THE EFFECT OF ANTICOAGULANTS ON DETERMINA- TIONS OF INORGANIC PHOSPHATE AND PROTEIN IN PLASMA BY OLIVER HENRY GAEBLER

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1 THE EFFECT OF ANTICOAGULANTS ON DETERMINA TIONS OF INORGANIC PHOSPHATE AND PROTEIN IN PLASMA BY OLIVER HENRY GAEBLER (From the Department of Laboratories, Henry Ford Hospital, Detroit) (Received for publication, August 16, 1932) Inorganic phosphate estimations are commonly carried out on serum from clotted blood, or on plasma from blood containing anticoagulants. Available data leave one in doubt as to whether the inorganic phosphate content in the two cases is identical and agrees with that of so called native plasma; that is, plasma obtained by chilling blood or collecting it in paraffined tubes and centrifuging before clotting occurs. Addition of oxalate to whole blood does not alter its inorganic phosphate content (1) and in this respect is preferable to defibrination, which is accompanied by a change of inorganic to organic phosphate (2). Moreover, Tolstoi (3) found good agreement between inorganic phosphate values for oxalate plasma and serum of human blood, the serum being separated after 3 hours. Likewise Buell(4) found agreement between the inorganic phosphate values for citrate plasma and native plasma of dog blood, and Wang and Felsher (5) found that citrate plasma and serum of sheep blood contained the same amount of inorganic phosphate. On the other hand, Buell found that oxalate plasma contained less phosphate than native plasma of dog blood. It is also known that in cell volume determinations hypertonic anticoagulants shrink the cells and dilute the plasma. Further evidence of this dilution is found in a fall in the serum proteins of defibrinated blood (6) on addition of 0.2 per cent of potassium oxalate. Finally, Barrenscheen, Doleschall, and Popper (1) found no agreement between inorganic phosphate values for native plasma and those for serum obtained from the same human blood samples within 2 hours. In eleven cases plasma gave the lower value six times, and the higher value 99

2 100 Anticoagulants and Plasma Phosphate five times, the differences being evenly distributed between and 0.94 mg. of inorganic phosphorus per 100 cc. If variations of this size occur, agreement between serum and anticoagulant plasma might simply lay both values open to question. The inorganic phosphate determination is used clinically in two connections in which small variations are important; namely, in determination of the inorganic phosphate curve after glucose administration, and in determination of calcium and phosphate products. In the former instance the fall in phosphate is small (7), and in the latter any error in the phosphate value is multiplied by the calcium value. The writer has therefore studied some of the variables involved in the estimation of phosphate (such as the effect of sodium fluoride, sodium citrate, potassium oxalate, or heparin), possible changes in the phosphate content of oxalate plasma during 5 hours, and differences between native plasma and oxalate plasma or native plasma and serum. A comparison was also made between the methods of Benedict and Theis (8) and Fiske and Subbarow (9). Determination of Phosphate in Presence of Fluoride In contrast with oxalate and citrate, fluoride interferes slightly with the color reaction used in the above methods for estimating phosphate when it is used in the usually recommended amount of 10 mg. per cc. of blood. In the presence of 4 times this amount of fluoride no color reaction whatever is obtained in one of the methods and very little in the other. This effect is easily overcome by addition of aluminum chloride. In Table I are recorded the results of an experiment with variable amounts of aluminum chloride and constant amounts of fluoride. The two compounds were added as solutions, replacing a part of the water required when the indicated amounts of phosphorus are determined in a total volume of 10 cc. The method of Fiske and Subbarow is more sensitive to fluoride interference than the other method, and therefore yields the more striking result. In Tube 4 (Table I) the interference with color development is still practically complete, while in Tube 5 it has been almost completely prevented. In the latter tube the ratio of aluminum to fluorine is the same as in the compound AlFs, hence the probable explanation of the above result is the formation of weakly ionized

3 0. H. Gaebler 101 aluminum fluoride. Comparison of Tubes 7 and 8 (Table I) shows that addition of aluminum chloride introduces no blank in either analytical method, while sodium fluoride introduces a small blank in the BenedictTheis method only. With smaller amounts of fluoride this becomes negligible, but some c. P. grades of sodium fluoride contain the substance responsible in amounts making them unfit for use. Experiments with fluoride added to blood serum gave results comparable with those abovecomplete interference with large amounts of fluoride, and complete prevention of this interference by aluminum chloride. Adding to the 5 cc. of trichloroacetic acid filtrate 1 cc. of a 20 per cent solution of TABLE Showing That Aluminum Chloride Prevents Interference of Fluoride in Colorimetric Determinations of Phosphate Tube No. NaF 41Cls. 6HzO mj _ ml. P taken I P found mg. m FiskeSubbarow P taken method P found m7. m Trace aluminum chloride (AICL~ 6H,O) in place of an equal amount of water is thus a simple means of preventing any interference due to fluoride. Comparison of Methods, and of Anticoagulant Plasma and Serum Values In Table II are shown the analytical results for anticoagulant plasma and serum of dog blood and human blood. 30 cc. samples of blood were drawn and discharged into two tubes, one of which contained an accurately measured portion of a concentrated solution of anticoagulant not greater than 1 per cent of the volume of blood added. Sodium fluoride was added as a solid. The solu

4 102 Anticoagulants and Plasma Phosphate tions were used on the assumption that the smaller amount of agitation required would minimize loss of COZ and changes in inorganic phosphate, but parallel tests carried out later indicate that results with solid anticoagulants are the same. The following anticoagulants were used: sodium fluoride, 10 mg. per cc. of blood; potassium oxalate, 2 mg. per cc. ; sodium citrate, 4 mg. per cc. ; and heparin, 0.4 mg. per cc. Sodium fluoride caused some hemolysis. The amount of the other anticoagulants is well below that which em&t TSBLE Comparison of Serum with Plasma Containing Various Anticoagulants method no. Inorganic P per 100 cc. Serum 1 mo mo Plasma Fiske Subbarow method mo II Anticoagulant Na fluoride < I K oxalate Na citrate I Heparin Na fluoride K oxalate SOWW of blood Dog L Human I will cause hemolysis, or interference with the color reaction used in determining phosphate. The tubes containing blood with and without anticoagulant stood side by side at room temperatures near 25 for 30 minutes, this being about the minimum time for separation of serum. They were then centrifuged for 5 to 10 minutes. Analyses by each method were carried out in duplicate. An examination of the results shows that there is good agreement between the two analytical methods, whether they are applied to serum or plasma

5 0. H. Gaebler 103 of normal dog blood or normal human blood having a fairly wide range of phosphate content. It is also apparent that the values for anticoagulant plasma are always decidedly lower than those for serum of the same blood sample. In the case of dog blood the difference is largest when sodium fluoride is used; potassium oxalate and sodium citrate in the amounts required have a smaller effect, and heparin lowers the plasma value very little. Unfortunately, heparin preparations contain material which is transmitted to trichloroacetic acid filtrates and causes slight turbidity in the determination. When the turbidity is removed by centrifuging there still remains a small blank which differs for the two analytical methods, probably because heparin preparations contain organic phosphate (10). Heparin is thus an undesirable anticoagulant in this connection. The few results which are recorded have had a blank of 0.2 to 0.3 mg. of inorganic phosphorus per 100 cc. of plasma subtracted. The sixth column of Table II shows the ratio of serum total nitrogen to plasma total nitrogen, both determined by the method of Howe (11). Although serum does not contain fibrinogen, it always contains more protein than anticoagulant plasma, except when heparin is used. The small amount of nitrogen in the heparin is entirely negligible in this connection; the fibrinogen nitrogen, if added to that of the serum, would increase all of the ratios recorded by an average of A comparison of the protein con tent of oxalate plasma and native plasma is recorded in the following section. Comparison of Serum and Oxalate Plasma with Native Plaslna In the following experiments an attempt was made to determine whether the difference found between serum and anticoagulant plasma is due entirely to changes produced by the anticoagulant, such as dilution of plasma by dehydration of the corpuscles, and altered distribution of inorganic phosphate between cells and plasma, or whether the serum values are also open to question. Serum and oxalate plasma were therefore compared with native plasma, obtained about 6 minutes after venepuncture, low temperature being used to delay coagu1ation.l Serum was separated 1 The following technique was used. 15 cc. conical centrifuge tubes were stoppered and placed in trunnions intended for 50 cc. tubes. The tubes

6 104 Anticoagulants and Plasma Phosphate after 30 minutes standing at room temperature, as in the preceding experiments, while oxalate plasma was separated after the same length of time, but with the difference that the specimen was placed in the refrigerator during this interval. The results are given in Table III. With a single exception, serum separated after 30 minutes contained more inorganic phosphate than native plasma of the same specimen, and oxalate plasma contained less than native plasma in every case. The difference between native plasma and serum did not vary as widely as in the Native plasma Comparison 7 TABLE III of Serum and Oxalate Plasma with Native Plasma. Inorganic P per 100 co. Native plasma oxa1ate plasma Difference mc7. mg. w?. T7. mg. mg $ o O Sobu;ocoedof Dog Human out on two series of bloods, one series The above analyses were carried serving for comparison of native plasma and serum, the other for comparison of native plasma and oxalate plasma. studies of Barrenscheen et al. (l), to which reference was made above, but in the present limited series of human bloods it varied more than the difference between oxalate plasma and native plasma. were held in the center of the trunnions by rubber bands passing over the stopper and around the lower end of the trunnion. The space between tube and trunnion was filled with water, and the outfit was then placed in a freezing mixture until the water surrounding the tube had frozen. Part of the blood drawn was discharged directly into such tubes and centrifuged for 5 minutes. Samples of plasma were then withdrawn and analyzed at once. The remainder of the blood drawn was allowed to clot, or was mixed with oxalate equivalent to 9.2 per cent final concentration.

7 0. H. Gaebler 105 Total nitrogen determinations were carried out on the oxalate plasma and native plasma of the last three blood samples in Table III. The nitrogen in oxalate plasma was 92.3 to 96.5 per cent of that in native plasma. On an average, the oxalate plasmas contained 5.0 per cent less nitrogen, and 7.2 per cent less inorganic phosphorus than the native plasmas. Stability of Oxalate Plasma at Low Temperature While the initial change in plasma composition which is caused by oxalate is a disadvantage, oxalate plasma also has a point in its TABLE Constancy of Inorganic Phosphorus Content of Oxalate Plasma in Contact with Corpuscles at 10 Specimen No. IV Inorganic P in 100 cc. oxalate plasma Min. after venepuncture ml. m7. mg. mg favor; namely, that its inorganic phosphate content remains unchanged for at least 5 hours when the oxalated blood is kept in the refrigerator at the usual temperatures of S10. This may be partly accounted for by the fact that both cold and oxalate inhibit glycolysis, with its attendant changes of acidity. The results in Table IV were obtained as follows: Freshly drawn samples of human blood were oxalated and divided among four tubes, one of which was centrifuged within 20 minutes, while the others were placed in the refrigerator and centrifuged after 1, 2, and 5 hours, respectively. The inorganic phosphate content of the plasma remained almost constant. This is important, since in determina

8 106 Anticoagulants and Plasma Phosphate tion of the phosphate curve following glucose administration the blood samples are drawn over a period of 23 hours, and unless each sample is analyzed separately, the specimens will stand a variable length of time before analysis. Effect of Varying the Amount of Oxalate Six samples of human blood were drawn, and each was discharged into two tubes containing potassium oxalate sufficient to make a final concentration of 0.2 per cent and 0.4 per cent respectively. The average inorganic phosphorus content of the plasma from blood with 0.4 per cent of oxalate was 0.16 mg. per 100 cc. below that containing 0.2 per cent. The quantity of oxalate should be controlled by measuring the amount of solution placed or evaporated in the tubes, and by adding a uniform amount of blood from the syringe. DISCUSSION Since the serum and oxalate plasma separated 30 minutes after the blood is drawn yield two different inorganic phosphate values, neither of which is identical with that of native plasma, it is in a sense unfortunate that serum and anticoagulant plasma have been used almost to the exclusion of whole blood in clinical studies involving the determination of phosphate. Determinations on whole blood are unaffected by an altered distribution of phosphate between cells and plasma, or by changes in plasma volume as the result of using anticoagulants. The precipitation and determination must, however, be carried out at once, for neither the whole blood nor the acid filtrate of it is stable as to its inorganic phosphate content (2, 10). The writer has observed that in trichloroacetic acid filtrates of human corpuscles the inorganic phosphate, calculated as phosphorus, per 100 cc. of corpuscles, increased 0.26 to 0.49 mg. in 5 hours. The stability of plasma filtrates, because of their negligible organic phosphate content, and the stability of oxalate plasma when kept in the refrigerator, even though it remains in contact with the cells, avoid the necessity of carrying out each determination separately. In comparative studies it should, however, be borne in mind that the phosphate content of oxalate plasma is definitely lower than that of serum or native plasma. The effect of fluoride in the amount usually used to inhibit gly

9 0. H. Gaebler 107 colysis is obviously too great to permit its use when plasma is to be obtained, even though its direct interference with the color reaction for phosphate is easily overcome. The possibility of stablizing the inorganic phosphate content of whole blood by means of fluoride has been partially investigated, since the writer finds that the increase of inorganic phosphate in laked defibrinated blood is decidedly less marked in the presence of fluoride. The changes in whole blood are, however, not completely inhibited by fluoride alone. CONCLUSIONS 1. Fluoride interferes with the color reaction used in the BenedietTheis and FiskeSubbarow methods for determination of inorganic phosphate. The interference is prevented by aluminum chloride. 2. Plasma obtained in 30 minutes, with fluoride, citrate, or oxalate as anticoagulants, always contains less inorganic phosphate than serum, and also less total nitrogen, despite the presence of fibrinogen. Dilution of the plasma with water drawn from the cells would seem to account for most of the fall in phosphate. 3. When native plasma is used as the standard, the inorganic phosphate content of oxalate plasma of human blood and dog blood is too low, and that of serum is too high. 4. The inorganic phosphate content of oxalate plasma left in contact with the cells at 10 remains unchanged for 5 hours. BIBLIOGRAPHY 1. Barrenscheen, H. Ii., Doleschall, F., and Popper, L., Biochem. Z., 177, 39 (1926). 2. Lawaczeck, H., B&hem. Z., 146, 351 (1924). 3. Tolstoi, E., J. Bid. Chem., 66, 157 (1923). 4. Buell, M. V., J. Biol. Chem., 66, 97 (1923). 5. Wang, C. C., and Felsher, A. R., J. Lab. and Clin. Med., 10,269 (1925). 6. Eisenman, A. J., J. Biol. Chem., 71, 587 (192627). 7. Hartman, F. W., and Foster, D. P., Am. J. Clin. Path., 2,289 (1932). 8. Benedict, S. R., and Theis, R. C., J. Biol. Chem., 61, 63 (1924). 9. Fiske, C. H., and Subbarow, Y., J. Biol. Chem., 66,375 (1925). 10. Kay, H. D., and Byrom, F. B., Brit. J. Exp. Path., 8, 240 (1927). 11. Howe, P. E., J. BioZ. Chem., 49,109 (1921).

10 THE EFFECT OF ANTICOAGULANTS ON DETERMINATIONS OF INORGANIC PHOSPHATE AND PROTEIN IN PLASMA Oliver Henry Gaebler J. Biol. Chem. 1932, 99: Access the most updated version of this article at Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts This article cites 0 references, 0 of which can be accessed free at #reflist1