5-Oxoprolinuria in hyperammonemic encephalopathy: Coincidence or worsening factor?

Accepted Manuscript 5-Oxoprolinuria in hyperammonemic Coincidence or worsening factor?

encephalopathy:

Guillaume Rousseau, Isabelle Signolet, Marie-Christine Denis, Juan Manuel Chao de la Barca, Rafaël Mahieu, Franck Letournel, Pascal Reynier, Gilles Simard PII: DOI: Reference:

S0009-9120(17)30839-1 doi:10.1016/j.clinbiochem.2017.09.025 CLB 9633

To appear in:

Clinical Biochemistry

Received date: Revised date: Accepted date:

21 August 2017 29 September 2017 29 September 2017

Please cite this article as: Guillaume Rousseau, Isabelle Signolet, Marie-Christine Denis, Juan Manuel Chao de la Barca, Rafaël Mahieu, Franck Letournel, Pascal Reynier, Gilles Simard , 5-Oxoprolinuria in hyperammonemic encephalopathy: Coincidence or worsening factor?. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Clb(2017), doi:10.1016/j.clinbiochem.2017.09.025

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ACCEPTED MANUSCRIPT Case Report 5-oxoprolinuria in hyperammonemic encephalopathy: Coincidence or worsening factor?

Guillaume Rousseaua, Isabelle Signoleta, Marie-Christine Denisa, Juan Manuel Chao de la

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Barcaa, Rafaël Mahieub, Franck Letournelc, Pascal Reyniera, Gilles Simarda,*

Department of Biochemistry and Genetics, University Hospital, Angers, France

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Department of Medical Intensive Care and Hyperbaric Medicine, University Hospital,

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Department of Neuropathology, University Hospital, Angers, France

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Angers, France

*Corresponding author:

Dr Gilles Simard, MD-PhD, Department of Biochemistry and Genetics, IBS University Hospital of Angers 4 rue Larrey, Angers, 49933 Cedex 9, France Tel: + 2 41 35 33 14 1

ACCEPTED MANUSCRIPT 1.

Introduction

Hyperammonemia is a common metabolic disorder in patients admitted to Intensive Care Units (ICU) and is associated with high morbidity and mortality rates [1]. This metabolic condition

reflects

an

imbalance

between

excessive ammonia

production and/or the

impairment of its elimination by the hepatic urea cycle, which may be directly primarily or

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secondarily affected [2]. Hyperammonemia occurs mainly in the event of chronic liver

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disease, but occasionally also in the absence of hepatic failure [1,2]. The physiopathology of

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hyperammonemia is complex and involves several metabolites, especially glutamine, a byproduct of ammonia formed by the amidation of glutamate and involved in the regulation of

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brain water (i.e. osmolytes) [3]. When over-synthetized, glutamine damages the central nervous system (CNS), which leads to swollen astrocytes and brain edema, as commonly

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observed in the case of hyperammonemic encephalopathy [4]. In the CNS, ammonia detoxification relies entirely on the availability of intra-astrocytic

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glutamate and the activity of glutamine synthase (GS), as long as urea cycle enzymes are not

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expressed in this tissue [5]. A constant level of glutamate in the astrocyte is therefore necessary to buffer the excess ammonia produced. Alpha-ketoglutarate and 5-oxoproline are intermediate metabolites of two pathways involved in glutamate metabolism: the Krebs and

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glutathione (GSH) cycles, respectively [6, 7]. Malnutrition may interfere with both these metabolisms due to altered protein turnover [8] and GSH synthesis [9], and may affect intracerebral glutamate disposal and, consequently, glutamine synthesis.

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ACCEPTED MANUSCRIPT 2. Case presentation A 76-year-old woman was referred to the geriatric clinic for an altered general condition combined with a 20kg (44lbs) weight loss over 6 months. The patient reported abdominal pain and vomiting, and her family mentioned an insufficient dietary intake due to loss of

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autonomy. The patient’s medical history included hypertension, type 2 diabetes mellitus and polymyalgia rheumatica.

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Malnutrition was confirmed by low albuminemia at 27.8 g/l (35–50 g/L) and an overall

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decrease in micronutrients, especially zinc at 0.27 mg/L (0.7-1.25 mg/L). On account of

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anemia with hemoglobin equal to 10.1 g/dL (11.8-15 g/dL) and a positive hemoccult test, the patient underwent a computed tomography scan with contrast medium injection, which

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highlighted an ileocecal tumor complicated by a subocclusive syndrome. It was decided that the patient required surgical care and oxycodone treatment was given for the pain. However,

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the patient’s neurological status rapidly deteriorated before surgery (no Glasgow Coma Scale

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available), with subsequent loss of consciousness, resulting in a transfer to the ICU. Upon admission, the patient required intubation and mechanical ventilation to ensure airway protection.

Clinical

observation

and

hemodynamic

measurements

revealed

apyrexia,

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hypotension (blood pressures at 81/44 mmHg), tachycardia (130 bpm) and an absence of hypoxia (oxygen saturation at 100%). Laboratory tests highlighted compensated metabolic acidosis with a significant anion gap (AG), acute renal failure, slight cytolysis with no evidence of cholestasis, and major inflammatory syndrome characterized by high levels of procalcitonin (Table 1).

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ACCEPTED MANUSCRIPT Table 1 Biochemical values before and after admission to the intensive care unit.

Creatinine (µmol/L)

Ten days before 57

Day of admission 166

Two days 143

Five days 68

16

30

30

29

23

16

8

-

20

111

61

43

18

114

-

146

Reference intervals: 49-90

Anion gap Reference intervals: 12-20

CO 2 (mmol/L) Reference intervals: 23-31

ASAT (IU/L) ALAT (IU/L)

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Reference intervals: 12-37

NH3 (µmol/L) Reference intervals: 18-50

242

47

125

39

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Procalcitonin (µg/L)

66

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Reference intervals: 11-43

Reference intervals: 0-0.25

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All parameters were determined using Architect C16000 (Abbott®), except for Procalcitonin (TRACE, Kryptor compact Brahms®)

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The main etiologies of non-traumatic coma were investigated and eliminated. Toxic causes were dismissed in the absence of any beneficial effects of administrating naloxone and

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flumazenil (antidotes to morphine and benzodiazepine, respectively) and through negative urinary drug screening. A cranial CT scan revealed no tumor or intracranial bleeding, while

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the analysis of cerebrospinal fluid (CSF) ruled out a CNS infection. Common metabolic factors involved in the etiology of a coma (i.e. hypoglycemia,

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hyperosmolar hyperglycemia and hypothyroidy) were investigated and dismissed. At the admission, prior to hyperammonemia (Table 1), together with an electroencephalogram and cerebral magnetic resonance imaging indicating metabolic encephalopathy, a plasmatic amino-acid chromatography was performed two days later, revealing a significant increase in glutamine at 1420 µoml/L with normal citrullinemia (Table 2). No orotic acid was detected. Moreover, main endogenous and exogeneous causes of high AG were also ruled out (normal lactate and toxicological screening). Urinary organic acid chromatography was performed, using gas chromatography- mass spectrometry, four days after the admission and highlighted 4

ACCEPTED MANUSCRIPT a sharp increase in 5-oxoproline (or pyroglutamic acid), up to 11350 µmol per mmol of creatinine (<70 µmol per mmol creatinine). Intravenous fluids, sodium bicarbonate and antibiotherapy were introduced. Renal and hepatic functions improved, however high AG and sepsis persisted. Despite intensive care and

digestive septic shock, six days after her admission to the ICU.

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normalization of the hyperammonemia, the patient remained unconscious and died from

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An autopsy was performed to clarify the etiology of the hyperammonemia. Hepatic biopsy

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showed significant macrovesicular steatosis (80% of hepatocytes) without cirrhosis. A genetic analysis of all the genes involved in the urea cycle (enzyme genes: CPS1, OTC, ASS1, ASL,

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ARG1, NAGS, CA5A) ruled out a primary deficiency (Necker’s Hospital, Paris). A colic

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biopsy showed mucinous adenocarcinoma. Histologic slides of brain biopsies performed on

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the frontal cortex showed an edema in the white matter and Alzheimer type II cells (Fig. 1).

Fig. 1 Histological slide of frontal cortex showing numerous Alzheimer type II astrocytes (black arrows; H&E staining).

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ACCEPTED MANUSCRIPT In addition, glutamine and 5-oxoproline concentrations were quantified in the CSF, the frontal cortex and in a serum collected ten days before the beginning of the coma and stored at -80°C. Results showed high levels of both glutamine and 5-oxoproline in CSF and the brain, with a slight increase in 5-oxoproline and the sum of glutamine and glutamate in the serum ten days

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earlier (Table 2).

Ten days before coma Plasma (reference intervals) 652 (486-670) 265 (2-4) 250 (<50)

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Table 2 Metabolic analysis performed on plasma, cerebrospinal fluid and autopsied brain tissue. Two days after coma

Post mortem

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Plasma Cerebrospinal Frontal cerebral (reference fluid (reference cortex(µmol/g)1 intervals) intervals) (normal control) Glutamine 1420 2720 1120 (µmol/L) (486-670) (486-670) (600) Glutamate 174 57.4 762 (µmol/L) (2-4) (2-28) (700) 5-oxoproline 4800 2240 284 (µmol/L) (<50) (<30) (50) Citrulline 23 (µmol/L) (<33) 1 : values of metabolites were compared to a mean value obtained through the analysis of

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autopsied frontal cerebral cortex from 2 patients free from neurological diseases. All parameters were quantified using ion pairing HPLC with positive electrospray liquid chromatography coupled with tandem mass spectrometry (AB Sciex) and using isotopic

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internal standards.

3. Discussion

Malnutrition associated with neoplasia was considered the reason for the hyperammonemia, after excluding the other main causes. Indeed, protein hypercatabolism produces excess ammonia [8] and the urea cycle may be impaired by zinc deficiency, zinc being an essential co-factor of certain urea cycle enzymes [10]. The slight increase in [glutamine-glutamate], previously observed (Table 2), and the Alzheimer type II cells discovered in the frontal 6

ACCEPTED MANUSCRIPT biopsies were consistent with pre-existing chronic hyperammonemia [11]. Malnutrition is also a risk factor for increased 5-oxoprolinemia, given that 5-oxoproline is formed in the GSH cycle, which takes place in most cells [12]. Its synthesis increases when GSH, formed by glutamate, glycine and cysteine, is depleted. Indeed, GSH exerts negative feedback on glutamyl cysteine synthetase, the first enzyme of the cycle, thereby regulating its own

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formation (Fig. 2). GSH depletion causes a loss of this feedback and the -glutamyl

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cyclotransferase pathway is preferred, increasing the synthesis of 5-oxoproline. Many factors

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can affect the cycle [13], and malnutrition is one of the main causes [9]. When overproduced, 5-oxoproline may lead to a high AG metabolic acidosis, which is often the first biological

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sign of this organic aciduria [14]. As shown in Table 2, a slight increase in 5-oxoproline was also already observed in our patient before the onset of consciousness disorders, highlighting

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GSH depletion.

Fig. 2 Two pathways of formation of glutamate in astrocytes: the Krebs and glutathione (GSH) cycles. (BCAAs: Branched-Chain Amino Acid, AT: Amino Transferase, NH3 : Ammonium)

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ACCEPTED MANUSCRIPT Most extra-hepatic organs, including the brain, lack a complete urea cycle. They therefore transform toxic ammonia to glutamine through the amidation of glutamate. The latter is a ratelimiting substrate and different pathways are involved in its metabolism to maintain a constant level. Alpha-ketoglutarate, an intermediary in the Krebs cycle, is a major source of glutamate via the alpha-ketoglutarate-linked amino transferases. The release of glutamate depends

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particularly on branched-chain amino acid (BCAA) transferases, and consequently on BCAA

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availability. Hyperammonemia, through pyruvate carboxylase stimulation, increases synthesis

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of alpha-ketoglutarate, which can produce more glutamate to buffer ammonia. Close to this main pathway, 5-oxoproline is identified as a minor source of glutamate via the 5-

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oxoprolinase reaction [6] (Fig. 2). In our case, the low level of BCAA due to malnutrition (data not shown) could probably limit the major pathway. Consequently, the formation of

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glutamate may involve other metabolisms to replenish astrocytes, notably the GSH cycle. Indeed, on the one hand, it has been established that malnutrition increases 5-oxoproline by

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depleting GSH [9]; on the other hand, the onset of sepsis and acute renal failure exacerbate intracerebral 5-oxoproline [15] (Table 2). If overproduced, 5-oxoproline may be involved in

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supplying alpha-ketoglutarate, releasing glutamate in astrocytes. The combination of these events could explain the level of glutamine in the brain, similar to the levels observed in cases

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of hepatic coma, and thereby also explain the origin of the patient’s neurological disorders [16]. In a context of malnutrition and produced in excess, 5-oxoproline could be seen as a worsening factor of hyperammonemic encephalopathy.

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ACCEPTED MANUSCRIPT 4. Conclusion Patients suffering from malnutrition, hyperammonemia and concomitant GSH depletion may develop encephalopathy, which can be underdiagnosed especially when ammonia levels are slightly increased. In predisposed individuals, metabolic analyses of urine, plasma and CSF

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may help reach a diagnosis, underlying precise mechanisms to ensure appropriate treatment

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for the encephalopathy.

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ACCEPTED MANUSCRIPT References

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[1] A.S. Clay, B.E. Hainline, Hyperammonemia in the ICU, Chest. 132 (2007) 1368–1378. [2] J. Häberle, Clinical and biochemical aspects of primary and secondary hyperammonemic disorders, Arch. Biochem. Biophys. 536 (2013) 101–108. [3] C.R. Bosoi, C.F. Rose, Brain edema in acute liver failure and chronic liver disease: similarities and differences, Neurochem. Int. 62 (2013) 446–457. [4] S.W. Brusilow, R. Traystman, Hepatic encephalopathy, N. Engl. J. Med. 314 (1986) 786–787. [5] A. Martinez-Hernandez, K.P. Bell, Norenberg, Glutamine synthetase: glial localization in brain, Science. 195 (1977) 1356–1358. [6] A.J. Cooper, T.M. Jeitner, Central Role of Glutamate Metabolism in the Maintenance of Nitrogen Homeostasis in Normal and Hyperammonemic Brain, Biomolecules. 6 (2016) 16. [7] A. Kumar, A.K. Bachhawat, Pyroglutamic acid: throwing light on a lightly studied metabolite, Current science. 102 (2012) 288–297. [8] I. De Blaauw, N.E. Deutz, M.F. Von Meyenfeldt, Metabolic changes in cancer cachexia-first of two parts, Clin Nutr. 16 (1997) 169–176. [9] K.M. Mogensen, J. Lasky-Su, A.J. Rogers, R.M. Baron, L.E. Fredenburgh, J. Rawn, M.K. Robinson, A. Massarro, A.M.K. Choi, K.B. Christopher, Metabolites Associated With Malnutrition in the Intensive Care Unit Are Also Associated With 28-Day Mortality: A Prospective Cohort Study, JPEN J. Parenter. Enteral Nutr. 41 (2017) 188– 197. [10] V.H. Roth, Influence of alimentary zinc deficiency on nitrogen elimination and enzyme activities of the urea cycle, J. Anim. Physiol. Anim. Nutr. 85 (2001) 45–52. [11] M.D. Norenberg, L.W. Lapham, The astrocyte response in experimental portal-systemic encephalopathy: an electron microscopic study, J. Neuropathol. Exp. Neurol. 33 (1974) 422–435. [12] A. Meister, Metabolism and functions of glutathione, Trends Biochem. Sci. 6 (1981) 231–234. [13] E. Mayatepek, 5-Oxoprolinuria in patients with and without defects in the gammaglutamyl cycle, Eur. J. Pediatr. 158 (1999) 221–225. [14] K. Myall, J. Sidney, A. Marsh, Mind the gap! An unusual metabolic acidosis, Lancet. 377 (2011) 526. [15] G. Biolo, R. Antonione, M. De Cicco, Glutathione metabolism in sepsis, Crit. Care Med. 35 (2007) S591-595. [16] J. Lavoie, J.F. Giguère, G.P. Layrargues, R.F. Butterworth, Amino acid changes in autopsied brain tissue from cirrhotic patients with hepatic encephalopathy, J. Neurochem. 49 (1987) 692–697.

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ACCEPTED MANUSCRIPT Highlights

Malnutrition is a risk factor of hyperammonemia and 5-oxoprolinuria.

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Hyperammonemia may be responsible for encephalopathy, even if it is marginally increased.

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5-oxoproline may be a source of glutamate in astrocytes, accelerating glutamine synthesis in the brain.

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Consider asking patients suffering from malnutrition for these parameters.

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