Potentiometric titration of unidentified serum constituents

Potentiometric Titration of Unidentified Serum Constituents l Edna B. Andrews and Adrian C. Kuyper From

the Department

of Physiological

Chemistry, Wayne Detroit, Michigan

Received

September

University

College

of Medicine,

13, 1951

INTRODUCTION

Information on the chemical composition of blood serum has been obtained principally by the application of specific methods for the determination of each constituent. The question arises as to what quantity of additional substances, undetected because of insufficient information regarding their chemical structures, may be present in blood. Two different methods have been used to estimate these unidentified substances. The one (1,2) consists of subtracting the total determined anions from the total determined cations and thus obtaining a value which has been interpreted to be undertermined anions. This procedure is subject to large error because of the relatively large amount of salt present in the blood, and its interpretation assumes the absence of unidentified cations. A second method for the determination of unidentified substances, that of potentiometric titration, has been performed with only a limited degree of precision, and the interpretation of the results, in most cases, has been complicated by the inclusion in the titration of large amounts of identified substances such as phosphate, bicarbonate, and protein (3,4). After the treatment of blood filtrate with alkaline copper sulfate, Perlzweig and Delrue (5) titrated from 5 to 8 mequiv. of organic acid between the pH limits of 2.3 and 8.0. When corrected for all identified substances, values determined by these two different methods vary from 0 to about 7 mequiv. of unidentified substances per liter of serum. r Thia paper is taken from a dissertation presented by Edna B. Andrews in partial fulfillment of the requirements for the degree of Doctor of Philosophy, Wayne University, 1950. The investigation was supported by the First Fellowship Award of the Wayne University Chapter of Sigma Xi and by Therapeutic Research Grant No. 553 of the American Medical Association. 106

BLOOD

SERUM

STUDIES

107

It was the purpose of this investigation to develop a precise titrimetric method for the determination of unidentified substances in relatively small samples of blood and to apply this to the quantitative analysis of sera of normal individuals. In its final form the method consists of the following steps: removal of carbonate and protein from the serum by adjustment to pH 4.7 and ultrafiltration, removal of phosphate from the ultrafiltrate by precipitation and filtration as the calcium salt, potentiometric titration of the filtrate between the limits of about pH 11.5 and 2.5 at constant temperature and ionic strength, and correction of the titration curve for the presence of remaining identified constituents. The resultant titration curve is that of unidentified substances. EXPERIMENTAL Fifty-ml. samples of blood were collected, allowed to clot, and centrifuged at 0”. To 20 ml. of serum were added sufficient hydrochloric acid to give a pH of 4.8, and sufficient 15% sodium chloride solution to adjust the ionic strength, immediately before titration, to 0.30. The serum was then ultrafiltered at 5’ through a cellophane membrane (6,7) which removed all protein as shown by negative salicylic acid, foam, biuret, and heat coagulation tests on the filtrate, but allowed small molecules to pass as shown by the quantitative filtration of maltose and by the normal values obtained for various constituents. To an 8-ml. aliquot of ultrafiltrate a sufficient volume of calcium chloride solution was added to adjust the ultrafiltrate immediately before titration to a calcium content of 60 mg.-‘%. The solution was placed in an apparatus which permitted aeration and f&ration in the presence of a carbon dioxide-free atmosphere. It was aerated with carbon dioxide-free air, made alkaline by the addition of 0.51 ml. of freshly prepared, carbonate-free, 0.30 N sodium hydroxide solution, and filtered through paper directly into the titration vessel. The addition of sodium hydroxide and the transfer of the titration vessel to the titration apparatus were performed with minimum exposure to room air. With the apparatus employed the volume of filtrate was 8.69 ml. The solution was titrated from a pH of approximately 11.5 to a pH of about 2.5 with a hydrogen electrode (8) at a constant temperature of 30” by the addition of small increments of a solution containing 0.05 N hydrochloric acid, 60 me.-‘% calcium as calcium chloride, and sufficient sodium chloride to give a total ionic strength of 0.35. Connection with the reference cell was through a renewable liquid junction having an inverted capillary opening. Potential readings were made to within 0.02 pH units with a Cambridge laboratory model pH meter. The titration curves thus obtained were corrected, first for the accumulation of free acid and base. A second correction was the subtraction of the titration curve of a salt solution which had been ultrafiltered and treated in the same way as blood serum. The titration curve of the salt solution regularly showed the presence of a substance titrating in the alkaline range. This substance is probably silicate, and although freshly prepared sodium hydroxide was used for the precipitation of calcium phosphate, this amounted to about 0.08 ml. of 0.05 N alkali in each titration, or about

108

EDNA

B.

ANDREWS

AND

ADRIAN

C.

KUYPER

0.5 mequiv./l. of serum. If the titration is performed by adding alkali to an acid ultrafiltrate, the amount of silicate titrated becomes larger and more variable and severely limits the accuracy of the titration. A third correction applied to the experimentally obtained titration curve was for the normal constituents of serum ultrafiltrate. The titrated solutions were analyzed quantitatively for glucose (9), lactic acid (lo), urea (ll), uric acid (12), creatinine (13), and amino acid nitrogen (14). The titration curves of all these substances, with the exception of the amino acids, were calculated (15) on the basis of the amounts present and their dissociation constants* as determined by titration under the same conditions as were used for the titration of the blood samples. Because it was impractical to determine the amount of each amino acid present in each blood serum, the corrections for their presence were based on the amino acid nitrogen content of the serum and on a composite titration curve constructed on the basis of values for their distribution in the normal individual (16); dissociation constants were either determined experimentally2 or taken from the literature (17). RESULTS

AND

DISCUSSION

For ease of presentation the unidentified substances in each of the nine sera studied are expressed in terms of the amounts titrated between the three pH limits, 3.0-6.0, 6.0-8.0, and 8.0-11.0 (Table I). Beyond the pH limits of about 3.0 and 11.0, values obtained for unidentified substances lose significance because of the relatively large corrections applied for free acid and base. With the exception of one low value which was obtained on a sample with a high lactic acid of 29 mg.-%, the unidentified substances titrating in the acid range are within the limits of 0.55 f 0.23 mequiv./l. of serum. About half of this variation may be accounted for .by a possible error in correction for hydrogen ion due to a maximum error of 0.02 in the pH reading. The small amount of unidentified substance is in part accounted for by traces of weak acids reported to be present in serum. Citric acid is present in a concentration of about 2 mg.-y0 (18). Since about one-half is removed by precipitation with calcium phosphate preliminary to the titration (19)) the titrated solutions correspond to a serum citrate of about 1 mg.%, or 0.15 mequiv./l. Other acids have been reported present in blood serum as follows: pyruvic acid, 0.08 mequiv. (20) ; guanidoacetic acid, 0.01 mequiv. (21); glucuronic acid, 0.05 mequiv. (22); acetone bodies 2 The pK’ values expressed with reference to a pH value of 4.00 for potassium acid phthalate are: lactic acid, 3.58; creatinine, 4.86; urea, 0.21; glucose, 11.91 f 0.06; uric acid, pK1,’ 5.25 f 0.03, and pK2,’ 10.02 f 0.09; glycine, 2.40 and 9.43; alanine, 2.40 and 9.61; arginine, 2.20 and 8.98; glutamine, 2.31 and 8.90; histidine, 1.8, 6.06, and 9.00; valine, 2.41 and 9.42.

BLOOD

SERUM

109

STUDIES

as p-hydroxybutyric acid, 0.05 mequiv. (23); and fumaric acid, (24) less than 0.05 mequiv./l. On the assumption that these acids are titrated to the extent of SO%, they account for about 0.31 mequiv. of the unidentified acid. This leaves only a small amount of somewhat doubtful significance, about 0.24 mequiv. of acid, to be accounted for in the average titration. The unidentified substances titrating in the pH range of 6.00-8.00 vary from 0.06 to 1.39 mequiv./l. of serum. Total identified substance titrating in this range is about 0.3 mequiv. of which about 0.2 is attribTABLE Distribution

of Serum

Constituents

I

Titrating

Between

All values are expressed as mequiv./l. pH 3-6 DOllOr

Unidentified

pH Total

Unidentified

A 0.35 1.87 B 0.80 2.68 C 0.52 1.94 D 0.08" 3.220 E 0.40 1.83 F 0.66 2.43 0.48 2.12 G H 0.76 2.43 I-lb 0.41 2.43 r-2* 0.54 2.60 0.55 2.22 Average a Excluded from average. b The ultrafiltrate from a single blood which first came through the ultratiter is I-2. Values for I-2 are excluded from the

pH

3.00

and 11 .O

of serum

6-8

pH 8-11 Total

Unidentified

Total

0.06

0.37

3.66

1.39 0.12

1.71

0.01 1.13

0.44 1.03 0.35 0.31

0.03 1.44 0.57 1.30

3.67 4.60 3.25

0.40

0.01

1.06 0.70 0.75 0.71

0.34 1.07 1.16 0.65

3.23 4.35 4.20 4.45 4.11

0.77

0.15 1.14 0.13 0.72

0.44 0.49 0.54

4.81

5.19

sample was collected in two portions. That designated I-l, the last portion is designated average.

utable to the amino acids glutamine and histidine. Variations in the amounts of these two substances reported in the literature (25-28), would not appreciably affect the total amino nitrogen content nor the correction for identified substances, They would, however, account for variations of about 0.15 mequiv. in unidentified substances. The remaining substances titrated in some of the samples cannot, be accounted for by hitherto recognized serum constituents. Carbon dioxide is titrated in this range; however, this was removed in preliminary treatment and the drift in potential characteristic of the titration of carbonate was not observed in the experimental samples.

110

EDNA

B.

4NDREWS

AND

ADRIAN

C.

KUYPER

Unidentified substances titrated in the alkaline range vary from 0.01 to 1.44 mequiv./l. of serum. This variation appears to be large, but since each value represents the difference between two values, each of which is at least several times as large as the difference, the variation loses significance. The principal identified substances titrating in this range are the amino acids, whose basic groups are titrated to the extent of about 85%, and glucose, which is titrated to the extent of about 11%. Because so many amino acids make up the amino nitrogen, and because their dissociation constants are of about the same magnitude, variations in their dist~bution would not have much influence on the corrections applied for these substances. The average value indicates that a small amount of unidentified substance is titrated. The average amount of ~identified substance titrating over the pH range 3-11 is 1.7 mequiv./l. Of this, about 0.4 mequiv. may be accounted for by normal constituents known to be present in very small quantities. It becomes apparent that the remainder, about 1.3 mequiv. is a relatively small value, if it is compared with the amounts of recognized constituents such as bicarbonate, amino acid, and lactic acid, 25, 3, and 1 mequiv., respectively, present in normal serum. Other reports on the amount of unidentified substance present in serum usually include substances which have here been included in the identified fraction. When corrected for these substances, the estimate of Perlzweig and Delrue (5) becomes 3-6 mequiv. titrating between the pH limits of 2.3 and 8.0. There appears to be no previous report on the unidentified substances titrating between the pH limits of 8 and 11. Values for unidentified acids obtained by subtracting the total anions from the total cations vary from 0 to about 9 mequiv./l. of blood serum (V)* SUMMARY

A procedure is presented for the potentiometric determination of unidentified serum constituents. A relatively high degree of precision is obtained by titrating an ultrafiltrate, at a constant ionic strength and temperature, and by correcting the titration curves for the presence of silicate and the normal constituents of serum. The unidentified constituents titrating between the pH limits of 3-6, 6-3, and 8-l 1, average 0.2, 0.5, and 0.6 mequiv./l. of serum, respectively.

BLOOD

SERUM

STUDIES

111

REFERENCES 1. OBERST, F. W., AND PLASS, E. D., J. Lab. Clin. Med. 26, 513 (194041). 2. ATCHLEY, D. W., AND BENEDICT, E. M., J. Clin. Inuest. 9, 265 (1930-31). 3. BR~HL, H., Biochem. 2. 266, 293 (1932). 4. GEX, M., AND SCHUHLER, H., Arch. phys. biol. 16, 68 (1942-43). 5. PERLZWEIG, W. A., AND DELRUE, G., Proc. Sot. Exptl. Biol. Med. 25,548 (1927). 6. KUYPER, A. C., ANDREWS, E. B., AND EIDT, G., Rev. Sci. Instruments 22, 218 (1951). 7. SEYMOUR, W. B., J. Biol. Chem. 134, 701 (1940). 8. HAMER, W. J., AND ACREE, S. F., J. Research Natl. Bur. Standards 33, 87 (1944). 9. BENEDICT, S. R., J. Biol. Chem. 92, 141 (1931). 10. BARKER, S. B., AND SUMMERSON, W., J. Biol. Chem. 138, 535 (1941). 11. BARKER, S. B., J. BioE. Chem. 162, 453 (1944). 12. BENEDICT, S. R., AND BEHRE, J. A., J. Biol. Chem. 92, 161 (1931). 13. FOLIN, O., AND WV, H., J. Biol. Chem. 38, 81 (1919). 14. HAMILTON, P. B., AND VAN SLYKE, D. D., J. Biol. Chem, 160, 231 (1943). 15. VAN SLYKE, D. D., J. Biol. Chem. 52, 525 (1922). 16. KREBS, H. A., Ann. Rev. Biochem. 19, 409 (1950). 17. COHN, E. J., AND EDSALL, J. T., Proteins, Amino Acids and Peptides as Ions and Dipolar Ions. The Reinhold Pub. Corp., New York, 1943. 18. KUYPER, A. C., AND MATTILL, H. A., J. Biol. Chem. 103, 51 (1933). 19. KUYPER, A. C., J. Biol. Chem. 123, 405 (1938). 20. FRIEDEMANN, T. E., HAUGEN, G. E., AND KMIECIAK, T. C., J. Biol. Chem. 157, 673 (1945). 21. HOBERMAN, H. D., J. Biol. Chem. 167, 721 (1947). 22. DEICHMANN, W. B., AND DIERKER, M., J. Bill. Chem. 163, 753 (1946). 23. WEICHSELBAUM, T. E., AND SOMOGYI, M., J. Biol. Chem. 140, 5 (1941). 24. MARSHALL, L. M., ORTEN, J. M., AND SMITH, A. H., J. Biol. Chem. 179, 1127 (1949). 25. HAMILTON, P. B., AND TARR, R. R., J. Biol. Chem. 163, 397 (1945). 26. PRESCOTT, B. A., AND WAELSH, H., J. Bill. Chem. 167, 855 (1947). 27. HIER, S. W., AND BERGHEIM, O., J. BioE. Chem. 163, 129 (1946). 28. KIRSNER, J. B., SHEFFNER, A. L., AND PALMER, W. L., J. Clin. Invest. 23, 716 (1949).