Determining Serum Protein-Bound Iodine RICHARD D. STRICKLAND and CLARA M. MALONEY Departrnenf of Biochemistry, Veterans Administration Hospital, Albuquerque,
b An improved method for determining protein-bound iodine in serum requires only 0.5 ml. o f serum, can be carried out in less than 3 hours, and has an error of less than 10%. The concentration of cerate is stabilized after the sample i s incubated b y the addition of mercuric mercury, so that final analysis can b e delayed without loss o f accuracy. A titrimetric determination of cerate i s theoretically preferable t o the conventional colorimetric determination. The effects o f varying the concentrations of the constituents of the working reagents are described. The range of concentration of proteinbound iodine in normal subjects is estimated a t 3.60 to 7.36 y per 100 ml.
M
OST m m o m
for determining submicrogram quantities of iodine depend upon measurement of the catalytic effect of iodine monochloride on the rate of the reaction between cerate and arsenite. As chloride and hydrogen ions also affect the rate of this reaction, their concentrations must be carefully controlled before reproducible measurements of iodine concentration can be made. The results of experiments to determine tke optimum concentrations of chloride and acid are given here. The chloric acid digestion method of Zak and coworkers (6-8) permits accurate adjustment of the acidity and chloride concentration of a sample. This method of digestion is preferable to the chromic acid method of Leipert (2) and the permanganate method of Riggs and Man (6) because it obviates the necessity for distilling the iodine. It is superior to the alkaline ashing method of Barker and Humphrey (1) because it is more rapid, the danger of cross contamination of samples is lessened, and $he final concentrations of acid and chloride are more easily controlled. Most procedures for serum proteinbound iodine require measurement of the concentration of cerate which remains in a sample a t once when the timed period of reaction has elapsed. It is more convenient to arrest the reaction by adding mercuric mercury to the reacting mixture (3). Mercury inhibits the catalysis by combining with halides. When a sample has been stopped in this way, the concentration of cerate does not change significantly for several hours. 1870
ANALYTICAL CHEMISTRY
N. M.
The erratic colorimetric behavior of cerate (4), as well as the large inherent error of determination resulting from the inverse relationship of the final concentration of cerate to the concentration of iodine, makes a titrimetric method preferable to the colorimetric method for determining residual cerate. A solution of the stable complex of ferrous iron and 2,2'-bipyridine is an excellent titrant for cerate. Both chloric and perchloric acids are powerful oxidizing agents and strong acids. The precautions given in the directions for their preparation and use must not be disregarded. Laboratory supervisors should make certain that persons working with these acids do not neglect the safety instructions. APPARATUS
Digestion Tubes. Determinations are facilitated by using special borosilicate glass tubes, which serve for centrifuging, digesting, and incubating the samples. Construct them by equipping test tubes (25 mm. wide X 80 mm. deep) with narrow necks (15 mm. wide X 80 mm. long). Graduate the tubes a t the 9-ml. level. Antibump Rods. Fuse a piece of glass tubing (4 mm. in outside diameter) to the end of a glass rod which is 3 mm. in diameter and 180 mm. long. Cut off the glass tube t o leave a shallow cup at the end of the rod. Fire polish the broken ends. Tube Support. Arrange nine 1-inch long sections of ll/,-inch copper tubing in a compact square by standing them side by side with their bores parallel. The square will have three tubes on a side with one tube in the center. Weld the tubes together in this position. Attach a short metal rod t o each corner tube to form legs which support the lower ends of the tubes l l / z inches above the ground.
Perchloric Acid. Use 72%, double vacuum distilled, lead-free perchloric acid (G. Frederick Smith Chemical Co.). Distill this acid, under reduced pressure, in a n all-glass apparatus. This distillation is no more hazardous than t h a t of any other strong acid, but it should be conducted in a n enclosed hood to forestall any damage which might result if the apparatus should collapse because of the vacuum. Potassium Chlorate. Dissolve 570 grams of potassium chlorate (Baker's analyzed reagent, J. T. Baker Chemical Co.) in 1000 ml. of boiling iodinefree Rater. Cool the solution to 0' C. and separate the crystals from the supernatant liquid by filtration through a sintered-glass funnel. Three crystallizations are required to obtain the iodinefree product, There is a 5.7% loss of potassium chlorate with each recrystallization; the amount of water used to dissolve the crystals should be adjusted accordingly for the successive recrystallizations. Sodium Chloride. Prepare SI saturated solution of sodium chloride (analytical reagent, Mallinckrodt Chemical Works) in boiling redistilled water. Continue boiling the solution until it is reduced to one half its original volume. Allow it to cool t o room temperature, then separate the crystals from the supernatant liquid by filtration through a sintered-glass funnel. Repeat this process twice to ensure an iodine-free product. Ninety grams of the salt must be used initially to obtain a yield of 10 grams from the last recrystallization. Other Chemicals. Ammonium tetrasulfatocerate (G. Frederick Smith Chemical Co.), sodium arsenite (analytical reagent, Mallinckrodt Chemical Works) , potassium dichromate (analytical reagent, Mallinckrodt Chemical Works), and sulfuric acid (analytical reagent, Mallinckrodt Chemical Works) can be used without purification. OBSERVATIONS
PURIFICATION O F CHEMICALS
The amount of trace iodine contamination varies greatly among different brands of reagents. The brand names which are given have proved satisfactory in practice; other brands with equally low iodine content may be substituted. Iodine-Free Water. Redistill water over alkaline potassium permanganate in all-glass yapparatus.
Concentration of Cerate. No departures from first-order behavior were observed in the rate of reduction of cerate when the concentration was varied &hin the range of 0.001 to O.1N. Samples which were 0.01N were convenient for titration. Concentration of Chromate. Chromate is used as a n oxidation-reduction indicator. Whenever the yellow chromate begins t o be reduced to the green chromous state during a diges-
tion, there is danger of iodine loss. The amount of chromate can be varied within rather wide limits without affecting the rate of catalysis (8). The chromate should be measured accurately, however, to prevent variations in the amount of arsenite excess; 0.5 X equivalent of chromate is easily visible in a sample, but will not leave excessive color after arsenite reduction. Concentration of Arsenite. Optimum results were obtained when arsenite was present in 7.5-fold excess over the cerate. Since 0.5 X equivalent of arsenite is reduced a t once by the chromate in a sample, 8.0 X equivalent must be added to a sample which will have a final volume of 10 ml., in order to allow the optimum excess when the cerate concentration is to be 0.01N. Concentration of Acid. Of the common acids (sulfuric, hydrochloric, nitric, perchloric, and acetic) only sulfuric acid suited the requirements of the analysis. Figure 1 shows the effect of concentrations of sulfuric acid ranging from 0.5 to 4.OM upon the first-order rate constant for the reduction of cerate by arsenite in the presence of 0.050 y of iodine (equivalent to 0.5-ml. portions of samples containing 10 y of iodine in 100 ml. of serum). When samples are digested, enough perchloric acid is formed by the decomposition of chloric acid to make a sample 0.8111 in perchloric acid after it has been diluted to 10 ml. The presence of this acid must be taken into ac-
count in the preparation of reagents. As the second hydrogen of sulfuric acid does not affect the rate of cerate reduction, compensation must be made on a mole for mole rather than an equivalent for equivalent basis, Concentration of Chloride. Concentrations of chloride ranging from l O - * X to 10-2M were tested to find the strength most suitable for iodine determination. The very low concentration ranges of chloride ryere too difficult to maintain a t controlled amounts, so that reproducible results were hard to obtain; nith the higher concentrations of chloride, a diminished rate of iodine catalysis was encountered. I n the higher ranges of chloride concentration, an over-all acceleration of the ceratearsenite reaction, which bore no relationship to iodine catalysis, was observed. Concentrations of chloride between and 10-3M gave excellent catalytic activity for the measurement of concentrations of iodine in the range of 0.02 to 0.20 y in 10 ml. A 4.0 x l O - 4 X concentration of chloride was chosen as best for use in the deterniination of serum protein-bound iodine. This concentration of chloride yields superior catalytic curves a t all acidities between 2 and 4M. It has been reported that bromide acts as a catalyst for the cerate-arsenite reaction. A sample of ammonium bromide which appeared to give good catalytic curves in conjunction with iodine lost all its apparent catalytic activity after it had been recrystallized
N
0, x X
Figure 1 . Effect of varying concentrations of sulfuric acid on rate of iodine-catalyzed cerate-arsenite reaction Reaction temperature 37" C.
Iodine concentration 0.05 y in 10 ml.
four times. It was concluded that chloride had been present as a contaminant in the original material. Determination of Cerate. Cerate in acid solution can be determined accurately by titration with a n aqueous solution of the stable complex of ferrous iron and 2,2'-bipyridine. S o n e of the substances present in a sample a t the time of titration interfere with the determination of cerate. Iodine Standards. Solutions of 3,sdiiodotyrosine, sodium iodide, and potassium iodate containing equivalent amounts of iodine gave identical results upon analysis. Potassium iodate was selected for use as a standard because the pure compound is easily obtainable. Interfering Substances. Traces of mercury, silver, fluoride, and cyanide inhibit the reaction between cerate and arsenite. The catalytic effect of osmium upon the reaction is well known; ruthenium and iron have a catalyzing effect. The effect of iron is small, b u t iron from the hemoglobin in a hemolyzed sample can cause significantly high results. PREPARATION OF-WORKING REAGENTS
Use iodine-free n-ater to prepare all reagents except where otherwise specified. Dilute Perchloric Acid. Dilute 100 ml. of purified Derchloric acid t o 1000 ml. wiih water.Chromate Solution. Dissolve 0.613 gram of potassium dichromate in water. Dilute t o 250 ml. Chloric Acid. Dissolve 250 grams of purified potassium chlorate in 450 ml. of boiling water. Stir the hot solution constantly while slowly adding 188 ml. of purified perchloric acid. (This should be done in a n area lvhich has been cleared of flammable material. A face shield and a rubberized apron should be worn, as some sputtering will occur.) Allow the mixture to cool, then place it in a deep freeze until its temperature is - 10"C. Filter out the precipitated crystals, using a sintered-glass funnel, while the acid is still cold. New sintered-glass funnels contain an inhibiting substance rvhich interferes with the determination of iodine. JJ7ash the funnel repeatedly, first by running hot nitric acid through it and finally with numerous small portions of chloric acid. Store the chloric acid in a glass-stoppered borosilicate glass reagent bottle. Keep it in a refrigerator. Arsenite Reagent. Place 6.50 grams of sodium arsenite (hraAs02) and 29 mg. of sodium chloride in a 1000-ml. volumetric flask. Dissolve the salts in 350 ml. of water. Add 500 ml. of cool 12.5M sulfuric acid. Dilute the mixture to 1000 ml. with water. Cerate Reagent. I n a 500-ml. volumetric flask, dissolve 31.6 grams of ammonium tetrasulfatocerate by adding 350 ml. of water and 83.5 ml. of concentrated sulfuric acid. Dilute to VOL. 29, NO. 12, DECEMBER 1957
1871
500 nil. and mix. Cool the flask under running water, then adjust the volume with water. Mercuric Nitrate Solution. Dissolve 1 gram of mercuric nitrate in 100 ml. of ordinary distilled water. Add just enough nitric acid t o prevent hydrolysis. Red Solution. Dissolye 1.3 grams of ferrous sulfate (FeSOd. 7Hz0) and 0.72 gram of 2,2'-bipyridine in ordinary distilled nater warmed t o 80' C. Cool the solution and dilute it t o 1000 ml. Iodine Stock Standard. Dissolve 168.5 mg. of reagent grade potassium iodate in water. Dilute t o 1000 ml. in a volumetric flask. This solution contains 10,000 y of iodine per 100 ml. Intermediate Standard. Dilute 10 ml. of the stock iodine standard t o 1000 ml. with water. This solution contains 100 y of iodine per 100 ml. Working Standards. Prepare standards containing 5 and 10 y per 100 ml. by diluting 5- and 10-ml. portions of the intermediate standard t o 100-ml. volumes.
move the tube from the hot plate a t once and add a drop of chloric acid to restore the yellow chromate color. Remove the tubes from the hot plate, allow them to cool until they can be held comfortably, then add 1 ml. of chloric acid to each. Return the tubes to the hot plate. Continue the digestion until yellow beads of condensed liquid form on the sides of the tubes, the yellow chromate color changes to redorange, and the volume of residual liquid in each tube is 1 ml. or less. Allow the tubes to cool to room temperature. Use a pipet to add 8 ml. of arsenite reagent to each tube. Wash
Table I. Precision of Protein-Bound Iodine Determination as Estimated by Duplicate Analyses of Human Sera
Iodine Found,
1872
ANALYTICAL CHEMISTRY
-{/loo
Serum Sample
I 3 88 7.50 4 30 5 80 6 90 4 80 5 34 4.60 4.80 5.97 7.00 6.10 13.70 4,13 6.30 6.30 6.65
1 2
3 4 5 6
PROCEDURE
Pipet 0.5 ml. of the serum to be analyzed into a digestion tube. Precipitate the protein by adding 5 ml. of dilute perchloric acid with a pipet having a large orifice, so that the rapid stream of acid will break the precipitating protein into fine particles. -Mix the acid and precipitate by rotating the tube. Centrifuge the tube a t 2500 r.p.m. for 5 minutes. Decant the supernatant acid and allow the inverted tube to drain upon a piece of filter paper for 5 minutes. Vipe the mouth of the tube nith filter paper. An additional 5 ml. of dilute perchloric acid may be used to wash the precipitate, but washing can be omitted without affecting the accuracy of the determination. Prepare a reagent blank and the standards in digestion tubes. Use 0.5 ml. of iodine-free water for the blank and 0.5-ml. portions of the 5- and 10-y working standards. Omit the protein precipitation and draining procedure described for the sample. T o the standard, blank, and sample tubes add 1 ml. of chromate solution and 5 ml. of chloric acid. Place a boiling rod in each tube. Use the boiling rod in the sample to break up the mass of protein precipitate. From this point, treat the blank, the standards, and the sample in precisely the same way. Conduct the digestion in a fume hood. Place the tubes in a supporting rack upon a n electrical hot plate set to "high." Allow the contents of the tubes to come to boiling. Whenever a tube foams excessively, remove it from the hot plate for a few moments. Continue the boiling until about 1 ml. of liquid remains in each tube. If the yellow chromate shows signs of becoming reduced to the green chromous state a t any time during the course of the digestion, there is a danger of iodine loss. When the color begins to change, re-
down the sides of the tubes with the stream of arsenite from the pipet while making the additions. Mix the contents of each tube thoroughly by lateral shaking. Make sure that none of the yellow digest remains entrapped in the cups of the boiling rods. Remove the boiling rods. As each rod is removed, be careful to shake out the drop of liquid that will be retained in its cup. The amount of liquid which will continue to adhere to the boiling rod (less than 0.02 ml.) can be neglected. Adjust the volume of liquid in each tube to 9 ml. with iodine-free water. Place the tubes in a 37" C. water
-
i9 10 11 12 13 14 15 16 17
111.
I1 3 60 7 25 4 10 5 80 6 69 4.il 5.12 4.30 4.50 5,94 7.20 6.10 13.50 4.13 6.18 6 05 6.50
Differences ( D ) between I and I1 0 28
D -Dm 0 12 0 09 0 04 0 16 0 05
0 25 0 20 0 00 0 21 0.09 0.22 0.30 0.30 0.03 0.20 0.00 0.20 0.00 0.12 0.25 0.15
( D - Om)' 0 0144 0 0081 0 0016 0.0256 0 0025 0,0049 0,0036 0.0196 0,0196 0,0169 0 . 0016 0,0256 0.0016 0.0256 0.0016 0.0081 0.0001
0.07 0.06 0 14 0.14 0.13
n
04
0.iC
0.04 0.16 0.04 0.09 0.01 0.16
Mean difference, D , 0.1810 Sum of ( D - 0,)' Standard deviation of differences (S. D.) AO.106 Maximum difference between duplicate samples (957, confidence level) 0.385 per 100 ml. 957c confidence limits = t X S. D. S.D. = Student's t for 17 samples (16 degrees of freedom) = 2.120 n = number of samples
-,
Table II. Accuracy of Iodine Determination as Estimated by Replicate Analyses of Standard Solutions of Potassium Iodate
Iodine Found in 5
-,/loo
111.
Difference from True Value, D
Iodine Found in 10 -,/I00 111.
Standard
0 2
Standard
5.1 5.4 5 1 5 0 4 9 5 1 4 7 4 9 5.0 4.6 4.8 4.7
0.01 0.18 0.01 0.00 0.01 0.01 0.09 0.01 0.00 0.18 0.04 0.09
10 4
0 1 0 4 0.1 0.0
0.1 0.1
0 3 0.1
0.0 0.4 0.2 0.3
Sum of D -0 7 0.59 Sum of D z Standard deviation of differences (S. D.) &O 224 Maximum error of accuracy ( 95yc confidence level) 10.493 Z(Dz)- (ZD)'/tt S.D. = n = number of samples
0.4
0.3 0.3
9;
9.7 10.3 9.8 9.3 10 2 10 4 10 1 10.4 9.6 9.5
0.3
0.2 0.5 0.2 0.4 0.1 0.4 0.4 0.5
0.16 0.09 0.09 0.09 0.04 0 25 0.04 0.16 0.01 0.16 0.16 0.25
-0.4 1.50
AO.369 AO.812 confidence level = t X S. D. Student's t for 12 samples (11degrees of freedom) = 2.201
bath for 15 minutes in order to bring their contents to constant temperature. At this time place a container of cerate reagent also in the water bath. Place a 1-ml. pipet in the cerate container. Incubate the samples for a fixed, accurately measured length of time after the cerate is added. The optimum incubation period depends upon the quality of the reagents; with good reagents, 15 or 20 minutes is satisfactory. Add 1 ml. of the warm cerate reagent to successive tubes a t 1-minute intervals, mixing the contents of each tube thoroughly immediately after the addition of cerate. The 1-minute interval is allowed to provide time for individual manipulation of the tubes in the later stages of the procedure. K h e n the incubation period has elapsed for a tube, transfer its contents to a 125-ml. Erlenmeyer flask to which has been added 2 drops of mercuric nitrate solution. Rinse the tube with three 5-ml. portions of iodine-free mater, transferring the nashings to the Erlenmeyer flask. The mercury inhibits the cerate-arsenite reaction by combining nith the iodine catalyst. so that the titration can be delayed for an hour or more n ithout impairing the results of the analysis. Mercuric nitrate must not be allowed to enter the digestion tubes. T i hen all samples have been transferred to the Erlenmeyer flasks, titrate them with red solution. During the course of a titration. the color of a sample changes progressively from cerate yellow through green and sky blue to red or purple a t the end point. The end point change requires no more than 0.0%ml. of red solution. CALCULATIONS
The amount of iodine in a sample can be established by comparing the volume of red solution used in this titration with the volumes used in titrating the standards, either graphically or by direct calculation. Use semilogarithmic paper to plot the volume of red solution against the concentration of iodine in each standard. Treat the blank as a standard containing zero concentration of iodine, Show the volumes of red solution on the logarithmic scale. The points should fall in a straight line. Direct calculation of the concentration of iodine in the sample is preferable to the graphical method. Divide the volume of red solut'ion used for a st'andard into the volume used for the reagent blank. Find the logarit'hm of the quotient. Divide the concentration in micrograms per 100 ml. of iodine in the standard by this logarithm. The result is a constant which is independent of the iodine concentration, varying only with differences in the reagents and (inversely) with the incubation time. Obt,ain t,he constant for the other standard in the same way. The two constant,s should be identical; where the$differ by more than 570, a det'ermina-
tional error should be suspected. Use the average of the two constants to calculate the iodine in the samples. Divide the volume used for the reagent blank by the volume of red solution used for a sample. Obtain the logarithm of this quotient. Multiply the logarithm by the average of the constants. The product is the amount of iodine in micrograms per 100 nil. of serum.
Table Ill, Concentration of ProteinBound Iodine in Sera from Clinically Normal Adults of Both Sexes ProteinBound Iodine,
Deviation from Mean, D
0 2
1 2 3 4 5
4.9 4.5 4.7 5.7 5.1
0.58 0.98 0.78 0.22 0.38
0.3364 0.9604 0.6084 0.0484 0.1444
6r 9 10
i
5.9 3.9 4.7 4.9 4.2
0.42 1.58 0.78 0.58 1.28
0.1764 2.4964 0 6084 0 3364 1 6384
11 12 13 14 15
5.8 4.9 7.0 5.9 6.6
0.32 0.58 1.52 0.42 1.12
0 0 2 0 1
16 17 18 19 20
4.7 6.9 5.7 6.0 6.3
0.78 1.42 0.22 0.52 0.82
0.6084 2.0164 0.0484 0.2704 0.6724
21 22 23 24 25
6 3 6 4 5 7 6 3 3.9
0 82 0 92 0.22 0 82 1.58
0 6724 0 8464 0 0484 0 6i24 2.4964
Serum Sample -,/100 111.
1024 3364 3104 1764 2544
Sum of D 2 19.8860 Standard deviation (S.D.) f 0 ,91 Standard error of mean (S.E.)f O .18 5.48 i 1.88 = 3.60 Range of normal concentrations to 7.36 -,/lo0 ml. (957c confidence limits): S. D. = n-1 S.E. = --S. D.
dn
n. = number of samples 95'-J'o confidence limits = 6 . D. X t Student's t for 25 samples (24 degrees o f freedom) = 2.064
DISCUSSION
Precision. T h e standard deviation of t h e differences between the results of duplicate determinations of 17 randomly selected samples v-as 1 0 . 1 0 6 y per 100 ml. (Table I). This allows a n error of precision a t the 9570 confidence level which does net exceed 0.385 y per 100 nil. for duplicate samples.
Accuracy. The accuracy of the determination was estimated by replicate analyses of potassium iodate standards containing 5 and 10 y of iodine per 100 ml. (Table 11). Twelve replicates were analyzed a t each concentration. The standard deviation a t the 5 y per 100 ml. concentration !vas A0.224, allon-ing an error of accuracy not to exceed 0.193 y per 100 ml. a t the 95% confidence level. The standard deviation a t the 10 y per 100-nil. concentration was h0.369, allowing an error of accuracy not to exceed 0.812 y per 100 ml. a t the 95% confidence level. Normal Range. The concentrat,ions of protein-bound iodine in samples of serum taken from 25 clinically normal adults were determined (Table 111). The mean concentration was 5.50 y per 100 ml. with a standard deviation of iO.91 y per 100 nil. Kit,hin 95% confidence limits, this allows a normal range of 3.60 t o 7.36 y per 100 ml. Time Required for Determination. A blank and tn-o standards should be prepared n-henever a group of samples is t o be analyzed. Preparation of t h e blank and standards requires 2 hours; a n additional 10 minutes should be allon-ed for each sample in the group. Washing Apparatus. When apparatus becomes contaminated with mercury (this happens when a n attempt) is made to analyze serum from a patient n-ho is receiving mercurial diuretics). it can be cleaned by washing with concentrated nitric acid and rinsing n i t h a dilute solution of sodium chloride. After the apparatus has been freed of mercury, it should be rinsed seven t,inics with iodine-free water to removo contaminating iodine.
LITERATURE CITED
(1) Barker,
S. B., Humphrey, 11. J..
Soley, 31. H., J . C2in. Invest. 30, 55 (1951). ( 2 ) Leipert, T., Biochem. 2. 261, 437 (1933". (3) AIeyer, Iloran, J. J., ANAL. Cmar. 24, 381 ( 1952) , ( 5 ) Riggs, D. S.,>Ian, E. B., J . Bioi. Chejji. 134, 193 (1940). (6) Z a k , B., Boyle, A . J., Ani. J . Glia. Patho/. 61, 260 (1952). ( 7 ) Zak, B., Koen, .i. AI., Boyle, 1.J.. Zbid., 23, 603 (1953). (8) Zak, B., Willard, H. H., Myers, G. B.. Boyle, .I,J.,As.41,. CHEM.24, 1345 (19521.
RECEIVED for review h-ovember 20, 1956. Accepted July 5, 1957. VOL. 29, NO. 12, DECEMBER 1957
1873
