Serum methylguanidine (MG) concentrations in patients on hemodialysis (HD) were determined by an improved enzymatic method. A significant correlation was found between MG (γ) and creatinine (χ) values in serum with a linear regression equation of γ=6.836χ-3.030 and a correlation coefficient of 0.705. The patients on HD could be divided into 3 groups based on Δ MG (the difference between the observed MG and the calculated MG from the above equation with measured creatinine):Δ MG≥1.37μmol/l, 1.37μmol/l>Δ MG>-1.37μmol/l, and Δ MG≤-1.37μmol/l. In about 70% of the patients with joint pain, infection and gastrointestinal bleeding, Δ MG was 1.37μmol/l or more. MG and Δ MG were significantly increased in patients on hemodialysis for 10 years or longer. We found overproduction of MG in those hemodialysis patients with inflammatory complications. The patients on hemodialysis for a long duration frequently complained of pain in various joints. In patients on HD for 10 years or longer duration, significant positive correlations were found between MG and the number of red blood cells, and between MG and hemoglobin and myoglobin levels. The generation of hydroxyl radicals in the presence of hydrogen peroxide and hemoglobin (or myoglobin) was observed by the electron spin resonance spectrometry (ESR) spin trapping technique. These findings suggest that the production of MG from creatinine in patients on HD is caused by the increase of blood heme protein level. The measurement of MG in serum of patients, on HD, may provide useful information concerning the generation of hydroxyl radicals.

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The erythrocyte membrane is altered in diabetes mellitus. We examined the effects of oxidative stress on the diabetic erythrocyte membrane. The erythrocyte membrane, prepared from diabetic or control subjects, was exposed to reactive oxidants generated from leukocytes, the resultant increase in lipid peroxide contents and the membrane damage were compared between diabetic and control erythrocyte membranes.
The glycation level in the erythrocyte was significantly higher in diabetic patients than in control subjects (mean±SD=418±72 versus 363±24nmol/10¹⁰ RBC:ρ <0.05). After incubation with activated leukocytes, the release of lactate dehydrogenase activity (mean±SD=905±283 versus 547±191 IU/10¹⁰ RBC:ρ <0.001) and lipid peroxide contents in the erythrocyte membranes (mean±SD=4.52±3.57 versus 1.93±0.77nmol/10¹⁰ RBC:ρ <0.02) were higher in the diabetic erythrocytes compared to control erythrocytes.
These findings showed that the diabetic erythrocyte membrane was vulnerable to lipid peroxidation, indicating the possible role of vascular complication in diabetes mellitus.

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Catecholamines (CA)[dopamine (DA) and norepinephrine (NE)] in the ventricular cerebrospinal (CSF) and lumbar spinal fluid obtained from 11 cases of Parkinson's disease and 3 control subjects were determined using a high performance liquid chromatography. Results were as follows:
1. The free DA concentration of ventricular CSF was decreased in Parkinson's disease compared to the control. However, there were no differences in free NE concentrations of ventricular CSF between the two groups.
2. There were no differences in free NE and DA concentrations of lumbar CSF between the two groups.
3. NE concentrations of the lumbar CSF showed a tendency to be higher than those of ventricular CSF, while the DA concentrations of ventricular CSF was much higher than that of lumbar CSF.
4. The administration of L-threo-DOPS and L-DOPA-DCI increased conjugated forms of NE and DA, resulting in the increase of total NE and DA concentrations of ventricular CSF.

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Selective enzymatic methods for measuring total and conjugated bilirubins in serum have been developed by using bilirubin oxidase (BOD). BOD can oxidize all bilirubin fractions to non-colored substances at pH 7.8 in the presence of anionic surfactant. On the other hand, BOD can oxidize only the conjugated bilirubin fraction at pH 5.5 in the presence of reagent components such as sodium fluoride and N-acetylcysteine, which can decrease the enzymatic reactivity to unconjugated bilirubin. The resulting decrease in absorbance at 450nm is linearly related to the concentration of bilirubin fractions in serum. These methods were found to have the satisfactory results with respect to the range of the measurement, the reproducibility, absence of interference by coexisting substances in serum and the stability of the reagents in practical use. The value obtained by the method for measuring the total bilirubin correlated well with those obtained by the conventional methods. The method for measuring the conjugated bilirubins was characterized to react only with conjugated bilirubins, but not with unconjugated and delta bilirubins, by its reactivity to the unconjugated bilirubin and the HPLC analysis. The measured value for the conjugated bilirubins by this method correlated well with that by the HPLC analysis. These methods were applicable to the fractional determination of bilirubin fractions in the neonatal serum. These findings suggest the usefulness of these new methods for the accurate measurement of bilirubin fractions present in serum.

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Total cholesterol (TC) and HDL-cholesterol (C) of human sera (n=134) from subjects with serum triglyceride (TG) levels from 40mg/dl to 5378mg/dl were measured by an improved HPLC system using a gel filtration column (TSK gel Lipopropak, 7.5mm×600mm/TOSOH Co. Tokyo) and an eluent (TSKeluent LP-1). The values were compared with TC measured by the automated enzymatic method (HITACHI 7070) and HDL measured by the precipitation method. By the HPLC method, TC and HDL-C levels as well as the HDL sizes could be simultaneously and accurately obtained from the HPLC patterns monitored by cholesterol for all subjects including severe hypertriglyceridemia (n=7), in which HDL could not be separated by the precipitation method. Although the TC levels obtained by the HPLC method were consistent with those obtained by the automated enzymatic method, HDL-C levels were slightly higher than those obtained by the precipitation method. The HDL of all the samples with discrepant values between the two methods were revealed to be large in size and rich in cholesterol. Therefore, HDL subclasses with a larger size of particle and higher amount of cholesterol than normal HDL were suggested to be precipitated selectively by the precipitation method. A negative correlation was obtained between the HDL-C level by the two methods and serum TG level, but correlations were improved by excluding the samples with discrepant HDL-C levels between the two methods.

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We found a significant discrepancy in serum glucose measurement between colorimetric assay (automated chemical analyzer) with glucose oxidase (GOD) and GOD electrode assay (instant analyzer) in a 70-year-old male patient with macroglobulinemia. The difference was 3.33mmol/l of glucose. The patient had IgM proteinemia and the serum precipitated in low ionic strength buffer of the colorimetric assay. The precipitate caused a rapid increase in optic absorbance resulting in a higher glucose concentration reading than the true concentration. This type of discrepancy is estimated to occur in 0.15% of the measurements (n=2000, 95% confidence interval 0.03-0.44%). Avoiding reading of initial elevated absorbance due to precipitation by delayed reading in the colorimetric assay was effective in obtaining reasonable measurement. We recommend laboratory analysts to find the best analytic condition.

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A reference method for the determination of triglyceride (TG) in serum is described. The method is based on the use of alkaline saponification to hydrolyze triglycerides, followed by an enzymatic determination of the generated glycerol. The principle of the proposed method is as follows. TG is hydrolyzed by alcoholic KOH. Glycerol released is determined by end point analysis, including glycerol kinase, pyruvate kinase, and lactate dehydrogenase reaction. Endogenous glycerol should be subtracted from the results. This method is mainly used for the evaluation of the accuracy of the results obtained by routine methods in the clinical laboratory, determination of assigned value for the routine available commercial reagent kits, and for the control materials of quality assurance, and for the survey materials of quality assessment. Analytical conditions of this method were constituted on the basis of the prior studies done by the old Committee on Reagents in the Kinki Sub-Group of the Analytical Section of JSCC during 1984-89.

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