravenous
`administration of amino acids mixture under the same starved condition gave rise
`to a significant elevation of blood ammonia level. Based on these results, it was
`concluded that hyperammonemia of adult-type citrullinemia could result from
`the accumulation of free ammonia which was produced from the catabolism of
`amino acids absorbed frcm the small intestine and surpassed the urea synthesis
`of defective urea cycle to flood into the blood. Furthermore.
`the rise of blood
`ammonia level at the night of the first fasting day suggested that
`the circadian
`rhythm of amino acid-carbohydrate metabolim might superimpcse on the process
`mentioned above.
`adult-type citrullinemia; hyperammonemia; diurnal
`fluctuation
`
`A 48-year-old man with adult-type citrullinemia has been described in our
`previous report (Yajima et al. 1981). He showedrepetitive episodes of encephalo-
`pathy and was diagnosed by quantitative estimation of amino acids and urea cycle
`enzymes in the liver.
`In this case, a regular diurnal fluctuation of the blood
`ammonia level was seen and the drip infusion of glutamate-arginine mixture and
`oral administration of citrate lowered the blood ammonia level. But
`the liver
`function tests of the patient became worse one month after the laparotomy for
`the evaluation of urea cycle enzymes and he clinically deteriorated to the state
`of general
`liver failure with jaundice and ascites. He died two months after the
`laparotomy. At the stage of general liver failure, citrate ingestion was ineffective
`and regular diurnal fluctuation of blood ammonia was nolonger seen.
`Received for publication, October 5, 1981.
`Dr. Yajima’s present address: The Third Department of Internal Medicine, Tohoku
`University School of Medicine, Sendai 980.
`213
`
`Par Pharmaceutical, Inc. Ex. 1023
`Par v. Horizon, IPR of Patent Nos. 9,254,278, 9,095,559, and 9,326,966
`Page 1 of 8
`
`

`

`214
`
`Y. Yajima et al.
`
`This paper discusses the mechanism of regular diurnal fluctuation of blood
`ammonia characteristic of this case from the data obtained in a starvation test,
`ammonia tolerance test,
`intravenous amino acids loading test and variation of
`blood ammonia during the period of general liver failure.
`
`MetHop AND SUBJECTS
`
`A case of adult-type citrullinemia and, as controls, five cases of decompensated liver
`cirrhosis were studied. Case | of control group was a 46-year-old male who had a mesocaval
`shunt operation for the esophageal varices and hypersplenism. Case 2 was a 53-year-old
`male in whom hepatic encephalopathy appeared following the rupture of the esophageal
`varices and thereafter encephalopathy of grade | to 2 according to Sherlock’s classification
`lasted. Case 3 was a 61-year-old female who was a decompensated cirrhotic patient with a
`triad of ascites, jaundice and encephalopathy and in whom lactulose administration of 60
`ml a day after the onset of encephalopathy lowered blood ammonia level. Case 4 was a 68-
`year-old female who had intractable ascites refractory to both strictsalt restriction and
`the use of potent diuretics during the three-months’ hospitalization. Case 5 was a 40-
`year-old male who was admitted to our hospital because of ascites and responded well to
`medical treatment. The ascites was not present at the time of blood ammonia determina-
`tion. Blood ammonia was measured by the modified method of Okuda and Fujii (1966)
`(normal range 40-80 ug/100 ml).
`
`RESULT
`
`Variation of blood ammonia levels in adult-type citrullinemia
`The effect of starvation. To prevent the hyperammonemia, protein intake was
`restricted to less than 50 g a day and theclinical course was observed. Symptoms
`of encephalopathy and asterixis occurred at night. The blood ammonia was
`measured four times a day,
`i.e., 9:00 a.m., 2:00 p.m., 8:00 p.m. and 11:00 p.m.
`during three consecutive days in order to elucidate the correlation between hyper-
`ammonemia and encephalopathy.
`It revealed that the level of blood ammonia
`depicted a gentle curve which had its peak between 8:00 p.m. and 11:00. p.m.
`Blood ammonia was then measured at 9:00 a.m. and 8:00 p.m. every day. The
`average of fasting levels was 120+38 yg/100 ml at 9:00 a.m. and 430+79 ug/100 ml
`at 8:00 p.m.
`
`Starvation
`
`froeayy
`
`“
`no -.
`“Ee
`-
`=R7
`senna a al
`3
`4
`
`2
`
`J
`
`level{Hg#100mi)
`Bloodammonia
`
`
`8 4
`
`00
`
`200
`
`Days
`
`Fig. 1. Variation of blood ammonia levels during starvation. The night of thefirst fasting
`day had a rise of blood ammonia level without taking any food, since then it decreased
`gradually to reach the lowest level in the morning of the third day. o,
`levels at 9:00
`a.m.; @,
`levels at 8:00 p.m.
`Second starvation trial was stopped on the third
`fasting day.
`
`Par Pharmaceutical, Inc. Ex. 1023
`Par v. Horizon, IPR of Patent Nos. 9,254,278, 9,095,559, and 9,326,966
`Page 2 of 8
`
`

`

`Diurnal Fluctuation of Blood Ammonia
`
`215
`
`In order to investigate the mechanism of the nocturnal rise of blood ammonia
`level the patient was put into a fasting state for four days. During the starvation
`period, water, electrolyte and 200g glucose were supplemented intravenously and
`the blood ammonia level was measured at 9:00 a.m. and 8:00 p.m. every day. The
`blood ammonia level rose in the night of the first fasting day, but since then it
`decreased gradually and reached the lowest level in the morning of the third fasting
`day. Butafter the night of the third day the blood ammonia level began to rise
`again. Another starvation trial under the same condition performed a week later
`showed the same result.
`
`The effect of intravenous administration of amino acids. Under the same con-
`dition as the previous two starvation trials, an amino acids mixture was adminis-
`tered intravenously in the night of the first and the second day and the morning of
`the third day. The amino acids mixture was a commercially available one which
`was synthesized after
`the constituent of
`the human milk. 500ml of a 5%
`solution of the amino acids mixture was administered by drip infusion for 2 hr
`and the blood ammonia level was measured immediately after the end of the in-
`fusion.
`In each occasion, the blood ammonia levels rose considerably by 1L00-
`200 wz/100 ml.
`In decompensated cirrhotic case (Case 4) with an ascites,
`three
`trials of amino acids infusion in the fasting state in the early morning had no
`influence on the blood ammonia levels.
`
`800
`
`600
`26 joo=Sap
`
`eo
`ES 400
`ae
`a
`5
`oOo
`
`150 [
`
`Control 4
`
`50.
`
`After
`
`200
`
`oO
`
`g2
`
`
`
`3
`
`o
`
`Before
`
`Days
`
`Fig. 2. The effect of the intravenous administration of amino acids mixture during the
`starvation period. Two broken lines indicate starvation trials without amino acids
`administration. @, blood ammonia levels after amino acids administration.
`
`Ammonia tolerance test. Ammonium chloride, 0.5 ¢/l0 kg body weight, was
`loaded orally and blood ammonia was measured every halfan hour. Blood ammonia
`had its peak at 60 min after loading and got to theinitial value 2 hr later (Fig. 3).
`
`Variation of blood ammonia level during the stage of general liver failure. The
`liver function tests of the patient became worse a month after the laparotomy
`for liver biopsy andclinically deteriorated to the state of generalliver failure with
`
`Par Pharmaceutical, Inc. Ex. 1023
`Par v. Horizon, IPR of Patent Nos. 9,254,278, 9,095,559, and 9,326,966
`Page 3 of 8
`
`

`

`216
`
`Y. Yajima et al.
`
`
`
`100 F
`
`0enSeeDSerananeneee!Ereeneeeeee
`
`o
`30
`60
`390
`120
`Time (min)
`
`Fig. 3. Ammonia tolerance test.
`
`BOO
`
`rt>
`
`
`=. 600es
`c =
`EO 400
`5S%:
`B~ 200
`=ao
`
`+— Intravenous hyperalimentation ——+
`
`0
`
`Fig. 4. Variation of blood ammonialevels in the stage of general liver failure.
`9:00 a.m.; @,
`levels at 8:00 p.m.
`
`o,
`
`levels at
`
`Days
`
`jaundice and ascites. He was not fed and was maintained with intravenous hyper-
`alimentation. At
`this stage,
`regular diurnal
`fluctuation of blood ammonia
`characteristic of this patient was no longer seen (Fig. 4).
`
`Variation of blood ammonia level in decompensated liver cirrhosis
`
`In the following five cases, protein intake was restricted to less than 50 ¢ a day.
`As a rule, blood ammonia was measured twice a day, 9:00 a.m. and 8:00 p.m. except
`Case 1.
`
`A case of mesocaval shunt (Case 1). Blood ammonia measurement was done
`twelve times serially over two days. There was no regularity observed in the
`variation of the blood ammonia level (Fig. 5).
`Cases of encephalopathy.
`In Case 2, the variation pattern was no longer diurnal
`but rather fluctuating with cycle of several days each.
`In Case 3, a trend of shght
`nocturnal rise of blood ammonia was seen before the onset of encephalopathy but
`the trend became unclear after the onset (Fig. 6).
`
`Par Pharmaceutical, Inc. Ex. 1023
`Par v. Horizon, IPR of Patent Nos. 9,254,278, 9,095,559, and 9,326,966
`Page 4 of 8
`
`400 -
`
`NH, Cl (05 9/10 kg B. W.)
`
`300 -
`
`See6oO
`ES 200+
`E ~
`og
`eekee
`
`a &
`
`2o
`
`O
`
`

`

`300
`
`200
`
`3=
`Ec!
`2 ¢
`50EO
`ES
`Bs
`a 2 100
`2oa
`
`Oo
`
`Diurnal Fluctuation of Blood Ammonia
`
`217
`
`Control
`
`|
`
`B:
`
`00
`
`12:00
`
`16:00
`
`800
`
`12:00
`
`16:00
`
`Hours
`
`Fig. 5. Diurnal fluctuation of the blood ammonia
`mesocaval anastomosis.
`
`levels in the cirrhotic patient with
`
`Control 2
`
`oe0
`
`200
`
`
`
`100}
`Encephalopathy
`. WUMMMM@MM@MlllMMlll
`300
`
`Control 3
`
`
`
`
`
`Bloodammonialevel(~g/!00mi)
`
`
`
`eo |Po4
`100+
`Encephalopathy
`Y
`5
`YU
`300
`
`
`
`{ Control 4
`
`“A
`
`100 +
`
`.
`
`(See 1 het
`
`L
`
`300 -
`
`Contral 5
`
`200 r
`
`100 r
`
`oObe
`\
`3
`5
`7
`
`Fig. 6. Variation of the blood ammonia levels in decompensated cirrhotic cases, ©, levels
`at 9:00 aom.; @,
`levels at 8:00 p.m.
`
`Days
`
`Par Pharmaceutical, Inc. Ex. 1023
`Par v. Horizon, IPR of Patent Nos. 9,254,278, 9,095,559, and 9,326,966
`Page 5of 8
`
`

`

`218
`
`Y. Yajima et al.
`
`blood
`seen in Fig. 6,
`In Cases 4 and 5, as
`Cases without encephalopathy.
`ammonia levels were lower than the levels of those cases with encephalopathy and
`depicted a gentle curve with a cycle of several to ten days.
`
`Discussion
`
`The contribution of ammonia to the pathogenesis of hepatic encephalopathy has
`been widely investigated (Walker and Schenker 1970; Schenker etal. 1974). Amino
`acids imbalance (Fischer et al. 1975) and short-chain fatty acid (Chen et al. 1970;
`Zieve et al. 1974) have recently been proposed as etiological
`factors
`in hepatic
`encephalopathy. Among them, ammonia is the most important in the treatment of
`hepatic encephalopathy in the sense that we can control
`it to some extent.
`In
`patients with portal-systemic shunt and experimental animals with hepatic damage
`or Eck fistula, meat intoxication or protein intoxication has been known (Nencki
`et al. 1896; Monguio and Krause 1934), and protein restriction is effective for lower-
`ing the blood ammonia level and improving the clinical course.
`There was no
`report, however, dealing with hepatic encephalopathy which referred to the varia-
`tion of blood ammonia level from the view point of diurnal fluctuation. The case
`with mesocaval shunt, one of the controls, showed no trends of blood ammonia to
`fluctuate diurnally and other four controls of decompensated cirrhosis also did not
`show the regular fluctuation pattern of blood ammonia.
`On the other hand,in thefield of congenital hyperammonemia, Batshaw et. al.
`(1975)
`reported a case of
`thirteen-year-old female with
`carbamyl phosphate
`synthetase deficiency in which nocturnal rise of blood ammonia was seen. Moser
`et al.
`(1967) reported two cases of argininosuccinic aciduria in which the blood
`ammonia levels were normal or slightly elevated in the fasting state but a marked
`elevation occurred postprandially.
`In thefields of adult-type citrullinemia, Suzuki
`et al. (1980) reported a case in which the variation of blood ammonia wasclosely
`associated with food ingestion.
`In our case, the measurement of blood ammonia
`over a two months period revealed rises of blood ammonia Jevel every night even
`when encephalopathy was not seen. These results suggested that postprandial
`elevation, or regular nocturnal rise, of blood ammonia should be a pathognomonic
`sign of the abnormality in urea cycle.
`Therefore,
`in the hyperammonemia caused by portal-systemic shunt, blood
`for the determination of ammonia need not necessarily be sampled in the fasting
`state in the morning andif adult-type citrullinemia is suspected, blood samples must
`be collected postprandially.
`The starvation test, performed to clarify the mechanism of the nocturnal rise
`of blood ammonia, showed a gradual decrease of blood ammonialevel, supporting
`the hypothesis that orally taken nitrogens are closely associated with the elevation
`of blood ammonia level.
`Incidentally, the blood ammonia levels in the night ofthe first fasting day were
`significantly elevated compared with those in the morning and re-elevation of
`levels began since the night of the third fasting day. As to the former, it was
`
`Par Pharmaceutical, Inc. Ex. 1023
`Par v. Horizon, IPR of Patent Nos. 9,254,278, 9,095,559, and 9,326,966
`Page 6 of 8
`
`

`

`Diurnal Fluctuation of Blood Ammonia
`
`219
`
`postulated that nocturnal rise of blood ammonia was not only caused by the meal
`but also can be influenced by the circadian rhythm of amino acid-carbohydrate
`metabolism regulated by the circadian rhythm of hormone secretion. The most
`probable explanation of the re-elevation of blood ammonia since the third day is
`the generation of free ammonia in the catabolism of endogenous amino acids.
`Intravenous administration of amino acids mixture under the same starved
`condition showed a remarkable elevation of ammonia in the adult-type citrullinemia
`but no effect in a decompensated cirrhotic patient. And, the ammonia tolerance
`test showed a peak at 60 min after ammoniumingestion, a little later than in
`cirrhosis. Murawaki et al.
`(1977)
`reported a case of 23-year-old male with
`ornithine transcarbamylase (OTC) deficiency in which the ammonia tolerance
`test showed a unique curve abruptly rising at 75 min after loading, which is
`obviously distinct from that seen in cirrhosis. But
`in two cases of adult-type
`OTC deficiency, reported by Short et al. (1973), both of them showed peaks at 30
`min,
`inconsistent with the case reported by Murawaki et al. (1977).
`As regards adult-type citrullinemia, in the case of Suzuki et al. (1980) and the
`case of Yamauchi et al.
`(1980),
`the results of ammonia tolerance test were
`reported.
`In both of them, ammonia tolerance curves rose slowly until 60 min,
`but it was not clear whether the value at 60 min was a peak value or not because
`the data after 60 min were not presented,
`In general, most of the cirrhotic cases
`have their peak values at 15 to 45 min (Conn 1960).
`It may be said that the
`patients with deficiency of urea cycle enzyme have their peaks later than the
`liver cirrhotics. But there is a question in diagnosing the deficiency of urea cycle
`enzyme from the ammonia tolerance test because some of the cirrhotics have their
`peaks at 60 or 90 min (Ohwada et al. 1977). On the other hand,
`the intravenous
`amino acids loading test is thought to be a useful test
`for
`the diagnosis of the
`deficiency of urea cycle enzyme.
`From these data, we proposed the following mechanismfor the diurnal fluctua-
`tion of the blood ammonia in our patient: Hyperammonemia of portal-systemic
`encephalopathy due to liver cirrhosis results from the generation of free ammonia
`by intestinal bacterial flora in the large intestine.
`It takes 4 hr on the average for
`non-digestible nitrogenous source to arrive at the large intestine (Read et al. 1980)
`and generate free ammonia and 50 hr on the average to be evacuated from the
`rectum (Cummingset al. 1976).
`It follows, therefore, that the nitrogenous source
`in a meal brings about the rise of blood ammonia with a time lag of 4 to 50hr.
`Because of this time lag, regular diurnal fluctuation of blood ammonia associated
`with meal ingestion is not seen in the portal-systemic encephalopathy. On the
`other hand, hyperammonemia of adult-type citrullmemia could result from the
`accumulation of free ammonia which was produced from the catabolism of amino
`acids absorbed from the small mtestine and surpassed the urea synthesis of defec-
`tive urea cycle to flood into the blood.
`in the night of the first fasting
`Furthermore, the rise of blood ammonia level
`day suggested that the circadian rhythm of amino acid-carbohydrate metabolism
`might superimpose the process mentioned above.
`
`Par Pharmaceutical, Inc. Ex. 1023
`Par v. Horizon, IPR of Patent Nos. 9,254,278, 9,095,559, and 9,326,966
`Page 7 of 8
`
`

`

`220
`
`Y. Yajima etal.
`
`Acknowledgment
`
`We are indebted to Prof. Y. Goto, The Third Department of Internal Medicine,
`Tohoku University School of Medicine,
`for his valuable advices and encouragement
`in
`preparing this paper.
`
`References
`
`!)
`
`2)
`
`3)
`
`4)
`
`5)
`
`6)
`
`7)
`
`8)
`
`9)
`
`10)
`
`13)
`
`14)
`
`15)
`
`16)
`
`17)
`
`18)
`
`19)
`
`Batshaw, M., Brusilow, S. & Walser, M. (1975) Treatment of carbamyl phosphate
`synthetase deficiency with ketoanalogues of essential amino acids. New Engl. J.
`Med., 292, 1085-1090.
`Chen, 8., Mahadevan, V. & Zieve, L. (1970) Volatile fatty acids in the breath of patients
`with cirrhosis of the liver.
`J. Lab. clin. Med., 75, 622-627.
`Conn, H.O. (1960) Ammonia tolerance in liver disease.
`J. Lab. clin. Med., 55, 855-
`871.
`Cummings, J.H., Jenkins, D.J.A. & Wiggins, H.S. (1976) Measurement of the mean
`transit time of dietary residue through the human gut. Guf, 17, 210-218.
`Fischer, E.J., Funovies, J.M., Aguirre, A., James, J-H., Keane, J.M. & Wesdorp, R.1.C.
`(1975) The role of plasma aminoacids in hepatic encephalopathy. Surgery, 78, 276-
`290,
`
`Monguio, J. & Krause, F. (1984) Uber die Bedeutung des NH, Gehaltes des Blutes fiir
`die Beurteilung der Leber Function. Klin. Wschr.. 13, 1142-1149.
`Moser, H.W., Efron, M.L., Brown, H., Diamond, R. & Neuman, ©.G. (1967) Argin-
`inosuccinic aciduria. Report
`of two cases
`and demonstration of intermittent
`elevation of blood ammonia. Amer. J. Med., 42, 9-26,
`Murawaki, Y., Yoshida, K., Hirayama, C., Nakao, T. & Nakaya, Y. (1977) Inherited
`hyperammonemia in adult. Acta hepatol. jap., 18, 856-863, (Japanese)
`Nencki, M., Pawlow, J.P. & Zaleski, J. (1896) Ueber den Ammoniakgehalt des Blutes
`und der Organe und die Harnstoffbildung bei den Séugethieren. Naunyn-Schmideberg’s
`Arch. exp. Path. Pharmak., 37, 26-51.
`(1977) Studies on ammonia
`Ohwada, Y., Koyama, K., Takagi, Y. & Anezaki, T.
`metabolism in the rat liver in biliary obstraction, Acta hepatol. jap., 18, 3338-340.
`(Japanese)
`(1966) Direct spectrophotometrical determination of blood
`Okuda, T. & Fujii, S.
`ammonia. Saishin-Igaku, 21, 622-627. (Japanese)
`Read, N.W., Miles, C.A., Fisher, D., Holgate, A.M., Kime, N.D., Mitchell, M-A., Reeve,
`A.M., Roche, T.B. & Wlaker, M.
`(1980) Transit
`time of a meal
`through the
`stomach, small intestine, and colon in normal subjects and its role in the pathogenesis
`of diarrhea, Gastroenterology, 79, 1276-1282.
`Schenker, 8., Breen, K.J. & Hoyumpa, A.M., Jr.
`current status. Gastroenterology, 66, 121-151.
`Short, E.M., Conn, H.O., Shodgrass, P.J., Campbell, A.G.M. & Rosenberg, L.E. (1973)
`Evidence for X-linked dominance of ornithine transcarbamylase deficiecy. New Engl.
`J. Med., 288, 7-12.
`Suzuki, Y., Yamaura, N., Nozawa, K., Akahane, Y., Kiyosawa, K., Nagata, A., Furuta,
`S. & Chiba, K. (1980) A case of adult-type congenital citrullinemia. Acta hepatol.
`jap., 21, 1215-1221. (Japanese)
`Walker, C.O. & Schenker, 8. (1970) Pathogenesis of hepatic encephalopathy —with
`special reference to the role of ammonia. Amer. J. clin, Nuér., 23, 619-632.
`Yamauchi, M., Kitahara, T., Fujisawa, K.. Kameda, H., Takasaki, $., Komori, R.,
`Saheki, T., Katsunuma, T. & Katsunuma, N. (1980) An autopsied case of hypercitrul-
`linemia in adult caused by partial deficiency of liver argininosuccinate synthetase.
`Acta hepatol. jap., 241, 326-334. (Japanese)
`Yajima, Y.. Hirasawa, T. & Saheki, T. (1981) Treatment of adult-type citrullinemia with
`administration cf citrate. Tohoku J. exp. Med., 134, 321-330.
`Zieve, F.J., Zieve. L.. Doizaki, W.M. & Gilsdorf, R.B. (1974) Synergism between
`ammonia and fatty acids in the production of coma;
`Implications for hepatic coma.
`J. Pharmacol. exp. Ther. 191, 10-16.
`
`(1974) Hepatic encephalopathy:
`
`Par Pharmaceutical, Inc. Ex. 1023
`Par v. Horizon, IPR of Patent Nos. 9,254,278, 9,095,559, and 9,326,966
`Page 8 of 8
`
`