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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 235, No. 6, June 1960 Phtea in U.S. A. Heme Synthesis in Iron-deficient Duck Blood* WOLFGANG VOGEL, DAN A. RICHERT, BURNETT Q. PIXLEY,~ AND MARTIN P. SCHULMAN From the Department of Biochemistry, State University of New York, Upstate Medical Center, Syracuse 10, New York (Received for publication, October 19, 1959) The mechanism by which iron is utilized for heme synthe- sis in isolated systems has been a subject of recent interest. Iron is part of the heme molecule and is utilized in the for- mation of heme by reacting with protoporphyrin. Thus iron participates as a substrate. The reaction of iron with proto- porphyrin appears to be enzymatic (14). Iron has also been implicated as a cofactor in the biosynthesis of &aminolevulinic acid (5, 6). The work to be described originated with the observation that heme synthesis in intact red cells of iron-deficient duck- lings could be stimulated severalfold by the addition of ferrous iron (7). When whole blood from iron-deficient ducklings was incubated with glycine-2-CY4, decreased amounts of C14-heme were formed; the addition of ferrous iron in vitro to the de- ficient blood restored heme synthesis to control values. Since the iron markedly stimulated heme synthesis in iron-deficient cells, exploration of this system seemed worthwhile in order to study the sites of reaction of iron in these processes. A stimulation of heme synthesis from glycine and other substrates by iron has been reported for normal erythrocytes (8, 9). The purpose of the present studies was a-fold: (a) to com- pare the rates of formation of heme and of PROTO’ from gly- tine and AML in iron-deficient and control blood of ducklings, and (b) to study the effects of iron deficiency on the biosyn- thesis of AML in washed particles of hemolysed red cells (10, 11). In previous studies it was possible to demonstrate that pyridoxal5’-phosphate was involved in AML synthesis by show- ing that red cells of vitamin BG-deficient ducklings incorporated glycine-2-W into heme at a reduced rate; the addition of py- ridoxal 5’-phosphate in vitro stimulated heme synthesis. On the other hand, normal amounts of heme were formed from AML by the deficient cells, and this was unaffected by the addition of the cofactor (12). By use of a similar technique in the present study, comparing PROTO formation from AML and glycine, iron has been implicated in AML biosynthesis; this is in agreement with the observations of Brown (5, 6). Although PROTO formation from glycine was depressed, its formation from AML was comparatively little affected by iron deficiency. Decreased amounts of heme were formed from both glycine and AML by iron-deficient blood, and these amounts * This investigation was aided by grants from the National Cancer Inst.itute of the National Institutes of Health, United States Public Health Service (PHS C-1852), and from the Di- vision of Biological and Medical Sciences, National Science Foundation (NSF-G7126). t Summer Fellow, supported in part by the Council on Foods and Nutrition, American Medical Association. 1 The abbreviations used are: AML, &aminolevulinic acid; PROTO, protoporphyrin; URO, uroporphyrin; COPRO, copro- porphyrin. could be increased by the addition of iron. AML synthesis by particles derived from iron-deficient blood was decreased, but the addition of iron was not stimulatory under our conditions. However, particles derived from iron-deficient cells which were preincuba.ted in the absence of iron lost synthetic activity, whereas those derived from cells preincubated with iron did not. Iron did not prevent the loss of AML synthesis when control cells were preincubated. Blood from iron-deficient ducklings contained increased num- bers of reticulocytes. This was in contrast to the decreased numbers of reticulocytes in various vitamin deficiencies. The number of reticulocytes must be considered since the rates of incorporation of glycine-2-Cl4 into heme in vitro is proportional to the reticulocyte count (13). EXPERIMENTAL Hemoglobin was determined by the method of Schultze and Elvehjem (14), and red blood cells were stained by the method of Manwell and Feigelson (15). Duck red cells were classed as immature and called reticulocytes (16) on the basis of nu- clear structure, nuclear shape, and cytoplasmic chromation (17). Jugular blood was collected in heparin under ether anesthesia. The iron-deficient diet consisted of 25 g of casein, 10 g of gelatin, 53.3 g of Argo corn starch, 3.5 g of Mazola corn oil, 0.1 g of inositol, 0.2 g of choline chloride, vitamins in the pro- portions used by Reid et al. (18), and 7.9 g of salt mixture (18), except that ferric citrate was omitted and anhydrous CaHP04 was used instead of the hydrated compound. Con- trol groups received the same diets supplemented with 0.02% iron as ferric citrate or ferrous ammonium sulfate. Day-old Peking ducklings were fed the diets and distilled drinking water ad libitum, and were usually maintained on the diets for 14 days. Incubation of Blood Samples (for Hemin and Porphyrins)- Since glycine is converted into heme more efficiently by whole cells whereas AML is utilized more efficiently by red cell he- molysates, these different systems were used with the two dif- ferent substrates. Hemolysates were prepared by replacing the plasma with an equal volume of cold water. Twelve milliliters of whole blood or red cell hemolysates (pooled samples when necessary) were incubated at 37” for 2 hours in air in a Dubnoff shaker with either 120 pmoles of glycine-2-Cl4 (18,500 c.p.m. per pmole) or AML-2 ,3-Cl4 (11,500 c.p.m. per pmole), respectively. Either 0.3 ml of 0.9% NaCl or 0.3 ml of iron (a fresh solution of ferrous ammonium sulfate 2 Labco casein was obtained from the Borden Company, and U.S.P. gelatin powder and reagent grade salts from Baker and Adamson, General Chemical Division, Allied Chemical and Dye Corporation. 1769 by guest on September 28, 2020 http://www.jbc.org/ Downloaded from

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  • THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 235, No. 6, June 1960

    Phtea in U.S. A.

    Heme Synthesis in Iron-deficient Duck Blood*

    WOLFGANG VOGEL, DAN A. RICHERT, BURNETT Q. PIXLEY,~ AND MARTIN P. SCHULMAN

    From the Department of Biochemistry, State University of New York, Upstate Medical Center, Syracuse 10, New York

    (Received for publication, October 19, 1959)

    The mechanism by which iron is utilized for heme synthe- sis in isolated systems has been a subject of recent interest. Iron is part of the heme molecule and is utilized in the for- mation of heme by reacting with protoporphyrin. Thus iron participates as a substrate. The reaction of iron with proto- porphyrin appears to be enzymatic (14). Iron has also been implicated as a cofactor in the biosynthesis of &aminolevulinic acid (5, 6).

    The work to be described originated with the observation that heme synthesis in intact red cells of iron-deficient duck- lings could be stimulated severalfold by the addition of ferrous iron (7). When whole blood from iron-deficient ducklings was incubated with glycine-2-CY4, decreased amounts of C14-heme were formed; the addition of ferrous iron in vitro to the de- ficient blood restored heme synthesis to control values. Since the iron markedly stimulated heme synthesis in iron-deficient cells, exploration of this system seemed worthwhile in order to study the sites of reaction of iron in these processes. A stimulation of heme synthesis from glycine and other substrates by iron has been reported for normal erythrocytes (8, 9).

    The purpose of the present studies was a-fold: (a) to com- pare the rates of formation of heme and of PROTO’ from gly- tine and AML in iron-deficient and control blood of ducklings, and (b) to study the effects of iron deficiency on the biosyn- thesis of AML in washed particles of hemolysed red cells (10, 11). In previous studies it was possible to demonstrate that pyridoxal5’-phosphate was involved in AML synthesis by show- ing that red cells of vitamin BG-deficient ducklings incorporated glycine-2-W into heme at a reduced rate; the addition of py- ridoxal 5’-phosphate in vitro stimulated heme synthesis. On the other hand, normal amounts of heme were formed from AML by the deficient cells, and this was unaffected by the addition of the cofactor (12). By use of a similar technique in the present study, comparing PROTO formation from AML and glycine, iron has been implicated in AML biosynthesis; this is in agreement with the observations of Brown (5, 6). Although PROTO formation from glycine was depressed, its formation from AML was comparatively little affected by iron deficiency. Decreased amounts of heme were formed from both glycine and AML by iron-deficient blood, and these amounts

    * This investigation was aided by grants from the National Cancer Inst.itute of the National Institutes of Health, United States Public Health Service (PHS C-1852), and from the Di- vision of Biological and Medical Sciences, National Science Foundation (NSF-G7126).

    t Summer Fellow, supported in part by the Council on Foods and Nutrition, American Medical Association.

    1 The abbreviations used are: AML, &aminolevulinic acid; PROTO, protoporphyrin; URO, uroporphyrin; COPRO, copro- porphyrin.

    could be increased by the addition of iron. AML synthesis by particles derived from iron-deficient blood was decreased, but the addition of iron was not stimulatory under our conditions. However, particles derived from iron-deficient cells which were preincuba.ted in the absence of iron lost synthetic activity, whereas those derived from cells preincubated with iron did not. Iron did not prevent the loss of AML synthesis when control cells were preincubated.

    Blood from iron-deficient ducklings contained increased num- bers of reticulocytes. This was in contrast to the decreased numbers of reticulocytes in various vitamin deficiencies. The number of reticulocytes must be considered since the rates of incorporation of glycine-2-Cl4 into heme in vitro is proportional to the reticulocyte count (13).

    EXPERIMENTAL

    Hemoglobin was determined by the method of Schultze and Elvehjem (14), and red blood cells were stained by the method of Manwell and Feigelson (15). Duck red cells were classed as immature and called reticulocytes (16) on the basis of nu- clear structure, nuclear shape, and cytoplasmic chromation (17). Jugular blood was collected in heparin under ether anesthesia.

    The iron-deficient diet consisted of 25 g of casein, 10 g of gelatin, 53.3 g of Argo corn starch, 3.5 g of Mazola corn oil, 0.1 g of inositol, 0.2 g of choline chloride, vitamins in the pro- portions used by Reid et al. (18), and 7.9 g of salt mixture (18), except that ferric citrate was omitted and anhydrous CaHP04 was used instead of the hydrated compound. Con- trol groups received the same diets supplemented with 0.02% iron as ferric citrate or ferrous ammonium sulfate. Day-old Peking ducklings were fed the diets and distilled drinking water ad libitum, and were usually maintained on the diets for 14 days.

    Incubation of Blood Samples (for Hemin and Porphyrins)- Since glycine is converted into heme more efficiently by whole cells whereas AML is utilized more efficiently by red cell he- molysates, these different systems were used with the two dif- ferent substrates. Hemolysates were prepared by replacing the plasma with an equal volume of cold water.

    Twelve milliliters of whole blood or red cell hemolysates (pooled samples when necessary) were incubated at 37” for 2 hours in air in a Dubnoff shaker with either 120 pmoles of glycine-2-Cl4 (18,500 c.p.m. per pmole) or AML-2 ,3-Cl4 (11,500 c.p.m. per pmole), respectively. Either 0.3 ml of 0.9% NaCl or 0.3 ml of iron (a fresh solution of ferrous ammonium sulfate

    2 Labco casein was obtained from the Borden Company, and U.S.P. gelatin powder and reagent grade salts from Baker and Adamson, General Chemical Division, Allied Chemical and Dye Corporation.

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  • 1770 Heme Synthesis Vol. 235, No. 6

    in 0.9% NaCl) was added so that the final concentration was 10 pg of iron per ml.

    Preparation of Hemin-For the determination of specific ac- tivities of hemin crystals, prepared by a modified Fischer (19) procedure, 2 ml of the above incubation mixture were processed as follows: 600 mg of commercial hemoglobin in 5 ml of 0.9% NaCl were added as heme carrier, and the solution was rapidly forced by syringe into 30 ml of a mixture (room temperature) of glacial acetic acid plus 0.5 ml of saturated NaCl contained in a conical centrifuge tube with a beaded rim. The tubes were covered with watch glasses to retard evaporation, im- mersed in a boiling water bath for 6 hours, allowed to cool to room temperature, and centrifuged. The packed hemin crys- tals were then washed and counted as previously described (12).

    This procedure was convenient, but was applicable only when the solution contained little or no free radioactive PROTO. Dresel and Falk (8) found that hemin crystallized in the pres- ence of free porphyrin was contaminated with the porphyrin; this could not be removed by washing the crystals. In this study it was found that the hemin was not freed from radio- active porphyrin even by recrystallization.

    The above procedure can be used with reasonable accuracy when glycine with low specific activity (18,500 c.p.m. per pmole) is the substrate, because glycine yields little radioactive PROTO. In a representative experiment, PROTO was responsible for about 3% of the radioactivity of the hemin crystals prepared from a 2 ml incubation mixture (plus hemoglobin carrier) con- taining 30 mg of heme, 20 mpmoles of radioactive heme, and 10 mpmoles of PROTO. When glycine with high specific ac- tivity (94,500 c.p.m. per pmole) was used as substrate, the contamination was 12% because PROTO of higher specific ac- tivity was adsorbed on the hemin crystals.

    This procedure cannot be used with accuracy when radio- active AML is used as the substrate because relatively less heme and more PROTO are formed (8). In a typical experi- ment 2 ml of an hemolysate incubated with AML resulted in the formation of 150 mpmoles of PROTO and only 7 mpmoles of heme. Even though more heme (60 mpmoles) was formed in the presence of added iron, 120 mpmoles of PROTO were still present in the incubation mixture resulting in highly con- taminated hemin crystals. In either case the total hemin sam- ple was contaminated with 40 to 50 mpmoles of radioactive PROTO.

    Separation of Hemin and Porphyrins-In order to obtain ac- curate values for hemin as well as an analysis of the free por- phyrins formed, the porphyrins were separated and measured by a modification of the method described by Dresel and Falk (8). In our hands the following procedure gave more precise separation than the original method and yielded homogeneous fractions. Ten milliliters of blood or hemolysed cells were mixed with 10 volumes of a mixture of ethyl acetate and acetic acid (3:l) and left in the refrigerator for at least 2 days to ensure complete precipitation of the proteins. The proteins were fil- tered on a sintered glass funnel and washed with another 10 volumes of ethyl acetate-acetic acid (3 : 1). The combined ethyl acetate-acetic acid extracts were washed twice with 50 ml of a saturated solution of sodium acetate and once with 50 ml of water to remove acetic acid. Small amounts of porphyrin went into the aqueous phase. They were recovered by ad- justing the combined sodium acetate and water washings to pH 3 and extracting with ethyl acetate. The latter was com-

    bined with the main ethyl acetate fraction containing heme and free porphyrins. The combined ethyl acetate solutions were extracted with 10 ml of 150/, HCl to remove all free por- phyrins. This was repeated until no more fluorescence was de- tected in the HCl extract.

    Hemin was isolated from the porphyrin-free ethyl acetate phase as follows: The ethyl acetate was added to 100 ml of water in a beaker and allowed to evaporate at room tempera- ture. The hemin accumulated in the water as a fine precipi- tate and was centrifuged, washed twice with ethyl acetate and twice with ethyl ether; it was plated from ethyl ether.

    The combined 15% HCl solution (containing URO, COPRO, and PROTO) was adjusted to pH 3 to 4 with 14% NHhOH (weight per volume) and extracted several times with peroxide- free ethyl ether, followed by CHCl,, until no more fluorescence was observed in the organic solvents. The combined ether- chloroform solution contained PROTO and COPRO, and the aqueous solution contained URO. Each was separated for analysis as follows: COPRO was extracted with 0.2% HCl. PROTO was then extracted from the ether-chloroform into 10 % HCl. URO was extracted into ethyl acetate after the pH of the aqueous solution was adjusted to pH 3 with HCl. It was then extracted from the ethyl acetate with 15’% HCl. Each porphyrin (COPRO, URO, and PROTO in 0.2, 10, and 15% HCl, respectively) was assayed in the Beckman spectropho- tometer and its concentration calculated according to Riming- ton and Sveinsson (20). The fractions were checked for purity by paper chromatography in lutidine-NH3-water (21) and found to be homogeneous.

    The PROTO values represent the differences between the amounts found before and after incubation with substrate. There was little difference in the amounts of PROTO found in unincubated samples and those observed after incubation without added substrate.

    The quantities of heme synthesized were calculated from:

    c.p.m/mg of hemin X total mg of hemin c.p.m/pmole of substrate X 8

    x 1000

    = mpmoles of hemin

    It was assumed that 8 moles of glycine or AML were incor- porated into 1 mole of heme. The total milligrams of hemin were calculated from the total amount of hemoglobin (original plus carrier) in the system by assuming that hemoglobin yields 3.94% hemin. Hemoglobin carrier was always added when he- min was isolated by the modified Fischer procedure, and omitted when hemin was prepared from an ethyl acetate solution.

    AML Biosynthesis-Washed insoluble particles of red cell he- molysates were prepared by the procedure of Laver et al. (lo), except that the entire insoluble mass was retained. It was suspended in 0.1 M phosphate buffer, pH 6.8, with which it was diluted to half the volume of the original blood sample. Two milliliters of the suspension, equivalent to 4 ml of blood, were incubated at 37” in 20-ml beakers with shaking (Dubnoff shaker) for 2 hours with 37 mg of glycine, 146 mg of sodium citrate.2Hz0, 50 mg of MgC12.2Hz0, 0.25 mg of pyridoxal 5’- phosphate .HzO, 1 mg of coenzyme A, 1 mg of DPN.4Hz0, 1 mg of oc-lipoic acid, 1.7 mg of glutamine, and sufficient buffer to bring the total volume to 6 ml. MgClz (lo), coenzyme A (10, ll), pyridoxal 5’-phosphate (10, II), and lipoic acid (11) were found by Laver et al. (10) and by Brown (11) to stimu- late AML formation by particles from chicken erythrocytes.

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  • June 1960 W. Vogel, D. A. Richer& B. Q. Pixley, and M. P. Xchulman

    FIG. 1 (left). Iron-deficient duck erythrocytes FIG. 2 (right). Normal erythrocytes from ducklings 2 weeks of age

    DPN and glutamine were included since Granick (22) found stimulatory effects from these on the formation of PROTO from glycine in chicken erythrocyte hemolysates. Pyridoxal 5’-phosphate and ol-lipoic acid were stimulatory in our prepara- tions. Omission of DPN, coenzyme A, or glutamine with or without inosine was without significant effect on the forma- tion of AML. Addition of 0.4 mg of thiamine pyrophosphate did not enhance AML synthesis. The use of 37 mg of gly- tine and 146 mg of sodium citrate per vessel represent nearly 3 times the concentrations used by Brown (6) with prepara- tions from chicken erythrocytes. These increased amounts of substrates yielded more AML.

    ducklings were dilated and enlarged, weighing about 60% more than the controls (1.18 g and 0.74 g per 100 g of body weight, respectively).

    Control and iron-deficient blood values usually were as fol- lows: hemoglobin, 10 to 11.5 and 4.0 to 5.5%, respectively; reticulocyte counts, 7 to 12 and 13 to 20%, respectively; total erythrocytes, 1.8 to 2.2 X lo6 and 1.4 to 1.8 X lo6 cells per mm3, respectively; and hematocrits 28 to 34 and 18 to 22%, respectively.

    The reaction was stopped by the addition of 2.5 ml of 20% trichloracetic acid, and AML was determined calorimetrically after condensation with acetylacetone (23) as modified by Gib- son et al. (24).

    RESULTS

    In 6 different iron-deficiency experiments, 2 ml of control whole blood (9.9% hemoglobin and 7.4% reticulocytes) incor- porated 136 mpmoles of glycine into heme per 2 hours. The addition of 7 pg of ferrous iron per ml stimulated this to 166. Deficient blood (3.4% hemoglobin and 15% reticulocytes) incorporated 63 mpmoles of glycine. Addition of iron stimu- lated this to 259 or approximately 4-fold. Thus the iron- deficient cells contained the enzymic machinery for making relatively large amounts of heme from glycine, but were un- able to do so until iron was added.

    Iron Dejiciency-The blood from iron-deficient ducklings con- Efect of Iron Dejiciency on Synthesis of Porphyrins and Heme tained more immature cells than the blood from control duck- from Glycine and AML-If iron were needed solely as a sub-

    lings of the same age (2 weeks). This is in agreement with strate for the formation of heme from PROTO, an accumula- the results reported by Smith and Medlicott (25) in rats. The tion of PROTO might be expected in the iron-deficient cells nucleus of an immature duck cell is rather large and diffuse, incubated with glycine. Large amounts of PROTO (but little

    and the cytoplasm takes up the blue stain. Red cells from heme) can and do accumulate when normal red cell hemolysates iron-deficient ducklings were smaller and sometimes poorly are incubated with AML. Iron-deficient cells (or hemolysates)

    shaped; some cells demonstrated poor staining qualities, and were therefore incubated with glycine or AML as substrate,

    caused difficulty in making accurate counts (Fig. I). The cells and the amounts of free porphyrins as well as heme were de-

    in Fig. 2 represent normal blood. The hearts of the deficient termined. The results are given in Table I.

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  • 1772 Heme Xynthesis

    TABLE I

    Vol. 235, No. 6

    Synthesis of porphyrins and heme from glycine and 6-aminolevulinic acid in iron-deficient blood

    Blood Substrate

    Iron deficienth Whole blood. ...... Whole blood Whole blood. ...... Whole blood. ......

    Glycine Glycine AML AML

    Hemolysate ........ Glycine Hemolysate ....... Glycine Hemolysate ....... AML Hemolysate ....... AML

    Control Whole blood ....... Whole blood ....... Whole blood ....... Whole blood .......

    Glycine Glycine AML AML

    Hemolysate ....... Glycine Hemolysate ....... Glycine Hemolysate ....... AML Hemolysate ....... AML

    Fe

    -

    + -

    +

    -

    + -

    +

    -

    + -

    +

    -

    + -

    +

    / Experiment la

    I Experiment 2

    I_ URO

    -c

    -

    -

    18d

    13

    - -

    3 3

    - 9 28 6 179

    34 834 16 28 498 344

    - -

    72 130 42 165

    24 786 41 19 594 313

    PROTO Heme

    6 31 3 210

    56 45 24 92

    0.8 2 0.2 4 936 11 559 300

    25 125 17 150 96 77 49 80

    6 20 4 40

    926 42 604 320

    Q In Experiment 1, 12 pmoles of AML were incubated instead of the usual 120 pmoles per 12 ml. a Red blood cell data on the Fe-deficient and control bloods, respectively, were as follows: Experiment 1: hemoglobin, 4.3 and 11.2%;

    hematocrit, 18 and 34; immature red cell count, 15 and 10%; and total red cell count, 1.34 X lo6 and 1.61 X lo6 per mm3. Experi- ment 2: hemoglobin, 4.0 and 12.0; immature red cell count, 22 and 10.

    c A dash means the values were two small to be measured. No measurements were made when there are blank spaces. d Unincubated Fe-deficient and control bloods contained 13 and 20 mpmoles of PROTO, respectively. Neither contained measurable

    amounts of URO and only control blood contained a measurable amount (10 mpmoles) of COPRO. These were subtracted from the total values found with incubated substrates so that the figures in the table represent net synthesis.

    TABLE II

    Effect of preincubation of iron-de$cient whole blooda with iron on protoporphyrin and heme synthesis from glycine

    Preincubation time

    v&in

    0

    30 60 90

    120

    PROTO Heme

    -Fe +Fe” -Fe I

    +Fe

    mpmoles/lO ml oj’ blood/Z hrs

    12 6 30 260 14 10 24 275 14 34 20 305 15 47 18 320 17 68 16 324

    a Hemoglobin, 5.7%; reticulocyte count, 19%. 6 Ten pg Fe per ml as ferrous ammonium sulfate.

    The amounts of URO and COPRO formed by control or iron-deficient bloods were so small that they could be ignored in the interpretation of the results. Low, rather than high, levels of PROTO were formed from glycine by the iron-de- ficient blood. The yields of both heme and PROTO were re- duced to one-fourth or less of the control values. The ad- dition in vitro of iron to the deficient cells restored heme synthesis, but for some unknown reason resulted in even lower free PROTO values. However, the synthesis of PROTO could be restored to control levels by preincubating the iron-de-

    ficient blood with iron in vitro (no glycine) and then incubat- ing the treated cells with glycine in the usual manner. Such an experiment is shown in Table II, the results of which suggest that iron hasa function in the synthesis of PROTO from glycine; however, the cells needed to be exposed to the iron for at least 60 minutes before a stimulatory effect was observed.

    There was no effect of iron deficiency on the amounts of PROTO formed from AML by red cell hemolysates (Table I) ; this suggests that iron functions in the biosynthesis of AML from glycine rather than somewhere between AML and PROTO. Al- though this is in agreement with similar conclusions by Brown (5, 6) it must be recognized that the results obtained with intact cells for glycine and with cell hemolysates for AML may not be directly comparable. Both systems can be compared directly in whole blood or hemolysates in Table I, and such comparisons support the previous conclusions except that iron deficiency causes some decrease in PROTO formation from AML as well as from glycine when intact cells are used. The results clearly demonstrate that: (a) iron-deficient intact cells make less PROTO as well as heme from glycine particularly, and (b) the cell-free system which converts AML to PROTO is not affected by an iron deficiency.

    E$ect of Iron Dejkiency on Synthesis of AML in Insoluble Parti- cles of Red Cell Hemolysates-A possible role for iron in the con- version of glycine to AML was studied directly by measuring AML formation in particulate preparations of red cell hemoly-

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  • June 1960 W. Vogel, D. A. Richert, B. Q. Pixley, and M. P. Schulman 1773

    sates obtained from iron-deficient ducklings. Brown (5) previ- ously reported that the addition in vitro of ferrous iron inhibited the formation of AML by fresh particulate preparations from normal chicken erythrocytes; however, the activity of his aged preparations which had fallen in 2 and 3 days to 17 and 14yo of their initial level, was again raised to 22 and 29 y. by the addition in vitro of ferrous ions. He suggested that iron might be involved in stabilizing a Schiff’s base formed between glycine and pyri- doxal 5’-phosphate.

    The results of these studies are given in Table III. In some experiments (Experiments 1, 2, 6, 7, 8) the amount of AML formed by the insoluble particles obtained from iron-deficient erythrocytes was lower than the amount synthesized by similar preparations obtained from control blood. In other experiments (3, 4, and 5) the raw data did not demonstrate such an effect. However, when the reticulocyte content of the samples were taken into account (by dividing the amount of AML synthesized by the percentage of reticulocytes in the sample), then the iron- deficient preparations consistently formed less AML than the corresponding control preparations. Average values for the 8 experiments were 23.5 and 9.4 mamoles of AML per 1% of re- ticulocytes per 4 ml of control and iron-deficient bloods, respec- tively. That AML synthesis takes place in reticulocytes is sup- ported by the observation that AML synthesis nearly parallels the reticulocyte count (see following section). In this connec- tion, Laver et al. (10) found that preparations made from normal chicken red cells were much less active than those made from reticulocytes produced by the administration of phenylhydrazine.

    Since the addition of iron to whole blood stimulated the incor- poration of glycine into heme, it might be anticipated that AML synthesis by iron-deficient cell preparations would also be stimu- lated by the addition of iron in vitro. Such an effect was not demonstrated directly. However, particulate fractions derived from cells which were preincubated in the absence of iron lost activity, whereas those which were preincubated in the presence of iron retained activity (Table IV). After 1 hour of incubation, the latter were 2 to 3 times as active as the former and also some- what more active than the nonpreincubated samples. In 3 other experiments not shown in the table, 6.6 and 6.1 (iron added) pg of AML per 4 ml of blood per 1% of reticulocytes were formed by particulate fractions obtained from cells which were not pre- incubated. After preincubation for 60 minutes the values were 5.1 and 10.9 (iron added), respectively. Control cells responded differently in as much as the preincubation with iron did not prevent the loss of activity which resulted from the incubation per se. So the added iron at least prevented the loss of activity for AML synthesis when the deficient cells were preincubated before preparation of the fractions, and may even have stimu- lated the activity. Since iron did not stabilize the control blood activity, it is possible that the values obtained after 60 minutes of incubation with iron resulted from a combination of a stimu- latory effect by the iron and a loss of activity from incubation per se. This, too, could explain the relatively small differences in AML synthesis between the nonincubated samples and those preincubated with iron for 60 minutes (Experiment 1, Table IV) as well as the large differences in AML formation between the samples preincubated with and without iron for 60 minutes.

    Since iron stimulates aconitase activity (26, 27), the effect of iron was tested with isocitrate replacing citrate as the source of succinyl coenzyme A. Iron stimulated AML synthesis both with this substrate and with citrate (Experiment 2, Table IV);

    TABLE III E$ect of iron cle$ciency on &aminolevulinic acid synthesis in

    insoluble fractions of erythrocyte hemolysates

    Control Iron deficient

    Experi merit

    2eticulo- cytes

    2eticulo- cytes

    1 2 3 4 5 66 7a 8”

    % f?%/[email protected]

    10 247 24.7 10 264 26.4 7 248 35.4 5 176 35.2 9 236 26.2

    15 156 10.4 10 104 10.4 6 117 19.5

    _-

    I

    _-

    T

    Aykpdml Ala/4 ml blood/l% reticulocytes

    [email protected]%oles

    54 2.4 208 9.5 244 16.3 192 19.2 300 15.0 44 2.2 52 3.5 66 7.3

    Avg. 9 193 23.5

    %

    22 22 15 10 20 20 15 9

    17 -

    145 9.4

    0 In the last 3 experiments 12 mg of glycine and 49 mg of citrate were used.

    TABLE IV Effect of preincubation (37”) of iron-dejicient whole blood with

    ferrous iron on &aminolevulinic acid synthesis activity nsoluble particles

    ‘reincuba- tion time

    min

    0 0

    30 30 60 60 90 90

    120 120

    0 0

    60 60

    120 120

    7 AML/4ml blood/l% reticulocytes

    1 P%c -

    IE -

    xperiment 1 Experiment 2 I

    .-

    Blood

    Citrate Citrate Isocitrate”

    m,.,moles/4 ml blood/Z hrs

    8.2. 12.0 9.3

    11.8 5.5

    14.7 4.2

    13.5 4.0

    12.8

    12.7 7.6 13.1 8.0

    9.5 8.0 27.4 26.2

    25.4 22.8 17.2 15.9 12.0 9.9

    Iron deficientb

    Controlc

    a n-Isocitrate was substituted for citrate which was routinely used in all other incubations.

    b Experiment 1: Hemoglobin, 8.7%; reticulocyte count, 19%. Experiment 2: Hemoglobin, 5.Oyo; reticulocyte count, 18%.

    c Hemoglobin, 11.5’$‘o; reticulocyte count, 9.3%.

    this indicates that the iron had some effect other than the simple restoration of aconitase activity in the iron-deficient blood.

    Iron was not stimulatory but slightly inhibitory when added directly to the particulate fractions. An insoluble preparation from 4 ml of iron-deficient blood formed 168, 168, 168, 152, and 136 mpmoles of AML after the addition of 0, 1, 4, 20, and 40 pg of iron per ml, respectively. The addition of either iron-defi-

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  • 1774 Heme Synthesis Vol. 235, No. 6

    cient or control plasma did not enhance the effectiveness of iron. In fact the addition of 1 ml of plasma actually inhibited the for- mation of AML by more than 50%. This was probably due to the presence of protein since a boiled plasma extract was without effect. Ferrous ascorbate produced a 31 y0 inhibition. Because of the possibility that the presence of citrate and phosphate made the added iron unavailable, succinate and ATP were substituted for citrate and Tris buffer was substituted for the phosphate. However, under these conditions much less AML was formed (28 mpmoles as compared to 180 mpmoles for the particles in- cubated in the usual phosphate-citrate medium), and the addi- tion of iron had no effect. Preincnbation of the particulate frac- tions with iron did not induce the same effect as preincubation of the whole blood. A fraction which formed 160 pg of AML, formed only 64 pg after it was preincubated with iron in isotonic KC1 and 68 pg when preincubated without added iron. To test the importance of the intact cell for the effectiveness of iron, red cells, hemolyzed by replacing the plasma with cold water, were preincubated with and without iron, and then fractionated. The fractions from these preparations formed only 80 mpmoles of AML (compared with 180 when the sample was not preincu- bated) whether iron was added or not. These results indicated that the iron-deficient system for synthesizing AML could be affected by iron only when it was added to the intact cells, but not to the isolated system.

    These results clearly demonstrate an effect of iron on the bio- synthesis of AML. The effect of preincubating iron-deficient cells with iron may be due to one of the following possibilities: (a) The enzyme system (including apoenzyme) needed for the biosynthesis of AML may be decreased in the iron-deficient cells, and the preincubation period with iron may stabilize or induce

    L 1 IO

    % RETICULOCYT:: FIG. 3. Effect of reticulocytes on the synthesis of &amino-

    levulinic acid, PROTO, and heme. For Lines 1 and 2, hemoly- sates of erythrocytes .with different reticulocyte counts were incubated with b-aminolevulinic acid-2.3-W and the PROTO and heme were separated and measured on the same samples of incubation mixtures. Synthesis of &aminolevulinic acid from glycine (Line 3) was measured in the insoluble particulate frac- tion prepared from erythrocyte hemolysates. Line 4, showing the conversion of glycine-2-C I4 to heme by whole blood, was reported previously (13).

    the formation of the enzyme, or (b) the inorganic iron must be converted to some organic-complex form before it can participate as a cofactor in AML biosynthesis, and that preincubation of the intact cell, but not the particles, supplies this form of iron.

    Relation of AML, Porphyrin, and Heme Synthesis to Reticulo- cyte Concentration-Washed red cells of blood, pooled from duck- lings 8 days old, were suspended in 28% albumin, as described by Allison and Burn (28), and centrifuged at 1500 r.p.m. for 10 minutes. The top third and bottom third portions of the cells were separated and suspended in enough 0.9% sodium chloride solution to adjust the hemoglobin concentration of each sample to 10%. These samples were then processed for AML, PROTO, and heme synthesis as described for whole blood.

    It was found that samples containing 13% reticulocytes formed only about half as much AML as the samples containing 25% reticulocytes (150 mpmoles of AML versus 275 mpmoles). This is shown in Line S, Fig. 3 which represents the average val- ues from 2 experiments. Line 4 represents the conversion of glycine to heme, which was reported previously to be propor- tional to the reticulocyte count (13). The mechanism for con- verting glycine to AML appears to be localized within the retic- ulocytes. The conversion of AML to PROTO (Line 2) was essentially independent of the reticulocytes, whereas heme for- mation from AML (Line 1) was only 75’% as efficient in the sample containing 13% reticulocytes as in the sample contain- ing 25%. Since the conversion of AML to PROTO was essen- tially the same in the samples containing dierent concentrations of reticulocytes, it is probable that Line 1 actually reflects the conversion of PROTO to heme. Apparently, the enzymes needed to convert AML to PROTO are present in all red cells, whereas the enzymes needed to convert glycine to AML occur predominantly if not exclusively in the reticulocytes. The con- version of PROTO to heme is faster in reticulocytes, but also takes place in other red cells. Hence a blood sample containing few reticulocytes would have little ability to convert glycine to either of the intermediates (AML or PROTO) or to the final heme. Such a blood sample could make both PROTO and heme from AML, but the rate of heme formation from PROTO would be less than that observed with a high reticulocyte concentration.

    SUMMARY

    1. The rate of heme synthesis from glycine-2-Cl4 by the blood cells obtained from iron-deficient ducklings was lower than that observed with normal ducks. Less, rather than more, free pro- toporphyrin accumulated under these conditions. The addition of ferrous iron in vitro stimulated the ability of the iron-deficient cells to synthesize heme from glycine.

    2. Only small amounts of heme were synthesized from &ami- nolevulinic acid in hemolysates of either iron-deficient or control red cells unless iron was added in vitro. However, much free protoporphyrin was formed and the quantities were essentially alike in iron-deficient and control samples. Addition of iron in- creased the amounts of heme and decreased the amounts of pro- toporphyrin.

    3. Less Saminolevulinic acid was synthesized from glycine by the insoluble particulate fractions prepared from iron-deficient red cells than from control cells. Activity was not restored by the addition of iron to the fractions.

    4. The above results indicated that iron deficiency had two effects on heme synthesis. It decreased the rate of synthesis of

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  • June 1960 W. Vogel, D. A. Richer& B. Q. Pixley, and M. P. Schulman 1775

    d-aminolevulinic acid from glycine and of heme from protopor- 12. SCHULMAN, M. P., AND RICHERT, D. A., J. Biol. Chem., 226,

    phyrin. 181 (1957).

    5. Preincubation of control cells with or without iron resulted 13. RICHERT, D. A., AND SCHULMAN, M. P., Am. J. Clin. Nu-

    in decreased formation of Caminolevulinic acid by particles pre- trition, ‘7, 416 (1959).

    14. SCHULTZE, M. O., AND ELVEHJEM, C. A., J. Biol. Chem., pared therefrom. Preincubation of iron-deficient cells without 106, 253 (1934). iron also resulted in decreased synthesis; however, when the cells 15. MANWELL, R. D., AND FEIGELSON, P., J. Lab. Clin. Med.,

    were preincubated with iron, there was no loss in S-aminolevu- 33, 777 (1948).

    16. KHABIR, P. A., AND MANWELL, R. D., J. Parasitol., 41, 595 linic acid synthetic activity. (1955).

    17. DAVIS, J. E., MCCULLOUGH. A. W., AND RIGDON. R. H.,

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  • Wolfgang Vogel, Dan A. Richert, Burnett Q. Pixley and Martin P. SchulmanHeme Synthesis in Iron-deficient Duck Blood

    1960, 235:1769-1775.J. Biol. Chem.

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