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1983 Vitamin B₆ activation of erythrocyte aspartate apotransaminase Brent A. Neuschwander-Tetri Yale University

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Recommended Citation Neuschwander-Tetri, Brent A., "Vitamin B₆ activation of erythrocyte aspartate apotransaminase" (1983). Yale Medicine Thesis Digital Library. 2984. http://elischolar.library.yale.edu/ymtdl/2984

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https://archive.org/details/vitaminbactivatiOOneus Vitamin B6 activation of erythrocyte

aspartate apotransaminase.

A Thesis Submitted to the

Yale University School of Medicine in Partial Fulfillment of the Requirements for

the Degree of Doctor of Medicine

1983.

by

Brent A. Neuschwander-Tetri

Thesis advisor: Lawrence R. Solomon M.D. jjjeci Ulo ■ T7/3 fY/9- $/£

Vitamin B6 metabolism and transport were investigated using activation of erythrocyte aspartate apotransaminase (EGOT) as a measure of intracellular availability of active cofactor forms of B6.

Questions asked here address the possibility that a form of B6 other than pyridoxal or pyridoxal-51-phosphate (PLP) is important in plasma and that entry of PLP into tissues may not involve mechanisms previously proposed. In plasma, two forms of vitamin B6, pyridoxamine-51-phosphate and pyridoxamine, were found to increase

EGOT activity to a greater extent than other vitamers. Consistent with previous findings, it v/as also found that the presence of plasma

inhibits EGOT activation by PLP. Further investigations of EGOT

activation by PLP v/ith two anion channel blocking agents suggest that

anion channels may play a role in PLP transport into the erythrocyte

only at high PLP concentrations. This is in contrast to previous

reports suggesting PLP entry into erythrocytes is entirely dependent

on anion channels.

11

ACKNOWLEDGEMENT

I would like to thank Dr. Lawrence Solomon for his guidance, patience, and support. I would also like to thank Connie Rutledge for advice and assistance in the laboratory. And I am ever grateful to Paivi for her patience now and in the future.

iii

I . INTRODUCTION

Over the past fifteen years, much attention has been focused on

Vitamin B6 metabolism and transport in vivo and in vitro in an attempt to discern on a cellular level how the entire organism utilizes this essential cofactor. Unresolved in the current literature are questions regarding the relative importance of the various forms of B6, the overall effect of the non-cofactor interaction of the aldehyde forms of B6 with plasma and intracellular proteins, and the mode of B6 transport into tissues.

The term "Vitamin Bs" is designated to include three different molecular forms and their phosphorylated derivatives known together as the B6 vitamers (IUPAC-IUB Commission on Biochemical Nomenclature

1973). These are: pyridoxine (PN), pyridoxine 5'-phosphate (PNP), pyridoxamine (PM), qyridoxamine 5'-phosphate (PMP), pyridoxal (PL),

and pyridoxal 5'-phosphate (PLP) (fig 1). Normal human plasma

concentrations of the six B6 vitamers as measured by chromatography

and fluorometric analysis (Lumeng et al 1930) are shown in Table 1.

It is generally recognized however that of the six vitamers, only PLP

and in some cases PMP are active cofactors at the enzymatic level

(Meister et al 1954). The majority of enzymes requiring B6 as a

cofactor are essential to amino acid metabolism. These include

transaminases, synthetases, hydroxylases, decarboxylases and

dehydratases. In addition, glycogen phosphorylase in muscle and

1

'3' o PYRIDOXIME PYRIDOXINE-51-PHOSPHATE (PN) (PNP)

(PM) (PMP)

H /O H /O

(PL) (PLP)

Figure 1. The B6 vitamers

£ H\ *N-CH2-PR0T. 0 h2n-ch2-prot. +

Figure 2. Schiff base formation.

Table 1. . B6 vitamer concentrations.

Total: 124 +/- 29nM (100%)

PLP : 77 nil (62%)

PN : 19 nH (15%)

PL : 17 nM (14%)

PH : 6 nM (5%)

PMP : 5 nM (4%)

delta-amino levulenic acid synthetase in the marrow require B6 for activation.

A deficiency of vitamin B6 is known to produce dermatitis, anemia, neuropathy, convulsions, renal and hepatic function changes,

decreased growth and decreased antibody formation (Sauberlich and

Canham 1980). Additionally, in the alcoholic patient, B6 deficiency

has been implicated in liver disease, peripheral neuropathy,

convulsions, and sideroblastic anemia (Hines and Cowan 1970, Bonjour

1980). In fact, even without exposure to alcohol, B6 deficiency has

been shown to produce fatty infiltration and cirrhosis of the liver

in monkeys (Greenberg 1964, Abe and Kishino 1982). There have also

been suggestions that B6 status is related to nephrolithiasis,

atherosclerosis, caries, altered hemostasis, and immunologic changes

(Greenberg 1964, Sauberlich and Canham 1980). Nonetheless, a precise

daily dietary requirement of vitamin B6 has not been established.

The vitamin B6 requirement has been estimated to be 1.5 mg of

3 / pyridoxine per 100 gm of dietary protein which translates into an RDA of 1.6-2.2 mg of PN per day depending on age and sex (Committee on

Dietary Allowances, 1980). No signs or symptoms of PN toxicity are presently recognized in adults. However, studies with beagles given

150mg/kg/day demonstrated neurological changes v/ithin two to six weeks (Hoover and Carlton 1981) and Unna1s (1940) early observations with rats showed 3gm/kg of PN produces convulsions and death within

24 hours.

Much of the interest in vitamin B6 pharmacology and metabolism originates from suggestions of its usefulness in therapy of certain vitamin B6-responsive disorders (i.e. dependency states). In addition, B6 therapy may be required for the treatment of simple deficiency states due to disorders of absorption and utilization.

Specifically, pyridoxine administration has been shown to be

helpful in certain gases of sideroblastic anemia, homocystinuria, and

drug induced defiencies (i.e. secondary to INH, hydralazine,

cycloserine, penicillamine, or oral contraceptive administration).

Additionally, it has been suggested that hepatic cell damage

secondary to alcohol abuse may be responsive to B6 administration

(Lumeng and Li 1974, Bonjour 1980) and that sickle cell disease

patients may benefit from pharmacological doses of B6 because of the

in vitro anti-sickling effect of pyridoxal and pyridoxal phosphate

(Zaugg et al 1977, Kark et al 1978). Thus, the importance of

elucidating the precise mechanisms of B6 metabolism and hov; they may

4 be advantageously manipulated is underscored.

Identification of the particular B6 vitamers which are most able

to produce a desired response is essential. Pyroxidine is the B6 vitamer presently used pharmacologically and orally administered PN

is well absorbed from the jejunem (Brain and Booth 1964). Although

Anderson (1980) argues that erythrocytes are a major site of PN

oxidation to PL, Lumeng, et al (1980) demonstrate that the liver is primarily responsible for PN metabolism resulting in a net rise in plasma concentrations of PL, PLP, and the inactive metabolite, pyridoxic acid. This group further reports that administration of

lOOmg of pyridoxine per day for three weeks to healthy adults results

in a steady state level within one week of a six to seven-fold rise

in plasma PLP concentration and a twelve-fold rise in PL

concentration. At the tissue level, orally administered PN produces

a sharp rise within hours in PL and PLP concentrations within the RBC

(Anderson et al 1971).

Important to the understanding of B6 vitamer activities

intracellularly and in plasma is the covalent binding of PL and PLP

to proteins. The reversible Schiff base formation by aldehydes with

primary amines at neutral pH's is a well described reaction first

recognized over a century ago (fig 2, Schiff 1864). Christensen

(1958) first proposed the binding of the aldehyde forms of B6, PL and

PLP, to the free amino groups of bovine serum albumin by Schiff base

formation. Subsequently, he and his coworkers were able to

5 demonstrate the binding of PLP to albumin in a molar ratio of two to one (Dempsey 1962). Borohydride reduction followed by protein hydrolysis revealed the binding by PLP to be with two epsilon-amino groups of lysine residues. These findings were later confirmed to be the case with human albumin (Lumeng et al 1974, and Anderson et al

1974). Lumeng et al (1974) also found that the globulins constitute a significant portion of plasma binding capacity in vitro at high PLP concentrations and that the total capacity of plasma to bind PLP is

in the range of 3 mM, which far exceeds the normal plasma concentration of this vitamer. Dempsey (1962) points out however

that at such supraphysiologic PLP concentrations, PLP binds nonspecifically to albumin in a ratio exceeding two to one. Thus, any calculation of the plasma PLP binding capacity at high PLP concentrations v/ill reflect binding potential not realized physiologically.

Although the formation of Schiff bases is well known to be

reversible, the reversibility of PLP binding to plasma proteins has been the source of some confusion. Christensen (1961) confirms the

reversibility of the PLP-albumin Schiff base formation in his initial

investigations. Lumeng, et al (1974), however, report tv/o

conflicting experimental findings regarding the reversibility of

Schiff base formation which they fail to resolve. They report the

results of a dialysis experiment which appear to show that PLP bound

to albumin is not dialyzable. A separate experiment in the same

paper convincingly demonstrates, however, that virtually all of the

6

PLP bound to albumin is hydrolyzable by alkaline phosphatase whether the alkaline phosphatase is free in solution or separated from the

PLP-albumin complex by a dialysis membrane. From the latter experiment they conclude that albumin bound PLP is indeed in equilibrium with free PLP in solution allowing membrane permeation by albumin bound PLP. However, without explanation, many of their subsequent conclusions (Lumeng et al 1980) are drawn by an inference from the first experiment that since PLP was found to be "non- dialyzable," it must therefore be irreversibly bound. For example, they state that Schiff base formation prevents PLP from being transported into erythrocytes. This may be true from a static viewpoint but may not be true in the actual dynamic equilibrium. The kinetics of PLP-albumin dissociation have not been adequately studied to establish this point. Furthermore, in a discussion of the fate of plasma PLP, they state that although PLP is found in the urine, it cannot arrive there by glomerular filtration since it's "not dialyzable." Clearly, the conceptual model derived from their dialysis experiment is a source of misdirection for themselves and perhaps others.

Pyridoxal phosphate is the B6 vitamer which activates B6 dependent enzymes and it is also the vitamer found in the highest concentration in plasma. However, it is unclear if the PLP in plasma enters cells to combine with apoenzymes or whether another plasma vitamer enters tissues and activates apoenzymes after conversion to an active form.

There remain two unexplained problems in the understanding of how

7 plasma PLP might enter cells. PLP is an organic phosphate and it is well recognized that such anions do not readily cross cell membranes without a specific transport mechanism. Such a mechanism has not been demonstrated for PLP. Also because the total plasma concentration of PLP is well below the plasma protein binding capacity for PLP, the amount of free PLP in solution may actually be

less than any other B6 vitamer. This may not affect PLP transport, but it questions the logic of emphasizing PLP as the principle B6 vitamer. Experiments directed at determining the relative enzyme

activation potentials for the B6 vitamers, the actual free PLP concentration in plasma, and the mechanism of PLP transport across cell membranes are thus essential to understanding the physiology of

b6-

Experiments studying B6 metabolism at a cellular level have been

carried out using cultured tumor cells (Pal and Christensen 1961),

% ) rabbit choroid plexus (Spector and Greenwald 1978), and erythrocytes.

Erythrocytes are the tissue upon which the majority of the literature

is based. Whether the findings with erythrocytes can be generalized

to the other cells of the body is not known; they remain nonetheless

the model tissue for these investigations.

The erythrocyte has several important properties affecting its

metabolism of vitamin B6. Three enzymes, a phosphatase, a kinase,

and an oxidase, have been identified in erythrocytes which appear to

play an essential role in B6 metabolism.

8

Yamada and Tsuji (1968) first demonstrated a membrane bound phosphatase able to cleave PLP to PL and inorganic phosphate.

Characterizing this enzyme, Lumeng and Li (1974) have shov/n that 80 mM phosphate is inhibitory to the phosphatase activity. They also suggested that acetaldehyde, the ethanol metabolite, may potentiate

the phosphatase. Their later work (Lumeng 1978) indicates that acetaldehyde may have this effect by displacing PLP from its protective protein binding. Mitchell et al (1976) also suggested from their in vivo studies in alcoholics that ethanol is responsible

for increased PLP degradation and by inference increased phosphatase activity. Many of the experiments in the literature which focus on

PLP transport into erythrocytes take advantage of the phosphate

inhibition of the phosphatase to examine the role of this enzyme in

Bs transport. The data derived from such experiments is analyzed with respect to alterations in the intracellular phosphorylated vitamer concentration. Unfortunately, it is also well documented

that phosphate stimulates red cell glycolysis. This results in

increased synthesis and decreased catabolism of ATP and other organic phosphates (Rose and Warms 1966, Beutler 1970, Warrendorf and

Rubinstein 1973). The role this plays in altering B6 metabolism has

not been investigated.

The two enzymes necessary for interconversion of the B6 vitamers

have been well studied. The enzyme pyridoxal kinase (EC 2.7.2.35)

uses as substrate either ATP or ADP and is capable of phosphorylating

PL, PM, and PN (McCormick et al 1961, Hamfelt 1967). It is

9 competitively inhibited by the substrate deoxypyridoxine (DOPM) which is itself slowly phosphorylated to DOPNP (McCormick and Snell 1961).

Furukawa (1981) has also shown that, at least with microbial kinase,

PL at high concentrations inhibits its activity by Schiff base formation at a non-active site. This inhibition, they note, is reversed by dialysis. Solomon and Hillman (1975) have investigated the in vivo regulation of pyridoxal kinase activity and find that orally administered PN induces increased kinase activity in young erythrocytes.

The oxidase (EC 1.4.3.5) is an flavin-dependent enzyme which oxidizes PMP and PNP to PLP (Wada and Snell 1961). The non- phosphorylated vitamers, PM and PN are minimally reactive substrates.

When Lumeng and Li (1974) measured the combined activities of the

kinase and oxidase by measuring intracellular PLP concentration after

incubation with PL, PN, or PNP, they found decreasing PLP content with increasing substrate concentrations. They concluded that the

kinase or oxidase or both are subject to substrate inhibition. The

investigations of the oxidase by Wada and Snell (1961) indicate the

absence of substrate inhibition or inhibition by non-phosphorylated

vitamers.

Whether there are mechanisms for vitamin B6 homeostasis is

unclear. The observed effects of substrates and products on the

phosphatase, kinase, and oxidase are suggestive that these enzymes

may play a role in maintaining homeostasis but this is presently

10 unresolved.

Another important property of erythrocytes is the ability of hemoglobin to bind considerable quanitities of PL and PLP by reversible Schiff base formation. This has much the same sequestering effect on PL and PLP intracellularly that albumin has extracellularly. Borohydride reduction and further analysis have demonstrated that PL forms a Schiff base preferentially with the terminal amino group of the alpha-chain of hemoglobin and PLP with the beta-chain (Benesch et al 1973). Oxygen dissociation curves of hemoglobin in the presence of PL and PLP indicate that the binding of

PL to hemoglobin lowers the p50 and PLP binding raises the p50

(Benesch et al 1973). This and the finding that PL or PLP binding inhibits sickle hemoglobin polymerization have raised the question of using B6 in sickle cell patients to prevent erythrocyte sickling

(Beutler et al 1972, Kark et al 1978). Although use of PN has not proven therapeutically fruitful, perhaps the use of other vitamers or modification of erythrocyte B6 metabolism would increase the effect

of hemoglobin binding in vivo.

Investigations in the current literature have examined some of

these issues. For most, the assumption is made that since the total

PLP content of plasma (including protein bound PLP) is the highest

among the vitamers and since it is the active coenzyme, then PLP must

by some means cross the cellular membrane. Because the erythrocyte

has a membrane bound phosphatase able to cleave extracellular PLP (in

11 addition to intracellular PLP) to PL and a kinase intracellularly v/hich is able to resynthesize PLP from PL and ATP, many investigations over the past two decades have focused on the question asking whether PLP is transported across the membrane intact or is cleaved and reformed intracellarly.

Suzue and Tachibana (1970) investigated this using a mixture of

14C-PLP and 32P-PLP. They found the intracellular ratio of 14C to

32P to be the same as the extracellular ratio and concluded from this that PLP must be transported intact across the cell membrane. They fail to elaborate on their reasoning but presumably if PLP were cleaved extracellularly and resynthesized intracellularly, the probability would be high that resynthesis would occur with non¬ radioactive phosphate. This v/ould cause an increase in the amount of

14C-PLP relative to 32P-PLP intracellularly which they did not observe.

Likewise, Spector and Greenwald (1978) attempted to show that

accumulation of PLP in rabbit cerebrospinal fluid is dependent on a

dephosphorylation-rephosphorylation mechanism. They found in vivo

that intravenous injection of the doubly-labeled [3H,32P]-PNP

resulted in accumulation of 3H-PLP in the CSF without accumulation of

32P-phosphate. They also report that the accumulation of PLP in CSF

is prevented by DOPN, the pyridoxal kinase inhibitor. This was

attributed to the failure to resynthesize PLP once PL had entered the

CSF. These experiments fails to discriminate, however, betv/een the

12 proposed mechanism of dephosphorylation prior to trans-membrane passage followed by rephosphorylation and the alternative that the phosphorylated vitamer may enter the CSF intact only to be subject to the activities of both a phosphatase and a kinase resulting in a net replacement of radioactive phosphate with non-radioactive phosphate.

Using erythrocytes, Lumeng et al (1974) attempted to demonstrate that cleavage of PLP to PL by the membrane bound phosphatase is necessary for its transport across the cell-membrane. In normal saline they found 22% to the PLP added to the medium was intracellular after incubation whereas 38% remained extracellular.

The remaining 40% of unrecovered PLP was presumed hydrolyzed by phosphatase. When the experiment was conducted with 80 mM phosphate to inhibit the phosphatase, 18% of the added PLP was still found

intracellularly after incubation. Extracellularly, 77% of the added

PLP was recovered leaving only 5% unrecovered thus indicating

significant phosphatase inhibition. From this data they concluded

that PLP cannot cross the erythrocyte membrane intact but that it must first be cleaved by phosphatase to PL. However, they fail to

account for the 18% of PLP that enters the cells with the phosphatase

"inhibited" versus the 22% with the phosphatase active. Indeed, this

can be accounted for in two ways: either 80 mM phosphate does not

fully inhibit phosphatase or another mechanism for transport of PLP

into erythrocytes obtains. In either case, their conclusion that

phosphatase activity is essential cannot be drawn with certainty from

their data. In fact, Maeda et al (1976) concluded from their similar

13

experiments that intact PLP enters erythrocytes by passive diffusion.

Using 160 mM phosphate buffer as an incubation medium, they

demonstrated increasing intracellular PLP concentrations over time with minimal loss of total PLP in the system. Furthermore, they

strengthen their argument for passive diffusion of PLP into

erythrocytes by demonstrating that the initial velocity of PLP uptake

is linear with respect to PLP concentration.

The studies by Suzue and Tachibana (1968) and Maeda (1976)

suggested PLP enters erythrocytes intact whereas the subsequent

studies by Spector and Greenwald (1978) and Lumeng et al (1974) were

not convincing that phosphatase activity is essential for transport.

Thus a need for further investigation is clearly indicated. One

mechanism by which PLP could enter tissues without prior cleavage is

by direct passage through membrane anion channels. This has been

investigated using anion channel blocking agents.

Cabantchik and Rothstein (1974) developed the use of

4,4!-diisothiocyano- 2,2'-stilbene disulfonic acid (DIDS) which they

found to be a non-penetrating, highly specific and reactive blocker

of erythrocyte anion channels. They later found that DIDS prevented

membrane permeation by PLP (Cabantchik et al 1975). Further

experiments characterizing the anion channel showed it is a single

protein isolable in membrane vesicles (Wolosin et al 1977). Because

DIDS binds to free amines, Cabantchik et al (1975) proposed that the

anion channel protein has a free amino group as part of its active

14

site. And because PLP also binds to free amino groups, this group conducted experiments to determine the effect of PLP on the anion channel. They found that like DIDS, PLP could also inhibit the flux

of anions across the membrane. Furthermore, they found that elution

of the PLP from its binding sites reversed the effect on anion flux

unless PLP was bound irreversibly to amines by borohydride reduction

(Rothstein et al 1976). Interestingly, in their studies of sickle

erythrocytes, Kark and Hicks (1930) found an increase in anion permeability of the membrane anion channels when compared to normal

cells. This finding raises questions regarding how flux through the

channels is regulated in the normal and abnormal erythrocyte and hov/

such regulatory mechanisms might affect passage of PLP through the membrane.

One of the major impediments to progress in the field of B6

physiology has been the development of an accurate means of measuring

vitamer concentrations in the intracellular and extracellular

environments. A number of different assays have been used including

chemical, fluorometric (Chauhan and Dakshinamurti 1979),

microbiological, and enzymatic. The direct chemical and fluorometric

measurements are inherently difficult because of the low vitamer

concentrations studied, the variable elution of PL and PLP from their

protein binding sites, and the instability of certain vitamers. The

microbiological techniques often lack specificity among vitamers or

are insensitive to vitamers requiring assay. As a means of assessing

the ability of a given vitamer to effect apoenzyme activation, the

15 enzymatic assays are quite useful. Erythrocytes contain readily measurable quantities of the PLP-dependent aspartate-oxaloacetate transaminase (EGOT, EC 2.6.1.1, Meister et al 1954, Walsh 1966).

This enzyme is found to be only 50-70% saturated by cofactor in normal adults (Solomon and Hillman 1978) making it useful for in vivo and in vitro supplementation studies. It has been found that PMP is also able to activate EGOT but that PMP and DOPNP inhibit EGOT activity (Meister et al 1954). Furthermore, Solomon and Hillman

(1975) demonstrated that orally administered PM can induce an increase in the total EGOT activity. Interestingly, they also observed a transient drop in EGOT activity within hours after oral PN or PLP administration. This was felt to be suggestive of reversible inactivation of the enzyme by non-specific Schiff base formation followed by redistribution to the active sites within a few days.

They have also shown that in spite of a rapid rise in intracellular

PL and PLP concentrations after an oral PN dose (Anderson et al

1971), the rise in EGOT activity occurs 27-48 hours later (Solomon and Hillman 1979). This last finding in itself raises questions regarding the kinetics of the vitamer reactions and interactions which will be addressed later.

Using the assay of EGOT as an index of PLP or PMP entry into tissues or entry of other vitamers followed by conversion to an active form, the experiments described here demonstrate that in plasma there is marked variation of EGOT activation by the various vitamers and that blocking the erythrocyte anion channels does not

16 V completely prevent entry of PLP into the cell.

17

II. EXPERIMENTAL

All reagents were obtained from Sigma Chemical Co. with the exception of 3-(N-morpholino)propanesulfonic acid (MOPS) which was obtained from Calbiochem Inc., 4-acetomido, 4-isothiocyanostilbene

2,21-disulfonic acid (SITS) and

4,4'-diisothiocyanostilbene-2,21-disulfonic acid (DIDS) which were obtained from ICN Pharmaceuticals.

Solutions of the B6 vitamers were made up to a concentration of approximately 45 mM, pH 7.40, and the actual concentrations were measured spectrophotometrically in the ultraviolet region (Storvick et al 1964). Aliguots of the solutions were stored frozen and thawed just prior to use. Solutions of 20 mM SITS and 0.5 mM DIDS in

isotonic saline were made up just prior to use and kept protected

from light at 0°C; ,

Freshly drawn heparinized blood from one of two healthy males was

separated by centrifugation at lOOOg for 10 min at 7°C. The buffy

coat was discarded and the RBC were washed with cold isotonic saline.

The RBC were either used directly or preincubated at 0°C. in isotonic

saline at a concentration of 30% (v/v) v/hich contained either 0.14 mM

SITS or 0.035 mM DIDS.

Incubation solutions of isotonic saline, 90% (v/v) heparinized

plasma, or bovine serum albumin (BSA; 6 gm/dl NS) with 10 mM glucose

18 were buffered with MOPS, pH 7.40, to a final MOPS concentration of

0.6 mM. Volumes of the vitamer solutions were added to give the final concentrations indicated in individual experiments. The values used for vitamer concentrations are calculated values based on the amount added to a given volumes of incubation solution. Thus they may not represent the actual free vitamer concentrations in the medium because of protein binding. In experiments employing SITS, a final concentration of 0.2 mM SITS in the incubation medium was used.

Either normal RBC, or RBC preincubated with BIDS or SITS were resuspended in the incubation solutions to a concentration of 10%

(v/v). The solutions were incubated at 37°C. in a shaking water bath and at timed intervals aliquots were removed, diluted with 7 volumes of ice cold isotonic saline, and centrifuged at 7°C. for 10 min at lOOOg. The pellet was washed with 90 volumes ice cold saline and recentrifuged under identical conditions.

EGOT activity was assayed as follows according to the method described by Karmen et al (1955) and modified by Solomon (1978). The

RBC were lysed by adding 30 ul of the washed RBC pellet to 3 ml of ice cold distilled water. After 10 min, 200 ul of the lysate was added to 2.6 ml of distilled water containing 33 mM d,1-aspartate,

0.067 mg/ml DPNH, and 67 U/ml malate dehydrogenase. This mixture was preincubated 10 min in a shaking water bath at 37°C. Total EGOT activity was measured by including PLP at a concentration of 0.5 mM in the preincubaton solution and preincubating for 30 min.

Thereafter, 200 ul of 100 mM alpha-ketoglutarate (in sodium phosphate

19 buffer, pH 7.5) was added and the consumption of DPNH was followed by- measurement of the decrease in optical density at a wavelength of 340

nm over 20-30 min at 37°C using a Gilford spectrophotometer with an

automatic chart recorder. Specific activity of EGOT is expressed as

Units/g Hgb where one Unit is defined as the loss of optical density

at 340 nm per minute divided by the extinction coeffecient for DPNH

at 340 nm of 6.22. Lysate hemoglobin concentration was determined by

the hemoglobincyanide method (van Kampen and Zijlstra 1961).

When values were determined in triplicate, the mean is displayed

graphically with the range of plus or minus one standard deviation.

The significance of differences between means was analyzed by the method of Student's t test.

20

Ill. RESULTS

To determine the relative abilities of the B6 vitamers to enter

tissues from plasma and activate B6 dependent enzymes, erythrocytes were resuspended in 90% plasma containing PLP, PMP, PM, and PN and

incubated for 6 hours. Aliquots removed at two hour intervals and

assayed for EGOT activation showed marked variation in the relative

abilities of the vitamers to effect GOT activation (fig 3). At 0.67 mM, PLP did not significantly increase GOT activation above control

levels. In contrast, the other vitamers were able to increase GOT

activation at concentrations of 0.67 mM with PMP being the most

active followed by PM. However, at a concentration of 3.3 mM, PLP

almost completely activated EGOT.

Since the total binding capacity of 90% plasma is probably in the

range of 2.7 mM (Lumeng et al 1974), a PLP concentration of 3.3 mM

would certainly provide a large amount of free PLP in solution.

Thus, it's not surprising that at such a high concentration, this

vitamer should be so active. Likewise, at 0.67 mM, the majority of

PLP will be bound to albumin (and perhaps globulin) leaving a

fraction free in solution that is quite small and unable to activate

EGOT to the extent that the other vitamers do at 0.67 mM.

These findings are consistent with those of Lumeng et al (1974)

and Anderson (1971) who found that the presence of plasma prevents

the entry of PLP into erythrocytes by virtue of binding to plasma

21

GOT (U/gHgb) plasma (v/v). 4.5 5.5 3.5 ~i Figure 3.ErythrocyteGOTactivationby B6vitamersin90% 5H ~i 1 0 / _- O A—

"principle" B6 vitamer because of its high plasma concentration.

Rather, this experiment suggests that physiologic levels of other B6 vitamers may enter tissues to a greater extent and effect a.poenzyme activation after conversion to active forms. Although PMP v/ould appear to be a strong possibility under these experimental conditions, it is found to constitute only 4% of the plasma B6 vitamers (Table 1). Thus an experiment with PMP, PM and PN at equimolar concentrations does not accurately reflect the physiologic ratios of these vitamer concentrations.

To confirm the theory that protein binding was responsible for the lack of GOT activation with 0.67 mM PLP in plasma, the previous experiment was performed in MOPS buffered isotonic saline. The > ) results (figs 4,5) clearly demonstrate that in the absence of plasma,

0.67 mM PLP effects GOT activation almost to the same extent as 3.3 mM PLP. Not surprisingly, PMP, PM and PN are as effective in activating GOT in saline as in plasma (figs 4,5). This confirms that plasma has no effect on these vitamers presumably because of their inability to form Schiff base adducts with plasma proteins.

The effect of inorganic phosphate on EGOT activation by PLP in plasma was investigated using a low phosphate concentration (5 mM)

and a moderately high concentration (20 mM). A higher concentration

23

buffered isotonicsaline. GOT (U/gHgb) Figure 4.ErythrocyteGOTactivationby E>6vitamersinMOPS Hours 24 3.3 mMPLP No B6 0.67 mMPM 0.67 mMPLP 0.67 mMP.MP 0.67 mMPN

PMP ineither90%plasma (v/v)orMOPSbufferedisotonicsaline after 4hoursincubation. Figure 5.Erythrocyte GOTactivationby1.0mMPLPor mM GOT (U/gHgb) Suspension Medium 25

of phosphate was not chosen because of the significant diminution of plasma volume this would require. Previous data (Solomon,

unpublished data) indicates that at 20 mM, phosphate inhibits

phosphatase activity by about 42%. At both concentrations however,

phosphate was found to have no effect on the activation of GOT by

either 3.8 mM PLP or 7.7 mM PLP (fig 6). This experiment would

suggest that under these conditions, phosphatase activity is either

not essential for transport of PLP into the erythrocyte at these

concentrations or that the residual phosphatase activity is

sufficient to mediate PLP uptake by dephosphorylation and duffusion

of PL.

To investigate an alternative mode of transport, the use of anion

channel blocking agents was studied. Preincubating erythrocytes in

0.035 mM DIDS resulted in significant alterations in apparent PLP

uptake from plasma. In the presence of 1.8 mM PLP there was no

significant difference distinguishable between the DIDS-treated cells

and normal cells. However, at the higher PLP concentration of 3.5

mM, further EGOT activation was noted in control cells but not in

DIDS-treated cells (fig 7). This experiment was repeated with DIDS

and v/ith another anion channel blocker, SITS, giving the same

results.

When the effects of DIDS and SITS on uptake of 11 mM PL and EGOT

activation were investigated, no differences between normal cells and

anion channel blocked cells v/ere found (fig 8). Interestingly, the

26

activation by3.85mMand7.7PLPin 76% plasmaafter4hours concentration arenot significant(p>0.2). incubation. Thedifferences betweenthemeansateachPLP GOT (U/gHgb) Figure 6.Effectof5mMand20phosphate onEGOT 8 “I 6-i 27 CZ3 5mMPi 123 NoPi UD 20mMPi Legend

plasma (v/v)after4 hoursincubationandPLactivationof EGOT.

Figure 7.EffectofDIDSonEGOTactivation byPLPin87% GOT (U/gHgb) 5.5 28 —-A —o PL PLP +DIDS PLP

87 5 plasma(v/v)after 4 hoursincubation.

Figure 8.EffectofDIDSonEGOTactivation byPLPandPLin GOT (U/gHgb) 8 7-\ 4 5~* // 0 // # PLP i 4 8 PLP (mM) PLP+DiDS 29 12 PL PL+DIDS activation of EGOT by 3.5 mM PL is significantly less than the activation by 1.8 mM PLP (fig 7). This is contrary to what would be expected if cleavage of PLP to PL were the mechanism responsible for

the non-anion channel dependent PLP transport.

The results of the previous experiments using DIDS and SITS

indicate anion channels may not be responsible for PLP transport at

PLP concentrations less than 1.3 mM. To further characterize the non-anion channel dependent PLP transport, an experiment was carried out comparing normal cells to DIDS treated cells using a suspension medium of bovine serum albumin (6 gm/dl) and glucose (10 mM) in

isotonic MOPS buffered saline. The results (fig 9) show that DIDS did not inhibit EGOT activation when the PLP concentration in the medium v/as 2.4 mM. In contrast, incubation with 4.8 mM PLP did not

further increase EGOT activation in DIDS treated cells. This

reproduces the observations made using plasma indicating that there

% l is not some previously unrecognized element of plasma such as free

amino acids which partakes in PLP transport.

30

buffered suspension medium ofbovineserumalbumin(6gm%)and glucose (10mM)inisotonic salineafter4hoursincubation.

Figure 9.EffectofDIDSonEGOTactivation byPLPinaMOPS GOT (U/gHgb) 0 24 6 B6 (mM) 31

IV. DISCUSSION

This study investigated several critical issues in vitamin B6 metabolism using activation of erythrocyte aspartate-oxaloacetate transaminase (EGOT) as an indicator of intracellular cofactor availability to vitamin B6 dependent enzymes. A comparison of the relative abilities of the various vitamers to enter the erythrocyte and effect EGOT activation demonstrated the inability of PLP to enter the erythrocyte from plasma whereas other vitamers, particularly PMP, were quite active.

Previous investigations of PLP entry into the erythrocyte have also shown that it is not readily transported across the cell membrane from plasma (Anderson et al 1971, Lumeng et al 1974). This observation which was confirmed here questions the importance generally accorded to PLP. The activities of the other vitamers as shown in figure 2 are in fact much greater than equimolar PLP. This raises the important question of which of these could play an important role in vivo.

Because of serum protein binding, plasma PLP is unable to enter erythrocytes in significant amounts in these experiments and also in the experiments of other investigators. It is unclear however whether these events accurately reflect events in vivo. One critical assumption is essential to the conclusions drawn from these experiments. To claim that PLP remains in plasma because it binds to

32 plasma proteins preferentially over intracellular or membrane binding sites requires an assumption that equilibrium is reached during the incubation period of these experiments. This has not been established. In fact, data of previous investigators suggest that

PLP may not reach equilibrium within hours but rather over days. For instance, in the experiments of Lumeng (1973), continued loss of PLP from plasma occurs during dialysis over the entire 24 hour period studied. Thus the time course of dissociation is quite slow.

Similarly, dialysis of liver cytosol over 24 hours liberated only 20% of hepatic cytosolic PLP from holoenzyme complexes. If it is assumed that all PLP is reversably bound to enzymes, then this dissociation is also quite slow. The in vivo investigations of Solomon and

Hillman (1979) clearly shov; an unexplained 24-48 hour delay between a single oral PN dose and a rise in EGOT activity. Solomon and Hillman

(1979) proposed that the initial drop in EGOT within hours of intramuscular PLP or, oral PN administration followed by a rise after days was due to non-specific inhibitory binding of PLP to the enzyme followed by redistribution to the active sites. Thus a delay of days is seen in the response of EGOT to increased PLP in several different experimental settings. One possible explanation for this is that PLP added to plasma, whether in vivo (from liver or by injection) or in vitro, requires days to dissociate from serum proteins and reach the active sites of apoenzymes. Thus, before more conclusions can be drawn regarding the thermodynamics of PLP binding, certain

assumptions regarding the binding kinetics clearly must be

33

substantiated or revised.

The investigations of membrane permeation by PLP using DIDS are

intriguing. Although Cahantchik (1975) reports that the anion channel blocking agent DIDS totally prevents PLP entry into erythrocytes, the experiments here demonstrate that there is a distinct component of PLP transport which is not affected by anion channel blockers. This effect is apparent at PLP concentrations in

the 1.0-1.5 mM range. This is well below the binding capacity of plasma for PLP which means that the actual amount of PLP free in solution is probably fairly low. Only when the plasma content of PLP

is near or above the binding capacity for this vitamer is DIDS blockable transport observed. In this case, the concentration of

free PLP in the medium is much higher and it is only then that the anion channels appear to play a role in transmembrane PLP passage.

Apparently, in the more physiologic situation of lov; PLP concentrations, a mechanism for PLP transport other than anion channels obtains. Investigation of the dephosphorylation-

rephosphorylation mechanism may help resolve this issue. Such experiments would best be done by inhibiting the phosphatase which is

essential to this mode of transport. The experiments conducted here

show that at 5 mM and 20 mM, phosphate has no effect on PLP

activation of EGOT in spite of its demonstrated inhibitory effect on

the phosphatase. Unfortunately, any results found using phosphate may be confused by the effect phosphate also has intracellularly of

34 raising concentrations of organic phosphate esters. A better inhibitor of the phosphatase is thus critically important to further examine this postulated mechanism.

Yet another possible mechanism of PLP transport might be coupling with amino acid transport. That membranes have specific mechanisms for amino acid transport has been well documented (Guidotti et al

1978). Felig et al (1973) have also shown that erythrocytes may act as a transport form of amino acids. One could hypothesize that amino acid uptake and PLP uptake by erythrocytes are interrelated, perhaps by Schiff base complexing between the two and transport of the adduct. However, the experiment described here using a medium of bovine serum albumin and glucose instead of plasma suggests that free amino acids found in plasma are not responsible for the non-anion channel dependent PLP uptake. On the other hand, no one has conclusively shov/n how PLP is released from hepatocytes and although it is proposed that it does so bound to albumin (Lumeng and Li 1980) the possibility that an amino acid-PLP Schiff base is the released form merits investigation. Although Gregory and Kirk (1978) once suggested that pyridoxyllysine may exert an anti-B6 activity, this claim has not been substantiated. Thus the possibility that amino acid-PLP adducts could play an important role physiologically or even pharmacologically deserves further attention.

35

V. CONCLUSION

Although a few questions are answered here, many more questions are raised. It is clear that under the experimental conditions employed, there is marked variability in the activation of a tissue enzyme, EGOT, by the various B6 vitamers. It is also clear that blockade of membrane anion channels can alter membrane permeability of PLP. But contrary to v/hat was previously found, this does not occur at PLP concentrations near the physiologic range.

Further elucidation of the mechanisms involved in PLP transport into tissues would thus be essential. Only with such knowledge can therapeutically advantageous manipulations of PLP transport be designed. That PLP is actually the B6 vitamer deserving this concentrated attention is, unfortunately, also unresolved. Although the experiments here; confirm the inability of PLP to enter tissues from plasma and further demonstrate that PMP and PM are quite active, a serious issue of kinetics of this interaction is questioned.

Thus, before any more theories regarding the behavior of PLP in plasma are asserted, conclusive experiments addressing the time course of the PLP-plasma interactions are imperative. Because the studies previously cited indicate that PLP may equilibrate with plasma over days instead of hours, it remains distinctly possible

that PLP is indeed the B6 vitamer which enters tissues from plasma to activate the B6-dependent enzymes. This certainly would not have

36 been demonstrated in these experiments or those of previous investigators because of v/hat may be grossly insufficient in vitro incubation periods.

37

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45

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