166 Thyroxine - The Journal of Biological Chemistry Thyroxine was then boiled with alkali. When pure...

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A METHOD FOR THE DETERMINATION OF THYROXINE IN THE THYROID* BY JESSICA P. LELANDt AND G. L. FOSTER (From the Department of Biological Chemistry, College of Physicians and Surgeons, Columbia University, New York) (Received for publication, November 19, 1931) Since it is now known (1, 2) that the thyroid gland contains at least one iodine compound other than thyroxine, it has become im- portant to know how much of t,he iodine of the gland is in the form of the physiologically active substance, thyroxine. A method for the estimation of thyroxine would be useful in assaying therapeutic preparations as well as in studies of thyroid physiology and pa- thology. Harington and Randall (3) have proposed a method for this purpose which is extremely simple technically, being merely a determination of the total iodine in the acid-insoluble fraction after partial hydrolysis of the gland with sodium hydr0xide.l Although their procedure undoubtedly allows a more satisfactory assay of thyroid preparations than was hitherto possible, nevertheless we feel that it is not sufficiently accurate for certain purposes. Our evidence for this belief will be given in this paper together with the details of a method which appears to determine more accurately the thyroxine in thyroid material. The method is based on the selective extraction of thyroxine by butyl alcohol after alkaline hydrolysis of the gland. Distribution ratios between butyl alcohol and 2 N sodium hydroxide were deter- mined for thyroxine, diiodotyrosine, and inorganic iodide, the three iodine compounds known to be present in an alkaline hydroly- sate of thyroid gland. The results which are shown in Table I * This work was aided by the Research Grant from the Chemical Founda- tion to this Department. t The data in this paper are taken from a thesis submitted by Jessica P. Leland in partial fulfilment of the requirements for the degree of Doctor of Philosophy in the Faculty of Pure Science, Columbia University. 1 See also the earlier work of Wilson and Kendall (4). 165 by guest on June 1, 2018 http://www.jbc.org/ Downloaded from

Transcript of 166 Thyroxine - The Journal of Biological Chemistry Thyroxine was then boiled with alkali. When pure...

A METHOD FOR THE DETERMINATION OF THYROXINE IN THE THYROID*

BY JESSICA P. LELANDt AND G. L. FOSTER

(From the Department of Biological Chemistry, College of Physicians and Surgeons, Columbia University, New York)

(Received for publication, November 19, 1931)

Since it is now known (1, 2) that the thyroid gland contains at least one iodine compound other than thyroxine, it has become im- portant to know how much of t,he iodine of the gland is in the form of the physiologically active substance, thyroxine. A method for the estimation of thyroxine would be useful in assaying therapeutic preparations as well as in studies of thyroid physiology and pa- thology. Harington and Randall (3) have proposed a method for this purpose which is extremely simple technically, being merely a determination of the total iodine in the acid-insoluble fraction after partial hydrolysis of the gland with sodium hydr0xide.l Although their procedure undoubtedly allows a more satisfactory assay of thyroid preparations than was hitherto possible, nevertheless we feel that it is not sufficiently accurate for certain purposes. Our evidence for this belief will be given in this paper together with the details of a method which appears to determine more accurately the thyroxine in thyroid material.

The method is based on the selective extraction of thyroxine by butyl alcohol after alkaline hydrolysis of the gland. Distribution ratios between butyl alcohol and 2 N sodium hydroxide were deter- mined for thyroxine, diiodotyrosine, and inorganic iodide, the three iodine compounds known to be present in an alkaline hydroly- sate of thyroid gland. The results which are shown in Table I

* This work was aided by the Research Grant from the Chemical Founda- tion to this Department.

t The data in this paper are taken from a thesis submitted by Jessica P. Leland in partial fulfilment of the requirements for the degree of Doctor of Philosophy in the Faculty of Pure Science, Columbia University.

1 See also the earlier work of Wilson and Kendall (4).

165

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166 Thyroxine

were obtained at room temperature. Determinations made at, 0’ were found to be practically identical, hence ordinary tempera- ture variations are not important. Furthermore, the ratios are practically the same from both N and 2 N sodium hydroxide. The effect of concentration of the iodine compounds on the ratios was determined in the case of diiodotyrosine and thyroxine where a lo- fold increase in concentration made little or no change in the ratios. In the case of potassium iodide the actual concentrations dealt with in the method to be described are so small that it seems unlikely that the distribution ratio would vary within the ranges of concentration encountered.

Any possible effect on the distribution ratios due to presence of the products of protein hydrolysis was ruled out by determining

TABLE I

Distribution Ratios of Thyrom’ne, Diiodotyrosine, and Potassium Iodide between Butyl Alcohol and 9 N NaOH

Substance concentraf~~t~iin B~OH Approximate concentration

concentration in NaOH at equilibrium

Thyroxine ..................... “ .....................

Diiodotyrosine ................. “ .................

KI .............................

92:8 0.0003 M in BuOH 95:5 0.003 “ “ “

2.5:97.5 0.004 “ “ NaOH 2.0:98.0 0.04 ‘I I‘ ‘I 7.6:92.4 0.007 “ “ “

the ratios for distribution of diiodotyrosine and thyroxine between butyl alcohol and the solution obtained on hydrolyzing 2 gm. of casein in 100 cc. of 2 N NaOH for 18 hours. Ratios of 2.2:97.8 and 94.5:5.5 were found for diiodotyrosine and thyroxine respec- tively, values which are not significantly different from those in Table I.

With these data, it was calculated that 99.0 per cent of the thyroxine, 0.27 per cent of the diiodotyrosine, and 1.64 per cent of the inorganic iodide would remain in the butyl alcohol layer after the following program of extraction.

Shake the 2 N sodium hydroxide solution containing the iodine compounds with an equal volume of butyl alcohol. Separate. Shake the aqueous layer again with a second equal volume of butyl alcohol. Separate. Combine the butyl alcohol extracts and wash

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J. P. Leland and G. L. Foster 167

them with an equal volume of N NaOH. Separate. Extract the sodium hydroxide washings with 0.5 volume of butyl alcohol. Combine the butyl alcohol fractions.

To compare theory with practice, known amounts of the pure substances dissolved in 100 cc. of 2 N NaOH were subjected to the above schedule of extraction and washing and the final butyl alcohol fractions ashed for determination of iodine. The results, shown in Table II, are in good agreement with the theory. The fact that an appreciable fraction of the inorganic iodide remains in the butyl alcohol does not seriously affect the thyroxine determination, for the total amount of iodide in a hydrolysate is small and the fraction of it which remains in the butyl alcohol is negligible.

TABLE II

Observed and Calculated Recoveries after Butyl Alcohol Extraction

Substance

Thyroxine Diiodotyrosine KI Thyroxine in pres-

ence of 8 times as much iodine in form of diiodo- tyrosine

Amount of 1% taken

mJ.

1.065 3.453 4.689 0.431 as thy-

roxine 3.453 as diiodo-

tyrosine

Expecteifon basis

distribution ratio

mg. per cent 1.058 99.0 0.007 0.19 0.077 1.65

0.433

- I Found by analysis

mg. per cent

1.039 97.6 0.010 0.29 0.082 1.68

0.425 0.425 ~ = 98.6 0.431

Hydrolysis of Thyroid Gland

The proposed method postulates hydrolysis of the thyroid sufficiently complete that the thyroxine shall be free from its com- bination in the gland. Since thyroxine is slowly destroyed by heating with alkali, it was necessary to find conditions which would give complete hydrolysis of the protein with minimum destruction of thyroxine. It was necessary to determine the most suitable conditions as to concentration of alkali, concent,ration of protein being hydrolyzed, and time of hydrolysis. The material used in this preliminary work was from a large and uniform lot of com- mercial desiccated thyroid. After hydrolysis under the various conditions, the solutions were extracted according to the scheme

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168 Thyroxine

mentioned above, and the final butyl alcohol extracts containing the thyroxine were ashed for determination of total iodine. The results of this preliminary search for the optimum conditions of hydrolysis are shown in Fig. 1.

Increasing yields of thyroxine were obtained up to 18 hours after which no further gain was apparent. Hydrolysis may be regarded as complete, therefore, at 18 hours but not before that time. Over longer periods of time the destruction of free thyroxine appears to be very slow.

The effect of different concentrations of alkali was found by hydrolyzing 2.5 gm. of desiccated thyroid with 100 cc. of N, 2 N,

and 4 N NaOH for 18 hours. The yields of thyroxine in terms of percentage of total iodine were respectively 16.8, 21.3, and 21.7.

FIG. 1. Curves showing yields of iodine in thyroxine fraction under different conditions and times of hydrolysis. Curve A, 5 gm. of thyroid (Burroughs Wellcome) hydrolyzed with 100 cc. of N NaOH; Curve B, 2.5 gm. hydrolyzed with 100 co. of NNILOH; Curve C, 2.5 gm. hydrolyzed with 100 cc. of 2 N NaOH; Curve D, 1.25 gm. hydrolyzed with 100 cc. of 2 N NaOH.

2 N alkali is thus shown to be a more effective hydrolyzing agent than N alkali. No significant further increase is apparent when 4 N alkali is used.

The yield of thyroxine varies somewhat with the proportion of gland substance to volume of alkali used in the hydrolysis. With 100 cc. of 2 N NaOH and 5, 2.5, 1.25, and 0.75 gm. of thyroid, the yields of iodine in the butyl alcohol fraction were respectively 20.6, 21.3, 23.1, and 22.8 per cent of the total iodine of the gland. The proportion 1.25 gm. to 100 cc. of 2 N alkali was chosen as the most suitable.

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J. P. Leland and G. L. Foster 169

Attempts were made to recover added thyroxine under varying conditions. In each case a known amount (about 1.5 mg.) of pure thyroxine dissolved in 0.1 N NaOH was added to the charge of thyroid substance before hydrolysis.

As shown in Table III the best recovery of added thyroxine was obtained when 2 N alkali was used and the proportion either of 2.5 or 1.25 gm. of thyroid material to 100 cc. of alkali. In an effort to discover why we were able to recover only 83 per cent, a study was made of the effect of boiling alkali on pure thyroxine (see Table IV).

TABLE III

Recovery of Added Thyroxine

Desiccated thyroid

gm. 5 2.5 2.5 1.25

NaOH, 100 cc. Added thyroxine recovered

per cent

69.0 75.8 83.3 83

TABLE IV

E$ect of Boiling Alkali on Pure Thyroxine

Time of boiling I

NaOH Recovery of thyroxine

hrs. N per cent

18 1 95.6 63 1 60.0 18 2 74.6

2 N alkali is seen to be much more destructive to pure thyroxine than N alkali, the recovery being only 74.6 per cent in one case, while it is 95.6 per cent in the other. This, without doubt, accounts for the recovery of only 83 per cent of thyroxine when added to desic- cated thyroid and hydrolyzed for 18 hours. We cannot explain why the recovery of added thyroxine when N alkali is used for hydrolyzing the gland is less than that found when 2 N is used (Table III) ; whereas in Table IV 2 N alkali is shown to be the more destructive to pure thyroxine. To determine whether the 8 per cent difference between the recovery of thyroxine alone and in the presence of gland material was due to some protective action given by the protein itself, thyroxine was added to casein which

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170 Thyroxine

was then boiled with alkali. When pure thyroxine was added to 1.25 gm. of Hammarsten casein and hydrolyzed for 18 hours with 100 cc. of 2 N NaOH, a recovery of 81 per cent was obtained.

Recovery of thyroxine after boiling 18 hours with 2 N sulfuric acid was less favorable than when alkali was used, only 62 per cent being recovered.

From the curves on Fig. 1 it can be seen that to obtain the high- est yield of thyroxine 2 N NaOH must be used as the hydrolytic agent in spite of the fact that serious destruction of the freed thyroxine is indicated from our work with the pure substance. While we are unable to furnish direct evidence t,hat the met,hod which we propose will give us more than 83 per cent of the thyroxine originally present, since we are unable to recover more than 83 per cent of the pure substance when added to thyroid material, we be- lieve that destruction is not nearly so great of t.he thyroxine built into the protein material. From the shape of the four curves in Fig. 1 destruction would seem to be negligible over long periods of time unless hydrolysis and destruction were taking place at exactly the same rate, which seems unlikely.

On the basis of these results, the conditions adopted for the method were as follows:

Method

1.25 gm. of desiccated thyroid are hydrolyzed for 18 hours with 100 cc. of 2 N NaOH in a 300 cc. short neck Kjeldahl flask, fitted with a reflux condenser and heated by a micro burner. (The use of a potassium thiocyanate bath is recommended to prevent over- heating of the bottom of the flask with subsequent breakage during the long hydrolysis.)

After cooling to room temperat’ure t.he hydrolysate is quantita- tively transferred to a 250 cc. separatory funnel and shaken gently for a few minut,es with an equal volume, 100 cc., of butyl alcohol which has been purified by distillation from NaOH. Violent shak- ing causes the formation of emulsions which require a long period of time for separation. After standing 1 hour the layers are sepa- rated, the aqueous layer being transferred to a second 250 cc. separatory funnel, and shaken a second time with 100 cc. of butyl alcohol. After standing again the layers are separated, and t.he first and second butyl alcohol fract’ions are filtered successively through

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J. P. Leland and G. L. Foster 171

glass wool into a 500 cc. separat’ory funnel. The funnels are washed with 5 cc. portions of butyl alcohol. The combined butyl alcohol fractions are shaken with an equal volume, 210 cc., of N

NaOH, allowed to stand, and separated. The NaOH layer is transferred to a second 500 cc. funnel and shaken with one-half its volume, 100 cc., of butyl alcohol, allowed to stand, and separated. The but’yl alcohol fractions are quantitatively transferred to a 500 cc. flask and evaporat.ed under reduced pressure to small volume. The residue is transferred quantitatively to a nickel dish and the flask washed with several small portions of butyl alcohol. 5 cc. of 50 per cent NaOH are added, t,he solution eva,porated on a hot plate until the butyl alcohol is entirely gone, and the residue ashed over a free flame. Care should be taken in ashing to use a low flame, in no case to allow the dish to become red-hot, and to con- sider the ashing complete when the melt has changt 3 from caramel to clear, all bubbling has ceased, and the bothom of the dish has become coated over with nickel oxide. Particles of carbon left in the melt need not be completely ashed as they do not interfere with the determination of iodine.

After cooling, the melt is dissolved in hot water and filtered hot through a Gooch crucible into a 125 cc. Erlenmeyer flask. After thorough washing with hot water (volume 50 to 60 cc.) 2 drops of a 1 per cent solution of sodium bisulfite are added, then 50 per cent HsS04 until acid to methyl orange with 3 drops in excess, and the solution boiled gently for a few minutes. 3 cc. of saturated bromine water are added, the solution boiled until colorless, and cooled under running water. 6 drops of 90 per cent phenol are added to remove t’he last traces of bromine, an excess of potassium iodide crystals is added, and the solution titrated with 0.01 N sodium thiosulfate with starch as an indicator. This strength of thiosulfate has been found to be &able if made up to contain 5 cc. of N NaOH per liter.

In a series of 52 such analyses the mean discrepancy between duplicat’es was 2.0 per cent,, the maximum discrepancy was 5.6 per cent.

Isolation of Pure Thyroxine

As a furt’her check on the validity of the method, we have attempted the isolation of pure thyroxine from the final butyl

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172 Thyroxine

alcohol fraction obtained as above from larger scale hydrolyses in which a specimen of partially purified thyroglobulin was used in- stead of desiccated thyroid. Butyl alcohol takes up a considerable amount of the black, tarry material formed during the alkaline hydrolysis. This tarry material must be removed before the thyroxine can be crystallized, but we were unable to remove it without serious loss of iodine. The best results were obtained in the following manner. The final butyl alcohol layer was evapo- rated under reduced pressure and the residue taken up in water. The hot solution was treated cautiously with barium hydroxide solution till the tar was precipitated, then quickly filtered or cent.rifuged. The clear, yellow, supernatant liquid was acidified just to the turning point of Congo red. The precipitate was col- lected and heated with 40 per cent barium hydroxide for a short time (about an hour) and thereafter the procedure of Harington (5) was followed. The yields of thyroxine obtained in this way were far from quantitative. In the best case 54 per cent of the total iodine in the butyl alcohol fraction was recovered as pure crystalline thyroxine.

A detailed account of this preparation is as follows: 100 gm. of thyroglobulin (570 mg. of 1~) were boiled 18 hours with 2000 cc. of 2 N NaOH. Since this experiment was of a preparative nature and not intended to be a quantitative separation, the hydrolysate was for the sake of simplicity extracted only once with an equal volume of butyl alcohol, from which, on the basis of the distribu- tion ratios, we should expect approximately 90 per cent of the thyroxine and 3 per cent of the diiodotyrosine to pass into the butyl alcohol layer. The butyl alcohol solution was washed with 500 cc. of N NaOH which should have reduced the diiodotyrosine to a negligible amount while removing only about 2 per cent of the thyroxine. The butyl alcohol was removed under reduced pres- sure and the residue taken up in 300 cc. of water. Analysis showed the presence of 105 mg. of iodine. The solution was heated to boiling and treated with saturated barium hydroxide solution till precipitation of the dark pigmented material seemed complete and then quickly filtered while hot. The filtrate, which contained 84 mg. of iodine, was made just acid to Congo red; whereupon all but 3 mg. of the iodine was precipitated. The precipitate was dis- solved in 100 cc. of water with the help of a few drops of ammonia.

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J. P. Leland and G. L. Foster 173

Barium hydroxide to make a 40 per cent solution was added and the solution was heated 1 hour on the steam bath and filtered. The precipitate was saved. The filtrate which contained 26 mg. of iodine was acidified to Congo red with HCl and the resulting pre- cipitate retreated with 40 per cent Ba(OH)2 in a smaller volume. The filtrate from the second crop of barium-insoluble material con- tained only 8 mg. of iodine and was discarded. The two crops of barium-insoluble material were combined and treated with NaOH and Na2S04, according to Haringt.on (5). On acidification of the alkaline filtrate from the barium sulfate, crude thyroxine separated in partly crystalline form. This was purified by dissolving in hot 0.5 per cent sodium carbonate solution and crystallizing out the sodium salt, and finally by crystallizing the free thyroxine from alcohol on acidification with acetic acid. The total yield was 89 mg. of thyroxine which analyzed 98 per cent pure, thereby account- ing for 54 per cent of the total iodine of the butyl alcohol fraction as crystallized thyroxine.

Comparison with Method of Harington and Randall

The method of Harington and Randall is based on the assump- tion that all of the thyroxine and none of the diiodotyrosine is present in the acid-insoluble fraction after partial hydrolysis by alkali. We cannot agree with this. If the acid-insoluble precipi- tate is collected and further hydrolyzed by boiling 18 hours with 2 N NaOH and the resulting solution fractionated with butyl alco- hol, it is found that about half the iodine remains in the aqueous layer; i.e., does not behave as does thyroxine. This fraction of the iodine is in organic combination, is not precipitated by acidifica- tion, and its solution gives a strong nitrous acid reaction; hence we conclude that it represents diiodotyrosine. A description of such an experiment follows.

100 gm. of desiccated thyroid were boiled 4 hours with 1 liter of N NaOH, cooled, and acidified to pH 5 with HCI, allowed to stand 6 hours, and filtered. The precipitate was dissolved in 110 cc. of 2 N NaOH. Analysis of 2 cc. aliquots of this solution showed the presence of 142 mg. of iodine in the acid-insoluble fraction. The solution was boiled 18 hours, and then extracted with butyl alcohol in the manner described. The combined butyl alcohol solutions were evaporated and taken up in 106 cc. of water. This solution

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174 Thyroxine

was found to contain only 70.6 mg. of iodine. Approximately half of Harington and Randall’s acid-insoluble iodine was in the form of thyroxine. The aqueous alkaline layer (volume 100 cc.) after the treatment with butyl alcohol, was acidified to Congo red with HzS04. No precipitate formed, but the solution gave a strong nitrous acid test. Analysis for inorganic iodide by the method previously described (6) showed the presence of only 5 mg. of iodine in this form; the rest, 66 mg., we conclude must have been present as diiodotyrosine.

The figures published by Harington and Randall (2) for t’he thyroxine content of eight samples of commercial thyroid prepara- tion indicate a much higher ratio of thyroxine iodine to total iodine than we found when we applied our method to a series of 52 human thyroids, t,he data of which are given in Table VI. When the same sample of thyroid was analyzed by both met’hods, the results were also quite different,. A sample of commercial thyroid gave by the method of Harington and Randall 45.4 per cent; by our method 23.3 per cent of the total iodine as t,hyroxine iodine. Another sample showed 48 per cent by the procedure of Harington and Randall and 23 per cent by ours.

Thyroxine Content of Normal Human Thyroids

The method was applied to the examination of a series of adult human thyroids, mainly from traumatic cases with sudden death. Histological examination of the glandswas made byDr. A. B. Gut- man of the Department of Medicine of this school, t’o whom we express our sincere thanks. Pieces of the glands removed for histological study (about 0.5 gm. of fresh tissue) are not included in the weights recorded in Table VI.

The glands were kept on ice from autopsy until collection could be made, an interval of from 1 to 3 days. They were then minced finely, dried at 78-80” in an electric oven for 18 hours, and ground to a uniformly fine consistency. They were defatted by extraction with petroleum ether in a Soxhlet apparatus for 9 hours. In a test sample analyses were made of the pet,roleum ether extract and of the desiccated thyroid being extract,ed. The loss of iodine by extraction was negligible.

The glands after defatting were brought to constant weight at 60” in the electric oven and then analyzed according to t,he method

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J. P. Leland and G. L. Foster 175

described above for thyroxine, 1.25 gm. samples being used except in the case of the smallest glands where 1 gm. samples were used. 0.25 gm. samples were used for the determination of total iodine.

Histological examination of the glands showed varying degrees of autolysis, due, no doubt, to the unavoidable delay in the han- dling of the glands. To determine whether the total iodine or the thyroxine content was affected by autolysis, three of the larger glands were divided into two parts after mincing. One-half was dried immediately while the second half was kept 1 week in the ice box (a length of time double the delay to which our glands were subjected) and then dried. Each half was defatted, brought to constant weight, and analyzed in the usual manner. The resu1t.s are shown in Table V.

TABLE V

E#ect of Autolysis of Gland on Total Iodine and Thy-oxine Iodine

Gland No.

44 52 54

Total 12, per cent

Before After

0.106 0.107 0.348 0.351 0.212 0.207

Thyroxine Iz, per cent of total

Before After

20.5 21.5 29.1 29.5 26.4 27.0

The close agreement between the pairs allows us to conclude that neither the total iodine nor the thyroxine content is affected by autolysis.

The detailed results of the analyses are shown in Table VI. We have not attempted to correct the results of thyroxine determina- tions for the presumable 15 per cent destruction of thyroxine during hydrolysis of the gland.

In the series of 52 human thyroids the mean content of total iodine was 0.174 per cent with a mean deviation of 0.066; whereas the percentage of the total iodine which was in the form of thy- roxine showed less scatt,er, the mean being 25.2 per cent with a mean deviation of 4.9.

SUMMARY

1. Thyroxine may be separated from the other iodine compounds which are present in an alkaline hydrolysate of the thyroid gland by extraction with butyl alcohol.

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J. P. Leland and G. L. Foster 179

2. On the basis of this finding, a method for the determination of thyroxine in desiccated thyroid gland is described.

3. There is unavoidable destruction of some of the thyroxine during the alkaline hydrolysis. The amount of this destruction as judged by recovery experiments is not more than 15 per cent of the total amount of thyroxine.

4. In a series of 52 human thyroids the mean thyroxine content (as iodine) was 25.2 per cent of the total iodine (without correction for the presumable 15 per cent destruction of thyroxine during hydrolysis).

BIBLIOGRAPHY

1. Harington, C. R., and Randall, S. S., J. Sot. Chem. Ind., 48,296 (1929). 2. Harington, C. R., and Randall, S. S., Biochem. J., 26,1032 (1931). 3. Harington, C. R., and Randall, S. S., Quart. J. Pharm. and Pharmaeol.,

2, 501 (1929). 4. Wilson, L. B., and Kendall, E. C., Am. J. Med. SC., 161, 79 (1916). 5. Harington, C. R., Biochem. J., 20,293 (1926). 6. Foster, G. L., and Gutman, A. B., J. Biol. Chem., 87,289 (1930).

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Jessica P. Leland and G. L. FosterTHE THYROID

DETERMINATION OF THYROXINE IN A METHOD FOR THE

1932, 95:165-179.J. Biol. Chem. 

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