THE ESTIMATION OF SERUM INORGANIC PHOSPHATE AND “ACID” AND “ALKALINE” PHOSPHATASE

ACTIVITY*

BY GEORGE Y. SHINOWARA, LOIS M. JONES,t AND HARRY L. REINHART

(From the Department of Pathology and the Laboratories of the University Hospital, College of Medicine, The Ohio State University, Columbus)

(Received for publication, November 17, 1941)

In 1930, Kay (6, 7) introduced a method for the quantitative determination of “alkaline” phosphatase activity of blood, employing sodium P-glycerophosphate as the substrate. Of the many modifications which have been introduced in which this substrate is employed, Bodansky’s (3, 4) method seems to have found the greatest usage because of its short incubation period and its accuracy. For our studies certain fundamental changes in the technique for the quantitative estimation of serum phosphatase activity and inorganic phosphate were necessary. Presented in this communication are procedures for the simultaneous deter- mination of serum inorganic phosphate and ‘(acid” and “alkaline” phosphatase activity. The estimation of all three tests may be made on 0.40 cc. of serum in the macrotechnique, 0.06 cc. in the microtechnique. Only one photometric calibration curve or one set of phosphorus standards for visual calorimetry is required.

The unit of phosphatase activity is defined here as follows: Each unit of serum phosphatase activity is equivalent to 1 mg. of phos- phorus as phosphate ion liberated during 1 hour of actual incuba- tion at 37” with a substrate containing sodium fl-glycerophosphate, hydrolysis not exceeding 10 per cent of the substrate; and at

* A portion of the expenses of this project was defrayed by a grant from the Comly Fund.

t The material on the microtechnique contained in this paper is part of a thesis submitted by Miss Lois M. Jones in partial fulfilment of the re- quirements for the degree of Master of Arts.

921

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922 Phosphatase Activity

optimum pH of the reaction mixture, for “acid,” 5.00 f 0.15 and for “alkaline,” 9.30 f 0.15. This definition is similar to that of Levene and Dillon (10) and that of Bodansky (4) for “alkaline” activity. However, it differs in the elimination from its calcula- tion of mathematical corrections for the interfering substances in the phosphorus determination and for variances in the incubation period, in its application to “acid” phosphatase, and in its estima- tion at optimum pH.

Reagents

The reagents for the determination of inorganic phosphate are those of Kuttner, Cohen, and Lichtenstein (8, 9) with suggestions by Raymond and Levene (11) and Bodansky (2). That these must be most carefully prepared cannot be overempha- sized. The molybdic acid reagent is made up daily by adding 1 part of 7.5 per cent sodium molybdate to 1 part of cold 10 N sul- furic acid with constant shaking. The dilute stannous chloride solution, prepared by adding 0.2 cc. of the stock reagent (6 gm. of SnClz.2Hz0 plus 10 cc. of concentrated HCl, kept in the refrigerator) to 100 cc. of cold distilled water, is used within 4 hours and is kept cold by means of an ice bath. Both of these solutions must be water-clear. For the “working” phosphate standard containing 2 y of P per cc., dilute 2 cc. of the stock phos- phate solution (0.4394 gm. of KH2POh in 1 liter of distilled water) to 100 cc.

The stock substrate is prepared as follows: Into a 500 cc, volu- metric flask introduce successively 15 cc. of petroleum ether (b.p. 2040°, J. T. Baker, Analyzed Special), about 400 cc. of water, 5.0 gm. of sodium P-glycerophosphate (c.P., Eastman, C3Hs(OH)2- NaP03.5+Hz0, mol. wt. 315)) and 4.24 gm. of sodium diethyl bar- biturate (c.P., Merck, mol. wt. 206.1). Bring the aqueous level up to the 500 cc. mark with distilled water. Transfer to a glass- stoppered bottle containing about an inch layer of petroleum ether. This is kept in the refrigerator.

For the preparation of ‘Lworking” substrate carefully pipette into a 100 cc. volumetric flask containing 5 cc. of petroleum ether 50 cc. of stock substrate. Add acid or alkali as indicated in Table I and bring the aqueous level up to the 100 cc. mark with

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Shinowara, Jones, and Reinhart 923

distilled water. Check the pH. These substrates are kept in the refrigerator.

Macrotechnique-Obtain 1 to 2 cc. of unhemolyzed serum. If postponement of the test is desirable, place the serum in the freezing unit of the refrigerator. No increase in activity is noted in serum stored in this manner; a less than 5 per cent decrease is obtained at the end of 7 days. (Th is is in contrast to Bodansky’s (4) findings.)

Add 1 part of serum (0.1 cc., minimum) to 10 parts of the proper substrate warmed to 37” (“total” phosphate). Incubate at this temperature for exactly 1 hour. 9 parts of 10 per cent trichloro- acetic acid are added to stop the enzyme action and to precipitate the proteins. At about the same time, to 1 part of serum (0.2 cc.,

TABLE I Acid or Base Added to Substrate

-___

Substrate Volume of serum used (see Table II) I

Acid or base

Alkaline, A 1.0 Macrotechnique 2.8 cc. 0.1 N NaOH ‘I B 0.5 “ 1.5 I‘ 0.1 ‘( “

1.0 Microtechnique ‘I C 0.6 “ 1.0 “ 0.1 I‘ “ ‘I D 0.1 Macrotechnique 0.2 “ 0.1 “ “

0.1 Microtechnique Acid Macro- and microtechnique 5.0 “ 1.0 “ HOAc

PH f0.05 at 25”

10.9 10.6

10.4 9.8

5.0

minimum) add 9 parts of trichloroacetic acid (inorganic phos- phate). Mix by inversion, and within 5 to 10 minutes after the addition of trichloroacetic acid centrifuge at about 2500 R.P.M. for 10 minutes. Decant the clear supernatant fluid into clean tubes.

To 1 cc. of “inorganic” protein-free fluid in a 25 cc. Erlenmeyer flask are added 5 cc. of 0.10 N NaOH. To 1 cc. or less of “total” phosphate protein-free fluid is added sufficient 0.05 N NaOH to make the total volume 6 cc. Add 2 cc. of molybdic acid with shaking to each flask as well as to a “blank” flask containing 6 cc. of water. Next add 2 cc. of dilute stannous chloride and make readings 6 minutes later. Since the color development begins with the addition of the reducing reagent, it is added to each of the flasks at definitely measured time intervals.

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924 Phosphatase Activity

Microtechnique-Where venipuncture is considered inadvisable, as in serial studies on infants and on small animals, capillary blood may be collected from a deep puncture wound by use of a capsule (Fig. 1). When a firm clot is formed, loosen it with a fine stylet wire. For centrifugation the capsule is packed with cotton in a tube the size of which depends upon the number of capsules and centrifuged for 10 minutes at 1500 R.P.M. With a Sahli hemo- globin pipette, 0.02 cc. of serum is immediately measured into each of two 12 X 75 mm. tubes (Kahn serological tubes) containing 0.2 cc. of water. The tests may be run at once or the tubes tightly corked and placed in a freezing tray.

One tube of diluted serum (“total” phosphate) is warmed to 37” in a water bath. To it is added 0.4 cc. of the proper substrate which has also been warmed to 37”. After 60 minutes, 0.4 cc. of

OAcn,DIAM.

FIG. 1. Blood-collecting capsule

10 per cent trichloroacetic acid is added. To a second tube (inor- ganic phosphate) is added 0.4 cc. of distilled water instead of substrate. 0.4 cc. of 10 per cent trichloroacetic acid is added to this tube at the same time it is to the tube containing substrate. The tubes are centrifuged for 5 minutes at 2000 R.P.M. The re- sulting supernatant fluid, which should be water-clear, is decanted into another tube.

0.5 cc. of protein-free fluid is placed in a 15 X 85 mm. test-tube (Kolmer serological tube) which is broad and permits rapid mixing while the reagents are being added. 0.7 cc. of 0.10 N sodium hydroxide is added, followed by 0.4 cc. of molybdic acid reagent. The tube is shaken during this addition and tapped afterwards to insure washing down any drops adhering to the sides of the tube. At the same time a “blank” is prepared by adding 0.4 cc. of

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Shinowara, Jones, and Reinhart 925

molybdic acid reagent to 1.2 cc. of double distilled water. When it is necessary to employ lesser quantities of protein-free fluid, owing to excessive phosphate concentration, the final volume before the addition of molybdic acid is made up to 1.2 cc. with 0.05 N sodium hydroxide. At this point the solution in all the tubes should have a volume of 1.6 cc. and should be water-clear, with no trace of color. Then, 0.4 cc. of cold, dilute stannous chloride is added to each tube with the same precautions as in the addition of the molybdic acid. As in the macrotechnique, readings are made 6 minutes later.

Calorimetry---The density of the color developed by the reduction of phosphomolybdic acid can be read on a photometer’ or a spectro- photometer. With the former, employ a filter with a maximum transmission at approximately 600 rnp. Construct a calibration curve by pipetting 0, 2, 4, and 6 cc. of the “working” phosphate standard solution. Make the total volume up to 6 cc. with dis- tilled water. Add 2 cc. of molybdic acid, followed by 2 cc. of stannous chloride. These will give readings for 0, 4, 8, and 12 y of P. If desired, more points on the curve may be determined. The macro curve is applicable to the micro phosphate technique. With the instrument employed in this investigation, the phos- phorus concentration-galvanometer unit curve, plotted on semi- logarithmic graph paper, is linear.

With a visual calorimeter, two standards containing 4 and 8 y of P, as well as a “blank,” are prepared. As in the photometric application, readings of the unknown are made 6 minutes after the addition of stannous chloride. Data from photometric studies on the relationship of time to color development show that satis- factory results can be obtained by comparison against 4 or 8 y standards, the color development time of which is not less than 4 minutes nor more than 8 minutes. In other words, about eight unknown readings may be made with one set of standards. The standards described above for the macroprocedure are employed for the micro modification. With concentrations under 12 y of P in the macroprocedure and 2.4 y of P in the microprocedure, errors

1 The Cenco-Sheard-Sanford photeIometer was employed in this investi- gation. The orange filter is standard equipment. No special accessories were required for the microtechnique. Readings were made on a 2 cc. volume contained in a 10 cc. fused cell, 1 cm. thick.

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926 Phosphatase Activity

due to deviation from Beer’s law are slight when comparisons are made against the closest standard.

Selection of Amounts of Serum and Protein-Free Fluid-There are two limitations in the selection of the proper volume of serum or protein-free fluid to be employed in the test: First, the hydrolysis should not exceed 10 per cent of the substrate; second, the con- centration of phosphate should not exceed 12 y of P in the macro- or 2.4 y of P in the microprocedure. The procedures described in detail above are applicable to serum having “acid” or “alkaline” phosphatase activity of less than 24 units or 48 units when half

TABLE II

Selection of Volumes of Serum and Protein-Free Fluid at Various “Acid” and “Alkaline” Phosphatase Activity Ranges; Calculation Factors

/ Range of phos- phatase activity Serum

-_____- units dume 0- 24 1.00* 8- 48 1.00

16- 96 0.50 32-192 0.50

160- 0.10

Inorganic phosphate..

Protein-free fluid Calculation factors

Macrotechnique Microtechnique _______ ~__~- -

du?ne

1.oot 0.50 0.50 0.25 0.25

1.00

2.00 2.04 4.00 4.08 8.00 8.16

16.00 16.32 80.00 81.60

1.00 2.04

* 0.50 cc. of serum to 5.0 cc. of substrate (macrotechnique); 0.02 cc. of serum to 0.4 cc. of substrate (microtechnique).

i 1 .OO cc. of protein-free fluid (macrotechnique) ; 0.50 cc. of protein-free fluid (microtechnique).

volumes of protein-free fluid are used. For sera having higher activities the various volumes to be employed are tabulated in Table II. Volume correction for a decreased amount of serum is niade with distilled water. Approximation of high “alkaline” phosphatase activity may be made by employing smaller quanti- ties of protein-free fluid than are indicated in Table II. When the range is established, the proper substrate and volumes of sera and protein-free fluid are selected and the test repeated, if accurate results are desired. For “acid” phosphatase activity, one sub- strate suffices for varying volumes of serum.

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Shinowara, Jones, and Reinhart 927

Calculations

The calculation factors in Table II are derived as follows:

Calculation factor = 100 cc. serum

A x;x&$

where A is the serum equivalent (in cc.) in the actual volume of protein-free fluid used in the color development, and B is the final volume of the color solution. In the macrotechnique, the latter is 10 cc.; in the microtechnique, 2 cc. The l/1000 is to convert micrograms to mg. Accordingly, the calculations for both macro- and microprocedures are greatly simplified.

Serum Inorganic and “Total” Phosphate-Photometry (micro- grams of P from the macro calibration curve), micrograms of P X calculation factor = inorganic or “total” phosphate in mg. of P per 100 cc.; visual calorimetry (micrograms of P in the macro standard), S/R X micrograms of P X calculation factor = inor- ganic or “total” phosphate in mg. of P per 100 cc.

Serum “Acid” or “Alkaline” Phosphatase Activity-“Total” minus inorganic = units per 100 cc.

Influence of Interfering Substances in Estimation of Inorganic Phosphate-Bodansky (2) found that errors up to 11 per cent in the estimation of inorganic phosphate can be attributed to varying quantities of glycerophosphate plus trichloroacetic acid in the protein-free filtrate. Application of his correction factors reduced this error to less than 2 per cent in phosphate concen- trations of 12 to 36 y of P. It was found in the investigation reported here that 0.90 cc. of 10 per cent trichloroacetic acid and 0.45 cc. of 10 per cent trichloroacetic acid plus 0.50 cc. of glycero- phosphate caused diminution in color intensity (range, 0.0 to 12 y of P) of approximately 7 and 4 per cent respectively. With lesser quantities of trichloroacetic acid plus glycerophosphate, the effects were correspondingly smaller.

By reducing the acidity of the protein-free fluid in either the macro- or microtechnique with dilute alkali, the effects of trichloro- acetic acid and of trichloroacetic acid plus glycerophosphate in the diminution of color development were completely negated, thereby eliminating the use of several calibration curves or correction

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928 Phosphatase Activity

tables. One calibration curve suffices, then, for the determination of serum inorganic phosphate as well as the “total” (inorganic plus hydrolysate) phosphate in both the macro- and microtech- niques . In solutions of known phosphate concentrations below

TABLE III pH and Serum or Plasma “Alkaline” Phosphatase Activity Obtained with

Sodium &Glycerophosphate Substrate, As Determined by Various Investigators

Investigators

Kay (6)

Roche (12)

Bodansky (4,

Belfanti, Contardi, and Ercoli (1)

Woodard, Twombly, and Coley (14)

* Alkaline polation.

-

1

- I jhosphatase activity was determined at pH 8.6 by inter-

Material tested Bllffer

Rat plasma Glycine + NaOH

Human plasma

Rabbit plasma

Horse serum Rabbit

serum Guinea pig

serum Human

serum Sodium diethyl

barbiturate Rabbit Sodium acetate $

serum sodium diethyl Horseserum barbiturate

Human serum

HCI + sodium di- ethyl barbitu- rate

NaOH + sodium diethyl barbi- turate

TWI Period pera. 3f hy- ture rolysir

"C. krs.

38 48

38

37

37

37

19

l-1 2

16

f-2

-

pH of reaction mixture

8.8-9.1 (Op- timum) “ “

‘I <‘

9.0 (Opti- mum)

8.6 (About)

9.7 (Opti- mum)

9.5 (Opti- mum)

8.2* (AP- proximate)

8.7* (AP- proximate)

12 y of P, the macrotechnique has an error of less than 1 per cent. In duplicate inorganic phosphate determinations on single serum specimens, the error is less than 3 per cent (on duplicate deter- minations on proteimjree fluid the error is even less). With the microprocedure the actual error on serum is less than 4 per cent.

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Shinowara, Jones, and Reinhart 929

Optimum pH for Serum “Acid” and “Alkaline” Phosphatuse Activity2-The pH of the reaction mixture in the determination of serum “alkaline” phosphatase activity, with sodium p-glycero- phosphate substrate, as reported by various investigators is tabu- lated in Table III. In respect to serum “acid” phosphatase activity with this substrate, Gutman and Gutman (5) have pre- sented conclusive evidence that the optimum pH is 5.0. Woodard and Higinbotham (13) determined serum “acid” phosphatase activity at pH 6.4 in an unbuffered sodium P-glycerophosphate substrate. In view of these slight but significant variances we have reinvestigated the serum phosphatase activity in both the

1. ” 5 b 7 8 9 IO II

pti SUBSTRATE 25°C.

FIG. 2. Titration curve of substrate (0.0206 M sodium diethyl barbiturate and 0.0160 M sodium @-glycerophosphate) ; buffer capacities, d(B)/d(pH).

“acid” and “alkaline” range, with particular reference to the latter.

In Fig. 2 the data obtained by the titration on sodium P-glycero- phosphate + sodium diethyl barbiturate are presented. Exami- nation of the curve demonstrates that the substrate is not a strong

2 A glass electrode electrometer was employed for all pH determinations. The instrument was checked at different temperatures with Clark and Lubs buffer solutions at pH 4, 7, 9, and 10, and counterchecked against another electrometer. In control studies it was found that no change in pH was encountered during the 1 hour hydrolysis period. No hydrolysis was de- tected when the various substrates were incubated without the presence of serum; likewise there was no significant change in the inorganic phosphate when serum was incubated in buffer solution (without sodium p-glycero- phosphate) at various pH.

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930 Phosphatase Activity

buffer especially on the alkaline side. The observed changes in pH obtained by adding serum to substrate are shown in Fig. 3. The pH of the substrate alone was determined at 25”; of serum-

o I PAW SERUM TO

pH SUBSTRATE

16 . \T;Ey2 SUBSTRATE.

AT 25-C. 1.2 . X I PARi SERUM m MINUS

,z.H SERUM- ”

$O;fRiS SUBSTFfATE.

8 * SUBSTRATE e

AT 37’C. 4. e 8

I--. . . 5 6 7 8 9 IO 11

pH SUBSTRATE 25-C.

FIG. 3. Scatter diagram showing differences in the pH of the substrate at 25” and of the serum-substrate at 37”.

5 b 7 8 9 10 pH SUBSTRATE-SERUM 37-C.

FIG. 4. pH-activity curves. Curve 1, normal human serum; Curves 2, 3, 5, and 6, sera from cases of obstructive jaundice; Curve 4, serum from a case of carcinoma of the prostate metastatic to the bone. 10 parts of substrate (0.0206 M sodium diethyl barbiturate and 0.0160 M sodium B-glyc- erophosphate) to 1 part of serum.

substrate, at 37”, the hydrolysis temperature for the estimation of serum phosphatase activity. It must be noted that approxi- mately 30 per cent of the apparent lowering in pH in the higher

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Shinowara, Jones, and Reinhart 931

TABLE IV

Serum “Alkaline” Phosphatase Activity at pH 8.6 to pH 10.0

The values below are in mg. of P hydrolyzed per 100 cc. in 1 hour; 0.0160 M sodium fl-glycerophosphate, 0.0296 M sodium diethyl barbiturate.

I-

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18 19 20

-

M NaOH in substrate

8.6 8.7 8.8 (a)

---

2.2 2.3 2.7 3.0 3.2 3.5 3.7 3.9 4.1 4.14.8 5.f 4.4 4.6 4.9 5.0 6.6 7.0 8.49.6 ll.C 9.0

!5.2 36.3

6.;

12.t

-

Range. ............ Mean ..............

9.0 9.1 9.2 (b)

7.1 7.9 7.! 6.7 7.4

11.0 11.0 13.0 13.8 14.i

9.3 9.4 (cl

--

3.4 4.5 4.7 6.3 5.8 5.5 5.7 6.9 6.9 7.9 7.: 8.4 9.0 7.5 8.6 2.6 3.8 .4.0 13.1 .4.2 !O.O 15.5

- 9.5

-

7.

13.

......................... . . 0.48-0.6E

......................... . 0.565

7 10.0

- 0.65 0.51 0.57 0.48 0.55 0.63 0.65 0.56 0.59

2.10.52 0.52 0.51 0.65 0.58 0.52 0.51

3.40.60 0.63 0.76t 0.74t

-___

.80

.90

.80

.82

.88

.80

.93

.80-0.93 8.85

* pH to the nearest 0.05; 1 part of serum to 10 parts of substrate. t Not included in the mean.

alkaline range is due to the difference in temperature. The effect of serum on the pH of the substrate decreases with the pH; at

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932 Phosphatase Activity

pH 5 the effect is negligible. When 1 part of either fresh or pre- served serum is added to 10 parts of substrate at pH 10.8 to 10.9 at 25”, the pH at 37’ of the final reaction mixture is between 9.2 and 9.4. The effect of adding 1 part of serum to 20 parts of sub- strate is appreciably less. Results obtained here point out the variations in pH of the final reaction mixture which can be ob- served due to even slight deviations in the pH of “alkaline” sub- strate or in the proportion of serum added.

It is evident from the six pH-activity curves in Fig. 4 that the serum “alkaline” phosphatase activity is highest in the pH range of 9.1 to 9.7, and that the slopes of the pH-activity curve on either side of this range are very steep. At approximately pH 7.5 and 10.5 there is very little “alkaline” phosphatase activity under the conditions of our experiments. The optimum pH for serum “acid” phosphatase activity as revealed by Curve 4 is about pH 5. This is in agreement with the work of Gutman and Gutman (5). A more detailed study in the pH range 8.6 to 10.0 of “alkaline” phosphatase activity is presented in Table IV. From the data on Sera 10 and 17 in Table IV and by interpolation of the curves in Fig. 4, it has been definitely shown that the optimum phosphatase activity is obtained when the substrate-serum reaction mixture has a pH of 9.3 f 0.15 at 37’. Experiments on eighteen sera (see Table IV) show that 48 to 65 per cent of the optimum “alkaline” phosphatase activity is obtained at pH 8.6 in sera of less than 15 units; in sera with higher activity the differences at the optimum and pH 8.6 become less. At pH 8.6 a deviation of 0.1 pH unit will cause a change in the phosphatase activity of about 14 per cent; at pH 9.3, a similar deviation will cause a change of only 3 per cent.

Results

The actual analytical error by either the macro- or microtech- nique for the estimation of phosphatase activity is less than 5 per cent in sera of normal or high activity. Normal values for “alka. line” phosphatase in adults range from 2.2 to 8.6 units. Results of our investigation of the serum LLacid” phosphatase, the details of which will be presented elsewhere, indicated a range of 0.0 to 1.1 units per 100 cc. in twenty healthy adult subjects and in 140 control patients. In cases of proved carcinoma of the prostate

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Shinowara, Jones, and Reinhart

metastatic to the bone, %cW phosphatase activity of 1.2 to 31.7 units was obtained.

SUMMARY

Micro and macro photometric techniques are described for se- rum inorganic phosphate and %cicP and “alkaline” phosphatase activity. Sodium /3-glycerophosphate is the substrate used. On thii basis the unit of phosphatase activity for both enzymes is comparable.

BIBLIOGRAPHY

1. Belfanti, S., Contardi, A., and Ercoli, A., Biochem. J., 29, 517 (1935). 2. Bodansky, A., J. Biol. Chem., 99, 197 (1932-33). 3. Bodansky, A., J. Biol. Chem., 101, 93 (1933). 4. Bodansky, A., Am. J. Clin. Path., Tech. Suppl., 1,Sl (1937). 5. Gutman, E. B., and Gutman, A. B., J. Biol. Chem., 136, 201 (1940). 6. Kay, H. D., J. Biol. Chem., 89, 235 (1930). 7. Kay, H. D., J. Biol. Chem., 89, 249 (1930). 8. Kuttner, T., and Cohen, H. R., J. Biol. Chem., 76, 517 (1927). 9. Kuttner, T., and Lichtenstein, L., J. Biol. Chem., 86, 671 (1930).

10. Levene, P. A., and Dillon, R. T., J. Biol. Chem., 88, 753 (1930). 11. Raymond, A. L., and Levene, P. A., J. BioZ. Chem., 79, 621 (1923). 12. Roche, J., Biochem. J., 26, 1724 (1931). 13. Woodard, H. Q., and Higinbotham, N. L., J. Am. Med. Assn., 116,

1621 (1941). 14. Woodard, H. Q., Twombly, G. H., and Coley, B. L., J. Clin. Inn., 16,

193 (1936).

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Harry L. ReinhartGeorge Y. Shinowara, Lois M. Jones and

ACTIVITYAND "ALKALINE" PHOSPHATASE

"ACID"INORGANIC PHOSPHATE AND THE ESTIMATION OF SERUM

1942, 142:921-933.J. Biol. Chem. 

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