DiclE TIP DERGiSi (JOURNAL OF MEDicAL SCHOOL) C:27 S:1 2000
ERYTHROCYTE
CARBONIC ANHYDRASE
ACTIVITY IN SMOKERS
Hayrullah YILMAZ MD 1
Abdurrahman~ENYiGiT MD 2
Mehmet CO~KUNSEL
MD 3
SUMMARY
Carbonic anhydrase (CA) isozymes are important enzymes for body metabolism
because it regulates pH in most tissues. The enzyme catalyzes the reversible hydration
of CO2 to HC03 - and W . Although several studies have been carried out on the
inhibitors of this enzyme, those on this effect of cigarette smoking are rare.
In the present study, in vitro effects of cigarette smoke on CA were investigated.
7;0 this end, the CA activity in Iyzed erythrocytes was measured in 30 smokers and 30
non-smokers. Also, in all the cases, urine and blood pH as well as K' and Na+ in blood
were investigated in order to determine side-effects that may occur based upon possible
enzyme inhibition.
The carbonic anhydrase activity was lower in active smokers than non-smokers.
The enzyme activities (EV) in smokers and non-smokers were measured 4350 1265
and 5345 1645, respectively. In terms of enzyme activity, a significant difference was
found between the two groups (p<0.001). But no abnormality was determined in blood
and urine pHs as well as blood Na+ and K' levels in both groups.
Currently, the pathophysiological
consequences of low CA activities in smokers
remain unclarified. Though CA enzyme inhibition occurs in some of active smokers, this
is likely to be compensated through other compensation mechanisms of the body.
However, it is possible that this inhibition should lead to a factor predisposed to
metabolic disorder, or to a disorder that can aggravate the existing disorder. In addition,
it is another subject-matter of investigation why enzyme inhibition does not occur in all
smokers. Further investigations are needed to answer all the questions.
Key Words: Carbonic anhydrase, cigarette smoke.
INTRODUCTION
Carbonic anhydrase (CA) has been a well characterized pH regulatory
enzyme in most tissues including erythrocytes (1-4). All three soluble isozymes
of CA in humans (CA I, 11,and Ill) are monometric, 29-kDa zinc metallo
enzymes that catalyze the reversible hydration of CO2 (4,5).
CO2 + H2O- H2C03- W + HC03 CA I is found primarily in erythrocytes (1,5). The human CA 11isozyme is
widely distributed. It has been identified in erythrocytes, brain, eye, kidney, etc.
Normally CA I and CA 11each contribute about 50 percent of the total activity
(5).
(1) Department
of Chemistry,
Faculty of Education Dicle University
(2,3) Department of Chest Diseases, Faculty of Medicine, Dicle University
DiYARBAKIR
DiYARBAKIR
114
Cigarette smoking has been implicated in the etiology of respiratory
diseases, cancer, and atherosclerosis (6). Furthermore, cigarette smoking is
known to affect several physiological processes including cardiovascular
regulation and energy metabolism (7).
There are several different studies on the effect of cigarette smoking on
enzymes (8,9). However, those carried out on its effect on CA are relatively few.
The activity of this enzyme was determined to change in a variety of diseases.
There is evidence that pathophysiological alterations such as diabetes mellitus
and hypertension are associated with variations in CA activity (10,11). The
process by various physiologic mechanisms leading to functional of CA is
currently under the investigation. In addition, although enzyme inactivation has
been determined in smokers, it is still unclear which substance is responsible
for this situation (10).
The present study was designed to investigate the effect of cigarette
smoking on CA activity in erythrocyte Iysates .
..
MATERIAL and METHODS
Subjects: The study was conducted in 60 healthy subjects of whom 30
smokers (16 males, 14 females) and 30 were non-smokers (17 female, 13
male). The smokers group included only those having smoked for at least 20
years. The mean age was found as 49.8 years (range 41-62) in smokers and
48 8 years (range 40-60) in non-smokers. In smokers, on average 33.7 13.6
pack-years was determined. The subjects were excluded if they reported a
history of arthritis, cirrhosis, hypertension, diabetes, or ischemic heart disease,
as they can influence CA enzyme levels.
In all cases, together with urine pH, K+, Na+ in blood, as well as HC03and pH in blood gases analysis were measured.
Processing of erythrocytes: The blood samples were centrifuged at
1500 rpm for 20 minute and the plasma and buff coat were removed. After the
paced red cells were washed with NaCI (0.9%) twice, the erythrocytes were
hemolyzed with cold water. The hemolysate was diluted with distilled water to
the 1a-fold volume, and this hemolysate was used to determine the CA
activity.
Determination of carbonic anhydrase activity: Carbonic anhydrase
activity was determined by following the hydration of CO2 according to the
method of Wilbur and Anderson (12). The enzyme unit was calculated
according to the formula: 1 U = ( to - te )/te , where to and te are the time (sec)
needed for the pH change without and with enzyme reactions, respectively.
Statistical analysis: For statistical analysis, Student's t test for
independent samples was employed.
RESULTS
Carbonic anhydrase activities(EU) are shown in table 1. Smokers had
significantly lower CA
activities in their erythrocyte than non-smokers
(p<0.001). Furthermore, 18% of the smokers was detected CA inhibition. But no
115
abnormality was determined in blood and urine pHs as well as blood Na+ and
K+levels in both groups.
Table 1. Carbonic anhydrase activities (EU)
Smokers (n=30)
Non-smokers (n=30)
4350
Mean
5345
3575-7850
2775-5250
Range
1265
SD
1645
Enzyme activities detected in all subjects is shown figure 1.
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Figure 1. Enzyme activities determined in all cases.
DISCUSSION
In the study, the erythrocyte CA activities were found lower in healthy
smokers than in the control persons. We conclude that cigarette smoking may
diminish CA activity.
It is generally recognized that CA controls the bulk of carbon dioxide
exchange between the blood and tissues as well as the regulation of proton and
other ion movements between cells and extracellular fluids (1,3,4). There is vast
literature on the chemistry and physiology of CA, with the mechanism of action
of sulfonamides and inorganic anions playing an important role (4,13-15).
Acetazolamide is the most common drug in this class of therapeutic agents
(16). Several other factors including physical exercise and thyroid hormone
have also been reported to influence CA activity. Tokuda et al described an
association of increased erythrocyte CA activity with repeated strenuous
116
aerobic exercise. Since CA plays a major role in CO2 removal. Tokuda et al.
attributed increased erythrocyte CA activity to adaptation to a recurrently
affected acid-base equilibrium (10). Inhibition of carbonic anhydrase causes
wasting of Na+, K+, and HC03- in the proximal tubules and represents a
pharmacologically induced proximal renal tubular acidosis (RTA) (16). In
proximal RTA, urine pH becomes <5.5. However, we did not find urine pH low
in any cases, including primarily smokers. This indicates that acidosis table that
is likely to develop, depending upon the enzyme inhibition that cigarette
smoking causes, is compensated through other tampon mechanisms. To CA
inhibition that cigarette smoking causes can be seen as a state that can be
predisposed to a metabolic disorder in patients in the late stages of chronic
obstructive pulmonary disease, which occurs depending upon cigarette, or that
can aggravate the existing disorder.
Tobacco smoke .contains large numbers of organic and inorganic
compounds (17). From inhaled cigarette smoke, a broad variety of these
substances is absorbed. Out of these, many could be taken into consideration
to explain an effect on CA activity. One of the best-known constituents of
cigarette smoke is nicotine. Nicotine as a respiratory stimulant forces CO2
elimination. So the decrease in CA activity in smokers could be caused by
adaptation to chronic influences on the acid-base balance, too (10).
Furthermore, nicotine induces a general sympathetic activation with elevated
catecholamine levels (7). Maybe, recurrently elevated catecholamine levels can
affect CA activity. However, alteration may occur in CA activation as a
complication of cigarette smoking at both lower doses and early periods.
Because, Abel et al. found in their study that in cases of 25 years old who
smoked at least one cigarette a day, CA activity was significantly lower with
respect to control group (10). Therefore, CA inhibition is a complication that
develop in all smokers. After inhalation, nicotine, because of its lipid solubility, is
quickly absorbed across the alveolar surface into pulmonary capillary blood,
causing a rapid increase in its levels in the arterial circulation (17). We
determined CA inhibition in 18 % of our cases who were smoking.
Although some plausible explanations for the reported effects of smoking
on CA can be brought forward, it remains unclear whether nicotine or other
components of cigarette smoke are responsible. The mechanisms underlying
the decrease in CA activity need further investigation (10).
Currently, the pathophysiological consequences of !cw CA activities in
smokers remain unclarified. The influence of smokiny habits on erythrocyte CA
activity may be important for designing future studies on carbonic anhydrase.
Also, prospective disorders that can develop in situations where this enzyme
inhibition is determined should be investigated in detail. Moreover, it is another
subject of investigation for the inhibition of this enzyme not to occur.
Acknowledgement: We thank to Or. Hasan Akbaym from Biostatistics
Department for his advice on the statistical analysis of the data.
~--
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117
6ZET
SiGARA i9EN OLGULARDA ERiTROsiT KARBONiK
ANHiDRAZ AKTiviTESi
Karbonik anhidraz (CA) isozimleri bir (fok dokuda pH'mn
dOzenlenmesinde rol oynadlklanndan vOcut metabolizmasl i(fin onemli
enzimlerdir. Enzim, C02'nin HC03- ve W'e reverzibl hidrasyonunu katalize
eder. Bu enzimin inhibitorleri ile ilgili farkll (fall~malar yapllml~ olmasma kar~m
sigaranm bu etkisi ile ilgili (fall~malar son derece azdlr.
Bu (fall~mada sigara dumanmm karbonik anhidraz Ozerindeki in vitro
etkileri ara~tlnldl. Bu ama(fla 30 aktif sigara i(ficisi ile kontrol grubu olarak
sigara i(fmeyen 30 olguda bu enzimin ol(fOmOnOyaptlk. Yine olgularda
muhtemel enzim inhibisyonuna bagll olu~abilecek yan etkileri saptamak
amaclyla tOmolgularda idrar ve kan pH'sl ile kanda K+ve Na+ (fah~lldl.
Aktif sigara i(ficilerinde CA aktivitesi, sigara i(fmeyenlerden dO~OktO.
Enzim aktivitesi (EU) sigara i(ficilerinde 4350 1265, i(fmeyenlerde ise
5345 1645 olarak hesaplandl. Her iki grup arasmda enzim aktivitesi a(flsmdan
anlamll farkllllk saptandl (p<0.001). Ancak her iki grupta da idrar ve kan
pH'lannda ve kan K+ve Na+degerlerinde anormallik tespit edilmedi.
GOnOmOzde sigara i(ficilerinde dO~Ok CA aktivitesinin patofizyolojik
sonu(flan tarn anla~llamaml~tlr. Aktif sigara i(ficilerinin bazllannda CA enzim
inhibisyonu olu~masma kar~m bu durumun vOcudun diger kompansasyon
mekanizmalan ile kompanse edilmesi muhtemeldir. Ancak bu inhibisyonun,
metabolik bozukluga predispoze bir faktor veya mevcut bozuklugu agreve
etmesi muhtemeldir. Aynca neden tOm sigara i(ficilerinde enzim inhibisyonunun
olu~madlgl da ara~tlrma konusudur. TOm bu sorulann (fozOmO i(fin ileri
(fall~malara ihtiya(f vardlr.
Anahtar Kelimeler: Karbonik anhidraz, sigara dumam.
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