Multiple Enzymatic Defects in Mitochondria

in Hematological Cells of

Patients with Primary Sideroblastic Anemia

YOSUKEAOKI, Department of Biochemistry, Jichi Medical School, Tochigi,Japan 329-04

A B S T RA C T Activities of mitochondrial enzymes inblood cells from 69 patients with primary sideroblasticanemia were determined to elucidate the pathogenesisof the disease. In erythroblasts of patients with primaryacquired type the activities of both 8-aminolevulinicacid synthetase and mitochondrial serine protease wereinevitably decreased. The susceptibility to the proteaseof apo-8-aminolevulinic acid synthetase prepared fromerythroblasts of patients with this type was within thenormal range, in contrast to that of pyridoxine-responsive anemia. The activities of mitochondrial en-zymes such as cytochrome oxidase, serine protease,and oligomycin-sensitive ATPase, except citrate syn-thetase, were usually decreased in mature granulo-cytes of the patients. Patients with hereditary sidero-blastic anemia also had decreased 8-aminolevulinicacid synthetase activity in erythroblasts, and decreasedserine protease activity in both erythroblasts andmature granulocytes. Mature granulocytes obtainedfrom patients with pyridoxine-responsive anemia be-fore therapy had decreased cytochrome oxidase ac-tivity, however, the activity increased to a normal levelwhen the patients were in remission. The activities ofother mitochondrial enzymes in mature granulocyteswere within normal range in these patients beforepyridoxine therapy. The activities of these mitochon-drial enzymes in lymphocytes were within normalrange in all groups of patients with primary sidero-blastic anemia.

Wesuggest that patients with primary acquired, andpossibly also those with hereditary sideroblasticanemia have impaired mitochondrial function in botherythroblasts and granulocytes. That only anemia is ob-served in these patients is because a functional ab-nomiality of mitochondria in erythroblasts is most im-portant because of the role of mitochondria in thefomiation of heme in erythrocyte development. In con-

Receivedfor publication 13 December 1979 and in revisedform 26 February 1980.

trast to these two types of sideroblastic anemia, only8-aminolevulinic acid synthetase in both erythroblastsand granulocytes seems to be impaired in patients withpyridoxine-responsive anemia.

INTRODUCTION

Patients with primary sideroblastic anemia show hypo-chromic or dimorphic anemia, elevated serum iron withhigh saturation of total iron binding capacity, anderythroid hyperplasia in the bone marrow with thepresence of a large number of ring sideroblasts.Ferrokinetic studies usually show accelerated plasmairon disappearance with impaired iron incorporationinto erythrocytes. Almost all features characteristicof this disease can be ascribed to a disturbance of hemesynthesis in erythroblasts (1, 2). In fact, the activity of8-aminolevulinic acid (ALA)' synthetase, the rate-limit-ing enzyme in heme synthesis is inevitably decreasedin erythroblasts of patients with primary sideroblasticanemia (2).

In the course of investigation ofthe regulation of ALAsynthetase activity in erythroblasts, a new serine pro-tease was found on the inner mitochondrial mem-brane of bone marrow cells including both erythro-blasts and granulocytes (3). An inverse relationship wasobserved between ALA synthetase activity and thisserine protease activity in erythroblasts (3). Therefore,the protease activity in erythroblasts of patients withprimary sideroblastic anemia was measured. Unex-pectedly, not only ALA synthetase activity but also theserine protease activity was inevitably decreased inerythroblasts of patients with primary acquired sidero-blastic anemia. Also the protease activity in maturegranulocytes obtained from patients with primary ac-quired sideroblastic anemia was decreased.

In this paper we studied several mitochondrial en-

' Abbreviations used in this paper: ALA, 8-aminolevulinicacid.

J. Clin. Invest. © The American Society for Clinical Investigation, Inc. * 0021-9738/80/07/0043/07 $1.00Volume 66 July 1980 43-49

43

zyme activities in erythroblasts, granulocytes, andlymphocytes obtained from patients with primarysideroblastic anemia. We found that multiple en-zymatic defects in mitochondria in both erythroblastsand granulocytes are characteristic to primary acquiredsideroblastic anemia and possibly to hereditary sidero-blastic anemia. On the other hand, only ALA synthe-tase in both erythroblasts and granulocytes seems tobe impaired in patients with pyridoxine-respon-sive anemia.

METHODS

Patients. 61 cases with primary acquired sideroblasticanemia, 6 cases with hereditary sideroblastic anemia, and 2cases with pyridoxine-responsive anemia were employedin this report. Samples from these patients were collected from34 hospitals in various parts of Japan between June 1972 andSeptember 1979. These patients were diagnosed on the basisof hypochromic or dimorphic anemia, hyperferremia withincreased saturation of iron binding capacity, erythroid hyper-plasia in the bone marrow, the presence of a large numberof ring sideroblasts (above 30%), and the absence of causativediseases such as leukemia, chronic inflammation, or malig-nancy. Diagnosis of six cases with hereditary sideroblasticanemia was based on family history, male sex, and early oc-currence of anemia. Every patient with these two types ofsideroblastic anemia was treated unsuccessfully with pharma-cological doses of pyridoxine. Two patients with pyridoxine-responsive anemia (classical form) have been described (4).

Cell separation. The erythroblast-rich fraction was pre-pared from aspirated bone marrow cells as described (4).Cellular components of bone marrow aspirate that were sus-pended in 20 ml of saline were filtered through four layersof gauze, and the filtrate was layered on a separating mediumconsisting of two parts (10 ml each), which were made bymixing appropriate amount of Lymphoprep (Nyegaad & Co.AS. Oslo, Norway) and saline. Specific gravity of each part was1.067 (upper) and 1.073 (lower). After centrifugation at 170 gfor 30 min at 4°C the cell layers formed between the sampleand upper part, and between upper and lower parts werecollected to obtain the erythroblast-rich fraction and the im-mature granulocyte-rich fraction, respectively.

Mature granulocytes and lymphocytes were prepared fromthe peripheral blood. Heparinized peripheral blood (15-20ml) was diluted with equal volumes of saline. It was layeredon the top of equal volumes of Lymphoprep, and centrifugedat 400 g for 30 min at 18°C. The layer formed between thesample and Lymphoprep was collected to obtain lympho-cytes. The precipitate that contained both erythrocytes andmature granulocytes was mixed with equal volumes of plasma,and was layered on the top of 8 ml of Lymphoprep. It wasleft at room temperature for 20-30 min, when almost allerythrocytes precipitated to the bottom and the layer wasformed between plasma and Lymphoprep. This layer was col-lected to obtain mature granulocytes.

Etnzyme assays. ALA synthetase activity was measured asdescribed (2). Cellular components of bone marrow as-pirates (5 x 106-5 x 107 of erythroblasts) were hemolyzedwith 4 vol of water for 10 min. The hemolysate was restoredisotonicity by adding 11.5% KCl solution, and was centri-fuged at 10,000 g for 10 min. The precipitate was washedonce by centrifugation, suspended in 10 ml of 1.15% KCIsolution containing 0.01 M potassium phosphate buffer, pH7.2, and sonicated for 1 min in ice water (20 kcycle/s, 120 W,model UR-150p, Tomy, Tokyo). From the precipitate that was

obtained by centrifuging the sonicated solution at 20,000 gfor 20 min ALA synthetase was extracted with 1 ml of 0.2%sodium deoxycholate (2). ALA synthetase activity was deter-mined using ['4C]succinyl-CoA as the precursor, and [14C]_ALA formed was isolated by Dowex (Dow Corning Corp.,Midland, Mich.) 50 x 8 Wcolumn chromatography to becounted by a liquid scintillation counter as described (2). 1 Uwas defined as the enzyme activity producing 1 nmol of ALAin 30 min under the conditions described (2).

The assay of the protease activity was performed as de-scribed (4). Blood cells (5 x 106_1 x 107) suspended in 3 mlof 0.85% NaCl containing 0.01 Mpotassium phosphate buffer,pH 7.5, were sonicated at 50 Wfor 15 s in a 50-ml centrifugetube (Branson Sonic Power Co. Danbury, Conn.; model W185), and then centrifuged at 20,000 g for 15 min. From theprecipitate, the protease was extracted with 1 ml of 0.5 Mpotassium phosphate buffer, pH 7.0, for 30 min at 37°C, andthe activity was measured using apo-ornithine transaminaseas substrate (3, 4). 1 U was defined as the amount of proteaseinactivating 50% of apo-ornithine transaminase in 30 minunder the conditions described (3, 4).

Cytochrome oxidase activity was measured by the methodof Smith (5). Blood cells (5-8 x 106) suspended in 1 ml ofsaline were sonicated at 50 Wfor 15 s in a 10-ml centrifugetube to be used as the enzyme solution. Reaction mixturescontained reduced cytochrome c, 90 nmol; potassium phos-phate buffer, pH 6.0, 225 imol; and enzyme solution, 0.2ml in a final vol of 3 ml. The control tube contained 1 mMpotassium cyanide in addition to the above components.Reactions were started by adding the enzyme solution, andinitial reaction rates were determined by measuring con-tinuously the decrease of absorbance at 550 nm at 25°C. 1 Uwas defined as the enzyme activity oxidizing 1 ,umol reducedcytochrome c/min under the conditions described above.The reaction was linear up to 3 x 106 of blood cells/3 ml ofincubation mixture, and the difference in duplicate analysiswas always within 10%.

Oligomycin-sensitive ATPase activity was measured by themethod of Kagawa (6). The enzyme solution was preparedby sonicating cells (1-1.5 x 107) suspended in 1 ml of 0.85%NaCl that contained 50 mMTris-HCl (pH 7.5) and 10 mMMgCl2 at 50 Wfor 15 s in a 10-ml centrifuge tube. Reactionmixtures contained ATP, 3 ,umol; MgCl2, 1.5 umol; phos-phoenol pyruvate, 2.5 ,umol; pyruvate kinase, 16 jLg; Tris-HCI (pH 7.4), 50 .mol; 2,4-dinitrophenol, 50 nmol; oligomy-cin A, 5 tLg; and enzyme solution, 0.2 ml in a final vol of 0.5ml. After incubation at 30°C for 15 min, reactions wereterminated by the addition of 0.1 ml of 50% trichloroaceticacid. After centrifugation inorganic phosphate was deter-mined in the supernate by the method of Lohmann andJendrassik (7). Oligomycin-sensitive ATPase activity wasdetermined by calculating ATPase activity measured withand without oligomycin. 1 U was defined as the enzyme ac-tivity releasing 1 ,umol inorganic phosphate/min under theconditions described above. Reactions were linear up to atleast 4 x 101 blood cells/0.5 ml of incubation mixture, and upto 25 min of incubation time. Differences in duplicate analysiswere always within 10%.

Citrate synthetase activity was measured according to themethod of Ochoa (8). Blood cells (5-7 x 106) suspended in1 ml of saline were sonicated at 50 Wfor 15 s in a 10-mlcentrifuge tube and 0.2 ml was used as the enzyme solution.The reaction mixture contained sodium malate, 10 ,umol;NAD, 0.3 umol; acetyl-CoA, 0.21 utmol; malate dehydro-genase from pig heart (Boehringer Mannheim GmbH, Mann-heim, West Germany), 0.5 Mg; Tris-HCl (pH 8.0), 75 ug; andenzyme solution, 0.2 ml in a final vol of 1.5 ml. Tubes con-taining the reaction mixture without sodium malate were

44 l. Aoki

employed as the control. The enzyme activity was meas-ured by monitoring continuously, at 340 nm, NADHproducedat 25'C. Reactions were linear up to 3 x 106 blood cells/1.5ml of incubation mixture, and differences in duplicate analy-sis were always within 5%. 1 U was defined as the enzymeactivity producing 1 ,umol NADH/min under the conditionsdescribed above.

RESULTS

Cell separation. The erythroblast-rich fraction ob-tained from bone marrow aspirate contained erythro-blasts (20-50%), lymphocytes (40-70%), and immaturegranulocytes (5-15%). The immature granulocyte-rich fraction consisted chiefly of immature granulo-cytes (85-90%) with a small number of lymphocytes(5-10%). Preparations of mature granulocytes obtainedfrom peripheral blood contained mainly mature gran-ulocytes (90-97%), and those of lymphocytes chieflylymphocytes (85-95%).

ALA synthetase activity in erythroblasts. 61 caseswith primary acquired type, 6 cases with hereditarytype, and 2 cases with pyridoxine-responsive typewere employed for the measurement of the ALA syn-thetase activity in erythroblasts. Every case showed de-creased ALA synthetase activity in erythroblasts asshown in Fig. 1.

Protease activity. The protease activity assayed by

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the method described above is considered to indicatethe activity of the new serine protease described (3, 4)from the following reasons. Firstly, the activity assayedby this method was completely inhibited by the addi-tion of a small amount of elastatinal (100 ,.g/ml), theinhibitor of both pancreas elastase and this protease(3, 9). Secondly, the enzyme solution used in these ex-periments showed no detectable elastolytic activity.Thirdly, elastase from swine pancreas (WorthingtonBiochemical Corp., Freehold, N. J.) exhibited a poorability to inactivate apo-ornithine transaminase ascompared with that of this new protease (17% ofthe protease).

Because the erythroblast-rich fraction contained asmall number of immature granulocytes and lympho-cytes, the protease activity in erythroblasts was calcu-lated considering relative contribution of differentcell populations to the protease activity as described(4). In contrast to the normal protease activity inerythroblasts of patients with pyridoxine-responsiveanemia, erythroblasts obtained from 18 cases withprimary acquired sideroblastic anemia and thosefrom three cases with hereditary sideroblastic anemiahad decreased protease activity as shown in Fig. 2.

The protease activity in mature granulocytes frompatients with primary sideroblastic anemia is shown inFig. 3. All except three cases with primary acquiredsideroblastic anemia had decreased protease activityin mature granulocytes. Mature granulocytes obtainedfrom three cases with hereditary sideroblastic anemia

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FIGURE 1 ALAsynthetase activity in erythroblasts of patientswith primary sideroblastic anemia. In the column of pyri-doxine-responsive anemia the symbol (0) shows the activitybefore therapy, and 0, that after treatment with pharmaco-logical doses of pyridoxal phosphate. Horizontal bars presentthe mean values.

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FIGuRE 2 The activity of the new serine protease in erythro-blasts of patients with primary sideroblastic anemia. Hori-zontal bars present the mean values.

Mitochondrial Defects in Blood Cells in Sideroblastic Anemia 45

TABLE ISusceptibility of apo-ALA Synthetase to Protease

Patients preparedlapo-ALA sy nthetase Susceptibility to protease

Norinal conitrols(n1 = 15) 0.94±0.18t

Pyridoxine-responsiveanemia (i = 2) 4.65+0.58

Primarv acquirecdsideroblastic anemia(Oi = 6) 0.83±0.12

Apo-ALA synthetase sollutionis were incubated at 37°C for 10mill in the presence of 120 U of protease to measure thedegree of iniactivationi by protease as described (4).

1 U of susceptibility to protease was definied as the degreeof iinactixation losing one-half of the enzyme activity in 30

imm under the conditions described (4).t Ieaii±SD.

Hereditary Pyridoxinetype responsive

type

FIGURE 3 The new serine protease activity in imiaturegranulocytes of patients with primary sideroblastic aniemia.Horizontal bars present the mean values.

also showed decreased protease activity. On the con-trary, mature granulocytes obtained from two caseswith pyridoxine-responsive anemia before therapy re-vealed the protease activity within normiial range.

Itnactivationi of apo-ALA sytithetase by the protease.Degrees of inactivation of the apo-ALA synthetase bythe protease that is considered to be engaged in theregulation of ALA synthetase levels in mitochondria inerythroblasts were examined by the method de-scribed (4). Results are shown in Table I. In contrastto the apo-ALA synthetase prepared from erythroblastsof patients with pyridoxine-responsive anemia, de-grees of inactivation by the protease of apo-ALA syn-thetase prepared from patients with primary acquiredsideroblastic anemia were almost the same as those ofnormal controls.

Cytochrome oxidase activity. Both mature granulo-cytes and lymphocytes in the peripheral blood wereemployed for the determination of cytochrome oxidaseactivity. Addition of catalase (10 nM) from bovineliver (Boehringer Mannheim GmbH) to the reactionmixture had no effect on the oxidation of cytochromiec by the enzyme solution, therefore, cytochrome cperoxidase does not play any roles in the oxidationof cytochrome c by the reaction system employedhere. Furthermore, the reaction mixture containing1 mMpotassium cyanide showed only a little oxi-dation of reduced cytochrome c, and that with 1 mnM

potassiuimi cyanide containing oxidized cytochromnec instead of the reduced form revealed no significantreduction of cvtochromiie c as judged by the changesin the absorbance at 550 nm. These results indicatethat the decrease in absorbance at 550 nm underthe conditions described in the Methods shows theactivity of cytochrome oxidase. Fig. 4 shows thecvtochrome oxidase activity in both mature granulo-cytes and lymphocytes obtained from patients withprimary sideroblastic aniemia. Every case with primaryacq(Iuired sideroblastic anemiiia had decreased cyto-chrome oxidase activity in mature granulocytes. Alsomature granulocytes obtained from two cases withpyridoxine-responsive anemia before treatmenitshowed decreasedl activity, however, the activity in-creased to the normiial level when the patients wentinto remission after treatment with pharmacologicaldoses of pyridoxal phosphate (Fig. 4). Lymphocytesobtained from both patients with primary acquiredtype and those with pyridoxine-responsive anemiarevealed the cvtochrome oxidase activity within nor-miial range.

Oligomitycin-.sen.sitive ATPa.se activity. Every casewith the primary ac(luired type had decreased oligo-mycin-sensitive ATPase activity in m-ature graniulo-cvtes as shown in Fig. 5. In contrast, the activity inimiature granulocytes obtained from two cases withpyridoxine-responsive anemia before treatment waswithin the normal range. The oligomycin-sensitiveATPase activity in lymphocytes obtained from bothpatients with primary acquired type and those withpyridoxine-responsive anemia before treatment waswithin the normal range.

Citriate si nlthetase activity. Citrate synthetase ac-

10a000 MEANtSD7,470*890

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46 V. Aoki

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FIGURE 4 Cytochrome oxidase activity in I

graniulocytes and lymphocytes obtained from pprimary sideroblastic anemia. In the column ofsymbol (0) shows the value of patients with primsideroblastic anemia, 0, that of patients withresponsive anemia before therapy, and 0, thaiwith pyridoxine-responsive anemia in remissionment with pharmacological doses of pyridoxa]Each horizontal bar presents the mean value (hoin the column of patients indicate the mean valuEwith primary acquired type, and MeantSD in tipatients shows that of patients with primary acq

tivity was measured in both mature granullymphocytes in the peripheral blood obt;both patients with primary acquired typewith pyridoxine-responsive anemia. Tubing the reaction mixture except both sodiand NAD hydrolyzed negligible amount(150 nmol) as measured by the changes in;at 340 nm. Fig. 6 shows the citrate synthetbin both mature granulocytes and lymphocpatients. Although some variations were

the citrate synthetase activity in both matucytes and lymphocytes obtained from palprimary acquired type was essentially wit]range. Also the activity in both mature gi

and lymphocytes obtained from two patpyridoxine-responsive anemia was withinrange as shown in this figure.

Changes of the enzyme activities byment. These enzyme activities in mitocho

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Mature granulocytes

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Normal Patients Normalcontrols controls

Patients

8.7* 6L0 FIGURE 5 Oligomycin-sensitive ATPase activity in both

mature granulocytes and lymphocytes obtained from patientswith primary sideroblastic anemia. In the column of patients

Patients the symbol (0) shows the value of patients with primary ac-

quired type, and 0, that of patients with pyridoxine-re-sponsive anemia before therapy. Horizontal bars and

)ot,h mature Mean-SD indicate the same as those in Fig. 4.atients with

patients, theary acquired measured before and after the treatment with pharma-t ofpatixnen- cological doses of pyridoxal phosphate (100-200 mg/d,by the treat- orally, 3-5 wk) in several patients with primary ac-

I phosphate. quired sideroblastic anemia. No significant alterations,rizontal bars of the activities were brought about by the treatment.as of patientsie column of

DISCUSSION

uired type.).

Primary sideroblastic anemia can be classified intolocytes and three types; primary acquired, hereditary, and pyri-ained from doxine-responsive. Besides these primary sideroblastic

and those anemias, secondary types such as those induced byes contain- drugs or alcohol, and those accompanying variousium malate diseases are recognized. Among primary sideroblasticof NADH anemias the most frequent type is the primary acquired

absorbance type. Almost all the symptoms of this disorder can bease activity ascribed to the impaired heme synthesis resulting frombytes of the the decreased ALA synthetase activity in erythroblasts

observed, (1, 2). In fact, every patient with primary sideroblasticre granulo- anemia, including that with primary acquired type,

tients with had decreased ALA synthetase activity in erythroblastshin normal as shown in Fig. 1. The decrease of this enzyme

ranulocytes activity in erythroblasts of the patients is consideredtients with not to be secondary to iron deposition in mitochondriathe normal but to be the primary defect in erythroblasts (1, 2).

Recently, we found a new serine protease in mito-the treat- chondria in both erythroblasts and granulocytes (3).

indria were The protease in erythroblasts is considered to be en-

Mitochondrial Defects in Blood Cells in Sideroblastic Anemia

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Patients Normalcontrols

FIGURE 6 Citrate synthetase activity in both ma

cytes and lymphocytes obtained from patients 1

sideroblastic anemia. In the column of patients(0) shows the value of patients with primary a(

and 0, that of patients with pyridoxine-resporbefore therapy. Horizontal bars and Mean+SDsame as those in Fig. 4.

gaged in the regulation of ALA synthetasmitochondria. However, protease activity iin erythroblasts of patients with primalsideroblastic anemia. Furthermore, the resi

susceptibility to the protease of apo-ALAprepared from erythroblasts of patients w

acquired sideroblastic anemia was within n

in contrast to that prepared from pyridoxineanemia suggest that the cause of the decresynthetase activity in erythroblasts differs itypes of sideroblastic anemia.

Besides the characteristic anemia that ission of the abnormality in erythropoietictients with primary acquired sideroblastic a

develop a decreased number of granulocperipheral blood (10, 11). In our series thethe patients with primary acquired typecreased numbers of granulocytes in theblood. As shown in Fig. 3, with three exc

protease activity in mature granulocyteswith primary acquired sideroblastic anen

iocytes creased. Furthermore, determination of the activitiesMEANtSD of mitochondrial enzymes such as cytochrome oxi-

12 5t 5.5 dase and oligomycin-sensitive ATPase in maturesot granulocytes revealed that also the activities of these

* mitochondrial enzymes were decreased in mature* granulocytes of the patients. On the other hand, the

activity of citrate synthetase that is located in mito-O chondria was within normal range in mature granulo-* cytes of the patients as shown in Fig. 6. Althougho the reason why certain enzymes in mitochondria are* impaired and others are not is remained to be clarified,

these results indicate that several components in mito-* chondria, but not all components are impaired in both

erythroblasts and granulocytes of patients with primary* acquired sideroblastic anemia. Since functions of

granulocytes such as phagocytosis or production of* superoxide are little influenced by the manipulation

impairing mitochondrial metabolism (12), we speculatethat defects of several mitochondrial enzymes in ma-

* ture granulocytes do not cause symptoms in patientswith primary acquired sideroblastic anemia. On thecontrary, mitochondria in erythroblasts are engaged inthe fundamental roles of the cell, i.e., heme synthesis.Therefore, the defects in mitochondria are more ap-parent in erythroblasts. That activities of mitochondrial

Patients enzymes such as cytochrome oxidase, oligomycin-sensitive ATPase, and citrate synthetase are within

ture granulo- normal range in lymphocytes of patients with primarywith primary acquired sideroblastic anemia are compatible with thei, the symbol clinical data that patients with this disease scarcelycquired type, develop immunological abnormalities. In contrast

indicate the to mitochondrial enzymes, activities of several cyto-somal enzymes in erythrocytes of patients with primaryacquired sideroblastic anemia were reported to bewithin normal range (13). Impaired synthesis of DNA

se levels in and RNA, and depression of protein synthesis ins decreased erythroblasts of patients with primary acquired sidero-ry acquired blastic anemia was reported by Wickramasinghe andults that the Hughes (14). They showed that ring sideroblasts at

synthetase an immature stage exhibited almost normal synthesis'ith primary of both nucleic acids and proteins, while at a moreLormal range mature stage the synthesis of both nucleic acids and-responsive protein was diminished. Therefore, the disturbances~ase of ALA of both nucleic acid synthesis and protein synthesis inin these two ring sideroblasts may stem from the mitochondrial

lesions. We speculate that defects in mitochondriathe expres- in both erythroblasts and granulocytes are the etiologyseries, pa- of primary acquired sideroblastic anemia.

Lnemia often The results that not only ALA synthetase activitywytes in the in erythroblasts but also the protease activity in both

majority of erythroblasts and granulocytes was decreased in pa-showed de- tients with hereditary sideroblastic anemia suggest

peripheral that mitochondrial defects in both erythroblasts andveptions the granulocytes may also be present in the hereditary type.of patients Patients with pyridoxine-responsive anemia also

nia was de- showed decreased ALA synthetase activity in erythro-

48 Y. Aoki

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blasts and decreased cytochrome oxidase activity inmature granulocytes. Furthermore, like ALA synthe-tase activity in erythroblasts, cytochrome oxidase ac-tivity in mature granulocytes increased to the normallevel after the treatment with pharmacological dosesof pyridoxal phosphate as shown in Fig. 4. These resultsseems to indicate that heme synthesis is equally im-paired in both erythroblasts and granulocytes of pa-tients with pyridoxine-responsive anemia beforetherapy. However, they showed normal proteaseactivity in both erythroblasts and mature granulocytes.Also activities of mitochondrial enzymes such asoligomycin-sensitive ATPase and citrate synthetase inboth mature granulocytes and lymphocytes werewithin normal range as shown in Figs. 5 and 6. Weconclude that only ALA synthetase in both erythro-blasts and granulocytes is impaired in patients withpyridoxine-responsive anemia.

ACKNOWLEDGMENTSI am grateful to Dr. K. Nakao, President of Jichi MedicalSchool, Professor F. Takaku, and Professor T. Yokoyama,Jichi Medical School, for interest and advice. I amalso gratefulto Professor T. Maekawa, The Third Department of Inter-nal Medicine, GummaUniversity, Gumma, Japan for help-ful discussions.

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siderocytes, and sideroblastic anemia. N. Engl. J. Med.292: 185-193.

2. Aoki, Y., G. Urata, 0. Wada, and F. Takaku. 1974. Meas-urement of 8-aminolevulinic acid synthetase activity inhuman erythroblasts.J. Clin. Invest. 53: 1326-1334.

3. Aoki, Y. 1978. Crystallization and characterization of anew protease in mitochondria of bone marrow cells.

J. Biol. Chem. 253: 2026-2032.4. Aoki, Y., S. Muranaka, K. Nakabayashi, and U. Ueda. 1979.

8-Aminolevulinic acid synthetase in erythroblasts of pa-tients with pyridoxine responsive anemia.J. Clin. Invest.64: 1196-1203.

5. Smith, L. 1965. Spectrophotometric assay of cytochromeoxidase. In Methods of Biochemical Analysis. D. Glick,editor. John Wiley & Sons, Inc., NewYork. II: 427-434.

6. Kagawa, Y. 1967. Preparation and properties of a factorconfering oligomycin sensitivity (FO) and of oligomycinsensitive ATPase (CFo-0F1). Methods Enzymol. X:505-510.

7. Lohmann, K., and L. Jendrassik. 1926. KolorimetrischePhosphorsaurebestimmungen im Muskelextrkt. Biochem.Z. 178: 419-426.

8. Ochoa, S. 1955. Crystalline condensing enzyme from pigheart. Methods Enzymol. I: 685-694.

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