j. biol. chem.-1927-kuttner-517-31

8/13/2019 J. Biol. Chem.-1927-Kuttner-517-31

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Theodore Kuttner and Harriet R. Cohen

SPINAL FLUIDCALCIUM IN PUS, PLASMA, ANDESTIMATION OF PHOSPHATE ANDCHLORIDE REAGENT. THE MICRO

MOLYBDIC ACID, STANNOUSMICRO COLORIMETRIC STUDIES: I. AARTICLE:

1927, 75:517-531.J. Biol. Chem.

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MICRO COLORIMETRIC STUDIES.I. A MOLYBDIC ACID, STANNOUS CHLORIDE REAGENT. THEMICRO ESTIMATION OF PHOSPHATE AND CALCIUM INPUS, PLASMA, AND SPINAL FLUID.

BY THEODORE KUTTNER* AND HARRIET R. COHEN.(From the Laboratories of the Mount Sinai Hospital, New York.)

(Received for publication, July 21, 1927.)During the course of certain biological and pharmacological in-vestigations in this laboratory, it was desirable to have at hand amethod for the estimation of small amounts of phosphorus, arsenic,and antimony. As is well known, when these substances in thepentavalent state are acted upon by a reducing agent in an acidmixture in the presence of molybdic acid, a blue solution results.

The intensity of color varies proportionately to the amount ofphosphorus, arsenic, or antimony present. This reaction has beenmade the basis of numerous quantitative methods for their esti-mation, but it is believed that until now the optimum condi-tions have not been established.It may be stated at the outset that we were looking for a stablesubstance which would act as reducing agent and produce themaximum intensity and stability of color in the cold without undueloss of time.A calorimetric method for phosphorus in which molybdic oxide is used,was published by F. Osmond (1) in 1337, in which the washed, precipitatedammonium phosphomolybdate is reduced with stannous chloride. Taylorand Miller (2), in 1914, used phenylhydrazine to reduce the ammoniumphosphomolybdate precipitated from an ashed sample. Hydroquinone wasemployed by Bell and Doisy (3) in their method for estimating phosphorusin blood. The reaction Icould be carried out with an excess of molybdateions and it was found unnecessary to isolate the phosphate as ammoniumphosphomolybdate. Briggs (4) modified the method by addition of a little

sodium sulfite to the reagent and carried out the reaction in an acidmedium, thus obtaining a better blue coloration with greater stability.Deniges (5), at about the same time as Briggs, used 4 drops of an ammo-* Eugene Meyer, Jr., Fellow.

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518 Micro Calorimetric Studies. Inium molybdate-sulfuric acid mixture and 2 drops of a freshly pre-pared stannous chloride solution, and wafited 10 minutes before comparingcolors. Stanford and Wheatley (6) have t#ested Briggs method and foundit to be accurate and the reaction to be trustworthy for quantitativeestimation of phosphorus, maximum intensity of color being reached n30 minutes. Benedict and Theis (7) have introduced a method, wherebyboiling the mixture of molybdic and sulfuric acidswith the phosphate com-pletes the reaction in 15 minutes. They retained the hydroquinone andsulfite reducing agent. Fiske and Subbarow (8) have recommended amino-naphtholsulfonic acid instead of hydroquinone. The 1,2,4- and the 1,4,6-acids are equally effective. They used sulfite as well as bisulfite of soda andobtained maximum intensity in 5 minutes in the cold.

Where sulfuric acid is substituted for the sulfite or bisulfite toprevent oxidation of the hydroquinone, as has been done occasion-ally, it is less effective and the solution becomes brown in a fewdays. In the present investigation many substances were testedwhich act as reducing agents. Among them were hydroquinone,diaminophenol, p-aminophenol, monomethyl-paminophenol, mon-ochlorohydroquinone, p-aminosaligenin edinol), 1, 2, 4- and 1,6, 8- aminonaphtholsulfonic acids, and stannous chloride. Thelast is by far the best when the concentrations of the reagents usedare properly regulated. The effect of varying concentrations wasstudied as well as certain conditions interfering with the maximumdevelopment of the color.

1. Molybdic-Sulfuric Acid and Stannous Chloride Reagents.A. Use of Sulfuric Acid in Method.-In agreement with Stan-ford and Wheatley it was found that in the Briggs method,

gradually increasing concentrations of sulfuric acid caused an in-crease in color until a maximum was reached, then with greaterconcentrations of the acid the color intensity decreased.In the present method the intensity as well as the purity of thecolor is regulated by varying the concentration of the reagents.The optimum acidity of sulfuric acid as shown by Fig. 1 for reduc-tion of the phosphomolybdic acid lies in a zone between 0.9 and1.05 N in the final mixture. Below this optimum acidity, molybdicacid is itself reduced; the lower the concentration of sulfuric acid,the greater the reduction of molybdic acid. An increase of acidityabove the optimum causesat first a retardation in the developmentof the color, then with further increase an inhibition of color pro-duction occurs. Thus at 1.1 N acidity, it requires about 2 minutes

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T. Kuttner and H. R. Cohen 519

N HzSO, XPer centcoloration.100

755035

I 0.9 0.8 0.7

.w-

FIQ. 1. Effect of increasing concentration of sulfuric acid. Zone la =decreased color production of 35, 50, and 75 per cent, also increase of tur-bidity with increase of acidity. Zone lb = delayed maximal color produc-tion, of 5 minutes at 5, to 2 minutes at 2. Zone 2 = maximal color produc-tion in 15 seconds. Zone 3 = production of additional color by reductionof Moos.

Zone 1 2 3Per cent sodium I Imolybdate. 0.63 0.65 0.70 0.73 0.75 0.80

Per centcoloration. 100807550350

FIG+. 2. Effect of increasing concentration of sodium molybdate. Zone1 = decreased coloration. Zone 2 = optimum for maximal color. Zone 3 =zone of additional color by reduction of MOOS.

Zone 1Per cent stannouschloride. 0.005 0.01 0.02 0.022Per centcoloration. 1: *-+

Fro. 3. Ef fect of increasing concentration of stannous chloride. Zone1 = decreased coloration. Zone 2 = optimum for maximal color. Zone3 = additional color by reduction of Moos.

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520 Micro Calorimetric Studies. Ito reach maximum intensity, at 1.2 N about 5 minutes, while at1.3 and 1.4 N the intensity of coloration is lessened to 75 and 50per cent respectively. In addition to this retarding effect anincreasing turbidity develops as the acidity is increased beyond1.2 N.The acidity of the final mixture in our method is about 1 Nsulfuric acid; that in the Briggs method is 0.27 N, in the Fiske andSubbarow procedure 0.5 N, in the Benedict and Theis methodabout 1.9 N, and in that of Roe and Kahn (9) about 1.4 N.B. Effect of Increasing Molybclic Acid Concentration.-A 7.5 percent solution of sodium molybdate was found convenient for thisstudy. The optimum concentration of sodium molybdate in thefinal mixture (as shown in Fig. 2) lies in a zone between 0.73 and0.75 per cent of sodium molybdate. Less than this amounteffects a decrease in color production, while above this concentra-tion the molybdic acid is reduced in direct proportion to theincreased amount.C. Stannous Chloride.-Stannous chloride, though used as areducing reagent in qualitative calorimetric analysis, has not foundfavor in quantitative work. This is perhaps because yellowishbrown molybdic oxide complexes are produced simultaneously withthe blue color, but in varying amounts, thus resulting in olive-green tints. This disturbing influence has been eliminated andthe use of sulfite found unnecessary. The optimal concentrationof stannous chloride lies in a zone between 0.02 and 0.022 per cent,as shown in Fig. 3. Stronger solutions reduce molybdic oxide aswell as phosphomolybdic acid.

2. Interfering Substances.Interfering substances can retard color production, inhibit itpartly or entirely, or develop a different color in addition. Thedegree of interference depends upon the quantity of the disturbingsubstance. For example, trichloroacetic acid begins to interferewith maximum color production only at concentrations above 4per cent in the final mixture. Hydrochloric acid has a tendency to

lessen color stability and retards and inhibits maximum coloration.Thus 2 N hydrochloric acid in the final mixture inhibits colorproduction. N hydrochloric acid retards and prevents maximumcoloration, while 0.5 N hydrochloric acid allows maximum produc-

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T. Kuttner and H. R. Cohen 521tion of the color but it soon fades. Nitric acid interferes above0.003 per cent, and with a large amount, for instance 1.5 per cent,a green coloration results. Tartaric acid interferes above 0.002per cent with inhibition of maximum color, and above 0.04 percent with reduction of molybdic acid itself. The effect of citricacid is twice as great. Hypochlorites interfere strongly. Up to0.00004 per cent there is no difference in intensity but a slightdifference in the tint, while at 0.00008 per cent a 20 per cent lossof color occurs, and there is a 50 per cent loss of color in the pres?-ence of 0.0004 per cent. Nitrites interfere strongly at concentrtGtions greater than 0.0001 per cent. Traces of copper or iron saltsdo not interfere.Sulfates interfere presumably by depressing the ionization of thesulfuric acid, the interference depending upon the quantity present.Salts of weak acids such as acetates, tartrates, and citrates alsointerfere, whereas acetic acid itself does not interfere, but causes-the development of purplish tints at concentrations above 5 percent.Silicates do not interfere at the optimum acidity, up to 4 or 5times the amount of phosphorus present.

3. General Considerations.The test is sufficiently sensitive to determine about 0.001 mg.of phosphorus, arsenic, or antimony in the pentavalent state in5 cc. of solution. To prevent misleading results, with so sensitivea method, it is obviously necessary to work with pure reagents.The blue color is produced practically instantaneously, withoutheating, the reaction being complete in 15 seconds when conditionsare right. The velocity of the reaction is about 5 to 10 timesgreater than in the Fiske and Subbarow method, about 40 timesthat of the Deniges procedure, and more than 100 times that of theBriggs or Benedict and Theis methods. The colors produced bythe other methods mentioned have a purplish tint, while thepresent method yields a better blue with greater intensity. Thereis no parallelism between the intensities and velocities of the

colors produced by the different methods. The estimation of thecomparative value of the colors is somewhat difficult be:ause ofthe difference in tint. In the new method the color is approxi-mately 3 to 4 times greater than that of the Benedict and Theis or

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522 Micro Calorimetric Studies. IDeniges methods, and about 12 to 15 times that of the Briggs orthe Fiske and Subbarow methods.

Based upon the reaction mentioned in this paper, methods forthe quantitative determination of phosphorus, arsenic, antimony,and calcium, etc., have been devised. Only methods for phos-phates and calcium will be considered in this paper.

4. Description of Methods.A. Method for Estimation of Phosphates.-The following solu-

tions are necessary and thus far they have remained unchangedafter severalmonths.1. Molybdic-Sulfuric Acid Mixture.-Dissolve 18.75 gm. ofsodium molybdate in 2.5 N sulfuric acid; or mix 1 volume of 7.5per cent sodium molybdate Kahlbaums Zur Analyse) with1 volume of 10 N sulfuric acid, and add 2 volumes of distilledwater. Store in a brown, glass-stoppered bottle.2. Stannous Chloride Stock Solution.-Dissolve 10 gm. in 25 cc.of concentrated hydrochloric acid. Store in a brown, glass-stop-pered bottle. Dilute 0.5 cc. of this stock solution to 100 cc. withdistilled water. The diluted reagent does not keep, and should befreshly prepared as needed.3. Standard Phosphate Stock Solution.-Dissolve 0.4394 gm. ofdried monopotassium phosphate in 1 liter of distilled water. Adda few drops of chloroform to prevent mold formation. 1 cc. =0.1 mg. of phosphorus. Make two standard phosphate solutionsby diluting 5 cc. and 10 cc. of the phosphate stock solution in 100cc. graduated flasks and fill to the mark with water. The solutionswill contain respectively 0.005 and 0.01 mg. of phosphorus per cc.The solution to be tested should contain between 0.4 and 1.2 mg.of phosphorus per 100 cc. 1) Place 2.5 cc. of the sample to be ex-amined in a test-tube graduated at 5 and 10 cc. The sample shouldcontain between 0.01 and 0.03 mg. of phosphorus. 2) Place2.5 cc.of each standard phosphate solution in two other similarly gradu-ated test-tubes, so that one tube contains 0.0125 mg. and the other0.025 mg. of phosphorus. 3) Add to each tube 2 cc. of the molyb-die-sulfuric acid reagent and mix. 4) Add simultaneously toeach of the three tubes 0.5 cc. of the diluted stannous chloridereagent. Insert rubber stoppers and mix without delay by in-verting the tubes once or twice.

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T. Kuttner and H. R. Cohen 523The color is produced immediately, and although the reaction is

complete in 15 seconds if conditions are right, it is advisable towait a full minute before comparison with the standards is made.Any type calorimeter can be used but it may be necessary to diluteal l solutions to 10 cc. if a plunger type is employed. The methodof procedure and calculation is carried out in the manner usual forthe type of calorimeter employed.

B. Method for Estimation of Calcium.-The estimation of cal-cium is similar to that of Roe and Kahn (9), the calcium beingprecipitated as phosphate and the phosphorus estimated colori-metrically. It differs however from theirs by the omission ofphenolphthalein and by the use of a standardized alkaline phos-phate solution for the precipitation. A phosphate standard ex-pressed in terms of calcium is also used in the present method.

Whether the concentration of alkali present for precipitationhas an influence on the character of the precipitate is being investi-gated, as well as the solubility of calcium phosphate. It appearsthat under certain conditions the precipitate does not consist oftertiary calcium phosphate Caa(PO& only, but of a mixture con-taining also dicalcium phosphate Ca2H,(PO&. The mixture mayvary in the proportion of each substance. The ratio of calcium tophosphorus in the tricalcic phosphate is approximately 4:2, inthe dicalcic phosphate 4 : 3. The latter contains 25 per cent morephosphorus with subsequent greater color production. It isobvious that a precipitate containing a mixture of the two wouldgive erroneous results when compared with a standard preparedwith pure calcium salt under different conditions.

The most favorable amount of calcium present for the test tobe described is a concentration of about 0.4 to 1.2 mg. per 100 cc.of solution.The following solutions are necessary:

I. Molybclate-Sulfuric Acid Mixture.2. Diluted Stannous Chloride Reagent.-Reagents 1 and 2 have

been described above under the phosphate method.3. 10 Per Cent Solution of Sodium Hydroxide Containing 1 Per

Cent of Basic Trisodium Phosphate.-Dissolve 1 gm. of the phos-phate in 50 cc. of distilled water and mix with 50 cc. of 20 per centsodium hydroxide (free from silica and calcium). If a precipitateforms it should be allowed to settle for 24 hours or a small portion

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524 Micro Calorimetric Studies. Imay be centrifuged in an hour for immediate use. In the Roe andKahn method the phosphate and hydroxide solutions are usedseparately and may therefore give rise to error in estimating thetrue calcium value.Q. 50 Per Cent Solution of Alcohol.-Dilute 55 cc. of 95 per centalcohol with water. Make faintly alkaline with calcium-freesodium hydroxide, litmus paper being used as indicator. Makeup to 100 cc.5. Two Standard Phosphate Solutions.-(A) Dilute 51.7 cc. ofthe phosphate stock solution described in the phosphate methodto 1000 cc. in a graduated flask. 2.5 cc. are equivalent to 0.025 mg.of calcium. (B) Dilute 51.7 cc. of the phosphate stock solutionin a graduated flask to 500 cc. 2.5 cc. are equivalent to 0.05 mg. ofcalcium.

Transfer 5 cc. of the solution of the sample to be analyzed to acentrifuge tube or Pyrex test-tube graduated at 5 and 10 cc. Thesolution should have a calcium content of about 0.02 to 0.06 mg.and an acidity of not more than 7 per cent trichloroacetic acid, aspreviously described. Add 1 cc. of the alkaline sodium phosphatemixture, mix, set aside for 1 hour, then centrifuge for 3 minutes.Discard the supernatant flu id, catching the last drop on a pieceof blotting paper. Allow to drain 1 to 2 minutes, then wipe therim of the tube, and wash twice with 5 cc. portions of the faintlyalkalinized alcohol, being careful in so doing also to rinse the sidesof the tube and to break up the phosphate precipitate. Drainand wipe the tube after each centrifuging as before. Add 2 cc. ofthe molybdic-sulfuric acid mixture to the tube and dissolve theprecipitate. Add 2.5 cc. of water and mix. If desired 4.5 cc. ofa diluted molybdate mixture can be used, prepared by diluting200 cc. of the molybdate-sulfuric acid mixture with 250 cc. ofdistil led water. Into one of two graduated test-tubes transfer2.5 cc. of calcium Standard A and 2.5 cc. of Standard B into theother; add 2 cc. of molybdic-sulfuric acid mixture to each, and mix.Add 0.5 cc. of the diluted stannous chloride reagent to each stand-ard as well as the unknown and without delay close with a rubberstopper and invert each tube once or twice. After about 1 minutecompare in a calorimeter. If the plunger type of calorimeter isused proceed in the same manner as described for phosphorus.

Described below is an adaptation of the method on a small

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T. Kuttner and H. R. Cohen 525scale, devised for the estimation of phosphates and calcium in0.1 to 0.2 cc. of blood plasma, spinal fluid, or pus. Calcium mayeven be determined if only 0.05 cc. of pus is available.The micro method was primarily devised for calcium determina-tionson the usually small amounts of pus obtained from the humanear in diseased conditions such as mastoiditis. Friesner andRosen (10) have shown that pus derived from bone destruction isrelatively high in calcium, and their investigation was the stimulusfor the development and use of the present micro method.

Pus, unlike plasma, is not a homogeneous fluid and may containdebris from bone or soft tissue. The calcium cannot be directlyisolated as oxalate, as Kramer and Tisdall (11) do in the case ofblood plasma, even if a sufficient quantity is available for the useof their method. In addit ion, the calcium isolated as oxalatefrom the ash of a small sample is too minute in amount tobe titrated with potassium permanganate. Less material is re-quired with the Van Slyke and Sendroy (12) gasometric method, inwhich 1 cc. of blood plasma is sufficient and even less may betaken. Roe and Kahn (9) use 2 cc. of plasma and estimatethe phosphorus by the Benedict and Theis (7) method fromthe precipitated tertiary calcium phosphate. This procedure wastested on ashed samples of small quantities of material. In theuse of this method, the exact matching of color depth with theDubosq calorimeter was prevented because of the somewhatvariable greenish tints in the unknowns. The influence of theadventitious yellow is increased and becomes more disturbing asthe depth of the liquid increases. This effect is lessened andminimized by the use of a dilu tion type of calorimeter such as themicro calorimeter (13) described in a previous paper. This in-strument also permits employment of smaller samples and lessreagent for the test.Ultimately it was found that phenolphthalein is the disturbingfactor, being adsorbed by the calcium precipitate, and the indica-tor is therefore omitted. Although the greenish tint is entirelyeliminated in the new method, the micro calorimeter is retainedbecause of convenience, equal accuracy, and other advantages.The old type of dilut ing tubes has been replaced by glass-stop-pered tubes1 of uniform bore, graduated to 180 on an etched

1 The colo&eter and glassware are manufactured by E. Leitz, Inc., 60East 10th Street, New York.

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526 Micro Calorimetric Studies. Iscale, each division on the scale equalling 0.02 cc. The use ofpermanent color standards has been found very convenient, andtheir preparation will be described in another paper. A pipettefurnished with a rubber nipple; and drawn to a fine bore at thelower end, replaces the old type, permitting the use of smallerdrops for dilut ion or a fine stream for rinsing. A narrow test-tube(12 X 120 mm.) graduated at 1 and 2 cc. has been found useful forthe precipitation of the protein.

Micro Estimation of Phosphates.Two permanent standards are used: A, equivalent to 0.0025 mg.of phosphorus, and B, equivalent to 0.005 mg.; or if desired, color

standards may be prepared from the standard phosphate solutions,but corresponding to these amounts.

Procedure.(1) Transfer 0.2 cc. of pus or blood plasma to the narrow test-

tube already described. (2) Add 7 per cent trichloroacetic acidto the 2 cc. maPk,2 close with a small rubber stopper, shake, andcentrifuge after a few minutes. (3) Transfer 0.5 cc. of, the clear&d colorless fluid, equivalent to 0.05 cc. of the original sample, tothe glass-stoppered, graduated, diluting tube of the micro colorim-eter. (4) Add 0.4 cc. of the molybdic-sulfuric acid solution andmix by sharply tapping the lower end of the tube. (5) Add 0.1 cc.of the diluted stannous chloride reagent, close with the glassstopper, and invert the tube at once.

The color is produced immediately, full intensity being reachedin 15 seconds under optimum conditions. After 1 minute, comparewith the permanent color standards or with freshly prepared stand-ards run through in the same manner simultaneously with theunknown. The unknown is compared with the proper standardsin the micro calorimeter by cautious dilution with small quantitiesof water with the aid of the special pipette. Any other calorimetermay be used if the unknown and standards are diluted to a suitablevolume.The method of computation using dark Standard B in the microcalorimeter is simple. Each division on the scale represents 0.1

* I f 0.1 cc. of material has been taken make up to the 1 cc. mark.

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T. Kuttner and H. R. Cohen 527mg. of phosphorus in 100 cc. of the original material taken foranalysis. For example, if the colors match exactly with themeniscus in the diluting tube at 56, then 56~ 10 equals 5.6 mg.of phosphorus in 100 cc. of the material taken.

If the color had been matched with the light standard eachdivision would represent 0.05 mg. per 100 cc. and the readingwould be divided by 20, thus 56+20 is 2.8 mg. When workingwith material relatively low in phosphorus and protein such asspinal fluid, for example, 0.2, 0.25, or 0.5 cc. is made up to 1 cc.only with the trichloroacetic acid. The reading is now dividedby 40,50, or 100 as the case may be.F = amount of original sample actually used for calorimetric comparison.S = amount of P or Ca in standard.R = reading of unknown on graduated scale.ThenPorCaper 100~. =F

For example, if F = 0.05 cc., R = 56, and S = 0.005 mg. of P orCa, then in 100 cc. original samplePorCa=Oq5X56X$ X 0.005= 5.6 mg. per 100 cc.

With the use of permanent standards .a correction should bemade by adding 1 per cent for every degree above 21C or sub-tracting below 21C.The smallest amount of phosphorus that can be determined inthis manner is 0.25 mg. in 100 cc.Micro Estimation of Calcium.

Two permanent standards are used : A, equivalent to 0.01 mg. ofcalcium, and B, equivalent to 0.005 mg. If desired, color standardsmay be prepared for calcium from the. standard phosphate solu-tions, but corresponding to these amounts. An accurately cali-brated 0.2 cc. pipette is used, graduated in hundredths and of abore sufficiently large for thick pus should such be encountered.Procedure.Transfer 0.1 cc. of pus or blood plasma with the above pipetteto a small platinum dish, rinse the pipette several times with water,

* 1 unit of the graduated scale.

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628 Micro Calorimetric Studies. Iand add the rinsings to the dish. Evaporate to dryness, cautiouslyburn off al l organic matter, add a drop or two of concentrated nitricacid, and again ignite in the usual manner. Care must be taken ofcourse to prevent spattering during the process, which can be com-pleted in about 10 minutes. After cooling, dissolve the precipitatewith 7 per cent trichloroacetic acid in successive small portionsof about 0.2 to 0.3 cc. each. The dish is rinsed down the sides witheach portion which is then transferred quantitatively to the narrowtest-tube, with the aid of the special pipette drawn to a fine boreat the lower end. The total volume should not be much over 1 cc.

The calcium may be precipitated from this solution or from 1 cc.of the deproteinized filtrate as obtained in the micro method forTABLE I.

Mg. of calcium per 100 cc.

Bloodserum............................... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Pus, empyema, chest. . . . . . . . . . . . .,. . . . . . . .I I 8 mo. later. . . . . . . . . .ic osteomyelitis.. . . . . . . . . . . . . . . . . . . . . . . . .I I . . . . . . . . . . . . . . . . . . . . . . . .

-Triohloro-Ashed. aceticacid.

-~11.5 9.510.0 8.010.0 8.512.5 10.445.6 32.014.6 13.819.8 17.413.5 12.2

-

I_-

-

Irichloro-aceticbhosphoricacid.

8.811.038.014.218.913.1

phosphate determination. In the latter case he results are some-what lower, but are generally within 10 per cent of those obtainedby the ashing method. When 0.5 cc. of syrupy phosphoric acidis added to the liter of 7 per cent trichloroacetic acid, the resultis better. This is shown in Table I.In either caseproceed as follows: 1) Add 0.2 cc. of the alkalinesodium phosphate mixture and set aside for 1 hour. Centrifuge 3minutes at high speed and discard the supernatant fluid, catchingthe last drop on a piece of blotting paper. Allow to drain 1 to 2minutes and wipe the rim of the tube. Wash twice with 1 cc. por-tions of the faintly alkalinized alcohol, rinsing the sidesof the tubefree from phosphates. Drain and wipe the rim of the tube aftereach centrifuging as before. When no colored indicator is used in

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T. Kuttner and H. R. Cohen 529the reagents it is difficult to see the slight precipitate, but this neednot cause concern. (2) Add 0.4 cc. of the molybdic-sulfuric acid re-agent and dissolve the precipitate, then add 0.5 cc. of distilled water.If desired, 0.9 cc. of a diluted reagent may be used in the proportionof 40 cc. of the molybdic-sulfuric acid to 50 cc. of distilled water.(3) Add 0.1 cc. of the diluted stannous chloride reagent, close thetube with a rubber stopper, and invert at once. After 1 minute,transfer the solution with the aid of the special pipette to the gradu-

TABLE II.Mg. of calcium per 100 CC. KEYTisdall.

Solution of CaCl~, Containing 10 mg. Ca 10.2per 100 cc. ........................... 9.9Solution of CaCle, containing 15.1 mg.1

15.2Ca per 100 cc. ........................ 14.8Blood serum (ashed). ...................... 11.4 ............................... ............................... 10.4L + 10 mg. as CaCla per 100 cc .... 20.2 ............................... 12.0 ............................... tetany. ... :. .......... ....... ........................ I ............................... 11.5Pus, empyema, chest. .... .... .... .... .... . 6.4 . .......... ......... .... 6.0 ............................ 45.2Spinal,fluid, infection .... .... .... .... .... .. 6.0

15.015.111.59.610.5

9.05.06.711.0

Micro-colori-metric.10.010.215.215.011.59.510.520.412.09.25.16.811.36.3.7.0

45.66.5

ated diluting tube of the calorimeter, and compare with thestandards.The colors are matched in the same manner as described forphosphorus. The computation is exactly similar, except that thefinal result must be multiplied by 2 if only 0.05 cc. of material hasbeen ashed.As shown in Table II the method is accurate and yields resultscomparable with those .of the Kramer and Tisdall (11) and theVan Slyke and Sendroy (12) methods.

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530 Micro Colorirnetric Studies. ISUMMARY.

A calorimetric method has been described for the estimation ofphosphorus and calcium, based on the selective reduction of phos-phomolybdic acid by stannous chloride when definite concen-trations of reagents are maintained.

Quantitative micro methods have been described for the estima-tion of phosphates and calcium in 0.1 to 6.2 cc. of material. Aslittle as 0.05 cc. of pus may be used if only this amount is available.

Newly designed glassware allowing greater accuracy has beenadded to the micro calorimeter (13). .A stannous chloride re-agent is described~ which is more stable than others heretoforeused.

Amounts of phosphorus and calcium can be estimated rangingfrom 0.25 mg. per 100 cc. to 36 mg. and upwards, each individualtest representing about 0.00125 to 0.009 mg.

The advantages are greater rapidity in development of color,and a better blue and more uu%nse coloratioti. The method ismore sensitive and permits the detection and determination of asmaller amount of phosphorus. and the use of less material thanother methods heretofore published.

BIBLIOGRAPHY.

1. Osmond, F., Bull. Sot. dim., 1887, x.X, 745.2. Taylor, A. E., and Miller, C. W., On the estimation of phosphorus inbiological material, J. Biol. Chem., 1914, xviii, 215.3. Bell, R. D., and Doisy, E. A., Rapid calorimetric methods for the,determination of phosphorus in urine and blood, J. Biol. Chem., 1920,xliv, 55.4. Briggs, A. P., A modification of the Bell-Doisy phosphate method, J:Bibl.. Chem., 1922, liii, 13.5. Deniges, G., Corn@. rend. Xoc. biol., 1921, lxxxiv, 875.6. Stanford, R. V., and Wheatley, A. H. M., The estimation of phosphoruscompounds in blood, Biochem. J., 1925, xix, 697.7. Benedict, S. R., and Theis, R. C., A modification of the molybdicmethod for the determination of inorganic phosphorus in serum,J. Biol. Chem., 1924, lxi, 63.8. Fiske, C.~ H;, and Subbarow, Y. , The calorimetric determination ofphosphorus, J. Biol. Chem., 1925, lxvi, 375.9. Roe, J. H., and Kahn, B. S., A calorimetric method for the estimation ofblood calcium, J. Biol. Chem., 1926, lxvii, 585.

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T. Kuttner and H. R. Cohen10. Friesner, I., and Rosen, S., An aid to diagnosis in msstoiditis, read atthe Academy of Medicine, New York, Otological Society meeting,March 11,1927.11. Kramer, B., and Tisdall, F. F., A simple technique for the determina-tion of calcium and magnesium in small amounts of serum, J. Biol.Chem., 1921, $vii, 475.12. Van Slyke, D. D., and Sendroy, J., Jr., Gasometric determination ofblood calcium, PTOC. Xoc. Exp. Biol. and Med., 1926-27, xxiv, 167.13. Kuttner, T., A new pocket calorimeter, J. Am. Med. Assn., 1915, lxv,245.

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