Major royal jelly proteins as markers of authenticity and quality of honey.

Until now, the properties of honey have been defined based exclusively on the content of plant components in the nectar of given plant. We showed that apalbumin1, the major royal jelly (RJ) protein, is an authentic and regular component of honey. Apalbumin1 and other RJ proteins and peptides are responsible for the immunostimulatory properties and antibiotic activity of honey. For the quantification of apalbumin1, an enzyme-linked immunosorbent assay (ELISA) was developed using polyclonal anti-apalbumin1 antibody. The method is suitable for honey authenticity determination; moreover it is useful for detection of the honey, honeybee pollen and RJ in products of medicine, pharmacy, cosmetics, and food industry, where presences of these honeybee products are declared. Results from the analysis for presence and amount of apalbumin1 in honeys will be used for high-throughput screening of honey samples over the world. On the basis of our experiments which show that royal jelly proteins are regular and physiologically active components of honey we propose to change the definition of honey (according to the EU Honey Directive 2001/110/EC) as follows: Honey is a natural sweet substance produced by honey bees from nectar of plants or from secretions of plants, or excretions of plant sucking insects, which honey bees collect, transform by combining with major royal jelly proteins and other specific substances of their own, deposit, dehydrate, store and leave in the honey comb to ripen and mature.

New homogeneous HDL-cholesterol assay without the influence of high TG sample using the selective detergent to lipoproteins.

Homogeneous HDL-cholesterol assays have been developed and used widely in routine analysis, but they have been reported to give inaccurate results in patients with hypertriglyceridemia. Recently, a new assay based on a new principle without the influence of triglycerides has also been developed and commercialized. We evaluated the basic performance of this new homogeneous HDL-cholesterol assay and compared it with the conventional polyethylene glycol/cyclodextrin-modified enzyme (PEGME) method using high-triglyceride (TG) samples (TG>8000 mg/L). For samples showing a discrepancy with the conventional method, other precipitation and ultracentrifugation (UC) methods were also used to confirm the values. This new homogeneous assay is based on the selective solubilizing effect of detergent on the different lipoproteins. First, non-HDL free cholesterol is consumed by enzyme and is cleared as a colorless reactant. Then. HDL-cholesterol is selectively solubilized by lipoprotein-specific detergent and reacted with the enzyme. As a result, the precision of this new homogeneous assay was good (CV<2%) over the wide range, and the measurement range was 0 to 2000 mg/L. This method correlated well with the PEGME method, which is a conventional method for normolipidemic samples (y=0.97x-3.1, r=0.994, n=424). It also correlated well with the UC method (y=0.99x+0.3, r=0.989, n=53). Fourteen high-TG samples showed different results from those obtained by the PEGME method. Among these samples, one contained abnormal lipoproteins (probably due to the influence of drug therapy) and gave a significantly different result from that obtained by the PEGME method. However, the values obtained by other methods (precipitation and ultracentrifugation) agreed well with those obtained by this new method. In conclusion, this method shows a good basic performance and is useful for high-TG samples without any interference. Therefore, it is considered to be very practical for a routine test.

New enzymatic assay for serum urea nitrogen using urea amidolyase.

We established an enzymatic assay for measurement of serum urea nitrogen using urea amidolyase (EC 3.5.1.45) from yeast species. The method is based on hydrolysis of urea by the enzyme. In this assay, we eliminated endogenous ammonium ion by use of glutamate dehydrogenase (EC 1.4.1.4). Then in the presence of urea amido-lyase, ATP, bicarbonate, magnesium, and potassium ions, ammonium ion was produced proportionally to urea concentration in serum. The concentra-tion of ammonium ion formed was determined by adding GLDH to produce NADP(+) in the presence of 2-oxoglutarate and NADPH. We then monitored the change of absorbance at 340 nm. The inhibitory effect of calcium ion on this assay was eliminated by adding glyco-letherdiamine-N, N, N', N'-tetraacetic acid to the reaction system. The with-in-assay coefficient of variations (CVs) of the present method were 1.80-3.76% (n = 10) at 2.8-19.0 mmol/L, respectively. The day-to-day CVs were 2.23-4.59%. Analytical recovery was 92-115%. The presence of ascorbic acid, bilirubin, hemoglobin, lipemic material, ammo-nium ion, or calcium ion did not affect this assay system. The correlation be-tween values obtained with the present method (y) and those by another enzy-matic method (x) was 0.997 (y = 1.02x - 0.10 mmol/L, Sy/x = 0.841, n = 100), with a mean difference of -0.18 +/- 0.86 mmol/L [(values by reference method - that of present method) +/- SD] using the Bland-Altman technique. J. Clin. Lab. Anal. 17:52-56, 2003.

Enzymatic assay for determination of bicarbonate ion in plasma using urea amidolyase.

BACKGROUND: One of the important buffering systems to maintain blood pH is carbonic acid-bicarbonate. Together with other clinical tests, the measurement of bicarbonate ion concentrations is widely used for the diagnosis of the acid-base balance. We developed a kinetic assay for measurement of bicarbonate ion in plasma using urea amidolyase (EC 3.5.1.45) from yeast species. We evaluated the analytical performance of the present enzymatic method and examined the relationship between bicarbonate ion concentrations by present method and with ABL 520 blood gas system. METHODS: Urea amidolyase catalyzes the reaction of bicarbonate ion with urea to rise to allophanate. We eliminated endogenous ammonium ion by the use of glutamate dehydrogenase (EC 1.4.1.4), and then monitored the production of ammonium ion in the presence of urea amidolyase, urea, ATP, potassium, and magnesium ions. Ammonium ion was produced proportional to the bicarbonate ion concentration and was determined by adding glutamate dehydrogenase to produce NADP(+) in the presence of 2-oxoglutarate and NADPH, and the change of absorbance at 340 nm was monitored. RESULTS: The within-assay and day-to-day assay coefficient variations (CVs) of the present method were 1.3-2.8% and 3.1-5.4%, respectively. The analytical recoveries were 90-110%. The presence of ascorbic acid, bilirubin, hemoglobin, lipemic material, hydrogen phosphate, dihydrogen phosphate, ammonium, or calcium ion did not affect this assay. The correlation coefficient between the values obtained by present method (y) and Radiometer ABL 520 blood gas system (x) was 0.983 (y = 1.029x-0.737 mmol/l, Sy/x = 0.764, n = 100), with a mean difference of 0.03 +/- 0.77 mmol/l [(values by reference method-that of present method) +/- S.D.] using the Bland-Altman technique.

Relation between iron content of serum ferritin and clinical status factors extracted by factor analysis in patients with hyperferritinemia.

OBJECTIVES: The aims of this study were to develop a new technique for determination of iron content of serum ferritin (ICF, micromol Fe/mg protein) and to investigate relations between ICF and clinical status in patients with hyperferritinemia. METHODS: ICF values were determined by a combination of immunoprecipitation of ferritin and direct colorimetric iron assay. One hundred fifty patients with hyperferritinemia were screened. Factor analysis of the results of 11 laboratory tests was applied to extract factors representing the clinical status of patients. Relations between the extracted factors and the ICF values or serum ferritin concentrations were assessed. RESULTS: Within-run coefficients of variation (CVs) of the ICF assay were <==5.7%. The mean ICF value of 150 patients was 0.423 micromol/mg (SD, 0.211 micromol/mg). Three factors representing clinical status were identified: inflammation, tissue cell damage, and body iron status. Serum ferritin level correlated with all three factors. In contrast, ICF correlated significantly only with the factor representing tissue cell damage (r = 0.293, p = 0.001), and this correlation was independent of inflammation and iron status (p = 0.008). CONCLUSIONS: ICF changes in response to tissue cell damage independent of inflammatory and body iron statuses, whereas serum ferritin changes in response to all three pathologic statuses.

Modification of fully automated total iron-binding capacity (TIBC) assay in serum and comparison with dimension TIBC method.

BACKGROUND: We previously reported the development of a fully automated assay for total iron-binding capacity (TIBC) in serum, using a multipurpose automated analyzer. However, this method requires four different reagents and is thus useful only with a limited number of available analyzers. We simplified our original assay and compared the analytical performance of the modified method with that of a commercial, fully automated TIBC assay (Dimension TIBC assay). METHODS: We simplified our original method to require only three reagents. Calibration was also altered and was performed with human transferrin standard solutions. An advantage of this method is that it does not require separation of excess unbound iron after the first step of transferrin saturation. Unbound iron is eliminated by formation of a complex with the chromogenic reagent ferrozine in the second step. Iron dissociated from transferrin by acidic pH reacts with ferrozine to form a colored complex in the final step, and the increase in absorbance at 570/660 nm is directly proportional to the TIBC measured. TIBC values were determined for 49 healthy individuals and 148 patients with this modified TIBC assay and with a commercial, fully automated TIBC method (Dimension clinical chemistry system), and calculation of TIBC based on the sum of the serum iron and unsaturated iron-binding capacity was performed for 97 patients. RESULTS: The within-run CVs for the modified TIBC assay and the Dimension TIBC assay were <4.8% and <2.4%, and the between-run CVs were 1.2% and 1.7%, respectively. The dilution curves were linear for TIBC values up to at least 180 micromol/L with both methods. TIBC values obtained by our method were linearly correlated with serum transferrin concentrations (r = 0.984; S(y/x) = 3.18 micromol/L; P <0.001). The correlation between the values obtained with the present method (y) and those obtained with the Dimension TIBC method (x) was y = 1.04x + 1.19 micromol/L (r = 0.985; S(y/x) = 2.47 micromol/L), and with the calculation method (x) was y = 1.18x + 2.62 micromol/L (r = 0.976; S(y/x) = 3.27 micromol/L). CONCLUSIONS: Our modified, fully automated TIBC assay performed similarly to the Dimension TIBC assay and is adaptable for use with many multipurpose automated analyzers.