INTRODUCTION

Delirium is a frequent complication among patients admitted to intensive care units (ICU) and is independently associated with increases in the durations of mechanical ventilation (MV) and ICU stay, long-term neuropsychological dysfunction, and mortality.^(1-3)

Several different mechanisms have been proposed to explain the development of delirium in ICU patients. It has been suggested that drug toxicity, inflammation and acute stress responses^(4-6) could affect several aspects of mental function.⁽⁷⁾ Other potential mechanisms include reductions of cerebral blood flow and oxygen extraction by the brain, disruptions of the blood-brain barrier, impairments of astrocyte function, and neuronal degeneration.^(8,9) The final common pathway that leads to delirium is probably the disruption of neurotransmission in the brain.

The cholinergic and serotoninergic pathways are the major neurotransmitter pathways that have been implicated in the development of delirium.^(5,10) The activation of cholinergic receptors modulates cognition, arousal, learning, and memory, which are the major brain functions that are affected by delirium. The synthesis of acetylcholine is vulnerable to different stressors, such as impairments in energetic metabolism and inflammation.^(11,12) Serotonin has a role in the arousal and sleep-wake cycles, and an increase in serotonin activity occurs in hepatic encephalopathy.^(13,14) Additionally, imbalances in the levels of the precursors of serotonin synthesis occur in delirium patients, which suggests a role of serotonin in the genesis of delirium.^(15,16) Pandharipande et al.⁽¹⁶⁾ demonstrated that either very low or very high levels of plasma tryptophan, a serotonin precursor, are independent risk factors for the occurrence of delirium in mechanically ventilated ICU patients. Cholinesterase activity has been proposed to have a role in the development of delirium in postoperative patients,^(17,18) but no definitive evidence suggests the roles of these neurotransmitters in the development of delirium in critically ill patients.

Therefore, we hypothesized that the plasma level of serotonin and acetylcholinesterase activity determined upon ICU admission would predict the occurrence of acute brain dysfunction in ICU patients. Additionally, because the metabolisms of both neurotransmitters can be altered by inflammation, we also hypothesized that these neurotransmitters would be differentially modulated in patients with sepsis-associated delirium.

METHODS

RESULTS

A total of 77 patients were included in the present study. There were 39 (50.6%) non-delirium and 38 (49.6%) delirium patients. The demographic and clinical variables are shown in table 1.

Table 1 - Demographic and baseline characteristics

Acetylcholinesterase activity and serotonin measurements and the occurrence of delirium

The serum acetylcholinesterase activities and serotonin levels were comparable in the delirium and non-delirium patients (Table 1). In the binary regression models, neither serum acetylcholinesterase activity nor serotonin level was independently associated with delirium. Additionally, there was no significant interaction between the biomarkers and sepsis (Table 2).

Table 2 - Binary regression analyses of the characteristics associated with the occurrence of delirium according to each biomarker

	OR (CI 95%)	p value
AChE	0.86 (0.48 - 1.5)	0.62
Sepsis	1.7 (0.26 - 12)	0.55
SOFA	0.98 (0.82 - 1.16)	0.83
Sedation	1.4 (0.35 - 5.5)	0.62
AChE* sepsis	0.84 (0.39 - 1.8)	0.66
Serotonin	2.9 (0.59 - 14)	0.18
Sepsis	57 (0.15 - 21)	0.18
SOFA	1.06 (0.88 - 1.2)	0.48
Sedation	1.03 (0.25 - 4.2)	0.96
Serotonin* sepsis	0.29 (0.046 - 1.8)	0.19

OR - odds ratio; CI - confidence interval; AChE - acetylcholinesterase enzyme; SOFA - Sequential Organ Failure Assessment. Hosmer and Lemeshow goodness-of-fit - AChE Χ² = 7.05, p = 0.53, serotonin Χ² = 11, p = 0.15.

* Indicates interaction between biomarker and sepsis.

Table 2 - Binary regression analyses of the characteristics associated with the occurrence of delirium according to each biomarker

Correlations of acetylcholinesterase activity and serotonin level with acute brain dysfunction and mortality

There were no significant correlations of acetylcholinesterase activity or serotonin level with the number of DCFDs (r = 0.003, p = 0.959 and r = 0.014, p = 0.317, respectively). Among the patients who developed delirium, there was a strong negative correlation between the acetylcholinesterase enzyme (AChE) level and the number of DCFDs (r = 0.838, p ≤ 0.001); patients with higher AChE levels spent fewer days alive without delirium or coma.

In the univariate analyses, sedation and MV use, age, SOFA score upon admission, the number of days in a coma, and the DCFDs were associated with mortality (Table 3). Thus, multivariate analysis was performed using age, SOFA score, the need for MV and the biomarkers. Only MV was found to be a risk factor for death in this sample (odds ratio - OR: 7.2, confidence interval 95% - CI95%: 1.03 - 50.9, p = 0.047).

Table 3 - Variables associated with mortality

	Univariate analysis			Multivariate analysis
	Survivors	Non-survivors	p value	OR (CI95%)	p value
	N = 57 (74%)	N = 20 (26%)
Age (years)	52 (30)	64 (29)	0.034*	0.96 (0.92 - 1.0)	0.058
Male	41 (71.9)	12 (60)	0.402	-	-
APACHE II	17.5 (13.5)	22.5 (16.75)	0.307	-	-
SOFA score on admission	7 (5.25)	9 (5.5)	0.042*	0.98 (0.01 - 1.19)	0.800
Emergency surgery patients	9 (15.8)	2 (10)
Elective surgery patients	9 (15.8)	5 (25)		-	-
Medical patients	39 (68.4)	13 (65)	0.587
Mechanical ventilation (yes)	30 (56.6)	17 (89.5)	0.011*	7.2 (1.03 - 50.9)	0.047*
Sedation use	32 (57.1)	18 (94.7)	0.002*	-	-
Mental status at enrollment
Comatose	25 (43.9)	6 (30)
Delirious	2 (3.5)	0	0.335	-	-
Normal	30 (52.6)	14 (70)
Reasons for admission
Cardiovascular	8 (14.3)	2 (10)
Postoperative	9 (16.1)	4 (20)
Neurological	11 (19.6)	5 (25)
Trauma	10 (17.9)	1 (5)
Respiratory distress (excluding sepsis)	10 (17.9)	3 (15)	0.198	-	-
Sepsis	1 (1.8)	2 (10)
Shock (excluding sepsis)	2 (3.6)	1 (5)
Digestive	4 (7.1)	0
Renal/metabolic	1 (1.8)	0
Other	0	2 (10)
Hospital length of stay (days)	18.5 (19.5)	18 (13.5)	0.981	-	-
Delirium (days)	1 (1)	1 (1)	0.535	-	-
Coma (days)	2 (6)	5 (7.5)	0.003*	-	-
Sepsis (yes)	21 (51.2)	14 (73.7)	0.159	-	-
Delirium/coma-free days	25 (6.25)	4 (17.25)	≤ 0.001*
AChE (µmol AcSCh/h mg protein)	1.77 (1.24)	1.25 (1.56)	0.155	1.14 (0.66 - 1.96)	0.648
Serotonin (ng/mL)	3.06 (0.86)	2.98 (1.08)	0.959	1.54 (0.77 - 3.07)	0.225

OR - odds ratio; CI - confidence interval; APACHE - Acute Physiology and Chronic Health Evaluation; SOFA - Sequential Organ Failure Assessment; AChE - acetylcholinesterase enzyme. Univariate analysis: the data are presented as the median (IQR) or number (percentage). Multivariate analysis: Hosmer and Lemeshow goodness-of-fit - Χ² = 4.11, p = 0.767.

* indicates a significant difference (i.e., p value ≤ 0.05).

Table 3 - Variables associated with mortality

DISCUSSION

In the present study, the plasma serotonin levels and acetylcholinesterase activities upon ICU admission were not associated with the occurrence of acute brain dysfunction or mortality. These findings oppose the hypothesis that neurotransmitter imbalance is a key factor in the pathogenesis of acute brain dysfunction.

The cholinergic system of the brain modulates attention, learning, memory, movement control, and other peripheral functions⁽²⁶⁾ and plays extensive roles in attention and consciousness. Acetylcholine focuses awareness by acting as a modulator of the signal-to-noise ratios of sensory and cognitive inputs. Irregularities in these brain functions can cause symptoms of both hypoactive and hyperactive delirium, including inattention, disorganized thinking, and perceptual disturbances.⁽¹¹⁾ Studies have demonstrated that higher serum AChE activity is associated with delirium in the post-operative period in elderly patients.^(12,27,28) In contrast, patients who develop postoperative delirium have also been described to exhibit lower AChE activity.^(17,18) Therefore, there is no consensus regarding whether acetylcholine is involved in the development of delirium at all or whether its serum biomarkers can serve as surrogate markers of brain dysfunction and disease severity.^(5,17) Despite the finding that the AChE activities upon admission did not predict acute brain dysfunction in the patients who were generally critically ill, we demonstrated that among the patients who developed delirium, the duration of the acute brain dysfunction was correlated with AChE activity. Thus, acetylcholine might play a role in the maintenance of brain dysfunction by interfering with the basic mechanisms of attention and memory. Additionally, cholinergic signaling protects striatal, hippocampal, and cortical neurons against the neurotoxicity induced by excitotoxic amino acids and other toxic insults. Despite these theoretical mechanisms, clinical trials have failed to demonstrate beneficial effects of cholinergic agonists in the treatment of delirium.^(29,30)

The cholinergic pathway acts as a predictor of the individual variation in the systemic inflammatory response to infection; thus, by modulating systemic inflammation, the cholinergic system can indirectly affect brain function.⁽³¹⁾ Lower plasma AChE activity has been associated with higher levels of proinflammatory markers during acute illness,^(32,33) and the metabolism of acetylcholine can be altered by inflammation.^(10,32,33) Additionally, similar to the peripheral cholinergic anti-inflammatory pathway, acetylcholine and nicotine⁽³⁴⁾ modulate lipopolysaccharide -induced tumor necrosis factor release from microglia through the activation of acetylcholine receptors. Thus, it is possible that the decrease in cholinergic neurons during systemic inflammation decreases the availability of an “anti-inflammatory” signal in the brain. Although it is plausible that sepsis could interfere with the relationship between acetylcholine and delirium, we were unable to demonstrate any interaction between sepsis, AChE activity and the occurrence of delirium.

Serotonin is considered to have a role in the development of delirium due to its relationships with thought, perception, arousal level, learning and memory.^(13-15) However, the role of serotonin in ICU delirium remains unclear. In addition to the evidence of the down-regulation of serotonin synthesis, there are also suggestions that serotonin levels are elevated during delirium.^(14-16) Serotoninergic syndrome presents with symptoms that are similar to those of delirium,^(14-16) and serotonin levels can be altered by drugs that are used in the ICU.^(14,35,36) Additionally, increases in serotonin levels have been related to cholinergic deficiencies in experimental models.⁽³⁷⁾ In mechanically ventilated patients, both extremely low and extremely high levels of tryptophan have been associated with increases in the risk of delirium. Nevertheless, it remains unclear whether the symptoms of delirium are associated with the production of the neurotoxic metabolites of tryptophan, fluctuations in serotonin and melatonin levels, or both of these processes.⁽¹⁶⁾ In contrast to these previous findings, our results do not support a role for serotonin in the prediction of the development of delirium.

There are some limitations to our study. First, this study may have lacked the statistical power to detect some clinically important associations. Because of the relatively small sample size, only a limited number of covariates could be incorporated into the regression models, but with a sample size of 38 patients per group, it was possible to identify a difference of 35% between the groups. Thus, although the negative results reported here could have been due to secondary to beta errors, we believe that a difference of less than 35% does not have biological significance in the development of the disease. Second, we examined only the baseline acetylcholinesterase activities and serotonin levels of samples acquired upon ICU admission. Examinations of the temporal patterns of these measurements may yield additional information. Additionally, the use of delirium duration and delirium-free days as outcome measures may lead to bias in cases of prolonged periods of coma because assessments of delirium cannot be performed in comatose patients.⁽³⁸⁾ Finally, we measured acetylcholinesterase activities and serotonin levels in the serum and not the cerebrospinal fluid or brain tissue, which precludes a definitive confirmation of a brain-specific effect. However, it has been demonstrated that blood serotonin levels are a reliable indicator of brain serotonin levels.⁽³⁹⁾ Additionally, serum acetylcholinesterase activity has been found to be involved in cognitive recovery following ischemia in an animal model, and serum acetylcholinesterase activity has been used to evaluate cognitive recovery in vascular dementia, suggesting that serum acetylcholinesterase activity reflects acetylcholine levels in the brain.⁽⁴⁰⁾

CONCLUSIONS

In conclusion, neither serum acetylcholinesterase activity nor serum serotonin level was associated with the occurrence of delirium or acute brain dysfunction in critically ill patients, and sepsis did not modify the relationships of these biomarkers with the occurrence of delirium.