During the past few years significant progress has been made in the use of bioluminescence and chemiluminescence in the research and clinical laboratories.There are several reasons for these developments.In the first place highly purified enzymes and chemicals have become commercially available at a reasonable price. Second, industry has developed and placed on the market inexpensive and highly sensitive photometers.Other reasons for the increased interest in luminescent systems include high specificity, speed of analysis and sensitivity, as low as 10-18 moles in some cases. In the present article we will review briefly the use of the firefly system for measuring ATP and the luminescent bacterial for NADH and NADPH.Through the use of other enzymes and coupling reagents it is possible to assay several hundred enzyme systems and their corresponding substrates, although most of these have not been completely researched for clinical applications. In the present paper we will not attempt a cmprehensive review of the literature. We refer the reader to a number of recent symposia and reviews on this subject1)-6).
For evaluating clinical chemistry control survey results, following parameters are introduced: proficiency of accuracy, proficiency of precision, relative accuracy and relative precision for each laboratories, and general evaluation indicators for evaluating the proficiency level of participant laboratories.These parameters are calculated based on an allowable error function E (x), which consists of statlonary error and proportional error of the assay.The parameters were applied to Kyushu Control Survey and Nagasaki Control Survey results, and were useful to detect individual laboratory error and progress of proficiency in Successive years.
Asimple and rapid high performance liquid chromatographic method for analysis of fluoresclmine-llbeled norepinephrine and dopamine in 2ml of human urine was devebped.About 10 urine samples could be analyzed in 8hours.Coefficient of vlriation for norepinephrine and doplmine for individual urine slmples were 6.5% (52.0±3.0 pmol/2ml, n=7) and 3.8% (1.97±0.08nmol/2ml, n=7), respectively, and those for the another urine were 2.9% (263±8.3 pmol/2ml, n=7) and 4.1% (2.55±0.10 nmol, /2mol n=7), respectively.There was no difference in overnight urinlry excretion rate of norepinephrine and dopamine between the normal and hypertensive groups.After 2 hr of upright posture, the excretion rate of norepinephrine in the patients with essential hypertension was significantly higher (P‹0.05) than that of the normal sublects.After a subsequent 2 hr recumbency, the norepinephrine excretion rate was also higher (P‹0.05) in the hypertensive patients. There were no significant differences in excretion rltes of dopamine between the two groups during any period.
The intestinal absorption of seaprose S(SAP), a microbial serme proteinase produced by A spergillus melleus, was studied using N-acetyl-L-tyrosine α-naphthyl ester (ATNE) as substrate.This substrate was highly susceptible to SAP, and with it, the minimum detectable concentration of SAP was about 0.01μg. In experiments on intestinal absorption, sodium dodecylsulfate (SDS) was added to the assay system at a final concentration of 0.1%, since at this concentration it inhibited almost of the endogeneous ATNE-hydrolytic activity in rabbit serum, but only about 50%of that of SAP, and thus permitted measurement of SAP concentration in blood. ATNE-hydrolytic activity of SAP in peripheral blood was detected in the range of intestinal administration of 10-100mg per kg of rabbit. The results of gel filtration on Sephadex G-200 of the serum after intestinal administ-Iration showed that ATNE-hydrolytic activity was present in void fraction, indicating that SAP after intestinal absorption was probably trapped by a-macroglobulin, since in theresult of gel filtration on Sephadex G-200 of the mixture of serum and SAP ATNE-hydolytic activity was also eluted in void fraction. These results sugges tthat SAP is absorbed in active formfrom intestine.
Theoretical considerations on enzymatic rate analysis for substrate determination were examined.In the case of enzymatic rate analysis, a dynamic range is decided by Km value of the using enzyme and allowable relative error. When allowable relative error is setted to one per-cent, a dynamic range is less than 1/100Km of the enzyme.Initial velocities are also determined by differential coefficient at zero time of exponential regression formula taken from the reaction time course and the results are in good agreement with the velocities derived from theoretical formula.These theoretical considerations were experimentally established in the system of ammonium ion assay with glutamate dehydrogenase.
lt was revealed that addition of AC-17 into human serum resulted in falsely elevated bitirubin values when determined as intensity of absorption at 600nm by routine laboratory procedure. As the cause of this false elevation in bi-irubirl value, the formation of chelate of AC-17 with heavy metals (Fe, Cu, etc.) existing as trace elements in the serum may be most important. Coincidentally, no false elevation in serum bilirubin value was recorded in same samples with AC-17 by means of the blank method using blank reagent containing large amount of cystein. This may be related to reduction of chelate compound by cystein.On the other hand, exact measurement of bilirubin in serum samples with AC-17 was achieved by a double wave length assay. In this assay, the difference in the intensity of absorption at 600nm and 520nm was constarlt irrespective of the amount of AC-17in serum samples.
FUT-175 (6-amidino-2-naphthyl 4-guanidino benzoate,) a new synthetic protease inhibitor, inhibits the enzyme activities of various proteases, such as Clr, Clesterase, thrombin, kallikrein, plasmin and trypsin. FUT-175 strongly inhibited complementmediated hemolysis via the classical and alternative pathway. The concentrations of FUT-175 required for 50% inhibition of complement-mediated hemolysis by the classical and alternative pathway were 6.9×10-8M and5.1×10-7M, respectively. The effects of FUT-175 on various immunological reactions in vivo were studied. The minimal effective dose of FUT-175 in systemic Forssman shock in guinea pigs were 12.5mg/kg i. p, and 50mg/kg p.o. In passive Arthus reactions in rats, the effective concentration were 25mg/kg i. p.and 250mg/kg p.o. FUT-175 also improved other immunological reactions, such as passive cutaneous anaphytaxis and delayed hypersensitivity. Furthermore, at a dose of 25mg/kg i. p.it strongly protected mice from death by endotoxin shock.
We preseht a new simple and rapid flame atomic absorption-micromethod of iron using a discrete nebulization technique.When 100-μl of supernate prepared from 50-μl of plasma or serum samples was inlected into the small Teflon funnel, a good sensitivity and precision (CV 2-4%) were obtained.To eliminate surely the interference of hemolysis in fresh samples, we used trichloroacetic acid (TCA)-ascorbate separation (no heating) in place of TCA heating system. Additionally, this separation brought the disappearance of the background absorption signals owing to another salts except sodium chloride in samples; accordingly, using only a physiological saline as a matrix of standard, plasma or serum iron concentration can be determined accurately without a deuterium background corrector.Results agree well with those by the proposed methods from the International Committee for Standardization in Hematology.In addition, this method is useful for pediatric samples, and we report reference intervals for full-term and premature infants, as well as adults.
Usefulness of the enzymatic method (combination of sphingomyelinase, alkaline phosphatase and chotine oxidase) for assay of sphingomyelin content in human plasma was confirmed by a comparison with TLC method.Of the plasma phospholipids, only sphingomyelin was hydrolyzed by this Sphingomyelinase. The ratios of choline Phosphoglyceride/choline phosphosphingoside determined by the enzymatic method agreed well with that determined by the TLC method.
A new enzymatic method is presented for the determination of serum free fatty acids (FFA), utilizing acyl-CoA synthetase (ACS), acyl-CoA oxidase (ACO) and peroxidase. The standard curve for the method is linear for FFA concentrations up to 2,000μmol/l.Within-run and between-run precision studies showed CV´s of≤1 and ≤ 2%, respectively. lnterference by bilirubin and lipemia are eliminated by use of potaSsium ferrocyanide and lipoProtein liPase resPectively.
Solid phase immunoassay of corticosterone was established using fluorescent tag labelled 2nd antibody and corticosterone conjugated polyacrylamide beads (lmmunobead®).The sensitivity of this assay was 40pg/tube.Light scattering from the beads was negligible in this assay conditbns.The most suitable protein to protect non-specific binding of materials to the assay tubes was highly purified gelatin because of its lower intensity of the back ground signal.