An algorithm is proposed for selection of data distribution type. Distribution models used in this method are normal, log-normal and power-normal (XP; P=-2-+3). Transformed data according to the distribution type are subjected to the iterative truucation and correction method, and normal range and skewness are calculated for each distribution type. The best fit distribution type is selected according to following criteria: (1) distribution which has small skewness enough by the skewness test (p=0.05),(2) simple distribution type, normal or log-normal distribution, and (3) distribution which has minimum skewnss. The distribution types of 33 chemistry and 8 hematology tests were analyzed by the present method, and distributions of some tests showed negative power distribution. Using computer simulation models, it is assumed that those types of distributions are obtained from mixed distribution models.
A method has been developed for the determination of free and antibody-bound insulin levels in the serum of patients with anti-insulin antibody. The assay procedure for the free insulin consists of antibody precipitation by polyethylene glycol (PEG, final 12.5%) and enzyme immunoassay (EIA) for immunoreactive insulin (IRI) in the supernatant. For the assay of total insulin (free insulin and antibody-bound insulin), serum was first acidified with HCI to dissociate bound insulin, and then PEG (final 9%) was mixed to precipitate antibody. After neutralization with NaOH, 10% BSA solution was added to compensate total protein concentration. Following centrifugation, IRI in the supernatant was measured using EIA kit. The standard solutions were prepared by dissolving lyophilized porcine insulin in human serum containing low level of IRI. The coefficients of variation of intra- and inter-assay of the free and total insulin were 5.9-19.2%, and the recovery tests were satisfactory (93.3-113.3%).
While determining polyamine levels in body fluids, unexpected values probably due to bacterial growth during storage are occasionally obtained. In this paper, we determined the effect of four common bacteria, namely, Escherichia coli, Streptococcus faecalis, Pseudomonas aeruginosa and Bacillus subtilis, which are often seen in urine samples, on various polyamine levels. Urines which were sterified by filtration through a 0.22?Em millipore filter, Sterifil D-G S, and then contaminated with Escherichia coli showed significantly high to tal polyamine values as compared to the corresponding sterified urines. This increase was mainly ascribed to free putrescine and cadaverine which were produced by Escherichia coli during storage. Acetylated polyamines were not increased in those samples. In contrast, the total polyamine levels, both free and acetylated polyamine levels, were found decreased in urine contaminated with Pseudomonas aeruginosa. In case of urine samples collected from patients, however, 8 out of 10 showed increase in free polyamine levels, and the other 2 samples did not show any change. Streptococcus faecalis and Bacillus subtilis had no effect on polyamine levels in urine. To avoid such changes in polyamine levels in urine due to bacterial growth during storage, polyamine, if possible, should be determined soon after collecting urine samples. Either sodium azide or toluene, if not evaporate during storage, is useful for determination of polyamine in 24 hour urine samples, or for storage of urine at 4 for 24 hours by preventing a factitious increase or decrease in the polyamine concentration due to bacteria in urine.
A simple high-performance liquid chromatographic method was established for detecting putrescine, cadaverine, spermidine and spermine in human urine. Polyamines were separated on an ion-exchange column with isocratic elution and measured by fluorometry cf the o-phthalaldehyde derivatives using 1, 6-diaminohexane as an internal standard. The recovery rate was 75% for cadaverine and nearly 100% for the other polyamines. The minimum detection limit of each polyamine was less than 0.5μmol/l in urine. The normal range (95% limit) of polyamines in 24-hr urine was 12.1-29.8μmol/day for putrescine, 1.1-27.0μmol/day for cadaverine, 4.7-16.8μmol/day for spermidine and less than 11.5μmol/day for spermine. The normal range (95% limit) in terms of polyamine/creatinine ratio was 8.3-22.7μmol/g for putrescine, 0.5-18.5μmol/g for cadaverine, 4.2-10.7μmol/g for spermidine and 0.1-7.8μmol/g for spermine.
The spot test for urinary 3-methoxy-4-hydroxy-mandelic acid (vanilylmandelic acid: VMA) combined with open-system chromatography was established and its separation mechanism was studied. Solutions of sample and standard were spotted on a filter paper, Whatman No.1, 15×15cm, and dried. A solution of diazotized 4-nitroaniline in 0.2 N hydrochloric acid was sprayed on the filter paper. The filter paper was placed in a dish containing 50ml of 70% ethanol in such a way that the spot was 1.5cm distant from the surface of the solvent. After development for about 40 minutes, the intensity of the colored spot was compared with of the adjacent standard. In the present method, the violet colored spot derived from VMA in the sample was separated from the abundunt spray reagent and co-existing uric acid and ascorbic acid. In this chromatography, the upward decrease of the content of ethanol in the filter paper was clear around the spotted line followed by gradual change. Consequently, the colored VMA spot was separated as a narrow band in the area of steep gradient of the ethanol content. The present method is selective and convenient enough to be applied to the first screening test for neuroblastoma and pheochromocytoma.
By applying a“hydrophobic”HPLC system, five of peptidic substances in the middle molecule range were separated from the hemofiltrate fluid of a dialysis patient with chronic uremia. This system was based on the hydrophobicity value of peptide hormones having molecular weight up to 5,000 daltons by using gradient elution with increasing acetonitrile concentration in the eluent. Many ultraviolet-absorbing solutes of the hemofiltrate detected on this system were classified as hydrophilics, low-hydrophobics and hydrophobics. Numerous peaks of the hydrophobic solutes could be detected only in the hemofiltrate but not in healthy subject urines. Both middle molecule crude fractions of low-hydrophobicity and hydrophobicity were separated by step-wise gradient elution and further separation of the middle molecule subfractions was achieved by isocratic elution. Each of five subfractions corresponded to a single middle molecule and was found to be peptidic substances.
We developed a new colorimetric method based on the oxidation and coloring reaction of leuco crystal violet (LCV) to determine hydrogen peroxide concentrations by the peroxidase-like activity of hemoglobin in the presence of hydrogen peroxide. The color intensity and hydrogen peroxide concentration were linear up to 1μM/ml. The minimum detectable concentration was 0.1μM/ml. The intra-assay coefficient of variation (CV) was 2.75% and the recovery rate was 104.0%.