Menadione Nicotinamide Bisulfite Is a Bîoactîve Source of ...

nutrient Requirements and Interactions
Menadione Nicotinamide Bisulfite Is a Bîoactîve
Source of Vitamin K and Niacin Activity for Chicks1
GEORGE W. ODÃœHO, THAU K. CHUNG2 AND DAVID H. BAKER3
Department of Animal Sciences and Division of Nutritional Sciences,
University of Illinois at Urbana-Champaign, IL 61801
ABSTRACT Young chicks were fed a vitamin Kdeficient
soybean concentrate basal diet containing
graded levels of menadione from menadione
nicotinamide bisulfite (MNB) or menadione dimethylpyrimidinol
bisulfite (MPB) to assess prothrombin time
as a function of menadione intake. Prothrombin time
decreased linearly as menadione dose increased from 0
to 400 /ig/kg. Multiple linear regression slope-ratio cal
culations indicated that both sources of menadione were
of equal potency. To assess niacin bioactivity of MNB,
graded doses of nicotinamide (0 to 5 mg/kg) from MNB
or nicotinamide were added to a niacin-deficient diet
based upon corn, corn gluten meal and vitamin-free
casein. Weight gain increased linearly as a function of
nicotinamide dose, and multiple linear regression
analysis of weight gain as a function of supplemental
nicotinamide intake revealed no significant differences in
slope between the two sources of nicotinamide. Using
excess doses, MNB was compared with MPB in acute
(single crop intubation) or chronic (fed in the diet for 14
d) toxicity trials. With a single menadione dose of 1600
mg/kg body wt, weight gain in the subsequent 14-d
period was reduced by MNB but not by MPB. Mortality
rates of 25 and 17% occurred for MPB and MNB,
respectively, at this dose level. Doses lower than 1600
mg/kg body wt caused neither morbidity nor mortality.
When provided in the diet for a 14-d feeding period,
menadione doses of 3000 mg/kg diet from MNB
reduced gain, feed intake, gain:feed ratio and blood he
moglobin concentration. Menadione doses of 6000 mg/
kg diet were required to produce morbidity of this type
when MPB was fed. The results suggest that MNB is
fully effective as a source of vitamin K and niacin ac
tivity, and only when doses exceed 1000 times the
chick's vitamin K requirement can morbidity or mortality
be demonstrated. J. Nutr. 123: 737-743, 1993.
INDEXING KEY WORDS:
•menadione nicotinamide bisulfite
•niacin •prothrombin time
•uitamin K
chicks
The biological activity of menadione salts or com
plexes as sources of vitamin K activity has been estab
lished in several chick studies. Frost et al. (1955)
reported that menadione sodium bisulfite complex
(MSEC)4 was three times more effective than mena
dione (2-methyl-l,4-naphthoquinone). Nelson and
Norris (1961) suggested that MSEC and phylloquinone
were equally active on a molar basis, and both
were more than twice as active as menadione.
Griminger (1965) and Dua and Day (1965) compared
MSEC and menadione dimethylpyrimidinol bisulfite
(MPB) and observed that MPB was more active than
MSEC in reducing prothrombin time of chicks. The
source of menadione evaluated herein, menadione
nicotinamide bisulfite (MNB), has never been
evaluated for its vitamin K (or niacin) activity.
Nicotinamide is known to be an effective source of
bioavailable niacin activity and has been used as such
for many years in both human and animal applica
tions. Studies by Childs et al. (1952) and Baker et al.
(1973) have suggested 20 to 30 mg niacin/kg diet as
the requirement for maximal growth of young chicks.
Baker et al. (1976) later reported that nicotinamide
was 23% more bioactive than nicotinic acid for chick
growth, although subsequent studies by Bao-Ji and
Combs (1986) and Ruiz and Harms (1988) were unable
to confirm this observation.
The objectives of this investigation were to assess
the vitamin K and niacin bioactivity of MNB (Fig. 1)
relative to known and established standards, i.e., MPB
for vitamin K activity and nicotinamide for niacin
activity. Both acute and chronic toxicity studies were
also conducted to establish a safety profile for MNB.
'Supported in part by Vanetta U.S.A., Inc., York, PA.
^Current address: 154 Clemenceau Avenue, #02-07 Haw Par
Centre, Singapore 0923.
3To whom correspondence should be addressed at 328 Mumford
Hall, 1301 W. Gregory Dr., Urbana, IL 61801.
4Abbreviations used: MNB, menadione nicotinamide bisulfite;
MPB, menadione dimethylpyrimidinol bisulfite; MSEC, menadione
sodium bisulfite complex.
0022-3166/93 $3.00 ©1993 American Institute of Nutrition. Received 14 September 1992. Accepted 5 November 1992.
737
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738 ODUHO ET AL.
FIGURE 1 Chemical structure of menadione
nicotinamide bisulfite.
MATERIALS AND METHODS
Animals and diets. All experiments were con
ducted using male chicks from the cross of New
Hampshire males and Columbian females (University
of Illinois Poultry Farm). On d 8 posthatching, after
food was withheld overnight, chicks were weighed
and allotted to experimental groups so that each
group had a similar average initial weight and weight
distribution. Diets and water were freely available.
Chicks were kept on a 24-h constant light schedule in
heated, thermostatically controlled batteries with
raised wired floors. All procedures were approved by
the University of Illinois Committee on Laboratory
Animal Care.
Vitamin K activity. Assay 1 was conducted to
determine the vitamin K (menadione) bioactivity of
MNB (45.7% menadione, 32% nicotinamide, Vanetta
SPA, Milano, Italy) when compared with MPB (45.5%
menadione, Vanetta SPA). From hatching to d 7
posthatching, chicks were fed a vitamin K-deficient
soybean concentrate diet (Table 1) estimated to
contain no more than 30 /¿gvitamin K/kg. On d 8
posthatching, duplicate groups of four male chicks
were assigned to the dietary treatments. Treatments
consisted of graded levels (0, 5, 25, 50 and 400 /¿g/kg)
menadione from MPB or MNB, which were added to
the vitamin K-deficient diet (Table 1) at the expense
of cornstarch. The NRC (1984) estimate of the dietary
vitamin K requirement of young chicks is 500 Mg/kg.
On d 22 posthatching, following the 14-d feeding
period, chicks were weighed and their feed intake
recorded, after which 1 mL of blood was drawn from
each chick by heart puncture. Blood from all chicks in
each pen was pooled and plasma was prepared.
Plasma prothrombin time was determined by the
method of Quick (1957), using sodium citrate as an
anticoagulant and acetone-dehydrated chicken brain
powder as a source of thromboplastin, prepared in our
laboratory using methodology described by Griminger
(1962 and 1965).
Ingredient1
TABLE 1
Composition of vitamin K and niacin assay diets
Cornstarch to
Dextrose
Corn
Corn gluten meal (60% protein)
Soybean protein concentrate (65%
protein)
Sucrose
Solka floe
Vitamin-K
assay diet2
100.00
35.40
20.00
g/100 g
3.00
Niacin
assay diet3
to 100.00
40.00
20.00
Vitamin-free casein (84% protein) 6.50
Corn oil 4.00 5.00
Mineral premix4 5.37 5.37
Vitamin premix (K or niacin-free)5 0.20 0.20
DL-choline chloride 0.20 0.20
all-rac-a-tocopheryl acetate (20 mg/kg) + +
Ethoxyquin (125 mg/kg) + +
Sulfathiazole 0.20
Amino acid premix6 1.60
DL-Methionine 0.30
1Cornstarch and dextrose obtained from A. E. Staley, Decatur,
IL; corn gluten meal (60% protein) and soybean protein concentrate
(65% protein) from Archer Daniel Midland, Decatur, IL; sucrose
from Holly Sugar, Colorado Springs, CO; solka floe from James
River, Berlin, NH; vitamin-free casein from U.S. Biochemicals,
Cleveland, OH.
Calculated analysis: 23% crude protein, 14.6 MJ/kg
metabolizable energy and

niacin bioactivity of MNB when compared with
nicotinamide (U.S. Biochemicals, Cleveland, OH). On
d 8 posthatching, quadruplicate groups of four male
chicks were assigned to the following dietary treat
ments: 0, 2.5, 5.0 and 30 mg/kg nicotinamide (the
latter a positive control) or 0, 2.5 and 5.0 mg/kg
nicotinamide from MNB. The NRC (1984) estimate of
the dietary nicotinic acid requirement of young
chicks is 27 mg/kg. On d 22 posthatching, chicks and
feed were weighed, and chick weight gain, feed intake
and feed efficiency were determined. Chicks that had
been fed 0 and 5.0 mg/kg nicotinamide (from both
MNB and nicotinamide) were killed by cervical dislo
cation at d 22 posthatching, and portions (0.25 g) of
liver tissue were removed and pooled by replicate.
Liver samples were homogenized and heated in a
boiling water bath for 5 min. Hepatic pyridine nucleotides
(NAD + NADH) were then determined by the
enzyme-cycling
(1969).
procedure of Nisselbaum and Green
Toxicity assays. Assay 3 was conducted to de
termine the toxic effects of an acute oral (crop intu
bation) dose of menadione from MPB (as a standard)
or MNB. Preliminary work in our laboratory had es
tablished that doses of MNB or MPB providing mena
dione at 500 mg/kg body weight or less caused neither
mortality nor morbidity. Assay 3 was therefore
designed to provide logarithmic doses of menadione,
starting at 100 mg/kg and working up to 1600 mg/kg
body weight. Following dosing at d 8 posthatching,
triplicate groups of four chicks at each dose level were
fed a 23% protein corn soybean meal starter diet5 for
a 14-d observation period.
Assay 4 was a 14-d chronic toxicity study involving
triplicate groups of four chicks at each menadione
dose level (0, 30, 300, 3000 and 6000 mg/kg diet) from
MPB or MNB. Additions were made to the cornsoybean
meal starter diet. At termination of the assay
on d 22 posthatching, blood was obtained by heart
puncture after which 1-mL portions from each chick
in a pen were pooled, resulting in three pooled
samples for each of the nine treatments. Blood hemo
globin was determined by the method of Crosby et al.
(1954).
Statistical analyses. Common-intercept multiple
linear regression was conducted for data in Assays 1
and 2 using the General Linear Model procedure of
SAS (1985). Data were first fitted to simple linear
regression equations to establish that Y-intercepts
were not different (P > 0.05) after which the multiplelinear
regression model was fitted
menadione biopotency assessment
to the data. For
in Assay 1, prothrombin
time (s) was regressed on supplemental
menadione intake (/¿g); for nicotinamide biopotency,
weight gain (g) was regressed on supplemental
nicotinamide intake (mg). The ratio of slopes (MNB:
MPB) gave estimates of menadione or nicotinamide
potency for MNB relative to MPB.
MENADIONE NICOTINAMIDE BISULFITE 739
TABLE 2
Prothrombin time of chicks fed diets with graded levels of
menadione from menadione dimethylpyrimidinol bisulfite
(MPB) or menadione nicotinamide bisulfite (MNB) (Assay I)1
menadioneUS/kg052550400ProthrombinMPBs3528252519time3MNB3533272417
'Values are means of duplicate groups of four male chicks at d
22 posthatching; average initial weight was 76 g.
The basal vitamin K assay diet (Table 1) was estimated to
contain 0.05) and this indicated that MPB and
MNB were equally efficacious in furnishing bioavailable
menadione.
Nicotinamide activity. Addition of graded doses of
nicotinamide or MNB below the niacin requirement
yielded linear (P < 0.01) responses in weight gain, feed
5Contained (g/100 g): corn, 52.8; dehulled soybean meal, 37.0;
alfalfa meal, 1.0; menhaden fish meal, 2.0; corn oil, 4.0; dicalcium
phosphate, 2.20; limestone, 1.00; DL-methionine, 0.20; NaCl, 0.40;
trace mineral premix (Mn, Fé,Zn, Cu, I, Se), 0.15; flavomycin
premix (4.4% flavomycin), 0.05; vitamin premix (retinyl acetate,
cholecalciferol, all-rac-a-tocopheryl acetate, menadione sodium bi
sulfite, vitamin B-12, niacin, D-Ca-pantothenate, riboflavin), 0.10;
choline chloride (60% choline), 0.10.
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740 ODUHO ET AL.
intake and gain:feed ratio (Table 3). Responses at 5
mg/kg nicotinamide were far lower (P < 0.01) than
those obtained when 30 mg/kg nicotinamide was sup
plemented. Hepatic NAD + NADH concentrations
were low in chicks fed the basal diet, but they in
creased (P < 0.05) upon adding 5 mg/kg nicotinamide
from either nicotinamide or MNB. Multiple linear
regression of weight gain (g) as a function of
nicotinamide intake (mg) resulted in a good fit (R =
0.98), with the MNB slope being 87% of the slope
value for nicotinamide (Fig. 2). Regression coefficients
(i.e., slopes), however, were not significantly different
(P > 0.05) between the two sources of nicotinamide.
Acute toxicity of menadione nicotinamide bi
sulfite. Single crop intubations of MPB or MNB at
menadione doses of 800 mg menadione/kg body
weight or lower did not cause mortality, nor was
growth depressed during the subsequent 14-d obser
vation period (Table 4). With a dose of 1600 mg/kg
body wt, however, 3 of 12 chicks (25%) receiving
MPB and 2 of 12 chicks (17%) receiving MNB died in
the first 2 d following dosing. This dose of menadione
from MNB but not MPB caused a depression in
weight gain, feed intake and gain:feed ratio during the
14-d post-dosing period.
Chronic toxicity of menadione nicotinamide bi
sulfite. Pharmacologie doses of MNB or MNB
provided in the diet at levels up to 6000 mg/kg mena
dione resulted in no mortality during the 14-d feeding
period. At 6000 mg/kg menadione, both sources of
menadione depressed (P < 0.05) gain, feed intake and
gain:feed, but only MNB depressed blood hemoglobin
concentration (Table 5). At 3000 mg/kg menadione,
MPB produced no morbidity, but MNB depressed all
measures of chick performance.
DISCUSSION
Menadione nicotinamide bisulfite
is an organic salt of two vitamins, menadione bi
sulfite and nicotinamide (Fig. 1). According to the
manufacturer (Vanetta SPA), MNB, a pale yellow
powder, has a melting point of 182°C,a pH range of 1
to 3.5 and a water solubility (25°C)of 19 g/L.
The one-stage prothrombin time chick bioassay
(Quick 1957) is considered the most sensitive assay
for biopotency of menadione compounds (Augustine
1985, Griminger 1965, Kindberg and Suttie 1989). In
our hands the prothrombin time assay worked far
better (i.e., better linearity) with thromboplastin iso
lated in our laboratory from chicken brain than was
the case with purchased rabbit thromboplastin. This
agrees with results reported by Griminger (1962).
Griminger (1965) and Griminger and Donis (1960)
used a vitamin K-assay diet similar to that used
herein, and their 21-d-old female chicks appeared to
be more deficient in vitamin K than was the case with
0.5
SupplementalNicotinamideIntake (mg/14d)
FIGURE 2 Multiple linear regression of weight gain (Y in
g) on supplemental nicotinamide intake [X in mg) from
nicotinamide (X, *) or MNB (X2, +): Y = 79.3 + 72.7 (±5.3)X\
+ 63.2 (±5.7)Xi, R = 0.98.
our unsupplemented male chicks. They used sulfaquinoxaline
(2 g/kg) to minimize gut synthesis of
vitamin K. We used 2 g/kg sulfathiazole in our
vitamin K-deficient diet, and this sulfa drug may have
been less effective than sulfaquinoxaline in
minimizing gut synthesis of the vitamin. In
Griminger's (1965) work, MSBC was 74% as effective
as MPB in furnishing bioavailable menadione. In our
study (Table 4), MPB and MNB were equally effective
in furnishing bioavailable menadione.
Based upon prothrombin times of 17 to 19 s at 400
Mg/kg menadione (Table 2), with normal prothrombin
times for chickens assumed to range from 12 to 25 s
(Smith and Russell 1984), it would appear that the
NRC (1984) estimate of 500 Mg/kg for a vitamin K
requirement of broiler chickens is a reasonable es
timate of the true requirement.
Growth bioassays in chicks work well to establish
niacin activity of test ingredients (Baker et al. 1973
and 1976, Jacob and Swenseid 1990), but if the test
sources contain tryptophan as well as nicotinic acid
or nicotinamide, it is difficult to separate niacin
responses from tryptophan responses (Baker 1986).
Our niacin assay diet contained corn, corn gluten
meal and casein. We assumed that nicotinic acid
would be only 20% available in corn and corn gluten
meal (Darby et al. 1975, Yen et al. 1977) and that
bioavailable tryptophan would be relatively low in
these ingredients as well (Yen et al. 1971). We were
therefore surprised that the niacin assay diet, when
adequately fortified with nicotinamide, would not re
spond to supplemental tryptophan. Regardless, the
diet responded markedly to nicotinamide addition,
and the fourfold growth response obtained to addition
of 30 mg/kg nicotinamide (Table 3) suggested that
lower doses of nicotinamide would result in linear
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Source0NANANAMNBMNBPooledSupplemental
MENADIONE NICOTINAMIDE BISULFITE 741
TABLE3
Performance of chicks fed diets with graded levels of nicotinamide (Assay 2)1
nicotinamideLevelmg/kg02.55.030.02.55.0SEMWeight
gain2731361732641081625Feed
intake2g/14
NAD NADH3nmol/g +
d1682272753642112575Gain:feed2g/kg43659962972551263016Hepatic liver140—215——24517
'Values are means of four replicate pens of four male chicks during the period 8 to 22 d posthatching; average initial weight was 75 g. For
hepatic NAD + NADH, only chicks fed the basal niacin assay diet and those with 5 mg/kg nicotinamide |NA) from NA and menadione
nicotinamide bisulfite (MNB) were sampled at d-22 posthatching.
2Linear {P < 0.01) response to NA and MNB; 30 mg/kg NA greater (P < 0.01) than all levels lower.
35 mg/kg NA or MNB greater [P < 0.05) than the basal diet.
responses. This was the case (Fig. 2) and the response
to nicotinamide from MNB was not significantly
different from the response to crystalline
nicotinamide.
The LD-50 (dose causing 50% mortality) of oral
menadione has been reported to be 500 mg/kg body
weight in mice (Molitor and Robinson 1940) and 804
mg/kg in chicks (Ansbacher et al. 1942). An oral dose
level of 750 mg/kg body weight was nonlethal for
chicks. In our acute toxicity study, oral menadione at
800 mg/kg body weight was nonlethal when mena
dione was provided as either MNB or MPB; even at
1600 mg/kg menadione, mortality was 25% or less
(Table 4).
Chronic toxicity studies involving menadione ap
parently have not been conducted in avians (NRC
TABLE 4
1987). Daily oral doses of 350 mg/kg body weight as
menadione over a 30-d period have been observed to
depress hemoglobin and blood erythrocyte count in
young rats (Molitor and Robinson 1940). Daily oral
doses of 500 mg/kg body weight were lethal to rats
(Molitor and Robinson 1940), while in this study a
dose level of 6000 mg/kg diet over 14 d (equivalent to
daily doses of approximately 1000 mg/kg body
weight) were nonlethal to young chicks. Avians thus
appear to be more tolerant than mammals to either
acute or chronic pharmacologie dose levels of mena
dione.
Although not apparent in mortality data, high
levels of MNB were not as well tolerated as high
levels of MPB in terms of morbidity criteria (Tables 4
Acute toxicity study: performance of chicks fed a corn-soybean meal diet with an acute oral dose of menadione dimethylpyrimidinol
bisulfite (MPB) or menadione nicotinamide bisulfite (MNB) (Assay 3/*
Menadione
dose2mg/kg
wt0100200400800 body
d247266244240
intakeMPB395381396403
MNB663656654667
MNB%00000
16003Pooled
211Feed 403MNB3903914163763813579Gain:feedMPB
615g/kg63819632639649630
591MortalityMPB 250000 17
SEMWeightMPB2492622502592692489gainMNBg/14
'Data represent means of three pens of four chicks during the period 8 to 22 d posthatching; average initial weight was 65 g.
2A single crop intubation was given on d 8 posthatching.
31600 mg/kg body wt of menadione from MNB depressed (P < 0.05) weight gain, feed intake and gain:feed.
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742 ODUHO ET AL.
TABLE 5
Chronic toxicity study: performance of chicks fed corn-soybean meal diets containing high levels of menadione from menadione
dimethylpyrimidinol bisulfate (MPB) or menadione nicotinamide bisulfite (MNB) (Assay 4)1
Menadione
dose2mg/kg030300
30002
60003Pooled
SEMWeight
gainMPB240239234
d244241240
219
218MNBg/14 1984Feed
intakeMPB358361
351
337
667647MNBS/kg680680690 649
71.6 58.9
337MNB359354348 3136Gain:feedMPB670662 6338HemoglobinMPB78.287.5 72.4MNBg/L86.983.269.0
55.87.2
'Values are means of three pens of four chicks during the period 8 to 22 d posthatching; average initial weight was 73 g.
2MNB depressed (P < 0.05) weight gain, feed intake, gain:feed and hemoglobin.
3Both MPB and MNB depressed [P < 0.05) gain, feed intake and gain:feed, but only MNB depressed (P < 0.05) hemoglobin. Gain and
hemoglobin depressions were greater {P < 0.05) for MNB than for MPB.
and 5). In an 8-d chronic toxicity study comparing
nicotinic acid and nicotinamide in chicks, Baker et al.
(1976) observed only a slight growth depression from
5000 mg/kg dietary nicotinamide, while 10,000 mg/
kg was markedly growth depressing (20,000 mg/kg
dietary nicotinic acid did not depress growth). It
seems probable that the greater morbidity caused by
MNB compared with MPB results from the combined
effects of menadione and nicotinamide in MNB.
The results of this investigation suggest that MNB
is fully active as a source of bioavailable vitamin K
and niacin activity. If used primarily as a source of
vitamin K activity, it is worthy of note that meeting
the vitamin K requirement from MNB would provide
only a small portion of the niacin requirement. Tox
icity studies revealed that MNB has a good safety
profile in that dietary levels of menadione well over
1000 times the chicks' minimal vitamin K re
quirement were required to cause morbidity. Because
menadione compounds may lose potency when stored
in vitamin or vitamin-trace mineral premixes
(Charles and Huston 1972) stability studies need to be
done comparing MNB to MPB.
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