Bert G. Wolthers

DIAGNOSIS OF CEREBROTENDINOUS XANTHOMATOSIS (CTX) AND EFFECT OF CHENODEOXYCHOLIC ACID THERAPY BY ANALYSIS OF URINE USING CAPILLARY GAS-CHROMATOGRAPHY

By means of capillary gas chromatography urine samples of patients with cerebrotendinous xanthomatosis (CTX) were investigated before and during treatment by oral administration of chenodeoxycholic acid. The occurrence of various conjugated bile alcohols, presumably glucuronides, was demonstrated, the major compound being 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 23 xi, 25-pentol. In the bile acid fraction norcholic acid and hydroxycholic acid were shown to be present in considerable amounts. In this way the presence of CTX can be demonstrated conclusively. After chenodeoxycholic acid therapy the excretion of both abnormal bile acids as well as of bile alcohols rapidly decreased within a few weeks, showing the effectiveness of the treatment. By early discovery and subsequent therapy it may be possible to prevent the onset of the detrimental symptoms such as mental deficiency, caused by the accumulation of cholestanol and cholesterol in CTX patients.

The determination of oxalic acid in plasma and urine by means of capillary gas chromatography

A gas chromatographic procedure for determining oxalate in plasma is described, in which the trimethylsilyl derivative of oxalate is analyzed on a 25-m capillary SE-30 column. In addition the effects of standing of whole blood or plasma and the effect of added glyoxalate on the oxalate concentration were studied. The present method offers good specificity and sensitivity and is easy to perform, in contrast to most methods hitherto described. The normal value of plasma oxalate was found to be 2.8 +/- 1.1 mumol/l (mean +/- 1 SD), which is close to the values obtained with in vivo tracer studies. Plasma oxalate values before and after haemodialysis are presented. By introducing a few minor modifications the method is also applicable to urine samples and in principle it should be possible to determine the glycollic acid concentration as well, both in urine and plasma.

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)

The concentration ratios of cholestanol/cholesterol in biological materials (serum, cerebrospinal fluid and tendon biopsy) were determined using a capillary gas chromatographic 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.

Bile acid therapies applied to patients suffering from cerebrotendinous xanthomatosis

Patients suffering from cerebrotendinous xanthomatosis, an inborn error of metabolism in bile acid synthesis, were given oral treatment with chenodeoxycholic acid, ursodeoxycholic acid, cholic acid and taurocholic acid. The effectiveness of the different therapies was evaluated by measuring the urinary excretion of 5 beta-cholestane-3 alpha,7 alpha,12 alpha,23,25-pentol, which should decrease, when the administered bile acid is able to suppress endogenous bile acid synthesis. From the results it is concluded that chenodeoxycholic acid and cholic acid activate the bile acid negative feedback mechanism, contrary to ursodeoxycholic acid and taurine conjugated cholic acid. Either cholic acid or chenodeoxycholic acid are the therapies of choice for the treatment of cerebrotendinous xanthomatosis. For various reasons the use of cholic acid is especially recommended.

CAPILLARY GAS-CHROMATOGRAPHIC DETERMINATIONS OF URINARY BILE-ACIDS AND BILE ALCOHOLS IN CTX PATIENTS PROVING THE INEFFECTIVITY OF URSODEOXYCHOLIC ACID TREATMENT

Urine samples and serum samples of a patient with cerebrotendinous xanthomatosis (CTX) were investigated by means of capillary gas chromatography, both before and during oral treatment with ursodeoxycholic acid (UDCA), and the results compared with those obtained during chenodeoxycholic acid (CDCA) therapy. The predominantly excreted bile alcohol, 5 beta-cholestane-3 alpha,7 alpha,12 alpha,23,25-pentol and two abnormal bile acids, i.e. 23-norcholic acid and 23-hydroxycholic acid were determined. In addition, the serum cholestanol/cholesterol ratio was determined. Whereas previous experiments demonstrated that the urinary excretion of 5 beta-cholestane-3 alpha,7 alpha,12 alpha,23,25-pentol and the abnormal bile acids decreased within a few weeks during CDCA therapy, the present study shows that their urinary excretions remain essentially the same during UDCA treatment. In contrast to the decrease in the serum cholestanol/cholesterol ratio during CDCA therapy, this ratio remains essentially the same during UDCA therapy. It is therefore concluded that, in contrast to CDCA therapy, UDCA treatment is not effective in the treatment of CTX.

DETECTION OF 3-BETA-HYDROXYSTEROID DEHYDROGENASE-DEFICIENCY IN A NEWBORN BY MEANS OF URINARY STEROID ANALYSIS

A urinary steroid excretion pattern of a 3-wk-old newborn, suffering from 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) deficiency, has been produced, employing capillary gas chromatography and subsequent mass spectrometric identification of the various excreted steroids. The diagnosis could be established, apart from the clinical symptoms, on the basis of a grossly elevated excretion of 16-OH-DHEA and 16-OH-pregnenolone, combined with mass spectrometric identification of the following steroids: 17-OH-preganolone, pregnanetriol, pregnanolone, pregnenetriol and 17-OH-pregnenolone.

ABNORMAL URINARY BILE-ACIDS IN A PATIENT SUFFERING FROM CEREBROTENDINOUS XANTHOMATOSIS DURING ORAL-ADMINISTRATION OF URSODEOXYCHOLIC ACID

The urinary bile acid profile, obtained by capillary gas chromatography, of a patient suffering from cerebrotendinous xanthomatosis and treated with ursodeoxycholic acid demonstrated, besides the occurrence of 23-norcholic acid and (23R)-hydroxycholic acid (as a consequence of this disease), six additional unknown bile acids and three known bile acids, viz. ursodeoxycholic acid, hyocholic acid and omega-muricholic acid. The structure of two of the unknown bile acids were elucidated and proven by organic syntheses. These were 23-norursodeoxycholic acid and 3 beta-ursodeoxycholic acid. The structures of three bile acids were tentatively elucidated as being 1 beta-hydroxyursodeoxycholic acid, 21-hydroxyursodeoxycholic acid and 22-hydroxyursodeoxycholic acid, and the possibility that the structure of the remaining bile acid is that of 5-hydroxyursodeoxycholic acid is discussed. Two of these bile acids (1 beta-hydroxyursodeoxycholic acid and 5-hydroxyursodeoxycholic acid) also occurred in urine of a healthy individual during oral ursodeoxycholic acid treatment, whereas 23-norcholic acid, 23-norursodeoxycholic acid, (23R)-hydroxycholic acid, 21-hydroxyursodeoxycholic acid and 22-hydroxyursodeoxycholic acid were only present in urine of the patient suffering from cerebrotendinous xanthomatosis. The metabolism of ursodeoxycholic acid, both in the normal state and in the cerebrotendinous xanthomatosis, is discussed.

Relationship between biliary lipid and protoporphyrin secretion; potential role of mdr2 P-glycoprotein in hepatobiliary organic anion transport

BACKGROUND/AIMSnErythropoietic protoporphyria, caused by ferrochelatase deficiency, leads to protoporphyrin accumulation in the liver. Therapeutic attempts to increase the secretion of this hydrophobic organic anion into bile are hampered by a lack of understanding of the secretory mechanism(s) involved. We have investigated biliary secretion of protoporphyrin in rats and mice, primarily targeted on the role of biliary lipids in this process.nnnMETHODSnGel permeation chromatography was applied to investigate the association of porphyrins with lipid fractions in bile. Secretion of endogenous porphyrins was studied in (GY mutant) rats and mdr2 P-glycoprotein deficient mice, under conditions of widely varying biliary lipid secretion rates.nnnRESULTSnGel permeation chromatography revealed that, in native human and rat bile, protoporphyrin associated with cholesterol/phospholipid vesicles upon elution with bile salt-free buffer. In contrast, the more hydrophilic coproporphyrin isomers I and III were found only in bile salt/organic anion hybrid particles and smaller aggregates. Interruption of the enterohepatic circulation in normal Wistar rat resulted in parallel decrease of endogenous protoporphyrin-, lipid-, and bile salt secretion, but did not alter the secretion of coproporphyrin I and III. Uncoupling of lipid- from bile salt secretion by sulfated taurolithocholate resulted in impaired secretion into bile of protoporphyrin only. Conversely, secretion of coproporphyrin I and III, but not that of protoporphyrin, was impaired in mutant Groningen Yellow rats with defective ATP-dependent hepatobiliary organic anion transport. In mice homozygous for a disruption of the mdr2 P-glycoprotein gene, resulting in complete absence of phospholipids in bile and strongly reduced cholesterol output, secretion of protoporphyrin was reduced by 90%, whereas that of coproporphyrin I and III was affected to a much lesser extent.nnnCONCLUSIONSnOur data demonstrate a close association between protoporphyrin and lipid secretion into bile, indicating that these processes are, at least functioning coupled. This finding implicates a role of mdr2 P-glycoprotein activity in hepatobiliary removal of the hydrophobic organic anion protoporphyrin. Hence, it may be speculated that protoporphyrin secretion can be influenced by drugs, diet or other means that affect biliary lipid secretion.

The diagnosis of 21-hydroxylase deficiency in a prematurely born infant on the basis of the urinary steroid excretion pattern

The urine of a 6-day-old prematurely born female infant (birth weight 1060 g) suspected of having a 21-OH-deficiency showed no steroid abnormalities on capillary GLC analysis. Using GC-MS tetrahydrocortisone (THE) and also 3 alpha, 17 alpha-dihydroxy-5 beta-pregnane-20-one (17-OH-Polone) were absent, but two androstanetriolone peaks were observed. In the urine collected on day 9 THE was absent, but a large amount of 3 alpha, 11 beta-dihydroxy-5-alpha-androstane-17-one (11-HA) was found by GC-MS to be contaminated by a small amount of 17-OH-Polone. The next urine specimen collected on the 22nd day while the child received cortisol therapeutically showed the characteristic steroid profile for the diagnosis 21-OH deficiency, large peaks of 17-OH-Polone, pregnanetriol (P3) and 11-keto-pregnanetriol (11-keto-P3). Over the next few weeks two other compounds were found to have been excreted in relatively large amounts, 3 xi, 16 xi, 17 xi, 20 xi-pregnanetetrol (16-OH-P3) and surprisingly also a 21-hydroxylated compound, namely 3 xi, 20 alpha, 21-trihydroxy-5-pregnene. These same two compounds were also found in the urine of another infant with suspected 21-OH deficiency. The urinary steroid excretion patterns characteristic for 21-OH deficiency are dependent on the maturity and age of the infant. In the prematurely born infant androstanetriolones appear in the urine before 17-OH-Polone. The occurrence of these different steroid excretion patterns is tentatively explained.

Improved gas chromatographic—mass fragmentographic assay for tetrahydroaldosterone and aldosterone in urine☆

A newly devised procedure for a simultaneous determination of urinary tetrahydroaldosterone and aldosterone is described. The procedure is based on deconjugation and acetalization, followed by extraction and derivatization of the urinary compounds to their trimethylsilyl ethers and subsequent gas chromatographic-mass fragmentographic detection. To evaluate the assay, aliquots of a urine sample of a healthy individual were analysed in multiplicate; a mean tetrahydroaldosterone concentration of 103 nmol/l and a within-sample, within-day- and day-to-day coefficient of variation of 1.8, 3.2 and 3.4%, respectively, were found. Determination of aldosterone in the same sample yielded a mean concentration of 25.3 nmol/l and the following coefficients of variation: 2.8% (within-sample), 3.8% (within-day) and 4.3% (day-to-day). The urinary excretion of tetrahydroaldosterone and aldosterone in 24-h urine portions was determined in twenty healthy individuals, aged 23-77 years; for tetrahydroaldosterone and aldosterone, an excretion of 94 +/- 66 nmol per 24 h and of 40 +/- 22 nmol per 24 h was found, respectively, in accord with the literature. An example of the usefulness of the described assay is given by establishing the cause of severe salt-wasting in an infant; a highly elevated tetrahydroaldosterone and aldosterone excretion was demonstrated, proving that the child suffered from unresponsiveness to aldosterone (pseudohypoaldosteronism).