2350
Anal. Chem. 1964, 56,2350-2351
Ultramicro Assay of Lactate by Fluorescence Microscopy Diane Hannon’ and Paul M. Quinton*’p2 Division of Biomedical Sciences, University of California, Riverside, California 92521, a n d Department of Physiology, University of California, School of Medicine, Los Angeles, California 90024
A method for the assay of lactate In sample volumes of less than 1 nL of blologlcal flulds has been devised using a microscope fluorometer. The procedure ls based upon the converslon of NAD to NADH by the enzymatic oxldatlon of lactate to pyruvate wlth lactate dehydrogenase (LDH). The lntenslty of the fluorescence of the NADH produced Is a llnear function of lactate concentratlon up to at least 100 mM.
The study of biological systems at diminishing levels of organization requires quantitative assays suitable for inconveniently small volumes of samples. In the assay of biological fluids, ultramicro methods of quantitating inorganic components have been achieved through the use of X-ray analysis (1-3). However, these techniques cannot be readily applied to organic compounds because characteristic X-rays of component atoms are of low energy and, more importantly, cannot be separated from other organic constituents that might be present. Since lactate is one of the most predominant organic ions in blood, extracellular fluid, and several secretory producta, an ultramicro method for its determination may be useful in any number of applications. For example, in our laboratory, the present assay was developed in order to determine lactate in a few nanoliters of sweat secretions from single glands. To our knowledge the minimal volume required for lactate determination by previously described techniques is about 1 p L (4). It is possible to reduce this required volume by 3 orders of magnitude by measuring the fluorescence properties of one of its products in the enzymatic oxidation of lactic acid with a microscope fluorometer. In this study we have shown that lactate concentrations can be accurately determined in nanoliter volumes of blood, saliva, and sweat.
EXPERIMENTAL SECTION Specimens of biological fluids were collected from normal, healthy adult volunteers. Blood was obtained by venapuncture in the absence of a tourniquet. Pure parotid saliva was obtained by placing a small plastic suction cup over the opening to Stenson’s duct. Sweat was collected either as (1)gross sweat collected in a plastic bag fitted over the torso during thermal stimulation or (2) micro sweat samples collected from single sweat glands after pharmacological stimulation with cholinomimetic agonists (5). Bulk samples and microsamples of standards and unknowns were assayed for lactate concentration using the same reagents. The reaction media was prepared by adding 100 pL of 1000 units/mL of stock beef heart LDH (Sigma No. 826-6) and 60 mg of disodium @-nicotinamideadenine dinucleotide (NAD; Sigma, grade 111) to 2.0 mL of 0.6 M glycine buffer containing 0.5 M hydrazine sulfate adjusted to pH 9.0. The final volume was brought to 6.1 mL by addition of distilled water. Standards were prepared by serial dilution of L-(+)-lactic acid (Sigma) dissolved in 0.6 M glycine buffer at pH 9.0. The assay reaction was initiated by adding one volume of sample to approximately 14 volumes of the above reagent solution. For the microassay, this mixture was formed by pipetting several microliters of reagent solution onto a plastic pedestal under Division of Biomedical Sciences, University of California. Department of Physiology, University of California.
mineral oil which had been equilibrated previously with the 0.6 M glycine buffer. When a specially constructed reagent micropipet under a dissecting microscope was used, one pipet volume of the reagent solution (ca. 25 nL) was pipetted onto the plastic surface under oil. The sample was added to the reagent solution with a second sample pipet made to deliver a volume approximately l/Idth of the above reagent pipet. The complete reaction mixture (reagent plus sample) was then taken up by slight suction into a constant bore glass capillary (68 pm id.; Glass Co. of America, Bargintown, NJ), expelled and taken up again several times to ensure complete mixing, and trapped between oil blocks in each end of the capillary. The two special pipets were constructed on a de Fonbrune microforge by using methods similar to those described previously (6,7).Constant volumes of all samples were ensured by using the same sample pipet to dispense all samples of standards and unknowns and the same pipette to dispense all aliquots of reagent solution. Instrumentation. Assays on bulk samples were carried out on a Beckman Model 25 spectrophotometerwith a 1-cm light path. The optical density of samples to ultraviolet light at 340 nm was recorded simultaneously against a blank containing the complete reagent solution plus a volume of perchloric acid (8% w/v) equal to the sample volume. Fluorometry of microsamples were carried out with a Zeiss Universal “R” microscope equipped with a mercury burner for epiillumination and a Zonax controller for shutter control. Constant bore capillaries containing a sample between oil blocks were immersed in water, and the sample was examined with a 40x water/oil immersion objective. The image and focal plane was standardized for each sample by centering the capillary containing the sample in the field of view with a micrometer and adjusting the focus to give the sharpest delineation of the inner walls of the capillary. Samples were excited to fluorescence for 1.5 s with UV light at 365 nm. Fluorescence was taken as the relative intensity of light emitted at a wavelengths above 395 nm. The fluorometer scale was always adjusted so that emission from the most concentrated standard gave an intensity reading of approximately 100 units. The unit of measurement is arbitrary and relative to this setting for each set of determinations. Intensities are given as “relative intensity units”. The slope of relation between concentration and these relative intensity units was calculated by linear regression analysis and used to determine the concentrations of unknowns.
RESULTS AND DISCUSSION During the past 20 years the most widely used and accepted methods for the assay of lactate have been based on the oxidation of lactate to pyruvate (8, 9). The reaction requires an enzyme, lactate dehydrogenase, and NAD. It is forced to completion by capturing the reaction product, pyruvate, with hydrazine in an alkaline medium. Under these conditions, the reduction of NAD by lactate leads to the production of a stochiometrically equivalent amount of NADH. The latter exhibits a native fluorescence when excited with ultraviolet light as well as a characteristic absorption at 340 nm. Since biological fluids are not innately fluorescent or absorptive at these wavelengths, monitoring the production of NADH photometrically provides a convenient method for determining lactate concentrations. The chemistry has the advantage of being straight forward, of requiring relatively few procedural steps, and of being highly specific since the reaction is enzyme dependent (10). While one of the first reports of enzymatic determination of lactate exploited the fluorescent properties
0003-2700/84/035~-2350$01.50/0 0 1984 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 56, NO. 13, NOVEMBER 1984
2351
Table I initial measured concn,
1
lo
t 10 20 30 40 50 60 MINUTES
!
I
I
’
I
2.0 3.0 4.0 5 0 6 0 7.0 HOURS
1 1
Figure 1. Stability of the reaction product is demonstrated by the plot of fluorescence (in relative intensity units) as a function of time for a standard 2-nL sample at 50 mM (upper trace) and at 25 mM lactate (lower trace). Each point represents a single measurement.
’
-I -0: E
2.5
F
2.0
-
1.5
-
1.0
.
z
s w I-
0
a _I
c
W
5LL 2 0.5 . J LL
05 io 15 20 UV SPECTROPHOTOMETER LACTATE CONC
(mM)
Figure 2. Concentration of lactate in sweat (0),blood (A),and saliva microfluorometry (ordinate) and UV spectrophotometry (abscissa). Values for sweat are reduced for plotting by a factor of XO.l. The solid line is the line of identity. The volume of samples used for microfluorometry was ca. 2 nL.
(e)as determined by
of NADH,most methods have relied upon the UV absorption characteristics of this reaction product. Even though the molar extinction coefficient of NADH is high at this wavelength, the path length in ultramicrosamplesis so short that this approach is of limited usefulness with microscope photometric instrumentation. In contrast, the fluorescence is sufficiently strong to be detectable in picoliter volumes at micromolar concentrations (11). The reaction time is short, and the fluorescent product is stable. Figure 1shows no change in fluorescence from a few minutes to over 4 h after initiation of the reaction. Stability for longer periods was not examined. The emitted fluorescent signal from microsamples was a linear function (r = 0.999) of lactate concentration in standards up to at least 100 mM. Relative errors of measurements were generally less than 2%. Since 100 mM is at least 2-3 times
vol, pL”
mM
predicted final concn, mM
final meas concn,
%
mM
recovery
25 50
12.0 12.0
16.7 21.4
16.2 20.0
97.0 93.5
25 50
10.4 10.4
15.1 19.9
14.2 18.8
94.0 94.5
25 50
14.2 14.2
18.8 23.5
18.0 21.8
95.7 92.8
50
11.8
21.2
20.6
97.2
25
14.1
18.8
19.0
101.0 95.7 av
a The recovery of lactate from biological samples was tested by adding either 25 or 50 WLof 200 mM lactate solution (column 1) to 1 mL of samples whose lactate concentrations had been determined previously by UV absorbance (column 2). The final concentration of lactate predicted after these additions (column 3) was compared to the concentrations actually measured by microfluorescence in these same samples (column 4) to give the % recovered (column 5).
the concentration normally found in biological samples, we did not investigate linearity in higher concentrations. When the same biological samples were assayed as bulk samples by UV absorption spectrometry and by the microfluorescence technique, the results were identical. Figure 2 shows that the lactate concentrations determined on the same samples of blood, saliva, and sweat absorption and fluorescence photometry fall on the line of identity (r = 0.999; slope = 1.0). Application of the microfluorescence technique to microsamples of sweat from single sweat glands gave lactate concentrations that were consistent with concentrations determined on bulk samples of sweat. Recovery of lactate added to samples was approximately 96%. When the concentration of lactate was determined by UV absorption and subsequently increased by adding approximately 5 or 10 mM lactate, the final lactate concentration determined by fluorescence agreed well with predicted values (Table I). In conclusion, a quantitative determination of lactate at biological concentrations can be carried out on ultrasmall L) of different biological fluids. The analysis volumes requires a minimum of micromanipulationsand uses standard microscope fluorometric instrumentation. Registry No. LDH, 9001-60-9; lactic acid, 50-21-5.
LITERATURE CITED ( 1 ) Quinton, P. M. Am. J . Physlol. 1978. 243 (3), F255-F259. (2) Lechene, C. Am. J . Physlol. 1977, 232(5), F391-F396. (3) Roinel, N.; de Rouffinac, C. “Scanning Electron Microscopy”; Joharl, 0. Ed.; AMF O’Hare: Chicago, 1982; pp 1155-1171. (4) Schon, R. Anal. Blochem. 1985, 12, 413-420. (5) BiJman,J.; Qulnton, P. M. Am. J . Physlol. 1984, 247, C3-CQ. (6) Qulnton, P. M. J . Appl. Physlol. 1978, 40 (2), 260-262. (7) Lechene. C. ”Microprobe Analysis as Applied to Cells and Tissues”; Hall, T., Echiin, P., Kaufmann, R. Eds.; Academic Press: London,
1974; pp 351-368.
(8) Lundholm, L.; Mohme-Lundholm, E.; Vamos,
N. Acta Physlol. Scand. 1863, 58,243-24s. (9) Loomls, M. J . Lab. Clln. Med. 1981, 57(6), 966-969. (IO) Friediand, I. M.; Dietrich, L. S. Anal. Blochem. 1981, 2 , 390-392. (11) Mroz, E.;Lechene, C. Anal. Biochem. 1980, 102, 90-96.
RECEIVED for review April 2, 1984. Accepted July 5, 1984. This work was supported by grants from Getty Oil Co., Gillette Co., and NIH (No. AM 26547).
