Changes in rectal epithelial cell proliferation and intestinal bile acids after subtotal colectomy in familial adenomatous polyposis.

Subtotal colectomy and ileorectal anastomosis in familial adenomatous polyposis patients can induce temporary regression of adenomas in the rectum. The mechanism for this phenomenon is unclear. We evaluated the effect of colectomy on rectal mucosal proliferation, in relation to changes in bile acid metabolism. Four familial adenomatous polyposis patients were studied before and 3-6 months after surgery, and eight others 7-22 years postoperatively. Within 6 months after surgery, the size of the proliferative zone of the colonic crypts was found to be reduced (P less than 0.05). The proliferative activity of total colonic crypts was not affected within this period. More than 7 years postoperatively, increased cell proliferation of total crypts (P less than 0.02), as well as mid (P less than 0.05) and basal (P less than 0.05) crypt compartments, were observed compared to shortly after colectomy. In duodenal bile, deoxycholic acid was absent shortly after operation, whereas several years after operation only a small fraction (2%) was present. Fecal secondary bile acid excretion diminished after colectomy and did not change several years postoperatively. In postoperative stools only, small proportions of ursocholic and ursodeoxycholic acids (about 5% each) were consistently found. As subtotal colectomy causes a temporary decrease in the length of the proliferative zone of rectal crypts toward a normal pattern, this may explain regression of rectal polyps. This temporary effect may be mediated, at least in part, by decreased amounts of cytotoxic secondary bile acids in the rectal lumen.

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.

Increased (23R)-hydroxylase activity in patients suffering from cerebrotendinous xanthomatosis, resulting in (23R)-hydroxylation of bile acids.

Patients suffering from cerebrotendinous xanthomatosis, an inborn error of metabolism in bile acid synthesis, excrete excessive amounts of 23-hydroxylated bile alcohols, 23-norcholic acid and 23-hydroxycholic acid into urine. In this study the configuration of this excreted 23-hydroxycholic acid was established as (23R)-hydroxycholic acid. Urine samples of two treated patients, receiving chenodeoxycholic acid, were investigated to see whether this administered bile acid was partly converted into 23-hydroxychenodeoxycholic acid. One patient was treated with ursodeoxycholic acid for 1 month and subsequently with chenodeoxycholic acid, and the urinary excretion of both (23R)-hydroxychenodeoxycholic acid and (23R)-hydroxyursodeoxycholic acid were followed. Indeed, all three patients excreted (23R)-hydroxylated chenodeoxycholic acid during oral treatment with chenodeoxycholic acid, and the patient treated with ursodeoxycholic acid excreted (23R)-hydroxylated ursodeoxycholic acid. During treatment with chenodeoxycholic acid the excretion of (23R)-hydroxychenodeoxycholic acid increases at first and later on decreases markedly. These findings suggest increased (23R)-hydroxylase activity in patients suffering from cerebrotendinous xanthomatosis, acting both on endogenously synthesized bile alcohols and on exogenously administered bile acids; during continuation of chenodeoxycholic acid treatment in an effective dose (750 mg/day) this enzyme activity gradually disappears.

A patient with adrenocortical carcinoma: characterization of its biological activity and drug resistance profile.

We describe a patient with a metastasized adrenocortical cancer who exhibited excessive production of both glucocorticoids and mineralocorticoids combined with suppressed androgen production. Unusual steroid metabolites found in the patient's urine have not been described previously in association with this tumor type. Investigation of the multidrug resistance phenotype in single-cell suspensions of the tumor revealed low expression of multidrug resistance protein but high expression of P-glycoprotein (Pgp) and lung resistance-related protein. Functional Pgp in these tumor cells was shown by the modulatory effect of PSC833 on daunorubicin accumulation. Mitotane, at a concentration achieved in this patient's plasma, completely reversed the Pgp-related resistance both in the Pgp-overexpressing KB8-5 cell line and in the patient's tumor cells. On the basis of these in vitro results, the patient was treated with a combination of multidrug resistance drugs (doxorubicin, vincristine, and etoposide) plus mitotane as a Pgp modulator. This treatment was ineffective, however. A chemosensitivity assay demonstrated that the tumor cells were highly resistant to the drugs used. The adrenocortical cancer cells expressed mutant p53, and no evidence for induction of apoptosis by these drugs was found.

Identification of 5-hydroxy-6-indolyl-O-sulfate in urine of patients with malignant melanoma.

It has previously been shown that enzymatically hydrolyzed urine of patients with malignant melanoma contains 5,6-dihydroxyindole (5, 6DHI ). In this study we describe the elucidation of the entire structure of urinary 5, 6DHI -conjugate. Differential hydrolysis of melanotic urine revealed that, in contrast to beta-glucuronidase, sulfatase can liberate 5, 6DHI from its conjugated form. 5, 6DHI -sulfate was synthetized by reacting 5, 6DHI with sulfur trioxide trimethylamine complex. Thin-layer chromatography (TLC) documented its close similarity to the Thorm ahlen -positive compound usually entitled "C." Gas chromatographic-mass spectrometric (GC-MS) analysis of methylated and subsequently hydrolyzed synthetic 5, 6DHI -sulfate showed that the synthetic product consisted of a mixture of 5-hydroxy-6-indolyl-O-sulfate and 6-hydroxy-5-indolyl-O-sulfate (with a certain amount of 5, 6DHI -disulfate). 5, 6DHI -sulfate was purified with use of DEAE-cellulose column chromatography from melanotic urine. Methylation of this conjugate with deuterated dimethylsulfate and subsequent GC-MS analysis of the hydrolyzed product provided evidence that 5, 6DHI from melanotic urine was almost exclusively sulfated in position 6. It was concluded (1) that 5, 6DHI is excreted as a 6-O-sulfate, and (2) that this compound is consistent with Thorm ahlen -positive compound "C".

Molecular heterogeneity of the fourth component of complement (C4) and its genes in vitiligo.

In view of evidence suggesting vitiligo is an autoimmune disease, we investigated whether vitiligo is associated with inherited deficiencies of the fourth (C4) and second (C2) component of complement and with certain human leukocyte antigens (HLA). Analysis of functional activities of C4 and C2 in sera of patients with vitiligo (n = 42) showed that 17% of them had a heterozygous C4 deficiency and 5% had a heterozygous C2 deficiency. In the normal control group (n = 30), 3% had a heterozygous C4 deficiency and none had a C2 deficiency. C4 typing by Western blot analysis showed the frequency of the C4A*Q0 allele in the vitiligo patient group to be close to normal. However, the frequency of one C4B*Q0 allele was three times higher, and that of two C4B*Q0 alleles five times higher in the vitiligo patient group than the reported frequencies in normal control groups. Southern blot analysis of Taq1 digests of DNA using C4 and 21-hydroxylase probes showed that two patients with two C4B*Q0 alleles had a deletion of a 21-OHA-C4B segment. In the other patients, having one or two C4B*Q0 alleles, these null alleles probably occurred due to a loss of C4 gene expression. HLA analysis did not show any allelic association of C4A*Q0 or C4B*Q0 with any HLA antigen in vitiligo, but confirmed the previous findings of a negative association with HLA-DR3 and a positive association with HLA-DR4. These results suggest that abnormalities of the C4B gene and the above-mentioned associations with HLA antigens may be some of the risk factors in vitiligo.

Alterations in cortisol metabolism in insulin-dependent diabetes mellitus: relationship with metabolic control and estimated blood volume and effect of angiotensin-converting enzyme inhibition.

11 beta-Hydroxysteroid dehydrogenase (11 beta HSD) catalyzes the interconversion of cortisol and its inactive metabolite, cortisone, and protects the mineralocorticoid receptor from activation by cortisol. Sodium and fluid retention is a well documented phenomenon in insulin-dependent diabetes mellitus (IDDM), but it is not known whether diabetes-associated alterations in cortisol metabolism contribute to its pathogenesis. Therefore, we evaluated some aspects of cortisol metabolism by measuring urinary metabolites of cortisol and cortisone in eight microalbuminuric and eight normoalbuminuric IDDM patients and eight matched control subjects. In both IDDM groups, the overnight excretion of tetrahydrocortisol (THF), allo-tetrahydrocortisol (allo-THF), and tetrahydrocortisone (THE) was lower than that in the control group (P < 0.05 to P < 0.01). Both the allo-THF/THF ratio, a parameter of 5 alpha/5 beta-reduction of cortisol, and the cortisol to cortisone metabolite ratio (THF+allo-THF/THE), which reflects the overall direction of the cortisol to cortisone interconversion, were lower in the IDDM groups (P < 0.05 to P < 0.01). In the combined subjects (n = 24), allo-THF, allo-THF/THF, and THF+allo-THF/THE were inversely correlated with hemoglobin A1c (r = -0.69, P < 0.001; r = -0.61, P < 0.01; and r = -0.58, P < 0.01, respectively). Upper arm segmental blood volume, estimated by an electrical impedance technique, was positively correlated with the cortisol to cortisone metabolite ratio in both the control subjects (r = 0.77; P < 0.05) and the IDDM patients in whom it was measured (r = 0.56; P < 0.05; n = 13), whereas the regression line was shifted leftward in IDDM (i.e. a lower ratio at the same blood volume; P < 0.03, by analysis of covariance). In seven microalbuminuric IDDM patients, the angiotensin-converting enzyme inhibitor, enalapril (10 mg daily for 6-12 weeks), resulted in a moderate further lowering of the cortisol to cortisone metabolite ratio (P < 0.05). The present data suggest a chronic hyperglycemia-related impairment in the reduction of corticoids to tetrahydro metabolites and an imbalance in 11 beta HSD. Altered 11 beta HSD activity is unlikely to be primarily responsible for the sodium and fluid retention in IDDM. Moreover, an additional mechanism of action of angiotensin-converting enzyme inhibition might be provided by an effect on 11 beta HSD activity.

The daily cortisol production reinvestigated in healthy men. The serum and urinary cortisol production rates are not significantly different.

We have measured the urinary cortisol production rate (uCPR) simultaneously with the serum cortisol production rate (sCPR) in four healthy men within a period of 3 days. uCPR, determined by isotope dilution of 11-oxoetiocholanolone was compared with sCPR, which was measured in three different ways (a, b, c). Blood was sampled at 10-min intervals for 24 h, and deconvolution analysis was applied to the cortisol concentrations. The daily serum cortisol production per liter, multiplied by the distribution volume yielded sCPR. The measurement methods are characterized as follows: a) the secretion and elimination terms were free; b) like method a, but with the input of the rate constants alpha and beta into the elimination function; c) the average 24-h cortisol concentration was multiplied by the metabolic clearance rate. uCPR was 25.4 +/- 4.7 [range: 21.3-31.4] micromol/(m2 x day), sCPR (method a) was 28.8 +/- 4.5 [range: 23.5-34.3] micromol/(m2 x day), sCPR (method b) was 27.9 +/- 8.1 [range: 18.5-37.7] micromol/(m2 x day), and sCPR (method c) was 29.3 +/- 4.8 [range: 22.7-33.2] micromol/(m2 x day). uCPR did not significantly differ from each of the 3 sCPR values (P > 0.30; > 0.46; and > 0.06, respectively). The patterns of the cortisol secretory rates in the present and previous studies do not necessarily represent the physiological process of the secretory bursts. We conclude that the estimated CPR, being 25-30 micromol/(m2 x day) [9-11 mg/(m2 x day)], can serve as a guideline for glucocorticoid replacement dose and that the urinary route to measure CPR is preferred because of its relative ease.

Measurement of the cortisol production rate in two sisters with 17 alpha-hydroxylase deficiency using [1,2,3,4-13C]cortisol and isotope dilution mass spectrometry.

[1,2,3,4-13C]cortisol was i.v. administered to two sisters aged 11 yr (patient I) and 3 yr (patient II) who suffer from 17 alpha-hydroxylase deficiency. This is the first time that the cortisol production rate (CPR) in patients with 17 alpha-hydroxylase deficiency has been measured with a stable labelled tracer using the urinary method. The urine was collected for 3 days. High-performance liquid chromatography (HPLC) of approximately 100 ml urine extracts was carried out to isolate the small amount of cortisol metabolites excreted. The cortisol metabolites were oxidized to 11-oxo-aetiocholanolone. The isotope dilution in the methyl oxime tert-butyldimethylsilyl ether derivatives was measured by selected ion monitoring gas chromatography/mass spectrometry (GC/MS). The CPR calculated from tetrahydrocortisone (THE) and the cortolones was 765 and 536 nmol/day, respectively in patient I. The CPR in patient II was only calculated from THE and was 62 nmol/day. If radioactive labelled cortisol had been used, much larger quantities of urine would have been needed for isolation of sufficient mass of metabolites, even then purification may have been difficult. Steroid profiling of 1 ml urine samples by GC and identification by GC/MS revealed high concentrations of pregnenolone, progesterone, 11 beta-hydroxy progesterone and corticosterone metabolites. Tetrahydrocorticosterone and 5 alpha-tetrahydrocorticosterone were found in urine at elevated excretions of 2.5 and 5.7, 0.9 and 2.0 mumols/24 h, in patients I and II respectively. No cortisol metabolites were detected by routine GC or GC/MS as the low amounts excreted co-eluted with the relatively abundant corticosterone metabolites.

Kinetics of intravenously dosed cortisol in four men. Consequences for calculation of the plasma cortisol production rate.

The kinetics of cortisol in the serum of 4 healthy men were studied following single i.v. doses of 2 and 0.8 mg of cortisol. The disappearance of cortisol was determined by blood sampling frequently over 2.5 h and analysing the apparently biexponential cortisol decay. The main results, shown as the mean (+/-SD), were: (a) the average distribution volume of cortisol at steady state (Vd,ss), which was 7.1 l/m2 body surface area. The extrapolated distribution volume (Vd,ext) was 8.4 l/m2, being 18% higher than the corresponding Vd,ss. (b) It was confirmed that plasma cortisol disappears biexponentially. Since the rapid phase remains unnoticed if cortisol is measured at an interval of 10 or more minutes, the obscured rapid-phase parameters can be found only if the known ratio of the two rate constants is used. (c) The fraction of cortisol, which during this fast phase irreversibly disappeared according to the two-compartment open model, was 5 to 8% larger than that found using the monocompartment model. (d) The half-life of the slow or beta phase was equal for the 2 and 0.8 mg experiments, namely t1/2(beta) = 66 +/- 18 min. The kinetics of cortisol in the same 4 men were also measured after an i.v. dose of radioactive cortisol (82 +/- 7 kBq 3H/m2). All urine was collected in 15 portions during the next 3 days, followed by measuring the cumulative radioactivity and analysing the triexponential increase of urinary radioactivity [1]. The main results with the urinary model were: (a) the half-life of cortisol elimination from the circulation was 40 +/- 11 min, (b) the maximal radioactivity (69 +/- 7% of the dose) in the first pool (liver) was found at 2 +/- 0.3 h, (c) the half-life of the cortisol metabolites in the body was 6.8 +/- 0.7 h. Forcing the measured cortisol concentrations in plasma to fit a monoexponential function, allowed us to compare the half-life of cortisol decay with that from the urinary model. It was found that these half-lives were similar with values between 30 and 40 min. Finally, the distribution volume has to be measured individually if a 24 h plasma cortisol profile is used for the calculation of the cortisol production rate.

Kinetics and metabolism of 11-deoxycortisol in a patient with congenital adrenal hyperplasia due to 11 beta-hydroxylase deficiency.

The kinetic features of 11-deoxycortisol (S) were studied in a 11 beta-hydroxylase deficient boy. After i.v. administration of 35 kBq [3H]S (11 pmol) together with 44 nmol [13C]cortisol all his urine was collected during the next 3 days. A recently reported kinetic model, by which the fate of radioactive cortisol (F) in the body can be described by analysis of only the urinary radioactivity, has been used to calculate the rate constants of S metabolism. The overall half-life of S in the circulation was 4.7 min, which is very close to a reported half-live of the rapid phase: 4.1 min determined from the plasma radioactivity. The time of maximal accumulation of S in the first metabolic pool--26 min is about one quarter of that found for F--109 +/- 20 min (n = 8). The half-live of the S metabolites in the body was 7.0 h, equal to that of F: 6.1 +/- 0.9 h (n = 8). Obviously S is taken up into the metabolic organs 4 times faster than F, but it is not metabolized faster. The production rates of S and F were 127 and 2.1 mumol/(m2*d), respectively, pointing to a severely deficient synthesis of F. However, from the urinary excretion of 3 alpha,21-dihydroxy-5 beta-pregnan-20-one in relation to 3 alpha,11 beta,21-trihydroxy-5 beta-pregnan-20-one it cannot be concluded that the synthesis of corticosterone was strongly impaired.

Synthesis and identification of twelve A-ring reduced 6 alpha- and 6 beta-hydroxylated compounds derived from 11-deoxycortisol, corticosterone and 11-dehydrocorticosterone.

The synthesis and identification of 12 A-ring reduced 6 alpha-(and 6 beta-)hydroxylated compounds derived from 11-deoxycortisol (S), corticosterone (B) and 11-dehydrocorticosterone (A) are reported here. These steroids were prepared in two steps from the corresponding 6 6 alpha-(and 6 beta-)hydroxy-4-pregnene-3-ones. Selective reduction of the 4,5 double bond yielded 12 6 alpha-(and 6 beta)hydroxy-5 alpha-(and 5 beta)pregnane-3,20-diones. Enzymatic reduction of these compounds with NADH and 3 alpha-hydroxysteroid dehydrogenase yielded the corresponding tetrahydro steroids. The steroids were characterized by high performance liquid chromatography (HPLC), gas chromatography mass spectrometry (GC and GC/MS) and in part by 1H-NMR. 6 beta OH-THS and 6 beta OH-5 alpha THS were identified by 1H-NMR. The structures of the two precursors, i.e. 6 beta OH-5 beta DHS and 6 beta OH-5 alpha DHS were confirmed by 1H-NMR using two-dimensional spectra. 6 alpha OH-THS was identified by comparing its HPLC, GC and MS data with those of the steroid obtained by enzymatic oxidation of the standard reference steroid 6 alpha OH-20 beta HHS to the corresponding 20-ketosteroid. The other steroids, e.g. 6 alpha OH-THB and 6 alpha OH-5 alpha THB were identified by using the proved sequence of elution of each of the epimer pairs on the normal phase HPLC column (5 alpha < 5 beta), and by the reversed order of elution of the same epimer pair as the methoxime-trimethylsilyl ethers on the GC column (5 alpha > 5 beta) and by the mass spectra, with the exception of 6 beta OH-THA.

New identified 15 beta-hydroxylated 21-deoxy-pregnanes in congenital adrenal hyperplasia due to 21-hydroxylase deficiency.

The identification of 3 new 15 beta-hydroxylated 21-deoxy-pregnanes in the urinary steroid profile of a 4-month-old girl with congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21OHD) is reported here. These steroids were identified by gas chromatography and gas chromatography-mass spectrometry as 3 alpha,15 beta,17-trihydroxy-5 alpha-pregnan-20-one (5 alpha II), 3 alpha,15 beta,17,20 alpha-tetrahydroxy-5 alpha-pregnane, and 3 alpha,15 beta,17,20 alpha-tetrahydroxy-5 beta-pregnane (20 alpha DH-II). Two other compounds in the urine, 3 beta,15 beta,17- trihydroxy-5 alpha-pregnan-20-one and 3 beta,15 beta,17-trihydroxy-5 beta-pregnan-20-one were also characterized. The identification of the former 3 steroids was obtained by comparing their methylene unit values and mass spectral data with the corresponding data of the standard steroids synthesized from 15 beta,17-dihydroxy-4-pregnene-3,20-dione. Seven other synthesized and identified 15 beta-hydroxylated steroids were 3 alpha,15 beta,17-trihydroxy-5 beta-pregnan- 20-one (II), 3 alpha,15 beta,17,20 beta-tetrahydroxy-5 beta-pregnane, 15 beta,17-dihydroxy-5 alpha-pregnane-3,20-dione, 15 beta,17-dihydroxy-5 beta-pregnane-3,20-dione, 3 alpha,15 beta-dihydroxy-5 alpha-androstan-17-one (15 beta OH-An), 3 alpha,15 beta-dihydroxy-5 beta-androstan-17-one (15 beta OH-Et) and 3 alpha,15 beta,17,20 beta- tetrahydroxy-5 alpha-pregnane. Of these the latter two have not been reported previously. This study supports the findings that 15 beta-hydroxylated steroids are common in the neonate and could play an important role in the diagnosis of CAH due to 21OHD, where II and the newly identified steroids from this investigation viz., 5 alpha II and 20 alpha DH-II appear the most important 15 beta-hydroxysteroid markers for this disease.

Optimization of a classical aromatase activity assay and application in normal, adenomatous and malignant breast parenchyma.

The tritium water release assay, originally described for the analysis of aromatase activity in placental tissue, was used to estimate aromatase activity in breast tissue samples. The lower activity in this tissue necessitates longer incubation times and thus optimization of the assay conditions. To prevent oxidative and proteolytic inactivation of aromatase, dithiothreitol and albumin were added to the incubation mixture. Extra NADPH, cofactor in the aromatase reaction, also improved reaction rate in placental incubations, but after approximately 120 min activity rapidly decreased. Inhibitors gradually produced during the incubation could explain this phenomenon. Quantitative gas chromatography-mass spectrometry (GC-MS) analyses of testosterone, oestradiol, oestrone and androstenedione after incubation with non-labelled androstenedione proved that a substantial amount of the substrate is converted into testosterone. Qualitative GC-MS steroid profiling of the incubation mixture demonstrated the presence of hydroxylated oestradiol and hydroxylated testosterone, produced during incubation, which could have caused partial aromatase inhibition. The adjusted assay was used to analyse 84 breast tissue samples, histologically classified as normal, adenoma or carcinoma. Aromatase activity was found in 56% of all samples and ranged from 0.6 to 26 pmol oestrogen/g protein per hour. Aromatase positivity was found in 80% of the normal samples, 56% of the adenoma samples and 48% of the carcinoma samples. Although carcinoma samples were less often aromatase positive than normal tissue samples (chi2 = 4.80; P < 0.050) there was no difference in absolute aromatase activity. Because no less than approximately 50% of the carcinomas contained aromatase activity and because of the non-routine character of the assay we conclude that it is justified to start aromatase inhibition therapy without previous knowledge of the aromatase status.

Effect of simvastatin in addition to chenodeoxycholic acid in patients with cerebrotendinous xanthomatosis.

The effects of combination therapy with chenodeoxycholic acid (CDCA) and simvastatin on serum cholestanol, low-density lipoprotein (LDL) cholesterol, and lathosterol levels were investigated in seven adult patients with cerebrotendinous xanthomatosis (CTX) who were on long-term treatment with CDCA. The patients were treated with a combination of CDCA 750 mg daily and an increasing dose of simvastatin from 10 mg to 40 mg daily for a period of 6 months. We found a significant effect of this combination therapy compared with CDCA alone in terms of decreasing the serum cholestanol and LDL cholesterol levels, particularly with a daily dose of 40 mg simvastatin. The mean cholestanol level decreased from 9.27 micromol/L (baseline) to 6.69 micromol/L (40 mg simvastatin), while the mean LDL cholesterol level decreased from 5.08 mmol/L (baseline) to 3.04 mmol/L (40 mg simvastatin). No side effects were reported, and there were no effects on the clinical condition, cerebral magnetic resonance imaging (MRI), visual evoked potentials, and electroencephalographic features. We conclude that a combination of 750 mg CDCA and 40 mg simvastatin daily is effective to further reduce serum cholestanol, LDL cholesterol, and lathosterol in adult CTX patients treated with long-term CDCA. Whether this combination treatment will be effective for the long-term prevention of neurological deterioration and atherosclerosis remains to be established.

A specific and sensitive determination of gamma aminobutyric acid in CSF and brain tissue by gas chromatography-mass spectrometry.

The determination of gamma-aminobutyric acid (GABA) in cerebrospinal fluid and brain extracts is described. Its heptafluorobutyryl-isobutanol derivative was measured both by electron impact and chemical ionization mass fragmentography using GABA-d6 as internal standard. The derivatization product is stable for several days. The method is sensitive (1 ng absolute in cerebrospinal fluid and 30 pg in standard GABA solutions) and specific, when chemical ionization mode is applied. Normal values of GABA are in rat brain extracts (1.40 +/- 0.32 mumol/g fresh weight) and human CSF (18.3 +/- 10.0 ng/ml).

Capillary gas chromatography of urine samples in diagnosing cerebrotendinous xanthomatosis.

A patient is described with many clinical features of cerebrotendinous xanthomatosis (CTX), but with only slightly elevated cholestanol/cholesterol concentration ratios in serum and xanthomatous tissue. However, with capillary gas chromatographic determinations of urinary bile acids and bile alcohols we demonstrated the typical biochemical abnormalities as seen in CTX patients. This article emphasizes the value of urinary capillary gas chromatography as a specific test to establish the diagnosis of CTX and to monitor the biochemical effectivity of the different treatment regimens.

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.

The determination of total cholesterol in serum by gas-liquid chromatography compared with two other methods.

A gas-liquid chromatographic method is described for the measurement of total cholesterol in serum. The method has been found to be simple, specific and precise. The results have been compared with the results of the enzymatic method of Röschlau, P., Bernt, E. and Gruber, W. (1974) (Z. Klin. Chem. Klin. Biochem. 12, 403) and with the results of an Auto-Analyzer method based on the manual method of Huang, T.C., Chen, C.P., Wefler, V. and Raftery, A. (1961) (Anal. Chem. 33, 1405) and Ness, A.T., Pastewka, J.V. and Peacock, A.C. (1964) (Clin. Chim. Acta 10, 229). The results of the gas-liquid chromatographic and of the enzymatic method show a high degree of correlation. The results of the Auto-Analyzer method are about 0.75 mmol/1 higher than those of the other two methods. The conclusion is drawn that the gas-liquid chromatographic method should be given consideration as a reference method for the measurement of total cholesterol in serum. It is a viable alternative for the generally accepted colorimetric reference method of Abell, L.L., Levy, B.B., Brodie, B.B. and Kendall, F.E. (1952) (J. Biol. Chem. 195, 357).

Volatile fatty acids in human peripheral and portal blood: quantitative determination vacuum distillation and gas chromatography.

In order to determine volatile fatty acids (VFA) in human blood, gas chromatographic analyses were performed after blood samples had been pre-treated by a vacuum distillation procedure with subsequent evaporation. Results of the reproducibility study, investigated by pre-treating five aliquots of one human serum sample showed C.V. values ranging from 7.4 to 18.2. Normal serum VFA values were determined in individual serum samples collected from healthy subjects and compared to those present in serum from patients undergoing gall-bladder surgery. The serum VFA values were comparable in the two groups. In the surgical patients, blood was also collected from the portal circulation. All portal serum VFA values, except that of iso-butyric acid, were higher than those found in the samples collected from the peripheral arm vein. VFA values were also determined in serum specimens obtained from blood collected from the arm vein of patients with extensive cirrhosis of the liver. The VFA values showed marked individual variations and were higher than those found in peripheral samples from healthy subjects and patients undergoing gall-bladder surgery, but were lower than those found in samples from the portal circulation from the surgical patients.