TITLE: Enzymatic Spectrophotometry Assay to Measure D-, L-, and Total Lactate in Canine Feces
AUTHORS: Amanda Blake, Jonathan Lidbury, Joerg Steiner, Jan Suchodolski

[DESCRIPTION]
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[STEPS]
?. [Aliquot feces]
Aliquot 120-130 mg feces into 1.5 mL microcentrifuge tube. Store at -80°C until deproteinization.
?. [Begin deproteinization. Add TEA buffer]
Make 0.1 M triethanolamine buffer (pH 9.15) using scale for accuracy (to the 0.01 mg). Then add 750 μL buffer to each aliquot. Vortex and place in refrigerator (-4°C) for three hours, vortexing every hour to ensure thorough mixing.
?. [Centrifuge #1]
Centrifuge samples at 13,000 x g for 5 minutes at 4°C. Carefully pipette 495 μL of supernatant into a new 1.5 mL microcentrifuge tube. Store supernates at -80°C overnight to provide ample time for next processing steps.
From empirical experience, the extra freeze-thaw cycles favorably increased protein pellet size while not affecting lactate concentration.
?. [Add TCA]
Thaw samples at room temperature for 20-40 minutes. Once thawed, add 10 μL of 6 M trichloroacetic acid to each sample. Vortex for 10 seconds and place immediately in ice bath for 20 minutes.
Performed this step and following steps (up to dilution with TEA) under a chemical fume hood with proper personal protective equipment.http://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=US&language=en&productNumber=T0699&brand=SIGMA&PageToGoToURL=http%3A%2F%2Fwww.sigmaaldrich.com%2Fcatalog%2Fproduct%2Fsigma%2Ft0699%3Flang%3Den
?. [Centrifuge #2]
Vortex samples again upon removing from ice bath, and centrifuge at 4,500 x g for 20 minutes at 4°C. After centrifugation, a protein pellet was noted in the bottom of each tube. Next, remove 400 μL of supernatant and place in a clean 2 mL microcentrifuge tube.
?. [Dilute samples]
Add 1600 μL 0.1 M triethanolamine buffer (pH 9.15) to each sample to achieve a neutral or alkaline pH (between 7 and 10). These deproteinized fecal extracts were either used immediately for lactate analysis or stored in -80°C for later use.
?. [Make standards]
Make standard dilutions with ultra-pure water and D- or L-lactate provided in enzymatic kit (D-/L-Lactate Enzymatic Kit, R-Biopharm Inc.)  ABC1Standard
Dilution
Concentration (g/L)
21
1:4
0.05175
32
1:10
0.0207
43
1:20
0.01035
54
1:40
0.005175
65
1:80
0.0025875
7Stock concentration = 0.207 g/L
ABC1Standard
Dilution
Concentration (g/L)
21
1:4
0.05175
32
1:10
0.0207
43
1:20
0.01035
54
1:40
0.005175
65
1:80
0.0025875
7Stock concentration = 0.207 g/L
?. [Make Master Mix]
Make master mix (MM) solution on an as needed basis depending on the number of samples. In a 15 or 50 mL tube, mix together (100 x n) µL solution 1, (20 x n) µL solution 2, and (2 x n) µL solution 3, with n equal to the total number of blanks, standards, and unknown samples.  AB1Solution Number
Description*
21
approx. 30 ml solution, consisting of: glycylglycine buffer, pH approx. 10.0; L-glutamic acid, approx. 440 mg
32
approx. 210 mg NAD, lyophilizate, reconstituted in 6 ml redist. Water
43
approx. 0.7 ml glutamate-pyruvate transaminase suspension, approx. 1100 U
54
approx. 0.7 ml D-lactate dehydrogenase solution, approx. 3800 U
65
approx. 0.7 ml L-lactate dehydrogenase solution, approx. 3800 U
7*Descriptions obtained from manufacturer protocol
AB1Solution Number
Description*
21
approx. 30 ml solution, consisting of: glycylglycine buffer, pH approx. 10.0; L-glutamic acid, approx. 440 mg
32
approx. 210 mg NAD, lyophilizate, reconstituted in 6 ml redist. Water
43
approx. 0.7 ml glutamate-pyruvate transaminase suspension, approx. 1100 U
54
approx. 0.7 ml D-lactate dehydrogenase solution, approx. 3800 U
65
approx. 0.7 ml L-lactate dehydrogenase solution, approx. 3800 U
7*Descriptions obtained from manufacturer protocol
?. [Plate your samples and incubate]
Pipette blanks, standards, and samples in duplicate onto 96-well plate and place on plate shaker for one minute. Shake at low enough speed that a plate cover is not needed. Then incubate the plate in plate incubator at 25°C for 15 minutes.  AB1Well type
Volumes
2blanks
122 µL MM, 100 µL water
3standards (D- and L-lactate)
122 µL MM, 100 µL standards 1-5
4unknown samples
122 µL MM, 100 µL fecal extract
5MM = master mix
AB1Well type
Volumes
2blanks
122 µL MM, 100 µL water
3standards (D- and L-lactate)
122 µL MM, 100 µL standards 1-5
4unknown samples
122 µL MM, 100 µL fecal extract
5MM = master mix
?. [A1 and D-LDH]
After incubation, take the first absorbance reading (A1) at 340 nm wavelength. Then immediately add 2 µL D-LDH to each well, shake plate for one minute, and then incubate at 25°C for 30 minutes.
?. [A2 and L-LDH]
Take second absorbance reading (A2) at 340 nm wavelength. Add 2 µL L-LDH to each well, shake for one minute, and incubate at 25°C for 30 minutes.
?. [Plate reader and settings]
Use path length adjustment settings in plate reader software to account for using a protocol originally designed for use with 1 cm diameter cuvettes. Set standards curve to quadratic and adjust lactate concentrations by subtracting the absorbance difference of the blank.
?. [A3]
Take third absorbance reading at 340 nm wavelength.
?. [Data processing]
Calculate standard curve and lactate concentrations using software above. Then, if D- or L-lactate concentrations are below their respective lower limits of quantification, adjust concentrations to their respective cutoff values. Ours were 0.002 g/L for D-lactate and 0.0007 g/L for L-lactate. Also adjust final lactate concentrations based on starting weight of feces, dilution factor, and dry matter content.