ILP z3185865

From CellBiology

Investigation of Delirium and Dementia: Focus on markers of oxidative stress

Final Kit Selection (updated with contact details)

Ready to order: iPF 2α -VI EIA Kits Cat # 516351-96

Supplier: Cayman Chemicals, the Australian distributor is Sapphire Bioscience,

Cat # 516351-96

http://www.sapphirebioscience.com/

Sensitivity: 2.7pg/mL

Price (AUD): $333 (excludes GST)

Delivery and handling: $20

Availability: Within 1-2 weeks.

Sapphire Bioscience Pty Ltd. Suite 1, 134 Redfern Street Redfern NSW 2016 Australia

Ph: +61 2 9698 2022 Fax: +61 2 9698 1022

Ready to order: Cat # 589320 8-hydroxy-2-deoxy Guanosine EIA Kit

Supplier: Cayman Chemicals, the Australian distributor is Sapphire Bioscience,

Cat # 589320 8-hydroxy-2-deoxy Guanosine EIA Kit

Sensitivity: 33 pg/mL

Price (AUD): $481 (excludes GST)

Delivery and handling: $20

Availability: Within 1-2 weeks.

Sapphire Bioscience Pty Ltd. Suite 1, 134 Redfern Street Redfern NSW 2016 Australia

Ph: +61 2 9698 2022 Fax: +61 2 9698 1022

Practise ELISA

Suggested samples to start with from refrozen box: • Patient 3, 6, 9, 10, 13, 15, 16, 18, 22, 25, 26. 27. 28, 33, 40, 31 • NOT 2 or 8 (they only have 1 other tube outside the refrozen box)

Practise ELISA - Cytochrome C ELISA Kit Cat. No. APT200

Mitochondrial Cytochrome C is a water-soluble protein that functions in the respiratory chain by interacting with redox partners of complex III and IV. It is also released from mitochondria to the cytosol in apoptosis.

Aim

To detect levels of Cytochrome C in the cerebrospinal fluid (CSF)and blood of patients with delirium compared with control patients with dementia.

Hypothesis

CSF and blood Cytochrome C levels of patients with delirium are higher than that of control group. Cytochrome C is released from mitochondria into the cytosol in response to apoptosis inducing agents.

Materials

- Cytochrome C ELISA Kit Cat No APT200 LOT VR 1351309

Containing: • Anti-cytochrome C antibody coated plate • Biotinylated Anti-Cytochrome C Monoclonal Antibody • Streptavidin-Enzyme Conjugate • Cytochrome C Standard • Assay Diluent • Wash Buffer • Substrate Solution • Stop Solution

- ELISA Plate Reader: Multiskan FC

Methods

CSF samples were collected by lumbar puncture under X-ray guidance using local anaesthetic but no sedation. Samples were stored at -80°C until required. Blood samples collected were sent to the laboratory on ice, centrifuged at 1000 r.p.m for 5 minutes and stored in 50 µL and 100 µL aliquots at -80°C. Cytochrome C levels (ng/mL), were measured by ELISA sandwich technique using a commercial kit. Blanks and standard dilutions (ten dilutions from 40ng/mL to 0.084ng/mL) were prepared in duplicate. From the patient samples 50µL of blood and CSF were pipetted in duplicate. At the end of the final incubation step, the plate was read by the Multiskan FC at an absorbance of 450nm. The absorbance readings from the standard dilutions were used to generate the standard curve. The absorbances from the sample duplicates were averaged and used to determine the concentration of Cytochrome C in each patient sample using the standard curve.

Results

Refer to standard curve.

Discussion

From the standard curve detectable results were obtained for three of the duplicates. This may indicate that levels of Cytochrome C in the samples were below the level that which can be detected by this kit (minimal detectable dose 0.31 ng/mL) or may be a consequence of human error.

Areas for improvement in conducting ELISAs in the future:

• Program ELISA plate reader earlier - colour fades with time.

• Practise pipetting- there was a discrepancy between several duplicates.

• More accuracy when pipetting blanks.



--Mark Hill 09:35, 16 May 2009 (EST) Basic project information added to your discussion page.

Oxidative Stress: Targets for ELISA

Free radicals, reactive oxygen species (ROS) and reactive nitrogen species (RNS), are molecules or molecular fragments containing one or more unpaired electrons and have high reactivity. Both species of free radicals are products of normal cellular metabolism. Endogenous processes that can generate ROS and RNS include mitochondrial respiration, cytochrome P-450 detoxification reactions, phagocytic oxidative bursts, and peroxisomal leakage (Chakravarti and Chakravarti, 2007). Mitochondrial oxidative phosphorylation generates the majority of free radicals in the cell and products of OS and NS accumulate with normal ageing (Mangialasche et al, 2009).


Oxidative stress (OS) and nitrosative stress (NS) are a result of dysregulation of the redox balance. This may be caused by deficiency of antioxidants and/or excess free radical production (Mangialasche et al, 2009). Acute exposure to relatively high levels of oxidants, especially in the presence of calcium, can uncouple oxidative phosphorylation and contribute to cytotoxicity via necrosis and/or apoptosis (through release of cytochrome c, Smac/DIABLO endonuclease G, Omi) (Beal, 2005). Free radical attack against lipids, proteins, sugars and nucleic acids leads to the formation of products that can be detected in tissues and fluids, including CSF (Mangialasche et al, 2009).

Isoprostanes

F2-isoprostane is a marker of lipid peroxidation derived from arachidonic acid. Lipid peroxidation is one of the major outcomes of free-radical mediated injury (Mangialasche et al, 2009). A number of studies have assessed lipid metabolism after traumatic or ischemic brain injury with a focus on biomarkers of lipid peroxidation. CSF F2-isoprostane had been found to increase early after total brain injury (TBI) in children and is correlated with neuron specific enolase, suggesting a role of oxidative stress in the evolution of secondary damage early after severe TBI (Varma et al, 2003). F2-isoprostane levels assessed by ELISA in ventricular CSF samples in adults with TBI, with a marked early peak in F2-isoprostane (day 1) (Bayir et al, 2004). F2-isoprostane was approximately 2-fold higher in males than females following TBI and CSF F2-isoprostane was also associated with hypoxemia (Bayir et al, 2004).

F2-isoprostane concentration has been found to be selectively increased in diseased regions of brain from patients who died from advanced Alzheimer’s Disease, where pathologic changes include amyloid beta amyloidogenesis, neurofibrillary tangle formation, and extensive neuron death (Montine et al, 2005). Increased cerebrospinal fluid (CSF) levels of F2-isoprostane also are present in patients with early AD (Montine et al, 2005).


Phospholipids in tissue (cell membrane) or plasma (lipoprotein)


--->


Rearrangement


--->


Reduction by cellular peroxidases


--->


Phospholipase A2.

8-hydroxy-2'-deoxyguanosine (8-OHdG)

In nuclear and mitochondrial DNA, 8-hydroxydeoxyguanosine (8-OHdG), an oxidized nucleoside of DNA, is the most frequently detected and studied DNA lesion (Wu et al, 2003). 8-OHdG is a commonly used biomarker for the measurement of endogenous oxidative DNA damage.

Fukudaa et al (2008) measured urinary and cerebrospinal fluid (CSF) levels of 8-OHdG in children with various forms of central nervous system (CNS) disorders (status epilepticus [SE], hypoxic–ischemic encephalopathy [HIE], CNS infections and chronic epilepsy) and compared with that of normal health children. It was found that urinary 8-OHdG levels in children with HIE and CNS infections were higher than in control subjects and that CSF 8-OHdG levels were higher in children with SE, HIE, and CNS infections than controls (Fukudaa et al, 2008).

Studies have demonstrated elevated 8-OHdG levels in the serum and cerebrospinal fluid (CSF) (Kikuchi et al., 2002), and urine (Sato et al., 2005) of patients with Parkinson's Disease (PD), indicating that 8-OHdG may be a useful biomarker in tracking PD progression

Kit Information

Lot number: 0411512 ID Number: TRB1496 Expiry Date: February 23, 2010. Experiment commenced 3/9/09. overnight incubation, plate read 4/9/09

Other markers

Lactate dehydrogenase

Lactate dehydrogenase (LDH) is an accepted marker for cellular damage and is released by all types of cells after loss of membrane integrity (Schmidt et al, 2003). Increases in cerebrospinal fluid (CSF) LDH levels have been noted in children with neurological disorders including hydrocephalus and raised intracranial pressure, with predominant isoenzymes being LDH-1, 2 and 3 (Nelson et al, 1975). Total LDH and isoenzyme LDH-1 has been found to be significantly higher in patients with Creutzfeldt-Jakob disease (CJD) compared with other dementias whereas the fractions of LDH-2 and LDH-3 were significantly lower in CJD patients (Schmidt et al, 2003).

ELISA Kit LDH
Description Sensitivity Supplier Cost
LDH A-like monoclonal antibody 100µg Assay Designs Stressgen NA
LDH B monoclonal antibody 100µg Assay Designs Stressgen NA
Human LDH isoenzyme 1 Sigma-Aldrich 203.29

Pyruvate dehydrogenase kinase - note commercial kits are not sensitive enough (only in microgram range)

Pyruvate dehydrogenase complex (PDHC) is a mitochondrial matrix enzyme complex that catalyses the oxidative decarboxylation of pyruvate to form the products acetyl CoA, NADH and carbon dioxide (Martin et al, 2005). This reaction is the bridge between anaerobic and aerobic cerebral energy metabolism. PDHC requires several cofactors and can be phosphorylated, and therefore inactivated, by pyruvate dehydrogenase kinase. PDHC enzyme activity is lost in selectively vulnerable neurons after cerebral ischemia and reperfusion (Pocernich & Butterfield, 2003). Inactivation of PDHC and acrolein may be responsible for the mitochondrial dysfunction and impaired cerebral energy metabolism associated with Alzheimer’s disease (AD) (Pocernich & Butterfield, 2003). Acrolein is a byproduct of lipid peroxidation that accumulates within the brain during AD. It binds to lipoic acid, a component of PDHC, thus decreasing activity of PDHC (Pocernich & Butterfield, 2003).


ELISA Kits:


http://www.caymanchem.com/app/template/Product.vm/catalog/516301/a/z

http://www.biocompare.com/ProductDetails/422176/Isoprostane-ELISA-Kit.html

http://www.biocompare.com/ProductListings/29413/iPF2-alpha-VI-Isoprostane-ELISA-EIA-Kits.html

http://www.biocompare.com/ProductListings/3194/PDK4-(pyruvate-dehydrogenase-kinase,-isozyme-4)-Antibodies.html?types=6-71548&sb=true

http://www.biocompare.com/ProductListings/3194/PDK1-(pyruvate-dehydrogenase-kinase,-isozyme-1)-Antibodies.html?types=6-71545&sb=true

Information for Isoprostane ELISA Kit

Kit: 8-Isoprostane. Lot Number: 0412445. ID Number:MEB20 Expiry: 02/12/09. Experiment Commenced: 19/08/09 (overnight incubation) Plate Read: 20/08/09.


Equilibrate pipette tip in ethanol. Add 100ul of standard to 900ul ultrapure water. Prepare standards according to diagram in kit. - Label 8 tubes 1-8. - Aliquot 900ul EIA buffer to tube 1, 600ul to tubes 2-8. - Serially dilute standard, remove 300ul from tube 1 to tube 2, mix thoroughly. Next remove 300ul from tube 2 and put into 3 and mix.... repeat until tube 8.

References

Bayir H, Marion DW, Puccio AM, Wisniewski SR, Janesko KL, Clark RS, Kochanek PM. Marked gender effect on lipid peroxidation after severe traumatic brain injury in adult patients. 2003 Aug;20(8):781-6.

Beal, M.F. (2005). Mitochondria take center stage in aging and neurodegeneration. Ann. Neurol. 58(4):495-505.

Chakravarti, B. & Chakravarti, D.N. (2007). Oxidative modification of proteins: age-related changes, Gerontology 3;128–139.

Fukudaa, M., Yamauchib, H., Yamamota, H., Aminsaqkab, M., Murakamia, H., Kamivamaa, Mivamotoa, Y., Koitabashia. (2008). The evaluation of oxidative DNA damage in children with brain damage using 8-hydroxydeoxyguanosine levels. Brain and Development. 30(2); 131-136.

Kikuchi, A., Takeda, A., Onodera, H., Kimpara, T., Hisanaga, K., Sato, N., Nunimura, A., Castellani, R.J., Perry, G. Smith, M.A., Itoyama, Y. (2002). Systemic Increase of Oxidative Nucleic Acid Damage in Parkinson’s Disease and Multiple System Atrophy. Neurobiology of Disease 9; 244–248.

Mangialasche, F., Polidori, M. C., Monastero, R., Ercolani, S., Camarda, C., Cecchetti, R., et al. Biomarkers of oxidative and nitrosative damage in alzheimer's disease and mild cognitive impairment. Ageing Research Reviews, In Press, Corrected Proof

Martin, E., Rosenthal, R. E., & Fiskum, G. (2005). Pyruvate dehydrogenase complex: Metabolic link to ischemic brain injury and target of oxidative stress. Journal of Neuroscience Research, 79(1-2), 240-247.

Montine TJ, Montine KS, McMahan W, Markesbery WR, Quinn JF, Morrow JD. (2005). F2-isoprostanes in Alzheimer and other neurodegenerative diseases. Antioxid Redox Signal. 7(1-2):269-75.

Nelson, P. V., Carey, W. F., & Pollard, A. C. (1975). Diagnostic significance and source of lactate dehydrogenase and its isoenzymes in cerebrospinal fluid of children with a variety of neurological disorders. Journal of Clinical Pathology, 28(10), 828-833.

Pocernich, C.B., Butterfield, D.A. Acrolein Inhibits NADH-Linked Mitochondrial Enzyme Activity: Implications for Alzheimer's Disease. (2003). Neurotoxicity Research. 5(7). 515-519.

Sato, S., Mizuno, Y, Hattori, N. (2005). Urinary 8-hydroxydeoxyguanosine levels as a biomarker for progression of Parkinson disease, Neurology 64 (2005), pp. 1081–1083.

Schmidt, H., Otto, M., Niedmann, P., Cepek, L., Schroter, A., Kretzschmar, H. A., et al. (2004). CSF lactate dehydrogenase activity in patients with Creutzfeldt-Jakob disease exceeds that in other dementias. Dementia and Geriatric Cognitive Disorders, 17(3), 204-206.

Varma S, Janesko KL, Wisniewski SR, Bayir H, Adelson PD, Thomas NJ, Kochanek PM. (2003). F2-isoprostane and neuron-specific enolase in cerebrospinal fluid after severe traumatic brain injury in infants and children. J Neurotrauma. (8): 781-6.