Erythrocyte membrane proteins from twenty-two patients with hereditary spherocytosis (HS) were analyzed by polyacrylamide gel disc electrophoresis in the presence of 0.1% SDS. Almost complete deficiency was found in a protein component, IVb, in four cases, three of which were siblings. A small but significant decrease in this component was noted in most of the other cases. Such a small decrease in IVb was also found in two cases of autoimmune hemolytic anemia and one case of congenital hemolytic anemia due to pyruvate kinase deficiency.
The IVb component was found to be most susceptible among the erythrocyte membrane proteins to the action of protease of leucocytes. It was unlikely, however, that the abnormalities in IVb were artifacts due to the leucocyte protease as judged from the following evidence. First, the deficiency in IVb was demonstrated even when eucocytes had been completely removed using a column of SE-cellulose before preparation and analysis of erythrocyte membrane. Second, mixing in equal amount the red cells from a normal subject and those from a patient with HS of IVb-deficient type resulted in the appearance of IVb peak of half size in the electrophorogram.
A hypothesis was presented that HS is composed of at least two types, of which IVb-deficient type is caused by membrane dysfunction due to the deficiency in IVb, while in the other type a small decrease in IVb may be secondary to an unknown primary defect. As compared to the other type, the IVb-deficient type showed rather milder clinical features and hematological disorders. Results of family examinations suggested that HS of IVb-deficient type may be inherited as a recessive character.

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Parachloromercuribenzenesulphonic acid (PCMBS), an agent which increases passive permeability of cations by reacting with sulfhydryl groups of the membrane, much less increases cation permeability in hereditary spherocytic (HS) red cells than normal red cells. Pronase which is thought to alterate the charged amino groups of the membrane, changes permeability of cations in the same degree with normal and HS red cells. Alteration of sulfhydryl groups of the membrane may be primarily involved in the changes of cation permeability in HS red cells.

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Considering a fact that the plasma membranes of mature human erythrocytes are more susceptible to the changes in plasma phospholipids, bile acid or LCAT activities, or their combinations, than to the simple increases in the plasma lipoprotein, influences of lysolecithin (LL) levels in the surrounding medium on the LL contents, morphology and some properties of the erythrocyte membranes were investigated as a model system for the pathological membrane alterations.
Washed erythrocytes were kept at 37° for 10 minutes in human plasma with varying LL concentration, ranging from 0.3-1.5 mM, prepared by either adding purified egg LL to normal plasma or preincubating normal plasma at 37° for 12-24 hours to convert a part of the plasma lecithin into LL under an action of the plasma LCAT. Scanning electron microscopic observations revealed that remarkable changes in the cell morphology occurred due to the membrane externalization (discocyte → echino-discocyte → echino-spherocyte → spherocyte), accompanied by the almost linear increase in the LL contents of the membrane up to saturation. Washing the normal erythrocytes with 4.5% serum albumin solution caused the appearance of cup-like cells with approximately one-half of the normal membrane LL content. In any case, no change in the content of the other membrane lipid was detected. As the changes in the membrane properties, characteristic changes in the osmotic fragility and definite decrease in the cell electrophoretic mobility were observed. Such membrane alterations proved to be not directly mediated by the changes in the intracellular ATP level which also should cause a similar morphological change.
Restoration of the membrane LL content to the normal, by washing the treated cells with normal plasma (or with albumin solution in the case of LL-rich cells), yielded the cells with normal biconcave disc shape. However, the osmotic fragility once reduced, due to the excess membrane LL, was not restored.
If phosphate-buffered saline was employed as the medium in place of human plasma, the concentrations of LL to cause these phenomena were reduced remarkably (to about 1/40-1/70), indicating that LL is distributed between the plasma proteins and erythrocyte membranes and only a small fraction of it is adsorbed onto the latter.
Possibility of occurrence of these kinds of phenomena in circulating blood under certain abnormal conditions was discussed, taking into consideration of the actual changes in the plasma LL concentrations in the cases of pancreatitis and some chronic liver diseases, and also changes in the plasma albumin concentration.
It was additionally found that no significant membrane alteration was noted in the cases of the erythrocytes of the rats with marked hyperlipemia, including the cases with ten-fold increase in plasma triglyceride in a genetic fatty rat strain and two-fold increase in cholesterol in nephroticrats produced experimentally.

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Oxygen affinities and 2,3-diphosphoglycerate (DPG) levels in red cells of various enzymo- and hemoglobinopathies were determined and the results were discussed in relation to the compensation mechanisms.
In enzymopathies (deficiencies of glucosephosphate isomerase, phosphofructokinase, phosphoglycerate kinase, diphosphoglyceromutase, and pyruvate kinase), among various intermediates of red cell glycolytic pathway only DPG levels showed very close correlations with oxygen affinities (r=+0.91).
While, in hemoglobinopathies (hemoglobin Bethesda, Rainier, and Seattle), there were no correlation between oxygen affinities and DPG levels, and their red cells reflected the features of the containing abnormal hemoglobins.
In common with enzymo- and hemoglobinopathies, marrow regulations seem to be in action compensating their tissue oxygen supply.

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Two NADH diaphorases, diaphorase I and II were isolated and purified from normal and congenital methemoglobinemic erythrocytes by column chromatography, and the relation between diaphorase activities and those of methemoglobin reduction was studied. Activities of diaphorase I for methemoglobinemic red cells were 80% and those of diaphorase II less than 5%, as compared with those for normal ones.
For both erythrocytes, normal and methemoglobinemic, cytochrome b₅ reductase activities were detected only in diaphorase II fractions.
The cytochrome b₅ reductase deficiency, might lead to the increase in methemoglobin through the decrease in the enzymatic cytochrome b₅ reduction and subsequent nonenzymatic reduction of methemoglobin by the reduced cytochrome b₅. In order to study the nature of enzyme deficiency in congenital methemoglobinemia, the diaphorase II fraction, obtained from methemoglobinemic cells was concentrated and examined. The methemoglobinemic and normal diaphorase II were without difference concerning Kms for the substrates (the dye and NADH), heat stability, pH response, and electrophoretic pattern.
The ratio of the diaphorase activity to the cytochrome b5 reductase activity was identical for the both enzymes. The production of an abnormal enzyme molecule by silent substitution could not be denied, but in this case the rate of synthesis of the normal enzyme might be decreased.

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Phosphofructokinase (PFK) defect has been demonstrated in erythrocyte as well as in muscle of patients with glycogenosis VII, which is characterized by a genetic defect of muscle type PFK. Pathogenesis of the shortened life span of the erythrocytes was studied by the determination of behaviors in glycolytic intermediates and nucleotides, glycolytic capacity and kinetic properties of PFK of the affected erythrocytes. Enzymatic analyses showed a 80% increase of fructose-6-phosphate and a 60% decrease of fructose-1, 6-diphosphate with a decline of triose phosphates and monoglyceric acids, being attributed to a block at the site of PFK in the glycolytic pathway. 2, 3-Diphosphoglyceric acid in the erythrocytes was decreased to 50% which resulted in the increased affinity of the hemoglobin for oxygen. The ATP content was slightly decreased with a three to four fold increase of AMP and ADP. The low rate of glycolysis in the erythrocytes was demonstrated in vitro by the reduced production of lactate from glucose. The pH dependency of the lactate production was observed in control and affected erythrocytes, and lactate production was 25%, 37% and 38% of normal at pH 6.7, 7.4 and 8.0, respectively. Concomitant mesurement of fructose-1, 6- diphosphate and glucose-6-phosphate in the erythrocytes revealed that the ratio of fructose-1, 6-diphosphate to glucose-6-phosphate was closely related to the rate of glycolysis, suggesting the significant contribution of residual PFK to the control of the glycolysis even in the affected erythrocytes. PFK in the deficient erythrocytes differed from normal in its kinetic properties: altered pH dependency and increased affinity for ATP. It may be concluded that the diminished life span of the PFK deficient erythrocytes is caused by the impairment of glycolysis due to the block at the site of PFK. Furthermore not only the low activity but also the kinetic aberration of the residual PFK might accout for the matabolic block in the affected erythrocytes.

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Quantitative and qualitative genetic defects of erythrocyte pyruvate kinase (PK) were characterized by electrophoresis, kinetics, stability test and immunologic study.
In quantitative defect, erythrocyte PK isozyme (PK-R₁ and PK-R₂) was not detectable but PK-M₂was present by compensatory mechanism. Severe hemolytic anemia may be caused by the low activity and instability of PK-M₂ and the loss of PK production in the mature erythrocyte and splenectomy revealed significant benefit with marked reticulocytosis although the patient has still hemolytic anemia. Cyanide which is toxic to the mitochondria, inhibited ATP production of PK deficient red cells with high reticulocyte count. It is suggested that there may be selective sequestration of reticulocytes by loosing the mitochondria which has an alternate ATP production (TCA cycle) in the spleen.
In qualitative defect, PK-Tokyo I, PK-Nagasaki and PK-Maebashi had high Km for phosphoenolpyruvate (PEP), low Vmax, urea instability and moderate hemolytic anemia but different electrophoretic mobilities in each other. PK-Sapporo and PK-Tsukiji had abnormal nucleotide specificity, high Km for PEP, high Vmax, urea instability and moderate to mild hemolytic anemia. PK-Tsukiji showed low affinity to ADP as well as PEP. The double defects may cause moderate hemolytic anemia despite of high Vmax. After splenectomy, the patient had normal red cell counts with reticulocytosis. Two cases of PK-Tokyo II in different family had low Km (PEP), low Vmax, fast moving bands on electrophoresis, stable enzyme and mild hemolytic anemia. Hemolytic anemia may be mild because of high affinity to PEP and stable enzyme although low Vmax. PK-Ube detected in a search for genetic polymorphism in healthy persons, had abnormal electrophoretic mobility but functionally normal, and no anemia. PK-Ube is considered to be heterozygous and there may be no defect of enzyme reacting site on the PK molecule.
In addition, there were three PK deficiencies in two acute granulocytic leukemias and an erythroleukemia, which showed normal function and electrophoresis and no hemolytic anemia or genetic evidence of PK deficiency. These cases may have secondary PK deficiency because of low enzyme production during erythroblast maturation in myeloproliferative disorders.

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(I) Human RBC-PK which appeared homogeneous on disc gel electrophoresis was prepared, and its specific activity was 192 U. SDS disc gel electrophoresis of this preparation also showed single band which suggested that the RBC-PK consisted of the subunit of homogeneous molecular weight. 6 M-urea acrylamidegel electrophoresis on this preparation was carried out to examine whether RBC-PK consists of the subunit heterogeneous on electric charge. However, the examination failed of success because of the technical problem.
(II) The PK activities which seemed likely normal L-M₂ hybrids on electrophoresis were isolated from rat kidney homogenate by P-cellulose chromatography. Five fractions, I, II III, IV and V in order of elution were almost identical immunologically (by anti-M₁ serum) to L-4, L-3·M₂-1, L-2·M₂-2, L-1·M₂-3 and M₂-4, respectively. On the other hand, artificial L-M₂hybrids was made up from L- and M₂-PK according to Cardenas et al. This preparation was chromatographically separated into fractions which seemed to correspond to L-4, L-3·M₂-1, L-2·M₂-2, L-1·M₂-3 and M₂-4, respectively. Acrylamide gel electrophoretic patterns of this preparation showed binominal distribution of PK activities according to the formula (L+M₂)⁴. Furthermore, electrophoretic migration of RBC-PK on the same experiment was almost equal to that of hybrid L-3·M₂-1. It was already reported that about 25% of RBC-PK activity was neutralized by anti-M₁ serum. Therefore, these results described above further extended progressively our hypothesis that RBC-PK could be hybrid form of L and M₂ subunit of PK isozymes.

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A new method for the determination of δ-aminolevulinic acid (ALA) synthetase activity in human erythroblasts has been developed. ALA synthetase in erythroblasts was partially purified so as to permit the use of ¹⁴C-succinyl-CoA as the precursor for the enzyme. The partially purified enzyme solution contained negligible activity of succinyl-CoA hydrolase. It also contained negligible or very low activity of succinyl-CoA synthetase and a-ketoglutarate dehydrogenation complex. After incubation at 37° for 30 min, ¹⁴C-ALA formed was isolated by Dowex 50W x 8 column. Radioactivity in the eluate from the column was proved by paperchromatography to be exclusively derived from IC-ALA. Only 20-30 % of the activity was lost in the course of enzyme purification. All the procedure requires only 4 hours. ALA synthetase activity in the erythroblasts of patients with several hematological disorders was determined by this new assay method. ALA synthetase activity in the erythroblasts of patients with iron deficiency anemia was normal or slightly elevated. Almost all cases of primary sideroblastic anemia showed decreased ALA synthetase activity. ALA synthetase froma case of primary acquired sideroblastic anemia had a Km for the pyridoxal phosphate 10 foldlarger than normal. A son of patient with congenital sideroblastic anemia showed decreased ALA synthetase activity. The enzyme activity was also decreased in the erythroblasts from patients with pyridoxine responsive anemia. However, the enzyme activity gradually increased in the course of treatment with pyridoxine. Apo-ALA synthetase was decreased in the two cases of pyridoxine responsive anemia.

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During acute and chronic hypoxia in peripheral tissues, erythrocyte metabolism, especially, the changes of 2, 3-Diphosphoglycerate (2, 3-DPG) and various enzyme activities in glycolysis were studied. Various factors known to influence on the erythrocyte metabolism were studied in vivo and vitro, and the following results were obtained.
1) It might be suggested that the plasma from patients with chronic renal disease contained unknown factors which enhanced erythrocyte glycolysis and therefore increased 2, 3-DPG levels.
2) The rate of 2, 3-DPG synthesis of red blood cells from normal adults was not altered by addition of thyroxine, triiodothyronine, and the plasma from hyperthyroidism. So it might be said that thyroid hormone had not the direct action on 2, 3-DPG formation.
3) 2, 3-DPG levels of red blood cell from hyperthyroidisms were higher than those of normal subjects and correlated with their basal metabolic rates.
4) Through incubation of erythrocyte from normal adults in a low glucose medium containing 1.7mM glucose, 2, 3-DPG decreased gradually and significantly.
5) After acute bleeding, erythrocyte 2, 3-DPG values took a few days to reach to the maximum level.
6) It might be suggested that the plasma from patients and rabbits with chronic anemia contained the factors which caused the elevation of 2, 3-DPG levels.
Judging from these results, also taking them into consideration with other reports we have known up to now, it seems to us that 2, 3-DPG has three separate effects on erythrocyte metabolism; (a) direct effect, i. e., effect to change the rates of pH, glucose, and so forth.(b) secondary effect, i. e., metabolic change (c) erythropoiesis.

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Human erythrocyte carbonic anhydrase isozyme B and C were measured by a specific and immunological method. The levels of carbonic anhydrase B and C were determined in normal subjects, patients with several diseases. The levels of carbonic anhydrase B showed a decrease in hyperthyroidism and lead -exposed workers, and increased in patients with chronic obstructive lung diseases and patients with epilepsies under treatment with acetazolamide. Closely negative correlations were observed between carbonic anhydrase B and T3 Resin Uptake or Protein Bound Iodine levels in hyperthyroidism. Carbonic anhydrase C levels were not so much changed in those cases.
Simultaneously, carbonic anhydrase B dependent esterase activity (active carbonic anhydrase B enzyme) was determined kinetically using the immunoadsorbent method in the abovepatients. The results were compared with the total enzyme protein (active and inactive carbonic anhydrase B enzyme), estimated by single radial immunodiffusion technique. In patients treated with acetazolamide, the specific activity of the carbonic anhydrase B (carbonic anhydrase B dependent esterase activity/total carbonic anhydrase B protein) decreased remarkably. Clinical significances of these isozymes in red cells were discussed.

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Intensive efforts for the standardization of enzyme assay were carried out in many laboratories, and these efforts were resolved themselves into two types of approaches for standardization. One of them is studies of kinetical properties of the enzymes. Another is studies of the relation between enzyme assay conditions and more precise clinical assessment. The first type of approaches were extensively studied in many countries, and GSCC1) and SSCC2) recommended the suitable assay condition for the several enzymes from this point of views. Recently we reported the relation between substrates and some kinetical properties of blood alkaline phosphatases (Al-P)3). In this report, differences of clinical assessment of Al-P in different substrate systems, and the differences of clinical assessment in combination between leucine amino peptidase (LAP) and Al-P in different measuring systems were studied.
Electrophoretically identified three types of sera, bone, hepatic and placental type dominant sera, were used in our experiments.
The assay conditions of Al-P, LAP and Al-P isoenzymes were summarized in TABLE I.
Fig. 1-a shows correlation of Al-P between PP-Carb and PNPP-DEA methods. The regression line with placental type Al-P was different from other sources of Al-P. This fact suggests that the placental type Al-P could be separated from the others by projecting new calculated Z axis. F or this reason, the ratio of Al-P measured with PP-Carb versus Al-P measured with PNPP-DEA were calculated and plotted in Fig. 2-a. From this ratio, placental type Al-P is significantly separated from the others statistically. Fig. 1-b shows correlation of Al-P between PP-Carb and PNPP-Ame method. Three regression lines obtained from the different sources of Al-P were almost identical. The ratio of Al-P measured with PP-Carb and with PNPP-Ame method were also calculated and plotted in Fig. 2-b. From this ratio, there was no difference among the three types of Al-P. So clinical appreciation of Al-P were almost same in this two methods.
For the purpose of elucidating the relation between enzyme assay condition and clinical

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Serum enzymes are derived from large organ such as liver, kidney, heart and skeletal muscles. A standardization of assay methods for serum enzymes is available not only to diagnose correctly the diseases of these organs, but also to compare with the data from each laboratory.
But a method to an enzyme is not suitable for enzymes with isozymes. Each isozyme has its own condition in preservation and assay. The Km and V_max is also proper to each isozyme. So the datum from one method must lead to suggest the disturbance of some organs, and to deny the disturbance of the others.
By using these difference in nature between isozymes, on the contrary, the source of serum enzyme can be searched after. In our labolatory the quantity and quality of serum enzymes have been studied in connection with the pathogenesis of muscular dystrophy1-6). This has necessarily led us to find the findings as follows. Serum LDH isozyme of Ducheme type changes with age, as a result of the progression of the disease. At an early stage its serum enzymes are derived mainly from white muscles. At the final stage these are derived mainly from red or heart muscles. For these studies only an assay method to LDH was not suitable.
In addition to muscular dystrophy mentioned above, recent reports indicate that increases of serum activities of CPK can also occur in some patients with such neurologic and psychiatric disorders as cerebral vascular disease and acute psychosis, and also that in these cases the composition of CPK is not BB type but MM type7). So some studies were made to elucidate by what mechanism a leakage of muscle enzyme into the serum occurs.
Using male rabbit 3 kg weighing, medial (sympathetic) or lateral (parasympathetic) area was electrically stimulated for 30 minutes. After a stimulation of sympathetic area, but not of parasympathetic area, serum enzyme activities of muscle type increased significantly in sera with a maximum at 6 to 18 hrs. Stimulation on sympathetic hypothalamus released such soluble sarcoplasmic enzymes as CPK, aldolase and LDH from white muscle to sera. These findings indicate more that individuals must be conditioned prior to sampling for LDH, CPK and aldolase assay, and also that the studies on pathogenesis of the above mentioned diseases go forward.

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For standardization of enzyme determinations, a great deal of efforts should be put forth from the following three standpoints; enzyme properties, clinical assessment and quality control. With regard to an approach from the third, the First International Symposium on Quality Control (ISQC, Jun. 1974, Tokyo) carried out a novel trial of enzyme surveys (LDH, Al-P), which was characterized by an experimental standpoint of view and a positive attitude to standardization, comparing with the other surveys previously conducted.
At present, a more detailed analysis was applied to the data of ISQC LDH survey with permission of Dr. M. Kitamura, chairman of the survey subcommittee. In this study, a new technique to reveal systemic errors from survey data was presented and several problems to be solved in the process of standardization were described.
Two steps of the statistical treatment of the data were done. The first of which was a truncation procedure, by which all the results outside ±2SD from the primary material were excluded, and a new mean and SD of the reduced material were calculated. This routine was repeated until the convergent mean and SD were obtained. Secondly, each datum was normalized against these truncated mean and truncated SD and a pair of values obtained with respect to two samples were plotted on a scattering chart.
At first, regarding the survey with each laboratory method, systemic deviations essentially attributable to individual methodology were revealed by means of this technique. Several groups of method were separated according to each specificity to isoenzymes, in the scattering chart concerning two samples (2, 6) and (5), whose principal ingredients were I, II and V isoenzymes, respectively (Fig. 2).
Since a projection to a new axis Z-1 gave the best discrimination, co-ordinates on this axis are considered to indicate systemic differences among several groups. Therefore, the degree of dispersion in each group on this axis seems to mean systemic errors from the reaction specificity inherent in individual methodology (systemic errors of the second sort).
On the other hand, the degree of dispersion in each group on Z-2 axis rectangular to Z-1 is referred to systemic deviations that exert the same effect (plus or minus) on the determinations of two samples (systemic errors of the first sort).
Youden introduced systemic (of the first sort) and random errors from a scattering chart with respect to two determinations, but systemic errors of the second sort have not been recognized and apt to be confused with random ones. We would like to emphasize that it is essential for standardization to reveal and analyze systemic errors, clearly discriminating between these two different significances of them. The projections to two axes Z-1 and Z-2 were proved to be effective to segregate two sorts of systemic errors and visualize several problems to be solved (Fig. 3, Fig. 4).
Next, systemic errors revealed from the survey with uniform kit method were proved to be mainly those of the first sort (Fig. 5, Fig. 6). This fact suggested that it is an essential process for standardization to elucidate various problems in the analytical steps of enzyme determinations containing the basic set-up of the conditions, for example, temperature, reaction time and so on.
1) A new analytical technique to reveal systemic errors from survey data was presented.
2) By using this, systemic errors of the different significance from those discussed by Youden were found out (systemic errors of the second sort).
3) It is an essential process for standardization to reveal and analyze two of systemic errors, clearly discriminating each other.
4) For this purpose, it is necessary to design and execute a more elaborate survey from an experimental standpoint of view.

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The stability of various enzymes in commercially available control sera was tested for suitability for use as standard materials and results showed a temperature-dependent increase in alkaline phosphatase (AL-P) activity in reconstituted lyophilized sera and a cold lability of LDH at 4°. Results obtained from further studies on the properties of AL-P and LDH in the reconstituted control sera were as follows;
1) Heterogeneity of AL-P isozymes in control sera gives the difficulty for standardizing the determination of AL-P activity.
2) Undialyzable substances other than albumin increase AL-P activity during storage. AL-P activity decreases with time when bovine serum albumin is added to the enzyme solution but is stabilized when human serum albumin added.
3) Treatment with lipoprotein lipase quenches a temperature-dependent increase in AL-P activity in reconstituted lyophilized sera. Desialidation, however, does not change the activity during storage.
4) Cold lability of LDH at 4° is observed in some control sera, in which contain LDH-4 and LDH-5 as the components of LDH.

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The usefulness of the separation of the amylase isozymes for the clinical test and the investigation for the standardization of the nomenclature of the individual isozyme were discussed.
The easiness of handling, the completeness of separation and the sensitivity for amylase activity were compared of the supporting media between cellogel membrane and thin layer polyacrylamide gel. Only two major components could be separated by cellogel membrane. However, it was impossible to separate the minor components by this method. As the incompleteness of separation of isozymes leads us to misjudgement, cellogel membrane was not suitable as the supporting medium for electrophoresis of amylase isozymes.
On the other hand, up to 8 amylase isozymes, were separated in serum and urine using a simple thin layer polyacrylamide gel electrophoresis. This must be the best supporting medium for electrophoresis of amylase isozymes, because of the completeness of separation which was the most important point for the clinical use.
There were recommendations for the nomenclature of isozymes from the Committee of Isozymes; isozymes should not be named according to the tissues in which they occur, but should be numbered consecutively beginning with number 1, and starting at the anodal end of the electrophoretic medium and continuing directly through the origin to the cathodal end. However these recommendations were not suitable in cases of amylase isozymes because the anodal end of the amylase isozymes changes according to the values of amylase activity of the sample and to the time of reaction. From these observations, it is proper to number consecutively beginning with number 1 starting from the isozyme of the least mobility but present in all normal subjects.

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The UV-method for the measurement of serum transaminsases was critically evaluated, with respect to condition of storage of reagents, grade of coupling enzymes, presence of cofactor, and absorbance linearity of instrument.
Those factors gave an obvious influence on the results of UV-procedure separately or in combination.
Above findings lead to the conclusion that the standard procedure for any enzyme analyses should be presented with the accummulation of the ditailed data on all of the assay conditions.

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