This paper is the fourth 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 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; Part 7. Certification of Four Reference Materials for the Determination of Enzymatic Activity of Gamma-Glutamyltransferase, Lactate Dehydrogenase, Alanine Aminotransferase and Creatine Kinase at 37 degrees C. A document describing the determination of preliminary upper reference limits is also in preparation. The procedure described here is deduced from the previously described 30 degrees C IFCC reference method. Differences are tabulated and commented on in Appendix 2.

We have previously reported that the normal membrane phospholipid organization is altered in sickled erythrocytes. More recently, we presented evidence of enhanced transbilayer movement of phosphatidylcholine (PC) in deoxygenated reversibly sickled cells (RSC) and put forward the hypothesis that these abnormalities in phospholipid organization are confined to the characteristic protrusions of these cells. To test this hypothesis, we studied the free spicules released from RSC by repeated sickling and unsickling as well as the remnant despiculated cells.The rate of transbilayer movement of PC in the membrane of deoxygenated remnant despiculated cells was determined by following the fate of "4C-labeled PC, previously introduced into the outer monolayer under fully oxygenated conditions using a PC-specific phospholipid exchange protein from beef liver. The rate of transbilayer movement of PC in the remnant despiculated cells was significantly slower than in deoxygenated native RSC and was not very much different from that in oxygenated native RSC or irreversibly sickled cells. The free spicules had the same lipid composition as the native cell, but were deficient in spectrin. These spicules markedly enhanced the rate of thrombin formation in the presence of purified prothrombinase (Factor Xa, Factor Va, and Ca2+) and prothrombin, indicating the exposure of a significant fraction of phosphatidylserine (PS) in the outer monolayer. This effect was not observed when the spicules in this assay were replaced by normal erythrocytes, deoxygenated native RSC, or a deoxygenated sample of RSC after repetitive sickling/unsickling.The results are interpreted to indicate that the destabilization of the lipid bilayer in sickled cells, expressed by the enhanced flip-flop of PC and the exposure of PS in the outer monolayer, occurs predominantly in those parts of the membrane that are in spicular form.

This paper is the fifth 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 6. Reference Procedure for the Measurement of Catalytic Concentration of Gamma-Glutamyltransferase; Part 7. Certification of Four Reference Materials for the Determination of Enzymatic Activity of Gamma-Glutamyltransferase, Lactate Dehydrogenase, Alanine Aminotransferase and Creatine Kinase at 37 degrees C. A document describing the determination of preliminary upper reference limits is also in preparation. The procedure described here is deduced from the previously described 30 degrees C IFCC reference method. Differences are tabulated and commented on in Appendix 3.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT • The influence of CYP2C19 on the kinetics and dynamics of omeprazole, lansoprazole and rabeprazole has been studied in Japanese subjects. • It has been suggested that subjects with *1/*1 genotype might need stronger acid suppression than *1/*2 and *2/*2 subjects. This suggestion comes from data in Japanese subjects and has not been confirmed in Caucasians. • Furthermore, a novel CYP2C19 mutation, *17, which mainly occurs in Caucasians has been discovered. This mutation has been associated with clinical failure, but its relevance for therapy with PPIs has not been studied yet. WHAT THIS STUDY ADDS • In this study, the influence of CYP2C19 on both the pharmacokinetics and dynamics in Caucasian subjects after single and repeated dosing has been investigated. • This is the first study showing that Caucasian subjects with *1/*1 and *1/*17 mutations need stronger acid‐inhibition. In this study three proton pump inhibitors (omeprazole, lansoprazole and pantoprazole, in different doses) were studied of which pantoprazole had not been studied before in this setting, not even in Japanese. AIMS To investigate the impact of CYP2C19 mutations *2‐*6 and *17 on acid‐inhibition and pharmacokinetics of lansoprazole (L15), omeprazole (O10, O20) and pantoprazole (P40) in Caucasians. METHODS CYP2C19 genotyping for *2–*6 and *17 mutations was assessed in subjects who were H. pylori negative in two randomized crossover trials. The influence of CYP2C19 mutations on single and repeated administration of L15 and O10 (study A) and O20 and P40 (study B) was investigated. Pharmacokinetics and the cumulative percentage of time with intragastric pH above 4 (% > pH 4) were assessed on day 1 and 6. RESULTS For study A CYP2C19 genotyping found five *1/*1, four *1/*2, one *1/*17 and one *2/*17. For study B the results were six *1/*1, two *1/*2, six *1/*17, one *2/*2 and one *2/*17. For all PPIs AUC was highest in *2/*2 and lowest in *1/*17. On day 1, all PPIs significantly increased percentage >pH 4 compared with baseline. *1/*1 genotype showed no significant acid‐inhibition after L15, O10 and O20. *1/*17 genotype showed no significant acid‐inhibition after O20 and P40. *1/*2 genotype showed significant acid‐inhibition after L15 and O10. On day 6, all four PPIs showed significantly increased acid‐inhibition. *1/*1 and *1/*17 showed a significantly increased percentage > pH 4 after treatment with O20 and P40. However, in *1/*1 subjects percentage > pH 4 was not significantly increased after L15 and O10. *1/*2 genotype showed a significant acid‐inhibitory effect after repeated dosing with L15 and O10. CONCLUSIONS Caucasian subjects with *1/*1 and *1/*17 genotype need stronger acid‐suppression therapy, especially during the first days of treatment or with on‐demand therapy.

This paper is the ninth in a series dealing with reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 °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 γ-glutamyltransferase; Part 7. Certification of four reference materials for the determination of enzymatic activity of γ-glutamyltransferase, lactate dehydrogenase, alanine aminotransferase and creatine kinase at 37 °C; Part 8. Reference procedure for the measurement of catalytic concentration of α-amylase. The procedure described here is derived from the previously described 30 °C IFCC reference method. Differences are tabulated and commented on in Appendix 1.