LIPOID-THIOCYANATE IN SERUM

BY JACK D. ROSENBAUM* AND PAUL H. LAVIETES

(Prom the Department of Internal Medicine, Yale University School of Medicine, New Haven)

(Received for publication, October 11, 1939)

When patients with transudates into the serous cavities are given thiocyanate, the concentration of thiocyanate in the transu- dates never reaches that in the serum (5). A portion of the thio- cyanate of the serum is restrained from diffusing through the capillaries in vivo, or through cellophane membranes in vitro. The present work demonstrates that this restraint is imposed by a lipoid-thiocyanate complex. The amount of thiocyanate so bound is large enough to permit some investigation into the nature of the combination, which is of special interest because of the evidence presented by Peters and Man (10) that a lipoid-chlorine complex may exist in native serum.

The possibility that thiocyanate in serum is combined with lipoid has been suggested by Lustig and Botstiber (6), but on the basis of very indirect and unconvincing evidence. Chlorides, bromides, and iodides have been demonstrated in large amounts in lipoid extracts of tissues (8, 9).

Methods

Inasmuch as the experiments performed varied considerably in type, only such methods as were employed in several groups of experiments will be described in his section. Additional tech- niques will be described together with the procedure of the par- ticular group of experiments in which they were used.

Thiocyanate-Proteins were precipitated from duplicate 2 cc. samples of serum by the addition of 2 cc. of 20 per cent trichloro-

* This article represents in part work done in fulfilment of the thesis requirement for the degree of Doctor of Medicine at Yale University School of Medicine.

663

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664 Lipoid-Thiocyanate in Serum

acetic acid. To 2 cc. of the filtrate an equal volume of ferric ni- trate reagent (50 gm. of Fe(NO&. 9H,O and 25 cc. of HNOB in a liter of water) was added. After 5 minutes were allowed for full development of color, the solution was compared with a freshly prepared standard of thiocyanate solution plus color reagent, with a Klett biocolorimeter. In analyses of protein-free ultrafiltrates the ferric nitrate reagent was added directly, since it was found that the omission of trichloroacetic acid from the standard solu- tions did not affect the intensity of the color developed. Analyses of sera containing known amounts of added sodium thiocyanate showed that the error of the method was never more than f3 per cent and rarely exceeded art 1 per cent, provided the concentra- tion of thiocyanate was 0.5 milliequivalent per liter or more. The addition of KCN, which was used as a preservative in some of the experiments, had no influence on the determination of SCN.

Water Content-Water content was taken as the loss of weight of an aliquot dried to constant weight at 95”. In some instances this determination was omitted and water content was calculated from protein concentration by the formula of Eisenman, Mac- kenzie, and Peters (3). When the serum of the same subject was being used repeatedly, water content was not determined for each sample of serum.

Preparation of Serum Containing Thiocyanate-Venous blood was obtained without stasis and without exposure to air. The blood was allowed to clot under oil and the serum was drawn after 20 minutes centrifuging. Measured amounts of dry sodium thiocyanate were added to the serum. In a few experiments serum was obtained from a subject who had previously ingested potas- sium thiocyanate, in which case the blood when drawn already contained thiocyanate.

UZtra$Ztration-Ultrafiltration was carried out across a cello- phane membrane1 by the method of Lavietes (4). In most ex- periments the serum had been exposed to air during the addition of thiocyanate, but the ultrafiltration was carried out anaerob- ically. When the thiocyanate had been ingested by the subject, however, the serum was introduced into the ultrafiltration chamber without any exposure to air, and the ultrafiltration completed

1 Cellophane No. 450 was used, not No. 300 as erroneously stated in the original description.

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J. D. Rosenbaum and P. H. Lavietes 665

without permitting the serum to come in contact with air at any point in the procedure. It was found that exposure to air did not affect the experimental results.

Ultrafiltration was usually conducted at room temperature (21&27”), but was occasionally carried out at 7” or 37”.

Thiocyanate was determined in serum and filtrate and occa- sionally in the substrate. It has been noted (4) that the con- centration of thiocyanate in the filtrate is independent of the proportion of serum water which has been filtered, so that com- parisons between serum and filtrate are not influenced by the extent to which ultrafiltration has progressed.

Experimental Procedures and Results

Ultrafiltration of Serum and Transudate-Thiocyanate-con- taining serum from two normal subjects was subjected to ultra- filtration on thirty-two occasions and serum from patients on twenty-two occasions. The concentration of SCN in serum ex- ceeded that in its ultrafiltrate in every instance; a similar, but smaller, discrepancy was noted between colloid-poor transudates and their ultrafiltrates.

Bound Thiocyanate-By means of a factor to correct for the Donnan effect, ionized SCN in serum was estimated from the concentration of SCN in its ultrafiltrate (all of which is assumed to be ionized) and subtracted from the total SCN of the serum to give bound or non-ionized SCN. The error which may be intro- duced into this calculation by using an improper correction ratio for the Donnan effect is small in relation to the amount bound. The factor 0.95 has been used in our calculation. Thus (SCN in water of serum) - (SCN in filtrate X 0.95) = SCN bound per liter of serum water.

The data for a few experiments in which different concentrations of SCN have been added to portions of the same serum are pre- sented in Table I. The proportion of SCN bound diminishes as the total concentration of SCN increases. The limit which bound SCN approaches as the total SCN concentration is raised was not established, but there were 9.3 milliequivalents bound in one experiment in which the serum of a normal subject was made to contain 86.4 milliequivalents. Bound SCN in the sera of the patients studied differed little from that in normal sera.

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666 Lipoid-Thiocyanate in Serum

These findings were confirmed by several experiments in which serum, in cellophane tubes, was dialyzed against physiological saline solution, after addition of NaSCN to either serum or dialy- sate. Furthermore, when SCN was added to one of two portions of the same serum and both were dialyzed against physiological saline solution, equilibrium was reached when the concentration of SCN was alike in the two samples of serum, 2.66 milliequivalents per liter of water, while that in the dialysate was only 2.10 milli- equivalents per liter. At this time Cl was 2 per cent higher in the dialysate than in serum water.

TABLE I

Ultrajiltration of Normal Sera

Experiment No. Total SCN of serum SCN of 6ltrate Bound SCN of BWUIU Hz0 -

?n.eq. per 1. 7n.q. Per 1. m.e*. per 1.

2.50 1.58 1.18 4.55 3.09 1.96 1.27 0.84 0.57 2.46 1.80 0.95 4.85 3.85 1.62 1.25 0.85 0.54 5.00 3.85 1.73

10.00 8.22 2.97 1.08 0.76 0.45

15.55 13.05 4.34 5.02 3.95 1.70

10.04 8.27 3.03

No appreciable restraint of SCN could be demonstrated when solutions of gelatin, recrystallized egg albumin, an emulsion of olive oil, or a commercial emulsion of neutral fat was used; nor was the capacity of serum to bind SCN enhanced by the addition of olive oil.

E$ect of Temperature on Thiocyanate Distribution-Simultaneous ultrafiltrations of the same SCN-containing serum were carried out at different temperatures. The amount of thiocyanate bound at each temperature was determined. The results, in Table II, reveal a constant and striking temperature effect. At 7” more SCN was bound than at room temperature (22-27”). In the last experiment 1 mg. of KCN was added per cc. of serum at the

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J. D. Rosenbaum and P. H. Lavietes 667

start to control the possible factor of bacterial growth. The effect of temperature is a reversible one, for if serum is kept in the refrigerator for 24 hours and then ultrafiltered at room tempera- ture, its binding power is the same as that of control serum kept at room temperature throughout the experiment. Moreover, successive samples of ultrafiltrate obtained from the same sample of serum at different temperatures show changes comparable to those recorded for simultaneous ultrafiltrations at different tem- peratures.

TABLE II

Iq’iuence oJ Temperature upon Thiocyanate Binding

Temperature Total SCN of serum

“C. m.f?q. per 1. watet 7n.q. per 1. water 7 13.5 2.50

37 13.5 1.60 7 1.06 0.33

37 1.06 0.19 7 6.64 2.25

22 6.64 1.79 7 10.70 2.79

25 10.70 1.90 7 10.70 3.43

25 10.70 2.93 7 8.54 2.60

25 8.54 1.97 7 3.35 1.14

27 3.35 0.83

E$ect of Hydrogen Ion Concentration on Thiocyanate Distribu- tion-In a series of experiments samples of the same SCN-con- taining serum were maintained at different hydrogen ion concen- trations during ultrafiltration, and bound thiocyanate determined (Table III). In the first group of experiments, one portion of serum was treat.ed with COZ, the other being retained as a control. Each sample was then transferred anaerobically into the ultra- filtration chamber. When SCN had been previously ingested, a third ultrafiltration was conducted in which anaerobic precau- tions were observed throughout. In the last two experiments the serum was brought approximately to pH 8.5 by the addition

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668 Lipoid-Thiocyanate in Serum

of KCN, and portions brought back to normal and acid reactions by adding HzS04 as indicated. Water equivalent in volume to the H&304 was added to the alkaline samples. The results indicate a small but constant increase in binding of SCN when pH is lowered slightly by increasing the CO2 tension. This effect is much more striking with the greater changes in reaction pro- duced in the last two experiments.

Lipoid Extraction of XCN-Containing Serum-Attempts to recover lipoid-SCN from petroleum ether extracts of serum dried

E$ect of pH u: pon Thiocyanate Binding Determined by UltraJiltration

Total SCN Bound SCN Conditions

m.eq. per 1. water me*. per 2. water 1.69 0.37 1.69 0.50 1.84 0.94 1.84 0.94 1.84 0.98 0.91 0.46 0.91 0.45 0.91 0.49 2.14 0.54 2.14 0.63 3.35 0.68 3.35 0.83 3.35 0.92

13.50 2.33 13.50 2.74 13.50 3.35

TABLE III

Control COz bubbled through serum Control Anaerobic CO2 bubbled through serum Control Anaerobic COe bubbled through serum Control CO2 bubbled through serum Alkaline (15 m.eq. KCN added) Neutral (same + 10 m.eq. HzSOd) Acid (same + 30 m.eq. HzS04) Alkaline (15 m.eq. KCN added) Neutral (same + 12 m.eq. HsSO4) Acid (same + 24 m.eq. HzSO~)

in vacua after freezing and from serum dried in vacua after being mixed with anhydrous sodium sulfate were unsuccessful.

The method of Peters and Man (lo), slightly modified (7), was employed for the extraction of lipoids from thirteen samples of SCN-containing serum and in each case SCN was found in the lipoid extract, although NaSCN could never be dissolved in petroleum ether in any det.ectable amount. In each experiment a second sample of the serum was subjected to ultrafiltration and total, ionized, and bound SCN was determined. The amount of

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Experiment No.

i

Total

?n.e*. per 1.

10 5

10

ii

10 10

-

J. 11. Rosenbaum and P. H. Lavietes 669

bound SCN as determined by ultrafiltration was compared with the amount of SCN found in the lipoid extract.

Although there was SCN in every lipoid extract, the differences between duplicate determinations were considerable, and the discrepancies between extracted lipoid-SCN and that found bound by ultrafiltration were even greater. It was impossible to draw any more exact conclusion from these experiments than that the amounts of SCN were of the order of magnitude of the non- diffusible fraction.

The method of Peters and Man (10) was next further modified in that the alcohol-ether extract was evaporated at atmospheric

TABLE IV

SCN in Lip&d Extract of Serum Compared with Bound SCN in Serum; Lipoid Extracts Evaporated with Nitrogen

Bound Lipoid

m.ep. per 1. m.eq. per 1.

1.8 1.3 2.1 1.8 3.3 2.7 1.8 2.8 1.8 2.2 2.8 3.0 2.7 2.3 2.8 2.6

pressure by the use of a stream of nitrogen. Agreement of dupli- cate determinations within 10 per cent was usually obtained by this procedure. The results of the extractions by this method, pre- sented in Table IV, indicate excellent agreement between the amount of SCN in the lipoid extracts and the bound SCN as determined by ultrafiltration of the same serum at room tempera- ture.

In the course of these experiments it was found that the results were not significantly affected by exposure of the dried alcohol- ether-soluble extract to air before extraction with petroleum ether. It was then found that equally good yields of lipoid-SCN could be obtained if a stream of air was substituted for the stream of nitrogen in removing the alcohol and ether from the extracts prior

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Lipoid-Thiocyanate in Serum

to reextraction with petroleum ether. The results were little, if any, influenced when the alcohol and ether were merely allowed to evaporate spontaneously. The technique adopted for further studies was simplified as warranted by these observations.

To 20 cc. of a mixture of 3 parts of 95 per cent alcohol to 1 part of ether in a 25 cc. volumetric flask are added 1.5 cc. of serum drop by drop with constant agitation. After thorough mixing, this is made to the mark with alcohol and ether, capped with tin-foil, and kept in the dark at room temperature for 10 to 24 hours. It is then again brought to the mark if there has been any evapora- tion, and filtered. 3 cc. aliquots of the filtrate are allowed to evaporate spontaneously in 30 cc. porcelain dishes and the residue is extracted with petroleum ether and transferred to 10 cc. round bottomed centrifuge tubes, one 4 cc. and four 1 cc. portions of petroleum ether being used, 10 drops of water are now added to the petroleum ether extract, and the tube capped with tin-foil and centrifuged at high speed for 10 minutes. The clear petroleum ether extract is then drawn off into a 15 cc. round bottomed Pyrex centrifuge tube and allowed to evaporate spontaneously below the boiling point of petroleum ether. The lipoids are then sus- pended in 2 cc. of 10 per cent trichloroacetic acid by forceful shaking wit’h the aid of a glass bead. The suspended fats are then removed by passage through No. 50 filter paper twice and thio- cyanate in the filtrate determined by calorimetry, a standard solu- tion containing 0.2 milliequivalent of NaSCN per liter of 10 per cent trichloroacetic acid being used for comparison.

Redistilled alcohol, ether, and petroleum ether were used at the beginning, but it was found subsequently that the results were unaffected by using the ordinary laboratory reagents. By the technique described above no SCN could be demonstrated in petroleum ether extracts of dry NaSCN, or of NaSCN plus butter fat or mineral oil. SCN was recovered quantitatively from mix- ture with butter fat by calorimetric determination in the filtrate of a trichloroacetic acid suspension, as used in the above procedure. Other control experiments demonstrated that double aliquots of alcohol-ether extract could be used without affecting the yields; also that delay of a few hours before the alcohol-ether-soluble matter is extracted with petroleum ether does not affect the results significantly but that delay of 3 to 6 days results in an increased

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J. D. Rosenbaum and P. H. Lavietes 671

yield. In separating the petroleum ether extracts, centrifugation at approximately 2000 R.P.M. was used. The yields were not decreased by more rapid centrifugation. Water added to the petroleum ether extract before centrifugation to facilitate subse- quent separation of the petroleum ether extract from the insoluble residue did not diminish the yield. When petroleum ether ex- tracts were shaken vigorously with water, however, the SCN was all transferred to the aqueous phase.

E$ect of Temperature on SCN Content of Lipoid Extracts- Aliquots of the same alcohol-ether extracts of serum were allowed to evaporate spontaneously at 7” and 25”. The yields of SCN from the subsequent petroleum ether extracts were much higher when the evaporation took place at 7” (Table V). The effect of

TABLE V

Lipoid-SCN and Temperature at Which Alcohol-Ether Extract Is Allowed to Evaporate

Experiment No.

1

2

Total Bound

?n.e*. per 1. 7n.q. per 1.

10 3.1 2.3

10 1.8 1.3

Temperature

“C.

7 25

7 25

temperature differences is found to be comparable to that on bound SCN as estimated by ultrafiltration (Table II). In Experi- ment 2 it was found that when aliquots were evaporated almost to the point of dryness at 25” and the evaporation completed at 7” the yield of lipoid-SCN rose to 1.6 milliequivalents per liter, midway between the yields at 7” and 25’. Transfer to a vacuum desiccator at room temperature at the same point in the procedure failed to increase the yield of lipoid-SCN, indicating that the reduced temperature does not act merely by inhibiting oxidation.

E$ect of Reaction on XCN Content of Lipoid Extracts-The addi- tion of 0.02 cc. of 0.1 N HzS04 or HCl to a 3 cc. aliquot of alcohol- ether extract (equivalent to 0.18 cc. of original serum) before evaporation causes the yield of lipoid-SCN to be doubled (Table VI). An equivalent amount of NaOH causes the yield to fall definitely below the controls (to which have been added 0.02 cc.

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672 Lipoid-Thiocyanate in Serum

of water). In the last two experiments additional samples were treated with varying amounts of acid. It was found with both HGSOJ and HCI that the highest yield was obtained when 0.1 N

acid was used and that the yield fell off sharply with 0.2 N acid and disappeared entirely when N acid was used. Control experi- ments showed that addition of NaCl and Na2S04 had no effect on lipoid-SCN recovery. Acidification of serum short of the point where precipitation of the proteins occurs prior to extraction with alcohol-ether mixture also causes increased recovery of lipoid-SCN.

TABLE VI

Effect on Recovery of Lipoid-XCN of Changing Reaction of Alcohol-Ether Extract

Lipoid-SCN in m.eq. per liter serum after addition* of

HZ0 H&301 HCl NaOH

1.5 2.3 1.3 1.4 3.3 0.7 2.2 3.4 1.9 2.2 3.6 2.2 2.2 3.2 2.4 4.9

* 0.02 cc. of water or of 0.1 N acid or alkali was added to 3 cc. of alcohol- ether extract (== 0.18 cc. of the original serum).

DISCUSSION

That SCN in serum is in some manner restrained from diffusion across semipermeable membranes both in vivo and in vitro seems to be established. The recovery of SCN from lipoid extracts of serum in amounts approximating non-diffusible SCN suggests that the mechanism by which SCN is bound consists of the formation of a lipoid-SCN combination in serum. The fact that bound SCN and lipoid-SCN are similarly influenced by temperature changes and also by changes in acidity lends strong support to this con- clusion.

There is considerable evidence that the lipoid-SCN complex is in equilibrium with inorganic SCN. Whether NaSCN is added to serum or to its dialysate, the distribution finally reached is found to be the same. The equilibrium can be disturbed by chang-

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J. D. Rosenbaum and P. H. Lavietes 673

ing the reaction or temperature of the serum. For a single serum there is a correlation between t,otal SCN and the amount bound. The combination is loose enough to be destroyed by precipitation of the lipoids together with the proteins of the serum by trichloro- acetic acid, since following such precipitation recovery of SCN from the filtrate is complete.

The presence of SCN in lipoid extracts of serum is of interest in connection with the reports of Spiegel-Adolph (ll), of Christensen and Corley (2), and of Christensen (1) that halides may be found in association with phospholipid derived from serum. Christensen and Corley (2) have confirmed the observation of Peters and Man (10) that chloride accompanies the lipoids obtained from serum by extraction methods, and have further shown that the phospholipid fraction is the one specifically involved. Christensen and Corley (2) and more recently Christensen (1) have expressed doubt that lipo-chloride combinations exist in native serum; but this possibility has not been excluded. In the case of SCN there can be no question regarding the presence of a bound form in serum both in vivo and in vitro, and apparently it is the serum lipoids that are involved in the combination. This is certainly suggestive evidence that the chemically similar halides may exist in the same form.

SUMMARY

In a series of 54 ultrblmrations and two dialyses of human serum containing thiocyanate there was in every case a higher concentra- tion of thiocyanate in the serum than in ultrafiltrate or dialysate.

Bound thiocyanate is in equilibrium with ionized thiocyanate of the serum. The amount bound was found to increase as the amount of thiocyanate added was increased, with a smaller frac- tion of the total bound at higher than at lower concentrations. Increases in temperature diminished thiocyanate binding by the serum. Increases in hydrogen ion concentration were found to increase thiocyanate binding.

Thiocyanate was demonstrated consistently in the petroleum ether extracts of serum containing thiocyanate, although sodium thiocyanate is completely insoluble in petroleum ether. The amount of lipoid-SCN extracted from a given serum sample agreed well with the bound SCN as determined by ultrafiltration. Fur-

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674 Lipoid-Thiocyanate in Serum

thermore, lipoid-SCN recoveries and bound SCN are similarly influenced by temperature changes and by variations of hydrogen ion concentration.

BIBLIOGRAPHY

1. Christensen, H. N., J. Biol. Chem., 129, 531 (1939). 2. Christensen, H. N., and Corley, R. C., J. Biol. Chem., 123,129 (1938). 3. Eisenman, A. J., Mackenzie, L. B., and Peters, J. P., J. Biol. Chem.,

116,33 (1936). 4. Lavietes, P. H., J. Biol. Chem., 120, 267 (1937). 5. Lavietes, P. H., Bourdillon, J., and Klinghoffer, K. A., J. Clin. Inv.,

16,261 (1936). 6. Lustig, B., and Botstiber, G., Biochem. Z., 220, 192 (1930). 7. Man, E. B., J. BioZ. Chem., 117, 183 (1937). 8. McLean, F. C., Arch. Innt. Med., 16, 92 (1915). 9. Oppenheimer, E., Arch. esp. Path. u. Pharmakol., 89, 29 (1921).

10. Peters, J. P., and Man, E. B., J. BioZ. Chem., 107, 23 (1934). 11. Spiegel-Adolph, M., Proc. Sot. Exp. BioZ. and Med., 36, 763 (1936).

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Jack D. Rosenbaum and Paul H. LavietesLIPOID-THIOCYANATE IN SERUM

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