Capillary gas chromatographic determination of cholestanol/cholesterol ratio in biological fluids. Its potential usefulness for the follow-up of some liver diseases and its lack of specificity in diagnosing CTX (cerebrotendinous xanthomatosis)

doi:10.1016/0009-8981(84)90119-0

Clinica Chimica Acta

Volume 137, Issue 3, 13 March 1984, Pages 305-315

https://doi.org/10.1016/0009-8981(84)90119-0 Get rights and content

Abstract

The concentration ratios of cholestanol/cholesterol in biological materials (serum, cerebrospinal fluid and tendon biopsy) were determined using a capillary gas Chromatographie method. The method was validated by gas chromatography-mass spectrometry. The ratio was determined in several groups of patients: (a) patients with cerebrotendinous xanthomatosis (in serum, cerebrospinal fluid and tendon biopsy), before and during chenodeoxycholic acid therapy, (b) patients receiving cholestyramine therapy (in serum), (c) patients suffering from various liver diseases (in serum) and (d) one patient before and after liver transplantation (in serum). It can be concluded that the cholestanol/cholesterol concentration ratio is a potentially useful parameter for monitoring liver diseases but is not specific for establishing the diagnosis of cerebrotendinous xanthomatosis.

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Therefore, determination of CHO is important from clinical point of view. Different analytical methods including gas chromatography (GC) (Salen and Grundy, 1973; Ishikawa et al., 1976), capillary GC (Koopman et al., 1984; Nikkilae et al., 1992; Salen et al., 1987), GC-mass spectrometry (GC–MS) (Seyama et al., 1976), high-performance liquid chromatography (HPLC) with fluorescence detection (Matsuoka et al., 1985a; Iwata et al., 1987a; Tsuruta et al., 1993a), and HPLC with UV detection (Kasama et al., 1987; Kim et al., 1991; Kuriyama et al., 1991; Hideka et al., 1990) have been reported for determination of CHO. These methods require complex procedures for precipitation of lipoprotein fractions which suffer from low specificity, instability of reagents and high cost.
In this work, a novel molecularly imprinted electrochemical sensor (MIES) has been fabricated based on electropolymerization of a molecularly imprinted polymer (MIP) onto a glassy carbon electrode (GCE) modified with gold-palladium alloy nanoparticles (AuPd NPs)/polydopamine film (PDA)/multiwalled carbon nanotubes-chitosan-ionic liquid (MWCNTs-CS-IL) for voltammetric and impedimetric determination of cholestanol (CHO). Modifications applied to the bare GCE formed an excellent biocompatible composite film which was able to selectively detect CHO molecules. Modifications applied to the bare GCE were characterized by scanning electron microscopy (SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (SEM). Under optimal experimental conditions, the sensor was able to detect CHO in the range of 0.1–60 pM and 1–50 pM by EIS and DPV, respectively. Moreover, the sensor showed high sensitivity, selectivity, repeatability, reproducibility, low interference and good stability towards CHO determination. Our records confirmed that the sensor was successfully able to the analysis real samples for determination of CHO.
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The development and maintenance of an enterohepatic circulation of bile acids are critical to health because it promotes normal bile flow and intraluminal bile acid concentrations that facilitate normal fat and fat-soluble vitamin absorption. Alterations in the synthesis of bile acids and their transport at the liver canalicular and basolateral membrane and ileal apical and basolateral membrane have significant impacts on the health of infants, children, and adults. Defects in bile acid synthesis lead to cholestatic liver disease or cirrhosis or neurodegenerative disease in older children and adults. Defects in membrane transport and function in the liver lead to conditions such as progressive familial intrahepatic cholestasis (PFIC) 1 to 3 and other related disorders. Defects in ileal bile acid transport lead to diarrhea with impacts on growth in infants and children and the health of adults.
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A useful multi-analyte blood test for cerebrotendinous xanthomatosis
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Molecular genetic testing confirmed the individual possessed a homozygous R474Q CYP27A1 gene mutation, previously reported to be a causative mutation for CTX [10]. 5α-Cholestanol can be elevated in a number of liver diseases and concerns have been raised regarding the specificity of this disease marker for CTX [11]. Although not routinely used for diagnosis, other markers elevated in CTX include cholesterol precursors such as 7-dehydrocholesterol and 8-dehydrocholesterol [12].
Cerebrotendinous xanthomatosis (CTX) is a rare genetic disorder of bile acid (BA) synthesis that can cause progressive neurological damage and premature death. Blood (normally serum or plasma) testing for CTX is performed by a small number of specialized laboratories, routinely by gas chromatography–mass spectrometry (GC–MS) measurement of elevated 5α-cholestanol. We report here on a more sensitive biochemical approach to test for CTX particularly useful for confirmation of CTX in the case of a challenging diagnostic sample with 5α-cholestanol that, although elevated, was below the cut-off used for diagnosis of CTX (10 μg/mL or 1.0 mg/dL).
We have previously described liquid chromatography–electrospray ionization-tandem mass spectrometry (LC–ESI-MS/MS) methodology utilizing keto derivatization to enable the sensitive quantification of plasma ketosterol BA precursors that accumulate in CTX. We have expanded this methodology to perform isotope dilution LC–ESI-MS/MS quantification of a panel of plasma ketosterol BA precursors, with internal standards readily generated using isotopically-enriched derivatization reagent.
Quantification of plasma ketosterol BA precursors (7α-hydroxy-4-cholesten-3-one, 7α,12α-dihydroxy-4-cholesten-3-one and 7α,12α-dihydroxy-5β-cholestan-3-one) in a single LC–ESI/MS/MS test provided better discrimination between a CTX-positive and negative samples analyzed (n = 20) than measurement of 5α-cholestanol alone.
Quantification of plasma ketosterol BA precursors provides a more sensitive biochemical approach to discriminate between CTX negative and positive samples. A multiplexed LC–ESI-MS/MS test quantifying a panel of plasma ketosterols, with simple sample preparation, rapid analysis time and readily available internal standards, can be performed by most clinical laboratories. Wider availability of testing will benefit those affected with CTX.
A blood test for cerebrotendinous xanthomatosis with potential for disease detection in newborns
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Citation Excerpt :
Elevated bile alcohol intermediate 5β-cholestane-3α,7α,12α,25-tetrol was previously detected in serum from a four-month-old CTX patient (1). There are concerns regarding the specificity of 5α-cholestanol as a marker for CTX as it can be elevated in other disorders (for example, sitosterolemia and liver disease) (40). Although 7αC4 can also be elevated in BA malabsorption and ileal resection (41), we previously reported that 7αC4 demonstrated improved utility over 5α-cholestanol as a circulating marker for CTX (21).
Cerebrotendinous xanthomatosis (CTX) is a rare, difficult-to-diagnose genetic disorder of bile acid (BA) synthesis that can cause progressive neurological damage and premature death. Detection of CTX in the newborn period would be beneficial because an effective oral therapy for CTX is available to prevent disease progression. There is no suitable test to screen newborn dried bloodspots (DBS) for CTX. Blood screening for CTX is currently performed by GC-MS measurement of elevated 5α-cholestanol. We present here LC-ESI/MS/MS methodology utilizing keto derivatization with (O-(3-trimethylammonium-propyl) hydroxylamine) reagent to enable sensitive detection of ketosterol BA precursors that accumulate in CTX. The availability of isotopically enriched derivatization reagent allowed ready tagging of ketosterols to generate internal standards for isotope dilution quantification. Ketosterols were quantified and their utility as markers for CTX was compared with 5α-cholestanol. 7α,12α-Dihydroxy-4-cholesten-3-one provided the best discrimination between CTX and unaffected samples. In two CTX, newborn DBS concentrations of this ketosterol (120–214 ng/ml) were ∼10-fold higher than in unaffected newborn DBS (16.4 ± 6.0 ng/ml), such that quantification of this ketosterol provides a test with potential to screen newborn DBS for CTX. Early detection and intervention through newborn screening would greatly benefit those affected with CTX by preventing morbidity and mortality.
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Capillary gas chromatographic determination of cholestanol/cholesterol ratio in biological fluids. Its potential usefulness for the follow-up of some liver diseases and its lack of specificity in diagnosing CTX (cerebrotendinous xanthomatosis)

Abstract

J Lipid Res

Steroids

J Lipid Res

J Lipid Res

Clin Chim Acta

Clin Chim Acta

Une forme cérébrale de la cholinesterinose généralisée

Cerebrotendinous xanthomatosis

Arch Neurol

Cholestanol deposition in cerebrotendinous xanthomatosis. A possible mechanism

Ann Intern Med

Biosynthesis of 5α-cholestane-3β-ol in cerebrotendinous xanthomatosis

J Clin Invest