Accepted Manuscript

Title: Role of hair pigmentation in drug incorporation into hair

Authors: Hyerim Yu, Won-Jun Jang, Jung-Hee Jang,

Byoungduck Park, Young Ho Seo, Chul-Ho Jeong, Sooyeun
Lee

PII: S0379-0738(17)30460-7
DOI: https://doi.org/10.1016/j.forsciint.2017.11.004
Reference: FSI 9048

To appear in: FSI

Received date: 17-8-2017

Revised date: 31-10-2017
Accepted date: 4-11-2017

Please cite this article as: Hyerim Yu, Won-Jun Jang, Jung-Hee Jang,
Byoungduck Park, Young Ho Seo, Chul-Ho Jeong, Sooyeun Lee, Role
of hair pigmentation in drug incorporation into hair, Forensic Science
International https://doi.org/10.1016/j.forsciint.2017.11.004

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Role of hair pigmentation in drug incorporation into hair

Hyerim Yu#,1, Won-Jun Jang#,1, Jung-Hee Jang2, Byoungduck Park1, Young Ho Seo1,

Chul-Ho Jeong*,1, Sooyeun Lee**,1

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1
College of Pharmacy, Keimyung University, 1095 Dalgubeoldaero, Dalseo-gu, Daegu

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42601, Republic of Korea

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2
Department of Pharmacology, School of Medicine, Keimyung University, Daegu 704-

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701, Republic of Korea

* U
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Corresponding author. Tel.: +82 53 580 6638; Fax: +82 53 580 5164.
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E-mail: chjeong75@kmu.ac.kr (C.H. Jeong).
**
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Corresponding author. Tel.: +82 53 580 6651; fax: +82 53 580 5164.

E-mail: sylee21@kmu.ac.kr (S. Lee).

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#
These authors contributed equally.
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Highlights

 The role of hair pigmentation in drug incorporation into hair was investigated.
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 Rats carrying hair with different melanin status were used.

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 Human cells representing the main pigmentary unit in hair were used.
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 Hair pigmentation is a facilitating factor for the incorporation of basic

compounds.

 The cell study provided insight into the drug incorporation process into hair.

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Abstract

Hair analysis has notably expanded its application as a bio-monitor for drug or toxicant

exposure. Hair pigmentation is proposed as a major factor affecting drug incorporation

into hair; however, the mechanisms underlying the incorporation of drugs into hair are

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still unclear. In the present study, the effect of hair pigmentation on drug incorporation

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into hair was examined using rats carrying hair with different melanin status and human

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cells (SK-Mel-28 cells, HaCaT cells and the co-cultured HaCaT cells with SK-Mel-28

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cells) representing the main pigmentary unit in hair. Tramadol, a synthetic opioid

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analgesic, was selected as a model drug. The distribution of tramadol and its phase I (O-
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desmethyltramadol [ODMT], N-desmethyltramadol [NDMT] and N,O-
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didesmethyltramadol [NODMT]) and phase II metabolites (ODMT-glucuronide and
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NODMT-glucuronide) was investigated in non-pigmented and pigmented hair from

Long-Evans rats. Moreover, the incorporation levels of ODMT and ODMT-glucuronide

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were compared in hair cells. The concentrations of tramadol and its phase I metabolites

were significantly higher in pigmented rat hair while those of phase II metabolites did
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not showed any consistent significant difference depending on the status of hair

pigmentation. ODMT was taken up to a greater extent than ODMT-glucuronide by SK-

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Mel-28 cells, HaCaT cells and the co-cultured HaCaT cells with SK-Mel-28 cells.
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Notably, the incorporated level of ODMT was higher in SK-Mel-28 cells than HaCaT
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cells and the concentration difference of ODMT was significantly larger than that of

ODMT-glucuronide. This study clearly demonstrated that hair pigmentation played a

role as a facilitating factor for the incorporation of basic compounds and provided

insight into the drug incorporation process into hair.

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Keywords: Drug abuse; Hair analysis; Hair pigmentation; Hair cell; Melanin; Tramadol.

1. Introduction

Hair analysis has notably expanded its application as a bio-monitor. In particular,

quantitative results for drugs or toxicants and their metabolites from the sectional

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analysis of hair strands provide information on the time and extent of an individual’s

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drug exposure, considering that the growth rate of hair is approximately 1 cm/month [1]

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[2] [3] [4]. In the field of forensic toxicology, not only repetitive drug exposure but also

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single drug use in crimes are demonstrated using hair analysis [2]. Recently, clinical

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interest in hair analysis has also increased. The metabolizing capacity of CYP2D6
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during pregnancy was investigated using the ratios of metabolite to parent drug in hair
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by month in the clinical field [5]. The carcinogen (2-amino-1-methyl-6-
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phenylimidazo[4,5-b]pyridine) levels in hair was proposed as a biomarker for cancer

risk [6]. Hair cortisol and dehydroepiandrosterone to cortisol ratio were also measured
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as biomarkers for chronic stress among peoples living with human immunodeficiency

virus [7].
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The suggested routes of drug incorporation are passive diffusion from blood into hair
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follicle cells, incorporation from surrounding tissues, sweat or sebum, and exposure to
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external contamination [8]. Previous studies reported that hair pigmentation is a major
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factor affecting drug incorporation into hair; however, it was not demonstrated

definitely but only by comparing the concentrations of drugs and metabolites in

pigmented hair with those in non-pigmented hair in several human or animal studies [9]

[10] [11]. Hair pigmentation is occurred by transferring melanin granules from hair bulb

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melanocytes to cortical keratinocytes, which is closely related to the hair growth cycle.

Namely, melanin is transferred mainly during anagen to the hair shaft cortex by the

interactions of melanocytes and adjacent keratinocytes. However, a drug incorporation

process related to the melanocyte-keratinocyte interactions has never studied previously

[12].

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The lipophilicity and basicity of drugs were also suggested to influence their

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incorporation into hair from blood stream. Lipophilic and uncharged molecules can

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easily cross membranes and exist in matrix cells. However, hydrophilic compounds or

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ions are not permeable across these membranes. With regard to basicity, the relatively
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basic compounds were found to be more permeable because of the protonation and
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deprotonation process related to the intercellular pH in hair cells and the pKa of drugs
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[1].
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In the present study, the effect of hair pigmentation on drug incorporation into hair was

examined using rats carrying hair with different melanin status and human cells
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representing the main pigmentary unit in hair. Tramadol (Figure 1), a centrally acting

synthetic opioid analgesic, was selected as a model drug. An animal experiment

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together with a cell-based experiment clearly demonstrated the role of hair pigmentation
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in drug incorporation into hair.

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2. Material and methods

2.1. Chemicals

Tramadol powder, ODMT, NDMT (1 mg/mL for each), tramadol-13C, d3, ODMT-d6 and

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codeine glucuronide-d3 (100 g/mL for each) were purchased from Sigma-Aldrich (St.

Louis, MO, USA). The powder forms of NODMT, ODMT-glucuronide and NODMT-

glucuronide were provided by TLC PharmaChem (Vaughan, Ontario, Canada) and that

of NODMT-d3 was obtained Toronto Research Chemicals Inc. (Toronto, Ontario,

Canada). All solvents and chemicals for liquid chromatography tandem-mass

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spectrometry (LC-MS/MS) were high performance LC grade.

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2.2. Animal experiment

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All procedures were approved by the Institutional Animal Care and Use Committee of

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the Biohealth Convergence Center (Daegu, Republic of Korea). Twelve male Long-
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Evans rats (140-160 g, Osan, Gyeonggi-do, Republic of Korea), which have both non-
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pigmented and pigmented hair on the body, were randomly assigned to two groups (n=6
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for each single and multiple administration group) for intraperitoneal injection of 20

mg/kg of tramadol. Before drug administration, areas of non-pigmented and pigmented

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hair in the dorsal region were shaved separately using an electric shaver for use as hair

color-matched calibrators in the analysis. Beginning on day 3 after shaving, tramadol

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was administered once per day for 1 day and for 2 weeks to rats in the single and

multiple administration groups, respectively. Six weeks after the initial treatment, newly
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grown non-pigmented and pigmented hair was shaved and collected. All shaved hair
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was stored at room temperature until analysis.

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2.3. Cell culture experiment

ODMT and ODMT-glucuronide were selected as representative compounds to examine

the incorporation of compounds into hair cells. The human melanoma cell line, the SK-

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Mel-28 cells (American Type Culture Collection, Manassas, VA, USA), and

immortalized human keratinocyte cell line, the HaCaT cells (American Type Culture

Collection), were cultured in Dulbecco’s modification of Eagle medium supplemented

with 10% fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin at

37°C in a humidified incubator with 5% CO2. The cells were seeded onto sets of 3 wells

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on a 6-well plate at a density of 1 × 105 cells/well and 7 × 104 cells/well, respectively.

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The cells were then treated with ODMT (2 μg/mL) and ODMT-glucuronide (2 μg/mL).

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After incubation for 48 h, the cells were harvested and the concentrations of ODMT and

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ODMT-glucuronide were determined using LC-MS/MS.

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A co-culture of SK-Mel-28 and HaCaT cells (Figure 2) was also prepared in triplicate.

SK-Mel-28 cells were seeded in a 6-well insert at a density of 1×105 cells/well and
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incubated for 24 h. Cells were then treated with medium containing ODMT (2 μg/mL)
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and ODMT-glucuronide (2 μg/mL) in 10% FBS-DMEM and incubated for 24 h. The

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insert containing the SK-Mel-28 cells was transferred to a 6-well plate containing

HaCaT cells that had been seeded at a density of 7×104 cells/well and incubated for 24 h.
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The co-cultured cells were incubated for further 48 h before the HaCaT cells were

harvested. The additional incubation time of 48 h was chosen since melanin in the co-
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cultured HaCaT cells was clearly visualized using Fontana-Masson staining after 48 h
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(Figure 3). Finally, the HaCaT cells were harvested and the concentrations of ODMT
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and ODMT-glucuronide in the cells were determined using LC-MS/MS.

2.4. Sample preparation for rat hair

Rat hair samples were decontaminated with methanol followed by distilled water and

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methanol again and then was dried at room temperature. Next, the hair was cut into

lengths approximately less than 1 mm, weighed accurately (c.a. 10 mg) and incubated

for 16 h in 2 mL of methanol. Ten microliters of a mixture solution of deuterated

internal standards (1 g/mL) were added prior to incubation. The extract was

evaporated to dryness under a nitrogen flow at 40◦C and reconstituted in 100 µL of a

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mixture solution of methanol and mobile phase A (1:1). The reconstituted sample was

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filtered with a 0.45-µm polyvinylidenefluoride microporous membrane and injected into

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a LC-MS/MS system.

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2.5. LC-MS/MS analysis for rat hair
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The analysis of rat hair was conducted using a fully validated a column-switching LC-
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MS/MS method, as described in our previous study (under review) using a 1260 Infinity
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LC system and 6460 triple quadrupole MS/MS (Agilent Technologies, Santa Clara, CA,

USA) coupled with a 1260 Infinity extra binary pump and degasser (Agilent
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Technologies). Separation was conducted with the Atlantis T3 column (100 × 2.1 mm

i.d., particle size 3 μm; Waters, Milford, MA, USA) after on-line sample enrichment
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with the XBridge BEH HILIC Sentry Guard Cartridge 130Å (20 × 4.6 mm i.d., particle

size 3.5 μm; Waters). The mobile phase consisted of ammonium formate (5 mM) and
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formic acid (0.1%) in water (A) and formic acid (0.1%) in acetonitrile (B). The gradient
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conditions were as follows: 0-1 min, 2% (B); 1-16 min, 2-80 % (B); 16-17 min, 80-95%
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(B); 17-19 min, 95% (B); 19-20 min, 95-2% (B) at a flow rate of 300 μL/min. The

column and autosampler temperatures were set at 40◦C and 5◦C, respectively, and the

injection volume was 30 µL. The limit of quantification was 1 pg/mg for all analytes.

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The MS system was operated using electrospray ionization in the positive ion mode.

Quantification of the analytes were conducted using the selected reaction monitoring

(SRM) mode (Table 1). The MS/MS conditions were optimized as follows: capillary

voltage, 4.5 kV; nebulization pressure, 45 psi; drying gas temperature, 300°C; drying

gas flow, 7 L/min; temperature of sheath gas, 250°C; sheath gas flow, 11 L/min; nozzle

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voltage, 0.5 kV.

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2.6. Determination of ODMT and ODMT-glucuronide in cells

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Adherent cells were washed with phosphate buffered saline, collected with ice-cold

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0.9% sodium chloride solution and centrifuged (13,000 g, 4°C, 5 min). After the
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supernatant was removed, 1 mL of a mixture solution of acetonitrile, methanol and
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water (40:40:20) was added to each cell pellet. Following centrifugation (13,000 g, 10
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min, 4°C), the supernatant was evaporated under a stream of nitrogen at 40°C and the

residue was reconstituted in 100 μL of methanol and mobile phase A (1:1) solution. The
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reconstituted sample was analyzed using the same method described in the 2.5. section.

Calibration curves were prepared in the reconstituting solution.

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3. Results and Discussion

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3.1. Animal experiment

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The concentrations of tramadol and the phase I metabolites were significantly higher in
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pigmented hair compared with non-pigmented hair (p<0.05) in both the single and

multiple administration groups. The mean concentrations of tramadol, ODMT, NDMT

and NODMT in non-pigmented hair from the single administration group were 0.25,

0.04. 0.31 and 0.03 ng/mg, respectively, while those in pigmented hair from the single

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administration group were 3.07, 2.05, 4.03 and 0.34 ng/mg, respectively. The mean

concentrations of tramadol, ODMT, NDMT and NODMT in non-pigmented hair from

the multiple administration group were 1.64, 0.26. 3.07 and 0.14 ng/mg, respectively,

while those in pigmented hair from the multiple administration group were 28.2, 3.85,

42.0 and 1.31 ng/mg, respectively (Figure 4).

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The concentration of NODMT-glucuronide was significantly higher in non-pigmented

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hair than pigmented hair (p<0.01) in the single administration group. However, the

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concentrations of phase II metabolites did not showed any consistent significant

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difference depending on the status of hair pigmentation in the multiple administration
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group (Figure 4).
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The significantly higher concentrations of tramadol and its phase I metabolites in

pigmented hair from Long-Evans rats indicated that hair pigmentation is closely related
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to the incorporation of basic compounds into hair. For less basic compounds such as the

phase II metabolites, the primary incorporation route might be simple passive diffusion
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from the blood to hair matrix cells rather than a melanin-mediated process. Previous

studies also reported the same observation. In the comparison of amphetamine and its
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non-basic analogue, N-acetylamphetamine, the concentration of amphetamine was

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higher in pigmented hair (6.44±1.31 ng/mg) than in non-pigmented hair (2.04±0.58

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ng/mg) from Long-Evans rats. However, no significant difference in the N-

acetylamphetamine concentration was observed [13]. In another study, the

concentrations of methamphetamine and amphetamine in pigmented rat hair were 2-10-

fold higher than in non-pigmented hair [14]. It was also reported that the concentration

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of phenobarbital, a weak acid, was similar between non-pigmented hair and pigmented

hair, whereas that of codeine, a weak base, was 15-fold higher in pigmented hair than in

non-pigmented hair from Long-Evans rats [15]. Although hair pigmentation was

suggested as a major determinant of drug incorporation into hair, other factors require

evaluation in further studies as drugs were also detected in the hair of albino animals

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[16].

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3.2. Cell culture experiment

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Regardless of cell types, the concentration of ODMT was higher than that of ODMT-

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glucuronide (p<0.01) (Figure 5A). In the co-culture, the highest level of melanin
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transfer from SK-Mel-28 cells to HaCaT cells was observed 48 h after the co-culture
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was established (Figure 3). Both compounds were detectable in the 48 h co-cultured
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HaCaT cells and the concentrations of ODMT was higher than that of ODMT-

glucuronide (p<0.05) (Figure 5B).

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The incorporation of compounds from blood vessels into hair cells is affected by several
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factors, including physicochemical properties such as the lipophilicity and basicity of

the compound, molecular weight and concentration gradient in matrix cells [1]. In
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particular, the difference between pKa of the compound and pH of the hair matrix cell is
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an important factor for drug incorporation. Because of the acidity of the hair matrix cell,
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compared with plasma (pH 7.4) [17] [18], more basic substances are more permeable

through the membrane by deprotonation or protonation processes [1]. In our study, the

concentrations of ODMT, with a higher pKa (strongest acidic; 9.62, strongest basic;

8.97, http://www.hmdb.ca/, [19]), were higher than those of ODMT-glucuronide in both

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SK-Mel-28 cells and HaCaT cells. Moreover, ODMT-glucuronide is very hydrophilic,

existing as a zwitter ion, with two pKa-values (strongest acidic; 9.62, strongest basic;

8.97 for ODMT, strongest acidic; 3.21, strongest basic; -3.7 for gluduronic acid,

http://www.hmdb.ca/, [19]), in plasma and thus be difficult to incorporated into the hair

cells. Notably, the concentration difference of ODMT between two cell types (2.4 fold)

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was significantly larger than that of ODMT-glucuronide (1.4 fold). Our results

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demonstrated that hair pigmentation played a role as a facilitating factor for the

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incorporation of basic compounds.

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To further understand the drug incorporation process, we used a co-culture system of
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SK-Mel-28 cells and HaCaT cells to mimic a melanin transfer route. The direct
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inoculation of melanosome, melanosome exocytosis and endocytosis of keratinocytes
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and cytophagocytosis of melanocyte dendrites are suggested as major routes for

melanosome transfer from melanocytes to keratinocytes [20]. Although direct binding

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between melanin and the compounds was not investigated in this study, it was presumed

that drugs or metabolites are incorporated during melanosome transfer from

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melanocytes to keratinocytes as well as directly through melanocytes or keratinocytes in

hair.
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4. Conclusion
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The present findings will contribute to further understanding the of drug incorporation

into hair. By simultaneously investigating the distribution of tramadol and/or its phase I

and II metabolites in non-pigmented and pigmented rat hair and different types of hair

cells, we clearly showed that hair pigmentation played a role as a facilitating factor for

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the incorporation of basic compounds. This study also provided insight into the drug

incorporation process into hair, including melanosome transfer from melanocytes to

keratinocytes.

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Acknowledgements

This research was supported by the Bio & Medical Technology Development Program

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of the National Research Foundation (NRF) funded by the Ministry of Science, ICT &

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Future Planning (NRF-2015M3A9E1028327) and by the Basic Science Research

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Program of NRF funded by the Ministry of Education (NRF-2016R1A6A1A03011325).
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Figure legends

Figure 1. Tramadol and its main metabolites

CYP3A4, cytochrome P450 3A4; CYP2B6, cytochrome P450 2B6; CYP2D6,
cytochrome P450 2D6; UGT1A8, UDP-glucuronosyltransferase 1A8; UGT2B7, UDP-

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glucuronosyltransferase-2B7

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Figure 2. Preparation of a co-culture of SK-Mel-28 and HaCaT cells

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Figure 3. Fontana-Masson staining (×680) of HaCaT cells (A) and co-cultured

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HaCaT cells (B). Arrows designate melanin.

Figure 4. Concentrations of tramadol and its metabolites in non-pigmented and

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pigmented hair from rats in single (A) and multiple (B) tramadol administration
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groups (n=6 for each group, *p<0.05, **p<0.01). Error bars represent standard
error of the mean. The concentrations of phase II metabolites are displayed as
A
multiplying by 100 for better visualization.
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Figure 5. Concentrations of ODMT and ODMT-glucuronide in HaCaT (A), SK-

Mel-28 (A) and co-cultured HaCaT (B) cells. (n=3 for each, *p<0.05, **p<0.01).
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Error bars represent standard error of the mean.

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Table 1. Selected reaction monitoring (SRM) transitions, retention times for each
analyte and internal standard

Compound name SRM transition, m/z (CE, V) tR (min)

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Tramadol 264.1→58.2 (14) 8.7
ODMT 250.1→58.2 (17) 7.3

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NDMT 250.1→44.2 (10) 8.7

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NODMT 236.1→44.2 (12) 7.4
ODMT-glucuronide 426.1→58.2 (37) 6.4
NODMT-glucuronide
Tramadol-13C, d3
412.2→44.2 (22)
268.1→58.2 (16) U 6.5
8.6
N
ODMT-d6 256.1→64.2 (17) 7.3
A
NODMT-d3 239.1→47.2 (10) 7.4
Codeine glucuronide-d3 479.2→303.1 (29) 6.3
M

tR, retention time; CE, collision energy.

ED
E PT
CC
A

19