l-Hcy-thiolactone·HCl, d,l-Hcy, l-methionine, N-acetyl-l-cysteine (NAC), horse spleen ferritin, dithiothreitol (DTT), tris-(2-carboxyethyl)phosphine (TCEP), o-phthaldialdehyde (OPA), monosodium phosphate, NaOH, sodium citrate, NaCl, CuCl2, FeCl2, FeCl3, vitamin C, sodium dodecyl sulfate (SDS), chloroform, methanol, HCl, trichloroacetic acid, were purchased from Sigma-Aldrich. Suprapure nitric acid, 65% and perchloric acid, 60% were from Merck. Reagents were prepared in MiliQ purified water.
Unaltered head hair was obtained from healthy individuals (females, n = 51, 0.5—84 years old; males, n = 30, 1.25—68 years old) recruited from the Poznań population. Shed hair was collected during regular daily combing. Their base was identified by the presence of the hair bulb and only those hairs with the bulb were used in experiments. A 1- or 3-cm segment of the hair shaft (~ 2 mg hair from 15 to 30 individual hairs) was used to quantify Hcy content. The age of a hair segment, estimated from its distance from the scalp, assuming an average hair growth rate of 1 cm/month (Nissimov et al. 2007), is indicated in figures. The terms “young hair” and “old hair” or “aged hair” define segments of long hair at their base (close to the scalp) and at the tip, respectively.
Sample preparation for hair S-Hcy-keratin and Hcy-keratin assays
Samples were prepared by a modification of previously described procedures as described below. The inter-assay and intra-assay variabilities for the quantification of various forms of Hcy were 7.3 and 11.5% (Jakubowski 2002, 2008, 2016).
To liberate Hcy from S-Hcy-keratin, hair (~ 2 mg) were treated in a 0.5 mL Eppendorf polyethylene tube with a hot solution containing 50 mM Na2HPO4, 20 mM NaOH, 25 mM DTT, 1% SDS (200 μL, 65 °C, 1 h). The extract was collected by centrifugation, hair were similarly extracted with a fresh solution the second time, and the extracts combined (400 μL). Control experiments show that this procedure liberates > 95% Hcy present in hair S-Hcy-keratin. The extracts (40 μL) were treated with DTT (4 μL, 0.25 M) and HCl (4 μL, 12 N) on a heat block at 100 °C for 30 min to convert the liberated Hcy to Hcy-thiolactone, which was then quantified by HPLC (Jakubowski 2016). Authentic Hcy was similarly processed as a standard for S-Hcy assays.
Hair pellets after extraction were saved for SDS-insoluble N-Hcy-keratin quantification.
Extracts (300 μL) of SDS-soluble Hcy-keratin were supplemented with 34 μL 100% trichloroacetic acid to precipitate keratin. Protein precipitates were collected by centrifugation, transferred to 1-mL Wheaton Gold Band ampoules, and hydrolyzed in 6 N HCl, 50 mM DTT (110 μL, 120 °C, 1 h). The hydrolysates were lyophilized on Labconco CentriVap (40 min, 70 °C), dissolved in 10 μL water, purified by two-dimensional TLC, and analyzed by HPLC.
Hair pellets containing SDS-insoluble Hcy-keratin were transferred to 1-mL Wheaton Gold Band ampoules and hydrolyzed in 6 N HCl, 50 mM DTT (110 μL, 120 °C, 1 h). The hydrolysates were lyophilized on a Labconco CentriVap (40 min, 70 °C), dissolved in 10 μL water, purified by two-dimensional TLC, and analyzed by HPLC.
The term “total Hcy-keratin’ refers to the content of all keratin-bound Hcy that is solubilized by acid hydrolysis (i.e., Hcy-keratin + S-Hcy-keratin). Hair (~ 2 mg) were hydrolyzed in 1-mL Wheaton Gold Band ampoules containing 6 N HCl, 50 mM DTT (100 μL, 120 °C, 1 h). The hydrolysates were lyophilized using a Labconco CentriVap, dissolved in 100 μL 0.5 M K2HPO4 on ice, and Hcy-thiolactone was purified by two-dimensional TLC. Alternatively, in some experiments Hcy-thiolactone was purified by a 5-min extraction of ice-cold chloroform/methanol (2:1, v/v, 400 μL). Organic layer containing Hcy-thiolactone (bottom), separated by a 1-min microcentrifugation at 4 °C, was collected and re-extracted with 150 μL 0.1 N HCl. Aqueous layer containing Hcy-thiolactone (top) was dried on a Labconco CentriVap (40 min, 70 °C), dissolved in 100 μL deionized water, and analyzed by HPLC.
Horse spleen ferritin, containing 0.49 mol N-Hcy/mol protein, was processed in parallel as a standard for N-Hcy (Jakubowski 2008, 2016). These procedures quantitatively liberate Hcy from N-Hcy-protein and convert the liberated Hcy to Hcy-thiolactone, which is then quantified by HPLC (Jakubowski 2002, 2008, 2016).
HPLC, detection, and quantification
Quantification of Hcy-thiolactone generated from N-Hcy-keratin and/or S-Hcy-keratin was carried out by as previously described (Chwatko and Jakubowski 2005; Jakubowski 2008, 2016). Briefly, a Beckman-Coulter System Gold Nouveau HPLC instrumentation with a manual injector (7725i Rheodyne, with 0.1 mL loop) and a Jasco 1520 fluorescence detector were used. Chromatograms were analyzed using a Gold Nouveau chromatography workstation software for Windows. Samples were injected onto a cation-exchange poly-sulfoethyl aspartamide column (35 × 2 mm, 5 μm, 300 Å) (PolyLC, Inc.), eluted isocratically with 30 mM NaCl, 10 mM sodium phosphate buffer (pH 6.6) at a flow rate 0.6 mL/min. The effluent was mixed in a three-way tee with 2.5 mM OPA in 0.25 M NaOH, delivered at a flow rate 0.3 mL/min, the mixture passed through a reaction coil (Teflon tubing, 0.3 mm I.D. × 3 m), and the fluorescence at 480 nm was recorded (excitation 370 nm). Hcy-thiolactone eluted at 3 min and each run was completed in 4 min.
Met-keratin and Hcy-keratin assays
Hair (~ 10 mg) were hydrolyzed in 1-mL Wheaton Gold Band ampoules containing 6 N HCl (100 μL, 120 °C, 1 h). The hydrolysates were dried out, dissolved in 50 μL 0.2 M sodium phosphate buffer (pH 7.4), reduced with 2 μL 0.25 M TCEP for 10 min, and supplemented with 10 μL 0.5 M NAC. Met and Hcy liberated from hair keratin were quantified by HPLC as previously described (Borowczyk et al. 2016). Briefly, a Hewlett-Packard (Waldbronn, Germany) 1100 Series system, controlled by HP ChemStation software, containing quaternary pump, auto-sampler, temperature control, vacuum degasser, and 1260 Series FL detector was used. Samples were injected on a reversed-phase PRP-1 column (150 × 4.6 mm, 5 μm; Hamilton, Energy Way, Reno, NV, USA) eluted at a flow rate 1 mL/min, 25 °C, with 0.01 M OPA, 0.1 M L − 1 NaOH (A) and acetonitrile (B) as follows: 0–8 min, 14–25% (B); 8–12 min, 25% (B), 12–14 min, 25–14% (B). Met and Hcy were identified by co-elution with the authentic standards, monitored by fluorescence from 0 to 7.2 min, exc. 348 nm, em. 438 nm for Met, and from 7.2 to 14 min, ex. 370 nm, em. 480 nm for Hcy. Elution times for Met and Hcy were 6.2 and 9.8 min, respectively.
Iron and copper assays
Hair samples were mineralized with 65% nitric acid (Merck) in a microwave oven (Mars 5 Digestion Microwave System, CEM Corporation). Iron and copper were quantified by flame atomic absorption spectrometry using a Zeiss AAS-3 spectrometer with deuterium background correction as previously described (Suliburska 2011). The accuracy of the assay was 94% for iron and 102% for copper as verified by certified reference materials (Human Hair NCS DC73347a, LGC Standards).
Demethylation of free Met to Hcy
Model reactions were carried out at 37 °C in Na-citrate buffer, pH 6.0, containing Met, copper or iron chloride salts, and vitamin C in a final volume of 400 μL under conditions specified in figure legends. Hcy generated from methionine was assayed by the conversion to Hcy-thiolactone, which was quantified by HPLC as previously described (Jakubowski 2016). Briefly, 5 μL aliquots of reaction mixtures were treated with 5 μL DTT, 10 μL water, and 10 μL 3 N HCl at 100 °C for 20 min. The assay mixtures were dried on a Labconco CentriVap, dissolved in 100 μL water, and analyzed by cation exchange HPLC. Authentic Hcy samples were similarly processed as standards. The inter-assay and intra-assay variabilities were 4.7 and 6.8% for Hcy, and 1.0 and 3.4% for Met, respectively.
The results are reported as mean ± standard deviation. Comparisons between two groups are analyzed by using two-sided Student’s test. Relationships between Hcy-keratin and hair age or metal content were fitted to exponential equations and analyzed by linear regression. The level of statistical significance was set to P < 0.05. Analyses were carried out by using Sigma Plot software.