Platelet and fibroblast monoamine oxidase in alcoholism

339

Psychiatry Research, 12, 339-347 Elsevier

Platelet

and Fibroblast

Earl Giller, Jr., James Necks, and Barbara Sherman

Monoamine Helen

Hall,

Oxidase Cathy

Stewart,

in Alcoholism Jerome

Schnitt,

Received Januar.v 18. 1984; revised version received May 22. 1984; accepted June I, 1984. Abstract. Monoamine oxidase (MAO) activity has been reported to be low in platelets (MAO B) and brain (MAO A and B) of some patients with alcoholism compared to control subjects. Whether the decreased platelet MAO activity found in alcoholism is secondary to the effect of alcohol or exists before alcohol abuse is not clear. The hypothesis that altered MAO A activity is determined by an abnormality in the genetic regulation of the enzyme can be tested by measuring MAO A activity in human fibroblasts cultured under controlled conditions. We first studied the kinetic parameters of platelet MAO B activity in patients hospitalized for treatment of alcoholism. Vmaxwas 38% lower in the patients (n q 14) than in normal controls (n = 22). but the enzyme affinity (Km) for the substrate tyramine was unchanged. Patients with the five lowest levels of platelet MAO activity had MAO activity measured from fibroblasts cultured from skin punch biopsies. Their fibroblast MAO activity was within the normal range, showing a dissociation between platelet MAO B and fibroblast MAO A activities and suggesting that MAO A activity is not low for genetic reasons in alcoholic subjects who do have low platelet MAO B activity. Key Words.

Monoamine oxidase, alcoholism, human fibroblasts.

The hypothesis that abnormal brain monoamine oxidase (MAO) activity in some psychiatric patients might be responsible for altered neurotransmitter metabolism, and also that such an MAO abnormality might be reflected throughout the body, has led to the study of MAO activity in platelets from psychiatric and control subjects. The data support the finding of altered platelet MAO activity in some patient populations, but it is not clear what characterizes these patients. The relationship of platelet and brain MAO is also uncertain (for review, see Fowler et al., 1982). Another enzyme parameter, the Michaelis constant (Km), a measure of substrate affinity, has been reported as low (Berrettini et al., 1977; Giller et al., 1980), normal (Murphy et al., 1976), and high (Belmaker et al., 1977) in platelet MAO in chronic schizophrenic patients but unchanged in alcoholic subjects (Fowler et al., 1981). It is now clear that the activity of the two types of MAO, A and B, vary across cell types and that factors which effect activity, whether genetic or environmental, may be different by isozyme and by type of cell. MAO activity may increase with age, perhaps accounting for the late onset of depression in some patients (Robinson et al., 197 I) and

Earl Giller, Jr.. M.D., Ph.D.. James Necks. M.D.. Helen Hdl. Cathy Stewart, Jerome Schnitt, M.D.. and Barbara Sherman, M.S.W.. are in Psychiatry/ Il6A. West Haven VAMC, West Haven. CTO6516. USA. (Reprint requests to Dr. E. Giller.) 0165-1781

X4 $03.00 0 1984 Elsevier Science Publishers

B.V

340

raising the possibility that high MAO may also be correlated with psychopathology. In clinical studies of MAO, a particularly strong finding is that alcoholic suicides did show lower brain MAO (Gottfries et al., 1975), while brain MAO values from nonalcoholic suicides have been reported to be similar to those of controls (Grote et al., 1974; Gottfries et al., 1975). MAO A is being reduced more than MAO B (Oreland et al., 1983). Alcoholic patients studied acutely or months after drinking show low platelet MAO (Takahashi et al., 1976; Brown, 1977; Wiberg et al., 1977; Major and Murphy, 1978; Sullivan et al., 1978a, 39786; Fowler et al., 1981; Alexopoulos et al., 1983; Schuckit, 1983), which may (Takahashi et al., 1976; Giller and Hall, 1983) or may not (Wiberg et al., 1977; Sullivan et al., 1978a, 1978b) eventually return to normal levels. In one study of schizophrenic patients, we found a negative correlation between alcohol consumption and platelet MAO activity (r = -0.53, p < 0.05) (Bierer et al., 1981). This finding raises the possibility that alcoholism may be one of the factors correlated with low platelet enzyme activity even in schizophrenic patients (Adler et al., 1980; Bierer et al., 198 1). It is still not clear whether low levels of platelet and brain MAO in alcoholic patients are indications of vulnerability to alcoholism or are secondary to chronic heavy ethanol ingestion. In addition to the many toxic metabolic effects of alcohol abuse, there is strong epidemiological evidence that a genetic predisposition exists for some forms of alcoholism (Goodwin et al., 1973; Cadoret and Gath, 1978; Cloninger et al., 1981). Recently, some metabolic abnormalities in neuropsychiatric disorders have been proposed as biologic markers that may be genetically associated with a disorder without necessarily being the etiologic abnormal gene product. Alternatively, the abnormality measured may reflect an underlying genetic mechanism requiring interaction with the environment for pathologic expression, as in phenylketonuria. Altered MAO activity may represent a vulnerability to neuropsychiatric disorders that cuts across diagnostic categories. In family studies, Buchsbaum et al. (1976) have found that subjects selected for low platelet MAO activity had more psychiatric difficulties in themselves and their families than subjects with high MAO activity. Some studies have found that relatives of alcoholic subjects had low levels of platelet MAO (Major and Murphy, 1978; Sullivan et al., 1979; Puchall et al., 1980; Alexopoulos et al., 1983). In studies using analyses of tissues taken directly from patients or cadavers, variations in tissue MAO activity may occur as a result of drugs, diet, or hormone levels. Recently, the discovery that skin fibroblasts express MAO (type A) activity (Roth et al., 1976) has allowed the measurement of this enzyme under culture conditions which control for these influences. Fibroblast MAO activity can be reliably determined in individual lines under standard culture conditions controlling for medium and passage number (Edelstein et al., 1978). Because the cells are grown in culture, hormonal and other metabolic variables can be controlled; e.g., the effects on MAO of the patient’s clinical state and exposure to drugs and medication can be eliminated. Fibroblast MAO A activity was found to be normal in schizophrenic (Groshong et al., 1978) and manic-depressive subjects (Breakefield et al., 1980). We extended this paradigm to measure fibroblast MAO activity from alcoholic subjects with low platelet MAO activity.

341

Methods Control Subjects. Control subjects were matched to the alcoholic subjects on age, sex (all male), and race (all Caucasian) (see Table I) and had no personal or family history of neuropsychiatric disorders, as determined by the Schedule for Affective Disorders and Schizophrenia (SADS) (Endicott and Spitzer, 1978) interview, and no abnormal alcoholrelated behavior by the Michigan Alcoholism Screening Test (MAST) (Seizer, 1971). Platelet and fibroblast control subjects were not the same individuals. For the platelet MAO measures, control and alcoholic subjects had no major medical illnesses, were not taking medication, and had normal hematocrits, red blood cell indices, and platelet counts. Fibroblast control subjects had no personal or family history of psychiatric disorder. Alcoholic Subjects. Patients admitted to an alcoholism unit for a 28-day inpatient program were invited to participate. Patients were evaluated with the SADS to exclude individuals with psychiatric disorders other than alcohol abuse. Alcoholism was rated by the MAST and a quantity/ frequency scale that records the amount of alcohol consumed in the past 30 days of drinking. Blood determinations of platelet MAO activity, complete blood cell counts (CBC), and liver function tests (SCOT, SGPT, alkaline phosphatase, bilirubin) were done.

Table 1. Platelet MAO-B alcoholic subjects. n

enzyme

parameters

(mean + SD) in control

Age Wars)

Vmax’ (nmoles/mg protein/hr)

and

Controls

22

43214

59.4 + 28.1

0.70 + 0.19

Alcoholics

14

40*

36.7 5 13.7

0.68 5 0.15

12

1. I = 3.24, df = 32.3 (unequal variance), p = 0.003.

Fibroblast Culture. The five alcoholic subjects with the lowest levels of platelet MAO activity were selected for biopsy. Skin biopsies (2-4 mm in diameter) were obtained from the ventral forearm or below the iliac crest under sterile conditions after an intradermal injection of 1% lidocaine for anesthesia (Edelstein et al., 1978). The dermis was stretched on a dissecting tray and divided into an upper (predominantly papillary) and lower (reticular) layer, by cutting 1 mm below the keratin layer, The papillary minced skin fragments were placed in a 60 mm polystyrene tissue culture dish under a glass coverslip in Dulbecco’s Modified Eagle’s Medium (Grand Island Biologicals) supplemented with 20% (vol) fetal calf serum (FCS). When fibroblasts reached confluency (at about 4 weeks), cells were subcultured at a I:5 ratio, as previously described by Edelstein et al. (1978). Cells were then grown as monolayers in medium with 5% FCS on polystyrene tissue culture dishes and flasks (Corning or Falcon) at 37OC in a humidified atmosphere of 95% air and 5% CO?. Medium used had been preincubated with 5% FCS for 2 days at 37°C. Since levels of MAO are affected by the conditions of cell growth, strictly controlled schedules of changes of the culture medium were followed before harvesting. The medium was changed at 5-day intervals and then 7 days before harvesting the cells. The cultures were washed four times with isosmotic buffer when harvested. The homogenates were prepared at a concentration of 1 to 5 mg protein per ml using 0.01 M potassium phosphate buffer, pH 7.4, as previously described (Giller et al., 1980) and stored at -70°C until assay. Platelet MAO Activity. Platelets were prepared from 7 ml of blood collected in a Vacutainer tube with 10.5 mg EDTA by the washout technique (Corash et al., 1978). Platelet pellets were stored for up to I month at -7OOC. The pellets were homogenized in 1 ml of 0.01 M sodium phosphate buffer,pH 7.4, by sonication on ice. IOJM sodium EDTA and 104Mascorbic acid

342 decreased

nonenqmatic

hrrakdo\sn enzyme

actI\ it!

concentrations(U.l-5

x IO-‘M

)foreach

[ I-t4C]-tyramine consists bath

of 80 ~1 of sonicate

at 37°C.

Richmond.

After

CA)

ml of water.

computed

Edelstein phosphate protein

pH

in a final fluid

(500:21,

with time and protein

two protein passages.

concentrations En/),me

maximal

curve

MA).

Buffer

(C’.,,,)

and

MO)and mixture

in a shaking

radioactivity

for each assay. and boiled

Michaelis

water

70 (Rio-Rad,

with two washes of

and

plots.

Enzyme blanks

(K,,,)

Enzyme

were units

MA0

A activity

were

was assayed

sonicated

and

0. I mM Reactiona

ml 5 V HCI.

were

toluene:liquifluor

concentration. in triplicate

acid.

carried

out

(New X0-600

mg protein

b!

potassium

England

Nuclear.

pg of homogenate a1 37°C

and

Mere extracted

dircctl!

into

reaction

was

England from

reported M

for 30 minutes Nuclear).

lor each fibroblas,

using homogenates

of product

and

product\

(Xew

Values

in 0.01

trvptamine

ascorbic

I>eamlnated

as previously

assayed

Ihe

line were deter-mined

two or more

separate

at

culture

minute.

Results Subjects.

Alcoholic

subjects

were

(+ SD) time of I8 f 14 days (range

assessed

7-60)

after

on admission, their

last

which drink.

occurred

None

was

at a mean in clinically

withdrawal. Their estimated mean (f SD) intake of alcohol over the most recent 30 days of drinking was 19.6 * 13.7 ounces of ethanol daily (range 3.4-52.0). Their mean MAST score was 35 + 8 (range 33-47). where a score over 5 is considered in the alcoholic range (Se17er. 1971). symptomatic

Platelet MAO Activity. Platelet MAO B activity, as indicated by apparent V,,,.,,(the derived maximal velocity), was significantly lower (38%~) in alcoholic than in control subjects (Table I). The Kmfor tyramine was not significantly different in alcoholic than in control subjects. Within the alcoholic population. there wah a significant correlation of platelet MAO activity with the number of days since last alcohol intake (r = 0.58, p = 0.03). There were no significant correlations between MAO and the following: MAST score (r -0.22), ounces of alcohol consumed over the past 30 drinking days (r 0.32). number of cigarettes (r = 0.23). or number of cups of coffee consumed per day (r -0. IO). There was no recovery of platelet MAO activity in this population at I5 weeks of apparent sobriety with mean (* SD) platelet MAO activity at 31.7 + 12.2 at entry and 3 1.4 f. 8.3 at week 15. Alcoholic subjects with a family history (first degree relationship) of alcoholism (n = 7) had platelet MAO B maximum activity ( I/ llldx= 34. I f 8.2) similar to alcoholic subjects without a family history ( V,,.,,= 32.2 + 12.X) (n = 5). The same was true for K,,,. q

q

I

was

en/pme

constant

f~-om I.ineacacer-8urkc

40 x IO-” .&f [
01 0. I ml.

units are pmolek

vial

Louis.

Bio-Rex

were eluted

were determined

\cloclty

(St.

K,,, and t!,ramine

The reaction

for 20 minutes

to each

For

different

hour.

c’i ‘mmole).

vols).

activity

fit program

7.4, containing

01 0.05

Sigma

(Boston.

on prewashed

added

1077).

at xvcn

was from

incubated

was

specific

Fibrohlast

volume

b>, addition

Nuclear

(0.5 x 2.0 cm). which

Homogenates

0.25

Iyramine

was placed

fluid

Murphy,

in duplicate

and with time to 40 minutes.

mg protein

Activity.

activity

columns

for apparent

(1978).

buffer.

scintillation linear

MAO

specific

stopped

protein

product

et al.

the mixture

for substrate

with a least-squares

Fibroblast

sample.

New England

of scintillation

with

Values

are nanomoles

WL~ determined

and 20 ~1 of substrate

exchange

ml

Standards

were similar.

final

cation

was linear

from

incubation,

Five

determined. activity

(SO c‘i. mole)

( I fonncli~ and

ot t!‘rarnInc

I ,,,:,,determinations.

q

343 Fibroblast MAO. The five alcoholic subjects with the lowest levels of platelet MAO B activity (Fig. 1) showed cultured fibroblast MAO A activity within the normal range of a control population (Fig. 2). Fibroblast and platelet enzyme activities were not correlated in these subjects, as has been found in other studies (Groshong et al., 1978; Ciller et al., 1980).

of platelet MAO V maxforalcoholic and control subjects

Fig. 1. Scatterplot

I

I

1

I

-.

.

I

.

ALCOHOLICS

4

(N=l4

.

CONTROLS 0

I

20

40

PLATELET (0)

indicates

subjects

MAO

in whom cultured

Fig. 2. Scatterplot (0) subjects 1

I

1

60

80

100

(nanomoles/mg

fibroblast

.

(N =22 1

1 Vmax

of fibroblast

)

a.

•N~oomam~

I

I

I

I

1

120

140

proteirdhr)

MAO was measured.

MAO activity for alcoholic

I

I

I

I

and control

I

0 00

0 0 ALCOHOLICS

(N=5)

”

“.“.

I

(0)

.

. .

CONTROLS(N=II) I

1

0

I

I

20 FIBROBLAST

Mean I+ SD1 enzyme activity

I

MAO

( pmoleslmg

was 53.2 + 9.9 for alcoholic

1

1

60

40

1 80

proteinlhr)

subjects and 38.2 -t 25.9 for controls

344 Discussion Other similar relevant studies have used paradigms based on a number of biological assumptions. These paradigms are: (1) cross-sectional (comparison of subject vs. control) or longitudinal (comparison of subject at different times); (2) inferential (using peripheral measures as an index of central nervous system mechanisms); or (3) genetic, using molecular genetic principles to assess whether a behavior is linked to other better defined (usually biological) parameters (which also involves the crosssectional paradigm). The assumptions inherent in these paradigms are that: (1) in cross-sectional comparisons, the populations differ in the dependent variable studied and in no other; (2) in longitudinal comparisons, that sufficient time has elapsed between the “sick” and “well” state for change to occur and that changes in other variables are not affecting the measure; (3) in inferring central nervous system (CNS) parameters, that peripheral measures are sufficient; and (4) in genetic studies of behavior, at times less mechanistically oriented, that concepts of molecular genetics hold true (which often also assumes that peripheral measures reflect CNS activity). The assumptions underlying these various paradigms are often confounded, making individual studies difficult to compare. Differences among studies of MAO and alcoholism, for example, may stem from differences among subjects in the amount of alcohol consumed or perhaps in other factors such as hormonal state (Zolovick et al., 1966; Southgate et al.. 1970; Parves and Parves, 1’373; Wyatt et al., 1973; Lyles and Callingham, 1974; Redmond et al., 1976; Murphy et al., 1977; Asaad and Clarke, 1978) iron deficiency (Symes et al., 1969, 197 1; Youdim et al., 1975) age (Robinson et al., 1971) or sex (Robinson et al., 1971). Longitudinal studies which differ in whether recovered alcoholic subjects do (Takahashi et al., 1976; Giller and Hall, 1983) or do not (Wiberget al., 1977; Sullivan et al., 1978a, 19786) show platelet MAO activity in the normal range differ in duration of abstinence. We measured MAO B in platelets (reflecting genetic and environmental factors) and MAO A in cultured fibroblasts (reflecting genetic differences with environmental factors constant). Our alcoholic subjects have lower platelet MAO B than age- and sex-matched controls (Table I), but the alcoholic subjects with the lowest platelet MAO B activity (Fig. 1) have fibroblast MAO A activity within the normal range (Fig. 2). These findings do not support the hypothesis that low MAO activity, at least MAO A, may predispose to alcohol abuse. This conclusion is supported by the positive correlation between platelet MAO activity and days of abstinence which suggests that MAO B may be lowered by alcohol intake; the fact that activity remained low after 15 weeks of sobriety may either mean the effect of chronic ethanol abuse is long-lasting or that platelet MAO B activity is low before alcohol abuse begins, as studies of relatives of alcoholics suggest (Major and Murphy, 1978; Sullivan et al., 1979; Puchall et al., 1980; Alexopoulos et al., 1983). It is possible that MAO A and B are affected differently by alcohol, but a recent study of brain MAO in alcoholic subjects found MAO A to be reduced more than MAO B (Oreland et al., 1983). While these data argue against the simple hypothesis that low MAO activity is a genetic predisposition to alcoholism, it still may be true that platelet MAO activity is a

345

helpful biological marker of vulnerability to psychopathology, regardless of the mechanism (genetic or environmental) (Bierer et al., 1984). The underlying biological mechanism may not be strictly genetic but due to variable responses of different individuals to other factors, whether exogenous or endogenous. Thus, the baseline (untreated) measure of platelet and/or fibroblast MAO, as well as the platelet MAO response to treatment and/ or fibroblast MAO sensitivity to factors tested in culture, may be helpful as biological markers in clinical work. Such measures may not be mechanistically important in understanding how the brain works, but may be clinically relevant to prognosis, diagnosis, and treatment response. Acknowledgments. We thank Janet Wojciechowski for technical assistance, Roland Lizotte, Vicki Fernicola, M.S.W., Robert Thompson, and Mary Ann Chomiak, P.A., for diagnostic assistance, and Sarah Murphy and Deborah Beauvais for manuscript preparation. The research reported was supported in part by VA Research Funds and the Council for Distilled Spirits.

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347

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