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Item does not contain fulltext A method for the determination of metadrenaline (MA), normetadrenaline (NMA) and 3-methoxytyramine (3-MT) in human urine using high-performance liquid chromatography followed by electrochemical detection (ECD) was validated primarily by comparing the results with those obtained by a gas chromatography-mass spectrometry (GC-MS) reference method. Correlation coefficients of 0.93, 0.94 and 0.91 were obtained for MA, NMA and 3-MT, respectively, in a group of healthy controls consisting of 30 women and 30 men. A systematic difference was detected only for 3-MT (-16%). Further tests of accuracy (linearity and recovery) and precision demonstrated that the described method must be considered to be a reliable approach to assess urinary metadrenalines in the diagnosis of phaeochromocytoma. At lower concentrations (MA, 248 nmol/L; NMA, 434 nmol/L; 3-MT, 402 nmol/L), within-assay coefficients of variation were close to 5% or less (5.3, 4.6 and 2.2%, respectively) and between-assay coefficients of variation were 8.9, 11.2 and 12.3%, respectively, for the same low levels. This raises the possibility that this method can also be applied to assess urinary free, unconjugated metanephrines. Sex differences were detected for MA and NMA excretion when expressed in nmol per 24 h and nmol/mmol creatinine, respectively, by both ECD and GC-MS methods.

In een dubbelblinde placebo gecontroleerde, vergelijkende studie werd aan 16 histamine intolerante patienten en aan 16 gezonde proefpersonen histamine toegediend. Het doel van de studie was het ontwikkelen van een relatief simpele en betrouwbare test voor het stellen van de diagnose histamine-intolerantie onder de aanname dat histamine-intolerante patienten, gastro-intestinaal, een reletief tekort aan het enzym diamine oxidase (DAO) hebben. Histamine werd zowel intracutaan als oraal toegediend. Bijwerkingen werden geregistreerd, de bloeddruk gemeten en bloed en urine verzameld. In het bloed werd de concentratie histamine en methylhistamine gemeten. In de urine werden N-methyl-imidazol-azijnzuur en N-methylhistamine bepaald. Noch intracutane toediening van histamine, noch orale toediening van histamine kan vooralsnog gebruikt worden als valide test om histamine-intolerantie te diagnostiseren, aangezien voor geen enkele parameter significante verschillen zijn gevonden tussen patienten en vrijwilligers.

Objectives: To evaluate Near-Infrared Analysis (NIRA) method for determining fecal fat, water and nitrogen. Design and methods: The results of fecal fat, water and nitrogen by NIRA were compared with results of van de Kamer and Acid Steatocrit (AS), Dumas and vacuum drying methods for fat, nitrogen and water respectively. Results of fat determining methods were also compared with total fecal energy as obtained by bomb calorimeter. Results: NIRA results correlated significantly (p

A method for the determination of metanephrine (MN, also known as metadrenaline), normetanephrine (NMN also known as normetadrenaline) and 3-methoxytyramine (3-MT) in human urine using high-performance liquid chromatography followed by electrochemical detection (ECD) was validated primarily by comparing the results with those obtained by a gas chromatography-mass spectrometry (GC-MS) reference method. Correlation coefficients of 0.93, 0.94 and 0.91 were obtained for MN, NMN and 3-MT, respectively. in a group of healthy controls consisting of 30 women and 30 men. A systematic difference was detected only for 3-MT (-16%). Further tests of accuracy (linearity and recovery) and precision demonstrated that the described method must be considered to be a reliable approach to assess urinary metanephrines in the diagnosis of phaeochromocytoma. At lower concentrations (MN, 248 nmol/L, NMN, 434 nmol/L; 3-MT, 402 nmol/L), within-assay coefficients of variation were close to 5% or less (5.3, 4.6 and 2.2%, respectively) and between-assay coefficients of variation were 8.9, 11.2 and 12.3%, respectively, for the same low levels. This raises the possibility that this method can also be applied to assess urinary free, unconjugated metanephrines. Sex differences were detected for MN and NMN excretion when expressed in nmol per 24h and nmol/mmol creatinine, respectively, by both ECD and GC MS methods.

A method for the determination of metadrenaline (MA). normetadrenaline (NMA) and 3-methoxytyramine (3-MT) in human urine using high-performance liquid chromatography followed by electrochemical detection (ECD) was validated primarily by comparing the results with those obtained by a gas chromatography mass spectrometry (GC MS) reference method. Correlation coefficients of 0.93, 0.94 and 0.91 were obtained for MA, NMA and 3-MT. respectively, in a group of healthy controls consisting of 30 women and 30 men. A systematic difference wits detected only for 3-MT (-16%). Further tests Of accuracy (linearity and recovery) and precision demonstrated that the described method must be considered to be a reliable approach to assess urinary metadrenalities in the diagnosis of phaeochromocytoma. At lower concentrations (MA, 248 nmol L; NMA. 434 nmol, L; 3-MT, 402 nmol/L), within-assay coefficients of variation were close to 5%, or less (5(.)3, 4(.)6 and 2(.)2%, respectively) and between-assay, Coefficients Of Variation were 8(.)9. 11(.)2 and 12(.)3%, respectively, for the same low levels. This raises the possibility that this method can also be applied to assess urinary free, unconjugated metanephrines. Sex differences were detected for MA and NMA excretion Mien expressed in nmol per 24h and nmol/mmol creatinine, respectively, by both ECD and GC MS methods.

OBJECTIVES To test whether insulin resistance in type 2 diabetes mellitus is associated with an altered overall setpoint of the 11 beta-hydroxysteroid dehydrogenase (11 beta HSD) mediated cortisol to cortisone interconversion towards cortisol, and to evaluate whether changes in insulin sensitivity induced by antecedent hyperinsulinaemia are related to changes in the 11 beta HSD setpoint. PATIENTS AND MEASUREMENTS The urinary ratio of (tetrahydrocortisol + allo-tetrahydrocortisol)/tetrahydrocortisone ((THF + allo-THF)/THE) and of free cortisol/free cortisone (UFF/UFE), as well as the plasma cortisol/cortisone ratio were measured in 8 male type 2 diabetic patients and 8 healthy male subjects without and after 24 h of insulin infusion. Insulin was infused at a rate of 30 mU/kg/h with blood glucose being clamped at euglycaemic levels in healthy subjects and at isoglycaemic levels in diabetic patients. Insulin sensitivity was assessed by measurement of whole body glucose uptake (M-value) during a 3-4 h euglycaemic clamp, directly after the 24 h insulin infusion and compared to the M-value on a control day, at least 1 week apart from the 24 h insulin infusion. RESULTS Despite impaired insulin sensitivity (M-value, 11.6 +/- 7.7 vs. 28.5 +/- 11.6 mu mol/kg/minutes, in type 2 diabetic and healthy subjects, respectively, P

Objectives. Liquorice abuse can lead to severe clinical complications, caused by its active compound 18 beta-glycyrrhetinic acid (18 beta GA). 18 beta GA inhibits dehydrogenase activity of 11 beta-hydroxysteroid dehydrogenase (11 beta HSD). This enzyme catalyses the interconversion between cortisol and cortisone and normally protects the mineralocorticoid receptor from being activated by cortisol. Diagnosing liquorice abuse can be notoriously difficult. The aim of our study was to develop an accurate and clinically applicable 18 beta GA urinary assay. Design. We developed a urinary 18 beta GA assay based on gas chromatography and mass spectrometry (GCMS) with sufficient sensitivity to detect 18 beta GA at low concentrations. The assay was validated in four volunteers consuming different amounts of liquorice. We applied its use in two patients with hypokalaemic hypertension and suppressed plasma renin activity and serum aldosterone, who were suspected of liquorice abuse. Results. The detection limit for 18 beta GA of the GC assay was 10 mu g L-1, which was lowered to 3 mu g L-1 by subsequent application of MS. In all volunteers, urinary 18 beta GA was detected during liquorice intake. Urinary 18 beta GA remained detectable until 5 days after stopping continued liquorice intake and until at least 51 h after ingestion of a single large amount. Urinary 18 beta GA was demonstrated in both patients, establishing a diagnosis of liquorice abuse. One patient showed changes in urinary cortisol metabolites, consistent with 11 beta HSD inhibition. Changes in cortisol metabolites were less pronounced in the other patient. Conclusion. Liquorice abuse can result in hypokalaemic hypertension with prolonged suppression of plasma renin activity and aldosterone concentration. This is caused by 18 beta GA-mediated inhibition of 11 beta HSD, resulting in activation of the renal mineralocorticoid receptor by cortisol. Urinary 18 beta GA measurement by GCMS is a useful aid in establishing liquorice abuse.

A sensitive and quantitative gas chromatographic assay for the determination of 18 beta-glycyrrhetinic acid (18 beta-GA), the main metabolite of glycyrrhizin after oral licorice consumption in human urine, has been developed and validated. For the extraction of 18 beta-GA from urine two Sep-Pak C-18 extractions, hydrolysis with Helix pomatia and three liquid-liquid extractions were performed, using 18 alpha-glycyrrhetinic acid (18 alpha-GA) as internal standard. Both 18 beta-GA and internal standard were converted into their pentafluorobenzyl- ester/ trimethylsilyl-ether derivatives and detected by flame ionization detection using a WCOT-fused-silica capillary column. Good quality control data were obtained in precision and accuracy tests. The detection limit of the gas chromatographic method was 10 mu g/l with a urine volume of 10 ml. A detection limit of 3 mu g/l was obtained by performing GC-MS. The GC method was used to monitor the urinary excretion of 18 beta-GA after licorice consumption by two healthy volunteers and a patient suspected of licorice abuse. Furthermore, it was shown that this GC assay enables to detect other metabolites related to licorice consumption. (C) 1999 Elsevier Science B.V. All rights reserved.

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] mu mol/(m(2).day), sCPR (method a) was 28.8 +/- 4.5 [range: 23.5-34.3] mu mol/(m(2).day), sCPR (method b) was 27.9 +/- 8.1 [range: 18.5-37.7] mu mol/(m(2).day), and sCPR (method c) was 29.3 +/- 4.8 [range: 22.7-33.2] mu mol/(m(2).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 mu mol/(m(2).day) [9-11 mg/(m(2).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.

We report a new mutation in the sterol 27-hydroxylase (CYP 27) gene in a Dutch family with cerebrotendinous xanthsmatosis: a G-->A transition in the splice donor site in intron 4. This mutation leads to skipping of exon 4. resulting in a loss of 66 amino acids in the CYP 27 enzyme molecule.

1. Stable urea isotopes can be used to study urea kinetics in humans, The use of stable urea isotopes far studying urea kinetic parameters in humans on a large scale is hampered by the high costs of the labelled material, We devised a urea dilution for measurement of the distribution volume, production rate and clearance of urea in healthy subjects and renal failure patients using the inexpensive single labelled [C-13]urea isotope with subsequent analysis by headspace chromatography-isotope ratio MS (GC-IRMS) of the [C-13]urea enrichment, 2. The method involves measurement of the molar percentage excess of [C-13]urea in plasma samples taken over a 4 h period after an intravenous bolus injection of [C-13]urea, During the sample processing procedure, the plasma samples together with calibration samples containing a known molar percentage excess of [C-13]urea are acidified with phosphoric acid to remove endogenous CO2, and are subsequently incubated with urease to convert the urea present in the plasma samples into CO2. The C-13 enrichment of the generated CO2 is analysed by means of GC-IRMS, This method allows measurement of the molar percentage excess of [C-13]urea to an accuracy of 0.02%. 3. Reproducibility studies showed that the sample processing procedure [within-run coefficient of variation (CV) 4. In clinical urea kinetic studies in a healthy subject and in a renal failure patient without residual renal function, reproducible values of the distribution volume, production rate and clearance of urea were determined using minimal amounts of [C-13]urea (25-50 mg). 5. because only low [C-13]urea enrichments are needed in this urea dilution method using GC-IRMS analysis, the costs of urea kinetic studies are reduced considerably, especially in patients with renal failure.

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.

11 beta-Hydrdxysteroid 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 isa well documented phenomenon in insulin-dependent diabetes mellitus (IDDM), but it is not known whether diabetes-associated alterations in cortisol metabolism contribute td its pathogenesis. Therefore, we evaluated some aspects of cortisol metabolism by measuring urinary metabolites of cortisol and cortisone in eight microalbuminuric and eight normoalbuminmuric 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

Infrared (IR) spectroscopy is used to analyze urinary calculus (renal stone) constituents. However, interpretation of IR spectra for quantifying urinary calculus constituents in mixtures is difficult, requiring expert knowledge by trained technicians. In our laboratory IR spectra of unknown calculi are compared with reference spectra in a computerized library search of 235 reference spectra from various mixtures of constituents in different proportions, followed by visual interpretation of band intensities for more precise semiquantitative determination of the composition. To minimize the need for this last step, we tested artificial neural network models for detecting the most frequently occurring compositions of urinary catculi. Using constrained mixture designs, we prepared various samples containing ammonium hydrogen urate, brushite, carbonate apatite, cystine, struvite, uric acid, weddellite, and whewellite for use as a training set. We assayed known artificial mixtures as well as selected patients' samples from which the semiquantitative compositions were determined by computerized library search followed by visual interpretation. Neural network analysis was more accurate than the library search and required less expert knowledge because careful visual inspection of the band intensities could be omitted. We conclude that neural networks are promising tools for routine quantification of urinary calculus compositions and for other related types of analyses in the clinical laboratory.

A simple and fast HPLC method for the determination of porphyrins in bile without extraction is described. Porphyrins were determined in bile from control subjects and from patients after orthotopic liver transplantation, including three patients with erythropoietic protoporphyria. It was found that: 1) coproporphyrin I is the predominant porphyrin in bile of controls, accompanied by some coproporphyrin III and protoporphyrin, whereas protoporphyrin mostly but not always is the predominant porphyrin in the bile of erythropoietic protoporphyria patients. In two of the three erythropoietic protoporphyria patients, the bile contained a hundred times more protoporphyrin than that of non-porphyric orthotopic liver transplantation patients. The third erythropoietic protoporphyria patient remained cholestatic and was unable to excrete sufficient amounts of protoporphyrin. 2) All investigated bile samples contained no secondary porphyrins derived from protoporphyrin, i. e. no deutero-, pempto-, or mesoporphyrin. Even when extracts of bile and serum were concentrated fifty to a hundred times, no traces of deutero-, pempto- and mesoporphyrin were detected. This complete absence of secondary porphyrins suggests that an enterohepatic circulation of dicarboxylic porphyrins from the distal gastrointestinal tract does not exist. 3) The HPLC chromatograms contain peaks from unknown compounds. No correlation between porphyrins and these compounds was found. Porphyrin profiles were followed in the bile of some orthotopic liver transplantation patients. Three episodes are recognizable. During the first three days after orthotopic liver transplantation there is a very high coproporphyrin excretion. There is then a lag of one to three weeks, in which no or very low porphyrin concentrations are detectable, followed by the restoration of normal biliary porphyrin patterns.

OBJECTIVES We developed an assay for delta-5-androstenediol (adiol) and delta-5-androstenediol-3-sulphate (adiol-3S) in serum and adiol and total adiol sulphate (adiol-S) in urine. DESIGN An analytical procedure using HPLC and gas chromatography-mass spectrometry was devised and tested for its reliability. MEASUREMENTS After addition of deuterated androstenediol as internal standard, serum and urine samples were extracted. Steroid sulphates were hydrolysed. The extracts and hydrolysates were purified on HPLC, adiol was derivatized using heptafluorobutyric anhydride and finally quantified by gas chromatography-mass spectrometry. RESULTS The assay is accurate and reproducible. The coefficient of variation (CV) for the determination of adiol in serum samples is 4% (intra-assay) and 9% (interassay) and for urine samples 3 and 8% respectively. The intraassay CV for the adiol-3S analyses is 5% for serum and 2% for urine samples while the interassay CV values for adiol-3S are 10% for serum and 7% for urine samples. The recovery of adiol and adiol-3S from serum and urine samples is 97%. CONCLUSIONS The developed assay meets the analytical demands needed for clinical applications.

OBJECTIVES We evaluated the role of delta-5-androstenediol (adiol) and its sulphates in health and endocrine diseases. DESIGN Serum and urine samples from healthy adult men and pre and post-menopausal women were analysed by gas chromatography-mass spectrometry to establish reference values. In patients who were either evaluated or treated for endocrine diseases, sequential serum samples were collected and analysed. PATIENTS Reference values were obtained from 24 healthy male, 23 premenopausal and 30 post-menopausal female volunteers. Adiol and delta-5-androstenediol-3-sulphate (adiol-3S) concentrations were determined in combination with other relevant steroids in patients with either pituitary (n = 5), adrenal (n = 2) or gonadal dysfunction (n = 1), or testicular carcinoma (n = 19). MEASUREMENTS After addition of deuterated adiol as internal standard, serum and urine samples were extracted. Steroid sulphates were hydrolysed. The extracts and hydrolysates were purified on HPLC, adiol was derivatized and finally quantified by gas chromatography-mass spectrometry. RESULTS The calculated reference ranges for adiol and adiol-3S concentrations in serum are respectively: in men 1.78-7.24 and 123-579 nmol/l, in premenopausal women 0.65-6.93 and 21.2-298 nmol/l and in post-menopausal women 0.29-2.90 and 6-1-184 nmol/l. Urinary values varied considerably. In the population with endocrine abnormalities serum adiol and adiol-3S concentrations were compared with other relevant steroids. CONCLUSIONS The wide concentration range of adiol and adiol-3S in urine makes analysis of these steroids in urine of little clinical value. Serum concentrations of adiol and adiol-3S are higher in men than in women. Premenopausal values are higher than post-menopausal. Adiol and adiol-3S in serum are significantly correlated in pre and post-menopausal women, r = 0.51 and r = 0.69 respectively, but not in men. In endocrine patients the serum concentrations of adiol show an ACTH or LH dependency in women; adiol correlates with cortisol, dehydroepiandrosterone or androstenedione and, in males, additionally with testosterone. However, in several situations adiol correlates with none of these steroids. Although adiol secretion can be stimulated by ACTH and LH, the level of serum adiol is also determined by other factors. Finally, in adrenal carcinoma serum adiol and adiol-3S may be used as tumour markers.

This report describes the synthesis of 6alpha,17,21- and 6beta,17,21-trihydroxypregn-4-ene-3,20-dione, 6alpha,7,21- and 6beta,11beta,21-trihydroxypregn-4-ene-3,20-dione, and-for the first time-that of 6alpha,21- and 6beta,21-dihydroxypregn-4-ene-3,11,20-trione. The former four compounds were prepared by 6-hydroxylation of 17,21-trihydroxypregn-4-ene-3,20-dione and 11beta,21-dihydroxypregn-4-ene-3,20-dione, respectively. This was achieved by autoxidation or by oxidation with 3-chloroperbenzoic acid, of the 3-methoxy-pregna-3,5-dienes of the latter two steroids. The yield of the 6beta-hydroxylated steroids, but not of their corresponding 6alpha-epimers, was higher using autoxidation than the peracid. The two 6-hydroxylated pregnenetriones were prepared from 6alpha,21-diacetoxy-11beta-hydroxypregn-4-ene-3,20-dione and 6beta,21-diacetoxy-11beta-hydroxypregn-4-ene-3,20-dione, respectively, by oxidation with pyridinium chlorochromate. The above-mentioned six steroids were identified and characterized by nuclear magnetic resonance, infrared, ultraviolet, high performance liquid chromatography, gas chromatography, and mass spectrometry.

Quantitative assessment of urinary calculus (renal stone) constituents by infrared analysis (IR) is hampered by the need of expert knowledge for spectrum interpretation. Our laboratory performed a computerized search of several libraries, containing 235 reference spectra from various mixtures with different proportions. Library search was followed by visual interpretation of band intensities for more precise semiquantitative determination of the composition. We tested partial least-squares (PLS) regression for the most frequently occurring compositions of urinary calculi. Using a constrained mixture design, we prepared various samples containing whewellite, weddellite, and carbonate apatite and used these as a calibration set for PLS regression. The value of PLS analysis was investigated by the assay of known artificial mixtures and selected patients' samples for which the semiquantitative compositions were determined by computerized library search followed by visual interpretation. Compared with that method, PLS analysis was superior with respect to accuracy and necessity of expert knowledge. Apart from some practical limitations in data-handling facilities, we believe that PLS regression offers a promising tool for routine quantification, not only for whewellite, weddellite, and carbonate apatite, but also for other compositions of the urinary calculus.

The identification of 3 new 15beta-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 3alpha,15beta,17-trihydroxy-5alpha-pregnan-20-one (5alphaII), 3alpha,15beta,17,20alpha-tetrahydroxy-5alpha-pregnane, and 3alpha,15beta,17,20alpha-tetra-hydroxy-5beta-pregnane (20alpha DH-11). Two other compounds in the urine, 3beta,15beta,17-trihydroxy-5alpha-pregnan-20-one and 3beta,15beta,17-trihydroxy-5beta-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 15beta,17-dihydroxy-4-pregnene-3,20-dione. Seven other synthesized and identified 15beta-hydroxylated steroids were 3alpha,15beta,17-trihydroxy-5beta-pregnan-20-one (II), 3alpha,15beta,17,20beta-tetrahydroxy-5beta-pregnane, 15beta,17-dihydroxy-5alpha-pregnane-3,20-dione, 15beta,17-dihydroxy-5beta-pregnane-3,20-dione, 15beta,17-dihydroxy-5beta-pregnane-3,20-dione, 3alpha,15beta-dihydroxy-5alpha-androstan-17-one (15betaOH-An), 3alpha,15beta-dihydroxy-5beta-androstan-17-one (15betaOH-Et) and 3alpha,15beta,17,20beta-tetrahydroxy-5alpha-pregnane. Of these the latter two have not been reported previously. This study supports the findings that 15beta-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., 5alphaII and 20alphaDH-II appear the most important 15beta-hydroxysteroid markers for this disease.

Patients with hereditary tyrosinaemia type I(HT) excrete large amounts of succinylacetone (SA) in urine. Owing to structural resemblance of SA to delta-aminolevulinic acid (ALA), SA inhibits the second enzyme in the pathway for haeme biosynthesis, porphobilinogen synthase, resulting in increased urinary ALA excretion. We investigated the relationship between urinary SA and ALA excretions of two patients with different forms of HT (late-infantile and juvenile). In both patients the urinary SA and ALA excretions showed a more or less inverse correlation. The patient with the early-infantile form of HT had a relatively greater increase in urinary SA and ALA excretions in comparison to the patient with the juvenile form of HT. A possible explanation for this unexpected inverse correlation between the urinary excretion of SA and ALA might be a lack of intramitochondrial glycine, a substrate for delta-aminolevulinic acid synthesis. It has been reported previously that high concentrations of SA reversibly and competitively inhibit the transport of glycine through membranes.

The kinetic features of 11-deoxycortisol (S) were studied in a 11beta-hydroxylase deficient boy. After i.v. administration of 35 kBq [H-3]S (11 pmol) together with 44 nmol [C-13]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 3alpha,21-dihydroxy-5beta-pregnan-20-one in relation to 3alpha, 11beta,21-trihydroxy-5beta-pregnan-20-one it cannot be concluded that the synthesis of corticosterone was strongly impaired.

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*QO 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.

[1,2,3,4-C-13]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-mu-mol/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.

The sterol composition of sera from patients with cerebrotendinous xanthomatosis (CTX) was investigated by gas chromatographic analysis of saponified extracts, using a polar (CP Wax 52CB) and an apolar (CP Sil 5CB) capillary column. Apart from already known sterols, the presence of increased amounts of 8-lathosterol (5-alpha-cholest-8(9)-en-3-beta-ol) and significant amounts of 8-dehydrocholesterol (cholesta-5,8-dien-3-beta-ol) were noticed. The latter compound has not been detected previously in human serum and possibly represents a hitherto unknown cholesterol precursor. The apparently elevated levels of DELTA-8-sterols in CTX serum suggests partial inhibition of migration of the 8,9 double bond to the 7,8 position in this condition. The concentration of 7-lathosterol, an indicator of cholesterol production rate, is also highly elevated in CTX serum and quickly returns to normal values after oral bile acid therapy. Determinations of serum lathosterol are not only useful in the follow-up of therapy of CTX patients, but also in the follow-up of hypercholesterolemic patients treated with either HMG-CoA reductase inhibitors or bile acid sequestrants.