THE DETERMINATION OF THYROXINE IN THYROID SUBSTANCE

BY NATHAN F. BLAU

WITH THE TECHNICAL ASSISTANCE OF ANDRE C. KIBRICK

(From the Johnston Livingston Fund for Experimental Biochemistry,

Department of Biochemistry, Cornell University Medical College, New York City)

(Received for publication, May 7, 1935)

In the chemical determination of thyroxine in thyroid sub- stance as hitherto proposed, it is essential first to hydrolyze the material and then to separate the hydrolysate into a thyroxine and a non-thyroxine-containing fraction. An analysis of either frac- tion for its iodine content will finally give the thyroxine value of the original material. Granted a satisfactory method for the determination of iodine, an investigation of the problem of thy- roxine determination resolves itself into a study of each of the two preliminary phases of the procedure as above stated. According to Leland and Foster (1) the process of hydrolysis is best accom- plished by boiling the desiccated gland substance with 2 N NaOH for 18 hours. The liberated thyroxine is then extracted from the alkaline solution by means of butyl alcohol. An accuracy of about 83 per cent was claimed for the procedure, since crystalline thy- roxine added to thyroid material or casein and then boiled could be recovered to this extent. The writer (2) has modified the extraction procedure of Leland and Foster so that it may be carried out more conveniently in an acid solution. It has also been shown that the modified procedure may yield values for thyroxine in thyroid substance, which are from 10 to 20 per cent higher than those obtained by the original Leland and Foster method. How- ever, of crystalline thyroxine which had been boiled for 18 hours with dried and defatted beef muscle in 2 N NaOH, only about 85 per cent could be extracted from the acidified solution. Since the thyroxine contained in a pure solution or that added to a cold

361

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352 Thyroxine in Thyroid Substance

alkaline solution of the hydrolytic products of protein can be ex- tracted quantitatively by either procedure, it follows that the chief fault of the Leland and Foster technique lies in the method of hydrolysis employed.

That some destruction of thyroxine occurs as a result of boiling with strong alkali is almost certain, as shown by the work of Leland and Foster. An effort was therefore made to find condi- tions for the hydrolysis of thyroid substance which would avoid the decomposition of thyroxine as far as possible. Various hydrolytic agents were used. Thyroxine in each hydrolyzed sample was extracted by means of butyl alcohol, with the procedure of Leland and Foster and of the author. In one series of experiments the results by the two procedures were also compared with the thy- roxine values obtained after adjusting the acidity of a portion of the thyroid solution to pH 5 and determining the acid-soluble iodine in the clear filtrate.

In this paper an account will be given of the work done with sodium hydroxide and with barium hydroxide as hydrolytic agents for thyroid material.

Hydrolysis of Thyroid Substance with Sodium Hydroxide-L25 gm. portions of thyroid powder (of fresh thyroid tissue, 4 to 5 times this weight was taken) were boiled with 100 cc. of 2 N NaOH for varying lengths of time. The boiling was stopped at 4 hour intervals and thyroxine determinations were made on aliquots of the solution diluted to a given volume with 2 N NaOH. The re- sults in Table I, taken from a larger series of experiments, show consistently higher thyroxine values obtained by extraction with butyl alcohol of an acidified solution. This is in agreement with previous findings (2). It may be noted, in addition, that in the parallel figures for thyroxine obtained by the acid and by the alkaline extraction methods, respectively, the greatest discrep- ancies occur during the early stages of hydrolysis. This would seem to indicate that in order to extract thyroxine from an acidified solution of thyroid substance it is not necessary to break down the thyroid protein as completely as is apparently required for the Leland and Foster procedure. Practically maximum amounts of thyroxine were extracted with butyl alcohol from the acidified solutions of the three thyroid preparations listed in Table I after only 8 hours of hydrolysis with 2 N NaOH. This is an important

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N. F. Blau 353

factor not merely from the standpoint of time saving, but in view of the destructive effect of boiling alkali upon thyroxine, also from the standpoint of the accuracy of the determination. The im- provement in this regard, as shown by tht recovery of added thyroxine, was, however, not significant. Nearly as much thy- roxine iodine was lost after 8 hours boiling with protein material in 2 N NaOH as after 18 hours, the average loss in a series of ex- periments amounting to about 15 per cent of the iodine taken. It was therefore decided to try some other alkali for setting free thyroxine from its combination in the gland.

TABLE: I

Comparative Yields of Thyroxine Extracted with Butyl Alcohol from All-saline and from Acid Solutions of Thyroid after Varying Periods of Hydrolysis

hrs.

4 8

12

16 20

P

--

-

with 2’ N NaOH

Thyroxine 1~ as per cent of total IZ

Desiccated pig thyroid; total 12. 2.66 mg. per gm.

Luther’s method

(a)

-

L2:d Foster’s method

(b)

Ratio @)/(a:

-

/

_-

L%d Foster’s method

(b)

Ratio P

(b)/(a)

iuthor’s method

(a)

1 1

Ratio m)/(a)

_-

23.4 17.3 74.0 28.5 21.6 75.7 11.4 8.2 72.0 25.7 18.6 72.3 32.7 26.2 80.0 12.4 10.2 82.3 25.9 21.3 82.3 32.2 26.7 83.0 11.9 10.2 85.7 24.0 21.1 88.0 31.3 26.2 83.0 12.1 11.1 92.5 23.5 20.1 85.6 27.5 25.2 91.6 12.1 11.1 92.5

Hydrolysis of Thyroid Substance with Ba.rium Hydroxide--Har- ington (3) has successfully employed barium hydroxide in a 10 per cent solution as a hydrolytic agent in connection with the isolation of thyroxine from desiccated thyroid. The high yield of crystalline material which he obtained led him to believe that thyroxine was more stable in a solution of barium hydroxide than in sodium hydroxide. Using barium hydroxide for the hydrolysis of the gland substance in conjunction with butyl alcohol extrac- tion, we have found that thyroxine determinations in such hydrol- ysates could be made more conveniently than in sodium hydrox-

-

I &sh pig thyroid; total I: 0.86 mg. per gm.

- I Fresh pathological human

thyroid: total 12, 1.36 mg. per gm;

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Thyroxine in Thyroid Substance

ide solutions of the gland. The period of hydrolysis could be shortened to 6 hours and the yield of thyroxine was as a rule greater by several per cent than that obtained by the Leland and Foster method. Preliminary experiments showed that the best results were obtained with 8 per cent Ba(OH)2.8Hz0 in the ratio of 50 cc. of the solution to 1.0 gm. of desiccated thyroid, or 5.0 gm. of fresh thyroid tissue. In order to minimize as far as possible the formation of an iodine-containing precipitate of insoluble barium salts, the warm solution at the end of the period of hydrolysis was diluted with water and 5 cc. of 10 per cent HCl to twice its original volume. An aliquot of the still alkaline solution was acidified with 1: 1 HCl to pH 3.5 to 4 and extracted with butyl alcohol as will be described.

Distribution Ratios of Thyroxine, Diiodotyrosine, and Potassium Iodide between Butyl Alcohol and Acid Barium Chloride Solution- Dried and defatted beef muscle or testicular powder was boiled for 6 hours with barium hydroxide in the proportion of 1.0 gm. of substance to 4 gm. of the alkali in 50 cc. of water. Weighed amounts of thyroxine, diiodotyrosine, and potassium iodide were dissolved in this in varying concentrations. Measured volumes of these solutions were transferred to separatory funnels and diluted with an equal volume of water. A few drops of brom-cresol green indicator were added and the solution was titrated with 1: 1 HCl to a pH of 3.5 to 4.0. The acid solution was shaken with an equal volume of butyl alcohol and allowed to stand for from 60 to 90 minutes. After separation of the two immiscible layers, their respective iodine contents were determined by the method of Kendall.

The distribution ratios between butyl alcohol and acid for thy- roxine and diiodotyrosine were found to be substantially the same as we had previously reported in connection with 2 N NaOH solu- tions of these substances acidified with H&SO+ namely 99.4:0.6 and 77.8~22.2 respectively. In contrast to our previous findings, however, for inorganic iodine the distribution ratio of 11.5:88.5 favors significantly the aqueous phase. We have also redeter- mined the distribution ratios of the three iodine compounds be- tween butyl alcohol and the washing solution consisting of 4 N

NaOH containing 5 per cent NatCOs. These have been found to be almost identical with the values previously reported.

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N. F. Blau

Relative Effectiveness of Barium Hydroxide and #odium Hydrox- ide As Hydrolytic Agents for Thyroid X&stance-Equal weights of a uniform batch of desiccated thyroid were hydrolyzed re- spectively with 8 per cent Ba(OH)2.8Hs0 and 0.5 N NaOH (1

gm. of substance to 50 cc. of alkali) for varying lengths of time. The course of hydrolysis, as shown in a representative experiment in Fig. 1, was followed by means of amino acid nitrogen determina-

114

CT

-AT

I)6 $3 210

FIG. 1. Showing comparison of progress of hydrolysis of desiccated thyroid gland boiled respectively with 0.5 N NaOH (50 cc. to 1.0 gm. of dry substance), with 8 per cent Ba(OH)2.8HzO (50 cc. of 1.0 gm. of dry sub- stance), and with 2 N NaOH (100 cc. to 1.25 gm. of dry substance). The abscissa represents time of hydrolysis in hours.; the ordinate, thyroxine iodine as per cent of total iodine and per cent of amino acid nitrogen set free. Curve AT represents thyroxine iodine in 0.5 N NaOH hydrolysates; Curve BT, thyroxine iodine in 8 per cent Ba(OH), hydrolysates; Curve CT, thyroxine iodine in 2 N NaOH hydrolysates; Curve AN, amino acid nitrogen in 0.5 N NaOH hydrolysates; Curve BN, amino acid nitrogen in 8 per cent Ba(OH)z hydrolysates; Curve CN, amino acid nitrogen in 2 N NaOH hydrolysates.

tions and of estimations of the amounts of thyroxine that could be extracted from the acidified solutions. With either criterion, it may be seen that barium hydroxide produces a more rapid rate of hydrolysis than an equivalent concentration of sodium hydrox- ide. It is especially noteworthy, however, that boiling with barium hydroxide renders the thyroxine iodine of the gland much more readily extractable than similar treatment with sodium hy-

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356 Thyroxine in Thyroid Substance

droxide. This apparently occurs independently of the amount of amino acid nitrogen set free and brings out the interesting fact that there is no definite relationship between the degree of protein breakdown, as indicated by amino acid nitrogen estimations, and the amount of thyroxine which may be found in the butyl alcohol extracts of the hydrolysates.

While there could thus be but little doubt of the greater ef- fectiveness of Ba(OH)z to liberate thyroxine from its combination in the gland, the question was further studied by comparison of the yields of iodine in the thyroxine fractions of barium hydroxide hydrolysates, as determined by the method to be described, with

TABLE II

Comparative Stability of Thyroxine (Approximately 1.0 Mg.) in Boiling 8 Per Cent Barium Hydroxide (60 Cc.) and in Boiling B N NaOH (186 Cc.)

Time of boiling, 6 hours.

Barium hydroxide

Thyroxine 1s from thyroxine and diiodotyrhsine (1:4)

Taken Recovered

mg. per cent 1.31 100.0 0.655 98.0

1.31 99.5

Taken Recovered

Tl. per cent 2.620 97.5 0.655 96.0

2.620 96.7 0.655 97.5 0.655 96.3

- 2 N NaOH

Thyroxine tlF;*rn thyroxine

Taken Recovered

wl. per cent 0.655 91.0 0.655 89.5

0.655 89.5 0.655 90.7

those obtained in the same gland material after varying periods of hydrolysis with 2 N NaOH. Fig. 1 shows that despite the some- what more rapid liberation of amino acid nitrogen by 2 N NaOH, in this medium, boiling for at least 16 hours is required in order to reach a maximum yield of thyroxine, which at best only approxi- mates that found in the 8 per cent Ba(OH)2 solution that has been boiled for 4 hours only.

Stability of Thyroxine Boiled in 8 Per Cent Barium Hydroxide for 6 Hours-In the results of the preceding experiment it may be noted that boiling the gland material with barium hydroxide for more than twice the length of time required for a maximum yield of

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N. F. Blau 357

thyroxine does not cause a loss of iodine in this fraction. Our own work and that of Leland and Foster seem to indicate that this is not true of ‘2 N NaOH hydrolysates. The difference, as may be seen from the results in Table II, lies in the greater stability of thyroxine in boiling barium hydroxide. The largest loss from this source is only 4 per cent and is not very significant. In contrast to this the recoveries of thyroxine boiled in 2 N NaOH for 6 hours are lower by several per cent.

Recovery of Crystalline Thyroxine Boiled with Protein Substance in 8 Per Cent Barium Hydroxide-Kendall (4) has stated that pure thyroxine is slowly altered chemically while it is boiled in a solution of gelatin, amino acids, or gum acacia with 5 per cent sodium hydroxide. Leland and Foster (1) reported a loss of 19 per cent when thyroxine was added to casein and then boiled for 18 hours. The effect of boiling barium hydroxide in the presence of protein material was therefore studied in a large series of ex- periments. After a period of hydrolysis of 6 hours the solutions were subjected to the extraction procedure to be described. As shown in Table III, with the proteins from three different sources, a loss of about 10 per cent is sustained in the amount of thyroxine iodine taken. While it is not altogether certain that a like loss of the bound thyroxine occurs when thyroid substance is subjected to hydrolysis with barium hydroxide, the values for the recoveries of added thyroxine (Table III) together with the data in Table II and in Fig. 1 would seem to suggest that the loss in native thy- roxine is no greater, but probably less than that shown in the present series.

Determination of Thyroxine-Place in a 300 cc. Erlenmeyer flask 1.0 gm. of desiccated thyroid (or 4 to 5 gm. of fresh thyroid tissue), 4 gm. of Ba(OH)2.8Hz0, and 50 cc. of hot distilled water. Shake, and if necessary heat gently to dissolve the barium salt. Add 2 cc. of butyl alcohol to prevent foaming and to promote smoother boiling. Connect the flask to a reilux condenser and heat to gentle boiling over a sand bath, or preferably in a hot water or steam bath, for 6 hours. Cool somewhat and pour the warm solution with as much of the precipitate it contains as pos- sible into a 100 cc. volumetric flask. Rinse the Erlenmeyer flask with two successive portions of 5 cc. of distilled water, adding each to the main bulk of the hydrolysate. Wash down the sides of

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358 Thyroxine in Thyroid Substance

the flask with 5 cc. of 10 per cent HCI and shake well. Add 2 cc. of butyl alcohol and continue shaking until all of the precipitate adhering to the sides of the flask will be inthe solution. This will occur readily and completely with the procedure outlined. Add the acid water-butyl alcohol mixture to the contents of the vol-

TABLE III

Recovery of Thyroxine Boiled for 6 Hours with 8 Per Cent Barium Hydroxide

Solution in Presence of Protein Material (1.0 Gm. of Dry Substance to 50 Cc. of Ba(OH)* Solution)

Thyroxine 1% taken Protein substance added Thyroxine Ia recovered

mu.

0.530 0.571 0.968 0.376 0.850 0.886 0.760 0.813 1.113 1.654* 0.979* 0.653* 0.979* 0.96 1.28 0.624 0.624 0.416 0.653 0.653 0.653

Powdered beef muscle “ I‘ I( “ 1‘ ‘I ‘I ‘I ‘1

Testicular powder I‘ ‘I ‘I ‘I I‘ I‘ ‘I ‘I “ (I I‘ ‘I ‘I “ ‘I “

Thyroglobulin Desiccated pig thyroid

I‘ ‘I ‘I I‘ “ ‘I “ ‘I “

Fresh human gland “ ‘I “ “ ‘I “

per cent

89.0 91.0 92.3 90.5 92.7 89.0 93.0 87.5 91.0 95.3 99.0 97.2 95.0 94.8 87.5 91.0 89.5 91.0 97.0 91.0 97.0

* Boiled with added diiodotyrosine.

umetric flask and fill the latter to the mark with distilled water washings of the flask used in the process of hydrolysis. The hydrolysate now consists of an alkaline solution containing a fine flocculent precipitate. Stopper the flask and shake well to obtain a uniform suspension of the solid particles and remove aliquots for

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N. F. Blau

the determination of total iodine and thyroxine iodine. 20 to 21 cc. will usually serve for the f0rmer.l

For the determination of thyroxine pipette 50 to 75 cc. of the hydrolysate into a 250 cc. separatory funnel, add 0.5 cc. of brom- cresol green indicator, and titrate the solution with 1: 1 HCl to a yellow color. With brom-phenol blue as an outside indicator, ad- just the pH to between 3.5 and 4.0. Shake the solution in the funnel with an equal volume of butyl alcohol. After allowing it to stand 1 to 2 hours or longer,2 draw off the aqueous layer as well as possible without removing much of any of the precipitate or scum which may be present at the bottom of the butyl alcohol. To the latter, remaining in the funnel, add an equal volume of 4 N NaOH containing 5 per cent anhydrous sodium carbonate.3 Shake well and allow to stand for 1 hour or longer until the layers are well separated. Draw off the alkaline layer, leaving any precipitate in the funnel with the butyl alcohol. Shake the latter with a volume of 4 N NaOH containing 5 per cent NazCO$ equal to that of one-half the volume of the butyl alcohol taken. Let stand an hour or longer. Draw off the washing fluid with as much of the precipitate as possible, taking care to avoid any loss of butyl alcohol. Occasionally, especially in the case of fresh thyroid tissue, there is at this stage a heavy precipitate in the upper stratum of the aqueous phase and a more accurate separa- tion may be effected by allowing the latter to run into a centrifuge

1 We have found the bulky precipitate of barium salts following alkaline fusion to interfere with the accurate determination of iodine. This precipi- tate could be removed most conveniently in the form of sulfate as follows: The melt in the crucible was dissolved with hot water and the solution and precipitate of barium carbonate transferred quantitatively to a 500 cc. Erlenmeyer flask. After cooling, methyl orange indicator and 5 cc. of 20 per cent NaHSOa were added and the solution made distinctly acid with a volume of 1:l sulfuric acid, 0.5 cc. in excess of that required to reach the turning point of the indicator. The solution was filtered hot through No. 5 Whatman filter paper and the precipitate washed well with hot water. The remainder of the procedure was carried out as described by Kendall, except that no phosphoric acid was used.

2 This is a convenient stage at which to interrupt the procedure at the end of the day, as the acid aqueous layer and the butyl alcohol may be allowed to remain in contact overnight.

*A 20 per cent solution of technical sodium hydroxide may be sub- stituted.

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360 Thyroxine in Thyroid Substance

tube with a small amount of the butyl alcohol layer and centrifug- ing. Having removed the washing fluid as nearly as possible, the butyl alcohol is distilled at reduced pressure and the residue trans- ferred to a nickel crucible for the determination of its iodine content.

In order to check the accuracy of the extraction procedure, known mixtures of thyroxine with diiodotyrosine were analyzed according to the directions given above. With concentrations corresponding to those likely to be encountered in the analysis of thyroid material the recoveries for thyroxine iodine, as shown in

TABLE IV

Recovery of Thyroxine from Mixtures of Thyroxine and Diiodotyrosine Dis- solved in 8 Per Cent Ba(OH)2 Hydrolysate of Testicular Powder

1.10 0.55

0.55 4.41 2.65 1.45 2.32

1.80 0.0

Approximate ratio of diiodotyrosine 12 to

thyroxine Ia

17: 1 17:l 17:l

4.6:1 3.3:1 3.7:1

6.0:1 1.7:1

-

_-

-

Thyroxine 1% taken

07 0.065 0.0323

0.0323 0.955 0.812

0.390 0.390 1.019

0.382

-

1

-

-

‘hyroxine Izrecovered

per cenl

Trace 105.0

98.0 95.0 99.5

102.0

99.7 99.8 99.8

98.0

Table IV, average close to 100 per cent. The effect of inorganic iodine seemed invariably too small to be measured.

Comparison with Method of Leland and Foster and with Separation of Thyroxine by Acid Precipitation-The figures in Table V were taken at random from a large series of analyses carried out over a period of more than a year. The material from human subjects consisted of goitrous glands removed after the usual preoperative regimen of iodine administration. The hydrolysis with 2 N NaOH was accomplished as directed by Leland and Foster. Acid precipi- tation was carried out in a volumetric flask at pH 4.5 to 5.0 and the thyroxine content calculated from the determination of the acid-soluble iodine in the clear filtrate, as suggested by Harington

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N. F. Blau

and Randall (5). The values obtained in the latter procedure are roughly 35 per cent higher than those found by the method of ex- traction with butyl alcohol.

Comparing the figures obtained in the barium hydroxide hy- drolysates, one finds somewhat higher thyroxine values by the proposed method (5 to 10 per cent) than by the author’s extraction procedure of 2 N NaOH solutions. This is in agreement with the finding that the use of barium hydroxide, as herein recommended,

TABLE V

Comparison of Thyroxine Values after Ba(OHj2 Hydrolysis with Those Obtained in 2 N NaOH H?l ldrolysates of Thyroid Substance

Pig thyroglobulin.. . Desiccated pig thyroid.. . Commercial thyroid powder.

‘I ‘I ‘I

Fresh pig thyroid.. . . “ “ “ . . .

Desiccated human thyroid..

Fresh human thyroid.. . “ ‘I ‘I

. I‘ ‘I “ ‘I ‘I I‘ . . ‘( C‘ I‘ .

- ‘I f ‘hyroxine 12 as per cent of total 1~ by dif- went methods of separating thyroxine IS

T : a

.t - Tl.

8.05 27.2 31.3 41.8 3.45 28.2 30.4 43.8 3.05 2.2 23.6 26.4 33.8 0.695 24.8 28.5 35.1 0.615 25.2 29.5 41.5 1.41 13.9 16.8 24.2

0.59 24.6 27.4 37.6 0.895 17.6 20.9 24.8 0.735 18.4 20.4 25.1 0.488 20.8 25.4 31.2 1.47 24.2 28.4 36.6

-

Total 12 2 N NaOH hydrolysate per gm.

8 per cent 3a(OH )z hydrol-

ysate

31.9 31.2

28.01 27.7 31.8 29.8

19.2 28.8 24.8

23.9 25.7 29.8

31.5 33.4

27.6 30.8 30.9 32.4

19.8 30.8 24.1

25.7 23.7 32.9

L-

is less destructive of thyroxine than 18 hours boiling with 2 N

NaOH. Of equally great interest is the relatively close agreement between the figures obtained by the two procedures applied to the separation of thyroxine from the barium hydroxide hydrolysates. Adjusting the acidity of these solutions to a pH of 4.5 to 5.0 results in yields of thyroxine differing from those after butyl alcohol ex- traction by only about 10 per cent. More often the discrepancy is much smaller. Great caution, however, is required in carrying

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362 Thyroxine in Thyroid Substance

out the procedure of thyroxine analysis by acid precipitation. For this reason agreement between duplicates is difficult of attainment. The pH of the acidified solution must not be allowed to vary be- yond a restricted range; and since the final thyroxine figure de- pends upon the often small difference between two iodine values, the determination of these, under the best of conditions subject to an error of about 3 per cent, must be executed with extreme care. Analyses of known mixtures of thyroxine and diiodotyrosine have shown that for accurate and readily reproducible results extraction of barium hydroxide hydrolysates with butyl alcohol is to be preferred.

On the other hand, the partition of iodine by means of acid precipitation has, because of its simplicity, much to recommend it where no very great precision is required, as for example, in the chemical assay of commercial thyroid preparations. A number of papers dealing with this important subject have been published during the last few years (6-9). The trend, as expressed by the workers in this field, seems to be fist, to ascribe greater importance to thyroxine content than to total iodine concentration as an index of therapeutic effectiveness, and second to favor the acid precipi- tation method as a more reliable means of standardization. For the latter, the procedure involving hydrolysis with sodium hydrox- ide, as described by Harington and Randall (5), has been used. That there are marked discrepancies between the results of this method and those obtained by the Leland and Foster procedure has been amply demonstrated. Applying the method of acid precipitation to hydrolysates with barium hydroxides of com- mercial thyroid powder, we have found that, observing the pre- cautions already mentioned, it is possible to obtain results which check closely those found after butyl alcohol extraction of the same solution. Neither procedure, however, gave consistent re- sults with thyroid material marketed in tablet form, the former yielding unduly high values for thyroxine, the latter unreasonably low ones. Experiments seemed to indicate that this was probably due to the tenacious adsorption of iodine compounds by the vehicle used in the manufacture of the tablets. A modification of the method will be necessary to overcome this difficulty.

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N. F. Blau

SUMMARY

1. It has been shown that for the extraction of thyroxine with butyl alcohol from an acidified solution of thyroid material, hy- drolysis with alkali need not be as complete as for similar extrac- tion at an alkaline reaction.

2. Barium hydroxide in an 8 per cent solution has been found to be a more effective agent for the hydrolysis of the thyroid sub- stance in connection with thyroxine determination than an equiva- lent concentration of sodium hydroxide, or even 2 N NaOH.

3. On the basis of the finding that it is possible to extract thy- roxine quantitatively from an 8 per cent solution of barium hydrox- ide by means of butyl alcohol, a method has been developed for the determination of thyroxine in thyroid powder and in fresh gland substance.

4. The advantage of the method is a shorter time required to yield results which are probably not over 10 per cent lower than the actual thyroxine content of the material analyzed.

5. Approximately 30 per cent of the total iodine in commercial thyroid powder and desiccated and fresh pig thyroids (summer glands) was found to be in the form of thyroxine.

BIBLIOGRAPHY

1. Lelsnd, J. P., and Foster, G. L., J. Biol. Chem., 96, 165 (1932). 2. Blau, N. F., J. Biol. Chem., 102,269 (1933). 3. Harington, C. R., Biochem. J., 20,293 (19%).

4. Kendall, E. C., Thyroxine, New York, 71 (1929). 5. Harington, C. R., and Randall, S. S., Quart. J. Pharm. and Pharmacol.,

2,501 (1929). 6. Kreitmair, H., 2. ges. exp. Med., 61, 202 (1928); Ergebn. Physicl., 30, 202

(1930). 7. Rotter, G., andMerz, M., Arch. exp. Path. u. Pharmakol., 166,649 (1932). 8. Rotter, G., and Soos, E., Arch. exp. Path. u. Pharmakol., 173, 614 (1933). 9. Cuny, L., and Robert, J., J. pharm. et chim., 18, 233 (1933).

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assistance of Andre C. KibrickNathan F. Blau and With the technical

SUBSTANCETHYROXINE IN THYROID THE DETERMINATION OF

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