Methods

Conclusions

Endogenous morphine-6-glucuronide (M6G) is present in the plasma of critically ill patients: validation of a specific anti-M6G antibody for clinical and basic research

Weinsanto I1, Mouheiche J1, Laux-Biehlmann A1, Delalande F2, Poisbeau P1, Van Dorsselaer A2, Schneider F3, Garnero P4, Goumon Y1

1CNRS UPR3212, Strasbourg, France, 2CNRS UMR7178, Strasbourg, France, 3Hôpitaux Universitaires, Strasbourg, France, 4Cisbio Bioassays, Codolet, France

Bibliography1. Laux-Biehlmann, A et al. "Endogenous morphine and its metabolites in

mammals: history, synthesis, localization and perspectives." Neuroscience233 (2013): 95-117.

2. Lewis, Susannah S et al. "Evidence that intrathecal morphine-3-glucuronide may cause pain enhancement via toll-like receptor 4/MD-2 and interleukin-1β." Neuroscience 165.2 (2010): 569-583.

3. Glattard, Elise et al. "Endogenous morphine levels are increased in sepsis: a partial implication of neutrophils." PLoS One 5.1 (2010): e8791.

4. Laux Biehlmann, Alexis et al. "Comparison of serum and lithium heparinateplasma for the accurate measurements of endogenous and exogenousmorphine concentrations." British journal of clinical pharmacology 74.2 (2012): 381-383.

We have successfully raised a specific anti-M6G rabbit polyclonal antibody.

This antibody was validated for ELISA and cross-reactivity was investigated using 30 compounds structurally related to M6G,

demonstrating high sensivity and selectivity.

We have shown that endogenous M6G can be found at higher levels than morphine in the

blood of morphine-naive critically ill patients.

We provide evidence that our ELISA procedure represents a powerful tool to

quantify endogenous M6G levels in human pathologies, and propose its use as a

pathological marker for sepsis.

Background and aimsAntibody production Antibody validation

Antibody validation

M6G couplingto KLH Immunization Purification M6G-specific ELISA

implementation

Sensitivity (LOD, LLOQ) and selectivity (vs related alkaloids) determination

Comparison with a previously validated LC-MS/MS quantification method

Measurement of endogenous M6G plasma levels in critically ill patients

Results

Plasma Morphine [nmol/L] Plasma M6G [nmol/L]

Values range Median 95% CI Values range Median 95% CI M6G-positive in LC-MS/MS

Healthy donors(n=8)

— — — — — — —

Morphine-treated(n=5)

108.7 — 640.7 288.4 13.3 — 563.1 324.6 — 2062.6 950.2 -1458.2 — 3358 100% (8/8)

Critically ill (n=26) 0.7 — 149.4 34.7 18.2 — 50.8 0.3 — 775.8 150.8 63.2 — 238.7 73% (19/26)

High sensitivity :

Limit of detection (LOD) = 0,15 nmol/L

Lower limit of quantification (LLOQ) = 0,64 nmol/L

High selectivity :

No or extemely low cross-reactivity with most tested compounds

Cross-reactivity with C6G and M3,6G (negligible morphine metabolites)

In the plasma of critically ill patients, endogenous M6Gand morphine levels are significantly elevated

In humans, morphine is metabolizedinto morphine-6-glucuronide (M6G) and

morphine-3-glucuronide (M3G) by the UDP-glucuronosyltransferases 1A and 2B present in the liver and in the brain [1]. M6G displays a greater

analgesic effect than morphine, whereas M3G has no affinity for the mu opioid receptor (MOR).

While morphine and M6G bind MOR, thus inducing analgesia, M3G and morphine seem to interact with the inflammation-transducing receptor TLR4, which may explain their proalgesic and proinflammatory

effects [2]. In addition to plants, synthesis of endogenous morphine (EM), M6G, and M3G occurs

in various mammalian tissues and cells, including humans [1]. Taken together, these data suggest that the activation of MOR and/or TLR4 is dependent of the morphine/M3G/M6G ratios and is crucial for the

modulation of pathophysiological processes.

Changes in the levels of endogenous morphine have been demonstrated in several pathologies

(e.g. infection, inflammation, or neurologic diseases) and they might be relevant as

pathological markers [1, 3-4]. While EM levels are detectable using ELISA, mass spectrometry (MS) analysis was, so far, the only approach to detect and quantify M6G. Our aim was to develop and to validate a specific ELISA able to quantify M6G in the plasma of healthy donors, morphine-treated,

and critically ill patients.