The Effect of Hair Color on the Incorporation of Codeine into Human Hair

The influence of melanin on the binding of xenobiotics in hair will impact the interpretation of drug concentrations determined by hair testing. The purpose of this study was to determine if codeine, as a model compound of abused drugs, would be incorporated into black, brown, blond, or red hair as a function of melanin concentration. Such data would assist in the interpretation of codeine concentrations in hair and help elucidate the potential influence of hair color on incorporation of drugs. Male and female Caucasians with black (n = 6), brown (n = 12), blond (n = 8), or red hair (n = 6) and non-Caucasians with black hair (n = 12) aged 21-40 years were enrolled in the study. Each subject was administered oral codeine phosphate syrup in a dosage of 30 mg three times a day for five days. Twenty-four hours after the end of the treatment period, a 30-mg codeine dose was administered and the subject's plasma area under the concentration time curve (AUC) for codeine was determined. Codeine and melanin were measured in the first 3 cm of hair closest to the vertex region of the scalp prior to and 1, 4, 5, 6, and 7 weeks after dosing. The quantitative and qualitative melanin profiles were determined for each subjects hair to provide an objective measure of hair color. The plasma concentrations of codeine were measured to eliminate differences in the bioavailability and clearance of codeine as factors that might account for the differences in codeine hair concentrations. The subjects were asked not to cut their hair in the vertex region of the scalp or to use any form of chemical treatment on their hair, but otherwise normal hygienic measures were permitted. The mean (± SE) hair codeine concentrations 5 weeks after dosing were 1429 (± 249) pg/mg in black hair; 208 (± 17) pg/mg in brown hair; 99 (± 10) pg/mg in blond hair; and 69 (± 11) in red hair pg/mg. In black hair, codeine concentrations were 2564 (± 170) pg/mg for Asians and 865 (± 162) pg/mg for Caucasians. Similar concentration relationships were observed at weeks 4, 6, and 7. A strong relationship between the hair concentrations of codeine and melanin (R2 = 0.73) was observed. Normalization of the codeine concentration with the melanin concentration reduced the hair color differences observed. These data demonstrate that the interpretation and reporting of hair test results for codeine are influenced by hair color. After this dosing protocol, the proposed federal guideline cutoff of 200 pg/mg of codeine would result in 100% of subjects with black hair and 50% of subjects with brown hair being reported as positive, and subjects with blond or red hair would be reported as negative. The incorporation of these drugs into hair should be studied carefully in humans to ensure the appropriate interpretation of drug concentration

A Novel Model of Intravital Platelet Imaging Using CD41-ZsGreen1 Transgenic Rats

<div><p>Platelets play pivotal roles in both hemostasis and thrombosis. Although models of intravital platelet imaging are available for thrombosis studies in mice, few are available for rat studies. The present effort aimed to generate fluorescent platelets in rats and assess their dynamics in a rat model of arterial injury. We generated CD41-ZsGreen1 transgenic rats, in which green fluorescence protein ZsGreen1 was expressed specifically in megakaryocytes and thus platelets. The transgenic rats exhibited normal hematological and biochemical values with the exception of body weight and erythroid parameters, which were slightly lower than those of wild-type rats. Platelet aggregation, induced by 20 μM ADP and 10 μg/ml collagen, and blood clotting times were not significantly different between transgenic and wild-type rats. Saphenous arteries of transgenic rats were injured with 10% FeCl₃, and the formation of fluorescent thrombi was evaluated using confocal microscopy. FeCl₃ caused time-dependent increases in the mean fluorescence intensity of injured arteries of vehicle-treated rats. Prasugrel (3 mg/kg, p.o.), administered 2 h before FeCl₃, significantly inhibited fluorescence compared with vehicle-treated rats (4.5 ± 0.4 vs. 14.9 ± 2.4 arbitrary fluorescence units at 30 min, respectively, n = 8, <i>P</i> = 0.0037). These data indicate that CD41-ZsGreen1 transgenic rats represent a useful model for intravital imaging of platelet-mediated thrombus formation and the evaluation of antithrombotic agents.</p></div

Biochemical parameters of CD41-ZsGreen1 transgenic rats.

<p>Biochemical parameters of CD41-ZsGreen1 transgenic rats.</p

Representative images of platelet-rich thrombus induced by 10% FeCl₃ in the saphenous artery of CD41-ZsGreen1 transgenic rats 30 min after FeCl₃ application.

<p>Male CD41-ZsGreen1 transgenic rats were anesthetized, the left saphenous artery was exposed, and a filter paper, presoaked with 10% FeCl₃ was attached for 3 min to induce thrombosis 2 h after administration of vehicle or prasugrel. Fluorescence was measured every 1 s for 25 min using a confocal laser microscope.</p

Analysis of the transgene structure in the genome of CD41-ZsGreen1 transgenic rats.

<p>(A) Schematic of transgene constructs and hybridization probe for Southern blot analysis. The start codon of <i>Cd41</i> was replaced by the ZsGreen1 fragment. (B) Southern blot analysis of tail DNA samples of CD41-ZsGreen1 transgenic rats. Genomic DNA isolated from the tail was digested with PstI, electrophoresed through an agarose gel, and transferred to a nylon membrane. The nylon membrane was hybridized to the ZsGreen1 probe to detect the 2.1-kb restriction fragment.</p

Representative ADP- and collagen-induced platelet aggregation tracings of CD41-ZsGreen1 transgenic rats.

<p>Citrated blood was collected from the abdominal aorta and centrifuged to obtain platelet-rich plasma. Platelet aggregation induced by ADP (20 μM) and collagen (10 μg/mL) was measured using light transmission aggregometry. WT: wild-type Sprague-Dawley rats, TG: CD41-ZsGreen1 transgenic rats, ADP: adenosine 5’-diphosphate.</p

Specific expression of green fluorescent protein in bone marrow megakaryocytes of CD41-ZsGreen1 transgenic rats.

<p>Bone marrow from the femur was excised, fixed with 10% buffered formalin, and embedded in paraffin. Sections were prepared and stained with DAPI or HE. Arrows in the HE photograph indicate megakaryocytes. WT: wild-type Sprague-Dawley rats, TG: CD41-ZsGreen1 transgenic rats, HE: hematoxylin-eosin, DAPI: 4′,6-diamidino-2-phenylindole.</p

Representative flow cytometric analysis of CD41-ZsGreen1 transgenic rats.

<p>EDTA-treated blood samples were withdrawn from the abdominal vein for flow cytometric analysis. The blood was mixed with phycoerythrin-labeled hamster anti-mouse CD61 antibody. Flow cytometric analyses were performed using a FACSCalibur. M1: ZsGreen1-positive platelets. WT: wild-type Sprague-Dawley rats, TG: CD41-ZsGreen1 transgenic rats.</p

Hematological parameters of CD41-ZsGreen1 transgenic rats.

<p>Hematological parameters of CD41-ZsGreen1 transgenic rats.</p