Lactate is an intermediate product of carbohydrate metabolism. Of the two forms of Lactate, D- and L-, the L- lactate is predominant isomer found in biological systems. L-lactate is formed during the anaerobic glycolysis by conversion of pyruvate to L-lactate by lactate dehydrogenase. Lactate level is an indicator for tissue oxygen demand and utilization. Abnormally high lactate levels are associated with diseases such as diabetes and lactate acidosis. Cell Technology’s Fluoro Lactate assay is a lactate oxidase-based method for detecting L-lactate in biological samples such as serum, plasma, blood, urine, and tissue extract.
In the assay, lactate oxidase (LOX) catalyzes the oxidation of L-lactate to pyruvate, along with the concomitant reduction of hydrogen peroxide (H2O2). The detection utilizes a non-fluorescent detection reagent, which is oxidized in the presence of horse radish peroxidase (HRP) and LOX to produce its fluorescent analog.
Cell Technology’s Fluoro Lactate assay provides a reliable, sensitive fluorimetric method for the quantification of lactate in biological samples such as serum, plasma, urine, and tissue extracts.
Figure.1 Standard curve of Lactate. 50 µl serial dilutions of lactate (Starting dose 40 µM in tubes) were added to the wells of 96-well fluorescent plate. 10 µL LOX was added to each well and incubated plate at 37ºC for 10 min. 50 µL of detection reagent was added and plate was read at Ex/Em=530/590 nm.
Table 1. An example showing serum L-lactate level. 50 μl diluted serum was added to the wells of 96 –well fluorescent plate. 10 μl LOX was added to each well and incubated plate at 37ºC for 10 min. 50 μl of detection reagent was added and plate was read at Ex/Em=530/590 nm.
Table 2. Spike and recovery experiments were performed to estimate % recovery of lactate. Serum (1:100) was spiked with lactate with the concentrations mentioned in the table above. The samples were processed as described in the protocol.
Table 3. Spike and recovery experiments were performed to estimate % recovery of lactate. Serum (1:100), heat-inactivated @ 56ºC, 30 min) was spiked with lactate with the concentrations mentioned in the table above. The samples were processed as described in the protocol.
- Highly effective and stable fluorescent assay for L-lactate.
- Simple and fast assay-add the reagent directly to your experimental samples. Plate can be incubated and read in 15-30 min.
- Works for serum, plasma and tissue extract.
|Hasegawa H., Fukushima T., Lee J., Tsukamoto K., Moriya K., Ono Y. and Imai K. (2003) Determination of serum D -lactic and L -lactic acids in normal subjects and diabetic patients by column-switching HPLC with pre-column fluorescence derivatization. Anal Bioanal Chem 377:886-891.|
|Kondoh Y., Kawase, M. and Ohmori S. (1992) Concentration of D-Lactate and its metabolic intermediates in liver, blood, and muscle of diabetic and starved rats. Res Exp Med 192: 407-414.|
|Lin, C. Y., Chen S. H., Kou G. H., Kuo C. M. (1999) An Enzymatic Microassay for Lactate. Concentration in Blood and Hemolymph. Acta Zoologica Taiwanica 10: 91-101.|
|McLellan, A. C., Phillips, S. A., and Thornally, P. J. (1992) Flourimetric assay of D-lactate. Anal Biochem 206: 12-16.|
|Scheijen J.L., Hanssen N. M., van de Waarenburg M. P., Jonkers D. M., Stehouwer C. D., Schalkwijk C. G. (2012) L(+) and D(-) lactate are increased in plasma and urine samples of type 2 diabetes as measured by a simultaneous quantification of L(+) and D(-) lactate by reversed-phase liquid chromatography tandem mass spectrometry. Exp Diabetes Res. 2012(doi:10.1155/2012/234812).|
|White R., Yaeger D., and Stavrianeas S. Determination of Blood Lactate Concentration: Reliability and Validity of a Lactate Oxidase-Based Method (2009) Int. J. Exerc Sci 2: 83-93.|
|Part # 7022||Lactate Standard 4mM, 500µl||2-8C|
|Part # 6004||Horseradish Peroxidase, 18.9 Units||2-8C|
|Part # 3011||5X Reaction Buffer, 25 mL||2-8C|
|Part # 4026||Detection Reagent, 1 Vial||-20C|
|Part # 6025||Reaction Enzyme Mix, 1 Vial - 1.1mL||-20C|