Automated determination of red cell methaemoglobin reductase activity by a continuous-flow system for screening hereditary methaemoglobinaemia.

A flow diagram for the automated determination of ferricyanide reductase activity in red blood cells was prepared in the modules from AutoAnalyzer AA I (Technicon Instruments Inc). Ferricyanide reductase assay can be substituted for assay of cytochrome b5 reductase (EC 1.6.2.2), which plays a major role in reducing methaemoglobin in erythrocytes, and is defective specifically in the erythrocytes of patients with hereditary methaemoglobinaemia. The effective sampling rate of the analysis is 30/h, and less than 0.05 ml of whole blood is required. Interference of haemoglobin with absorption by potassium ferricyanide at 420 nm is effectively exculded by dialysis. This automated method was compared with the accepted diaphorase method, and it distinguished clearly the ferricyanide reductase activity of cord bloods from that of adult bloods. The activity of the blood from a patient with hereditary methaemoglobinaemia was only residual. It is suggested that the method is useful as a mass screening test for hereditary methaemoglobinaemia.

A case of transient hyperphosphatasaemia of infancy associated with a probable allergic disorder.

Serum alkaline phosphatase (ALP) activity was found to be grossly elevated (8594 U/L) in a 2-year-old female child, returning towards normal during the subsequent 2 months. Electrophoresis revealed two bands of ALP activity; isoenzyme analysis identified the cathodic band as being of bone origin and the anodic band as sialylated liver ALP. Whilst the aetiology of transient hyperphosphataemia remains unclear a probable allergic disorder appears to be a contributory factor in this patient.

Phylogenic study of denaturation of lactate dehydrogenase isoenzymes from different species by high and low temperature.

We studied the influence of storage at different temperatures on lactate dehydrogenase (EC 1.1.1.27; LD) isoenzymes from different tissues and different species, and analysed biochemical and biophysical mechanisms of denaturation during storage. Isoenzymes obtained from tissue extracts of mammals, poultry, reptiles. amphibians and fish were shown to have their own denaturation ranges at low temperatures by post-treatment assays and transition temperature analysis. These ranges were between -10 and -20 degrees C for most vertebrate LD isoenzymes. Circular dichroism analysis indicated that the denaturation of LD isoenzymes was probably caused by a change in the hydrophobic interactions in the molecule. At higher temperatures, LD-1 isoenzyme was more thermostable than LD-5 from the same animal species, except for rats, the LD-5 activity of which was more thermostable than the LD-1 activity. These findings indicate that variable effects of storage of samples and reference materials at low temperatures should be considered, and that it is necessary to establish LD isoenzyme standards for animal clinical laboratory investigations.

[A predictive and prognostic study on two cases of transient hyperphosphatasemia of infancy].

We presented two cases with transient hyperphosphatasemia (TH) of infancy, whose serum alkaline phosphatase (EC 3.1.3.1, ALP) activity showed markedly increased and the atypical isoenzyme fractions were seen by electrophoresis. These isoenzymes migrated into normal bone ALP region (alpha 2-beta globulin fraction) and fast liver ALP region (fast alpha 2 globulin fraction). From various investigation such as, heat stability, inhibition test by amino acid, neuraminidase treatment and Triton X-100 treatment, former fraction seemed to derive from bone ALP and later fraction from liver ALP. From our study, increment of the activity of alpha 2-beta gl fraction was in advance one month before maximum ALP activity stage, and fast alpha 2 gl fraction followed increasing 3 weeks after that. On the other hand, decreasing of fast alpha 2 gl fraction showed a shorter delay than alpha 2-beta gl fraction. These results suggest that a differential exchange of sugar chain or an impaired clearance of the enzyme from circulation was possibly occurred. It seemed to be important to increase a study of such a predictive and prognostic change of ALP activity and isoenzyme fraction in TH cases.

High-performance liquid chromatographic determination of plasma ascorbic acid in relationship to health care.

We have developed a simple reversed-phase high-performance liquid chromatographic method for determining plasma ascorbic acid level and studied the relationship between its plasma concentration and fruit and vegetable intake and plasma dopamine-beta-hydroxylase activity. The samples were pretreated by precipitating the proteins and injected onto the column. Elution with a methanol gradient in sodium phosphate buffer was carried out by monitoring the absorbance at 265 nm, and the peak corresponding to ascorbic acid was well separated from other peaks of reagents used for pretreatment and from plasma endogenous components. The proposed method correlated well with the conventional dichlorophenol-indophenol method. Mean levels of ascorbic acid in normal human plasma were 0.86 +/- 0.36 mg/dl for males (twenty subjects, 19-28 years old) and 1.01 +/- 0.30 mg/dl for females (twenty subjects, 19-21 years old). There was good correlation between plasma ascorbic acid levels and dopamine-beta-hydroxylase levels, reflecting activities of daily living, but no correlation was found between these levels and dietary consumption of vegetables or fruits.

Selective determination of lactate dehydrogenase isoenzyme 1 in serum after inhibition by 1,6-hexanediol.

A rapid selective method for measuring the activity of lactate dehydrogenase isoenzyme LD-1 in serum by using 1,6-hexanediol as an inhibitor of the M-subunit was developed. Hexanediol was added to serum at a final concentration of 0.7 mol/l. After incubation at 30 degrees C for 15 min, the activity was measured with an automatic analyser. The inter-assay coefficient of variation was 6.9% for the lactate dehydrogenase isoenzyme LD-1 measurement. The results obtained from the sera of 100 patients analysed by the proposed selective method and by the conventional electrophoretic method, respectively, showed an excellent correlation. This selective method was used to determine the lactate dehydrogenase isoenzyme LD-1 activity of sera from patients with acute myocardial infarction, and the results were correlated well with those obtained by the immunological, Roch Isomune method. Addition of 1,6-hexanediol did not affect the measurement of activities of other enzymes such as alkaline phosphatase, gamma-glutamyltransferase, aspartate aminotransferase and alanine aminotransferase.

New variant of cytochrome b5 reductase deficiency (b5RKurashiki) in red cells, platelets, lymphocytes, and cultured fibroblasts with congenital methemoglobinemia, mental and neurological retardation, and skeletal anomalies.

A Japanese man with cytochrome b5 reductase (b5R) deficiency in various blood cell lineages (red cells, platelets, and lymphocytes) and in cultured fibroblasts demonstrated congenital methemoglobinemia associated with mental and neurological retardation, and various skeletal anomalies, such as spondylosis deformans and finger joint deformations, which have never been described in association with this enzyme deficiency. Cytochrome b5 reductase deficiency was most severe in red cells (0.3-4%) and less marked in platelets (13-27%), lymphocytes (18-31%), and fibroblasts (50%). The present case appears to be a new variant of cytochrome b5 reductase deficiency (b5RKurashiki).

Biochemical and enzymological study of lactate dehydrogenase isoenzymes from commercial quality control sera and several animal tissue sources.

We assayed the isoenzymes of lactate dehydrogenase (EC 1.1.1.27) in commercial quality control sea and several animal tissue extracts, using electrophoresis. We compared the Km values and activation energies of the isoenzymes, in order to find suitable animal tissue sources with a similar isoenzyme profile to that of human serum lactate dehydrogenase. Some of the control sera contained only one isoenzyme fraction corresponding to porcine heart isoenzyme-1 or chicken heart isoenzyme-1, which showed essentially no changes of enzyme activity as a function of pyruvate substrate concentration. Other control sera, which contained isoenzymes from human red cells haemolysates, or from animal tissue extracts with a human serum matrix, showed significant changes of enzyme activity was a function of substrate concentration, and showed different Km values and activation energies from those of human serum. Of the serum and tissue samples from several animal sources, rat heart and kidney extracts showed the greatest similarity to human serum, with respect to the electrophoretic pattern and the Km, pH optimum and activation energy of lactate dehydrogenase isoenzymes.

Polymerase chain reaction test for differentiation of five toxin types of Clostridium perfringens.

In order to avoid the use of experimental animals, the polymerase chain reaction (PCR) method was applied to differentiate Clostridium perfringens into five toxin types. Twenty-two out of 23 strains tested produced the toxin(s) corresponding to the toxin gene(s) identified by PCR, and vice versa. Consequently, the gene typing was consistent with conventional typing by animal tests. Twenty-five strains were identified as types different from original ones by the PCR method as well as a toxin neutralization test. These findings suggest that the PCR method, which is easy and timesaving, is applicable to identify the toxin types of C. perfringens as an alternative to animal tests, and that beta-, epsilon- and iota-toxin genes might be lost by long-term preservation. The reasons why the strains lost the genes are discussed.

Met-form hemoglobins in long-term stored ACD blood.

On passing ACD blood stored for a year at 4 degrees C through a column of Sephadex G-75, the surviving erythrocytes were separated from hemolyzed cells and plasma. About 18% of stored erythrocytes survived a year of storage, and were spherocytic. The concentration of total ferric hemoglobins in the surviving erythrocytes was about 75% of that in the supernatant. The met-form hemoglobins in the erythrocytes and hemolytic solutions were further analyzed by isoelectric focusing electrophoresis, and (alpha 2+ beta 3+)2 and (alpha 3+ beta 2+)2 valency hybrids were found in addition to methemoglobin. Since the activity of NADH cytochrome b5 reductase was extremely low in the surviving erkythrocytes, the difference in proportion of ferric hemoglobins (valency hjybrids + methemoglobin) in these two fractions might be due simply to the difference in pH between intracellular and extracellular fluids, which is largely reflected in the oxidation rate of hemoglobin. The course of hemoglobin oxidation seems to be through hemoglobins such as (alpha 2+ beta 3+)2 and (alpha 3+ beta 2+)2 to methemoglobin.

Protective effect of glycerol against the increase in alkaline phosphatase activity of lyophilized quality-control serum.

We studied the effectiveness of glycerol or ethylene glycol in preventing the increase in alkaline phosphatase activity of lyophilized or refrigerated quality-control serum after reconstitution or repeated freezing and thawing. Control serum reconstituted completely from the lyophilized state with subsequent storage at -20 degrees C showed a considerable decrease in alkaline phosphatase activity immediately after thawing, and a gradual increase in activity on allowing it to stand at room temperature. Adding glycerol or ethylene glycol to the reconstituted serum obviated these changes in activity, glycerol being more effective than ethylene glycol. Reconstituted serum with added glycerol maintained maximum activity before refrigeration during either storage for 30 days or on repeated freezing and thawing. Practical applications of this glycerol-supplemented control serum are discussed.

Activity of lactate dehydrogenase isoenzymes LD1 and LD2 in serum as determined by using an inhibitor of the M-subunit.

To measure activities of lactate dehydrogenase (EC 1.1.1.27) isoenzymes LD1 and LD2 in serum, we developed a method involving 1,6-hexanediol as specific inhibitor of the M-subunit. Addition of hexanediol, 0.6 mol/L, to five LD isoenzyme fractions purified from human liver and heart homogenates resulted in complete loss of activities of LD4 and LD5, and partial loss of LD2 and LD3. The activity of LD1, which is composed of the H-subunit only, was not affected. In studying what conditions would allow only the activities of LD1 or LD1 + LD2 to be expressed in serum, we found that the respective activities could be determined by treatment with hexanediol, 0.75 mol/L and 0.55 mol/L, respectively. Results of binding experiments and analytical-recovery tests supported the effectiveness of analyses with this inhibitor in determination of LD1 and LD1 + LD2 activities in serum. Results by this proposed inhibition method correlated well with those by the conventional electrophoretic method for determination of LD1 activity, but LD1 + LD2 activities by the inhibition method were a little less than those by the electrophoretic method, requiring some correction.

Amylase in thyroid tissues from normal and various thyroid diseases. Biochemical and immunohistochemical study.

Amylase activity was measured in thyroid tissues of various thyroid diseases and was analysed electrophoretically. Normal thyroid tissues contained significant amounts of amylase (mean +/- SD; 2.71 +/- 1.15 IU/g of tissue), and their amylase isozyme was composed of a majority of salivary type isoamylase and other peculiar isoamylase. The statistical decrease of amylase activities in tissues of Graves' disease under hyperthyroidism, thyroid carcinoma, and most of thyroid adenomas were found (Graves' disease; 1.04 +/- 0.41, carcinoma; 1.49 +/- 1.10, adenoma (except five cases with high activity); 0.88 +/- 0.49 IU/g tissue). Five of 18 cases of adenoma showed strikingly higher amylase activity in their tissues. Electrophoretical patterns of amylase isoenzymes in these five adenoma tissue were different from those of normal thyroid tissues. The cellular localization of amylase in the normal thyroid tissues and the adenoma tissues was also demonstrated immunohistochemically.

NADH-cytochrome b5 reductase in platelets and leukocytes with special reference to normal levels and to levels in carriers of hereditary methemoglobinemia with or without neurological symptoms.

Normal levels of NADH-cytochrome b5 reductase activity in platelets, lymphocytes and granulocytes were determined. The homogenate of each cell was treated with Triton X-100 after incubation with lipoprotein lipase. The reductase was extracted very well from the cells by this treatment. Moreover, the assay of the reductase activity in the cells became accurate and reproducible by the treatment. The reductase level of each cell was also determined in cases of hereditary methemoglobinemia. It was normal in the case of the disease without mental retardation, and low with mental retardation. This latter case might be due to the deficiency of cytochrome b5 reductase in the whole tissue.

Congenital methaemoglobinaemia due to NADH methaemoglobin reductase deficiency: successful treatment with oral riboflavin.

A Japanese family with congenital methaemoglobinaemia is described. The family pedigree was compatible with autosomal recessive type of inheritance. The increased methaemoglobin concentration was ascribed to the red cell NADH diaphorase deficiency associated with the almost complete lack of one of the two peaks of the diaphorase activity as separated by DEAE Sephadex column chromatography. The NADH diaphorase and NADH methaemoglobin reductase deficiency was limited to the red cells. The methaemoglobin content in the blood of the propositus was 17.8% and isoelectric focusing analysis on a polyacrylamide gel plate showed that the haemoglobin consisted of 65.2% oxyhaemoglobin (alpha 2+ beta 2+)2, 29.6% half-oxidized forms, 20.9% (alpha 3+ beta 2+)2 and 8.7% (alpha 2+ beta 3+)2, and 3% full-oxidized methaemoglobin (alpha 3+ beta 3+)2. Oral administration of riboflavin 120 mg/d resulted in a gradual but significant decrease in the level of the met-form haemoglobins in parallel with a gradual increase in the red cell flavin content. Riboflavin is considered to be effective by activating the NADPH diaphorase (NADPH flavin reductase) system and appears to be useful for the treatment of congenital methaemoglobinaemia.

Cold lability of lactate dehydrogenase isoenzymes and the effective preparation of reference material for clinical laboratory use.

To evaluate the storage of samples and enzyme reference materials, and to improve the commutability for inter-laboratory surveillance of activity values of lactate dehydrogenase (LD; EC 1.1.1.27) in clinical laboratory medicine and in animal veterinary medicine, we studied the electrophoretic patterns and cold lability of LD isoenzymes from tissue sources of some common vertebrate species and also from human serum sources. Among many isoenzymes from these sources, only rat LD fractions showed similar electrophoretic patterns to those of human sera, and rat LD-1 fraction was relatively cold-stable. Total LD and isoenzyme LD-1 activities in routine laboratory samples and quality-control sera were measured using eight kinds of commercially available LD assay kit, including lactate and pyruvate substrate systems. Coefficients of variation between these assay kits were markedly reduced when the activities were calculated using the partially purified rat LD-1 fraction as an enzyme reference material, compared with the activities calculated using the factor indicated in each assay kit. In the regression analysis, the intercept and slope were calculated for the regression equations obtained from 12 pairs of these assay kits. The values obtained from a small amount of human serum and control serum samples were within the 95% confidence regions of those from larger amounts of human serum samples by using the present rat LD-1 standard for measuring LD-1 activities with lactate substrate. It was evident that a cold-stable and homogeneous LD isoenzyme as an enzyme reference material might contribute to accurate measurement of activities in heterogeneous samples for inter-laboratory quality-assurance surveys in both human and animal clinical laboratory use.

Lack of effects of thyroid hormones on human erythrocyte acetylcholine esterase.

Effects of thyroid hormones and their metabolites such as L-T1, L-T2, L-T3 and L-T4 on human erythrocyte acetylcholine esterase were studied. The activity of the enzyme of intact erythrocytes was not affected by these hormones, though studied under various conditions. The physiological significance of the binding of these hormones to erythrocyte membranes remains unclear. Our results indicate that the acetylcholine esterase is not a suitable enzyme for cytochemical bioassay for thyroid hormones.