Dietary nitroprusside alleviates atherosclerosis in hypercholesterolemic Japanese quail (Coturnix coturnix japanica)

Camp. Eiochem. Physiol. Vol. I IZA, No. 1. pp. 151-154, 1995 Copyright Q 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0300-9629/95 $9.50 + 0.00

Pergamon 0300-9629(95)00076-3

Dietary nitroprusside alleviates atherosclerosis in hypercholesterolemic Japanese quail (Coturnix co turnix japonica > C. H, Hill,* E. P. Pullman,* B. Starch@

and J. C. H. Shih*

*Department of Poultry Science, North Carolina State University, Raleigh, NC 276957608, U.S.A.; and YUniversity of Texas Health Center at Tyler, Tyler, TX 75701, U.S.A. Male Japanese quail (Cotuvnix cotuvnix japonica), susceptible to cholesterol-induced atherosclerosis, were rendered bypercholesterolemic by feeding a diet containing 0.5% cholesterol in two experiments. Half the animals also received a dietary supplement of sodium nitroprusside ranging in concentration from 0.005% to 0.015%. After 10 weeks on the diets, serum was obtained for cholesterol analysis, the animals were killed, and the aortae removed and examined for the presence of atherosclerotic lesions. The number of animals having lesions and the severity of the lesions was reduced in a dose dependent manner among those animals receiving nitroprusside. Serum cholesterol was also reduced in response to increasing levels of dietary nitroprusside. These tindings indicate that, in this model, dietary nitroprusside, a source of nitric oxide, can reduce the appearance and severity of atherosclerotic lesions in the aorta. Key words: Nitric

oxide; Atherosclerosis; Japanese quail; Cholesterol; Hydroxy proline; Desmosine; Resistance; Susceptibility. Comp. Biochem.

Physiol.

Hypercholesterolemia;

112A, 1.51-154, 1995.

Introduction There is a growing body of evidence which suggests that nitric oxide (NO), the endothelium derived relaxing factor originating from the metabolism of L-arginine, could play a role in the development of atherosclerosis. Naruse et al. (1994) reported that inhibition of NO synthesis increased the lesion surface area of descending thoracic aortae of cholesterol-fed rabbits. Wang et al. (1994) found that dietary arginine prevented atherosclerosis in the coronary arteries of hypercholesterolemic rabbits. Casino et al. (1993) concluded that hypercholesterolemia in humans results in a defect in the bioactivity of NO. Several studies using rabbits as the experimental model have indicated CorrespondenceIO: C. H. Hill, Department

that endothelium-dependent vasodilation is impaired in hypercholesterolemia (Verbeuren et al., 1986, 1990; Jayakody et al., 1987; Ragazzi et al., 1989; Simonsen et al., 1991). In order to investigate the effect of an NO donor on the development of atherosclerosis, studies have been conducted using male Japanese quail (Coturnix coturnix japonica) which have been bred for either increased susceptibility or increased resistance to cholesterolinduced atherosclerosis (Shih et al., 1983; Casale et al., 1992).

Materials and Methods

of Poultry Science, Box 7608, N.C. State University, Raleigh, NC 27695-7608, U.S.A. Tel. 919-515-2692; Fax 919-5152625. Received 29 November 1994; revised 8 February 1995; accepted 16 February 1995.

In these studies, male Japanese quail obtained from the colony maintained at North Carolina State University were used. The quail were fed a diet of natural ingredients from the day of hatching until they were 6-8 weeks of age, the age of sexual maturity in this species. At that time, the diet was changed to one supporting

151

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C. H. Hill et al.

egg production and containing 0.5% added cholesterol. Previous work has indicated that neither line of quail will develop atherosclerotic lesions unless they are fed cholesterol. Sodium nitroprusside, a source of NO, was fed at the indicated levels to appropriate lots of quail. The quail were killed by decapitation after receiving the experimental diets for 10 weeks. At that time, blood was obtained for cholesterol analysis, and the aortae removed for scoring for the extent of atherosclerotic lesions, and analysis for desmosine and hydroxyproline. Cholesterol was determined by the method of Zlatkis et al. (1953) after an overnight fast. The method of scoring for atherosclerotic lesions was that used by Shih et al. (1983) 0 representing no lesions and l-4 representing increasing areas of aortae affected. A section of each aorta was removed for histological examination for elastin and collagen, and a 3 mm punch was removed for amino acid analysis. The elastin stain was a modified Verehoeff’s (Roman et al., 1967) and the collagen stain was Gamori’s tri chrom (Luna, 1968). For amino acid analysis, a sample of the aorta was hydrolyzed at 100°C in 6 N HCl for 24 hr, evaporated to dryness, and analyzed for hydroxyproline with a Beckman 6300 analyzer. For desmosine determination, 2 ~1 of each sample were analyzed in duplicate by radioimmunoassay (King et al., 1980; Starcher and Scott, 1992). In the first experiment, 24 resistant and 24 susceptible quail were used. Twelve of each line were fed nitroprusside at a level of 0.025%, while the remaining 12 served as controls. In the second experiment, 60 resistant and 60 susceptible quail were used. Fifteen quail from each line were fed each diet which consisted of a control and nitroprusside at levels of 0.005%, O.OlO%, and 0.025%. In this experiment, the aortae were further evaluated histologically and chemically for their content of elastin and collagen.

The results of the cholesterol analyses were evaluated for statistical significance by analysis of variance. In the first experiment, differences were evaluated by Duncan’s multiple range test. Because of unequal numbers of quail in the various lots in the second experiment, the means were separated by least square means. The General Linear Models Procedure of SAS was used for this analysis. The percentages positive for atherosclerotic lesions were converted to arcsin values for analysis, and Duncan’s multiple range test was used to evaluate the significance of the differences observed.

Results From the results of the first experiment (Table l), it is apparent that the presence of nitroprusside in the diet resulted in a decrease in the number of aortae positive for atherosclerosis as well as a decrease in the extent of the lesions as indicated by the reduced score among the susceptible quail. These reductions were accompanied by a significant decrease in serum cholesterol concentration. Among the resistant quail, only one aorta was positive for atherosclerosis, and serum cholesterol concentration was marginally reduced among those fed nitroprusside. In the second experiment (Table 2), results are given for each replicate of seven or eight birds started in the experiment. As the nitroprusside content of the diet was progressively increased, the number of aortae positive for atherosclerosis decreased among the susceptible quail, falling from 73.3% to 6.7% at the highest level of nitroprusside fed. The extent of the lesions, as indicated by the scores, also fell as the dietary level of nitroprusside was increased. In this experiment, dietary nitroprusside progressively decreased serum cholesterol concentration in both the susceptible and the resistant quail. Statistical analysis indicated that this decrease was significant. Furthermore, the analysis revealed that the differences in serum cholesterol

Table 1. Effect of sodium nitroprusside on serum cholesterol and atherosclerosis NO*

Control Serum cholesterol$ mS/dl Nr positive for Atherosclerosis Atherosclerosis Score

sust

REST

SUSt

REST

713 f 85”

231 _+17b

273 f 37b

188 f 17b

11/12

O/l2

219

l/10

A$ 1.50

Rll 1.64

A§ 0

BB -

A§ 0.22

*Sodium nitroprusside fed at 0.025%. jSUS = susceptible, RES = resistant to atherosclerosis. fMean + SEM. All quail sampled. §Total score/total no. animals. ?Total score/no. animals positive. Means followed by different superscripts differ significantly (P < 0.01).

B? 1

A§ 0.20

B4 2

Nitric Table 2. Effect of sodium

153

oxide and atherosclerosis

nitroprusside

on serum cholesterol

and atherosclerosis

RES*

sus*

Rep I

Not,% 0 0.005 0.010 0.025

1123 k 873 f 736k 653k

0 0.005 0.010 0.025

617 3/8 l/7 O/8

194 290 54 76

(7) (8) (7) (8)

Serum cholesterol, 1037 * 107 861 k 167 674 + 179 560 i 129

Number 518 216 117 t/7

AY 1.33 0.57 0.18 0.07

0 0.005 0.010 0.025

Rep

Rep 2 mg/dl$ (8) (6) (7) (7)

725 658 577 449

*SUS = susceptible, RES = resistant to atherosclerosis. tSodium nitroprusside. fMean f SE, ( ) indicates number. $Reps combined. BTotal score/total no. animals. j/Total score/no. animals positive. Figures followed by different superscripts differ significantly

values between the two lines of quail were significant, that the decline in these values with increasing dietary nitroprusside was linear, not quadratic, in both lines, but that the slopes and intercepts of the regression of serum cholesterol on dietary nitroprusside levels were significantly different between the two lines. Analyses of the aortae for desmosine and hydroxyproline in Experiment 2 (Table 3) did not reveal any differences among treatments or between lines. Histological examination of the aortae also failed to detect any differences related to treatment or strain. The lack of treatment effects may be a reflection of the relatively mild lesions observed at this early stage of development of atherosclerosis.

Discussion The results of these experiments clearly demonstrate that atherosclerosis in the suscepTable 3. Desmosine and hydroxyproline content of quail fed levels of nitroprusside

sus* NO.?%

DMS

HPS

nmol/3 mm biopsy 0 0.005 0.010 0.025

3.47 k 0.66 3.56 & 1.34 3.56 + 0.81 3.80+0.81

221 & 52 202 k 47 191 f 32 212+37

of aortae

RES* DM: HP1 punch5 3.08 f 0.55 3.26 + 0.86 4.01 + 0.73 3.30kO.75

+ 55 & 27 & 48 + 54

positive for atherosclerosis 73.3%” O/8 35.7%“b O/7 14.3%b O/8 6.7%b O/5 Atherosclerosis score sus RI1 1.82 1.60 0.83 1.oo

165 k 31 185 + 32 218 f 61 211+21

*SUS = susceptible, RES = resistant to atherosclerosis. YSodium nitroprusside. $DM = desmosine, HP = hydroxyprohne. §Means + SD. n = total number/treatment as in Table

2.

I

Rep 2 (8) (7) (8) (5)

674 * 179 (7) 686+ 75 (8) 525 k 102 (6) 493,35 (7)

217 l/8 O/6 O/7

13.3% 6.7% 0 0

RES AT 0.13 0.07 0 0

RII 1.00

I .oo

(P < 0.05).

tible strain of quail can be alleviated by the addition of nitroprusside to the diet. A marked reduction in serum cholesterol concentration accompanied the reduction in atherosclerosis in the treated birds. Nitroprusside is a source of NO which has been identified as the active component of an endothelium-derived relaxing factor (Ignarro et al., 1987; Palmer et al., 1987). To the author’s knowledge, this is the first report indicating that dietary nitroprusside would alleviate atherosclerosis and hypercholesterolemia. Naruse et al. (1994) reported that oral administration of an antagonist of NO synthase increased serum cholesterol. This effect is evidently a reflection of intestinal function since Cayette et al. (1994) found that subcutaneous administration of an NO antagonist did not change the serum lipid profile. Wang et al. (1994) found that dietary arginine, a substrate for NO synthase, alleviated atherosclerosis in the hypercholesterolemic rabbit without affecting serum cholesterol concentration. Further experiments are needed to determine whether the quail would respond to dietary arginine in the same manner. The mechanism underlying the effect of nitroprusside on serum cholesterol is unknown. It seems possible that decreased absorption or increased secretion of cholesterol is the basis for this effect, but increased catabolism of cholesterol cannot be ruled out. It has been shown that nitric oxide synthase exists in the intestinal tract of guinea-pigs and rats (Young et al., 1992; Nichols et al., 1993) and that nitric oxide can

C. H. Hill et al.

154

influence intestinal function in the rat (Tamai and Gaginella, 1993). Whether or not nitric oxide functions in quail intestines is not known. Because the quail model used in these experiments is based on cholesterol-induced atherosclerosis, the most likely explanation for the reduction in atherosclerotic lesions by nitroprusside is the reduction in serum cholesterol. However, NO has other anti-atherosclerosis influences. For instance, in tissue culture, the proliferation of smooth muscle cells is inhibited by NO donors (Garg and Hassid, 1989) as is platelet adhesion (Bath et al., 1991). It is possible that all of these factors play a role in the alleviation of cholesterol-induced atherosclerosis in the Japanese quail.

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