Published: Journal of Analytical Toxicology, Volume 23, Number 2, March/April 1999, pp.134-135.

A Safer Method for the Measurement of Plasma Thiocyanate
C.J. Vesey, H. McAllister, and R.M. Langford

To the Editor:
The low levels of thiocyanate (SCN) normally present in body fluids increase with chronic exposure to cyanide. Thus, the measurement of plasma SCN is important in studies of cigarette smokers (1), for monitoring patients during lengthy infusions with sodium nitroprusside (SNP) (2), and in surveys of populations consuming cyanogenic plant foods (3).

The most sensitive colorimetric procedures for measuring SCN (and also cyanide) employ various modifications of the König synthesis of pyridine dyestuffs. SCN is first converted to cyanogen halide with bromine or chloramine T. On addition of a mixture of pyridine and a primary amine, a colored product is obtained. However, pyridine has a most unpleasant odor and is potentially toxic, and the primary amines may be carcinogenic. As a result, they have been replaced by the less noxious isonicotinic and barbituric acids, respectively (4). More recently, 1,3-dimethylbarbituric acid has been used in place of barbituric acid because of the more rapid reaction and formation of an end-product which is more highly colored (5,6). Lundquist et al. (7) have employed these two reagents for the measurement of SCN in urine and plasma. They first isolated the SCN from an alkaline solution of the sample on an ion-exchange resin, eluted it with a solution of sodium perchlorate, and used hypochlorite as the chlorinating agent instead of chloramine T.

Classically, trichloroacetic acid (TCA) has been used to release SCN bound to albumin and produce a protein-free extract of plasma for assay (8). A 10% (w/v) solution of TCA is typically used, and the precipitate is removed by filtration. However, we found that mixing plasma with nine times its volume of 5% (w/v) TCA precipitates the protein in a form that can be readily removed by centrifugation with good recovery of SCN (9). We have therefore adapted the colorimetric assay for use with 5% (w/v) TCA extracts of plasma.
Duplicate 200-µL samples of plasma, free of hemolysis, were transferred to 2-mL microcentrifuge tubes, and 1.8 mL of 5% TCA (prepared from AnalaR trichloroacetic acid) was added to each. Following vortex mixing, the tubes were left to stand for 10–15 min and then centrifuged at 12,000 ¥ g for 5 min. A chlorinating solution was prepared by dissolving 7 g of Na3PO4•12H2O (AnalaR) and 1.5 mL of NaOCl (hypochlorite solution with approximately 14% [w/v] available Cl2) in a volume of 250 mL with purified H2O. This chlorinating solution (1.5 mL) was added to 1-mL samples of each supernatant and to 1-mL duplicates of an appropriate SCN standard in 5% TCA. This was followed by the addition of 300 µL of the colorimetric reagent, which contained 1.85 g NaOH (AnalaR), 2.86 g isonicotinic acid (Aldrich), and 3.5 g 1,3-dimethylbarbituric acid (Aldrich) in a volume of 100 mL with purified H2O. The solution was mixed thoroughly and allowed to stand in the dark for 10 min, and the absorbance of the blue solution was measured at 607 nm with a Cecil 2010 spectrophotometer fitted with a microsipette system.

Figure 1 shows a typical standard curve for SCN in 5% TCA (such standards are stable for at least 1 year when stored at 5°C). This shows that the relationship between absorbance and SCN concentration is nonlinear above 50 µmol SCN/L, and TCA extracts with higher SCN values need to be diluted with appropriate volumes of 5% TCA. In Figure 2 plasma SCN results obtained with this procedure are compared with those obtained with our automated method, which uses pyridine and 1,4-phenylene diamine and which, in turn, was shown to give good agreement with an automated ferric nitrate assay (9).

Lundquist et al. (7) found that SNP had a negative effect on the measurement of SCN. We investigated this possibility with the described method by adding various amounts of NH4SCN in a volume of 100 µL to 10 2.4-mL samples of fresh plasma. The samples were then each divided into two volumes of 1.14 mL, and 60 µL of a 1 g/L solution of SNP was added to one of each pair to give a concentration of 50 mg SNP/L (well above the level that would be present in the plasma of a patient infused long-term with SNP). Instead of the SNP solution, 60 µL of water was added to each of the remaining tubes. The assay results for these spiked plasma samples are shown in Table I and demonstrate that SNP has no inhibitory effect on the measurement of SCN by our method. However, it will be noted that 8 out of the 10 samples containing SNP show slightly higher values than the corresponding controls. A paired t-test indicated that the differences were not significant at the 5% level (p = 0.076). The small differences may be explained by a reaction with plasma (10) that releases some of the cyanide from the SNP molecule; the mean of the positive differences is equivalent to 0.72% of the cyanide in the added SNP.

In conclusion, we have shown that a method that employs less hazardous reagents than established colorimetric procedures for measuring SCN gives entirely satisfactory results when modified for use with 5% TCA extracts of plasma. The whole procedure takes about 30 min and thus would appeal to clinical chemistry laboratories receiving occasional requests for a thiocyanate determination. Such requests may come from cardiac or renal units when patients have received lengthy infusions of SNP during or following heart surgery or for malignant hypertension, so it is noteworthy that this hypotensive agent has no significant effect on the assay even when present at a much higher concentration than would normally be present in such samples.

C.J. Vesey, H. McAllister, and R.M. Langford
Anaesthetic Laboratory
St. Bartholomew’s Hospital
London, England EC1A 7BE

References

1. C.J. Vesey. Thiocyanate and cigarette consumption. In Smoking and Arterial Disease, R.M. Greenhalgh, Ed. Pitman Medical, Bath, England, 1981, pp 107–117.
2. C.J. Vesey and P.V. Cole. Blood cyanide and thiocyanate concentrations following long-term therapy with sodium nitroprusside. Brit. J. Anaesth. 57: 148–155 (1985).
3. J. Cliff, P. Lundquist, H. Rosling, B. Sorbo, and L. Wilde. Thyroid function in a cassava-eating population affected by epidemic spastic paraparesis. Acta Endocrinologica 113: 523–528 (1986).
4. S. Nagashima. Spectrophotometric determination of cyanides with sodium isonicotinate and sodium barbiturate. Anal. Chim. Acta 99: 197–201 (1978).
5. J.C.L. Meussen , E.J.M. Temminghoff, M.G. Keizer, and I. Novozamsky. Spectrophotometric determination of total cyanide, iron-cyanide complexes, free cyanide and thiocyanate in water by a continuous-flow system. Analyst 114: 959–963 (1989).
6. A.J.A. Essers, M. Bosveld, R.M. van der Grift, and A.G.J. Voragen. Studies on the quantification of specific cyanogens in cassava products and introduction of a new chromogen. J. Sci. Agric. 63: 287–296 (1993).
7. P. Lundquist, B. Kagedal, and L. Nilsson. An improved method for the determination of thiocyanate in plasma and urine. Eur. J. Clin. Chem. Clin. Biochem. 33: 343–349 (1995).
8. K.F. Stoa. Studies on thiocyanate in serum with some supplementary investigations in saliva, urine, and cerebrospinal fluid. Univ. Bergen Medical Yearbook, Vol. 2. A.S. John Griegs Boktrykkeri, Bergen, Norway, 1957, p 17.
9. C.J. Vesey and C.J.C. Kirk. Two automated methods for measuring plasma thiocyanate compared Clin. Chem. 31: 270–274 (1985).
10. C.J. Vesey, M. Stringer, and P.V. Cole. Decay of nitroprusside. 1. In vitro. Brit. J. Anaesth. 64: 696–703 (1990).

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