The development of an artificial stool usable for the surveillance of faecal haemoglobin testing.

Background Faecal occult blood testing is an important diagnostic tool for the detection of colorectal cancer. However, it has not been standardized due to the absence of suitable specimens for surveillance. Methods We developed a ready-to-use artificial stool made from rice flour. This new artificial stool homogeneously contains not only human haemoglobin A0 (HbA0) but also glycerol as an internal standard material. After the collection of the artificial stool into a buffer, the haemoglobin concentration in dispersed solution was measured using a method based on the peroxidase like activity of haemoglobin. The glycerol concentration was measured using a commercially available triglyceride measurement kit. Results With regard to the haemoglobin stability, the decrease in the level of human haemoglobin in the artificial stool was <2% when it was stored at -80℃ for four months, -20℃ for two weeks, and 5℃ for two days. The artificial stool was easily collected with the collecting tubes of a commercially available faecal haemoglobin test kit. The weight of the collected artificial stool could be calculated by measuring the concentration of glycerol in the extracting solution of the collected stool sample. The haemoglobin concentrations could be adjusted based on their collection weights. Conclusions The artificial stool has a paste-like consistency and contains both haemoglobin and glycerol homogeneously. Furthermore, the measured haemoglobin concentration could be determined based on the collected stool weight, which was directly related to the glycerol concentration. These features make it a useful material for the surveillance of faecal occult blood testing.

Is there a relation between triglyceride concentrations in very low density lipoprotein and the index of insulin resistance in nondiabetic subjects?

BACKGROUND: Serum very low density lipoprotein (VLDL) levels increase during the early stages of insulin resistance; therefore, determination of VLDL levels would be useful for evaluating the progression of metabolic syndrome and diabetes mellitus. The aim of this study was to clarify the clinical utility of triglyceride in VLDL (VLDL-TG) level, determined using a homogeneous assay kit (Shino-test Corporation, Tokyo, Japan), as an index of insulin resistance. METHODS: We enrolled 74 subjects in this study (diabetic subjects, n = 42; nondiabetic subjects, n = 32). The levels of VLDL-TG, remnant-like lipoprotein particle cholesterol, preheparin lipoprotein lipase mass, and other biochemical markers were determined. RESULTS: VLDL-TG levels were significantly higher in the diabetic group (1.04 ± 0.84 mmol/l vs. 0.64 ± 0.42 mmol/l, P < 0.01) than in the nondiabetic group. In the nondiabetic group, VLDL-TG was significantly correlated with the homeostasis model assessment of insulin resistance (HOMA-IR), the index for insulin resistance (r = 0.513, P = 0.003). VLDL-TG levels, but not TG levels, were higher in the highest quartile (HOMA-IR) of the nondiabetic group. CONCLUSION: VLDL-TG level was a useful early marker for insulin resistance, especially in nondiabetic subjects. The homogeneous VLDL-TG assay is a simple, low-cost method for determining insulin resistance.

Semiquantitative analysis of apolipoprotein A-I modified by advanced glycation end products in diabetes mellitus.

BACKGROUND: Apolipoprotein A-I (Apo A-I), the major component of high-density lipoprotein (HDL), is modified by reactive α-oxoaldehydes, such as methylglyoxal (MG) and glycolaldehyde (GA), and these modifications affect the function of Apo A-I. GA- and MG-modified Apo A-I serum levels were semiquantitatively evaluated in diabetic patients to elucidate the association of each protein with diabetes and to determine its appropriateness as a serum marker of diabetes. METHODS: We enrolled 44 subjects in this study (diabetic subjects, n = 24; nondiabetic subjects, n = 20). GA- and MG-modified Apo A-I levels in serum were determined by sandwich enzyme-linked immunosorbent assay (ELISA) by using anti-GA or anti-MG antibody and anti-Apo A-I antibody. RESULTS: The GA-modified Apo A-I levels did not significantly differ between the diabetic and nondiabetic subjects (1.00 ± 0.38 vs. 0.96 ± 0.22). However, the MG-modified Apo A-I levels in the diabetic subjects were significantly higher than those in the nondiabetic subjects (1.33 ± 0.52 vs. 0.90 ± 0.20). In addition, MG-modified Apo A-I levels correlated with the glycated hemoglobin (HbA1c) levels, HDL-cholesterol levels, and the homeostasis model assessments of insulin resistance, which are indicators of insulin resistance. CONCLUSION: The MG-modified Apo A-I level may be an indicator of diabetic dyslipidemia and insulin resistance.

Phosphoglucomutase activity as a novel biomarker in patients with acute myocardial infarction.

BACKGROUND: Phosphoglucomutase (PGM), a key enzyme in cellular glucose utilization and energy homeostasis, has been reported to show a relationship with oxidative stress. However, the clinical importance of PGM activity has not been investigated in patients with ischemic heart disease (IHD). The aim of the present pilot study was to clarify whether PGM activity has potential as a cardiovascular risk predictor in patients with IHD. METHODS AND RESULTS: The levels of serum PGM activity in 237 patients with IHD (63 patients with acute myocardial infarction (AMI) and 174 patients with stable effort angina pectoris (EAP)) were evaluated. PGM activity was compared with levels of various myocardial, thrombosis, and inflammatory biomarkers on admission. PGM activity in the AMI group was significantly increased relative to that in the EAP group on admission (AMI, 55.5 µmol·min(-1)·L(-1) (U/L); EAP, 14.4 U/L (P<0.001)), and was observed to increase in parallel with well-established myocardial markers (P<0.001). Moreover, PGM activity and the lipid, thrombosis, and inflammatory biomarkers in the AMI group were higher than those in the EAP group. CONCLUSIONS: PGM activity increased with levels of myocardial, thrombosis, and inflammatory biomarkers in patients with AMI, and might be useful in diagnostic applications during the acute phase in patients with AMI.

[Study of mycological examination methods in clinical laboratories--specimen pretreatment and isolation].

We performed a comparative study of the effects of centrifugation, large amounts of inoculum and incubation temperature with regard to recovery of Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus from fungal suspensions in order to identify optimal processing methods for mycological examination of clinical specimens. The number of fungal colonies, except for Candida spp., isolated from respiratory specimens, and the duration of incubation needed to isolate pathogenic fungi from clinical specimens were also analyzed retrospectively. There was a difference in the number of recovered colonies, with or without centrifugation, between inoculum sizes of 10 microl and 50 microl, but no differences were observed in the results obtained under two sets of centrifugation conditions: 2,000 x g for 15 minutes and 3,000 x g for 20 minutes. Candida albicans and Aspergillus fumigatus developed more rapidly at 35 degrees C than at 27 degrees C in the first 24 hours of incubation, while Cryptococcus neoformans formed a larger colony at 27 degrees C than at 35 degrees C. One to three colonies of Aspergillus spp. and Cryptococcus spp. were isolated from respiratory specimens in 73% and 50% of cases, respectively. The required incubation period was six days for isolation of 65 Aspergillus spp. strains from respiratory specimens, while 14 days was needed for isolation of 46 dermatophyte strains. Based on these results, we recommend a pretreatment of centrifugation and a large quantity of inoculum for respiratory specimen processing, as well as an incubation period of at least 7 days and 21 days for internal and dermatological specimens, respectively.

Hyperlipidemia and hyperinsulinemia in the spontaneous osteoarthritis mouse model, STR/Ort.

Recent studies have focused on the association between primary osteoarthritis and dyslipidemia. STR/Ort mice have unique characteristics including osteoarthritis and hyperlipidemia, and may be a useful model for investigating the effect of dyslipidemia on the underlying mechanism of primary osteoarthritis. However, little is known about the hyperlipidemic properties of STR/Ort mice. In this study we investigated hyperlipidemia and lipotoxicity in STR/Ort mice. STR/Ort mice have human hyperlipidemic patient-like symptoms such as hypercholesteremia, hypertriglyceridemia, hyperinsulinemia, insulin resistance, dysregulation of NEFA, and low serum adiponectin. Excess triglyceride accumulation in the liver of STR/Ort mice was not observed even when they exhibited hyperinsulinemia. This information may be useful for researchers investigating lipid metabolism and primary osteoarthritis using STR/Ort mice.

With a novel evaluation method comparison of 2 homogeneous assay kits for high density lipoprotein cholesterol.

BACKGROUND: 2 homogeneous assay kits, Determiner L HDL-C from Kyowa Medex, Co., Ltd. (Tokyo, Japan) (KWM) and Cholestest NHDL from Daiichi Pure Chemical, Co., Ltd. (Tokyo, Japan) (DIC), for HDL-C are widely used in laboratory tests worldwide. Meanwhile, it was reported that free cholesterol (FC) in HDL is not measured with one of these kits. We devised a novel evaluation method and analyzed the 2 kits in detail. METHODS: Using HPLC, the residual amounts of cholesterol in reaction mixtures were compared before and after reaction with homogeneous reagents. Healthy sera were reacted with homogeneous reagents or with heated, non-enzyme reagents from each kit. The FC and total cholesterol in each lipoprotein were continuously measured with HPLC. RESULTS: With KWM, the HDL-C was measured >95%.With DIC, all FC in sera was eliminated in the first reaction, and FC in HDL was not measured. Moreover, the peak X that was assumed to be a part of HDL was separated with DIC, and the cholesterol in the peak X was not measured. CONCLUSIONS: 2 HDL-C assay kits were compared using a novel evaluation method. KWM was good overall. DIC was found to have 2 problems: 1) FC in HDL was not measured, and 2) the cholesterol in peak X that was assumed to be a part of HDL was not measured.

[Educational program in the Medical Science Course, Kitasato University School of Allied Health Sciences].

The aim of education in the Medical Laboratory Science course, Kitasato University School of Allied Health Sciences, is to bring up train students who have Kitasato spirit, for careers in laboratory medicine of hospital or scientific staff of medical companies or as researchers. General and enlightening education concerning "Kitasato spirit" and professional education composed of major subjects was carried out in the first and during the 2nd and two third of 3rd grade, respectively. Medical practice and research training were alternatively carried out for 6 months between November of the 3rd year and November of the 4th year, in order to gain practical experience. Two problem-based learning (PBL) tutorial courses, "Infectious Diseases Course" and "Team Medical Care--Interprofessional Collaborations" were also carried out at the end of the 3rd and beginning of the 4th years, respectively, in order to convert a memory to knowledge. Team medical care course enrolls 1000 students at the School of Allied Health Sciences, Medicine, Nursing, Pharmacy and Kitasato College Applied Clinical Dietetics Course, is now one of special courses available at our university. This attempt is thought to result in a way of thinking that recognizes the importance of co-operation as a team member and personal contributions to actual team medical care.

[Investigation of susceptibility testing methods for micafungin: comparison of two microdilution methods].

As there is not yet a standardized in vitro susceptibility test of micafungin (MCFG), we evaluated the methods of such testing, focusing on the judgment method of MIC, based on the National Committee for Clinical Laboratory Standards (NCCLS) M27-A2, M38-A, the proposed standards of The Japanese Society for Medical Mycology (JSMM) for yeast (JSMM-Y) and for filamentous fungi (JSMM-F) against Candida spp. and Aspergillus spp. The judgment of MIC value was performed spectrophotometrically and visually in both (NCCLS and JSMM) assays. Only the spectrophotometric MIC judgment against Aspergillus spp. in the NCCLS assay used two end points: 80% inhibitory concentration (IC80) of the growth control and 50% inhibitory concentration (IC50). The end point for the visual judgment against Aspergillus spp. in the NCCLS assay was determined to be no growth from the small clumps of altered hyphae in the microtiter plate. The other MIC judgments used an IC80 end point. The MICs of MCFG for Candida spp. were 4mug/ml and 0.0078-0.0313mug/ml). However, the MICs using the IC50 end point and those by JSMM assay agreed with the result of the visual assessment. Therefore, we recommend the JSMM assay, the NCCLS assay using the IC50 end point or the novel visual judgment for the susceptibility testing of MCFG against Aspergillus spp.

High glucose augments arginase activity and nitric oxide production in the renal cortex.

To clarify the interaction between arginase and nitric oxide (NO) production in the kidney with normal and high glucose levels, renal cortical slices from male Sprague-Dawley rats were incubated in Hank's solution containing various concentrations of L-norvaline (Nval; an arginase inhibitor), 500 U/mL superoxide dismutase, and either 5 mmol/L (normal) or 20 mmol/L (high) glucose (n = 5 per group). Incubation with Nval increased renal cortical NOX (nitrite + nitrate) production dose-dependently, indicating competition between arginase and NO synthase (NOS) for the substrate (L-arginine). In the basal condition without Nval, high glucose also increased NO(X) production to a rate 3 times that observed during incubation with normal glucose (P < .01). This effect of high glucose was not altered by Nval. Rather, the effects of high glucose and Nval were additive, indicating that the activity of NOS per se is enhanced by high glucose. Direct assay of arginase and NOS activities confirmed stimulation of both enzymes under the high glucose condition (P < .05, P < .01, v normal glucose, respectively). However, high glucose did not change the amount of L-arginine present in renal cortical slices. These data reveal that arginase competes with NOS for L-arginine in the renal cortex, and that high glucose increases the activity of both enzymes without affecting the amount of substrate. These results suggest that increased NOS activity, rather than altered substrate availability, may be the principal factor underlying increased NO synthesis in diabetic kidneys.