1) a standardized manufactured liquid formula was designedI0J containing components that were shown to challenge pp TG metabolism: saturated fat, sucrose, and ethanol;I2-l6 (2) this oral metabolic tolerance test (oMTT) was applied to a homogeneous group of 113 healthy male volunteers of similar age (25.0 f 0.3 years) and body mass index (BMI = 22.4 f 0.4 kg/m2)11 to avoid any variance in TG response by these ~ariables.5-'7-~9 0 This work was supported by the Stifterverband f i r die Deutsche Wissenschaft by the award of an H.-and L.-Schilling-Professorship to J. Schrezenmeir, and by the Institut Danone fir Erniihrung.353

A reliable simple reversed-phase liquid chromatographic method for the routine determination of ascorbic acid in plasma and urine with ultraviolet detection is described. This method enables the complete separation of the ascorbic acid peak from others with a recovery of above 95% within 8 minutes. The method can be used for analysing multiple samples within a day. In addition, the storage conditions and stability of ascorbic acid in plasma and urine were investigated. Samples of plasma and urine can be stored on ice in darkness for at least 60 min without reduction of ascorbic acid concentration. Prepared samples can be stored in darkness at 4 degrees C for at least 120 min and in liquid nitrogen for 42 days.

As a system for study, the isolated human polymorphonuclear leukocyte combines the advantages of a quasi-non-invasive preparation with a nearly complete complement of enzymes of carbohydrate and energy metabolism. However, small sample volumes and, in some cases, very low enzyme activities make high demands on sample processing, storage, and performance of continuous measurements, if the enzyme activities are to be measured with acceptable reproducibility. In the presented study several aspects of homogenization, storage, and continuous measurement were scrutinized, to identify critical steps and consider ways of optimizing the method. Polymorphonuclear leukocytes were separated from the blood of healthy subjects by sedimentation and density gradient centrifugation. After ultrasonic homogenization, 13 enzymes of glycolysis and gluconeogenesis, the tricarboxylic acid cycle, and glycogen metabolism were determined photometrically. The variation of several conditions showed: 1. The duration of exposure to ultrasound for the homogenization of polymorphonuclear leukocytes has no influence over a wide range of time.2. Addition of the detergents Triton X-100 and deoxycholic acid, as well as the SH-group protector dithiothreitol, to the homogenizing medium increased the measured activities of only a few enzymes.3. Considerable inaccuracy was encountered-when the suspension was divided into parts for homogenization with different additives; such splitting of the suspension should therefore be performed only when necessary, as in the determination of reference values (e. g. protein or DNA content of the cell suspension).4. Twenty four-fold determination of enzyme activities from one homogenate resulted in precisions between 4.5% (citrate synthase) and 14.4% (transketolase), which is satisfactory for the low activities (as low as 1 U/l) in the homogenate. 5. The reproducibility of enzyme activities, measured in homogenates of polymorphonuclear leukocytes from different blood samples drawn simultaneously, was only slightly worse than that of the continuous measurement method itself. Thus, the precision of the measurement of enzyme activity seems to be the main determinant of the overall method. In conclusion, the described procedure of separation, homogenization, and enzyme measurement in human polymorphonuclear leukocyte meets the requirements of biochemical or clinical trials and can be recommended for clinical metabolic studies.

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