PRECIPITATION BY TRICHLOROACETIC ACID AS A SIMPLIFICATION IN THE DETERMINATION OF

ZINC IN BLOOD AND ITS COMPONENTS

BY FREDERIC L. HOCH* AND BERT L. VALLEEt

(From the Department of Biology, Massachusetts Institute of Technology, Cambridge, and the Laboratory of Clinical Physiology, McLean Hospital, Waverley,

Massachusetts)

(Received for publication, July 6, 1949)

Recent studies have established the importance of zinc as a biological constituent in human blood. Normal zinc levels for human whole blood, plasma, erythrocytes, and leucocytes have been established (1) ; significant deviations from the norm occur in leucemia and pernicious anemia, and, possibly, in sickle-cell anemia (24). Zinc concentration is considered an expression of the carbonic anhydrase content of erythrocytes (5).

The method employed for zinc analysis in these earlier studies was accurate for samples of biological material containing between 2 and 30 y of zinc, with a standard error of about f10 per cent (6). Its usefulness for the study of blood is limited by the costly and specialized equipment necessary and by the small number of samples which can be analyzed daily.

These objections to the earlier method have now been obviated by pro- tein precipitation with trichloroacetic acid, making zinc quantitatively available for measurement with diphenylthiocarbazone. Zinc determina- tions, after ashing at 500” (6), have also been made on all samples so studied.

Procedure

Glassware was cleaned as previously described (6). Water from a Barnstead still’ was used throughout; repeated tests showed it to be zinc- free.

Reagents- 1. Diphenylthiocarbazone (Eastman Kodak). A solution of 20 mg. in

200 ml. of Ccl,, c.P., stored at 4-6” in the dark; dilutions are made as re- quired.

2. Buffer solution. 1100 gm. of Na&&03, c.P., 180 gm. of CH&OONa,

* Postdoctorate Research Fellow of the United States Public Health Service. t Senior Research Fellow of the Committee on Growth of the National Research

Council, supported by the American Cancer Society. 1 Manufacturer’s catalogue No. SMH-5, Barnstead Still and Sterilizer Company,

Boston. 296

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296 DETERMINATION OF BLOOD ZINC

c.P., and 20 gm. of KCN, c.P., are dissolved in about 2000 ml. of zinc-free HzO. The pH is adjusted to 5.5 by the addition of glacial acetic acid, with use of a Beckman pH meter for control, and the volume brought to 4000 ml. The buffer is extracted with dithizone in a large Squibb separatory funnel, and all the zinc present in the reagents is thus removed.

3. Tartrate solution. 20 per cent KNaC4H406*4Hz0, c.P., in distilled water; zinc is extracted as above.

4. Concentrated NH,OH. NHhOH, c.P., is distilled from an all Pyrex still. Since ammonia extracts zinc, forming a tetra-coordinated complex ion, Zn(NH&++, even from Pyrex glassware, it must be freshly prepared at weekly intervals, at least, and stored at low temperature.

5. Chlorophenol red. 0.1 gm. per cent in zinc-free distilled HSO. 6. 6 N HCI. Distilled in an all Pyrex still from 6 N HCI, c.P., and

stored in clean Pyrex containers. 7. Trichloroacetic acid. Recrystallized and stored at room tempera-

ture in a dark paraffin-sealed bottle. 200 ml. of a 20 per cent solution in distilled Hz0 are made up as needed.

Preparation of Biological Materials-Human plasma, erythrocytes, and leucocytes were stored in clean Pyrex containers at 4-6”. Aliquots were prepared for zinc analysis by two methods: (1) trichloroacetic acid precipi- tation of proteins with subsequent analysis of the supernatant fluid, and (2) ashing in platinum crucibles at 500” as described in the earlier method (6).

Plasma-Plasma was used undiluted. Lot A was human plasma ob- tained by centrifugation of fresh whole blood. Lot B was reconstituted dried human plasma.

Erythrocytes-Homogeneous samples are necessary, and since it is dif- ficult to obtain such samples of intact erythrocytes, the following procedures were adopted:

Red cells were separated from fresh titrated whole blood by centrifuga- tion; the citrate had previously been extracted with dithizone for zinc. An arbitrary quantity of erythrocytes was spun down in a 15 ml. graduated Pyrex tube, and the volume of packed cells was noted. The small amount of plasma still present was removed. The cells were then washed and spun down three times with a zinc-free isotonic NaCl solution, lyzed with distilled water, transferred to a 200 ml. volumetric flask, and made up to volume with water. In this fashion, homogeneous samples were obtained, the total erythrocyte content of which was known. Several such erythrocyte solutions were prepared containing from 0.141 ml. to 0.232 ml. of red blood cells per ml. of solution.

Alcohol-chloroform precipitation of hemoglobin (7) was used as an al- ternative method for obtaining homogeneous erythrocyte lysates. This

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F. L. HOCH AND B. L. VALLEE 297

hemoglobin-free erythrocyte lysate contains zinc as carbonic anhydrase (8), while most of the erythrocyte protein has been removed. This prep- aration was analyzed.

Leucocytes-250 ml. of human leucocytes were obtained by pouring off the buffy coats of titrated blood after the removal of plasma.2 This suspension contained large numbers of erythrocytes, which were removed by lysis with water. The resulting leucocyte suspension was allowed to stand for 3 weeks at 4”. On microscopic examination the cells had completely disintegrated. Nevertheless, due to coagulation, particulate matter was still present. This suspension was diluted 1: 1 with water and dispersed by stirring, and aliquots were used for analysis. Because of the nature of the solution, these aliquots were not homogeneous.

Method

2 ml. samples of the stock solution were measured into 15 ml. numbered Pyrex centrifuge tubes, and 1 ml. of zinc-free 6 N HCl was delivered into a correspondingly numbered set of Pyrex test-tubes.

To each centrifuge tube, 2 ml. of 20 per cent trichloroacetic acid (TCA) was added, immediately following which a white precipitate formed. The samples were incubated at 90” in a water bath for 5 minutes, with frequent stirring until the precipitates were finely divided. The tubes were cooled and centrifuged at 3000 R.P.M. for 20 minutes. The clear supernatant fluids were decanted into their correspondingly numbered test-tubes and placed in the water bath at 90”.

This procedure, at first, was repeated on the precipitate from one to four times, 2 ml. of 10 per cent TCA being used in all precipitations after the first. A triple precipitation was finally adopted. 2 ml. of 20 per cent TCA were used in the first precipitation, and 2 ml. of 10 per cent TCA were used in the second and third precipitations.

Following incubation for 1 hour at 90”, the combined brownish superna- tants were decanted into 125 ml. Squibb separatory funnels. The test- tubes were washed three times with hot distilled HzO, which was added to the contents of the separatory funnels.

2 ml. of tartrate solution were placed in the funnels, and the pH was adjusted to approximately 5.5 by adding sufficient amounts of concentrated zinc-free NH40H, with use of chlorophenol red as an indicator. The appearance of a red-violet color was the end-point. 50 ml. of buffer solu- tion were then added. Both a reagent blank and a measured amount of zinc standard solution were analyzed with the daily run of biological samples.

*We are indebted to Dr. Robert Pennell of Sharp and Dohme, Glenolden, Penn- sylvania, for the generous supply of human leucocytes.

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298 DETERMINATION OF BLOOD ZINC

Extraction of zinc with dithizone in carbon tetrachloride was performed as described (6). Carbon tetrachloride solutions were transferred to vol- umetric flasks and made up to volume. Further dilutions, when necessary for calorimetry, were made directly in the Coleman cuvettes.

Calorimetry-The samples were read on a Coleman universal spectro- photometer, model 14, with a minimum of delay after extraction. Dithi- zone has two absorption maxima, at 625 and at 440 mp. The absorption of zinc dithizonate at 625 rnp is negligible. Zinc dithizonate has an absorption maximum at 525 rnp and at this wave-length dithizone has an

TABLE I

Determination of Ratio of Optical Density of Dithizone at 6.26 rnp to That at 6%V mp; Random Dilutions of Dithizone in CCL

pbs. 625

pbs. 525 R

0.740 0.288 0.372 0.960 0.368 0.555 0.870 0.980 0.760 1.150 0.770 0.513 0.328

Mean. . . . . . . S.D.......................

0.180 0.075 0.100 0.225 0.094 0.142 0.215 0.243 0.182 0.276 0.190 0.131 0.087

4.11 3.84 3.72 4.26 3.92 3.91 4.05 4.03 4.17 4.17 4.05 3.91 3.78

3.98 f0.16

appreciable absorption. Table I shows the ratio (R) of the optical densities of dithizone solutions at 625 rnp to those at 525 rnp. This ratio was ob- served to vary between 3.5 and 4.0 for different batches of dithizone, and was determined routinely with each set of zinc analyses.

The zinc content of each sample is determined from the following equa- tions. Calculations were carried out as previously described (6) and are here presented again for convenience.

DXK Zn = E:$ x loo

- V

where Zn = zinc in y in entire sample, l$$k = Ei$ - (E$$/R), where

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F. I,. HOCH AND B. L. VALLEE 299

E$t is the optical density observed at 525 rnp, and E$t is the optical density at 625 rnp. D = dilution of the original extracted volume of dithizone solution. v = original extracted volume of dithizone solution. K = a calibration constant, which was determined from the equation

A series of zinc standards was extracted with dithizone and read on the Coleman spectrophotometer. By substituting in equation (2) K was found to be 62.6 f 1.6 (Table II).

TABLE II Determination of Calibration Constant, K

Zinc pbs. 525

Bobs. 625

pbs. 625 R

pr. 525

Zn=K jfo’.

52.5

79Yl 52.7 35.2 23.4 16.6 10.4 6.95 4.64 3.10

-

-

1.450 0.890 0.222 1.228 64.4 0.950 0.565 0.141 0.809 65.1 0.646 0.377 0.094 0.552 63.8 0.440 0.251 0.063 0.377 62.2 0.289 0.166 0.042 0.247 63.3 0.202 0.112 0.028 0.174 59.9 0.132 0.074 0.019 0.113 61.5 0.088 0.051 0.013 0.075 61.9 0.059 0.032 0.008 0.051 60.8

Mean...... 62.6 SD.. . . . . . . f1.6

Results

Standards-Thirty-seven consecutive samples of ZnC& standards, gravi- metrically determined as 4 y of zinc, were analyzed by the dithizone method. The results ranged from 3.6 to 4.8 y of zinc with a mean of 4.2 y of zinc and with a standard deviation of ~0.31 y.

Plasma-Analysis of a stock of fresh human plasma (Table III) by triple TCA precipitation gave a zinc content of 2.4 f 0.45 y per ml. Anal- ysis of the same stock by dry ashing at 500” gave 1.6 f 0.42 y of zinc per ml. The stock of reconstituted human plasma, when analyzed by triple TCA precipitation, contained 3.6 f 0.65 y of zinc per ml. while by dry ashing it contained 3.9 f 0.99 y of zinc per ml. (Table III). 2 ml. samples of plasma were used in the TCA precipitation in both these series. When larger plasma samples (5 ml.) were analyzed by the same procedure,

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TABLE III

Zinc Anulysis of Human Plasma by Triple TCA Precipitation and by Ashing

__-- TCA precipitation

I Dry ashing

Total Zn Zn per ml. 1 Sample / Total Zn / Znperml.

Mean.... S.D..

ml.

2 2 2 2 2 2 2 2 2 2 2 2 2 2

Fres plasma; L

Y Y

4.6 2.3 4.6 2.3 3.8 1.9 3.1 1.6 3.6 1.8 6.1 3.0 4.1 2.1 5.4 2.7 4.4 2.2 5.6 2.8 4.5 2.2 5.7 2.9 5.7 2.8 6.0 3.0

2.4 f0.45

Reconstituted plasm

3 2 7.3 3.7 3 2 7.5 3.7 3 2 6.5 3.3

3 2 6.3 3.1 3 2 5.0 2.5 3 2 5.2 2.6 3 2 7.0 3.5 3 2 6.5 3.3 3 2 7.7 3.8 3 2 8.8 4.4 3 2 6.0 3.0 3 2 7.9 4.0

3 2 8.1 4.0 3 2 9.5 4.8 3 2 9.1 4.5

3 2 6.0 3.0 3 2 7.7 3.9

Mean.... S.D..

3.6 ho.65

300

tot

- Ia:

- ,A

n&l.

2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 5 5

: Lot B

Y Y

3.9 1.9 4.0 2.0 1.7 0.9 3.5 1.7 3.4 1.7 3.6 1.8 2.0 1.0 2.3 1.2 3.4 1.7 2.4 1.2 4.0 2.0 4.8 2.4 3.5 1.7 3.0 1.5 1.5 0.8 9.1 1.8 9.6 1.9

I-

I 2 2 2 2 5 5 5 5 5 r

8 5

10 10

___- -

4.9 4.9 8.2 4.1 8.7 4.4 8.8 4.4

11.3 5.6 20.1 4.0 19.9 4.0 23.5 4.7 16.0 3.2 19.1 3.8 17.7 3.7 21.6 4.3 18.5 3.7 19.0 1.9 16.5 1.7

3.9 f0.99

~- -

1.6 ho.42

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F. L. HOC11 .\SD IS. I,. \‘.\I,LP:E 301

which employs a total of 4 ml. of 20 per cent TCA, the zinc was incompletely recovered. The residual zinc was recovered from the protein residues by ashing. The ashed protein residues of 2 ml. samples contained no zinc.

When amounts of zinc from 2 to 20 y were added as ZnCk to 2 ml. fresh plasma samples, complete zinc recovery was achieved by routine triple TCA precipitation.

The effect of variation in the number of TCA precipitations is shown in Table IV. 2 ml. samples of fresh plasma were used. Single TCA pre-

TABLE IV

Zinc Analysis of Fresh Human Plasma by Single, Double, and Quadruple TCA Precipitation on 8 Ml. Samples

Single precipitation Double precipitation /( luadruple precipitation ._~._~ ..-.~~-.__--- -

Total Zn ..~ ~_._----~- -~-- .-~~--

7

3.2 3.5 6.0 4.9 3.3 3.1 3.2 3.4

.--.- --.- __ ----. Mean......................... S.D.....,.......,..............

I

Zn per ml.

Y

1.6 1.8 3.0 2.4 1.7 1.5 1.6 1.7

Y

5.7 6.6 4.5 5.0 3.7 5.0 2.9 2.8 3.1 3.4 4.1 3.7

Y

2.8 3.3 2.3 2.5 1.8 2.5 1.5 1.4 1.5 1.7 2.1 1.8

1.9 2.1 f0.49 ho.56

$?J deviation from triple precipi- tation mean (Table III). , -25

-.~ Total Zn h per ml. Total Zn

Y

6.3 4.9 4.3 5.0 4.8 5.4

-15 -

Zn per ml.

Y

3.1 2.5 2.1 2.5 2.4 2.6

---~~ -. ._

2.5

+5

cipitation (2 ml. of 20 per cent TCA) resulted in 75 per cent zinc recovery as compared to triple precipitation, and double TCA precipitation (3 ml. of 20 per cent TCA) gave 85 per cent zinc recovery. Quadruple TCA precipitation gave zinc values 5 per cent above the triple precipitation values. When the protein residues from a single TCA precipitation were merely washed twice with 2 ml. of water and the supernatants combined as usual, no additional zinc was recovered.

Erythrocytes-An erythrocyte solution containing 0.141 ml. of packed red blood cells per ml. of solution, when analyzed by triple TCA precipita- tion, contained I .7 f 0.22 gm. of zinc per ml. of solution, corresponding

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302 DETERMINATION OF BLOOD ZINC

TABLE V

Zinc Analysis of Erythrocyte Lysate Containing 0.141 Ml. of Packed Red Blood Cells per Ml. of Sample, Determined by TCA Precipitation

and by Ashing

TCA precipitation --

Sample ~_.-.-___

ml.

2 2 2 2 2 2 2 2 2 2 2

Mean................ S.D.. . . . . . . . . .

-

1 1 1 1 1

Mean................ --

2 2 2 2 2

Mean................

1 1

---- Mean................ S.D..................

h of pre- :ipitations

3 3 3 3 3 3 3 3 3 3 3

-- 3 3 3 3 3

Total Zn !n per ml.

-t Y

3.6 1.8 3.4 1.7 4.0 2.0 3.6 1.8 2.4 1.2 3.4 1.7 3.6 1.8 3.5 1.8 3.9 1.9 3.2 1.6 2.8 1.4

1.7 f0.22

2.6 2.6 1.7 1.7 1.5 1.5 2.3 2.3 1.7 1.7

3.8 1.9 3.9 1.9 2.3 1.2 4.6 2.3 3.8 1.9

1.8

2.1 1.9

2.1 1.9

2.0

2.0

Dry ashing

Sample

ml.

2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 5 5 1 1

Total Zn

7

2.6 2.3 1.8 2.3 2.2 2.2 2.1 3.2 3.7 3.5 2.8 3.1 3.5 2.8 4.7 8.6 6.2 3.4 3.9

-

- h per ml. --

7

1.3 1.2 0.9 1.2 1.1 1.1 1.1 1.6 1.8 1.8 1.4 1.5 1.8 1.4 2.4 1.7 1.2 3.4 3.9

to 12.1 y of zinc per ml. of packed red blood cells. Ashing of aliquots of this solution gave a value of 1.7 f 0.7 y of zinc per ml. of solution (Table V).

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F. L. HOCH AND B. L. VALLEE 303

The effect of varying the size of the sample and the number of TCA pre- cipitations is also shown in Table V.

Table VI shows the results of analysis of a hemoglobin-free erythrocyte lysate by TCA precipitation and by ashing.

TABLE VI

Zinc Analysis of Hemoglobin-Free Erythrocyte Lysate

Method of analysis

TCA, 1 precipitation

TCA, 2 precipitations

TCA, 3 precipitations

Dry ashing

I- -~

Total 7% Zn per ml.

Y Y

9.1 4.5 9.2 4.6 9.1 4.6

10.5 5.2 10.3 5.2

9.9 4.9 10.4 5.2

9.8 4.9 10.3 5.2 10.5 5.2

Ml. Sample)

TABLE VII

Zinc Analysis of Leucocyte Suspension by Triple TCA Precipitation and by Ash&g

ml.

2 2 2 2 2 2 2 2

10 10

-- I

-- ___--

Mean. .......... S.D ..............

TCA precipitation Dry ashing

Total Zn Zn per ml. Total Zn Zn per ml.

Y Y

6.9 3.5 6.5 3.3 8.2 4.1

10.3 5.1 6.2 3.1 6.5 3.2 7.9 4.0 5.4 2.7

---- .-

7 Y

6.5 3.3 7.3 3.6 6.6 3.3

15.5 7.7 9.6 4.8

14.7 7.3 4.3 2.2

11.3 5.6 25.2 2.5 24.7 2.5

-__---- _- --- 3.6 4.3

f0.7 f1.8

Leucocytes-Triple TCA precipitation gave 3.6 f 0.7 y of zinc per ml. of suspension; ashing gave 4.3 X!Z 1.8 y of zinc per ml. (Table VII).

DISCUSSION

Before any data obtained with the TCA precipitation could be evaluated, contamination of reagents and glassware had to be controlled by measure-

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304 DETERMINATION OF BLOOD ZINC

ments of “blanks.” The blank was composed of all chemicals in the pro- portions and quantities used in the analysis of the biological samples, with randomly selected glassware. A “standard” was designed to check the reproducibility of experimental conditions from day to day, and the extrac- tion of a 4 y standard was taken as evidence that no variation in the stand- ard solution, the extraction procedure, or the calorimetry had occurred. The standard in thirty-seven consecutive samples was 4.2 f 0.31 y.

When the supernatant from the TCA-precipitated proteins is shaken with dithizone in CC14, the CC14 phase is emulsified in the HZ0 phase. It collects only incompletely into a separate phase after a period of several hours. Under these conditions quantitative extraction of zinc is not pos- sible. Incubation of the supernatant with 1 ml. of 6 N HCl prior to ex- traction results in complete separation of the two phases. Presumably, inadequate phase separation is due to protein degradation products of high molecular weight, which are further reduced in size by HCI.

We have substituted chlorophenol red for methyl red, which was used previously in this technique (6). Methyl red is extracted into the Ccl, phase and its calorimetric absorption may cause positive errors in zinc determination. Moreover, the methyl red range lies between pH 4.4 (red) and 6.0 (yellow), and the “peach color” supposed to occur at pH 5.5 is not distinct and is subject to observational error. On the contrary, chlorophenol red is not CC&-soluble and abruptly changes from yellow to violet at pH 5.5, the critical pH of the procedure.

The results of the analyses of blood components by triple TCA precipi- tation show excellent recovery of zinc. The standard deviations for the TCA series and for ashing controls are comparable. It is apparent that the standard deviations of the ashing series are greater, in all instances but one, than those obtained by TCA precipitation. The consistently nar- rower spread of the TCA values indicates better reproducibility. Still better reproducibility could be obtained in both the TCA and the ashing methods when larger samples were analyzed, but for ease of handling and for economy of materials small samples seemed preferable.

The mean values obtained by these two methods are not significantly different statistically, except in the case of fresh human plasma, for which the mean value by triple TCA precipitation is significantly higher than the ashing mean. We are unable to explain the discrepancy in this one series. Contamination was excluded as a source of error, but it is possible that zinc was lost from the ashed samples by volatilization.

That all the zinc was recovered by TCA precipitation is further demon- strated by the complete recovery of known amounts of zinc added to plasma samples, and by the absence of zinc from the protein residues after triple acid precipitation. It is realized that these series presented are small for

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P. L. HOCH 9ND IS. L. VALLEE 305

statistical evaluation. However, the means and standard deviations for plasma and erythrocytes are in good agreement with the larger series previously obtained by ashing (1).

With single precipitation only 75 per cent, and with double precipitation 85 per cent, of the total zinc in 2 ml. samples was recovered. Quadruple precipitation, however, resulted in no appreciably greater zinc recoveries than triple precipitation. When 5 ml. samples were triply precipitated with the same amounts of TCA as used on 2 ml. samples, not all the zinc was recovered; the deficit could be demonstrated, by ashing, to be in the protein residue. It is, therefore, evident that a total of 4 ml. of 20 per cent TCA is not adequate to split off all the zinc from 5 ml. samples of the type of biological substrate employed in these studies. For 2 ml. samples, triple precipitation is optimal.

In the case of the hemoglobin-free erythrocyte lysate, 2 ml. samples treated by double acid precipitation showed zinc values identical with similar triply precipitated samples, or ashed samples. These samples had a smaller amount of protein residue than the other materials handled.

The data presented point to a possible relationship between the amount of TCA necessary for complete zinc liberation and the protein content of the sample. Though these data were not specifically designed to answer this question, it would seem that this is indicated, on one hand, by the smaller amount of TCA necessary to liberate zinc from samples of low protein content (hemoglobin-free erythrocyte lysate), and, on the other hand, by the incomplete recovery of zinc from larger samples.

The greatest variation in results is noted in both the TCA-precipitated and the ashed leucocyte samples, although the means are comparable. This stock solution was noted to contain undissolved material, and was not homogeneous, giving aliquots that were not strictly comparable. They were, nevertheless, utilized to demonstrate complete zinc recovery from white blood cells by the TCA method.

It has been shown that zinc exists in blood components in several states (3), and it is noteworthy that this TCA procedure will liberate zinc quanti- tatively from its various forms of combination in plasma, erythrocytes, and leucocytes. This may be of value in elucidating the manner of binding of zinc in zinc proteins.

As has been pointed out, the ashing technique requires expensive plat- inum crucibles and muffle furnaces. The time-consuming nature of the procedure, as well as the expense, limits its general applicability to clinical and research work. The TCA method requires no specialized equipment. It is more reproducible, and therefore more accurat,e, than the ashing procedure. Double the daily number of analyses, or more, can be per- formed without additional effort.

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306 DETERMINATION OF BLOOD ZINC

SUMMARY

1. A method for zinc analysis by TCA precipitation applicable to ery- throcytes, leucocytes, and plasma is presented.

2. The technique is accurate for samples containing as little as 1 y of zinc.

3. The procedure is more accurate than dry ashing and is more rapid; it is well suited for general clinical use.

We wish to express our gratitude to Dr. John R. Loofbourow for the advice and encouragement he has given so unstintingly.

BIBLIOGRAPHY

1. Vallee, B. L., and Gibson, J. G., 2nd, J. Biol. Chem., 176, 445 (1948). 2. Vallee, B. L., J. Clin. Invest., 27,559 (1948) 3. Vallee, B. L., and Altschule, M. D., Blood, 4,398 (1949). 4. Vallee, B. L., and Gibson, J. G., 2nd, Blood, 4, 455 (1949). 5. Vallee, B. L., Lewis, H., Altschule, M. D., and Gibson, J. G., 2nd, Blood, 4, 467

(1949). 6. Vallee, B. L., and Gibson, J. G., 2nd, J. Biol. Chem., 176,435 (1948). 7. Tsuchihashi, M., Biochem. Z., 140, 63 (1923). 8. Keilin, D., and Mann, T., Biochem. J., 34, 1163 (1940).

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Frederic L. Hoch and Bert L. ValleeAND ITS COMPONENTS

DETERMINATION OF ZINC IN BLOOD SIMPLIFICATION IN THE

TRICHLOROACETIC ACID AS A PRECIPITATION BY

1949, 181:295-306.J. Biol. Chem. 

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