[Blood glucose measurement].

For all diabetic patients in which the blood glucose is unstable, self-monitoring of blood glucose (SMBG) is effective. Especially, the patient who insulin supplies are necessary is indispensable SMBG in spite of tyep 1 and/or type 2 diabetes mellitus. SMBG measuring devices of looked forward nothing noninvasive type entered the practical stage. For example, the patient who requires the frequent measurement can be applied to alarms of the hypoglycemia unawareness, etc. from present performance, because blood glucose monitoring in the any time possible, though changing in taking in SMBG measuring device of the conventional invasive type, is not possible. And, it is possible to utilize in health care and protective measurement of the lifestyle related disease, etc., because the daily observation of the blood glucose is carried out in the long term. The function of data assist center, etc. is expected these this data for processing through the internet, etc. and follow of the result.

IFCC recommendation on reporting results for blood glucose.

In human beings, glucose is distributed like water between erythrocytes and plasma. The molality of glucose (amount of glucose per unit water mass) is the same throughout the sample. Different water concentrations in calibrator, plasma, and erythrocyte fluid can explain some differences that are dependent on sample type, methods requiring sample dilution, and direct reading biosensors detecting molality. Different devices for the measurement of glucose detect and report fundamentally different analytical quantities. The differences exceed the maximum allowable error of glucose determinations for diagnosing and monitoring diabetes mellitus, and they complicate the treatment. The goal of the International Federation of Clinical Chemistry, Scientific Division, Working Group on Selective Electrodes (IFCC-SD WGSE) is to reach a global consensus on reporting results. The document recommends harmonizing to the concentration of glucose in plasma (with the unit mmol/l), irrespective of sample type or technology. A constant factor of 1.11 will convert measured concentration in whole blood to the equivalent concentration in plasma.

Relationships of glucose concentrations in capillary whole blood, venous whole blood and venous plasma.

We studied the difference in glucose levels between capillary and venous whole blood during 75-g oral glucose tolerance test (OGTT) in 75 healthy subjects. Capillary and venous whole blood glucose values were measured by HK-G6PD method after deproteinization. The post-loaded glucose levels in capillary blood were significantly higher than those in venous blood, and the mean values of capillary and venous difference at 30, 60, 90, 120 and 180 min were 1.37, 1.40, 1.07, 0.95 and 0.52 mmol/l, respectively, with the maximum difference at 60 min. No correlation was found in the magnitude of the differences in glucose between capillary and venous blood specimens. We determined the inaccuracy of six self-monitoring blood glucose devices relative to the reference method using venous plasma, venous whole blood and capillary whole blood from 31 diabetic patients. The differences of mean values of venous whole blood and capillary whole blood, and venous whole blood and venous plasma, and capillary whole blood and venous plasma were 9.6%, 11.3% and -3.2%, respectively. The range of bias and Sy/x were 0.31-1.06 mmol/l and 0.71-1.07 mmol/l, respectively, compared to the reference method using venous plasma.

Correlation between histological differentiation and DNA-synthesizing enzymes in rat colorectal tumors induced with 1, 2-dimethylhydrazine.

Thymidylate synthase and thymidine kinase are key enzymes involved in de novo and salvage pathways for pyrimidine nucleotide synthesis. Colorectal carcinogenesis induced with 1,2-dimethylhydrazine in rats enhanced mRNA expression levels of both enzymes, resulting in the increase of both enzyme activities and bromodeoxyuridine-immunoreactive S-phase cells. Poorly and well differentiated adenocarcinomas of the colorectum showed the relative elevation of activities of thymidylate synthase and thymidine kinase, respectively. These results indicate that the relationship between de novo and salvage pathways for pyrimidine nucleotide synthesis may depend on the histopathological grades of cell differentiation.

Effects of 5-fluorouracil derivative UFT on thymidylate synthetase and thymidine kinase in rat colorectal tumors.

BACKGROUND: Thymidylate synthetase and thymidine kinase are key enzymes involved in de novo and salvage pathways for pyrimidine nucleotide synthesis, respectively. MATERIALS AND METHODS: Weekly injections of 1,2-dimethyl-hydrazine induced high incidence of colorectal adenocarcinomas in rats. RESULTS: An increased activity of thymidylate synthetase was found in the poorly differentiated adenocarcinomas of the chemically induced rat colorectal tumors. Six-week oral administration of 1-(2-tetrahydrofuryl)-5-fluorouracil in combination with uracil (UFT) reduced the total number of colorectal tumors, with the reduction of thymidylate synthetase activity in the poorly-differentiated type, though the mRNA expression of thymidylate synthetase and thymidine kinase differed little between the groups with or without UFT treatment. CONCLUSIONS: These results indicate that the long-term oral administration using UFT suppresses colorectal carcinogenesis and the growth of the poorly-differentiated type tumors.

Serum des-gamma-carboxy prothrombin levels determined by a new generation of sensitive immunoassays in patients with small-sized hepatocellular carcinoma.

OBJECTIVE: Des-gamma-carboxy prothrombin (DCP), also called protein induced by vitamin K absence or antagonist II (PIVKA-II), is a tumor marker complementary to AFP for the diagnosis of hepatocellular carcinoma (HCC). Currently available immunoassays for DCP are not sensitive enough to detect HCC at an early stage. Recently, two new immunoassays with enhanced sensitivity were developed. The aim of this study was to assess the diagnostic values of the new methods in patients with small-sized HCC. METHODS: Coded serum samples obtained from 36 patients with small-sized and single-nodular HCC (< or = 3 cm in diameter) and 49 patients with posthepatitic cirrhosis not carrying HCC were analyzed. DCP levels were determined in three different ways: 1) conventional EIA; 2) a new immunoassay using the electrochemiluminescence (ECLIA) detection system; and 3) a new immunoradiometric assay (IRMA). Lectin-reactive profiles of AFP (AFP-L3) were also determined. RESULTS: In 36 patients with small-sized HCC, the rates of abnormal values obtained by the conventional, ECLIA, and IRMA methods were 2.7%, 27.8%, and 16.7%, respectively. An ROC analysis of the two new methods (ECLIA vs IRMA) revealed a better performance by the ECLIA method (p < 0.05). The true positive rate of AFP-L3 was 22.2%, whereas a combination assay of ECLIA for DCP and AFP-L3 resulted in a 41.7% sensitivity with a specificity of 90%. CONCLUSIONS: Compared with the conventional method, the sensitivity in detecting small-sized HCC was increased in the two new DCP immunoassays (ECLIA and IRMA). The overall performance as evaluated by an ROC analysis was significantly better in ECLIA than in IRMA.

Effects of long-term administration of UFT plus leucovorin on colorectal tumors induced with 1,2-dimethylhydrazine in rats.

Thymidylate synthase and thymidine kinase are key enzymes involved in the de novo and salvage pathways for pyrimidine nucleotide synthesis, respectively. Thymidylate synthase is inhibited by 5-fluorodeoxyuridine monophosphate, forming an inactive ternary complex with intracellular folate. We investigated the effects of 1-(2-tetrahydrofuryl)-5-FU plus uracil (UFT) with or without leucovorin on 1,2-dimethylhydrazine-induced rat colorectal carcinomas. Thirty-week administration of UFT with or without leucovorin markedly suppressed both colorectal carcinogenesis and tumor growth, resulted in the increase of thymidylate synthase inhibition and the decrease of thymidine kinase activity in the tumor cells. These results indicate that the combination of UFT with leucovorin could be useful in the development of pre- and post-operative adjuvant chemotherapy programs.

Effects of a low dose leucovorin with 5-fluorouracil derivative on colorectal tumors induced with 1,2-dimethylhydrazine in rats.

Thymidylate synthase, which is a key enzyme involved in the de novo pathway for pyrimidine nucleotide synthesis, is inhibited by 5-fluorodeoxyuridine monophosphate, forming an inactive ternary complex with intracellular folate. We investigated the effect of a 5-fluorouracil derivative (UFT) with or without low dose leucovorin on the number of 5-fluorodeoxyuridine monophosphate binding sites, thymidine kinase activity and intracellular folate concentration in 1,2-dimethylhydrazine-induced rat colorectal carcinomas. A 10-day administration of UFT with or without leucovorin enhanced the thymidine kinase activity and the number of 5-fluorodeoxyuridine monophosphate binding sites, with an increase of thymidylate synthase mRNA expression. Thymidylate synthase inhibition was slightly increased as the intracellular folate concentration increased. These results indicate that thymidylate synthase inhibition increases when the intracellular folate is exogenously supplemented and maintained at an adequate concentration.

Additive effects of medroxyprogesterone acetate and 5-fluorouracil derivative on 7,12-dimethylbenz[a]anthracene-induced rat mammary tumors.

Chronic oral administration of 1-(2-tetrahydrofuryl)-5-fluorouracil in combination with uracil suppressed thymidylate synthetase (TS) gene expression followed by reduction of TS activity in rat mammary tumors induced with 7,12-dimethylbenz[a]anthracene. Addition of medroxyprogesterone acetate (MPA) to the anticancer drug caused an additional decrease in TS and thymidine kinase activities in the tumor growth and restoration of bone loss. These results suggest that the simultaneous administration of MPA and anticancer drugs causes increased inhibition of mammary tumor growth and also diminishes the bone loss.

Effects of tamoxifen on mammary tumors and bone in 7,12-dimethylbenz-(a)anthracene-treated rats.

We investigated the effects of tamoxifen on the growth of 7,12-dimethylbenz(a)anthracene induced rat mammary tumors, the activity of thymidylate synthetase and thymidine kinase (key enzymes involved in de novo and salvage pathways for pyrimidine nucleotide synthesis), and also their gene expression. The effects on immunohistochemistry using bromodeoxyuridine in the tumors and bone mineral density of the femur in rats were also studied. Chronic administration of tamoxifen markedly reduced the expression of thymidylate synthetase mRNA, followed by a reduction in enzyme activity and S-phase cells in the mammary tumors, and significantly enhanced the bone mineral density. Tamoxifen not only attenuated bone loss in aging but also enhanced bone volume in mammary tumor-bearing rats in which tumor growth was suppressed via both the de novo and salvage pathways for pyrimidine nucleotide synthesis.

Effects of macrophage- and granulocyte-colony stimulating factors on thymidylate synthase and thymidine kinase activity in rat hematopoietic cells.

The effects of macrophage (M) and granulocyte (G) colony-stimulating factors (CSFs) on the activity of thymidylate synthase and thymidine kinase, which are involved in de novo and salvage pathways for pyrimidine nucleotide synthesis, were investigated in the hematopoietic cells of rats treated with cyclophasphamide. Thymidine kinase activity, but not that of thymidylate synthase, was markedly enhanced in these cells by M- and G-CSF treatment (p < 0.05 and p < 0.01). G-CSF directly, and M-CSF indirectly stimulate myeloid cells and lead to S-phase predominantly via the salvage pathway for pyrimidine nucleotide synthesis. The present study indicates that these CSFs can be effective inducers of complete remission in acute leukemias when employed together with chemotherapy.

Additional effects of medroxyprogesterone acetate on mammary tumors in oophorectomized, estrogenized, DMBA-treated rats.

Although hormone replacement therapy not only relieves vasomotor symptoms but also reduces cardiovascular disease and osteoporosis, long-term estrogen therapy increases the risk of endometrial and/or mammary cancer. We investigated the effects of conjugated estrogens with or without medroxyprogesterone acetate in oophorectomized, 7,12-dimethylbenz(a)anthracene-treated rats. Chemically induced mammary carcinogenesis was completely suppressed by the simultaneous oophorectomy, but conjugated estrogens replacement with or without medroxyprogesterone acetate markedly stimulated mammary carcinogenesis in the ovariectomized rats. The chronic administration of conjugated estrogens and medroxyprogesterone acetate markedly reduced the activities of thymidylate synthetase and thymidine kinase and bromodeoxyuridine-immunoreactive (S-phase) cells in mammary tumors. These results indicate that the treatment using conjugated estrogens with or without medroxyprogesterone acetate may promote the mammary carcinogenesis in postmenopausal women but the chronic administration of medroxyprogesterone acetate may alter the development of established mammary cancer.

Effects of a new clerodane diterpenoid isolated from propolis on chemically induced skin tumors in mice.

Propolis is a resinous material gathered by honey bees from the buds and bark of certain trees and plants, and used inside their hives. Characteristic components of propolis are many kinds of flavonoid aglycones. The methanol extract of a Brazilian propolis was fractionated by HPLC, and a tumoricidal substance was isolated and characterized as a new clerodane diterpenoid (PMS-1) with a molecular formula of C20H32O3 (MW: 320). We investigated the effects of PMS-1 on skin tumorigenesis and the development of skin tumors induced by 7,12-dimethylbenz(a)anthracene application on mouse back skin. It was tentatively concluded that PMS-1 reduced the incidence of skin tumors by inhibition of DNA synthesis in a de novo pathway, and suppressed the growth of the tumors by decreasing DNA synthesis in a salvage pathway.

Effects of estrone with or without medroxyprogesterone acetate on the femur in aged rats.

In an attempt to define the efficacy of hormone replacement therapy in postmenopausal osteoporosis, we investigated the effects of estrone with or without medroxyprogesterone acetate on the bone mineral density of the femur of aged rats, of which plasma levels of luteinizing hormone (LH), follicle stimulating hormone (FSH) and estradiol were markedly reduced in comparison with those of younger rats. Although additional sex hormones had little effect on the plasma levels of gonadotropins, chronic administration of estrone significantly enhanced the bone mineral density of femur in aged animals.

Accuracy evaluation and hematocrit effect of commercial ionized calcium analyzers using Japanese serum reference material.

The serum reference material for concentration measurement of ionized calcium in blood has been established by our research group in Japan. The reference method used is the reference standard cell (RSC:IFCC/WGSE, Covington-Umemoto Cell) system. Here we present the results of the evaluation of accuracy using the serum reference material and the hematocrit effect of the nine commercial ionized calcium analyzers being used in routine measurements by Japanese round robin test program. The ionized calcium concentration and pH of two concentration levels of the reference material were analyzed in triplicate measurements according to the standard procedures designated by manufacturers. The effect of hematocrit in ionized calcium concentration measurement was tested using plasma and whole blood with hematocrit values of 35%, 50% and 65%, respectively. The range of bias of ionized calcium values were -0.14 approximately +0.09 mmol/L, -0.12 approximately +0.09 mmol/L for ionized calcium, respectively. The effect of hematocrit in ionized calcium concentration measurement was strongly observed in GEM Premier, CAI-101 and GASTAT-2 analyzers.

Serum thymidine kinase as a tumor marker of colorectal carcinogenesis induced with 1,2-dimethylhydrazine in rats.

Thymidine kinase is the key enzyme in salvage pathway for pyrimidine nucleotide synthesis. High activities of thymidine kinase have been found in rapidly proliferating normal, neoplastic tissues and sera from patients with various diseases including lymphoma, leukemias and small cell lung cancer. We investigated the serum thymidine kinase activities and the colorectal carcinomas in rats treated with 1,2-dimethylhydrazine. The positive correlations between serum thymidine kinase activities and numbers of total and large tumors were observed. These findings suggest the measurement of serum thymidine kinase activity may be clinically valuable in an early stage of the colonic carcinogenesis.

[Calibration and standardization in enzyme immunoassay].

Calibration is performed using multipoints of the calibrator in enzyme immunoassay (EIA). Calibration curves in EIA are frequently represented by non-linearity. Therefore, calibration in EIA is fitted with suitable functions. These functions are demonstrated with logistic curve, logit-log conversion, spleen function, etc. Consequently, the calibration procedure is indicated for the manufacturer's instruction, and also the calibrator is prepared as a commercial available reagent kit. To minimize EIA measurements among laboratories, and among methods, standardization with a common suitable reference material is needed. Reference materials for EIA, international standards are prepared by the expert committee of WHO. These standards must be transferred to national standards, manufacturer's standards and calibrators of commercial available reagent kits. Finally, the measured value from the routine method is maintained with commutability and traceability.

Pathological and enzymatic features of 1,2-dimethylhydrazine induced colorectal carcinomas in rats.

Well- and moderately well-differentiated colorectal adenocarcinomas accounted for 86% of all tumors induced by 1,2-dimethylhydrazine in rats, and were distributed throughout the colorectal tract. Poorly differentiated carcinomas, 14% of all tumors, were markedly restricted to the proximal half of colon, i.e., 94% of the poorly differentiated type was found in the proximal colon and caecum. Thymidylate synthetase and thymidine kinase, key enzymes in the de novo and salvage pathways, respectively, for pyrimidine nucleotide synthesis were found to be reduced and elevated, respectively, with increasing cellular differentiation. These results suggest that biochemical differences in colorectal tumors may be associated with differences in tumor frequency, distribution, and histological type.