1 DETERMINATION OF BLOOD CYANIDE AND ITS ...

DETERMINATION OF BLOOD CYANIDE AND ITS ROLE IN PRODUCING INCAPACITATION IN FIRE VICTIMS D.A. Purser Department of Inhalation Toxicology, Huntingdon Research Centre plc Presented at the Royal Society of Chemistry (Analytical Division) meeting "Analytical aspects of biological safety evaluation" Huntingdon, 6th June, 1984

INTRODUCTION I would like to tell you about some work that I have been doing with HCN and since you are principally analytical chemists, I intend to concentrate on the methodological aspects of the work. However, since HCN is known to be a lethal gas and the work involves exposing animals, in fact primates, I think it is important for me to explain the background to the work and exactly why it was done.

FIRES AND THE HOME OFFICE The reason for my interest in HCN is that it is one of the toxic gases found in fires. Now the Home Office has kept detailed records of fire deaths and injuries over the last 30 years, and analysis of these statistics in the mid 1970's led to the conclusion that not only were a ,large proportion of fatal and non-fatal fire casualties being reported in the category of "Overcome by smoke and toxic gases" rather than by heat or burns, but that there was a four-fold increase in the category between 1955 and 1971 (1), an increase which has continued over the last decade so that now approximately half of all fatal casualties and a third of all non-fatal casualties of fires in dwellings, themajority caused by fires in furniture and bedding, are reported as being over-come by smoke and toxic gases (2) This has occurred despite the fact that the numbers of burn casualties have decreased and the total annual numbers of fires have remained approximately constant over this period of time. Another point that has emerged is that it is not so much the lethality of fire products that is important (since all fires are potentially lethal) but, how victims become incapacitated during the early stages of fires and are prevented from escaping. As part of their work is attempting to understand this trend and find ways of reducing deaths and injury in fires the Fire Research Station asked me to carry out animal exposures to low, sub-lethal levels, of common fire gases and combustion products in an attempt to understand the mechanisms whereby victims become incapacitated in fires. IRRITANTS AND NARCOTICS What we have found is that there are 2 basic kinds of toxic products in fire atmospheres.

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1.

Those that incapacitate by causing irritation making the eyes sting and water and causing a burning sensation in the mouth, nose and chest.

2.

Those that incapacitate by causing narcosis, making the victim confused and sleepy, then unconscious and leading ultimately to death if exposure is prolonged.

Now in the second category of narcotic gases only 2 seem to be important, despite the large number of chemical products in fire atmospheres, and these are CO and HCN. CO is always present in fires and is the main narcotic product in most fires, but where nitrogen containing materials such as wool, polyacrylontrile or polyurethane foam are involved cyanide can be formed and substantial concentrations have been detected in experimental fires. Perhaps more importantly a recent pathological study of fire victims in the Strathclyde region of Scotland revealed significant quantities of cyanide in the blood of 78% of victims and in some 31% of cases cyanide was present at potentially incapacitating levels (3). Although HCN is known to be lethal at high levels less is known about how relatively low levels cause incapacitation. Also it is difficult to work back from blood levels in victims after exposure to determine the likely exposure during the fire and how this might have affected the victim’s ability to escape (since cyanide was also usually present in the blood) It was for these reasons that we decided to examine the physiological effects of low level HCN exposure in primates. The method used was to expose the animals for 30 minutes to low level HCN gas mixtures via a face mask, while measuring effects upon the heart, brain and respiration. During these experiments I did some preliminary measurements of blood cyanide uptake, taking venous samples and sending the whole blood to the Department of Pathology at Glasgow University for Cyanide analysis by gas-chromatography-mass spectroscopy (GC-MS).

PRELIMINARY FINDINGS What we found was rather unexpected as Figure 1 shows. Initially, the cyanide was taken up rapidly into the blood, but then it leveled off despite the fact that the animals were still inhaling the gas. It seems that some equilibrium was set up, whereby at the peak levels the cyanide was leaving the blood at the same rate as the uptake. There are three possible explanations for these findings as follows: 1.

The cyanide was being excreted by the lungs in the exhaled breath. This seems unlikely since HCN is extremely soluble in non-acidified aqueous media (4), however it is possible that metabolic acidosis may have resulted in a blood pH 9f less than 7.

2.

Detoxification of cyanide by rhodonase to thiocyanate in the liver.

3.

Temporary binding of cyanide in the blood followed by gradual diffusion into the tissues.

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But whatever mechanism was responsible one would expect a steady decline in blood cyanide when the exposure was finished and cyanide uptake ceased, and the half-life in man is thought to be 1-2 hours. However, this did not apparently occur in our monkeys since the level hardly changed over an hour. This seemed odd so I suspected an anomaly. Possibly the venous blood sample was stale, and unrepresentative, since peripheral circulation is poor during cyanide uptake, or possibly the blood samples had deteriorated in the post. (Since strange things can sometimes happen to cyanide in stored blood and cadavers). CURRENT STUDY I therefore decided to repeat the experiment, taking arterial blood from an indwelling catheter and analysing the blood at HRC as well as in Scotland by GC-MS. The method I decided to use was to extract the cyanide into NaOH and estimate the levels by means of a. cyanide ion selective electrode.

EXTRACTION The extraction method used was that described by Egekeze and Oehme C.J. (5) 3 mls of blood was placed in a gas washing tube with 10 ml water. 2 mls of 9 molar H2SO4 was added to digest the sample and liberate the HCN. A sintered bubbler was quickly inserted after the H2SO4 and air was drawn over the sample to wash out the HCN. The tube was placed in water heated to 900°C to promote the extraction which wascontinued for 15 minutes. The air extract was scrubbed in a second trap containing lead acetate to remove sulphide (which interfers with the CN- electrode), and then trapped in a third tube containing 6 ml 10-1 NaOH. 3 ml of blood was thus extracted into 6 ml NaOH. This solution was then read directly with the electrode. STANDARD CURVE Before estimating unknown samples it was necessary to develop a standard curve for the electrode response to CN- and then to determine the recovery of CN- from the blood samples. The CN- measurements were made by using the pH input of a Radiometer Digital Acid Base Analyser type pHM72C in conjunction with a Radiometer FlO42CN cyanide electrode and a Calomel (K401) reference electrode. NaCN standards were made up in 10-1 NaOH and the CN- then estimated in terms of pCN by setting the NaOH 0 CN- solution to a reading of 7.000 and l-4 M CN to 9.300.

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MEASUREMENT The samples were placed in a darkened tube under constant stirring conditions and a reading was taken after 2 minutes. In constructing the curve standards were read sequentially starting with NaOH 0 and working up to l0-4 M CN- after which the electrode was washed for 2 x 2 minutes in 2 changes of NaOH before the procedure was repeated. At the beginning the end of each set of measurements the zero was set to pH 7.000 and the gain adjusted to give a reading of 9.300 in l0-4 M CNBy this means it was possible to construct the standard curve shown in Figure 2 which is the mean of 12 occasions each of 3-5 sets of readings. Although the standard error bars are so small that they are within the diameter of the dots, and the method obviously works, this gives rather a false impression of the problems we had with the electrode reading which was subject to considerable (and unpredictable) drift, so that adjustments were necessary before and after each set of standards were measured. This drift meant that there were errors in readings unknowns by as much as 0.1 pCN units, and the inaccuracy is made worse by the logarithmic response curve, so that a 0.1 pCN error could mean an approximate 10% error in estimations of CN content. To overcome this it was necessary to read unknowns in conjunction with bracketed standards, and to adjust the reading by the mean deviation of the bracket standards from the standard curve. In addition each set of unknowns and bracketing standards were read 3 times and the mean of the corrected readings taken as the result. By means of this rather tedious procedure I was able to obtain consistent and reasonably accurate results. SPIKED BLOOD RECOVERY To determine the recovery of CN- from blood, 3 ml samples were spiked with 100 µl CN, taken through the extraction procedure and read generating the curve shown in Figure 3. At the top end the recovery is approximately 75%, while at the bottom it is apparently in excess of 100%. This is due to the background level of CN- in blood which can be extracted from the un-spiked sample at 3.63 µM/l. This is slightly higher than that found by Anderson and Harland at Glasgow (1) in control men at 2.9 µm/l by CC-MS. However this is not a bad agreement considering that our curve becomes non-linear at these very low levels making accurate estimations difficult. If the extract curve is corrected for background CN- the recovery of the spiked dose becomes constant over the range and the 2 lines run parallel, with a mean recovery of 77%, and all estimates of unknowns were corrected assuming this recovery. ANALYTICAL RESULTS The tables shows our results together with those from the same samples analysed by CC-MS in the Department of Anaesthesiology of Glasgow University by Dr. RonWatson. What we have measured here is the CN- content of the 5 ml NaOH solutions, which were posted to Glasgow in sealed polypropylene syringes after CN- electrode measurements. Blood CU- was

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estimated by multiplying by 2 to allow for 3 ml of blood and adjusting for an assumed 77% recovery. The first 3 sets show an excellent agreement between the 2 methods, although in the last 3 the CCMS estimates came out a little lower than the electrode results for some reason. However I think this agreement between the 2 methods is generally good and it gave me confidence that the electrode gave valid results, and in particular that we were not suffering from interfering compounds which can effect electrode estimations.

RESULTS FROM ANIMALS That then was how we analysed the blood, but what did the results tell us? Firstly I must show you this slide from previous work (Figure 4) which illustrates what happens when an animal is exposed to HCN at l00-200 ppm. HCN as well as being a narcotic is a respiratory stimulant, so you tend to get a positive feedback situation, the more HCN the animal takes up the more it breathes, so the rate of uptake increases until the animal becomes semiconscious and depressive effects on the brain cause a reduction in respiration and thus in uptake. On might therefore, expect blood uptake to be 'front loaded' increasing more rapidly at first. However at low HCN concentrations (