Microdetermination of lactic Acid in Urine MAKEPE4CE U. TSAO, M. L. BAUMANN, ~ N SHIRLEY D WARK Department of Pediatrics and Communicable Diseases, Gnirersity of Michigan, Ann -4rbor. .\fich. This work was initiated to fill a need for a reliable method for the determination of lactic acid in urine samples of limited quantity, as in the metabolic study of infants. The method described requires a small sample, allows a number of samples to be run simultaneously, and gives a standard deviation of 5.5% for a single run, which is sufficiently precise for biological studies. Ten to 40 micrograms of lactic acid may be determined. Acetaldehyde, the oxidation product of lactic acid under the conditions employed, is isolated from substances interfering with the extremely sensitive color reaction. An apparatus devised to achieve this end has possible application to problems of similar nature.
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HE procedures for the determination of lactic acid in urine published prior to 1927 were inadequate, according to Jervell (6). Because those described since then (8, 11, 12) do not include a relatively simple and accurate method, this investigation is reported. The method described is Ixtaed on the extremely sensitive color reaction of acetaldehyde, an oxidation product of lactic acid, in sulfuric acid with phydrosydiphenyl (3). The procedure for blood lactic acid (1, 9) when directly applied t.o urine samples results in inhibition of color or atypical colors. TOavoid these difficulties, the acetaldehyde as it is produced from lactic acid should be separated before the tlevelopnient of color. The acetaldehyde is further osidized if it is allowed to remain in contact n.ith the oxidant ( 7 ) ; therefore, it is essential to accomplish the separation a t maximum siieed. \Vith these requirements in mind, an apparatus has been constructed which achieves in one step the Oxidation of lactic acid with ceric sulfate and the rapid transfer of the acetaldehyde thus obtained into sulfuric acid. This also makes possible development of the color in the same photometer tube. APPARATUS
sulfate is removed by soaking in approximately 50% sulfuric acid solution, after first wishing thoroughly in tap water. The rods are again thoroughly rinsed in tap water and allowed to dry in a dust-free cabinet. Such measures have been found necessary only for the first cleaning. After use in the procedure, the rods are merely rinsed in running tap water, soaked in 50% sulfuric acid for 1 hour or longer, and again rinsed very thoroughly in running tap water. The use of tap water alone appears to be justified, as no improvement was obtained with additional rinsing with glass-redistilled n.ater, while still-distilled water gave high blanks. Occasional rods were found to give consistently l o er~ values than the others and were subsequently eliminated from use. I t xas found practical to run the same standard solution repeatedly on all rods and select for permanent use those which gave maximum transfer with 2.5 hours’ diffusion. The uniformity of these rods is reflected in the results in Table I. Search is still in progress to find a satisfactory substitute that can be fabricated from commercial parts.
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Pipets. Kirk 0.2-ml. micropipets, caliI Chi. brated “to contain.” Constriction-type 0.35Figure 1. ml. pipet calibrated “to deliver.” Automatic Schema t i c syringe pipets: one 5-ml. syringe adjusted Cross Secto deliver 0.9 ml., and one 1-nil. syringe adtional View justed to deliver 0.1 ml. of Steel Diffusion Rod. Borosilicate glass from Mold for laboratory glassware is pulverized and sifted Fusion of through 30-mesh and 60-mesh sieves; the porDiffusion tion coarser than 30-mesh is further ground. Rod A mixture of 3 oarts (bv volume) of 30- to 60mesh glass po&der, ’1“part of 60-mesh and finer glass powder, and 3 parts of 60-mesh Alundum (Norton Co., Worcester, Mass.), is prepared. -4 steel mold (Figure 1)is lined with several layers of tissue paper, and a borosilicate glass rod 5 mm. in diameter and 50 mm. long is placed in it. The rod is held in the center, and the mixture of powder is poured into the space around the rod with occasional gentle tapping. This assembly is heated and rotated occasionally in an electric furnace a t approximately 650’ C., removed, and cooled, and the fused material is forced from the mold. A shallow borosilicate glass cup about 12 mm. in diameter and 8 mm. in inside diameter is fused to one end of the rod. The other end is then fused onto a 150-mm. glass rod, which will fit into a single-hole, No. 3 rubber stopper. This should be so adjusted that, when inserted into an Evelyn photometer tube (22 X 180 mm.), the cup is suspended about 60 mm. from the bottom of the tube. Before use these rods must be thoroughly cleaned. This has been most efficiently accomplished by soaking them in hot ceric sulfate solution (see Reagents) until all the charred material left on the rods appears to have disappeared. The excess ceric
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Figure 2. Schematic Drawing of Assembled Apparatus Diffusion rod in place in tube Infrared heater C. Sheet-metal cover with holes to accommodate tubes D. Sheet-metal shield E. Tin foil F. Sheet-metal tube supporter ring 6. Crockery jar A. B.
722
723
V O L U M E 2 4 , NO. 4, A P R I L 1 9 5 2 Table 1.
Reproducibility
Lactic . i d , 31-2.A 14.6 1 R .
