Mass spectrometry of urinary ß2 microglobulin oligomer in patients with proteinuria.

Urinary ß2 microglobulin (ß2-m) is a marker of renal tubule dysfunction; however, ß2-m might become degraded under acidic conditions. To confirm the degradation and consequent deactivation of ß2-m under acidic conditions, we used matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) to detect the levels and forms of ß2-m in the urine samples of patients with high proteinuria (n = 21) and healthy subjects (n = 6). ß2-m was purified in crude form using immunoprecipitation. A signal of 11.74 kDa, corresponding to the molecular weight of ß2-m, was detected in all samples. In addition, several high-molecular-weight proteins were detected in a patient as integrals of the intensity at 11.74 kDa. These results indicate that posttranslational modifications of ß2-m might be involved in the pathological process of proteinuria. Therefore, MS can be used for monitoring proteinuria and predicting the risk of progression.

[Clinical verification of serum procalcitonin measurement in sepsis].

To evaluate the clinical application of measuring procalcitonin (PCT) level for diagnosis of bacterial sepsis in patients with and without systemic inflammatory response syndrome(SIRS), we studied the relationship between blood culture (BC) and serum PCT level in clinical 207 cases. In addition, we evaluated the time courses of PCT and other inflammatory markers: tumor necrosis factor-alpha (TNF-alpha), interleukin 6 (IL-6), E-selectin, WBC count and C-reactive protein (CRP) in 5 bacterial septic patients with SIRS. Serum PCT showed sensitivity of 41% and specificity of 61%, while BC showed specificity of 88%. In 27 BC-positive cases, serum PCT was significantly elevated in gram-negative bacterial sepsis. We observed 11 cases with BC(+) and serum PCT below 0.5 ng/ml. Major causes of these discrepancies were probably due to gram-positive bacterial infection, local bacterial infection or pretreatment with broad-spectrum antibiotics. In contrast, 10 cases with BC(-) and serum PCT over 10 ng/ml were presumably due to some cytokine elevation caused by virus infection or collagen diseases. In 5 cases studied for inflammatory markers, TNF-alpha level elevated earlier than the others and followed by PCT, IL-6, WBC, CRP, and E-selectin. It was suggested that the measurement of serum procalcitonin in septic patients is clinically useful marker to diagnose gram-negative bacterial septic patients with SIRS.

Genetic polymorphisms of enzymes related to oral tegafur/uracil therapeutic efficacy in patients with hepatocellular carcinoma.

Oral tegafur/uracil therapy has been indicated for patients with hepatocellular carcinoma (HCC) and is often used as a single-agent treatment. However, how the treatment efficacy is related to 5-fluorouracil (5-FU) metabolic enzymes is unclear. We investigated genetic polymorphisms of the 5-FU metabolic enzymes in Japanese patients with HCC. We examined two genetic polymorphisms of the metabolic enzymes cytochrome P450 2A6 (CYP2A6) and dihydropyrimidine dehydrogenase (DPD) in 58 Japanese hepatitis C virus-seropositive HCC patients. To measure efficacy, we investigated genetic polymorphisms of the variable number of tandem repeats (VNTRs) of thymidylate synthase (TS) and classified the genotypes as high or low expression types. The frequency of the CYP2A6*4 allele (no-activity allele) among 58 HCC patients was 0.233 and a homozygous genotype (*4/*4) was found in five patients. The heterozygous genotype (T/C) of DPYD*9 (T85C) was detected in eight patients and the frequency of the DPYD*9 allele among 58 HCC patients was 0.069. Of 58 patients, 42 were classified as high expression type and 16 as low expression type for TS VNTR. Fifteen of these 16 patients appeared to have normal CYP2A6 metabolic activity and 13 of these 15 patients likely had normal DPD metabolic activity. Only 13 of 58 HCC patients (22.4%) tested may respond positively to treatment with oral tegafur/uracil. Therefore, when administering oral 5-FU in patients with HCC, it is important to consider three genetic polymorphisms (CYP2A6, DPYD, and TS) associated with 5-FU metabolic enzymes.

[Relationship between glycated albumin (GA) and glycated hemoglobin (A1c) in 255 patients with liver diseases using cross-sectional laboratory data].

To investigate how liver disease alter the serum glycated proteins as markers of diabetic control, we studied serum GA, A1c and especially GA/A1c ratio in 255 patients having over 35IU/L in ALT(transaminase) compared with those of 829 type 2 diabetes mellitus (DM) in cross sectional manner. 255 patients with liver diseases were divided into 69 patients with biopsy proven liver cirrhosis (LC), 66 patients with chronic hepatitis(CH) and 120 patients with fatty liver(FL) diagnosed by abdominal echography. The mean GA/A1c ratio (+/-SD) was significantly higher (p<0.0001) in LC group(3.71+/-1.03) than the other groups (3.03+/-0.45 for CH, 3.05+/-0.42 for DM), while the mean GA/A1c ratio in FL group was significantly lower(2.74+/-0.31) (p<0.0001)) than that of DM groups. In LC group the GA/A1c ratio increased significantly depending upon serum albumin and/or platelet reductions. The GA/A1c ratio was significantly correlated with the other laboratory data such as serum albumin, cholinesterase, total cholesterol levels and weakly correlated with serum hemoglobin level. We also followed the serum levels of GA and A1c and the GA/A1c ratio during about 13 months (5 times blood collections) in 18 patients enrolled in this study. Resultantly the coefficient of variation of GA/A1c ratio was the smaller than the others(GA, A1c). The ROC curve of GA/A1c ratio for LC versus FL group was the most reliable between four groups and the cut-off value for LC versus FL was 2.94. Theses results suggest that GA/A1c ratio could be an useful marker for different diagnosis when facing patients with abnormal serum ALT level in a clinical setting.

Structural and glycation site changes of albumin in diabetic patient with very high glycated albumin.

BACKGROUND: Glycated albumin (GA) has been utilized to monitor mid-term glycemic control, and reflects the status of blood glucose more rapidly and effectively than hemoglobin A(1c) (HbA(1c)). To examine the relationship between GA level and structural changes or glycation sites of albumin, we analyzed pre- and post-treatment samples from a diabetic patient with extraordinary increase of GA. METHOD: A female diabetic patient with poor glycemic control had a GA >94% and was treated with intensive insulin therapy to decrease blood glucose. We analyzed changes in fluorescence derived from tryptophan (Trp) and advanced glycation end product (AGE) of albumin isolated/purified from pre- and post-treatment samples. To determine the sites of glycation of albumin, samples were carboxymethylated and digested by Glu-C endoprotease, and peptides were analyzed using liquid chromatography/mass spectrometry. RESULTS: GA level decreased almost linearly and reflected the improved glycemic state well. Trp-related fluorescence of pre- and post-treated samples did not change while AGE-related fluorescence increased depending on GA level. Ten major glycation sites were detected in the pre-treatment sample, while 3 major glycation sites were detected in post-treated samples. CONCLUSIONS: GA level reflects the status of blood glucose more rapidly than HbA(1c). Since GA level was related to AGE-related fluorescence and number of glycation sites, it might be a good marker for not only glycemic control of diabetic patients but also structural and functional changes of albumin.

In vivo metabolic activity of CYP2C19 and CYP3A in relation to CYP2C19 genetic polymorphism in chronic liver disease.

To study whether chronic liver disease (CLD) and genetic polymorphism affect the hepatic activity of cytochrome P450 (CYP) isoforms, we compared in vivo CYP2C19 and CYP3A activities using 3-hour omeprazole hydroxylation index (plasma concentration ratio of omeprazole to its 5-hydroxylated metabolite; a higher index indicates lower CYP2C19 activity) and partial formation clearance of cortisol to 6beta-hydroxycortisol (CL(cortisol-->6beta-HC)) in 31 CLD patients (9 with chronic hepatitis; 22 with cirrhosis comprising 20 Child-Pugh type A, 1 type B, and 1 type C) and 30 healthy subjects with different CYP2C19 genotypes. The mean (+/-SEM) omeprazole hydroxylation index in CLD patients with homozygous extensive metabolizer (EM) genotype (*1/*1, n = 8), heterozyous EM (*1/*2, n = 11; *1/*3, n = 6) genotypes and poor metabolizer (PM) genotypes (*2/*2, n = 3; *3/*3, n = 3) were 17.15 +/- 2.12, 20.02 +/- 2.63, and 26.04 +/- 3.15, respectively, which were significantly higher compared with control subjects with the corresponding CYP2C19 genotypes (0.81 +/- 0.09, 1.55 +/- 0.20, and 15.5 +/- 1.52). CLD patients with PM genotype had significantly (P < .05) higher omeprazole hydroxylation indexes than did those with homozygous EM genotype, and those with heterozygous EM genotypes had intermediate values. The mean CL(cortisol-->6beta-HC) decreased significantly (P < .001) in CLD patients compared with control subjects (1.19 +/- 0.12 versus 2.26 +/- 0.24 mL/min). Multiple regression analysis showed that CLD, serum albumin level, and CYP2C19 genotype correlated significantly (P < .05) with the omeprazole hydroxylation index, whereas no significant correlation was observed between CL(cortisol-->6beta-HC) and other variables, except CLD. Because CLD and genetic polymorphism of CYP2C19 act additively to reduce CYP2C19 activity, genotyping these patients may be of value in averting adverse reactions of drugs that depend on CYP2C19 for elimination.

Genetic polymorphisms of cytochrome P450 in patients with hepatitis C virus-associated hepatocellular carcinoma.

BACKGROUND: The carcinogenic process can be modulated by exposure to endogenous or environmental substance(s) acting as carcinogens or protocarcinogens. Polymorphic enzymes of cytochrome P450 (CYP) that play a role in detoxication/toxication of such substances via metabolization may account for the interpatient variability of clinical course in cancers such as hepatocellular carcinoma (HCC). Many CYP genetic polymorphisms, which may change enzyme activity, are known to exist in Japanese. The aim of the present study was to compare the frequencies of CYP polymorphisms between hepatitis C virus (HCV)-related HCC patients and healthy subjects. METHODS: Seven mutant alleles and related genotypes of CYP in 44 HCV-positive HCC patients were chosen as follows: *1C heterozygous, *1C homozygous and *1F homozygous for CYP1A2, *4A homozygous for CYP2A6, *2A or *3 heterozygous, *2A or *3 homozygous and *2A and *3 heterozygous for CYP2C19, and *10/*5 homozygous for CYP2D6. These mutant alleles have been reported to change the CYP enzyme activity in Japanese. The frequencies of the mutant alleles and genotypes were then compared with those reported in healthy Japanese. RESULTS AND CONCLUSION: There is no statistically significant difference in genetic mutant alleles between the two groups, except for the genotype of CYP2A6*4A homozygous. The frequency of this genotype in the HCC patients (0.144) is significantly higher than that in healthy Japanese (0.034; P < 0.05; odds ratio 3.36). The clinical significance related to HCC is unknown. Further evaluation of CYP2A6*4A (deletion type) in HCV-related HCC patients is required.

Genetic and immunological analyses of patients with increased serum butyrylcholinesterase activity and its C5 variant form.

Recent evidence has denied genetic abnormality as a mechanism of the C5 variant of butyrylcholinesterase (BChE) and proposed the binding of an unknown protein with the C4 component. The present study aimed to evaluate whether the coding sequences and non-translated sequences of the BChE gene at exons 1 to 4, 3q are structurally different in subjects having elevated BChE with and without the C5 variant phenotype. We also attempted to identify the unknown protein associated with the C5 variant and measured the BChE-specific activity in the C5 variant with an enzyme-linked immunosorbent assay (ELISA) using anti-BChE monoclonal antibody. We investigated five subjects, four of whom had elevated plasma BChE (three C5-positive [C5(+)] and one C5-negative [C5(-)]) and one control with a normal plasma BChE level. Direct DNA sequencing of the BChE gene revealed no relevant genetic mutations and no abnormal migrations in the genes of all five subjects. Precipitation of the patients' sera with anti-human immunoglobulin A (IgA), -IgG, -IgM, anti-human albumin antibodies had no effect on the BChE activity. The measured BChE activity in C5(+) was 30 to 54% higher than the activity calculated from BChE protein content. The present results suggest that the C5(+) phenotype is not associated with any genetic abnormality in the CHE1 locus, and BChE-specific activity is enhanced in the C5(+) variant. However, the exact nature of the unknown protein related to the C5(+) phenotype remains unclear.