Vit C - ANY body works with it ? (Nov/15/2005 )

thnx for the previous answers . i shall chk up the links .

i am wrking on a prjt which involves chking Vit C levels in serum . I am using MPA - meta phosphoric acid to ppt the prtns , and also to provide the acidic env for the development of the colour later on during the reaction [ when i add the dye 2,6- dichlorophenol indo phenol ] .
However since the last few times , the sample turns very turbid , before i can start taking the readings , and this is proving to be very difficult to get the correct results because its a follow up study . i don't think there is anything wrong with pipetting error since my blank and std show me constant readings as before .
Has anyone worked with another protocol for serum vit C levels . please let me know so that i can modify whatever i may be doing wrong .
thnx .

thnx for the previous answers . i shall chk up the links .

i am wrking on a prjt which involves chking Vit C levels in serum . I am using MPA - meta phosphoric acid to ppt the prtns , and also to provide the acidic env for the development of the colour later on during the reaction [ when i add the dye 2,6- dichlorophenol indo phenol ] .
However since the last few times , the sample turns very turbid , before i can start taking the readings , and this is proving to be very difficult to get the correct results because its a follow up study . i don't think there is anything wrong with pipetting error since my blank and std show me constant readings as before .
Has anyone worked with another protocol for serum vit C levels . please let me know so that i can modify whatever i may be doing wrong .
thnx .

hi milica , sorry for the delay and thnx for the reminder .

ok the protocol i use is estimation of Vitamin C from the PLASMA ..
i guess that was the biggest mistake in my first email .. not serum but plasma . i collect about 5 ml blood in heparinized vials [ or sod. citrate or EDTA bulbs .. but generally with EDTA we tend to get the turbidity that i was talking of . it struck me after i used heparin vial for the same patient on his next reading .]
anyhow so u take it in the vial with a good anti coagulant , and then centrifuge it for abt 5 mins .. i put it at 2500 or 3000 for 5 minutes so that there is enough plasma .

then we collect 2 ml of plasma , and add to it 3 ml of 3g% MPA [ metaphosphoric acid ] that is to deproteinize the solution [ it is very good , however now when you spin it .. or centrifuge it make sure you do it for a good 10 mintues .. what i do is spin it for 10 mins , then take 2 ml of this de proteinized solution again and spin it for a min to get rid of any protein which could interfere later on . now to this 2ml of solution i add 0.3 ml D/w , O.2 ml Sodium citrate solution and 1.0 ml of the dye which is 2,6-dichlorophenoindophenol .. i make a stock sloution of the dye 0.025M by dissolving 100mg of the powder in 100ml D/w the soltion is stable for several months in the refrigerator . for a wrking solutin i dilute it to a 1:10 concentration .. well so when you add this dye to ur solution it turns pink from the initial Blue .
The Blank is prepared with 0.5ml D/w , 1.2ml MPA , 0.8ML Sod . citrate and again 1 ml of wrking DYE . the reading of ur blank shud b close to 0.70 0r 0.65.
the std is prepared by taking 0.1ml of stck std Vit C 40mg/dl , 1 ml Dw and 3.9 ml MPA , mixing and then taking 2 ml of the wking std , add to it 0.5 ml sod. citrate and 1.0 ml dye .
the blank shud have the highest reading.

for a correction once u take readings add excecc of vit c powder and subtract the reading you now get .
finally . [corrected rdging for blank - correc rdging for sample / corect rdging for B - coreec Rdgin for Std ] * 2 .
I HOPE this helps .. it shud work actually . but just make sure the deproteinization and the entire expt is done within an hr , cuz vit C IS UNSTABLE .
best of luck , and let me know.

dazank
PS : incase this is not what you need ... i have a feeling you won't since u are dealing with tissues . , let me know . i can look up in the book we have in lab to see what protocol do they use for SERUM .



i am wrking on a prjt which involves chking Vit C levels in serum . I am using MPA - meta phosphoric acid to ppt the prtns , and also to provide the acidic env for the development of the colour later on during the reaction [ when i add the dye 2,6- dichlorophenol indo phenol ] .
However since the last few times , the sample turns very turbid , before i can start taking the readings , and this is proving to be very difficult to get the correct results because its a follow up study . i don't think there is anything wrong with pipetting error since my blank and std show me constant readings as before .
Has anyone worked with another protocol for serum vit C levels . please let me know so that i can modify whatever i may be doing wrong .
thnx .
[/quote]

Hi!
First of all,I will be very thankful if u send me a protocol of your method to determine vit.C concentration in serum, if that procedure ever worked.
My lab is working with determ.of vit.C in different tissues.To determine concentr. we use one very old(from 1942)but very precise procedure: TCA filtrate of blood is shaken with norit and filtered-this is very boring part of the procedure because it is time wasting!!!That's why I asked u to tell me procedure u use for vit.C oxidation.
Then norit filtrate is treated with 2.4-dinitrophenylhydrazine and thiourea(thiourea produces mildly reducing medium).Color is produced by adding 85% sulfuric acid.
I hope I helped u.
Milica
[/quote]

[quote name='dazank' date='Nov 25 2005, 04:33 AM' post='32395']
hi milica , sorry for the delay and thnx for the reminder .

ok the protocol i use is estimation of Vitamin C from the PLASMA ..
i guess that was the biggest mistake in my first email .. not serum but plasma . i collect about 5 ml blood in heparinized vials [ or sod. citrate or EDTA bulbs .. but generally with EDTA we tend to get the turbidity that i was talking of . it struck me after i used heparin vial for the same patient on his next reading .]
anyhow so u take it in the vial with a good anti coagulant , and then centrifuge it for abt 5 mins .. i put it at 2500 or 3000 for 5 minutes so that there is enough plasma .

then we collect 2 ml of plasma , and add to it 3 ml of 3g% MPA [ metaphosphoric acid ] that is to deproteinize the solution [ it is very good , however now when you spin it .. or centrifuge it make sure you do it for a good 10 mintues .. what i do is spin it for 10 mins , then take 2 ml of this de proteinized solution again and spin it for a min to get rid of any protein which could interfere later on . now to this 2ml of solution i add 0.3 ml D/w , O.2 ml Sodium citrate solution and 1.0 ml of the dye which is 2,6-dichlorophenoindophenol .. i make a stock sloution of the dye 0.025M by dissolving 100mg of the powder in 100ml D/w the soltion is stable for several months in the refrigerator . for a wrking solutin i dilute it to a 1:10 concentration .. well so when you add this dye to ur solution it turns pink from the initial Blue .
The Blank is prepared with 0.5ml D/w , 1.2ml MPA , 0.8ML Sod . citrate and again 1 ml of wrking DYE . the reading of ur blank shud b close to 0.70 0r 0.65.
the std is prepared by taking 0.1ml of stck std Vit C 40mg/dl , 1 ml Dw and 3.9 ml MPA , mixing and then taking 2 ml of the wking std , add to it 0.5 ml sod. citrate and 1.0 ml dye .
the blank shud have the highest reading.

for a correction once u take readings add excecc of vit c powder and subtract the reading you now get .
finally . [corrected rdging for blank - correc rdging for sample / corect rdging for B - coreec Rdgin for Std ] * 2 .
I HOPE this helps .. it shud work actually . but just make sure the deproteinization and the entire expt is done within an hr , cuz vit C IS UNSTABLE .
best of luck , and let me know.

dazank
PS : incase this is not what you need ... i have a feeling you won't since u are dealing with tissues . , let me know . i can look up in the book we have in lab to see what protocol do they use for SERUM .





Thanks for your answer!
Sorry for bother u again,but I don't know what does the oxidation of vit.C?
U need to convert ascorbic acid to dehydroascorbic acid, right?
Because english is not my mother language,I'm not sure what is D/w(distillated water,maybe?).

P.S. we'll try this method probably next Thurstday and I'll let u know.

Milica

[quote name='Milica' date='Nov 25 2005, 03:58 AM' post='32420']
[quote name='dazank' date='Nov 25 2005, 04:33 AM' post='32395']
hi milica , sorry for the delay and thnx for the reminder .

ok the protocol i use is estimation of Vitamin C from the PLASMA ..
i guess that was the biggest mistake in my first email .. not serum but plasma . i collect about 5 ml blood in heparinized vials [ or sod. citrate or EDTA bulbs .. but generally with EDTA we tend to get the turbidity that i was talking of . it struck me after i used heparin vial for the same patient on his next reading .]
anyhow so u take it in the vial with a good anti coagulant , and then centrifuge it for abt 5 mins .. i put it at 2500 or 3000 for 5 minutes so that there is enough plasma .

then we collect 2 ml of plasma , and add to it 3 ml of 3g% MPA [ metaphosphoric acid ] that is to deproteinize the solution [ it is very good , however now when you spin it .. or centrifuge it make sure you do it for a good 10 mintues .. what i do is spin it for 10 mins , then take 2 ml of this de proteinized solution again and spin it for a min to get rid of any protein which could interfere later on . now to this 2ml of solution i add 0.3 ml D/w , O.2 ml Sodium citrate solution and 1.0 ml of the dye which is 2,6-dichlorophenoindophenol .. i make a stock sloution of the dye 0.025M by dissolving 100mg of the powder in 100ml D/w the soltion is stable for several months in the refrigerator . for a wrking solutin i dilute it to a 1:10 concentration .. well so when you add this dye to ur solution it turns pink from the initial Blue .
The Blank is prepared with 0.5ml D/w , 1.2ml MPA , 0.8ML Sod . citrate and again 1 ml of wrking DYE . the reading of ur blank shud b close to 0.70 0r 0.65.
the std is prepared by taking 0.1ml of stck std Vit C 40mg/dl , 1 ml Dw and 3.9 ml MPA , mixing and then taking 2 ml of the wking std , add to it 0.5 ml sod. citrate and 1.0 ml dye .
the blank shud have the highest reading.

for a correction once u take readings add excecc of vit c powder and subtract the reading you now get .
finally . [corrected rdging for blank - correc rdging for sample / corect rdging for B - coreec Rdgin for Std ] * 2 .
I HOPE this helps .. it shud work actually . but just make sure the deproteinization and the entire expt is done within an hr , cuz vit C IS UNSTABLE .
best of luck , and let me know.

dazank
PS : incase this is not what you need ... i have a feeling you won't since u are dealing with tissues . , let me know . i can look up in the book we have in lab to see what protocol do they use for SERUM .





Thanks for your answer!
Sorry for bother u again,but I don't know what does the oxidation of vit.C?
U need to convert ascorbic acid to dehydroascorbic acid, right?
Because english is not my mother language,I'm not sure what is D/w(distillated water,maybe?).

P.S. we'll try this method probably next Thurstday and I'll let u know.

Milica
[/quote]

hey milica ,
not a prob . yeah D/w is distilled water and yes , ascorbic acid reduces the dye to a colourless leuco compound "EVENTUALLY " that means if u used a higher conc of Vit C in ur tube [ like do a std curve and use a conc of 10mg or more , the dye would turn colourless.]
yeah so basically u r converting the the ascorbic acid to dehydroascorbate . since Vit C is an anti oxidant it itself performs the function of reducing something else , in this case the dye .
your MPA is providing the correct , necessary pH for this , since it has to be an acidic environment to facilitate the transfer of the protons.