Metabolism of vitamin B6 in the I-strain mouse

ARCHIVES

OF

BIOCHEMISTRY

AND

Metabolism 1. Absorption,

BIOPHYSICS

of Vitamin

Excretion, R. RAINES

Department

147, 588-601 (1971)

B6 in the

and Conversion BELL

of Vitamin

AND BETTY

of Food Science, University

I-Strain

of Illinois,

Mouse

to Enzyme

Co-factor’

E. HASKELL Urbana, Illinois

61801

Received July 27, 1971; accepted September 21, 1971 The I-St strain mouse is an inbred strain whose elevated requirement for vitamin Bg and whose acute sensitivity to audiogenic seizures is confirmed in experiments described here. We have compared vitamin Be metabolism in the I strain mouse to that in a control strain, the C57B1/6J, to determine whether abnormalities can be detected in absorption, excretion, or in the ability to convert vitamin Be to the major co-factor form, pyridoxal phosphate. We have also compared the two strains with regard to the activity of glutamic acid decarboxylase, a pyridoxal phosphate enzyme in brain which may regulate neuroirritability. No physiologically significant differences between the two strains were observed in absorption of the vitamin or in the ability to convert dietary vitamin Be to enzyme co-factor. Both the activity of glutamic acid decarboxylase and the degree of saturation of the enzyme with pyridoxal phosphate were similar in the two strains. When mice were fed a vitamin Bsdeficient ration highm protein, increased urinary losses of vitamin Be were observed in I strain mice as compared to C57 mice. This difference in excretion of vitamin Be is explored in detail in an accompanying paper.

Unknown metabolic defects in humans which result, in elevated requirements for vitamin BE2 have been the subject of intensive research, (for recent, reviews, see Refs. (1) and (2)) but similar phenomena which occur in laboratory animals have been investigated only in the case of the I/St strain3 mouse (3, 4). This inbred mouse is acutely sensitive to vitamin Be depletion. 1 This work was supported by a research grant from National Science Foundation and by fellowships from National Science Foundation and from the U. S. Public Health Service. Experiments described here are contained in the Ph.D. thesis of Roma C. Raines (University of California, Davis, CA, 1970). A preliminary report of this work has appeared (Raines, R. C. and Haskell, B. E., Fed. Proc. 29,823 (1970). 2 Vitamin Bg is used as a generic term for the three forms of the vitamin: pyridoxal, pyridoxine, and pyridoxamine. 3 The I/St mouse is referred to hereinafter as the I strain mouse; the C57B1/6J, as the C57 strain

It dies in convulsions after a brief period of vitamin Bs depletion sufficient to produce only moderate weight loss in a control strain (3). The cause of the profound growth failure of the vitamin BG-depleted I strain mouse and its susceptibility to audiogenic seizures is unknown. A particularly puzzling feature of vitamin BB deficiency in the I strain mouse is that tissue stores of vitamin BB are not, reduced in proportion to the obvious severity of the deficiency, as indicated by weight loss, skin lesions, and audiogenic seizures (4). In fact, the total vitamin Bs content of liver and brain from a vitamin BG-depleted I strain mouse is not significantly different from that of a vitamin Bedepleted C57Blj6J mouse3 (4). The C57 mouse is commonly used as a control strain in biochemical, genetic, and nutritional studies (5). This mouse is slow to lose weight, on a vitamin Bs-deficient, diet. Hyperirritability and audiogenic seizures

588

589

BG METABOLISM

have never been observed in this straineven in animals so severely depleted that death eventually occurs (3, 6). Lyon has shown that the vitamin Be requirement of the I strain mouse, as indicated by growth response, is satisfied by 2 mg pyridoxine per kilogram of diet (3). This is about twice the normal requirement (7). But to describe the I strain mouse simply as an animal with an elevated requirement for vitamin B6 is to underestimate the complexity of the problem. The I strain mouse is strangely intolerant of a high protein diet. Fed an 18% casein diet supplemented with ample vitamin Bs , the I strain mouse grows as well as the C57 mouse (3). But when the vitamin B, content of the diet remains constant and the protein content is increased to 3070, growth depression occurs (3). It is well known that increasing the protein content of the diet increases the requirement for vitamin Bs (8). The surprising thing, however, is that growth depression in the I strain mouse fed a 30 % casein diet is not reversed by increasing the vitamin B, content of the diet-not even by a supplement of 200 mg pyridoxine per kilogram diet (3). The I strain mouse is, therefore, not an animal with a higher requirement for vitamin Bs in the usual sense and thus offers a challenging problem in abnormal vitamin Bs metabolism. Lyon’s group has carried out extensive studies on the relationship of vitamin Bs nutrition to glycogen metabolism in the I/FnLn strain mouse (9-11) and has shown that the phosphorylating enzyme, phosphorylase b kinase, is missing in I strain muscle (12). There is no published data which suggest an obvious relationship between phosphorylase b kinase deficiency and the acute sensitivity to vitamin B, depletion which is characteristic of the I strain mouse. Whether phosphorylase b kinase deficiency in the I strain mouse is genetically linked or dissociable from vitamin Bs sensitivity is unknown. Aside from Lyon’s work, there has been no systematic exploration of vitamin Be metabolism or of the enzymology of vitamin B,-catalyzed reactions in the I strain mouse.

In principle, abnormal vitamin B6 metabolism in the I strain mouse might be due to: (1) failure to absorb the vitamin at a normal rate, (2) increased excretion of the vitamin, (3) failure to convert vitamin (usually administered as pyridoxine) to the major cofactor form4, pyridoxal phosphate, at a normal rate, (4) increased degradation of the vitamin to 4-pyridoxic acid, (5) impaired binding of a pyridoxal phosphate enzyme to its co-factor, or (6) increased turnover of one or more enzymes whose co-fact)or is pyridoxal phosphate. This paper considers the first three possibilities : absorption, excretion, and conversion of vitamin to the enzyme co-factor. We have compared enzyme preparations from I strain and C57 strain tissue with regard to activity and properties of the two enzymes required to convert vitamin to cofactor. These enzymes are pyridoxal phosphokinase (ATP: pyridoxal-5’-phosphotransferase EC 2.7.1.35) and pyridoxine phosphate oxidase (pyridoxamine-5’-phosphate: oxygen oxidoreductase (deaminating) EC 1.4.3.5). In addition, we have carried out preliminary studies on the relationship of glutamic acid decarboxylase activity in brain to the audiogenic seizures which occur in vitamin Be-depleted I strain mice. Glutamic acid decarboxylase (L-glutamate l-carboxylyase EC 4.1.1.15) is a pyridoxal phosphate enzyme which may regulate neuroirritability (13). MATERIALS

AND METHODS

Chemicals. Pyridoxine.HCl labeled with tritium by an exchange procedure was a product of Amersham-Searle, Arlington Heights, IL. It was lyophilieed to dryness five times from a small volume of distilled water to remove labile tritium. The final specific activity was 1.69 Ci/mmole. All other chemicals were purchased from commercial sources and were used without further purification. Animals and diet. I/St mice were purchased from Leone11 C. Strong Research Foundation, Inc., San Diego, CA; C57B1/6J mice were purchased from Jackson Laboratories, Bar Harbor, 4 Pyridoxal phosphate is the co-factor in all known vitamin Bs-catalyzed reactions except transamination. In transamination, either pyridoxamine phosphate or pyridoxal phosphate is active.

590

BELL

AND

ME. Mice were fed either a stock diet (Strong Mouse Blox, Shame1 Milling Co., East Concord, NY) or a purified diet with or without a supplement of vitamin Bg . Unless indicated otherwise, the purified diet contained (g/100 g diet) : vitaminfree casein, 18; corn oil, 8; glucose, 66.5; salt mix (14), 6; and vitamin Be-free vitamin mix (14), 1.5. One hundred grams of diet supplied the following amounts of minerals and vitamins: CaCol , 1.80 g; K2HPOd, 2.10 g; CaHP04 , 0.36 g; N&l, 1.00 g; FeSOn.7 H20, 150 mg; MgSOn.7Hz0, 750 mg; KI, 1.8 mg; 1.5 mg; ZnCOs , 4.8 mg; CuS04.5Hz0, chloride, 7.5 mg; MnSOd,HaO, 14 mg; choline inositol, 37.5 mg; ascorbic acid, 1.75 mg; calcium pantothenate, 3.75 mg; niacin, 2.25 mg; thiamine. HCl, 2.25 mg; menadione, 1.87 mg; p-aminobenzoic acid, 750 pg; riboflavin, 750 pg; folic acid, 45 rg; biotin, 19 pg; vitamin Blz, 2rg; vitamin E, 9 IU; vitamin D, 112 ICU; vitamin A, 1136 IU. In certain experiments, the vitamin-free casein content of the diet was increased to 30yo and glucose reduced to 54.5yo. All other components of the diet remained the same. The purified diet contained zero to 5Opg vitamin Bs per kilogram as estimated by microbiological assay with 8. carlsbergensis no. 4228 (ATCC 9080) (15). Animals were caged individually in wirebottomed cages in an air-conditioned laboratory maintained at 72-74°F. They had free access to water and to diet. Tissue for enzyme assays was obtained from mice which were decapitated, exsanguinated, and rapidly dissected. Tissue was homogenized and assayed immediately or was stored at -15” until analyzed. Absorption experiments. Two pair of female I strain and C57 strain mice matched by age and weight were fasted for 20 hr and administered 0.1 ml of an aqueous solution of tritiated pyridoxine. HCl by stomach tube. Labeled vitamin (1.69 Ci/ mmole) was diluted with nonradioactive pyridoxine.HCl to the desired specific activity (357 pCi/mmole in Exp. 1; 330 pCi/pmole in Exp. 2). Animals received a 4-pg dose of the vitamin in Exp. 1, a 5.6~pg dose in Exp. 2). After 2 hr animals were decapitated, exsanguinated, and dissected. Tissues were analyzed for tritium after oxidation to tritiated water in a Packard Tri-Carb Sample Oxidizer, Model 305 (Packard Instrument Co., Downers Grove, IL). The tritiated water was transferred quantitatively to a scintillation vial and combined with 15 ml of scintillation fluid consisting of naphthalene, 100 g; l,Cdioxane, 730 ml; toluene, 135 ml; absolute methanol, 35 ml; 2,5-diphenyloxazole, 5 g; and 1,4-bis-2 (4-methyl5-phenyloxazolyl)benzene, 0.3 g (16). Samples were counted in a Packard Tri-Carb Scintillation Counter, Model 3320. The efficiency of count for

HASKELL tritium, as determined by the channels-ratio method (17), was 55-58%. Excretion experiments. Four adult male I strain and C57 strain mice were administered a physiological dose of tritiated pyridoxine.HCl (3.36 pg, sp act 1.69 Ci/mmole) by subcutaneous injection. The animals were maintained in stainless-steel metabolism cages with free access to water and diet while continuous urine collections were carried out. For the first 29 days, mice were fed stock diet supplying 4 mg vitamin Bg per kilogram diet. For the next 19 days, the mice were fed a purified vitamin Ba-free diet containing 30% casein. Urine was collected daily. Aliquots were combined with scintillation fluid and counted as described above. Pyridoxal phosphokinase. Assays for pyridoxal phosphokinase were carried out as described by McCormick et al. (18) on tissues homogenized in 9 vol of cold 0.25 M sucrose. The enzyme assay mixtures contained 1.25 rmoles pyridoxal, 1.25 amoles ATP, 130 pmoles potassium phosphate buffer, pH 6.25, 25 nmoles ZnSOa.7HzO and O-120 ~1 enzyme (O-12 mg tissue) in a final volume of 2.5 ml. Preliminary experiments showed that the relationship of product formation to enzyme concentration was linear in the range of concentrations routinely used in assays (19). Enzyme assays were incubated for 1 hr at 37”. The reaction was stopped by heating in a boiling water bath for 3 min. Product formation with pyridoxal as substrate was measured by assaying aliquots of the reaction mixture for pyridoxal phosphate by an improved apotryptophanase method (20). With pyridoxine or pyridoxamine as substrate, product formation was measured after enzymatic oxidation of pyridoxine phosphate and pyridoxamine phosphate to pyridoxal phosphate with pyridoxine phosphate oxidase purified from rabbit liver (21). Pyridoxine phosphate oxidase. Assays for pyridoxine phosphate oxidase were carried out on tissues homogenized in 4 vol of 0.02 M potassium phosphate buffer, pH 7.0. The homogenates were centrifuged at 18,000 g for 30 min at zero to 4”. The supernatant fraction, which contained 97% of the activity in the whole homogenate (19), was assayed for pyridoxine phosphate oxidase activity according to the phenylhydrazine procedure of Wada and Snell (21). Unless otherwise noted, assays were carried out with 200-590~1 enzyme (40-100 mg tissue) and 0.3 pmoles pyridoxine phosphate in 0.2 M Tris buffer, pH 8 (final volume 3.5 ml). Preliminary experiments showed that product formation was linear with increasing enzyme concentration in the range routinely used in assays (19). After incubation at 37” for 30 min with shaking, the reaction was stopped by the addition

,591

BG METABOLISM of 0.3 ml 100% (w/v) trichloroacetic acid (21). The precipitate was sedimented by centrifuging, and pyridoxal phosphate was determined colorimetrically by the phenylhydrazine method (21). Pyridoxine phosphate oxidase was purified from livers of I strain and C57 strain mice according to the procedure described by Wada and Snell for the isolation of the enzyme from rabbit liver (21). Carried through the alumina C-gamma step, the procedure resulted in a 24-fold purification of the I strain liver enzyme and a 17.fold purification of the C57 liver enzyme. Vitamin Bg Gz tissues. Total vitamin Bg in brain and liver of mice fed a 3Oyc casein diet was determined by microbiological assay with S. carlsbergensis no. 4228 (15) after acid hydrolysis of the tissue in 0.055 N HCl for 5 hr at 121” (22). To determine free and phosphorylated forms of vitamin BF, in mouse liver, vitamin Be was extracted with dilute perchloric acid as described by Bain and Williams (23). Since S. carlsbergenesis responds only to nonphosphorylated forms of in neutralized vitamin Be, the free vitamin perchloric acid extracts was determined by microbiological assay prior to hydrolysis; total vitamin Be by microbiological assay after hydrolysis as described above. The phosphorylated forms of vitamin Be were separated by ion-exchange chromatography (23). Each fraction from the column was hydrolyzed (22), and the vitamin Be was estimated microbiologically. The recovery of total vitamin Bs applied to the ion-exchange column was 92yc for I strain liver and 82yc for C57 strain liver. The pyridoxal phosphate content of I strain and C57 strain mice maintained on stock diets was determined by assaying neutralized perchloric acid extracts of mouse tissue by a modified apotryptophanase method (20). Glutamic acid decarboxylase. Mitochondrial lysates of mouse brain were assayed for glutamic acid decarboxylase according to the method of Susz et al. (24) in the reaction vessels described by Roberts and Simonsen (25). Lysis of mitochondria according to the method of Susz et al. (24) releases the glutamic acid decarboxylase which requires pyridoxal phosphate as a co-factor. A second glutamic acid decarboxylase whose properties differ from the pyridoxal phosphate enzyme is released from mitochondrial preparations only after more vigorous treatment (26). To determine the extent of saturation of glutamic acid decarboxylase preparations with pyridoxal phosphate, assays were carried out with and without the addition of 0.1 rmole pyridoxal phosphate to reaction vessels. Although Susz et al. (24) recommended adding pyridoxal phosphate early in the isolation procedure to stabilize

glutamic acid decarboxylase, we found that pyridoxal phosphate could be omitted without loss of activity in enzyme preparations held at 04” for 24 hr (19). Assays routinely were carried out within 12 hr. RESULTS

Absorption. To determine whether tho higher vitamin B, requirement of the I strain mouse was due to defective absorption, the following experiments were carried out. Two pair of unanesthetized I and C57 strain mice matched by age and weight were administered 4-5.6 pg tritiated vitamin Be by stomach tube. (For details, see Materials and Methods.) These doses approximate tho daily vitamin Bs requirement of the adult mouse which is about 3 kg per day (7). Animals were killed after 2 hr and the tissues analyzed for tritium with the results shown in Table I. The amount of tritium remaining in the intestine was approximately the same in the two strains, indicating that I and C57 mice absorbed the vitamin with equal efficiency. Total recovery of the radioisotope and tissue distribution were similar in the two strains. Excretion. To determine whether the I strain mouse requires more vitamin B6 because it excretes more of the vitamin, four adult stock-fed males were administered a physiological dose of tritium-labeled pyriTABLE ABSORPTION

I

OF [3H]P~~~~~~~~~ STRAIN MICEQ

C57 AND

IN

I

70 Intubated dose Tissue

Intestine Liver Blood Other tissues Total

C57 strain

I strain

Exp. 1

Exp. 2

Exp. 1

Exp. 2

9.96 31.51 6.15 17.64

8.59 21.91 6.43 15.15

10.36 23.87 5.60 27.61

11.54 22.24 6.08 20.25

65.26

52.08

67.44

60.11

a Adult female I strain and C57 strain mice were administered a physiological dose of tritiated pyridoxine.HCl (Exp. 1, 4 rg; Exp. 2, 5.6 rg) by stomach tube. After 2 hr the animals were killed and dissected. Tritium was recovered from tissues after oxidation to tritiated water (see Materials and Methods).

592

BELL

AND TABLE

URINARY

LOSSES

OF RADIOISOTOPE

yO Injected dose recovered in urine

Stock diet

Vitamin diet

BG-free

O-5 days 6-10 days 11-19 days 2@29 days 30-39 days 40-48 days

HASKELL II

AFTER INJECTION [aH]P~~~~~~~~~G

OF A PHYSIOLOGICAL

es7 Exp. 1

Exp. 2

28.63 3.42 1.82 1.25 0.47 0.58

16.85 3.20 1.84 1.37 0.44 0.25

DOSE

OF

I AWage

22.74 3.31 1.83 1.31 0.46 0.42

Exp. 1

Exp. 2

Average

9.93 2.86 1.51 1.15 0.83 0.81

8.63 3.36 1.82 1.25 0.71 0.73

9.33 3.11 1.66 1.20 0.77 0.77

a Two adult male C57 and two adult male I strain mice were injected with 3.36 pg[sH]pyridoxine.HCl (27.6 &i) and maintained in metabolism cages with free access to stock diet and water while urine was collected daily. After 29 days, animals were shifted to a vitamin Be-deficient diet containing 30% protein.

doxine . HCl (3.36 ~g, 27.6 &i), by subcutaneous injection:. Urine was collected daily, and the tritium recovery was determined by counting a suitable aliquot in a scintillation counter. Feces were not collected, since losses of vitamin B6 in feces have been shown to be negligible (27). Table II shows that the I strain mouse retained more radioactive vitamin B6 during the first 5 days after injection than did the C57 strain, indicating that the elevated vitamin Bg requirement of the I strain mouse is not due to a lowered kidney threshold for the vitamin. The rate of loss between Day 6 and Day 29 is similar for the two strains. After 29 days, I strain and C57 strain mice were shifted to a purified vitamin Bs-fret diet containing 30% casein. Since a high protein intake is known to increase the requirement for vitamin Be (8), this diet might be expccted to accentuate any abnormalities of vitamin B, metabolism in the I strain mouse. Under these dietary conditions, the rate of loss of radioisotope in the urine is greater in the I strain mouse than in the C57 mousea phenomenon of interest in connection with possible differences in oxidation of vitamin Bs in the two strains (See second paper of this series (28).) Tissue levels of vitamin B6. We carried out experiments to determine whether the increased urinary loss of vitamin BB in I strain mice fed a vitamin BG-free diet containing 30% casein was reflected in lower tissue stores of the vitamin. When weanling male

I strain and C57 strain mice were maintained on a purified diet containing 30% casein with or without vitamin Be for 28 days, mean total weight gain was lower in the I mouse than in the C57 mouse. This was true whether the animals were fed the vitamin Be-supplemented or the deficient ration (see Table III). However, t’he differences were not statistically significant5. Mean values for total vitamin B, in liver were lower in I strain than in C57 strain animals fed the supplemented diet; they were higher in I strain than in C57 strain animals fed the deficient diet. In neither case was the difference statistically significant when results were corrected for differences in liver weight (Table III). Pgridoxal phosphokinase. We began our study of vitamin Be-metabolizing enzymes with a comparison of pyridoxal phosphokinase activity in enzyme preparations from I strain and C57 stlain mice. A single enzyme, pyridoxal phosphokinase, converts all three forms of the vitamin-pyridoxal, pyridoxine, and pyridoxamine-to the corresponding 5’ phosphate esters (18). A summary of the transformations of vitamin B, in animal tissue is shown in Fig. 1; for a recent review, see Ref. (2). Although pyridoxal phosphokinase activity is detectable in every organ investigated, brain is one of the richest sources (18). The ability of the brain enzyme to phosphorylate vitamin Bc was of particular interest in 6 Student’s

t-test.

5!)3

Be METABOLISM TABLE

WEIGHT GAIN

AND

III

VITAMIN Bs CONTENT

OF HIGH-PROTEIN

TISSUE

OF C57 AND

+ Vitamin B6 No. animals in group Total wt gain in 28 days (g)

Total vitamin Bg Brain (pg/g tissue) Brain (pg/organ) Liver (pg/g tissue) Liver kg/organ)

es7

3.76 1.47 13.20 13.39

f f

1.00 0.35

f

1.88

f

2.22

MICE FED

A

- Vitamin Be I 7 9.30 f 1.19

7 12.30 h l.os*

I STRAIN

DIE~U

3.62 1.35 10.36 7.47

CS1 5 7.60 * 0.99

f

0.50

2.59 f

0.44

f

0.18

0.97 f

0.16

f f

2.06” 1.58”

3.52 f 3.34 f

0.95

1.01

I 6 5.40 f 0.99

2.14 0.80 4.44 2.48

f f f f

0.29 0.14 1.44 0.84

a I strain and C57 strain mice were fed a purified diet containing 30% casein with or without a supplement of 10 mg pyridoxine.HCl per kilogram diet for 28 days. The total vitamin Bs content of Gssue was determined by microbiological assay (see Materials and Methods). * Mean f the standard deviation. c Although t,he total vitamin Ba per liver was lower in I strain mice than in C57 mice when both strains were fed the supplemented ration (p < O.OOl), the vitamin B, content per gram liver did not differ significantly in the two strains.

connection with the sensitivity of the vitamin B,-depleted I strain mouse to audiogenic seizures. (Experiments to determine whether phosphate esters of vitamin B6 are hydrolyzed prior to entering brain are described elsewhere (29).) A defective brain kinase might reduce the ability of the I strain mouse to form pyridoxal phosphate at the normal rate and might account for the audiogenic seizures which are a common feature of vitamin Bg deficiency in this strain. To determine whether a relationship exists between hyperirritability and pyridoxal phosphokinase activity, weanling male animals of both strains were maintained on a purified diet with or without vitamin Bs (200 mg pyridoxine.HCl per kilogram diet) for 18 days. By Day 18 on the experimental diet,, all of the deficient I strain mice exhibited wild running and leaping in response to a mild noise stimulus; one died in convulsions. By contrast, no hyperactivity or convulsions were observed in the C57 strain mice fed the same ration. Tissue from animals killed on Day 18 were analyzed for pyridoxal phosphokinase activity with the results shown in Table IV. Mean values for pyridoxal phosphokinase activity consistently were lower in I strain tissue than in C57 strain tissue. However, a statistically significant difference was observed only when mice fed the supplemented ratio were com-

pared. Pyridoxal phosphokinase activity was lower (p = 0.05) in livers of I strain mice fed ample vitamin B, than in livers of C57 strain mice fed the same ration. To find out whether the afinity of pyridoxal phosphokinase for its three substrates differs in the two strains, Michaelis constants were determined according to the procedure of Lineweaver and Burk (30). Table V shows that enzyme preparations from I strain and C57 strain tissue do not differ in the value of the K, for pyridoxal, the only substrate whose product can be measured without substantial error. Because no satisfactory method exists for measuring pyridoxine phosphate and pyridoxamine phosphate directly, both could be assayed only after enzymatic oxidation to pyridoxal phosphate. Thus, each determination with pyridoxine or pyridoxamine as substrate represents the results of a threestep assay: (1) phosphorylation of pyridoxine or pyridoxamine by the kinase, (2) oxidation of the product of the kinase reaction to pyridoxal phosphate, and (3) estimation of pyridoxal phosphate by its ability to restore activity to apotryptophanase. (For details, see Materials and Methods.) A two-fold difference in K, is within the error observed in duplicate determinations. Although the results of our determinations show differences in the K, of the I strain

594

BELL

AND

HASKELL

4 PYRIOOXIC ACID ALOEHYOE OXIOASE PYRIOOXINE *

--------) PYRIDOXINE OXIDASE

PHOSPHATASE

PYRIOOXAMINE TRANSAMINASE c_.----- -_. \’ 25’ T \) PYRIOOXAL c------PYRIOOXAMINE * A PYRIDOXINE PHOSPHATE OXIOASE PHOSPHATASE

PHOSPHATASE PYRIOOXAL PHOSPHDKINASE (+ATP)

PYRIOOXAL PHOSPHOKINASE (+ATP)

” PYRIDOXINE PHOSPHATE

U PYRIOOXINE PHOSPHATE OXIOASE

TRANSAMINASES

PYRIOOXAL ::::e-Z-Z-Z-‘, PHOSPHATE PYRIOOXINE PHOSPHATE OXIOASE

PYRIOOXAL PHOSPHDKINASE (+ATP) ” PYRIOOXAMINE PHOSPHATE

FIG. 1. Metabolic transformations of vitamin Be Major pathways in the metabolism of vitamin Be are indicated by solid arrows. Pyridoxal phosphate, the major co-factor form of vitamin Be , can be formed by two routes: (1) by phosphorylation of pyridoxal (18) or (2) by conversion of pyridoxine and pyridoxamine to the corresponding 5’ phosphate esters (18) and oxidation of the phosphate esters to pyridoxal phosphate (21). A single enzyme, pyridoxal phosphokinase, converts all three forms of the vitamin-pyridoxal, pyridoxine, and pyridoxamine--to the corresponding phosphate (18). It is probable that a single enzyme, pyridoxine phosphate oxidase, also catalyzes the oxidation of pyridoxine phosphate and pyridoxamine phosphate to pyridoxal phosphate (21). Pyridoxal phosphate functions as co-factor in all known vitamin Be reactions except transamination (2). In transamination, both pyridoxal phosphate and pyridoxamine phosphate are active. Reactions indicated above by broken lines have been demonstrated to occur in animal tissue but their physiological significance in connection with vitamin Bg interconversions is questionable (2). Pyridoxal phosphate is formed from pyridoxamine phosphate by transamination. However, the importance of this pathway as a means of increasing tissue pools of pyridoxal phosphat,e is unknown (2). The phosphate esters of pyridoxal, pyridoxine, and pyridoxamine are hydrolyzed by phosphatases (2). Pyridoxal (but not pyridoxine or pyridoxamine) is oxidized t.o 4-pyridoxic acid by aldehyde oxidase (6). The three forms of vitamin BG , their phosphate esters, and 4-pyridoxic acid are excreted in urine (23).

and C57 enzymes for pyridoxine and pyridoxamine, these are of doubtful significance in view of the imprecision of the assay. In any case, the differences invariably favor the ability of the I strain mouse to utilize small amounts of vitamin Bs , since the K, values are smaller for the I strain than for the C57 strain. We conclude that our results show no differences in pyridoxal phosphokinase properties which explain hyperirritability or the abnormal vitamin B6 requirement of the I strain mouse. Pyridoxine phosphate oxidase. Pyridoxine phosphate oxidase, an enzyme whose activity is concentrated in liver, converts pyridoxine phosphate and pyridoxamine

phosphate to pyridoxal phosphate (21). To determine whether activity of pyridoxine phosphate oxidase differs in liver of I strain and C57 strain mice, weanling male mice were fed a purified diet with or without ample vitamin B6 (10 mg/kg diet) for 30 days. Liver was analyzed for pyridoxine phosphate oxidase activity with the results shown in Table VI. Activity of pyridoxine phosphate oxidase is significantly higher (p < 0.001) in livers of vitamin Be-supplemented I strain mice than in livers of C57 strain mice fed the same diet. In both strains, activity falls during vitamin B, depletion. The decrease in activity is greater in vitamin B6-depleted I strain mice (28 % less than I

595

Be METABOLISM TABLE PYRIDOXAL

Diet

PHOSPHOKINASE I STRAIN

TABLE

IV ACTIVITY MICE~

IN C57 AND

V

PROPERTIES

OF PYRIDOXAL PHOSPHOKINASE I STRAIN AND C57 STRAIN MICE

FROM

I strain

C57 strain Brain

Purified + Bs Purified - Be

689 f 50 (14) 631 f 50

775 f 114b (9)” 747 f 49 (10)

(16) Liver

Purified + I& Purified - BG

801 f 119 (9) 646 f 90 (10)

527 f 3gd (14) 492 f 35

Pyridoxal c57 I Pyridoxamine c57 I Pyridoxine c57 I

3.08 2.90

39 32

0.44 0.44

37 49

6.45b 2.35

57 36

3.0Sb 2.26

47 115

10.006 5.83

83 123

6.16b 3.07

104 77

(16)

(1Weanling male I strain and C57 strain mice were fed a purified diet with or without a supplement of 200 mg pyridoxine.HCl per kilogram diet for 18 days. Tissues were assayed for pyridoxal phosphokinase activity with pyridoxal as substrate (see Materials and Methods). Activity expressed as nmoles pyridoxal phosphate formed in 60 min/g wet weight tissue. b Mean f standard deviation. c Number of animals in each group. d Significantly lower when compared to liver of C57 strain mice fed a vitamin BG-supplemented diet (p = 0.05).

strain controls) than in vitamin B6-depleted C57 mice (13 % less than C57 controls). To determine whether pyridoxine phosphate oxidase in I strain and C57 strain tissue differs in its affinity for substrate, Michaelis constants were determined with the results shown in Table VII. No differences were observed in Michaelis constants for brain preparations from the two strains. Nor did the two strains differ in the Michaelis constants of the liver enzyme with pyridoxamine phosphate as substrate. (Assays were carried out at pH 8.0; see Table VII.) However, the K, of the I strain mouse liver enzyme for pyridoxine phosphate was more than five times greater than that of the C57 liver enzyme for the same substrate. Pyridoxine phosphate oxidase was purified from livers of both strains (for details, see Materials and Methods) and the K, determinations repeated. Assays were carried out at the optimum for each substrate, pH 9.5 for pyridoxamine phosphate and pH 8.0 for

a I’,., is expressed in millimicromoles of phosphorylated vitamin Bg derivatives formed per minute per gram wet weight of tissue. b Duplicate determinations of the Km with pyridoxamine and pyridoxine as substrates gave s.smuchasa2-folderror becauseof thecumbersome three-step assay used. (See text and Materials and Methods.)

pyridoxine phosphate. Under these condi. tions, the Michaelis constants of the I strain enzyme exceeded by ii-fold those of the C57 enzyme with either pyridoxamine phosphate or pyridoxine phosphate as substrate (see Table VII). Growth on the three forms of vitamin B6. To assess the physiological significance of the observed differences in pyridoxine phosphate oxidase in I strain tissue, we turned to growth experiments. The higher K, values suggested that the I strain mouse might be handicapped in converting vitamin to cofact’or when dietary supplies of the vitamin were limited. On the other hand, higher total oxidase activity-observed both in Be-deficient and in B,-supplemented I strain animals-might compensate for the reduced affinity of the I strain enzyme for its substrates. We therefore tested irk vivo the ability of the I strain mouse to form pyridoxal phosphate at a normal rate by the following experiment : We maintained weanling, male I strain and C57 strain mice on purified diets supplemented with growthlimiting amounts of pyridoxal, pyridoxine, and pyridoxamine. We reasoned that a physiologically significant defect in the ability

596

BELL

AND

HASKELL

TABLE PYRIDOXINE

OXID~SE

PHOSPHATE

VI

ACTIVITY

nmoles

IN C57 AND I STRAIN MOUSE LIVERY

pyridoxal

phosphate

formed/g

C57strain Purified

diet + Be

Purified

diet -

tissue

I

954.4 f 11.4”,” (8)” 828.2 z!z 43.8 (8)

Be

wet weight

in 30 min strain

1109.6 f 17.4cs d (5) 798.6 f 37.5 (5)

a Weanling C57 and I strain mice were fed a purified diet with or without a supplement of 10 mg pyridoxine.HCl per kilogram diet for 30 days. Assays for pyridoxine phosphate oxidase were carried out with pyridoxine phosphate as substrate. Product formation was determined calorimetrically with phenylhydrazine. (For details, see Materials and Methods.) b Mean f standard deviation. c Significantly higher in vitamin BG-supplemented animals than in animals of the same strain fed the deficient diet. For C57 strain, p = 0.02; for I strain, p < 0.001. d Significantly higher in I strain mice fed the supplemented diet than in C57 mice fed the same diet (p < 0.001). 8 Number of animals in each group. TABLE MICHAELIS

CONSTANTS

FOR

PYRIDOXINE

VII

PHOSPHATE

OXIDASE FROM c57 AND 1 STRAIN

TISSUE Pyridoxine

A. Brain supernatant” C57 strain I strain B. Liver supernatant? h C57 strain I strain C. Purified liver enzyme” C57 strain I strain

phosphate

4.44 x 10-B M 2.92 X lo- M

Pyridoxamine

phosphate

3.00 x lo+ M 5.64 X 1O-6 M

6.75 X W6 M 36.6 X UF6 M

8.96 x lo-6 10.20 x lo-

4.10 x lo-’ M 15.7 x 1o-5 M

2.91 X lo- M 11.5 x lo-B M

M

M

(1Substrate inhibition occurs at pyridoxine phosphate concentration greater than 0.1 mM, therefore, significance and are not reported here. values for V,,, have little physiological b Assays for pyridoxine phosphate oxidase with pyridoxamine phosphate as substrate were carried out at pH 8.0 with the liver supernatant enzyme; at pH 9.5 (the optimum for this substrate) with the purified enzyme. c Pyridoxine phosphate oxidase was purified 24-fold from I strain liver; 17-fold from C57 strain liver. The purification scheme was that of Wada and Snell (21) for pyridoxine phosphate oxidase from rabbit

liver. of the I strain oxidase to form co-factor should result in differences in the rate of growth on the three forms of the vitamin. For example, the I strain mouse should grow better on pyridoxal (which requires only phosphorylation for conversion to co-factor) than on pyridoxine or pyridoxamine which require both phosphorylation and oxidation. To insure that the level of pyridoxine, pyridoxamine, and pyridoxal in the purified

diet actually was growth limiting (but neither low enough to produce acute deficiency nor high enough for normal growth), two additional groups of mice were fed either a diet containing no added vitamin BB or ample vitamin Ba . Growth curves for I strain and C57 strain mice maintained on diets containing no vitamin B, , limiting pyridoxine, pyridoxamine, or pyridoxal(l.22 pmoles/kg diet) or ample vitamin BB (48.6

Bg METABOLISM

pmoles pyridoxine per kilogram diet) are shown in Fig. 2A and 2B. When I strain mice were fed limiting amounts of pyridoxal, pyridoxamine, and pyridoxine, the animals grew equally well on all three forms of the vitamin (Fig. 2A). The same was true of the C57 strain (Fig. 2B). A marked difference between the two strains was observed, however, in growth on diets containing either no vitamin B, or growth-limiting amounts of the vitamin. After 30 days on a diet containing no vitamin B, , I mice actually weighed slightly less than at the beginning of the experiment;

12 18 Days on Diet

FL+ 0

I

/ 12

Days

1 18

1 24

I

30

on Diet

FIG. 2. Growth of I strain and C57 strain mice on three forms of vitamin Be . Weanling I strain (Fig. 2A) and C57 strain (Fig. 2B) mice were maintained on purified diets containing no vitamin Bg , excess vita.min Be (48.6 Hmoles pyridoxine per kilogram diet), or growth-limiting amounts (1.22 pmoles/kg diet) of pyridoxine (PN), pyridoxal (PL), or pyridoxamine (PM) for 30 days.

597

C57 mice subjected to the same treatment gained about 5 g. I mice fed the purified ration containing suboptimal amounts of pyridoxine, pyridoxal, or pyridoxamine gained only about half as much weight (4666%) as C57 mice fed the same diet. The poor growth in the I strain mouse occurred despite a daily food intake which consistently was slightly higher than the C57 mouse. The incidence of vitamin B6 deficiency symptoms was greater in I mice fed a ration limiting or deficient in vitamin Bs as shown in Table VIII. I mice fed the purified diet containing ample vitamin B6 grew as well as C57 mice fed the same ration and were completely free of deficiency symptoms. Phosphorylated vitamin B, in liver. Growth data suggested that pyridoxine phosphate oxidase in the I mouse functions as efficiently as does the C57 enzyme in converting its substrates to pyridoxal phosphate. To confirm that the I mouse has normal ability to oxidize pyridoxine phosphate and pyridoxamine phosphate, we decided to check for possible accumulation of these two substrates in I strain tissue. Pyridoxine phosphate normally occurs in animal tissue only in trace amounts (23). It is reported to be an inhibitor of certain vitamin Be enzymes (31) and has no known co-factor role (2). Its only metabolic fates are hydrolysis to free ppridoxine and oxidation to pyridoxal phosphate. The presence of abnormal amounts of this metabolite in the tissue of I strain mice grown on limiting amounts of vitamin Bs should be readily detectable and of significance in connection with pyridoxine phosphate oxidase function. Pyridoxamine phosphate, however, can be formed either by phosphorylation of pyridoxamine or by transamination of pyridoxal phosphate. The significance of an elevation of pyridoxamine phosphate in animal tissue would be difficult to assess, since it is not known whether pyridoxamine phosphat’e form.ed by transamination reactions remains tightly enzyme bound or whether it’ is available as a substrate for pyridoxine phosphate oxidase (2). Phosphorylated and free form.s of vitamin Be were estimated in tissue of both strains with the results shown in Table IX. No pyridoxine phosphate could be detected in I

598

BELL

AND

HASKELL

TABLE VIII INCIDENCE OF VITAMIN ~~~~~~~~~~~~~ SYMPTOMS IN C57 AND I STRAIN MICE FED DIETS CONTAINING AMPLE, GROWTH-LIMITING OR No VITAMIN Bea % Animals Diet supplement (pm&s/kg diet)

Skin

lesions

c57

hmple (48.6 Limiting (1.22 Limiting (1.22 Limiting (1.22 None

pyridoxine rmoles) pyridoxine pmoles) pyridoxal wmoles) pyridoxamine pmoles)

Greasy I

c57

afflicted

or rough

coat

Hyperactivity

I

c57

I

0

0

0

0

0

0

0

40

0

80

0

40

0

45

0

67

0

56

0

56

0

56

0

45

0

60

25

100

0

100

a Weanling male C57 and I strain mice were fed a purified diet containing ample vitamin Be, no vitamin Be, or growth-limiting amounts of pyridoxal, pyridoxine, and pyridoxamine for 30 days. (See growth curves in Fig. 2A and B.) The number of animals in each group was as follows: Ample pyridoxine, 8 C57, 10 I; limiting pyridoxine, 7 C57, 10 I; limiting pyridoxal, 8 C57, 9 I; limiting pyridoxamine, 8 C57, 9 I; no vitamin Be, 8 C57, 10 I. TABLE TOTAL AND PHOSPHORYLATED VITAMIN GROWTH-LIMITING

IX

B6 IN LIVER OF C57 .~ND I STRAIN MICE FED AMOUNTS OF PYRIDOXINE” c57

rig/g

Total vitamin Be Free vitamin Bg Pyridoxamine phosphate Pyridoxal phosphate Pyridoxine phosphate

tissue

3420 294 1272 1228 Trace

I rig/organ

3078 264 1145 1105 Trace

rig/g

tissue

3218 359 1987 639 0

rig/organ

3478 388 2146 690 0

a Vitamin Bc was isolated from the tissues of I strain and C57 strain mice which had been fed growthlimiting amounts of pyridoxine (1.22 pmoleslkg diet) for 30 days. For details of analytical procedures, see Materials and Methods. Values shown above are averages for tissue from two mice of each strain.

liver; a trace could be detected in C57 strain liver. Despite the apparently greater deficiency of the I strain mouse maintained on limiting pyridoxine, as indicated by growth failure, skin lesions, coat condition, and hyperirritability, the total vitamin Be content of I strain and C57 strain liver did not differ. Glutamic acid decarboxylase. Lower pyridoxal phosphate levels in brain have been suggested as a cause of the sensitivity of the I strain mouse to audiogenic seizures (4). Since there had been no direct investigation of this possibility, we compared the activity of glutamic acid decarboxylase in I strain

strain

and C57 strain mice which had been fed a purified diet with or without a supplement of 10 mg pyridoxine. HCl per kilogram diet, for 35 days. Glutamic acid decarboxylase is a pyridoxal phosphate enzyme which converts glutamic acid to gamma-aminobutyric acid, a compound which may function as an inhibitory neurotransmitter (13). Table X shows that there is no difference between the two strains in the activity of glutamic acid decarboxylase preparations from mice fed deficient or supplemented rations. Enzyme preparations from both deficient and supplemented tissue were incompletely saturated with pyridoxal phosphate, as indi-

599

Be METABOLISM TABLE GLTJTAMIC

ACID

DECARBOXYLASE

ACTIVITY

IN

- PLP

Purified + Be

OF

I

STRAIN

AND

C57

STRAIN

MICE”

Glutamicacid decarboxylase activity (rmoles CO*/30 min/g wet weight tissue)

Diet

c57 Purified - Bs

X BRAIN

0.81 f 0.W (11) 1.32 f 0.26 (11)

+ PLP

c

2.16 f

0.34d

38.0 f

7.6

1.92 f

0.33d

68.6 f

9.5

2.39 f

0.36d

35.6 f

3.32

2.14 f

0.26d

66.0 f

I Purified - Be Purified + Be

0.85 f

0.14c (9) 1.40 f 0.28 (9)

14.0

0 Glutamic acid decarboxylase assays were carried out on brains from I strain and C57 strain mice which had been fed a purified 30y0 casein diet with or without 10 mg/kg pyridoxine.HCl for 35 days. The degree of saturation with pyridoxal phosphate (PLP) was determined by assaying samples with or without the addition of 0.1 rmole pyridoxal phosphate per flask. b Mean f the standard deviation. Number in group in parentheses. c Glutamic acid decarboxylase activity in C57 and I strain BG-deficient mice is significantly lower than in the corresponding vitamin Be-supplemented controls when assayed without PLP (p < 0.001). d Glutamic acid decarboxylase activity for both strains is significantly higher (p =
cated by the increase in activity which occurred when pyridoxal phosphate was added to the reaction mixture. However, the degree of saturation with pyridoxal phosphate was the same (about 40 %) in enzyme preparations from deficient I strain mice and from deficient C57 strain mice. When enzyme preparations from supplemented mice of the two strains were compared, the degree of saturation with pyridoxal phosphate in both cases was about 70%. DISCUSSION

Our data confirm the observation of other investigators (3, 4) that the I strain mouse suffers rapid growth failure and acute symptoms of vitamin Be deficiency when maintained on diets containing growth-limiting or no vitamin B,. What is the metabolic defect in the I strain mouse which creates an unusual degree of dependency on a high dietary intake of vitamin Bei) Our data provide no answer to this question. Nevertheless, our experiments exclude several important possibilities and establish the extent to which certain aspects of vitamin B6 metabolism in the I strain mouse are normal. 1. We have excluded the possibility that.

malabsorption is the cause of vitamin Be deficiency in the I strain mouse. Experiments with tritiated pyridoxine show that absorption is equally efficient in I strain and C57 strain animals. 2. We have shown that the higher vitamin B6 requirement in the I strain mouse is not due to more rapid excretion of the vitamin in the period immediately after injection. On the contrary, the initial retention of an injected dose of tritiated vitamin B6 is higher in the I strain mouse than in the C57 strain. Higher initial retention of a test dose of vitamin B6 is a characteristic of a vitamin B6-deficient animal (32, 33). However, both I strain and C57 strain mice used in excretion experiments were healthy adults which had been maintained on a rich natural diet supplying 4 mg vitamin B, per kilogram diet (about 12 pg/day). To conclude that the higher initial retention of tritiated vitamin B, in the I strain mouse indicates decreased tissue stores of the vitamin is not consistent with other data. For example, Lyon et al. (4) have reported that tissue stores of vitamin B, in I strain and C57 strain mice fed stock diets do not differ. Also, we have determined the pyridoxal phosphate content of tissues

600

BELL

AND HASKELL

from I strain and C57 strain mice maintained on our stock diet and find no difference between the two strains. We have no explanation for the increased retention of vitamin Bc by stock-fed I strain mice during the 5-day period immediately after administration of the radioisotope. 3. We have shown that the acute growth failure and hyperirritability observed in the I strain mouse fed a vitamin Be-free ration is not due to failure to form co-fact’or from vitamin at a normal rate. This interesting idea seemed worth exploring because other investigators had shown that brain stores of pyridoxal phosphate are more greatly reduced in vitamin B6-deficient I strain mice than in vitamin Be-deficient C57 mice (4). Also, the ratio of pyridoxamine phosphate to pyridoxal phosphate differs in the two strains. In I strain mice fed a complete ration, liver contains more pyridoxamine phosphate and less pyridoxal phosphate than does liver from C57 mice fed the same ration (4). Although these observations suggested the interesting possibility that I strain mice might lack the ability to convert the vitamin (usually supplied as pyridoxine) to pyridoxal phosphate at a normal rate, our data fail to dem.onstrate that this is so. We have compared the activity and substrate affinity of pyridoxal phosphokinase and pyridoxine phosphate oxidase, the two enzymes required to convert vitamin B6 to co-factor. We find no differences between the two strains in pyridoxal phosphokinase activity or in substrate affinity which would account for the higher vitamin BB requirement of the I strain mouse. With regard to pyridoxine phosphate oxidase, the K, of the I strain enzym.e for its substrates is four times greater than that of the C57 strain enzyme. However, total activity of pyridoxine phosphate oxidase in I strain liver consistently is higher than in C57 strain liver. We doubt that the higher Michaelis constants of the I strain mouse enzyme for its substrates constitutes a defect that is physiologically significant, since I strain mice grow equally well on limiting amounts of pyridoxal, pyridoxamine, and pyridoxine. These results are consistent with other data which indicate that normal animals utilize the three forms of the vitamin with equal efficiency (34, 35).

Were pyridoxine phosphate oxidase activity impaired in I strain mice, one might expect its substrates-pyridoxine phosphate and pyridoxamine phosphate-to accumulate in I strain tissue. No pyridoxine phosphate was detectable in the livers of I strain animals which had been fed a diet containing limiting amounts of pyridoxine; trace amounts were found in the livers of C57 mice fed the same ration. Although the livers of I strain mice fed a diet limiting in pyridoxine contained more pyridoxamine phosphate and less pyridoxal phosphate than did livers of C57 mice fed the same ration, the higher pyridoxamine phosphate level in I strain liver is not necessarily a consequence of impaired pyridoxine phosphate oxidase function. Pyridoxamine phosphate can be formed by several enzymatic pathways (see Fig. 1). The altered ratios of pyridoxamine phosphate to pyridoxal phosphate in I strain liver (3: 1 as compared to 1: 1 for C57 tissue) may simply reflect the current balance of transamination. 4. Other investigators had suggested that the lower pyridoxal phosphate levels in brains of vitamin B6-deficient I strain mice might be the cause of the audiogenic seizures which occur in this strain (4). It was postulated that low pyridoxal phosphate levels might depress the activity of glutamic acid decarboxylase, a pyridoxal phosphate enzyme which may control neuroirritability. Our data show that the activity of glutamic acid decarboxylase-and its degree of saturation with pyridoxal phosphate-are the same in the hyperirritable vitamin B6-depleted I strain mouse as in the convulsionresistant C57 mouse. 5. We have observed that the response of the I strain mouse to the stress of a 30% casein diet is different from that of the C57 mouse. Urinary losses of vitamin Be in the I strain mouse are almost twice as great as in the C57 mouse when both strains are maintained on a vitamin B6-free, 30 % casein diet (Table II). However, an attempt to demonstrate that tissue stores of total vitamin Be are lower in I strain mice fed a 30% casein diet than in C57 strain mice fed the same ration gave negative results. We found no statistically significant difference between the two strains in the vitamin B, content of

Bs METABOLISM

tissue from mice maintained for 28 days on a 30 % casein diet with or without a vitamin Bs supplement (Table III). In general, our data suggest that the abnormally high requirement of the I strain mouse for vitamin B6 does not stem from a defect in absorption, in kidney threshold, or in conversion of vitamin to enzyme cofactor. The possibility that degradation of the vitamin to 4-pyridoxic acid may differ in the two strains is explored in an accompanying paper (25). ACKNOWLEDGMENT The skillful technical assistance Holzer is gratefully acknowledged.

of Mrs.

Mary

REFERENCES 1. MIJDD, S. H., Fed. Proc. 30,970 (1971). 2. SNELL, E. E., AND HASKELL, B. E., in ‘LComprehensive Biochemistry” (M. Florkin and E. H. St,otz, eds), Vol. 21, p. 47. Elsevier, Amsterdam (1970). 3. LYON, J. B., JR., WILLIAMS, H. L., AND ARNOLD, E. A., J. Nutr. 66,261 (1958). 4. LYON, J. B., JR., BAIN, J. A., AND WILLIAMS, H. L., J. Biol. Chem. 337, 1989 (1962). 5. GREEN, E. L. (ed), “Handbook on Genetically Standardized Jax Mice,” 2nd ed., p. 21. Jackson Laboratory, Bar Harbor, Maine (1968) . 6. BELL, R. R., AND HASKF,LL, B. E., unpublished observations. 7. National Research Council, “Nutrient Requirement of Laboratory Animals,” No. 10, p. 44. Publ. 990, National Academy of Sciences, Washington (1962). 8. Food and Nutrition Board, National Research “Recommended Dietary AllowCouncil, ances,” 7th rev. ed., p. 46. Publication 1694, National Academy of Sciences, Washington (1968). 9. LYON, J. B., JR., AND PORTER, J., Biochim. Biophys . Acta 68,248 (1962). 10. LYON, J. B., JR., AND PORTER, J., J. Biol. Chem. 238, 1 (1963). 11. DANFORTH, W. H., AND LYON, J. B., JR., J. Biol. Chem. 239,4047 (1964). 12. LYON, J. B., JR., PORTER, J., AND ROBERTSON, M., Science 166, 1550 (1967). 13. ROBERTS, E., AND KURIYAMA, K., Brain Res. 8, 1 (1968).

601

14. SWENERTON, H., AND HURLEY, L. S., personal communication. 15. HASKELL, B. E., AND SNELL, E. E., Methods Enzymol. 18A, 512. 16. “Operation Manual for Packard Model 300 Tri-Carb Sample Oxidizer” (Manual No. 2069), Section 111-3. Packard Instrument Co., Downers Grove, Illinois. 17. DAVIDSON, J. D., AND FEIGELSON, P., Int. J. Appl. Radiat. Isotop. 2, 1 (1957). 18. MCCORMICK, D. B., GREGORY, M. E., AND SNELL, E. E., J. Biol. Chem. 236,2076 (1961). 19. RAINES, R. C., “A Comparison of Vitamin Bg Metabolism in I strain and C57 strain mice,” Ph.D. Thesis, University of California, Davis, California (1970). 20. HASKELL, B. E., AND SNELL, E. E., Anal. Biochem., in press. 21. WADA, H., AND SNELL, E. E., J. Biol. Chem. 236, 2089 (1961). 22. RABINOWITZ, J. C., AND SNELL, E. E., Anal. Chem. 19, 277 (1947). 23. BAIN, J. A., AND WILLIAMS, H. L., in “Inhibition in the Nervous System and GammaAminobutyric Acid” (E. Roberts, ed.), p. 275. Macmillan (Pergamon), New York (1960). 24. Susz, J. P., HABER, B., AND ROBERTS, B., Biochemistry 6, 2870, (1966). 25. ROBERTS, E., AND SIMONSEN, D. B., Biochem. Pharmacol. 12,113 (1963). 26. HARER, B., KURIYAMA, K., AND ROBERTS, E., Brain Res. 22, 105 (1970). 27. Cox, S. H., MURRAY, A., AND BOONE, I. U., Proc. Sot. Exp. Biol. Med. 109,242 (1962). 28. BELL, R. R., BLANCHARD, C. A., AND HASKELL, B. E.,Arch. Biochem. Biophys. 147,602 (1971). 29. KEFER, J., BELL, R. R., BLANCHARD, C. A., AND HASKELL, B. E., manuscript in preparation. 30. LINEWEAVER, H., AND BURK, D., J. Amer. Chem. Sot. 66, 658 (1934). 31. SCHREIBER, G., AND HOLZER, H., in “Methods of Enzymatic Analysis” (H. U. Bergmeyer, ed.), p. 606. Academic Press, New York (1965). 32. JOH.4NSSON, S., LINDSTEDT, S., AND REGISTER, U. Amer. J. PhysioZ. 210, 1086 (1966). 33. VAUGHAN, D. A., AND WINDERS, R. L., Metabolism 16, 676 (1966). 34. SARMA, P. S., SNIZLL, E. E., AND ELVEHJE:M, C. A., J. Biol. Chem. 166, 55 (1946). 35. WAIB~~L, P. E., CRAVENS, W. W., AND SNELL, E. E., J. Nutr. 48,531 (1952).