Conclusions

Urine at a cotinine cut off of 5nmol/ml had a higher sensitivity (61.4% > 55.7%) and higher specificity (68.5% > 56.8%) than serum at a serum cotinine cut off of 10nmol/ml.As shown inTable XIX and Table XX.

Likewise, urine at a cotinine cut-off of 5nmol/ml had a higher positive predictive value (65.5% > 55.7%) and higher negative predictive value (64.5% > 56.8%) than serum at a serum cotinine cut-off of 10nmol/ml.As shown inTable XIX and Table XX.

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Table XIX. Serum and Urine Cotinine Sensitivity, Specificity and Predictive values table.

Parameters Smoked :

Serum Urine

Do not Smoke : Serum Urine

Total :

Serum Urine

High Serum Cotinine High Urine Cotinine

( a ) 88

97

( b ) 70

51

( a + b ) 158

150

Low Serum Cotinine Low Urine Cotinine

( c ) 70

61

( d ) 92

111

( c + d ) 162

170

Total : Serum Urine

( a + c ) 158

158

( b + d ) 162

162

( a + b + c + d ) 320

320

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Table XX.Sensitivity, Specificity and Predictive values of serum and urine cotinine assays.

Parameters Serum Percentage detected

Urine Percentage detected Sensitivity = a/a+c 88 x 100

88 +70

55.7% 97 x 100 97 +61

61.4%

Specificity = d /b+d 92 x 100 92 + 70

56.8% 111 x 100 111 + 51

68.5%

Positive Predictive value a/a+b

88 x 100 88 + 70

55.7% 97 x 100 97 + 51

65.5%

Negative Predictive value = d/c+d

92 x 100 92 +70

56.8 % 111 x 100 111 + 61

64.5%

lxxxvi CHAPTERFIVE DISCUSSION

5-1 DISCUSSIONIn carrying out this study among the undergraduate students of

Ahmadu Bello University a total of three hundred and twenty (320) participants were recruited, 158 of whomwere Subjects (smokers comprising 157 males with one female) and 162 of whom were Controls (nonsmokerscomprising 159 males with 3 females). Despite employing non- probability sampling method of selection combined with accidental and snowballing methods care was taken to match subjects with controls for socio-demographic characteristics.

There were no significant differences in means of ages, height and weekly wages of subjects and controls. The distribution of accommodation pattern is identical in both groups in which case higher numbers of participants in each group reside in apartments shared by 2-5 occupantswith fewer participants occupying self-contained apartments.Noteworthy however is that more subjects were able to afford the more expensive self-contained apartments compared to controls, majority of whom were only able to afford the cheaper costs of shared apartments.

Some significant differences were however observed in some socio-demographic parameters but these are not expected to have significant impacts on the aim and objectives of this study. For instance, there were significant differences in the means of weight and BMI of subjects and controls.

The gender distribution showed a near absence of female participants among the subjects,thus necessitating the selection of a control population that will match the gender distribution observed among the subject population. This gender discrepancy was due to the

lxxxvii

fact that female smokers rarely smoke in public and those that were identified by friends were not willing to be part of the study for various reasons including suspicion and distrust. This observed difference is in agreement with findings of the study conducted by Emeroleet al amongstudents ofImo State University where it was discovered that the prevalence of smoking is 25% among males and 2% among females.⁷⁵

It was observed that most subjects started smoking in their teenage and were mostly recruited via peer pressures. This is in agreement with the report of the U.S Surgeon General of 2012 which noted that 88% of those who smoke started by age 18yrs while 99% started by age 26year.⁷⁶This study recorded 85% before age 20yrs and 97% by age 25yrs.

This study showed that in terms of duration and venue of exposure to secondhand smoke the controls were actually less exposed to other people’s cigarette smoke compared to subjects which contributed to the higher serum and urine cotinine levels observed in subjects and further increases the risk of adverse health consequences in subjects.

Considering the fact that this study focused mainly on healthy individuals it is not surprising to discover that majority of the participants reported no symptoms. Cough was the most reported symptom and mostly by subjects. Shortness of breath was next reported to cough and exclusively by subjects. Peptic ulcer (dyspepsia) was reported next to shortness of breath but mostly by controls. Other symptoms less reported were asthma, mouth sores, skin allergy and eating disorder in order of decreasing frequencies. At this early stage in the life of subjects who reported symptoms it is rather too premature to exclusively attribute their

symptoms to their smoking habits. These symptoms should rather serve as indicators for further

lxxxviii

follow up of such individuals for long-standing adverse health consequences of cigarette

smokingor encourage them to quit smoking at this early stage which would make them regain or attainthe health status of a never smoker after some years of quitting.

The Smoking Index showed most of the subjects were light or mild smokers, few were moderate smokers while heavy smokers were negligible in numbers. This trend was also

apparent with Fagerstrom’s Test for Nicotine Dependence (FTND) in which case majority of the subjects either had no or low nicotine dependence, few had low-to-moderate or moderate nicotinedependence while those with high nicotine dependence were quite negligible.These observations can be attributed to the fact that most of these subjects are still financially dependent on their sponsors and can only afford few sticks of cigarettes a day due to financial constraints.

Physical examination for nicotine stain showed about half of the subjects had nicotine stains while none of the controls had. Finger staining was the highest recorded, followed by tooth staining and least staining recorded on the lips. Some subjects had stains at multiple sites.

Arterial Oxygen Saturation (SPO2) using the pulse oximeter showed that the mean arterial SPO2 is greater in controls but the difference is not statistically significant. However, the Perfusion Index (PI) with a higher mean in controls showed a significant difference when compared with the mean in subjects. These findings are supportive of the findings reported by Miyang Jean et al and Igor Mexander et al who respectively noted that there was no significant difference in SPO2 of smokers and nonsmokers after single exposure to cigarette smoke unlike findings of vascular events where “cutaneous blood flow-index” was significantly reduced in

lxxxix

cigarette smokers, thus highlighting an increased risk to a generalized microvascular dysfunction.^77,78 The arterial SPO2 finding is a reflection of the fact that the gas exchange mechanism of the lung function is still largely preserved in the huge functional reserve capacity of the lungs especially at this early stage of the smoking habits of these youths.

The means of the Systolic and Diastolic blood pressures were observed to be higher in subjects than that of controls and were statistically significant. This is in agreement with the findings of Umar and Samaila who reported increased prevalence of hypertension with tobacco smoking.⁷⁹ Also supported by findings of Grappelli et al and Marcos et al who both noted persistent BP increase in heavy smokers.^80,81The findings ofPrimatestaet al also supported higher BP in smokers than in nonsmokers.⁸²The likely explanation to my findings is that due to chronic inflammatory effects of the various toxic substances in cigarette smoke on vascular endothelium resulting in various cardiovascular events including hypertension.

Laboratory investigations revealed that the means of the liver enzymes (AST,ALT and ALP) were all higher in subjects than in controls but the differences were not statistically significant. Similar study conducted by Wannamethee and Shaper revealed that cigarette smoking was significantly associated with increased levels of alkaline phosphatase (ALP) but was inversely associated with increased aspartate amino transferase (AST) after adjustment for alcohol intake, body mass index and physical activity.⁸³Their study population was very large and also in middle-aged men (4,595 men aged 40 –59 yr). They however concluded that

smoking does not cause liver injury, but may enhance the effects of alcohol on liver cells injury in heavy drinkers. This conclusion therefore may explain the finding of no statistically

xc

significantdifference in liver function tests between subjects and controls in this study.

Serum electrolytes showed that the mean serum levels of sodium and chloride were higher in subjects while the mean serum levels of potassium and bicarbonate were

lower in them(subjects) but these differences were not statistically significant. This is in agreementwith similar study conducted by Emad-Aldin et al which revealed that serum sodium waselevated in smokers while serum potassium was reduced in smokers compared to

nonsmokers.⁸⁴Just as observed in this study they also observed no statistically significant differences between subjects and controls even though their study population size was 60 – participants (30 – subjects and 30 - controls) unlike in this study with 320 – participants.

The means of Urea, Creatinine and Creatinine Clearance were lower in subjects compared to controls but all differences were not statistically significant. However, similar studies carried out by Munzir et al and Halimiet al both showed contrary findings where the former revealed elevated serum creatinine and urea in smokers and the latter revealed elevated creatinine clearance also insmokers.^85,86The studies of Munzir et al was actually conducted on a smaller population size of 80 – participants (40 – subjects and 40 – controls) unlike this study that engaged 320 – participants, even though both studies were conducted on younger populations. Halimi et al who observed elevated creatinine clearance in subjects also noted that acute smoking reduced glomerular filtration rate in never smokers or occasional smokers but not in habitual smokers. He also noted that chronic smoking is not associated with impaired renal function in normal subjects. Therefore, the finding of no

statistically significant difference in renal functions of healthy subjects and controls in this study

xci is supported by the findings ofHalimi et al.

The correlation between self-reported smoking status and cotinine assay in body fluids is shown by a finding of higher levels in the mean serum and urine levels of cotinine in subjects compared to controls with statistically significant differences between the two groups of participants.The reason for these findings was because subjects were more exposed to cigarette smokes than controls. This finding is in agreement with findings of Bassey and Udoh whoreported serum cotinine to be higher in active smokers than in passive or nonsmokers.⁸⁷ Urine cotinine levels were also found to be higher in smokers than in nonsmokers in a study carried out by NiimaBabhadiasharet al on correlation between cigarette smoking and urine cotinine levels in gastric cancer patients.⁸⁸

In the assessment of serum cotinine levels relative to degree of cigarette consumption an inverse or perhaps a non-linear relationship was observed as the mean of serum cotinine levels in heavy smokers (SI > 300) is lower than that in moderate smokers (SI of 100 - 300) which in turn is lower than that inmild /light smokers (SI < 100). The possible explanation for this finding is the possibility of abias in the reporting of the true degree of cigarette consumption by subjects in which casethere is a possibility of under-reporting in the heavy-smokers category or over-reporting in thelight-smokers category.⁸⁹ There is also the possibility of intake of foods or food productswith high nicotine contents especially in the light smokers category.^90,91 The verysmallnumber of subjects in the heavy-smokers category, unlike in the light-smokers, may bestatistically insufficient to give an expected direct relationship of higher degree of cigarette

xcii

consumption with higher serum cotinine levels. Moreover, mean body fluids cotinine levels in 3 heavy smokers in comparison to that of 39 moderate smokers or 116 mild smokers is statistically unrealistic.

My study population was largely made up of males who were all blacks. The black race and male gender are also factors or attributes associated with higher plasma cotinine levels according to the findings of Wagenknecht et al and Gan et al respectively.They attributed the higher serum cotinine levels in blacks and males to the activity of the CYP2A6 which is slower in blacks due to genetic variation and faster in females asestrogen induces CYP2A6.^92,93,94

In the assessment of urine cotinine levels relative to degree of cigarette consumption findings were exact replica of the findings in the assessment of serum cotinine relative to degree of cigarette consumption. Confounding factors can also be explained as in the case of serum cotinine above.⁸⁹These inverse relationships were paradoxical and almost similar to the findings of Niima et al who found no significant relationship with urine cotnine and average number of cigarette smoked or duration of cigarette smoking.⁸⁸

In determining which of the two body fluids cotinine assay best correlates with self -reported cigarette smoking status it was revealed that more subjects just as more controls were appropriately classified as True Positives and True Negatives respectively with urine cotinineassay than with serum cotinine assay. On the contrary, more subjects and more controls were inappropriately classified as nonsmokers (False negatives) and as smokers (False

Positives) respectively with serum cotinine assay than with urine cotinine assay. Hence, the urinecotinine assay best correlates with self-reported smoking status. This finding is in

xciii

agreementwith the findings of Jarvis et al.⁹⁵ The possible explanation for this finding is that nicotine/cotinine in serum is distributed over a larger volume into various body fluids and tissues, it is more protein-bound and its availability is more influenced by serum pH and its ionization.^31,32 Urine cotinine is however less affected by these factors thus making urinary cotinine more stable and more reliable for assessing exposure to cigarette smoke.^43,44

The correlation between serum and urine cotinine levels is shown inthe findings of a positive correlation (r = 0.320) between the two body fluids and with both body fluids showing inverse correlation with the degree of cigarette consumption. However, the correlation

demonstratedbetween serum and urine cotinine assays is not a perfect correlation because the degree of correlation at ‘r’ value of 0.320 is medium (i.e 0.30 < r< 0.50). This is in contrast with the study carried out by Mitoli Raja et al on Diagnostic Methods for Detection of Cotinine Level in Tobacco Users where a high correlation was demonstrated between the body fluids – saliva, blood and urine.⁹⁶ The possible explanation for thismedium positive correlationmight bebased on the fact that pH, ionization and Protein-binding have unequal influences on serum andurine cotinine levels with more influences observed on serum cotinine level.

A determination of the Sensitivity, Specificity and Predictive values of both body fluids cotinine assays revealed that, at a serum cotinine cut off of 10ng/ml and urine cotinine cut offof 5ng/ml, there is a higher sensitivity and higher specificity with higher positive predictive and higher negative predictive values with urine cotinine assay than with serum cotinine assay.

This is in agreement with the study carried out by Mitoli Raja et al on Diagnostic Methods for Detection of Cotinine Level in Tobacco Users.⁹⁶They demonstrated a higher specificity in urine

xciv

compared to blood with an added advantage of its noninvasiveness. The possible explanation for this finding is that urine is less affected by pH or binding proteins that exert significant influences on serum cotinine, thus making urine cotinine more stable and more reliable.