|
|
| Prof. Paul Holvoet information |
|
| E-mail address |
paul.holvoet@med.kuleuven.be |
| Home address |
Domeinstraat 15 |
| City |
3010 Leuven |
| Country |
Belgium |
| Day phone |
/ |
| Mobile phone |
3216347149 |
| Fax |
3216347114 |
| Lipid peroxidation Product analized |
OXLDL |
| Method name |
ELISA |
| Standardization Method |
MERCODIA standard |
| Detection Limit of this method (in your experience) |
10U/L |
Typical Measured Levels
(please specify for each type of sample and say whether samples are control
/ disease / stressed etc.): |
|
| 1. Sample type |
EDTA plasma control |
| Normal level measured |
50 U/L or lower |
| 2. Sample type |
EDTA plasma CHD patient |
| Normal level measured |
70 U/L or higher |
| 3. Sample type |
|
| Normal level measured |
|
| 4. Sample type |
|
| Normal level measured |
|
| 5. Sample type |
|
| Normal level measured |
|
| 6. Sample type |
|
| Normal level measured |
|
| 7. Sample type |
|
| Normal level measured |
|
| Sample Handling Procedures: |
|
Sample extraction / preparation
(e.g. temp, aqueous/organic extraction, internal standard, etc) |
Rapid
centrifugation to remove blood platelets |
Antioxidants / stabilizers included
(amounts/ concentrations) |
NO |
Sample storage
(temp, under N2, acceptable storage period) |
-80°C |
| Assay Protocol (please expand box as necessary) |
ELISA |
Procedure variations
(please specify any possible variations to the procedure, for example for
different types of sample) |
/ |
| Known Artefacts / Interfering Substances |
Several freezing and thawing samples |
| Additional Comments |
|
| References: |
|
| 1. |
Clin Chem. 2006 Apr;52(4):760-4. |
| 2. |
|
| 3. |
|
| Is this protocol adapted from a published protocol? |
YES |
| Keywords about Your work (please separate words with ","
; for example: "microbiology, stress, oxydative stress") |
"oxydative stress, inflammation, metabolic
syndrome, atherosclerosis" |
| Institute Address |
Atherosclerosis and Metabolism Unit
Katholieke Universiteit Leuven
Herestraat 49, PB 705
3000 Leuven
Belgium |
| Dr. Milan Stefek information |
|
| E-mail address |
exfastfk@savba.sk |
| Home address |
Dubravska cesta 9 |
| City |
Bratislava |
| Country |
Slovak Republic |
| Day phone |
+421-2-59410667 |
| Mobile phone |
+421-2-54775 928 |
| Fax |
/ |
| Lipid peroxidation Product analized |
LOOH determination |
| Method name |
Ferric thiocyanate assay |
| Standardization Method |
CumOOH (Cumene hydroperoxide) |
| Detection Limit of this method (in your experience) |
2 µM CumOOH |
Typical Measured Levels
(please specify for each type of sample and say whether samples are control
/ disease / stressed etc.): |
|
| 1. Sample type |
DOPC Liposomes |
| Normal level measured |
2-50 µM |
| 2. Sample type |
|
| Normal level measured |
|
| 3. Sample type |
|
| Normal level measured |
|
| 4. Sample type |
|
| Normal level measured |
|
| 5. Sample type |
|
| Normal level measured |
|
| 6. Sample type |
|
| Normal level measured |
|
| 7. Sample type |
|
| Normal level measured |
|
| Sample Handling Procedures: |
Aliquots (1 ml) of the incubation mixtures were extracted with 2 ml portions
of ice-cold mixture CHCl3:MeOH (2:1, v/v) containing BHT (0.05%). Lipid
hydroperoxide content was determined by the thiocyanate method according
to Mihaljevic et al. 1996 by sequentially adding CHCl3:MeOH (2:1, v/v) mixture
(1.4 ml) and the thiocyanate reagent (0.1 ml) to the extract (1 ml). The
reagent was prepared by mixing equivalent volumes of methanolic solution
of KSCN (3%) and ferrous-ammonium sulphate solution (45 mM in 0.2 mM HCl).
After the mixture had been left at ambient temperature for at least 5 min,
the absorbance at 500 nm was recorded. The lipid peroxide value was determined
using a calibration curve prepared with standard cumene hydroperoxide. |
Sample extraction / preparation
(e.g. temp, aqueous/organic extraction, internal standard, etc) |
ice-cold mixture CHCl3:MeOH (2:1, v/v) |
Antioxidants / stabilizers included
(amounts/ concentrations) |
BHT (0.05%) |
Sample storage
(temp, under N2, acceptable storage period) |
No storage recommended |
| Assay Protocol (please expand box as necessary) |
Aliquots (1 ml) of the incubation mixtures were extracted with 2 ml portions
of ice-cold mixture CHCl3:MeOH (2:1, v/v) containing BHT (0.05%). Lipid
hydroperoxide content was determined by the thiocyanate method according
to Mihaljevic et al. 1996 by sequentially adding CHCl3:MeOH (2:1, v/v) mixture
(1.4 ml) and the thiocyanate reagent (0.1 ml) to the extract (1 ml). The
reagent was prepared by mixing equivalent volumes of methanolic solution
of KSCN (3%) and ferrous-ammonium sulphate solution (45 mM in 0.2 mM HCl).
After the mixture had been left at ambient temperature for at least 5 min,
the absorbance at 500 nm was recorded. The lipid peroxide value was determined
using a calibration curve prepared with standard cumene hydroperoxide. |
Procedure variations
(please specify any possible variations to the procedure, for example for
different types of sample) |
/ |
| Known Artefacts / Interfering Substances |
Oxygen-deaerated solvents are needed / excessive iron |
| Additional Comments |
Molar absorptivity of the ferric thiocyanate complex: 58,440 M-1 cm-1 |
| References: |
|
| 1. |
Mihaljevic, B.; Katusin-Razem, B.; Razem, D. The reevaluation of the
ferric thiocyanate assay for lipid hydroperoxides with special considerations
of the mechanistic aspects of the response. Free Radic. Biol. Med. 1996,
21,53–63 |
| 2. |
2. Rackova L, Snirc V, Majekova M, Majek P, Stefek M. Free radical scavenging
and antioxidant activities of substituted hexahydropyridoindoles. Quantitative
structure-activity relationships. J Med Chem. 2006, 49:2543-8 |
| 3. |
|
| Is this protocol adapted from a published protocol? |
Yes |
| Keywords about Your work (please separate words with ","
; for example: "microbiology, stress, oxydative stress") |
"lipid hydroperoxide, ferric thiocyanate, liposomes" |
| Institute Address |
Institute of Experimental Pharmacology
SLOVAK ACADEMY OF SCIENCES
Dubravska cesta 9
SK-841 04 Bratislava
Slovak Republic |
| Prof. Manuel Portero-Otin information |
|
| E-mail address |
manuel.portero@cmb.udl.es |
| Home address |
c/Montserrat Roig,2 |
| City |
Lleida |
| Country |
Spain |
| Day phone |
973702408 |
| Mobile phone |
/ |
| Fax |
973702426 |
| Lipid peroxidation Product analized |
malondialdehyde-lysine |
| Method name |
GC/MS |
| Standardization Method |
Double standardization method is used: First, external standardization
with authentic standards.
Second, interna standardization with deuterium labelled malondialdehyde |
| Detection Limit of this method (in your experience) |
Minimum amount of
protein needed: 250 micrograms
Minimum product detected: 10 micromol/mol lysine. Around 1 pmol on
column
|
Typical Measured Levels
(please specify for each type of sample and say whether samples are control
/ disease / stressed etc.): |
|
| 1. Sample type |
human brain cortex |
| Normal level measured |
260±31 micromol/mol lysine |
| 2. Sample type |
rat kidney cortex |
| Normal level measured |
127 micromol/mol lys |
| 3. Sample type |
human spinal cord |
| Normal level measured |
290 micromol/mol lys |
| 4. Sample type |
human fibroblasts |
| Normal level measured |
50 micrmol/mol lys |
| 5. Sample type |
|
| Normal level measured |
|
| 6. Sample type |
|
| Normal level measured |
|
| 7. Sample type |
|
| Normal level measured |
|
| Sample Handling Procedures: |
|
Sample extraction / preparation
(e.g. temp, aqueous/organic extraction, internal standard, etc) |
Homogenized in a buffer containing 180mM KCl, 5 mM 3-[N-morpholino]propanesulfonic
acid, 2mM ethylenediaminetetraacetic acid (EDTA), 1mM diethylenetriaminepentaacetic
acid and 1µM butylated hydroxyl toluene, 10 ?g/ml aprotinin, 1mM
phenylmethylsulfonyl
fluoride, pH 7,3 (Potter-Eljeveim device,at 4ºC).
Samples containing 0.75-1 mg of protein are delipidated using chloroform:methanol
(2:1 v/v), and proteins are precipitated by adding
10% trichloroacetic acid (final concentration) and subsequent centrifugation.
Protein samples are reduced overnight with 500 mM NaBH
(final concentration) in 0.2M borate buffer, pH 9.2, containing 1 drop
of hexanol as an anti-foam reagent. Proteins are then reprecipitated by
adding 1ml of 20% trichloroacetic acid and subsequent centrifugation.
The following isotopically labelled internal standards are then added:[2H8]Lysine
(d8-Lys; CDN Isotopes) and [2H8]MDAL (d8-MDAL), prepared as described
|
Antioxidants / stabilizers included
(amounts/ concentrations) |
see above |
Sample storage
(temp, under N2, acceptable storage period) |
-80ºC 2-4 months
|
| Assay Protocol (please expand box as necessary) |
The samples are hydrolysed at 155oC for 30 min in 1ml of 6N HCl, and then
dried in vacuo. The N,O-trifluoroacetyl methyl ester derivatives
of the protein hydrolysate are prepared as previously described .
GC/MS analyses are carried out on a Hewlett-Packard model 6890 gas chromatograph
equipped with a 30m HP-5MS capillary column (30m x 0.25mm x 0.25 ?m) coupled
to a Hewlett-Packard model 5973A mass selective
detector (Agilent, Barcelona, Spain). The injection port is maintained at
275oC; the temperature program was 5 min at 110oC, then 2oC/min to 150oC,
then 5oC/min to 240oC, then 25oC/min to 300oC, and finally hold at 300oC
for 5 min. Quantification was performed by external standardisation using
standard curves constructed from mixtures of deuterated and non-deuterated
standards. Analytes are detected by selected ion-monitoring GC/MS. The ions
used are: lysine and d8-lysine, m/z 180 and 187, respectively; and MDAL
and d8-MDAL, m/z 474 and 482, respectively. The amounts of products were
expressed as the ratio ?mol
of MDAL/mol lysine. |
Procedure variations
(please specify any possible variations to the procedure, for example for
different types of sample) |
Adaptation is needed for fatty tissues, as well as
for low abundant proteins. In those cases, extensive delipidation is a strong
requisite. |
| Known Artefacts / Interfering Substances |
None so far |
| Additional Comments |
|
| References: |
|
| 1. |
Requena, J.R., Fu, M.X., Ahmed, M.U., Jenkins, A.J., Lyons, T.J., Baynes,
J.W. and Thorpe, S.R. (1997) Biochem. J. 322, 317-325 |
| 2. |
Requena, J.R., Chao, C.C., Levine, R.L. and Stadtman E.R.
(2001)
Proc. Natl. Acad. Sci. USA 98, 69-74 |
| 3. |
|
| Is this protocol adapted from a published protocol? |
YES |
| Keywords about Your work (please separate words with "," ; for
example: "microbiology, stress, oxydative stress") |
mitochondria, ageing, proteasome,
neurodegeneration |
| Institute Address |
IRBLLEIDA
c/Montserrat Roig,2
25008 Lleida
SPAIN |
| Prof. Darko Modun information |
|
| E-mail address |
dmodun@bsb.mefst.hr |
| Home address |
A.B.Simica 9 |
| City |
Split |
| Country |
Croatia |
| Day phone |
385 21 557 851 |
| Mobile phone |
385 98 892 373 |
| Fax |
385 21 465 073 |
| Lipid peroxidation Product analized |
LOOH (lipid hydroperoxides) |
| Method name |
FOX (2) |
| Standardization Method |
/ |
| Detection Limit of this method (in your experience) |
/
|
Typical Measured Levels
(please specify for each type of sample and say whether samples are control
/ disease / stressed etc.): |
|
| 1. Sample type |
Heparinized plasma |
| Normal level measured |
5 µM |
| 2. Sample type |
|
| Normal level measured |
|
| 3. Sample type |
|
| Normal level measured |
|
| 4. Sample type |
|
| Normal level measured |
|
| 5. Sample type |
|
| Normal level measured |
|
| 6. Sample type |
|
| Normal level measured |
|
| 7. Sample type |
|
| Normal level measured |
|
| Sample Handling Procedures: |
Fresh samples analysis |
Sample extraction / preparation
(e.g. temp, aqueous/organic extraction, internal standard, etc) |
/ |
Antioxidants / stabilizers included
(amounts/ concentrations) |
/ |
Sample storage
(temp, under N2, acceptable storage period) |
/ |
| Assay Protocol (please expand box as necessary) |
Plasma lipid hydroperoxides measurement by an automated xylenol orange
method. Anal Biochem. 2004 Feb 1;325(1):158-63. |
Procedure variations
(please specify any possible variations to the procedure, for example for
different types of sample) |
/ |
| Known Artefacts / Interfering Substances |
/ |
| Additional Comments |
/ |
| References: |
|
| 1. |
Obad A, Valic Z, Palada I, Brubakk Ao, Modun D, Dujic Z. Antioxidant pretreatment
and reduced arterial endothelial dysfunction after diving. Aviat Space Environ
Med 2007; in press. |
| 2. |
Modun D, Music I, Vukovic J, Brizic I, Katalinic V, Obad
A, Palada I, Dujic Z, Boban M. The increase in human plasma antioxidant
capacity after red wine consumption is due to both plasma urate and wine
polyphenols. Atherosclerosis. 2007;in press. |
| 3. |
Boban M, Modun D, Music I, Vukovic J, Brizic I, Salamunic I, Obad A, Palada
I, Dujic Z. Red wine induced modulation of vascular function: separating
the role of polyphenols, ethanol and urates. J Cardiovasc Pharmacol 2006;47:695-701. |
| 4. |
Modun D, Music I, Katalinic V, Dujic Z, Boban M. Glycerol and ethanol
in red wine are responsible for urate-related increase in plasma antioxidant
capacity. Clin Chem. 2006;52:785-7. |
| 5. |
Katalinic V, Modun D, Music I, Boban M. Gender differences in antioxidant
capacity of rat tissues determined by 2, 2'-azinobis (3- ethylbenzothiazoline
6-sulfonate; ABTS) and ferric reducing antioxidant power (FRAP) assays.
Comp Biochem Physiol C Toxicol Pharmacol. 2005;140:47-52. |
| 6. |
Katalinic V, Milos M, Modun D, Music I, Boban M. Antioxidant effectiveness
of selected wines in comparison with (+)-catehin. Food Chem. 2004;86:593-600. |
| Is this protocol adapted from a published protocol? |
Yes |
| Keywords about Your work (please separate words with "," ; for
example: "microbiology, stress, oxydative stress") |
oxidative stress, antioxidants, red wine, endothelium, human |
| Institute Address |
University of Split School of Medicine, Soltanska 2, 21 000 Split, Croatia |
|
webmail
RBI Home 
search
