a rapid and sensitive method for the determination of l-α-glycerophosphate in animal tissues

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ANALYTICAL BIOCHEMISTRY 3. 3y(i462 (1962) A Rapid and Sensitive Method for the Determination of L- a-Glycerophosphate in Animal Tissues1 EDWARD I. CIACCIO From the Merck Institute for Therapeutic Research, Rahway, New Jersey Received October 6, 1961 In the course of investigating the metabolism of n-a-glycerophosphate in normal and tumor tissues (l-3) a rapid, precise, and sensitive method for the determination of n-a-glycerophosphate has been developed and has been used in this laboratory for the last three years. This method utilizes the reaction: L-a-glycerophosphate + DPN -+ dihydroxyacetone phosphate + DPNH + H’ catalyzed by n-n-glycerophosphate dehydrogenase and involves the spec- trophotometric determination of DPNH. A method depending upon the measurement of the DPNH formed at equilibrium was suggested by Bublitz and Kennedy (4) and modified by Wieland and Suyter (5). Although recent modifications (6, 7) have improved the sensitivity of the original procedure, the long incubation periods needed, the possible in- stability of the enzyme under the conditions used (6, 8), and the extra handling involved, left room for improvement. The quantitative assay method described in this paper utilizes the rate of reaction rather than the estimation of the total DPNH formed at equilibrium. In addition to increasing the sensitivity from lo- to lOOO-fold over the previously published methods, the procedure is simplified to a rapid analytical method that requires no preincubation and thereby permits large num- bers of determinations to be handled conveniently. As most enzymic assays involve the determination of the total product formed or substrate utilized at equilibrium rather than the reaction rate (9, lo), the analysis of substrates of other dehydrogenases by rate measurements is also discussed. The application of this method to the analysis of the L-cY-glycerophos- phate content of a number of vertebrate tissues is described and the 1 This work was supported by the Cancer Chemotherapy National Service Center, National Cancer Institute, under the NationaI Institutes of Health Contract #SA- 43-pH-1886. 396

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Page 1: A rapid and sensitive method for the determination of l-α-glycerophosphate in animal tissues

ANALYTICAL BIOCHEMISTRY 3. 3y(i462 (1962)

A Rapid and Sensitive Method for the Determination of

L- a-Glycerophosphate in Animal Tissues1

EDWARD I. CIACCIO

From the Merck Institute for Therapeutic Research, Rahway, New Jersey

Received October 6, 1961

In the course of investigating the metabolism of n-a-glycerophosphate in normal and tumor tissues (l-3) a rapid, precise, and sensitive method for the determination of n-a-glycerophosphate has been developed and has been used in this laboratory for the last three years. This method utilizes the reaction:

L-a-glycerophosphate + DPN -+ dihydroxyacetone phosphate + DPNH + H’

catalyzed by n-n-glycerophosphate dehydrogenase and involves the spec- trophotometric determination of DPNH. A method depending upon the measurement of the DPNH formed at equilibrium was suggested by Bublitz and Kennedy (4) and modified by Wieland and Suyter (5). Although recent modifications (6, 7) have improved the sensitivity of the original procedure, the long incubation periods needed, the possible in- stability of the enzyme under the conditions used (6, 8), and the extra handling involved, left room for improvement. The quantitative assay method described in this paper utilizes the rate of reaction rather than the estimation of the total DPNH formed at equilibrium. In addition to increasing the sensitivity from lo- to lOOO-fold over the previously published methods, the procedure is simplified to a rapid analytical method that requires no preincubation and thereby permits large num- bers of determinations to be handled conveniently. As most enzymic assays involve the determination of the total product formed or substrate utilized at equilibrium rather than the reaction rate (9, lo), the analysis of substrates of other dehydrogenases by rate measurements is also discussed.

The application of this method to the analysis of the L-cY-glycerophos- phate content of a number of vertebrate tissues is described and the

1 This work was supported by the Cancer Chemotherapy National Service Center, National Cancer Institute, under the NationaI Institutes of Health Contract #SA- 43-pH-1886.

396

Page 2: A rapid and sensitive method for the determination of l-α-glycerophosphate in animal tissues

L-wGLYCEROPHOSPHATE DETERMINATION 397

data compared with those originally obtained by Leva and Rapoport (11) using a semimicro calorimetric method.

MATERIALS AND METHODS

Reagents

Diphosphopyridine Nucleotide. A 0.1 M solution of P-diphosphopyri- dine nucleotide (Sigma 99-100%) was prepared and kept frozen when not in use.

Glycerophosphate Dehydrogenase. Crystalline rabbit muscle a-glycero- phosphate dehydrogenase (Boehringer-Calbiochem) was diluted to 0.5 mg/ml with distilled water; the diluted solution was stable in the frozen state for at least a month.

wGlycerophosphate Sodium Salt (98oJo), Na&,H, (OH) ,PO,*6H,O (Eastern Chemical Co., Newark, N, J.). Polarimetric measurements (12) of this preparation indicated it was an equal mixture of D and L isomers. The content of L-Lu-glycerophosphate.6H,O was assumed to be 49%.

Hydrazine-Buffer Solution, p1-I 9.8. A stock solution containing 1.1 M (5.5%) hydrazine hydrate (K 6s K Laboratories), 0.2 M (1.5%) glycine, and 0.02M MgCI, was adjusted to pH 9.8 with KOH.

Other materials were obtained from commercial sources and used without further purification.

Instrumentation

Either a Cary recording spectrophotometer, Model 11, or a Beckman DU spectrophotometer connected to a recorder was used to measure the change in optical density at 340 rnp. The rate of reduction of DPNH during the first twenty seconds of the reaction was found to give the most precise and sensitive standard curve. Where a recorder is not avail- able the total increase in optical density over the first twenty seconds of the reaction can be measured manually with little decrease in sensi- tivity or precision. In the system described, equilibrium was approached in approximately five minutes at room temperature (20-23’C).

Analytical Procedure

Into a cuvette of l-cm light path and 3-ml capacity were added 1.8 ml of the hydrazine buffer solution, 0.1 ml of DPN, 0.1 ml of cY-glycero- phosphate dehydrogenase, and sufficient distilled water to give a total volume of 3.0 ml after consideration of the amount of standard or test solution to be added. The spectrophotometer was adjusted, after which the standard or test solution was rapidly added and stirred. This required three to five seconds. The optical density following the addition of sub-

Page 3: A rapid and sensitive method for the determination of l-α-glycerophosphate in animal tissues

rnA moles/ml L-a-GLYCEROPHOSPHATE CONCENTRATION

FIG. 1. Effect of increasing amounts of rrcr-glycerophosphate on rate of DPNH _ formation. Standard AnaIysis System, optical density change: tnangies, tangent to initial rate (measured over 20 set); circles, total change at 20 sec. Gary Recording Spectrophotometer Model 11; chart speed, I”/min with slide wire to give O-l.0 optical density unit on full scale.

398 CIACCIO

strate was recorded. Standard curves were determined by measuring the change in the initial twenty seconds of the tracing.

Preparation of Test Samples

Tissues for analysis were removed quickly from anesthetized (Nem- butal) animals and immediately frozen in a dry ice-ethanol mixture. This material while still in the frozen state was blotted dry, weighed, and quickly homogenized in a measured volume of cold 5% (0.3M) trichloroacetic acid, using 2 ml of acid per gram of tissue. Either a Waring Blendor or a Lourdes homogenizer gave satisfactory homog- enates. The mixture was centrifuged at 2000 X g and neutralized, and the supernatant fluid was analyzed.

In in vitro experiments involving the metabolism of L-a-glycerophos- phate, the reaction was stopped at the appropriate time with 0.1 ml of 4.8M trichloroacetic acid per 3 ml of reaction mixture. After centri- fuging, the supernatant fluid was analyzed. Neutralization was not required where 0.2 ml or less of sample was added per 3 ml of reaction mixture.

RESULTS AND DISCUSSTON

Figure 1 illustrates the relationship between the amount of L-W

Page 4: A rapid and sensitive method for the determination of l-α-glycerophosphate in animal tissues

L-wGLYCEROPHOSPHATE DETERMINATIOX 399

glycerophosphate in the reaction mixture and the rate of optical density increase. As there is a decrease in the rate even during the first twenty seconds, two graphs were constructed, one utilizing the initial rate as measured by a tangent drawn to the curve, and the other utilizing the increase in optical density over the first twenty seconds of the recording. The measurements of the total change during the first twenty seconds are proportional to the initial rates, as well as more convenient to measure, and give a linear standard curve from 2 to over 40 mpmoles L-a-glycerophosphate,/ml solution. The standard twenty-second curve, as shown in Fig. 1, has been reproduced with little variation in the course of many analyses in this laboratory. A precision of t5% was con- sistently obtained for the values on each standard curve.

We have also determined standard curves after five minutes of in- cubation, at which time the production of DPNH approached equilib- rium. At the same concentrations of a-glycerophosphate shown in Fig. 1, a standard curve measured at equilibrium is not reproducible from day to day and often does not go through the origin. This may be due to instability of the a-glycerophosphate dehydrogenase or of the end product DPNH (6, 8) or possibly to a drift in the spectrophotometer. At higher substrate concentrations where maximal sensitivity is not re- quired, the equilibrium method will however give satisfactory results.

The standard curve shown was confirmed by using known amounts of enzymically synthesized L-a-glycerophosphate. As there was good agreement between the synthesized LW and the racemic a-glycerophos- phate (calculated as 49% ~-a), it is evident that the n-a-isomer is not interfering in the test system.

In order to assure sufficient DPN in the analytical system the final concentration was kept in excess of 2 X 10e3 M. This is necessary in view of the high Michaelis constant of a-glycerophosphate dehydrogenase with respect to DPN, of from 7 to 4 X 10w4M (6, 8). The hydraeine containing buffer at pH 9.8 serves two purposes: (1) dihydroxyacetone phosphate is trapped by hydrazine as well as destroyed by the high pH, thus pulling the reaction to the right, and (2) the equilibrium of the reaction at the high pH also favors the DPN reduction and concomitant glycerophosphate oxidation, while at a neutral pH glycerophosphate formation would be favored.

The enzymic purity of the glycerophosphate dehydrogenase used in this assay was checked by substituting lactate, malate, propanediol phos- phate, glycerol, succinate, fructose-1,6-diphosphate, ethanol, pyruvate, and glyceraldehyde-3-phosphate as substrate sources. No reduction of DPN was obtained with any of these compounds and, therefore, it was assumed that this method could be utilized for the quantitative analysis nf b-a-glpccrophosphate in tissue extracts,

Page 5: A rapid and sensitive method for the determination of l-α-glycerophosphate in animal tissues

400 cIAcc10

In order to test the possibility that inhibitory substances in the sample might interfere with the assay, tissues were analyzed with and without added quantities of L-a-glycerophosphate. Inhibitory substances have never been detected in vertebrate tissue extracts. It is possible that cinnamates, which are known constituents of plant tissues and are in- hibitors of glycerophosphate dehydrogenase,2 would interfere in the assay of these materials.

Table 1 illustrates typical recovery data obtained when L-a-glycero-

TABLE 1 RECOVERY OF ADDED L-a-GLYCEROPHOSPHATE~

Amt. L-S-GP added (m~moles/ml)

Recovered from rat kidney homogenate

mpmoles/ml %

150.0 149.0 99 75.0 75.3 101 30.0 30.6 102 15.0 15.6 104 7.5 6.9 92

0 L-cr-Glycerophosphate was added to a 10% rat kidney homogenate. The endogenous values were subtracted from figures given .

phosphate was added to a kidney homogenate before precipitation with trichloroacetic acid. The endogenous amount of L-a-glycerophosphate in the homogenate (16.1 m~moles/ml) was subtracted from the values shown. These results a.re typical of recovery studies routinely observed with other tissues. Bulbitz and Kennedy (4) found that trichloroacetic acid apparently agglutinated a-glycerophosphate to the precipitated pro- tein and recommended metaphosphoric acid as a deproteinizing agent. We have never observed a loss in L-a-glycerophosphate recovery with the use of trichloroacetic acid; however, trichloroacetate is an inhibitor of a-glycerophosphate dehydrogenase2 at concentrations of 0.1 M or higher. Where such levels may be approached in the analysis system, the sub- stitution of metaphosphoric acid is recommended.

This method has been used to analyze a large number of normal and tumor tissues for n-a-glycerophosphate endogenously present (1) or produced by various metabolic reactions (2, 3). In Table 2 a few of the endogenous values obtained are compared to those reported by Leva and Rapoport (11) using a nonenzymic calorimetric method.

A number of other substrates have been analyzed in this laboratory by means of enzymic rate reactions. In addition to L-a-glycerophosphate,

* Ciaccio, E. I., unpublished data

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401

TABLE 2 L-WGLYCEROPHOSPHATE CONTENT OF VARIOUS TISSUES

~moles/gm w-et weight

Tissue anslyzed Enaymic method0 Chemical methodb

Rat liver (fed) 1.5 (0.9-2.1) 1.4 Rat liver (fasted) 0.6 (0.3-0.85) 0.7 Rat kidney 0.3 (0.1-0.55) 0.4 Rat brain 0.5 (0.36-0.75) 0.4

a Averages are of five to six determinations consisting of pools of two to three animals each; ranges are given in parentheses.

b From Leva and Rapoport (11).

dihydroxyacetone phosphate is conveniently determined by means of the oxidation of DPNH by a-glycerophosphate dehydrogenase in pH 7.5 tri- ethanolamine buffer (13). A sensitivity limit of 2 mpmoles dihydroxy- acetone phosphate/ml test solution has been obtained. Malic and lactic

acids have been analyzed by methods identical to those described for L-a-glycerophosphate except that the appropriate crystalline enzymes, namely, malic and lactic dehydrogenases,” were substituted for the (Y- glycerophosphate dehydrogenase. Similarly, pyruvate and oxalacetate have been determined by measuring the rate of oxidation of DPNH in

triethanolamine buffer at pH 7.5.

SUMMARY

A simple and rapid enzymic method has been described for the analysis of L-a-glycerophosphate which is sensitive down to 2 mpmoles L-a-glycerophosphate/ml solution. The use of measurements of the rates of reactions in place of determinations of the products at equilib- rium in the analysis of L-a-glycerophosphate as well as other substrates shortens the time periods involved, requires less handling, increases the sensitivity many fold, and decreases the possibility of errors due to unstable enzymes, substrates, or products.

REFERENCES

1. CMCCIO, E. I., AND ORANGE, J. B., Abstracts of Bmerican Chemical Society Meeting, Div. of Biological Chem., September 13-18, 1960, p, 27C.

2. CL~CCIO, E. I., AND KELLER, D. L., Federation Proc. 19, 34 (1960). 3. CIACCIO, E. I., KELLER, D. L., AND BOXER, G. E., Biochim. et Bio~hys. Acta

37, 191 (1960). 4. BUBLITZ, C., AND KENNEDY, E. P., J. Biol. Chem. 211, 951 (1954). 5. WIELAND, O., AND SUYTER, M., Biochem. Z. 329, 320 (1957). 6. BOLTVALIK, J. J., AND NOLL, H., And. &o&em. 1, 269 (1960).

’ Obtained from Worthington Biochemical Corporation, Freehold, New Jersey.

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402 CiAcc20

7. HOHORST, H. J., KREUTZ, F. H., AND B~CHER, TH., Biochem. 2. 332, 18 (1959). 8. YOUNG, H. L., AND PACE, N., Arch. Biochem. Biophys. 75, 125 (1958). 9. DEVLIN, T. M., Anal. Chem. 31, 977 (1959).

10. GLOCK, G. E., AND MCLEAN, P., Biochem. J. 61, 381 (1955). 11. LEVA, E., AND RAPOPORT, S., J. BioZ. Chem. 149, 47 (1943). 12. BAER, E., in “Biochemical Preparations,” Vol. II, p. 31. Wiley, New York, 1952. 13. BEISENHERZ, G., B&HER, TH., AND GARBADE, K. H., in “Methods in Enzymology”

(S. P. Colowick and N. 0. Kaplan, eds.), Vol. I, p. 391. Academic Press, New York, 1955.