Abstract

7-dehydrocholesterol (7-DHC) and cholesterol (CHOL) are biomarkers of Smith-Lemli-Opitz Syndrome (SLOS), a congenital autosomal recessive disorder characterized by elevated 7-DHC level in patients. Hair samples have been shown to have great diagnostic and research value, which has long been neglected in the SLOS field. In this study, we sought to investigate the feasibility of using hair for SLOS diagnosis. In the presence of antioxidants (2,6-ditert-butyl-4-methylphenol and triphenylphosphine), hair samples were completely pulverized and extracted by micro-pulverized extraction in alkaline solution or in n-hexane. After microwave-assisted derivatization with N,O-Bis(trimethylsilyl)trifluoroacetamide, the analytes were measured by GC-MS. We found that the limits of determination for 7-DHC and CHOL were 10 ng/mg and 8 ng/mg, respectively. In addition, good linearity was obtained in the range of 50–4000 ng/mg and 30–6000 ng/mg for 7-DHC and CHOL, respectively, which fully meets the requirement for SLOS diagnosis and related research. Finally, by applying the proposed method to real hair samples collected from 14 healthy infants and two suspected SLOS patients, we confirmed the feasibility of hair analysis as a diagnostic tool for SLOS. In conclusion, we present an optimized and validated analytical method for the simultaneous determination of two SLOS biomarkers using human hair.

Graphical abstractSupplementary key wordsAbbreviations: 7-DHC (7-dehydrocholesterol), BHT (2,6-Di-tert-butyl-4-methylphenol), CHOL (cholesterol), DHCR7 (7-dehydrocholesterol reductase), KOH (Potassium hydroxide), LLE (liquid-liquid extraction), LLOQ (lower limit of quantification), LOD (limit of detection), MPE (Micro-pulverized extraction), RSD (relative standard deviation), SLOS (Smith-Lemli-Opitz syndrome), TFA (trifluoroacetic acid), TPP (triphenylphosphine)Cholesterol (CHOL) is a ubiquitous biomolecule for human and animal function serving as an utmost important precursor of steroid hormones, bile acids, and cell membrane (1Schade D.S. Shey L. Eaton R.P. Cholesterol review: a metabolically important molecule.). 7-dehydrocholesterol (7-DHC) is the penultimate metabolite in the Kandustch-Russell pathway for CHOL synthesis, catalyzed by an enzyme, 7-dehydrocholesterol reductase (DHCR7) (2DeBarber A.E. Eroglu Y. Merkens L.S. Pappu A.S. Steiner R.D. Smith-Lemli-Opitz syndrome.).Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive disorder of CHOL biosynthesis, caused by the deficiency of DHCR7. Consequently, high 7-DHC plasma levels (at least 10 times higher than healthy individuals) and low CHOL plasma levels (59–1790 μg/ml of SLOS patients versus 429–2743 μg/ml of healthy children (3Diagnosis of Smith-Lemli-Opitz syndrome by gas chromatography/mass spectrometry of 7-dehydrocholesterol in plasma, amniotic fluid and cultured skin fibroblasts.)) were described as a characteristic biochemical manifestation and the diagnostic criteria of SLOS (4Smith D.W. Lemli L. Opitz J.M. A newly recognized syndrome of multiple congenital anomalies., 5Smith-Lemli-Opitz syndrome: pathogenesis, diagnosis and management.). Due to the irreplaceable role of CHOL, wide-ranging and even fatal clinical symptoms are frequently reported, including polydactyly, behavioral and cognitive deficits, specific facial features, organ growth retardation, and immune and endocrine malfunction (5Smith-Lemli-Opitz syndrome: pathogenesis, diagnosis and management., 6Smith-Lemli-Opitz syndrome: phenotype, natural history, and epidemiology.). Various phenotypes also bring great difficulty to the clinical suspicion and diagnosis of SLOS. SLOS incidence rate ranges from 1/20,000 to 1/60,000, with the highest prevalence in the population of European descent, and the carrier frequency is 1%–2% (6Smith-Lemli-Opitz syndrome: phenotype, natural history, and epidemiology., 7Lazarin G.A. Haque I.S. Evans E.A. Goldberg J.D. Smith-Lemli-Opitz syndrome carrier frequency and estimates of in utero mortality rates.). With the increasing number of cases reported in Asia in recent years (8Gao C. Duan J. Zhang P. Gao Y. Zhang Y. Wang Y. et al.[Clinical and genetic analysis of a Chinese pedigree affected with Smith-Lemli-Opitz syndrome]., 9Tamura M. Isojima T. Kasama T. Mafune R. Shimoda K. Yasudo H. et al.Novel DHCR7 mutation in a case of Smith-Lemli-Opitz syndrome showing 46,XY disorder of sex development.), SLOS may be more common than originally thought (10Nowaczyk M.J. Waye J.S. Douketis J.D. DHCR7 mutation carrier rates and prevalence of the RSH/Smith-Lemli-Opitz syndrome: where are the patients?.).Early intervention and treatment can improve the condition of newborns (11Donoghue S.E. Pitt J.J. Boneh A. White S.M. Smith-Lemli-Opitz syndrome: clinical and biochemical correlates.), so it is critical to have an efficient and simple diagnostic method. The metabolome of conventional matrices (i.e., plasma, serum) sometimes can be dynamic, resulting in a relatively unstable composition depending on maternal condition, dietary or circadian variations. In contrast to the conventional matrices (i.e., plasma, serum), hair, as a complementary matrix, can reflect the biochemical level of infants in the prenatal stage and avoid the interference of diet after birth on the analysis results (12Sallabi S.M. Alhmoudi A. Alshekaili M. Shah I. Determination of Vitamin B3 Vitamer (Nicotinamide) and Vitamin B6 Vitamers in human hair using LC-MS/MS.). This is because once analytes are incorporated into hair, they can remain unchanged without fluctuations in content caused by degradation or metabolism (13Suwannachom N. Thananchai T. Junkuy A. O'Brien T.E. Sribanditmongkol P. Duration of detection of methamphetamine in hair after abstinence.). Hair can also be simply stored for a long time at room temperature and no strict anticorrosion measures are required during preservation or transportation procedure (12Sallabi S.M. Alhmoudi A. Alshekaili M. Shah I. Determination of Vitamin B3 Vitamer (Nicotinamide) and Vitamin B6 Vitamers in human hair using LC-MS/MS., 14Gottardo R. Fanigliulo A. Sorio D. Liotta E. Bortolotti F. Tagliaro F. Monitoring compliance to therapy during addiction treatments by means of hair analysis for drugs and drug metabolites using capillary zone electrophoresis coupled to time-of-flight mass spectrometry.). The sample collection of hair can be convenient (without the need of trained professionals) and less invasive (12Sallabi S.M. Alhmoudi A. Alshekaili M. Shah I. Determination of Vitamin B3 Vitamer (Nicotinamide) and Vitamin B6 Vitamers in human hair using LC-MS/MS.), which is more friendly to newborns, especially those who are sick. Moreover, the analysis of hair has a much wider window of detection and could show the presence of analytes in individual’s body from a few weeks up to a year (12Sallabi S.M. Alhmoudi A. Alshekaili M. Shah I. Determination of Vitamin B3 Vitamer (Nicotinamide) and Vitamin B6 Vitamers in human hair using LC-MS/MS.), which is meaningful for the biochemical level monitoring of some children and adult patients.GC-MS has been reported to be a tool for diagnosis of SLOS and other cholesterolopathies, which benefits from its excellent peak resolution, no influence of matrix effects on ionization, higher chromatographic efficiency, and relatively lower cost of instrument (15Jezela-Stanek A. Siejka A. Kowalska E.M. Hosiawa V. Krajewska-Walasek M. GC-MS as a tool for reliable non-invasive prenatal diagnosis of Smith-Lemli-Opitz syndrome but essential also for other cholesterolopathies verification., 16Lutjohann D. Bjorkhem I. Friedrichs S. Kerksiek A. Lovgren-Sandblom A. Geilenkeuser W.J. et al.First international descriptive and interventional survey for cholesterol and non-cholesterol sterol determination by gas- and liquid-chromatography-Urgent need for harmonisation of analytical methods.). However, the published analytical methods for CHOL in hair still have a series of problems such as nonstandardized hair weighing and homogenization (17Serra M. Matabosch X. Ying L. Watson G. Shackleton C. Hair and skin sterols in normal mice and those with deficient dehydrosterol reductase (DHCR7), the enzyme associated with Smith-Lemli-Opitz syndrome., 18Masukawa Y. Tsujimura H. Imokawa G. A systematic method for the sensitive and specific determination of hair lipids in combination with chromatography.), time-consuming extraction step (e.g., overnight soaking, 16 h ultrasound etc.) (17Serra M. Matabosch X. Ying L. Watson G. Shackleton C. Hair and skin sterols in normal mice and those with deficient dehydrosterol reductase (DHCR7), the enzyme associated with Smith-Lemli-Opitz syndrome., 19Son H.H. Lee D.Y. Seo H.S. Jeong J. Moon J.Y. Lee J.E. et al.Hair sterol signatures coupled to multivariate data analysis reveal an increased 7beta-hydroxycholesterol production in cognitive impairment., 20Ryu H.K. Jung B.H. Kim K.M. Yoo E.A. Woo J.T. Chung B.C. Determination of cholesterol in human hair using gas chromatography-mass spectrometry.), lack of necessary antioxidation measures (17Serra M. Matabosch X. Ying L. Watson G. Shackleton C. Hair and skin sterols in normal mice and those with deficient dehydrosterol reductase (DHCR7), the enzyme associated with Smith-Lemli-Opitz syndrome., 18Masukawa Y. Tsujimura H. Imokawa G. A systematic method for the sensitive and specific determination of hair lipids in combination with chromatography., 19Son H.H. Lee D.Y. Seo H.S. Jeong J. Moon J.Y. Lee J.E. et al.Hair sterol signatures coupled to multivariate data analysis reveal an increased 7beta-hydroxycholesterol production in cognitive impairment., 20Ryu H.K. Jung B.H. Kim K.M. Yoo E.A. Woo J.T. Chung B.C. Determination of cholesterol in human hair using gas chromatography-mass spectrometry.), inappropriate internal standard (IS) (17Serra M. Matabosch X. Ying L. Watson G. Shackleton C. Hair and skin sterols in normal mice and those with deficient dehydrosterol reductase (DHCR7), the enzyme associated with Smith-Lemli-Opitz syndrome., 18Masukawa Y. Tsujimura H. Imokawa G. A systematic method for the sensitive and specific determination of hair lipids in combination with chromatography., 19Son H.H. Lee D.Y. Seo H.S. Jeong J. Moon J.Y. Lee J.E. et al.Hair sterol signatures coupled to multivariate data analysis reveal an increased 7beta-hydroxycholesterol production in cognitive impairment.), relatively incomplete method validation data (17Serra M. Matabosch X. Ying L. Watson G. Shackleton C. Hair and skin sterols in normal mice and those with deficient dehydrosterol reductase (DHCR7), the enzyme associated with Smith-Lemli-Opitz syndrome., 18Masukawa Y. Tsujimura H. Imokawa G. A systematic method for the sensitive and specific determination of hair lipids in combination with chromatography., 19Son H.H. Lee D.Y. Seo H.S. Jeong J. Moon J.Y. Lee J.E. et al.Hair sterol signatures coupled to multivariate data analysis reveal an increased 7beta-hydroxycholesterol production in cognitive impairment., 20Ryu H.K. Jung B.H. Kim K.M. Yoo E.A. Woo J.T. Chung B.C. Determination of cholesterol in human hair using gas chromatography-mass spectrometry.), and long extraction or derivatization times (up to more than 18 h) leading to low throughput (17Serra M. Matabosch X. Ying L. Watson G. Shackleton C. Hair and skin sterols in normal mice and those with deficient dehydrosterol reductase (DHCR7), the enzyme associated with Smith-Lemli-Opitz syndrome., 18Masukawa Y. Tsujimura H. Imokawa G. A systematic method for the sensitive and specific determination of hair lipids in combination with chromatography., 20Ryu H.K. Jung B.H. Kim K.M. Yoo E.A. Woo J.T. Chung B.C. Determination of cholesterol in human hair using gas chromatography-mass spectrometry.).In addition, at present, there is still a lack of analysis methods for 7-DHC in hair. In the only publication using hair as the sample for 7-DHC analysis, Serra et al. (17Serra M. Matabosch X. Ying L. Watson G. Shackleton C. Hair and skin sterols in normal mice and those with deficient dehydrosterol reductase (DHCR7), the enzyme associated with Smith-Lemli-Opitz syndrome.) found that the level of 7-DHC (570–780 ng/mg) in the hair of DHCR7-deficient mice (n=2) was hundreds of times higher than that of the healthy mice (＜1 ng/mg) with decreased CHOL levels, which was consistent with the trend in SLOS patients’ plasma. However, the levels of 7-DHC and CHOL in human hair have not been studied, and the reference values of 7-DHC and CHOL in the hair of SLOS patients and healthy individuals have yet to be established.This study aimed at developing a reliable, simple, and efficient GC-MS method, for simultaneous determination of 7-DHC and CHOL in human hair as an aid for the diagnosing and monitoring of SLOS. The hair decontamination, weighing, and homogenization strategies were carefully compared and optimized. Micro-pulverized extraction (MPE) and microwave-assisted derivatization were conducted for increasing analytical throughput. Antioxidant steps were also performed to prevent the oxidative degradation of the analytes and two deuterated ISs were adopted for accuracy as recommended (16Lutjohann D. Bjorkhem I. Friedrichs S. Kerksiek A. Lovgren-Sandblom A. Geilenkeuser W.J. et al.First international descriptive and interventional survey for cholesterol and non-cholesterol sterol determination by gas- and liquid-chromatography-Urgent need for harmonisation of analytical methods.). Furthermore, this method was applied to authentic hair samples of healthy and suspected infants to describe possible differences in the two biomarkers’ levels between these groups.Materials and methodsChemicals and reagents

Potassium hydroxide (KOH) and ethanol were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). 7-DHC, 7-DHC-d7 (7-DHC-25,26,26,26,27,27,27-d7), CHOL, CHOL-d7 (cholest-5-en-25,26,26,26,27,27,27-d7), 2,6-Di-tert-butyl-4-methylphenol (BHT), triphenylphosphine (TPP), and all other chemicals and reagents used were purchased from Merck (Darmstadt, Germany).

Neonatal hair samples were provided by Obstetrics and Gynecology Hospital Affiliated to Fudan University and Shanghai Children’s Hospital, Shanghai Jiao Tong University. Informed oral consent was obtained from all volunteers’ guardians in accordance with the Declaration of Helsinki principles. All related experimental and samples collection procedures in this study were approved by the ethical committee of Obstetrics and Gynecology Hospital Affiliated to Fudan University, Shanghai, China.

Preparation of standard solutions

7-DHC (1 mg/ml) and CHOL (10 mg/ml) were diluted in isopropanol to a final concentration of 800 μg/ml and 1200 μg/ml respectively as standard solutions. 7-DHC-d7 and CHOL-d7 were dissolved in isopropanol and finally diluted to the concentration of 40 μg/ml and 60 μg/ml respectively as IS. All solutions were stored at −40°C and prepared daily.

5 mg/ml BHT and 12.5 mg/ml TPP were prepared in ethanol and used as the antioxidant solution. KOH solution (1 M) was obtained by dissolving 560 mg KOH in 10 ml of 80% ethanol solution and used as an alkaline reagent in the hydrolysis step.

Instrumentation

MPE was conducted in a Bead Ruptor (JinXin, JXFSTPRP-6K, ShangHai, China) with temperature control function. Microwave-assisted derivatization was performed in a Haier MZC-2070M1 household microwave (ShangHai, China). Ultrapure water was prepared using a Millipore Milli-Q purification system (Bedford, MA). GC-MS was performed by an Agilent 7890B gas chromatograph connected with an Agilent 7000D triple quadrupole mass spectrometer (Agilent, Palo Alto, CA). The data acquisition software was Agilent MassHunter Quantitative Analysis B.09.00.

Sample collectionHair collection and storage steps followed internationally accepted guidelines (21Cooper G.A.A. Kronstrand R. Kintz P. Society of Hair Testing guidelines for drug testing in hair., 22Salomone A. Tsanaclis L. Agius R. Kintz P. Baumgartner M.R. European guidelines for workplace drug and alcohol testing in hair.). In short, hair strands were collected from the posterior vertex of the head of the infants and stored in paper envelopes at dry place at room temperature, protected from light after collection. During the collection process, blunt scissor was carefully cleaned, and the personnel wore gloves to avoid contamination of the samples. Hair samples proportionate to the thickness of a pencil were collected close to the scalp from multiple sites within the posterior vertex region of the head (21Cooper G.A.A. Kronstrand R. Kintz P. Society of Hair Testing guidelines for drug testing in hair.). Newborns’ hair will not be affected by factors such as cosmetic treatments and sun exposure, but visible stains (such as meconium) should be avoided during collection.Sample preparation

Hair samples were washed successively with water, then with acetone (5 ml/15 mg, 2 min under vortex agitation), which were then dried at room temperature with gentle air flow and cut into 1–2 mm long pieces.

50 μl of antioxidant solution (5 mg/ml BHT and 12.5 mg/ml TPP in ethanol) and IS solution (40 μg/ml 7-DHC-d7, 60 μg/ml CHOL-d7, 50 μl for each kind, equal to 200 ng/mg 7-DHC-d7, 300 ng/mg CHOL-d7 in hair) were added in a 2 ml MPE tube with ceramic beads. Afterward, aliquots of 10 mg hair and 850 μl of the extraction solvent (1 M KOH in 80% ethanol) were added into the tube. The samples were placed on a Bead Ruptor system and pulverized at −30°C using the following conditions: speed: 21 m/s; time: 40 s; dwell: 30 s. This procedure was repeated 15 times to completely pulverize the hair. After pulverization, the mixture was centrifuged at 10,000 g for 5 min, and then an aliquot of 100 μl (equal to 1 mg hair sample) of the supernatant was transferred to another tube. 900 μl of n-hexane (n-hex) as the liquid-liquid extraction solvent was added. The mixture was vortexed for 3 min and then centrifuged at 10,000 g for 5 min. The organic phase was transferred into a glass tube and the residue obtained after evaporation was redissolved in 60 μl of the derivatization reagent N,O-Bis(trimethylsilyl)trifluoroacetamide. After vortexing for 10 s, the tube was sealed and microwave-assisted derivatized at medium-high power (460 W) for 3 min. Then the derivatized sample was completely transferred for GC-MS analysis and an aliquot of 1 μl was injected.

GC-MS analysis

The electron energy of GC-MS was 70 eV and the ion source temperature was 320°C. Each sample (1 μl) was injected in split mode (10:1) at 240°C and separated through a HP-5MS capillary column (30 m × 0.25 mm × 0.25 μm) (Agilent Technologies, Palo Altro, CA) using helium as carrier gas with a flow rate of 1 ml/min. The initial temperature of GC oven was held at 240°C for 1 min and subsequently ramped at 20°C/min to 280°C and held for 17 min. 7-DHC and CHOL were determined based on peak area using two qualifier ions (m/z = 325, 351) and three qualifier ions (m/z = 129, 329, 368), respectively. All of the ions were monitored in the selected ion monitoring mode. Agilent Chemstation was applied for data collection, processing, and GC-MS control.

Method validationPreparation of blank samples1/10 aliquot of 10 mg hair alkaline digested sample was considered as blank sample after being repeatedly extracted (3 times, by 900 μl n-hex) until 7-DHC and CHOL were not detected or detected below their lower limits of quantification (LLOQ). The residual levels of the two analytes were monitored by the standard spiked method, before and after the repetitive extraction as described elsewhere (23Dong Z. Wang C. Zhang J. Wang Z. A UHPLC-MS/MS method for profiling multifunctional steroids in human hair., 24Schaffer M. Cheng C.C. Chao O. Hill V. Matsui P. Analysis of cocaine and metabolites in hair: validation and application of measurement of hydroxycocaine metabolites as evidence of cocaine ingestion.).Specificity

Specificity experiments were carried out using six blank hair samples from different healthy infant sources obtained by repeated extraction, to evaluate any possible interference from endogenous compounds.

Linearity and sensitivity

A calibration curve for each compound was prepared by spiking blank matrix with standard solutions to achieve the following final concentrations: 50, 100, 200, 400, 800, 1600, and 4000 ng/mg for 7-DHC; 30, 100, 300, 500, 1000, 3000, and 6000 ng/mg for CHOL. The two calibration curves of 7-DHC and CHOL were obtained using least-squares linear regression, with a 1/x2 weighting factor.

Limit of detection (LOD) and LLOQ were determined with blank matrix, spiked with different concentrations of 7-DHC and CHOL. LOD was defined as the lowest concentration of analyte having an S/N≥3. LLOQ was the lowest result of meaningful data with an S/N≥10, while maintaining CV within 20% of the nominal concentration.

Precision and accuracy

Accuracy and precision were evaluated at four levels including LLOQ (50 ng/mg for 7-DHC and 30 ng/mg for CHOL) and three QC concentrations (100, 1200, 3200 ng/mg for 7-DHC, and 100, 1800, 4800 ng/mg for CHOL). Intraday precision was assessed in replicate determinations (n = 5) of QC samples in one run. Interday precision was assessed in replicate determinations (n = 5) of the three QC samples on three independent days. Accuracy was assessed by comparing the calculated concentrations to their respective theoretical values and precision was recorded by relative standard deviation (RSD).

Extraction recovery rates

Extraction recovery were assessed by calculating the percentages of peak area ratios of extracted samples to those of nonextracted counterparts (representing 100% recovery), which were prepared by direct N,O-Bis(trimethylsilyl)trifluoroacetamide derivatization in dry, neat form at the three nominal concentrations (LQC, MQC, and HQC, n = 5).

Carryover

Carryover was evaluated by injecting five replicates of HQC sample (3200 ng/mg 7-DHC and 4800 ng/mg CHOL) followed by methanol. The calculated concentration of 7-DHC and CHOL in the methanol was used to assess the carryover.

Dilution integrity

High concentration spiked samples (10,000 ng/mg for 7-DHC and 10,000 ng/mg for CHOL) were prepared as described above. After MPE, 100 μl of supernatant were transferred and diluted 5 times and 10 times with blank hair samples, respectively, before subsequent preparations. Five replicate samples were analyzed for each dilution level, and the dilution integrity was assessed by calculating the precision and accuracy.

Stability

Stability of three QC concentrations (100, 1200, 3200 ng/mg for 7-DHC, and 100, 1800, 4800 ng/mg for CHOL) was evaluated under four different conditions. Short-term stability was determined after placing QC samples at room temperature for 3 h. Postpreparative stability was determined after placing pretreated QC samples in the autosampler (25°C) for 24 h. When evaluating the freeze-thaw stability, QC samples were frozen at −40°C for 24 h, then thawed and kept in the dark at room temperature for 30 min. The freeze-thaw cycle was repeated three times. Long-term stability was evaluated after storing QC samples at −40°C for 30 days.

Application to real samplesThe 7-DHC and CHOL level in the hair samples of 14 healthy newborns (seven males, seven females) and two suspected SLOS patient (two females) were analyzed. In terms of specific clinical symptoms, these two infant girls were diagnosed with SLOS typical symptoms of syndactyly of the second and the third toes (left foot, Fig. 1). These patients were suspected by clinicians to be SLOS undiagnosed patients. The hair collection time of all newborns was the day of delivery to minimize the influence of external factors. Because the patients’ guardian refused to provide blood, genetic test could not be conducted.