This paper is the sixth in a series dealing with reference procedures for the measurement of catalytic activity concentrations of enzymes at 37degreesC and the certification of reference preparations. Other parts deal with:Part 1. The Concept of Reference Procedures for the Measurement of Catalytic Activity Concentrations of Enzymes; Part 2. Reference Procedure for the Measurement of Catalytic Concentration of Creatine Kinase; Part 3. Reference Procedure for the Measurement of Catalytic Concentration of Lactate Dehydrogenase; Part 4. Reference Procedure for the Measurement of Catalytic Concentration of Alanine Aminotransferase; Part 5. Reference Procedure for the Measurement of Catalytic Concentration of Aspartate Aminotransferase; Part 7. Certification of Four Reference Materials for the Determination of Enzymatic Activity of gamma-Glutamyltransferase, Lactate Dehydrogenase, Alanine Aminotransferase and Creatine Kinase at 37degreesCA document describing the determination of preliminary upper reference limits is also in preparation. The procedure described here is deduced from the previously described 30degreesC IFCC reference method (1). Differences are tabulated and commented on in Appendix 1.

This paper is the eighth in a series dealing with reference procedures for the measurement of catalytic activity concentrations of enzymes at 378C and the certification of reference preparations. Other parts deal with: Part 1. The concept of reference procedures for the measurement of catalytic activity concentrations of enzymes; Part 2. Reference procedure for the measurement of catalytic concentration of creatine kinase; Part 3. Reference procedure for the measurement of catalytic concentration of lactate dehydrogenase; Part 4. Reference procedure for the measurement of catalytic concentration of alanine aminotransferase; Part 5. Reference procedure for the measurement of catalytic concentration of aspartate aminotransferase; Part 6. Reference procedure for the measurement of catalytic concentration of g-glutamyltransferase; Part 7. Certification of four reference materials for the determination of enzymatic activity of g-glutamyltransferase, lactate dehydrogenase, alanine aminotransferase and creatine kinase at 378C. The procedure described here is deduced from the previously described 308C IFCC reference method. Differences are tabulated and commented on. Clin Chem Lab Med 2006;44:1146–55. Schumann et al.: IFCC primary reference procedures 1147 Article in press uncorrected proof Table 2 Conditions for the measurement of a-amylase. Temperature 37.08C"0.18C Wave length 405 nm "1 nm Band width F2 nm Light path 10.00 mm "0.01 mm Incubation time 60 s Delay time 180 s Measurement interval 180 s Readings (measurement points) G6 Table 1 Concentrations in the final complete reaction mixture for the measurement of a-amylase. N-2-Hydroxyethylpiperazine-N9-ethanesulfonic acid 50 mmolOl pH (378C) 7.00"0.03 4,6-Ethylidene(G1)-4-nitrophenyl(G7)-a-(1TM4)-D-maltoheptaoside 5 mmolOl Sodium chloride 70 mmolOl Calcium chloride 1 mmolOl a-Glucosidase (378C) 135 mkatOl (8100 UOl)* Volume fraction of sample 0.0323 (1:31) *Uninhibited catalytic concentration. Note: The indicated catalytic concentration of a-glucosidase shall be measured in the final complete reaction mixture if 9 gOl (154 mmolOl) sodium chloride solution is used as the sample (no inhibition by the sample matrix). Note: Besides the substances listed in Table 1, the final complete reaction mixture contains 0.1 gOl albumin, which is originally a component of Solution 3. The presence of albumin in the Reaction Solution and consequently in the final complete reaction mixture stabilizes the a-glucosidase.

This paper is the seventh in a series dealing with reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 degrees C and the certification of reference preparations. Other parts deal with: Part 1. The Concept of Reference Procedures for the Measurement of Catalytic Activity Concentrations of Enzymes; Part 2. Reference Procedure for the Measurement of Catalytic Concentration of Creatine Kinase; Part 3. Reference Procedure for the Measurement of Catalytic Concentration of Lactate Dehydrogenase; Part 4. Reference Procedure for the Measurement of Catalytic Concentration of Alanine Aminotransferase; Part 5. Reference Procedure for the Measurement of Catalytic Concentration of Aspartate Aminotransferase; Part 6. Reference Procedure for the Measurement of Catalytic Concentration of Gamma-Glutamyltransferase. A document describing the determination of preliminary reference values is also in preparation. The certification of the catalytic activity concentrations as determined by the recently elaborated IFCC primary reference methods at 37 degrees C of four enzyme preparations, namely IRMM/IFCC 452 (gamma-glutamyltransferase), IRMM/IFCC 453 (lactate dehydrogenase 1), IRMM/IFCC 454 (alanine aminotransferase) and IRMM/IFCC 455 (creatine kinase) is described. Homogeneity data were derived from previous results. Stability was assessed using recently obtained data as well as data from previous stability studies. The collaborative study for value assignment was performed under a strict quality control scheme to ensure traceability to the primary reference method. Uncertainty of the materials was assessed in compliance with the Guide to the Expression of Uncertainty in Measurement. The certified values obtained at 37 degrees C are 1.90 microkat/l +/- 0.04 microkat/l (114.1 U/l +/- 2.4 U/l), for gamma-glutamyltransferase, 8.37 microkat/l +/- 0.12 microkat/l (502 U/l +/- 7 U/l), for lactate dehydrogenase 1, 3.09 microkat/l +/- 0.07 microkat/l (186 U/l +/- 4 U/l), for alanine aminotransferase and 1.68 microkat/l +/- 0.07 microkat/l (101 U/l +/- 4 U/l), for creatine kinase. The materials are intended for internal quality control as well as for the evaluation of test systems as required by recent European Union legislation. Furthermore, the materials can be used to transfer accuracy from a reference method to a routine procedure provided the procedures exhibit the same analytical specificity and the certified materials are commutable.

Uncertainties of four enzyme-CRMs that have recently been certified in a co-operation between the IRMM and the International Federation for Clinical Chemistry were estimated. Estimation was based on the sum of the uncertainties of characterization, homogeneity and stability. Data from the certification collaborative study were used to estimate laboratory uncertainties, which form the basis for the uncertainty of characterization. Estimations for the uncertainty of homogeneity were derived from classical homogeneity studies. The estimations of uncertainty of stability caused the most difficulties. Realistic uncertainties fitting the needs of customers while being derived from measurement data based on theoretical considerations were obtained.

BACKGROUNDA reference measurement procedure is needed to demonstrate the traceability of results of urea measurements in human serum. We developed a measurement procedure using the principle of isotope dilution gas chromatography/mass spectrometry.METHODS[(13)C,(15)N(2)]Urea as internal standard was added to a serum sample and equilibrated with endogenous nonlabeled urea. For the preparation of calibrators, the same amount of labeled urea was mixed with known amounts of nonlabeled urea. The serum samples were treated with ethanol to remove proteins by precipitation. The labeled and nonlabeled urea of the samples was converted into a trimethylsilyl derivative of 2-hydroxypyrimidine. The gas chromatography/mass spectrometry system was adjusted to monitor m/z 153 and 168 for the nonlabeled urea derivative and m/z 156 and 171 for the isotopically labeled analogs. The results of the determination were calculated from peak ratios by a hyperbolic calculation function based on the theory of isotope dilution analysis.RESULTSThe procedure was applied to control samples and patient samples and evaluated with respect to its trueness and precision. The standard uncertainty of the results was 0.47-1.72%.CONCLUSIONSThis reference measurement procedure allows values to be assigned to controls and calibrators that are traceable to the primary urea reference material of NIST and, therefore, to the Système International unit "mole" with a low degree of uncertainty. This procedure provides a tool for the highly accurate determination of urea in control materials as well as in patient sera.

OBJECTIVESA new method for the quantitative determination of 17alpha-ethinylestradiol-17beta (EE2) in serum is presented here based on the principle of isotope dilution mass spectrometry (IDMS) with [13C]EE2 as internal standard. The technique was used to determine the concentration profiles of EE2 in the serum of female subjects who had taken oral contraceptives with different progestin components. The method has proved to be very reliable with respect to trueness, specificity, precision and detection sensitivity and offers considerable advantages compared with the immunological methods of measurement used to date.STUDY DESIGNForty-seven female volunteers took two different oral contraceptives containing EE2 combined with different progestins in accordance with a cross-over design. After the administration of 30 microg EE2 combined with 75 microg gestodene (EE2/GSD) or 150 microg desogestrel (EE2/DES), blood samples were taken from the subjects on certain days and in certain previously specified cycles in the course of 12 h after medication.RESULTS AND CONCLUSIONSThe biometric analysis of the results showed that the concentration profiles of EE2 were in their statistics, significantly equivalent after the administration of either of the two oral contraceptives. The sometimes contradictory results found in former studies after the administration of the different contraceptives were presumably due to the methodological shortcomings of the radioimmunological measurement technique. With the use of the highly accurate and specific technique of IDMS it can now be unequivocally established that the different progestins in the tested oral contraceptives have no influence on the bioavailability of EE2 (area under EE2 serum concentration curves, as usually defined in pharmacokinetics).

Suramin, a polysulphonated naphthylurea used in the treatment of human African trypanosomiasis (HAT), is known to cause adrenocortical insufficiency in doses exceeding the quantity used for treatment of HAT. We have previously reported that Trypanosoma brucei rhodesiense infection causes a combined central and peripheral adrenal insufficiency. To evaluate whether suramin therapy acts as an additional adrenotoxic factor, we assessed adrenocortical function in 72 patients suffering from HAT at different times during treatment with either suramin or melarsoprol by a rapid adrenocorticotropic hormone test. We found a significantly diminished peak cortisol response to stimulation in the acutely ill patients (P=0.001), indicating impaired adrenocortical function, as well as a high incidence of partial adrenocortical insufficiency (27%). During and after trypanocidal therapy the incidence of partial adrenal insufficiency gradually declined (to 25% and 18% respectively). Stimulated peak cortisol levels did not differ significantly between patients receiving suramin and those given melarsoprol. No correlation was found between serum suramin concentration and the cortisol response to stimulation (r=0.09, P=0.47). Thus we conclude that suramin in trypanocidal doses neither causes nor worsens the adrenocortical dysfunction observed in Rhodesian HAT.

ZusammenfassungDie Vorgaben für die erforderliche Präzision und die Richtigkeit quantitativer Analysen in der klinischen Chemie sollten sich letztendlich an den medizinischen Erfordernissen orientieren, die sich aus den biologischen Streuungen der einzelnen Meßgrößen ableiten

African sleeping sickness (SS) is a severe, potentially lethal parasitic disease. The treatments of choice are the antiparasitic agents suramin, which is adrenotoxic, and/or melarsoprol. We evaluated the functional integrity of the hypothalamic-pituitary-adrenal (HPA) axis of patients with SS before, during, and after therapy with suramin and/or melarsoprol, in two sequential stages. First, we employed the standard adrenocorticotropic hormone (ACTH) 1-24 stimulation test (250 micrograms i.v.) to assess the maximal adrenocortical responsiveness of 69 patients with SS and 38 normal controls. We demonstrated paradoxically subnormal cortisol responses before suramin therapy [net cortisol response 60 min after stimulation: 10.5 +/- 2.9 (mean +/- SE) vs. 17.5 +/- 1.0 micrograms/dl for controls, p = 0.004], with 27% of the patients falling within the adrenal insufficiency range (stimulated cortisol concentration < 20 micrograms/dl). These responses subsequently and unexpectedly improved with suramin and/or melarsoprol therapy. Second, we performed a human corticotropin-releasing hormone (hCRH) test (100 micrograms i.v.) in 68 additional patients with SS and 14 control subjects to examine whether the glucocorticoid deficiency observed was primary and/or secondary. Compared to controls, the ACTH and cortisol responses to hCRH were blunted (ACTH after 60 min: 29 +/- 7 vs. 58 +/- 8 pg/ml in controls, p = 0.014; cortisol: 15.2 +/- 1.5 vs. 19.6 +/- 0.7 micrograms/dl, p = 0.018), suggesting the presence of secondary adrenal insufficiency. There was improvement of both ACTH and cortisol responsiveness to hCRH with therapy, with cortisol recovery occurring before ACTH, suggesting an additional primary component of adrenal dysfunction in these patients.(ABSTRACT TRUNCATED AT 250 WORDS)

In internal and external quality assessment of clinical chemical analyses, the use of reference method values is prescribed in the Federal Republic of Germany as a measure of accuracy control. In the field of lipid analyses, reference methods are available for measuring total cholesterol and total glycerol in control sera. The reference measurement procedures are based on the highly accurate analytical principle of isotope dilution mass spectrometry. A very detailed protocol that ensures a high level of reliability must be followed when reference measurement procedures are carried out. The introduction of reference methods as a basis for accuracy control has greatly limited the unacceptable use of inaccurate routine methods.

respond to metoclopramide, domperidone, ranitidine, amantadine, levodopa, carbamazepine, clonidine, prazosin, haloperidol, methysergide, or metoprolol.He improved slightly with the application of scopolamine patches, which had to be stopped because of side-effects.Intramuscular chlorpromazine (25 mg) would sometimes prevent attacks for up to a week.He was prescribed baclofen 10 mg, three times daily, after which his hiccups ceased for five days.When they recurred, baclofen dose was increased to 20 mg three times daily which has been successful, with only an occasional solitary hiccup.When he ran out of tablets for two days, the hiccups returned with their former ferocity.He has now remained under control on this medication for three months, has gained weight, and is feeling well for the first time in 30 years.The family history suggests transmission by an autosomal dominant gene.

The determination of thyroid hormones is widely used for the diagnosis and therapy control of thyroid disorders. In particular, thyroxine in serum is one of the most frequently determined endocrine parameters. Unfortunately, the results obtained by the use of different commercial test kits vary significantly, and until now there has been no means to decide whether a particular enzyme or radioimmunoassay kit yields accurate results or not. It seemed, therefore, necessary to develop definitive or reference methods for the measurement of thyroxine and to apply this technique for the assessment of target values in control sera for internal and external quality control. In the present investigation, an analytical protocol using the isotope dilution mass spectrometry technique is described which is herewith proposed as a definitive method for the measurement of thyroxine in human serum. The procedure consists of the following steps. (i) Equilibration of endogenous thyroxine in a serum sample with 100 ng [13C2]thyroxine. (ii) Isolation of the thyroid hormones by using a cation exchange resin. (iii) Formation of the methyl ester by reaction with methanolic hydrochloric acid. (iv) Purification of the methyl ester by column chromatography on Sephadex LH-20. (v) Formation of the N,O-bistrifluoroacetyl derivative with trifluoracetic anhydride. (vi) Selected ion monitoring of fragment ions of the thyroxine and the [13C2]thyroxine derivatives at m/z 870 and 872 using a magnetic sector field mass spectrometer with electron impact ionization combined with a capillary gas chromatography column. This method is now used to assign target values in a German quality control scheme. The precision of the method is of the order of 1–2% (coefficient of variation).

In the years 1977 to 1981, 14 quality-control surveys for the determination of steroid hormones were performed in cooperation with the Deutsche Gesellschaft für Klinische Chemie. Hereby the laboratories participating could in each case analyze the following steroids: aldosterone, cortisol, oestradiol-17 beta, oestriol, progesterone, and testosterone. In the light of the results an investigation was made as to whether, in the course of time, an improvement in the accuracy or in the precision of the determinations had been attained, and to what extent the determinations depend on the qualities of the test material. A clear improvement in the accuracy of the results of the analyses could only be ascertained for oestradiol-17 beta. For aldosterone and cortisol, values were found in pool-plasma whose medians were significantly above the definitive values. An improvement in precision could be noted especially with oestradiol-17 beta and to lesser degrees with cortisol and oestriol. The kind of test material--plasma which contained only the hormones to be analyzed on the one hand, and, on the other hand, pool-plasma, which also contained all endogenous hormones--had no influence on the precision of the results from various laboratories. Low concentrations of the individual steroids led--on the basis of the methodological principle of radioimmunoassays--in almost all cases to a reduced interlaboratory precision in regard to values. The accuracy of the analysis values was considerably impaired only with aldosterone and oestradiol-17 beta by low concentrations: the medians here were in part twice as high as the definitive values.