Zinc and Inflammation in Sepsis

The purpose of the proposed project is to investigate measures of zinc status in relation to oxidative stress and inflammation in patients with sepsis. We hypothesise that zinc depletion can modulate inflammatory responses, leading to increased oxidative stress and mitochondrial dysfunction.

Sepsis is a severe infection is the leading cause of death in critically ill patients \[1\]. Zinc deficiency impairs overall immune function and resistance to infection \[2\]. In vitro exposure of monocytes to lipopolysaccharide (LPS) leads to decreased cellular zinc content \[3\] and zinc redistribution has been shown in human volunteers in response to LPS \[4\]. Zinc depletion occurs in hospitalized patients including those with infections, the elderly, alcoholics, trauma or burns \[5-8\], conditions which are common among critically ill patients with sepsis. In a mouse model of sepsis, zinc depletion prior to sepsis resulted in more inflammation and more severe organ injury and increased mortality \[9\]. In patients with sepsis, early feeding with zinc resulted in faster recovery of organ function compared with control \[10\]. Zinc status is likely to be compromised in the critically ill and that zinc depletion may affect inflammatory responses and recovery.

Although zinc is not an antioxidant itself, it binds to metallothionein \[11\] and zinc supplementation decreases oxidative stress \[12\]. Oxidative stress has been consistently reported in patients with sepsis \[13-15\]. We have recently shown that protection of mitochondrial function with antioxidants can reduce organ damage in rats \[16\]. Pentraxin-3 is an inflammatory marker which is regulated in part by antioxidants and plays a key role in innate immunity \[17\].

The consequences of zinc deficiency may relate, in part, to its effects on nuclear factor NFκB, a transcription factor crucial to the signalling networks involved in sepsis \[18\]. Higher NFκB activity is associated with increased mortality in patients with sepsis \[19,20\]. It is likely that compromised antioxidant defences and inflammation occurs as a consequence of zinc deficiency.

We propose to measure plasma zinc and metallothionein mRNA status in relation to inflammatory markers including key cytokines, pentraxin-3, markers of oxidative stress and antioxidant status in patients with sepsis.

Following ethical approval consent will be sought from either the patient, or assent from a near relative. Successive patients admitted to the Intensive Care Unit at Aberdeen Royal Infirmary who fulfil the following criteria for sepsis, given below, within a 24h time window, will be included.

* Clinical suspicion or evidence of acute infection
* SIRS defined by two or more of the following:

  1. Core temperature \<36 degrees C or \>38 degrees C
  2. Tachycardia; heart rate \> 90 beats/min.
  3. Tachypnoea; respiratory rate \> 20 breaths/min or mechanical ventilation
  4. White blood count \>12 x 109/l or \<4 x 109/l

In this pilot study we will recruit 20 patients with sepsis and 20 critically ill patients with no clinical suspicion of sepsis. Patients will be excluded if they are \<16 years, pregnant or lactating, HIV positive, receiving corticosteroids or other known immuno-modulatory drugs (including statins), have cancer or autoimmune disorders or if consent/assent is refused.

Blood samples will be obtained on Days 1,2, 5, 10 and 14 of the ICU stay. Peripheral whole blood will be sampled from an indwelling arterial line and plasma will be separated for analysis of plasma zinc using atomic absorption spectroscopy. Plasma interleukin-6 (IL-6) and pentraxin-3 (PTX3) will be measured using enzyme immunoassay and total antioxidant capacity and lipid hydroperoxides will be measured colourimetrically \[17\]. Peripheral blood mononuclear cells (MNC) will be separated using single density gradient centrifugation, RNA extracted and metallothionein mRNA measured using qPCR \[21\]. Nuclear extracts of MNC will be prepared and nuclear factor kappa B (NFκB)activation measured using an enzyme immunoassay \[17\].

Eligibility Criteria:
Patients with sepsis

Inclusion Criteria:

* admitted to ICU with clinical suspicion or evidence of acute infection PLUS two or more of the following:

  1. Core temperature \<36 degrees C or \>38 degrees C
  2. Tachycardia; heart rate \> 90 beats/min.
  3. Tachypnoea; respiratory rate \> 20 breaths/min or mechanical ventilation
  4. White blood count \>12 x 10 9/l or \<4 x 10 9/l

     Control patients
* admitted to ICU with no clinical evidence or suspicion of infection

Exclusion Criteria:

* under 16 years old
* pregnant or lactating
* HIV positive
* receiving corticosteroids or other known immuno-modulatory drugs
* treated with statins in the last month
* have cancer
* have autoimmune disorders
* consent/assent is refused