289
Clinica Chimica Acta, 0 Elsevier/North-Holland
76 (1977) 289-292 Biomedical Press
SHORT COMMUNICATION _.._~~_ CCA
848
1
FLUORIMETRIC ACID
G. CIJRZON Deparlmcnl (U.K.)
* and
DETERMINATION
B.D.
November
FATTY
KANTAMANENI
of Neurochemistry,
(Received
OF PLASMA UNESTERIFIED
2nd,
Institule
ol:Neurology,
Queen
Square,
London,
WC1
1976)
Summary A reliable and simple method is described for the determination of plasma unesterified fatty acid. The method involves extraction by Dole’s reagent, washing with dilute sulphuric acid and determination by the decrease of fluorescence of a dilute solution of 7-hydroxy-4-methylcoumarin. Results on rat and human plasma are in good agreement with those obtained by titration.
Introduction Plasma unesterified fatty acid (UFA) has been determined by methods involving extraction into organic solvents and either titration [l] or formation of metal soaps and subsequent metal determination [2-61. The titration methods are accurate but time consuming and usually require large volumes of plasma though a micro-modification has been described [7]. Metal soap methods require little plasma but suffer from many sources of error [4,8]. The original Dole titration method has been improved by removing interfering carboxylic acids from the organic extract by,washing with dilute sulphuric acid (91. Also the laborious titration step has been avoided by instead allowing the extracted UFA to alter the pH of a weak solution of a buffer and measuring this change calorimetrically using an indicator with a pK close to that of the buffer [lO,ll]. These methods, however, have disadvantages because of limited sensitivity or because extinction changes are linear over only a small concentration range and they have been used relatively infrequently in recent years. This paper describes a reliable and convenient method for plasma UFA deter-
* To
whom
correspondence
should
be addressed
mination involving extraction, nation using a dilute solution
washing with of a fluorescent
dilute sulphuric indicator.
acid and determi-
Method
Principle Unesterified fatty acids are extracted from plasma with Dole’s solution and heptane + water added to givcl two phases. Interfering substances are removed from the organic phase by washing with dilute sulphuric acid and lJFA detcrmined by measuring the decrease in the> fluorescence of an added solution of 7-hydroxy-‘I-methylcoumarin (P-methyl umhelliferonc) in ethanol/lz-hcptalle.
Reagents 1. Dole’s
solution:
isopropyl
alcohol/!z-heptane/0.5
M H,SO,
(40 : 10 : 1,
v/v). 2. Fluorescent indicator. Stock solution: 20 mg 7-hydroxy-4-mcthylcoumarin (R.N. Emanuel, London) in 10 ml ethanol. Working solution: 0.3 ml of stock solution + 8 ml 2.5% w/v sodium barbitone in water. 0.1 ml of this is added to 10 ml ethanol + 20 ml rz-heptane immediately before it is required. 3. Fatty acid standards: 0.5 mM and 1.0 mM palmitic acid in n-heptane.
Procedure 2.0 ml of Dole’s
solution is added to 0.4 ml heparinised plasma (or 0.2 ml plasma + 0.2 ml water if UFA > 1.0 mequiv./l is expected) in a 12-ml glass stoppered round bottomed tube, mixed by Vortex, and left for 10 min. 0.4 ml of each standard (in tz-heptane) + 2.0 ml Dole’s solution and blanks consisting of 0.4 ml water + 2.0 ml Dole’s solution are similarly treated. Then 1.2 ml n-heptane + 0.8 ml water is added to unknowns and blanks and 0.8 ml nheptane + 1.2 ml water added to standards. The mixtures are shaken by hand for 2 min, left for 10 min and briefly centrifuged. 1.0 ml of the organic upper phase is withdrawn taking care not to disturb the aqueous phase and shaken by hand for 5 min in a 12-ml glass stoppered centrifuge tube with 1.0 ml of 0.025% v/v H,SO,. After centrifugation 0.3 ml of the organic phase is carefully removed and added to 0.45 ml indicator solution, rapidly mixed and a steady stream of N, passed for about ten seccnds. Fluorescence is then read in microcells using a Farrand spectrophotofluorometer. The indicator is added and Nz passed through one sample at a time and fluorescence read immediately (activation = 365 nm, fluorescence = 455 nm; 7-54 and 3-73 filters). Results
Fluorescent indicator. 7-Hydroxy-4-methylcoumarin was chosen as fluorescence was intense and decreased linearly with increasing UFA concentration. r!t the concentration used linearity was obtained over a convenient range of UFA concentration. Linearity. Fluorescence decreased linearly as UE’,L\ concentration increased up to 1.0 mequiv./l (Fig. 1). Reproducibility. Multiple determinations on a single plasma gave 0.85 i 0.02 mequiv./l (IZ = 8, value i on
Clinica Chimica Acta, 0 Elsevier/North-Holland
76 (1977) 289-292 Biomedical Press
SHORT COMMUNICATION _.._~~_ CCA
848
1
FLUORIMETRIC ACID
G. CIJRZON Deparlmcnl (U.K.)
* and
DETERMINATION
B.D.
November
FATTY
KANTAMANENI
of Neurochemistry,
(Received
OF PLASMA UNESTERIFIED
2nd,
Institule
ol:Neurology,
Queen
Square,
London,
WC1
1976)
Summary A reliable and simple method is described for the determination of plasma unesterified fatty acid. The method involves extraction by Dole’s reagent, washing with dilute sulphuric acid and determination by the decrease of fluorescence of a dilute solution of 7-hydroxy-4-methylcoumarin. Results on rat and human plasma are in good agreement with those obtained by titration.
Introduction Plasma unesterified fatty acid (UFA) has been determined by methods involving extraction into organic solvents and either titration [l] or formation of metal soaps and subsequent metal determination [2-61. The titration methods are accurate but time consuming and usually require large volumes of plasma though a micro-modification has been described [7]. Metal soap methods require little plasma but suffer from many sources of error [4,8]. The original Dole titration method has been improved by removing interfering carboxylic acids from the organic extract by,washing with dilute sulphuric acid (91. Also the laborious titration step has been avoided by instead allowing the extracted UFA to alter the pH of a weak solution of a buffer and measuring this change calorimetrically using an indicator with a pK close to that of the buffer [lO,ll]. These methods, however, have disadvantages because of limited sensitivity or because extinction changes are linear over only a small concentration range and they have been used relatively infrequently in recent years. This paper describes a reliable and convenient method for plasma UFA deter-
* To
whom
correspondence
should
be addressed
mination involving extraction, nation using a dilute solution
washing with of a fluorescent
dilute sulphuric indicator.
acid and determi-
Method
Principle Unesterified fatty acids are extracted from plasma with Dole’s solution and heptane + water added to givcl two phases. Interfering substances are removed from the organic phase by washing with dilute sulphuric acid and lJFA detcrmined by measuring the decrease in the> fluorescence of an added solution of 7-hydroxy-‘I-methylcoumarin (P-methyl umhelliferonc) in ethanol/lz-hcptalle.
Reagents 1. Dole’s
solution:
isopropyl
alcohol/!z-heptane/0.5
M H,SO,
(40 : 10 : 1,
v/v). 2. Fluorescent indicator. Stock solution: 20 mg 7-hydroxy-4-mcthylcoumarin (R.N. Emanuel, London) in 10 ml ethanol. Working solution: 0.3 ml of stock solution + 8 ml 2.5% w/v sodium barbitone in water. 0.1 ml of this is added to 10 ml ethanol + 20 ml rz-heptane immediately before it is required. 3. Fatty acid standards: 0.5 mM and 1.0 mM palmitic acid in n-heptane.
Procedure 2.0 ml of Dole’s
solution is added to 0.4 ml heparinised plasma (or 0.2 ml plasma + 0.2 ml water if UFA > 1.0 mequiv./l is expected) in a 12-ml glass stoppered round bottomed tube, mixed by Vortex, and left for 10 min. 0.4 ml of each standard (in tz-heptane) + 2.0 ml Dole’s solution and blanks consisting of 0.4 ml water + 2.0 ml Dole’s solution are similarly treated. Then 1.2 ml n-heptane + 0.8 ml water is added to unknowns and blanks and 0.8 ml nheptane + 1.2 ml water added to standards. The mixtures are shaken by hand for 2 min, left for 10 min and briefly centrifuged. 1.0 ml of the organic upper phase is withdrawn taking care not to disturb the aqueous phase and shaken by hand for 5 min in a 12-ml glass stoppered centrifuge tube with 1.0 ml of 0.025% v/v H,SO,. After centrifugation 0.3 ml of the organic phase is carefully removed and added to 0.45 ml indicator solution, rapidly mixed and a steady stream of N, passed for about ten seccnds. Fluorescence is then read in microcells using a Farrand spectrophotofluorometer. The indicator is added and Nz passed through one sample at a time and fluorescence read immediately (activation = 365 nm, fluorescence = 455 nm; 7-54 and 3-73 filters). Results
Fluorescent indicator. 7-Hydroxy-4-methylcoumarin was chosen as fluorescence was intense and decreased linearly with increasing UFA concentration. r!t the concentration used linearity was obtained over a convenient range of UFA concentration. Linearity. Fluorescence decreased linearly as UE’,L\ concentration increased up to 1.0 mequiv./l (Fig. 1). Reproducibility. Multiple determinations on a single plasma gave 0.85 i 0.02 mequiv./l (IZ = 8, value i on
