Improved synthesis of glycine, taurine and sulfate conjugated bile acids as reference compounds and internal standards for ESI-MS/MS urinary profiling of inborn errors of bile acid synthesis.

Bile acid synthesis defects are rare genetic disorders characterized by a failure to produce normal bile acids (BAs), and by an accumulation of unusual and intermediary cholanoids. Measurements of cholanoids in urine samples by mass spectrometry are a gold standard for the diagnosis of these diseases. In this work improved methods for the chemical synthesis of 30 BAs conjugated with glycine, taurine and sulfate were developed. Diethyl phosphorocyanidate (DEPC) and diphenyl phosphoryl azide (DPPA) were used as coupling reagents for glycine and taurine conjugation. Sulfated BAs were obtained by sulfur trioxide-triethylamine complex (SO3-TEA) as sulfating agent and thereafter conjugated with glycine and taurine. All products were characterized by NMR, IR spectroscopy and high resolution mass spectrometry (HRMS). The use of these compounds as internal standards allows an improved accuracy of both identification and quantification of urinary bile acids.

[Using purified conjugated bilirubin as a calibrator for bilirubin measurement].

OBJECTIVES: To improve the accuracy and precision of the determination of bilirubin, especially direct bilirubin (DB), and the standardization of that as well. METHODS: Purified conjugated bilirubin (Bc) and ditaurobilirubin(DTB) and their diazo products were subjected to absorption spectrum analysis. The diazo reaction characters of their calibration solutions were compared by the method of Doumas J-G(TB & DB). RESULTS: Bc, DTB and their azopigments were found to have the similar absorption spectra with the same lambda max. Their TB standard curves almost superposed together all over. Although the slopes of their DB standard curves were not markedly different ((YBc = 0.00366X + 0.00933, rBc2 = 0.9977, P < 0.01; YDTB = 0.00391X + 0.00023, rDTB2 = 0.9987, P < 0.01; Pb1-b2 > 0.05, n1 = n2 = 5), the DB value measured for Bc differed from that for DTB(n = 5, P < 0.05). In addition, the calibrators made from Bc based different matrices, such as HSA, BSA and human serum, were significantly different in DB/Bc, but no difference was seen among the concentrations. Furthermore, the DB values determined for DTB or Bc increased linearly with the corresponding concentrations, respectively, with no difference between the slopes (YBc = 0.8300XBc + 1.9463, rBc2 = 0.9977, P < 0.01; YDTB = 0.8853XDTB-0.0251, rDTB2 = 0.9986, P < 0.01; n1 = n2 = 5, Pb1-b2 > 0.05). CONCLUSIONS: The results demonstrate that the diazo reaction characters of Bc are identified with those of DTB. However, under the condition of DB, Bc reacts differently from DTB. This study also indicates that as a calibrator of DB based human serum, Bc has the similar constant effect of HCl as serum samples do, so it is a more reliable calibrator to eliminate the matrix effects.

The European acamprosate trials: conclusions for research and therapy.

In an excellent methodological approach, the European acamprosate study project showed that acamprosate increases sobriety times. In one randomized prospective study (n = 260) comparing acamprosate and placebo, with a 1-year treatment phase and 1-year follow-up phase, the authors found that acamprosate is effective only in Lesch type I and type II patients. To investigate the possible influence of diagnostic subgrouping, we applied the Lesch typology in a co-work with the main researchers of the UK study. The UK results concerning acamprosate's effects in the types do not mirror the Vienna results, but the numbers of type I and type II patients, retrospectively found as included in the UK centers, were too small for any conclusions. The distribution of the types points to the fact that too many type III and IV patients had been included to give acamprosate the chance to be effective. Following our typology and also these studies, we developed special treatment approaches. For relapse prevention studies, the cumulative abstinence duration together with the Lesch typology seems to be promising.