Chapter 4  |  References
 
CHAPTER 5

VERIFICATION OF CYANIDE EXPOSURE--AN AUTOMATED MICRODISTILLATION ASSAY FOR CYANIDE IN BLOOD

 
5-1. Background

a. Cyanide (CN-)is an extremely poisonous and fast acting compound that is rapidly absorbed in the blood. Its primary toxic action is the inhibition of cytochrome oxidase. Although detoxication occurs at a relatively fast rate, frequently the organism is overwhelmed and expires within minutes due to lack of cellular respiration.

b. Various methods have been used for CN- detection in blood.1,2 Most of them involve prolonged specimen preparation using diffusion or bubbling procedures, both of which require larger blood volumes to achieve desired sensitivity than the automated fluorometric method described here.3 The CN- assay methods provide direct measurement of plasma free CN- and the stabilization of total CN- in blood. Samples for both plasma free CN- and blood total CN- are assayed directly without prior isolation of CN-, by a completely automated method requiring only 16 minutes from sampling to readout.

5-2. Materials and methods

a. Reagents for free CN- assay.

(1) Phosphate buffer, pH 7.4, 0.05 mole (M). Add 250.0 ml of 0.2 M potassium dihydrogen phosphate (KH2PO4) and 197.5 ml of 0.2 M NaOH to a 1 liter (L) volumetric flask. Dilute to 1 L with deionized water.

(2) First and second diluents: phosphate buffer, pH 7.4, 0.05 M containing 1.0 ml Brij 35 per L.

(3) Recipient solution: phosphate buffer, pH 7.4, 0.05 M containing 0.1 ml Brij 35 per L.

b. Reagents for total CN- assay.

(1) First diluent: Triton X-100, 0.5 percent. Dissolve 5.0 ml of Triton X-100 in 900 ml of physiological saline and dilute to 1 L.

(2) Second diluent: Sulfuric acid, 0.5 percent. Add 5.0 ml of concentrated sulfuric acid (H2SO4) to 500 ml of deionized water. Dilute to 1 L with deionized water and mix.

(3) Recipient solution: Sulfuric acid, 0.25 percent. Add 2.5 ml of concentrated H2SO4 to 500 ml of deionized water. Dilute to 1 L and mix. Add 0.1 ml of Brij 35 and mix.

c. Reagents for free and total CN- assay.

(1) Glycine-sodium chloride stock solution. Dissolve 77.4 grams (g) glycine and 58.6 g NaCl in deionized water. Dilute to 1 L with deionized water and mix. Keep refrigerated.

(2) Glycine buffer, pH 10. Add 63.0 ml of stock glycine-sodium chloride solution to 850 ml deionized water. Adjust pH to 10.0 with 1.0 M NaOH. Dilute to 1 L with deionized water. Refrigerate when not in use and prepare fresh weekly.

(3) Chelate: Potassium bis (5-sulfoxino) palladium (II). Dissolve 120 mg chelate in 1 L deionized water and add 1 ml Brij 35. Refrigerate when not in use and prepare fresh weekly. Prepare according to Hanker, et. al.4

(4) Magnesium chloride. Dissolve 12 g magnesium chloride (MgCl2) 6H2O in 1 L deionized water and add 1 ml Brij 35. Refrigerate when not in use and prepare fresh weekly.

(5) Isotonic saline. Dissolve 9.0 g NaCl in 1 L deionized water.

(6) CN- stock solution, 0.04 M (1040
µg/ml). Dissolve 196 mg of sodium cyanide (NaCN) and dilute to 100 ml with 0.1 M NaOH. Refrigerate when not in use and prepare fresh monthly.

(7) CN- standards. Dilute appropriate volumes of CN- stock solution with 0.01 M NaOH. Refrigerate when not in use and prepare fresh weekly.

(8) 4-Dimethylaminophenol (4-DMAP), 0.5 g/dl. Dissolve 0.05 g 4-DMAP in 10 ml deionized water. Refrigerate when not in use and prepare fresh weekly.

(9) Brij 35 and Triton X-100. These surfactants are obtained from Sigma Chemical Company, St. Louis, MO.

d. Apparatus. The analytical system consists of the following modules from Technicon Instruments Corporation, Tarrytown, NY.

(1) Sampler IV.

(2) Proportioning pump IV.

(3) Dialyzers, one each, 6-, 12-, and 24-inch lengths with type C membranes.

(4) Silicon oil heating bath at 116 oC.

(5) Fluoronephelometer III with Corning Glass filters excitation-#5970 with maximum transmission at 370 nm and emission-#4308 and #3389 with maximum transmission at 470 nm and sharp cut off below 400 nm.

(6) Flatbed recorder.

(7) An Eppendorf Model #5412 microcentrifuge.

e. Analyses manifold (fig 5-1).

(1) The manifold is constructed using the apparatus listed above.

(2) Standards, plasma, and blood specimens are sampled with one saline wash cup between each at 60/hour resulting in an effective sampling rate of 30/hour. In the free CN- assay, plasma is mixed with and dialyzed against pH 7.4 phosphate buffer. In the total CN- assay, the blood is mixed with saline containing Triton X-100 which hemolyzes the erythrocytes and then is acidified with the addition of 0.5 percent sulfuric acid. The sample stream is dialyzed against 0.25 percent sulfuric acid. Additional air is introduced in the recipient stream before distillation.

(3) The distillation assembly is constructed of glass- to-glass fittings joined with heat shrink tubing. After distillation, the liquid and vapor phases are separated in the manifold trap (#116-0110) and a portion of the latter segments the glycine buffer stream where the CN- is absorbed.

(4) An aliquot of the solution is added to the chelate stream where the CN- demasks the non-fluorescent potassium bis (5-sulfoxino) palladium (II). The resulting 8-hydroxy-5-quiniline sulfonic acid coorditates with magnesium to form the fluorescent chelate.

f. Sample collection.

(1) Blood should be collected with syringes or VacutainersTM containing heparin or EDTA. Immediately transfer 250 µ l to a 0.5 ml sample cup containing 5 ml of 4-DMAP. Cap, mix and set aside for total CN- assay. Immediately centrifuge 1 ml of blood for 1 minute at 15,000 rpm, quickly remove plasma and sample for free CN-. CN- bound to plasma albumin is unrecoverable.

(2) After the free CN- analyses are completed, change to the total CN- reagents and perform the total CN- assay on the blood specimens previously set aside.

g. Fluorometer adjustment. All modules are powered up and the manifold filled with reagents.

(1) Set Sample Aperture to Position C.

(2) Set Standard Calibration control to 0.00.

(3) Set Baseline control to 0.00.

(4) Set Reference Aperture to Position 3.

(5) Set Function switch to the Reference Position.

(6) Adjust the light pipe to give a 15 chart units reading on the recorder strip chart.

(7) Reset Function switch to No Damping.

(8) Reset Sample and Reference Apertures and Standard Calibration for desired sensitivity (table 5-1).

(9) Adjust baseline to 0 chart units.

h. Standardization. Standards are prepared by diluting the stock CN- solution with 0.01 M NaOH. The concentration range of standards should cover the expected CN- range of the samples. The full set of standards is assayed at the beginning and end of the analyses of the unknowns. An intermediate standard should be sampled periodically as well to provide a measure of precision and correction for gain associated with continuous flow methods.

i. Precision and recovery. Appropriate concentrations of CN- were prepared by adding
µ l volumes of stock CN- to 0.01 M NaOH and to heparinized blood containing 20 µl/ml of 4-DMAP. Each CN- concentration was analyzed 12 times during one working day.

5-3. Results

a. Standardization. Linear curves are obtained for each range of CN- aqueous concentrations when mM values are plotted versus fluorescence on cartesian coordinate graph paper. Blood CN- readings fall within the 95 percent confidence limits of the aqueous standards.

b. Precision. The highest coefficient of variation for aqueous CN- concentrations ranging from 4000
µM to 1 µ M is 5.8 percent and occurs at the lowest concentration measured. Fluorescence units were used to establish the precision in table 5-2.

c. Recovery. The correlation of added versus measured CN- in blood is excellent (r=0.999). Recovery and other statistical data are presented in table 5-3.

d. Stabilizing total CN- in blood.

(1) CN- added to blood is rapidly bound to Hgb and to plasma proteins. Subsequent analyses show the loss of recoverable CN- with time.

(2) Hgb is converted to methemoglobin when 4-DMAP is added to blood.5 Methemoglobin is further converted to cyanmet-hemoglobin in the presence of CN-. In this form the CN- is stable for at least 3 hours at ambient room temperature and at least for a week when refrigerated.

5-4. Discussion

a. The automated system for the determination of free and total CN- in blood incorporates dialysis, distillation, absorption, and the production of a fluorescent chelate.

b. The introduction of additional air in the recipient stream before distillation increases the air-liquid surface area and enhances the volatilization of CN- into the air segmented liquid stream.

c. A minimum amount of the surfactant (Brij 35) is added to the recipient stream to prevent foaming in the manifold trap after distillation. The distillation-absorption assembly is an all glass construction, eliminating contact with plastic tubing, which would result in reaction and loss of CN-.

d. The wide range of sensitivities is achieved by interchanging dialyzer sizes and adjusting the sample aperture of the fluorometer. Changes in flow rates, predilution of samples, or reagent concentration changes are unnecessary.

e. Thiocyanate and thiosulfate, which interfere in many CN-methods, do not affect the CN- measurement.

f. Plasma-free CN- levels are obtained by combining rapid techniques for plasma separation, to minimize loss of CN-, followed by rapid automated analysis.

g. In the total CN- analysis, a fraction of the endogenous Hgb is oxidized to methemoglobin by the addition of 4-DMAP to the blood specimen. The procedure described in this report results in the production of 60 percent methemoglobin within 15 to 30 minutes and stabilizes blood CN- for at least 3 hours at room temperature.


Table 5-1. Conditions for measuring three concentration ranges of CN-









Apertures





Range

Dialyzer
(inches)


CN-
mM

S

R

Standard calibration

I

6

< 4000

2

4

~ 0.5

II

12

< 200

3

4

~ 1.2

III

24

< 10

4

4

~ 4.0

Table 5-2. Precision of CN- measurements in aqueous solution






Fluorescence units





Range

CN-
mM

X

SD

CV%

I

4000

90.79

0.61

0.7

3000

71.61

0.61

0.9



2000

49.03

0.60

1.2



1000

26.03

0.38

1.5



500

13.35

0.10

0.8



400

10.75

0.11

1.0













II

200

90.62

0.66

0.7

100

42.83

1.37

3.2



50

20.99

0.67

3.2



40

16.35

0.78

4.8



20

7.35

0.25

3.4













III

10

89.97

1.59

1.7

5

45.70

0.97

2.1



2

14.89

0.48

3.2



1

6.48

0.38

5.8



Table 5-3. Precision and recovery of CN- from blood in vitro






CN-

Measured

mM



Range

Added CN-
(mM)

X

SD

CV%

Recovered
(%)


I

2500

2511.40

48.08

1.9

100.5

1000

1063.90

18.04

1.7

106.4



500

513.30

7.94

1.6

102.7















II

200

202.70

2.57

1.3

101.4

50

50.50

0.84

1.6

101.0



20

18.64

0.46

2.7

93.1















III

10

10.12

0.38

3.8

101.2

5

5.10

0.18

3.5

102.0



2

2.16

0.05

2.3

108.0



Figure 5-1. Flow diagram of manifold for assay of blood total CN- and plasma free CN->

(Illustration not yet available)


Footnotes

1 Feldstein, M. and N.C. Klendshoj. "The Determination of Cyanide in Biological Fluids by Microdiffusion Analysis," J. Lab. and Clin. Med. 44 (1954), pp. 166-170.

2 Lundquist, P., H. Rosling, and B. Sorbo. "Determination of Cyanide in Whole Blood, Erythrocytes, and Plasma," Clin. Chem. 31 (1985), pp. 591-595.

3 Groff, W.A., Sr., F.W. Stemler, A. Kaminskis, H.R. Froehlich, and R.P. Johnson. "Plasma Free Cyanide and Blood Total Cyanide: A Rapid Completely Automated Microdistillation Assay," Clin. Toxicol. 23 (1985), pp. 133-163.

4 Hanker, J.S., A. Gelberg, and B. Witten. "Fluorometric and Colorimetric Estimation of Cyanide and Sulfide by Dimasking Reactions of Palladium Chelates," Anal. Chem. 30 (1958), pp. 93-95.

5 Kiese, M. and N. Weger. "Formation of Ferri-hemoglobin With Aminophenols in the Human for the Treatment of Cyanide Poisoning," Eur. J. Pharmacol. 7 (1969), pp. 97-105.


Chapter 4  |  References
 
 
 
 
 

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