Comparison of Serum Beta Carotene and Retinol in Smokers and Non-smokers

INTERNATIONAL JOURNAL OF AGRICULTURE & BIOLOGY
1560–8530/2003/05–4–452–454
http://www.ijab.org
Comparison of Serum ?-carotene and Retinol in Smokers and
Non-smokers

RIFFAT TAHIRA, A. REHMAN† AND S.A. NAGRA‡
Institute of Biochemistry and Biotechnology, University of Punjab–Lahore, Pakistan
†Oil Seed Technology Lab, AARI, Faisalabad– Pakistan
‡Institute of Chemistry, University of Punjab, Lahore– Pakistan

ABSTRACT

The study under report was an attempt to evaluate the effect of smoking on serum ?-carotene and retinol. A total of 50 men
aged 20-25 years were selected and grouped as smokers and non-smokers (25 each). Subjects included as smoker were
smoking 8-10 cigarettes per day for the last 3-4 years. Serum was prepared from 5 mL venous blood from the donors. Serum
?-carotene and retinol were determined using trifluoro acetic acid (TFA) method. Proteins were precipitated with 95% ethanol.
Beta-carotene and retinol were extracted into light petroleum and followed by spectrophotometric analysis to study the
intensity of the yellow color present due to carotenes. After evaporating the light petroleum, the residue was dissolved in
anhydrous chloroform and mixed with TFA. The intensity of the blue color produced, gave the absorbance for retinol. The
data obtained was statistically analysed for descriptive statistics and comparison of means using independent sample t-test,
through SPSS 400. Statistical appraisal of the data did not indicate a significant difference in the serum ?-carotene of smokers
and non-smokers but a significant (P<0.05) difference for retinol between smokers and non-smokers was observed.

Key Words: ?-carotene; Retinol; Smokers

INTRODUCTION
bilirubin, albumin, DNA repair enzymes and methionine

sulphoxide reductase.

Smoking is described as the single largest preventable

?-carotene is a precursor of retinol in human body.
cause of death. Tobacco use is very common in Pakistan;
The beneficial effects of ?-carotene are mainly due to its
34% of men and 12.5% of women, use some form of
high pro-vitamin activity and its antioxidant nature (Hilbert
tobacco on regular basis (National Health Survey of
& Mohsenin, 1996). They can inactivate the free radicals
Pakistan, 1998). Tobacco use is a major risk factor for
and singlet oxygen by a process termed as quenching
diseases of the heart and blood vessels, chronic bronchitis
(Masci, 1991). They also inhibit the lipid peroxidation in
and emphysema, cancers of the lung, larynx, oral cavity,
membrane but only at low O2 concentration. Retinol is a fat
esophagus, pancreas and bladder and other problems such as
soluble vitamin and in the serum, circulates largely in the
respiratory infections and stomach ulcers.
form of a 1:1 complex of retinol and retinol binding protein

Cigarette smoke causes the production of free radicals
(RBP). Vitamin A plays an important role in growth,
in human body. A free radical is any molecule that contains
reproduction, growth of healthy skin and epithelial cells of
one or more unpaired electrons e. g., superoxide (O.2) and
mucosal linings, cellular differentiation, glycoprotein
hydroxyl ion (O.H-) (Dubick & Keen, 1991). Free radicals
synthesis, membrane stabilization and the immune response.
are extremely reactive and can cause cell injury and death.
The data on any aspect of smoking with respect to serum
Polyunsaturated fatty acids in cell membranes, proteins such
concentrations of ?-carotene and retinol is very scanty. The
as enzymes and membrane ion transporters and DNA are
main objective of the study was to evaluate the effect of
the cell components, which are damaged by these reactive
smoking on serum ?-carotene and retinol.
species. Each free radical can initiate a series of chain

reactions, which continues and damages the cell
MATERIALS AND METHODS
constituents. These species disappear from the body

following reactions with other free radicals or more
Selection of study samples. A total of 50 volunteer young
importantly, due to the actions of the antioxidant system.
men aged 20-25 years, were selected for the study, and
These radicals play a significant role in the disease process,
divided into two groups i. e., Group A: Smokers (n=25) and
because they cause the destruction or depletion of
Group B: Non-smokers (n=25). Subjects included as
antioxidants during their neutralization (Al-Senaidy et al.,
smokers were those consuming 8-10 cigarette/day for the
1997). The antioxidant system consists of superoxide
last three years.
dismutase, glutathione peroxides, metal binding proteins,
Collection of blood samples. Venous blood (5 mL) was
vitamin E, Vitamin C, ?-carotene, vitamin A, uric acid,
drawn with the help of pyrogen free, disposable syringe,

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SERUM ?-CAROTENE AND RETINOL IN SMOKERS AND NON-SMOKERS / Int. J. Agri. Biol., Vol. 5, No. 4, 2003
between 9-11 A.M. The blood samples were free from
Table I. Absorbance values of carotene working
haemolysis and were kept protected from light.
standards of different concentrations and calculation of
Preparation of serum. Blood was transferred to centrifuge
factor A1
tube and centrifuged at 2500 rpm for 7-10 min. The

supernatants (serum) were separated with the help of 3 mL
Sr. No.
Conc. of carotene
A450 of carotene
Calculations
venoject tube, stored at 4°C and analyzed for ?-carotene and
working standards
working standards
Conc./Obs X 3
(mg/L)
retinol within 72 h.
1
0.5
0.17
0.5/0.17 x 3 = 8.8
Determination of ?-carotene and retinol in serum. Serum
2
1.0
0.3
1/0.3 x 3= 10
?-carotene and retinol were determined using TFA method.
3
2.0
0.59
2/0.59 x 3 = 10
Concentration of serum ?-carotene. It was calculated by
4
3.0
0.85
3/0.85 x 3 = 10.5
5
4.0
1.1
4.0/1.1 x 3 = 10
the following formula:










Average = 10.04 = A
A
1
450 of Unknown
Serum ?-carotene (mg/L) = ------------------X Concentration of Standard X 3





A
Table II. Absorbance values of different retinol working
450 of Standard

Absorbance of each working standard (carotene) at
standards of different concentrations and calculation of
450 nm having different concentration is given in Table I.
factor A2
By putting this value to the formula finally the serum

carotene value was calculated as:
Sr.
Conc. of
A450 of retinol
Calculations
Serum Carotene = A
No.
retinol working
working
Conc./Obs x 3/2
450 of Unknown X A1
Concentration of serum retinol. Formula applied to
standards
standards
(mg/L)
calculate the serum retinol concentration was:
1
0.4
0.043
0.1/0.043 x 3/2 = 13



A’620 of Unknown
2
0.8
0.11
0.8/0.11 x 3/2 = 10.9
Serum Retinol (mg/L) = ------------------- X Concentration of Standard X 3/2
3
1.2
0.16
1.2/0.16 x 3/2 = 11.2

A’620 of Standard
4
1.6
0.215
1.6/0.215 x 3/2 = 11.2

To calculate of each retinol working standard was







Average = 11.8 = A2
used:











A’620 = A620 – F . A450,
Table III. Comparison of serum ?-carotene in smokers
Where, A620 = Absorbance of each retinol working standard
and non-smokers by t-test
at 620 nm, A450 = Absorbance of each carotene working

standard at 450 nm, F = Calculated as A620/A450
Statistical parameters
Smokers
Non-smokers

After substituting the value of F, A’620 of each retinol
Mean 134.4
156.1
working standard was determined and given in the Table II.
S. E.
15.7
12.6
Substituted the average value A
Minimum 30.0
60.0
2 to the formula to calculate
the serum retinol concentration.
Maximum 281
281
P (t-test)
0.283
Non-significant
Statistical analysis. The data thus obtained was subjected to

statistical analysis for descriptive statistics and comparison
Table IV. Comparison of serum retinol between
of mean using independent sample t-test, using SPSS 400.
smokers and non-smokers by t-test


RESULTS AND DISCUSSION
Statistical parameters
Smokers
Non-smokers

Mean 28.9
46.9

Very little data is available on any aspect of smoking
S. E.
4.0
5.0
under local conditions therefore interpretation of the results
Minimum 11.0
13.0
becomes difficult.
Maximum 82.0
90.0
?
P (t-test)
0.05
Significant at P < 0.05
-carotene. Statistical appraisal of the data (Table III) did

not indicate a significant difference in the serum ?-carotene
Retinol. Retinol is not a strong antioxidant as its precursor
of smokers and non-smokers. These findings are not in
?-carotene (Hilbert & Mohsenin, 1996), but retinol has
agreement with Singh et al. (1994), Street et al. (1994) and
many other vital functions. Our results (Table IV) showed
Al-Senaidy et al. (1997). They had reported low serum and
low levels of retinol in smokers and normal levels in non-
plasma carotenes in smokers and associated it with high
smokers. Statistical analysis revealed a significant (P<0.05)
lipid peroxidation. However, our results are in line with
difference between smokers and non-smokers. Our findings
those of Berr et al. (1998). They reported higher levels of ?-
are fairly in line with those of Singh et al. (1994), who
carotene in female smokers due to consumption of fruits and
reported significantly (P<0.05) low serum retinol levels in
milk. Dietry history of our study samples indicated
smokers. Faruque et al. (1995) and Al-Senaidy et al. (1997)
reasonable intake of fruits (Table V). This probably explains
also reported low serum retinol levels in smokers but these
the normal level of ?-carotene as observed in the present
low were not statistically significant. Low level of serum
study.
retinol in smokers may be due to a marginal intake of

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TAHIRA et al. / Int. J. Agri. Biol., Vol. 5, No. 4, 2003
Table V. Comparison of various factors influencing the
Berr, C., C. Coudary, C.K. Bonithon, A.M. Roussel, F. Mainard and A.
antioxidant status between smokers and non-smokers
Alperovitch, 1998. Demographic and cardiovascular risk factors in
relatio to antioxidant status; the EVA study. Int. J. Vitam. Nutr. Res.,

68: 26–35
Parameters Smokers
Non-smokers
Dubick, M.A. and C.L. Keen, 1991. Influence of nicotine on tissue trace
No. of subjects
25
25
element concentrations and tissue antioxidant defense. Biol. Trace
Average intake of fruits
Frequent
High
Elem. Res., 31: 97–109
Average intake of tea
Seldom
Seldom
Faruque, M.A., M.R. Khan, M.H. Rahman and F. Ahmed, 1995.
Average intake of cigarettes
8-10
0
Relatioship between smoking and antioxidant nutrient status. British
Average intake of milk
Seldom
High
J. Nutr., 73: 625–32
Hilbert, J. and V. Mohsenin, 1996. Adaptation of lung antioxidants to

cigarette smoking in humans. Chest, 110: 916–20
vitamin A, which is generally present in the society.
Marangon, K., B. Herbeth, E. Lecomte, A.D. Paul, P. Grollier, Y.
Smoking perhaps shifts this from margin to deficiency
Chancerelle, Y. Artur and G. Siest, 1998. Diet, antioxidant status and
smoking habits in French men. American J. Clin. Nutr., 67: 231–9
(National Health Survey of Pakistan, 1998). ?-carotene is
Masci, D., 1991. Antioxidant defense system; the role of carotenoids.
the precursor of retinol, and it is used in quenching of free
American J. Clin. Nutr. Suppl., 53: 1945–2005
radicals produced by cigarette smoke, thus sufficient
National Health Survey of Pakistan, 1998. Pakistan Medical Research
amount is not available for conversion into retinol
Council, Islamabad–Pakistan
Singh, R.B., M.A. Njaz, T. Bishnoi, J.P. Sharma, S. Gupta, S.S. Rastooi, R.
(Zondervan et al., 1996).
Singh, R. Begum, H. Chibo and Z. Shoumin, 1994. Diet, antioxidant

vitamins, oxidative stress and risk of coronary artery disease: the
CONCLUSION
Peerzada prospective study. Acta. Cardiol., 49: 453–67
Street. D.A., G.W. Comstock, R.M. Salkeld, W. Scheup and M.J. Klao,

1994. Serum antioxidants and myocardial infarction. Are low levels

Based on the results of present study, it is suggested
of carotenoids and ?-tocopherol risk factors for myocardial
that dietary/supplementary intake of ?-carotene and retinol
infarction. Circulation, 90: 1154–61
should be increased for smokers.
Zondervan, K.T., M.C. Ocke, H.A. Smit and J.C. Seidell, 1996. Do dietry
and supplementary intakes of antioxidants differ with smoking

status. Intl. J. Epidemiol., 25: 70–79
REFERENCES


(Received 13 August 2003; Accepted 20 September 2003)
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smoking on serum levels of lipid peroxides and essential fat soluble

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