A Comparison of Sodium Hydroxide and Sodium Sulfide Digestion

Journal of Analytical Toxicology, Vol. 24, January/February 2000

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A Comparison of Sodium Hydroxide and Sodium Sulfide Digestion of Mouse Hair in the Recovery of Radioactivity Following Systemic Administration of [3H]-Nicotine and [3H]-Flunitrazepam

David J. Claffey, Peter R. Stout, and James A. Ruth* University of Colorado Health Sciences Center, Molecular Toxicology and Environmental Health Sciences Program, 4200 East Ninth Avenue, Box 0238, Denver, Colorado 80262

I Abstract [ Pigmented (C57BI) and nonpigmented (balb/c) mice, 25 days of age, were treated intraperitoneally with [aH]-nicotine (4 mg/kg, 555 dpm/ng) or [3H]-flunitrazepam (1 mg/kg, 2200 dpm/ng) daily for three days. After 21 days, shaved back hair was digested at 37~ for 24 h with either 1M sodium hydroxide or 1M sodium sulfide. With both drugs, sodium sulfide extraction removed the same amount of radioactivity as sodium hydroxide from nonpigmented hair. However, sodium sulfide removed significantly more radioactivity from pigmented hair than did sodium hydroxide. In pigmented hair, sodium sulfide solubilized 35% and 74% of the flunitrazepam- and nicotine-associated radioactivity, respectively. Of this, 12% and 43%, respectively, could be partitioned into ethyl acetate. Microscopic examination of residual pellets after digestion demonstrated a more thorough dissolution of the hair shaft with sodium sulfide with only melanosomes remaining. The results demonstrate the significant interaction of flunitrazepam and nicotine with melanins and the utility of sodium sulfide in increasing drug recovery.

Introduction

We recently reported that radiolabeled serum constituents such as 45Ca 2§ 3601-, [14C]-urea, and [35S]-cysteine deposited into mouse hair after systemic administration are remarkably stable to 24-h pH 6 phosphate buffer extraction, with a significant portion of the radioactivity in each case remaining with the hair shaft (1). We have also demonstrated that after systemic administration of [3H]-cocaine, [aH]-nicotine, and [.~H]-flunitrazepam to pigmented mice, only a small portion of the radioactivity deposited in the hair could be removed by methanol wash, extended phosphate buffer extraction, or en-

�9 Aulhor to whom corresp<)ndence should be addressed. Email [email protected].

zymatic digestion of the hair (< 0.4 ng/mg of pigmented hair < 0.1 ng/mg of nonpigmented hair) (2). Digestion of the hair i~ 1M sodium hydroxide was required to solubilize significa~ t radioactivity. Of the radioactivity in solution, which shoul~ represent free drug in the case of nicotine and flunitrazepa~ no more than 43% could be extracted into ethyl acetate. TI~ data reflect the strong interaction of drugs with melanin cop ponents in hair and in partially solubilized hair. Numero~ other authors have provided evidence for such strong dru~ melanin interactions in vitro (3-10).

In an effort to determine a method for maximal recovery 0 base-stable drugs from hair matrix, sodium sulfide was exa~ ined as an agent for protein digestion. Sodium sulfide, lil~ sodium hydroxide, provides a very basic solution for protein h~ drolysis. It also has the ability to disrupt protein disulfi~ bonds that crosslink structural keratins (11) and possibly pr0 tein components in melanosomes that can occlude deposit~ drugs. In this study, both sodium hydroxide and sodium sulfid were employed to digest hair from pigmented and nonpil mented mice following treatment with radiolabeled nicoti~ and flunitrazepam. The use of radioactive drugs allows for. complete mass balance of deposited drug to be establishJ This is especially valuable in the measurement of drug that m~ have been sequestered within digest residues. Twenty-five-d~ old mice were employed because they are entering a second riod of synchronized hair growth (12). Both drugs used in till study are of current interest: nicotine poses a general heal~ risk and flunitrazepam is used as a date-rape drug. Both a~ generally chemically stable to aqueous base.

Materials and Methods

Male balb/c (nonpigmented) and C57B1 (eumelanin-pil mented) mice, 23 days old, were purchased from Jackson L~

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oratories (Bar Harbor, ME) and maintained on a 12-h light/dark cycle with food and water ad libitum in the UCHSC Animal Care Facility, an AALAC-approved facility. For radiotracer studies, the animals were caged separately in biohazard-isola- tion cabinets. [Methyl-3H]-flunitrazepam (87 Ci/mmol) and [N-methyl-3H]-nicotine (85 Ci/mmol) were purchased from New England Nuclear Corp. (Boston, MA). [3H]-Toluene (2.36 x 106 dpm/mL) was manufactured by Packard Instrument Co. (Downers Grove, IL). Inorganic chemicals (Aldrich Chemical Co., Milwaukee, WI) were ACS grade.

Nicotine was prepared for injection in 0.9% saline. Fluni- trazepam was prepared in water/propylene glycol/dimethyl sul- foxide (49.5%:49.5%:1%, v/v/v). Final desired drug concentrations were adjusted to a nontoxic, pharmacologi- cally active range (13,14) with unlabeled compounds and in- jected intraperitoneally daily for three days to four mice of each strain. Nicotine was administered at 4 mg/kg (555 dpm/ng) (13), and flunitrazepam was administered at I mg/kg (2200 dpm/ng) (14). Each drug was administered to four mice of each strain in separate experiments. After 21 days the animals were sacrificed by exposure to carbon dioxide. Hair was shaved from the backs with electric clippers.

Hair samples (10 rag) were placed in I mL of 1M sodium hydroxide or 1 mL of 1M sodium sulfide solution. The sodium hydroxide and sodium sulfide digests were allowed to stand at 37~ for 24 h with periodic shaking for the first 3 h. The digests Were centrifuged at 14,000 rpm for 10 rain, and the radioactivity in the supernatant and pellet fractions was determined by liquid scintillation counting using Fisher Scintisafe Plus TM

50% scintillation cocktail and a Packard 1500 liquid scintillation COunter. All radioactivity measurements were corrected for quenching using drug-free hair controls and tritiated toluene (10 IJL). Counting efficiency was in the order of 5% for the melanosome fractions and 20% for the supernatant fractions. In separate experiments, the sodium hydroxide and sodium sulfide digests were extracted with ethyl acetate followed by liquid Scintillation counting of the organic phase to determine the partitioning of radiolabeled species.

Pellets were prepared for light microscopy by mounting a 5-mL aliquot of each sample on a slide with Permount. Spec-

0.3

0.25

0.2- [ ~Sodium sulil~ didst ;~0.15 ; i �9 Sodium hydroxide �9 ; digest

0.11 : . . . . . . . . . . . .

0,05

0 Flunltrazepam Nicotine

Figure 1. Comparison of the total amount of drug-associated radioactivity extractable (mean + SEM, n = 4) from nonpigmented (balb/c) mouse hair for each of the digestion methods

imens were examined at 600- or 1200-fold magnification and photographed on a Nikon Microphot-FX microscope.

Statistical analysis of data was accomplished using a two- sample Student t-test assuming equal variance. For statistical tests, the r level was set at 0.01.

0 �9

7-

6-

44

3

2

1

0

[] Sodium sulfide digest

,(, �9 Sodium hydroxide digest

T

Flunltrazepam Nicotine

Figure 2. Comparison of the total amount of drug-associated radioactivity extractable (mean + SEM, n = 4) from pigmented (C57BI) mouse hair for each of the digestion methods. (* indicates sodium sulfide significantly dif- ferent from sodium hydroxide at p < 0.01 level)

0, f . . . . . . . . . 1 0.35 . . . . . . . . . . . . . . . . . .

0.3 t . . . . . . . . . . . . . . ~0.25 . . . . . . . . . . . . _ Z _ _ _ ~ . _ ~ Z Fill Sodium eulfid e

~ 0.2]- . . . . . . . . ~ l ~ i [ extract ] . . . . . . . . . . - W - - F " ' "

0.1 t . . . . . . . . . o.o . . . . . . . . . . .

Flunltrozepam Nicotine

Figure 3. Distribution (mean • SEM, n = 4) of drug-associated radioactivity between the pellet and sodium sulfide solution after digestion of nonpigmented (balb/c) mouse hair.

5 -

5

4 - �9 sulfide 1 extract

3 �9 Pell{ t _J

2

1

0 Flunltrazepam Nicotine

Figure 4. Distribution (mean + SEM, n = 4) of drug-associated radioactivity between the pellet and sodium sulfide solution after digestion of pigmented (C57BI) mouse hair.

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Results

The removal of radioactivity associated with nicotine and flunitrazepam from hair from nonpigmented mice by sodium hydroxide and sodium sulfide digestion is shown in Figure 1. The two digestion methods did not significantly differ in the amount of drug-associated radioactivity solubilized from nonpigmented hair, with approximately 0.02 ng of flunitrazepam and 0.2 ng of nicotine per milligram of hair being extracted. However, for pigmented hair, sodium sulfide digestion solubilized approximately fivefold more radioactivity for each drug than sodium hydroxide (Figure 2). The total levels of each drug obtained from pigmented hair were over 10-fold greater than those obtained from nonpigmented.

Figure 3 illustrates the extent of solubilization of radioactivity by sodium sulfide digestion of hair from nonpigmented mice. With flunitrazepam, approximately 85% of the radioactivity in the hair was present in the sodium sulfide digest solution. More than 95% of the radioactivity associated with nicotine was extractable by sodium sulfide. However, consid-


erably less drug was solubiiized from pigmented hair: 74% for nicotine and 35% for flunitrazepam (Figure 4).

Organic extracts of the basic digests were made in an effort to determine the extent to which the solubilized drugs could be partitioned into ethyl acetate. As Figure 5 demonstrates, vir- tually no flunitrazepam-associated radioactivity and only 15% of solubilized nicotine-associated radioactivity in nonpig- rnented hair could be extracted into ethyl acetate. In Figure 6, it can be seen that 12% of flunitrazepam-associated radioactivity and 43% of nicotine-associated radioactivity could be extracted into ethyl acetate from the sodium sulfide digests of hair of pigmented animals.

Figure 7 shows the results of light microscopy studies on pigmented hair sediments fo|iowing digestion. In Figure 7A, an untreated hair from a control C57 (pigmented) mouse is shown. Desiccated medullary cells (m) containing melanosomes

0.9,

0"6 1-5 ~ - ~ ~0. I ocetate wash

~0.4 [ mSodlum -,ulflde aoluUon

0.3

0.2

0.1 ~ L ~

FlunRrazepam Nlcotlns

Figure N. Amount of drug-associated radioactivity extradable (mean • SEM, n = 3) with ethy$ acetate from the isolated sodium sulfide solution after digestion of nonpigmented (balb/c) mouse hair.

3

2

4 ) �9 Ethyl acetate wash

�9 Sodium oulflde soluUon

Flunltrazepam NlcoUne Figure 6. Amount of drug-associated radioactivity extractable (mean + SEM, n = 3) with ethyl acetate from the isolated sodium sulfide solution after digestion of pigmented (C57BI) mouse hair.

Figure 7. A, Light microscopic image (600x) of normal pigmented (C57BI) mouse hair. Bar represents a distance of 50 ram. "m" indicates the remains of medullary cells, dried as the hair shaft develops. "As" indicates air space between medullary cells. B, Light microscopic image (600x) of the residue following sodium hydroxide digestion of pigmented (C57BI) mouse hair. C, Light microscopic image (1200x) of the residue following sodium sulfide digestion of pigmented (C57BI) mouse hair.

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and air spaces (As) are marked with arrows. In Figure 7B, the residue from sodium hydroxide digestion of hair from C57 mice is shown. Cell structures are no longer visible, but tightly aSsociated melanosomes are seen. In Figure 7C, the residual material following sodium sulfide digestion of C57 hair is shown. Only loosely associated melanosomes can be found.

Discussion

The age and strain of mice used in this study afford an ex- cellent model for comparison of the effectiveness of sodium hy- Adroxide and sodium sulfide in the recovery of drugs from hair. t 23 days of age, mice undergo a synchronized period of an-

agen (12). This ensures that hair was growing at the time of dosage, allowing for maximal drug deposition. The two strains of mice differ in pigmentation. C57 mice are predominately eumelanin pigmented with little pheomelanin (12), whereas balb/c mice are nonpigmented. This has allowed for the com- Parison of pigment effects on deposition. The role of pigmentation in the accumulation of drugs in hair is a widely observed phenomenon (2,6,9,15-20). This study has backed-up these ~ ndings, with between 12- and 150-fold more radioactivi~ eing recovered from pigmented hair than from nonpigmented

hair. The use of isotopes in this study allows an exact mass balance

of drug in extracts and hair residues to be established, but does not easily allow a quantitative determination of the distribution of parent drug and metabolites deposited in the hair. However, it has been demonstrated that nicotine is de- Posited in hair mainly as unmetabolized parent drug (21). We have confirmed this by autoradiographic demonstration of nicotine and flunitrazepam deposition in melanosomes of hair follicles within 10 rain of systemic administration to a pigmented animal (2). The deposition would thus most likely represent parent drug because this is very early in the metabolic time course of both drugs. Cirirnele et al. (22) sug- gested that the metabolite 7-aminoflunitrazepam is deposited in hair at higher concentrations than flunitrazepan~. Their results were obtained by gas chromatography-mass spectrometry measurements and may reflect a differential extent of extraction of parent drug and metabolite from hair. The labeling Pattern of the isotope we employed would not distinguish flunitrazepam and 7-aminoflunitrazepam, and both may be Present. However, other authors (23) have noted that concentrations of parent drug in hair often exceed metabolite concentrations.

The inability of sodium hydroxide and sodium sulfide to COmpletely solubilize drug-associated radioactivity from hair in Pigmented mice is consistent with a tight association of the drugs with melanin. Both compounds result in a highly basic Solution, which can support protein hydrolysis. The enhanced ability of sodium sulfide to solubilize deposited radioactivity ap- Pears consistent with the additional chemical reactivity of sodium sulfide toward disulfide crosslinks in proteins (11). This differential action is supported by microscopic examination of hair residues following digestion. The tightly packed as-

sociation of melanosomes in a tubular array following sodium hydroxide digestion suggests the presence of an intervening protein matrix retaining the shape of a hair. The freely dis- persed melanosomes following sodium sulfide digestion are consistent with a much more thorough dissolution of the hair matrix.

The inability of ethyl acetate extraction to remove solubilized radioactivity from the sodium sulfide digest is consistent with drug association, possibly covalent, with degraded water-soluble melanin components. The conversion of the drugs to acidic derivatives water soluble at pH 12 either chemically or metabolically is also a possibility.

The results of this study demonstrate that sodium sulfide digestion is much more efficient in solubilization of deposited drug-associated radioactivity from pigmented hair than is sodium hydroxide extraction. However, a significant portion of the released compounds may still be chemically associated with melanin fragments. This study indicates that sodium sulfide digestion of hair is a more efficient method of recovery of base-stable drugs from hair than the commonly used sodium hydroxide digest.

Acknowledgment

This work was supported by NIH grant DA09545.

References

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19. R.W. Reid, F.L. O'Connor, A.G. Deakin, D.M. Ivery, and J.W. Crayton. Cocaine and metabolites in human graying hair: pigment relationship. J. Toxicol. Clin. Toxicol. 34" 685-690 (1996).

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Manuscript received February 19, 1999; revision received April 27, 1999.

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A Comparison of Sodium Hydroxide and Sodium Sulfide Digestion

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