by the rate at which it disappears, since the proportion excreted

Transcription

1 228 J. Physiol. (I940) 98, I2.oI5-34: DETERMINATION OF THE METABOLIC RATE OF ALCOHOL BY M. GRACE EGGLETON1 From the Department of Pharmacology, University College, London, and the Institute of Physiology, Cardiff (Received 25 January 1940) THE rate at which alcohol is oxidized in the body is measured approximately by the rate at which it disappears, since the proportion excreted is small and regular; and the rate at which it disappears is equal to the body weight multiplied by the rate of change of concentration in the whole body. In man, however, the rate of change of concentration can be measured only in the blood. Hence, it is of importance to know the relation between the concentration in the blood and the concentration in the whole body at the same moment. If this relationship is not reasonably constant, the extent and time relations of any variation must also be known. The proportion of the body in alcohol equilibrium with the blood (i.e. the mean concentration in the whole body expressed as a fraction of the blood concentration) has been designated r by Widmark [1932] and is derived from the change in blood alcohol concentration in the following manner (see Fig. 2). The straight line joining the experimental points is extrapolated back to zero time, giving the theoretical initial concentration in the blood (CO): the amount of alcohol injected and the weight of the subject are measured; then alcohol injected (g.) r=_ body weight (kg.) x CO (mg./g.) The metabolic rate of alcohol is, therefore, r multiplied by the rate of decrease in blood alcohol concentration (designated fi by Widmark). This value r, and the metabolic rate calculated from it, are intimately dependent on the line extrapolated from the blood alcohol curve and therefore on the regularity of the experimental points from which that 1 Working under a grant from the Rockefeller Foundation to the Maudsley Mental Hospital.

2 METABOLIC RATE OF ALCOHOL 229 curve is constructed. Moreover, the assumption is implicit that the blood is in equilibrium with the same proportion of the body tissues throughout the experiment, and it might be expected that the same individual should show the same value of r during different experiments performed within a short interval of time. In most of the work published, in which Widmark's [1922] micro-method of alcohol estimation is used, these conditions are not fulfilled. Irregularity in the experimental points of the blood alcohol curve renders extrapolation of any straight line drawn through them a dubious matter, and, possibly because of this, the value of r for any individual varies within quite wide limits, e.g in man [Bernhard & Goldberg, 1935] and in the dog [Widmark, 1933]. Some factors, other than analytical, affecting the regularity of the decrease in blood alcohol concentration have been suggested by various workers. It seems likely that. analysis of venous blood gives less regular results than that of arterial blood, presumably because the circulation in some tissues is less effective than in others, and since these tissues (e.g. muscle) are not using alcohol, blood which has lingered there will contain a higher concentration of alcohol than blood in the general circulation which has passed through the liver. The same explanation, extended possibly to the spleen, covers the observations of Nyman & Palml6w [1934], who found that in subjects who had been completely resting, renewed activity actually produced a slight increase in the concentration of alcohol in the blood instead of the expected decrease with the passage of time. In addition, the decrease in the concentration of alcohol in the blood is a result not only of its metabolism but also of its excretion. In the present series of experiments, excretion in the urine accounted as a rule for l-2 % of the alcohol injected, occasionally rising as high as 3j % and no correction in the value of fi was considered necessary. In view of the importance of this factor r in the measurement of the metabolic rate of alcohol, experiments were designed to test the validity of its derivation from the change in blood alcohol concentration, and its constancy in any one individual. METHODS Cats were used as experimental animals, and were given only water for the 18 hr. previous to experiment. They were anaesthetized with nembutal, given intraperitoneally (0-65 c.c./kg.), and thereafter maintained at constant body temperature C. Alcohol was usually injected intravenously in 5, 10 or 20 % solution in saline into the jugular

3 230 M. G. EGGLETON vein at a rate varying from mg./min. Blood samples were removed from the carotid artery. The blood, collected in tightly stoppered tubes with oxalate, was placed immediately at 00 C., centrifuged within a few hours, and the plasma returned to the refrigerator until analysis the following day. If kept in tightly stoppered small vessels, no appreciable loss of alcohol occurred during several days at 00 C. Muscles were removed after ligature, ground immediately (after being weighed) with a measured volume of cold 10 % trichloroacetic acid, and the mixture left at 00 C. in a stoppered centrifuge tube until centrifuged before analysis. In some cases they were first frozen in liquid air on removal from the body, a procedure always followed in the case of liver, which was removed at the end of an experiment. Analysis of the alcohol content was then made on 1 c.c. plasma or on 1 c.c. acid filtrate of the tissues, in the following manner. The method used was based on that described by Newman [1936], but altered in certain details. In essence, the alcohol is distilled under reduced pressure from the plasma mixed with anhydrous Na2SO4 into a standard K2Cr2O7- H2S04 mixture, the excess of K2Cr2O7 being titrated with thiosulphate. In practice, standard conditions are found essential at many points for accurate recovery. (1) An excess of anhydrous Na2SO4 is used to prevent frothing (a crude preparation should be used, since the A.R. material supplied by British Drug Houses Ltd. is not sufficiently finely dispersed for the purpose), and the plasma is added to it only shortly before analysis. (2) The tube connecting the distillation flask with the receiving tube is both dry and tilted downwards towards the receiving vessel. (3) The distillation flask is maintained at C. at a pressure of mm. Hg for min., and the connecting tube then gently warmed before the vacuum is broken. An ordinary reading lamp on an adjustable stand suffices for this purpose. With a battery of six sets of apparatus connected with the same vacuum pump, the whole six estimations could be completed and the apparatus reassembled in 1j- 1 hr. No special apparatus is required beyond 25 c.c. Pyrex Erlenmeyer flasks and c.c. Pyrex collecting tubes, together with suitable two-way taps on each for adjusting the pressure. Rubber corks are used and the connecting tube is joined in the middle with ordinary rubber tubing. The receiving flask contains 5 c.c. N/20 K2Cr2O7 in 50 % H2SO4 and is titrated against N/40 thiosulphate of which 1 c.c. is equivalent to mg. alcohol. The technique described is adequate for determination of mg. alcohol/100 c.c. plasma. The recovery of alcohol, from watery solution or plasma, is complete, and the discrepancy between

4 METABOLIC RATE OF ALCOHOL duplicate determinations is rarely greater than 2 mg./100 c.c. The presence of trichloroacetic acid in the distilling flask does not affect the estimation. RESULTS The relation between alcohol concentration in the tissues and in blood plasma Following an intravenous injection of alcohol into the body, equilibrium between muscles and blood is reached only after about 30 mn. (Fig. 1). The time course of the alcohol concentration in the blood suggests, however, that equilibrium in the whole body is not complete for 100 ~ ' lni. Time after end of intravenous injection of alcohol Fig. 1. The rate of equilibration between skeletal muscle and blood plasma following an intravenous injection of alcohol as determined by direct analyses of the two tissues. The 100 % value is taken as the average equilibrium value obtained 1-6 hr. after an injection. over an hour (Fig. 2); this difference may well be due in part to the delay which is known to occur in the establishment of equilibrium between the blood and cerebro-spinal fluid [Abramson & Linde, 1930]. When equilibrium has been reached, all the tissues analysed, with the exception of fat, show a concentration of alcohol % of that in the blood plasma (Table I). If liquid air was not used in killing the tissue, a considerably lower value (55-65 %) was obtained with liver, as recorded in previous literature. No such difference was observed in the case of muscle. This

5 232 M. G. EGGLETON is to be expected in view of the discovery that at least 90 % of the metabolism of alcohol occurs in the liver and none in the muscles [Lundsgaard, 1937]. 300 f+ Cat 3i26 kg. C6=2-35 r X O-64 C 4-r- p C; +Ci Sp 5 _ P4200- C; 0 8 ~~~~~~+ co ~~~~~~~~ ta 1l _ 4p.m g. alcohol intravenously Fig. 2. The alcohol concentration in blood plasma following an intravenous injection of alcohol showing Widinark's method of determining the factor r and thus the metabolic rate. Equilibrium between tissues and blood is not complete until ca. 1i hr. after the end of the injection. TABLE I. The relation of the concentration of alcohol in tisues to that in plasma after equilibrium is established. Ratio of concentration of alcohol in tissue to that in plasma Standard Tissue (per g. of tissue) error Skeletal muscle 0-74 Liver ± ±002 Kidney *01 Intestine *01 Spleen 0*8, 0-81 Brain 0* Fat 0*16 +0*03 No. of observations

6 METABOLIC RATE OF ALCOHOL 233 The low value for fat, corroborating previous workers, was unchanged whether the alcohol was estimated directly by distilling the fat under reduced pressure, or by previous extraction with water or trichloroacetic acid and distillation of the watery extract. It is stated in the literature [Smith & Stewart, 1932] that bone also contains little alcohol as compared with the blood, and the varying proportions of bone and fat in different individuals would readily explain the widely differing values of r observed by the indirect method. With a view to deternining the constancy of r during any one experiment, muscles were removed at varying times. The ratio of the concentration of alcohol in muscle and plasma remained appreciably unchanged for 6 hr. or so after equilibrium had been reached, and was also unaffected by the absolute concentration of alcohol in the body (Table II). A similar constancy of the ratio of the concentration of TABLE II. The relation of the concentration of alcohol in muscle to that in plasma, (A) at varying times after the injection, and (B) at varying concentrations of alcohol. A Ratio of concentration of Time after alcohol in muscle to that alcohol injection in plasma hr. (per g. of tissue) (6) (5) A (5) 3-4 0O (8) 4-5 0*76 (1) < (i) 6-7 0* (8) L I G R A R B Ratio of concentration of \ Alcohol alcohol in muscle to that \ A concentration in plasma mg./100 C.C. (per g. of tisue) *75 + 0*02 (10) ±001 (16) *02 (8) -W1," alcohol in both brain and liver tissue to that in the blood during 1-12 hr. following a dose of alcohol was observed by Harger, Hulpieu & Lamb [1937] in dogs. Assessment of the metabolic rate of alcohol from changes in the plasma alcohol concentration From these direct analyses, one would expect the values obtained by indirect assessment of r to lie between 02 and 0O8, lean muscular individuals showing a higher value than obese ones. This is in fact the

7 234 M. G. EGGLETON case. Average figures obtained by Widmark [1933] were 0-68 for men and 0 55 for women. In the present series of cats, r varied from 0-48 (a very large, fat cat) to 0X78 (a young, muscular one) with an average value of 0* These values are not strictly comparable with those of previous workers since the terms fi and r are used in a slightly different manner from that introduced by Widmark.,, the decrease in alcohol Cat 2-5 kg. r ks r =O050 p = 0f00285 p = CB 280 c, o uo Fig C 4 I-1 1I I 12 1v-"" g. alcohol 3-8 g. alcohol intravenously intravenously p I I p.m. The concentration of alcohol in blood plasma following two separate intravenous injections of alcohol, showing the identity of r in the two cases. concentration in the blood, is expressed as mg./c.c. plasma/min. instead of mg./g. blood/min., and r is the proportion of the body in equilibrium with the blood plasma instead of with the whole blood. Alcohol concentration is % higher in the plasma than in whole blood (in man; [Miles, 1922]) and the value of r as used in this series of animals is, therefore, possibly % lower than that of Widmark. If the large variations of r in any one individual noted by other workers are real, the question of the time factor of this variation becomes of importance, for obviously if r varies from hour to hour the whole method of assessment of metabolic rate from changing concentration of alcohol in the blood is suspect. In twenty animals (nineteen cats, one I s.e. of mean is given here and elsewhere in the paper.

8 METABOLIC RATE OF ALCOHOL 235 dog) r was determined twice, a second alcohol injection being given after the first value of r had been obtained, as shown in Fig. 3. In no pair of values on the same animal was a difference greater than observed. The second determination was higher than the first in nine animals, lower in seven, and the same in four: the mean difference between the second and the first value in each of the animals being The standard deviation from the mean of the difference between any pair of determinations is , and it seems unlikely that the errors involved in the whole experimental technique would be less than this. Assessment of the metabolic rate of alcohol by constant infusion The reliability of the assessment of the metabolic rate of alcohol from the observed value of fi and inferred value of r was subjected to a further test. The procedure shown in Fig. 2 was adopted in the first half of the experiment, so that the metabolic rate could be calculated in the usual way; a constant intravenous infusion of alcohol was then given over a period of hours, at a rate calculated to equal approximately the aetabolic rate. The infusion apparatus used was a 20 c.c. syringe operated by a screw, driven by a geared motor. In the first experiment, in which 1x9 g. alcohol/kg. was injected into a 2-62 kg. cat, r was and, in the hour preceding the constant infusion, at which time the plasma alcohol concentration was 149 mg./100 c.c. The calculated metabolic rate was thus 186 mg./kg./hr. The infusion which followed (5.5 c.c./ hr. of 8'5 % alcohol in saline) was 179 mg./kg./hr. After 3 hr., the plasma alcohol concentration had risen by only 2 mg./100 c.c. This difference is within the limits of experimental error of the method, but supposing it to be real and the additional 2 mg./100 c.c. distributed throughout the body, then 20 mg./kg. of the alcohol infused remained unmetabolized during 3 hr., or 7 mg./kg./hr. The metabolic rate, therefore, lay between 172 and 179 mg./kg./hr., and the metabolic rate assessed from the decrease in plasma alcohol concentration was 4-8 % higher than the true value. In a second experiment of the same nature, the plasma alcohol concentration rose from mg./100 c.c. over a period of 31 indicating the accumulation of about 6 mg./kg./hr. During this period, hr., 154 mg./kg./hr. had been infused, and the metabolic rate lay, therefore, between 148 and 154 mg./kg./hr. Values of 0 59 for r and for fi obtained earlier in the experiment yielded a value of 141 mg./kg./hr. In this case, therefore, the assessed metabolic rate was 4-8 % lower than the true value. In view of the errors, both physiological and chemical,

9 236 M. G. EGGLETON involved in such experiments as these, the results may be accepted as evidence that the value obtained by indirect assessment of metabolic rate is a fair approximation to the true value. DIscusSION The average ratio of the concentration of alcohol in the tissues examined (other than fat) to that in the plasma is about 075, and the average value of r, assessed from changes in plasma alcohol concentration, 059. It has been assumed that this latter value is what one might expect for the whole body, since the concentration of alcohol at any time is considerably less in bone and fat than in the other tissues; but no attempt has so far been made to correlate the two values quantitatively. This cannot be done with any accuracy, but an approximate calculation made from the data in Table I and the average proportion of the different tissues in the human body [Vierordt, 1893] yields a value of 06. This figure is also of the same order as that obtained by Widmark [1932] for men, (when due allowance is made for the difference in concentration of alcohol in plasma and whole blood), from changes in the concentration of blood alcohol. Observed values lower than 06 are readily understandable, for the body weight can be increased very considerably by the simple addition of fat, but higher values, as e.g. the maximum observed of 078, are more difficult to interpret. This particular value was obtained (by two separate determinations) in a 2 kg. kitten, and the two other values encountered above 07 (072 and 074) occurred also in young lean cats. Two possible factors may be concerned in the production of these high values. In the first place, the proportionate weight of the different tissues varies with age, and on the balance, tissues which contain a higher concentration of alcohol may predominate in the young. In the second place, the cats used (13) for the tissue analyses in Table I were larger than the average, and since fat is deposited to a greater or less extent in all tissues, the ratios obtained may be lower than would have been the case if young lean cats had been used. On the whole, the evidence available suggests that the values obtained by indirect assessment of r are compatible with the directly determined concentrations of alcohol in the tissues. Although the evidence presented here suggests that r is a constant in any individual (apart from gross variations in body weight), a study has not been made of possible long term variations. It is difficult to imagine that the proportions of different tissues change materially in an animal of constant weight, or that variations in the value of r could

10 METABOLIC RATE OF ALCOHOL occur in the absence of such changes. Yet Widmark [1932] has noted that the raised metabolism of alcohol present in fever is due entirely to an increase in the value of r. He gave no details of possible changes in body weight, but the increase in r was too large (0-12 in one subject) to be readily accounted for by loss of body fat in the course of a few days. In the present state of knowledge, therefore, the possibility of relatively large changes in r occurring in normal healthy individuals and caused by some factor or factors unknown, cannot be dismissed. SUMMARY 1. A method is described for the estimation of alcohol in 1 c.c. of plasma or trichloroacetic acid filtrate of tissue, yielding full recovery, with an error of + 1 mg./100 c.c. 2. Following an intravenous injection of alcohol into cats under nembutal anaesthesia, direct determinations on muscle and plasma indicate that equilibrium between them is established only after 30 min. From the shape of the blood alcohol-time curve, it would appear that complete equilibrium throughout the body is established only after 1-1 hr. 3. When equilibrium is established, most tissues in the body contain % of the concentration of alcohol present in the plasma. Fat contains only %. 4. This equilibrium value between muscle and plasma is unaffected by the absolute concentration of alcohol, or by the passage of time (6 hr. or so). 5. If the alcohol concentration in the whole body, expressed as a fraction of that in the plasma (which is a modification of Widmark's factor r) is estimated indirectly it is found to remain constant in any one animal, though it varies widely from one animal to another ( in the present series), presumably owing to the widely varying proportion of fat in the body. 6. Assessment of the metabolic rate of alcohol from the modified factor r and the observed decrease in the concentration of alcohol in the plasma agrees, within the limits of experimental error, with the direct measurement of metabolic rate (constant infusion method). 237 PH. XCVIII. 16

11 238 M. G. EGGLETON REFERENCES Abramson, L. & Linde, P. [1930]. Arch. int. Pharmacodyn. 39, 325. Bernhard, C. G. & Goldberg, L. [1935]. Acta. med. wcand. 86, 152. Harger, R. N., Hulpieu, H. R. & Lamb, E. B. [1937]. J. biol. Chem. 120, 689. Lundsgaard, E. [1937]. C.R. Lab. Carltberg, Ser. chim. 22, 333. Miles, W. R. [1922]. J. Pharmacol. 20, 265. Newman, H. W. [1936]. J. Pharmacol. 56, 278. Nyman, E. & Palmlow, A. [1934]. Skand. Arch. Phy8iol. 68, 271. Smith, G. & Stewart, C. P. [1932]. Brit. med. J. 1, 89. Vierordt, H. [1893]. Daten und TabeUen fiur Mediciner. Jena: G. Fischer. Widmark, E. M. P. [1922]. Biochem. Z. 131, 473. Widmark, E. M. P. [1932]. Die theoretiwchen G-rundlagen und die praktische Verwendbarkeit der gerichtlichmedizini8chen Alkoholbetimmung. Berlin: Urban and Schwarzenberg. Widmark, E. M. P. [1933]. Biochem. Z. 267, 128.