Effects by Heme, Insulin, and Serum Albumin on Heme and Protein Synthesis in Chick Embryo Liver Cells Cultured in a Chemically Defined Medium, and a Spectrofluorometric Assay for Porphyrin Composition*

Primary chick embryo liver cells, which had been previously cultured in Eagle's medium containing 10% fetal bovine serum, had the same characteristics (inducibility of delta-aminolevulinic acid synthetase and synthesis of plasma proteins) when cultured in a completely defined Ham F-12 medium containing insulin. Insulin was active in the physiological range; 2 to 3 nM were sufficient to increase the induced delta-aminolevulinic acid synthetase to 50% of the maximum effect obtained with a saturating amount of insulin (30 nM). Serum albumin added to the Ham-insulin medium caused protoporphyrin but not uroporphyrin, generated in the cultured liver cells, to be transferred to the medium. As little as 10 mug of human serum albumin per ml caused the transfer of one-half of the protoporphyrin. Bovine serum albumin was only about 1/30 as effective. A spectrofluorometric method and calculation procedure are described for quantitation, in the nanomolar range, of total porphyrin and the percentage of this that is protoporphyrin or uroporphyrin plus coproporphyrin. The method is satisfactory for the measurement of porphyrins generated by 1 mg wet weight of cells in culture in 20 hours. Heme (0.1 to 0.3 muM), when added to the medium as hemin, human hemoglobin, or chicken hemoglobin, specifically inhibited the induction of delta-aminolevulinic acid synthetase by one-half. This high sensitivity for heme was observed under conditions in which the defined medium was free of serum and where a chelator of iron was added to the medium to diminish the synthesis of endogenous heme. Heme endogenously generated from exogenous delta-aminolevulinic acid also inhibited the induction; chelators of iron prevented this inhibition. The migration of heme from the mitochondria to other portions of the cell is discussed in terms of the affinities of different proteins for heme. A hypothesis of a steady state of liver heme metabolism, controlled by the concentration of "free" heme, is presented. The different effects of heme on the synthesis of a number of proteins are summarized.

Primary chick embryo liver cells, which had been previously cultured in Eagle's medium containing 10% fetal bovine serum, had the same characteristics (inducibility of 6-aminolevulinic acid synthetase and synthesis of plasma proteins) when cultured in a completely defined Ham F-12 medium containing insulin. Insulin was active in the physiological range; 2 to 3 nM were sufficient to increase the induced b-aminolevulinic acid synthetase to 50% of the maximum effect obtained with a saturating amount of insulin (30 nM).
Serum albumin added to the Ham-insulin medium caused protoporphyrin but not uroporphyrin, generated in the cultured liver cells, to be transferred to the medium. As little as 10 Kg of human serum albumin per ml caused the transfer of one-half of the protoporphyrin.
Bovine serum albumin was only about %O as effective.
A spectrofluorometric method and calculation procedure are described for quantitation, in the nanomolar range, of total porphyrin and the percentage of this that is protoporphyrin or uroporphyrin plus coproporphyrin.
The method is satisfactory for the measurement of porphyrins generated by 1 mg wet weight of cells in culture in 20 hours.
Heme (0.1 to 0.3 wM), when added to the medium as hemin, human hemoglobin, or chicken hemoglobin, specifically inhibited the induction of b-aminolevulinic acid synthetase by one-half. This high sensitivity for heme was observed under conditions in which the defined medium was free of serum and where a chelator of iron was added to the medium to diminish the synthesis of endogenous heme. Heme endogenously generated from exogenous &aminolevulinic acid also inhibited the induction; chelators of iron prevented this inhibition.
The migration of heme from the mitochondria to other portions of the cell is discussed in terms of the affinities of different proteins for heme. A hypothesis of a steady state of liver heme metabolism, controlled by the concentration of "free" heme, is presented. The different effects of heme on the synthesis of a number of proteins are summarized.
In 1963 (1) a method was developed in this laboratory to culture primary chick embryo liver cells in Eagle's basal medium supplemented with 10% fetal bovine serum. A variety of chemicals, drugs, and steroids, acting on the cells in this medium was found to induce a marked (lo-to 30-fold) increase in porphyrins, the increase being caused primarily by an increase in b-aminolevulinic acid synthetase activity (2, 3). Depending on the chemical, the induction was at the level of transcription or was post-transcriptional (4). Discussions of the induction mechanisms have been presented in several recent controlling an unknown post-transcriptional step of the induction of the enzyme. Heme was found not to change the rate of decay of the enzyme activity in the cultured cells in the presence of acetoxycycloheximide (4). The purified enzyme activity from chick embryo liver mitochondria was inhibited by hemin but only at much higher hemin concentrations (K, = 35 PM).' Hayashi et al. (9) have postulated that in the rat, hemin may also inhibit the transport into mitochondria of newly synthesized d-aminolevulinic acid synthetase. However, in chick embryo liver no evidence has been found that hemin inhibits this postulated transport (Tomita et al. (lo), Strand et al. (ll), see also Footnote 2). Moreover, our recent experiments have demonstrated a decrease in the amount of immunoprecipitable &aminolevulinic acid synthetase after the treatment of cells with hemin.' On the basis of this evidence the decrease in &aminolevulinic acid synthetase activity caused by heme may be equated with a decrease in the amount of this enzyme caused by heme.
In this paper we report on a method of culturing chick embryo liver cells in a completely chemically defined medium of modified Ham's F-12 (12) supplemented with insulin. This defined medium has made two investigations possible: (a) an inquiry into which components of fetal bovine serum, added to Eagle's basal medium, are required for maximal induction of &aminolevulinic acid synthetase. (b) A reinvestigation of the amount of the added hemin required to inhibit the induction of d-aminolevulinic acid synthetase. The absence of fetal bovine serum in the defined medium eliminated problems of the binding of exogenous hemin to serum proteins. The endogenous formation of heme by the cells was decreased by adding, to the defined medium, chelators of iron to block heme synthesis at the ferrochelatase step. By means of these modifications, the concentration of exogenous hemin which would inhibit by 50% the induction of d-aminolevulinic acid synthetase in the cells could be determined.
We have used porphyrin accumulation as an indirect measure of &aminolevulinic acid synthetase. To quantitate the porphyrins accumulated by the cells and in the medium, a fluorescence assay was developed of sufficient sensitivity to determine porphyrins in the picomole range. This assay made possible the finding that human serum albumin in the medium caused the transport of protoporphyrin out of the cells. The method also distinguished protoporphyrin from uroporphyrin (plus coproporphyrin), and this made possible the recognition and quantification of the specific effect of chlorinated hydrocarbons on causing umporphyrin formation (13).   Table I).

Actual Procedure and Calculations
We shall now detail the steps in the procedure and the calculations to determine the percentage of protoporphyrin and the total porphyrin in a mixture.
1. For determination of the porphyrins in the 1 ml of culture medium, 0.1 ml of the medium is pipetted into 0.4 ml of perchloric acid/methanol contained in the Kimble test tube used as a cuvette. If protein is present in the medium the test tube is centrifuged to pellet the protein.
The emission spectrum of the supernatant layer is scanned from 580 to 680 nm. 2. A "blank" curve of the medium is made and is subtracted from the first curve.
3. The ratio of the band maxima (max, at 602 to 608 nm/maxt at 654 to 662 nm) is calculated and referred to Fig. 3, Column I of the nomogram.
4. The nomogram is read across to Column 2 to obtain the percentage of total porphyrin that is protoporphyrin. By reading across to Column 4, a "corresponding factor" is obtained. 5. From the fluorescence curve, the FU at max, is determined. This FU value is multiplied by the "corresponding factor" in Column 4 and again by 10 to give picomoles of total porphyrin in the 1 ml of medium. 6. For the determination of the porphyrin in the cells on the coverslip, the remainder of the medium in the vial is removed, and 0.5 ml of perchloric acid/methanol is added to the coverslip in the vial. in the 600 to 680 nm region may be due to substances other than porphyrins in, or added to the medium. The non-porphyrin fluorescence may be recognized by the shape of the fluorescence curve or by the fact that the ratio of the two fluorescence peaks may be greater than 1.3 (Fig. 3 pmtoporphyrin by 20% and an underestimate in total porphyrin by 10%.

Factors in Defined and Serum-containing Culture Media for Optimizing
Induction for &Aminoleuulinic Acid Synthetase The increase of b-aminolevulinic acid synthetase caused by inducing chemicals, e.g. allylisopropylacetamide, and the consequent porphyrin accumulation require continuing RNA and protein synthesis in the primary cultured chick embryo liver cells (3). In this paper we used the criterion of maximum induction of porphyrin accumulation as a measure of the ability of substances of the medium to support hepatocyte protein synthesis.
Defined Medium-The completely defined medium consisted of Ham F-12 free of lipids, pyruvate, and trace metals but supplemented with bovine insulin (0.2 rg/ml). The lipids and pyruvate were found not to be essential. Sufficient amounts of the trace metals were presumably supplied as contaminants.
The cells cultured in this medium exhibited the same characteristics as those cultured in the presence of Eagle's medium containing 10% fetal bovine serum: induction of the synthesis of d-aminolevulinic acid synthetase by various chemicals, drugs, and steroids; inhibition by heme of the synthesis of d-aminolevulinic acid synthetase; and the synthesis and secretion of plasma proteins into the medium (18).
Functions of Fetal Bovine Serum-Several functions were noted. (a) The proteins of the serum protected the cells against damage by the enzymes, trypsin and Varidase, which were used to separate the liver cells in preparing the cell inoculum. The effect of fetal bovine serum was seen in the satisfactory attachment of the cells to the coverslip, with a cell inoculum of 3 x 10' cells per ml of medium, when the medium contained 10% fetal bovine serum, but the attachment was poorer in the presence of 3% fetal bovine serum. Once the cells had become attached, then Ham-insulin without fetal bovine serum was sufficient to support the cell culture. It is important to note that the addition of serum was not essential for cell attachment since cells could attach in the absence of fetal bovine serum if the cell suspension was washed twice with Eagle's minus calcium and magnesium to remove the trypsin and Varidase, and Ham-insulin then added to the cells; however, this procedure required that 2 to 4 times more cells be used to yield an equivalent cell density on the coverslip. In general, better attachment and heavier growth was always obtained on Falcon plastic surfaces than on glass. (b) Another function of fetal bovine serum was to increase the protoporphyrin production to a level equivalent to that obtained when certain amino acids and vitamins were added to enrich the Eagle's medium and make it equivalent to the Ham. No individual amino acid, but rather a combination of them increased porphyrin production. Of the vitamins, B,, alone increased porphyrin production slightly, but the combination of vitamins was somewhat better. (c) In addition, the fetal bovine serum supplied a component with insulin-like activity which could be replaced by low concentrations of insulin. Insulin Effect to Increase Porphyrin Production-A number of investigators have reported that, depending on the cell type and clone used, insulin could replace the requirement of serum entirely or in part to support cell function or cell multiplication (19-21). However, multiplication of HeLa or L cells in suspension did not require serum or insulin.
In the case of chick embryo liver cells, insulin added to Ham F-12 medium but not to.Eagle's medium, completely replaced the requirement of serum for maximum porphyrin production. Commercial insulin (Sigma) was equally as effective as purified bovine insulin free of glucagon (courtesy of Dr. Lyman Craig, Rockefeller University).
Addition of insulin (0.2 rg/ml) to the Ham medium in the presence of allylisopropylacetamide increased the induction of &aminolevulinic acid synthetase as shown by the increase in protoporphyrin yield 2-to 5.5-fold (Fig. 4). Plasma protein synthesis was increased by the insulin almost 2-fold; over-all plasma protein synthesis comprises about 50% of the total liver protein synthesis. 8 The effect of insulin to increase the protoporphyrin yield (Fig. 4) was maximal at 8 to 15 nM, 50% of the maximum was at 2 to 3 nM, and even 0.3 to 1 nM caused a doubling over the control value. The physiological range of insulin in the hepatic portal vein of the rat is about 1 nM (22). Although 6aminolevulinic acid synthetase was increased by induction in the presence of insulin there was no effect of insulin on the conversion of S-aminolevulinic acid to porphyrin. Similar dose response curves were obtained in the presence of allylisopropylacetamide and the iron chelator desferrioxamine (760 PM). The dose response curves for insulin to increase plasma protein synthesisa were similar to those for increasing protoporphyrin accumulation.
Insulin appeared to be more effective when glucose was absent from the medium, i.e. the half-maximal increase of protoporphyrin required 0.3 to 2 nM insulin. However, lower concentrations of insulin (0.01 nM) have been reported to be sufficient for half-maximal stimulation of glucose oxidation to COI, or for the conversion of glucose to lipid in the liver cells or fat cells of the rat (23).
Other Hormones-These were also tested for their effect on the accumulation of porphyrins in the presence of allylisopropylacetamide (20 PM) plus desferrioxamine (760 PM) and insulin (30 nM porphyrin yield about 40%, and ovine prolactin (2 &ml) enhanced it 10 to 20%.
No effect on protoporphyrin accumulation was observed with reduced insulin (0.2 to 2 &ml), reduced at pH 7 with 0.1 M mercaptoethanol for 2 days under N,. Nor was there an effect of N*,OO'-dibutyryl adenosine 3':5'-monophosphate (10 to 30 @g/ml), cortisol (2 to 10 @g/ml), or thyroxin (1 to 5 &g/ml) in the presence or absence of insulin. In the presence of insulin the following hormones were slightly inhibitory at 10 &ml but not at 1 pg/ml: porcine glucagon, ovine follicle-stimulatirig hormone, ovine thyrotropin, and the prostaglandins. Prostaglandin F,a was least and prostaglandin F,cr was most inhibitory.
None of these hormones damaged the cells, for they did not cause a loss of porphyrins from the cells as judged by measurements of the ratio of porphyrin in the cells to porphyrin in the medium.
The 60% inhibitory effect of hemin (0.1 Kg/ml) on the induction of porphyrin by allylisopropylacetamide was not overcome by insulin (0.2 &ml) whether the hemin or insulin was added first and a 2-hour period intervened.
This result suggests that hemin and insulin act independently and that hemin does not affect the insulin receptor site on the surface of the cell membrane.

Effect of Serum Albumins on Pmtoporphyrin Accumulation
To study the effects of other constituents of fetal bovine serum as they affect protoporphyrin yield, we chose to test the effect of the major protein constituent of fetal bovine serum, serum albumin.
Effect of Different Serum Albumins, Added to Ham-insulin Medium, on Protoporphyrin Yield-These are listed in Table  II. A maximum change of ;t30% in protoporphyrin yield was obtained, depending on the particular preparation of serum albumin and its species of origin. With defatted, purified monomer (half-cystinyl) human serum albumin (100 &ml) (24), an increase of 30% in protoporphyrin yield was observed, a commercial untreated preparation (Mann, 100 ag/ml) caused an inhibition of 30% in protoporphyrin yield, and a crystallized preparation, Pentex (Miles), even at 1000 pg/ml did not affect the yield. Bovine serum albumin (Armour, 1000 H/ml), also did not affect the yield. Fetal bovine serum (30 pi/ml), i.e. 3% serum, decreased the protoporphyrin yield by 30%, but fetal bovine serum heated at 50" for 30 min was not inhibitory. One explanation for these results may be that variations in protoporphyrin are due to differences in impurities adhering to or accompanying the different preparations of serum albumin. Release of Protoporphyrin from Cells by Serum Albumin-A second effect of serum albumin was to cause protoporphyrin generated in the cells to be released into the medium (Table  II). (In the presence of desferrioxamine, the porphyrin formed is all protoporphyrin.) The effect was most marked with purified human serum albumin where even 10 wg/ml caused one-half of the protoporphyrin to leave the cells. In the absence of serum albumin, the ratio of protoporphyrin in the control cells to that in the medium was 3:l. Hemin (0.1 fig/ml) added to the medium in the absence of serum albumin did not affect this ratio (Table  II, Experiment 2, last line), although this hemin concentration was sufficient to decrease the protoporphyrin formed to 40% of the control. The loss of protoporphyrin from the cells to the medium in the presence of human serum albumin was not due to cell damage because the total protoporphyrin formed was greater by 30% than the control, i.e. in absence of human serum albumin. Also supporting the idea that cell damage is not involved is the experiment (Table   II, Experiment 2) with human serum albumin (Mann, 100 pg/ml); the human serum albumin caused inhibition of protoporphyrin synthesis yet produced a protoporphyrin ratio (cell to medium) similar to that of the defatted human serum albumin.
This result suggests that the property of human serum albumin involved in transport of protoporphyrin out of the cells is not related to the inhibitory properties of certain human serum albumin preparations (e.g. Mann human serum albumin).
Fetal bovine serum in a concentration of 3% (v/v) of the medium effectively caused the release of protoporphyrin from the cells. However, on the basis of serum albumin content, fetal bovine serum was l/20 as effective and bovine serum albumin alone was l/30 as effective as human serum albumin.
The protoporphyrin distribution between cells and media in the presence of human serum albumin was independent of the kind of inducer, whether allylisopropylacetamide or secobarbital, and of their concentrations (5 to 50 &ml). It was independent of the source of the d-aminolevulinic acid, whether generated in the cell by &aminolevulinic acid synthetase or from exogenous Saminolevulinic acid. Neither the presence nor absence of the chelators, desferrioxamine or (Ca + Mg)-EDTA affected the protoporphyrin distribution caused by human serum albumin.
Human serum albumin caused the release of protoporphyrin rather than uroporphyrin from the cells. In the presence of Aroclor-1254 the cells accumulated mainly uroporphyrin (13). In the presence of human serum albumin, uroporphyrin remained in the cells as indicated by the high ratio of 3.2 of porphyrin in cells to medium (Table II, Experiment  3). The decrease in ratio from 4.4 to 3.2 caused by human serum albumin was probably due to the release of the small amount of protoporphyrin made by the cells.

Control by Heme of Heme Synthesis
Repression by Hemin of Synthesis of &Aminolevulinic Acid Synthetase-It had previously been found (3) that the addition of 2 pM hemin to the medium was required to inhibit the synthesis of induced porphyrins by 50% in the primary chick embryo liver cells cultured in the presence of 10% fetal bovine serum. The significance of this value was difficult to assess because of the following considerations: serum was present which had some affinity for hemin; the serum was contaminated with variable amounts of hemoglobin heme; in the cell inoculum were red cells which tended to hemolyze; and in addition heme was produced within the hepatocytes.
We succeeded in controlling these complications by growing the cells in a defined medium devoid of serum, by perfusing the chick embryo livers to remove red cells before preparing the hepatic cell suspension, and by adding high concentrations of chelators of iron (14) to diminish the endogenous level of heme production.
With these modifications it now became possible to repress the synthesis of d-aminolevulinic acid synthetase with l/10 to l/20 of the previous heme concentration.
As seen in Fig. 5 (arrows) the activity was repressed to one-half when the medium contained 0.1 to 0.2 pM hemin. The activity was followed by determining the amount of protoporphyrin accumulated in the intact cells in culture. After leaving the cells for 2 hours in Ham-insulin containing desferrioxamine (500 kg/ml), the medium was changed to 1 ml of the same composition, and to this was added allylisopropylacetamide (21 or 210 pM) plus increasing concentrations of hemin. The hemin was dissolved in a 1:l (v/v) mixture of 10 mM KOH/ethanol, and 2 pl was added to 1 ml of the medium.
Arrows indicate the hemin concentration that inhibited protoporphyrin yield by 50% of the maximal.
The basal protoporphyrin formed is 0.1 to 0.2 nmol. below 1 PM in the defined medium, the experiments (see "Repression by Hemin of Synthesis of &Aminolevulinic Acid Synthetase") were repeated with the use of, as source of soluble heme, supematants of adult human and chick embryo red cell lysates and adult human hemoglobin crystallized by the modified Drabkin procedure as described in Ref. 25. It was found that these preparations, in terms of heme concentration, were as inhibitory as hemin itself. The hemoglobins became denatured in the medium and released hemin. After the 20-hour incubation, denatured globin was observed as a deposit on the coverslip, even in the absence of hepatocytes. It is of interest that administration to rats of hemin, ferric hemoglobin, ferric heme albumin, but apparently not hemoglobin, was found by Marver et al. (26) to block the allylisopropylacetamide-stimulated increase of both &aminolevulinic acid synthetase activity and of microsomal cytochromes. In our experiments in which no serum or globin or hemopexin (27) was present in the culture medium, hemin appeared to get into the cells as judged by the hemin causing a decreased induction of b-aminolevulinic acid synthetase. Repression of Induction of &Aminolevulinic Acid Synthetase by Endogenous Heme Synthesized from Added b-Aminolevulinic Acid-As seen in Table III   Acid Synthetase Activity-In order to show more directly that hemin inhibition of protoporphyrin accumulation was due to the repression of the synthesis of 6aminolevulinic acid synthetase, the enzyme activity was measured dir&y in homogenates of cells grown with and without hernia. Levulinate (10 mM) was added to the assay mixture to block the activity of C-aminoievulinic acid dehydratase so that b-aminolevulinic acid accumulated and was determined colorimetrically.
In this experiment cells were cultured on plastic dishes in a final culture medium of Ham-insulin containing allylisopropyiacetamide (35 FM)  growth hormone or prolactin in the medium containing insulin (0.2 pglml). It is of interest that these three hormones which increased porphyrin production appear to be related, The We found that the requirement for fetal bovine serum could be replaced by additional amino acids, vitamins, and insulin. The effect of insulin appears to be anabolic.
It promoted several cell functions: the increase in 6-aminolevulinic acid synthetase in response to inducers, the increased synthesis and secretion of plasma proteins,' and the increase in amino acid incorporation into general proteins; concomitantly the monolayer colonies appeared healthier, with more cell extensions. In the presence of inducers, insulin, in the physiological range of i nq increased porphyrin production up to 5.5-fold (e.g. Fig. 4). Porphyrin production was enhanced somewhat more by added decreased the enzyme activity to 1.9 or about one-half the control activity. The corresponding 50% inhibition of protoporphyrin synthesis (Fig. 5) required 0.1 to 0.2 ~.LM of hemin. These and similar experiments together with those of Sassa and Granick (4) suggest that inhibition by hemin of protoporphyrin accumulation reflects the inhibition by hemin of the synthesis of b-aminolevulinic acid synthetase. Furthermore, the decrease in activity after hemin treatment correlates with a decrease in amount of immunoprecipitable &aminolevulinic acid synthetase. 1 Production in Presence of Allylisopropylacetamide-As seen in Fig. 5, protoporphyrin production was inhibited by 50% when hemin at 90 to 210 nM (arrows) was present in the culture Effect of Exogenous Hemin in Repressing Protoporphyrin NH,-terminal region of growth hormone exhibits insulin-like activity (30), and prolactin is immunologically similar to growth hormone. None of the other polypeptide hormones tested, nor thyroxin, cyclic adenosine 3':5'-monophosphate, and prostaglandins enhanced porphyrin production, to mouse fibroblasts, microvilli had increased on the cell surface, while simultaneously the uptake of uridine, leucine, and glucose was increased. The insulin effect may become most The general anabolic effect of insulin may in part be explained by insulin causing an increase in the surface area of the plasma membrane as a result of an increase in microvilli formation.
Insulin receptors have been shown by Cuatrecasas to be present on membranes of liver and fat cells (23). Evans et al. (31) have found that within 1 hour after insulin was added medium.
An inhibitory effect of hemin was detectable at concentrations as low as 50 nM. (None of the concentrations of hemin used in these experiments quenched the porphyrin fluorescence in the perchloric acid/methanol solvent.) In the presence of higher concentrations of allylisopropylacetamide (e.g. 212 PM), more exogenous heme (210 nM) was required to inhibit protoporphyrin accumulation by 50% than at the lower concentrations of allylisopropyIacetamide (21 FM). These results may be explained by assuming that allylisopropylacetamide at higher concentrations was causing a more effective destruction of heme in the cells (6, 7, 29). Therefore, to compensate for this loss of heme, higher concentrations of apparent when the surface area of the cells becomes limiting for metabolism, as when the cells become confluent. Griffiths (32) observed that insulin stimulated protein synthesis and glucose uptake in WI-38 human cells when the surface areas of the cells exposed to the medium were minimal, a condition similar to that in our induction experiments.

Effect of Serum Albumin in Causing Release of Protoporphyrin from Hepatocyte
This effect was most marked with human serum albumin and appeared to depend on the presence of one high affinity site (Kd = 10 nM) on the human serum albumin that is identical for heme and protoporphyrin (33,34). Heme was suggested to bind through the two propionic acid side chains. In the presence of as little as 10 pg of human serum albumin added to 1 ml of the defined medium we found that one-half of the protoporphyrin migrated out of the hepatocytes or approximately 1 mol of protoporphyrin per mol of human serum albumin. Bovine serum albumin, with a much weaker binding site, was only l/20 to l/30 as effective in causing protoporphyrin to be released from the cells.
Fluorescence microscope examination of the hepatocytes indicated that protoporphyrin generated in the cells was localized in the cytosol, and uroporphyrin (Table II) mainly in the nucleus. Uroporphyrin was not released by the action of exogenous serum albumin into the medium.
Studies by others have indicated that although serum albumin is produced and secreted by hepatocytes, this protein does not appear to re-enter the cells (35,36). The release of lipophilic protoporphyrin, but not hydrophilic uroporphyrin, from the cells most probably occurs in a manner similar to one that has been postulated for serum albumin-fatty acid transport exchange across the plasma membrane.
The release of protoporphyrin from the cells by human serum albumin provides a simple and highly sensitive assay for use in the study of the mechanism of action of serum proteins for transfer of lipophilic anionic molecules across the plasma membrane of the hepatocyte.

Control of Liver Heme Metabolism by Heme
We had shown before that &aminolevulinic acid synthetase was the rate-limiting enzyme in the biosynthesis of heme. The enzyme had a relatively short half-life of 3 hours in the chick embryo liver cells in culture (5) and 1 hour in adult rat liver (37). A major control on the enzyme was by heme acting as a repressor at the post-transcriptional level, presumably on the synthesis of this enzyme (4). The repression of the enzyme by exogenous hemin was as rapid as the inhibition of the synthesis by acetoxycycloheximide, and the rate of decay of activity of the enzyme in the presence of acetoxycycloheximide was not changed by the addition of hemin (4). The rapid repression by hemin indicated that the hepatic cell was readily permeable to hemin.
By culturing the chick embryo liver cells in a completely defined Ham-insulin medium and by blocking the formation of endogenous heme with a chelator of iron, it became possible to determine several parameters of liver heme metabolism and formulate a hypothesis on the steady state control of heme in the liver cell.
Repression by Hemin and K,-The present experiments showed by two independent methods that hemin is a highly effective repressor. At a concentration of exogenous hemin of 0.1 pM, the synthesis of d-aminolevulinic acid synthetase was repressed' by one-half, a value which we define as K, ( FIG. 6. The affinities of cell constituents and hemonroteins for heme, and the control of' heme metabolism by heme. The heavy arrows indicate main paths for heme. As yet there has been no demonstration of acceleration by heme of translation of proteins (E) in liver.