Alcohol dehydrogenase activity in the human tissues

BIOCHEMICAL

MEDICINE

31, 1-9 (1984)

Alcohol Dehydrogenase Activity in the Human Tissues MAYSOON M. N. M. SALEEM,* YASSARY. AL-TAMER,* LADISLAV SKURSKY ,* AND ZAKARIA AL-HABBAL? *Department

of Medical

Biochemistry University

and tDepartment of Mosul, Mosul.

of Surgery. Iraq

College

of Medicine.

Received September 7. 1982

Alcohol dehydrogenase (Alcohol:NAD oxidoreductase,EC 1.1.1.1) (ADH’) has been considered to be almost exclusively confined to the liver of humans and animals (1,2), although some ADH has also been found in other organs of animals and humans (3-5). There have been attempts to make ADH activity in blood serum a biochemical parameter for diagnosis of hepatocellular damage, since it was assumed that the enzyme in serum originated from liver cells (6). An ADH activity assay has not been introduced into clinical practice because the determination is not sensitive enough to detect ADH in sera of healthy individuals (6). Progress in designing more sensitive methods (7-9) revived interest in diagnostic utilization of serum ADH. Two recent studies on clinical material (10,ll) seem to prove that ADH activity in serum really could be a useful diagnostic tool since they show remarkable rises in liver diseases. Diagnostic value of serum ADH activity is based essentially on indirect evidence: (a) on a good correlation of the activity value and clinical condition and (b) on the known fact that the liver is the main locale of ADH in the body (2). A direct proof that the ADH enzyme found in serum really stems from liver has not yet been accomplished, although it is known that the isoenzyme spectrum of ADH in various organs might be different (2). Electrophoretic separation of ADH in serum has not yet been undertaken because of the very minute amount of the enzyme in serum and its low molecular activity (1) which makes the existing techniques of detection not sensitive enough. In this paper we collected more data on ADH distribution in various organs of the human body which could be considered possible sources of serum enzyme. In some cases ADH activity is studied on both the ’ Abbreviation

used: ADH. alcohol dehydrogenase. 1

0006-2944/84 $3.00 Copyright 0 I!%4 by Academic Press, Inc. All rights of reproduction in any form reserved.

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SALEEM

ET AL.

normal and pathological condition of organs. In others ADH activity investigated only in pathological or in normal conditions. MATERIALS

is

AND METHODS

Chemicals. All chemicals were of an analytical grade (AR) quality. Tissue samples. Biopsy specimens of 31 apparently normal healthy human tissues obtained from patient during operations (the samples of organs were considered healthy and were obtained from well-functioning tissues). Autopsy specimens of various human tissues were obtained immediately after removal of the samples from the cadavers of 29 healthy individuals succumbed to accidents. The specimens were stored at - 20°C until ready for extraction. Biopsy specimens of 76 various pathological condition were used in this study. Tissue extracr. Human tissue was cut into small pieces, collected on filter paper, weighed, and homogenized ice cold with an equal volume of 0.1 M phosphate buffer, pH 7.6 (6). The homogenate was centrifuged to give clear or only opalescent supematant. The supematant was collected and used as the solution for ADH activity measurement either directly or after appropriate dilution. Analytical methods. These were performed at 30°C on Unicam SP 1800 spectrophotometer with a Unicam AR 50 Recorder. The ADH activity was determined by the conventional method (12). A modified new sensitive method (9,13) which permits determination of the “pyrazole insensitive portion” of the total activity was followed. The concentration of soluble protein in the tissue extract was determined by Biuret method (14). RESULTS

Experimental results on liver tissues are summarized in Tables 1 and 2. Results of all other samples of human tissues are included in Table 3 (A and B). The pyrazole insensitive portion of the total activities has not been included in the tables. The value of this parameter varied between 2 and 55% of the total activity in various organs and seems to be scattered randomly. Examined samples of fat, breast tissue, peritoneal membrane, and skin tissue did not have pyrazole insensitive portion. Figure I shows a histogram of the ADH activity of various healthy human tissues. DISCUSSION

1. ADH Activity in Normal and Pathological Liver ADH activity in livers of various species has been tested by many authors (1,2). It is not possible to compare ADH activities found in human liver in this study with those previously published since the methods used were different. We tried to compare both total and specific activities in a series of healthy and diseased livers. Table I shows a considerable

ALCOHOL

300

: .-: ‘;;

DEHYDROGENASE

3

ACTIVITY

-

zoo-

: x c > 2 0 $ a

loo-

0 ;; F

Adrenal gland

Kidney

I31

13)

spleen (31

thyrond 13)

heart (3)

tot

breast

tissue

tissue

(31

12)

placenta I121

muscle (71

skin tissue

broln

(31

(3)

peritoneum membrance (31

FIG. 1.

decrease of ADH activity in pathological samples which supports the assumption that increased ADH activity found in sera of patients who suffer from liver diseases (10,ll) stems really from the liver. This conclusion is further strengthened by the results shown in Table 2, which is concerned with four individuals from whom parallel samples of liver tissues and serum were taken. The serum ADH activity is approximately indirectly proportional to that of the liver. The real relationship of ADH in healthy and diseased livers is not so straightforward, and final clarification will require more observation. Table 1 shows the total activity which is remarkably lower in moderately affected livers (chronic cholecystitis) and is more so in seriously damaged tissue (parenchymal liver disease). In terms of specific activity, the value of ADH activity in pathological liver is much less sensitive, especially if the method used is the conventional one (6). The results are in good agreement with the findings of Li and Magnes (15). The specific activity seems to be practically constant in all three types of liver samples included in Table 1. It remains to be seen whether this could be explained as a result of edema (swelling of the diseased liver causing increase of the organ’s total weight) or whether it is due to the disappearance of ADH from the organ. following protein

_~_.

AND SPECIFIC ---.~.

ACTIVITY

TABLE _---~

IN HEALTHY

1 AND

17.7 -e 1.4 4.0 r 1.9

1.6 t 0.2 0.8 t 0.4

Total ADH activity U/mg wet tissue __ .-__ ..l_ll-~ .-New method” Old method’ .~ 33.7 t 5.0 3.3 2 0.5

--~-I__

ADH LIVER

SAMPLES

liver

IO.9 t 0.2 5.7 + 0.5

10.2 jl 0.2

Ratio of two methods’

disease. 2 cases of parrnchymal

297 + 62 27.2 + 5.9 164 rt 70 30.9 2 13.4 ___ ._~_..____..~.

Specific ADH activity U/g of soluble protein _____I_-. __~ -___-New method” Old methodh -. ~31.8 I 4.8 337 i 55

PATHOLOGICAL

” ’ Methods described in Ref. 9 and 6, respectively. ’ Ratio of activity values obtained by the new method (9) and the old one (6). ,’ Operating surgeon classified the liver as completely healthy looking. The collection comprises: 3 cases of uncomplicated cirrhosis. I case of cirrhosis with gall bladder disease. 3 cases of hepatomegaly of unrelated cause possibly due to parenchymal liver disease.

No, of Characteristics of liver diseases cases __-~._I__ .__.~.. -____ Healthy individuals 8 Chronic cholecystitis (Healthy looking liver”) 4 Parenchymal liver disease’ 9

___

TOTAL

m 37 5: 2 P r

e

ALCOHOL

RELATION

OF

DEHYDROGENASE

ACTIVITY

5

TABLE 2 ADH ACTIVITY IN SERA AND LIVER OF FOUR INDIVIDUALS .---

Total ADH activity” Diagnosis

Age/sex

Normal liver from gall bladder operation 30/m Liver cirrhosis 35/f Parenchymal liver disease 55/m Liver cirrhosis and gall bladder disease 45/m ___” Measured by new method (9).

In serum (Uil) 0.55 3.20 5.70 12.50

In liver tissue Wmg wet wt) 30.50 5.60 4.70 2.40 --~

leaking out (leaving the specific ADH activity more or less unchanged), or both or something else. It is interesting to note that in severe pathological cases of parenchymal disease, the ratio of activities obtained by the two applied methods is almost as constant as it is in the two former groups (healthy liver and cases of chronic cholecystitis) but with approximately half of its value. 2. ADH Activity

in Other Tissues

None of the tissues tested were found to be lacking ADH activity completely although in some of the samples listed in the lowest part of Tables 3A and B they would have been barely detected by the conventional method (6). All the samples were assayed by the new method (9) giving high values since the method forces the enzyme to work more rapidly (16). The comparison of our data with those previously published (4,5,17) is again not possible: However the order of organs according to the magnitude of the total ADH activity does not differ much from published data (2,3) whenever the comparison is possible since some previously examined organs, e.g., lungs (3), were not available to us. On the other hand, we were able to examine the ADH activity in some tissues which have not been previously reported and reexamined others because the sensitive method used for ADH activity determination gave higher values than the conventional one. An example of such a tissue is adrenal gland tissue (see Tables 3A and B) which seems to be the richest source when compared with gastric mucosa of which we had only one sample and which is known to be rich in ADH (4). It is tempting to speculate that comparatively high ADH activity in the normal adrenal gland tissue (only approximately 130 times less than liver) could be related to steroid hormone metabolism. Pathological conditions have apparently influenced ADH activity in all organs (Table 2). To draw clear-cut qualitative conclusions by their com-

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ET AL.

TABLE

3A

ADH ACTIVITY

IN SAMPLES OF NORMAL HUMAN TISSUES(ORDERED BY THE MAGNITUDE OF ITS TOTAL VALUE) IN COMPARISON WITH ACCESSIBLE PATHOLOGICAL CASES WHENEVER POSSIBLE ACTIVITY MEASURED BY THE NEW METHOD (REF. 9)

ADH activity

Type of tissues and medical data

No. of cases and sources”

Total activity (U/g wet wt)

Specific activity (Ui g soluble protein)

Adrenal gland : normal

3a

260

11.7

5.8 e 0.3

Kidney : normal Hydronephrosis disease Chronic calculus pyeloneprotic disease

3a 4b

180 t 40.9 156.6 e 141

4.3 k 0.9 2.0 _’ 0.7

4b

156.2 ? 115.5

2.2 + 1.5

Spleen : normal Spleenomegaly (due to anemia)

3a 2b

90.5 2 61.0 2

20 16.0

1.2 t 0.2 I .o ‘-c 0.03

Thyroid gland : normal Non-toxic goiter (simple and nodular) Toxic goiter (simple and nodular)

3a

90.5 t

9.6

3.7 _f 0.4

20 b

29.0 k

18.4

0.5 2 0.2

5b

50.0 -c 29.4

0.8 t 0.1

Heart : normal

3a

46.8 t

0.7 It 0.1

Fat tissue : normal Benign lipoma (Extraperitoneal) Liposarcomal (abdominal wail)

3a 3b 3b

37.6 i 1.8 30.9 k 32 24.9 IL 17.6

1.7 k 0.6 2.1 k 2.08 1.0 -e 0.2

Female breast tissue : normal Carcinoma Fibreadenoma, lymphoadenoma

2b 3b 3b

23.8 k 1.6 26.7 k 7.8 34.0 -c 11.8

1.7 t- 0.6 3.6 -c 0.4 3.0 + 0.6

Placenta : normal deliveries (diabetic patient)

12 b 1

20.5 t 6.4 40.0

1.0 ” 0.2

7(3a,4b)

19.9 t

7.2

1.3 k 0.3

2b

133.4 5

19.7

12.0 -c 1.0

Skin tissue (subcutaneous from hand)

3b

17.5 t

5.0

1.8 -+ 0.4

Brain : normal

3a

11.5 k

I.1

0.4 ‘- 0.02

Peritoneal membrane : normal Malignancy of peritoneum

3b lb

1.0

0.7 -+ 0.1 1.6

Skeletal muscle : normal Muscular dystrophy (enlarged heart)

” a = Autopsy specimen. b = Biopsy specimen.

t

7.0 + 207

7.2

’

I

.

ALCOHOL

DEHYDROGENASE TABLE

TISSUES

OF WHICH

ONLY

3B

SAMPLESWERE AVAILABLE

PA~THOLWICAL

_ ~.--~-Type of tissues and medical data Stomach :chronic atrophic gastritjs Mucosa only (hypertroic gastritis) Prostate benign hyperplasia Gall bladder calculous cholecystitis Calculous cholecystitis and cholangitis

No. of cases and sources0

7

ACTIVITY

--~

4b lb

ADH

-

activity

Total activity (U/g wet wt)

Specific activity (U/g soluble protein)

27.4 + 16.8

1.3 t 0.02

266.4

1.8

3b

22.0 t

6.0

1.3 ? 0.4

4b

43.0 5 21.6

1.5 ? 0.5

lb

62

3.4

Metastase, abdominal

2b

Duodenum :carcinoma duodenum

2b

192.0 2 14.1 35.0 f

5.7

3.6 ” 3.9 2.3 2 0.1

’ a = Autopsy specimen, b = Biopsy specimen.

parison is not possible because of the small number of cases and their biological diversity. The impact of these conditions on ADH activity is demonstrated by marked fluctuations of standard deviation. The cases of thyroid goiter showed a marked decrease of both total and specific ADH activity. The possible explanation could be that the goiterous tissue produces less ADH than the healthy ones. Contrary to this phenomenon are cases of muscular dystrophy although they were only very few. The ADH activity remarkably increased under these conditions opening a question of the importance of ADH for the heavily altered muscular tissues. From the individual cases of malignancy of the peritoneal membrane (tumor in the abdominal wall tissue) that also showed dramatic increase of ADH over the normal peritoneum, one could assume that the primary tumor was that of liver or other ADH-rich tissue. One of the thirteen examined placenta had markedly different ADH activity from the twelve others (Table 3A). The clinical record does not reveal any abnormality neither in the pregnancy nor in delivery so it would be interesting to know whether further examination of ADH activity for this tissue differs in some pathological conditions. The total ADH content of the human body can not be assayed yet with full reliability. Data collected in Table 4 indicate that from the total activity contained in tested organs the liver contains up to 98.8%.

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ET AL.

TABLE 4 DISTRIBUTIONOF ADH ACTIVITY IN VARIOUS HUMAN ORGANS Average weight Organs of organs (g)” ____--.~~-.-~~ ~~~- ~-. __. Liver 1,800 a Spleen 300a Heart 300b Kidney 300 a Thyroid gland 30 a Adrenal gland 10 a Brain 1,500 b Placenta 600a Muscle 15,000 b Skin 1,500 b Fat 3,000 b Breast tissue 2,000 b Peritoneal membrane 1,500 b --

IUiwhole weight of the organ

% of the total activity

6,066,OOO 27,250 14.040 54,000 3.620 2,600 17.175 12,282 318,400 31.536 131.600 59.375

98.800 0.045 0.025 0.090 0.006 0.004 0.030 0.020 0.520 0.050 0.220 0.096 0.020

~-__

12.528

’ a = Reference (18). b = Judged from personal communications pathologists.

of anatomists and

There are, of course, many other tissues which have not been investigated yet (bone, bone marrow, lung, intestine, stomach, retina, prostate, etc.) which may also contain some ADH activity. If those were included in the calculation, the relative portion of the total ADH contained in liver would be only moderately lower. The above data support the calculatory argument (19) that approximately %% of the total ADH activity is confined to the liver. This preliminary calculation, although incomplete, leads to the assumption that any large increase of ADH activity in serum is most probably due solely to the leakage of the enzyme from the liver. SUMMARY

Total and specific alcohol dehydrogenase activity has been compared in homogenates of 19 different types of human tissues from different sources. ADH activities were detected in tissues which have not been tested yet, e.g., thyroid gland, adrenal gland, fat tissue, skin tissue, peritoneal membrane, breast tissue, duodenum, and gall bladder. Healthy and pathological human tissue differ in their ADH activity. The percentage of the total activity has been estimated in each tested organ in relation to the total activity of the whole body. ACKNOWLEDGMENTS The authors are thankful to the Department of Surgery, and Forensic Medicine for supplying specimens of various human tissues. The research was supported by the College of Medicine, University of Mosul.

ALCOHOL

DEHYDROGENASE

ACTIVITY

9

REFERENCES 1. Branden, C. I., Jornvall, H., Ekhmd, H., and Furugren. B., in “The Enzymes” (P. D. Boyer, Ed.), 3rd Ed., Vol. 1I, Part A, p. 104. Academic Press, New York, 197.5. 2. Pietruszko, R., in “Biochemical Pharmacology of Ethanol” (E. Majchrowicz. Ed.), Vol. 56, p. 1. Plenum Press, New York, 1975. 3. Smith, M., Hopkinson, D. A., and Harris, H., Ann. Hum. Genet. 35, 243 (1972). 4. Hempel, J. D., and Pietruszko, R. Alcoholism, 3, 96 (1979). 5. Smith, M., Hopkinson, D. A., and Harris, H.. Ann. Hum. Genet. 34, 251 (1971). 6. Schimasue, A., Murakami. M.. and Tsunokura, T.. Hiroshima J. Med. Sci. 21, 131 (1972). 7. Tamaoki, H., Mizushima, H., Takei, S., Minato. S., and Nakanishi. K.. Ann. Sanhyo Res. Lnb. 25, 85 (1973). 8. Carter, E. A., and Isselbacher, K. J., Proc. Sot. Exp. Biol. Med. 155, 290 (1977). 9. Skursky, L., Kovar, J., and Stachova, M., Anal. Biochem. 99, 65 (1979). 10. Kovaf, J., Neubauerova, J., and Broinek, P. &s. Lt+k. et-s. 119, 929 (1980). II. Khayrollah, A.. Al-Tamer, Y. Y., Taka, M., and Skursky. L., Ann. C/in. B&hem. 19, 35 (1982). 12. Dalziel, K., Acra Chim. Sand. 11, 397 (1957). 13. Skursky, L., and Khayrollah, A., Drug Alcohol Depend. 6, 187 (1980). 14. Hoffman, W. S., in “The Biochemistry of Clinical Medicine” W. S. Hoffman (Ed.). 4th ed., p. 41 Year Book Medical Publishers, (1973). 15. Li, T. K., and Magnes, L. J., Biochem. Biophys. Res. Comm. 63, 202 (1975). 16. Kovar, J.. and Kucera, I., Eur. J. Biochem in press. 17. Krasner. J., Tischler, F., and Yaffe. S. J.. J. Med. 7, 323 (1976). 18. Warwick, R., and Williams, P. L., “Gray’s Anatomy.” 35th ed. pp. 599, 718. 1316, 1378. Len’ ans Green, New York, 1973. + . 19. Khayrollah, A., Thesis. College of Medicine, University of Mosul, Mosul, Iraq. (1980).