PERENNIAL CLAIMS OF “ENDOGENOUS ALCOHOL” OR ALCOHOL-LIKE SUBSTANCES*

by

R o l l a N.

I. Scientific Studies Published, 1859-1934 In 1859, Hutson Ford published a paper, “O n the Presence of Alcohol in Normal Blood and Tissues . . . (1 ). $ Fresh animal blood or hashed tissue, in quantities rang­ing from 280 g. to 36 kg., were mixed with w ater and distilled. H e reported tha t the distillates, after purification and concentra­tion to a small volume, reduced chromic acid solution and evolved an inflammable vapor when boiled. He estimated the alcohol content of this concentrate from its specific gravity. T he results ranged from 0.2 to3.5 mg. % . As to w hether this alcohol was present antem ortem , he said:

“T he existence of alcohol in the distillates of fresh blood and tissues a t the earliest mom ent when this sub­stance can be procured by distillation was thus dem onstrated, but we must by no means conclude tha t the portion of alcohol obtained in any of these cases actually existed in the blood or tissue during life. I t must have been formed after the interruption of respiration and the circulation precedent to death, and also after the removal of the matters distilled, by the continuation of a nor­mal process. . . . Alcohol, therefore, cannot be supposed to be normally existent in the blood a t any time, for we must assume tha t it is oxidized as fast as formed.” §

D uring the 75 years following Ford’s 1859 paper, at least a score of investigations on the “norm al” alcohol present in dis­

*T he first p a rt of the m aterial in this paper was presented in a symposium on alcohol, honoring the sixtieth birthday of Pauli I. Tuovinen, Alkoholiky- symys, 1963, No. 2, Helsinki, Finland,

f ln d ia n a University School of M edicine, Indianapo­lis, Indiana

JFifty-seven years after the first publication of his investigation, Ford republished it in the Journal of Physiology.

§These sentences occur in F o rd ’s description of his “ Exp. X X X II” and have probably been over­looked by many readers.

HARGERf

tillates from body materials were published. Table I summarizes 18 of these studies and includes the concentration of “endogenous” alcohol reported. Several of the investiga­tors described the isolation of a material from their distillates of body materials which supported combustion, reduced chromic acid with the formation of aldehyde or organic acid, and yielded a positive iodoform re­action. M aignon (5) stated that he con­verted the alcohol or acetic acid formed into ethyl acetate, ethyl butyrate, and cacodyl. No “normal” alcohol was found by Lenoble and Daniel (10) or by Kridelka and Bohet (16 ), but they used the Nicloux dichromate reduction method on distillates from only2.5 to 5.0 ml. of blood or spinal fluid, which procedure would probably not detect less than about 10 mg. % of alcohol.

In their 1932 paper (18), Gettler et al reported an experiment in which they steam-distilled 28 kg. of pigs’ brain, and then repeatedly redistilled and purified this distillate to give a final volume of 5 ml. Using an ingenious micro-distillation ap­paratus, they isolated from this 5 ml. a droplet (about 20 mg.) of a clear fluid, which boiled at 78°C, had the carbon content of ethanol, and was converted into ethyl iodide and ethyl benzoate. The press hailed these findings as settling the question of endogenous alcohol in the affirmative and perhaps also proving that ethanol is a necessary body substance. G ettler’s group have always been meticulous experimenters, and I have no doubt that the droplet iso­lated from the 28 kg. of pigs’ brain was anhydrous alcohol as they claimed. How­ever, this does not exclude the possibility of postmortem fermentation or artifact for­mation of alcohol, as we shall see later.

II . Scientific Studies Published, 1935-1965In 1930, Dr. Anna Goss and myself began

a study of “endogenous” alcohol, analyzing our distillates by a micro-dichromate re­duction method (20), but we approached

182

R. N . HARGER 183

T a b l e I

Reported Values for “Endogenous” Alcohol (1879-1934)

“ Alcohol”Investigator(s) Year Analytical M ethod Concentration

m g.%

Bechamp (2) 1879 Volatile acid from reaction w ith K 2Cr207 Present*Nicloux (3) 1900 Dichrom ate reduction 0.2-1.4Landsberg (4) 1904 D ichrom ate reduction 3.0-9.0M aignon (5) 1905 D ichrom ate reductions 1.2-11.4Reach (6) 1907 Zeisel alkoxy 3.0Pringsheim (7) 1908 D ichrom ate reduction 7.4-14.1Schweisheimer (8) 1912 D ichrom ate reduction 3.0-3.6Taylor (9) 1913 W t. of p-nitro benzoate ester 2.4Lenoble and Daniel (10) 1919 D ichrom ate reduction NoneK uhn (11) 1924 Interferom eter 0.6-5.1H eilner (12) 1924 D ichrom ate reduction 3.0-8.4Aoki (13) 1925 Dichrom ate reduction 2.0-3.0M cNally et al. (14) 1927 D ichrom ate reduction 0.9-5.2G ettler and T iber (15) 1929 Volatile acid from reaction with K2Cr2C>7 0.6-2.3K ridelka and Bohet (16) 1929 Dichrom ate reduction NoneBock (17) 1931 I nterferom eter 1.5-11.3G ettler et al. (18) 1932 Zeisel alkoxy 0.4-4.0Carlson e t al. (19) 1934 Dichrom ate reduction 3.0

*Bechamp gave no quantitative figures for fresh body materials.

the problem from a somewhat different angle than had been used previously. We first confirmed the findings of Ford (1) and Pringsheim (7) that delay between death of the animal, or taking samples from living animals, and steam-distillation of the body material causes a marked rise in the amount of dichrom ate-reducing material in the distillate. For this reason we held this time interval to a minimum in our experi­ments. Next, we found tha t if one continues the steam-distillation beyond the point where all preformed alcohol would have been removed, the volatile reducing sub­stance continues to come over in the dis­tillate in quantities almost as great as at the beginning. This means tha t the m ajor part of the volatile reducing m aterial can not be preformed alcohol. Finally, we found that a tenfold concentration of the distillates by redistillation, first after acidification and then after adding silver nitrate and alkali, caused a great reduction in the volatile re­ducing substance, with loss of only about 13% of 1 mg. portions of ethanol added to 100 g. controls.

As a result of these findings, our pro­cedure was to steam-distill 100 g. portions of hashed tissue or 50 g. portions of blood, collecting two successive fractions of dis­tillate each having a volume equal to the weight of tissue or volume of blood used. These distillates were purified and concen­trated tenfold, as described above. We then calculated the maximum endogenous alcohol

values as A-B, where A represents the weight of dichromate reduced by fraction 1 and B the weight of dichrom ate reduced by fraction 2, both expressed as ethanol. The study was published in 1935 (21). O ur results for maxim um endogenous alcohol, expressed as m g.% , were: blood, 0.0 to 0.027; brain, 0.049 to 0.119; liver, 0.085 to 0.221; kidney, 0.044 to 0.142; and muscle, 0.037 to 0.227. We concluded,

“T he normal concentration of ethanol,if any, is very much smaller than hasbeen reported by most previous in­vestigators.”

Four years after our paper appeared, Dr. Gettler’s pupil, Dr. C. J. Umberger, re­ported a study (22) confirming our find­ing tha t prolonged distillation of body m a­terials continues to bring over alcohol, or an alcohol-like substance, in very slowly diminishing quantities, long after the re­moval of any preformed alcohol. For de­termining alcohol in tissues, Umberger em­ployed the volatile acid method of Gettler and Tiber (15), and for blood he developed a micro-alkoxy procedure (23) in which the resulting alkyl iodide is reacted with bromine to form IBr. O n adding w ater the IBr forms H IO 3. K I is then added and the iodine liberated is titrated with N /100 thiosulfate. Both analytical methods are more specific for alcohol than is dichro­m ate reduction. Umberger also found that ammonia was liberated from tissues or blood

184 C H E M IC A L T E S T S

T a b l e II

F ractionf

No. 1 No. 2 No. 3 No. 4 No. 5 No. 6

Effect of Prolonged Distillation on Yield of “Normal Alcohol”* Results Expressed as mg.% “alcohol”

Harger and Goss K 2C r20 7 in 17N

H 2SO 4 ; excess K.2Cr207 titra ted

Liver

1.361.071.36 3.75J

U mberger K 2C r207 in 5N H 2SO 4 ; acetic

acid formed distilled and

titrated

Blood

9.25.88.94.63.51.7

Umberger Alkoxy reaction

with 70% H I; alkyl iodide H IO s; K I

added and liberated iodine titrated

Blood

4.02.51.7

^Selected from d ata published by H arger and Goss (21) and U m berger (22)fT h e volume of each fraction of distillate was: H . & G ., 1 m l. per g. of tissue, analyzed directly; U .,

d ichrom ate m ethod, 8 m l. p er m l. of blood; U ., alkoxy m ethod, blood heated to dryness in a stream of C O 2, then w ater added before the next analysis.

JV ery rapid distillation

when these were distilled, the quantity of am m onia formed being proportional to the quantity of alcohol-like substance evolved. Table I I shows the results of prolonged di- tillation of body materials obtained in two experiments by U m berger and one by H arger and Goss.

U m berger also steam-distilled 3 kg. of sheep’s blood, discarded the first 3 L. of distillate, and purified and concentrated the second 3 L. to a final volume of 2 ml. H alf of this concentrate was analyzed by his micro-alkoxy method and the result indicated 0.07 mg.% of alcohol in the blood. T he rem ainder of the concentrate yielded typical color reactions for ethanol.* In a 1954 publication (26), Umberger s ta ted :

“Experim ental work on the so-called norm al alcohol (22) dem onstrated that the ethyl alcohol isolated from normal tissues by Gettler, Niederel and Bene- ditti-Pilcher (18) was produced in the tissue distillation process. T he fact that the norm al alcohol increased with the increase in am monia liberated during the distillation led to the postulation tha t the alcohol was formed by hydro­lysis of compounds containing the amino linkage.” (Numbers refer to references in the present paper.)

*In the present paper we have presented U m berger’s study in some detail because, so far, his only pub­lication of it is his 1939 thesis (21). However, we have briefly reviewed p a r t of it in certa in pub­lications (24, 25).

From the findings of ourselves and U m ­berger, it is evident tha t one can augment several fold the “endogenous” alcohol of body materials simply by increasing the volume of distillate to be analyzed. Distilla­tion to dryness will probably produce the same result.

W ith the advent of the alcohol dehydro­genase (A D H ) enzyme method for deter­mining ethanol, a new tool was available for investigating the “endogenous” alcohol problem. This method requires only 5-50 micros of alcohol, and it is almost specific for ethanol. In the usual procedure employing 0.02 ml. of blood, the reading for non­alcoholic subjects is zero. However, using up to 100 times the usual aliquot of blood, the following findings of “endogenous” al­cohol have been reported for blood: Bucher and Redetzki (27 ), 0.24 m g.% ; Marshall and Fritz (28), “a very small fraction of 1 mg. per cent” ; Redetzki and Johanns- meier (29 ), 0.15 m g.% ; and Lundquist and Wolthers (30), 18 analyses with 9 subjects, ave. 0.12 m g.% , range, 0.035 to 0.26 mg.% (one subject, 0.55 m g.% ).

M uch higher levels of “endogenous” al­cohol were reported by McManus et al, in1960 (31), who used a novel method of separating the volatile materials from tissues. They homogenized equal parts of the tissues and water, lyophilized it (freeze-drying under high vacuum ), and condensed the distillate at a low temperature. The dis­tillate was then analyzed by an ADH method. By this procedure they found

R. N . HARGER 185

“endogenous” alcohol values ranging from 1.1 to 6.7 m g.% , averaging 3.8 mg.% . A product having the properties of ethyl 3.5- dinitrobenzoate was formed from an in­gredient of the distillate. I t would be in­teresting to see if further “endogenous” alcohol could be evolved by adding water to the freeze-dried residue and analyzing the second distillate, which is analogous to the procedures used by ourselves and Um- berger.

A review, “Endogenous Ethanol,” giving a somewhat incomplete presentation of the literature, was published by Lester in 1961 (32). While he devoted considerable space to the findings of Gettler et al. (18) and McManus et al. (31), he did not mention the im portant studies of Umberger (22) and of Lundquist and Wolthers (30). We had published two reviews of Umberger’s investigation in 1956 and an abstract of the paper by Lundquist and Wolthers appeared in a 1959 issue (33) of the journal in which Lester’s review was published. Lester’s review does give the endogenous alcohol values re­ported by H arger and Goss (21), but omits our “m axim um ” reservation and says nothing about our results on analyzing successive distillation fractions from body materials. From his survey of the literature Lester concluded tha t normal blood probably con­tains 2 to 3 mg.% of ethanol. H e even speculated on the rate of formation of endogenous alcohol required to maintain these concentrations of blood-alcohol and concluded:

“As a normal level in human beings of between 20 and 30 mg. per liter (2 and 3 m g.% ) seems not unlikely, 1.6 to 2.4 g. of ethanol may be produced per hour, accounting for some 14 to 20 per cent of the basal energy requixe- ment.”

This line of reasoning means that the body of a 70 kg. abstainer would m anu­facture, daily, a quantity of ethanol equal to the alcohol in 97 to 146 ml. (3.3 to 5.0 U.S. fl. oz.) of a beverage containing 50% of alcohol by volume!

Three years previously, Lundquist and Wolthers (30) had made a similar calcula­tion from their data and arrived at an hourly production in the body of only 0.15 g. of ethanol, which they thought may be due to intestinal fermentation.

In 1962 Lester published a second paper entitled, “Concentration of A pparent En­dogenous Ethanol” (34 ), which was sub­mitted for publication just six months after

his review paper. The second paper indi­cates that, in this interval of time, his esti­mate of the maximum level of “endogenous” alcohol had dwindled to one-twentieth of his previous value. In this second paper Lester described a research by himself in which he calculated the level of “normal” alcohol in hum an blood from analysis of alveolar air, employing the commonly accepted ratio of 2100:1 for the distribu­tion of alcohol between blood and alveolar air (35). Seven ml. samples of alveolar were analyzed for alcohol content by means of gas-liquid chromatography, using an in­strument equipped with a hydrogen flame ionization detector which Lester stated would read as little as 0.002 /tg. of ethanol. The reported “endogenous” alcohol levels of his 25 subjects, expressed as m g.% , were: ten, zero; seven, 0.05; five; 0.10; and three, 0.15. Lester stated:

“I t is concluded that ethyl alcohol may be present in humans in concentrations up to 1.5 mg. per liter of blood (0.15 m g.% ) but whether this alcohol is of endogenous origin is unresolved; even if such formation were endogenous, its fraction of the basal metabolic rate would not exceed 1 per cent.”

Eriksen and Kulkarni (37) also employed gas chromatography to analyze the breath of non-drinking hum an subjects. They col­lected the breath samples in glass vessels cooled in liquid air, and introduced 5 ^L of the condensed w ater into the column of a chrom atograph fitted with a hydrogen- flame detector. Since one liter of breath, as exhaled, contains about 37 /xh of water (38), the 5 (j.L of water analyzed would represent about 135 ml. of breath. The chromatograms which they obtained cor­responded to those of both ethanol and methanol. The quantities reported of each alcohol per liter of breath were: m ethanol, 0.06 to 0.49 /xg., ave. 0.25; ethanol, 0.01 to 1.11 fig., ave. 0.32. If the breath used was alveolar air, the calculated blood levels, using the 2100:1 ratio, would be: 0.013 to0.103 mg.% for methanol, and 0.002 to0.234 mg.% for ethanol.

One possible source of the m ethanol would be fruits containing pectin, the methoxy group of which can yield m ethanol on hydrolysis. Thus Amerine states that wine may contain up to 150 mg.% of methanol (39), and Western and Ozburn (40) reported traces of m ethanol in apples, oranges, celery, and carrots.

1 8 6 C H E M IC A L T E S T S

Lundquist and W olthers suggested (30) th a t the source of the “norm al” blood- ethanol reported by them might be en­terogenous, and not endogenous, due to the action of microorganisms in the gut. Eriksen and K ulkarni mentioned the same mechanism to possibly account for the m eth­anol which they reported in breath.

In 1965 Axelrod and Daly (41) reported th a t the pituitary gland of several species of animals contains an enzyme which can transform S-adenosyl methionine (a methy- lating agent in the body) to m ethanol plus S-adenosyl homocysteine and suggested that this enzyme may account for the methanol found in breath by Eriksen and Kulkarni.

As for the present status of “norm al” blood-alcohol, Table I I I summarizes our own results and those of six subsequent investigations. T he values reported in TableI I I simply mean tha t ethanol, if present a t all in the subjects tested, cannot exceed the reported levels and may be much less, or even zero. We should not forget tha t neither the ADH method nor gas chrom ato­graphy is absolutely specific for ethanol. Also, artifact formation of ethanol is not excluded in some of the steps leading to the final readings with the ADH method. Since 10 of the 25 subjects, whose breath was analyzed by Lester, tested zero for ethanol, this would raise the question w hether the word normal should be applied to the ethanol reported for the other 15 subjects.

We m ight m ention tha t the maxim um values for “norm al” blood-alcohol reported by the investigators listed in Table I I I are of about the same order of m agnitude as the prevailing values for “norm al” lead, arsenic, and mercury in the body, and it is generally agreed tha t the presence of traces of these elements in body tissues and fluids is not normal bu t accidental.

II I . Claims of Fantastic Levels of “Norm al” Alcohol

The levels of “endogenous alcohol” listed in Table I I I are all too low to be of medicolegal significance and are of aca­demic interest only. However, during the past 25 years, there have been interm ittent reports of incredibly high concentrations of alcohol-like substances in the blood of humans and animals in the absence of the adm inistration of alcohol. These results, if true, would cast grave doubt on the validity of chemical tests for intoxication. We will briefly review five of these reports:

1. Leake, Swim, and McCawley, 1940. In a letter to the J.A .M .A . (42), these authors told of a case where analysis of postmortem blood by a dichromate method showed a considerable amount of alcohol, although no history of drinking could be obtained. This led them to experiment with rabbits, and they reported “alcohol” levels up to 150 mg.% where the animals has been subjected to asphyxiation. They sug­gested tha t accumulated lactic and pyruvic acids might cause some of these acids to distill from the blood and produce false results. They mentioned experiments “still in progress.”

These claims were promptly challenged by Heise (43) and myself (44) in letters to the editor of the J.A .M .A ., pointing out that the boiling points of these two acids are too high for them to distill in appre­ciable amounts and stating that the alleged results were contrary to all previously published work.

In our laboratory we then ran alcohol analyses on the blood of 50 rabbits used for pregnancy tests which were killed by drowning instead of being chloroformed. Not one of these bloods yielded the slightest evidence supporting the claims of Leake

Investigator (V)

T a b l e I I I

Low Blood “Endogenous” Alcohol Values Reported 1935-1962

Year Analytical M ethod

H arger and Goss (21) 1935

Bucher and Redetzki (27) 1951M arshall and Fritz (28) 1953Redetzki and Johannsm eier (29) 1956L undquist and W olthers (30) 1958 Lester (34)Eriksen and K ulkarni (37)

19621965

Dichrom ate reduction using two successive distillation fractions*'

“ Endogenous”Alcohol

mg.%

0.0-0.027

ADH . 024 AX)H Very small fraction of 1.0ADH „ , n°r}5ADH 0.035-0.26 (one, 0.55)Gas chrom atography using alveolar air 0.0-0.15Gas chrom atography using water __condensed from breath 0.002-0.234

R. N . HARGER 187

et al. We delayed publishing our results until the complete paper by Leake et al. had appeared, but it was never published. O ur rabbit-drowning experiments were fi­nally briefly described in a 1949 paper (45).

2. Pansini et al., 1952. (46). These Italian investigators published a series of papers on “norm al” alcohol in hum an subjects. They used the Friedemann-Klaas alkaline dichromate method, and the values which they reported were apparently the normal blanks by this method. They claimed up to 4.6 mg.% of “endogenous” alcohol, which claims are much more modest than those of the other authors mentioned in this section but far higher than the ADH blanks.

3. Plesso and Fuskon, 1957 (47) re­ported tha t the “endogenous” alcohol of schizophrenics may reach 2-41 m g.% , as compared with 9 m g.% , for control sub­jects.

4. Certhoux and Ram et, 1962 (48). These authors claimed tha t 15 out of 25 patients receiving tranquilizers, but no al­cohol, showed blood-“alcohol” levels rang­ing from about 60 mg.% to over 100 mg.% . They used the Cordebard method (49) for blood-alcohol, which is official in France.

Subsequently, Le Breton repeated this type of study, also using the Cordebard method (50), and analyzed blood from 15 patients who had been taking tranquilizers continuously for a long time. T he analytical findings were zero for 9 subjects and below10 mg.% for the remaining 7. According to Le Breton, these values represent the normal blank for alcohol-free subjects and refute the claims of Certhoux and Ramet.

5. Breazeale, 1965. (51). The author, E. A. Breazeale, operated a serological laboratory in Arizona, and his paper ap­peared in a recently launched journal, Lex et Scientia. The details and alleged find­ings of his “research” somewhat duplicate those of Leake et al. (42) twenty-five years earlier.

Breazeale, too, reported a case with “no history of drinking” where chemical analy­sis showed a high level of blood-“alcohol.” The case represented a pedestrian fatality. Breazeale stated tha t he analyzed heart blood from the victim by the H arger acid dichromate method (20) and that the result indicated a blood-“alcohol” level of 193 m g.% . H e then theorized tha t this “false” blood-alcohol finding m ight be due to stress, associated with over-production

of adrenalin. He proceeded to perform three types of experiment to test his hypo­thesis. His experimental methods and alleged results follow:

I. Reduction of dichromate by adrena­lin. H e added 0.1 cc. of Parke-Davis Ad­renalin Solution to a standard acid-dichro- m ate solution, and reported a reduction of dichromate equal to tha t produced by 1.5 mg. of ethanol. Perusal of the label on the Parke-Davis bottle should have enabled any chemist to predict th a t this fluid would cause some reduction of dichromate, be­cause the label states tha t each cc. of this fluid contains 1 mg. of adrenalin, 5 mg. of chloretone, and 1 mg. of sodium bisulfite. The last two are added as preservatives. Adrenalin is a secondary alcohol joined to di-hydroxy benzene, chloretone is trichlor- tertiary butyl alcohol, and bisulfites are well known reducing compounds. Adrenalin (M.P. 216° C.) would not distill in any blood-alcohol method even if it were present in the utterly impossible concentration of0.1 mg. per cc.

II. Alleged formation of a volatile re­ducing substance on addition of a trace of adrenalin to blood. In this experiment Breazeale mixed 0.08 cc. of Parke-Davis Adrenalin Solution with 5 cc. of blood. After this treated blood had stood for 1 hour a t 25 'C ., it was analyzed for alcohol by the H arger method, and the re­sult he reported was a reduction of dichro­mate corresponding to 150 mg.% of ethanol Breazeale found no reducing substance in the untreated blood.

III . '‘Alcohol1’ production in rabbits subjected to stress. Samples of heart blood from three rabbits were analyzed by the H arger method and the results showed no alcohol. O ne day later, these rabbits were placed on animal boards and restrained with cords, and a revolver was discharged near the animal’s head just prior to taking the sample of heart blood. Analysis of these three samples of heart blood by the H arger method showed, according to Breazeale, dichromate reduction equivalent to tha t produced by 170, 150, and 130 mg.% of ethanol, respectively.

As mentioned earlier, we found in 1925 that stress associated with drowning did not form any volatile reducing substance in the blood of rabbits. In 1961, Vidoni and Redenti (52) reported no alcohol, or alcohol-like substance, in the blood of rab­bits subjected to fatal burning while under

188 C H E M IC A L T E S T S

Epival narcosis. Although these results pretty well refuted the alleged findings of Breazeale in his experiment I I I , we decided to waste some time and a few rabbits in repeating all three of Breazeale’s “experiments.” For our alcohol analyses we used not only the original H arger method but also the later modification (53) where the blood pro­teins are precipitated in the distilling flask.

In our hands, Breazeale’s three “experi­ments” yielded the following results:

I. O ne-tenth cc. of Parke-Davis Ad­renaline Solution was added to 5 cc. of w ater and the total fluid was analyzed by the H arger method. There was a small re­duction of dichrom ate, equal to tha t pro­duced by 0.23 mg. of ethanol. This is less than one-sixth of the value reported by Breazeale.

II . This experiment by Breazeale was duplicated in all details. O ur analyses showed no reducing substance whatever, before, or after, the addition of 0.08 cc. of Parke-Davis Adrenaline Solution of 5 cc. of blood.

I I I . We used six rabbits. H eart blood samples were taken during the control period, and one day later, just after dis­charging a revolver near the anim al’s head. T he sound of the gunshot caused the rabbits to lurch violently against their re­straining cords, and they appeared greatly excited. T he results of our analyses of the six samples of blood taken during the con­trol period, and the six samples taken dur­ing the stress period, are given below. Any reduction of dichrom ate is expressed as m g.% of ethanol.

A. Original Harger method.Control period: 0 to 5.9; Ave. 2.1.Stress period: 0 to 5.0; Ave. 1.8.

B. M odified Harger method.Control period: 0 to 3.4; Ave. 2.1.Stress period: 0 to 2.0; Ave. 1.1.

All of these results are well within the range of the customary blank which we obtain when analyzing alcohol-free blood by our method.

Just how such monstrous errors could have been made by Breazeale in 1965 and by Leake et al. in 1940, we cannot say. In Breazeale’s case, we may never know, because we understand tha t he passed away a few m onths after his paper appeared in Lex et Scientia. T he editors of this journal evidently did not ask any one familiar with this field to review Breazeale’s paper prior to its publication.

R e f e r e n c e s

1 Ford, W . H . : “ Elliott Soc. N atural History of Charleston,” S. Carolina, 1859; J. Physiol. 34: 430, 1906.

2 Bechamp, J . : Com pt. rend. 89 : 573, 1879.3 Nicloux, M .: Compt. rend. 130 : 857, 1900.4 Landsberg, G .: Z. physiol. Chem. 41: 505, 1904.5 M aignon, F .: Compt. rend. 140: 1063, 1905.6 Reach, F .: Biochem. Z. 3: 333, 1907.7 Pringsheim, J . : Bichem. Z . 12: 158, 1908.8 Schweisheimer, W .: Deutsch. Arch. hlin. M ed.

109: 281, 1913.9 Taylor, A. E .: / . Biol. Chem. 15: 217, 1913.

10 Lenoble, E. and Daniel, F .: Bull. m em. soc. med. hop. Paris 43 : 809, 1919.

11 K uhn, G .: Arch. exp. Path. u. Pharmacol. 103: 295, 1924.

12 H eilner, E .: M unch, med. Wchnschr. 71: 1422, 1924.

13 Aoki, M .: ] . Biochem. (Japan) 5: 329, 1925; Ibid. 6: 307, 1925.

14 McNally, W. D ., Embree, H . C ., and Rust, C. A .: J. Biol. Chem. 74: 219, 1927.

15 G ettler, A. O ., and T iber, A .: Arch. Path. Lab. M ed. 3: 220, 1927.

16 K ridelka, L. and Bohet, M .: J. pharm. Belg. 11: 415, 1929.

17 Bock, J . C .: / . Biol. Chem. 93 : 654, 1931.18 G ettler, A. O ., Niederl, J . B., and Benedetti-

Pilcher, A. A.: ] . Am . Chem. Soc. 54: 1484, 1932.

19 Carlson, A. J ., K leitm an, N. M uehlberger,C. W ., M cLean, F. C ., Gullicksen, H ., and Carlson, R . B .: “ Studies on the Possible Intoxi­cating Effect of 3.2 Per Cent Beer.” Univ. of Chicago Press, 1934, p. 6.

20 Harger, R. N .: J. Lab. Clin. M ed. 20:746, 193521 H arger, R. N ., and Goss, A. L .: A m . J . Physiol.

112: 374, 1935.22 Um berger, C. J . : “ A Study of the Ethyl Al­

cohol Normally Present in Blood.” Thesis, New York University, New York, 1939.

23 G ettler, A. O ., and Umberger, C. J .: J. Biol. Chem. 143: 633, 1942.

24 Harger, R . N ., and Hulpieu, H. R .: “ The Pharmacology of Alcohol,” Chapter 2 in Al­coholism, Ed. by Thompson, G. N ., Spring­field, 111., Chas. C. Thomas Pub. Co., 1956, pp. 105-106.

25 H ulpieu, H . R ., and H arger, R . N .: “ The Al­cohols,” C hapter 6 in Pharmacology of M edicine, 2nd. Ed., Ed. by Drill, V . A., New York, M cGraw-Hill, 1956, pp. 202-203.

26 Gonzales, R. A., Vance, M ., H elpem ,M. and Um berger, C. J . : “ Legal Medicine Pathology and Toxicology,” 2nd. Ed., New York, Appleton Century-Crofts, 1954, p. 1112.

27 Bucher, T . and Redetzki, H .: K in. Wchnschr. 29: 615, 1951.

28 M arshall, E. K ., and Fritz, W. F .: / . Pharmacol. Exptl. Therap. 109: 431, 1953.

29 Redetzki. H ., and Joliannsm eiei, K . : Archil' Toxicol. 16: 73, 1956.

30 Lundquist, F. and W olthers, J .: Acta Pharmacol. 14: 265, 1958.

31 M cM anus, I. R ., Contag, A. O ., and Olson, R. E .: Science 131: 102, 1960.

32 Lester, D .: Quart. J. Studies Ale. 22: 554, 1961.33 Abstract: Quart. J. Studies A le. 20 : 362, 1959.34 Lester, D .: Q uart. J . Studies Ale. 23: 17, 1962.35 Carlson, C. J ., Fridem ann, T . E ., Je tte r, W. W.,

and M uehlberger, C. W .: “ Evaluating Chemical Tests for Intoxication,” Chicago, National Safety Council, 1953, pp. 12-13.

36 Plesso, G. I. and Fusov, V. S .: Sbornik Nauch. No. 13, 254, 1957; Chem. Abs. 53 : 20466-f, 1959.

R. N . HARGER 189

37 Eriksen, S. P ., and Kulkarni, A. B.: Science, 141: 639-640, 1963.

38 H arger, R . N ., Forney, R_ B., and Barnes,H . B.: ] . Lab. Clin. M ed. 36: 306-318, 1950.

39 Amerine, A .: Scientific M onthly 77: 250, 1953.40 W estern, O . C., and O zburn, E. E .: V . S.

Naval M ed. Bull., 49 : 574, 1949.41 Axlerod, J ., and Daly, J . : Science, 150: 892-893,

1965.42 Leake, C. D ., Swim, J . B., and McCawley,

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53 H arger, R . N .: “ Ethyl Alcohol,” Chap. 4 in “ Toxicology, Mechanisms and Analytical M ethods,” Ed. by Stewart, C. P ., and StoLman,A., Academic Press, New York, 1961, pp. 129-132.

DISCU SSION

Dr. Heise: O ne can get a filtrate of the blood and plenty of it in the urine. By using the urine, which does not carry on all of the proteins and carbohydrates and all of the other gadgets that are in the blood, one can get a filtrate which shows practically no alcohol of any kind, but there is another little factor to this. Leonardi wrote that people who have normally as much as 20 milligrams per hundred cc. of normal al­cohol will go up as high as 44 when they get up in an airplane or get up to about1,000 feet altitude. Now in my own plane, I went up there with a load of w ater first of all so I would get plenty of specimens and concentrated this material. I can assure you there was no alcohol. I will adm it I was high but not that way, and I think it is time we give endogenous alcohol a decent burial.

Dr. Redetzki: I think it should not be construed that this minimum alcohol which is found with the enzymatic method is really truly ethyl alcohol. I have my doubts with this kind of concentration which we have to use. Going down to the limit of the ethanol and getting a little reduction of D PN , I doubt that we can truly say that this is ethyl alcohol because we know that there are quite a few substances which are in the blood which can be concentrated just as well. So, I don’t w ant to imply that this is really truly alcohol. I t m ight be just below tha t value, even far below it.

D r. H arger: You perhaps know that the gentleman who just spoke is one of the dis­coverers of ADH, so you are getting informa­tion, as we say in this country, “from the horse’s m outh.”