This study was performed to clarify the relationship between diagnosis of acuti myocardial infarction (AMl) and level of creatine phosphokinase (CPK) activity. We studied a patient with AMl (case preserltation). ln order to obtain the time course of CPK activity, the serial serum samples were assayed for the total CPK and for one of its isoenzymes, CPKMB.Six hours after the admission, the ratio of CPK-MB to total CPK was 18% and after 1 week, it returned to normal range. Seven weeks later the patient was discharged. We next discussed the serum level of CPK-MB in AMl group (18 patients) and in non-AMl group (41 patients). The value of CPK-MB/total CPK in AMl was 2.5 fold compared with non-AMl within 24 hours and 2.2 fold within 24 to 48 hours after theadmission. We lastly investigated the relationship between the elapsed time after chest pain and the level of CPK activity.The CPK-MB activity diagnostically increased within 2 hours following onset of chest pain and markedly within 6 to 12 hours.The elevated level of more than 7% of CPK-MB/total CPK correlated well with diagnosis of AMl. From these results, it has been definitely shown that subjects can bediagnosed as AMl in an early phase by the determination of CPK-MB in serum.
In a patient with lgA (K) myelomatosis, total calcium level in the serum was found to be relatively high although ionized calcium was normal and albumin was low (total calcium, 2.5 mmol/l; ionized calcium, 1.15 mmol/l; albumin, 26g/l). By a study of calcium-binding capacity of serum protein fractions obtained with the gel chromatographic method of Toffaletti et al.(1977), extent of calcium-binding capacity of the lgA (K) monoclonal component purified from the patient's serum was 1.22×10-2 mmol calcium per 1 g Protein whereas those of albumin and globlin from normal subjects were 1, 05×10-2 and 0.44×10-2mmol/g respectively. Accordingly, the lgA (K) monoclonal component of the patient is the globulin which has almost the same affinity for calcium as albumin.
A simple high-performance liquid chromatographic method is developed for the fluorimetric determination of forphenicinol in human serum. Forphenicindl is converted to a fluorescent compound by alkaiine ferricyanide oxidation and the compound is then separated within 8 min on a reversed-phase column, Lichrosorb RP-18 with isocratic elution ushg an ammonia buffer (pH 8.5). The fluorescence of the compound is monitored at excitation and emission wavelengths of 385 and 500 nm, respectively.This method permits the sensitve quantification of forphenicinol in serum from men administered forphenichol in clinical investigations.The detection limit of forphenicinol is 1.0 nmol/ml in serum, corresponding to 8 pmol (1.6 ng) in a 100-μl injection volume.
A rapid flat gel isoelectric focusing method has been developed for the determination of VLDL apolipoprotein (apo) E isoform patterns. Isoelectric focusing in 5% polyacrylamide flat gel with 8M urea and 2.8% pharmalyte (PH 4-6.5)(Pharmacia) was carried out at 3000 V and 4° for 1 hr under a constant power of 30 W, using a flat bed apparatus FBE 3000 (Pharmacia) and an electrophoresis constant power supply ECPS 3000/150 (Pharmacia).The separation of apo E isoform bands was good, and isoelectric points were determined 5.95 for apo E4, 5.81 for apo E3 and 5.68 for apo E2 in our focusing system. We analyzed apo E isoform patterns in our population (n=123) using this focusing method. The results obtained were as follows; 1) The apo E phenotype frequencies were 0.0% for E2/2, 6.5% for E3/2, 71.6% for E3/3, 0.8% for E4/2, 19.5% for E4/3 and 1.6% for E4/4, indicating that Japanese have a higher frequency of ε3 allele and a lower frequency of ε2 allele than either German or Americans. 2) Two phenotypes, apo E3/3 and E3/2 were differentiated on the basis of the apo E2/E3 ratios. The ratio was 0.38±0.02 for group E3/3 and 1.12±0.04 for group E3/2. No overlap was observed between the two groups. The cut-off point between the two groups was assumed to be approximately 0.9 in our focusing system. This method is useful for the analysis of apo E isoform patterns.
For cholecystokinin (CCK)-specific radioimmuoassay (RIA), CCK-33 and CCK-8 were iodinated by 1251-Bolton-Hunter (BH) reagent. Both RIAs using 125I-BH-CCK-33 and 1251-BHCCK-8 were identical in sensitivity and specificity. On the other hand, the RIA using 125I-BHCCK-33 allowed us to measure CCK in plasma without prior extraction of plasma samples, while extraction of plasma was required for RIA using 125I-BH-CCK-8. Histidine residues of synthetic human parathyroid (hPTH) 1-34 were iodinated by chloramine T for N-terminal PTH RIA. The specific radioactivity of the 125I-hPTH1-34 was 110μCi/μg and immunological activity was almost maintained by the iodination.
Rat liver lysosomes were solubilized by treatments of detergents, hydrolyzing enzymes, sonication, freezing-thawing and hypotonicity with a sucrose solution. We investigated the effects of these treatments on the release of four enzymes, acid phosphatase (AcP), N-acetyl-β-D-glucosaminidase (NAG), β-glucuronidase (β-Glu) and acid deoxyribonuclease (AcDN), from the lysosomes. These four enzymes were solubilized to a similar extent by Triton X-100, a nonionic detergent. β-Glu and AcP were released more easily than NAG and AcDN by the treatment of cholic acid, an anionic detergent. In the case of trypsin digestion, the ratios of released AcDN and NAG were higher than those of released β-Glu and AcP. β-Glu was solubilized from the lysosomes in a ratio higher than the other enzymes by lipase digestion. In the treatment of sonication, there was little difference in the ratio of solubilization among these four enzymes. The treatment of freezing-thawing caused little release of NAG and AcDN but it had an apparent solubilizing effect on β-Glu and AcP in which their solubilization extents increased with an increase in freezing-thawing times. β-GIu was most easily solubilized by the treatment of hypotonicity with a sucrose solution. The present results indicate that β-Glu is one of the enzymes which are easily released from rat liver lysosomes by various solubilizing treatments.
The high-performance liquid chromatographic method for the determination of 7-methylguanine and 1-methyladenosine, the degradation products of ribonudeic acids, in urine was described.The urine samples were at random specimens of normal and cancer patients.One ml of 6N hydrochloric acid was added to 10 ml of the urine.The mixture was filtered through millipore membrane (0.45μm) and an aliquot of the filtrate was injected onto the high-performance liquid chromatograph equipped with cation exchange particle and ultraviolet photometric detector.The separation was performed with phosphate buffer solution (pH 4.5).The peaks of 7-methylguanine, 1-methyladerlosine, and creatinine were successfully detected and determined.when compared to normal control values, the ratio of the base or nucleoside to creatinine increased significantly in the urine of patients.
Here we report an effect of monobasic potassium phosphate (KH2PO4) on the elution of dihydroquinazolinium compound from high-performance liquid chromatography, which is useful for the determination of ornithine aminotransferase activity. A low concentration of KH2PO4 remarkably affected the elution time and the pattern of dihydroquinazolinium. At the concentration between 3 and 5 mM, it was eluted as a sharp peak and separated from other peaks, otherwise it showed a broad peak or a peak which overlapped with other Deaks.
A method for enzyme immunoassay of thyroid stimulating hormone (TSH) has been developed.TSH was conjugated with glucose oxidase (GOD) according to Nakane´s method, and the separation of free and bound fractions after immune reaction was achieved by using double antibody solid phase bead (DASP-bead).The working curve for TSH standards is satisfactory to recognize TSH concentration as 0.05μu/assay tube.