Ameliorative Role of Threshold Level of Bilirubin in Newborn Jaundice and Efficacy of Noni Fruit Extract on Phenyl Hydrazine Induced Jaundice Rat

 

U.S. Mahadeva Rao1*, B. Arirudran 1 and S. Subramanian2

1Department of Bio-Chemistry, S.R.M. Arts and Science College, Kattankulathur-603203

2Department of Biochemistry, University of Madras, Guindy Campus, Chennai-600 025

 

ABSTRACT:

The aims of the present work were to elucidate the role of bilirubin as a free radical scavenger in unconjugated hyperbilirubinemic newborns. Bilirubin above an optimum level is toxic to human systems and is extcreted in urinary and through gastrointestinal tract. It is observed that serum bilirubin up to 85µmole/l (5mg/dl) has a free radical scavenger activity and exceeding 200µmole/l (12mg/dl) is highly pro-oxidant. Phototherapy is the accepted therapeutic management of jaundiced newborns and has been shown to augment the oxidative stress. The ethanolic extract of Noni fruit administered orally at a dose of 3mg/ml is found to diminish the oxidative stress in erythrocytes of phenylhydrazine –induced unconjugated hyperbilirubinemic jaundiced rats, treated with phototherapy, which ultimately reduces the bilirubin level without inducing additional damages.

 

KEYWORDS: Bilirubin, free radical scavenger, Pro-oxidant, Phototherapy, jaundice, Oxidative stress, Noni

 

 

INTRODUCTION:

Bilirubin is derived from the breakdown of heme proteins which are present in hemoglobin, myoglobin and certain heme containing enzymes. Three fourth of bilirubin comes from hemoglobin catabolism. One gram of hemoglobin results in the production of 580µmole/l (34mg) of bilirubin. A normal term newborn produces about 6-10mg/kg/ day of bilirubin.

 

Bilirubin, a linear tetrapyrrole is the degradative product of hemoproteins. The heme ring released from hemeprotein is metabolized by heme oxygenase to produce carbon monoxide, free iron and green biliverdin. Biliverdin reductases (BVR) reduce bilirubin to yellow bilirubin, utilizing NADPH.

 

The hydrophobic bilirubin is converted to bilirubin-diglucuronide in the liver by uridine diphospho glucuronate glucuronosyl transferase to facilitate its excretion into the bile. The elevated bilirubin level > 85µmole/l (>5mg/dl) is obvious in the appearance of jaundice, especially in newborn of unconjugated hyperbilirubinemic condition.1, 2

 

The transient newborn jaundice, otherwise called as “Physiologic jaundice”, is a physiological predominantly unconjugated hyperbilirubinemia with clinical jaundice, affecting about half of all human neonates during first five days of life.

It results from increased bilirubin production and delayed maturation of liver UDP-glucuronyl transferase activity. Maternal - fetal Rh blood group incompatibility and hereditary hyperbilirubinemia syndromes exaggerate the condition, which, if untreated, can lead to kernicterus+. [+ Kernicterus is an encephalopathy  associated with degeneration and yellow pigmentation of basal ganglia and other nerve cells in the spinal cord and brain3.

 

High risk infant should be identified so that they can be kept under close surveillance, and any treatable condition promptly managed. The following risk factors place infants at high risk:

·        Asphyxia

·        Trauma

·        Metabolic disorders, e.g. Hypoglycemia, acidosis

·        Sepsis

·        Intestinal obstructions

·        Intrauterine infections

·        Enzyme deficiency, e.g. G6PDH deficiency

·        Hypothyroidism.

 

Though there are several specific preventive measures such as phenobarbitone, agar-agar, tin-protoporphyrin etc., by considering relative degree of side effects, the phototherapy is considered as commonest form of accepted treatment and is effective in almost all cases, if high intensity phototherapy is used.

 

At highly elevated level > 200µmole/l (>12mg/dl), unconjugated bilirubin impairs the antioxidant system of the erythrocyte, augments oxidative stress i.e., elevates reactive oxygen species (ROS) formation, induces morphological alteration and loss of phospholipids symmetry of RBC4 , disturbs the membrane transport systems of erythrocytes5 , and can cross the blood-brain-barrier (B-B-B) causing kernicterus6 .The currently available phototherapy devices  such as fluorescent tubes, halogen spotlights, and fileroptic blankets, though having many disadvantages, like, high heat production, unstable broad wavelength light output etc., it is regarded as the accepted modality for newborn jaundice management. Recent literature also reveals that phototherapy is an oxidative stress and can cause lipid peroxidation7-9.

 

Evidence also support that bilirubin acts as antioxidant by scavenging peroxyradicals, protects cell against complement-mediated anaphylaxis, myocardial ischemia, pulmonary fibrosis10 etc. Antioxidant activity of bilirubin is anticipated by cycling between bilirubin and biliverdin. Bilirubin interacts with ROS, neutralizes its toxicity and transforms to biliverdin, which is then subsequently reduce by BVR to regenerate bilirubin. It is obscure to rationalize on the exact mechanism of, how bilirubin interacts with ROS to reduce its toxicity and, on the other hand, at elevated levels, increases ROS formation. Elevated bio-availability of ROS, in turn, disturbs membrane redox potential, lipid and protein oxidation4, 5 etc. NADPH, the major product of PPP of erythrocyte2 is also expected to render a crucial role in the bilirubin-ROS interaction.

 

The present work has investigated the FRSA of optimum level of bilirubin, by assessing the mode of bilirubin-biliverdin conversion and measuring BVR activity under normal and elevated levels of bilirubin in the blood of newborns. The antioxidant status is further ascertained through venous blood by assaying the MDA, total thiols, GSH, ascorbic acid, SOD, and albumin. G6PDH and 6-PGDH of PPP have been assayed in newborn jaundice patients, before and after phototherapy to assess the production efficiency of NADPH. The level of TK has been assayed to explore the thiamin status of newborns. The effects of ethanolic extract of Noni fruit to diminish the detrimental side effects of jaundice in experimental rats treated with phototherapy have also been studied.

 

MATERIALS AND METHODS:

Antioxidant status in jaundiced newborns before and after phototherapy:

The study was carried out on 30 full term jaundiced newborns with appropriate weight, delivered normally in the labour room of Mahatma Gandhi Memorial Hospital, Addis Ababa, Ethiopia, and East Africa. These infants received continuous phototherapy except during feeding, cleaning and sampling. It was   ensured that the jaundice was non-hemolytic by Van Den Bergh diazotised sulfanilic acid test. Bilirubin levels were monitored for all infants at 24 hours interval. All the parameters estimated before and after phototherapy. These were also assessed in cord blood samples of 20 newborns as control that didnot develop jaundice in the neonatal period. Three ml of venous blood was collected aseptically and hemolysate was prepared by osmotic shock method served as s source of enzymes of RBC4.

 

Biochemical parameters assayed in neonates:

Hemoglobin level of this hemolysate was estimated by Drabkin and Austin [11] method and diluted to 1%g using distilled water, which was subsequently used for the following estimations:

 

MDA was estimated using the principal that lipid products react with thiobarbituric acid to give a red chromogen whose absorbance was read at 548nm spectrophotometerically12. Total thiols were estimated by Ellman reaction in which 5-5’ dithiols-2-nitrobenzoic acid react with total sulfydryl groups and measured spectrophotometrically at 420nm13. GSH was analyzed using the method of Beutler et al.,14.

 

Ascorbic acid was determined using the property of its oxidation to dehydroascorbic acid in presence of Cu2+, which then reacts with 2,4-dinitrophenyl hydrazine (DNPH) to form a red bis-hydrazone having absorbance maximum at 529nm15.

 

SOD levels were estimated in the hemolysate by the method of Misra and Fridovich based on inhibition of auto-oxidation of epinephrine to adrenochrome at pH 10.216.

 

Albumin was determined in plasma using the dye binding method of Doumas and Biggs17. The interaction of bilirubin and biliverdin with free radical was studied using potassium superoxide (KO2) dissolved in acetonitrile containing dicyclohexane -18-crown-6-ether and characterized by  UV-Vis  spectrophotometer (spectrascanUV2600). The superoxide solution- crown ether complex was prepared18, 19 by weighing KO 2 and quickly adding to the equivalent moles of the crown ether in acetonitrile and stirred in sealed condition for 30mintes.

 

The biliverdin concentration in the serum was measured by previously described method20 and bilirubin concentration was estimated by spectrophotometric method21. The spectroscopic method was correlated with Malloy and Evelyn’s22 method and the correlation coefficient were found to be 0.9.

 

The NADPH- dependent BVR activity was assayed at 37C0 for 5 minutes23.  The incubation mixture was contained 60µM NADPH dissolved in 10 µM potassium phosphate buffer (pH 7.4) and 13 µM of biliverdin.

 

G6PDH activity was assayed in the hemolysate utilizing the method of Begrmeyer24 . The assay mixture contained 0.05M triethanol amine buffer (pH 7.5), 0.003M NADP+ and 0.004M glucose-6-phosphate. The activity of 6-PGDH was measured according to the previously described method25. The reaction mixture contained 0.04M glycerol -glycine buffer (pH 9), 0.1M MgCl2, 0.04M 6-phosphogluconate and 0.003M NADPH. The activity of TK was also determined by standard method26. The assay mixture contained 0.01M glycyl-glycine buffer (pH 7.5), 0.02M ribulose-5-phosphate, 0.02M ribose-5-phosphate, 0.003M NADH, 0.1ml of α-glycerophosphate dehydrogenase (0.01mg/ml).

 

Noni fruit Extract preparation for animal model study:

The FRSA of the Noni fruit was measured with DPPH assay27 . The ethanolic extract of Noni fruit   was prepared by standard method28. An ethanolic solution of DPPH (100mM) was incubated with an ethanolic solution of the Noni fruit extract (0.50- 4mg/dl) and the absorbance was monitored spectrophotometrically at 520nm. The concentration (IC0.20) of the test compound that induced a decrease of 0.20 in absorbance during a 30 minute absorbance was taken as measure of antioxidant potency. It was used as the standard dose for administration to the rats. The herbal extract was administered orally for 3 days (300mg/kg b.w/rat/day).

 

Experimental Animals:

The animals’ experiments were designed and conducted according to the ethical norms approved by Ethiopian Government and Institutional Animal Ethics Committee (IAEC) for the investigation of experimental pain conscious animals. Before beginning the experiments, the rats were allowed to acclimatize to animal house condition for a period of one week. Throughout the experimental period, the rats were fed with balanced pellet diet with composition of 5% fat, 21% protein, 55% nitrogen-free extract, and 4% fiber (w/w) with adequate mineral and vitamin for the animals. Diet and water were provided ad libitum.

 

Experimental design:

Adult albino rats of Wister strain of both sexes (weighing 180±20g) were fed with basal diet and water and maintained under standard laboratory conditions. Rats were induced jaundice by feeding orally with aqueous solution of 1% phenyl hydrazine29 once daily for 3 days. The rats were later given phototherapy (with 420-450nm lamp) for 1 hour with an interval period of 30 minutes with (group IV)   and without herbal extract (group I) against control rats. Group II rats received phenyl hydrazine along with ethanol. The group III rats were given extract only after phenyl hydrazine administration.

i.e. Groups of rats receiving different treatment is summarized as follows:

Group I- Administrated with phenylhydrazine and then treated by phototherapy;

Group II- Administered with phenylhydrazine and ethanol supplement;

Group III- Administered with phenylhydrazine and crude ethanolic extract of Noni fruit (300mg/kg b.w/rat/day).

Group IV- Administered with phenylhydrazine and crude extract of Noni fruit along with phototherapy treatment.

 

Biochemical parameters:

The erythrocyte membrane was isolated from the hemolysate, following standard protocol5 and oxidative damage was assayed by standard markers. Lipids peroxidation (LPO), a marker of cellular damage was assayed by malondialdehyde formation30 and expressed as nmole MDA formed/mg protein and estimated as described previously31. The reaction mixture contained 20% trichloroacetic acid, 0.76% thiobarbituric acid and 0.05M Tris-buffer. Rats were then sacrificed by cervical dislocation and livers were excised and homogenized in 0.25M sucrose containing 1mM EDTA. 4-Hydroxynonenal (HNE) adducts formed during ROS-mediated damage, were also studied by immunoblotting technique in treated and control rats32. Proteins (albumin) content was estimated by the method of Lowry et al.33.

 

Statistical analysis:

The values were expressed as mean ± SD for six rats in each group. All the data were analyzed with SPSS/7.5 student software. Hypotheses testing method included one way analysis of variance (ANOVA) followed by post hoc performed with Least Significant Difference (LSD) test. The p values of less than 0.001 were considered to indicate statistical significance.

RESULTS AND DISCUSSION:

Neonate Study:

All the 30 newborn patients in the study group receiving phototherapy ranging from 2-4 days and majority received in 3 days or more. The levels of bilirubin and albumin in plasma and their ratio when compared between controls and in patients before and after phototherapy are depicted in Table 1. The erythrocyte levels of MDA (indicator of oxidative stress) and antioxidants like GSH, total thiols, ascorbic acid and SOD in three study groups are tabulated in Table 2.

 

From the above data, it is revealed that the levels of MDA rose markedly after treatment of newborns with phototherapy. Elevated values of MDA could be due to increased generation of ROS as a result of phototherapy. Since bilirubin acts as a photo-sensitizer and becomes energized by phototherapy, the energy, thus gained, is subsequently transferred to molecular oxygen, thereby generating singlet oxygen and other ROS. These oxygen species, in turn, can oxidize many other important biomolecules including membrane lipids, as well as bilirubin itself. As free bilirubin is mainly responsible for this, the ratio of bilirubin to albumin is important34. Ostrea et al studied cord blood cells, which were exposed to phototherapy in the presence of bilirubin and resulted in a significant increase in concentration of TBARS, diene conjugation and hemolysis35, probably suggesting the red cell membrane lipid peroxidation and hemolysis, secondary to the phototherapy.

 

In Table 2, the RBC’s GSH levels depleted after phototherapy and similarly total thiol levels were also depleted. It is understood from literature that jaundice produce an oxidative stress as witnessed by fall in the levels of cellular GSH, glutathione peroxidase (GPx) and SOD in various studies36, 37 .

 

From Table 2, it is further explained that the ascorbic acid levels showed a marked decline after phototherapy, but SOD levels in hemolysate exhibited a rise. The elevation of SOD could be due to its induction to counter the effect of enhanced oxidative stress. Plasma albumin levels decreased after phototherapy. Phototherapy, therefore, results in oxidative damage to RBC as indicated by marked fall in FRSA.

 

The interaction of bilirubin and biliverdin with superoxide radical:

In the reaction of bilirubin with superoxide, it is obvious to note that the bilirubin interacts, can quench the superoxide radical (O2.-), as observed by depletion of absorption maximum at 440nm. A stable product with absorption maximum at 450nm also appears. After keeping the solution in dark for a day, the product absorbing at 540nm is diminished and absorption around 435nm is increased.  Along with other products, absorbing in the same region, a part of bilirubin might have been regenerated.

 

 

Table 1: Plasma bilirubin, biliverdin, and albumin levels, bilirubin-albumin ratio and BVR activity in controls and in jaundiced newborns (pre and post-phototherapy).

Content

Controls

Before phototherapy

After phototherapy

Bilirubin (mg/dl)

1.41± 0.23

14.92±1.91*

8.61±1.4**

Biliverdin (mg/dl)

0.441±0.031

0.451±0.027*

2.45±0.176*

Albumin (g/dl)

3.61±0.09

3.55±0.46

2.99±0.19*#

Bilirubin-Albumin ratio  ( x 10-3)

0.51± 0.11

6.11±0.72*

4.72±0.5***

BVR(nmole bilirubin/min/mg protein)

8.31±0.415

____

0.917±0.21*

*P<0.001 as compared to controls; **P<0.001 as compared between two study groups; # P< 0.01 as compared between two study groups.

 

Table 2.  Levels of MDA, Total Thiols, GSH, Ascorbic acid and SOD in controls and newborn jaundiced patients (Pre and Post-phototherapy)

Content

Controls

Before phototherapy

After phototherapy

MDA(nM/gHb)

3.41±0.92

4.58±0.71*

5.58±0.82* **

Total Thiols (µM/gHb)

62.13±6.92

50.01±9.63*

44.97±7.73* **

GSH (µM/gHb)

31.01±3.74

15.76±1.62*

12.72±2.01* **

Ascorbic acid (mg/gHb)

0.901±0.11

0.541±0.07*

0.401±0.09* **

SOD (EU/gHb)

1592.07±391.19

2567.63±432.61*

3011.92±476.56* **

*P<0.001 as compared to controls; **P< 0.001 as compared between two study groups.

 

Table 3: Measurement of activity of G6PDH, 6-PGDH, TK of PPP in erythrocyte of newborn jaundiced patients against age-matched control.

Subject

G6PDH (nmole of NADPH produced/min/mg protein)

6-PGDH(nmole of NADPH produced/min/mg protein)

TK(µmole of NADH oxidized/min/mg protein)

Controls

5.91±0.19

0.567±0.019

0.291±0.011

Neonate patients

( Before phototherapy)

5.01±0.72

0.418±0.006*

0.222±0.025*

Neonate patients

( After  phototherapy)

4.36±0.03

0.328±0.10

0.136±0.018

*P<0.001 as compared to controls

 

 

But in the reaction of bilirubin with superoxide, it is observed that the characteristic absorption of biliverdin at 640nm decreases and simultaneously in the lower wavelength of the absorption spectrum, a red shift with a maximum at around 440nm is observed.  This shifting of absorption maxima of biliverdin indicates the formation of bilirubin after immediate interaction with O2.- radical. After keeping the biliverdin –O2.- solution in dark for 1 day, the absorption due to biliverdin comes lack with some signature of presence of low concentration of bilirubin. Thus, the outcome of interaction of bilirubin and biliverdin with O2.-  hypothesize that both bilirubin and biliverdin can possess FRSA  in vitro.

 

Table 1 show that biliverdin concentration is higher in jaundiced newborns with proportionate decline in bilirubin concentration. The fall of biliverdin concentration in age matched control newborns can be accounted by their elevated BVR activity. The ROS generated in the system is reduced by the endogenous bilirubin present.  So the activity of the redox cycle of bilirubin-biliverdin conversion is found to be normal in the newborns taken as control, maintaining the normal bilirubin level.

 

At mild elevated level of unconjugated bilirubin, the BVR activity is decreased compared to the control group. The ROS generated at mild elevated level possibly lowers the BVR activity and diminishes bilirubin production. Thus the bilirubin-biliverdin interconversion is thus distributed in this group. The bilirubin present may not be adequate to counteract the total ROS generated at that level and gets transformed to bilirubin, resulting in slight increase of bilirubin levels.

 

At excess elevated level of unconjugated bilirubin, the bilirubin level is low and zero BVR activity is found. Thus, the bilirubin is incapable to reduce the ROS generated in the neonate patients during marked increase of bilirubin level. From Table 1, thus it infers that bilirubin can possess FRSA in the newborns of serum bilirubin level up to 85µmole/l (5mg/dl), above which it becomes a pro-oxidant.

 

Table 3 explain that G6PDH activity remains unaltered in the newborn jaundice patients compared to the control, suggesting the supply of NADPH remain unaltered before phototherapy. However, the enzyme activity, such as 6-PGDH and TK, is low in jaundiced newborns. The activity of the G6PDH, 6-PGDH and TK are severely affected after phototherapy. The absorption maximum of bilirubin and vitamin B2 are nearly identical (440-540nm). Phototherapy causes photoisomerization [9] of bilirubin, accompanied by photo degradation of vitamin B2. The loss of activity of G6PDH can be related to the indirect effect of reduction of GR activity, an FAD containing enzyme [4]. The GR recycles NADPH to NADP+, providing the substrate for G6PDH. This recycling is prevented during phototherapy, as NADPH cannot be recycled to NADP+ , and thus G6PDH activity is reduced after the phototherapy.

 

During phototherapy, the rate of conversion of glucose-6-phosphate to 6-phosphogluconolactone, and further to 6-phosphogluconate is decreased. i.e., Hampering the activity of 6-PGDH, along with TK. The diminished activity of the TK reflects the vitamin B1 deficiency of the newborns.  G6PDH deprived cells are facing risk for glycation of protein which, in turn, increases the cellular damage4, 5, 34. Thus, an inverse correlation has been found between the activity of G6PDH and the fragility of RBC. The PPP along with GSH and its related enzymes such as, G6PDH, 6-PGDH   and TK, protect the erythrocyte against fragility and hemolysis [2, 4]. Thus, from the above study, it is suggested that, the phototherapy disturbs the enzymes of PPP and augments the vulnerability of erythrocyte from hemolysis, further thus increasing the oxidative stress in the newborns.

 

Animal model study:

Table 4 depicts the results of the FRSA of ethanolic extract of Noni fruit tested in vitro, by using DPPH and suggest that the extract is a potent antioxidant38. The IC0.20 value of the extract is found to 3mg/ml. The herbal extract of 300mg/kg b.w. /day has been fed to the experimental animals.

 

Table 4: Percentage of DPPH scavenged by Noni fruit extract

No. Observations

Noni fruit extract conc. (mg/ml)

DPPH scavenged (%)

1

0.50

1.9

2

1.00

9.7

3

1.50

28.3

4

2.00

34.6

5

2.50

39.8

6

3.00

42.4

7

3.50

49.6

8

4.00

53.7

 

p=0.004

 

Figure 1.  Percent of LPO inhibition in the erythrocyte membrane of experimental rats by Noni fruit extract

 

From figure 1, it is portraited the percent inhibition of LPO, i.e. MDA formation, in the erythrocyte membrane of the albino rats receiving treatment. The group of rats receiving Noni fruit crude extract after jaundice induction has shown declined LPO, compared to the rats receiving phototherapy only. Crude herbal extract probably scavenges the ROS generated at the high bilirubin level, diminishes its detrimental effect and induces the FRSA of the bilirubin itself.

 

CONCLUSION:

From the above studies, we can put forward that, the optimum concentration of bilirubin in blood may act as an antioxidant (FRSA) and excess concentration of the same acts as pro-oxidant. It is further hypothesized that the phototherapy may elevate the risk of oxidative stress as well as lipid peroxidation and hence recommended to be used with care and caution in the neonatal jaundiced patients.

 

ABBREVIATION:

BVR- biliverdin reductase; DPPH-2,2-diphenyl-1-picrylhydrazyl; FRSA-free radical scavenging activity; G6PDH-glucose-6-phosphate dehydrogenase; HNV-4-hydroxynonenal; LPO- Lipid peroxidation; MDA-malondialdehyde; NADPH-reduced nicotinamide adenine dinucleotide  phosphate;  6-PGDH- 6-Phosphogluconate dehydrogenase;  PPP- Pentose Phosphate Pathway; B-B-B- blood brain barrier;  ROS- reactive oxygen species;  TK- transketolase;  GR- glutathione reductase;  GSH-reduced glutathione

 

REFERENCES:

1.       Text book of Pediatrics (Behrman R E, Vanghen V C, Nelson W E and Saunders W B, eds), Philadelphia, USA, (1992) 14th edn:  476

2.       Murray R K Harper's Illustrated Biochemistry, McGraw Hill, USA (2003) 26th edn, 270-285

3.       Kaplan M, Muraca M, Hammerman C, Rubaltelli F F, Vilei M T, Vreman H J and Stevenson D K Pediatrics  (2002) 110, e47

4.       Mazumder S, Sarkar U and Sengupta D. Indian J Exp Biol  (1995) 33: 303-305

5.       Mazumder S, Sarkar U and Sengupta D Indian J Exp Biol (2000) 38 :91-94

6.       Wennberg R P, Ahlfors C E, Bhutani V K, Johnson L H and Shapiro S M Pediatrics(2006)   117:474-485

7.       Kiran Dahiya, A.D.Tiwari, Vijay Shankar, Simmi Kharb, Rakesh Dhankhar,. Indian J. Cli. Biochem. (2006):21(1) 157-160

8.       N Nag, S Halder, R Chaudhuri, S Adhikary and S Mazumder,   Indian Journal of Biochemistry and Biophysics( 2009) 46:73-78.

9.       Bader D, Kugelman A, Blum D E, Riskin A and Tirosh E. IMAJ (2006)   8: 12-16

10.     Greenberg D A Proc Natl Acad Sci (2002) 99: 15837-15839

11.     Drabkin DL, Austin JH    J. Biol.  Chem. (1932) 98:719-733

12.     Kumar, R., Seth, R.K., Sekhon, M.S. and Bhargava, J.S., Burn. (1995) 21: 96-97..

13.     Ellman, G.L.. Arch. Biochem. Biophys.  (1959). 82: 70-77.

14.     Beutler, E., Duron, O. and Kelly, B.M. J. Lab. Clin. Med. (1963). 61: 882-888.

15.     Mc Cormick, D.B. and Greene, H.L Vitamins, In: Teitz Textbook of Clinical Chemistry Eds. Burtis,CA and Ashwood, ER.. W.B. Saunders Company, Philadelphia,(1999) 3rd ed   999-1028.

16.     Doumas, B.T. and Biggs, H.G.).  Chem. (1972) 7:175-188.

17.     Misra, HP. and Fridovich, I.  J. Biol. Chem. (1972) 247: 3170-3175.

18.     Sun J, Schulman S G and Perrin J H Anal Chem Acta  (1997) 338: 1-2

19.     Valentine J S and Curtis AB  J Am Chem Soc (1975)  97: 224-226

20.     Larson E A, Evans G T and Watson CJ  J Lab Clin Med (1947) 32: 481-488

21.     Varley H, Gowenlock A H and Bell M Practical Clinical Biochemistry, 5th edn, W H Med Book Ltd. London (1984) 1024-1027

22.     Malloy H T and Evelyn K A J. Biol Chem. (1937) 119: 481-90

23.     Cascone O, Frydman R B, Ferrara P, Tomaro M L and Rosenfeld J Eur J Biochem (1989) 179, 123-130

24.     Lohr C W and Waller H D  In: Methods in enzymology (Bergmeyer H ed), Academic Press, New York (1974),636

25.     Yamada K and Shimazono W J Biochem  (1962) 51: 242-245

26.     Horecker B L and Symrniotis P Z In: Methods in enzymology (Colowick S P, Kaplan N O, eds), Academic Press, New York(1955)  Vol. 1:  371

27.     Mellors A and Tappel A L J Biol Chem  (1966) 241: 4353-4356

28.     Liu Y, Shi J and Langrish T A G Chem Eng J (2006) 120: 203-209

29.     Huang W, Zhang J, Chua S S, Qatanani M, Han Y, Granata R and Moore D D Proc Natl Acad Sci (USA) (2003) 100: 4156-4161

30.     Gallucci M T, Lubrano R, Meloni C, Morosetti M, Manca di Villahermosa S, Scoppi P, Palombo G, Castello M A and Casciani C U Clini Nephrol(1999) 52:239-245

31.     Esterbaur H, Schauer R J and Zollner H  Free Radic Biol Med (1991) 11: 81-128

32.     Yoritaka A, Hattori N,  Uchida K, Tanaka M,  Stadtman E R and Mizuno Y Proc Natl Acad Sci(1996)   93:2696-2701

33.     Lowry O H and Lopez J A J Biol Chem (1946) 162:421-428

34.     Jain S K Free Radic Biol Med  (1998) 24:197-201

35.     Ostrea, E.M. Jr., Cepeda, E.E., Fleury, C.A. and Balun, J.E  Acta. Pediatr.  (1985) 74:378-381

36.     BracciR,TalluriB and BerniS. Acta. Paediatr. Scan(1988) 77 (3): 349-356.

37.     Turgut, M., Basaran, O., Cekmen, M., Karatas, F., Kurt, A. and Aygun, A.D.  J. Paediatr. Child Health. (2004). 40 (11): 633-637.

38.     Mahadeva Rao.US and Subramanian S.  J. Med. Chem. Res (2009) 18:433-446

 

Received on 18.05.2011

Accepted on 29.06.2011     

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Research J. Pharmacology and Pharmacodynamics. 3(4): July –August, 2011, 196