Indian Journal of Experimental 8 tu10gy Vol. 39, May 2001, pp. 441 -446

Sub chronic toxicity studies of lactulose in rats

V Baskaran, K Narasimha Murthy*, Mahadevamma, S Vishwanatha & B R Lokesh Department of Biochemistry and Nutrition, Central Food Technological Research Institute, Mysorc 570 013, India

Fax: 0821-516308. E-mail: director@cptri .rcn.nic.in

Received 8 March 2000; revised 15 Januwy 2001

Lactulosc has profound health benefits by way of increasing bifidobacterial Oora in the intestine of infants thereby protecting them against enteric infection , constipation and systemic encephalopathy. In the present study to assess the sub chronic toxicity of lactulosc syrup, the rats were fed on a basal feed supplemented with lactulosc syrup at 0.5, 1.0, 2.0 and 5.0 % for a period of 21 weeks. Monitoring of food consumption, gain in body weight and physical observations did not reveal any treatment-related toxicity in any of the group of rats. Terminal autopsy also did not reveal any signs of toxicity. Further, no significant alterations in relative organ weight, serum biochemistry and urinalysis were observed up to I% lactulose supplementation level. The results suggest that supplementation of lactulose in the diet docs not produce any toxicity at the doses tested.

Lactulose (0-fructopyranosyi-D-fructofuranose), a disaccharide, is a keto analog of lactose. It is present in very small quantities in certain foods 1 and heat treated milk and milk products2

·3 to the extent of 0.07 to 0. I%.

Although lactulose was synthesized as early as in 1930 by Montogomery and Hudson4

, its nutritional significance as a bifidogenic factor was not known until late fifties5

. Since then, lactulose has been used as a bifidogenic factor in various food formulae3

·6

·.

With currently available technology, lactulose can be produced from agricultural byproducts7 and from lactose8

. At present, the amount of lactulose that is being used globally in the preparation of nutritional and pharmaceutical products is estimated to be at 6000 tonnes per annum9

. Lactulose has been approved as a special food material in Japan for health maintenance and protection against enteric disorders.t 0

. Nagendra et 16 II h d h' h . f . . a . · ave reporte 1g er retention o llllcronutnents,

lower caecal pH and increased growth of bifidobacte1ial flora in caecum of rats and faeces of infants fed with lactulose. Further, Baskaran et al. 12

·13

have reported that lactulose supplementation in infant milk food formula does not alter the protein quality (nutritional aspect) of the product nor cause any ill effects (short-term toxicity) in rats and its safety is similar to that of sucrose 14

• Lactulose has also been used successfully in the treatment of systemic encephalopath/5

. Since lactulose is non caloric and does not exhibit any carcinogenic properties 12

·, it has a potential application in speciality foods like infant

*Corrcsponctcnt author

foods and ice creams. Lactulose also has additional health benefits due to its ability to create an acidic environment within the colon for growth of bifidogenic bacteria6

.

We have already developed a milk based infant food formula containing lactulose as a bifidogenic factor 16

and unraveled its nutritional aspects and carried out short-term toxicity trials in rats and in infants. Nevertheless, there is paucity of data on the long-term feeding effect of lactulose. Therefore, the present study was undertaken to assess the toxicity (oral) of lactulose syrup (50% strength) at different doses for 2 I weeks in laboratory rats.

Materials and Methods

Test substance Lactulose syrup (containing 50% lactulose) was

prepared by the alkaline isomerization method8 which involved treating lactose solution (15° Brix) with calcium hydroxide (0.04 M). This resulted in 30% isomerization of lactose to lactulose. The mixture thus obtained was subsequently cooled to room temperature and clarified using a cation (Amberlite-IR C50, BDH) and an anion (Indiana-80 BDH) exchange resins separately. The clarified syrup was concentrated to 55° Brix using a falling film evaporator (Turbo-film votator, USA), at 40-45°C and 25" Hg (vacuum) and allowed to stand at room temperature for 12 hr. The quiescent storage resulted in separation of lactulose from the unreacted lactose. After removal of the unreacted lactose through crystallization at 4 °C, syrup

442 INDIAN J EXP BIOL, MAY 2001

containing 50% lactulose was obtained. The lactu lose content of the syrup was estimated spectrophotometrically (Shimadzu, UV, 160 A) according to the method of Nagendra and Yen kat Rao8

.

Animals and diets Six week old male albino rats (Wistar-CFT strain)

obtained from the Institute Animal House facility were statistically grouped by randomized design and assigned to five groups of 8 animals each. They were housed individually in stainless steel cages with screen bottom in a room maintained at 25 ± 2°C temperature, a relative humidity of 45-70% and under 12:12 hr light and dark cycle. The basal diet fed to the animals consisted of casein 18%; sucrose (cane sugar) 10%; mineral mixture 2% (ref. 17); vitamin mixture 1% (ref. 18); groundnut oil 9% and corn starch to make up the final component to 100%. Lactulose syrup was incorporated in the diet at 0.5, 1.0, 2.0 and 5.0% levels. Fresh batch of diets were prepared each week and stored at 4°C. Food and water were given ad libitum. The dosages were selected based on the findings of our

I. d' " 13 d d' ear 1er stu 1es -· an are accor mg to the recommended toxicity testing procedures of FDA 19.

Daily food intake was monitored and weekly body weights were recorded. During the course of the experiment all the animals were carefu lly monitored for any signs of toxicity. At the end of the experiment, all the animals were humanely killed under light ether anaesthesia.

Clinical chemisfly Blood samples were collected from 6 rats each from

a given group at the end of 21 weeks feeding trial, under light ether anaesthesia and the serum was separated. The following parameters were analyzed spectrophotometrically. Serum glucose was measured by the glucose oxidation method of Huggets and Nixon20 and albumin was quantitated according to dye binding method of Cooper21 . Urea nitrogen and globul in were estimated according to the procedure of Wybenga et al. 22

, while creatinine was estimated by the Falin's method by reacti ng it with alkaline picrate solution according to the procedure of Oser23. Serum was analyzed for various enzymes, viz. glutamic oxaloacetic transaminase (GOT) (aspartate amino transferase); glutamic pyruvate trans::~minase (GPT) (alanine amino transferase); alkaline phosphatase (ALP) and acid phosphatase (ACP) according to the method of Reitman and Frankel24

. Total protein was measured by the Lowry's procedure25

.

Urinalysis During the last week of the experiment, urine

samples were collected over a period of 24 hr from each rat and examined for physical appearance, refractive index, pH, glucose, albumin, ketone bodies and bile salts employing standard procedures26.

Organ weight and histopathology At the end of 21 weeks experiment, the animals were

autopsied and the vital organs, viz. li ver, kidney, spleen, heart, brain, caecum and testes were excised, weighed and organ/body weight ratio~ were calculated. Portions of these organs were fixed i buffered I 0% formalin and 5 IJm thick paraffin sections were stained with haematoxylin and eosin according to Lillie27 and microscopic examinations were carried out.

Haematology Blood sa1 pies collected out of 6 rats from each

group were examined for haemoglobin concentration according to the cyanmethaemoglobin method28

. The peripheral counts of red blood cells (RBCs), total white blood cells (WBCs) and differential count of WBCs were carried out according to the method of Bharucha et a/.29

, while packed cell volume (PCV) was measured using micro capillaries supplied with the microhaemarocrit centrifuge (Thomas Scientific Co., USA).

Statistical design of the data (ANOV A) was carried out with 6 rats per group, according to the method pf Snedecor and Cochran30.

Results and Discussion Dietary feeding of lactulose to rats at different levels

for 21 weeks did not produce any signs of toxicity or mortality. However, rats fed with lactulose beyond 2.2glkg body wt/day level experienced mild diarrhoea as evidenced by semi sol id stool. The rats however were free from diarrhoea wi th in 3 to 5 hr of feeding lactulose. These observations are in agreement with those who fed rats with non-digestible disaccharide/maltulose especially ~ t high levels of . . 31·33 Tl I I . mcorporat10n 1e resu ts are a so 111 agreement with the findings of sub chronic toxicity (oral) studies using polysaccharides like hydroxymethyl cellulose and hydroxypropyl cellulose34

Supplementation of lactulose syrup to basal diet at different levels did not signi ficantly affect the food intake. The total lactulose consumed calculated based on the total average food intake per rat in 21 weeks expressed as g/kg body wt./day ranged from 1.1 to 11 .3

BASKARAN eta/.: SUB CHRONIC TOXICITY STUDIES OF LACTULOSE IN RATS 443

for lactulose supplementation of 0.5-5.0% (Table 1). Although there was a slight reduction in food intake at the higher lactulose supplementation levels, it did not reflect adversely on the overall growth rate. The results

are in agreement with the reports of Elder35 and Me Collister et al. 36 in experimental rats fed with cellulose or its derivatives.

There were no marked differences in the absolute or

Parameters

Table 1-Growth rate and feed effic iency of rats fed on lactulose supplemented diet

[Values are mean ± SE of 8 rats (ANOV A)]

Lactulose quantum in diet(%) 0.0 0.5 1.0 2.0

Lactulose intake g/kg b.wtlday 0.0 1.1 2.2 4.0

Initial body weight (g) 89±4 89 ± 3 89 ± 4 89 ± 4

5.0

11.3

89 ± 3

Increase in body weight (g in 21 week) 340 ± 20.5 335 ± 22.5 320 ± 18.5 330 ± 22.5 336 ± 18.5

Av .Food consumption (g/kg b.wtlday) 51.2 ± 2.5 44.9 ± 2.3 43 .3 ± 1.8 39.8 ± 3. 1

Feed efficiency ratio (FER) 0.25±0.0 1 0.30±0.01 0.30±0.01 0.32±0.01

Table 2-Absolute and relative organ weight of rats fed on lactulose supplemented diet Values are mean weight ± SE of 8 rats

Organs Absolute organ weight (g) at lactulose level(%) in feed 0.0 0.5 1.0 2.0

40.7 ± 2.9

0.28±0.01

5.0

Liver 12.24 ± 1.01 11.71 ± 0.8 1 11 .68 ± 0.68 12.69 ± 0.58 12.97 ± 0.69

Kidney 2.28 ± 0.16 2.27 ± O.Q7 2. 19±0. 10 2.34 ± 0.15 2.36 ± 0.10

Heart 1.21 ±0.13 1.11 ± 0.03 1.05 ± 0.06 1.12 ± 0.05 1.16 ± 0.04

Spleen 0.86 ± 0.11 0.65 ± 0.06 0.73 ± 0.07 0.79 ± 0.06 0.75 ± 0.05"

Brain 1.72 ± 0.07 1.72 ± 0.07 1.79 ± 0.02 1.74 ± 0.03 1.71 ± 0.05

Testes 3.46 ± 0.19 3.2 1 ±0.14 3.46 ± 0.16 3.31 ± 0.09 2.10 ± 0.82

Caecum 7. 10±0.1 1 7.08 ± 0.07 7.28 ± 0.09 7.88 ± 0.14" 9.23 ± 0.22"

Relative organ weight (g/1 OOg body weight)

Liver 2.85 ± 0.1 1 2.76 ± 0.11 2.81 ± 0.08 3.07 ± 0.25 3.05 ± 0. 13

Kidney 0.54 ± 0.03 0.54 ± 0.03 0.52 ± 0.02 0.57 ± 0.25 0.56 ± 0.03

Heart 0.28 ± 0.01 0.26 ± 0.0 1 0.25 ± 0.03 0.27 ± 0.02 0.27 ± 0.02

Spleen 0.20 ± 0.02 0. 15 ± 0.01 0. 18 ± 0.02 0.19 ± 0.02 0.20 ± 0.02

Brain 0.4 1 ± 0.02 0.4 1 ± 0.02 0.43 ± 0.02 0.42 ± 0.02 0.4 1 ± 0.03 Testes 0.82 ± 0.03 0.77 ± 0.06 0.84 ± 0.05 0.78 ± 0.05 0.78 ± 0.04 Caecum 1.70±0. 12 1.68 ± 0.07 1.77 ± 0.04 1.92 ± 0.04" 2. 17 ± 0.12"

"P<0.05 (ANOV A)

Table 3-Haematological profile of rats fed on diet supplemented with lactulose [Values are mean ±SE of 6 rats]

Parameters

Hb (g/d l)

RBCx 106/).ll

WBCx l03/).ll

~f PCV (%)

0.0

13.8 ± 0.3

8.9 ± 1.0

13.1 ± 7. 1

23.0 ± 1.3

78.8 ± 1.6

2.6 ± 0.4

1.0 ± 0. 1

37.6 ± 1.1

0.5

13.4 ± 0.3

9.6 ± 0.8

14.1 ± 6.9

24.8 ± 1.5

72.2 ± 2.4

2.4 ± 0.7

1.1 ± 0.1

37.8 ± 0.4

Lactulose quantum in die t (%) 1.0 2.0

14. 1 ±0.4

9.9 ± 0.7

11.8 ± 9.5

22.2 ± 1.5

75 .8 ± 1.4

1.6 ± 0.4"

0.9 ± 0.2

37.6 ± 0.9

14.5 ± 0.4

10.4 ± 0.8"

14.6 ±15.8

20.2 ± 1.6

78.0 ± 1.4

2.0 ± 0.3

1.3±0.4

37.8 ± 0.6

5.0

14.1 ±0.4

9.0 ± 0.5

12.8 ± 20.6

26.0 ± 3.6

72.4 ± 1.9"

3.4 ± 0.7

1.6 ± 0.3

37.4 ± 0.9

Hb-Haemoglobin , RBC-Red blood cells, WBC-White blood cells, DC-Differential count, N-Neutrophil , L-Lymphocyte, M­Monocyte, E- Eosinophil , PCV-Packed cell volume. "P<0.05 (ANOVA)

444 INDIAN 1 EXP BIOL, MAY 200 1

Table 4-Scrum biochemical constituents and enzymes in rats fed with lactulose supplemented diet [Values are mean± SE of 6 rats]

Parameters Lactulose guantum in diet(%) 0.0 0.5 1.0 2.0 5.0

Glucose (mg/d l) 96.0 ± 6.2 97.2 ± 5.6 95.8 ± 4.2 97.3 ± 5.2 98.6 ± 5.2

Albu min(g/d l) 3.7 ±0.1 3.9±0.1 4.2 ± 0.4 4.6 ± 0.8 3.8 ± 0.6

13.9± 1. 1 13.8 ± 2.0 13.6 ± 1.8 14.2 ± 2.6 14.4 ± 2.8

Globulin (g/dl) 2.8 ±0. 1 2.6 ± 0.2 3. 1 ± 0.2 3.2 ± 0.3 3.0 ± 0.2

Total protein (g/dl) 7.2 ±0. 1 7.8 ± 0.8 8.0 ± 1.0 7.6± 1.5 8.2 ± 1.4

Creatinine (mg/dl) 1.1 ± 0.02 1.2 ± 0.0 1 1.4 ± 0.04 1.1 ± 0.01 1.3 ± 0.20"

GOT (~J.mol/min/ml ) 0.66 ± 0.0 1 0.72 ± 0.0 1 0.68 ± 0.01 0.75 ± 0.02 0.79 ± 0.02"

GPT (~J.mol/min/ml ) 0.23 ± 0.0 1 0.26 ± 0.01 0.28 ± 0.02 0.31 ± 0.02 0.33 ± 0.02"

ALP+ 0.013 ±0.00 1 0.0 15±0.001 0.0 15 ±0.00 1 0.0 16 ±0.001 0.0 18 ±0.002"

ACP+ 0.015 ±0.00 1 0.0 15 ±0.00 1 0.0 18 ±0.002 0.020 ±0.002 0.01 8 ± 0.00 1

GOT -Glutamic oxaloacetic transaminase, GPT -Glutamic pyruvic transaminase, ALP-Alkaline phosphatase, ACP- Acid phosphatase +expressed as ~J.mol p-nitrophenol formed /min/mg creatinine

"P<0.05 (ANOVA)

Table 5- Urinalysis of rats fed with lactu lose supplemented diet [Values arc mean± SE of 6 rats]

Lactu lose guantum in diet(%) Parameters 0.0 0.5 1.0 2.0 5.0

72.6 ± 3.0

6.4 ± 1.0

285.4 ± 4.0

Volume (ml/day/kg b.wt)

pH at 24 hr.

Rl at 24 hr.

Glucose

Albumin

Ketone bodies

Bile salts

77.2 ± 4.5

7.6 ± 1.0

273.5 ± 2.0

75.9 ± 3.5

6.2 ± 1.0

275.2 ± 4.5

64.3 ±4.0

6.8 ± 2.0

279.2 ± 3.5

65.8 ± 2.5

6.4 ± 1.0

280.8 ± 3.5

- indicates not detected (ANOVA), Rl- Refractive index measured at ambient temperature (26±2°C).

the relati ve weights of various vital organs in the lactulose treated rats compared to control (Table 2). However, significant increase (P<0.05) in the caecum weight of rats fed with lactulose at 2 and 5% levels could be attributed to compensatory hypertrophy which is considered to be an adopti ve mechanism rather than a pathological one37

• Increase in caecum weight in rats fed with diet containing ketopolysaccharides has also been reported38

. This effect is commonly observed with poorly digested starches and dextrins39

·8 and other

fermen table carbohydrates40.

Haematology and Histopathology The results of the various haematological parameters

are presented in Table 3. No significant differences were observed in different haematological indices except for a significant increase in red cell count at 2% lactulose level and decrease in monocyte differential

count at 1% lactulose level when compared to controls. Such changes have been attributed to normal biological variations and are presumably not caused by the compound itselr'. Me Collister et al. 36 have also reported similar findings when feed ing rats on cellulose derivatives up to 3%.

Gross examination and even microscopic examination of various vital organs on autopsy did not reveal any abnormal ities.

Clinical chemistry Various parameters studied under clinical chemistry

revealed no major treatment related changes except for slight elevation (p<0.05) in the activities of serum enzymes like GOT, GPT, ALP and creatin ine at the highest lactulose supplementation level (5%) (Table 4). Excepting for the above changes, all the other values for serum biochemical constituents and enzymes were

BASKARAN eta/.: SUB CHRONIC TOXICITY STUDIES OF LACTULOSE IN RATS 445

comparable with those of the control group and were well within the normal limits documented for laboratory rats 42

.

Urinalysis No significant difference was observed with respect

to different physico-chemical characteristics for urinalysis among different experimental groups as compared to controls (Table 5). However, the urine pH tended to remain low in all the experimental rats.

In conclusion, the results of the present study clearly show that lactulose up to 5 % level in the diet do not cause any toxicity in rats as evidenced by parameters such as growth, organ weights, histology, haematology, clinical biochemistry and urinalysis. However, rats fed on higher levels of lactulose were prone to temporary diarrhoea and enlargement of caecum. Based on our findings 0.5% lactulose supplementation in infant food formulas is quite safe and is sufficient to induce growth of beneficial microflora in the caecum of rats and in human subjects which is equivalent to a concentration of l.lg/kg body wt/day for rats.

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