Indian Journal of Experimental Biology Vol. 37, January 1999, pp. 32-36

Hepatic pyridine nucleotides content in rat-A better indicator for determining available niacin values of friod

Rahul Behl* & A D Deodhar

Animal Biochemistry Division, National Dairy Research Institute, Kamal 13200 I, India

Received 10 March 1997; revised 21 September 1998

After 10 days depletion period with niacin free diet, weanling rats were repleted with either reference diets containing niacin at four levels (2, 4, 8 and 12 mg/kg diet) or test diets containing test material at two levels as source of niacin. Gains in body weight and hepatic pyridine nucleotides content increased with increase in niacin intake. The cOiTelatioIl coefficient for hepatic pyridine nucleotides content and niacin intake was 0.98, whereas for weight gain and niacin intake was 0.89. Dried skim milk and pearl millet were taken as test materials. Dried skim milk with most of its niacin in free form showed 98% niacin equivalent to be bioavailable whereas in pearl millet niacin was in bound fOim and bioavailability equivalent was only 48 per cent.

Determining the value of food as a source of niacin (nicotinic acid and amide) is complicated by dietary tryptophan being a second source of niacin and thus the indication of availability of niacin in various foods differs widely. It has long been known that large proportion of niacin in various sources remains in chemically bound forms . Experiments with rats and some other animal species have indicated that this bound niacin has low or negligible biological value l

-4 .

Assessment of bioavailability in these studies was based on the change in growth rate when niacin depleted rats were given niacin from test materia ls. Such criterion is non-specific as several intrinsic and extrinsic factors influence the growth rate of the animal5

• To assess the true bioavai lability, a more specific criterion reflecting biochemical and functional association of niacin at the cellular level is needed6

. In view of this, the present study has been undertaken to t st bioassay procedure in which hepatic pyridine liLlcleotides concentration of young rats was considered as response measure for assessmg biologically available niacin values from test materials . Dried skim milk (DSM) and pearl millet (Bajra:

·Present add ress: Animal Genetics Division, National Bureau of Animal Genetic Resources, P.B. No. 129, Makrampur Campus, G.T. Bypass Road, Kamal 132 001 , India

Pennisetum typhoides) were taken as test materials.

Materials and Methods Samples of dried skin milk (DSM) were

obtained from the Experimental Dairy, NDRl , India. Pearl milkt was purchased from local market, cleaned by removing foreign substances, powdered to uniform fineness and subsequently used for analysis and diet formulati on.

Chetnicals--Glllcose-6-phosphate, sodium-pyruvate, 2-oxygluterate (di sodium salt) and enzymes alcohol dehydrogenase, gl llcose-6-phosphate dehydro~enase , lactate dehydrogenase and glutamate dehydrogenase for enzymatic estimation of pyridine nl'cleotides 'were purchased from Sigma Chemical Co., USA. All other chemicals were of the reagent grade .

Determination of niacin equivalent-In the first phase niacin equivalent of test materials was deterrmned by measuring vitamin content and precursor amino acid tryptophan in these materials. Niacin was detcnnined microbiologically using Lactobacillus planwrum ATCC 8014 as test organism as descri bed by Freed7

• For total niacin, extraction wa~ done wi th 1 N H2S01 whereas water was used for extraction of free niaCin. Standards (0.01 to 0.1 j..lg n iacin) as well as samples (containing about 0.025 to 0.05 ~!g niacin), in

BEHL & DEODHAR: HEPATIC PYRIDINE NUCLEOTIDE--INDICATOR FOR NIACIN VALUE 33

triplicate were made 5 ml with water and mixed with 5 ml of niacin assay medium (7.3 g dehydrated medium/IOO ml). Tubes were plugged, sterilized and inoculated with a drop of suitably washed and diluted inoculum. Tubes were incubated at 37°C for 72 hr and lactic acid produced was titrated against 0.05 N NaOH with bromothymolblue (0.1 %) as external indicator.

For tryptophan determination standards (10 to 200 )lg) and samples (in duplicate) containing about 50 to 100 )lg tryptophan were made upto a final volume of I ml with water and mixed with 9 mt of p-dimethylaminobenzaldehyde reagent (0 .3% w/v in 21.1 N H2S04) . Tubes were left overnight. Blue colour was developed by adding I drop of 0.2% w/v NaN02 and keeping at room temperature for 30 min. Absorbance was measured at 590 nm (Spice and Chamber, 1949).

For determining niacin equivalent, tryptophan to niacin conversion ratio, 60: 1 was used ". The results are presented in Table 1. Although the chemical evaluation oftest material was suggestive of bioavailability, adequacy of recommended dietary allowance for nutrient can not be critically assessed merely from such assessmentl2. It was felt equally essential to have accurate information on its bioavailability with some bioassay.

Bioassay procedure-Twenty-one days old weanling male albino rats (Wistar strain) weighing 33.7±1.5 g were considered for the experiment. Fifty four rats were depleted of niacin by feeding niacin free basal diet for W days. The basal diet consisted of casein, 35 ; gelatin, 60; zein, 58; starch, 345 ; groundnut oil , 30; vitamin mix (retinyl palmitate (250,000 ill/g), 30; cholcalciferol, 2.78; DL-tocoferyl acetate (500 ill/g), 120; G­pantothenate, 12; thiamine-HCI, 4; riboflavin, 5; pyridoxine-HCl, 9.4; Cobalamix premix (0.1 % w/v in manitol) , 2.0; menadione, 2; choline chloriue, 1,000 and starch to 5,000 parts) , 5; salt mix (NaCl,

69.65; Kl, 0.395; KH2PO., 194.5; MgSO •. 7H20, 58.63; CaC03, 190.7; FeS04.7H20, 13.5; MnS04.H20, 2.005 ; ZnS04.H20 , 0.274; CuSO., 0.238 and CaCI2.6H20, 0.011 parts), 40; an amino acid supplement and sucrose to 1,000 parts . Amino acid supplement was calculated to bring the estimated level of digestible tryptophan in basal diet to 0.77 g/kg4. One group of six rats was sacrificed at the beginning of repletion (0 day) to obtain baseline data for hepatic pyridine nuc1eotides content. Forty-eight rats were randomized to different repletion test diets with six rats per group housed individually in anodized aluminium cages.

Rats were repleted for niacin by giving reference or test diets for next 19-days. Reference and test diets based on DSM and pearl millet were formulated in which graded amounts of niacin were included as pure vitamin or test material. Protein . content of the diet was kept constant by decreasing the amount of casein or zein in basal diet depending on the amount and quantity of protein present in test material.

Four groups were given reference diets containing 2, 4, 8 and 12 mg niacin/kg diet, respectively. DSM groups were given diets containing 28 and 56 g DSM (3 and 6 mg niacin equivalent)lkg diet. In case of pearl millet groups diets containing 89.6 and 179.2 g pearl millet (5 and 10mg niacin equivalent)lkg diet was given . During repletion period, animals were fed ad libitum and had free access to water. Food intake was recorded daily. Body weight was recorded every third day.

Tissue collection and Pyridine nucleotide estimation-At the end of the experiment, animals were sacrificed, liver was excised, washed with physiological saline, blotted and quickly frozen in liquid nitrogen. Frozen liver samples were powdered in pestle and mortar with the aid of

Table I-Niacin contents of test materials [Values are mean±SE of6 observations]

Sample Total niacin Free niacin

DSM (mg/l 00 g) 1.246±.046 I . I 76±O.053 (94.4)-

Pearl millet 2.980±0.052 0.810±0.022 (mg/ IOO g) (27.2)

·Values given in parentheses arc percent of total niacin value

Tryptophan

566.5± 13.25

156.2±6.61

Niacin equivalent

I 0.69±0.22 I

5.58±0.144

34 INDIAN J. EXP. BIOL. , JANU ARY 1999

liquid nitrogen and used for estimation of pyridine nucleotides by enzymatic method of Klingenberg9

•

Oxidised pyridine nucleotides, viz. NAD and NADP were extracted with HCl04 (0.6 N); alcohol ic KOH (0.5 N) was used for extraction of reduced pyridine nucleotides viz. NADH and NADPH as described earlier9

.

. NAD was estimated uSing alcohol dehydrogemse enzyme system whereas glucose-6-phosphate dehydrogenase enzyme system was used for NADP estimation . Reduced pyridine nucleotides; NADH and NADPH were estimated uSing lactate dehydrogenase and glutamate dehydrogenase enzyme systems respectively. Increase in absorbance for oxidised pyridine nucleotides or decrease in absorbance for reduced pyridine nucleotides (E) at 340 nm in 5 min . was measured and amount of particular pyridine nucleotide was calculated (E=6.22 )lmole-'cm-'t

Statistical analysis- Data were analysed made according to Bliss and White 'O for correlation coefficient and regression.

Results and Discussion The study was conducted on niacin-depleted rats

as experimental mode l. Such type animals respond to dietary vitami ns even at the level of nutritional relevance more favourably than normal ones l2

•

Niacin defi ciency was observed after ten days deplet ion period and was characterized by poor growth, very low gain in body weight, 0.38 g/day as aga inst 2 to 3 g/day observed in normal rats. Beside ') this, hepatic pyr 'dine nucleotide levels showed marked depletion (Table 2). Signi ficant decrease in the level of urinary N'-methyl­niacinamide, metabolic end product of niacin, by

tenth day in niacin deficient rats has been reported.4

Niacin depleted rats were repleted by feeding test diets with graded amounts of niacin from test materials or pure vitamin for the period of 19 day. Gains in body weight increased wi th increase in the niacin intake during the repletion period with the increase in dosage of niacin from 2 to i 2 mg per kg diet (Table 3). The body weight gain criterion is often considered non-specific6

•

In order to obtain a more specific response criterion reflecting biochemical and functional association of vitamin at cellular level, pyridine nucleotides, viz. NAD, NADH, NADP and NADPH contents were detennined In liver in different groups of rats receiving graded amount of vitamin from standard as well as test material. This was considered appropriate since no niacin dependent enzyme activity has so far been reported to be affected during defici ency. Pyridine nucleotide co-enzymes are loosely bound to apoenzymes unlike many other coenzymes of B­vitamins and exist in the cellular pool. It was hypothesized that niacin nutritional status would better reflect In the pyridine nucleotide pool size. Util ity of such criterion has been mooted by Bender and Bender5

, who suggested that tissue concentration of NAD(P) would gi ve precise estimate of niacin nutritional status. Data on these co-enzymes of niacin, obtained at differen t levels of niacin intake are given in Table 2. When compared with base line data on nucleotide contents at the beginning of repletion (Oth day), a steady increase was vldent of all pyridine nucleotides . Recently, hepatic NAD levels have also been shown to increase. linearly when

Table 2-Hepatic NAD. A DH , NA DP and ADPH contents in d iffe rent g roups of rats fed either depletion or repletion di et [Val ues are mean:tSE of 6 observat IOns]

Niac in Intake (p g)

Depletion d iet 159 .9±3.28 322.6±8.44

707 .:' ±23.R4 I075.9±34 .. 44

270.3±3. IG 526.8 ± 14.32 430.9 ±6 .72 8 :67±S .07

NAD

0.3 09 ±.OI 6 0.357 ±v.005 O.397±0.OO6 0.495 ±O.OOE O.6 12±0.O09 O. 381±0.007 0.449 ±0 .003 0369 ±O 006 0.44 1±0.0 11

Pyrid ine _nucleotide U.Jmol~slg liver) NADif ~AOP NADPH

0.1 82 ±O.O 13 0.042 ±.OO I 0 .025 :to.OUI 0.226 ±0.009 0.046±0.O02 0. 029.t0 .002 , O.266±0.00 I 0.05 9±0.OQ2 0 .034±0.002 0 ,296±OOI2 O.072±O. OO I 0.043±0 002 O.3 3 1±0.012 0 093±0.v02 .O50±O.001 0.240±0.005 0.058±0.002 0 037 ±O.OOI 0.272 ±0.009 0.069 ±0.OU2 0. 042 ±0.002 0.223 ±O.OOi 0.047 ±O. OOI 0.028 ±O.OOI 0.275±0.009 0.06410 .001 0.039 ±O.O02

BEHL & DEODHAR: HEPATIC PYRIDINE NUCLEOTIDE-INDICATOR FOR NIACIN VALUE 35

Table 3- Food and niacin intake, and weight gain in different groups of rats fed either reference or test diets [Values are mean±SE of6 observations]

Experimental diet Feed consumed Niacin Weight of animals (g) Weight

(g) intake Before After niacin After niacin gain (g) niacin depletion repletion

(I-Ig) depletion (day 0) (days 19)

2 mg niacinlkg diet 80.0 ± 1.6 159.9 ±3 3 33 .5 ± 1. 8 35 .3±34 44.1±3.1 8.8 ±.0.9 4 mg ni acinlkg diet 80.7±2.1 322.6±8.4 33 .5± 1.5 34.3±3.5 52.6±4.5 18.3±1.1 8 mg niacinlkg diet 88 .4±2.9 7073±23.8 34.3± 1.5 36.7±3.6 58 .5±4.1 21.8±.6 12 mg niaci nlkg diet 89 .6±2.9 1075.9±344 35.0±1. 1 37.2± 1. 8 61.9±1.8 24.7±0.2 28 g DSMlkg diet 90.6±4.5 270 .3±3.2 56 g DSM kg diet 88 .1±1.7 526.8±14.3 89.6 g pearl millet! kg diet 86 .2±1 .5 430.9±6.7 179.6 g pearl millet! kg diet 83.7± 1.8 836.7±5 .1

weanling Fisher-344 rats were given large supplements of niacin at different levels compared with controls given niacin-sufficient diets )).

To find the degree of correlation by determining correlation coefficient (Tab le 4), it was observed that there was strong positive correlation ranging between 0.96 to 0.99 in case of pyridine nucleotide compared with weight gain (r = 0.89). This supported the contention that pyridine nucleotides content in liver is valid and useful to assess niacin nutritional status and bioavailability of vitamins. Apparently, measurements of co-enzyme forms of vitamin were more dependable than rather non-speci fic criterion such as growth rate.

Data for these parameters were fitted by least square model of linear response to both standard and test material through a common zero. Regression line for linear response of total pyridine nucleotide content in liver to nial:in intake is presented in Fig. 1. Relative availability of niac in equivalent from test material was determined by interpolating the amount of niacin that would have given the same value for the response criterion . Relative bioavailability of niacin equivalent assessed by taking pyridine nucleotides content as response measure was 98 and 48% for DSM and pearl millet, respectively. However, relative bioavailability determined using weight gain as response measure (fig. not presented) was 108.2 and 65% for DSM and pearl millet, respectively which are much higher estimates compared to the values determined taking hepatic pyridine nucleotide content as response measure.

32.2±.9 33 .5 ± I. 7 47 .5±1.9 14.0±.8 34.0±1.7 34.0± 1.8 54.3±1 .8 20.3±1 .0 33 .2± 1.5 35 .9± 1.9 494±1.7 13.5±.2 34.1 ± 1.9 35 .8±2 .6 55.1±2.1 19.3±0.6

Table 4--Correlation of ci ifferent response measures to niacin intake

Parameter Correlation coefficient (r)

0.89 Weight gain Hepatic contents of pyrid ine nucleotide:

NAD NADH NADP NADPH

0.99 0.96 0.98 0.98

Total hepatic pyridine nucleotides content 0.98

1.4 0: ILl 1.3 > :::; 1.2

'" "- 1.1 Ul U.I 9 1.0 I-0

0 .9 U.I -' u

0.9 :::> 2

0.7

U.I 0.6 2 a 0.5 -0: >- 0 .4 Q..

0.3 ~ 0 0.2 E ~ 0.1 -

0

a

•

-----. STANDARD

,"--- -i> DRIED SKIM MILK

G--- ' -<> PEARL MILLE T

200 400 600 800 1000 1200

NIACIN INTAKE (JIg) , I

2.53 4.93

DRIED SKIM MILK INTAKE (g)

o 7~2 11~9 PEARL MILLET INTAKE (g)

Fig . I-Regression lines of linear response of hepatic pyridi ne nucleotides content to niac in intake, from standard diet, with pure niacin and test diets , containing DSM and pearl millet as source of niacin eql!ivalent in rat passing through common zero.

36 rNDlAN J. EXP. BIOL., JANUARY 1999

It is clear from our study that to assess the true niacin potential of food material, bioassay involving hepatic pyridine nucleotide content as response measure is more valid and accurate as compared to presently l:sed methods involving chemical analysis and bioassays involving weight gam as response measure.

Acknowledgment

Rahul Behl gratefully acknowledges the fellowship awarded by NDRI, Kamal during the course of this study.

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