A Historical and Experimental Study of the Breath/Blood Alcohol Ratio

Transcription

1 A Historical and Experimental Study of the Breath/Blood Alcohol Ratio A. W. Jones1, B. M. Wright2-3 and T. P. Jones Although breath alcohol analysis has been used for the determination of blood alcohol concentration for more than 40 years, the relationship between the breath and blood alcohol concentrations is still uncertain. For this reason, while the blood alcohol level is now almost universally accepted as a reliable indicator of intoxication, breath is still frequently regarded as only suitable for a screening test. The obvious advantages in convenience and acceptability of breath testing have nevertheless led some countries to adopt it as a substantive test, and most other countries would do so if only the relationship between the breath and blood levels were more clearly established and shown to be sufficiently constant for practical purposes. HISTORY OF THE BREATH/BLOOD ALCOHOL RATIO When examining the history of breath alcohol analysis one must remember that it has probably never been studied for its intrinsic scientific interest. The object has always been to find a simple and convenient way to determine the blood alcohol concentration. For this reason the work done and the conclusions drawn have often been influenced by considerations which were scientifically irrelevant though thought to be important from a forensic or commercial point of view. Workers in this field have seldom had sufficient knowledge of physiology or physical chemistry to appreciate fully the complexity of the problem. As a result, although there is a massive literature going back 100 years, very little of it is of any real value. Therefore in summarizing the literature we shall consider only the most significant contributions to the subject. Although Dubowski (9) has traced the history of breath alcohol analysis as far back as 1874, its first practical use was by Bogen in 1927 (2). Bogen also made some comparisons between urine and breath and concluded that the breath/urine ratio was fo r m e r ly of the University of Wales Institute o f Science and Technology, Cardiff, U.K., now at the Department of Alcohol Studies, Karolinska Institute, Stockholm, Sweden. ^Clinical Research Centre, Northwick Park Hospital, Harrow, U.K. 3The investigations described in this paper have been carried out over a period o f 4 years. During this time we have had the benefit of consultations with many colleagues at the University of Wales Institute o f Science and Technology and at the Clinical Research Centre for which we are most grateful. We are indebted to the Department of Medical Illustration of the Clinical Research Centre for preparing the figures. 509

2 510 A. W. Jones, B. M. Wright, et al. about 1 : The first serious quantitative studies of the breath/blood relationship, however, were made by Liljestrand and Linde (27) in Although their work is regularly quoted and the breath/blood ratio they arrived at is similar to that used today, the limitations of technique available at that time make their findings of very little evidential value. Nevertheless, they had a clear appreciation of the underlying physico-chemical and physiological factors involved, and laid the foundation of all the later work by putting forward the following basic concepts: 1. The breath sample must be of alveolar air, as defined by Haldane and Priestley (18) i.e. collected after at least 500 ml of breath have been discarded, to avoid dilution with dead space air and give a substantially constant C 02 concentration. 2. Breath is equilibrated with the blood in the alveoli at about 37 C but its alcohol content, when collected from the mouth, is less than it was in the alveoli, due to re-equilibration at a lower temperature in the upper respiratory tract, so that the actual alcohol content will depend on expired air temperature. Harger, in 1931 (19) in a communication to the American Chemical Society, suggested an entirely new approach. The report, which is very brief, reads in part as follows: Previous attempts to estimate the concentration o f alcohol in the body by analysing the breath have given quite erratic results. This is probably because the breath analysed was not always air from the alveoli or air cells, in which exchange o f oxygen and carbon dioxide between the blood and the lungs takes place. By the new method the alcohol and carbon dioxide content o f the breath are determined simultaneously. Since the carbon dioxide content o f the air is constant, this gives a means o f estimating the alveolar alcohol in any sample o f breath. Although it was based on the nonphysiological assumption that the C 02 content of alveolar air is constant, and ignored cooling of the breath in the upper respiratory tract, which reduces its alcohol but not its C 02 content, Harger s method came into wide use in the form of an instrument called the Drunkometer. A full account of this instrument was published in 1938 (20). Meanwhile in 1934, Haggard and Greenberg (16) had repeated Liljestrand and Linde s work and, in an apparently faultless study, made detailed determinations of the air/water and air/blood alcohol partition ratios. Unfortunately, for some reason that has never been satisfactorily explained, they obtained completely erroneous results. They concluded, quite differently from the air/water studies of all previous workers, that the breath/blood alcohol ratio was 1: 1300 at 34 C and not 1 : 2000 at 31 C as suggested by Liljestrand and Linde. Harger and his associates also believed the breath/blood ratio to be about 1 : 2000 and they vigorously attacked Haggard and Greenberg s findings. In 1941 Haggard and Greenberg (17) revised their previous findings and put forward a ratio of 1 : 1600 at 34 C. In the same year Greenberg and Keator (15) described a new breath alcohol device, the Alcometer, which analysed alveolar air for alcohol and took no account of C 02. There then followed a prolonged and acrimonious controversy between the Yale School, represented by Haggard and Greenberg, and the Indiana School, represented by Harger and his associates, about the use of the C 02/alcohol ratio and the correct figure for the breath/blood ratio for alveolar air. In 1950 Harger, Raney, Bridwell and Kitchel (22) published an extensive study of the in vitro air/water and air/blood partition ratios for alcohol and obtained air/water results that agreed well with those of all previous observers except Haggard and Greenberg. Their air/blood results also differed somewhat from those of Haggard and Greenberg. In the same year Harger,

3 Breath/Blood Alcohol Ratio 511 Forney and Barnes (21) published the results of a study of alcoholic breath by four methods, alveolar air, rebreathed air, alveolar air using the alcohol/c02 ratio, and mixed expired air using the same method. They also measured the temperature of expired air, and noted that it rose from 31 to 35 during prolonged expiration, which was in accordance with the findings of a number of previous investigators. Their results for a simple analysis of alveolar air, defined by them as breath delivered after 600 ml had been discarded, showed a considerable scatter with a mean breath/blood ratio around 1 : 2100 at 34 C. The results for rebreathed air, which they considered to be essentially the same as alveolar air, were similar. Breath/blood correlations for alveolar air and mixed expired air, using the C 02 method, were also similar, but none of the correlations would now be considered good enough for anything but a screening test. The controversy ended in 1953 when a special committee appointed by the U.S. National Safety Council issued the following statement: The basic principle governing the operation o f the three presently used breath alcohol methods (the Drunkometer, the Intoximeter, and the Alcometer) is the constant ratio existing between the concentration o f alcohol in the alveolar air and the blood. Available information indicated that this alveolar air/blood ratio is approximately 1 : However, since each method involves different procedures, different empirical factors are involved in the calculation o f concentrations o f alcohol in the blood in each o f the methods. The Drunkometer C 02 method was finally abandoned about 1960, after Smith (34) made a vigorous attack on the C 02 principle and showed that reliance on the fixity of the alveolar C 02 produced more errors than it avoided. The Intoximeter, which originally operated on the same principle as the Drunkometer, changed over to the use of alveolar air and still survives. It has also given its name to a portable gas chromatograph made by the same company. The Alcometer was succeeded by the Breathalyzer which survives to the present day. It has been frequently copied and has become universally accepted throughout the world and has given its name as a generic term for all breath alcohol analysers in the U.K. The Breathalyzer This instrument was developed by Borkenstein in about 1950, although a full description was not published until 1961 (3). It used a new and original photo-electric principle, as well as a new method of collecting end-expired air, both of which have remained unchanged, except for some minor details, to the present day. In view of its great practical importance the instrument is worth considering in some detail. The subject blows through a warmed flexible tube into a heated metal cylinder with a free piston which is raised until his breath escapes through side ports. When he stops blowing, the piston drops a short way, closes the ports and trapes a volume of 52.5 ml. When the piston is released the breath sample is bubbled through a potassium dichromate and sulphuric acid solution in a glass ampoule. The decolorisation of the solution by the alcohol is measured photo-metrically, and the result can be read off on a scale calibrated in blood alcohol units. Borkenstein accepted unquestioningly the correctness and fixity of the 1 : 2100 ratio and his instrument was designed specifically the conform to it, even the cylinder volume of 52.5 ml being l/40th of It was

4 512 A. W. Jones, B. M. Wright, et al. therefore always difficult to explain why, in practice, the instrument gave substantial variations in the breath/blood alcohol ratio and also tended to read on the average 10-15% low. When considering the sources of error in the Breathalyzer it is essential to realize that, although ostensibly calibrated a priori, the Breathalyzer is in fact calibrated a posteriori by means of an equilibrator, because the analytical performance of the instrument is influenced by a number of factors which cannot be precisely controlled, and each instrument s sensitivity has to be adjusted during manufacture while its calibration must be checked for each ampoule. The equilibrators used are based on the method, employed by Harger, of bubbling air through a mixture of alcohol and water of a suitable concentration at a controlled temperature. Wright (37) criticised this method on two grounds: (a) it is very difficult, if not impossible, to produce complete equilibration by bubbling; (b) it is impossible to avoid the production of a liquid aerosol, due to the bursting of the bubbles, and the subsequent evaporation of the aerosol can lead to super-saturation, or the droplets can even be carried over into the analyser. In practice, the first criticism, to some extent, tends to be cancelled by the second, but the performance of an equilibrator of this kind is critically dependent on its design, and on the rate of gas flow through it. Moreover, with a temperature coefficient of 6.5% per C (12) the solution and the gas need to be temperaturecontrolled to better than 0.5 C to obtain even ± 5% accuracy. Commercial equilibrators commonly operate at 34 C, which can lead to appreciable condensation of the vapour in the head space and connecting tubes if the device is used in a cool room. Moreover, the air is usually supplied from a hand bulb, and is therefore at room temperature. Wright therefore advocated, instead, the use of dynamic mixing of alcohol, water and air in known proportions to produce artificial alcoholic breath for absolute calibration purposes. The question of calibration, however, concerns only the analytical accuracy of the Breathalyzer; it is equally important to consider its ability to collect a suitable breath sample. Wright pointed out that, although the Breathalyzer contained an effective end-expired breath sampler, the type of sample obtained depended on the reliability of the operator and the co-operation of the subject; since the sample could be taken after as little as 100 ml had been discarded or after a full expiration, it could be anything from dead space air to an end vital capacity sample. In 1950 Harger et al (21) pointed out that, since final equilibration takes place in the mouth, at temperatures which can vary from 32 to 35 C, the high temperature coefficient of the partition ratio could produce variations of ± 10% in the breath/blood ratio. Wright (37) suggested that, as a result of this temperature effect, since it was known that the breath temperature rose continuously during expiration, the breath alcohol concentration would also be expected to rise in the same way. Using a special flame ionisation detector without a column, he showed that it did appear to do so, and that the rate of rise differed between subjects, so that it could produce variations between subjects in the breath/blood ratio, even if the same discard volume were used. Also in 1962, Kitagawa and Wright (26) described a new breath analyser which was based on the same fundamental sampling principles as the Breathalyzer, but designed to be cheaper to make and easier to use. This analyzer incorporated an automatic device which compelled the subject to discard 500 ml of breath before the instrument would operate, and made use of a special detector tube (25) to estimate the alcohol content of the breath. The Breathalyzer has been widely used in scientific work on the effects of

5 Breath/Blood A Icohol Ratio 513 alcohol on human personality and behaviour, especially in relation to driving (4), so it is unfortunate that the exact calibration of the instruments used has usually not been known. Nearly all early studies of the reliability of the Breathalyzer were vitiated by presenting no evidence of the reliability of the blood analysis, by not taking duplicate breath or blood samples, and by not taking sufficient care to make blood/breath comparisons only when absorption and distribution of alcohol were complete, so that there was no arterio-venous difference. In 1964 the first fully satisfactory study of the accuracy and precision of the Breathalyzer was made by Begg, Hill and Nickolls (1). The blood analyses were blind duplicated and no comparisons were made until serial breath analyses showed a steadily falling level, indicating that the absorption and distribution were complete. Begg et al found the Breathalyzer to read, on the average, about 13% low and to have 1% confidence limits of ± 16% for the estimation of the blood alcohol from the breath (38). These results were in line with those of most previous investigators, but for the first time they were unequivocal. At the same time it must be noted that, although their observations of precisions are probably valid for all instruments of the same type, (subject to operator performance, which may have considerable importance), the 13% figure is valid only for the particular instrument they used. At the Indiana Conference in 1965, Enticknap and Wright (11) described the results of another attempt to determine the in vivo breath/blood ratio, using the Kitagawa-Wright analyser. The instrument was calibrated on artificial alcoholic breath generated by dynamic mixing to conform to the 1 : 2100 ratio at 34 C. Thus the precision and accuracy of the breath method were established as an integral part of the experiment. Breath samples were taken in duplicate, one before and one after each blood sample. Blood samples were taken from indwelling intra-arterial or intravenous cannulae and were analysed blind in duplicate. Only two subjects were studied, each on two occasions, but it was possible to draw the following conclusions: (a) both subjects had mean breath/blood ratios well below 1 : 2100, one being 1 : 2370 and the other 1 : 2580; (b) these ratios were consistently different for the two subjects; (c) both subjects showed sudden changes in breath and blood alcohol concentrations which caused temporary changes in the ratio. In spite of the relatively (by modern standards) imprecise methods of blood and breath analysis, these sudden deviations were probably real because the duplicates of both breath and blood showed good agreement. Enticknap and Wright (11) therefore concluded that the ratio was more variable and lower than commonly supposed, and suggested a mean figure of 1 : 2370 on the best available evidence, which they considered was that of Begg et al (1). At the same conference, Fox et al. (14) gave the results of a meticulous breath/ blood comparison, using the Breathalyzer, and found it to read about 12% low, in spite of great care being taken to sample after a full expiration. In 1966 Payne et al. (30) created considerable confusion by publishing the results of a breath/blood correlation study which appeared to show a non-linear relationship, the breath alcohol falling relative to the blood at higher blood levels, and concluded that, due to the wide variations in the breath/blood relationship breath analysis was virtually excluded as a method of determining the blood alcohol. It was pointed out editorially however (5), that this sweeping statement was counter to almost all the evidence adduced by other workers. Their findings have never been confirmed and may have been due to the use of an unreliable infra-red analyser for breath Ȧt the Freiburg Conference in 1969 Franklin (13) published the results of

6 514 A. W. Jones, B. M. Wright, et al. careful studies of the Breathalyzer in which she found the ratio to be 1 : 2300, and Noordzij (29) at the same conference, using the Ethanograph, which works on the same principal as the Breathalyzer, found the ratio to be 1 : Up to this time, however, all observations could be criticised on the ground that the chemical analytical methods used were too imprecise to determine the ratio with sufficient certainty to establish that the difference between 2100 and 2300 was really significant. In 1966 Curry, Walker and Simpson (8) described a method of blood alcohol analysis by gas chromatography, using an internal standard, and the modern era of blood analysis, with standard deviations from 1 to 2 mg began, but it was not until 1969 that similar precision was obtained with breath alcohol analysis on a practical scale, when Penton and Forrester published their account of the Gas Chromatograph Intoximeter (32). In 1972 a meeting was convened at Indiana University to consider whether the 1 : 2100 ratio should be revised. After a prolonged argument, the following statement was issued: The basic principles governing the design o f breath alcohol instruments is that a physiological relationship exists between the concentration o f alcohol in expired alveolar air and in the blood. Available information indicates that 2.1 litres o f expired alveolar air contain approximately the same quantity o f alcohol as 1 millitre o f blood. Continued use o f this ratio in clinical and legal applications is warranted. This rather colourless statement was a compromise between the advocates of the 2100 and 2300 figures. The use of 2.1 litres instead of 2100 indicated that the ratio was only reliable to two significant figures, and the word approximately indicated that the question was still open. The question is now, however, virtually settled since Dubowski (10), using a very precise infra-red analyser and equally precise blood analysis, and assuming a 1 : 2100 ratio, found the breath to under-read the blood by about 12%, and Noordzij, Harris and Breen have respectively reported similar findings in this volume. The present work was begun in 1970, the original object being to repeat all the previous classical experiments, using the more precise and rapid methods of alcohol analysis now available to make a definitive determination of the breath/blood ratio in vivo and in vitro, and to try to explain the apparent deficiency and variability of the alcohol content of the breath. EXPERIMENTAL STUDIES Analytical M ethods Alcohol in water or blood. This was determined by gas chromatography, using a Perkin-Elmer flame-ionisation instrument, the water or blood being diluted 1:10 with a n-propanol internal standard solution, as described by Curry et al. (8), using a peak-height ratio for the determination. Under closely controlled conditions, taking and analysing the samples in the same laboratory, the standard deviation (SD) of duplicate samples at the 100 mg/100 ml level was 0.4 mg/100 ml for aqueous samples, and 1.3 mg/100 ml for venous blood samples taken from subjects. Repeated analysis of the same blood sample had SD of 0.3 mg/100 ml.

7 Breath/Blood A Icohol Ratio 515 Accuracy was checked with commercially available standard solutions, and also with made-up standards, and was better than 1.3%. Since we were concerned almost entirely with ratios, the absolute accuracy of our methods was not very important, however. Alcohol in air or breath. This was also determined by gas chromatography, using a Mark II Gas Chromatograph Intoximeter (GCI). In this instrument (32) the subject breathes through a mouth piece which leads to a sampling valve and then to an outlet nozzle, the whole being kept at a 100 C inside the case of the instrument. The outlet nozzle can be fitted with a bag, usually of 1.5 litres capacity, to which in turn a whistle can be connected that indicates when the bag has been filled. Air samples can be injected directly into the sampling valve which takes a volume of 0.25 ml. Alcohol-in-air standards. The precision of the analysis was repeatedly checked with alcohol-in-air head space samples prepared by static equilibration, as follows. Solutions of alcohol in water of various known concentrations were held in conical flasks with thick rubber caps immersed in a thermostatically controlled, stirred, water bath at 25 C ± At this temperature there is less danger of loss of alcohol by condensation on the neck or cap while sampling. It was found that the alcohol concentration in the gas phase came to a steady level in about ten minutes and forty 5 ml samples could be taken before the concentration had fallen by 1%. Air/water partition ratio. The alcohol concentration in the gas phase of the equilibrator was checked by re-determining the air/water partition ratio by a new absolute method. A measured volume (about 1 ml) of an alcohol solution of known concentration was placed in a 1 or 2 litre flask and equilibrated at a known temperature. A portion of the solution was then removed and re-analysed. In this way the amount of alcohol lost to the air in the flask was determined and the partition ratio at that temperature could be calculated. The partition ratio at 25 C, determined in this way, was found to agree well with that of Harger (21), which in turn agrees with those of all earlier workers except Haggard and Greenberg (16, 17). It was therefore felt that it was satisfactory to use air samples produced by static equilibration for calibrating the GCI and to determine the partition ratios at other temperatures by analysis of head-space samples. Multiple analyses of air samples prepared in this way, taken with a warmed 5 ml syringe and injected into the sampling valve of the GCI, has a SD of 0.5 mg/100 ml at a concentration approximately equivalent to blood levels of 100 mg/100 ml. Blood/breath correlation studies. Having established the accuracy and precision of our methods we used them to make a number of breath/blood correlations, using healthy young male volunteers in the laboratory. The subjects were asked to blow into the GCI for as long as possible, and only when the sound of the whistle showed that the flow was slackening was the sample taken. The exact volume discarded was not measured, but it was never less than 2 litres, and in some cases an end-vital Capacity sample was taken. Capillary blood samples were taken between two breath samples once absorption and equilibration had been shown to be complete by a steady fall in breath alcohol. The results of 78 such comparisons on 15 subjects are shown in Figure 1. Three points will be noted: 1. The line of best fit is not 1 : 2100, but 1 : 2300, which is very close to the figure suggested by authors such as Enticknap and Wright (11), by Franklin (13) and by Noordzij (29). 2. The correlation between breath and blood, though good, is not as close as would be expected from the known precision of the air and blood analyses. With SDs of 0.5 mg for alcohol-air mixtures and 1.3 mg for blood, the SD of an estimate of the blood alcohol concentration from the breath

8 516 A. W. Jones, B. M. Wright, et al. should be 1.55 mg but in fact it is 3.4 mg. 3. The mean SD of paired breath samples was 0.85 mg, the range being from 0.6 to 1.1 for different subjects. Figure 1 CAPILLARY BLOOD ALCOHOL CONCENTRATION MG/lOOml Breath/Blood correlation study on 15 male subjects, using gas chromatography for both breath and blood. These results suggested that the doubts about the precision and accuracy of the determination of blood alcohol by breath analysis, even with such a precise method, were well founded and called for a careful re-examination of the theoretical and practical basis of the procedure. We therefore decided, as a first step, to re-check the in vitro air/blood ratio. In vitro Studies o f the A ir/b lood R atio Methods. Equilibration was carried out as for the air/water standard and the blood and air alcohol concentrations were determined as already described. Most of the work was done on small blood samples kindly supplied by the Welsh Regional Blood Transfusion Unit. Alcohol was added to the samples to produce blood alcohol concentrations usually of the order of 100 m g/100 ml.

9 Breath/Blood A Icohol Ratio 517 The mean air/blood ratio at 34 C was 1 : 2164 (SD 43) for men and 1 : 2208 (SD 45) for women. The difference of 2% between the sexes, which was of high statistical significance, was presumably due to the well-known difference in haematocrit between men and women. The mean of all the subjects is 1 : We also determined, in 42 subjects, the temperature coefficient for the ratio from 20 to 40 C. We found it to be nearly constant at 6.3% per C between 30 and 40, which agrees well with Harger s figure of 6.5% (21). Our mean ratio at 34 was, however, 6% lower than Harger s figure of 1 : This we believe can be largely accounted for by the fact that we used heparin as an anti-coagulant while he used 0.5% sodium fluoride which we have found to raise the vapour pressure of alcohol at this temperature by 5.1%. It therefore seemed that, although the discrepancy between the in vitro and in vivo breath/blood ratios at 34 C was rather less than had appeared, it was still substantial. Therefore we decided to make a detailed study of the changes in alcohol concentration in the breath during expiration, in relation to expired volume and breath temperature. In vivo Studies o f Breath/Blood Ratio Methods. Breath alcohol concentration was measured with the GCI as already described. Volume was measured with a Wright Respirometer (36) connected to the outlet of the GCI, using both the original mechanical respirometer and also an electronic version (7) with which volume could be recorded on a 2-channel potentiometric recorder. Breath temperature was measured with a thermistor inserted in the mouth-piece of the GCI and connected to the recorder. The thermistor was calibrated and frequently checked by immersion in three stirred water baths thermostatically controlled at 32, 34 and36 C±.05. The connections from the respirometer and thermistor to the recorder were taken through switches which were arranged to disconnect them when the sampling button was pressed, so that the exact volume and temperature at the moment of sampling were recorded. A typical record obtained in this way is shown in Figure 2. Procedure. To overcome the difficulty of a continually falling blood alcohol level, and to avoid the need for possibly erroneous back calculations, the breath alcohol level in a sample taken at the end of a full Vital Capacity (VC) was taken as 100%, and the level after lesser expirations was expressed as a percentage of that in an immediately preceding or following end-vc sample. Samples could be taken and read at two minute intervals so that no significant error due to time lag resulted. Using this technique it was possible to obtain alcohol expirograms from a number of subjects. From these it appeared that although the breath alcohol level was flattening off it had not really reached a constant level by the end of even a full VC expiration and never reached the level predicted from the temperature of the breath at the moment of sampling and the blood alcohol level at the time. It was therefore decided to use the re-breathing technique of Harger, Forney and Baker (23). In this procedure, the subjects were asked to take a moderate inspiration and then breathe in and out of a plastic bag of about 4 litres capacity. The bag was kept warm in an electrically heated outer bag, but taken out for use. At the end of re-breathing the subject expired into the bag which was then discharged into a modified Drunkometer. Professor Harger kindly loaned us one of his heated outer bags

10 518 A. W. Jones, B. M. Wright, et al. TIME IN SECONDS Figure 2 R ecord o f 3 successive full expirations from one subject. The temperature and volume records were switched o ff simultaneously at the moment o f sampling. but we modified his technique. Instead of taking the inner plastic bag out for use, we kept it in the outer bag where its temperature was maintained at 40 C ± 3. This slight elevation of temperature seemed unlikely to influence the breath temperature because the heat capacity of dry air is very small compared with that of the respiratory tract. On the other hand, loss of alcohol due to water condensation in the bag, though slight, was, we felt, to be avoided if possible. F igu re 3 Correlation between rebreathed air and blood alcohol levels obtained by equilibration in vitro in 11 subjects.

11 Breath!Blood A Icohol Ratio 519 In the second modification the subject expired directly into the GCI after taking his last breath from the bag, instead of sampling from the bag. In this way, the breath temperature could be recorded, which was not possible using Harger s original method. The first observation we made was that the re-breathed air alcohol concentration (RBAC) was, as already noted, substantially higher than that of an end-vc sample. Secondly, when re-breathed air was compared with air equilibrated over a sample of the subject s blood taken at the same time and held at the same temperature, the two alcohol levels were found to agree very closely (Figure 3). We therefore decided to call the RBAC 100%, and to do expirograms with up to five re-breathings to allow us to see the complete pattern of alcohol change during the procedure. To measure the volume expired during re-breathing, a second (mechanical) respirometer was inserted in the bag. Each sample taken after less than five rebreathings was paired with a five re-breathing sample, as was done previously with the end-vc samples. Figure 4 shows the results on six subjects where breath temperature and alcohol level are plotted against expired volume. Volumes above about 5 litres were, of course, only reached by re-breathing, as shown by the discontinuity in the curve at this point. A ^ A Breath alcohol concentration as % of rebreathed air level Breath alcohol concentration corrected for temperature c o o» AA A A % A O A A O "EXPIRED"VOLUME - LITRES Figure 4 Alcohol expirogram for 6 subjects.

12 520 A. W. Jones, B. M. Wright, et al. The breath temperature rises very rapidly to about 34 and then more slowly, to reach a plateau at about 35 after 2 or 3 re-breathings and shows intersubject variations of up to 1 C at the same expired volume. The breath alcohol also rises rapidly at first and is still rising at the end of a full VC expiration and does not flatten out until after 2 or 3 re-breathings. The smaller, undifferentiated dots show the alcohol concentration that would be expected if it depended solely on temperature, as suggested by Liljestrand and Linde (27). The points were obtained by calculation from the observed temperature and the 100% level, using a temperature coefficient of 6.3% per degree. It can be seen that the alcohol level lags behind the expected level and does not reach it until both curves flatten out after 2 or 3 re-breathings. DISCUSSION OF THE HISTORICAL SURVEY Our historical survey of breath alcohol analysis shows that there have been good reasons to doubt the validity of blood alcohol estimates obtained by breath analysis, and that, in the early years, its reliability was considerably overestimated. In view, however, of the very high blood alcohol levels (up to 150 m g/100 ml) that were tolerated in those days, no injustice is likely to have been caused by relying solely on breath analysis. Moreover, since the introduction of the Breathalyzer, the blood alcohol has usually been underestimated by 10-15% which has been a further precaution against injustice. Unfortunately, however, this argument has been, and still is being, used to ignore, or even deny (10), this constant error, with the result that there have been no previous systematic investigations of its origin. Similarly, the desire of the Courts for a simple yes or no answer has led to a reluctance to do repeated breath analysis, or both breath and blood alcohol analyses in forensic cases. Thus, the non-reproducibility of breath analysis and its relatively poor agreement with blood analysis have not been apparent. The curious Haggard and Greenberg, (16,17) and Payne et al. (30) episodes have also contributed to reasonable doubts about the validity of breath analysis, though recent studies, including our own, with more reliable instruments and more careful observers have amply confirmed that the determination of the BAC from breath is possible with a precision and accuracy which, though not quite as good as obtained with direct blood analysis in the laboratory, are quite adequate for practical purposes. DISCUSSION OF THE EXPERIMENTAL STUDIES In vitro Studies Our fresh determination of the air/water partition ratio for alcohol using a new technique agrees well with the results of previous workers. As it depends only on analysis of the liquid phase, it makes no assumptions about the reliability of gas analysis and so can be validly used to check the reliability of our air-alcohol standards. Our redetermination of the air/blood ratio in vitro at various temperatures is only the fourth that has ever been made. Of these, that of Liljestrand and Linde (27) was too hampered by technical difficulties to be of much evidential value. In particular, they obtained a ratio of 1 : 2000 at 31 which is about 20% too high for that temperature. The common habit (24) of quoting their results as supporting this ratio is not therefore justifiable.

13 Breath/Blood AIcohol Ratio 521 Haggard and Greenberg s results (16, 17) must, of course, be disregarded, but our excellent agreement with Harger s figures (21), allowing for the salting out effect of sodium fluoride, makes us feel that further work in this field is probably unnecessary. Our observation of the difference in the in vitro ratio between men and women shows that variations in haematocrit, as suggested by Payne et al, (31) must contribute to variations in the breath blood ratio in vivo because the alcohol in the breath is derived from blood by equilibration while blood analysis is normally on a weight/ volume or volume/volume basis. Hitherto the lack of precise analytical methods and the other causes of variation in the ratio have made haematocrit effects relatively unimportant (39), but now that breath analysis is becoming much more precise it must be pointed out that, where there is a discrepancy between breath and blood from this cause, the breath estimate is to be preferred as it depends on the partial pressure of alcohol in the tissues which presumably controls its effect on the brain (39). Conversely, this source of error could be eliminated if breath analysis were carried out by direct head space chromatography on whole blood. However, apart from technical problems, this would have to involve a change in legislation since all standards are at present based on weight/volume or volume/volume analysis. Breath/Blood. Correlation Study As already noted, our observation that the best estimate of the breath/blood ratio is 1 : 2300 and not 1 : 2100 is now commonplace, as is our observation that the variability of the ratio is substantially greater than can be accounted for by the analytical errors of breath and blood analysis. Our figure of 0.5 mg for the SD of air-alcohol analysis may seem low, but, considering the much more reliable standard and much more precise analyser that we used, it is not so much lower than the figure of 1.3 mg found by Britt and Borkenstein using a Breathalyzer and Alcoholic Breath Simulator in 1965 (6). The low and variable SD for repeated breaths is interesting and confirms the observation of Enticknap and Wright (11) that the breath/blood ratio can sometimes change without any change in breath concentration, and that individual variations in the stability of the breath concentration occur. The Alcohol Expirogram Our demonstration of the changes that occur in the alcohol level during expiration and their relation to volume and temperature (Figure 4) seem to us to be the most valuable result of our work. It is easy to see that the alcohol level in any single breath is lower than predicted by the accepted theory, and why, with the Breathalyzer, where the subject is free to discard a volume varying from a few hundred ml to several litres, the results should be so variable. The Kitagawa-Wright, with its fixed discard volume of 500 ml, is little better because the alcohol level at this volume is still rising sharply and variations of up to 10% for different subjects are still occurring. It is not until after two or three rebreathings that the variation falls to within ± 3% of the mean level and the level flattens out. Theory o f the Alcohol Expirogram The classical theory of Lindjestrand and Linde (27) postulated that alcohol in breath behaved very like C 02, so that it was only necessary to obtain an alveolar air sample

14 522 A. W. Jones, B. M. Wright, et al. to be able to determine the alcohol concentration in the blood, provided that allowance was made for re-equilibration at a lower temperature during expiration. Harger s observations (21) that the average temperature of expired air was 34, which gave an in vitro ratio of 1 : 2100, and that the in vivo ratio was also about 1 : 2100, led to the adoption of this ratio as the correct one. In fact, as Figure 4 shows, the alcohol level does not reach that predicted for the breath temperature until after two or three rebreathings. This cannot be attributed to a failure of temperature equilibration, however, since the breath from the alveoli is at a higher temperature and a failure of temperature equilibration would lead to a higher and not a lower breath alcohol level than expected. We have therefore to look for a mechanism which causes alcohol to be lost from the breath during expiration, to a variable extent, decreasing with volume expired and only reaching its proper level after rebreathing. We believe that this mechanism lies, as suggested by Forster (12), in the respiratory dead space. This consists of the conducting airways of the respiratory tract and extends from the lips to the respiratory bronchioles. It is called dead because gas exchange through its surface between blood and air is very slow compared with that in the alveoli. The concept of alveolar air therefore is that it is the fraction of the breath that emerges after washing out the anatomical dead space, which has a volume of about 200 ml. Haldane and Priestley (18) showed that the CO2 level reached a plateau after 500 ml had been discarded, at the end of a normal expiration, i.e. during quiet breathing. More recent studies, using a mass spectrometer (10), have shown that up to 65% of the vital capacity may need to be discarded before the C 02 level becomes stable, if the expiration follows a deep inspiration. Figure 5 shows a typical C 0 2 expirogram obtained by mass spectrometry and it will be seen that, at first glance, it closely resembles the alcohol expirogram. In fact, however, there are two important differences. The C 02 level starts at zero, whereas the alcohol level, even at a 100 ml expired volume is over 90% of the predicted level for the breath temperature, and the alcohol level, as already noted, rises continually throughout one single breath. These differences can be shown to be due to the great differences in the solubility of C 02 and alcohol in water and the much higher concentration of C 02 in the blood and breath. The partition ratio for C 02 is 1 : 1.5 at 34 (33) which is nearly 1,400 times as high as that of alcohol, and its concentration in breath is 200 times as high as that of alcohol at a blood level of 100 m g/100 ml. In addition, C 02 is carried in the blood in the red cells, so that virtually no interchange takes place with plasma or other body fluids. Alcohol, on the other hand, diffuses freely through all body fluids including the layer of mucus covering the surface of the dead space. This layer is about 5 microns thick and covers a surface of about 6000 cm2 (35) so it has a volume of about 3 cm3. This can absorb about half the alcohol from six litres of breath while it can only absorb the C 0 2 from about 20 ml. It therefore seems that the loss of alcohol from breath during expiration can be explained quite simply as follows. In the fully equilibrated state the dead space mucus will contain alcohol at the same partial pressure as in the rest of the body but, during inspiration, air with no alcohol in it passes over the mucus and must extract alcohol from it. If this is not replaced immediately from the blood it will be replaced from the expired air whose alcohol level will be thereby lowered. This is a simple and attractive hypothesis, but two conditions must be satisfied before it can be accepted. The first is that the alcohol lost to the mucus cannot be completely replaced from the blood stream between inspiration and expiration. The blood supply to the mucosa is rela

15 Breath/Blood Alcohol Ratio 523 G o u ld Inc., Instrument Syste m s Division Figure 5 CO2 expirogram by mass spectrometry. Reproduced with permission from K. Dubowski, (10). tively good, though in no way comparable to that of the alveoli, the blood being about 50 microns from the surface instead of about 1 micron as in the alveoli, and with an enormously smaller surface/volume ratio. Only very rough estimates of the time taken for complete replacement to occur can be made by theoretical calculations so we carried out two simple experiments. The first showed that, after rebreathing, it took three breaths of fresh air to restore the breath alcohol to its normal level. The second showed that, if a subject held his breath with the mouth closed for thirty seconds after a deep inspiration, the alcohol level in an ordinary expired sample reached the rebreathed air level. From these two experiments it seems that it takes much longer than the normal interval between inspiration and expiration for the alcohol abstracted from the mucus during inspiration to be replaced. It also suggests that the effect of rebreathing is, in part, to allow time for the mucosal alcohol to be replaced from the blood as well as directly from the breath. The relative importance of these two factors could be determined by varying the duration and frequency of rebreathing, but we have not yet investigated this point. The second requirement is that alcohol must evaporate from the mucosa more rapidly than water so that its concentration in the mucus will fall. It can be shown by simple calculation from the partition ratio and the saturation level of water vapour in air that the alcohol/water ratio in air at body temperature is about eight times that in water, so there is no doubt on this point. In the light of this theory it is easy to see the reason for the difference between the C 02 and alcohol expirograms. C 02 starts from zero because air in the anatomical dead space has no contact with red cells and so acquires no C 02. Alcohol on the other hand does not, because almost complete equilibration with the mucosa of the dead space takes place very rapidly. It must be remembered that most of the surface of the dead space is in the smaller bronchi where the temperature is nearly 37 and the surface/volume ratio is relatively high.

16 524 A. W. Jones, B. M. Wright, et al. C 02 will only reach a plateau after a 500 ml discard, however, if the expiration, expiration follows on the end of a normal tidal expiration, as in Haldane and Priestley s original description (18). When, as is usually the case with breath sampling for alcohol, a deep breath is taken first, many more alveoli are opened up and some of these, due to ventilation/perfusion defects, may not have become fully equilibrated for C 02. The result is an apparent increase in the dead space, known as the physiological dead space so that, as noted earlier, several litres may need to be discarded before a stable C 02 level is obtained. This effect does not operate with alcohol because it equilibrates instantaneously in the alveoli and, as we have seen, very rapidly with any tissue fluid. Even a so-called unperfused alveolus must have some tissue fluid supply with which alcohol can equilibrate. The physiological dead space for alcohol is therefore actually in the anatomical dead space in the form of the mucus layer and it is evident that it carries a chronic alcohol debt that can only be fully repaid if inspiration of fresh air is stopped either by holding the breath or by rebreathing. If we accept the dead space theory as a more or less complete explanation of the excretion of alcohol in breath the air/blood ratio in vitro can be seen to be of no importance, except as mentioned above, because the final determination of the breath /blood ratio in vivo takes place in the dead space. The observed breath/blood ratio in vivo must therefore be governed almost entirely by the condition of the dead space mucus. Such factors as the ascularity of the mucosa, the thickness of the mucus, the body temperature, patterns of breathing, and the temperature and humidity of the inspired air are what must be taken into account when trying to explain the variations in breath blood ratio which are observed. If anything is to be done, apart from instrumental improvements, to improve the reliability of breath testing, this is the field in which research is required. Such research is now relatively easy to carry out, especially with the aid of a mass spectrometer, because it does not require blood analysis which can be replaced by an analysis of rebreathed air. REFERENCES 1. Begg, T. B., Hill, I. D., and Nickolls, L. C., Breathalyzer and Kitagawa-Wright Methods of measuring Breath Alcohol. British Medical Journal i, 9 (1964). 2. Bogen, E., Drunkenness, Quantitative Study of Acute Alcohol Intoxication. Journal American Medical Association 89, 1508 (1927). 3. Borkenstein, R. F. and Smith, H. W. The Breathalyzer and its Application. Medicine, Science and the Law 1, 13 (1961). 4. Borkenstein, R. F., Crowther, R. F., Shumate, R. P., Ziel, W. B. and Zylman, R., The Role o f the Drinking Driver in Traffic Accidents. Indiana University, Bloomington, Indiana, U.S British Medical Journal: Editorial: Distribution o f Alcohol in the Body, i, 184 (1966). 6. Britt, B. J. and Borkenstein, R. F., Reproducibility o f Breathalyzer using an Alcoholic Breath Simulator: In, Alcohol and Traffic Safety: Proceedings o f the Fourth International Conference on Alcohol and Traffic Safety, Bloomington, Indiana University, 1966, p Cox, L. A., Almeida, A. P., Robinson, J. S. and Horsley, J. K., An Electronic Respirometer. British Journal o f Anaesthesia 46, 302 (1974). 8. Curry, A. S., Walker, G. W. and Simpson, G. S., Determination of Ethanol in Blood by Gas Chromatography, Analyst 91, 742 (1966). 9. Dubowski, K. M., Measurement of Ethyl Alcohol in Breath. In Laboratory Diagnosis o f Diseases caused by Toxic Agents. F. W. Sunderman and F. W. Sunderman Jr. (Eds.). W. H. Green, St. Louis, Mo Chapter Dubowski, K. M., Biological Aspects of Breath Analysis, Clinical Chemistry 20, 294 (1974). 11. Enticknap, J. B. and Wright, B. M., In-Vivo Determination of the Breath-blood Ratio. In,

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