systematic review of the relationship between the 3-hydroxycotinine/cotinine ratio and cigarette...

REVIEW

Systematic review of the relationshipbetween the 3-hydroxycotinine/cotinine ratioand cigarette dependence

Oliver West & Peter Hajek & Hayden McRobbie

Received: 17 January 2011 /Accepted: 30 April 2011 /Published online: 20 May 2011# Springer-Verlag 2011

AbstractRationale Individual differences in the rate of nicotinemetabolism (RNM) could be related to dependence and successin stopping smoking. A range of studies have examined RNMmeasured by the ratio of trans-3′-hydroxycotinine and cotininein body fluids (the ratio). A systematic review of this literatureis needed to draw conclusions and identify gaps in evidence.Objective The aim of this study is to review evidence on theassociation of the ratio to cigarette dependence and its role inindividual tailoring of smoking cessation pharmacotherapy.Results We reviewed 27 studies of the ratio related to itsreliability, validity, and relationship to dependence. The ratio is areasonably accurate proxy for RNM. There is little evidence thatthe ratio is related to questionnaire measures of dependence,though the existing data are limited and the ratio has been linkedto smoking at night and to some aspects of smoking topography.The ratio is also only weakly associated with cigaretteconsumption. Its relationship to the severity of withdrawalsymptoms seems also weak at best, but limited data exist. Onestudy suggests the ratio predicts outcome of unaided quitting.Importantly, the ratio seems to predict responses both to NRTand bupropion, and thus could guide pharmacotherapy.Conclusions The evidence that the ratio is related tosmoking behaviours and to cigarette dependence is limited,but the ratio seems to influence treatment response to twostop smoking medications. Further studies of the relation-ship between the ratio and cigarette dependence and trialsof ratio-guided pharmacotherapy are needed.

Keywords Dependence .Metabolism . Nicotine . Smoking

There are considerable individual differences in the rateof nicotine metabolism (Benowitz et al. 1982). Thesecould in theory influence such factors as cigaretteconsumption, puffing behaviour, latency to onset ofwithdrawal symptoms, and reinforcement derived fromsmoking. Such effects would in turn make plausible thehypothesis that the rate of nicotine metabolism (RNM) iscausally related to cigarette dependence. (Note that asimilar acronym, NMR, has been used for ‘nicotinemetabolite ratio’ and ‘nicotine metabolic rate’). In addi-tion, fast metabolisers might underdose on nicotinereplacement treatment (NRT), meaning RNM could guidemedication choice and dose.

There exists a slowly increasing volume of literature onthis potentially important issue, with sometimes contradic-tory results and no firm generally accepted conclusions interms of how the ratio relates to cigarette dependence. Weset out to provide the first comprehensive review of therelevant findings to see what inferences can be derived andto identify possible directions of future research. Below wedescribe the methods of measuring the RNM and reviewthe literature examining the relationship between the mostpracticable and widely used measure of RNM and cigarettedependence.

Measuring the rate of nicotine metabolism

The key requirement for any progress in this area is anaccurate measure of RNM. There are two main approachesavailable. The most accurate and reliable techniqueinvolves intravenous administration of a known dose of

O. West (*) : P. Hajek :H. McRobbieUK Centre for Tobacco Control Studies, Wolfson Institute ofPreventive Medicine, Barts and The London School of Medicineand Dentistry, Queen Mary University of London,London E1 2AD, UKe-mail: [email protected]

Psychopharmacology (2011) 218:313–322DOI 10.1007/s00213-011-2341-1

nicotine followed by assessments of plasma nicotine levelsat regular intervals for several hours. A much simplermethod involves measuring the ratio of two nicotinemetabolites—cotinine (COT) and its primary metabolite,trans-3’-hydroxycotinine (3HC)—derived from a saliva,blood, or urine sample (Benowitz et al. 2003, Dempsey etal. 2004). Assessing the ratio of a metabolite to its parentcompound is a standard method for determining the speedof drug metabolism (Dempsey et al. 2004). Higher ratiosindicate faster metabolism.

The reason the ratio of cotinine to nicotine is not usedin this context is due to the relatively short half-life ofnicotine (approximately 2 h; (Benowitz et al. 1982)). Thecotinine/nicotine ratio therefore depends substantially onthe time since last cigarette and the smoking pattern overthe past few hours. In contrast to this, cotinine and 3HCboth have comparatively long half-lives; 16 h and 5–6 h,respectively (Benowitz and Jacob 1994), and when 3HC isgenerated directly from cotinine, it has the same half-lifebecause the elimination of 3HC is formation limited. The3HC/COT ratio is thus fairly stable over time (Dempsey etal. 2004).

The hepatic enzyme exclusively responsible for themetabolism of cotinine to 3HC, cytochrome P450 2A6(CYP2A6), also metabolises nicotine to cotinine (Messinaet al. 1997). The ratio of 3HC/COT (hereafter ‘the ratio’) istherefore considered to be an accurate marker of CYP2A6activity, and thus RNM.

Genotyping CYP2A6 variants is an appealing approachfor determining RNM, although only about 60% of thevariance in RNM is thought to be heritable (Swan et al.2005, Swan et al. 2009). Heritability coefficients for otheraspects of smoking, such as Fagerstrom Test for NicotineDependence (Heatherton et al. 1991) scores, smokingpersistence, and cessation are typically above 50% (Munafoand Johnstone 2008). Over 90 CYP2A6 alleles have beenidentified (see http://www.cypalleles.ki.se/cyp2a6.htm for a

regularly updated list), but these alone do not explain thevariability in CYP2A6 activity. The ratio is thus consideredto be a better indicator of the actual individual speed ofnicotine metabolism than a single genetic marker, as itaccounts for a wider range of influences both genetic andenvironmental (Ray et al. 2009).

Methods

Search strategy

We set out to identify and analyse existing empirical studieswhich examine the reliability and validity of the ratio andits relationship to markers of cigarette dependence. Oursearch strategy is described in Table 1. Titles and abstractsof papers were searched for mention of the 3HC/COT ratioin any context. The full text of 39 papers was subsequentlyread. Citations from these papers were searched foradditional studies of the 3HC/COT ratio, which identifiedfive papers (none of which was appropriate for this review).The 27 papers that reported on the validity, reliability, orrelationship of the ratio to measures of dependence wereincluded in our review.

Results

Table 2 provides an overview of the 27 studies discussed inthis review.

Validity and reliability

Several studies have assessed the relationship of theplasma, saliva, and urine ratios to different metabolismand clearance parameters (Lea et al. 2006, Dempsey et al.2004, Benowitz et al. 2006, Swan et al. 2009, Malaiyandi et

Table 1 Search details

Databases searched Medline, PsycInfo, and Embase

Search terms All fields within each database searched for: nicotine and cotinineand hydroxycotinine.

Number of records identified 419 across all databases; 276 unique records

Number of records identified from other sources 5

Number of records screened 281

Number of records excluded (reasons for exclusion) 242 (did not report on the 3HC/COT ratio)

Number of full-text articles assessed 39

Number of full-text articles excluded (reasons for exclusion) 12 (did not relate the 3HC/COT ratio to cigarette dependence)

Number of full-text articles included in review 27

314 Psychopharmacology (2011) 218:313–322

http://www.cypalleles.ki.se/cyp2a6.htm

Table 2 Characteristics and main findings of studies included in review

Paper 3HC/COT ratio methods Main findings concerning 3HC/COT ratio

Al Koudsi et al.(2006)

Plasma ratio obtained after intravenous infusions of NICand COT, N=222

No effect of *21 CYP2A6 variant on the ratio

Benowitz et al.(2003)

Urinary ratio obtained from ad libitum smoking N=72 Significant correlation between ratio and CPD(r=.33)

FTND not associated with ratio

Benowitz et al.(2006)

Plasma and urinary ratios obtained after intravenousinfusions of NIC and COT, N=278

Clearance of intravenously administeredNIC correlated with plasma (r=.62) and urinary(r=.47) ratios.

Individuals with at least one *2, *4, *9, or *12CYP2A6 allelic variant had significantly slowerratios than wild types.

Berlin et al. (2007) Plasma ratio obtained from ad libitum smoking prior toNRTsmoking cessation trial, N=120 (adolescent sample)

CPD and time to first cigarette not associatedwith ratio

Dempsey et al.(2004)

Plasma and saliva ratios obtained after oral dosing of NICand COT, N=62

Significant correlation between both plasma andsaliva ratios and oral clearance of NIC: r=.79and r=.78, respectively

Derby et al. 2008 Urinary ratio obtained from ad libitum smoking, N=585 Multivariate regression found CPD to predict ratio(reg coef 0.116, p<.05).

Ho et al. (2008) Plasma and ratio obtained after oral dosing of NIC, N=280 Individuals with at least one *17 or *23 CYP2A6allelic variant had significantly slower ratios thanwild types

Ho et al. (2009) Plasma ratio obtained from ad libitum smoking prior toNRT smoking cessation trial, N=646

When using nicotine gum or placebo gum, slowermetabolisers more likely to stop smoking thanfaster metabolisers (data combined across activeand placebo conditions; not presented separately).

Individuals with at least one *4, *9, *17, *20, *27,*28, or *35 CYP2A6 allelic variant had significantlyslower ratios than wild types.

No effect of *12, *24, *25, or *26 variants on the ratio

*1B homozygotes had significantly faster ratios thanwild types.

CPD, FTND, and CDS not associated with ratio

Johnstone et al.(2006)

Plasma ratio from ad libitum smoking prior to NRTsmokingcessation trial, N=545 (only treatment failures included)

Individuals with at least one *2, *9, or *12 CYP2A6allelic variant had significantly slower ratios thanwild types.

*1B homozygotes had significantly faster ratios thanwild types.

CPD significantly positively associated with ratio: β=.16

FTND and Horn-Russell not associated with ratio

Kandel et al. (2007) Urinary ratio obtained from ad libitum smoking, N=904 CPD and FTND not associated with ratio.

FTND item ‘Which cigarette would you most hate to giveup?’ (‘The first of the morning’ or ‘Another one’)significantly associated to ratio, among whites, notAfrican Americans, Hispanics, or Asians

Kim et al. (2005) Salivary ratio obtained from pregnant smokers at ∼26timepoints, N=16.

Ratio significantly lower in those who smoked ≤10 CPD,relative to ≥20 CPD

Lea et al. (2006) Salivary ratio obtained from regular smokers, N=6 Ratio not affected by time of day of sampling.Within-subject variation in ratio is 26%.

CPD not associated with ratio

Lerman et al. (2006) Plasma ratio obtained from ad libitum smoking prior toNRT smoking cessation trial, N=480

When using nicotine patch, slower metabolisers more likelyto stop smoking than faster metabolisers

Faster metabolisers using nicotine patch had higher cravinglevels than slower metabolisers 1 week into quit attempt. Noeffect of ratio on craving among users of nicotine nasal spray

Psychopharmacology (2011) 218:313–322 315

Table 2 (continued)


Other withdrawal symptoms not associated with ratio ineither patch or spray condition

Significant correlation between ratio and CPD (r=.12)


Lerman (2010) Plasma ratio obtained from ad libitum smoking priorto NRT smoking cessation trial, N=471


A significant benefit of 24 weeks versus 8 weeks of nicotinepatch use on cessation outcome was found only amongslower metabolisers; however, treatment by metabolisergroup (slow versus fast) interaction was not significant.

Malaiyandi et al.(2006)

Plasma ratio from regular smokers, N=152 Individuals with at least one *2, *7, *9, or *12 CYP2A6allelic variant had significantly slower ratios than wildtypes.

CPD not associated with ratio

Moolchan et al.(2009)

Plasma ratio obtained from ad libitum smoker prior toNRT smoking cessation trial, N=85 (adolescent sample)

Ratio explained 6.7% of variance in mean puff volumeacross total sample.

For males, ratio positively associated with mean puffvolume,explaining 19.2% of variance, and negatively associatedwith puff number and mean puff duration, explaining23.7%and 10.1% of respective variances. No significant effectsamong females

Mooney et al. (2008) Urinary ratio from regular smokers at baseline and during‘waitlist’ ad libitum and reduction phases of controlledstudy. Total n=123 (subset in ‘waitlist’ phase=47)

Ratio generally stable during ad libitum smoking; smallvariation when reducing smoking and using NRT

Significant correlation between ratio and CPD (r=.19, totalsample; r=.39, waitlist sample) and waking at night tosmoke (r=.23, total sample; r=.49, waitlist sample)

Mwenifumbo et al.(2007)

Plasma ratio obtained after oral dosing of NIC. Totaln=190 (current smokers=94)

Ratio obtained from ad libitum smoking and after oraldosingof NIC correlated with each other (r=.85, 1.5 h after oralNIC; r=.96, 4.5 h after oral NIC).

CPD significantly higher in smokers from the fastest ratioquartile, relative to the 2nd fastest and slowest, but notsecond slowest, quartiles.

Mwenifumbo et al.(2008a)

Plasma ratio obtained after oral dosing of NIC. Totaln=281 (current smokers=138)

Individuals with at least one *9, *12, *17, *23, *24, *26, or*28 CYP2A6 allelic variant had significantly slower ratiosthan wild types.

No effect of *14, *20, or *25 variants on the ratio.

Mwenifumbo et al.(2008b)

Plasma ratio obtained after intravenous infusion of NIC.Total n=163 (current smokers=27)

*1B homozygotes had significantly faster ratios than wildtypes, while possessing only one *1B variant had noeffect on the ratio.

Patterson et al. (2008) Plasma ratio obtained from ad libitum smokers priorto bupropion smoking cessation study, N=414

Significant interaction between ratio and treatment(buproprion vs. placebo); only those in the fastestmetabolising quartile benefited from the bupropion. Whenon placebo, chances of quitting decreased as ratio quartileincreased; 32% of slowest ratio quartile stopped smokingcompared with 10% of fastest quartile.

Craving and withdrawal symptoms not associated with ratio

CPD and FTND not associated with ratio

Piper et al. (2008) Plasma ratio obtained from ad libitum smoking priorto NRT smoking cessation trial, N=480

WISDM ‘craving’, ‘cue exposure’, ‘negative reinforcement’,‘positive reinforcement’, and ‘tolerance’ subscales notassociated with ratio. Remaining eight WISDM subscalesnot administered in this study

Rubinstein et al.(2008)

Plasma ratio obtained from ad libitum smoking,N=20 (adolescent sample)

Some individual withdrawal symptoms and total withdrawalsymptoms greater in ‘faster metabolisers’ (fastest ratio


al. 2006, Mooney et al. 2008). These include the rate ofclearance of orally and intravenously administered nicotine,and the rate at which nicotine is cleared exclusively viametabolism to COT, all measured by regular sampling ofnicotine and COT plasma concentrations. Associationsbetween the different types of ratio have been assessed(Dempsey et al. 2004, Benowitz et al. 2006), although nosingle study measured all three. Effects on the ratio ofdifferent storage techniques and changes in smokingbehaviour have also been reported (Lea et al. 2006,Mooney et al. 2008).

The plasma and saliva ratios correlate with the clearance oforally administered nicotine; r=.79 and .78, respectively(Dempsey et al. 2004). Urine and plasma ratios correlate withthe clearance of intravenously administered nicotine; r=.47and .62, respectively (Benowitz et al. 2006). The plasma ratioderived from ad libitum smoking correlates very closely withthe ratio derived from orally administered nicotine; r=.96(4.5 h post-dosing) and .85 (1.5 h post-dosing), (Mwenifumboet al. 2007). Finally, the plasma ratio is closely associated withthe ratio in saliva and urine; r=.88, and .70, respectively(Benowitz et al. 2006, Dempsey et al. 2004). These resultsindicate that the ratio is a good, but not perfect, proxy for therate of nicotine metabolism. As RNM is likely to beinfluenced by enzymes other than CYP2A6, (Hukkanen etal. 2005), the correlation between clearance of nicotine andthe ratio is unlikely to be perfect.

There is no significant variation in the saliva ratio whensamples are stored either for 7 days compared with 24 h, orat room temperature compared with −20°C (Lea et al.2006). The saliva ratio is not affected by the time of day thesample was taken (i.e. morning versus evening (Lea et al.2006)). Furthermore, the ratio does not seem to varysubstantially within individuals across several measurestaken over one (Lea et al. 2006) (saliva ratio) and8 (Mooney et al. 2008) (plasma ratio) weeks of ad libsmoking (coefficients of variation=38% and 35%, respec-tively). However, in smokers deliberately reducing theircigarette consumption while using NRT over a 12-weekperiod, a significant variation in the plasma ratio wasobserved during the first 4 weeks. RNM increased duringthis 4-week period. There is no obvious reason why thiswould result from substituting nicotine from cigarettes withnicotine from NRT provided the nicotine intake remainedthe same, so it is likely that the increase was due to achange (probably a reduction) of the habitual nicotineintake. After this, cigarette consumption seemed to havebeen maintained at a new steady level, and the variability inthe ratio diminished (Mooney et al. 2008). The totalvariation in the plasma ratio is lower than in the urine ratio(Swan et al. 2009). This is believed to be due to a strongerinfluence of environmental factors on the urine comparedwith the plasma ratio, as well as individual differences inthe in renal clearances of cotinine and 3HC (Swan et al.

Table 2 (continued)


quartile, n=5) than remainder of sample

No effect of ratio on craving

CPD significantly higher in faster metabolisers

Self-reported ‘level of addiction’ significantly correlatedwith ratio (r=.5).

Adolescent versions of FTND and HONC not associatedwith ratio

Schnoll et al. (2009) Plasma ratio obtained from ad libitum smoking priorto NRT smoking cessation trial, N=585


Craving and withdrawal symptoms not associated with ratio

Significant correlation between ratio and CPD (r=.11)


Strasser et al. (2011) Plasma ratio obtained from ad libitum smokers priorto NRT smoking cessation trial, N=109

Ratio explained 51% of variance in total puff volumeper cigarette.


Swan et al. (2009) Plasma and urinary ratios obtained after intravenousinfusions of NIC and COT, N=276

Heritability estimates for plasma and urinary ratios: 49.4%and 42.0%, respectively

Plasma and urine ratios significantly correlated with eachother (r=.70).

No effect of *1B CYP2A6 variant or gene duplication onratio


2009) In particular, the urine ratio might be affected bydietary factors that impact urine flow rate and urine pH—two factors that affect clearance of COT and 3HC throughthe kidneys (Swan et al. 2009).

Nine studies have measured the ratio of individuals with atleast one variant CYP2A6 allele. Individuals with two copies ofthe *1 allele (wild type) are assumed to have 100% CYP2A6activity (Benowitz et al. 2006). Wild-type homozygotes havelower ratios (i.e. slower metabolism) than those who have atleast one of the following variants: *2 (Johnstone et al. 2006,Malaiyandi et al. 2006, Benowitz et al. 2006); *4 (Benowitz etal. 2006, Ho et al. 2009, Malaiyandi et al. 2006); *7(Malaiyandi et al. 2006)*9 (Benowitz et al. 2006, Ho et al.2009, Johnstone et al. 2006, Malaiyandi et al. 2006,Mwenifumbo et al. 2008a); *17 (Ho et al. 2009, Ho et al.2008, Mwenifumbo et al. 2008a); *20 (Ho et al. 2009) *23(Mwenifumbo et al. 2008a, Ho et al. 2008); *27 (Ho et al.2009); *28 (Ho et al. 2009, Mwenifumbo et al. 2008a); and*35 (Ho et al. 2009). Effects of variants *12, *24, and *26 areless clear, with some studies finding these variants reduced theratio (*12, (Benowitz et al. 2006, Malaiyandi et al. 2006,Johnstone et al. 2006); *24, (Mwenifumbo et al. 2008a); *26(Mwenifumbo et al. 2008a), while other studies did not (*12,(Ho et al. 2009); *24, (Ho et al. 2009); *26, (Ho et al. 2009)).

Having one *1B variant was associated with higherratios (faster metabolism) in some cases, yet not for others(Mwenifumbo et al. 2008b). Another study found nodifferences in ratios between data pooled across wild-typehomozygotes and those with one *1B variant, comparedwith individuals homozygous for *1B or with an addi-tional CYP2A6 gene (Swan et al. 2009). Three studiesobserved higher ratios among those homozygous for *1B,compared with wild-type *1A homozygotes (Ho et al.2009; Johnstone et al. 2006; Mwenifumbo et al. 2008b),although a fourth found no such effect (Mwenifumbo etal. 2008a). Other dose–response effects have beenobserved. For example, in one study, individuals with a*1/*17 variant combination had approximately 50%CYP2A6 activity, while those with *17/*17 had only∼15% activity (Mwenifumbo et al. 2008a).

Among a sample of individuals of either wild-typehomozygotes, or *2, *4, *7, or *9 alleles, genotypecorrelated with the ratio, r=.39 (Malaiyandi et al. 2006).When those with another variant, *12, were added to thisanalysis, the relationship increased to .46 (Malaiyandi et al.2006). Variants *14 (Mwenifumbo et al. 2008a), *20(Mwenifumbo et al. 2008a), *21 (Al Koudsi et al. 2006),and *25 (Mwenifumbo et al. 2008a, Ho et al. 2009) havenot been shown to affect the ratio.

Overall, several genotypes have been identified thatpromote reduced RNM, as measured by the ratio. Data formany genotypes are quite limited or unclear, which is inpart due to the very low prevalence of variant alleles.

In conclusion, these data suggest that the ratio—despitesome limitations—is a reasonably accurate marker ofRNM, insofar as it records information similar to othervalid methods, and indicates the relevant genotypes.Environmental effects and individual variability have asmall influence on its capacity to indicate CYP2A6 activity.Non-metabolic clearance of nicotine, renal excretion, is alsonot captured by the ratio. Reliability is acceptable providedsmokers have not recently substantially altered theirsmoking behaviours. The slight differences between thethree sources of the ratio are not cause for concern,although the urine ratio does appear less reliable. Thechoice of sampling method should be informed by practicalissues.

Cigarette consumption

A positive association between cigarettes per day (CPD)and the ratio was observed in 9 of 15 studies (Benowitz etal. 2003, Derby et al. 2008, Kim et al. 2005, Lerman et al.2006, Mooney et al. 2008, Mwenifumbo et al. 2007,Schnoll et al. 2009, Rubinstein et al. 2008, Johnstone etal. 2006). When found, the relationship was overall notstrong (r’s ranged from .1 to .19), although two highercorrelations were reported, with r’s of .33 (Benowitz et al.2003) and .38 (Mooney et al. 2008). Of the six studies thatreported nonsignificant findings (Berlin et al. 2007, Kandelet al. 2007, Lea et al. 2006, Malaiyandi et al. 2006,Patterson et al. 2008, Ho et al. 2009), one had a very smallsample size (n=6 (Lea et al. 2006)), and one comprised asample of adolescents, where the smoking pattern may nothave yet stabilised (Berlin et al. 2007). Several of therelevant studies do not report their results in a way whichwould allow a meta-analysis, but the overall pattern ofresults suggests a significant, but weak, association.Cigarette consumption is generally not closely associatedwith nicotine intake (Fidler et al. 2008) or with cigarettedependence, especially in samples of smokers seekingtreatment (e.g. Russell et al. 1993). Nicotine intake percigarette varies widely due to differences in puff frequencyand depth of inhalation. In this context, the finding thatthere is an association between the two variables, albeitweak, is encouraging.

Questionnaire measures of dependence

Of the studies that have measured the relationship of theratio with the most popular questionnaire measure ofdependence, the Fagerstrom Test of Nicotine Dependence(FTND), none have found a significant association (Benowitzet al. 2003, Ho et al. 2009, Johnstone et al. 2006, Kandelet al. 2007, Lerman et al. 2006, Patterson et al. 2008,Schnoll et al. 2009, Strasser et al. 2011). No relationship


was found with other measures of smoking behaviour,including Cigarette Dependence Scale (Ho et al. 2009),Horn-Russell Questionnaire (Johnstone et al. 2006),modified Fagerstrom Tolerance Questionnaire (Rubinsteinet al. 2008), Hooked On Nicotine Checklist (Rubinstein etal. 2008); and the Wisconsin Index of Smoking Motives(Piper et al. 2008)).

Two studies looked at individual FTND items. In one,the question ‘Which cigarette would you most hate to giveup?’ (‘The first of the morning’ or ‘Another one’) wassignificantly related to the ratio, although only amongwhites and not among African Americans, Hispanics, andAsians (Kandel et al. 2007). Time to first cigarette was notrelated to the ratio in a sample of adolescent smokers(Berlin et al. 2007).

Self-reported ‘level of addiction’ was found to correlatewith the ratio (r=.5) in a sample of 20 adolescents(Rubinstein et al. 2008). Frequency of waking at nightand smoking, which is considered a marker of cigarettedependence, also correlated with the ratio in a sample ofadult smokers; r=.23 (Mooney et al. 2008). After 12 weeksof smoking reduction, the ratio and smoking at night werere-measured, and the correlation increased to .49.

Lack of a strong relationship between some of theexisting questionnaire measures of dependence and the ratiodoes not necessarily suggest a limited utility of the ratio.The standard questionnaires are actually very weak meas-ures of the construct in that they either show no relationshipto success or failure of quit attempts in smokers seekingtreatment or explain only a small proportion of the variance(Baker et al. 2007, West 2005). The relationship of the ratioto success or failure of quit attempts, covered in a latersection, represents a much stronger test of its potentialutility.

Smoking topography

Two studies have measured smoking topography along-side the ratio. Among adolescents, the ratio explained6.7% of the variance in mean puff volume across theentire sample (Moolchan et al. 2009). For males, the ratiowas positively associated with mean puff volume, explain-ing 19.2% of the variance, and negatively associated withpuff number and mean puff duration, explaining 23.7%and 10.1% of the respective variances. No significanteffects were found among females. In adults, fastermetabolisers had greater total puff volumes, and the ratioexplained 51% of the variance in total puff volume (othertopography indices were not reported in this study)(Strasser et al. 2011). The evidence from the adult sample,in particular, suggests faster metabolisers might inhalemore deeply in order to sustain desired blood levels ofnicotine.

Craving and withdrawal

In one study, faster metabolisers had higher levels ofcraving than slower metabolisers 1 week into a quit attemptwhen using nicotine patch (Lerman et al. 2006), while asimilar study did not replicate this finding (Schnoll et al.2009). Craving was not associated with the ratio amongabstaining smokers who used nicotine nasal spray orbupropion during a quit attempt (Lerman et al. 2006,Patterson et al. 2008). No other withdrawal symptoms wereassociated with the ratio across these three studies.Examining the ratio in participants using placebo wouldhave helped elucidate if the negative finding was aconsequence of pharmacotherapy reducing individual dif-ferences in tobacco withdrawal. There were no placebogroups in the patch or nasal spray studies, and data were notreported separately for active and placebo groups in thebupropion study.

Among adolescent light smokers (n=20;1–6 cigarettesper day) who underwent a 24-h enforced abstinence, fastermetabolisers (those who fell into the top quartile of theratio) had higher levels of several individual withdrawalsymptoms and overall withdrawal ratings than slowermetabolisers (Rubinstein et al. 2008), but did not differ incraving.

Success in stopping smoking

The association between the ratio and the outcome of a quitattempt presents the best test of the hypothesis that the ratiois related to cigarette dependence. No study has set out toexamine explicitly the relationship between the ratio andthe outcome of an unassisted quit attempt, but five studiesreported associations of the ratio with outcomes of quitattempts assisted by pharmacological treatments. Despitethe fact that the relationship of the ratio to success or failurein stopping smoking is not known, the main focus of all thestudies was the treatment response according to RNM,rather than the relationship of the ratio per se to outcome.However, one study reported the association between theratio and quit rate in participants using placebo medication.In the existing studies, smokers were grouped post hocaccording to their RNM using different approaches, detailsof which are summarised in Table 3.

Slower metabolisers were more likely to be abstinent atthe end of a standard course of treatment in all three studiesusing nicotine patches (Lerman et al. 2010, Lerman et al.2006, Schnoll et al. 2009). One study that comparednicotine gum with placebo gum (Ho et al. 2009) reportedthat slower metabolisers were more likely to quit than fastermetabolisers, but as data were collapsed across activemedication and placebo conditions, it is not clear whetherthis was due to differing medication responses across levels


of RNM, or the ratio is related to outcome regardless ofmedication. In other words, slower metabolisers could findquitting less difficult, or they could be more responsive tothe nicotine gum. One study compared extended (24 weeks)versus standard (8 weeks) nicotine patch use and found asignificant effect of extended treatment only among slowermetabolisers; however, the treatment by metaboliser group(slow versus fast) interaction was not significant (Lerman etal. 2010). No association was found between the ratio andsuccess in quitting among nicotine nasal spray users oracross pooled data from combined patch and nasal spraysamples (Lerman et al. 2006). One explanation why slowermetabolisers had better chances of quitting with nicotinepatch but not nasal spray could be due to elevated nicotinelevels in this group when using the patch. The capacity totitrate nicotine dose with the nasal spray might havemasked any effect of RNM (Johnstone et al. 2006).

In a study comparing bupropion with placebo (Pattersonet al. 2008), there was an interaction between the ratio andtreatment. Only those in the fastest metabolising quartilebenefited from the drug. Among smokers on placebo, thechances of quitting decreased as the ratio quartile increased,with a notable difference between the slowest (32%abstinent) and the fastest quartiles (10% abstinent). Insummary, slow nicotine metabolism seems to increase theefficacy of nicotine patches, and there is some evidencefrom one trial that faster metabolisers find quitting moredifficult.

Discussion

The existing data suggest that the 3HC/COT ratio is a valid,though not perfectly accurate, indicator of the rate ofnicotine metabolism (RNM). There is little evidence thatthe ratio is related to some widely used indices of cigarettedependence, though the data are limited. The ratio is relatedto some aspects of smoking behaviour, albeit weakly.

The ability of the ratio to predict the outcome of a quitattempt—perhaps the most clinically useful measure of

dependence—has been assessed in only one drug trial.Among smokers who received placebo, the likelihood ofquitting decreased with each increase in ratio quartile.Smokers with high ratio (fast metabolisers) seem to bemore likely to benefit from bupropion, while those with lowratio may derive more benefit from nicotine patches andpossibly also from nicotine gum, but not from nicotinenasal spray.

The fact that the ratio is unrelated to the establisheddependence questionnaires and yet, in one study at least,showed a relationship to outcome of a quit attempt maymean that it may reflect aspects of dependence which areindependent of behavioural elements of smoking. The weakassociation between the ratio and cigarette consumptioncould be due in part to smokers’ ability to titrate theirnicotine dose by varying the number of puffs and depth ofinhalation. This enables smokers to achieve differentnicotine concentrations from similar number of cigarettes(Benowitz 2001, Kozlowski et al. 2001). Indeed, there issome evidence that faster metabolisers puff on theircigarettes with more intensity. Future studies should includeeffect sizes and report their findings in a format whichallows their inclusion in meta-analyses.

The most interesting finding so far concerns theinteraction between the ratio and two stop smokingmedications. The better response to nicotine patch amongslower metabolisers could be because they accumulatehigher and therefore more therapeutic plasma nicotinelevels. Regarding bupropion, there is no intuitive mecha-nism through which RNM might mediate the effects of thismedication. One explanation could be that faster metabo-lisers simply are more dependent smokers, for whom aneffective medication is needed in order to increase thechances of quitting to a degree comparable to slowmetabolisers not taking a medication. The existing data onthe effect of individual differences in the ratio on cravingand other withdrawal symptoms in abstaining smokers areinconsistent and possibly influenced by effects of pharma-cotherapy. There are more negative than positive findings,but the issue has not been well examined so far.

Table 3 Details of studies looking at interactions between the ratio and stop smoking medications

Study Intervention Ratio groups

Lerman et al. (2006) Nicotine nasal spray versusnicotine patch

Grouped into quartiles: all quartiles compared with one another

Patterson et al. (2008) Bupropion versus placebo Grouped into quartiles: Slowest quartile compared separately against eachof the other quartiles

Ho et al. (2009) Nicotine chewing gum versus placebo Grouped into quartiles: Slowest quartile compared with the combined otherthree quartiles

Schnoll et al. (2009) Nicotine patch Same as Ho et al. (2009)

Lerman (2010) Standard versus extendednicotine patch

Same as Ho et al. (2009)


The existing studies raise an intriguing possibility thatthe ratio could be used as a diagnostic tool to guideintensity of treatment, with faster metabolisers offered moreintensive support and bupropion, if their better reaction tothe drug is confirmed, while slower metabolisers are offerednicotine patches. No data on interaction between the ratioand reactions to varenicline are currently available, and thisis an obvious area for further research. A trial comparingratio-guided and random allocations of NRT and bupropionis needed to examine whether the ratio assessment canimprove the efficacy of these two pharmacotherapies.

Acknowledgements We thank Caryn Lerman and Neal Benowitzfor their helpful comments on an earlier draft of this manuscript.

Conflicts of interest PH and HM have undertaken research andconsultancy for manufacturers of smoking cessation medications. OWhas no conflicts of interest to declare.

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