Effect of genetic variation on the fatty acid-binding properties of human serum albumin and proalbumin.

In the circulation, non-esterified fatty acids are transported by albumin which also facilitates their removal from donor cells and uptake into receptor cells. We have studied whether genetic variations in the albumin molecule can affect its in vivo fatty acid-binding properties. The fatty acids bound to 25 structurally different variants and to their wildtype counterparts, isolated from heterozygous carriers, were determined gas chromatographically. The variants were proalbumins, albumins with single amino acid substitutions and glycosylated or truncated albumins. In eight cases the total amount bound to the variants was diminished (0.4-0.8-fold), and in seven cases the load was increased to 1.3 or more of normal. Twenty-one fatty acids were quantitated, and for 19 alloalbumins significant deviations from normal were found. Usually, changes in total and individual fatty acid binding were of the same type, but several exceptions to this rule was found. The glycosylated albumin Casebrook showed the largest changes, the total load and the amount of bound palmitate was 8.6 and 14 times, respectively, the normal. The most pronounced changes and the majority of cases of increased binding were caused by molecular changes in domain III. Mutations in domain I, II and the propeptide resulted in smaller effects, if any, and these were often reductions in binding.

Detection of circulating cancer cells with von hippel-lindau gene mutation in peripheral blood of patients with renal cell carcinoma.

Mutations of the von Hippel-Lindau (VHL) tumor suppressor gene have been detected in up to 60% of sporadic clear cell renal carcinomas (RCCs). Even patients with RCCs believed to be curable with radical nephrectomy sometimes develop distant metastasis 5-10 years after surgery, suggesting hematogenous circulation of cancer cells. Useful tumor markers have not yet been established for RCC. To detect patients at high risk of metastasis after surgery, we developed a highly sensitive and specific nested reverse transcription-PCR method using VHL gene mutation to detect circulating cancer cells. We screened 29 sporadic clear cell RCCs from patients for mutations of the VHL gene by direct sequencing. We next examined blood samples from patients with the VHL gene mutation using mutation-specific nested reverse transcription-PCR. Somatic mutations were detected in 20 of 29 (69.0%) sporadic clear cell RCCs. The VHL gene mutations were detected in peripheral and/or renal venous blood from 15 of 20 (75%) patients. The mutations were detected in the peripheral blood in 2 of 17 (11.8%) patients before surgery, 6 of 16 (37.5%) patients within 24 h after surgery, 3 of 16 (18.8%) patients on day 7 after surgery, and 2 of 11 (18.2%) patients on day 30 after surgery. In seven of nine (77.8%) patients, mutations were detected in renal venous blood during surgery. These findings indicate the presence of circulating cancer cells with VHL gene mutation. Although much larger studies are needed to determine the clinical significance, our study shows that this technique is feasible for detecting circulating RCC cells.

Neuropeptide Y innervation in the spinal nucleus of bulbocavernosus of the rat.

Immunoelectron microscopy combined with a retrograde tracing technique was carried out to examine the synaptic interaction between neuropeptide Y (NPY)-like immunoreactive (LI) axon terminals and spinal motoneurons innervating bulbocavernosus muscles. Cell bodies and proximal dendrites of these motoneurons were frequently found to receive synaptic inputs from NPY-LI axon terminals.

Reference limits of bone and liver alkaline phosphatase isoenzymes in the serum of healthy subjects according to age and sex as determined by wheat germ lectin affinity electrophoresis.

Wheat germ lectin affinity electrophoresis was employed for quantitating the bone and liver isoenzymes of alkaline phosphatase (EC 3.1.3.1) in serum and for determining the reference limits of each isoenzyme activity in 488 healthy individuals. Bone phosphatase activity was detected even after bone growth, accounting for 60-70% of the total activity. An increase in bone phosphatase activity occurred in older females, but there was a decrease in older males. Liver phosphatase activity gradually increased with age in both sexes, males showing higher activity than females at all ages. Wheat germ lectin affinity electrophoresis of serum alkaline phosphatase is a simple and useful method for quantitating bone and liver alkaline phosphatase activities.

The molecular abnormality of albumin Niigata: 269 Asp----Gly.

A slow albumin variant was isolated from the serum of a patient with bisalbuminemia. Reverse-phase peptide mapping revealed a single altered peak when tryptic digests of the normal and variant albumin were compared. After rechromatography and amino acid analysis, a sequence of Tyr-Ile-Cys-Glu-Asn-Gln-Gly-Ser was obtained for the mutant peptide, while a sequence of Tyr-Ile-Cys-Glu-Asn-Gln-Asp-Ser was obtained for the normal peptide. This establishes the mutation as 269 Asp----Gly and the new albumin has been named albumin Niigata.

Effect of sulfaphenazole on tolbutamide distribution in rabbits: analysis of interspecies differences in tissue distribution of tolbutamide.

The effect of sulfaphenazole on the distribution of tolbutamide was examined by comparing the change in the steady-state volume of distribution (Vdss) determined from in vivo plasma elimination with the tissue-to-plasma concentration ratio of various tissues (Kp) in rabbits; this effect was compared with that previously reported in rats. In rabbits, the Kp values of six tissues studied (i.e., brain, heart, spleen, small intestine, muscle, and skin) increased in the presence of sulfaphenazole ; except for brain, lung, and adipose tissue, the tissue-to-plasma unbound concentration ratio (Kp,f) of other tissues did show a significant decrease. This suggested that both the tissue and plasma protein binding of tolbutamide were affected by sulfaphenazole and that the increase in Kp was due mainly to the displacement of plasma protein binding of tolbutamide by sulfaphenazole , which was greater than that of tissue binding, while no change in Kp was due to a parallel change in both the plasma protein binding and tissue binding of tolbutamide. In both rabbits and rats, the Vdss calculated from plasma concentration versus time curve was very close to that calculated from the Kp values and volumes of various tissues in the presence and absence of sulfaphenazole , respectively. The interspecies difference of the effect of sulfaphenazole on the tissue distribution of tolbutamide between rabbits and rats was elucidated from both in vivo tissue distribution and in vitro plasma protein binding studies.

A rapid method for measuring serum amyloid A protein by latex agglutination nephelometric immunoassay.

Serum amyloid A protein (SAA) is a sensitive acute phase reactant. We developed a method for the rapid measurement of human SAA in serum by kinetic nephelometry of anti-SAA antibody-coated latex agglutination. Measurement takes less than 6 min using an automated analyser. Standardization of the assay employs SAA-enriched high density lipoprotein as the primary standard. The values determined by our new method and by conventional enzyme-immunoassay showed good agreement (r = 0.988). The normal range was 0.17-10.0 mg/L [mean(SD)]. This rapid method should prove useful in clinical laboratories.

Bisalbumin (fast and slow type) induced by human pancreatic juice.

To clarify the relationship of pancreatic juice to bisalbumin, we examined the effects of human pancreatic juice and pancreatic proteolytic enzymes on human serum albumin. Electrophoresis showed that a fast-moving band and an increased component of alpha 1-globulin appeared after incubation of serum with pancreatic juice. These components were shown to be albumins by immunofixation and immunoelectrophoresis. The alpha 1-component increased after incubation of serum with trypsin, and was confirmed as a slow-type albumin by immunoelectrophoresis. Fast-type albumin was observed after incubation of serum with chymotrypsin, and carboxypeptidases A and B. In all patients with pancreatic ascites, the fast- and slow-type albumins were seen in serum and ascitic fluid. Bisalbumins appeared in ascites at higher concentrations than in serum, and disappeared after surgical or conservative treatment. The results demonstrate that human pancreatic juice can produce fast- and slow-type albumins by proteolytic enzymes, and they are seen in patients with pancreatic ascites.

Three families with fast bisalbuminaemia.

We describe three cases of familial (SH, AY, and TM) fast-type bisalbuminaemia identified from 300 000 electrophoretic strips screened during the past six years. The immunological antigenicity and chemical composition of the isolated fast and normal albumins were essentially indistinguishable in all three cases. Detailed analyses on one of them (TM) by circular dichroism and fluorescent spectra measurements indicated that there was a marked change in the environment of the single tryptophan residue of the fast albumin, suggesting an alteration in the tertiary structure of the molecule. It is likely that this abnormality of the tertiary structure modulated (perturbed) the distribution of electric charges on the protein surface and thus changed its electrophoretic mobility.

Reference values of serum and urine creatinine, and of creatinine clearance by a new enzymatic method.

A new, totally enzymatic procedure for the determination of creatinine in serum and urine, using creatinine amidohydrolase, creatine amidinohydrolase, sarcosine oxidase and formaldehyde dehydrogenase is described. The assay was adapted to a discontinuous analyser with each analysis requiring only 20 microL of serum or 3 microL of urine. Analytical recovery of creatinine in serum and urine averaged 100.6%. Within-run and between-run precision studies gave coefficients of variation of 1.1% and 1.8%, respectively, for a serum with mean values of 83 mumol/L (9.4 mg/L) creatinine. Creatinine concentrations in serum and urine were measured by this procedure, in Japanese children and adults. The reference intervals for serum creatinine concentrations in adults were 55-96 mumol/L (6.2-10.9 mg/L) in men and 40-66 mumol/L (4.5-7.5 mg/L) in women, and for urine, 9.46-19.01 mmol/day (1070-2150 mg/day) in men and 6.75-10.61 mmol/day (764-1200 mg/day) in women. The reference intervals of creatinine clearance were 88.0-176.4 mL/min in men and 75.7-173.0 mL/min in women.

A new enzymatic method for the determination of inulin.

A new enzymatic method for the determination of inulin in plasma and urine, using inulase (EC 3.2.1.7), fructokinase (EC 2.7.1.4), phosphoglucoisomerase (EC 5.3.1.9) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) is described. The assay is based on the hydrolysis of inulin or Inutest (INutest which is the injectable form of inulin), by inulase and the determination of fructose released. The assay was linear up to 2 g/L of Inutest. The within-batch and between batch coefficients of variation were 2.3% and 2.2%, respectively. Recovery of added Inutest from plasma and urine was 98-102%. There was no interference from glucose (27.7 mmol/L), fructose (1.7 mmol/L) or mannose (1.7 mmol/L). When inulin clearance (using this method) and thiosulphate clearance were compared in 37 patients the inulin clearance was 9.3 mL/min (12%) lower than the thiosulphate clearance. We conclude that this enzymatic method is a simple and specific method.

Effects of modified low density lipoprotein and hypoxia on the expression of endothelial leukocyte adhesion molecule-1.

OBJECTIVES: Expression of endothelial leukocyte adhesion molecule (ELAM-1 or CD62E) plays a role as an early event of atherogenesis. It is well known that interleukin-1 (IL-1) expresses ELAM-1 on vascular endothelial cells. We have examined pathological factors that induce ELAM-1 expression on cultured endothelial cells. METHODS: Examined factors were native low density lipoprotein (LDL), oxidized LDL, glycated LDL, hypoxia, and IL-1. Peroxidation of LDL was performed by ultraviolet radiation. Hypoxia was reproduced by adding a hypoxic cell-culture medium that was deoxygenated by use of a vacuum pump and nitrogen gas. Endothelial cells were harvested from a porcine aorta and were allowed to proliferate to be subconfluent in slide chambers. Expression of ELAM-1 was evaluated by counting the number of cells that were characterized by positive staining with the immunohistochemical technique. RESULTS: Without any stimulants, about 6.9% of the endothelial cells expressed ELAM-1. Weakly oxidized LDL (12 pmol/micrograms protein) significantly expressed ELAM-1 (14.8%) after an incubation period of 1 hour. Glycated LDL induced significant expressions (12.6%) in a fructosamine concentration of 65 pmol/micrograms protein. A one-hour incubation with a hypoxic culture medium expressed ELAM-1 in 16.3% of the cells. Native LDL did not cause any significant increases in the percentage. IL-1 expressed ELAM-1 in 30% of the cells even with as low a concentration as 3.1 U/ml. CONCLUSIONS: The present study shows that not only IL-1 but also weakly oxidized LDL, glycated LDL, and hypoxia may be possible factors that cause the expression of ELAM-1.

[The improvement of colorimetric determination of lipid peroxide in human serum].

The present work details an improved application of kit to the measurement of lipid peroxides in human serum. Kits based on the reaction of lipid hydroperoxides with a leucomethylene blue derivative in the presence of hemoglobin. Overall sensitivity was increased by taking samples 5 times larger than those indicated in product literature for the system. It was necessary, however, to compensate actual data for protein error. To do this, the following empirically obtained formula was employed: A = a/(-3.45x + 98) x 100, where A (nmol/ml) indicates compensated data for lipid peroxides, a (nmol/ml) indicates the data for the actual measurement of lipid peroxides, and x (g/dl) indicates the total protein. We obtained what we considered to be satisfactory results by the above formula, as it allowed us to observe that lipid peroxides in fresh human serum undergo a change, momentarily.

[Calculation of normal range using a nonlinear least squares method].

We developed a method to calculate the normal range of laboratory tests using nonlinear least squares method. Our method decomposes a histogram of laboratory test into constituent distributions assuming these constituents to be gaussian distributions. The proposed method has been applied to the histograms of serum total protein and serum lactate dehydrogenase, and good fittings were obtained assuming the histograms consist of 2 and 3 constituents respectively. Accuracy and precision were calculated for the stimulated data of known distribution pattern with contamination of abnormal values to examine the robustness of our method. With the contamination rate of 10%, accuracy less than 1% and precision less than 3% have been shown for the sample size more than 2,000. And the estimation was not markedly affected by the contamination rate up to 50%. Based on the parameters estimated by our method, we proposed a new measure for laboratory tests named abnormality index, which gives a probability to be abnormal.

[Measurements of prostatic acid phosphatase and prostate specific antigen by fully automated enzyme immunoassay system--its analytical and clinical evaluation].

Analytical and clinical evaluations were made on the measurement of prostatic acid phosphatase (PAP) and prostate specific antigen (PA) by a fully automated enzyme immunoassay system. Results concerning reproducibility, recovery and sensitivity were good. PAP values by this method correlated well with those obtained by radioimmunoassay. PA values by this method were higher than those obtained by other enzyme immunoassays, although the correlation coefficient was high. PAP, PA and gamma-Seminoprotein (gamma-Sm), another prostatic tumor marker, were all poorly correlated to one another. Normal upper value, 2 SD of 720 healthy males was 1.2 ng/ml for PAP, and 3.7 ng/ml for PA. Positive ratios of these tests in 31 patients with prostatic carcinoma were high at advanced stages, and low at early stages. ROC (receiver operating characteristics) curve analysis on patients with prostatic carcinoma and benign prostatic hypertrophy indicated that PAP and PA were more effective than gamma-Sm for the differential diagnosis of prostatic carcinoma, and that the clinical cut off was 2.0 ng/ml for PAP, 7.4 ng/ml for PA and 4.0 ng/ml for gamma-Sm.

[Use of heart rate variability and pulse wave velocity for diagnosing cardiovascular autonomic neuropathy].

Heart rate (HR) and arterial pulse wave velocity (PWV) were used for the evaluation of cardiovascular autonomic neuropathy. Data were analyzed from 30 patients with diabetes mellitus (aged from 13 to 75 years). Twenty healthy male subjects (aged from 22 to 44 years) were analyzed for computing normal values as well. After 15 minutes rest, electrocardiogram (ECG) of lead II and plethysmogram of finger tip were simultaneously recorded for each subject. The recording was first done in supine position for 120 seconds and subsequently in upright position for 40 seconds. HR was computed on the basis of consecutive pairs of R wave of the ECG. PWV was estimated by transit time from R wave peak to initial rise of pulse wave. Mean and coefficient of variation (CV) were obtained from the HR in the supine and upright positions respectively, and also from the PWVs. The influence of changing the position was evaluated by percentage of the difference (increasing rate). All the parameters were statistically tested for the difference between the patients with neuropathy and those without neuropathy. As the result, two parameters, that is, the CV of HR in the supine position and the increasing rate of PWV were found to be useful for diagnosing the cardiovascular autonomic neuropathy (p less than 0.05).

[Evaluation of serum protein fractions using nonlinear least squares method].

We developed a method to evaluate the contents of serum protein fractions using nonlinear least squares approach. Parameters that determined the distributions of fractions of a densitogram were estimated assuming that each fraction obeys Gaussian distribution. One hundred test results of patients and one hundred and fourteen simulated patterns were analyzed with the proposed method and the traditional method. For patient data, the traditional method tended to give higher values in beta-globulin fraction and lower values in alpha 2-globulin and gamma-globulin fractions compared with our method. For the simulated data, the traditional method also tended to give higher values in beta-globulin fraction and lower values in gamma-globulin fraction, whereas our method gave the correct values.

[Serum cholesterol levels in normal subjects taking into consideration the accuracy of the measuring system].

Serum cholesterol levels were determined in 5,843 normal subjects aged zero to seventy years. The accuracy of our assay method was checked by Standard Reference Material 909 and Certified Reference Serum, both of which were supplied by the National Bureau of Standards. Reference values of serum cholesterol were confirmed for each age group. That of male subjects in the 15-20 year age group was within a range of 109-203 mg/dl, and that of female subjects in the 20-25 year age group was within a range of 133-215 mg/dl. Therefore, among normal subjects, the above-mentioned age groups had the lowest serum cholesterol levels. Serum cholesterol levels increased with age in both male and female subjects. The upper limit of cholesterol levels was 248 mg/dl for males in the 50-60 year age group and 284 mg/dl for females in the same age group. We observed the necessity of paying consideration those changes which occur with aging, in the determination of reference values of serum cholesterol. Our findings also showed that serum cholesterol levels remained nearly constant in male subjects of all age groups over a period of 25 years. However, we found mean levels in female subjects in the 50-70 year age groups to be significantly elevated, when compared with those observed in persons in the same age group of 25 years previous. We also found that the mean cholesterol level in girls aged 12 years was higher than that of boys the same age.

[The measurement of antioxidant activity in human plasma using cumene hydroperoxide].

We describe a new method using cumene hydroperoxide to determine antioxidant activity (AO) in human plasma. We used a kit (Determiner LPO: Kyowa Medex Co., LTD. Tokyo Japan) for the determination of lipid peroxides in plasma or serum. 30 microliters 1 of sample was mixed with 70 microliters 1 of cumene hydroperoxide (50 nmol/ml) and incubated at 30 degrees C for 120 min before analysis. Samples were mixed with 1.0 ml of reagent-I (Determiner LPO) and incubated at 30 degrees C for 5 min. Then 2.0 ml of reagent-II (Determiner LPO) was added and incubated at 30 degrees C for 10 min, at which time the absorbance at 675 nm was measured. AO were calculated using the following formula: AO nmol/ml = 35 nmol/ml-(Es-Eb)/(Estd-Eb) x 35 nmol/ml (Es = sample abs., Eb = blank abs., Estd = standard abs.). Within-run precision for plasma AO was 2.3%. AO in plasma samples stored for 4 h at 4 degrees C was decreased by 1 nmol/ml. After 3 h at room temperature, AO was decreased by the same amount. Because this method measured ascorbic acid, alpha-tocopherol, glutathione peroxidase and quercetin as antioxidant compounds, we were able to measure antioxidant activity in human plasma. Our reference values were calculated from the volunteers group which consisted of 172 students and 82 soldiers. The reference intervals for plasma AO by this procedure were 15.4-20.9 nmol/ml.

[A case of congenital mesoblastic nephroma in adulthood].

A 20-year-old man was referred to our hospital with a complaint of asymptomatic macrohematuria. A diagnosis of a right renal tumor was made after several radiographic examinations and right radical nephrectomy was performed. Histopathologically the tumor was a congenital mesoblastic nephroma. Congenital mesoblastic nephroma is a relatively rare renal tumor predominantly of childhood. Occurrence in adults is exceedingly rare and 18 cases have been reported to date.