Determination of Lactose in Biological Materials &I. G. HOROWITZ, H. &I. D.AVIDSON, F. D. HOWARD, L-niversity of Oregon, Eugene, Ore. The determination of lactose in biological materials is often difficult. The specific problem at hand was that of determining small changes in lactose concentration in metabolizing niammary gland homogenates. S o method in the literature was sufficientl? sensitive, specific, and convenient. Three methods were tested. i diderential fermentation method employing >casts was found useful as a check, but was too cumbersome for routine w o r k .
S
TL'DIES in progress in this laboratory on the mechanism of
lactose synthesis in mammary gland homogenates have Iiosed problems in the determination of lactose. Others ( 2 , 7 ) have expressed the view that methods for determining this sugar :ire unsatisfactory when applied to complex mixtures. It is niandat,ory to have a t hand :I method which is specific, sensitivr, :idconvenient if possible -1review of the literature revealed a number of methods for determining lactose based on the following: reducing power before and after acid hydrolysis ( 8 , l l ), nianometric determination of galactose and/or glucose before and after hydrolysis ( 1 4 ) , diffeiw~tialfermentation (3, 10, l 6 ) , paper chromatography ( 5 ) , and color reaction with alkali and methylamine ( 6 ) . In the authors' hands, as in others' ( 7 ) , the use of methods employing acid hydrolysis led to spurious results. Presumably polysaccharides of low molecular weight are present, which interfere. I n homogenates containing only glucose and lactose it was found impractical to remove glucose by yeast adsorption ( 4 ) or fc.rmentation and t,o determine lactose by the change in reducing power. Lactose seemed to interfere with the adsorption of glucose by yeast, and there were always present small but inconstant amounts of nonlactose reducing substances after fermentation. -1further complicat~ionwas introduced by the fact that the concentrations of lactose involved were so low as to dictate minimum dilution due to deproteinization procedures. Under such conditions all nonsugar reducing substances were difficult to remove from mammary gland preparations. Paper chromatography was found to be very useful for qualitative explorations, but the quantitative methods were deemed too tedious and exacting i n c~\-pc~rinients ahere many determinations n-ere to be made. Thus it seemed desir:~bleto test : i i d :id:ipt other methods to the neetl~.
F. J . REITHEL
AND
.A manometric method involving the use of lactase was satisfactory if certain interfering substances were removed. The most rapid and convenient method is colorimetric. The lack of satisfactory methods for lactose analysis has impeded progress and cast doubt upon many investigations in the field of lactose metabolism. The present paper offers details and modifications of t w o methods previously used and one new method.
A manifold was used, so that
sly or more tubes could be handled a t one time. The tubes !%erenow heated a t 55" in a serological nater bath for 30 * 0.5 minutes and cooled in tap water. Color development n-as estimated with a Klett-Summerson photoelectric colorimeter, using the 54 filter. Readings taken 15 minutes after withdrawal from the water bath were nearly identical with those made a t 2 minutes.
typical standard curve 1s illustrated by the values in Table I. Such values were found to be highly reproducible. Recovery of lactose from homogenates was excellent if the folIon ing interfering suhstances, when present, were removed: monosaccharides, maltose, crllohiose, sugar phosphates, and calcium and barium ions.
Table I.
Determination of Lactose
Lactose, Mg. 0.25 0 50 1.00 2.00 3.00
Reading Klett 10 32
87 191 288
MANOMETRIC METHOD
Principle. In developing this method an attempt was made to circumvent the difficulties inherent in accurately determining true sugar reducing power and to avoid dilution attendant upon deproteinization. Winzler ( I ; ) has shown that it is possible to determine glucose manometrically by yeast fermentation i n the presence of sodium azide. This procedure was used to determine the glucose produced when lactose was hydrolyzed in the presence of lactase. I n order to achieve complete hydrolysis it was found imperative to use a lactase-lactose ratio of about 0.5, maintain a pH of 6.8 during incubation, and remove oxygen from the Polutions and incubate in the absence of oxygen.
COLORIMETRIC 31ETHOD
Principle. Compounds Containing the 1,4 glucosidic linkage ieact with alkali and methylamine to yield a pink color ( I ) . Recently it was shown (6) that this can be made the basis of a quantitative method. The follom-ing modification was found to he much more convenient and somen-hxt more sensitire.
Apparatus and Reagents. A Warburg respirometer was used to measure the carbon dioxide evolved during fermentation. Sodium succinate buffer, 0.15 M, pH 4.5. Sodium azide, 0.06 M. A suspension of baker's yeast washed five times with distilled water, 75 mg per ml. Lactase A concentrate (Rohm & Haas). Procedure. A 15 0-ml. sample of a l0Yc mammary gland homogenate was centrifuged and the supernatant was aerated with nitrogen for several minutes. Three 4.0-ml. aliquots were taken; one served as a blank, to the second were added 20 mg. of enzyme, and to the third were added 20 mg. of enzyme and 5.0 mg. of lactose. Bfter dilution to 8.0 ml. each solution was adjusted to pH 6.8. -4fter incubation for 1 hour at 37" 0.5-ml. aliquots were taken for analysis.
Reagents. Methylamine hydrochloride, 1.6%. Sodium hydroxide, 5.4 S. Procedure. T o each of a series of Klett tubes was added 0*.3ml. of reagent made by adding 2 volumes of 1.6% methylamine hydrochloride to 1 volume of 5.4 N sodium hydroxide The samples containing lactose were added and the volumes were adjusted to 5.0 ml. with distilled water. Each tube was fitted with a cork through which passed a section of 7-mm. outside diameter capillary tubing. Through the test solutions was bubbled a stream of nitrogen purified by passage over hot copper. At the end of 10 to 15 minutes the corks were firmly seated in the tubes, and the capillaries were withdravm above the 5-rnl. mark and closed off by means of a small clamp.
37s
ANALYTICAL CHEMISTRY
376 Table 11. Determination of Lactose in Mammary Gland Homogenates Homogenate Analyzed, hll. 0.50 0.50 0.50 0.50 0.50 0.50
Lactose Added, Mg.
Lactose Mg./G.’ Tissue
...
17.3 11.24
0.50 0.50 0.50 0.50
... ... ...
Lactose Recovered, Mg. .
.
I
0.55 0.45 0.50 0.50
Each series of determinations included: yeast blank, glucose standard, lactase blank, homogenate plus lactase (basal lactose value), and homogenate and lactase and added lactose (recovery). Into the body of a Warburg flask were introduced 1.0 ml. of sodium succinate buffer, 1.0 ml. of sodium azide, and the sample to be analyzed. -4total volume of 3.0 ml. was used. Into the side arm was pipetted 0.50 nil. of washed yeast suspension. After temperature and nitrogen equilibration the organisms were tipped into the body of the flask and readings were taken until carbon dioxide evolution ceased, usually about 25 minutes. Characteristic results are shown in Table 11. Several sugars were tested for interference. Euperiments similar to those above were set up with test solutions containing sugars at about the same concentration and in addition to lactose. Lactose recovery in the presence of sucrose and maltose was near loo%, but low recoveries were found in the presence of melibiose, glycogen, xylose, and cellobiose. This phenomenon, to be described more fully in another communication, is due to the effect of these sugars on yeast in the presence of azide and is not due to inhibition of lactase action. DIFFERENTIAL FERMEYTATION METHOD
Principle. This method is based upon the observation that ,S. bayanus (NRRL 966) fermented only glucose, S. carlsbergensis (NRRL 379) fermented glucose and galactose in a mixture of these two sugars n-ith lactose, and S. fragilis fermented all three. I
